MXPA00011770A - Pyrrole substituted 2-indolinone protein kinase inhibitors - Google Patents

Abstract

The present invention relates to novel pyrrole substituted 2-indolinone compounds and physiologically acceptable salts and prodrugs thereof which modulate the activity of protein kinases andtherefore are expected to be useful in the prevention and treatment of protein kinase related cellular disorders such as cancer.

Description

aaa PROTEIN KINASE INHIBITORS OF 2-INDOLINONE REPLACED BY PIRROL RELATED APPLICATIONS This application is related to, and claims priority of, the United States of America Provisional Patent Application Serial Number 60 / 087,310, filed May 29, 1998, and the Provisional Patent Application 10 of the United States of America with serial number 60 / 116,106, filed on January 15, 1999, both of which are incorporated by reference as if they were fully stipulated herein.

INTRODUCTION The present invention relates in general to organic chemistry, biochemistry, pharmacology and medicine. More particularly, it refers to novel 2-indolinone compounds substituted by pyrrole, and their physiological salts Acceptable and prodrugs, which modulate the activity of protein kinases ("PKs"), and therefore, are expected to exhibit a healthy effect against disorders related to the abnormal activity of protein kinase.

BACKGROUND OF THE INVENTION The following is offered only as information from > - »-» "* - ----" *? • * » 2, and is not admitted as a prior art for the present invention. Protein kinases are enzymes that catalyze the phosphorylation of hydroxyl groups on the protein, tyrosine, serine and threonine residues. The consequences of this seemingly simple activity are staggering: cell growth, differentiation and proliferation, that is, virtually all aspects of cell life in one way or another depend on the activity of the cell kinase. protein. In addition, the abnormal activity of protein kinase has been linked to a large number of disorders, from relatively non-life threatening diseases, such as psoriasis, to extremely virulent diseases, such as glioblastoma (brain cancer). 15 Protein kinases can conveniently be broken down into two classes, tyrosine protein kinases (PTKs), and serine-threonine kinases (STKs). One of the main aspects of the activity of PTK is its involvement with the receptors of the factor of growth. The growth factor receptors are cell surface proteins. When they bind to a growth factor ligand, the growth factor receptors are converted to an active form that interacts with the proteins on the inner surface of a membrane cellular. This leads to phosphorylation on Z5 residues « 3 receptor tyrosine and other proteins, and to the formation within the cell of complexes with a variety of cytoplasmic signaling molecules that, in turn, effect numerous cellular responses, such as cell division 5 (proliferation), cell differentiation, cell growth , expression of metabolic effects towards the extracellular environment-m, etc. For a more complete discussion, see Schlessinger and Ullrich, Neuron, 9: 303-391 (1992), which is incorporated as a reference, including the drawings, as if stipulate completely in the present. Growth factor receptors with PTK activity are known as receptor tyrosine kinases ("RTKs"). They comprise a large family of transmembrane receptors with diverse biological activity. In the present, at least nineteen (19) different subfamilies of RTKs have been identified. An example of these is the subfamily designated as the "HER" RTKs, which include EGFR (epithelial growth factor receptor), HER2, HER3 and HER4. These RTKs consist of a glycosylated ligand binding domain extracellular, a transmembrane domain, and an intracellular cytoplasmic catalytic domain that can phosphorylate tyrosine residues on proteins. Another subfamily of RTK consists of the insulin receptor (IR), the growth factor receptor type insulin I (IGF-1R), and the receptor related to the receptor s. of insulin (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II form a heterotermer of two subunits and two glycosylated β-subunits entirely extracellular that cross the cell membrane and that contain the tyrosine kinase domain. A third subfamily of RTK is referred to as the platelet-derived growth factor receptor ("PDGFR") group, which includes PDGFR, PDGFRβ, CASFIR, c-kit and c-fms. These receptors consist of glycosylated extracellular domains composed of variable numbers of immunoglobulin-like cycles and an intracellular domain, wherein the tyrosine kinase domain is interrupted by unrelated amino acid sequences. Another group that, due to its similarity to the PDGFR subfamily, is sometimes assumed to be the last group, is the subfamily of the fetus liver kinase receptor ("flk"). It is believed that this group is composed of the fetal-kinase-1 kinase receptor-domain kinase insert (KDR / FLK-1), flk-lR, flk-4 and tyrosine kinase 1 type fms-1 (flt-1 ). An additional member of the tyrosine kinase growth factor receptor family is the subgroup of the fibroblast growth factor receptor ("FGF"). This group consists of four receptors, FGR1-4, and seven ligands, FGF1-7. Although they are still not well defined, it seems that the% receptors consist of a glycosylated extracellular domain containing a variable number of immunoglobulin-like cycles, and an intracellular domain in which the tyrosine kinase sequence is interrupted by regions of unrelated 5 amino acid sequences. Yet another member of the tyrosine kinase growth factor receptor family is the subgroup of the vascular endothelial growth factor receptor ("VEGF"). VEGF is a dimeric glycoprotein similar to PDGF, but has different biological functions and specificity of white cells in vivo. In particular, it is currently thought that VEGF plays an ntial role in vasculogenesis and angiogenesis. A more complete listing of the RTK subfamilies known is described in Plowman et al., DN &P, 7 (6): 334-339 (1994), which is incorporated by reference, including any drawings, as if fully stipulated herein. In addition to the RTKs, there is also a family of PTKs entirely intracellular, termed "non-receptor tyrosine kinases" or "cellular tyrosine kinases". This last designation, abbreviated as "CTK", will be used herein. The CTKs do not contain extracellular or transmembrane domains. In the present, more than 24 have been identified CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack). The Src subfamily appears to be the largest group of CTKs up to now, and includes Src, Yes, Fyn, Lyn, Lck, Bik, Hck, Fgr and Yrk. For a more detailed discussion of the CTKs, see Bolen, Oncoaene, 8: 2025-2031 (1993), which is incorporated by reference, including any drawings, as if fully stipulated herein. Serine / threonine kinases, STKs, such as CTKs, are predominantly intracellular, although there are a few receptor kinases of the STK type. STKs are the most common of cytosolic kinases; that is, the kinases that perform their function in the part of the cytoplasm different from the cytoplasmic organelles and the cytoskeleton. The cytosol is the region inside the cell where many of the cells' intermediate metabolic and biosynthetic activity occurs; for example, it is in the cytosol that the proteins are synthesized on the ribosomes. RTKs, CTKs and STKs have all been implicated in a large number of pathogenic conditions, including, in a significant way, cancer. Other pathogenic conditions that have been associated with PTKs include, without limitation, psoriasis, liver cirrhosis, diabetes, angiogenesis, restenosis, eye diseases, rheumatoid arthritis and other inflammatory disorders, immune disorders, such as autoimmune disease, cardiovascular disease, such as atherosclerosis. , and a variety of kidney disorders. > 7 With respect to cancer, two of the main advanced hypotheses to explain the excessive cell proliferation that drives tumor development are related to functions that are known to be regulated by the protein kinase. That is, it has been suggested that malignant cell growth results from a break in the mechanisms that control cell division and / or differentiation. It has been shown that the protein products of a number of proto-oncogens are involved in the transduction pathways of signals that regulate cell growth and differentiation. These proto-oncogene protein products include extracellular growth factors, PTK receptors for transmembrane growth factor (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above. 15 In view of the apparent link between cellular activities related to protein kinase and a wide variety of human disorders, it is not surprising that a great effort is being made in an attempt to identify ways to modulate the activity of protein kinase. .

Some of these have involved biomimetic approaches using large molecules in patterns on those involved in real cell processes (eg, mutant ligands (U.S. Patent Application Number 4,966,849), receptors and antibodies soluble (U.S. Patent Application Number 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 (International Publications Nos. WO 94/03427, WO 92/21660, WO 91/15495, WO 94/14808; United States of America Number 5,330,992; Mariani et al., Proc. Am. Assoc. Cancer Res., 35: 2268 (1994)). In addition to the above, attempts have been made to identify small molecules that act as protein kinase inhibitors. For example, bis-monocyclic, bicyclic and heterocyclic aryl compounds (PCT Publication Number WO 92/20642), vinylene-azaindol derivatives (PCT Publication Number WO 94/14808), and l-cyclopropyl-4-pyridylquinolones (U.S. Patent Number 5,330,992) have been described as tyrosine kinase inhibitors. Styryl compounds (U.S. Patent No. 5,217,999), pyridyl substituted by styrene compounds (U.S. Patent No. 5,302,606), quinazoline derivatives (European Patent Application Number 0 566 266 Al) , selenaindoles and selenides (PCT Publication Number WO 94/03427), tricyclic polyhydroxy compounds (PCT Publication Number WO 92/21660), and benzylphosphonic acid compounds (PCT Publication Number WO 91/15495) have been all described as PTK inhibitors useful in the treatment of cancer.

COMPENDIUM OF THE INVENTION We expect our own efforts to identify small organic molecules that modulate the activity of protein kinases and that, therefore, are useful in the treatment and prevention of disorders involving abnormal kinase activity. protein, have led us to the discovery of a family of novel 2-indolinone compounds substituted by pyrrole that exhibit protein kinase modulating activity and, therefore, are expected to have a salutary effect against disorders related to abnormal activity of the protein kinase; it is these compounds that are the subject of this invention. Therefore, the present invention relates in general to novel 2-indolinones substituted by pyrrole that modulate the activity of receptor tyrosine kinases (RTKs), non-receptor tyrosine protinase kinases (CTKs), and kinases of serine / threonine protein (STKs). In addition, the present invention relates to the preparation and use of pharmaceutical compositions of the disclosed compounds, and their physiologically acceptable salts and prodrugs, in the treatment or prevention of disorders triggered by protein kinase, such as, a example manner and not limitation, cancer, diabetes, liver cirrhosis, cardiovascular disease, such as atherosclerosis, angiogenesis, immunological disease, such as autoimmune disease, and kidney disease. The terms "2-indolinone", "indolin-2-one" and "2-oxindole" are used interchangeably herein to refer to a molecule having the chemical structure: A "pyrrole" refers to a molecule that has the chemical structure: "2-indolinone substituted by pyrrole" and "3-pyrrolidenyl-2-indolinone" are used interchangeably herein to refer to a chemical compound having the general structure shown in Formula 1. A "pharmaceutical composition" is refers to a mixture of one or more of the compounds described herein, or physiologically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism. A "prodrug" refers to an agent that becomes the parent progenitor in vivo. Prodrugs are often useful, because in some situations, they may be easier to administer than the parent drug. For example, they may be bioavailable by oral administration, while the parent drug is not. The prodrug may also have a better solubility in pharmaceutical compositions on the parent drug. An example, without limitation, of a prodrug, would be a compound of the present invention that is administered as an ester (the "prodrug"), to facilitate transmission through a cell membrane, wherein the solubility in water is detrimental to mobility, but then metabolically hydrolyzed to the carboxylic acid ,. the active entity, once inside the cell, where the solubility in water is beneficial. A further example of a prodrug could be a short polypeptide, for example, without limitation, a polypeptide of 2 to 10 amino acids, linked through a terminal amino group with a carboxyl group of a compound of this invention, wherein the polypeptide is hydrolyzes or is metabolized in vivo to release the active molecule. A "pyrrole aldehyde" refers to a molecule that has the chemical structure: As used herein, a "physiologically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism, and does not abrogate the biological activity and properties of the compound administered. An "excipient" refers to an inert substance added to a pharmaceutical composition to facilitate > 13 additionally the administration of a compound. Examples, without limitation, of the excipients, include calcium carbonate, calcium phosphate, different sugars and types of starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. 1. CHEMISTRY A. Structural General Characteristics In one aspect, the present invention relates to 2-10 indolones substituted by pyrrole which, in addition to being otherwise optionally substituted both on the pyrrole and on the 2-indolinone portion of the compound, are necessarily substituted on the pyrrole fraction with one or more hydrocarbon chains, which are themselves substituted with at least one polar group. Physiologically acceptable salts and prodrugs of the claimed compounds are also within the scope of this invention. A "hydrocarbon chain" refers to a group Alkyl, alkenyl or alkynyl, as defined herein. A "polar" group refers to a group in which the nuclei of the atoms covalently linked to each other to form the group, do not share the electrons of the covalent bonds that unite them equally; that is, the cloud of electrons is denser around one atom than another.

This results in one end of the covalent bonds being relatively negative, and the other end being relatively positive; that is, there is a negative pole and a positive pole. Examples of the polar groups include, without limitation, hydroxyl, alkoxy, carboxyl, nitro, cyano, amino, ammonium, amido, ureido, sulfonamido, sulfinyl, sulfonyl, phosphono, morpholino, piperazinyl and tetrazolo. Although they do not wish to be bound by any particular theory, the applicants this time believe that polar groups can interact electronically, for example, but without limitation, through hydrogen bonds, Van der Walls forces and / or ionic bonds. (but not the covalent link), with the amino acids in an active PTK site. These interactions can help the molecules of this invention to bind to an active site with sufficient tenacity to interfere with, or prevent the natural substrate from entering the site. Polar groups can also contribute to the selectivity of the compounds; that is, a polar group may have higher affinity for a PTK binding domain than other polar groups, such that the compound containing the first particular polar group is more potent than the compounds containing the other polar groups. Accordingly, one aspect of the present invention relates to compounds having the following chemical structure: V fifteen R is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, Hydroxyl, alkoxyl, C-carboxyl, 0-carboxyl, acetyl, C-a ido, C-thioamido, sulfonyl and trihalo-ethanesulfonyl. R2 is selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, aryl, heteroaryl and heteroalicyclic. R3, R4, R5 and R6 are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, sulfinyl, sulfonyl, S-25 sulfonamido, N-sulfonamido, trihalomethanesulfonamido, carbonyl, C-carboxyl, O-carboxyl, C-amido, N-amido, cyano, nitro, halogen, O-carbamyl, N-carbamyl, O-thiocarbamyl, N -thiocarbamyl, amino and -NR11R12. R11 and R12 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl and, in combination, a five or six membered heteroalicyclic ring. R3 and R4, R4 and R5, or R4 and R5, may be combined to form a six-membered aryl ring, a methylenedioxyl group or an ethylenedioxyl group. R7 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxyl, O-carboxyl, sulfonyl and trihalomethanesulfonyl. R8, R9 and R10 are independently selected from the group consisting of hydrogen, alkyl, tri-haloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, carbonyl, C-carboxyl, 0-carboxyl, cyano, nitro, halogen, O-carbamyl, N-carbamyl, 0-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and -NR R, in the understanding, however, that at least one of R8, R9 or R10 is a group having the formula - (alkx) Z. Alk-L is selected from the group consisting of alkyl, alkenyl or alkynyl. Z is a polar group. As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon, including straight chain and branched chain groups. Preferably, the alkyl group has from 1 to 20 carbon atoms (whenever a numerical range is mentioned herein, for example, "1 to 20", it means that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms More preferably, it is a medium-sized alkyl having 1 to 10 carbon atoms, more preferably, is a lower alkyl having 1 to 4 carbon atoms The alkyl group may be substituted or unsubstituted When substituted, the substituent groups are preferably one or more individually selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxyl , alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxyl, O-carboxyl , nitro, silyl, amino and -NR1: LR12, co n R11 and R12 as defined above. A "cycloalkyl" group refers to an all-monocyclic or condensed carbon ring (i.e., rings that share an adjacent pair of carbon atoms), wherein one or more of the rings does not have a fully pi-electron system conjugate. Examples, without limitation, of the cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, ada antano, cyclohexadiene, cycloheptane and cycloheptatriene. A cycloalkyl group can be substituted or unsubstituted. When substituted, the substituent groups are preferably one or more individually selected from the alkyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, cyano, halogen, carbonyl, thiocarbonyl, C-carboxyl, O-carboxyl, O-carbamyl, N-carbamyl, C-amido, N-amido , nitro, amino and -NR1: Lr12, with R11 and R12 as defined above. An "alkenyl" group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms, and at least one carbon-carbon double bond. An "alkynyl" group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms, and at least one carbon-carbon triple bond. An "aryl" group refers to an all-monocyclic or fused ring polycyclic carbon group (ie, rings that share adjacent pairs of carbon atoms), which has a fully conjugated pi-electron system. Examples, without limitation, of the aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group can be substituted or unsubstituted. When substituted, the substituent groups are preferably one or more selected from halogen, trihalomethyl, alkyl, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, cyano, nitro, carbonyl, thiocarbonyl, C-carboxyl, O-carboxyl , 0-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NR1: LR12, with R11 and R12 as defined herein. As used herein, a "heteroaryl" group refers to a monocyclic or fused ring (i.e., rings that share a pair of adjacent atoms), which has one or more atoms selected from the group on the ring. It consists of nitrogen, oxygen and sulfur and, in addition, it has a fully conjugated pi-electron system. Examples, without limitation, of the heteroaryl groups, are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole. The heteroaryl group may be substituted or unsubstituted. When substituted, the substituent groups are preferably one or more selected from alkyl, cycloalkyl, halogen, trihalomethyl, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, C-carboxyl , O-carboxyl, sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and -NR1: LR12, with R11 and R12 as defined above. A "heteroalicyclic" group refers to a monocyclic or fused ring group having in the ring one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a fully conjugated pi-electron system. The heteroalicyclic ring can be substituted or unsubstituted. When substituted, the substituent groups are preferably one or more selected from alkyl, cycloalkyl, halogen, trihalomethyl, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, cyano, nitro, carbonyl, thiocarbonyl, C-carboxyl, or carboxyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido, amino and -NR1: LR12, with R11 and R12 as defined above. A "hydroxyl" group refers to an -OH group. An "alkoxy" group refers to both an -O-alkyl group and an -O-cycloalkyl group, as defined herein.

An "aryloxy" group refers to both an -0-aryl group and an -O-heteroaryl group, as defined herein. A group "mercapto" refers to a group -SH. A "thioalkyl" group refers to both an S-alkyl group and an S-cycloalkyl group, as defined herein. A "thioaryl" group refers to both an S-aryl group and an S-heteroaryl group, as defined herein. A "carbonyl" group refers to a group -C (= 0) -R ", wherein R" is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (linked through an ring carbon), and heteroalicyclic (linked through a ring carbon atom), as defined herein. An "aldehyde" group refers to a carbonyl group, where R "is hydrogen A" thiocarbonyl "group refers to a group -C (= S) -R", with R "as defined herein. "C-carboxyl" group refers to a group -C (= 0) 0 -R ", with R" as defined herein An "O-carboxyl" group refers to a group -0C (= 0) R ", with R" as defined herein An "ester" group refers to a group -C (= 0) 0 -R ", with R" as defined herein, except that R "can not be hydrogen An "acetyl" group refers to a group -C (= 0) CH3. A "carboxylic acid" group refers to a C-carboxyl group, where R "is hydrogen A" halogen "group refers to fluorine, chlorine, bromine or iodine A" trihalomethyl "group refers to a -CX3 group , wherein X is a halogen group as defined herein A "trihalomethanesulfonyl" group refers to group X3CS (= 0) 2-, with X as defined above. A "cyano" group refers to a group -C = N. A "sulfinyl" group refers to a group -S (= 0) -R ", wherein, in addition to being as defined above, R" may also be a hydroxyl group. A "sulfonyl" group refers to a group -S (= 0) 2R ", wherein, in addition to being as defined above, R" may also be a hydroxyl group. A "methylenedioxyl" group refers to a group -0CH20-, wherein the two oxygen atoms are bonded to the adjacent carbon atoms. An "ethylenedioxyl" group refers to a group -0CH2CH20-, wherein the two oxygen atoms are bonded to the adjacent carbon atoms. An "S-sulfonamido" group refers to a group -S (= 0) 2NR "I-LR" L, I, with R 11 and R 1"as defined herein. An "N-sulfonamido" group refers to a group -NR1: LS (= 0) 2R12, with R11 and R12 as defined herein. An "O-carbamyl" group refers to a group -OC (= 0) NR 11 R 12, with R 11 and R 12 as defined herein. A "N-carbamyl" group refers to a group R120C (= 0) NR?: L, with R11 and R12 as defined herein. An "O-thiocarbamyl" group refers to a group -OC (= S) NR1: LR12, with R11 and R12 as defined herein. A group "N-thiocarbamyl" refers to a group R120C (= S) NR1: L-, with R11 and R12 as defined herein. An "amino" group refers to a group -NR1: LR12, wherein R11 and R12 are both hydrogen. A "C-amido" group refers to a group -C (= 0) NR11R12, with R11 and R12 as defined herein. An "N-amido" group refers to a group R12C (= 0) NR11- with R11 and R12 as defined herein. An "ammonium" group refers to a group - + NHR1: LR12, wherein R11 and R12 are independently selected from the group consisting of alkyl, cycloalkyl, aryl and heteroaryl. A "ureido" group refers to a group -NR1: LC (= 0) NR12R13, with R11 and R12 as defined herein, and R13 defined as R11 and R12. A "guanidino" group refers to a group -RNC (= N) NR12R13, with R11, R12 and R13 as defined herein. An "amidino" group refers to a group -R1: LR12NC (= N), with R11 and R12 as defined herein. A "nitro" group refers to a group -N02. A "phosphonyl" group refers to a group -0P (= 0) 20R ", with R" as defined herein. A "morpholino" group refers to a group that has the chemical structure: A "piperazinyl" group refers to a group that has the chemical structure: A "tetrazolo" group refers to a group that has the chemical structure.

B. Preferred Structural Features It is a currently preferred feature of this invention that R1 is hydrogen. It is also a currently preferred feature of this invention that R is hydrogen. In the same way, it is a currently preferred feature of this invention, that R7 is hydrogen. It is a presently preferred feature of this invention that the above three limitations exist in the same molecule; that is, that, in a compound of this invention, R1, R2 and R7 are hydrogen. It is also currently preferred that R3, R4, R5 and R6 are selected from the group consisting of hydrogen, unsubstituted lower alkyl, lower alkyl substituted with a group selected from the group consisting of hydroxyl, halogen, C-carboxyl substituted with a group selected from the group consisting of hydrogen and unsubstituted lower alkyl, amino, or -NR 11 R 12; unsubstituted lower alkyl, alkoxy, lower alkoxy substituted with one or more halogen groups, lower alkoxy substituted with a group consisting of unsubstituted aryl, or aryl substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, hydroxyl unsubstituted lower alkyl, alkoxy, halogen, amino, unsubstituted lower alkyl-S-sulfonamido, or -NR1: 1R12, unsubstituted aryl or aryl substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, lower alkyl unsubstituted alkoxy, lower alkoxy substituted with one or more halogen groups, lower alkoxy substituted with a group selected from the group consisting of unsubstituted aryl, or aryl substituted with one or more groups independently selected from the group consisting of lower alkyl unsubstituted, hydroxyl, lower alkyl-al unsubstituted coxyl, halogen, amino, unsubstituted lower alkyl-S-sulfonamido, or -NR 12 R 12, hydroxyl, amino, unsubstituted lower alkyl sulfonamido, C-carboxyl substituted with a group selected from the group consisting of hydrogen or unsubstituted lower alkyl, morpholino, -NR1: LR12, trihalomethyl, aryl, aryl substituted with one or more groups independently selected from the group consisting of hydroxyl, halogen, trihalomethyl, amino, -NR11R12, sulfonamido, C-carboxyl substituted with a group selected from of the group consisting of hydrogen or unsubstituted lower alkyl, unsubstituted lower alkyl or substituted lower alkyl with a group selected from the group consisting of hydroxyl, halogen, C-carboxyl substituted with a group selected from the group consisting of hydrogen or unsubstituted lower alkyl, amino or -NR1: LR12, unsubstituted heteroalicyclic, eteroalicyclic one or more groups independently selected from the group consisting of halogen, hydroxyl, unsubstituted lower alkyl, unsubstituted lower alkylcarbonyl, hydroxyl, unsubstituted lower alkyl-alkoxy, or alkoxy substituted with one or more halogen groups, unsubstituted aryloxy, substituted aryloxy with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, trihalomethyl, halogen, hydroxyl, amino or -NR1: 1R12, mercapto, unsubstituted lower alkylthioalkyl, unsubstituted thioaryl, thioaryl substituted with one or more selected groups from the group consisting of halogen, hydroxyl, amino or -NR1: LR12, C-carboxyl substituted with a group selected from the group consisting of hydrogen and unsubstituted lower alkyl, unsubstituted lower alkyl, O-carboxyl, lower alkyl- S-unsubstituted sulfonamido, nitrounsubstituted lower alkyl-C-amido, lower alkyl-unsubstituted N-amido, amino and -R1: LR12. In other currently preferred embodiments of this invention, R3, R4, R5 and R6 are independently selected from the group, which consists of hydrogen, halogen, unsubstituted lower alkyl, lower alkyl substituted with one or more groups selected from the group consisting of in hydroxyl, halogen, C-carboxyl substituted with a group selected from the group consisting of hydrogen or unsubstituted lower alkyl, amino or -NR1: LR12, lower alkyl-unsubstituted alkoxy, lower alkyl-alkoxy substituted with one or more halogen groups , unsubstituted aryloxy, aryloxy substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, lower alkyl substituted with one or more halogen, hydroxyl, lower alkyl-unsubstituted alkoxy, halogen, amino or -NR 11 R 12, S groups -sulfonamido, wherein R11 and R12 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl, unsubstituted aryl, aryl substituted with one or more groups independently selected from the group consisting of halogen, unsubstituted lower alkyl, lower alkyl substituted with one or more halogen groups, lower alkyl-unsubstituted alkoxy, amino or - NR 11 R 12, unsubstituted heteroaryl, heteroaryl substituted with one or more groups independently selected from the group consisting of unsubstituted lower alkyl, lower alkyl substituted with one or more halogen groups, lower alkyl-unsubstituted alkoxy, hydroxyl, halogen, amino or -NR 1: LR12, unsubstituted heteroalicyclic, heteroalicyclic substituted with one or more groups independently selected from the group consisting of halogen, hydroxyl, unsubstituted lower alkyl, lower alkyl substituted with one or more halogen groups, lower alkyl-unsubstituted alkoxy, amino, or -NR1 : 1"R12, lower alkyl-O-ca unsubstituted carboxyl, C-amido, wherein R 11 and R 12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, and unsubstituted aryl, and, N-amido, wherein R 11 and R 12 are independently selected from the group which consists of hydrogen, unsubstituted lower alkyl, and unsubstituted aryl. It is a presently preferred feature of this invention that one of R8, R9 and R10 is - (alkx) Z, while the other two are independently selected from the group consisting of hydrogen, hydroxyl, unsubstituted lower alkyl, unsubstituted lower alkenyl, unsubstituted lower alkynyl, unsubstituted lower alkyl-alkoxy, lower alkoxy substituted with one or more halogen groups, unsubstituted arylalkoxy, amino, -NR12Ri2, halogen, C-carboxyl substituted with a group selected from the group consisting of hydrogen or unsubstituted lower alkyl unsubstituted lower alkyl-O-carboxyl, unsubstituted lower alkyl-C-amido, unsubstituted lower alkyl-N-amido, acetyl, unsubstituted lower alkyl-S-sulfonamido, unsubstituted aryl or substituted aryl with a group selected from the group consisting of in halogen, hydroxyl, lower alkyl-unsubstituted alkoxy, alkoxy substituted with one or more halogen groups, C- carboxyl substituted with a group selected from the group consisting of hydrogen or unsubstituted lower alkyl, unsubstituted lower alkyl-O-carboxyl, amino, unsubstituted lower alkyl-S-sulfonamido and -NR 11 R 12. It is a presently preferred feature of this invention that R8 and R10 are selected from the groups consisting of hydrogen and unsubstituted lower alkyl. It is also a currently preferred feature of this invention that alkx is an unsubstituted lower alkyl group. In yet another currently preferred feature of this invention, Z is selected from the group consisting of hydroxyl, amino, -NR "L1R12, quaternary ammonium, C-carboxyl substituted with a group selected from the group consisting of hydrogen or unsubstituted lower alkyl, C-amido substituted with groups selected from the group consisting of hydrogen and lower alkyl unsubstituted, morpholino, piperadynyl, tetrazolo and phosphonyl A further presently preferred feature of this invention is that alk-j ^ is an unsubstituted lower alkyl group of two to four carbon atoms, and Z is a carboxylic acid. of this invention, that R9 is alk1Z In the same way, it is a presently preferred feature of this invention, that R11 and R12 are independently selected from the group comprising hydrogen, unsubstituted lower alkyl, hydroxyl, unsubstituted lower alkyl-alkoxy, unsubstituted lower alkylcarbonyl, unsubstituted lower alkyl-O-carboxyl and acetyl. Ally preferred of this invention, R1, R2, R3, R4, R5, R6 and R7 are hydrogen, R8 and R10 are methyl, and R9 is -CH2CH2C (= 0) OH. It is also a presently preferred embodiment of this invention that R1, R2, R3, R4, R5, R6, R7 and R8 is hydrogen, R10 is methyl, and R9 is -CH2CH2C (= 0) OH. In still another currently preferred embodiment of this invention, R7 is selected from the group consisting of: hydrogen, unsubstituted lower alkyl, and lower alkyl substituted with a group selected from the group consisting of unsubstituted cycloalkyl, unsubstituted aryl, and aryl substituted with a group selected from hydroxyl, unsubstituted lower alkyl-alkoxy, and halogen. It is also a currently preferred embodiment of this invention that Z is selected from the group consisting of -C (= 0) NR 11 R 14, wherein R 13 and R 14 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, alkyl lower substituted with a group selected from the group consisting of amino and -NR1: LR12, unsubstituted aryl, aryl substituted with one or more groups selected from the group consisting of halogen, hydroxyl, lower alkyl-unsubstituted alkoxy, and trihalomethyl , unsubstituted heteroaryl, unsubstituted heteroalicyclic, and, in combination, a five-membered or six-membered unsubstituted heterocyclic, and -NR1: LR12, wherein R11 and R ~ 2 are independently selected from the group consisting of unsubstituted lower alkyl, and , combined, an unsubstituted heteroalicyclic ring of five members or six members. Yet another currently preferred embodiment of this invention is that R7 is selected from the group consisting of unsubstituted lower alkyl, lower alkyl substituted with one or more groups selected from the group consisting of unsubstituted cycloalkyl, unsubstituted aryl, aryl substituted with one or more groups independently selected from the group consisting of halogen and lower alkyl-unsubstituted alkoxy, and unsubstituted lower alkylcarboxyalkyl, and Z is selected from the group consisting of unsubstituted C-carboxyl, and lower alkyl-C-carboxyl unsubstituted Finally, it is a currently preferred embodiment of this invention, that R3, R4, R5 and R6 are independently selected from the group consisting of hydrogen, halogen, unsubstituted lower alkyl, lower alkyl substituted with one or more hydroxyl groups, unsubstituted lower alkoxy , unsubstituted aryl, aryl substituted with one or more unsubstituted lower alkoxy groups, and -S (0) 2NR 11 R 12, R 5 is hydrogen, R 6 is -NR 11 R 12, and R 11 and R 12 are independently selected from the group consisting of hydrogen, lower alkyl unsubstituted, and, in combination, a five-membered or six-membered unsubstituted heteroalicyclic ring. The chemical formulas referred to herein may exhibit the phenomena of tautomerism and structural isomerism. For example, the compounds described herein can adopt an E or Z configuration around the double bond connecting the 2-indolinone moiety with the pyrrole moiety, or they can be a mixture of E and Z. This invention encompasses any structural isomeric form or tautomeric, and mixtures thereof, which possess the ability to modulate the activity of RTK, CTK and / or STK, and is not limited to any isomeric structural or tautomeric form. 2. SYNTHESIS / COMBINATION LIBRARIES A further aspect of this invention is a combination library of at least ten 3-pyrrolidinyl-2-indolinone compounds that can be formed by the reaction of oxindoles of structure 2 with aldehydes of structure 3. wherein R1 to R10 have the meanings stipulated above. As used herein, a "combination library" refers to all compounds formed by the reaction of each compound of one dimension with a compound in each of the other dimensions in a multi-dimensional array of compounds. In the context of the present invention, the array is two-dimensional, and one dimension represents all the oxindoles of the invention, and the second dimension represents all the aldehydes of the invention. Each oxindole can be reacted with each and every aldehyde, in order to form a 3-pyrrolidinyl-2-indolinone compound. All 3-pyrrolidinyl-2-indolinone compounds formed in this way are within the scope of the present invention. Also within the scope of the present invention are the smaller combination libraries formed by the reaction of some of the oxindoles with all the aldehydes, all the oxindoles with some of the aldehydes, or some of the oxindoles with some of the aldehydes. The oxindole in the above combination library is preferably selected from the group consisting of oxindole itself and substituted oxindoles, such as, without limitation, 6-bromo-oxindole, 5-hydroxyoxindole, 5-methoxyoxindole, 6-methoxyoxindole, 5-phenylenedosulphonyloxyindole, 4- [2- (2-isopropylphenoxy) -ethyl] oxindole, 4- [2- (3-isopropylphenoxy) -ethyl] oxindole, 5-fluoro-oxindole, 6-fluoro-oxindole, 7-fluoro -oxindole, 6-trifluoromethyloxindole, 5-chloro-oxindole, 6-chloro-oxindole, indole-4-carboxylic acid, 5-bromo-oxindole, 6- (N-acetamido) -oxindole, 4-methyloxindole, 5-methyloxyindole, 4-methyl-5-chloro-oxindole, 5-ethyloxyindole, 6-hydroxyoxindole, 5-acetyloxindole, oxindole-5-carboxylic acid, 5-methoxyoxindole, 6-ethoxyoxindole, 5-amino-oxindole, 6-amino-oxindole, 4 - (2-N-morpholinoethyl) oxindole, 7-azaoxindole, oxindole-4-carabamic acid t-butylester, oxindole-6-carbamic acid t-butylester, 4- (2-carboxyethyl) oxindole, 4-n-butyloxindole , 4,5-dimethoxyoxindole, 6- (methanesulfonamido) ox indole, 6- (benzamido) -oxindole, 5-ethoxyoxindole, 6-phenyloxyindole, 6- (2-methoxyphen-1-yl) oxindole, 6- (3-methoxyphen-1-yl) oxindole, 6- (4-methoxyphen) -1-yl) oxindole, 5-aminosulfonyloxyindole, 5-isopropylamino-sulphonyloxyindole, dimethylaminosulfonyloxyindole, 5- (n-morpholinosulfonyl) oxindole and 4- (2-hydroxyethyl) oxindole. The aldehyde in the above combination library is preferably selected from the group consisting, without limitation, of 3- (5-formyl-2,4-dimethyl-lH-pyrrol-3-yl) propionic acid, - (5-formyl-4-methyl-lH-pyrrol-3-yl) propionic acid, 3- (2-benzyl-5-formyl-2,4-dimethyl-lH-pyrrol-3-yl) propionic acid, 3 - (5-formyl-1-methoxycarbonyl-methyl-2,4-dimethyl-lH-pyrrol-3-yl) propionic acid, 3- (5-formyl-1,2,4-trimethyl-lH-pyrrol-3-yl) ) propionic, 3- [5-formyl-1- (3-methoxybenzyl) -2,4-dimethyl-lH-pyrrol-3-yl] propionic acid methyl ester, 3- (l-cyclohexylmethyl-5-methyl ester) -formyl-2, 4-dimethyl-lH-pyrrol-3-yl) propionic acid methyl ester of 3- [l- (2,2-dimethyl-propyl) -5-formyl-2,4-dimethyl-lH-pyrrole -3-i1] propionic, 1,3, 5-trimethy1-4- (3-morpholin-4-yl-3-oxopropyl) -lH-pyrrole-2-carbaldehyde, 3- (5-formyl-1, 2, 4-trimethyl-lH-pyrrol-3-yl) -N- (2-morpholin-4-yl) ethyl) propionamide, 3- (5-formyl-1-, 2, 4-trimethyl-lH-pyrrol-3-yl ) -Nf nylpropionamide, 1,3,5-trimethyl-4- (3-oxo-3-piperidin-1-yl-propyl) -lH-pyrrole-2-carbaldehyde, 1,3,5-trimethyl-1-4- (3-oxo) -3-pyrrolidin-1-yl-propyl) -lH-pyrrole-2-carbaldehyde, 3- (5-formyl-1,2,4-trimethyl-lH-pyrrol-3-yl) -N- (4-methoxy) phenyl) propionamide, 3- (5-formi1-1,2, 4-trimethyl-lH-pyrrol-3-yl) -N- (4-methoxy-phenyl) propionamide, N- (4-fluoro-phenyl) -3- (5-formyl-1-, 2, 4-trimethyl-lH-pyrrol-3-yl) propionamide, 3- (5-formyl-1,2,4-trimethyl) -lH-pyrrol-3-yl) -N- (4-trifluoromethyl-phenyl) propionamide, 3- [5-formyl-1- (3-methoxy-benzyl) -2,4-dimethyl-lH-pyrrole-3-acid -yl] propionic, 3- (l-cyclohexylmethyl-5-formyl-2,4-dimethyl-lH-pyrrol-3-yl) -propionic acid, 3- [1- (3-fluorobenzyl) -5- methyl ester formyl-2, 4-dimethyl-lH-pyrrol-3-yl] propionic, 3- (l-benzyl-5-formyl-2,4-dimethyl-lH-pyrrol-3-yl) propionic acid, 3-methyl ester - [l- (4-flurobenzyl) -5-formyl-2,4-dimethyl-lH-pyrrol-3-yl] propionic acid, 3- [1- (4-fluorobenzyl) -5-formyl-2, 4- dimethyl-lH-pyrrol-3-yl] propionic acid, 3- [l- (3-fluorobenzyl) -5-formyl-2,4-dimethyl-lH-pyrrol-3-yl] propionic acid, 3, 5-dimethyl- 4- (3-morpholin-4-yl) propyl) -lH-pyrrole-2-carbaldehyde, 4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrole-2-carbaldehyde, 5-formyl-2 acid , 4-dimethyl-lH-pyrrole-3-carboxylic acid, 3,5-dimethyl-4- (4-methyl-piperazin- 1-carbonyl) -1H-pyrrole-2-carbaldehyde, 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-dimethylaminoethyl) amide. Another aspect of this invention provides a method for the synthesis of a 3-pyrrolidinyl-2-indolinone of Formula 1, which comprises reacting an oxindole of Formula 2 with an aldehyde of Formula 3 in a solvent, preferably in a the presence of a base. Examples of the oxindoles of Formula 2 which can be reacted with an aldehyde of Formula 3 to give the 3-pyrrolidinyl-2-indolinones of Formula 1, are oxindole itself, and substituted oxindoles, such as, without limitation , 6-bromo-oxindole, 5-hydroxyoxindole, 5-methoxyoxindole, 6-methoxyoxindole, 5-phenylaminosulfonyloxyindole, 4- [2- (2-isopro-phenephenoxy) -ethyl] oxindole, 4- [2- (3-isopropylphenoxy) ethyl] oxindole, 4- [2- (4-isopropylphenoxy) ethyl] oxindole, 5-fluoro-oxindole, 6-fluoro-oxindole, 7-fluoro-oxindole, 6-trifluoromethyloxindole, 5-chloro-oxindole, 6-chloro oxindole, indole-4-carboxylic acid, 5-bromo-oxindole, 6- (N-acetamido) -oxindole, 4-methyloxindole, 5-methyloxindole, 4-methyl-5-chloro-oxindole, 5-ethyl oxindole, 6-hydroxyoxindole , 5-acetyloxyindole, oxindole-5-carboxylic acid, 5-methoxyoxindole, 6-methoxyoxindole, 5-amino-oxindole, 6-amino-oxindole, 4- (2-N-morpholinyl) oxindole, 7-azaoxindole, t-butylester of oxindole 4-carbamic acid, oxindole-5-carbamide t-butylester ico, 4- (2-carboxyethyl) oxindole, 4-n-butyloxyindole, 4,5-dimethoxyoxindole, 6- (methanesulfonamido) oxindole, 6- (benzamido) oxindole, 5-ethoxyoxindole, 6-phenyloxyindole, 6- (2- methoxyphen-1-yl) oxindole, 6- (3-methoxyphenyl-1-yl) oxindole, 6- (4-methoxyphenyl-1-yl) oxindole, 5-aminosulfonyloxyindole, 5-isopropylaminosulfonyloxyindole, dimethylaminosulfonyloxyindole, 5- (N-morpholine sulfonyl) ) oxindole, and 4- (2-hydroxyethyl) oxindole. Examples of the aldehydes of structure 3 that can be reacted with the oxindoles of structure 2 are, without limitation, 3- (5-formyl-2,4-dimethyl-lH-pyrrol-3-yl) propionic acid, 3- (5-formyl-4-methyl-lH-pyrrol-3-yl) propionic acid, 3- (l-benzyl-5-formyl-2,4-dimethyl-lH-pyrrol-3-yl) propionic acid, 3- (5-formyl-1-methoxycarbonylmethyl-2,4-dimethyl-lH-pyrrol-3-yl) propionic acid, 3- (5-formyl-1,2,4-trimethyl-1H-pyrrol- 3-yl) propionic acid, 3- [5-formyl-1- (3-methoxybenzyl) -2,4-di: methyl-lH-pyrrol-3-yl] propionic acid methyl ester, 3- [l-cyclohexylmethyl] methyl ester -5-formyl-2,4-dimethyl-lH-pyrrol-3-yl] propionic acid methyl ester 3- [l- (2, 2-dimethylpropyl) -5-formyl-2,4-dimethyl-lH-pyrrole -3-i1] propionic, 1,3, 5-trimethy1-4- (3-morpholin-4-yl-3-oxo-propyl) -lH-pyrrole-2-carbaldehyde, 3- (5-formyl-1, 2, 4-trimethyl-lH-pyrrol-3-yl) -N- (2-morpholin-4-yl-ethyl) ropionamide, 3- (5-formyl-1-, 2, 4-trimethyl-lH-pyrrole-3 -il) -N-fen ilpropionamide, 1,3, 5-trimethyl-4- (3-oxo-3-piperidin-1-yl-propyl) -lH-pyrrole-2-carbaldehyde, 1,3,5-trimethyl-1-4- (3-oxo) -3-pyrrolidin-1-yl-propyl) -lH-pyrrole-2-carbaldehyde, 3- (5-formyl-1,2,4-trimethyl-1H-pyrrol-3-yl) -N- (4-methoxyphenyl) propionamide, 3- (5-formyl-1,2,4-trimethyl-lH-pyrrol-3-yl) -N- (4-methoxy-nyl) -propionamide, N- (4-fluorophenyl) -3- (5-formyl- 1, 2, 4-trimethyl-lH-pyrrol-3-yl) propionamide, 3- (5-formyl-1,2,4-trimethyl-lH-pyrrol-3-yl) -N- (4-trifluoromethylphenyl) propionamide , 3- [5-formyl-1- (3-methoxybenzyl) -2,4-dimethyl-lH-pyrrol-3-yl] propionic acid, 3- (l-cyclohexylmethyl-5-formyl-2,4-dimethyl acid -lH-pyrrol-3-yl) propionic acid, 3- [l- (3-fluorobenzyl) -5-formyl-2,4-dimethyl-lH-pyrrol-3-yl] propionic acid methyl ester, 3- (l- -benzyl-5-formyl-2,4-dimethyl-lH-pyrrol--yl) propionic acid, 3- [1- (4-fluorobenzyl) -5-formyl-2,4-dimethyl-lH-pyrrole-3-methyl ester; 3-yl] propionic acid, 3- [1- (4-fluorobenzyl) -5-formyl-2,4-dimethyl-lH-pyrrol-3-yl] propionic acid, 3- [l- (3-fluorobenzyl) - 5-formyl-2, 4-dimethyl-lH-pyrrol-3-yl] propionic, 3,5-dimethyl-4- (3-morpholin-4-yl-propyl) -lH-pyrrole-2-carbaldehyde, 4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrole-2-carbaldehyde, 5-formyl- 2,4-dimethyl-lH-pyrrole-3-carboxylic acid, 3,5-dimethyl-4- (4-methylpiperazine-1-carbonyl) -1H-pyrrole-2-carbaldehyde, 5- (2-dimethylaminoethyl) -amide formyl-2, 4-dimethyl-lH-pyrrole-3-carboxylic acid. The reaction can be carried out in the presence of a base. The base can be an organic or inorganic base. If an organic base is used, it is preferably a nitrogen base. Examples of organic nitrogen bases include, but are not limited to, di-isopropylane, trimethylamine, triethylamine, aniline, pyridine, 1,8-diazabicyclo [5.4. l] undec-7-ene, pyrrolidine and piperidine. Examples of the inorganic bases are, without limitation, ammonia, hydroxides, phosphates, carbonates, bicarbonates, bisulfates and alkali metal or alkaline earth metal amides. The alkali metals include lithium, sodium and potassium, while alkaline earth metals include calcium, magnesium and barium. In a presently preferred embodiment of this invention, when the solvent is a protic solvent, such as water or alcohol, the base is an inorganic base of alkali metal or alkaline earth metal, preferably an alkali metal or alkaline earth metal hydroxide. It will be clear to those skilled in the art, based on the known general principles of organic synthesis, and on the disclosures herein, which basis would be most appropriate for the contemplated reaction. The solvent in which the reaction is carried out may be a protic or aprotic solvent, preferably it is a protic solvent. A "protic solvent" is a solvent that has hydrogen atoms covalently bonded to the oxygen or nitrogen atoms, which makes the hydrogen atoms appreciably acidic, and therefore able to be "shared" with a solute through of the hydrogen bond. Examples of the protic solvents include, without limitation, water and alcohols. An "aprotic solvent" can be polar or non-polar, but in any case, it does not contain acidic hydrogens, and therefore, it is not capable of hydrogen bonding with solutes. Examples, without limitation, of the non-polar aprotic solvents are pentane, hexane, benzene, toluene, methylene chloride and carbon tetrachloride. Examples of the polar aprotic solvents are chloroform, tetrahydrofuran, dimethyl sulfoxide and dimethylformamide. In a presently preferred embodiment of this invention, the solvent is a protic solvent, preferably water or an alcohol, such as ethanol. The reaction is carried out at temperatures higher than room temperature. The temperature is generally from about 30 ° C to about 150 ° Cf preferably from about 80 ° C to about 100 ° C, more preferably from about 75 ° C to about 85 ° C, which is about the boiling point of the ethanol . "Approximately" means that the temperature range of preference is within 10 degrees Celsius of the indicated temperature, more preferably within 5 degrees Celsius of the indicated temperature and, more preferably, within 2 degrees Celsius of the indicated temperature. Accordingly, for example, "about 75 ° C" means 75 ° C + 10 ° C, preferably 75 ° C ± 5 ° C, and more preferably 75 ° C + 2 ° C. 3. BIOCHEMISTRY / PHARMACOTERAPY Another aspect of this invention relates to a method for modulating the catalytic activity of a protein kinase by contacting a protein kinase with a compound of this invention or a physiologically acceptable salt or prodrug thereof. As used herein, "protein kinase" refers to receptor tyrosine protein kinase (RTKs), non-receptor or "cellular" tyrosine kinase (CTKs), and serine-threonine kinases (STKs). The term "method" refers to the manners, means, techniques and procedures for performing a given task, including, but not limited to, the manners, means, techniques and procedures known to, or readily developed from, ways, means, techniques and procedures known to practitioners of chemical, pharmaceutical, biological, biochemical and medical techniques. As used herein, the term "modulation" or "modular" refers to the alteration of the catalytic activity of the RTKSs, CTKs and STKs. In particular, modulate refers to the activation of the catalytic activity of the RTKs, CTKs and STKs, preferably to the activation or inhibition of the catalytic activity of the RTKs, CTKs and STKs, depending on the concentration of the compound or the salt to which the RTK, CTK or STK is exposed, or more preferably, to the inhibition of the catalytic activity of the RTKs, CTKs and STKs. The term "catalytic activity", as used herein, refers to the rate of tyrosine phosphorylation under the influence, directly or indirectly, of the RTKs and / or CTKs, or the phosphorylation of serine and threonine under the influence , direct or indirect, of the STKs. The term "contacting", as used herein, refers to putting a compound of this invention and a white protein kinase together, such that the compound can affect the catalytic activity of the protein kinase, since either directly, that is, by its interaction with the kinase itself, or indirectly, that is, by its interaction with another molecule on which the catalytic activity of the kinase depends. This "contact" can be carried out "in vitro", that is, in a test tube, a Petri dish or the like. In a test tube, the contact may involve only one compound and one protein kinase of interest, or it may involve whole cells. The cells can also be maintained or cultured in cell culture dishes, and can be contacted with a compound in that environment. In this context, the ability of a particular compound to affect a protein kinase-related disorder, i.e., the IC50 of the compound, defined below, can be determined before using the compounds in vivo with more complex living organisms. For cells outside the organism, there are multiple methods, and are well known to those skilled in the art, to put protein kinases in contact with the compounds, including, but not limited to, direct cell microinjection, and numerous carrier techniques. transmembrane A further aspect of this invention is that modulation of the catalytic activity of protein kinases can be performed using a compound of this invention in vitro or in vivo. "in vitro" refers to procedures performed in an artificial environment, such as, for example, without limitation, in a test tube or culture medium. As used herein, "in vivo" refers to procedures performed within a living organism, such as, without limitation, a mouse, rat or rabbit. A still further aspect of this invention is that the protein kinase whose catalytic activity is being modulated by a compound of this invention is selected from the group consisting of receptor protein tyrosine kinases, cellular tyrosine kinases, and kinases. of serine-threonine. It is an aspect of this invention that the receptor protein kinase, whose catalytic activity is modulated by a compound of this invention, is selected from the group consisting of EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRQ !, PDGFRβ, CSFIR, C-Kit, C -fms, Flk-IR, Flk4, KDR / Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R. In addition, it is an aspect of this invention that the cellular tyrosine kinase, whose catalytic activity is modulated by a compound of this invention, is selected from the group consisting of Src, Fr, Btk, Csk, Abl, ZAP70, Fes / Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Bik, Hck, Fgr and Yrk. Another aspect of this invention is that the serine-threonine protein kinase, whose catalytic activity is modulated by a compound of this invention, is selected from the group consisting of CDK2 and Raf, a pharmaceutical composition of a compound of this invention with A pharmaceutically acceptable carrier is still another aspect of this invention. This pharmaceutical composition may also contain excipients. A method for the treatment or prevention of a disorder related to protein kinase in an organism, which comprises administering a therapeutically effective amount of a compound, salt or prodrug that is a 3-pyrrolidenyl-2-indolinone of the present invention to the organism , is another aspect of this invention. As used herein, "protein kinase-related disorder", "protein kinase-driven disorder" and "abnormal protein kinase activity" all refer to a condition characterized by an inappropriate protein kinase catalytic activity, that is, low, or more commonly high, wherein the particular protein kinase can be an RTK, a CTK or an STK. Inappropriate catalytic activity can occur as the result of either: (1) the expression of protein kinase in cells that do not normally express protein kinases, (2) increased expression of protein kinase leading to proliferation, differentiation and / or or undesired cell growth, or (3) decreased expression of protein kinase leading to undesired reduction in cell proliferation, differentiation and / or growths. An over-activity of a protein kinase refers to the amplification of the gene that encodes a particular protein kinase, or to the production of a protein kinase activity level that can be correlated with a disorder in proliferation, differentiation and / or cell growth (ie, as the protein kinase level increases, the severity of one or more of the symptoms of the cell disorder increases). The sub-activity, of course, is the inverse, where the severity of one or more symptoms of a cellular disorder increases as the activity level of the protein kinase decreases. As used in the present, the terms "prevent", "preventing" and "prevention" refer to a method to prevent an organism from acquiring a disorder related to protein kinase in the first place. As used herein, the terms "treat", "treating" and "treatment" refer to a method for alleviating or abrogating a cellular kinase-mediated protein disorder, and / or their combined symptoms. With respect particularly to cancer, these terms simply mean that the life expectancy of an individual affected with a cancer will be increased, or that one or more of the symptoms of the disease will be reduced. The term "organism" refers to any living entity comprised of at least one cell. A living organism can be as simple as, say, a single eukaryotic cell, or as a complex like a mammal, including a human being. The term "therapeutically effective amount," as used herein, refers to the amount of the compound being administered that will alleviate to some degree one or more of the symptoms of the disorder being treated. With reference to the treatment of cancer, a therapeutically effective amount refers to the amount that has the effect of: (1) reducing the size of the tumor, (2) inhibiting (ie, slowing to some degree, preferably stopping) tumor metastasis, (3) inhibit to some degree (ie, slow down to some degree, preferably stop) tumor growth, and / or, (4) alleviate to some degree (or, preferably, eliminate) one or more symptoms associated with cancer. It is an aspect of this invention that the above-referenced protein kinase-related disorder is selected from the group consisting of a receptor tyrosine protein kinase-related disorder, a tyrosine kinase cell disorder, and a kinase-related disorder. of serine-threonine.

In yet another aspect of this invention, the above-referenced protein kinase-related disorder is selected from the group consisting of an EGFR-related disorder, a PDGFR-related disorder, an IGFR-related disorder and a flk-related disorder. . The above-referenced protein kinase-related disorder is a cancer selected from the group consisting of squamous cell carcinoma, sarcomas, such as Kaposi's sarcoma, astrocyte a, glioblastoma, lung cancer, bladder cancer, colorectal cancer, Gastrointestinal cancer, cancer of the head and neck, melanoma, ovarian cancer, prostate cancer, breast cancer, small cell lung cancer and glioma in a further aspect of this invention. The above-referenced protein kinase-related disorder is selected from the group consisting of diabetes, a hyperproliferation disorder, von Hippel-Lindau disease, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, an inflammatory disorder and angiogenesis in yet another aspect of this invention. Additional disorders that can be treated or prevented using the compounds of this invention are immunological disorders, such as autoimmune disease (AIDS) and cardiovascular disorders, such as atherosclerosis. It is an aspect of this invention that a chemical compound that modulates the catalytic activity of a protein kinase can be identified by contacting the cells expressing this protein kinase with a compound, salt or prodrug that is a 3-pyrrolidylidene. 2-indolinone of the present invention, and then monitor these cells to determine an effect. "Monitoring" means observing or detecting the effect of contacting a compound with a cell that expresses a particular protein kinase. The observed or detected effect may be a change in the cellular phenotype, in the catalytic activity of a protein kinase, or a change in the interaction of a protein kinase with a natural binding component. The techniques to observe or detect these effects are well known in the material. The aforementioned effect is selected from a change or an absence of change in a cellular phenotype, a change or an absence of change in the catalytic activity of the protein kinase, or a change or an absence of change in the interaction of the protein kinase with a natural binding component in a final aspect of this invention. A "cell phenotype" refers to the external appearance of a cell or tissue, or to the biological function of the cell or tissue. Examples, without limitation, of a cellular phenotype are cell size, cell growth, cell proliferation, cell differentiation, cell survival, apoptosis, and nutrient recovery and use. These phenotypic characteristics can be measured by techniques well known in the art. A "natural binding component" refers to a polypeptide that binds to a particular protein kinase in a cell. The natural binding components may have a role in the propagation of a signal in a signal transduction process mediated by protein kinase. A change in the interaction of the natural binding component with the protein kinase may be manifested as a higher or lower concentration of the protein kinase complex / natural binding component, and as a result, an observable change in the capacity of the protein kinase. to mediate signal transduction. It is also an aspect of this invention that a compound described herein, or its salt or prodrug, could be combined with other chemotherapeutic agents for the treatment of the diseases and disorders mentioned above. For example, a compound, salt or prodrug of this invention could be combined with alkylating agents, such as fluorouracil (5-FU) alone or in an additional combination with leucovorin.; or other alkylatagents, such as, without limitation, other pyrimidine analogs, such as UFT, capecitabine, gemcitabine and cytarabine, alkyl sulfonates, for example, bisulfan- (used in the treatment of chronic granulocytic leukemia), improsulphan and piposulfan; aziridines, for example, benzodepa, carbocuone, meturedepa and uredepa; ethylene imines and methylmelamines, for example, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; and nitrogen mustards, for example, chlorambucil (used in the treatment of chronic lymphocytic leukemia, primary macroglobulinemia, and non-Hodgkin's lymphoma), cyclophosphamide (used in the treatment of Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer , ovarian cancer, lung cancer, Wilm's tumor and rhabdomyosarcoma), estramustine, ifosfamide, novembriquine, prednimustine and uracil mustard (used in the treatment of primary thrombocytosis), non-Hodgkin's lymphoma, Hodgkin's disease and cancer. ovary); and triazines, for example, dacarbazine (used in the treatment of soft tissue sarcoma). In the same way, one would expect that a compound, salt or prodromate of this invention have a beneficial effect in combination with other anti-metabolite anti-metabolism agents, such as, without limitation, folic acid analogs, for example, methotrexate (used in the treatment of water lymphocytic leukemia, choriocarcinoma, mycosis fungiodes, breast cancer, cancer of the head and neck and osteogenic sarcoma) and pteropterin; and purine analogs, such as mercaptopurine and thioguanine, which find use in the treatment of acute granulocytic, acute lymphocytic and chronic granulocytic leukemias. A compound, salt or prodrug of this invention could also be expected to be effective in combination with chemotherapeutic agents based on natural products, such as, without limitation, vinca alkaloids, for example, vinblastine (used in the treatment of breast cancer and testicular), vincristine and vindesine; epipodophyllotoxins, for example, etoposide and teniposide, both of which are useful in the treatment of testicular cancer and Kaposi's sarcoma; antibiotic chemotherapeutic agents, for example, daunorubicin, doxorubicin, epirubicin, mitomycin (used for the treatment of cancer of the stomach, cervix, colon, breast, bladder and pancreatic), dactinomycin, temozolomide, plica icine, bleomycin (used in the treatment of cancer of the skin, esophagus and genitourinary tract); and enzymatic chemotherapeutic agents, such as L-asparaginase. In addition to the forego one would expect that a compound, salt or prodrug of this invention have a beneficial effect used in combination with the platinum coordination complexes (cisplatin, etc.); substituted ureas, such as hydroxyurea; methylhydrazine derivatives, for example, procarbazine; adrenocortical suppressors, for example, mitotane, aminoglutethimide, and hormones and hormone antagonists, such as adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate); Estrogens (For example, diethylstilbesterol); antiestrogens, such as tamoxifen; androgens, for example, testosterone propionate; and aromatase inhibitors (such as anastrozole). Finally, one would expect that the combination of a compound of this invention to be particularly effective in combination with mitoxantrone or paclitaxel for the treatment of solid tumor cancers or leukemias, such as, without limitation, acute myelogenous (non-lymphocytic) myelogenous leukemia. A presently preferred compound of this invention, which could be expected to have a beneficial effect in combination with one or more of the above chemotherapeutic agents, is 3- [2, 4-dimethyl-5- (2-oxo-1, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrole-3-i1] ropionic acid.

DETAILED DESCRIPTION OF THE INVENTION 1. BRIEF DESCRIPTION OF THE TABLES Table 1 shows the chemical structures and biological activity of some exemplary compounds of this invention. The numbers of compounds correspond to the number of examples in the Examples section. That is, the synthesis of Compound 1 in Table 1 is described in Example 1. The bioassays used are described in detail below. The results are reported in terms of IC50, the micromolar concentration (μM) of the compound being tested that causes a 50 percent change in the activity of the white PTK, compared to the activity of the PTK in a control that does not no test compound has been added. In a specific manner, the results shown indicate the concentration of a test compound necessary to cause a 50 percent reduction in white PTK activity. The compounds presented in Table 1 are exemplary only, and should not be construed to limit the scope of this invention in any way.

TABLE 1 TABLE 2 Table 2 shows the chemical structures of some additional compounds of this invention. As in Table 1, the numbers of compounds correspond to the numbers of Examples. The general description of the above bioassays also applies to the bioassays shown in Table 2. 2. INDICATIONS / WHITE DISEASES Protein kinases, whose catalytic activity is modulated by the compounds of this invention, include tyrosine protein kinases, of which there are two types, receptor tyrosine kinases (RTKs) and cellular tyrosine kinases (CTKs), and the serine-threonine kinase (STKs). Signal transduction mediated by RTK is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of intrinsic tyrosine protein kinase activity, and phosphorylation. In this way, binding sites for intracellular signal transduction molecules are created, and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (eg, cell division, metabolic effects on the micro -Extracellular environment, etc.). See, Schlessinger and Ullrich, 1992, Neuron 9: 303-391. It has been shown that the tyrosine phosphorylation sites on the growth factor receptors function as high affinity binding sites for the SH2 domains (src homology) of the signaling molecules. (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). Several intracellular substrate proteins have been identified that are associated with RTKs. They can be divided into two main groups: (1) substrates that have a catalytic domain, and (2) substrates that lack that domain, but that serve as adapters and are associated with catalytically active molecules. Songyang et al., 1993, Cell 72: 767-778. The specificity of the interactions between the receptors and the SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. The differences in the binding affinities between the SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on the particular receptors are consistent with the differences observed in their substrate phosphorylation profiles. Songyang et al., 1993, Cell 72: 767-778. These observations suggest that the function of each RTK is determined not only by its pattern of expression and ligand availability, but also by the arrangement of the downstream signal transduction pathways that are activated by a particular receptor. Therefore, phosphorylation provides an important regulatory step that determines the selectivity of the signaling pathways recruited by specific growth factor receptors, as well as by the receptors of differentiation factors. STKs, which are primarily cytosolic, affect the internal biochemistry of the cell, often as a down-line response to a PTK event. STKs have been implicated in the signaling process that initiates DNA synthesis, and the subsequent mitosis, leading to cell proliferation. Accordingly, signal transduction of protein kinases results, among other responses, in cell proliferation, differentiation, growth and metabolism. Abnormal cell proliferation can result in a broad set of disorders and diseases, including the development of neoplasia, such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders, such as leukemia, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy, and other disorders related to angiogenesis and / or uncontrolled vasculogenesis. A precise understanding of the mechanism by which the compounds of this invention inhibit protein kinases for the purpose of practicing the present invention is not required. However, although we do not intend here to bind ourselves to any particular mechanism or theory, it is believed that the compounds interact with the amino acids in the catalytic region of the protein kinases. Protein kinases usually have a bi-lobed structure, where ATP seems to attach to the cleft between the two lobes in a region where amino acids are conserved between protein kinases. It is believed that inhibitors of protein kinases are linked by non-covalent interactions, such as hydrogen bonding, van der Waals forces, and ionic interactions, in the same general region where the aforementioned ATP binds with protein kinases. . More specifically, it is thought that the 2-indolinone component of the compounds of this invention is fixed in the general space normally occupied by the adenine ring of ATP. The specificity of a particular molecule for a particular protein kinase can then be presented as the result of additional interactions between the different substituents on the 2-indolinone core and the specific amino acid domains for the particular protein kinases. Accordingly, different indolinone substituents can contribute to preferential binding with particular protein kinases. The ability to select the active compounds at different binding sites of ATP (or other nucleotide), makes the compounds of this invention useful for directing any protein to said site. The compounds disclosed herein, therefore, may have utility as in vitro assays for these proteins, as well as exhibit therapeutic effects in vivo through their interaction with these proteins. In another aspect, the protein kinase, whose catalytic activity is modulated by contact with a compound of this invention, is a tyrosine protein kinase, more particularly a receptor tyrosine protein kinase. Among the receptor tyrosine protein kinases whose catalytic activity can be modulated with a compound of this invention, or a salt thereof, are, without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRa, PDGFRβ , CSFIR, C-Kit, C-fms, Flk-IR, Flk4, KDR / Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R. The tyrosinei protein kinase whose catalytic activity is modulated by contact with a compound of this invention, or a salt or prodrug thereof, can also be a cellular tyrosine protein (CTK) or non-receptor protein kinase. Therefore, the catalytic activity of the CTKs, such as, without limitation, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Bik, Hck, Fgr and Yrk, can be modulated by contact with a compound or salt of this invention. Still another group of protein kinases to which their catalytic activity can be modulated by contact with a compound of this invention, are the serine-threonine protein kinases, such as, without limitation, CDK2 and Raf. In another aspect, this invention relates to a method for the treatment or prevention of a disorder related to protein kinase, by administering a therapeutically effective amount of a compound of this invention, or a salt or a prodrug thereof, to an organism. It is also an aspect of this invention that a pharmaceutical composition containing a compound of this invention, or a salt or prodrug thereof, is administered to an organism, for the purpose of preventing or treating a disorder related to protein kinase. Accordingly, this invention relates to compounds that modulate the protein kinase signal transduction, affecting the enzymatic activity of the RTKs, CTKs and / or STKs, thereby interfering with the signals transduced by these proteins. More particularly, the present invention relates to compounds that modulate the signal transduction pathways mediated by RTK, CTK and / or STK, as a therapeutic approach to cure many kinds of solid tumors, including, but not limited to, carcinomas, sarcomas, including Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma, and myoblast a.

The treatment or prevention of non-solid tumor cancers, such as leukemia, is also contemplated by this invention. Indications may include, but are not limited to, brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreatic cancers, colon cancers, blood cancers, lung cancers and bone cancers. Additional examples, without limitation, of the types of disorders related to an inappropriate activity of the protein kinase wherein the compounds described herein may be useful for preventing, treating and studying, are cell proliferative disorders, fibrotic disorders and the metabolic disorders. Cell proliferative disorders, which can be prevented, treated or further studied by the present invention, include cancer, proliferative disorders of blood vessels, and proliferative disorders of mesangial cells. Proliferative disorders of blood vessels refer to disorders related to abnormal vasculogenesis (blood vessel formation) and angiogenesis (extension of blood vessels). Although vasculogenesis and angiogenesis play important roles in a variety of normal physiological processes, such as embryonic development, corpus luteum formation, wound healing, and organ regeneration, they also have a pivotal role in the development of cancer. , where they result in the formation of new capillaries needed to maintain a live tumor. Other examples of disorders of blood vessel proliferation include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and eye diseases, such as diabetic retinopathy, where the new capillaries in the retina invade the vitreous humor, bleed and cause blindness. Two structurally related RTKs have been identified that bind VEGF with a high affinity: the tyrosine receptor type fms 1 (fit-1) (Shibuya et al., 1990, Oncogene, 5: 519-524; De Vries et al., 1992; Science, 255: 989-991) and the KDR / FLK-1 receptor, also known as VEGF-R2. It has been reported that vascular endothelial growth factor (VEGF) is a specific mitogen of endothelial cells, with activity promoting endothelial cell growth in vitro. Ferrara and Henzel, 1989, Biochein. Biophys. Res. Comm. , 161: 851-858; Vaisman et al., 1990, J. Biol. Chem., 265: 19461-19566. The information stipulated in the applications of the United States of America with serial numbers 08 / 193,829, 08 / 038,596 and 07 / 975,750 strongly suggests that VEGF is not only responsible for the proliferation of endothelial cells, but also is the regulator Primary of normal and pathological angiogenesis. See in general, Klagsburn and Soker, 1993, Current Biolosy. 3 (10) 699-702; Houck et al., 1992, J. Biol. Chem., 267: 26031-26037. Normal vasculogenesis and angiogenesis have important roles in a variety of physiological processes, such as embryogenic development, wound healing, organ regeneration and female reproductive processes, such as the development of follicles in the corpus luteum during ovulation , and placental growth after pregnancy. Folkman and Shing, 1992, J_. Biological Chem .. 267 (16): 10931-34. Uncontrolled vasculogenesis and / or angiogenesis has been associated with diseases, such as diabetes, as well as malignant solid tumors that rely on vascularization for growth. Klagsburn and Soker, 1993, Current Bioloqy. 3 (10): 699-702; Folkham, 1991, J. Nati. Cancer Inst., 82: 4-6; Wiedner et al., 1991, New Engl. J. Med .. 324: 1-5. The role of VEGF in the proliferation and migration of endothelial cells during angiogenesis and vasculogenesis indicates an important role for the KDR / FLK-1 receptor in these processes. Diseases such as diabetes mellitus (Folkman, 1998, in Xlth Conqress of Thrombosis and Haemostasis (Verstraeta, et al., Editors), pages 583-596, Leuven University Press, Leuven), and arthritis, as well as malignant tumor growth, can result from uncontrolled angiogenesis. See, for example, Folkman, 1971, N. Enal. J. Med., 285: 1182-1186. The receptors to which VEGF specifically binds are an important and powerful therapeutic target for the regulation and modulation of vasculogenesis and / or angiogenesis, and a variety of severe diseases involving abnormal cell growth caused by these processes. Plowan et al., 1994, DN & amp;; P, 7 (6): 334-339. More specifically, the highly specific role of the KDR / FLK-1 receptor in neurovascularization makes it a target of choice for therapeutic approaches for the treatment of cancer and other diseases that involve the uncontrolled formation of blood vessels. . Accordingly, one aspect of the present invention relates to compounds capable of regulating and / or modulating the tyrosine kinase signal transduction, including the signal transduction of the KDR / FLK-1 receptor, in order to inhibit or promote angiogenesis and / or vasculogenesis, ie, compounds that inhibit, prevent, or interfere with the signal transduced by KDR / FLK-1 when activated by ligands, such as VEGF. Although it is believed that the compounds of the present invention act on a receptor or other component along the signal transduction pathway of tyrosine kinase, they can also act directly on tumor cells resulting from uncontrolled angiogenesis. Although the nomenclature of the human and murine counterparts of the generic "flk-I" receptor differ, in many respects they are interchangeable. The murine receptor, Flk-1, and its human counterpart, KDR, share a sequence homology of 93.4 percent within the intracellular domain. In the same manner, murine FLK-I binds to human VEGF with the same affinity as mouse VEGF, and according to the foregoing, is activated by the ligand derived from any species. Millauer et al., 1993, Cell, 72: 835-846; Quinn et al., 1993, Proc. Nati Acad. Sci. USA, 90: 7533-7537. FLK-1 is also associated with, and subsequently phosphorylates with human tyrosine RTK substrates (eg, PLC-? Or p85) when co-expressed in 293 cells (human embryonic kidney fibroblasts). The models that rely on the FLK-1 receiver, therefore, are directly applicable to the understanding of the KDR receiver. For example, the use of the murine FLK-1 receptor in methods that identify compounds that regulate the murine signal transduction pathway is directly applicable to the identification of compounds that can be used to regulate the signal transduction pathway. human, that is, that it 'regulates the activity related to the KDR receptor. Therefore, chemical compounds identified as KDR / FLK-1 inhibitors in vitro can be confirmed in suitable models in vivo. It has been shown that both the live mouse model and the rat animal model are of excellent value for the examination of the clinical potential of the agents acting on the signal transduction pathway induced by KDR / FLK-1. Accordingly, in one aspect, this invention relates to compounds that regulate, modulate and / or inhibit vasculogenesis and / or angiogenesis, affecting the enzymatic activity of the KDR / FLK-1 receptor, and interfering with the signal transduced by KDR. / FLK1. In another aspect, the present invention relates to compounds that regulate, modulate and / or inhibit the signal transduction pathway mediated by KDR / FLK-1 as a therapeutic approach for the treatment of many classes of solid tumors, including, but not limited to, glioblastoma, melanoma and Kaposi's sarcoma, and carcinoma of the ovary, lung, breast, prostate, pancreatic, colon and epidermoid. In addition, the data suggest that the administration of compounds that inhibit the signal transduction pathway mediated by KDR / Flk-1 can also be used in the treatment of hemangioma, restenosis and diabetic retinopathy. A further aspect of this invention relates to the inhibition of vasculogenesis and angiogenesis by other receptor-mediated pathways, including the pathway comprising the flt-1 receptor.

The signal transduction mediated by receptor tyrosine kinase is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of intrinsic tyrosine protein kinase activity, and autophosphorylation. In this way, binding sites are created for the intracellular signal transduction molecules, which leads to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response, for example, effects of cell division and metabolism in the extracellular micro-environment. See, Schlessinger and Ullrich, 1992, Neuron, 9: 1-20. The close homology of the intracellular regions of KDR / FLK-1 with that of the PDGF-β receptor (50.3 percent homology), and / or the related flt-1 receptor, indicates the induction of overlapping signal transduction pathways . For example, for the PDGF-β receptor, it has been shown that members of the src family (Twamley et al., 1993, Proc.Nat.Accid.Sci.A.90: 7696-7700), phosphatidylinositol-3 '- kinase (Hu et al., 1992, Mol Cell. Biol., 12: 981-990), phospholipase c? (Kashishian and Cooper, 1993, Mol. Cell. Biol., 4: 49-51), ras-GTPase activating protein (Kashishian et al., 1992, EMBO J .. 11: 1373-1382), PTP-ID / syp (Kazlauskas et al., 1993, Proc. Nati, Acad. Sci. USA, 10 90: 6939-6943), Grb2 (Arvidsson et al., 1994, Mol.

Cell. Biol., 14: 6715-6726), and Shc and Nck adapter molecules (Nishimura et al., 1993, Mol.Cell. Biol .. 13: 6889-6896), bind to regions involving different autophosphorylation sites. See generally, Claesson-Welsh, 1994, Prog. Growth Factor Res., 5: 37-54. Accordingly, it is possible that the signal transduction pathways activated by KDR / FLK-1 include the ras pathway (Rozakis et al., 1992, Nature, 360: 689-692), PI-3 '- kinase, and pathways mediated by src and by plc ?. Each of these routes may have a critical role in the angiogenic and / or vasculogenic effect of KDR / FLK-1 on endothelial cells. Accordingly, a still further aspect of this invention relates to the use of the organic compounds described herein to modulate angiogenesis and vasculogenesis, as these processes are controlled by these routes. Conversely, disorders related to shrinkage, contraction or closure of blood vessels, such as restenosis, are also involved and may be treated or prevented by the methods of this invention. Fibrotic disorders refer to the abnormal formation of extracellular matrices. Examples of fibrotic disorders include liver cirrhosis and mesangial cell proliferative disorders. Liver cirrhosis is characterized by an increase in the constituents of the extracellular matrix that result in the formation of a hepatic scar. An increased extracellular matrix that results in a hepatic scar can also be caused by a viral infection, such as hepatitis. Lipocytes seem to play an important role in liver cirrhosis. Other fibrotic disorders involved include atherosclerosis. Mesangial cell proliferative disorders refer to disorders caused by the abnormal proliferation of mesangial cells. Mesangial proliferative disorders include different human renal diseases, such as glomerulonephritis, diabetic nephropathy and malignant nephrosclerosis, as well as disorders, such as thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. The RTK PDGFR has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993, Kideny International 43: 47S-54S. Many cancers are cellular proliferative disorders, and as noted above, protein kinases have been associated with cellular proliferative disorders. ThereforeIt is not surprising that protein kinases, such as, for example, members of the RTK family, have been associated with the development of cancer. Some of these receptors, such as EGFR (Tuzi et al., 1991, Br. J. Cancer 63: 227-233, Torp et al., 1992, APMIS 100: 713-719) HER2 / neu (Slamon et al., 1989, Science 244 : 707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7: 627-633) are overexpressed in many tumors and / or are persistently activated by autocrine cycles. In fact, in the most common and severe cancers, these receptor over-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci., 111: 119-133, Dickson et al., 1992, Cancer Treatment Res. 61: 249-273, Korc et al., 1992, J. Clin.Invest.90: 1352-1360) and autocrine cycles (Lee and Donoghue, 1992, J. Cell. Biol. 118: 1057-1070, Korc. and collaborators, supra, Akbasak and Suner-Akbasak et al., supra) have already been demonstrated. For example, EGFR has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, cancer of the head and neck, lung cancer and bladder cancer. HER2 has been associated with cancer of the breast, ovary, gastric, lung, pancreas and bladder. PDGFR has been associated with glioblastoma and melanoma, as well as cancer of the lung, ovary and prostate. RTK c-met has also been associated with malignant tumor formation. For example, c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic, gastric and hepatocellular carcinomas and lymphomas. Additionally, c-met has been linked to leukemia. The over-expression of the c-met gene has also been detected in patients with Hodgkins disease and Burkitts disease. IGF-IR, in addition to being involved in nutritional support and type II diabetes, has also been associated with several types of cancer. For example, IGF-I has been implicated as an autochthonous growth stimulant for several types of tumors, for example, human breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin. Invest., 84: 1418 -1423) and small lung tumor cells (Macauley et al., 1990, Cancer Res., 50: 2511-2517). In addition, IGF-I, although it is integrally involved in the normal growth and differentiation of the nervous system, also seems to be an autochthonous stimulant of human glycogen. Sandberg-Nordqvist et al., 1993, Cancer Res. 53: 2475-2478. The importance of IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, ieloid cells, chondrocytes and osteoblasts (cells of stem from the bone marrow)) are stimulated by IGF-I to grow. Goldring and Goldring, 1991, Eukaryotic Gene Expression, 1: 301-326. In a series of recent publications, Baserga suggests that IGF-IR plays a central role in the mechanism of transformation and, as such, could be a preferred target for therapeutic interventions for a broad spectrum of human malignancies. Baserga, 1995, Cancer Res., 55: 249-252, Baserga, 1994, Cell 79: 927-930, Coppola et al., 1994, Mol. Cell. Biol .. 14: 4588-4595.

STKs have been implicated in many types of cancer, including notoriously breast cancer (Canee, et al., Int. J. Cancer, 54: 571-77 (1993)). The association between the abnormal activity of the protein kinase and the disease is not restricted to cancer. For example, RTKs have been associated with diseases such as psoriasis, diabetes mellitus, Ls endometriums, angiogenesis, atheromatous plaque development, Alzheimer's disease, von Hippel-Lindau disease, epidermal hyperproliferation, neurodegenerative diseases, age-related macular degeneration. and hemangiomas. For example, EGFR has been indicated in the healing of corneal and dermal wounds. Defects in Insulin-R and IGF-1R are indicated in type II diabetes mellitus. A more complete correlation between the specific RTKs and their therapeutic indications is stipulated in Plowman et al., 1994, DN &P 7: 334-339. As noted above, not only the RTKs, but the CTKs, including, but not limited to, src, abl, fps, yes, fyn, lyn, lck, bik, hck, fgr and yrk (reviewed in Bolen et al., 1992 , FASEB J., 6: 3403-3409) are involved in the proliferative and metabolic signal transduction pathway, and therefore, one might expect, and have been shown, to be involved in many disorders mediated by PTK that are It addresses the present invention. For example, it has been shown that the mutated src (v-src) is an oncoprotein (pp60v_src) in chickens. Moreover, its cellular homologous, the proto-oncogene pp60c-srcI) transmits oncogenic signals from many receptors. Over-expression of EGFR or HER2 / neu in tumors leads to the constitutive activation of pp60c-src, which is characteristic of malignant cells, but is absent in normal cells. On the other hand, mice deficient in the expression of c-src exhibit an osteopetrophic phenotype, indicating a key role of c-src in the function of osteoclasts, and a possible involvement in related disorders. In a similar manner, Zap70 has been implicated in T cell signaling, which may be related to autoimmune disorders. STKs have been associated with inflammation, autoimmune disease, immune responses, and hyperproliferation disorders, such as restenosis, fibrosis, psoriasis, osteoarthritis, and rheumatoid arthritis. Protein kinases have also been implicated in the implantation of embryos. Accordingly, the compounds of this invention can provide an effective method for preventing this implantation of embryos, and therefore, may be useful as birth control agents. Finally, it is currently suspected that both RTKs and CTKs are involved in hyperimmune disorders.

Another aspect of the invention is a method for identifying a chemical compound that modulates the catalytic activity of one or more of the protein kinases discussed above. The method involves contacting cells that express the desired protein kinase, with a compound of this invention (or its salt or prodrug), and monitoring the cells to determine any effect the compound has on them. The effect can be any change or absence of observable change, either at a glance or through the use of instrumentation, in a cellular phenotype. The change or absence of change in the monitored cell phenotype can be, for example, without limitation, a change or absence of change in the catalytic activity of the protein kinase in the cells, or a change or absence of change in the interaction of the protein kinase with a natural binding component. Examples of the effect of a number of example compounds of this invention on several PTKs are shown in Tables 1 and 2, and in the section on Biological Examples, below. The compounds and data presented should not be construed to limit the scope of this invention in any way.

. PHARMACEUTICAL COMPOSITIONS AND USE A compound of the present invention, a prodrug thereof or a physiologically acceptable salt of the compound or its prodrug, can be administered as such to a human patient, or it can be administered in pharmaceutical compositions wherein the above materials are mixed with suitable carriers or excipients. Techniques for drug formulation and administration can be found in "Remington's Pharmacological Sciences," Mac Publishing Co., Easton, PA, latest edition.

Routes of Administration As used herein, "administering" or "administration" refers to the delivery of a compound, salt or prodrug of the present invention, or of a pharmaceutical composition containing a compound, salt or prodrug of this invention, to an organism, for the purpose of preventing or treating a disorder related to protein kinase. Suitable routes of administration may include, without limitation, oral, rectal, transmucosal or intestinal administration, or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal or intraocular injections. The preferred routes of administration are oral and parenteral. Alternatively, the compound can be administered in a local rather than systemic manner, for example, by injection of the compound directly into a solid tumor, often in a depot or sustained release formulation. In addition, the drug can be administered in a targeted drug delivery system, for example, in a liposome coated with a tumor-specific antibody. The liposomes will be targeted and will be selectively recovered by the tumor.

Composition / Formulation The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, and ulizing, encapsulating, entrapping or lyophilizing processes. . Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner, using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. The appropriate formulation depends on the route of administration chosen. For injection, the compounds of; The invention can be formulated in aqueous solutions, preferably in physiologically compatible regulators, such as Hanks' solution, Ringer's solution, or physiological saline regulator. For transmucosal administration, appropriate penetrants are used so that the barrier is permeated in the formulation. These penetrants are generally known in this field. For oral administration, the compounds may be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. These vehicles make it possible for the compounds of the invention to be formulated as tablets, pills, pills, dragees, capsules, liquids, gels, syrups, pastes, suspensions and the like, to be ingested orally by a patient. Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the granule mixture, after adding other suitable auxiliaries, if desired, to obtain tablets or dragee cores. Useful excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol, cellulose preparations, such as, for example, corn starch, wheat starch, rice starch and potato starch, and other materials, such as gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium caboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinylpyrrolidone, agar, or alginic acid. A salt, such as sodium alginate, can also be used. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidione, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and solvents or mixtures of suitable organic solvents. Dyes or pigments can be added to tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push fit capsules may contain the active ingredients mixed with a filler, such as lactose, a binder, such as starch, and / or a lubricant, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers can be added in these formulations as well. To be administered by inhalation, the compounds to be used in accordance with the present invention are conveniently supplied in the form of an aerosol spray, using a pressurized pack or a nebulizer and a suitable propellant, for example, without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosing unit can be controlled by providing a valve to supply a measured quantity. You can formulate capsules; and cartridges, for example, of gelatin, for use in an inhaler or insufflator, containing a powder mixture of the compound and a suitable powder base, such as lactose or starch. The compounds can also be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in a unit dosage form, for example, in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulation materials, such as suspending, stabilizing and / or dispersing agents. Pharmaceutical compositions for parental administration include aqueous solutions of a water-soluble form, such as, without limitation, a salt, of the active compound. Additionally, suspensions of the active compounds can be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate and triglycerides, or materials such as liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain stabilizers and / or suitable agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form to be constituted with a suitable vehicle, eg, sterile, pyrogen-free water, before use. The compounds can also be formulated in rectal compositions, such as suppositories or retention enemas, using, for example, conventional suppository bases, such as cocoa butter or other glycerides. In addition to the formulations described above, the compounds can also be formulated as depot preparations. These long acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly), or by intramuscular injection. A compound of this invention can be formulated for this route of administration with suitable polymeric or hydrophobic materials (eg, in an emulsion with a pharmacologically acceptable oil), with ion exchange resins, or as a sparingly soluble derivative, such as, without limitation, a sparingly soluble salt. A non-limiting example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase, such as a VPD cosolvent system. VPD is a solution of 3 percent weight / volume of benzyl alcohol, 8 percent weight / volume of non-polar surfactant Polysorbate 80MR, and 65 percent weight / volume of polyethylene glycol 300, filled to volume in ethanol absolute. The VPD cosolvent system (VPD: D5W) consists of VPD diluted 1: 1 with a 5 percent dextrose solution in water. This cosolvent system dissolves hydrophobic compounds well, and it itself produces a low toxicity after its systemic administration. Naturally, the proportions of this cosolvent system can be varied considerably without destroying its solubility and toxicity characteristics. In addition, the identity of the components of the cosolvent can be varied: for example, other non-polar surfactants of low toxicity can be used in Polysorbate 80 MR, the fractional size of the polyethylene glycol can be varied, other biocompatible polymers can replace the polyethylene glycol, example, polyvinylpyrrolidone, and other sugars or polysaccharides can replace dextrose. In an alternative way, other delivery systems for the hydrophobic pharmaceutical compounds can be employed. Liposomes and emulsions are well known examples of carriers or carriers for hydrophobic drugs. In addition, certain organic solvents, such as dimethyl sulfoxide, may be employed, although often at the cost of increased toxicity. Additionally, the compounds can be delivered using a sustained release system, such as in semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Different sustained release materials have been established, and are well known to those skilled in the art. Sustained-release capsules, depending on their chemical nature, can release the compounds for a few weeks to more than 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for stabilizing the protein may be employed.

The pharmaceutical compositions herein may also comprise suitable solid or gel phase carriers or excipients. Examples of these carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycols. Many of the protein kinase modulator compounds of the invention can be provided as physiologically acceptable salts, wherein the claimed compound can form the negatively or positively charged species. Examples of salts wherein the compound forms the positively charged fraction include, without limitation, quaternary ammonium (defined elsewhere herein), salts, such as hydrochloride, sulfate, carbonate, lactate, tartrate, maleate, succinate , wherein the nitrogen atom of the quaternary ammonium group is a nitrogen of the selected compound of this invention, which has reacted with the appropriate acid. Salts wherein a compound of this invention forms the negatively charged species include, without limitation, the sodium, potassium, calcium and magnesium salts formed by the reaction of a carboxylic acid group in the compound with an appropriate base (e.g., hydroxide) sodium (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca (OH) 2), etc.).

Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount sufficient to achieve the intended purpose, i.e., the modulation of protein kinase activity or the treatment or prevention of a disorder related to protein kinase. More specifically, a therapeutically effective amount means an amount of the compound effective to prevent, alleviate or lessen the symptoms of the disease, or prolong the survival of the subject being treated. The determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from cell culture assays. Then, the dosage can be formulated for use in animal models, in order to achieve a circulating concentration range that includes the IC50 determined in the cell culture (i.e., the concentration of the test compound that achieves a mean-maximum inhibition of the activity of the protein kinase). Then this information can be used to determine more precisely the useful doses in humans. The toxicity and therapeutic efficacy of the compounds described herein can be determined by conventional pharmaceutical methods in cell cultures or in experimental animals, for example, by determining the IC 50 and the LD 50 (both of which are discussed in any another part of the present) for an object compound. The data obtained from these cell culture assays and animal studies can be used in the formulation of a range of dosages for use in humans. The dosage may vary depending on the dosage form used and the route of administration used. The exact formulation, the route of administration and the dosage can be chosen by the individual physician in view of the patient's condition (see, for example, Fingí et al., 1975, in "The Pharmacological Basis of Therapeutics", Chapter 1, page 1). The amount and range of dosage can be adjusted individually to provide plasma levels of the active species that are sufficient to maintain the kinase-modulating effects. These levels in plasma are referred to as the minimum effective concentrations (MECs). The MEC will vary for each compound, but it can be estimated from the in vitro data, for example, the concentration necessary to achieve 50 to 90 percent inhibition of a kinase can be asserted using the assays described herein. The dosages necessary to achieve the minimum effective concentration will depend on the individual characteristics and the route of administration. HPLC assays or bioassays can be used to determine plasma concentrations. The dosage intervals can also be determined using the value of the minimum effective concentration. The compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10 to 90 percent of the time, preferably between 30 and 90 percent, and most preferably between 50 and 90 percent by time. hundred. In cases of local administration or selective recovery, the effective local concentration of the drug may not be related to the plasma concentration, and other methods known in the art may be employed to determine the correct dosage amount and range. The amount of a composition administered, of course, will depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Packaging If desired, the compositions may be presented in a package or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. For example, the package may comprise metal or plastic sheet, such as a blister pack. The package or dosing device may be accompanied by instructions for its administration. The package or dispenser may also be accompanied by a notification associated with the container in a form prescribed by a governmental agency that regulates the manufacture, use or sale of pharmaceutical products, whose notification reflects the approval by the agency of the form of the compositions or of human or veterinary administration. This notification, for example, may be from the label approved by the U.S. Food and Drug Administration for prescription drugs, or an insert of the approved product. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier, placed in an appropriate container, and labeled for the treatment of an indicated condition can also be prepared. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes and the like. 6. SYNTHESIS The compounds of this invention, as well as the 2-oxindoles and precursor aldehydes, can be easily synthesized using techniques well known in the chemical field. It will be appreciated by those skilled in the art, that other synthetic routes are available to form the compounds of the invention, and that the following is offered by way of example and not limitation.

A. General Synthetic Procedure The following general methodology can be used to prepare the compounds of this invention: Appropriately substituted 2-oxindole (1 equivalent), appropriately substituted aldehyde (1.2 equivalents), and piperidine (0.1 equivalents) are mixed with ethanol (1-2 milliliters / millimole of 2-oxindole), and then the mixture is heated at 90 ° C for 3 to 5 hours. After cooling, the reaction mixture is concentrated and acidified to a pH of 3. The precipitate that forms is filtered, washed with water to a pH of 7, and then with cold ethanol, ethyl acetate and / or hexane. , and dried under vacuum to give the white compound. The product can optionally be further purified by chromatography.

B. 2-oxindoles The following examples are representative of the syntheses of the 2-oxindole precursors for the compounds of this invention. These 2-oxindoles will form the claimed compounds by their reaction with an appropriately substituted pyrrole aldehyde, using the above general synthetic procedure, or the procedures exemplified in section C, below. It should be understood that the following syntheses should not be interpreted as limiting, either with respect to the synthetic approach or to the oxindoles whose synthesis is exemplified. 5-Amino-2-oxindole The 5-nitro-2-oxindole (6.3 grams) was hydrogenated in methanol over 10 percent palladium on carbon to give 3.0 grams (60 percent yield) of the title compound as a solid. White. 5-Bromo-2-oxindole The 2-oxindole (1.3 grams) in 20 milliliters of acetonitrile was cooled to -10 ° C, and 2.0 grams of N-bromosuccinimide were slowly added with stirring. The reaction was stirred for 1 hour at -10 ° C, and for 2 hours at 0 ° C. The precipitate was collected, washed with water, and dried to give 1.9 grams (90 percent yield) of the title compound. 4-Methyl-2-oxindole Diethyl oxalate (30 milliliters) in 20 milliliters of dry ether was added with stirring to 19 grams of potassium ethoxide suspended in 50 milliliters of dry ether. The mixture was cooled in an ice bath, and 20 milliliters of 3-nitro-o-xylene in 20 milliliters of dry ether were added slowly. The thick dark red mixture was heated to reflux for 0.5 hours, concentrated to a dark red solid, and treated with 10 percent sodium hydroxide, until almost all of the solid dissolved. The dark red mixture was treated with 30 percent hydrogen peroxide, until the red color changed to yellow. The mixture was treated alternately with 10 percent sodium hydroxide and 30 percent hydrogen peroxide, until the dark red color was no longer present. The solid was filtered, and the filtrate was acidified with 6N hydrochloric acid. The resulting precipitate was collected by vacuum filtration, washed with water, and dried under vacuum to give 9.8 grams (45 percent yield) of 2-methyl-6-nitrophenylacetic acid as a white solid. The solid was hydrogenated in methanol over 10 percent palladium on carbon to give 9.04 grams of the title compound as a white solid. 7-Bromo-5-chloro-2-oxindole 5-chloro-2-oxindole (16.8 grams) and 19.6 grams of N-bromosuccinimide were suspended in 140 milliliters of • w * * 98 acetonitrile, and refluxed for 3 hours. Thin layer chromatography (silica, ethyl acetate) at 2 hours of reflux showed 5-chloro-2-oxindole or N-bromosuccinimide (Rf 0.8), product (Rf 0.85), and a second product (Rf 0.9). ), whose proportions did not change after another hour of reflux. The mixture was cooled to 10 ° C, the precipitate was collected by vacuum filtration, washed with 25 milliliters of ethanol, and sucked to dry for 20 minutes in the funnel, to give 14.1 grams of the wet product (56 percent yield). The solid was suspended in 200 milliliters of denatured ethanol, and washed in paste by stirring and refluxing for 10 minutes. The mixture was cooled in an ice bath at 10 ° C. The solid product was collected by vacuum filtration, washed with 25 milliliters of ethanol and dried under vacuum at 40 ° C to give 12.7 grams (51 percent yield) of 7-bromo-5-chloro-2-oxindole. 5-Fluoro-2-oxindole 5-fluoroisatin (8.2 grams) was dissolved in 50 milliliters of hydrazine hydrate, and refluxed for 1 hour. Then the reaction mixtures were poured into ice water. The precipitate was then filtered, washed with water, and dried in a vacuum oven to provide the title compound. 25 5-Nitro-2-oxindole 2-Oxindole (6.5 grams) was dissolved in 25 milliliters of concentrated sulfuric acid, and the mixture was maintained at -10 ° C to -15 ° C while 2.1 milliliters of nitric acid were added dropwise. in vaporization. After the addition of the nitric acid, the reaction mixture was stirred at 0 ° C for 0.5 hour, and poured into ice water. The precipitate was collected by filtration, washed with water and crystallized from 50 percent acetic acid. The crystalline product was then filtered, washed with water, and dried under vacuum to give 6.3 grams (70 percent) of 5-nitro-2-oxindole. 5-Iodo-2-oxindole 2-Oxindole (82.9 grams) was suspended in 630 milliliters of acetic acid with mechanical stirring, and the mixture was cooled to 10 ° C in an ice water bath. Solid N-iodosuccinimide (175 grams) was added in portions for 10 minutes. After the addition was completed, the mixture was stirred for 1 hour at 10 ° C. The suspended solid, which had always been present, became very thick at this time. The solid was collected by vacuum filtration, washed with 100 milliliters of 50 percent acetic acid in water, and then with 200 milliliters of water, and sucked dry for 20 minutes in the funnel. The product was dried under vacuum to give 93.5 grams (36 percent) of 5-iodo-2-oxindole containing approximately 5 percent of 2-oxindole "> r »100 by proton nuclear magnetic resonance. 5-Methyl-2-oxindole 5-Methylisatin (15.0 grams) and 60 milliliters of hydrazine hydrate were heated from 140 ° C to 160 ° C for 4 hours. Thin layer chromatography (ethyl acetate: hexane, 1: 2, silica gel) showed no remaining starting material. The reaction mixture was cooled to room temperature, poured into 300 milliliters of ice water, and acidified to a pH of 2 with 6N hydrochloric acid. After After standing at room temperature for 2 days, the precipitate was collected by vacuum filtration, washed with water, and dried under vacuum to yield 6.5 grams (47 percent yield) of 5-methyl-2-oxindole. 5-Bromo-4-methyloxindole and 5/7-dibromo-4-methyloxindole The 4-methyl-2-oxindole (5 grams) in 40 milliliters of acetonitrile was treated with 7.26 grams of N-bromosuccinimide, and stirred at room temperature. environment for 4 hours. Thin layer chromatography (ethyl acetate: hexane, 1: 2, silica gel) showed a mixture of 5-bromine (Rf 0.3) and 5.7 dibromo (Rf 0.5) as products. Another 7.26 grams of N-bromosuccinimide was added, and the mixture was stirred for an additional 4 hours. The solid was collected by vacuum filtration, washed with 20 milliliters of acetonitrile, and dried to give a 1: 1 mixture of mono and dibromo compounds. He The filtrate was concentrated and chromatographed on silica gel (ethyl acetate: hexane (1: 2)) to give 1.67 grams of 5-bromo-4-methyl-2-oxindole as a beige solid. The remaining 1: 1 mixture of solids was recrystallized twice from glacial acetic acid to give 3.2 grams of 5,7-dibromo-4-methyl-2-oxindole as a light orange solid. The filtrates from this material were passed by chromatography as above to give 0.6 grams of 5-bromo-4-methyl-2-oxindole and 0.5 grams of 5,7-dibromo-4-methyl-2-oxindole. 6-Fluoro-2-oxindole Sodium hydride (2.6 grams) and 14.5 grams of dimethyl malonate were stirred and heated at 100 ° C in 160 milliliters of dimethyl sulfoxide for 1 hour. The mixture was cooled to room temperature, 7.95 grams of 2,5-difluoronitrobenzene were added, and the mixture was stirred for 30 minutes. The mixture was then heated at 100 ° C for 1 hour, cooled to room temperature and poured into 400 milliliters of a saturated solution of ammonium chloride. The mixture was extracted with 200 milliliters of ethyl acetate, and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was crystallized from methanol to give 24.4 grams (80 percent yield) dimethyl 4-fluoro-2-nitrophenylmalonate as a white solid, Rf 0.2 in thin layer chromatography (ethyl acetate: hexane, 1: 6 , Silica gel). The filtrate was concentrated and chromatographed on a silica gel column (ethyl acetate: hexane, 1: 8) to give an additional 5.03 grams of dimethyl 4-fluoro-2-nitrophenylmalonate, for a total of 29.9 grams ( 96 percent yield). The dimethyl 4-fluoro-2-nitrophenylmalonate (5.0 grams) was refluxed in 20 milliliters of 6N hydrochloric acid for 24 hours. The reaction was cooled, and the white solid was collected by vacuum filtration, washed with water and dried to give 3.3 grams (87 percent yield) of 4-fluoro-2-nitrophenylacetic acid, Rf 0.6 in layer chromatography. thin (ethyl acetate: hexane, 1: 2, silica gel). The 4-fluoro-2-nitrophenylacetic acid (3.3 grams) dissolved in 15 milliliters of acetic acid was hydrogenated over 0.45 grams of 10 percent palladium on carbon at 60 psi of H2 for 2 hours. The catalyst was removed by filtration, and washed with 15 milliliters of methanol. The combined filtrates were concentrated and diluted with water. The precipitate was collected by vacuum filtration, washed with water, and dried, to give 1.6 grams (70 percent yield) of 6-fluoro-2-oxindole, Rf 0.24 in thin layer chromatography. The filtrate was concentrated to give a purple solid with a nuclear magnetic resonance spectrum similar to the first culture. Chromatography of the purple solid (ethyl acetate: hexane, 1: 2, silica gel) gave a second culture of 6-fluoro-2-oxindole as a white solid.

-Aminosulfoni1-2-oxindole To a 100-milliliter flask loaded with 27 milliliters of chlorosulfonic acid, 13.3 grams of 2-oxindole were added slowly. The temperature of the reaction was kept below 30 ° C during the addition. After the addition, the reaction mixture was stirred at room temperature for 1.5 hours, heated at 68 ° C for 1 hour, cooled, and poured into water. The precipitate was washed with water and dried in a vacuum oven to give 11.0 grams of 5-chlorosulfonyl-2-oxindole (50 percent yield), which was used without further purification. 5-Chlorosulfonyl-2-oxindole (2.1 grams) was added to 10 milliliters of ammonium hydroxide in 10 milliliters of ethanol, and stirred at room temperature overnight. The mixture was concentrated, and the solid was collected by vacuum filtration to give 0.4 grams (20 percent yield) of the title compound as a white solid. 5-Methylaminosulfonyl-2-oxindole A suspension of 3.38 grams of 5-chlorosulfonyl-2-oxindole in 10 milliliters of 2M methylamine in tetrahydrofuran, was stirred at room temperature for 4 hours, during which time a white solid formed. The precipitate was collected by vacuum filtration, washed twice with 5 milliliters of water, and dried under vacuum at 40 ° C overnight to give 3.0 grams (88 percent yield) of 5-methylaminosulfonyl-2-oxindole . 5- (4-Trifluoromethylphenylaminosulfonyl) -2-oxindole A suspension of 2.1 grams of 5-chlorosulfonyl-2-oxindole, 1.6 grams of 4-trifluoromethylaniline, and 1.4 grams of pyridine in 20 milliliters of dichloromethane was stirred at room temperature for 4 hours. hours. The precipitate that formed was collected by vacuum filtration, washed twice with 5 milliliters of water, and dried under vacuum at 40 ° C overnight, to give 2.4 grams of the crude product containing some impurities by layer chromatography. thin. The crude product was passed through chromatography on silica gel, eluting with ethyl acetate: hexane (1: 2), to give 1.2 grams (37 percent yield) of 5- (4-trifluoromethylphenyl-aminosulfonyl) -2-oxindole . 5- (Morpholinosulfonyl) -2-oxindole A suspension of 2.3 grams of 5-chlorosulfonyl-2-oxindole and 2.2 grams of morpholine in 50 milliliters of dichloromethane was stirred at room temperature for 3 hours. The white precipitate was collected by vacuum filtration, washed with ethyl acetate and hexane, and dried under vacuum at 40 ° C overnight to give 2.1 grams (74 percent yield) of 5- (morpholinosulfonyl) -2 -oxindol. 6-Trifluoromethyl-2-oxindole Dimethyl sulfoxide (330 milliliters) was added to 7.9 grams of sodium hydride, followed by dropwise addition of 43.6 grams of diethyl oxalate. The mixture was heated at 100 ° C for 1 hour, and cooled to room temperature. 2-Nitro-4-trifluoromethyl toluene (31.3 grams) was added, the reaction was stirred for 30 minutes at room temperature, and then heated at 100 ° C for 1 hour. The reaction was cooled and poured into a mixture of saturated aqueous ammonium chloride, ethyl acetate and hexane. The organic layer was washed with saturated ammonium chloride, water and brine, dried, and concentrated to give dimethyl 2- (2-nitro-4-trifluoromethyl) malonate. The diester was dissolved in a mixture of 6.4 grams of lithium chloride and 2.7 milliliters of water in 100 milliliters of dimethyl sulfoxide, and heated at 100 ° C for 3 hours. The reaction was cooled and poured into a mixture of ethyl acetate and brine. The organic phase was washed with brine, dried with sodium sulfate, concentrated and chromatographed on silica gel (10 percent ethyl acetate in hexane). The fractions containing the product were evaporated to give 25.7 grams of methyl 2-nitro-4-trifluoromethyl-phenylacetate. The methyl 2-nitro-4-trifluoromethylphenylacetate (26 milligrams) was hydrogenated over 10 percent palladium on carbon, and then heated at 100 ° C for 3 hours. The catalyst was removed by filtration, and the solvent was evaporated to give the title compound. - (2-Chloroethyl) oxindole The 5-chloroacetyl-2-oxindole (4.18 grams) in 30 milliliters of trifluoroacetic acid in an ice bath was treated with 4.65 grams of triethylsilane, and stirred at room temperature for 3 hours. The mixture was poured into 150 milliliters of water, and the precipitate was collected by vacuum filtration, washed with 50 milliliters of water, and dried to give 2.53 grams (65 percent yield) of 5- (2-chloroethyl) -2-oxindol as a solid reddish-brown. 5-Methoxycarbonyl-2-oxindole The 5-iodo-2-oxindole (17 grams) was refluxed with 2 grams of palladium diacetate, 18.2 grams of triethylamine, 150 milliliters of methanol, 15 milliliters of dimethyl sulfoxide, and 2.6 grams of DPPP in an atmosphere saturated with carbon monoxide. After 24 hours, the reaction was filtered to remove the catalyst, and the filtrate was concentrated. The concentrate was passed through chromatography on silica gel (30 percent ethyl acetate in hexane). The fractions containing the product were concentrated and allowed to stand. The precipitated product was collected by vacuum filtration to give 0.8 grams (7 percent) of the title compound as a white solid. 4-Carboxy-2-oxindole A solution of trimethylsilyldiazomethane in hexane (2M) was added dropwise to a solution of 2.01 grams of 2-chloro-3-carboxy-nitrobenzene in 20 milliliters of methanol at room temperature until no longer presented no release of additional gas. The excess of trimethylsilyl diazomethane was quenched with acetic acid. The reaction mixture was dried by rotary pump, and the residue was further dried in a vacuum oven overnight. The product (2-chloro-3-methoxycarbonyl-nitrobenzene) was sufficiently pure for the next reaction. Dimethyl malonate (6.0 milliliters) was added to an ice-cold suspension of 2.1 grams of sodium hydride in 15 milliliters of dimethyl sulfoxide. The reaction mixture was then stirred at 100 ° C for 1 hour, and then cooled to room temperature. 2-Chloro-3-methoxycarbonyl-nitrobenzene (2.15 grams) was added to the above mixture in one portion, and the mixture was heated at 100 ° C for 1.5 hours. Then the reaction mixture was cooled to room temperature, and poured into ice water, acidified to a pH of 5, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated to give 3.0 grams of dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate. The dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate (3.0 grams) was refluxed in 50 milliliters of 6 N hydrochloric acid overnight. The mixture was concentrated to dryness, and refluxed for 2 hours with 1.1 grams of tin (II) chloride in 20 milliliters of ethanol. The mixture was filtered through Celite, concentrated, and chromatographed on silica gel (ethyl acetate: hexane: acetic acid) to give 0.65 grams (37 percent yield) of 4-carboxy-2-. oxindole as a white solid. 5-Carboxy-2-oxindole 2-Oxindole (6.7 grams) was added to a stirred suspension of 23 grams of aluminum chloride in 30 milliliters of dichloroethane in an ice bath. Chloroacetyl chloride (11.3 grams) was added slowly, and hydrogen chloride gas was evolved. After ten minutes of stirring, the reaction was heated from 40 ° C to 50 ° C for 1.5 hours .. Thin layer chromatography (ethyl acetate, silica gel) showed no remaining starting material. The mixture was cooled to room temperature, and poured into ice water. The precipitate was collected by vacuum filtration, washed with water and dried under vacuum to give 10.3 grams (98 percent) of 5-chloroacetyl-2-oxindole as a white solid. A suspension of 9.3 grams of 5-chloroacetyl-2-oxindole was stirred in 90 milliliters of pyridine at 80 ° C at 90 ° C for 3 hours, and then cooled to room temperature. The precipitate was collected by vacuum filtration, and washed with 20 milliliters of ethanol. The solid was dissolved in 90 milliliters of 2.5N sodium hydroxide, and stirred at 70 ° C to 80 ° C for 3 hours. The mixture was cooled to room temperature, and acidified to a pH of 2 with 0.5N hydrochloric acid. The precipitate was collected by vacuum filtration, and washed thoroughly with water, to give the crude 5-carboxy-2-oxindole as a solid dark brown. After standing overnight, the filtrate produced 2 grams of 5-carboxy-2-oxindole as a yellow solid. The crude brown product was dissolved in hot methanol, the insoluble material was removed by filtration, and the filtrate was concentrated to give 5.6 grams of 5-carboxy-2-oxindole as a sc >.brown lido. The combined yield was 97 percent. 5-Carboxyethyl-2-oxindole The 5-cyanoethyl-2-oxindole (4.02 grams) in 10 milliliters of water containing 25 milliliters of concentrated hydrochloric acid was refluxed for 4 hours. The mixture was cooled, water was added, and the resulting solid was collected by vacuum filtration, washed with water, and dried, to give 1.9 grams (44 percent yield) of the title compound as a yellow solid. 5-Iodo-4-methyl-2-oxindole To 2 grams of 4-methyl-2-oxindole in 40 milliliters of glacial acetic acid in an ice bath, 3.67 grams of N-iodosuccinimide were added. The mixture was stirred for 1 hour, diluted with 100 milliliters of 50 percent acetic acid in water, and filtered. The resulting white solid was dried under a high vacuum to give 3.27 grams (88 percent yield) of the title compound as a white solid. 5-Chloro-4-methyl-2-oxindole A suspension of 3.0 grams of 4-methyl-2-oxindole was stirred in 50 milliliters of acetonitrile at room temperature, while 3.3 grams of N-chlorosuccinimide was added in portions. Then trifluoroacetic acid (1 milliliter) was added. The suspension was stirred at room temperature for 3 days, during which time, the solid was always present. The white solid was collected by vacuum filtration, washed with a small amount of cold acetone, and dried overnight in a vacuum oven at 40 ° C, to give 2.5 grams (68 percent) of 5-chloro- 4-methyl-2-oxindole. 5-Butyl-2-oxindole Triethylisilane (2.3 grams) was added to 2 grams of 4-butanoyl-2-oxindole in 20 milliliters of trifluoroacetic acid at room temperature, and the solution was stirred for 3 hours. The reaction was poured into ice water to give a red oil, which solidified after standing. The solid was collected by vacuum filtration, washed with water and hexane, and dried, to give 1.7 grams (91 percent yield) of the title compound as a white solid. 5-Eti1-2-oxindole To 5-acetyl-2-oxindole (2 grams) in 15 milliliters of trifluoroacetic acid in an ice bath, 1.8 grams of tretylsilane was added slowly; then the reaction was stirred at room temperature for 5 hours. 1 milliliter of triethylsilane was added, and the stirring was continued overnight. The reaction mixture was poured into ice water, and the resulting precipitate was collected by vacuum filtration, washed copiously with water, and dried under vacuum to give 1.3 grams (71 percent yield) of the title compound as a solid. yellow. 5-Morpholin-4-ethyl-2-oxindole The 5-chloroethyl-2-oxindole (2.3 grams), 1.2 milliliters of morpholine, and 1.2 milliliters of di-isopropylethylamine were heated overnight at 100 ° C in 10 milliliters of sulfoxide. of dimethyl. The mixture was cooled, poured into water, and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated. The residue was passed through chromatography on silica gel (50 percent methanol in chloroform) to give 0.9 grams (31 percent) of the title compound as a white solid. 5- (4-Methoxycarbonylbenzamido) -2-oxindole A mixture of 82.0 milligrams of 5-amino-2-oxindole and 131.0 milligrams of 4-methoxycarbonylbenzoyl chloride in pyridine was stirred at room temperature for 3 hours and poured into water frost. The precipitate was filtered, washed with water, and dried in a vacuum oven, to give 138.0 milligrams of 5- (4-methoxycarbonylbenzamido) -2-oxindole (yield 81 percent). - (4-Carboxybenzamido) -2-oxindole The 5- (4-methoxycarbonylbenzamido) -2-oxindole (0.9 grams) and 0.4 grams of sodium hydroxide in 25 milliliters of methanol were refluxed for 3 hours. The mixture was concentrated, water was added, and the mixture was acidified with 6N hydrochloric acid. The precipitate was collected by vacuum filtration to give 0.75 grams (87 percent) of the title compound as a white solid. 5-Methoxy-2-oxindole Chloral hydrate (9.6 grams) was dissolved in 200 milliliters of water containing 83 grams of sodium sulfate. The solution was heated to 60 ° C, a solution of 11.4 grams of hydroxylamine hydrochloride in 50 milliliters of water was added, and the mixture was maintained at 60 °. In a separate flask, 6.4 grams of 4-anisidine and 4.3 milliliters of concentrated hydrochloric acid in 80 milliliters of water were heated at 80 ° C. The first solution was added to the second, and the mixture was refluxed for two minutes, after which it was cooled slowly to room temperature, and then cooled in an ice bath. The tanned precipitate was collected by vacuum filtration, washed with water, and dried in vacuo to give 8.6 grams (85 percent yield) of N- (2-hydroximino-acetyl) anisidine. The concentrated sulfuric acid (45 milliliters) containing 5 milliliters of water, was heated to 60 ° C, and 8.6 grams of N- (2-hydroxyaminoacetyl) anisidine were added in one portion. The stirred mixture was heated at 93 ° C for 10 minutes, and then allowed to cool to room temperature. The mixture was poured 500 grams of ice, and extracted three times with ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate, and concentrated to give 5.1 grams (65 percent yield) of methoxy-isatin as a dark red solid. The 5-methoxy-isatin (5.0 grams) and 30 milliliters of hydrazine hydrate were heated to reflux for 15 minutes. The reaction mixture was cooled to room temperature, and 50 milliliters of water was added. The mixture was extracted 3 times with 25 milliliters of ethyl acetate each time, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to give a yellow solid. The solid was stirred in ethyl acetate, and 1.1 grams of insoluble material was removed by vacuum filtration and stored. This material was 2-hydrazinocarbonylmethyl-4-anisidine. The filtrate was concentrated and chromatographed on silica gel, eluting with ethyl acetate: hexane (1: 1), to give 0.7 grams of 5-methoxy-2-oxindole as a yellow solid. The 1.1 grams of 2-hydrzinocarbonylmethyl-4-anisidine was refluxed for 1 hour in 20 milliliters of IN sodium hydroxide. The mixture was cooled, acidified to a pH of 2 with concentrated hydrochloric acid, and extracted 3 times with 25 milliliters of ethyl acetate each time. The organic extracts were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated to give 0.8 grams of 5-methoxy-2-oxindole as a yellow solid. The combined yield was 1.5 grams or 33 percent. 7-Azaoxindole The 3, 3-dibromo-7-azaoxindole (2.9 grams) was dissolved in a mixture of 20 milliliters of acetic acid and 30 milliliters of acetonitrile. To the solution was added 6.5 grams of zinc powder. The mixture was stirred for 2 hours at room temperature. The solid was filtered from the mixture, and the solvent was evaporated. The residue was formed into a paste with ethyl acetate. The ethyl acetate solution containing the insoluble solid was passed through a short column of silica gel. The collected ethyl acetate solution was evaporated, and the residue was dried under vacuum to give 1.8 grams (91 percent yield) of 7-azaoxindole-acetic acid salt. 5-Dimethylaminosulfonyl-2-oxindole A suspension of 2.3 grams of 5-chlorosulfonyl-2-oxindole in 10 milliliters of 2M dimethylamine in methanol was stirred at room temperature for 4 hours, at which time a white solid formed. The precipitate was collected by vacuum filtration, washed with 5 milliliters of IN sodium hydroxide, and 5 milliliters of IN hydrochloric acid, and dried under vacuum at 4 ° C overnight to give 1.9 grams (79 percent yield). ) of 5-dimethylaminosulfonyl-2-oxindole. 6-Feni1-2-oxindole. Dimethyl malonate (10 milliliters) in 25 milliliters of dimethyl sulfoxide was added dropwise to 3.5 grams of sodium hydride suspended in 25 milliliters of dimethyl sulfoxide, and the mixture was heated to 100 °. C for 10 minutes. The mixture was cooled to room temperature, and 4.7 grams of 4-fluoro-3-nitrobiphenyl in 25 milliliters of dimethyl sulfoxide was added. The mixture was heated at 100 ° C for 2 hours, cooled, and quenched with 300 milliliters of a saturated solution of ammonium chloride. The mixture was extracted three times with ethyl acetate, and the combined organic layers were washed with water and brine, and evaporated to give, as a yellow solid, the crude dimethyl 3-nitrobiphenyl-4-malonate. The crude dimethyl 3-nitrobiphenyl-4-malonate was refluxed in 30 milliliters of 6N hydrochloric acid for 24 hours. The precipitate was collected by filtration, washed with water and dried to give 4.5 grams of 3-nitrobiphenyl-4-acetic acid as a cream colored solid. Iron powder (2.6 grams) was added all at once to 4.5 grams of 3-nitrobiphenyl-4-acetic acid in 40 milliliters of acetic acid. The mixture was refluxed for 2 hours, concentrated to dryness, and taken up in ethyl acetate. The The solids were removed by filtration, and the filtrate was washed twice with IN hydrochloric acid and brine, and dried over anhydrous sodium sulfate. The filtrate was concentrated to give 3.4 grams (93 percent yield) of 6-phenyl-2-oxindole as a light tan solid. 6-2-Methoxyphenyl) -2-oxindole Tetrakis (triphenylphosphine) palladium (1 gram) was added to a mixture of 5 grams of 2-methoxyphenylboronic acid, 6.6 grams of 5-bromo-2-fluoronitrobenzene, and 300 milliliters of 2 M sodium carbonate solution in 50 milliliters of toluene and 50 milliliters of ethanol. The mixture was refluxed for 2 hours, concentrated, and the residue was extracted twice with ethyl acetate. The ethyl acetate layer was washed with water and brine, then dried, and concentrated to give a dark green oil, which solidified upon standing, of crude 4-fluoro-2'-methoxy-3-nitrobiphenyl. Dripping dimethyl malonate (14 milliliters) was added dropwise to 2.9 bouquets of sodium hydride suspended in 50 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 15 minutes, and cooled to room temperature. The crude 4-fluoro-2'-methoxy-3-nitrobiphenyl was added in 60 milliliters of dimethyl sulfoxide, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of sodium chloride, and extracted twice with ethyl acetate. The extracts were combined, washed with saturated ammonium chloride, water and brine, dried over anhydrous sodium sulfate and concentrated to give crude dimethyl 2'-methoxy-3-nitrobiphenyl-4-malonate as a yellow oil. The crude dimethyl 2'-methoxy-3-nitrobiphenyl-4-malonate was heated at 100 ° C in 50 milliliters of 6N hydrochloric acid for 24 hours, and cooled. The precipitate was collected by filtration, washed with water and hexane, and dried to give 9.8 grams of the 2'-methoxy-2-nitrobiphenyl-4-acetic acid as a light tan solid. Iron powder (5 grams) was added in one portion to 9.8 grams of 2'-methoxy-3-nitrobiphenyl-4-acetic acid in 50 milliliters of glacial acetic acid, and heated at 100 ° C for 3 hours. The reaction mixture was concentrated to dryness, sonicated in ethyl acetate, and filtered to remove the insolubles. The filtrate was washed twice with IN hydrochloric acid, water and then brine, dried over anhydrous sodium sulfate and concentrated. The residue was chromatographed on silica gel in ethyl acetate: hexane (1: 2) to give 5.4 grams of 6- (2-methoxyphenyl) -2-oxindole as a pink colored solid. 6- (3-Methoxyphenyl) -2-oxindole Tetrakis (triphenylphosphine) palladium (0.8 grams) was added to a mixture of 5 grams of 3-methoxyphenylboronic acid, 5 grams of 5-bromo-2-fluoro-nitrobenzene, and 11 milliliters of a 2 M sodium carbonate solution in 100 milliliters of toluene. The mixture was refluxed for 2 hours, diluted with water and extracted with ethyl acetate. The ethyl acetate was washed with saturated sodium bicarbonate and brine, and then dried and concentrated to give an oily solid. The solid was passed through chromatography on silica gel (ethyl acetate: hexane) (1: 6) to give 4.3 grams (77 percent yield) of 4-fluoro-3'-methoxy-3-nitrobiphenyl. Dimethyl malonate (9.7 milliliters) was added dropwise to 2.0 grams of sodium hydride suspended in 50 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 35 minutes and cooled to room temperature. 4-Fluoro-2'-methoxy-3-nitrobenphenyl (4.2 grams) was added in 50 milliliters of dimethyl sulfoxide, and the mixture was heated at 100 ° C for one hour. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted twice with ethyl acetate. The extracts were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 3'-methoxy-3-nitrobiphenyl-4-malonate as a pale yellow solid. Crude dimethyl 3 '-methoxy-3-nitrobiphenyl-4-malonate was heated at 110 ° C in 45 milliliters of 6 N hydrochloric acid for 4 days, and then it cooled down. The precipitate was collected by filtration, washed with water and hexane, and dried to give 5.3 grams of 3'-methoxy-2-nitrobiphenyl-4-acetic acid as a light tan solid. 3'-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 grams) was dissolved in methanol, and hydrogenated over 0.8 gram of 10 percent palladium on carbon for 3 hours at room temperature. The catalyst was removed by filtration, washed with methanol, and the filtrates were combined and concentrated to give a tan solid. The solid was chromatographed on silica gel in ethyl acetate: hexane: acetic acid (33: 66: 1) to give 3.0 grams of 6- (3-methoxyphenyl-2-oxindole as a pink solid. -methoxyphenyl) -2-oxindole Tetrakis (triphenylphosphine) palladium (1 gram) was added to a mixture of 5 grams of 4-methoxyphenylboronic acid, 6.6 grams of 5-bromo-2-fluoronitro-benzene, and 30 milliliters of a solution of 2 M sodium carbonate in 50 milliliters of toluene and 50 milliliters of ethanol The mixture was refluxed for 2 hours, concentrated, and the residue was extracted twice with ethyl acetate.The ethyl acetate layer was washed with ethyl acetate. water and brine, dried, and concentrated to give a solid oily brown.The solid was passed through chromatography on silica gel (5 percent ethyl acetate in hexane) to give 4-fluoro-4'-methoxy- Crude 3-nitrobiphenyl as a pale yellow solid, Dimethyl malonate (10 milliliters) was added dropwise to 2.0 grams of hr. Sodium idruro suspended in 60 milliliters in dimethyl sulfoxide. The mixture was heated at 100 ° C for 10 minutes, and cooled to room temperature. Crude 4-fluoro-2'-methoxy-3-nitrobiphenyl (5.2 grams) in 50 milliliters of dimethyl sulfoxide was added, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of sodium chloride, and extracted 3 times with ethyl acetate. The extracts were combined, washed with saturated ammonium chloride, water and brine, dried over anhydrous sodium sulfate, and concentrated to give crude dimethyl 4'-methoxy-3-nitrobiphenyl-4-malonate as a yellow oil. . The crude dimethyl 4-methoxy-3-nitrobiphenyl-4-malonate was heated at 100 ° C in 60 milliliters of 6N hydrochloric acid for 15 hours, and cooled. The precipitate was collected by filtration, washed with water and hexane, and dried to give 7.2 grams of crude 4'-methoxy-3-nitrobiphenyl-4-acetic acid as a light tan solid. Iron powder (3.6 grams) was added in one portion to 7.2 grams of 4'-methoxy-3-nitrobiphenyl-4-acetic acid in 50 milliliters of glacial acetic acid, and heated at 100 ° C overnight. The reaction mixture was concentrated to dryness, sonicated in ethyl acetate, and filtered to remove the insolubles. The filtrate was washed twice with IN hydrochloric acid and brine, dried over anhydrous sodium sulfate, and concentrated to give 2.7 jramos of 6- (4-methoxyphenyl) -2-oxindole as a pink colored solid. 6- (3-Ethoxyphenyl) -2-oxindole Tetrakis (triphenylphos) palladium (0.8 grams) was added to a mixture of 4.2 grams of 3-ethoxyphenylboronic acid, 5.0 grams of 5-bromo-2-fluoronitrobenzene and 22 milliliters of a 2 M sodium carbonate solution in 50 milliliters of toluene and 50 milliliters of ethanol. The mixture was refluxed for 2 hours, concentrated, water was added and the mixture was extracted twice with ethyl acetate. The ethyl acetate layer was washed with water and brine, then dried, and concentrated. The residue was chromatographed on silica gel, 5 percent ethyl acetate in hexane) to give 5.3 grams (90 percent yield) of 4-fluoro-3'-ethoxy-3-nitrobiphenyl as a yellow oil. Dripping dimethyl malonate (11.4 milliliters) was added dropwise to 4.0 grams of sodium hydride suspended in 20 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 10 minutes, and then cooled to room temperature. Crude 4-fluoro-3'-ethoxy-3-nitrobiphenium (5.3 grams) in 25 milliliters of dimethyl sulfoxide was added, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted three times with ethyl acetate. The extracts were combined, washed with water and brine, and then dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 3'-ethoxy-3-nitrobiphenyl-4-malonate as a yellow oil. The crude dimethyl 3'-ethoxy-3-nitrobiphenyl-4-malonate was heated at 100 ° C in 60 milliliters of 6N hydrochloric acid for 4 days, and then cooled. The precipitate was collected by filtration, washed with water and hexane, and dried to give 4.7 grams of the crude 3'-ethoxy-3-nitrobiphenyl-4-acetic acid as a light tan solid. Iron powder (2.4 grams) was added in one portion to 4.6 grams of the 3'-ethoxy-3-nitrobiphenyl-4-acetic acid in 40 milliliters of glacial acetic acid, and refluxed for 2 hours. The reaction mixture was concentrated to dryness, repeatedly treated with ethyl acetate and filtered to remove the insolubles. The filtrate was washed twice with IN hydrochloric acid and brine, and then dried over anhydrous sodium sulfate, and concentrated to give 3.5 grams (91 percent yield) of 6- (3-ethoxyphenyl) -2-oxindole as a solid light brown. 6-Bromo-2-oxindole. Dimethyl malonate (13 milliliters) was added dropwise to 2.7 grams of sodium hydride suspended in 20 milliliters of dimethyl sulfoxide. The mixture is heated. { or at 100 ° C for 10 minutes, and then cooled to room temperature. 5-Bromo-2-fluoronitrobenzene (5.0 grams) was added in 25 milliliters of dimethyl sulfoxide, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted 3 times with ethyl acetate. The extracts were combined, washed with saturated ammonium chloride, water and brine, dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 4-bromo-2-nitrophenylmalonate as a pale yellow oil. The 4-bromo-2-nitrophenylmalonate and crude dimethyl was heated at 110 ° C in 40 milliliters of 6N hydrochloric acid for 24, and then cooled. The precipitate was collected by filtration, washed with water, and dried to give 5.3 grams (89 percent yield) of 4-bromo-2-nitro-phenylacetic acid as a white solid. The 4-bromo-2-nitrophenylacetic acid (0.26 grams), 0.26 grams of zinc powder, and 3 milliliters of 50 percent sulfuric acid in 5 milliliters of ethane were heated at 100 ° C overnight. The reaction mixture was filtered, diluted with a little acetic acid, concentrated to remove the ethanol, diluted with water, and extracted twice with ethyl acetate. The combined extracts were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give 0.19 grams (90 percent yield) of 6-bromo-2-oxindole as a yellow solid.

-Aceti1-2-oxindole The 2-oxindole (3 grams) was suspended in 1,2-dichloroethane, and 3.2 milliliters of acetyl chloride were slowly added. The resulting suspension was heated at 50 ° C for 5 hours, cooled, and poured into water. The resulting precipitate was collected by vacuum filtration, washed copiously with water, and dried in vacuo to give 2.9 grams (73 percent yield) of the title compound as a tan solid. 5-Butanoyl-2-oxindole To 15 grams of aluminum chloride suspended in 30 milliliters of 1,2-dichloroethane in an ice bath were added 7.5 grams of 2-oxindole, and then 12 grams of butanoyl chloride. The resulting suspension was heated at 50 ° C overnight. The mixture was poured into ice water, and extracted 3 times with ethyl acetate. The combined ethyl acetate layers were washed with brine, dried over sodium sulfate, and concentrated to dryness to give a tan solid. The solid was passed through chromatography on silica gel (50 percent ethyl acetate in hexane), to give 3 grams (25 percent) of the title compound as a yellow solid. 5-Cyanoethyl-2-oxindole Potassium cyanide (2.0 grams) was added to 15 milliliters of dimethyl sulfoxide, and heated to 90 ° C. 5-Chloroethyl-2-oxindole (3.0 grams) dissolved in 5 milliliters of dimethyl sulfoxide was added slowly with stirring, and the reaction was heated at 150 ° C for 2 hours. The mixture was cooled, poured into ice water, and the precipitate was collected by vacuum filtration, washed with water, dried and then chromatographed on silica gel (5 percent methanol in chloroform), to give 1.2 grams (42 percent yield) of the title compound. 6-Morpholin-4-yl-2-oxindole The 6-amino-2-oxindole (2.2 grams), 4.0 grams of 2, 2'-dibromoethylether, and 7.9 grams of sodium carbonate, were refluxed in 20 milliliters of ethanol overnight, concentrated and diluted with 50 milliliters of water. The mixture was extracted three times with 50 milliliters of ethyl acetate, and the organic extracts were combined, washed with 20 milliliters of brine, dried over anhydrous sodium sulfate, and concentrated to dryness. The solid was chromatographed on a column of silica gel (ethyl acetate: hexane (1: 1) containing 0.7 percent acetic acid), to give 1.2 grams (37 percent yield) of the title compound as a solid beige. 6- (3-Trifluoroacetylphenyl) -2-oxindole 3-aminophenylboronic acid (3.9 grams), 5 grams of 5-bromo-2-f luoronitrobenzene, 0.8 grams of tetrakis (triphenylphosphine) palladium, and 23 milliliters of a bicarbonate solution of 2 M sodium in 50 milliliters of toluene, were refluxed under nitrogen for 2.5 hours. The reaction mixture was poured into 200 milliliters of ice water, and the mixture was extracted three times with 50 milliliters of ethyl acetate. The combined organic layers were washed with 50 milliliters of water and 20 milliliters of brine, dried over anhydrous sodium sulfate, and concentrated to give 9.7 grams (92 percent yield) of 2-fluoro-5- (3-aminophenyl) ) nitrobenzene as a dark brown oil. Trifluoroacetic anhydride (5.4 milliliters) was added slowly to a stirred solution of 9.7 grams of 2-fluoro-5- (3-aminophenyl) -nitrobenzene and 5.3 milliliters of triethylamine in 50 milliliters of dichloromethane at 0 ° C, and the mixture was stirred for an additional 20 minutes. The mixture was concentrated, and the residue was chromatographed on a column of silica gel (10 percent ethyl acetate in hexane) to give 8.6 (65 percent yield) of 2-fluoro-5- (3- trifluoroacetamidophenyl) nitrobenzene as a pale orange oil, which solidified upon standing. Dripping dimethyl malonate (9.6 milliliters) was added dropwise to a stirred suspension of 3.2 grams of 60 percent sodium hydride in mineral oil in 40 milliliters of anhydrous dimethyl sulfoxide under nitrogen. The mixture was stirred for 10 minutes, and 2-fluoro-5- (3-trifluoroacetamido-phenyl) nitrobenzene in 20 milliliters of dimethyl sulfoxide was added. The resulting dark red mixture was heated at 100 ° C for 2 hours. The reaction was quenched by pouring in 100 milliliters of a saturated solution of ammonium chloride, and extracted twice with 50 milliliters of ethyl acetate. The organic phase was washed with 50 milliliters of each of saturated ammonium chloride solution, water and brine, dried over anhydrous sodium sulfate, and concentrated to a yellow oil. The oil was chromatographed on a column of silica gel (ethyl acetate: hexane (1: 4)) to give 4.4 grams (50 percent yield) of 2- [2-nitro-4- (3-trifluoroacetamidophenyl) ) phenyl) -dimethyl malonate as a pale yellow solid. The dimethyl 2- [2-nitro-4- (3-trifluoroacetamidophenyl) -phenyl] malonate (4.4 grams) was refluxed overnight in 50 milliliters of 6N hydrochloric acid. The reaction mixture was cooled to room temperature, and the solids were collected by vacuum filtration, washed with water, and dried in vacuo to yield 2.7 grams (73 percent yield) of 2- [2-nitro] 4- (3-trifluoroacetamidophenyl) phenyl] acetic acid. 2- [2-Nitro-4- (3-trifluoroacetamidophenyl) -phenyl] acetic acid (100 milligrams), and 50 milligrams of iron powder in 3 milliliters of acetic acid were heated at 100 ° C for 2 hours. The reaction mixture was concentrated, and the residue was sonicated in 5 milliliters of ethyl acetate. The insoluble solids were removed by vacuum filtration, and the filtrate was washed with IN hydrochloric acid, water and brine, dried over anhydrous sodium sulfate, and concentrated to give 10 milligrams (14 percent yield) of the title compound as a solid pink color.

B. Aldehydes 5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid Tertiary butyl 3-oxobutyrate (158 grams, 1 mole) in 200 milliliters of acetic acid was dissolved in a 3-ounce round bottom flask. necks of 500 milliliters, equipped with a thermometer, addition funnel and mechanical agitator. The mixture was cooled in an ice bath at about 10 ° C. Sodium nitrite (69 grams, 1 mole) was added for 75 minutes, keeping the temperature below 15 ° C. The cold bath was removed, and the mixture was stirred for 30 minutes, and then allowed to stand for 3.5 hours to give tertiary butyl 2-hydroxyimino-3-oxobutyrate. The ethyl 3-oxobutyrate (130 grams, 1 mole) was dissolved in 400 milliliters of acetic acid, in a 2-liter 3-necked round bottom flask, equipped with a thermometer, an addition funnel, mechanical stirrer, and placed in an oil bath. Zinc powder (50 grams, 0.76 moles) was added, and the mixture was heated to 60 ° C with stirring. The tertiary butyl 2-hydroxyimino-3-oxobutyrate solution prepared above was added slowly, maintaining the temperature of the reaction mixture at about 65 ° C. Then more zinc powder (4 x 50 grams, 3.06 moles) was added, adding the last portion after all the t-butylester had been added. At the end of the additions, the temperature was 64 ° C. The temperature was increased to 70-75 ° C, stirred for one hour, and then poured into 5 liters of water. The floating gray precipitate was collected by vacuum filtration, and washed with 2 liters of water to give 354 grams of a wet raw product. The crude product was dissolved in 1 liter of hot methanol, and filtered hot to remove the zinc. The filtrate was cooled, upon which a precipitate formed. The precipitate that was collected by vacuum filtration was dried to give 118 grams of the product. The filtrate was placed in the refrigerator overnight, upon which additional product was precipitated. A total of 173.2 grams of 4-ethyl ester of 2-tert-butyl ester of 3,5-dimethyl-lH-pyrrole-2,4-dicarboxylic acid was obtained. The 4-ethyl ester of 2-tert-butyl ester of 3,5-dimethyl-lH-pyrrole-2,4-dicarboxylic acid (80.1 grams, 0.3 moles), and 400 milliliters of trifluoroacetic acid, were stirred for 5 minutes in a bottom flask 3-neck round 2 liters, equipped with mechanical stirrer, and heated to 40 ° C in an oil bath. The mixture was then cooled to -5 ° C, and triethyl orthoformate (67.0 grams, 0.45 moles) was added all at once. The temperature was increased to 15 ° C. The mixture was stirred for about 1 minute, removed from the cold bath, and then stirred for 1 hour. The trifluoroacetic acid was removed by rotary evaporation, and the residue was placed in the refrigerator, where it solidified. The solid was dissolved by heating and pouring in 500 grams of ice. The mixture was extracted with 800 milliliters of dichloromethane to give a red solution and a brown precipitate, both of which were stored. The precipitate was isolated and washed with 150 milliliters of a saturated solution of sodium bicarbonate. The dichloromethane phase was also washed with 150 milliliters of sodium bicarbonate. Then the dichloromethane solution was washed three more times with 100 milliliters of water. The dichloromethane solution was evaporated to dryness. The remaining dark residue was recrystallized twice from ethyl acetate containing Darco carbon black, to give golden yellow needles. The chestnut precipitate was recrystallized from 350 milliliters of ethyl acetate which also contained Darco, to give a yellow-red solid. All the recrystallized solids were combined and recrystallized from 500 milliliters of ethanol to give 37.4 grams (63.9 percent) of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester as yellow needles ( pf 165. 6 - 166.3 ° C, lit. 163 - 164 ° C. The residues obtained after the evaporation of the mother liquors of ethyl acetate and ethanol, they were combined and recrystallized from 500 milliliters of ethanol, to give a second culture (10.1 grams) or product, as dirty yellow needles. 5-Formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid ethyl ester (2 grams, 10 mmol) was added to a solution of potassium hydroxide (3 grams, 53 mmol) dissolved in methanol (3 milliliters) and water (10 milliliters). The mixture was refluxed for 3 hours, cooled to room temperature, and acidified with 6N hydrochloric acid to a pH of 3. The solid that formed was collected by filtration, washed with water and dried in an oven. under vacuum overnight, to give 1.6 grams (93 percent) of 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid. ? H NMR (300 MHz, DMS0-d6) ?: 12.09 (s, br, 2H, Nh &; COOH), 9.59 (s, ÍH, CHO), 2.44 (s, 3H, CH3), 2.40 (s, 3H, CH3). -formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid 2-dimethylaminoethyl) A mixture of 5-formyl-2,4-dimethyl-lH-pyrrole-3-carboxylic acid (1.67 grams, 10 mmol) in dimethylformamide (10 milliliters), was added benzotriazol-1-yloxytris (dimethylamino) -phosphonium hexafluorophosphate (BOP reagent, 6 grams, 13.5 mmol), followed by 3 milliliters of di-isopropylethylamine. After stirring for 5 minutes, 1 milliliter of N, N-dimethylethylenediamine was added, and the mixture was stirred at room temperature for 24 hours. To the reaction mixture was added 25 milliliters of IN sodium hydroxide and 25 milliliters of brine. After stirring for 30 minutes, the reaction mixture was poured into water (100 milliliters), and extracted (3 x 200 milliliters) with 10 percent methanol in dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, and evaporated using a rotary evaporator. The remaining residue was purified by chromatography (silica gel column, 5-10 percent methanol in dichloromethane) to give 1 gram (42 percent) of 5-formyl-2-acid (2-dimethylaminoethyl) amide. , 4-dimethyl-lH-pyrrole-3-carboxylic acid.

X H NMR (360 MHz, DMSO-d 6) d: 11.77 (s, ΔH, NH), 9.53 (s, ΔH, CHO), 7.34 (t, J - 5.6 Hz, ΔH, CONH), 3.27 (m, 2H, CONCI ^ CH,), 2.37 (t, J - 6.8 Hz, 2H, CONCHjCH,), 2.35 (s, 3H, CH,), 2.3 (s, 3H, CH,), 2.17 (s, 6H, 2 X CH ,). MS m / z 238.3 [M + l] *. 3, 5-Dimeti-1-4- (4-methyl-piperazine-1-carbonyl) -lH-pyrrole-2-carboxaldehyde To a mixture of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ( 1.67 grams, 10 millimoles) in dimethylformamide (10 milliliters), was added benzotriazole-i-yloxytris (dimethylamino) -phosphonium hexafluorophosphate (BOP reagent, 6 grams, 13.5 mmol), followed by 3 milliliters of di-isopropylethylane. After stirring for 5 minutes, 2 milliliters of 1-methylpiperazine was added, and the mixture was stirred at room temperature for 24 hours. Then 25 milliliters of IN sodium hydroxide and 25 milliliters of brine were added to the reaction. After stirring for 30 minutes, the reaction mixture was poured into water (100 milliliters), and extracted (3 x 200 milliliters) with 10 percent methanol in dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, and evaporated on a rotary evaporator. The residue that remained was purified by chromatography (silica gel column, 5-10 percent methanol in dichloromethane) to give 1 gram (40 percent) of 3,5-dimethyl-4- (4-methylpiperazine-1). carbonyl) -lH-pyrro1-2-carboxaldehyde.

* H NMR (360 MHz, DMS0-d6) d: 1 * ÍH, CHO), 3.14 (br m, 4H, 2xCH2), (S, -3H, CH,), 2.17 (s, 3H, CH,), 2.1- »MS The 249 [MI *.

C. Examples - Synthesis of substituted 2-indolinones or pyrrole The following syntheses of the representative compounds of this invention are shown by way of example only, and should not be construed to limit the scope of this invention to the synthetic approach or to the compounds comprising this invention.

Example 1 3- [5-Chloro-2-oxo-l, 2-dihydroindol-3-ylidene-methyl) -4-methyl-iH-pyrrol-3-yl] -propionic acid 4- (2-carboxyethyl) - 2-formyl-3-methylpyrrole (4.5 grams), 4.2 grams of 5-chloro-2-oxindole, and 2.9 milliliters of piperidine in 50 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in acetone, and the yellow precipitate was filtered, washed with cold ethanol, 2N aqueous hydrochloric acid, and water at a pH of 6, then dried in a vacuum oven overnight to give 7.2 grams of the compound of the title (88 percent) as a yellow solid. * HNMR (360 MHz, DMS0-d6): d 13.31 (S, br, ÍH, NH-1 '), 12.06 (s, br, ÍH, COOH), 10.88 (s, br, ÍH, NH-1), 7.93 (d, J « 1. 88Hz, ÍH, H-4), 7.75 (a, ÍH, H-vim'lo), 7.19 (d, J «3.1 Hz, ÍH, H-2 '), 7.1 (dd, b, J - 1.88,8.40 Hz, ÍH, H-6), 6.84 (d, J = 8.40 Hz, ÍH, H-7), 2.65 (t, J - 7.44 Hz, 2H, CH-CH.COOH), 2.46 (t, J »7.44 Hz, 2H, CH, CH, C0OH), 2.28 (s, 3H, CH,). Example 2 3- [5- (6-Methoxy-2-oxo-l 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid 4- (2-carboxyethyl) - 2-formyl-3-methylpyrrole (190 milligrams), 163 milligrams of 6-methoxy-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated at 90 ° C for 3 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 140 milligrams of the title compound (43 percent) as a tan solid.

JHNMR (360 MHz, DMSO-dd): d 13.1 (s, br, ÍH, NH-1 '), 12.04 (s, br, ÍH, COOH), 10.76 (s, br, ÍH, NH-1), 7.63 (d, J = 8.2.9Hz, ÍH, H-4), 7.46 (s, ÍH, H-vini? Q), 7.07 (d, J - 3.03 Hz, ÍH, H-2 '), 6.55 (dd, J - 2.32, 8.29Hz, ÍH, H-5), 6.43 (d, J = 2.32 Hz, ÍH, H-7), 3.74 (s, 3H, OCH,), 2.63 (t, J =. 7.31 Hz, 2H, CHjCHjCOOH), 2.45 (t, J - 7.31 Hz, 2H, CH ^ COOH), 2.23 (s, 3H, Oi,); MS m / z (Relative Strength,%) 327 ([M + l] *, 100).

Example 3 3- [5- (5-Chloro-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid 3- (2- carboxyethyl) -2,4-dimethyl-5-formylpyrrole (220 milligrams), 147 milligrams of 5-chloro-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated at 90 ° C for 3 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 172 milligrams of the title compound (50 percent) as a solid. Brown.

* HNMR (360 MHz, DMSO-d6): d 13.42 (s, br, ÍH, NH-1 '), 12.03 (s, br, ÍH, COOH), 10.80 (s, br, ÍH, NH-1), 7.87 (d, J - 2.06Hz, ÍH, H-4), 7.67 (s, ÍH, H-Villile), 7.06 (dd, J »2.06, 8.3Hz, ÍH, H-6), 6.83 (d, J »8.3 Hz, ÍH, H-7), 2.64 (t, J * 7.6 Hz, 2H, CHjCH, COOH), 2.34 (t, J = 7.6 Hz" 2H, CH, CH, COOH), 2.29 (s, 3H, CH,), 2.27 (s, 3H, CH,); MS m / z (relative intensity - *) 345 ([M + l] *, 64).

EXAMPLE 4 3- [4-Methyl-5- (2-oxo-1,2-dichloroindo-3-ylidemethyl) -lH-pyrrol-3-yl] -propionic acid. Sodium metal (1.5 grams) was placed in a flask. 3-neck round bottom, 3 liters, equipped with a thermometer, reflux condenser and mechanical stirrer, and placed in an oil bath. Absolute ethanol (1 liter) was added with stirring. When the sodium was dissolved, 350 grams of pentan-2,4-dione were added all at once, and then 310 grams of ethyl acrylate were added for 30 minutes. The mixture was refluxed for 2.5 hours, and then allowed to cool to room temperature overnight. Glacial acetic acid (3 milliliters) was added, and the solvent was removed by rotary evaporation. The residue was filtered through a pad of diatomaceous earth, and distilled in a film cleaned at 0.1 millimeters. The distillate was made distilled using a 25.4 centimeter vacuum-jacketed Vigreux column to give 518 grams of ethyl 5-acetyl-4-oxohexanoate, boiling point: 84-92 ° C at 0.2-0.7 millimeters.

To a 5-liter three neck flask, equipped with a thermometer and mechanical stirrer, and heated on a steam bath, were added 350 grams of ethyl 5-acetyl-4-oxohexanoate, 329 grams of ethyl aminomalonate hydrochloride, 133 grams of sodium acetate, and 1.2 liters of acetic acid. The mixture was heated at 99 ° C for 37 minutes. At 62 ° C, the release of carbon dioxide was already rapid. After a total of 35 minutes at 99 ° C, the evolution of C02 gas became very slow. After another hour, the mixture was cooled, the sodium chloride was removed by vacuum filtration, and the solvent was evaporated. The residue was mixed with 1 liter of cold water. The precipitate was collected by vacuum filtration, washed with 400 milliliters of water, and dissolved in 1 liter of 95 percent hot ethanol. The solution was treated with 20 grams of Darco G-60, filtered hot, and cooled to room temperature. The crystalline solid was collected by vacuum filtration, washed twice on the filter with 200 milliliters of 50 percent ethanol, and dried under vacuum at 70 ° C to give 265 grams (64 percent yield) of 2- ethoxycarbonyl-4- (2-ethoxycarbonylethyl) -3,5-dimethylpyrrole. The filtrate was refrigerated overnight to give another 53.1 grams (yield of 11.9%) of the product, for a total yield of 75.9 percent. 2-Ethoxycarbonyl-4- (2-ethoxycarbonylethyl) -3,5-dimethylpyrrole (285 grams), and 3500 milliliters of ethyl ether were placed in a three-neck, five-liter flask equipped with a mechanical stirrer, a reflux condenser, and an addition funnel, and cooled in an ice bath. Sulfuryl chloride (435 grams) was added dropwise over 145 minutes. As the addition proceeded, the mixture became cloudy and green, and then cleared. At the end of the addition, the mixture was clear and slightly yellow. The mixture was stirred for an additional hour, and then heated to reflux for 1 hour. The mixture was cooled on a rotary evaporator, diluted with 1,500 milliliters of ether, and rotary evaporated again. The dilution and evaporation were repeated again. The residue was added to 8 liters of water containing 802 grams of acetic acid and 535 grams of sodium hydroxide. The mixture was briefly heated to 85 ° C, and then allowed to cool overnight with stirring. The aqueous layer, which contained solids, was separated and extracted with 800 milliliters of ether. The solids and the ether layer were added to 2.5 liters of water, containing 300 grams of sodium carbonate, stirred for 1 hour, and filtered to remove a small amount (approximately 7 grams) of a solid. Sulfuric acid (137 grams) was added to the mixture, and the resulting precipitate was washed twice with 250 milliliters of water, and dried under vacuum to give 56.4 grams of the product. Sulfuric acid (92 grams) was added to the filtrate, and the resulting precipitate was washed twice with 0.5 liters of water, and dried under vacuum to give 220 grams of the product, for a total of 276.4 grams (86.8 percent yield) of 2-carboxy-5-ethoxycarbonyl-3- (2-ethoxycarbonylethyl) -4-methylpyrrole. The 2-carboxy-5-ethoxycarbonyl-3- (2-ethoxycarbonyl-ethyl) -4-methylpyrrole (50.5 grams), and 400 milliliters of a 10 percent sodium hydroxide solution, were heated to 180 ° C in a Parr autoclave for 90 minutes. This process was repeated four more times, until a total of 252.5 grams of 2-carboxy-5-ethoxycarbonyl-3- (2-ethoxycarbonylethyl) -4-methylpyrrole had been treated. The five solutions were combined and rotated evaporably to a volume of approximately 1.8 liters of a thick black residue. The mixture was cooled to 10 ° C in a water bath, and 50% sulfuric acid was slowly added to maintain the temperature at <; 20 ° C, until the pH was 2. Ethylether (1,400 milliliters) was added, the mixture was filtered, and the precipitate was saved. The precipitate was extracted in a Soxhlet extractor with 500 milliliters of ether. The combined ether layers were washed with 250 milliliters of water, followed by 150 milliliters of water. The combined water layers were back-extracted with 150 milliliters of ether. All the ether layers were rotary evaporated, and the residue was dried to give 123.5 grams of 3- (2-carboxyethyl-4-methylpyrrole.

The 3- (2-carboxyethyl) -4-methylpyrrole (123 grams) was mixed with 1,500 milliliters of ethyl ether and 250 milliliters of methanol in a magnetically stirred receiving flask. A separate, 3-liter 3-neck round bottom flask was equipped with a magnetic stirrer, a distillation head, and a condenser leading to the inlet of the receiving flask, and heated in a water bath. In the 3 liter flask were placed 240 grams of Diazald dissolved in 1,800 milliliters of ethyl ether, and a solution of 73 grams of potassium hydroxide dissolved in 360 milliliters of 95 percent ethanol, and 112 milliliters of water. The 3-liter flask was stirred and heated to 65-75 ° C in a water bath, and the diazomethane-ether mixture was distilled in the stirred flask for about 2.5 hours. Ethylether (200 milliliters) was added to the 3 liter flask, and the distillation continued until complete. The receiving flask was stirred for another 30 minutes, and then 10 milliliters of acetic acid were added. The mixture was extracted twice with 500 milliliters of water, then twice with 200 milliliters of saturated sodium bicarbonate. The ether layer was dried over anhydrous sodium sulfate, and distilled to leave a dark fluid residue. The residue was distilled twice through a Vigreux column of 10.16 centimeters, and once through a Vigreux vacuum-jacketed column of 25.4 centimeters to give 108 grams (80.6 percent yield) of 3- (2-ethoxycarbonylethyl) - 4-methylpyrrole. Boiling point: 108-113 ° C to 0.5 mm. Dimethylformamide was charged to a 500 milliliter, 3-necked round bottom flask equipped with a mechanical stirrer, a thermometer, and a dropping funnel, and kept under a nitrogen atmosphere. The flask was cooled to 0 ° C and 58.4 milliliters of phosphorus oxychloride were added dropwise over 80 minutes. Dichloroethane (280 milliliters) was added, and the mixture was allowed to warm to room temperature, and then cooled to -10 ° C. 3- (2-Methoxycarbonyl-ethyl) -4-methylpyrrole (55.7 grams) dissolved in 80 milliliters of dichloroethane was added dropwise over 1 hour, and the mixture was stirred for another 35 minutes. The mixture was rotary evaporated to < 30 ° C. The fluid residue was poured into 2,700 milliliters of an ice cold 2 N sodium hydroxide solution. The resulting solution was heated at 88 ° C for 20 minutes, and then kept at this temperature for an additional 30 minutes. The solution was cooled to room temperature, and extracted with 200 milliliters of ethyl ether. The aqueous solution was cooled to 0 ° C, and acidified to a pH of 3.5, slowly adding approximately 1,350 milliliters of 5 N hydrochloric acid. The yellow precipitate was collected by vacuum filtration, washed four times with 100 milliliters of water, and dried in a vacuum oven at room temperature to give 54.4 grams (90.2 percent yield) of crude 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole.

The crude material was placed in a reflux mixture of 425 milliliters of ethanol and 700 milliliters of ethyl ether, and filtered hot to remove an insoluble residue, which was retained. The filtrate was placed in the freezer, and the resulting precipitate was collected by vacuum filtration, and washed with 50 milliliters of ether. The filtrate was used to extract the insoluble residue again, it was filtered hot, and put in the freezer. The resulting precipitate and the first precipitate were combined to give 26.1 grams of 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole as a chestnut powder, m.p. 149.0 - 150.3 ° C. The filtrate was combined with the filtrate from the previous preparation, and concentrated to give 43 grams of a tan solid. The solid was placed in a reflux mixture of 500 milliliters of ether and 100 milliliters of ethanol, and filtered. The filtrate was treated with refluxing Norit, and filtered hot again. The filtrate was placed in the freezer to give 3 additional cultures of 4- (2-carboxyethyl) -2-formyl-3-ethylpyrrole, 7.7 grams, m.p. 148-151 ° C, 3.2 grams, m.p. 128-134 ° C, and 4.1 grams, m.p. 148.2-150.0 ° C. The 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (9.0 grams), and 6.0 grams of 2-oxindole in 50 milliliters of ethanol, were heated to 70 ° C in a 3-necked round bottom flask, of 250 milliliters, equipped with a thermometer, a reflux condenser, and magnetic stirrer. When most of the solids were dissolved, 4.5 grams of piperidine were added slowly, and the mixture was refluxed for 4 hours. Acetic acid (12 milliliters) was added slowly, resulting in a copious precipitate. The mixture was refluxed for 5 minutes, cooled to room temperature, and the precipitate was collected by vacuum filtration, and washed with 30 milliliters of ethanol. The precipitate was washed at reflux in 30 milliliters of ethanol, cooled to room temperature, collected by vacuum filtration, washed with 20 milliliters of ethane, and dried in vacuo to give 11.9 grams (80 percent yield). ) of 3- [4- (2-carboxyethyl) -3-methylpyrrol-2-methylindenyl] -2-indolinone, SU6663, as an orange solid.

* HNMR (360 MHZ, DMS0-d6): d 13.29 (s, br, ÍH, NH-1 '), 12.05 (s, br, ÍH, COOH), 10.78 (s, br, ÍH, NH-1), 7.73 (d, J = 7.43Hz, HH, H-4), 7.61 (s, HH, H-Vinilq, 33 { D> gn 2 15 Hz, HH, H-2 '), 7.10 (t , J - 7.43 Hz, ÍH, H-6), 6.97 (t, J - 7.43 Hz, ÍH, H-5), 6.85 (d, J- 7.43 Hz, ÍH, H-7), 2.64 (t, J - 7.38 Hz, 2H, CHjCHjCOOH), 2.46 (t, J- 7.38 Hz, 2H, CH2CHJCOOH), 2.25 (s, 3H, CH,); MS m / z (relative strength t%) 297 ([M + l] *, 100).

Example 5 3- [2,4-Dimethyl-5- (2-oxo-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid 2-4-dimethyl-5-ethoxycarbonyl -3- (2-ethoxycarbonyl-ethyl) -pyrrole (1.07 kilograms) and 3.2 liters of 5 N sodium hydroxide were mechanically stirred in a 12-liter three-necked round bottom flask equipped with a reflux condenser and a addition funnel, and heated in an oil bath. The mixture was refluxed for 3 hours, after which the internal temperature was 96 ° C, all solids were dissolved, and thin layer chromatography showed that hydrolysis was complete. The heating bath was removed, and the mixture was cooled to 50 ° C in a water bath. 12N hydrochloric acid (approximately 1.3 liters) was added slowly. After approximately 50 percent of the acid was added, gas evolution started, and the temperature reached 60 ° C. As more acid was added, the gas evolution increased, and a yellow precipitate formed. The final pH was adjusted to 3.5 with hydrochloric acid. The mixture was cooled in an ice bath at 8 ° C. The solids were collected by vacuum filtration, washed twice with 0.5 liters of distilled water, and dried for 48 hours in a vacuum oven at 55-60 ° C, to give 677 grams (101 percent yield) of 3- (2-carboxyethyl) -2,4-dimethylpyrrole. lHNMR (d4-DMSO) d 11.9 (s, ÍH, COOH), 9.9 (s, ÍH, NH), 6.2 (s. ÍH, aromatic), 2.5 (t, 2H, CH2), 2.2 (t, 2K, CH ,), 2.0 (s, 3H, CH,), 1.9, (8, 3H, .CH,); MP 134-136 ° C.

Dimethylformamide (28.5 grams) in 250 milliliters of dichloromethane in a 1-liter three-necked round bottom flask equipped with a magnetic stirrer, a thermometer and a dropping funnel was cooled in an ice-salt bath to -1 ° C. Phosphorus oxychloride (59.3 grams) was placed in the dropping funnel, and added slowly to the reaction mixture. The funnel was flooded with 25 milliliters of dichloromethane to ensure all of the phosphorus oxychloride. The maximum temperature reached by the mixture was 5 ° C. The mixture was stirred for 15 minutes, at which time the temperature was -3 ° C. Solid 3- (2-carboxyethyl) -2,4-dimethylpyrrole (32.6 grams) was added in portions over 15 minutes. The maximum temperature reached by the mixture was 7 ° C. The reddish black mixture was stirred for a further 30 minutes, and then heated to reflux for 1 hour. The mixture was cooled to 15 ° C, and 300 milliliters of water were added, leading to a vigorous reaction, during which the temperature was increased. The mixture was stirred and cooled to 22 ° C, and the layers were separated and stored. The organic layer was extracted with 100 milliliters of water, and the aqueous layers were combined and washed with 50 milliliters of dichloromethane. The organic layers were discarded. The aqueous layer was adjusted to a pH of 11, with approximately 180 milliliters of 10 N sodium hydroxide. The temperature was increased to 40 ° C. The mixture was stirred for 30 minutes, at which time the temperature was 27 ° C. The mixture was acidified to a pH of 2, with approximately 120 milliliters of 10 N hydrochloric acid, which increased the temperature to 30 ° C. Ethyl acetate (150 milliliters) was added, and the mixture was stirred to extract the product. During the stirring, a considerable amount of black solid appeared on top of the water layer. The ethyl acetate layer was separated, and the aqueous layer and the solid were extracted twice with 100 milliliters of ethyl acetate. The solid still present was collected by vacuum filtration, washed thoroughly with water, and dried under vacuum at 40 ° C to give 12 grams (31 percent yield) of 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole as a brown black solid . Thin layer chromatography (dichloromethane: acetic acid, 95: 5, silica gel) showed a spot at Rf 0.7, and a colored spot at the origin. The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, and evaporated to obtain a brown chestnut solid, which was dried under vacuum at 40 ° C to give 21 grams (55 percent yield, 86 percent total yield) of 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole, of identical appearance to the above solid, by thin layer chromatography. Alternatively, dimethylformamide (124 milliliters) in 750 milliliters of dichloromethane, in a 5-liter, three-necked round bottom flask, equipped with a mechanical stirrer, a thermometer, and a dropping funnel, was cooled in a bath of ice-salt at -9 ° C. Phosphorus oxychloride (114 milliliters) was added quickly by means of the dropping funnel, which was flooded in the reaction mixture with 50 milliliters of dichloromethane. The maximum temperature reached by the mixture was -4 ° C. Solid 3- (2-carboxyethyl) -2,4-dimethylpyrrole (133.6 grams) was added in portions over 20 minutes. The maximum temperature reached by the mixture was 3 ° C. The dark reddish mixture was heated to reflux for 1 minute, and then cooled to -1 ° C. The mixture was cooled to 1 ° C, and 800 milliliters of ice water was added quickly. The maximum temperature reached was 15 ° C. The organic layer was separated and discarded. The aqueous layer was slowly adjusted to a pH of 12-13 with approximately 800 milliliters of 10 N potassium hydroxide, adding ice to control the temperature. The temperature was increased to 37 ° C. The mixture was stirred for 90 minutes at room temperature, at which time the thin layer chromatography showed only a trace of light colored material at the origin, with the product at Rf 0.3. The mixture was cooled to 0 ° C. The mixture was acidified to a pH of 3 with approximately 600 milliliters of 10 N hydrochloric acid, with ice added to control the temperature. The maximum temperature reached was 10 ° C. The mixture was stirred for 1 hour in cold. The solid was collected by vacuum filtration, washed 4 times with 100 milliliters of water, and dried under vacuum at 50-60 ° C, to give 140.6 grams (90 percent yield) of 3- (2-carboxyethyl) -2, 4-dimethyl-5-formylpyrrole as a chestnut solid. lHNMR (dj-DMSO): d 12.0 (s, ÍH, COOH), 11.3 (s, ÍH, NH), 9.4 (s, ÍH, CHO), 2.6 (t, 2H, CH2), 2.3 (t, 2H, CH,), 2.2 (s, 3H, CH,), 2.1 (s, 3H, CH,). MP 145-147 ° C.

The 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (18.2 grams), and 11.7 grams of 2-oxindole, were dissolved in 100 milliliters of ethanol, heating in a 250 milliliter round bottom flask, equipped with a magnetic stirrer and a reflux condenser in an oil bath. Pyrrolidine (7.0 grams) was added, and the reaction mixture was refluxed for 2 hours, at which time a large amount of chestnut black solid was present. Thin layer chromatography (ethyl acetate: ethanol: acetic acid, 96: 2: 2, silica gel) showed the ace of oxindole starting material. 8 milliliters of acetic acid were added, and the mixture was refluxed for 15 minutes. The thick mixture was diluted with 50 milliliters of ethanol, and cooled to 10 ° C. The solid was collected by vacuum filtration, and washed with 50 milliliters of ethanol. The solid was stirred in 125 milliliters of refluxing ethanol for 10 minutes, cooled to 10 ° C, collected by vacuum filtration, and washed with 50 milliliters of ethanol. The product was dried overnight at 45 ° C under vacuum, to give 25.5 grams (88 percent yield) of 3- [2,4-dimethyl-3- (2-carboxyethyl) pyrrol-5-methylinde-nil] -2-indolinone as an orange solid. Alternatively, a mixture of 3- (5-formyl-2,4-dimethyl-lH-pyrrol-3-yl) -propionic acid (10 grams, 51 mmol), 2-oxindole (6.5 grams, 49 mmol) , and sodium hydroxide (40 grams, 58 mmol) dissolved in 50 milliliters of water, was stirred at 50 ° C for 4 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was acidified to a pH of 3, with 12 N hydrochloric acid. The solid that was precipitated was collected by vacuum filtration, washed with 10 milliliters of water, and dried under vacuum overnight. The crude solid paste was washed with hot ethanol twice. The solid was then collected by vacuum filtration, washed with 10 milliliters of ethanol, and dried under vacuum to give 13.8 grams (91 percent) of 3- [2,4-dimethyl-5- (2-oxo- 1, 2-dihydro-indol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid. lHNMR (360 MHz, DMSO-d6): d 13.38 (s, br, ÍH, NH-1 '), 12.05 (s, br, ÍH, COOH), 10.70 (s, br, ÍH, NH-1), 7.69 (d, J = 7.39Hz, ÍH, H-4), 7.53 (s, ÍH, H-vinyl), 7.06 (t, J - 7.39 Hz, 1H, H-6), 6.95 (t, J- 7.39 Hz , ÍH, H-5), 6.85 (d, J = .7.39 Hz, ÍH, H-7), 2.63 (t, J - 7.45 Hz, 2H, CH, CH, COOH), 2.34 (t, J = 7.45 Hz, 2H, CHjCH, C0OH), 2.28 (s, 3H, CH,), 2.24 (s, 3H, CH,); MS m / z relative intensity,%) 31.1 ([M + l] *, 100).

Example 6 3- [5- (5-Bromo-2-oxo-l, 2-dihydroindol-3-ylidene-methyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid 2-Oxindole (53.3) grams) was suspended in 640 milliliters of acetonitrile, and the mixture was cooled to 7 ° C in an ice bath with mechanical stirring. Solid N-bromosuccinimide (74.8 grams) was added in portions over 20 minutes. After approximately one third of the N-bromosuccinimide had been added (for 5 minutes), the temperature was increased to 12 ° C. The addition was stopped until the temperature of the mixture had dropped to 10 ° C. The addition was resumed keeping the temperature below 12 ° C. After the addition was complete, the mixture was stirred for 1 hour at 10 ° C, and then for an additional 1 hour, during which time, the mixture was allowed to warm to room temperature. The precipitate was collected by vacuum filtration, washed with 80 milliliters of ethanol, and sucked to dry for 20 minutes in the filtration funnel to give the product containing 6.4 percent 2-oxindole by HPLC. The solid was suspended in 1440 milliliters of denatured ethanol, and washed in paste by stirring and refluxing for 5 minutes, during which time most of the solid dissolved. The mixture was cooled in an ice bath at 13 ° C. The solid product was collected by vacuum filtration, washed with 80 milliliters of ethanol, and dried under vacuum to give 57.7 grams (68.0 percent) of 5-bromo-2-oxindole containing 1.13 percent 2-oxindole by HPLC. Washing in paste with 30 percent less ethanol gave a better yield (88 percent), but contained more 2-oxindole (1.76). lHNMR (360 MHz, DMS0-d6): d 10.44 (s, br, ÍH, NH-1), 7.32- 7.36 (m, 2H), 6.76 (d, J - 8.50 Hz, ÍH, H-7), 3.5 (s, 2H, CH,); MS / z 212.1 / 214.1 (M * / [M + 2] *). 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90 milligrams), 106 milligrams of 5-bromo-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 2N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 120 milligrams (64 percent) of the title compound as a solid. Brown.

'HNMR (360 MHZ, DMS0-d6): d 13.31 (s, br, ÍH, NH-1'), 12.06 (a, br, ÍH, COOH), 10.90 (s, br, ÍH, NH-1), 8.06 (s, br, ÍH, H-4), 7.75 (s, ÍH, H-vinild »7.23 (d, br, J« 8.50 Hz, ÍH, H-6), 7.19 (d, J - 2.84 Hz, ÍH, H-2 '), 6.80 (d, br, J - 8.50 Hz, ÍH, H-7), 2.65 (t, J- 7.65 Hz, 2H, CH, CH, C00H), 2.46 (t, J - 7.65 Hz, 2H, CHjCH, C0OH), 2.28 (s, 3H, CH,); MS m / z 375.1 / 377.2 (M * / [M + 2] *).

Example 7 3- [5- (5-iodo-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] -propionic acid 2-oxindole (82.9 grams) it was suspended in 630 milliliters of acetic acid and the mixture was mechanically stirred and cooled to 10 ° C in an ice water bath. Solid N-iodosuccinimide (175 grams) was added in portions for 10 minutes. After the addition was completed, the mixture was stirred for 1 hour at 10 ° C. The suspended solid that was always present became very thick at this time. The solid was collected by vacuum filtration, washed with 100 milliliters of 50 percent acetic acid in water, and then with 200 milliliters of water, and sucked to dry for 20 minutes in the filtration funnel. The product was dried under vacuum to give 93.5 grams (36 percent) of 5-iodo-2-oxindole containing approximately 5 percent of 2-oxindole by nuclear magnetic resonance of protons.

* HNMR (360 MHz, DMSO-d6): d 10.45 (s, ÍH, NH-1), 7.49 (s, 1H, H-4), 7.48 (d, J "8.10 Hz, ÍH, H-6), 6.64 (d, J- 8.10 Hz, ÍH, H-7), 3.46 (s, 2H, CH, -3); MS m / z 258 [M-l] *. 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90 milligrams), 130 milligrams of 5-iodo-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 2N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 162 milligrams (77 percent) of the title compound as a solid. Brown.

XHNMR (360 MHz, DMS0-d6): d 13.30 (s, br, 1H, NH-1 '), 12.06 (s, br, 1H, COOH), 10.88 (s, br, 1H, NH-1), 8.18 (s, br, ÍH, H-4), 7.73 (s, ÍH, H - vinyl9 (7.40 (d, br, J = 8.03 Hz, ÍH, H-6), 7.19 (d, J »2.94 Hz, ÍH, H-2 '), 6.69 (d, br, J - 8.03 Hz, ÍH, H-7), 2.65 (t, J- 7.40 Hz, 2H, CHjCH, COOH), 2.46 (t, J = »7.40 Hz, 2H, CHjCHjCOOH), 2.28 (s, 3H, CH,) / MS m / z 423 [M + 1] *.

Example 8 3- [4-Methyl-5- (4-methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid Diethyl oxalate (30 milliliters ) in 20 milliliters of dry ether with stirring, to 19 grams of potassium ethoxide suspended in 50 milliliters of dry ether. The mixture was cooled in an ice bath, and 20 milliliters of 3-nitro-o-xylene in 20 milliliters of dry ether were added slowly. The thick dark red mixture was refluxed for 0.5 hour, concentrated to a dark red solid, and treated with 10 percent sodium hydroxide, until almost all of the solid dissolved. The dark red mixture was treated with 30 percent hydrogen peroxide until the red color changed to yellow. The mixture was treated alternately with 10 percent sodium hydroxide and 30 percent hydrogen peroxide until the dark red color was no longer present. The solid was filtered, and the filtrate was acidified with 6 N hydrochloric acid. The resulting precipitate was collected by vacuum filtration, washed with water, and dried in vacuo to give 9.8 grams (45 percent yield) of the acid. -methyl-6-nitrophenylacetic as a white solid. The solid was hydrogenated in methanol over 10 percent palladium on carbon to give 9.04 grams of 4-methyl-2-oxindole as a white solid.

XHNMR (360 MHz, DMS0-d6): d 10.27 (s, br, ÍH, NH-1), 7.06 (t, J = 7.71 Hz, ÍH, H-6), 6.74 (d, J - 7.73 Hz, H -5), 6.63 (d, J - 7.73 Hz, ÍH, H-7), 3.36 (s, 2H, CH,), 2.18 (s, 3H, CH,). 4- (2-Carboxyethyl) -2-formyl-3-methylpyrrole (90 milligrams), 74 milligrams of 4-methyl-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated to. 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6-N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 80 milligrams (52 percent) of the title compound as a tan solid.

'HNMR (360 MHz, DMSO-d6): d 13.33 (s, br, 1H, NH-1'), 10-84 (s, br, 1H, H-1), 7.54 (s, 1H, H-Vinyl) *, 7.12 (d, J -2-0 HZ, 1H, H-2 '), 7.01 (t, J «7.75 Hz, 1H, H-6), 6.79 (d, J = 7.75 Hz, H-5) , 6.74 (d, J - 7.75 Hz, ÍH, H-7), 2.64 (t, J -7-S5 Hz, 2H, CHjCHjCOOH), 2.57 (s, 3H, CH3), 2.42 (t, J - 7.65 Hz , 2H, CH, CH, COOH), 2.19 (s, 3H, CH,), MS / z (-relative intensity,%) 311 ([M + l] *, 100).

EXAMPLE 9 3- [4-Methyl-5- (5-methyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -iH-pyrrol-3-yl] propionic acid 5-Methylisatin (15.0 grams), and 60 milliliters of hydrazine hydrate were heated at 140-160 ° C for 4 hours. Thin layer chromatography (ethyl acetate: hexane 1: 2, silica gel) showed no remaining starting material. The reaction mixture was cooled to room temperature, poured into 300 milliliters of ice water, and acidified to a pH of 2 with 6N hydrochloric acid. After standing at room temperature for 2 days, the precipitate was collected by filtration. vacuum, washed with water, and dried under vacuum to give 6.5 grams (47 percent yield) of 5-methyl-2-oxindole. lHNMR (360 MHz, DMS0-d6): d 10.20 (s, br, ÍH, NH-1), 6.99 (s, 1H, H-4), 6.94 (d, J- 8.11 Hz, ÍH, H-6), 6.68 (d, Ja 8. 11 Hz, 1H, H-7), 3.39 (s, 2H, CH, -3), and 2.22 (s, 3H, CH, -5).

The 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90 milligrams), 74 milligrams of 5-methyl-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours. hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 65 milligrams (42 percent) of the title compound as a solid. Brown.

'HNMR (360 MHz, DMS0-d6): d 13.30 (s, br, ÍH, NH-1'), 12.05 (s, br, 1 H, COOH), 10.67 (s, br, ÍH, NH-1) , 7.57 (s, 2H, H-'Vi'tilo H-4), 7.12 (d, J- 2.65 Hz, 1H "H-2 '), 6.91 (d, J - 7.82 Hz, ÍH, H-6) , 6.74 (d, J - 7.82 Hz, 1H, H-7), 2.65 (t, J = 6.94 Hz, 2H, CHjCHjCOOH), 2.46 (t, J at 6.94 Hz, 2H, CHjCH, COOH), 2.30 (s) , 3H, CH,), 2.25 (s, 3H, CH,); MS m / z (relative intensity.%) 311 ([M + l] *, 100).

EXAMPLE 10 3- [5- (5,6-Dimethoxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] -propionic acid 4- (2- carboxyethyl) -2-formyl-3-methylpyrrole (90 milligrams), 97 milligrams of 5,6-dimethoxy-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 2 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 104 milligrams (58 percent) of the title compound as a solid. Brown.

* HNMR (360 MHz, DMSO-d6): d 13.19 (s, br, ÍH, NH-1 '), 12. 05 (s, br, 1 H, COOH), 10.53 (s, br, ÍH, NH-1), 7.46 (s, ÍH), 7.41 (3, ÍH), 7.02 (s, ÍH, H-2 '), 6.45 (s, ÍH), 3.74 (s, 3H, OCH,), 3.70 (s, 3H, OCH,), 2.59 (t, J - 7.43 Hz, 2H, CH, CH, COOH), 2.44 (t, Ja 7.43 Hz, 2H, CH, CH, COOH) , 2.22 (s, 3H, CH,); MS / z 357 [M + l] *.

Example 11 3- [5- (6-Chloro-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid 4- (2-carboxyethyl) acid -2-formyl-3-methylpyrrole (200 milligrams), 167.6 milligrams of 6-chloro-2-oxindole, and 166 microliters of piperidine in 2 milliliters of ethanol, were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 246 milligrams (74 percent) of the title compound as a solid. Brown. lHNMR (360 MHZ, DMSO-d6): d 13.22 (s, br, ÍH, NH-1 '), 12.09 (s, br, 1 H, COOH), 10.95 (s, br, ÍH, NH-1), 7.78 (d, J = 7.95 Hz, ÍH, H-4), 7.66 (s, ÍH, H-vinyl), 7.18 (d, J - 2.64 Hz, ÍH, H-2 '), 7.01 (dd, J - 1.90, 7.95 Hz, ÍH, H-5), 6.86 (d, J at 1.90Hz, ÍH, H-7), 2.65 (t, J - 7.14Hz, 2H,, 2.45 (t, J at 7.14 Hz, 2H , CH, CH, COOH), 2.26 (s, 3H, CH,).

EXAMPLE 12 3- [4- (2-Carboxyethyl) -3-methyl-lH-pyrrol-2-ylmethylene] -2-oxo-2,3-dihydro-lH-indole-5-carboxylic acid methyl ester was refluxed 5-iodo-2-oxindole (17 grams) with 2 grams of palladium diacetate, 18.2 of triethylamine, 150 milliliters of methanol, 15 milliliters of dimethyl sulfoxide, and 2.6 grams of DPPP in an atmosphere saturated with carbon monoxide. After 24 hours, the reaction mixture was filtered to remove the catalyst, and the filtrate was concentrated. The concentrate was chromatographed on silica gel using 30 percent ethyl acetate in hexane. The fractions containing the product were concentrated and allowed to stand. The product was precipitated and collected by vacuum filtration to give 0.8 grams (7 percent) of 5-methoxycarbonyl-2-oxindole as a white solid.

'HNMR (360 MHz, DMSO-d6) d 10.70 (s, br, ÍH, NH-1), 7.83 (dd, J-1.77, 8.29 Hz, ÍH, H-6), 7.77 (s, br, ÍH, H-4), 6.89 (d, J »8.29 (Hz, ÍH, H-7), 3.80 (s, 3H, COOCH, -5), 3.51 (s, 2H, CH, -3).

The 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.6 milligrams), 88.6 milligrams of 5-methoxycarbonyl-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol, were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 123 milligrams (69 percent) of the title compound as a solid. yellow.

'HNMR (360 MHz, DMSO-d6): d 13.27 (s, br, ÍH, NH-1'), 12.0 (s, vbr, 1H, COOH), 11.16 (s, br, ÍH, NH-1), 8.36 (s, br, ÍH, H-4), 7.80 (s, ÍH, H-Vim'lq, 7.40 (dd, J - 1.80, 8.14 Hz, ÍH, H-6), 7.20 (d, J - 2.91 Hz, ÍH, H-5 '), 6.96 (d, J = 8.14 Hz, ÍH, H-7), 3.84 (s, 3H, COOCH,), 2.66 (t, J == 7.55 Hz, 2H, CH2CH, COOH), 2.46 (t, J = 7.55 Hz, 2H, CHjCH;, COOH), 2.30 (s, 3H, CH,); MS m / z (relative intensity%) 355 ([M + l] *, 100) .

Example 13 3- [4- (2-Carboxyethyl) -3-methyl-lH-pyrrol-2-ylmethylene) -2-0x0-2, 3-dihydro-lH-indole-5-carboxylic acid 2-oxindole (6.7) grams) was added to a stirred suspension of 23 grams of aluminum chloride in 30 milliliters of dichloroethane in an ice bath. Chloroacetyl chloride (11.3 grams) was added slowly, and hydrogen chloride gas was evolved. After ten minutes of stirring, the reaction was heated at 40-50 ° C for 1.5 hours. Thin layer chromatography (ethyl acetate, silica gel) showed no remaining starting material. The mixture was cooled to room temperature, and poured into ice water. The precipitate was collected by vacuum filtration, washed with water, and dried under vacuum to give 10.3 grams (98 percent) of 5-chloroacetyl-oxindole as a white solid.

A suspension of 9.3 grams of 5-chloroacetyl-oxindole was stirred in 90 milliliters of pyridine at 80-90 ° C for 3 hours, and then cooled to room temperature. The precipitate was collected by vacuum filtration, and washed with 20 milliliters of ethanol. The solid was dissolved in 90 milliliters of 2.5 N sodium hydroxide, and stirred at 70-80 ° C for 3 hours. The mixture was cooled to room temperature, and acidified to a pH of 2 with 0.5 N hydrochloric acid. The precipitate was collected by vacuum filtration, and washed thoroughly with water to give the crude 5-carboxy-2-oxindole as a dark brown solid. After standing overnight, the filtrate produced 2 grams of 5-carboxy-2-oxindole as a yellow solid. The crude brown product was dissolved in hot methanol, the insoluble material was removed by filtration, and the filtrate was concentrated to give 5.6 grams of -carboxy-2-oxindole as a chestnut solid. The combined yield was 97 percent, HNMR (360 MHZ, DMSO-d6) d 12.56 (s, br, ÍH, C00H-5), . 70 (s, ÍH, NH-1), 7.82 (dd, J = 1.57, 7.79 Hz, ÍH, H-6), 7. 74 (s, br, ÍH, H-4), 6.87 (d, J- 7.79 Hz, ÍH, H-7), ^. .. 3 . 53 (s, 2H, CHj-3). MS m / z (i ntensi dad rel ati va%) 178 ([M +? -] \ 100). 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.6 milligrams), 88.6 milligrams of 5-carboxy-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven. The crude product was purified by chromatography on a silica gel column using ethyl acetate-hexane-acetic acid as eluent, to give 51 milligrams (30 percent) of the title compound as a yellow solid. 1KNMR (360 MHz, DMSO-d6): d 13.27 (s, br, ÍH, NH-1), 12.28 (s, vbr, 2H, 2XC00H), 11.11 (s, br, ÍH, NH-1), 8.34 ( d, J = 1.36Hz, ÍH, H-4), 7.78 (s, ÍH, H-VÍM'l?), 7.40 '(dd, J = 1.36, 8.20 Hz, ÍH, H-6), 7.19 (d , J »3.07 Hz, ÍH, H-5 '), 6.93 (d, J = 8.20 Hz, ÍH, H-7), 2.65 (t, J - 7.56 Hz, 2H, CH, CH, COOH), .2.46 (t, J -7.56 Hz, 2H, CH2CH, COOH), 2.29 (s, 3H, CH,); MS m / z 341.0 [M + l] *.

EXAMPLE 14 3- [4-Methyl-5- (2-oxo-5-sulfamoyl-1-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl]] propionic acid To a 100 milliliter flask charged with 27 milliliters of chlorosulfonic acid, 13.3 grams of 2-oxindole were added slowly. The temperature of the reaction was kept below 30 ° C during the addition. After the addition, the reaction mixture was stirred at room temperature for 1.5 hours, heated at 68 ° C for 1 hour, cooled, and poured into water. The precipitate was washed with water and dried in a vacuum oven to give 11.0 grams of 5-chlorosulfonyl-2-oxindole (50 percent yield), which was used without further purification. The 5-chlorosulfonyl-2-oxindole (2.1 grams) was added to 10 milliliters of ammonium hydroxide in 10 milliliters of ethanol, and stirred at room temperature overnight. The mixture was concentrated, and the solid was collected by vacuum filtration to give 0.4 grams (20 percent yield) of 5-aminosulfonyl-2-oxindole as a white solid. -. rHNMR (360 MHz, DMS0-d6); d 10.67 (s, ÍH, NH-1), 7.63 - 7.66 (m, 2H, H-4.6), 7.13 (s, 2H, 5-S0, NH,), 6.91 (d, J = • 8.04 Hz , ÍH, H-7), and 3.56 (3, 2H, CH, -3); MS m / z '(relative intensity,%) 211 ([M-l]', 100).

The 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.6 milligrams), 106 milligrams of 5-aminosulfonyl-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours. hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 132 milligrams (70 percent) of the title compound as a solid. yellow. lHNMR (360 MHz, DMSO-d6); d 13.28 (s, br, ÍH, NH-1 '), 12.0 (s, vbr, ÍH, COOH), 11.15 (s, br, ÍH, NH-1), 8.20 (d, J = .60Hz, ÍH, H-4), 7.73 (3, ÍH, H-vinho), 7.59 (dd, J = 1.60, .17 Hz, ÍH, H-6), 7.22 (d, J - 2.85 Hz, ÍH, H-2 ' ), 7.10 (s, H, NH,), 6.98 (d, J - 8.17 Hz, ÍH, H-7), 2.67 (t, J = 7.41 Hz, H, CHjCH, COOH), 2.46 (t, J « 7.41 Hz, 2H, CH, CH, COOH), 2.29 (s, 3H, CH,).

Example 15 3- [4-Methyl-5- (5-methylsulfamoyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid A suspension of 3.38 grams of 5- chlorosulfonyl-2-oxindole in 10 milliliters of 2 M methylamine in tetrahydrofuran was stirred at room temperature for 4 hours, at which time a white solid was present. The precipitate was collected by vacuum filtration, washed twice with 5 milliliters of water, and dried under vacuum at 40 ° C overnight to give 3.0 grams (88 percent yield) of 5-methylaminosulfoni1-2-oxindole .

XHNMR (300 MHz, DMSO-d6): d 10.87 (s, br, ÍH, NH-1), 7.86 (3, br, ÍH, 5-SO, NHCH,), 7.61 (d, J = 7.80 Hz, H-6), 7.32 (d, J w 4.67 Hz, ÍH, H-4), 6.97 (d, Ja 7.80 Hz, ÍH, H-7), 2.53 (s, 2H, CH2-3), and 2.36 ( s, 3H, 5-SO, NHCH,); MS / z (relative strength v%) 226 (M *, 100).

The 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.6 milligrams), 113 milligrams of 5-methylaminosulfonyl-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven to give 163 milligrams (83 percent) of the title compound as a solid. yellow.

'HNMR (360 MHz, DMSO-d6): d 13.30 (s, br, ÍH, NH-1'), 12.0 (s, vbr, ÍH, COOH), 11.19 (s, br, ÍH, NH-1), 8.18 (d, J = 1.64Hz, ÍH, H-4), 7.80 (s, ÍH, H-vinyl), 7.53 (dd, J - 1.64, 8.17 Hz, 1H, H-6), 7.23 (d, J - 2.80 Hz, ÍH, H-2 '), 7.13 (q, J = 5.15Hz, ÍH, NHCH,), 7.02 (d, J - 8.17 Hz, ÍH, H-7), 3.84 (s, 3H, COOCH ,), 2.66 (t, J at 7.54 Hz, 2H, CH, CH, COOH), 2.47 (t, J = 7.54 Hz, 2H, CHjCHjCOOH), 2.41 (d, J = 5.15Hz, 3H, NCH,), 2.30 (s, 3H, CH3); MS m / z 390 [M + 1] *.

Example 16 Acid 3-. { 3- [4- (2-carboxyethyl) -3-methyl-lH-pyrrol-2-ylmethylene] -2-oxo-2,3-dihydro-lH-indol-5-yl} -propionic 5-chloroacetyl-2-oxindole (4.18 grams) in 30 milliliters of trifluoroacetic acid in an ice bath, treated with 4.65 grams of triethylsilane, and stirred at room temperature for 3 hours. The mixture was poured into 150 milliliters of water, and the precipitate was collected by vacuum filtration, washed with 50 milliliters of water, and dried to give 2.53 grams (65 percent yield) of 5- (2-chloroethyl) -2-oxindol as a solid reddish-brown. Potassium cyanide (2.0 grams) was added to 15 milliliters of dimethyl sulfoxide, and heated to 90 ° C. 5-Chloroethyl-2-oxindole (3.0 grams) dissolved in 5 milliliters of dimethyl sulfoxide was slowly added with stirring, and the reaction was heated at 150 ° C for 2 hours. The mixture was cooled, poured into ice water, and the precipitate was collected by vacuum filtration, washed with water, and dried to give the crude product. The crude material was chromatographed on silica gel using 5 percent methanol in chloroform to give 1.2 grams (42 percent yield) of the title compound. The 5-cyanoethyl-2-oxindole (4.02 grams) in 10 milliliters of water containing 25 milliliters of concentrated hydrochloric acid was refluxed for 4 hours. The mixture was cooled, water was added, and the resulting solid was collected by vacuum filtration, washed with water, and dried to give 1.9 grams (44 percent yield) of 5-carboxyethyl-oxindole as a yellow solid.

NHTM (360 MHZ, DMSO-d6): d 12.00 (s, br, 1H, 5-CH, CH, COOH), 10.21 (s, 1H, NH-1). 7.05 (s, 1H, H-4), 6.99 (d, J = 8.68 Hz, 1H, H-6), 6.69 (d, J-8.68 Hz, 1H, H-7), 3.40 (s, 2H, CH , -3), 2.74 (t J "7.44 Hz, 2H, 5-CH, CH, COOH), and 2.46 (t, J = 7.44 Hz, 2H, -CHjCHjCOOH). 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.6 milligrams), 102.6 milligrams of 5-carboxyethyl-2-oxindole and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours . The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight. The crude solid was purified by chromatography on a silica column, eluting with ethyl acetate-hexane-acetic acid, to give 121 milligrams (66 percent) of the title compound as a yellow solid. 'HNMR (360 MHz, DMS0-d6): d 13.30 (d, J »2.38Hz, ÍH, NH-1'), 12.0 3 (s, vbr, 2H, 2xC00H), 10.68 (s, br, ÍH, NH -1), 7.63 (s, ÍH, H-4), 7.59 (s, ÍH, H-VÍnÜO, 7.12 (d, J = 2.64 Hz, 1H, H-2 '), 6.96 (dd, J = 1.22, 7.93Hz, ÍH, H-6), 6.75 (d, J -'7.93 Hz, ÍH, H-7), 2.81 (t, J = 7.75Hz, 2H, CH, CH, C00H), 2. 65 (t, Ha 7.75Hz, 2H, CH, CH, C00H), 2.55 (t, J = 7.75Hz, 2H, CH, CH, COOH), 2.46 (t, J- 7.42 Hz, CH, CH, C00H) , and 2.26 (s, 3H, CH,).

EXAMPLE 17 3- [5- (5-Ethyl-1-2-oxo-1,2-dihydro-indo-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] -propionic acid 2-oxindole (3 grams) was suspended in 1,2-dichloroethane, and slowly treated with 3.2 milliliters of acetyl chloride. The resulting suspension was heated at 50 ° C for 5 hours, cooled, and poured into water. The resulting precipitate was collected by vacuum filtration, washed copiously with water, and dried in vacuo to give 2.9 grams (73 percent yield) of 5-acetyl-2-oxindole as a tan solid. The 5-acetyl-2-oxindole (2 grams) in 15 milliliters of trifluoroacetic acid in an ice bath was treated slowly with 1.8 grams of triethylsilane, and then stirred at room temperature for 5 hours. 1 milliliter of triethylsilane was added, and the stirring was continued overnight. The reaction mixture was poured into ice water, and the resulting precipitate was collected by vacuum filtration, washed copiously with water, and dried under vacuum to give 1.3 grams (71 percent yield) of the title compound as a solid. yellow. lHNMR (360 MHz, DMSO-d6): d 10.25 (s, br, NH-1), 7.03 (s, ÍH, H-4), 6.97 (d, J »8.05 Hz, ÍH, H-6), 6.69 (d, J = 8.05 Hz, 1H, H-7), 3.40 (s, 2H, CH, -3), 2.51 (q, J - 7.69 Hz, 2H, CH, CH, -5), and 1.12 (t , J - 7.42 Hz, 3H, CH, CH, -5). 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.6 milligrams), 80.5 milligrams of 5-ethyl-2-oxindole, and 75 microliters of piperidine in 2 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated, the residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight. The crude solid was purified by chromatography on a column of silica gel, eluting with ethyl acetate-hexane-acetic acid to give 52 milligrams (32 percent) of the title compound. lHNMR (360 MHz, DMSO-d6): d 13.31 (3, br, ÍH, NH-1 '), 12.0 4 (s, vbr, ÍH, COOH), 10.66 (s, ÍH, NH-1), 7.59 ( s, 2H, H-4 and H-vinyl), 7.11 (d, J- 3.29 Hz, ÍH, H-2 '), 6.94 (d, J = • 7.85Hz, ÍH, H-6), 6.75 (d , J at 7.85 Hz, ÍH, H-7), 2.65 (t, J = 7.66Hz, 2H, CH, CH, COOH), 2.57 (q, J 7.83Hz, 2H, CH, CH,), 2.46 ( t, J = 7.66Hz, CH, CH, COOH), 1.20 (t, J-7.83, 3H, CH, CH,), 2.26 (s, 3H, CH ,;, MS m / z 325 [M + l] *.

Example 18 3- [5- (5-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] -propionic Chloral hydrate (9.6 grams) was dissolved in 200 milliliters of water containing 83 grams of sodium sulfate. The solution was heated to 60 ° C, a solution of 11.4 grams of hydroxylamine hydrochloride in 50 milliliters of water was added, and the mixture was maintained at 60 ° C. In a separate flask, 6.4 grams of 4-anisidine and 4.3 milliliters of concentrated hydrochloric acid in 80 milliliters of water were heated to 80 ° C. The first solution was added to the second, and the resulting mixture was refluxed for 2 minutes, cooled slowly to room temperature, and then cooled in an ice bath. The tanned precipitate was collected by vacuum filtration, washed with water, and dried in vacuo to give 8.6 grams (85 percent yield) of N- (2-hydroximinoacetyl) anisidine. The concentrated sulfuric acid (45 milliliters) containing 5 milliliters of water, was heated to 60 ° C, and 8.6 grams of N- (2-hydroximinoacetyl) anisidine were added in one portion. The stirred mixture was heated at 93 ° C for 10 minutes, and then allowed to cool to room temperature. The mixture was poured into 500 grams of ice, and extracted 3 times with ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate, and concentrated to give 5.1 grams (65 percent yield) of 5-methoxy-isatin as a dark red solid. 5-Methoxy-isatin (5.0 grams) and 30 milliliters of hydrazine hydrate, they were heated to reflux for 15 minutes. The reaction mixture was cooled to room temperature, and 50 milliliters of water was added. The mixture was extracted 3 times with 25 milliliters of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to give a yellow solid. The solid was stirred in ethyl acetate, and 1.1 grams of the insoluble material was removed by vacuum filtration, and stored. This material was 2-hydrazino-carbonylmethyl-4-anisidine. The filtrate was concentrated and chromatographed on silica gel, eluting with ethyl acetate: hexane, 1: 1, to give 0.7 grams of 5-methoxy-2-oxindole as a dirty yellow solid. The ll grams of 2-hydrazinocarbonylmethyl-4-anisidine were refluxed for 1 hour in 20 milliliters of 1 N sodium hydroxide. The mixture was cooled, acidified to a pH of 2 with concentrated hydrochloric acid, and extracted 3 times. with 25 milliliters of ethyl acetate. The organic extracts were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated to give 0.8 grams of 5-methoxy-2-oxindole as a dirty yellow solid. The combined yield was 1.5 grams or 33 percent.

'HNMR (360 MHz, DMS0-d6): d 10.13 (s, ÍH, NH-1), 6.84 (s, ÍH, H-4), 6.72 (d, J, 8.68 Hz, ÍH, H-6), 6.69 (d, J - 8.68 Hz, ÍH, H-7), 3.68 (s, 3H, 0CH, -5), 3.41 (s, 2H, CH, -3). MS m / z (relative intensity,%) 163 ([M + l] *, 100). 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90 milligrams), 82 milligrams of 5-methoxy-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated at 95 ° C for the night. The reaction mixture was cooled and concentrated. The residue was suspended in 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 110 milligrams (67 percent) of the title compound like a solid chestnut. • lHNMR (360 MHz, DMSO-d6): d 13.38 (S, br, ÍH, NH-1 '), 12.03 (3, vbr, ÍH, COOH), 10.57 (s, ÍH, NH-1), 7.63 ( s, ÍH, H-vinyl), 7.42 (d, J »2.46Hz, ÍH, H-4), 7.12 (d, J - 3.08 Hz, ÍH, H-2 '), 6.74 (d, J - 8.26Hz , ÍH, H-6), 6.75 (dd, J »2.46, 8.26 HZ, ÍH, H-7), 3.77 (s, 3H, OCH,), 2.65 (t, J» 7.40Hz, 2H, CH, CH, COOH), 2.46 (t, J »7.40Hz, CH2CH2COOH), 2.27 (s, 3H, CH,); MS m / z (relative intensity,%) 327 ([M + l] *, 100).

EXAMPLE 19 3- [5- (5-Bromo-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid 3- (2- carboxyethyl) -2,4-dimethyl-4-formylpyrrole (97.5 milligrams), 106 milligrams of 5-bromo-2-oxindole, and 75 microliters of piperidine in 3 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 171 milligrams (88 percent) of the title compound. like an orange solid. 1HNMR (360 MHZ, DMS0-d6): d 12.04 (s, vbr, ÍH, COOH), 10.80 (s, br, ÍH, NH-1), 8.0 (d, J «2.06Hz, ÍH, H-4) , 7.67 (s, ÍH, H-vinylO), 7.19 (dd, J - 2.06, 8.40 Hz, ÍH, H-6), 6.79 (d, J - 8.40 Hz, ÍH, H-7), 2.65 (t, J - 7.63 Hz, 2H, CH2CH2COOH), 2.35 (t, J - 7.63 Hz, 2H, CH, CH, COOH), 2.29 (s, 3H, CH,), 2.27 (s, 3H, CH,); MS m / z (relative intensity,%) 389 ([M + l] *, 100). 3- [5- (5-Iodo-2-oxo-l "2-dihydro-indido-3-ylidene-methyl) -2,4-dimethyl-iH-pyrrol-3-yl] -propionic acid. 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (97.5 milligrams); 130 milligrams of 5-iodo-2-oxindole, and 75 microliters of piperidine in 3 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N Acid Hydrochloric Acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 155 milligrams (71 percent) of the title compound. like an orange solid.

HNMR (360 MHz, DMSO-d6): d 13.41 (s, br, ÍH, NH-1 '), 12.03 (s, br, ÍH, COOH), 10.79 (s, br, ÍH, H-1), 8.12 (d, J = 1.70 Hz, ÍH, H-4), 7.65 (s, ÍH, H-'Vinilq, 7-3β (dd / J at 1.70, 7.93 Hz, ÍH, H-6), 6.79 (d, J - 7.93 Hz, ÍH, H-7), 2.64 (t, J = 7.76 Hz, 2H, CH, CH, COOH), 2.34 (t, J = 7.76 Hz, 2H, CH, CH, COOH), 2.29 ( s, 3H, CH,), 2.27 (s, 3H, CH,); MS m / z (relative i-ntensiaad -%) 437 ([M + l] *, 100).

EXAMPLE 20 3- [5- (5-Iodo-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid 3- (2- carboxyethyl) -2,4-dimethyl-5-formylpyrrole (97.5 milligrams), 130 milligrams of 5-iodo-2-oxindole, and 75 microliters of piperidine in 3 milliliters of ethanol were stirred at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 155 milligrams of the title compound (71 percent) like an orange solid. lHNMR (360 MHz, DMSO-d6) d 13.41 (s, br, ÍH, NH-1 '), 12.03 (s, br, ÍH, COOH), 10.79 (s, br, ÍH, NH-1), 8.12 ( d, J - 1.70 Hz, ÍH, H-4), 7.65 (s, ÍH, H-vinyl), 7.36 (dd, J at 1.70, 7.93 Hz, ÍH, H-6), 6.79 (d, J at 7.93 Hz, ÍH, H-7), 2.64 (t, J -7.76 Hz, 2H, CHjCH2COOH), 2.34 (t, Ja 7.76 Hz, 2H, CH, CH, COOH), 2.29 (s, 3H, CH,), 2.27 (s, 3H, CH,). MS m / z (i relative strength.) 437 ([M +?] \ Io).

Example 21 3- [2,4-Dimethyl-5- (4-methyl-2-oxo-l, 2-dihydroindol-3-ylidene-methyl) -lH-pyrrol-3-yl] -propionic acid 3- ( 2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (97.5 milligrams), 74 milligrams of 4-methyl-2-oxindole, and 75 microliters of piperidine in 3 milliliters of ethanol were heated at 95 ° C for 5 hours . The reaction mixture was cooled and concentrated. The residue was suspended in 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 60 milligrams (37 percent) of the title compound. as a green solid.

JHNMR (360 MHz, DMS0-d6): d 13.41 (s, br, ÍH, H-1 '), 12.03 (s, br, ÍH, COOH), 10.72 (a, br, ÍH, NH-1), 7.50 (s, 1H, H-Vipil), 7.01 (t, j 7.82 Hz, 1H, H-6), 6.79 (d, J, 7.82 'Hz, H-5), 6.74 (d, J »7.82 Hz, ÍH, H-7), 2.64 (t, J - 7.76 Hz, 2H, CH2CH2COOH), 2.56 (s, 3H, CH,), 2.34 (t, J at 7.76 Hz, 2H, CHjCH, COOH), 2.29 (3 , 3H, CH,), 2.18 (s, 3H, CH,); MS / z (iritensiaacL elativa%). 325 ([M + 1] *, 100).

EXAMPLE 22 3- [2,4-Dimethyl-5- (5-met-il-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid 3- (2 carboxyethyl) -2,4-dimethyl-5-formylpyrrole (97.5 milligrams), 74 milligrams of 5-methyl-2-oxindole, and 75 microliters of piperidine in 3 milliliters of ethanol were heated at 95 ° C for 5 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 104 milligrams (64 percent) of the title compound. as a yellow solid. lHNMR (360 MHZ, DMSO-d6): d 13.38 (s, br, ÍH, NH-1 '), 12.02 (s, br, ÍH, COOH), 10.57 (3, br, ÍH, NH-1), 7.52 (s, br, 1H, H-4), 7.50 (s, ÍH, H-vinyl), 6.87 (d, J - 7.86 Hz, 1H, H- 6), 6.73 (d, J- 7.86 Hz, ÍH, H-7), 2.63 (t, J - 7.49 Hz, 2H, CH, CH, COOH), 2.34 (t, J at 7.49 Hz, 2H, CH, CH, C00H), 2.29 (s, 3H, CH,) , 2.28 (s, 3H, CH,), and 2.24 (s, 3H, CH,); MS m / z (intensity .. elativa ....%) 325 ([M + l] *, 66).

Example 23 3- [5- (6-Hydroxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid 3.26 grams of 6-methoxy -2-oxindole in 60 milliliters of dichloromethane was cooled to -2 ° C, and 40 milliliters of a 1 M boron tribromide solution in dichloromethane were added dropwise. The reaction mixture was stirred in an ice bath for one hour, and then at room temperature for 1 hour. It was then poured into ice water, and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, concentrated, the precipitate that formed was filtered, and then dried in a vacuum oven overnight to give 2.46 grams of the 6-hydroxypropionate. 2-oxindole (86 percent yield).

XHNMR (360 MHz, DMS0-d6): d 10.13 (s, ÍH, NH-1), 9.22 (s, 1H, 0H-6), 6.93 (d, J at 7.76 Hz, ÍH, H-4), 6.27 -6.31 (m, 2H, H-5.7), and 3.29 (s, 2H, CH, -3); MS / z 150 [M + l] *.

The 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (82 milligrams, 63 milligrams of 6-hydroxy-2-oxindole, and 48 microliters of piperidine in 2 milliliters of ethanol were heated to 90 ° C. for 2 days The reaction mixture was cooled, concentrated, and purified by silica gel column chromatography, eluting with ethyl acetate-hexane-acetic acid, to give 55 milligrams (40 percent) of the title compound like a solid dark brown. lHNMR (360 MHZ, DMS0-d6): d 13.10 (s, br, ÍH, NH-1 '), 12.0 (s, vbr, ÍH, COOH), 10.51 (s, br, ÍH, NH-1), 9.41 (s, ÍH, OH), 7.44 (d, Ja 7.83, ÍH, H-4), 7.29 (s, ÍH, H-vinyl), 6.37 (dd, J = 2.16, 7.83 Hz, ÍH, H-5) , 6.33 (d, Ja 2.16 Hz, ÍH, H-7), 2.62 (t, J at 7.75 Hz, 2H, CH, CH, COOH), 2.32 (t, J at 7.75 Hz, 2H, CH, CH, COOH ), 2.25 (s, 3H, CH,), 2.20 (S, 3H, CH,); MS m / z 325 [M + 1] *.

Example 24 3- [5- (6-Methoxy-2-oxo-1,2-dihydroindo-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid 3- (2- carboxyethyl) -2,4-dimethyl-5-formylpyrrole (97.5 milligrams), 82 milligrams of 6-methoxy-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated at 95 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 2N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 130 milligrams (76 percent) of the title compound. as a yellow solid. 1HNMR (360 MHz, DMSO-d6): d 13.15 (a, br, ÍH, NH-1 '), 11.75 (s, vbr, ÍH, COOH), 10.65 (s, br, ÍH, NH-1), 7.58 (d, J = 8.27, ÍH, H-4), 7.29 (s, ÍH, H-Vinyl), 6.37 (dd, J to 2.26, 8. 27 Hz, ÍH, H-5), 6.33 (d, J - 2.26 Hz, ÍH, H-7), 3.74 (s, 3H, OCH,), 2.62 (t, J at 7.67 Hz, 2H, CH, CH , COOH), 2.33 (t, J at 7.67 Hz, 2H, CHjCHjCOOH), 2.26 (s, 3H, CH,),. and 2.22 (s, 3H, CH,); MS m / z (relative intensity,%) 341 ([M + l] *, 100).

Example 25 3- [5- (6-Hydroxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid 4- (2-carboxyethyl) acid -2-formyl-3-methylpyrrole (543 milligrams), 450 milligrams of 6-hydroxy-2-oxindole, and 450 microliters of piperidine in 10 milliliters of ethanol, were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 2N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give a tan solid. lHNMR (360 MHz, DMSO-d6): d 13.05 (s, br, ÍH, NH-1 '), 10.60 (s, br, ÍH, NH-1), 9.4 (s, ÍH, OH), 7.49 (d , J = 8.08, 1H, H-4), 7.35 (s, ÍH, H-vinyl), 7.02 (d, J = 3.22 Hz, ÍH, H-2 '), 6.38 (dd, J = 2.28, 8.08 Hz , ÍH, H-5), 6.32 (d, J = 2.28 Hz, ÍH, H-7), 2.62 (t, J - 7.67 Hz, 2H, CHjCHjCOOH), 2.44 (t, J = 7.67 Hz, 2H, CH , CH, COOH), 2.21 (s, 3H, CH,); MS / z1 relative intensity,%) 313 ([M + l] *, 60).

EXAMPLE 26 3- [5- (6-Hydroxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid 3,5-dimethoxybenzylester 3- [5- (6-Hydroxy-2-oxo-l, 2-dihydroindol-3-ylidemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid (100 milligrams), 56 milligrams of chloride of 3, 5-dimethoxybenzyl, and 207 milligrams of potassium carbonate in 2 milliliters of anhydrous dimethylformamide were heated at 90 ° C overnight. The reaction mixture was cooled, poured into water, and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by chromatography on a column of silica gel, eluting with ethyl acetate-hexane-acetic acid, to give 39 milligrams of the title compound as a tan solid. lHNMR (360 MHz, DMSO-d6): d 13.06 (s, br, IH, NH-1 '), 10.60 (S / br, IH, NH-1), 9.4 (s, br, IH, OH), 7.49 (d, J = 8.03, ÍH, H-4), 7.35 (s, ÍH, H-vinild, 7.01 (d, J- 3.08 Hz, ÍH, H- 2 '), 6.47 (d, J = 2.29Hz, 2H, aromatic), 6.42 (t, J = 2.29Hz, ÍH, aromatic), 6.38 (dd, J- 2.15, 8.03 Hz, ÍH, H-5), 6.33 (d, J -2.15 Hz, ÍH, H- 7), 5.01 (s, 2H, CH, -Ph), 3.70 (s, 6H, 2xOCH,), 2.59-2.72 (m, 4H, CH2CH, COOH), 2.20 (s, 3H, CH,).

Example 27 Acid 3-. { 5- [3-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-iH-pyrrol-3-yl} -propionic tetrakis (ina trifenilfos) palladium (0.7 g) to a mixture of 5 grams of 3-methoxyphenyl-boronic acid, 3.8 grams was added 5-bromo-2-fluoronitrobenzene, and 11 milliliters of a solution of sodium carbonate 2 M in 100 milliliters of toluene. The mixture was refluxed for 2 hours, diluted with water, and extracted with ethyl acetate. The ethyl acetate was washed with saturated sodium bicarbonate and then with brine, dried, and concentrated to give an oily solid. The solid was chromatographed on silica gel using ethyl acetate: hexane (1: 6) to give 4.3 grams (77 percent yield) of 4-fluoro-3 • -methoxy-3-nitrobiphenyl. Dimethyl malonate (9.7 milliliters) was added dropwise to 2.0 grams of sodium hydride suspended in 50 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 35 minutes, and then cooled to room temperature. 4-Fluoro-2'-methoxy-3-nitrobiphenyl (4.2 grams) was added in 50 milliliters of dimethyl sulfoxide, and the mixture was heated at 100 ° C for 1 hour. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted twice with ethyl acetate. The extracts were combined, washed with brine, dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 3'-methoxy-3-nitrobiphenyl-4-malonate as a pale yellow solid. The crude 3 '-methoxy-3-nitrobiphenyl-4-malonate was heated at 110 ° C in 45 milliliters of 6 N hydrochloric acid for 4 days, and cooled. The precipitate was collected by filtration, washed with water and ethane, and dried to give 5.3 grams of the 3'-methoxy-2-nitrobiphenyl-4-acetic acid as a light tan solid. 3'-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 grams) was dissolved in methanol, and hydrogenated over 0.8 gram of 10 percent palladium on carbon for 3 hours at room temperature. The catalyst was removed by filtration, washed with methanol, and the filtrates were combined and concentrated to give a tan solid. The solid was chromatographed on silica gel in ethyl acetate: hexane: acetic acid, 33: 66: 1 to give 3.0 grams (75% yield based on 4-fluoro-3'-methoxy-3-nitrobiphenyl) ) of 6- (3-methoxyphenyl) -2-oxindole as a pink solid.; HNMR (360 MHz, DMS0-d6): d 10.39 (s, br, ÍH, NH), 7.35 (t, J »7.85Hz, ÍH), 7.26 (d, J at 7.78Hz, ÍH), 7.19 (dd) , J - 1.22, 7.8HZ, ÍH), 7.13-7.16 (m, ÍH), 7.09-7.1 (m, ÍH), 7.01 (d, J - 1.48Hz, ÍH), 6.90-6.93 (m, ÍH), 3.8 (s, 3H, 0CH3), 3.49 (s, 2H, CH2); MS m / z (relative intensity "%) 240.0 ([M + l] *, 100).3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (117 milligrams), 120 milligrams of 6- (3-methoxyphenyl) -2-oxindole and 3 drops of piperidine in 3 milliliters of ethanol were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 190 milligrams (91 percent) of the title compound like a solid chestnut.

XHNMR (360 MHz, DMSO-d6): d 13.38 (s, br, ÍH, NH-1 '), 12.04 (s, br, ÍH, COOH), 10.79 (s, br, ÍH, NH-1), 7.77 (d, J = 8.05, ÍH, H-4), 7.58 (s, ÍH, H-vinylO), 7.27 (dd, J = 1.49, 8.05 Hz, ÍH, H-5), 7.09 (d, Ja 1.49 Hz , ÍH, H-7), 6.89-7.59 (m, 4H), 3.81 (s, 3H, OCH,), 2.65 (t, J = 7.62 Hz, 2H, CH, CH2C0OH), 2.35 (t, J- 7.62 Hz, 2H, CH, CH2COOH), 2.30 (s, 3H, CH,), and 2.27 (s, 3H, CH,); MS m / z ("relative intensity,%) 417 ([M + l] *, 75).

Example 28 3- [5- (6-Bromo-2-oxo-i "2-dihydroindol-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] -propionic acid. Dimethyl malonate was added dropwise ( 13 milliliters) to 2.7 grams of sodium hydride suspended in 20 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 10 minutes, and cooled to room temperature. 5-Bromo-2-fluoronitrobenzene (5.0 grams) was added in 25 milliliters of dimethyl sulfoxide, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted three times with ethyl acetate. The extracts were combined, washed with saturated ammonium chloride, water and brine, dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 4-bromo-2-nitro-phenylmalonate as a pale yellow oil. The crude dimethyl 4-bromo-2-nitrophenyl malonate was heated at 110 ° C in 40 milliliters of 6 N hydrochloric acid for 24 hours, and cooled. The precipitate was collected by filtration, washed with water, and dried to give 5.3 grams (89 percent yield) of 4-bromo-2-nitrophenylacetic acid as a white solid. The 4-bromo-2-nitrophenylacetic acid (0.26 grams), 0.26 grams of zinc powder, and 3 milliliters of 50 percent sulfuric acid in 5 milliliters of ethanol was heated at 100 ° C overnight. The reaction mixture was filtered, diluted with a little acetic acid, concentrated to remove the ethanol, diluted with water, and extracted twice with ethyl acetate. The combined extracts were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give 0.19 grams (90 percent yield) of 6-bromo-2-oxindole as a yellow solid. 1HNMR (360 MHZ, DMS0-d6): d 10.45 (a, br, ÍH, NH-1), 7.14 (d, J- 7.89 HZ, ÍH, H-4), 7.09 (dd, J-1.53, 7.89 Hz , ÍH, H-5), 6.93 (d, J - 1.53 HZ, ÍH, H-7), and 3.43 (s, 2H, CH2-3); MS m / z (relative intensity,%) 210 ([M-2] \ 100) and 212 (M *. 100). 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90 milligrams), 106 milligrams of 6-bromo-2-oxindole, and 3 drops of piperidine in 3 milliliters of ethanol were heated at 90 ° C for 4 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to yield 172 milligrams (92 percent) of the title compound. as a yellow solid. j HNMR (360 MHz, DMSO-d6): d 13.22 (a, br, ÍH, NH-1 '), 12. 04 (a, br, ÍH, COOH), 10.92 (a, br, ÍH, NH-1), 7.73 (d, J = 8.37, 1H, H-4), 7.67 (3, ÍH, H-vTnilo), 7.18 (d, J - 3.22 Hz, 1H, H-2 '), 7.14 (dd, J = 1.33, 8.37 Hz, ÍH, H-5), 6. 99 id, J = 1.33 Hz, 1H, H-7 ), 2.64 (t, J = 7.39 Hz, 2H, CH, CH, COOH), 2-46 (t, J at 7.39 Hz, 2H, CH, CH, COOH), 2.25 (s, 3H, CH,); MS (APCl) w./z 375.0 [M + l] *.

Example 29 Acid 3-. { 5- [6- (3-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-iH-pyrrol-3-yl} -propionic 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.5 milligrams), 120 milligrams of 6- (3-methoxyphenyl) -2-oxindole, and 3 drops of piperidine in 3 milliliters of ethanol, were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 195 milligrams (97 percent) of the title compound. like a solid chestnut.

XHNMR (360 MHz, DMSO-d6): d 13.29 (s, br,] .H, NH-1 '), 12.07 (s, br, 1H, COOH), 10.88 (s, br, 1H, NH-1) , 7.82 (d, J - 7.77, ÍH, H-4), 7.65 (3, ÍH, H-yinÜC *, 7.27 (dd, J = 1.41, 7.77 Hz, 1H, H-5), 7.09 (d, J - 1.41 Hz, ÍH, H-7), 6.89-7.36 (m, 5H), 3.82 (s, 3H, OCH,), 2.65 (t, J- 7.55 Hz, 2H, CH, CH, COOH), 2.47 ( t, J = 7.55 Hz, 2H, CH, CH, C0OH), 2.27 (a, 3H, CH,); MS (APCl) m / z 401 [Ml] *.

Example 30 Acid 3-. { 5- [6- (3-ethoxyphenyl) -2-oxo-? / 2-dihydroindol-S-ili enmeti ^ -s ^ - imethyl-iH-pyrrol-S-ilj-propi ^ i ^ Tetrakis (triphenylphosphine) was added ) palladium (0> 8 grams) to a mixture of 4.2 grams of 3-ethoxyphenylboronic acid, 5.0 grams of 5-bromo-2-fluoronitrobenzene, and 22 milliliters of a 2M sodium carbonate solution in 50 milliliters of toluene and milliliters of ethanol. The mixture was refluxed for 2 hours, and then concentrated. Water was added, and the mixture was extracted twice with ethyl acetate. The combined ethyl acetate layers were washed with water and brine, and then dried and concentrated. The residue was chromatographed on silica gel using 5 percent ethyl acetate in hexane to give 5.3 grams (90 percent yield) of the crude 4-fluoro-3'-ethoxy-3-nitrobiphenyl as a yellow oil. . Dripping dimethyl malonate (11.4 milliliters) was added dropwise to 4.0 grams of sodium hydride suspended in 20 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 10 minutes, and cooled to room temperature. The crude 4-fluoro-3'-ethoxy-3-nitrobiphenyl (5.3 grams) in 25 milliliters of dimethyl sulfoxide was added, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled, quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted three times with ethyl acetate. The extracts were combined, washed with water and brine, dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 3'-ethoxy-3-nitrobiphenyl-4-malonate as a yellow oil. The crude dimethyl 3'-ethoxy-3-nitrobiphenyl-4-malonate was heated at 100 ° C in 60 milliliters of 6 N hydrochloric acid for a total of 4 days, and cooled. The precipitate was collected by filtration, washed with water and hexane, and dried to give 4.7 grams (77 percent yield, based on 5-bromo-2-fluoronitrobenzene) of 3'-ethoxy-3-nitrobiphenyl-4 -acetic raw as a light tan solid. Iron pieces (2.4 grams) were added in one portion to 4.6 grams of the 3'-ethoxy-3-nitrobiphenyl-4-acetic acid in 40 milliliters of jlacial acetic acid, and the mixture was refluxed for 2 hours. The reaction mixture was concentrated to dryness, repeatedly treated with ethyl acetate, and filtered to remove the insoluble material. The filtrate was washed twice with 1N hydrochloric acid, then with brine, dried over anhydrous sodium sulfate, and concentrated to give 3.5 grams (91 percent yield) of 6- (3-ethoxyphenyl) -2-oxindole as a light chestnut solid. HNMR (360 MHz, DMS0-d6): d 10.4 (s, br, ÍH, NH), 7.33 (t, J = 8.4Hz, ÍH, H-3 '), 7.35 (d, J = 7.77Hz, ÍH) , 7.19 (dd, J -1.3, 7.66HZ, ÍH), 7.13 (d, J - 7.69Hz, ÍH), 7.07-7.08 (m, ÍH), 7.0 (s, br, ÍH), 6.9 (dd, J = 2.82, 8.08Hz, ÍH), 4.08 (q, J = 7Hz, 2H, OEt), 3.49 (s, 2H, CH2), 1.34 (t, J - 7Hz, 3H, OEt); MS m / z (-retinitive intensity -t%) 254.2 ([M + l] *, 100).

The 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (97.6 milligrams), 127 milligrams of 6- (3-ethoxyphenyl) -2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol are they were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 227 milligrams of the title compound (ca. cent) as a solid chestnut. lHNMR (360 MHz, DMSO-d6): d 13.38 (s, br, 1H, NH-1 '), 12.06 (s, br, 1H, COOH), 10.81 (a, br, 1H, NH-1), 7.77 (d, J = 7.97, ÍH, H-4), 7.58 (s, ÍH, H-vinyl) »? .26 (dd, J = 1.35, 7.97 Hz, ÍH, H-5), 7.08 (d, J = 1.35 Hz, ÍH, H-7), 6.87-7.36 (m, 4H), 4.09 (q, J - 7.0 Hz, 2H, CH, CH,), 2.65 (t, J = 7.54 Hz, 2H, CH2CH2COOH) , 2.47 (t, J = 7.54 Hz, 2H, CH, CH, COOH), 2.30 (s, 3H, CH,), 2.26 (a ,, 3H, CH,), 1.34 (t, J = 7.0 Hz, 3H , CHjCH,); MS m / z (intensity. Idti va,%) 431 ([M + l] *, 21).

Example 31 Acid 3-. { 5- [6- (3-Ethoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-lH-pyrrol-3-yl} -propionic 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (90.5 milligrams), 127 milligrams of 6- (3-ethoxyphenyl) -2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol, they were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 200 milligrams (96 percent) of the title compound. like a solid chestnut.

: HNMR (360 MHz, DMSO-d6): d 13.29 (s, br, ÍH, NH-1 '), 12.07 (s, vbr, ÍH, COOH), 10.88 (s, br, ÍH, NH-1), 7.82 (d, J = 7.97, ÍH, H-4), 7.65 (a, ÍH, H-vinyl), 7.23 (dd, to 1.20, 7. 97 Hz, HH, H-5), 7.08 (d, J = 1.20 Hz, HH, H-7), 6.87-7.36 (m, 5H), 4.09 (q, J = 6.98Hz, 2H, CH, CH, ), 2.65 (t, J = 7.47 Hz, 2H > CH, CH, COOH), 2.47 (t, J = 7.47 Hz, 2H, CH, CH, COOH), 2.27 (s, 3H, CH,), 1.34 (t, J = 6.98Hz, 3H, Ctf, CH,).

Example 32 3- [2,4-Dimethyl-5- (2-oxo-6-phenyl-1,2-dihydroindol-3-ylidemethyl) -lH-pyrrol-3-yl] -propionic acid tetrakis (triphenylphosphine) palladium (0.8 grams) to a mixture of 3.1 grams of benzeneboronic acid, 5 grams of 5-bromo-2-fluoronitrobenzene, and 22 milliliters of a 2M sodium carbonate solution in 50 milliliters of toluene and 50 milliliters of ethanol. The mixture was refluxed for 2 hours, concentrated, and the residue was extracted twice with ethyl acetate. The ethyl acetate layer was washed with water and brine, dried, and concentrated to give a yellow oil. The oil was chromatographed on silica gel using 5 percent ethyl acetate in hexane to give 4.75 grams (96 percent yield) of 4-fluoro-3-nitrobiphenyl as a yellow oil.

Dimethyl malonate (10 milliliters) in 25 milliliters of dimethyl sulfoxide was added dropwise to 3.5 grams of sodium hydride suspended in 25 milliliters of dimethyl sulfoxide, and the mixture was heated at 100 ° C for 10 minutes. The mixture was cooled to room temperature, and 4.7 grams of 4-fluoro-3-nitrobiphenyl in 25 milliliters of dimethyl sulfoxide was added. The mixture was heated at 100 ° C for 2 hours, cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride. The mixture was extracted three times with ethyl acetate, and the combined organic layers were washed with water and brine, and evaporated to give a yellow oil, crude dimethyl 3-nitrobiphenyl-4-malonate. The crude dimethyl 3-nitrobiphenyl-4-malonate was refluxed in 30 milliliters of 6N hydrochloric acid for 24 hours. The precipitate was collected by filtration, washed with water, and dried to give 4.5 grams (80 percent, based on 4-fluoro-3-nitrobiphenyl) of 3-nitrobiphenyl-4-acetic acid as a cream colored solid. Iron bits (2.6 grams) were added all at once to 4.5 grams of! 3-Nitrobiphenyl-4-acetic acid in 40 milliliters of acetic acid. The mixture was refluxed for 2 hours, concentrated to dryness, and taken up in ethyl acetate. The solids were removed by filtration, and the filtrate was washed twice with 1N hydrochloric acid and brine, and dried over anhydrous sodium sulfate. The filtrate was concentrated to give 3.4 grams (93 percent yield) of 6-phenyl-2-oxindsl as a light tan solid.

JHNMR (360 MHz, DMSO-d6): d 10.4 (s, br, 1H, NH-1), 7.57-7.6 (m, 2H), 7.42-7.46 (m, 2H), 7.32-7.37 (m, ÍH) , 7.27 (d, J = 7.7, ÍH, H-4), 7.19 (dd, J »1.6, 7.7Hz, ÍH, H-5), 7.01 (d, J = 1.6Hz, 1H, H-7), 3.49 (s, 2H, CH,), - MS m / z (relative intensity /%) 210 ([M + l] *, 100). 3- (2-carboxyethyl) -2,4-dimethyl-5-ormilpyrrole (97.6 milligrams), 105 milligrams of 6-phenyl-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated to 90 ° C. overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to yield 138 milligrams (71 percent) of the title compound. like a solid chestnut.

HNMR (360 MHz, DMSO-d6): d 13.38 (a, br, ÍH, NH-1 '), 12.05 (3, br, ÍH, COOH), 10.81 (a, br, ÍH, NH-1), 7.78 (d, J = 7.84, ÍH, H-4), 7.58 (s, ÍH, H-vinyl), 7.25-7.63 (m, 6H), 7.09 (s, br, ÍH, H-7), 2.64 (t , J - 7.71 Hz, 2H, CHjCHjCOOH), '2.35 (t, J = 7.71 Hz, 2H, CHjCHjCOOH), 2.30 (a, 3H, CH,), and 2.26 (3, 3H, CH,); MS m / z (relative intensity.%) 387 ([M + l] *, 100).

EXAMPLE 33 Acid3- [4-methyl-5- (2-oxo-6-phenyl-1,2-dihydro-indol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic 4- (2-carboxyethyl) ) -2-formyl-3-methylpyrrole (90.5 milligrams), 105 milligrams of 6-phenyl-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to yield 146 milligrams (78 percent) of the title compound. like a solid chestnut. 1HNMR (360 MHz, DMSO-d6): d 13.29 (a, br, ÍH, NH-1 '), 12.01 (s, vbr, ÍH, COOH), 10.89 (a, br, ÍH, NH-1), 7.83 (d, J = 7.92, ÍH, H-4), 7.65 (a, 1H / H-vimlo), 7.30-7.65 (m, 5H), 7.16 (d, J = 2.83 Hz, ÍH, H- 2 ') , 7.28 (dd, J = 1.58, 7.92 Hz, ÍH, H-5), 7.09 (d, J = 1.58Hz, ÍH, H-7), 2.66 (t, J = 7.76 Hz, 2H, CH, CH, COOH), 2.45 (t, J - 7.76 Hz, 2H, CH, CH, C00H), and 2.27 '(a, 3H, CH,); MS m / z (relative intensity%) 373 ([M + l] *, 100).

Example 34 Acid 3-. { 5- [l6- (4-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-iH-pyrrol-3-yl} -propionic Tetrakis (triphenylphosphine) palladium (1 gram) was added to a mixture of 5 grams of 4-methoxyphenylboronic acid, 6.6 grams of 5-bromo-2-fluoronitrobenzene, and 30 milliliters of a 2M sodium carbonate solution. in 50 milliliters of toluene and 50 milliliters of ethanol. The mixture was refluxed for 2 hours, concentrated, and the residue was extracted twice with ethyl acetate. The ethyl acetate layer was washed with water and brine, dried, and concentrated to give an oily brown solid. The solid was chromatographed on silica gel using 5 percent ethyl acetate in hexane to give the crude 4-fluoro-4'-methoxy-3-nitrobiphenyl as a pale yellow solid. Dimethyl malonate (10 milliliters) was added dropwise to 2.0 grams of sodium hydride suspended in 60 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 10 minutes, and cooled to room temperature. Crude 4-fluoro-2'-methoxy-3-nitrobiphenyl (5.2 grams) in 50 milliliters of dimethyl sulfoxide was added, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted three times with ethyl acetate. The extracts were combined, washed with saturated ammonium chloride, water and brine, dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 4'-methoxy-3-nitrobiphenyl-4-malonate as a yellow oil. .

The crude 4'-methoxy-3-nitrobiphenyl-4-malonate was heated at 100 ° C in 60 milliliters of 6 N hydrochloric acid for 15 hours, and cooled. The precipitate that formed was collected by filtration, washed with water and hexane, and dried to give 7.2 grams of the crude 4'-methoxy-3-nitrobiphenyl-4-acetic acid as a light tan solid. Iron pieces (3.6 grams) were added in one portion to 7.2 grams of 4 • -methoxy-3-nitrobiphenyl-4-acetic acid in 50 milliliters of glacial acetic acid, and they were heated at 100 ° C overnight. The reaction mixture was concentrated to dryness, sonicated in ethyl acetate, and filtered to remove the insolubles. The filtrate was washed twice with 1N hydrochloric acid, then with brine, dried over anhydrous sodium sulfate, and concentrated to give 2.7 grams (54 percent yield, based on 5-bromo-2-fluoronitrobenzene) of 6 g. - (4-methoxyphenyl) -2-oxindole as a pink colored solid.

XHNMR (360 MHz, DMSO-d6): d 10.38 (s, br, ÍH, NH-1), 7.52 (d, J-9Hz, 2H,), 7.23 (d, J = 7.3Hz, ÍH, H-4 ), 7.14 (d, d, J = 1.38, 7.3Hz, ÍH, H-5), 7.0 (d, J = 9Hz, 2H), 6.96 (d, J = 1.38Hz, ÍH, H-7), 3.78 (a, 3H, OCH,), 3.47 (a, 2H, CH,); MS m / z (relative intensity,%) 214.0 ([M + l] *, 100). 4- (2-carboxyethyl) -2-f-ormyl-3-methylpyrrole (90.5 milligrams), 120 milligrams of 6- (4-methoxyphenyl) -2-oxindole, and 3 drops of piperidine in 2 milliliters of ethanol, they were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water a pH of 6, and dried in a vacuum oven overnight to give 118 milligrams (59 percent) of the title compound as a solid chestnut . 'HNMR (360 MHz, DMS0-d6): d 13.26 (s, br, ÍH, NH-1'), 10.83 (s, br, ÍH, NH-1), 7.78 (d, J = * 8.07 Hz, ÍH , H-4), 7.61 (s, ÍH, H-vinylO), 7.56 (d, J - 8.97 Hz, 2H), 7.22 (dd, J = 1.44, 8.07 Hz, ÍH, H-5), 7.13 (d , J »3.09 Hz, ÍH, H- 2 '), 7.04 (d, J» 1.44 HZ, ÍH, H-7), 7.0 (d, J> 8.97 Hz, 2H), 3.79 (a, 3H, OCH ,), 2.65 (t, J- 7.54 Hz, 2H, CH, CH2COOH), 2.44 (t, J = 7.54 Hz, 2H, CH2CH, COOH), and 2.27 (s, 3H, CH,); MS m / z. (relative intensity%) 404 ([M + l] *, 100).

Example 35 Acid 3-. { 5- [6- (4-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-lH-pyrrol-3-yl} -propionic 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (98 milligrams), 120 milligrams of 6- (4-methoxyphenyl) -2-oxindole, and 3 drops of piperidine in 2 milliliters of ethanol were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 118 milligrams (57 percent) of the title compound. like a solid chestnut. lHNMR (360 MHz, DMSO-d6): d 13.35 (a, br, ÍH, NH-1 '), 12.02 (a, br, ÍH, COOH), 10.75 (s, br, ÍH, NH-1), 7.73 (d, J = 6.75Hz, ÍH, H-4), 7.56 (d, J »9.01Hz, 2H), 7.54 (s, ÍH, H- v? ml < 3, 7.21 (dd, J- 1.59, 6.75 Hz, ÍH, H-5), 7.04 (d, J = 1.59Hz, ÍH, H-7), 7.01 (d, J = 9.01 Hz, 2H), 3.79 (a, 3H, OCH,), 2.65 ( t, J at 7.54 Hz, 2H, CH2CH2COOH), 2.35 (t, J - 7.54 Hz, 2H, CH2CH2COOH), 2.29 (a, 3H, CH,), and 2.26 (s, 3H, CH,); MS m / z (relative intensity%) 417 ([M + l] *, 100).

Example 36 Acid 3-. { 5- [2-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-lH-pyrrol-3-yl} -propionic Tetrakis (triphenylphosphine) palladium (1 gram) was added to a mixture of 5 grams of 2-methoxyphenylboronic acid, 6.6 grams of 5-bromo-2-fluoronitrobenzene, and 30 milliliters of a 2M sodium carbonate solution in 50 grams. milliliters of toluene and 50 milliliters of ethanol. The mixture was refluxed for 2 hours, concentrated, and the residue was extracted twice with ethyl acetate. The combined layers of ethyl acetate were washed with water and brine, dried, and concentrated to give a dark green oil, which solidified on standing to give crude 4-fluoro-2'-methoxy-3-nitrobiphenyl. Dripping dimethyl malonate (14 milliliters) was added dropwise to 2.9 grams of sodium hydride suspended in 50 milliliters of dimethyl sulfoxide. The mixture was heated at 100 ° C for 15 minutes, and cooled to room temperature. The crude 4-fluoro-2'-methoxy-3-nitrobiphenyl was added in 60 milliliters of dimethyl sulfoxide, and the mixture was heated at 100 ° C for 2 hours. The reaction mixture was cooled and quenched with 300 milliliters of a saturated solution of ammonium chloride, and extracted twice with ethyl acetate. The extracts were combined, washed with saturated ammonium chloride, water and brine, dried over anhydrous sodium sulfate, and concentrated to give the crude dimethyl 2'-methoxy-3-nitrobiphenyl-4-malonate as a yellow oil. . The crude 2'-methoxy-3-nitrobiphenyl-4-malonate was heated at 100 ° C in 50 milliliters of 6 N hydrochloric acid for 14 hours, and cooled. The precipitate was collected by filtration, washed with water and hexane, and dried to give 9.8 grams of the 2'-methoxy-2-nitrobiphenyl-4-acetic acid as a light tan solid. Iron bits (5 grams) were added in one portion to 9.8 grams of 2'-methoxy-3-nitrobiphenyl-4-acetic acid in 50 milliliters of glycolic acetic acid, and the mixture was heated at 100 ° C for 3 hours. The reaction mixture was concentrated to dryness, sonicated in ethyl acetate, and filtered to remove the insolubles. The filtrate was washed twice with 1N hydrochloric acid, water and brine, dried over anhydrous sodium sulfate, and concentrated. The residue was chromatographed on silica gel using ethyl acetate: hexane, 1: 2, to 5.4 grams (69 percent yield, based on 5-bromo-2-fluoronitrobenzene) of 6- (2-methoxyphenyl) - 2-Oxindole as a pink colored solid. lHNMR (360 MHz, DMSO-d6): d 10.32 (s, br, ÍH, NH), 7.29- 7.34 (m, ÍH), 7.19-7.25 (m, 2H), 7.08 (d, J = 8Hz, 1H, H-4), 6.97-7.02 (m, 2H), 6.91 (d, J => 1.05Hz, ÍH, H-7), 3.8 (s, 3H, OCH,), 3.47 (s, 2H, CH2); MS m / z (relative intensity%) 239.8 (100, [M + l] *). 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (217 milligrams), 239 milligrams of 6- (2-methoxyphenyl) -2-oxindole, and 3 drops of piperidine in 2 milliliters of ethanol, were heated to 90 ° C at night. The reaction mixture was cooled and concentrated. The residue was suspended in 6 N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 348 milligrams (86 percent) of the title compound. like a solid chestnut.

* HNMR (360 MHz, DMSO-d6): d 13. 29 (s, br, ÍH, NH-1 '), 11. 59 (s, br, ÍH, COOH), 10. 78 (s, br, ÍH, NH-1), 7. 75 (d, J 8.13Hz, ÍH, H-4), 7.62 (s, ÍH, H-vinylc), 7.0-7.34 (m, 7H), 3.76 (a, 3H, OCH,), 2.56 (t, J - 7.46 Hz, 2H, CHjCHjCOOH), 2.46 (t, "J - 7.46 Hz, 2H, CH, CHjCOOH), and 2.27 (a, 3H, CH,); MS m / z (relative intensity,%) 401 ([M + l] *, 100).

Example 37 Acid 3-. { 5- [6- (2-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-iH-pyrrol-3-yl} -propionic 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (234 milligrams), 239 milligrams of 6- (2-methoxyphenyl) -2-oxindole, and 3 drops of piperidine in 2 milliliters of ethanol , they were heated at 90 ° C overnight. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight. The crude solid was purified by chromatography on a column of silica gel, eluting with ethyl acetate: hexane, 1: 1, containing 0.1 percent acetic acid, to give 182 milligrams (44 percent) of the title compound as a solid chestnut lHNMR (360 MHz, DMS0-d6): d 13.38 (a, br, ÍH, NH-1 '), 12.0 (a, br, ÍH, COOH), 10.7 (a, br, ÍH, NH-1), 7.71 (d, Ja 7.74Hz, ÍH, H-4), 7.55 (s, ÍH, H-vinilcJ, 7.0-7.33 (m, 6H), 3.76 (a, 3H, OCH,), 2.65 (t, J = 7.6 Hz, 2H, CH, CH, C00H), 2.35 (t, J = 7.6 Hz, 2H, CH, CH, COOH), 2.3 (s, 3H, CH,), and 2.26 (s, 3H, CH,); MS (APCl neg) m / z (relative intensity,%) 415 ([Ml], 100).

EXAMPLE 38 3- [2,4-Dimethyl-5- (6-morpholin-4-yl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid tin chloride dihydrate (225 grams) to a solution of 2,4-dinitrophenylacetic acid (22.6 grams) in ethanol (450 milliliters). The mixture was heated at 90 ° C for 10 hours. The reaction mixture was cooled and basified to a pH of 11 with 12M sodium hydroxide. The solids were removed by filtration, and the filtrate was concentrated. The residue was treated with ethanol (300 milliliters). The insolubles were filtered and washed with ethanol (60 milliliters five times). The combined methanol washings were evaporated and dried in vacuo to give 15 grams of 6-amino-2-oxindole as a chestnut powder. lHNMR (360 MHz, DMSO-d6): d 10.03 (s, br, NH), 6.78 (d, J = 8.55Hz, ÍH, H-4), 6.09-6.11 (m, 2H), 4.95 (s, br , 2H, NH,), 3.22 (a, 2H, H-3); MS (+ APCI) m / z (relative intensity -,%) 147 ([M-l] *, 100).

The 6-amino-2-oxindole (2.2 grams), 4.0 grams of 2, 2'-dibromoethylether, and 7.9 grams of sodium carbonate, were refluxed overnight in 20 milliliters of ethanol, concentrated and diluted with 50 milliliters of water. The mixture was extracted three times with 50 milliliters of ethyl acetate, the organic extracts were combined, washed with 20 milliliters of brine, dried over anhydrous sodium sulfate, and concentrated to dryness. The solid was chromatographed on a column of silica gel, eluting with ethyl acetate: hexane, 1: 1, containing 0.7 percent acetic acid, to give 1.2 grams (37 percent yield) of 6- (morpholine) -4-yl) -2-oxindole as a beige solid.

'XHNMR (360 MHz, DMSO-d6): d 10.2 (a, br, ÍH, NH-1), 7.02 (d, J = 7.87Hz, ÍH, H-4), 6.47 (dd, J-2.11, 7.87 Hz, ÍH, H- 5), 6.37 (d, J = 2.11HZ, ÍH, H-7), 3.69-3.72 (m, 4H), 3.32 (a, 2H, CH,), 3.01-3.04 (m, 4H); MS m / z (Relative intensity%) 219 ([M + l] *, 100). 3- (2-carboxyethyl) -2,4-dimethyl-5-formylpyrrole (3.3 grams), 4 grams of 6- (morpholin-4-yl) -2-oxindole, and 1.8 milliliters of piperidine in 60 milliliters of ethanol , they were heated at 90 ° C for 7 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight. The resulting solid was purified by chromatography on a column of silica gel, eluting with ethyl acetate-hexane-acetic acid, to give 2.78 grams (38 percent yield) of the title compound as a tan solid. lHNMR (360 MHZ, DMSO-d6): d 13.13 (a, br, ÍH, NH-1 '), 12.02 (s, br, ÍH, COOH), 10.57 (a, br, ÍH, NH-1), 7.52 (d, J at 8.46Hz, ÍH, H-4), 7.32 (a, ÍH, H ^ ini] 0), 6.58 (dd, J «1.99, 8.46Hz, ÍH, H-5), 6.41 (d, J = 1.99Hz, ÍH, H-7), 3.71-3.74 (m, 4H), 3.06-3.09 (m, 4H), 2.62 (t, J - 7.57 Hz, 2H, CH, CH, COOH), 2.33 ( t, J = 7.57Hz, 2H, CH, CH, COOH), 2.26 (a, 3H, CH,), and 2.21 (a, 3H, CH,); MS m / z (relative intensity,%) 396 ([M + l] *, 100).

Example 39 3- [5- (5-Chloro-4-methyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid A suspension of 3.0 grams of 4-methyl-2-oxindole was stirred in 50 milliliters of acetonitrile at room temperature, while 3.3 grams of N-chlorosuccinimide were added in portions. Then trifluoroacetic acid (1 milliliter) was added. The suspension was stirred at room temperature for 3 days, during which time the solids were always present. The white solid was collected by vacuum filtration, washed with a small amount of cold acetone, and dried overnight in a vacuum oven at 40 ° C to give 2.5 grams (68 percent) of 5-chloro-4 -methyl- 2-oxindole. lHNMR (360 MHz, DMSO-d6): d 10.38 (s, br, ÍH, NH), 7.19 (d, J = 8Hz, lH, aromatic), 6.64 (d, J = 8Hz, ÍH, aromatic ^ 3.46 (s , 2H ', H-3) », 2.19 (a, 3H, CH,). 3- (2-Carboxyethyl) -2,4-dimethyl-5-formylpyrrole (98, 202 milligrams), 91 milligrams of 5-chloro-4-methyl-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated at 90 ° C for 4 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 100 milligrams of the title compound.

* HNMR (360 MHz, DMS0-d6): d 13.47 (a, br, ÍH, NH-1 '), 12.03 (s, br, ÍH, COOH), 10.83 (s, br, ÍH, NH-1), 7.61 (a, ÍH, H ?? nilo), 7.14 (d, J = 8.17 Hz, ÍH, aromatic), 6.74 (d, J = 8.17 Hz, ÍH, aromatic), 2.64 (3, 3H, CH,), 2.64 (t, J - 7.62 Hz, 2H, CHjCHjCOOH), 2.34 (t, J - 7.62 Hz, 2H, CH, CH, COOH), 2.3 (s, 3H, CH,), and 2.20 (s, 3H, CH,).

Example 40 Acid 3- [5- (5-Chloro-4-methyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic The 4- (2-carboxyethyl) -2-formyl-3-methylpyrrole (91 milligrams), 92 milligrams of 5-chloro-4-methyl-2-oxindole, and 2 drops of piperidine in 2 milliliters of ethanol were heated at 90 ° C for 4 hours. The reaction mixture was cooled and concentrated. The residue was suspended in 6N aqueous hydrochloric acid. The precipitate was filtered, washed with water at a pH of 6, and dried in a vacuum oven overnight to give 95 milligrams of the title compound.

'HNMR (360 MHz, DMSO-d6): d 13.36 (s, br, ÍH, NH-1'), 11.98 (s, br, ÍH, COOH), 10.92 (s, br, ÍH, NH-1), 7.68 (s, ÍH), 7.19 (d, j - 7.i4Hz, H, aromatic, 7.17 (S> 1H) # 6-75 (d, J = 7.14 Hz, ÍH, aromatic), 2.66 (s, 3H "CH, -4), 2.66 (t, J = 7.51 Hz, 2H, CH, CH, COOH), 2.45 (t, J at 7.51 Hz, 2H, CHjCHjCOOH), 2.21 (s, 3H, CH,); m / z (relative intensity _,%) 345 ([M + l] *, 100).

EXAMPLE 41 3- [2,4-Dimethyl-5- (2-oxo-l, 2-dihydroindol-3-ylidemethyl) -iH-pyrrol-3-yl] -propionic acid, sodium salt A suspension of 8 grams of the acid 3- [2,4-dimethyl-5- (2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] propionic in 60 milliliters of water, was added to 0.98 grams of hydroxide sodium in 10 milliliters of water. The mixture was stirred at room temperature for 30 minutes, and filtered. The filtrate was frozen and lyophilized to give 8 grams of the title compound. Alternatively, a suspension of 117 grams of 318-005 in 470 milliliters of water was added to 16.57 grams of sodium hydroxide in 74 milliliters of water. The mixture was stirred at room temperature for 15 minutes, and filtered. The filtrate was added to 210 milliliters of ethanol, and the resulting precipitate that formed was collected by suction filtration. After drying, a total of 106 grams of the title compound was obtained. lH R (360 MHz, DMSO-d6): d 13.34 (a, br, 1H, NH-1 <) 10'82 (s, br, 1H, NH-1), 7.65 (d, J at 7.52Hz, 1H, H-4), 7 5 < •• 1H, H-vinyl), 7.04 (t, J at 7.52 Hz, 1H, H-6), 6.93 (t, J - 7-52 HZ, 1H, H-5), 6.85 (d, J at 7.52 Hz, 1H, H-7), 2.55 (t, J - 6.95 Hz, 2H, CH, CH, COOH), 2.28 (s, 3H, CH,), 2.24 (s, 3H, CH,), '. 1.99 (t / J ß 6-95 RZf 2H? CHjCH C00H) # Example 42 3- [3,5-Dimethyl-4- (3-morpholin-4-ylpropyl) -lH-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one Step 1: To a suspension of the acid 3 - (2,4-dimethyl-lH-pyrrol-3-yl) -propionic acid (10 grams, 60.8 mmol) in 60 milliliters of dichloromethane was added with 1, 1'-carbonyldiimidazole (11.6 grams, 71.8 mmol), followed by morpholine (5.5 milliliters, 60.8 millimoles), and N, N-di-isopropylethylamine (Hunig's base, 10 milliliters, 60.8 millimoles). The dark red reaction mixture was stirred at room temperature overnight, and poured into ice water. The organic layer was washed with brine until the wash had a pH of about 6, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified on a column of silica gel, eluting with dichloromethane-methanol (92: 2), to give 13.84 grams (96 percent) of 3- (2,4-dimethyl-lH-pyrrol-3-yl. ) -1-morpholin-4-yl-propan-l-one. Step 2: To a suspension of lithium aluminum hydride (2.67 grams, 70 millimoles) in tetrahydrofuran (100 milliliters), a solution of 3- (2,4-dimethyl-1H-pyrrol-3-yl) was added dropwise. ) -1-morpholin-4-yl-propan-l-one (13.84 grams, 59 mmol) in tetrahydrofuran (50 milliliters). The reaction mixture was stirred at 80 ° C for 1 hour, and cooled in an ice bath. Ice was added to the reaction mixture slowly, until the evolution of gas ceased. A few drops of 2N sodium hydroxide were added, and the reaction mixture was stirred at room temperature for 30 minutes. Then the reaction mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give 10.37 grams (79 percent) of 4- [3- (2, 4-dimethyl-1H-pyrrole-3. -il) -propyl] -morpholine as a light brown oil, which was used without further purification. Step 3: To an ice-cold solution of N, N-dimethylformamide (5.5 milliliters, 70 mmol) in dichloromethane (30 milliliters), phosphorus oxychloride (6.5 milliliters, 70 millimoles) was added dropwise. When the addition was complete, the reaction mixture was stirred at room temperature for 15 minutes, after which a solution of 3,5-dimethyl-4- (3-morpholin-4-yl-propyl) -lH- was added dropwise. pyrrole-2-carboxaldehyde (10.37 grams, 46.6 mmol) in dichloromethane (20 milliliters) at 0 ° C. The final reaction mixture was refluxed at 60 ° C for 4 hours, and then cooled in an ice bath. Ice was slowly added to the reaction mixture, followed by the addition of 2 N sodium hydroxide, until a pH of 12 was reached. The reaction mixture was stirred at room temperature for 30 minutes, and then extracted with ethyl acetate. ethyl. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was purified on a column of silica gel, eluting with dichloromethane-methanol-ammonium hydroxide (9.5: 0.5 ), to give 4.57 grams (39 percent) of 3,5-dimethyl-4- (3-morpholin-4-yl-propyl) -iH-pyrrole-2-carbaldehyde as a dark red oil: HNMR (360 MHz, DMSO-d,) d 11.34 (s, br, ÍH, NH-1), 9.40 (s, ÍH, CHO-2), 3.55 (t, J- 4.68 Hz, 4H, O (CH, CH2), NCH, CHjCHj- 4), 2.28-2.34 (m, 6H, 0 (CHjCH,) jNCH, CH, CH, -4), 2.21 (t, 2H, J »7.10 Hz, 2H, 0 (CHjCHj) 2NCHaCHjCHj-4) CH, -3), 2.19 (s, 3H, CH, -5), 2.14 (a, 3H, CH, -3), 1.51 (quint., J at 7.10 Hz, 2H, 0 (CHjCHj) JNCHJCHJCHJ-4), MS m / z (intensity -relative,%) 251 ([M + l] * -, 100).

Step 4: A mixture of 1,3-dihydroindol-2-one (133 milligrams, 1.0 mmol), 3,5-dimethyl-4- (3-morpholin-4-yl) -propyl) -lH-pyrrole-2- carboxaldehyde (250 milligrams, 1.0 mmol), and 3 drops of pyrrolidine in 2.0 milliliters of ethanol, was refluxed at 90 ° C for 4 hours, and then cooled to room temperature. The precipitate was filtered, washed with cold ethanol and hexane, and dried in a vacuum oven overnight to give 308.9 milligrams (85 percent) of 3- [3,5-dimethyl-4- (3-morpholine- 4-yl-propyl) -lH-pyrrole-2'-yl-ethylene] -1,3-dihydroindol-2-one as a yellow solid: rHNMR (300 MHz, DMS0-dt) d 13.37 (s, ÍH, NH-1 '), 10.71 (s, ÍH, NH-1), 7.68 (d, Ja 7.47 Hz, ÍH, H-4), 7.53 ( s, 1H, H- • V '^ M, 7.06 { dt, J = 7.47 Hz, lH, H-6), 6.94 (dt, J at 7.74 Hz, ÍH, H-5), 6.84 (d, J at 7.47 Hz, ÍH, H-7), 3.55 (t, J - 4.37 Hz, 4H, 0 (CH, CH2), NCH, CH, CH, -4 '), 2.40 (t, J at 7.31 Hz, 2H, 0 (CH, CH,), NCH, CH, CH, -4 '), 2.31 (t, J = 4.37 Hz, 4H, 0 (CH, CH,), NCH, CH, CH2-4'), 2.28 (a, 3H, CH, -3 '), 2.23 (s, 3H, CH3-5'), 2.23 (t, J = 7.31 Hz, 2H, 0 (CH, CH,), NCH, CH5CH, -4 '), 1.56' (quint., J = 7.31 Hz, 2H, 0 (CH, CH2), NCH, CH, CHj-4 '), MS m / e (relative intensity' /%) 365 (M * ', 100).

Example 43 5-Bromo-3- [3,5-dimethyl-4- (3-morpholin-4-yl-propyl) -lH-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one A mixture of 5-bromo-l, 3-dihydroindol-2-one (212 milligrams, 1.0 mmol), 3,5-dimethyl-4- (3-morpholin-4-yl-propyl) -lH-pyrrole-2-carbaldehyde (250 milligrams, 1.0 mmol), and 3 drops of pyrrolidine in 2.0 milliliters of ethanol, was refluxed at 90 ° C for 4 hours, and then cooled to room temperature. The precipitate was filtered, washed with cold ethanol and hexane, and dried in a vacuum oven overnight to give 399.8 milligrams (90 percent) of 5-bromo-3- [3,5-dimethyl-4- ( 3-morpholin-4-yl-propyl) -lH-pyrrol-2-ylmethylene] -l, 3-dihydroindol-2-one as a red solid. lHNMR (300 MHz, DMS0-dβ) d 13.43 (s, ÍH, NH-1 '), 10.81 (s ΔH, NH-1), 7.99 (d, J = 2.07 Hz, ΔH, H-4), 7.66 ( a, ÍH, H-vinyl), 7.18 (dd, J = 2.07, 7.58 Hz, lH, H-6), 6.79 (d, J at 7.58 Hz, ÍH, H-7), 3.55 (t, J = 4.39 Hz, 4H, 0 (CH, CH2) 2NCH2CH, CH2-4 '), 2.40 (t, J = 7.32 Hz, 2H, 0 (CH, CH,), NCH, CH, CHJ-4'), 2.31 (t , J = 4.39 Hz, 4H, 0 (CH, CH,) 2NCH, CH, CH, -4 '), 2.29 (a, 3H, CH, -3'), 2.26 (a, 3H, CH, 5 ') , 2.23 (t, J = 7.32 Hz, 2H, O (CH, CH,), NCH, CH, CH2-4 '), 1.56 (quint., J = 7.32 Hz, 2H, .0 (CH, CH,) , NCH, CH., CH, -4 '), MS m / e (relative intensity,%) 443 (M *', 1.00), 445 ([M + 2] * ', 100).

Example 44 3- [3,5-Dimethyl-4- (3-morpholin-4-yl-propyl) -lH-pyrrol-2-ylmethylene] -6-phenyl-1,3-dihydroindol-2-one Using the procedure of Example 2, an 88 percent yield of the title compound was obtained as a yellow solid: 'HNMR (300 MHz, DMS0-dβ) d, 13.37 (a, ÍH, NH-1'), 10.81 (a, 1H, NH-1), 7.77 (d, J-8.20 Hz, 1H, H-4) , 7.62 (d, J - 7.39 Hz, 2H, H-2 », 6«), 7.58 (3, 1H, H-vini.lo,, 7.44 (t, J - 7.39 Hz, 2H, H-3", 5 »), 7.32 (t, br, J - 7.39 Hz, ÍH, H-4"), 7.26 (dd, J = 1.49, 8.29 HZ, ÍH, H-5), 7.09 (d, J - 1.49 Hz, ÍH, H-7), 3.56 (t, J - 4.48 Hz, 4H, O (CH, CH,), NCH, CHjCHj-4 '), 2.41 (t, J - 7.18 HZ, 2H, 0 (CHjCHj), NCHjCHjCía-4 ') < 2.31 (t, J - 4.48 Hz, 4H, 0 (CHjCHj), NCH, CH, CH, -4'), 2.29 (a, 3H, CH, -3 '), 2.26 (s , 3H, CH, - 5 '), 2.24 (t, J = 7.18 Hz, 2H, O (CH, CH,), NCE:! CH, CH2-4'), 1.56 (quint., J = 7.18 Hz, 2H, O (CH, CHa) 2NCHjCH_jCHj - 4 '), MS m / e (iní nsTdad' 'relative ",%) 441 (M *', 100).

Example 45 3- [3,5-Dimethyl-4- (3-morpholin-4-yl-propyl) -lH-pyrrol-2-ylmethylene] -6- (2-methoxy-enyl) -1,3-dihydroindol-2 -one Using the procedure of Example 2, an 86 percent yield of the title compound was obtained as a yellow solid: lHNMR (300 MHz, DMSO-df) d 13.37 (a, ÍH, NH-1 '), 10.72 (a, ÍH, NH-1), 7.71 (d, J «7.79 Hz, ÍH, H-4), 7.55 (a, ÍH, H- 7.27-7.34 (m, 2H), 6.98-7.10 (m, 4H), 3.76 (a, 3H, OCH, -2"), 3.56 (t, J - 4.50 Hz, 4H, O (CH, CH2):? NCH2CH2CH, -4 '), 2.41 (t, J - 7.12 Hz, 2H, O (CHjCHj) jNCH2CH, CH, - 4'), 2.21-2.31 (m, 6H, OIJJCHJJJJNCHJCHJCÜJ ^ ' ), 2.29 (a, 3H, CH, -. 3 '), 2.25 (a, 3H, CH, -5 <), 1.57 (quint., J «7.12 Hz, 2H, O (CH2CH2), NCHjCH, CH ,-4 ' ) , MS m / e (.intensity ..relive -,%) 471 (M * '"100).

EXAMPLE 46 3- [3,5-Dimethyl-4- (3-morpholin-4-yl-propyl) -lH-pyrrol-2-ylmethylene] -6- (3-methoxyphenyl) -1,3-dihydroindol-2- Ona Using the procedure of Example 2, an 87 percent yield of the title compound was obtained as a yellow solid: 'HNMR (300 MHz, DMSO-d,) d 13.38 (s, 1H, NH-1'), 10.80 (s, ÍH, NH-1), 7.76 (d, J = 7.93 Hz, ÍH, H-4) , 7.57 (s, ÍH, H-vinyl), 7.35 (t, J = 8.08 Hz, ÍH, H-5"), 7.26 (dd, J = 1.73, 7.93 Hz, ÍH, H-5), 7.18 (d , br, J = 8.08 Hz, ÍH, H-4"), 7.13 (t, br, J = 1.94 Hz, ÍH, H-2"), 7.08 (d, J = 1.73 Hz, ÍH, H- 7) , 6.90 (dd, J = 1.94, 8.08 Hz, ÍH, H-6"), 3.81 (s, 3H, OCH, - 3"), 3.56 (t, J = 4.38 Hz, 4H, O (C CH,) , NCH, CHjCHj-4 '), 2.41 (t, J = 7.19 Hz, 2H, 0 (CH, CHj) jNCH, CH, CH, -4'), 2.31 (t, J = 4.38 Hz, 4H, 0 ( CH, CH,), NCH, CH, CSj-4 '), 2.29 (3, 3H, CH, -3'), 2.26 (a, 3H, CH, -5 '), 2.24 (t, J = 7.19 Hz , 2H, 0 (CH, CH2), NCH2CHjCH, -4 '), 1.56 (quint., J at 7.19 Hz, 2H, O (CH, CH2) jNCHjCIijCHj-4'), MS m / e (relative intensity .. %) 471 (m *.? Oo).

EXAMPLE 47 3- [3,5-Dimethyl-4- (3-morpholin-4-yl-propyl) -lH-pyrrol-2-ylmethylene] -6- (4-methoxyphenyl) -1,3-dihydroindole-2- Ona Using the method of Example 2, a yield of 52 percent of the title compound was obtained as a yellow solid: XHNMR (300 MHz, DMSO-d,) d 13.35 (a, ÍH, NH-1 '), 10.77 (a, ÍH, NH-1), 7.72 (d, J at 7.97 Hz, ÍH, H-4), 7.55 (d, J = 8.57 Hz, 2H, H-2", 6"), 7.54 (a, ÍH, H-vinyl), 7.20 (dd, J = 1.35, 7.97 Hz, ÍH, H-5), 7 ..04 (d, J »1.35 Hz, ÍH, H-7), 6.99 (d, J = 8.57 Hz, ÍH, H-3 ', 5"), 3.78 (a, 3H, OCH, -4") , 3.55 (t, J - 4.57 Hz, 4H, 0 (Ca, CHj) jNCHaCHjCHj-4 '), 2.40 (t, J - 6.97 Hz, 2H, O (CH, CHJ) JNCH, CH, CÜJ-4') , 2.30 (t, J - 4.57 Hz, 4H, 0 (CHJCÜJ), NCHJCHJCHJ-4 '), 2.28 (s, 3H, CH, -3'), 2.24 (s, 3H, CH, 5 '), 2.23 ( t, J = 6.97 Hz, 2H, 0 (CH, CH,), Nq, CH, CH, -4 '), 1.55 (quine, J = 6.97 Hz, 2H, O (CH, CH,), NCH, CH , CH, -4 '), MS m / e (relative intensity,%) 471 (M', 100).

Example 48 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one Using Steps 1, 2 and 3 of Example 1, obtained a 63 percent yield of 4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrole-2-carboxaldehyde as a dark red oil: 1HNMR (360 MHz, DMSO-d,) d 11.33 (a, br, H, NH-1), 9.40 (a, ÍH, CHO-2), 2.30 (t, J - 7.42 Hz, 2H, (CH,) 5NCH2CH, CH2-4), 2.18 (s, 3H, CH, -3), 2.15 (t, J - 7.42 Hz, 2H, (CH,): NCHjCH, CH, - 4), 2.14 (a, 3H, CH3-5), 2.10 (3, 6H, (CH,), NCH, CH, CH, - 4), 1.47 (quint ., J = »7.42 Hz, 2H, (CH,), NCH, CHjCH, -4), MSTITIZ (relative intensity -,%) 208 ([M + l] * ', 100).

Using Step 4 of Example 1, a 52 percent yield of the title compound was obtained as a yellow solid: 'HNMR (360 MHz, DMSO-d,) d 13.38 (a, ÍH, NH-1'), 10.70 (a, ÍH, NH-1), 7.68 (d, J at 7.54 Hz, ÍH, H-4), 7.53 (a, ÍH, H-vinyl), 7.06 (t, J at 7.54 Hz, ÍH, H- 6), 6.94 (t, J = 7.54 Hz, ÍH, H-5), 6.85 (d, J, 7.54 Hz, ÍH, H-7), 2.38 (t, J at 7.25 Hz, 2H, (CH,) jNCH2CHjCHj-4 ') f 2.27 (a, 3H, CH, -3'), 2.23 (a, 3H, CH, -5 '), 2.17 (t, J - 7.25 Hz, 2H, (CH,), NC? ?, CHaCH2-4 '), 2.11 (s, 6H, (CH,) jNCHjCH, CHj-4'), 1.52 (quint., J = 7.25 Hz, 2H, (CH), NCHjCHjCH, -4 '), MS m / z (relative intensity -d -,%) 323 (M * -, 100).

Example 49 5-Bromo-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one Using the procedure of Example 2, a 71 percent yield of the title compound as a red solid: lHNMR (360 MHz, DMSO-d,) d 13.42 (s, ÍH, NH-1 '), 10.81 (a, ÍH, NH-1), 7.98 (d, J-1.89 Hz, ÍH, H-4), 7.66 (a, ÍH, H-vinilt)), 7.17 (dd, J-1.89, 8.23 Hz, ÍH, H-6), 6.79 (d, J = 8.23 Hz, ÍH, H-7), 2.38 (t, J - 7.23 Hz, 2H, (CH,), NCH, CH, CH, - 4 '), 2.27 (a, 3H, CH, -3'), 2.25 (s, 3H, CH, -5 '), 2.16 (t, J = 7.23 Hz, 2H, (CH,) 2NCHaCH, CH, -4 '), 2.10 (s, 6H, (CH,), NCH, CH: CH, - 4'), 1.51 (quint., J at 7.23 Hz, 2H, (CH,) 2NCH, CH, CH, -4 '), MS m / z (eVative interest,%) 401 ([Ml] * -, 100) and 403 ([ M + l] * -, 100).

EXAMPLE 50 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6-phenyl- ?, 3-dihydroindol-2-one Using the procedure of Example 2, obtained an 83 percent yield of the title compound as an orange solid: 'VHNMR (360 MHz, DMSO-d,) d 13.38 (a, ÍH, NH-1'), 10.81 (a, 1H, NH-1), 7.77 (d, J at 7.82 Hz, ÍH, H-4) , 7.62 (d, J = 7.59 Hz, 2H, H-2", 6"), 7.58 (s, ÍH, HV.i? Lljc), 7.44 (t, J = 7.59 Hz, 2H, H-3", 5"), 7.32 (t, J = 7.59 Hz, ÍH, H-4"), 7.27 (dd, J = 1.11, 7.82 Hz, ÍH, H-5), 7.09 (d, J = 1.11 Hz, ÍH, H-7), 2.39 (t, J = 7.18 Hz, 2H, (CH,), NCH2CH2CHa-4 '), 2.29 (a, 3H, CH, -3'), 2.25 (3, 3H, CH, -5 '), 2.17 (t, J = 7.18 Hz, 2H, (CH,), NCH2CH2CH2- 4'), 2.11 (s, 6H, (Qi,), NCH, CH, CH, -4 '), 1.53 (quint ., J = 7.18 Hz, 2H, (CH,) jNCH, CH2CHj-4 '), MS m / z (i: ntdnsi'uaa re > a- iv? I,%) 399 (M * \ 100).

Example 51 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6- (2-methoxyphenyl) -1,3-dihydroindol-2-one Using the procedure of Example 2, a yield of 83 percent of the title compound was obtained as a yellow solid: XHNMR (360 MHz, DMSO-d,) d 13.38 (s, ÍH, NH-1 '), 10.72 (s, ÍH, NH-1), 7.70 (d, J at 8.06 Hz, ÍH, H-4), 7.55 (s, ÍH, H-vinylc), 7.28-7.36 (m, 2H, H-4", 5"), 7.14 (d, J - 8.32 Hz, ÍH, -H-6"), 7.04 (dd, J »1.21, 8.06 Hz, ÍH, H-5), 6.99 (d, J = 7.42 Hz, ÍH, H-3"), 6.99 (d, J = 1.21 Hz, ÍH, H-7) 3.76 (s, 3H, 0CH, -2"), 2.39 (t, J - 7.24 Hz, 2H, (CH,), NCH, CH2CH2-4 '), 2.28 (s, 3H, CH, -3'), 2.25 (s, 3H, CH, -5 '), 2., 18 (t, J = 7.24 Hz, 2H, (CH,) jNCH, CH2CH, -4') r, 2.11 (a, 6H, (CE,) 2NCH2CH2CH, - 4 ' ) , 1. 53 (quint., J - 7.24 Hz, 2H, (CH,) 2NCH, CH, CH2-4 '), MS m / z (tnt &relative nsity -,%) 429 (M * \ 100).

Example 52 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6- (3-methoxyphenyl) -i, 3-dihydroindol-2-one Using the procedure of Example 2, a yield of 83 percent of the title compound was obtained as a red solid: 'HNMR (360 MHZ, DMSO-d,) d 13.38 (a, ÍH, NH-1'), 10.80 (s, IH, NH-1), 7.60 (d, J-8.06 Hz, ÍH, H-4) , 7.57 (a, ÍH, H- vinyl), 7.35 (t, J - 8.15 Hz, ÍH, H-5 »), 7.26 (dd, J = 1.39, 8.06 Hz, 1H, H-5), 7.19 (d , br, J - 8.15 Hz, H-6"), 7.13 (m, ÍH, H-2"), 7.09 (d, J »1.39 Hz, ÍH, H-7), 6.90 (dd, J - 2.57, 8.15 Hz, ÍH, H-4"), 3.81 (a, 3H, 0CH, -3"), 2.39 (t, J - 7.17 Hz, 2H, (CH), NCH, CH, CH, -4 ') , 2.29 (s, 3H, CH, -3 '), 2.25 (3, 3H, CH, -5'), 2.17 (t, J »7.17 Hz, 2H, (CH), NCHjCH, CH, -4 ' ), 2.11 (a, 6H, (CHj) jNCHjCH, CH, -4 '), 1.53 (quint., J = 7.17 Hz, 2H, (CH,), NCHjCH, CH, -4'), MS m / z (relative intensity,%) 429 (M * '"100).

Example 53 3- [4- (3-Dimetiaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6- (4-methoxyphenyl) - ?, 3-dihydroindol-2-one Using the procedure of Example 2, a yield of 83 percent of the title compound was obtained as a tan solid: 'HNMR (360 MHz, DMSO-d,) d 13.35 (a, ÍH, NH-1'), 10.77 (a, ÍH, NH-1), 7.73 (d, J = 7.82 Hz, ÍH, H-4) , 7.56 (d, J = 8.83 Hz, 2H, H-2-, 6"), 7.54 (a, ÍH, H-vinyl), 7.20 (dd, J at 1.64, 7.82 Hz, ÍH, H-5), 7.04 (d, J - 1.64 Hz, H-7), 7.00 (d, J - 8.83 Hz, 2H, H-3 », 5), 3.78 (s, 3H, OCH, -4"), 2.39 (t, J = 7.24 HZ, 2H, (CH,) jNCHjCHjCHa, -4 '), 2.28 (a, 3H, CH, -3'), 2.25 (s, 3H, CH, -5 '), 2.17 (t, J- 7.24 Hz, 2H, (CH,) jNCH.! CH, CH, -4 '), 2.11 (s, 6H, (CHJ), NCH1CHaCHj-4'), 1.52 (quint., J - 7.24 Hz, 2H, ( CHj), NCHjCHaCHj-4 '), MS m / z (elastic intensity -t% j 42'9 ^ t 100) # Example 54 5-Chloro-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one Using the procedure of Example 2, a yield of 53 percent of the title compound as a chestnut solid: 'HNMR (360 MHz, DMSO-d,) d 13.43 (a, ÍH, NH-1'), 10.84 (s, ÍH, NH-1), 7.87 (d, J »1.85 Hz, ÍH, H-4), 7.66 (s, 1H, H-vinyl), 7.05 (dd, J at 1.85, 8.15 Hz, ÍH, H-6 ), 6.83 (d, J = 8 15 Hz, ÍH, H- 7), 2. 36 -2. 45 (m, 4H, (CH,) ¡NCHjCHjCH, ^ '), 2. 30 (s, 6H, (CH,) 2NCH2CH2CHj-4 '), 2.28 (s, 3H, CH, -3'), 2 '.26 (s, 3H, CH, -5'), 1.58 (quint., J = 7.52 Hz, 2H, (CH,) 2NCH2CH2CH2-4 '), MS m / z (intensity-relative,%) 357 ([Ml] *, 100).

Example 55 6-Chloro-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one Using the procedure of Example 2, a yield of 77 percent of the title compound: MS 357 [Ml] +. Example 56 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -5-methoxy-l, 3-dihydroindol-2-one Using the procedure of Example 2, a yield of 77 percent of the title compound: MS 353 [M] +. Example 57 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6-methoxy-1,3-dihydroindol-2-one Using the procedure of Example 2, a yield of 74 percent of the title compound. Example 58 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -5-methyl-1,3-dihydroindol-2-one Using the procedure of Example 2, a yield of 45 percent of the title compound: MS The 337 [M] +. Example 59 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -4-methyl-1,3-dihydroindol-2-one Using the procedure of Example 2, a yield of 99 percent of the title compound: lHNMR (300 MHz, DMSO-d,) d 13.45 (a, br, ÍH, NH), 10.78 (s, br, 1H, NH), 7.50 (a, ÍH, H-vinyl), 6. 98 (t, J «8.1 Hz, ÍH, H-6), 6.76 (t, J at 8.1 Hz, 2H, H-5 &H-7), 2.88 (m, 2H, CH,), 2.64 (s , 6H, 2x H,), 2.56 (s, 3H, CH,), 2.43 (t, J = 7.4 Hz, 2H, CHj), 2.29 (s, 3H, CH,), 2.19 (a, 3H, CH,), 1.65-1.75 (m, 2H, CH,), MS The 337 [M] *.

Example 60 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -4- (2-hydroxy-ethyl) -1,3-dihydroindol-2-one Using the procedure of Example 2, a yield of 98 percent of the title compound was obtained. Example 61 3- (4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -2-oxo-2,3-dihydro-lH-indol-5-sulfonic acid amide Using the procedure of Example 2, a yield of 59 percent of the title compound was obtained: lHNMR (300 MHz, DMSO-d,) d 13.41 (a, ÍH, NH-1 '), 11.12 (a, ÍH, NH-1), 8.16 (d, J - 1.78 Hz, ÍH, H-4), 7.66 (a, ÍH, H- vinyl), 7.55 (dd, at 1.78, 8.18 Hz, ÍH, H-6), 7.11 (a, br, 2H, H, NSO, -5), 6.98 (d, J » 8.18 Hz, ÍH, H-7), 2.47-2.50 (m, 2H, (CHJ) JNCHJCHJQ? J-4 '), 2.41 (t, J - 7.37 Hz, 2H, (CH,) 2NCHaCH2CHj-4'), 2.36 (a, 6H, (CH,) JNCHJCHJCHJ-4 '), 2.30 (s, 3H, CHj-3'), 2.28 (s, 3H, CH, -5 '), 1.61 (quint., J = 7.37 Hz , 2H, (CH,) 2NCH, CHaCH, -4 '). . . .

EXAMPLE 62 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -2-oxo-2,3-dihydro-1H-indole-5-sulphonic acid isopropylamide Using the procedure of Example 2, a 64 percent yield of the title compound was obtained: MS 444 [M] +. Example 63 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -5- (morpholin-4-sulfonyl) -l, 3-dihydroindol-2-one Using the procedure of Example 2, a yield of 90 percent of the title compound was obtained. MS The 472 [M] +. EXAMPLE 64 3- [4- (3-Dimethylaminopropyl) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -2-oxo-2,3-dihydro-1H-indol-5-sulfonic acid dimethylamide Using the procedure of Example 2, a yield of 92 percent of the title compound was obtained: lHNMR (300 MHz, DMSO-d,) d 13.5 (s, br, ÍH, NH), 12.21 (s, br, ÍH, NH), 8.18 (d, J - 1.8 Hz, ÍH, H-4), 7.84 (s, ÍH, H- vmyl), 7.44 (dd, J at 1.8, 8.4 Hz, ÍH, H-6), 7.05 (d, J »8.4 Hz, ÍH, H-7), 2.59 (s, 6H, 2xCH,), 2.59-2.64 (m, 2H, CH,), 2.44 (3, 6H, 2xCH,), 2.38-2.44 (m, 2H, CHa), 2.31 (s, 6H, 2xCH,), 1.59-1.69 (m, 2H, CH2), MS The 430 [M] *. 7. BIOLOGICAL EVALUATION It will be appreciated that, in any given series of compounds, a spectrum of biological activity will be provided. In its presently preferred embodiments, this invention relates to novel 2-indolinones substituted by pyrrole, which demonstrate the ability to modulate the activity of RTK, CTK and STK. The following tests are used to select compounds that demonstrate the optimum degree of the desired activity.

A. Assay Procedures The following in vitro assays can be used to determine the level of activity and the effect of the different compounds of the present invention on one or more of the protein kinases. Similar assays can be designed along the same lines for any protein kinase, using techniques well known in the art. The cellular / catalytic assays described herein are performed in an ELISA format. The general procedure is as follows: a compound is introduced to cells expressing the test kinase, either naturally or in a recombinant manner, for a selected period of time, after which, if the test kinase is a receptor, a ligand that is known to activate the receptor is added. The cells are lysed, and the lysate is transferred to the wells of an ELISA plate previously coated with a specific antibody that recognizes the substrate of the enzymatic phosphorylation reaction. Components that are not a substrate of the cell lysate are washed, and the amount of phosphorylation on the substrate is detected with an antibody that specifically recognizes phosphotyrosine, compared to control cells that were not contacted with a test compound. The cellular / biological assays described herein measure the amount of DNA formed in response to the activation of a test kinase, which is a general measure of a proliferative response. The general procedure for this assay is as follows: a compound is introduced into the cells expressing the test kinase, either naturally or recombinantly, for a selected period of time, after which, if the test kinase is a receptor, a ligand that is known to activate the receptor is added. After incubation at least overnight, a DNA marker reagent is added, such as bromodeoxyuridine (BrdU) or 3H-thymidine. The amount of labeled DNA is detected with either an anti-BrdU antibody, or by measuring the radioactivity, and compared with the control cells that did not make contact with a test compound. Cell / Catalytic Assays Enzyme linked immunosorbent assays (ELISA) can be used to detect and measure the presence of protein kinase activity. The ELISA can be conducted according to known protocols, which are described, for example, in Voller et al., 1980, "Enzyme-Linked Immunosorbent Assay", in Manual of Clinical Immunology, 2nd edition, edited by Rose and Friedman, pages 359-371, Am. Soc. of Microbiology, Washington, DC The disclosed protocol can be adapted to determine the activity with respect to a specific protein kinase. That is, preferred protocols for conducting ELISA experiments for specific protein kinases are provided below. However, the adaptation of these protocols to determine the activity of a compound for other members of the RTK family, as well as for CTKs and STKs, is well within the scope of the knowledge of the experts in this field. FLK-1 assay An ELISA assay is run to measure the kinase activity of the FLK-1 receptor, and more specifically, the inhibition or activation of TK activity on the FLK-1 receptor. Specifically, the following assay can be conducted to measure the kinase activity of the FLK-1 receptor in cells genetically engineered to express Flk-1. Materials and Reagents a. Corning 96-well ELISA plates (Corning, Catalog No. 25805-96), b. Cappel anti-rabbit goat IgG (Catalog No. 55641), c. PBS (Gibco, Catalog No. 450-1300EB), d. TBSW regulator (50 mM Tris (pH 7.2), 150 mM NaCl, and 0.1 percent Tween 20), e. Supply of ethanolamine (10 percent ethanolamine (pH 7.0), stored at 4 ° C). F. HNTG regulator (HEPES 20mM regulator (pH of 7. 5), 150mM NaCl, 0.2 percent Triton X-100, and 10 percent glycerol), g. EDTA (0.5 M (pH of 7.0) as a 100X supply), h. Sodium orthovanadate (0.5 M, as a 100X supply), i. Sodium pyrophosphate (0.2 M, as a 100X supply), j. 96-well V-bottom polypropylene plates NUNC (Applied Scientific, Catalog No. AS-72092), k. NIH3T3 C7 # 3 cells (cells expressing FLK-1), 1. DMEM with high glucose L-glutamine IX (Catalog No. 11965-050), m. FBS, Gibco (Catalog No. 16000-028), n. L-glutamine, Gibco (Catalog No. 25030-016), or. VEGF, PeproTech, Inc. (Catalog No. 100-20) (maintained as 1 microgram / 100 microliter supply in Milli-Q dH20, and stored at -20 ° C), p. Antiserum anti-FLK-1 purified by affinity, q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley et al. 1990, Cancer Research 50: 1550-1558), r. Goat anti-mouse IgG-POD grade EIA (BioRad, Catalog No. 172-1011), s. Solution of 2, 2-azino-bis (3-ethylbenz-thiazolin-6-sulfonic acid (ABTS)) (citric acid 100mM (anhydrous), 250mM Na2HP04 (pH 4.0), 0.5 milligrams / milliliter of ABTS (Sigma, Catalog No. A-1888)), the solution should be stored in the dark at 4 ° C until it is ready to be used, t. H202 (30 percent solution) (Fisher, Catalog No. H325), u. ABTS / H202 (15 milliliters of ABTS solution, 2 microliters of H202) prepared 5 minutes before use, and left at room temperature, v. 0.2 M HCl supply in H20, w. Dimethyl sulfoxide (100 percent) (Sigma, Catalog No. D-8418), e and. Trypsin-EDTA (Gibco BRL, Catalog No. 25200-049). Protocol 1. Corning 96-well ELISA plates are coated with 1.0 micrograms per well of Cappel anti-rabbit IgG antibody in Na2C03 O.lM, pH 9.6. The final volume is brought to 150 microliters per cavity. The plates are coated overnight at 4 ° C. Plates can be maintained for up to two weeks when stored at 4 ° C. 2. Cells are cultured in a culture medium (DMEM, supplemented with 2.0 mM L-glutamine, 10% FBS) in suitable culture dishes, until confluent at 37 ° C, 5% C02. 3. The cells are harvested by trypsinization and seeded in 96-well round bottom polystyrene Corning 25850 cell plates at 25,000 cells / well in 200 microliters of culture medium. 4. The cells are cultured at least one day at 37 ° C, 5% C02. 5. Wash the cells with D-PBS IX. 6. Add 200 microliters / well of starvation medium (DMEM, 2.0 mM l-glutamine, 0.1% FBS). They are incubated overnight at 37 ° C, C02 at 5 percent. 7. Compounds are diluted 1:20 in 96-well polypropylene plates, using the starvation medium. Dimethyl sulfoxide is diluted 1:20 to be used in the control wells. 8. The starvation medium is removed from the 96-well cell culture plates, and 162 microliters of fresh starvation medium is added to each well. 9. Add 18 microliters of a dilution of compounds diluted 1:20 (from Step 7) to each well, plus the dilution of dimethyl sulfoxide at 1:20 to the control wells (+/- VEGF), for a final dilution of 1: 200 after the cellular stimulus. The final dimethyl sulfoxide is 0.5 percent. The plate is incubated at 37 ° C, C02 at 5 percent, for two hours. 10. Unbound antibody is removed from the ELISA plates by inverting the plate to remove the liquid. Wash three times with TBSW + 0.5% ethanolamine, pH 7.0. Tap the plate on a paper towel to remove excess liquid and bubbles. 11. The plates are blocked with TBSW + 0.5% ethanolamine, pH 7.0, 150 microliters per well. The plate is incubated thirty minutes while stirring on a microtiter plate shaker. 12. The plate is washed 3 times as described in Step 10. 13. 0.5 micrograms / well of affinity purified anti-FLU-1 polyclonal rabbit antiserum is added. The final volume is brought to 150 microliters / well with TBSW + 0.5% ethanolamine, pH of 7.0. The plate is incubated for 30 minutes with shaking. 14. 180 microliters of starvation medium is added to the cells, and cells are stimulated with 20 microliters / 10.0 mM sodium orthovanadate cavity, and 500 nanograms / milliliter of VEGF (resulting in a final concentration of 1.0 mM orthovanadate of sodium and 50 nanograms / milliliter of VEGF per well) for eight minutes at 37 ° C, C02 at 5 percent. Negative control wells receive only starvation medium. 15. After eight minutes, the medium should be removed from the cells, and washed once with 200 microliters / well of PBS. 16. Cells are lysed in 150 microliters / well of NHTG with stirring 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 lysate, pipette up and down while the wells are scraped. 19. The plate is washed three times as described in Step 10. 20. The ELISA plate is incubated with 0.02 micrograms / well of UB40 in TBSW + 0.5% ethanolamine. The final volume is brought to 150 microliters / well. Incubate with shaking for 30 minutes. 21. The plate is washed three times as described in Step 10. 22. The ELISA plate is incubated with 1: 10,000 horseradish peroxidase conjugated with goat anti-mouse IgG grade EIA diluted in TBSW + 0.5% ethanolamine. cent, pH of 7.0. The final volume is brought to 150 microliters / well. Incubate with agitation for thirty minutes. 23. The plate is washed as described in Step 10. 24. 100 microliters of an ABTS / H202 solution is added to the well. Incubate for ten minutes with shaking. 25. Add 100 microliters of 0.2 M HCl to a final concentration of 0.1 M HCl to stop the color development reaction. It is stirred for 1 minute at room temperature. The bubbles are removed with a slow stream of air, and the ELISA plate is read on an ELISA plate reader at 410 nanometers. EGF-Chimeric Receptor Assay HER2 in Whole Cells The activity of HER2 kinase is measured in whole EGFR-NIH3T3 cells as described below: Materials and reagents a. EGF: supply concentration: 16.5 ILM, EGF 201, TOYOBO, Co., Ltd., Japan. b. 05-101 (UBI) (a monoclonal antibody that recognizes an extracellular domain of EGFR). c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see Fendley et al., supra). d. Detection antibody: horseradish peroxidase conjugate with goat anti-rabbit IgG, TAGO, Inc., Burlingame, CA. and. TBST regulator: Tris-HCl, pH 7.2 50 mM 150 Mm NaCl Triton X-100 0.1 f. Supply HNTG 5X: HEPES 0.1 M NaCl 0.75 M Glycerol 50% Triton X-100 1.0% g. Supply ABTS: 100 mM citric acid Na2HP04 250 mM HCl, conc. 0.5 mM ABTS * 0.5 mg / ml * (2,2 '-azinobis (3-ethylbenzo-thiazole-sulphonic acid)). The solution is kept in the dark at 4 ° C until used. h. Supply reagents: 100 mM EDTA, pH 7.0 Na3V04 0.5 M Na4 (P207) 0.2 M Procedure ELISA plate pre-coating 1. Coat ELISA plates (Corning, 96 wells, Catalog No. # 25805-96) with antibody 05-101 at 0.5 micrograms per well in PBS, 100 microliters of final volume / well, and stored overnight at 4 ° C. Coated plates are good for up to 10 days when stored at 4 ° C. 2. On the day of use, the coating regulator is removed, and replaced with 100 microliters of blocking buffer (Carnation Instant Non-Calcium Dry Milk at 5 percent in PBS). The plate is incubated, stirring at room temperature (about 23 ° C to 25 ° C) for 30 minutes. Just before use, the blocking regulator is removed, and the plate is washed 4 times with TBST regulator. Cell Seeding 1. An NIH3T3 cell line that overexpresses a chimeric receptor containing the EGFR extracellular domain and the intracellular kinase domain HER2 can be used for this assay. Plates are selected that have a confluence of 80 to 90 percent for the experiment. The cells are trypsinized and the reaction is stopped by the addition of 10 percent fetal bovine serum. The cells are suspended in a DMEM medium (10 percent CS DMEM medium), and centrifuged once at 1,500 rpm, at room temperature for 5 minutes. 3. The cells are resuspended in the seeding medium (DMEM, 0.5 percent bovine serum, and the cells are counted using trypan blue.) Viability above 90 percent is acceptable. DMEM (0.5 percent bovine serum), in a density of 10,000 cells per well, 100 microliters per well, in a 96-well microtiter plate.The seeded cells are incubated in 5 percent C02 at 37 ° C for approximately 40 hours Test Procedures 1. Seed cells are checked for contamination, using an inverted microscope.The drug supply (10 milligrams / milliliter in dimethyl sulfoxide) is diluted to 1:10 in a DMEM medium, then transfer 5 microliters to a TBST well for a final drug dilution of 1: 200, and a final dimethyl sulfoxide concentration of 1 percent.The control wells receive only dimethyl sulfoxide and are incubated at C0. 2 to 5 percent at 37 ° C for 2 hours. 2. The EGF ligand is prepared: the supply EGF is diluted in DMEM, so that, when transferring 10 microliters of diluted EGF (1:12 dilution), a final concentration of 100 nM is obtained. 3. Fresh enough HNTG * is prepared for 100 microliters per well, and placed on ice. HNTG * (10 milliliters): Supply HNTG 2.0 ml Milli-Q H20 7.3 ml EDTA, 100 mM, pH 7.0 0.5 ml Na3V04 (0.5 M) 0.1 ml Na4 (P207) (0.2 M) 0.1 ml 4. After 120 minutes of incubation with the drug, the ligand SGF prepared to the cells is added, 10 microliters per well, to a final concentration of 100 nM. Control wells receive DMEM only. They are incubated with agitation, at room temperature, for 5 minutes. 5. Remove the drug, EGF, and DMEM. The cells are washed twice with PBS. The HNTG * is transferred to the cells, 100 microliters per well. They are placed on ice for 5 minutes. Meanwhile, the blocking regulator is removed from another ELISA plate, and washed with TBST as described above. 6. With a pipette tip surely adapted to a micropipettor, cells are scraped off the plate, and the cell material is homogenized by aspirating and repeatedly dosing the lysis regulator HNTG *. The lysate is transferred to a coated, blocked and washed ELISA plate. It is incubated by shaking at room temperature for one hour. 7. The lysate is removed and washed 4 times with TBST. The freshly diluted anti-Ptyr antibody is transferred to the plate ELISA in 100 microliters per well. It is incubated by shaking at room temperature for 30 minutes in the presence of anti-Ptyr antibody (dilution of 1: 3000 in TBST). 8. Remove the anti-Ptyr antibody, and wash 4 times with TBST. Transfer the freshly diluted anti-rabbit IgG TAGO antibody to the ELISA plate in 100 microliters per well. It is incubated by shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: dilution of 1: 3000 in TBST). 9. Remove the TAGO detection antibody, and wash 4 times with TBST. Transfer the freshly prepared ABTS / H202 solution to the ELISA plate, 100 microliters per well. It is incubated by stirring at room temperature for 20 minutes. (ABTS / H202 solution: 1.0 microliter of H202 at 30 percent in 10 milliliters of ABTS supply). 10. The reaction is stopped by adding 50 microliters of H2S045N (optional), and O.D. at 410 nanometers. 11. The maximum phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of the phosphotyrosine content for the wells containing extract is then calculated, after subtraction of the negative controls. PDGF-R Assay All cell culture medium, glutamine, and fetal bovine serum can be purchased from Gibco Life Technologies (Grand Island, NY), unless otherwise specified. All cells are grown in a humid atmosphere of 90-95 percent air, and 5 to 10 percent C02 at 37 ° C. All cell lines are routinely subcultured twice a week, and are negative for mycoplasma, determined by the Mycotect method (Gibco). For ELISA assays, cells (U1242, obtained with Joseph Schlessinger, NYU) are grown to an 80 to 90 percent confluence in the culture medium (MEM with 10 percent FBS, NEAA, 1 mM NaPyr, and GLN 2 mM), and plated in tissue culture plates of 96 wells in 0.5 percent serum, in 25,000 to 30,000 cells per well. After incubation overnight in a medium containing 0.5 percent serum, the cells are changed to a serum-free medium, and treated with the test compound for 2 hours in an incubator with 5 percent C02, at 37 ° C. The cells are then stimulated with ligand for 5 to 10 minutes, followed by lysis with HNTG (20 mM Hepes, 150 M NaCl, 10 percent glycerol, 5 mM EDTA, 5 mM Na3V04, 0.2 percent Triton X-100, and NaPyr 2 mM). Cell lysates (0.5 milligrams / well in PBS) are transferred to ELISA plates previously coated with receptor-specific antibody, and which had been blocked with 5 percent milk in TBST (50 mM Tris-HCl, pH 7.2, 150 NaCl mM, and Triton X-100 0.1 percent) at room temperature for 30 minutes. The lysates are incubated with shaking for one hour at room temperature. The plates are washed with TBST four times, and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. The excess anti-phosphotyrosine antibody is removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody is added to the ELISA plate for 30 minutes at room temperature, followed by rinsing with TBST four times more. ABTS (100 mM citric acid, 250 mM Na2HP04, and 0.5 milligrams / milliliter of 2, 2 '-az ino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) plus H202 (1.2 milliliters of 30% H202) is added. for 10 milliliters of ATBS) to the ELISA plates to initiate color development. The absorbance at 410 nanometers with a reference wavelength of 630 nanometers is recorded approximately 15 to 30 minutes after the addition of ABTS. RECEPTOR IGF-1 assay The following protocol can be used to measure the level of phosphotyrosine on the IGF-1 receptor, which indicates the activity of receptor tyrosine kinase IGF-1. Materials and Reagents a. The cell line used in this assay is 3T3 / IFG-1R, a cell line genetically designed to overexpress the IGF-1 receptor. b. NIH3T3 / IGF-1R is grown in an incubator with 5 percent C02 at 37 ° C. The culture medium is DMEM + 10 percent FBS (heat inactivated) + 2 mM L-glutamine. c. Anti-IGF-lR 17-69 antibody purified by affinity. d. D-PBS: KH2P04 0.20 g / 1 KH2P04 2.16 g / 1 KCl 0.20 g / 1 NaCl 8.00 g / 1 (pH 7.2) e. Blocking Regulator: TBST plus 5 percent milk (Carnation Instant Instant Skim Milk). F. TBST Regulator: 50 mM Tris-HCl 150 mM NaCl (pH 7.2 / 10 N HC1) Triton X-100 0.1% The TBS delivery solution (10X) is prepared, and Triton X-100 is added to the regulator during dilution. g. HNTG regulator: 20 mM HEPES 150 mM NaCl (pH 7.2 / HC1 IN) Glycerol 10% Triton X-100 0.2% Prepare the supply solution (5X), and store at 4 ° C. h. EDTA / HC1: 0.5 M, pH 7.0 (NaOH) as a 100X supply. i. Na3V04: 0.5 M as a 100X supply, and the aliquots are stored at 80 ° C. j. Na4P207: 0.2 M as a 100X supply. k. Insulin-1 growth factor of Promega (Cat # G5111). 1. Rabbit polyclonal anti-phosphotyrosine antiserum. m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat. No. 4520, Lot No. 1802): Tago, Inc., Burlingame, CA. n. ABTS solution (2, 2 '-azinobis (3-ethylbenzothiazolinesulfonic acid)). 100 mM citric acid Na2HP04 250 mM (pH 4.0 / 1 N HCl) ABTS 0.5 mg / ml The ABTS solution should be kept in the dark and at 4 ° C. The solution should be discarded when it becomes green. or. Hydrogen peroxide: the 30 percent solution is kept in the dark and at 4 ° C. Procedure All the following steps are conducted at room temperature, unless specifically indicated otherwise. All ELISA plate washes are performed by rinsing the plate with tap water three times, followed by a rinse with TBST. The plate is dried with paper towels. Cell Seeding: Cells, grown in a tissue culture dish (Corning 25020-100) to a confluence of 80 to 90 percent, are harvested with trypsin-EDTA (0.25 percent, 0.5 milliliter / D-100, GIBCO) . 2. Cells are resuspended in fresh DMEM + 10% FBS + 2 mM L-glutamine, and transferred to a 96-well tissue culture plate (Corning, 25806-96) at 20,000 cells / well ( 100 microliters / well). They are incubated for 1 day, then the medium is replaced by a medium free of serum (90 microliters) and incubated in 5% C02 and at 37 ° C overnight. Coating and Blocking the ELISA Plate: 1. Coat the ELISA plate (Corning 25805-96) with anti-IGF antibody at 0.5 micrograms / well in 100 microliters of PBS for at least 2 hours. 2. The coating solution is removed, and replaced with 100 microliters of blocking buffer, and stirred for 30 minutes. The blocking regulator is removed, and the plate is washed just before adding the lysate. Test Procedures: 1. The drugs are tested under serum-free conditions. 2. Dilute the drug supply (in 100 percent dimethyl sulfoxide) to 1:10 with DMEM in a 96-well polypropylene plate, and transfer 10 microliters / well of this solution to the cells to achieve dilution end of the drug of 1: 100, and the final concentration of dimethyl sulfoxide of 1.0 percent. The cells are incubated in 5 percent C02 at 37 ° C for 2 hours. 3. Prepare the fresh cell lysis regulator (HNTG *) HNTG 2 ml EDTA 0.1 ml Na3V04 0.1 ml Na4 (P207) 0.1 ml H20 7.3 ml 4. After the incubation of the drug for two hours, transfer 10 microliters / well of 200nM IGF-1 ligand in PBS to the cells (the final concentration is 20 nM), and incubated in 5 percent C02 at 37 ° C for 10 minutes. 5. The medium is removed, and 100 microliters / well of HNTG * are added, and stirred for 10 minutes. You see the cells under the microscope, to see if they are properly lysed. 6. A 12-channel pipette is used to scrape the cells from the plate, and the lysate is homogenized by repeated aspiration and dosing. Transfer all of the lysate to the ELISA plate coated with antibody, and stir for 1 hour. 7. Remove the lysate, wash the plate, transfer the anti-pTyr (1: 3,000 with TBST), 100 microliters / well, and stir for 30 minutes. 8. Remove the anti-pTyr, wash the plate, transfer the TAGO (1: 3,000 with TBST), 100 microliters / well, and stir for 30 minutes. 9. Remove the detection antibody, wash the plate, and transfer the fresh ABTS / H202 (1.2 microliters of H202 for 10 milliliters of ABTS), 100 microliters / well to the plate, to initiate color development. The OD is measured at 410 nanometers with a reference wavelength of 630 nanometers in the Dynatec MR5000. EGFR Assay The activity of EGF receptor kinase in cells genetically engineered to express human EGF-R can be measured as described below: Materials and Reagents a. Ligand EGF: supply concentration = 16.5 μM, EGF 201, TOYOBO, Co., Ltd., Japan. b. 05-101 (UBI) (a monoclonal antibody that recognizes an extracellular domain of EGFR). c. Antibody-antiphosphotyrosine (anti-Ptyr) (polyclonal). d. Antibody detection: horseradish peroxidase conjugate with goat anti-rabbit IgG, TAGO, Inc., Burlingame, AC. and. TBST regulator: Tris-Hcl, pH 7 50 mM NaCl 150 mM Triton X-100 0.1 Supply of HNTG 5X: HEPES 0.1 M NaCl 0.75 M Glycerol 50 Triton x-100 1.0% Supply ABTS: Citrus Acid 100 M Na3V04 250 Mm HCl, conc. 4.0 pH ABTS * 0.5 mg / ml The solution is stored in the. darkness at 4 ° C until used. h. The supply reagents are: 100 nM EDTA pH 7.0 Na3V04 0.5 M Na4 (P207) 0.2 M Procedure ELISA plate pre-coating 1. Coat the ELISA plates (Corning, 96 wells, Catalog # 25805-96) with antibody 05- 101 at 0.5 micrograms per well in PBS, 150 microliters in final volume / well, and stored overnight at 4 ° C. Coated plates are good for up to 10 days when stored at 4 ° C. 2. On the day of use, the coating regulator is removed, and replaced with blocking buffer (Carnation Instant Non-Calcium Dry Milk at 5 percent in PBS). The plate is incubated, shaken at room temperature (from about 23 ° C to 25 ° C) for 30 minutes. Just before use, the blocking regulator is removed and the plate is washed 4 times with TBST regulator. Seed Cells 1. The NIH 3T3 / C7 cell line can be used (Honegger et al. Cell 51: 199-209, 1987) for this assay. 2. Plates are selected that have a confluence of 80 to 90 percent for the experiment. The cells are trypsinized and the reaction is stopped by the addition of a 10 percent CS DMEM medium. The cells are suspended in DMEM medium (CS percent DMEM 10 percent), and centrifuged once at 1,000 rpm at room temperature for 5 minutes. 3. The cells are resuspended in the seeding medium (DMEM, 0.5 percent bovine serum), and the cells are counted using trypan blue. Viability above 90 percent is acceptable. The cells are seeded in the DMEM medium (0.5 percent bovine serum) at a density of 10,000 cells per well, 100 microliters per well, in a 96-well microtiter plate. The seeded cells are incubated in 5 percent C02 at 37 ° C for approximately 40 hours.

Test Procedures 1. Seed cells are checked for contamination using an inverted microscope. The supply of the test compounds (10 milligrams / milliliter in dimethyl sulfoxide) is diluted to 1:10 in a DMEM medium, then 5 microliters are transferred to a test well to test a dilution of the compounds and the drug. : 200, and a final concentration of dimethyl sulfoxide of 1 percent. The control wells receive only dimethyl sulfoxide. It is incubated in 5 percent C02 at 37 ° C for 1 hour. 2. The IGF ligand is prepared: the supply EGF is diluted in DMEM, so that after transferring 10 microliters of diluted EGF (dilution of 1:12), a final concentration of 25 nM is reached. 3. Prepare 10 milliliters of fresh HNTG * sufficient for 100 microliters per well, where HNTG * comprises: supply of HNTG (2.0. Milliliters), Milli-Q H20 (7.3 milliliters), EDTA, 100 mM, pH 7.0 ( 0.5 milliliter), Na3V04 0.5 M (0.1 milliliter) and Na4 (P207), 0.2 M (0.1 milliliter). 4. It is placed on ice. 5. After two hours of incubation with the drug, the prepared EGF ligand is added to the cells, 10 microliters per well, to produce a final concentration of and is stirred at room temperature for 5 minutes. 6. Remove the test compound, EGF, and DMEM. The cells are washed twice with PBS. The HNTG * is transferred to the cells, 100 microliters per well. It is placed on ice for 5 minutes. Meanwhile, the blocking regulator is removed from another ELISA plate, and washed with TBST as described above. 7. With a pipette tip surely adapted to a micropipettor, cells are scraped off the plate, and the cell material is homogenized by aspirating and repeatedly dosing the lysis regulator HNTG *. The lysate is transferred to a coated, blocked and washed ELISA plate. It is incubated stirring at room temperature for 1 hour. 8. The lysate is removed and washed 4 times with TBST. The freshly diluted anti-Ptyr antibody is transferred to the plate ELISA in 100 microliters per well. It is incubated by shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (dilution of 1: 3000 in TBST). 9. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transfer the freshly diluted anti-rabbit IgG TAGO 30 antibody to the ELISA plate in 100 microliters per well. It is incubated by shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: dilution of 1: 3000 in TBST). 10. Remove the detection antibody and wash 4 times with TBST. Transfer the freshly prepared ABTS / H202 solution to the ELISA plate, 100 microliters per well. It is incubated at room temperature for 20 minutes. ABTS / H202 solution: 1.2 microliters of H202 at 30 percent in 10 milliliters of ABTS supply. 11. The reaction is stopped by the addition of 50 microliters of H2S045N (optional), and the O.D. at 410 nanometers. 12. The maximum phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of the phosphotyrosine content is then calculated for the wells containing extract, after subtraction of the negative controls. Met Autophosphorylation Assay This assay determines the tyrosine kinase activity of Met by the analysis of tyrosine protein kinase Met levels on the Met receptor. Reagents a. HNTG (5X supply solution): Dissolve 23. 83 grams of HEPES and 43.83 grams of NaCl in approximately 350 milliliters of dH20. The pH is adjusted to 7.2 with HCl or NaOH, 500 milliliters of glycerol and 10 milliliters of Triton X-100 are added, mixed, dH20 is added up to 1 liter of total volume. To make 1 liter of working solution IX, add 200 milliliters of 5X supply solution to 800 milliliters of dH20, check and adjust the pH as necessary, and store 4 ° C. b. PBS (Regulated Solution with Dulbecco's Phosphate), Gibco, Catalog # 450-1300EB (solution IX). c. Blocking Regulator: in 500 milliliters of dH20, 100 grams of BSA, 12.1 grams of Tris-pH of 7.5, 58.44 grams of NaCl, and 10 milliliters of Tween-20, and diluted to 1 liter of total volume are placed. d. Kinase Regulator: To 500 milliliters of dH20 are added 12.1 grams of TRIS (pH of 7.2), 58.4 grams of NaCl, 40.7 grams of MgCl2, and 1.9 grams of EGTA, and brought to a total volume of 1 liter with dH20 . and. PMSF (phenylmethylsulfonyl fluoride), Sigma, Catalog Number # P-7626, up to 435.5 milligrams, 100 percent ethanol is added to a total volume of 25 milliliters, vortexed. f. ATP (Bacterial Source), Sigma, Catalog # A-7699, powder is stored at -20 ° C, to reconstitute the solution for use, dissolve 3.31 milligrams in 1 milliliter of dH20. g. Anti-phosphotyrosine conjugated with RC-20H HRPO, Tansduction Laboratories, Catalog # E120H. h. Pierce l-StepMR Turbo TMB-ELISA (3, 3 ', 5,5'-tetramethylbenzidine, Pierce, Catalog # 34022). i. H2SO4, add 1 milliliter concentrate (18 N) to 35 milliliters of dH20. J. TRIS HCl, Fischer, Catalog # BP152-5, to 121.14 grams of the material, add 600 milliliters of MilliQ H20, adjust the pH to 7.5 (or 7.2) with HCl, and bring the volume up to 1 liter with MilliQ H20. k. NaCl, Fischer, Catalog # S271-10, a 5M solution is formed. 1. Tween-20, Fischer, Catalog # S337-500. m. Na3V04, Fischer, Catalog # S454-50, to 1.8 grams of the material are added 8 milliliters of MilliQ H20, the pH is adjusted to 10.0 with HCl or NaOH, boiled in a microwave, cooled, the pH is checked, repeated the procedure until reaching a stable pH of 10.0, MilliQ H20 is added to a total volume of 100 milliliters, aliquots of 1 milliliter are made, and stored at -80 ° C. n. MgCl2, Fischer, Catalog # M33-500, a 1 M solution is formed. HEPES, Fischer, Catalog # BP310-500, to 200 milliliters of MilliQ H20, 59.6 grams of the material are added, the pH is adjusted to 7.5, the. volume up to a total of 250 milliliters, is filtered sterile. p. Albumin, Bovine (BSA), Sigma, Catalog # A-4503, 30 grams of the material are added sterile distilled water to bring the total volume to 300 milliliters, and stored at 4 ° C. q. TBST regulator: to approximately 900 milliliters of dH20 in a 1 liter graduated cylinder, add 6.057 grams of TRIS and 8.766 grams of NaCl; when they dissolve, the pH is adjusted to 7.2 with HCl, 1 milliliter of Triton X-100 is added, and it is brought to a total volume of 1 liter with dH20. r. Goat anti-rabbit IgG purified by affinity (whole molecule), Cappel, Catalog # 55641. s. Anti-h-Met rabbit polyclonal IgG antibody (C-28), Santa Cruz Chemical, Catalog # SC-161. t. Transiently transfected EGFR / Met chimeric cells (EMR) (Komada et al. Oncogene, 8: 2381-2390 (1993).) Sodium Carbonate Regulator (Na2C04, Fischer, Catalog # S495): to 10.6 grams of the material, they add 800 milliliters of MilliQ H20; when dissolved, the pH is adjusted to 9.6 with NaOH, brought to a total volume of 1 liter with MilliQ H20, filtered, and stored at 4 ° C. Procedure All the following steps are conducted at room temperature, unless specifically indicated otherwise. All washing of the ELISA plate is rinsing four times with TBST.

EMR Lysis This procedure can be performed the night before or immediately before the beginning of the capture of the receiver. 1. The lysates are rapidly thawed in a water bath at 37 ° C with an oscillating movement, until the last crystals disappear. 2. The cell granule is smoothed with HNTG IX containing PMSF lmM. 3 milliliters of HNTG are used per dish of 15 centimeters of cells. Half of the calculated volume of HNTG is added, the tube is vortexed for 1 minute, the remaining amount of HNTG is added, vortexed for another minute. 3. The tubes are equilibrated, centrifuged at 10,000 x g for 10 minutes at 4 ° C. 4. The supernatants are pooled, an aliquot is removed to determine the protein. 5. The pooled sample is rapidly frozen in a dry ice / ethanol bath. This step is performed regardless of whether the lysate is stored overnight, or if it is to be used immediately following the determination of the protein. 6. Determination of the protein is performed using the standard bicinchoninic acid (BCA) method (Pierce Chemical BCA Assay Reagent Kit, Catalog # 23225). ELISA procedure 1. Corning 96-well ELISA plates are coated with 5 micrograms per well of goat anti-rabbit antibody in carbonate buffer for a total well volume of 50 microliters. They are stored overnight at 4 ° C. 2. The unbound goat anti-rabbit antibody is removed, inverting the plate to remove the liquid. 3. 150 microliters of blocking regulator is added to each well. They are incubated for 30 minutes with shaking. 4. Wash 4 times with TBST. Dry the plate with a paper towel to remove excess liquid and bubbles. 5. Add 1 microgram per well of rabbit anti-Met antibody diluted in TBST for a total well volume of 100 microliters. 6. The lysate is diluted in HNTG (90 micrograms of lysate / 100 microliters). 7. Add 100 microliters of diluted lysate to each well. Stir for 60 minutes. 8. Wash 4 times with TBST. Dry with a paper towel to remove excess liquid and bubbles. 9. 50 microliters of IX lysate regulator is added per well.

. Compounds / extracts are diluted 1:10 in kinase IX regulator in a 96-well polypropylene plate. 11. Transfer 5.5 microliters of the diluted compound to the wells of the ELISA plate. They are incubated at room temperature with stirring for 20 minutes. 12. Add 5.5 microliters of 60 μM ATP solution per well. Negative controls do not receive ATP. They are incubated for 90 minutes, with shaking. 13. Wash 4 times with TBST. Dry the plate with a paper towel to remove excess liquid and bubbles. 14. 100 microliters are added per well of RC20 (dilution of 1: 3000 in block regulator). They are incubated for 30 minutes with shaking). 15. Wash 4 times with TBST. Dry the plate with a paper towel to remove excess liquid and bubbles. 16. 100 microliters are added per well of Turbo-TMB. Incubate with agitation for 30 to 60 minutes. 17. 100 microliters per well of 1M H2SO4 is added to stop the reaction. 18. Read the assay on the ELISA reader Dynatech MR7000. Test filter = 450 nanometers, reference filter = 410 nanometers.

Src Biochemical Assay This assay is used to determine the src protein kinase activity, by measuring the phosphorylation of a biotinylated peptide as the reading. Materials and Reagents: a. Yeast transformed with src (Sugen, Inc., Redwood City, California). b. Cell lysates: The yeast cells expressing src are granulated, washed once with water, re-granulated, and stored at -80 ° C until used. c. Biotinylated EEEYEEYEEEYEEEYEEEY of the N terminus is prepared by conventional procedures well known to those skilled in the art. d. Dimethyl sulfoxide: Sigma, St. Louis, MO. and. 96-well ELISA plate: 96 well washed Plcica Corning, modified flat bottom, Corning, Catalog # 25805-96. F. V NUNC 96-well bottom polypropylene plate for the dilution of compounds: Applied Scientific, Catalog # A-72092. g. Vecastain Reagent ELITE ABC: Vector, Burlingame, CA. h. Anti-src (327) mab: Schizosaccharo-myces Pombe is used to express the recombinant Src (Superti-Furga et al., EMBO J., 12: 2625-2634, Superti-Furga et al., Nature Biochem .. 14: 600- 605). The S is cultivated Pombe strain SP200 (h-s leul.32 ura4 ade210) as described, and transformations of the pRSP expression plasmids are made by the lithium acetate method (Superti-Furga, supra). Cells are cultured in the presence of 1 μM thiamine to suppress expression from the nmtl promoter, or in the absence of thiamine to induce expression. i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may be used instead). j. TMB-ELISA turbo peroxidase substrate: Pierce Chemical. Regulatory Solutions a. PBS (Regulated Serum with Dulbecco Phosphate): GIBCO PBS, GIBCO, Catalog # 450-1300EB. b. Blocking Regulator: 5 percent skim milk (Carnation) in PBS. c. Carbonate regulator: Na2C04 from Fischer, Catalog # S495, is filled up to a 100 nM supply solution. d. Kinase Regulator: 1.0 milliliter (from a 1M supply solution) of MgCl2, 0.2 milliliter (from a 1M supply solution) of MnCl2, 0.2 milliliter (from a 1M supply solution) DTT, 5.0 milliliter (from a 1M supply solution) of HEPES, 0.1 milliliters of TX-100, is brought up to a total volume of 10 milliliters with MilliQ H20. and. Lysis regulator: HEPES 5.0 (from a 1M supply solution), 2.74 milliliters of NaCl (from a 5M supply solution), 10 milliliters of glycerol, 1.0 milliliters of TX-100, 0.4 milliliters of EDTA (a from a 100 mM supply solution), 1.0 milliliters of PMSF (from a 100 mM supply solution), 0.1 milliliters of Na3V04 (from a 0.1 M supply solution), is brought up to a total volume of 100 milliliters with MilliQ H20. F. ATP: Sigma, Catalog # A-7699, is filled up to a 10 mM supply solution (5.51 milligr/ milliliter). F. TRIS-HC1: Fischer, Catalog # BP 152-5, at 660 milliliters of MilliQ H20 121.14 grof the material are added, the pH is adjusted to 7.5 with HCl, and it is brought to a total volume of 1 liter with MilliQ H20. h. NaCl: Fischer, Catalog # S271-10, is filled up to a 5M supply solution with MilliQ H20. i. Na3V04: Fischer, Catalog # S454-50, 80 milliliters of MilliQ H20, add 1.8 grof the material, adjust the pH to 10.0 with HCl or NaOH, boil in a microwave, cool, check the pH, repeat the pH adjustment, until the pH remains stable after the heating / cooling cycle, it is brought up to a total volume of 100 milliliters with MilliQ H20, aliquots of 1 milliliter are formed, and stored at -80 ° C. j. MgCl2: Fischer, Catalog # M33-500, is filled up to a 1M supply solution with MilliQ H20. k. HEPES: Fischer, Catalog # BP 310-500, 200 milliliters of MilliQ H20, 59.6 grof the material are added, the pH is adjusted to 7.5, it is brought up to a total volume of 250 milliliters with MilliQ H20, it is filtered sterile (1M supply solution). 1. TBST regulator: At 900 milliliters of dH20 6.057 grams of TRIS and 8.766 grams of NaCl are added, the pH is adjusted to 7.2 with HCl, 1 milliliter of Triton X-100 is added, it is brought up to a total volume of 1 liter with dH20. m. MnCl2: Fer, Catalog # M87-100, is taken to a 1M supply solution with MilliQ H20. n. DTT: Fer, Catalog # BP172-5. or. TBS (Regulated Serum with TRIS): to 900 milliliters of MilliQ H20 6.057 grams of TRIS and 8.777 grams of NaCl are added, it is brought up to a total volume of 1 liter with MilliQ H20. p. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 milliliters of Kinase Reagent, 200 micrograms of GST-, is brought to a final volume of 8.0 milliliters with MilliQ H20. q. EEEYEEYEEEYEEEYEEEY labeled with biotin: The peptide delivery solution is formed (1mM, 2.98 milligrams / milliliter in fresh water just before use. r. VEBLASTAIN REAGENT ELITE ABC: To prepare 14 milliliters of working reagent, add 1 drop of reagent to 15 milliliters of TBST, and invert the tube several times to mix. Then add 1 drop of reagent B. Place the tube in an orbital shaker at room temperature, and mix for 30 minutes. Procedures: ELISA plate preparation coated with src 1. The ELISA plate is coated with 0.5 microgram / well of anti-src monoclonal antibody in 100 microliters of sodium carbonate buffer at a pH of 9.6, and is maintained at 4 ° C during the night. 2. Wells are washed once with PBS. 3. Block the plate with 0.15 milliliters of 5 percent milk in PBS for 30 minutes at room temperature. 4. Wash the plate 5 times with PBS. 5. Add 10 microliters / well of yeast lysates transformed with src diluted in lysis buffer (total volume of 0.1 milliliters per well). (The amount of the lysate may vary between batches). The plate is stirred for 20 minutes at room temperature. Preparation of ELISA plate coated with phosphotyrosine antibody. 1. 4G10 plate: 0.5 micrograms / well of 4G10 are coated in 100 microliters of PBS overnight at 4 ° C, and blocked with 150 microliters of 5 percent milk in PBS for 30 minutes at room temperature. KINASE ASSAY PROCEDURE 1. The unbound proteins are removed from the plates, and the plates are washed 5 times with PBS. 2. Add 0.08 milliliters of kinase reaction mixture per well (containing 10 microliters of 10X kinase regulator), and biotin-EEEYEEYEEEYEEEYEEEY 10 μM (final concentration) per well, diluted in water. 3. Add 10 microliters of the diluted compound in water containing 10 percent dimethyl sulfoxide, and pre-incubate for 15 minutes at room temperature. 4. The kinase reaction is started by adding 10 microliters / well of 0.05 mM ATP in water (final 5 μM ATP). 5. The ELISA plate is shaken for 15 minutes at room temperature. 6. The kinase reaction is stopped by adding 10 microliters of 0.5 M EDTA per well. 7. Transfer 90 microliters of supernatant to an ELISA plate coated with blocked 4G10. 8. Incubate for 30 minutes with shaking at room temperature. 9. Wash the plate 5 times with TBST.

. It is incubated with ELTA ABC vectastain reagent (100 microliters / well) for 30 minutes at room temperature. 11. The wells are washed 5 times with TBST. 12. It is developed with Turbo TMB. Ick Biochemical Assay This assay is used to determine lck protein kinase activities by measuring the phosphorylation of GST- as the reading. Materials and Reagents: a. Yeast transformed with Schizosaccharomyces Pombe lck is used to express the recombinant Lck (Superti-Furga, et al., EMBO J, 12: 2625-2634, Superti-Furga, et al., Nature Biotech., 14: 600-605). The S is cultivated Pombe, strain SP200 (h-s leul.32 ura4 ade210) as described, and transformations are made with the pRSP expression plasmids by the lithium acetate method (Superti-Furga, supra). Cells are cultured in the presence of 1 μM thiamine to induce expression. b. Cell lysates: The yeast cells expressing Ick are granulated, washed once in water, granulated again, and stored frozen at -80 ° C until used. c. GST-: DNA encoding the GST-C fusion protein for expression in bacteria, obtained from Arthur Weiss of the Howard Hughes Medical Institute at the University of California, San Francisco. The transformed bacteria are grown overnight with shaking at 25 ° C. GST-f is purified by glutathione affinity chromatography, Pharmacia, Alameda, CA. d. Dimethyl sulfoxide: Sigma, St. Louis, MO. and. 96-well ELISA plate: 96-well Corning plate, easy to wash, modified flat bottom, Corning, Catalog # 25805-96. f. NUNC 96-well V-bottom polypropylene plates for the dilution of compounds: Applied Scientific, Catalog # AS-72092. g. Purified anti-GST rabbit antiserum: Amrad Corporation (Australia), Catalog # 90001605. h. Goat anti-rabbit IgG-HRP: Amersham, Catalog # V010301. i. Anti-mouse sheep IgG (H + L): Jackson Labs., Catalog # 5215-005-003. j. Anti-Lck monoclonal antibody (3A5): Santa Cruz Biotechnology, Catalog # sc-433. k. UBI 05-321 monoclonal anti-phosphotyrosine (UB40 can be used instead). Regulatory solutions: a. PBS (Regulated Serum with Dulbecco's Phosphate) solution IX: GIBCO PBS, GIBCO, Catalog # 450-1300EB. b. Blocking Regulator: 100 grams of BSA, 12.1 grams of TRIS (ph of 7.5), 58.44 grams of NaCl, 10 milliliters of Tween-20, is brought to a total volume of 1 liter with MilliQ H20: c. Carbonate regulator: Na2C04 from Fischer, Catalog # S495, is taken to a 100 mM solution with MilliQ H2 ?. d. Regulator Kinase: 1.0 ml (from a supply solution 1M) MgCl2, 0.2 ml (from a supply solution 1M) MnCl2, 0.2 ml (from a supply solution 1M) DTT, 5.0 milliliters (from a 1M supply solution) of HEPES, 0.1 milliliters of TX-100, is brought to a total volume of 10 milliliters with MilliQ H20. and. Lysis Buffer: HEPES 5.0 (from a supply solution 1M), 2.74 ml of NaCl (from a solution supply 5M), 10 ml of glycerol, 1.0 ml TX-100, 0.4 milliliters of EDTA (to from a 100 mM supply solution), 1.0 milliliters of PMSF (from a 100 mM supply solution), 01 milliliters of Na3V04 (from a 0.1 M supply solution), is brought up to a total volume of 100 milliliters with MilliQ H20. F. ATP: Sigma, Catalog # A-7699, is taken to a 10 mM supply solution (5.51 milligrams / milliliter). g. TRIS-HC1: Fischer, Catalog # BP 152-5, 600 milliliters of MilliQ H20, 121.14 grams of material are added, the pH is adjusted to 7.5 with HCl, it is brought to a total volume of 1 liter with MilliQ H20. h. NaCl: Fischer, Catalog # S271-10, is taken to a 5M supply solution with MilliQ H20. i. Na3V04: Fischer, Catalog # S454-50, 80 milliliters of MilliQ H20, add 1.8 grams of the material, adjust the pH to 10.0 with HCl or NaOH, boil in a microwave, cool, check the pH, repeat the pH adjustment until the pH remains stable after the heating / cooling cycle, it is brought to a total volume of 100 milliliters with MilliQ H20, aliquots of 1 milliliter are made, and stored at -80 ° C. j. MgCl2: Fischer, Catalog # M33-500, is taken to a 1M supply solution with MilliQ H20. k. HEPES: Fischer, Catalog # BP 310-500, at 200 milliliters of MilliQ H20, 59.6 grams of material are added, the pH is adjusted to 7.5, it is brought up to a total volume of 250 milliliters with MilliQ H0, it is filtered sterile ( 1M supply solution). 1. Albumin, Bovine (BSA), Sigma, catalog # A4503, to 150 ml MilliQ H20, were added 30 grams of material is brought to a total volume of 300 ml with MilliQ H20, filtered through a filter of 0.22 microns, it is stored at 4 ° C. m. Regulator TBST: 900 milliliters of dH20 were added 6.057 grams TRIS and 8.766 grams of NaCl, pH adjusted to 7.2 with HCl, 1 ml of Triton X-100 is added, brought to a total volume of 1 liter dH20. n. MnC12: Fischer, Catalog # M87-100, is taken to a 1M supply solution with MilliQ H20. 0. DTT: Fischer, Catalog # BP172-5. p. TBS (Regulated Serum with TRIS): to 900 milliliters of MilliQ H20 6.057 grams of TRIS and 8.777 grams of NaCl are added, it is brought up to a total volume of 1 liter with MilliQ H20. q. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 milliliter of kinase buffer, 200 micrograms of GST-C, is brought to a final volume of 8.0 milliliters with MilliQ H20. Procedures: Preparation of ELISA plate coated with Lck 1. 2.0 micrograms / well of sheep anti-mouse IgG is applied in 100 microliters of sodium carbonate buffer with a pH of 9.6 at 4 ° C overnight. 2. Wash well once with PBS. 3. The plate is blocked with 0.15 milliliters of blocking buffer for 30 minutes at room temperature. 4. Wash the plate 5 times with PBS: 5. Add 0.5 micrograms / well of anti-lck (mab 3A5) in 0.1 milliliters of PBS at room temperature for 1 to 2 hours. 6. Wash the plate 5 times with PBS. 7. Add 20 micrograms / well of yeast lysates transformed with lck diluted in lysis buffer (total volume of 0.1 milliliters per well). The plate is stirred at 4 ° C overnight to prevent loss of activity. Preparation of the ELISA plate coated with phosphotyrosine antibody. 1. UB40 plate: 1.0 micrograms / well of UB40 in 100 microliters of PBS overnight at 4 ° C, and blocked with 150 microliters of blocking buffer for at least 1 hour. KINASE ASSAY PROCEDURE 1. The unbound proteins are removed from the plates, and the plates are washed 5 times with PBS. 2. Add 0.8 milliliters of kinase reaction mixture per well (containing 10 microliters of 10X kinase buffer, and 2 micrograms of GST-C per well diluted with water). 3. Add 10 microliters of the compound diluted in water containing dimethyl sulfoxide to. 10 percent, and pre-incubate for 15 minutes at room temperature. 4. The kinase reaction is started by adding 10 microliters / well of 0.1 mM ATP in water (final 10 μM ATP).

. The ELISA plate is shaken for 60 minutes at room temperature. 6. The kinase reaction is stopped by adding 10 milliliters of 0.5 M EDTA per well. 7. Transfer 90 milliliters of the supernatant to an ELISA plate coated with blocked 4G10 from section B above. 8. Incubate with agitation for 30 minutes at room temperature. 9. Wash the plate 5 times with TBST. 10. Incubate with anti-GST rabbit antibody at a dilution of 1: 5000 in 100 microliters of TBST for 30 minutes at room temperature. 11. The wells are washed 5 times with TBST. 12. Incubate with goat anti-rabbit IgG-HRP at a dilution of 1: 20,000 in 100 microliters of TBST for 30 minutes at room temperature. 13. The wells are washed 5 times with TBST. 14. Developed with Turbo TMB. Test that measures the phosphorylating function of RAF The following test reports the amount of phosphorylation catalyzed by RAF of its MEK white protein, as well as the white MAPK of MEK. The sequence of the RAF gene is described in Bonner et al., 1985, Molec. Cell. Biol., 5: 1400-1407, and is easily accessible in multiple gene sequence data banks. The construction of the nucleic acid vector and the cell lines used for this portion of the invention are fully described in Morrison et al., 1988, Proc. Nati Acad. Sci. USA, 85: 8855-8859. Materials and Reagents 1. Sf9 cells (Spodoptera frugiperda), GIBO-BRL, Gaithersburg, MD. 2. RIPA regulator: Tris 20 M / HCl, pH 7.4, 137 mM NaCl, 10 percent glycerol, 1 mM PMSF, 5 milligrams / liter protein A, 0.5 percent Triton X-100. 3. Thiorredoxin-MEK fusion protein (T-MEK): The expression and purification of T-MEK is performed by affinity chromatography according to the manufacturer's procedures. Catalog # 350-01 and R 350-40, Invitrogen Corp., San Diego, CA. 4. His-MAPK (ERK 2): His-tagged MAP is expressed in Blue XLl cells transformed with the pUC18 vector encoding His-MAPK. His-MAPK is purified by Ni affinity chromatography. Catalog # 27-4949-01, Pharmacia, Alameda, CA, as described herein. 5. Anti-mouse sheep IgG: Jackson Laboratories, West Grove, PA, Catalog # 515-006-008, Lot # 28563. 6. RAF-1 Protein Kinase Specific Antibody: URB2653 from UBI. 7. Coating regulator: P.BS, phosphate-regulated serum, GIBO-BRL, Gaithersburg, MD. 8. Washing regulator: TBST (Tris 50 M / HCl, pH 7.2, 150 mM NaCl, 0.1% Triton X-100). 9. Blocking regulator: TBST, 0.1% ethanolamine, pH 7.4. 10. Dimethyl sulfoxide: Sigma, St. Louis, MO. 11. Kinase regulator (KB): 20 mM HEPES / HCl, pH 7.2, 150 mM NaCl, 0.1 percent Triton X-100, 1 mM PMSF, 5 milligrams / liter protein A, 75 mM sodium orthovanadate, 0.5 mM DTT and 10 mM MgCl2. 12. ATP mixture: 100 mM MgCl 2, 300 mM ATP, 10 mCi V33P ATP (Dupont-NEN) / milliliter. 13. Stop Solution: 1 percent phosphoric acid, Fisher, Pittsburg, PA. 14. Cellulose Phosphate Filter Mats Wallac, Wallac, Truku, Finland. 15. Filter wash solution: 1 percent phosphoric acid, Fisher, Pittsburg, PA. 16. Tomtec plate harvester, Wallac, Turku, Finland. 17. Wallac beta plate reader # 1205, Wallac, Turku, Finland. 18. 96-well NUNC V-bottom polypropylene plates for compounds, Applied Scientific, Catalog # AS- 72092. Procedure All the following steps are conducted at room temperature, unless specifically indicated otherwise. 1. ELISA plate coating: The ELISA wells are coated with 100 milliliters of antisera purified by sheep anti-mouse affinity (1 milligram / 100 milliliters of coating buffer) overnight at 4 ° C. ELISA plates can be used for two weeks when stored at 4 ° C. 2. Invert the plate and remove the liquid. 100 milliliters of blocking solution are added, and inte for 30 minutes. 3. The blocking solution is removed, and washed 4 times with washing buffer. Dry the plate with a paper towel to remove excess liquid. 4. Add 1 milligram of antibody specific for RAF-1 to each well, and inte for 1 hour. Wash as described in Step 3. 5. The lysates are thawed from Sf9 cells infected with RAS / RAF, and diluted with TBST to 10 milligrams / 100 milliliters. 10 milligrams of diluted lysate are added to the wells, and inted for 1 hour. The plate is shaken during intion. Negative controls do not receive a lysate. Lysates of Sf9 insect cells infected with RAS / RAF are prepared after the cells are infected with recombinant baculoviruses at an MOI of 5 for each virus, and harvested 48 hours later. The cells are washed once with PBS, and lysed in RIPA regulator. The insoluble material is removed by centrifugation (5 minutes at 10,000 x g). The aliquots of the lysates are frozen in dry ice / ethanol, and stored at -80 ° C until used. 6. Unbound material is removed, and washed as previously illustrated (Step 3). 7. Add 2 milligrams of T-MEK and 2 milligrams of His-MAEPK per well, and adjust the volume to 40 milliliters with kinase regulator. Methods for purifying T-MEK and MAPK from cell extracts are provided herein as an example. 8. The compounds are pre-diluted (10 milligram / milliliter dimethyl sulfoxide supply solution), or the extracts 20 times in TBST plus 1 percent dimethyl sulfoxide. 5 milliliters of the compounds / extracts previously diluted are added to the wells described in Step 6. They are inted for 20 minutes. The controls do not receive a drug. 9. The kinase reaction is initiated by the addition of 5 milliliter of ATP mixture, the plates are shaken on an ELISA plate shaker during intion.

. The kinase reaction is stopped after 60 minutes by adding 30 milliliters of stop solution to each well. 11. Place the phosphocellulose mat and the ELISA plate on the Tomtec plate harvester. The filter is harvested and washed with the filter wash solution according to the manufacturer's recommendation. The filter mats are dried. The filter mats are sealed, and placed in the fastener. The fastener is inserted into the radioactive detection apparatus, and the radioactive phosphorus is quantified on the filter mats. Alternatively, aliquots of 40 milliliters can be transferred from the individual wells of the assay plate to the corresponding positions on the mat of the phosphocellulose filter. After air drying the filters, the filters are placed in a tray. The tray is gently swung, changing the wash solution at 15 minute intervals for 1 hour. The filter mats are air dried. The filter mats are sealed and placed in a suitable holder to measure the radioactive phosphorus in the samples. The fastener is inserted into a detection device, and the radioactive phosphorus is quantified on the filter mats. Inhibition Assay of CDK2 / Cyclin A This assay analyzes the protein kinase activity of CDK2 on an exogenous substrate. Reagents: A. Regulator A: (Tris 80 mM) (pH of 7.2), MgCl 40 mM), 4.84 grams of Tris (FW = 121.1 grams / mol), 4.07 grams of MgCl2 (FW = 203.31 grams / mol) dissolved in 500 milliliters of H20. The pH is adjusted to 7.2 with HCl. B. Histone Hl Solution (0.45 milligrams / milliliter of Histone Hl), and 20 M HEPES, pH 7.2: 5 milligrams of Histone Hl (Boehringer Mannheim) in ll.lll milliliters OF 20 mM HEPES, pH 7.2 (477 milligrams of HEPES (FW = 238.3 grams / mol) dissolved in 100 milliliters of ddH20, stored in aliquots of 1 milliliter at -80 ° CC ATP solution (60 μM ATP, 300 micrograms / milliliter of BSA, 3 mM DTT): 120 microliters of 10 mM ATP, 600 microliters of BSA in 10 milligrams / milliliter for 20 milliliters, stored in aliquots of 1 milliliter at -80 ° CD CDK2 solution: cdk2 / cyclin A in 10 mM HEPES, pH 7.2, 25 mM NaCl, 0.5 mM DTT, 10 percent glycerol, are stored in aliquots of 9 microliters at -80 ° C. Protocol 1. Inhibitor solutions are prepared at three times the desired final assay concentration in ddH20 / 15 percent by volume DMSO 2. 20 microliters of inhibitors are dosed to the wells of the 96-well polypropylene plates (or 20 m icroliters of 15 percent dimethyl sulfoxide for positive and negative controls). 3. The Histone Hl solution (1 milliliter / plate), the ATP solution (1 milliliter / plate plus 1 aliquot for the negative control), and the CDK2 solution (9 microliters / plate) are thawed. The CDK2 is kept on ice until it is used. Aliquots of the CDK2 solution are appropriately made to avoid repeated freeze-thaw cycles. 4. Dilute 9 microliters of CDK2 solution in 2. 1 milliliter of Regulator A (per plate). They mix. 20 microliters are dosed in each well. 5. Mix 1 milliliter of histone Hl solution with 1 milliliter of ATP solution (per plate) in a 10 milliliter screw cap tube. 33P ATP is added to a concentration of 0.15 μCi / 20 microlitres (0.15 μCi / well in the assay). Mix carefully to avoid frothing the BSA. 20 microliters are added to the appropriate wells. The plates are mixed on the plate agitator. For the negative control / mix the ATP solution with an equal amount of 20 mM HEPES, pH 7.2, and add? 3P ATP to a concentration of 0.15 μCi / 20 microliters of solution. 20 microliters are added to the appropriate wells. 6. Let the reactions proceed for 60 minutes. 7. 35 microliters of TCA at 10 percent is added to each well. The plates are mixed in the plate agitator. 8. 40 microliters of each sample are placed on P30 filter mat squares. The mats are allowed to dry (approximately 10 to 20 minutes). 9. Filter mats are washed 4 x 10 minutes with 250 milliliters of 1 percent phosphoric acid (10 milliliters of phosphoric acid per liter of ddH20) . 10. The filter mats are counted with the beta plate reader. Cellular / Biological Assays Incorporation of BrdU Induced by PDGF Materials and Reagents: (1) PDGF: PDGF B / B human, 1276-956, Boehringer Mannheim, Germany. (2) BrdU Marker Reagent: 10 mM, in PBS (pH 7.4), Catalog No. 1 647 229, Boehringer Mannheim, Germany. (3) FixDenat: fixation solution (ready to use), Catalog No. 1 647 229, Boehringer Mannheim, Germany. (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (6) PBS Wash Solution: PBS IX, pH 7.4 (Sugen, Inc., Redwood City, California). (7) Albumin, Bovine (BSA): powder of fraction V, A-8551, Sigma Chemical Co., USA. (8) 3T3 cell line genetically engineered to express human PDGF-R Protocol (1) Cells are seeded at 8,000 cells / well in DMEM, 10 percent CS, 2 mM Gln, in a 96-well plate.

The cells are incubated overnight at 37 ° C in 5 percent C02. (2) After 24 hours, the cells are washed with PBS, and then the serum is depleted, in a serum-free medium (CS 0% DMEM with 0.1% BSA) for 24 hours. (3) On day 3, the ligand (PDGF, 3.8 nM, prepared in DMEM with 0.1 percent BSA) and the test compounds are added to the cells simultaneously. Negative control wells receive serum-free DMEM with 0.1 percent BSA only, and positive control cells receive the ligand (PDGF), but no test compound. The test compounds are prepared in serum-free DMEM with ligand in a 96-well plate, and serially diluted for 7 test concentrations. (4) After 20 hours of ligand activation, the diluted BrdU label reagent is added (1: 100 in DMEM, 0.1% BSA) and the cells are incubated with BrdU (final concentration = 10 μM for 1.5 hours. (5) After incubation with the labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel, FixDenat solution (50 microliters / well) is added and the plates are incubated at room temperature for 45 minutes. minutes on a plate shaker (6) The FixDenat solution is completely removed by decanting and tapping the inverted plate on a paper towel, adding milk (5% dehydrated milk in PBS, 200 microliters / well) as a blocking solution, and the plate is incubated for 30 minutes at room temperature on a plate shaker (7) The blocking solution is removed by decanting, and the wells are washed once with PBS, anti-BrdU-POD solution is added (dilution of 1 100 in PBS, 1 percent BSA) (100 microliters / well), and the plate is incubated for 90 minutes at room temperature on a plate shaker. (8) The conjugate antibody is completely removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. (9) The TMB substrate solution (100 microliters / well) is added, and incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection. (10) The absorbance of the samples is measured at 410 nanometers (in the "double wavelength" mode with a filter reading at 490 nanometers, as a reference wavelength) on a Dynatech ELISA plate reader. Incorporation Assay of BrdU Induced by EGF Materials and Reagents (1) EGF: Mouse EGF, 201, Toyobo, Co., Ltd., Japan. (2) BrdU Marker Reagent: 10 mM, in PBS (pH 7.4), Catalog No. 1 647 229, Boehringer Mannheim, Germany. (3) FixDenat: fixation solution (ready to use), Catalog No. 1 647 229, Boehringer Mannheim, Germany. (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (6) PBS Wash Solution: PBS IX, pH 7.4 (Sugen, Inc., Redwood City, California). (7) Albumin, Bovine (BSA): powder of fraction V, A-8551, Sigma Chemical Co., USA. (8) 3T3 cell line genetically engineered to express human EGF-R. Protocol (1) Cells are seeded at 8,000 cells / well in percent CS, 2 mM Gln in DMEM, in a 96-well plate. The cells are incubated overnight at 37 ° C in 5 percent C02. (2) After 24 hours, the cells are washed with PBS, and then the serum is depleted in serum-free medium.

(CS DMEM at 0 percent with BSA at 0.1 percent) for 24 hours. (3) On day 3, the ligand (EGF, 2 nM, prepared in DMEM with 0.1 percent BSA) and the test compounds are added to the cells simultaneously. Negative control wells receive serum-free DMEM with 0.1 percent BSA only, and positive control cells receive the ligand (EGF), but no test compound. The test compounds are prepared in serum-free DMEM with ligand in a 96-well plate, and serially diluted for 7 test concentrations. (4) After 20 hours of ligand activation, the diluted BrdU label reagent is added (1: 100 in DMEM, 0.1 percent BSA), and the cells are incubated with BrdU (final concentration = 10 μM for 1.5 hours. (5) After incubation with the labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. The FixDenat solution (50 microliters / well) is added, and the plates are incubated at room temperature for 45 minutes on a plate shaker. (6) The FixDenat solution is completely removed by decanting and tapping the inverted plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 microliters / well) is added as a blocking solution, and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting, and the wells are washed once with PBS. The anti-BrdU-POD solution (1: 100 dilution in PBS, 1 percent BSA) (100 microliters / well) is added, and the plate is incubated for 90 minutes at room temperature on a plate shaker. (8) The conjugate antibody is completely removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. (9) The TMB substrate solution (100 microliters / well) is added, and incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection. (10) The absorbance of the samples is measured at 410 nanometers (in the "double wavelength" mode, with a filter reading at 490 nanometers, as a reference wavelength), in a Dynatech ELISA plate reader . Incorporation of BrdU Powered by Her2 Induced by EGF Materials and Reagents (1) EGF: Mouse EGF, 201, Toyobo, Co., Ltd., Japan. (2) BrdU Marker Reagent: 10 M, in PBS (pH of 7. 4), Catalog No. 1 647 229, Boehringer Mannheim, Germany. (3) FixDenat: fixation solution (ready to use), Catalog No. 1 647 229, Boehringer Mannheim, Germany. (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (6) PBS Wash Solution: PBS IX, pH 7.4, made at home. (7) Albumin, Bovine (BSA): powder of fraction V, A-8551, Sigma Chemical Co., USA. (8) 3T3 cell line designed to express a chimeric receptor that has the extracellular domain of EGF-R, and the intracellular domain of Her2. Protocol (1) Cells are seeded at 8,000 cells / well in DMEM, 10 percent CS, 2 mM Gln, in a 96-well plate. The cells are incubated overnight at 37 ° C in 5 percent C02. (2) After 24 hours, the cells are washed with PBS, and then the serum is depleted in a serum-free medium (CS 0% DMEM with 0.1% BSA) for 24 hours. (3) On day 3, the ligand (EGF = 2 nM, prepared in DMEM with 0.1 percent BSA) and the test compounds are added to the cells simultaneously. Negative control wells receive serum-free DMEM with 0.1 percent BSA only, and positive control cells receive the ligand (EGF), but no test compound. The test compounds are prepared in serum-free DMEM with ligand in a 96-well plate, and serially diluted for 7 test concentrations. (4) After 20 hours of ligand activation, the diluted BrdU label reagent is added (1: 100 in DMEM, 0.1 percent BSA), and the cells are incubated with BrdU (final concentration = 10 μM) for 1.5 hours . (5) After incubation with the labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. The FixDenat solution (50 microliters / well) is added, and the plates are incubated at room temperature for 45 minutes on a plate shaker. (6) The FixDenat solution is completely removed by decanting and tapping the inverted plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 microliters / well) is added as a blocking solution, and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting, and the wells are washed once with PBS. The anti-BrdU-POD solution (1: 100 dilution in PBS, 1 percent BSA) (100 microliters / well) is added, and the plate is incubated for 90 minutes at room temperature on a plate shaker. (8) The conjugate antibody is completely removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. (9) The TMB substrate solution (100 microliters / well) is added, and incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection. (10) The absorbance of the samples is measured at 410 nanometers (in the "double wavelength" mode, with a filter reading at 490 nanometers, as a reference wavelength), in a Dynatech ELISA plate reader . Enzyme of BrdU Incorporation Induced by IGF1 Materials and Reagents (1) Ligand IGF1: human, recombinant, G511, Promega Corp, USA. (2) BrdU Marker Reagent: 10 mM, in PBS (pH 7.4), Catalog No. 1 647 229, Boehringer Mannheim, Germany. (3) FixDenat: fixation solution (ready to use, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated to peroxidase, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Catalog No. 1 647 229, Boehringer Mannheim, Germany. (6) PBS Wash Solution: PBS IX, pH 7.4 (Sugen, Inc., Redwood City, California). (7) Albumin, Bovine (BSA): powder of fraction V, A-8551, Sigma Chemical Co., USA. (8) 3T3 cell line genetically designed to express the human IGF-1 receptor. Protocol (1) Cells are seeded at 8,000 cells / well in DMEM, 10 percent CS, 2 mM Gln in a 96-well plate.

The cells are incubated overnight at 37 ° C in 5 percent C02. (2) After 24 hours, the cells are washed with PBS, and then the serum is depleted in a serum-free medium (CS 0% DMEM with 0.1% BSA) for 24 hours. (3) On day 3, the ligand is added (IGF1 = 3.3 nM, prepared in DMEM with 0.1 percent BSA), and the test compounds to the cells simultaneously. Negative control wells receive serum-free DMEM with 0.1 percent BSA only, and positive control cells receive the ligand (IGF1), but no test compound. The test compounds are prepared in serum-free DMEM with ligand in a 96-well plate, and serially diluted for 7 test concentrations. (4) After 16 hours of ligand activation, the diluted BrdU label reagent is added (1: 100 in DMEM, 0.1 percent BSA), and the cells are incubated with BrdU (final concentration = 10 μM) for 1.5 hours. (5) After incubation with the labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution (50 microliters / well) is added, and the plates are incubated at room temperature for 45 minutes on a plate shaker. (6) The FixDenat solution is completely removed by decanting and tapping the inverted plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 microliters / well) is added as a blocking solution, and the plate is incubated for 30 minutes at room temperature on a plate shaker. (7) The blocking solution is removed by decanting, and the wells are washed once with PBS. The anti-BrdU-POD solution (dilution 1: 100 in PBS, 1% BSA) (100 microliters / well) is added and the plate is incubated for 90 minutes at room temperature on a plate shaker. (8) The conjugate antibody is completely removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel. (9) The TMB substrate solution (100 microliters / well) is added, and incubated for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection. (10) The absorbance of the samples at 410 nanometers (in the "double wavelength" mode with a filter reading at 490 nanometers, as a reference wavelength) is measured in a Dynatech ELISA plate reader. FGF-Induced BrdU Incorporation Assay This assay measures FGF-induced DNA synthesis in 3Tc7 / EGFr cells expressing endogenous FGF receptors. Materials and reagents: (1) FGF: human FGF2 / bFGF (Gibco BRL, No. 13256-029). 2. BrdU Marker Reagent (10 mM PBS (pH 7.4), Boehringer Mannheim, Catalog No. 1 647 229). 3. Fixing Solution Fixdenat (Boehringer Mannheim, Catalog No. 1 647 229). 4. Anti-BrdU-POD (mouse monoclonal antibody conjugated with peroxidase, Boehringer Mannheim, Catalog No. 1 647 229). 5. TMB (tetramethylbenzidine, Boehringer Mannheim, Catalog No. 1 647 229). 6. PBS Wash Solution, pH 7.4 (Sugen, Inc.). 7. Albumin, bovine (BSA), fraction V powder (Sigma Chemical Co., Catalog No. A-8551). Procedure 1. Designed cell line 3T3: 3T3c7 / EGFr. 2. The cells are seeded at 8,000 cells / well in DMEM, 10 percent CS, and 12 mM Gln in a 96-well plate. They are incubated for 24 hours at 37 ° C in 5 percent C02. 3. After 24 hours, the cells are washed with PBS, then the serum is depleted in a serum-free medium (0 percent DMEM, 0.1 percent BSA) for 24 hours. 4. The ligand (FGF2 (1.5 nM in DMEM with 0.1 percent BSA) is added, and the test compound simultaneously.The negative control wells receive serum-free DMEM with 0.1 percent BASA only, and the wells Positive controls receive the ligand FGF2, but no test compound.The test compounds are prepared in serum-free DMEM with ligand in a 96-well plate, and serially diluted to make seven (7) test concentrations. After 20 hours, the diluted BrdU label reagent (1: 100, BrdU: DMEM, 0.1% BSA, final concentration is 10 μM) is added to the cells, and incubated for 1.5 hours. The traces of the material are removed with a paper towel, FixDenat (50 microlitres / well) is added, and incubated at room temperature for 45 minutes on a plate shaker 7. The Fixdenat solution is removed. of blockade (dehydrated milk at 5 percent in PBS ( 200 microliters / well)), and incubate for 30 minutes at room temperature on a plate shaker. 8. The blocking solution is decanted, the wells are washed once with PBS. The anti-BrdU-POD solution (dilution of 1: 100 in PBS, 0.1% BSA) is added, incubated for 90 minutes at room temperature on a plate shaker. 9. The conjugated antibody is decanted, the wells are rinsed 5 times with PBS. The plate is dried by inverting on a paper towel and lightly tapping. 10. Add the TMB solution (100 microliters / well), incubate for 20 minutes at room temperature on a plate shaker, until the color development is sufficient for photometric detection. 11. The absorbance at 410 nanometers is measured on a Dynatech ELISA plate reader using the "double wavelength" mode, with a filter at 490 nanometers. EGFR Biochemical Test This assay measures the in vitro kinase activity of EGFR using ELISA. Materials and Reagents 1. Corning 96-well ELISA plates (Corning, Catalog No. 25805-96). 2. SUMOl anti-EGFR monoclonal antibody (Biochemistry Lab, SUGEN, Inc.). 3. PBS (Regulated Serum by Dulbecco Phosphate, Gibco, Catalog No. 450-1300EB). 4. TBST Regulator Molecular Weight Reagent Working Quantity per liter Tris 121.14 50 mM 6.057 g NaCl 58.44 150 mM 8.766 g Triton X-100 NA 0.1% 1.0 ml Blocking Regulator: Reagent Molecular Weight Concentration Quantity per Work 100 ml Carnation 5% Instant Skim Milk 5.0 g PBS NA NA 100 ml 6. Cellular lysate A431 (Screening Lab, SUGEN, Inc.). 7. TBS regulator: Reagent Molecular Weight Concentration Working Quantity per liter Tris 121.14 50 mM 6.057 g NaCl 58.44 150 mM 8.766 g 8. TBS + 10% DMSO Reagent Molecular Weight Concentration Working Quantity per liter Tris 121.14 50 mM 1.514 g NaCl 58.44 150 mM 2.192 g DMSO NA 10% 25 ml 9. 5 '- Adenosine triphosphate (ATP, equine muscle, Sigma, Catalog No. A-5394). A 1.0 mM solution in dH20 is prepared. This reagent should be formed immediately before use, and kept on ice. 10. MnCl2. A 1.0 M delivery solution in dH20 is prepared. 11. Phosphorylation mixture of ATP / MnCl2.

Reagent Solution Quantity by Concentration Supply 10 ml of work ATP 1.0 mM 300 μl 30 μM MnCl. 1.0 M 500 μl 50 mM dH20 9.2 ml This reagent should be prepared immediately before use, and kept on ice. 12. V NUNC 96-well bottom polypropylene plates (Applied Scientific, Catalog No. AS-72092). 13. Ethylenediaminetetraacetic acid (EDTA). The 200 mM working solution is prepared in dH20. It is adjusted to a pH of 8.0 with 10 N NaOH. 14. Polyclonal rabbit anti-phosphotyrosine serum (Biochemistry Lab, SUGEN, Inc.). 15. Peroxidase conjugate with goat anti-rabbit IgG (Biosource, Catalog No. ALI0404). 16. ABTS 2, 2 '-azino-bis (3-ethylbenzthiazoline-5-sulfonic acid), Sigma, Catalog No. A-1888). Reagent Molecular Weight Concentration Amount of Work per liter Citric acid 192.12 100 mM 19.21 g Na2HP04 141.96 250 mM 35.49 g ABTS NA 0.5 mg / ml. 500 mg First, two ingredients are mixed in approximately 900 milliliters of dH20, the pH is adjusted to 4.0 with phosphoric acid. ABTS is added, covered, allowed to settle for approximately 0.5 hours, and filtered. The solution should be kept in the dark at 4 ° C until it is ready to be used. 17. 30 percent hydrogen peroxide solution (Fisher, Catalog No. H325). 18. ABTS / H202. 15 milliliters of ABTS solution and 2.0 microliters of H202 are mixed. Prepare 5 minutes before use. 19. 0.2 M HCl Procedure 1. Coat 96-well Corning ELISA plates with 0.5 micrograms of SUMOl in 100 microliters of PBS per well, and store overnight at 4 ° C. 2. Remove the unlinked SUMO from the wells by inverting the plate to remove the liquid. Wash once with dH 0. Tap the plate on a paper towel to remove excess liquid. 3. 150 microliters of Blocking Regulator are added to each well. They are incubated for 30 minutes at room temperature with shaking. 4. Wash the plate 3 times with deionized water, then once with TBST. The plate is tapped on a paper towel to remove excess liquid and bubbles. 5. The lysate is diluted in PBS (7 milligrams of lysate / 100 microliters of PBS). 6. 100 microliters of diluted lysate is added to each well. Stir at room temperature for 60 minutes. 7. The plates are washed as described in 4 above. 8. Add 120 microliters of TBS to the ELISA plate containing the captured EGFR. 9. Dilute the test compound to 1:10 in TBS, in 96-well polypropylene plates (ie, 10 microliters of the compound plus 90 microliters of TBS.) 10. 13 microliters of the diluted test compound are added to the plate. ELISA: Control wells (wells that do not receive any test compound), add 13.5 microliters of TBS + 10 percent dimethyl sulfoxide. 11. Incubate for 30 minutes with shaking at room temperature. 12. 15 microliters of phosphorylation mixture are added directly to all wells, except the negative control well, which does not receive ATP / MnCl2 (the final well volume should be approximately 150 microliters with 3 μM ATP / 5 mM MnCl2). of final concentration in each well). They are incubated for 5 minutes with shaking. 13. After 5 minutes, the reaction is stopped by adding 16.5 microliters of 200 mM EDTA (pH 8.0) to each well, stirring continuously. After all the EDTA has been added, it is stirred for 1 minute. 14. Wash 4 times with deionized water, twice with TBST. 15. 100 microliters of anti-phosphotyrosine (dilution of 1: 3000 in TBST) per well is added. Incubate for 30 to 45 minutes at room temperature, with shaking. 16. Wash as described in the previous 4. 17. Add 100 microliters of peroxidase conjugate with goat anti-rabbit IgG Biosource (dilution of 1: 2000 in TBST) to each well. They are incubated for 30 minutes at room temperature, with shaking. 18. Wash as described in the previous 4. 19. 100 microliters of solution is added ABTS / H202 to each well. 20. Incubate for 5 to 10 minutes with shaking. Any bubbles are removed. 21. If necessary, the reaction is stopped with the addition of 100 microliters of 0.2 M HCl per well. 22. The assay is read in the Dynatech MR7000 ELISA reader. Test Filter: 410 nanometers. Reference filter: 630 nanometers. PDGFR Biochemical Assay This assay measures the in vitro kinase activity of PDGFR using ELISA. Materials and Reagents Unless otherwise noted, the preparation of the working solution of the following reagents is the same as that for the previous EGFR biochemical assay. 1. Corning 96-well ELISA plates (Corning, Catalog No. 25805-96). 2. Monoclonal Antibody 28D4C10 anti-PDGFR (Biochemistry Lab, SUGEN, Inc.). 3. PBS (Regulated Serum by Dulbecco Phosphate, Gibco, Catalog No. 450-1300EB). 4. TBST controller. 5. Blocking Regulator 6. NIH 3T3 cell lysate expressing PDGFR-β (Screening Lab, SUGEN, Inc.). 7. TBS controller. 8. TBS + 10 percent dimethyl sulfoxide 9. 5 'Adenosine triphosphate (ATP, equine muscle, Sigma, Catalog No. A-5394). 10. MnCl2. 11. Phosphorylation mixture of kinase regulator Reagent Quantity by Concentration Solution Supply 10 ml of work T Trriiss 1 1 MM 250 μl 25 mM M NaCl 200 μl 100 mM MnCl2 1 M 100 μl 10 mM TX-100 100 mM 50 μl 0.5 mM 12. NUNC 96-well V-bottom polypropylene plates (Applied Scientific, Catalog No. AS-72092). 13. Ethylenediaminetetraacetic acid (EDTA). 14. Polyclonal rabbit serum cinti-phosphotyrosine (Biochemistry Lab, SUGEN, Inc.). 15. Conjugate of peroxidase with goat anti-rabbit IgG (Biosource, Catalog No. ALI0404). 16. 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS, Sigma, Catalog No. A-1888) 17. 30% hydrogen peroxide solution (Fisher, Catalog No. H325). 18. ABTS / H202 19. HCl 0.2 M Procedure 1. Coat the 96-well Corning ELISA plates with 0.5 micrograms of 28D4C10 in 100 microliters of PBS per well, and store overnight at 4 ° C. Remove the unbound 28D4C10 from the wells, inverting the square to remove the liquid, wash once with dH20, tap the plate on a paper towel to remove excess liquid, 3. Add 150 microliters of Regulator from Blockade to each well. Incubate for 30 minutes at room temperature with shaking. 4. Wash the plate 3 times with deionized water, then once with TBST. The plate is tapped on a paper towel to remove excess liquid and bubbles. 5. The lysate is diluted in HNTG (10 micrograms of lysate / 100 microliters of HNTG). 6. 100 microliters of diluted lysate is added to each well. Stir at room temperature for 60 minutes.

. The plates are washed as described in 4 above. 8. Add 80 microliters of working kinase regulator mixture to the ELISA plate containing the captured PDGFR. 9. Dilute the test compound to 1:10 in TBS in 96-well polypropylene plates (i.e., 10 microliters of compound + 90 microliters of TBS). 10. Add 10 microliters of diluted test compound to the ELISA plate. To the control wells (the wells that do not receive any test compound), 10 microliters of TBS + 10 percent dimethyl sulfoxide are added. 11. Incubate for 30 minutes with shaking at room temperature. 12. Add 10 microliters of ATP directly to all wells, except for the negative control well (the final volume of the well should be approximately 100 microliters with 20 μM ATP in each well). They are incubated for 30 minutes with shaking. 13. After 20 minutes, the reaction is stopped by the addition of 10 microliters of 200 mM EDTA (pH 8.0) to each well). 14. Wash 4 times with deionized water, 2 times with TBST. 15. Add 100 microliters of anti-phosphotyrosine (dilution of 1: 3000 in TBST (per well), incubate for 30 to 45 minutes at room temperature, with shaking 16. Wash as described in 4 above. 100 microliters of peroxidase conjugate are added with goat anti-rabbit IgG Biosource (dilution of 1: 2000 in TBST) to each well. Incubate for 30 minutes at room temperature, with shaking. 18. Wash as described in the previous 4. 19. 100 microliters of ABTS / H202 solution is added to each well. 20. Incubate for 10 to 30 minutes with shaking. The bubbles are removed. 21. If necessary, the reaction is stopped with the addition of 100 microliters of 0.2 M HCl per well. 22. The assay is read in the Dynatech ELISA reader MR700: test filter: 410 nanometers; Reference filter: 630 nanometers. FGFR Biochemical Assay This assay measures the in vitro kinase activity of the Myc-GyrB-FGFR fusion protein using ELISA. Materials and Reagents 1. HNTG • T 297 Reagent Weight MoConcentration Amount per Conc. Of lecular Supply 5x Liter work 1 > HEPES 238.3 100 mM 23.83 g 10 mM NaCl 58.44 750 mM 43.83 g 150 mM Glycerol NA 50% 500 ml 10% Triton X - 100 NA 5% 10 ml 1.0% To make a liter of 5x supply solution, dissolve ether and NaCl in approximately 350 milliliters of dH20, adjust the pH to 7.2 with HCl or NaOH (depending on the HEPES that is used, add glycerol, Triton X-100, and then dH20 up to the volume 2. PBS (Regulated Serum with Dulbecco's Phosphate, Gibco, Catalog # 450-1300EB9 15 3. Blocking Regulator 4. Reagent Kinase Regulator Molecular Weight Concentration Supply Concentration workload lx HEPES (pH 7.2) 238.3 500 mM 50 mM MnCl. 203.32 20 Mm 2 mM MgCl2 200 Mm 10 mM Triton X-100 1% 0.1% DTT 380.35 5 Mm 0.5% mM 5. Phenylmethylsulfonyl fluoride (PMSF, Sigma, Catalog No. P-7626). • i 298 Working solution: 100 mM in ethanol. 6. ATP (bacterial source, Sigma, Catalog No. A- 7699). 3.31 milligrams per milliliter of 5 MilliQ H20 are used for a 6 mM supply concentration. 7. Anti-phosphotyrosine monoclonal antibody conjugated with biotin (clone to 4G10, Upstate Biotechnology Inc., Catalog No. 16-103, Serial No. 14495). 8. Vectastain reagent Elite ABC (avidin 10 peroxidase conjugate, Vector Laboratories, Catalog No. PK-6 100). 9. ABTS solution. 10. 30 percent hydrogen peroxide solution (Fischer, Catalog # H325). 15 11. ABTS / H202. 12. 0.2 M HCl. 13. TRIS-HC1 (Fischer, Catalog No. BP 152-5). A 1.0 mM solution is prepared in MilliQ H20, the pH is adjusted to 7.2 with HCl. 20 14. NaCl (Fisher, Catalog No. S271-10). A 5 M solution is prepared in MilliQ H20. 15. MgCl2 (Fischer, Catalog No. M33-500). A 1 M solution is prepared in MilliQ H20. 16. HEPES (Fisher, Catalog No. BP310-500). 25 Prepare a 1 M solution in MilliQ H20, se < t 299 adjusts the pH to 7.5, filters sterile. 17. TBST controller. 18. Sodium Carbonate Regulator (Fischer, Catalog No. S495). 5 Prepare a 0.1 M solution in MilliQ H20, adjust the pH to 9.6 with NaOH, filter. 19. Dithioerythritol (DTT, Fischer, Catalog No. BP172-25). A 0.5 mM working solution is prepared in 10 MilliQ H20 just before use. Store at -20 ° C until used, and any excess is discarded .. 20. MnCl2. 21. Triton X-100. 22. Goat anti-rabbit IgG (Cappel). 15 23. GST of affinity-purified anti-rabbit GyrB (Biochemistry Lab., SUGEN, Inc.). Procedure All the following steps are conducted at room temperature, unless otherwise indicated. 20 1. Corning 96-well ELISA plates are coated with 2 micrograms of goat anti-rabbit antibody per well in Carbonate Regulator, such that the total volume of the well is 100 microliters. It is stored overnight at 4 ° C. 25 2. The goat anti-rabbit antibody is removed. 300 linked by inverting the plate to remove the liquid. The plate is tapped on a paper towel to remove excess liquid and bubbles. 3. Add 150 microliters of 5 Blocking Regulator (5% Fat Low Fat in PBS) to each well. It is incubated with agitation on a microtiter plate shaker for 30 minutes. 4. Wash 4 times with TBST. The plate is tapped on a paper towel to remove the excess liquid and bubbles. 5. 0.5 micrograms of rabbit antibody against anti-GyrB is added per well. The antibody is diluted in DPBS to a final volume of 100 microliters per well. It is incubated with agitation on a plate stirrer. microtiter at room temperature for one hour. 6. Wash 4 times with TBST as described in Step 4. 7. Add 2 micrograms of COS / FGFR from cell lysate (source Myc-GyrB-FGFR) in HNGT to each well, to give a final volume of 100 microliters per well. It is incubated with agitation on a microtiter plate shaker for 1 hour. 8. Wash 4 times with TBST as described in Step 4. 25 9. Add 80 microliters of kinase regulator lx per well. 10. Dilute the test compound to 1:10 in 1x kinase regulator + 1% dimethyl sulfoxide in a 96-well polypropylene plate. 11. Transfer 10 microliters of the solution of the diluted test compound, and from the control wells from the wells of the polypropylene plate to the corresponding wells of the ELISA plate, and incubate with agitation on a plate stirrer. microtiter for 20 minutes. 12. Add 10 microliters of 70 μM ATP diluted in kinase buffer to the positive control and test wells (the final ATP concentration is 7 μM / well). 10 microliters of kinase regulator lx are added to the negative control wells. They are incubated with agitation on a microtiter plate shaker for 15 minutes. 13. The kinase reaction is stopped by the addition of 5 microliters of 0.5 M EDTA to all wells. 14. Wash 4 times with TBST as described in Step 4. 15. Add 100 microlitres of biotin-conjugated a-phosphotyrosine monoclonal antibody (b4G10) diluted in TBST to each well. It is incubated with agitation on a microtiter plate shaker for 30 minutes. 16. The ABC vestastain reagent is prepared. 1 drop of Reagent A is added to 15 milliliters of TBST. Mix by inverting the tube several times. Add 1 drop of reagent B and mix again. 17. Wash 4 times with TBST as described in Step 4. 18. Add 100 microliters of ABC HRP reagent to each well. It is incubated with agitation on a microtiter plate shaker for 30 minutes. 19. Wash four times with TBST as described in Step 4. 20. Add 100 microliters of solution ABTS / H202 to each well. 22. Incubate for 5 to 15 minutes with shaking. The bubbles are removed. 23. If necessary, the reaction is stopped by the addition of 1 microliter of 0.2M HCl / well. 24. The assay is read on a Dynatech MR7000 ELISA Plate Reader; Test filter: 410 nanometers; Reference filter: 630 nanometers. FLK-1 Biochemical Assay This assay evaluates flk-1 autophosphorylation activity in vitro using ELISA. Materials and Reagents 1. Tissue culture dishes of 15 centimeters. 2. Flk-1 / NIH cells: NIH fibroblast line overexpressing Clone 3 of human flk-1 (SUGEN, Inc., obtained from MPI, Martinsried, Germany). 3. Culture medium: DMEM plus 10 percent heat inactivated FBS and 2 mM glutamine (Gibco-BRL). 4. Medium of starvation: DMEM plus FBS inactivated by 0.5 percent heat, 2 mM glutamine (Gibco-BRL). 5. Corning 96-well ELISA plates (Corning, Catalog No. 25805-96). 6. Monoclonal antibody L4 or E38 specific for flk-1, purified, by affinity chromatography of protein-A agarose (SUGEN, Inc.). 7. PBS (Dulbecco Phosphate Regulated Serum) Gibco, Catalog No. 450-1300EB). 8. HNTG (see BIOCHEMICAL FGFR for preparation). 9. The BCA protein determination kit is punctured. 10. Locking regulator. 11. TBST (ph of 7.0) 12. Kinase Regulator 13. Kinease Stop Solution: 200 mM EDTA 14. Biotinylated 4G10, specific for phosphotyrosine (UBI, Catalog No. 16-103). 15. AB Case (Vecotor Laboratories, Catalog No. PK 4000). 16. Dimethyl sulfoxide 17. NUNC 96-well V-bottom polypropylene plates (Applied Scientific, Catalog No. AS-72092). 18. Turbo-TMB (Pierce) 19. Turbo-TMB Stop Solution: HB2S04 1 M 20. ATP (Sigma, Catalog No. A-7699 21. 20% Dimethyl Sulfoxide in TBS (pH of 7.0). Procedure Preparation of Cell Culture and Lysate 1. Cells are seeded in the culture medium, and cultured for 2 to 3 days to a confluence of 90 to 100 percent at 37 ° C and at 5 percent C0. Passage # 20 is not exceeded. 2. The medium is removed, and the cells are washed twice with PBS. They are lysed with HNTG lysis regulator. All the lysates are collected, and vortexed for 20 to 30 seconds. 3. The insoluble material is removed by centrifugation (5 to 10 minutes at approximately 10,000 xg). 4. The protein concentration is determined using the BCA kit. 5. The lysate is divided into aliquots of 1 milligram, and stored at -80 ° C. Assay Procedure 1. Corning 96-well ELISA plates are coated with 2 milligrams / well of purified L4 (or E 38) in 100 microliters of PBS. They are stored overnight at 4 ° C. 2. Unbound proteins are removed from the wells, inverting the plate to remove the liquid. Wash once with dH20, tap the plate on a paper towel to remove excess liquid. 3. The plates are blocked with 150 microliters of blocking regulator per well. They are incubated for 45 to 60 minutes with shaking at 4 ° C. 4. The blocking regulator is removed, and the ELISA plate is washed three times with dH20, and once with TBST. The plate is tapped on a paper towel to remove excess liquid. 5. The lysate is diluted in PBS to give a final concentration of 50 micrograms / 100 milliliters. 100 microliters of diluted lysate is added to each well. They are incubated with shaking at 4 ° C overnight. 6. Unbound proteins are removed from the wells by inverting the plate. They are washed as in Step 4. 7. 80 microliters of kinase buffer is added to the wells (90 microliters to the negative control wells). 8. The test compounds are diluted (usually 10 times) in the wells of a polypropylene plate containing 20 percent dimethyl sulfoxide in TBS. 9. 10 microliters of the diluted compounds are added to the ELISA wells containing immobilized flk-1, and shaken. Control wells do not receive compounds. 10. From the ATP lmM supply, a 0.3 mM ATP solution in dH20 is prepared (alternatively, a kinase regulator can be used). 11. Add 10 microliters of 0.3 mM ATP to all wells, except negative controls. They are incubated for 60 minutes at room temperature with shaking. 12. After 1 hour, the kinase reaction is stopped by adding 11 microliters of 200 mM EDTA. They are stirred for 1 to 2 minutes. 13. Wash the ELISA plate 4 times with dH20, and twice with TBST. 14. Add 100 microliters of 1: 5000 biotinylated 4G10: TBST to all wells. They are incubated for 45 minutes with stirring at room temperature. 15. While the above is being incubated, 50 microliters of solutions A and B of the ABC kit are added to 10 milliliters of TBST. These solutions should be combined approximately 30 minutes before use. 16. Wash the plates as in Step 4. 17. Add 100 microliters of the preformed A and B complex to all wells. They are incubated for 30 minutes with stirring at room temperature. 18. Wash the plates as in Step 4. 19. Add 100 microliters of turbo-TMB. They are stirred at room temperature for 10 to 15 minutes. 20. When the color in the positive control wells reaches an absorbance of approximately 0..35-0.4, the reaction is stopped with 100 microliters of the turbo-TMB stopping solution. 21. The plates are read on the Dynatech MR7000 ELISA reader; Test filter: 450 nanometers; Reference filter: 410 nanometers. HUV-EC-C Assay The following protocol can also be used to measure the activity of a compound against PDGF-R, FGF-R, VEGF, aFGF or Flk-1 / KDR, all of which are naturally expressed by HUV cells. -EC. DAY O 1. HUV-EC-C cells are washed and trypsinized (endothelial cells of the human umbilical vein) (American Type Culture Collection, Catalog No. 1730 CRL). Wash with Dulbecco's phosphate buffered serum (D-PBS, obtained in Gibco BRL, Catalog No. 14190-029) twice to approximately 1 milliliter / 10 cm2 of the tissue culture flask. They are trypsinized with 0.5% trypsin-EDTA in nonenzymatic cell dissociation solution (Sigma Chemical Company, Catalog No. C-1544). The 0.05 percent trypsin is made by diluting 0.25 percent trypsin / 1 mM EDTA (Gibco, Catalog No. 25200-049) in the cell dissociation solution.

Trypsinize with about 1 milliliter / 25-30 cm2 of the tissue culture flask for approximately 5 minutes at 37 ° C. After the cells are separated from the flask, an equal volume of test medium is added, and transferred to a 50 milliliter sterile centrifuge tube (Fisher Scientific, Catalog No. 05-539-6). 2. The cells are washed with approximately 35 milliliters of test medium in the 50 milliliter sterile centrifuge tube, by addition of the test medium, centrifuged for 10 minutes at approximately 200 × g, the supernatant is aspirated, and the suspend with 35 milliliters of D-PBS. The washing is repeated twice more with D-PBS, the cells are resuspended in approximately 1 milliliter of assay medium / 15 cm2 of the tissue culture flask. The test medium consists of an F12K medium (Gibco BRL, Catalog No. 21127-014) and 0.5 percent heat-inactivated fetal bovine serum. The cells are counted with a Coulter Counter® (Coulter Electronics, Inc.), and the assay medium is added to the cells to obtain a concentration of 0.8-1.0 x 105 cells / milliliter. 3. Add the cells to the 96-well flat bottom plates, at 100 microliters / well, or 0.8-1.0 x 104 cells / well, incubate for approximately 24 hours at 37 ° C, with 5 percent C02.

DAY 1 1. Double test compound titrations are formed in separate 96-well plates, generally 50 μM down to 0 μM. The same assay medium as mentioned on day 0, Step 2 above is used. Titrations are made by adding 90 microliters / well of the test compound to 200 μM (4 times the final well concentration) to the upper well of a particular plate column. Because the delivery test compound is usually 20 mM in dimethyl sulfoxide, the drug concentration of 200 μM contains 2 percent dimethyl sulfoxide. A diluent made in 2 percent dimethyl sulfoxide is used in the test medium (F12K + 0.5 percent fetal bovine serum) as a diluent for the titrations of the test compound, in order to dilute the test compound, but Keep the concentration of dimethyl sulfoxide constant. This diluent is added to the remaining wells of the column at 50 microliters / well. 60 microliters of the 120 microliters of the dilution of the 200 μM test compound are taken in the upper well of the column, and mixed with the 60 microliters of the second well of the column. 60 microliters are taken from this well, and mixed with the 60 microliters of the third well of the column, and so on, until the double titrations are finished. When the next well is mixed with the latter, 60 microliters of the 120 microliters of this well are taken and discarded. The last well is left with 60 microliters of dimethyl sulfoxide diluent / medium as a control containing no test compound. 9 columns of the titled test compound are made, sufficient to triplicate the wells, each for: (1) VEGF (obtained in Pepro Tech Inc., Catalog No. 100-200), (2) endothelial cell growth factor ( ECGF) (also known as acid fibroblast growth factor, or aFGF) (obtained from Boehringer Mannheim Biochemica, Catalog No. 1439 600), or, (3) human B / B PDGF (1276-956, Boehringer Mannheim, Germany), and a test medium control. The ECGF comes as a preparation with sodium heparin. 2. 50 microliters / well are transferred from the dilutions of the test compound to the 96-well assay plates containing the 0.8-1.0xl04 cells / 100 microliters / well of the HUV-EC-C cells from day 0, and incubated for about 2 hours at 37 ° C, with 5 percent C02. 3. In triplicate, add 50 microliters / well of 80 micrograms / milliliter of VEGF, 20 nanograms / milliliter of ECGF, or a control of the medium for each condition of the test compound. As with the test compounds, the concentrations of the growth factor are 4 times the desired final concentration. The test medium is used from Day O, Step 2, to make the concentrations of the growth factors. They are incubated for approximately 24 hours at 37 ° C, with 5% C02. Each well will have 50 microliters of dilution of the test compound, 50 microliters of growth factor or medium, and 100 microliters of cells, which is calculated for a total of 200 microliters / well. Accordingly, the 4X concentrations of the test compound and the growth factors become IX when everything has been added to the wells. DAY 2 1. Add 3H-thymidine (Amersham, Catalog No. TRK-686) in 1 μCi / well (10 microliters / well of a solution of 100 μCi / milliliter made in RPMI medium + heat-inactivated fetal bovine serum) 10 percent), and incubated for approximately 24 hours at 37 ° C, with 5 percent C02. The RPMI is obtained in Gibco BRL, Catalog No. 11875-051. DAY 3 1. The plates are frozen overnight at -20 ° C. DAY 4 The plates are thawed and harvested with the 96-well plate harvester (Tomtec Harvester 96®) on filter waxes (Wallac, Catalog No. 1205-401), counts are read on a Wallac liquid scintillation counter BetaplateMR Live Animals Models Xenoinse Animal Models The ability of human tumors to grow as xenografts in athymic mice (eg, Balb / c, nu / nu) provides a useful model in vivo to study the biological response to tumor therapies humans. Due to the first successful xenotransplantation of human tumors in athymic mice (Rygaard and Povlsen, 1969, Acta Pathol, Microbial, Scand 77: 758-760), many different human tumor cell lines have been transplanted (for example, breast melanomas, lung, genitourinary, gastrointestinal, head and neck, glioblastoma, bone, and motile), and have been successfully cultured in hairless mice. The following assays can be used to determine the level of activity, specificity, and effect of the different compounds of the present invention. Three general types of assays are useful to evaluate the compounds: cellular / catalytic, cellular / biological and in vivo. The purpose of cell / catalytic assays is to determine the effect of a compound on the ability of a TK to phosphorylate tyrosines on a known substrate in a cell. The purpose of cell / biological assays is to determine the effect of a compound on the biological response stimulated by a TK in a cell. The purpose of the in vivo assays is to determine the effect of a compound in an animal model of a particular disorder, such as cancer.Suitable cell lines for subcutaneous xenograft experiments include C6 cells (glioma, ATCC # CCL 107); A375 cells (melanoma, ATCC # CRL 1619), A431 cells (squamous cell 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, and any other test kinase. The following protocol can be used to perform xenograft experiments: Female nude mice (BALB / c, nu / nu) are obtained from Simonsen Laboratories (Gilroy, CA). All animals are kept under clean conditions in micro-insulating cages with Alpha-dri beds. They receive food for sterile rodents and water to taste. Cell lines are cultured in an appropriate medium (e.g., MEM, DMEM, Ham's FIO, or Ham's F12 plus 5-10 percent fetal bovine serum (FBS), and 2 M glutamine (GLN)). All cell culture media, glutamine and fetal bovine serum are purchased from Gibco Life Technologies (Grand Island, NY), unless otherwise specified. All cells are grown in a humid atmosphere of 90 to 95 percent air, and 5 to 10 percent C02 at 37 ° C. All cell lines are routinely subcultured twice a week, and are negative for mycoplasma, determined by the Mycotect method (Gibco). Cells are harvested at or near confluence with 0.5 percent trypsin-EDTA, and granulated at 450 x g for 20 minutes. The granules are resuspended in sterile PBS or in medium (without FBS) to a particular concentration, and the cells are implanted in the posterior flank of the mice (8 to 10 mice per group, 2-10 x 10 6 cells / animal ). Tumor growth is measured for 3 to 6 weeks using venier gauges. Tumor volumes are calculated as a product of length by width by height, unless otherwise indicated. The P values are calculated using Student's t test. The test compounds in 50-100 microliters of excipient (dimethyl sulfoxide, or VPD: D5W) can be delivered by intraperitoneal injection in different concentrations, starting generally on day one after implantation. Tumor Invasive Model The following model of tumor invasion has been developed, and can be used to evaluate the therapeutic value and the efficacy of the compounds identified by selectively inhibiting the KDR / FLK-1 receptor. Procedure 8-week old hairless mice are used (females) (Simonsen Inc.) as experimental animals. The implantation of the tumor cells can be performed in a laminar flow hood. For anesthesia, a cocktail of xylazine / ketamine (100 milligrams / kilogram of ketamine, and 5 milligrams / kilogram of xylazine) is administered intraperitoneally. An incision is made of the midline to expose the abdominal cavity (approximately 1.5 centimeters in length) to inject 107 tumor cells in a volume of 100 microliters of the medium. The cells are injected, either into the duodenal lobe of the pancreas, or under the serosa of the colon. The peritoneum and muscles are closed with a continuous 6-0 silk suture, and the skin is closed using wound clips. The animals are observed daily. Analysis After 2 to 6 weeks, depending on the observations of the animals, the mice are sacrificed, and local tumor metastases are cut and analyzed in different organs (lung, liver, brain, stomach, spleen, heart, muscles) ( measurement of tumor size, degree of invasion, immunochemistry, determination of in situ hybridization, etc.). Measurement of Cellular Toxicity Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect. A measure of the effectiveness and cellular toxicity of a compound can be obtained, determining the therapeutic index, that is, IC50 / LD50. The IC50, the dose required to achieve 50 percent inhibition, can be measured using conventional techniques, such as those described herein. LD50, the dosage that results in 50 percent toxicity, can also be measured by conventional techniques (Mossman, 1983, J. Immunol. Methods, 65: 55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol. Methods, 64: 313, Decker and Lohmann-Matthes, 1988, J. Immunol. Methods, 115: 61), or by measuring the lethal dose in animal models. Compounds with a large therapeutic index are preferred. The therapeutic index should be greater than 2, preferably at least 10, and more preferably at least 50. B. Examples - Biological Activity Examples of the in vitro potency of the compounds of this invention are shown in Tables 1 and 2. The data show that the compounds are generally very potent against a variety of in vitro PTKs. The compounds also maintain excellent activity when tested in vivo. For example, several compounds of the present invention, when administered orally, exhibit markedly reduced average size of C6 glioma tumors subcutaneously implanted in mice. However, compound 5 was notoriously superior to the other compounds of this invention. In one experiment, C6 human glioma cells were implanted subcutaneously (3 x 10 6 cells, n = 10-20 animals / group) on the hind flank of female BALB / c nu / nu mice on day 0. Oral administration of the Compounds 3, 5, 19 and 20 in aqueous labolla at 200 milligrams / kilogram / day, started one day after implantation. Tumor growth was measured using vernier calipers, and tumor volumes were calculated as the product of length times width times height.

As shown in the following graph, all the compounds tested show a marked inhibition compared to the vehicle control only. However, compound 5 clearly, and considering the close structural similarity of the tested compounds, stands out in a surprising way from the rest. That is, while compounds 3, 19 and 21 are clustered around 40 to 45 percent inhibition of tumor growth on day 18 after implantation, compound 5 inhibits tumor growth by 80 to 85 percent at that point.

Days after implantation The unexpected efficacy of compound 5 in vivo, particularly after oral administration, is further demonstrated when compared to compound 65: &1 Compound 65 manifests almost one order of magnitude more in vitro potency than compound 5 (no show the data). However, when tested interperitoneally in mice against two different tumor cell lines, SF763T and SF767T, compound 5 is from slightly (a greater inhibition of 5 percent at 21 days) to notoriously (a greater inhibition of 14 percent at 21 days) more effective than compound 65. The difference in activity between compound 5 and compound 65 is even greater when the two compounds are administered orally. The oral efficacy of compound 65 and several of its analogs, compounds 66-69, is shown graphically below: £ 2 As can be seen in the graph, compound 65, administered orally at 200 milligrams / kilogram / day, shows an inhibition of approximately 65 percent of C6 tumors at 18 days after subcutaneous implantation in mice, which is clearly superior to its own analogues, which average an inhibition of approximately 14 to 16 percent of tumor growth. Surprisingly, however, the oral efficacy of compound 65 is still markedly lower than that of compound 5, which, as noted above, demonstrates an 80 to 85 percent inhibition at the same time in the same tumor model. When compared to compound 70, compound 5 shows K? S (inhibition constants) significantly smaller (ie, higher potency) against FGF-R1 (1.2 for compound 5, versus 19.49 for compound 70) and PDGFR ( the data is not shown).

. The unexpected superiority of compound 5 is further demonstrated when its oral efficacy is compared to that of a close analog, compound 71: 71 Despite the structural similarity, when tested orally at 200 milligrams / kilogram / day against C6 human melanoma tumors subcutaneously implanted in BALB / c nu / nu mice, at 18 days after implantation, compound 71 shows only a 57 percent inhibition of tumor growth (data not shown), comparing again with the 80 to 85 percent inhibition demonstrated by compound 5.

Based on the surprising prior efficacy of compound 5 when administered orally, compound 5 is currently a preferred embodiment of this invention.

CONCLUSION Accordingly, it will be appreciated that the compounds, methods and pharmaceutical compositions of the present invention are effective in modulating protein kinase activity, and therefore, are expected to be effective as therapeutic agents against disorders related to RTK, CTK and STK One skilled in the art would readily appreciate that the present invention is well suited to realize the objects and obtain the purposes and advantages mentioned, as well as those inherent herein. The molecular complexes and methods, procedures, treatments, molecules, specific compounds described herein, are currently representative of the preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Experts in the field will think about changes in them and in other uses, which are encompassed within the spirit of the invention, as defined by the scope of the claims. It will be readily apparent to one skilled in the art that variations, substitutions and modifications to the invention disclosed herein may be made without departing from the scope and spirit of the invention. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are hereby incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as incorporated by reference. The invention described in an illustrative manner herein may be practiced suitably in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Accordingly, for example, in each case, in the present, any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with any of the other two terms. The terms and expressions that are used are used as terms of description and not limitation, and there is no intention that, in the use of these terms and expressions, any equivalents of the characteristics shown and described, or portions of the same, but it is recognized that different modifications are possible within the scope of the claimed invention. Therefore, it should be understood that, although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concepts disclosed herein, and these Modifications and variations are considered within the scope of this invention, as defined by the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also described by the same in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as being selected from the group consisting of bromine, chlorine and iodine, the proclamations that X is bromine, and that X is bromine and chlorine are completely described. Other embodiments are within the following claims.

Claims (24)

1. A 2-indolinone substituted by pyrrole that has the chemical structure: wherein: R1 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, hydroxyl, alkoxy, C-carboxyl, 0-carboxyl, acetyl, C-amido, C-thioamido, sulfonyl and trihalomethanesulfonyl. R2 is selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, aryl, heteroaryl and heteroalicyclic. R3, R4, R5 and R5 are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, sulfinyl, sulfonyl , S-sulfonamido, N-sulfonamido, trihalomethanesulfonamido, carbonyl, C-carboxyl, O-carboxyl, C-amido, N-amido, cyano, nitro, halogen, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl , amino and -NR1: LR1

2. R11 and R12 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl and, in combination, a five or six membered heteroalicyclic ring. R3 and R4, R4 and R5, or R4 and R5, may be combined to form a six-membered aryl ring, a methylenedioxyl group or an ethylenedioxyl group. R7 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C -carboxyl, O-carboxyl, sulfonyl and trihalomethanesulfonyl. R, R and Rx? are independently selected from the group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxyl, alkoxy, aryloxy, mercapto, thioalkyl, thioaryl, sulfinyl, sulfonyl, S-sulfonamido, N- sulfonamide, carbonyl, C-carboxyl, 0-carboxyl, cyano, nitro, halogen, O-carbamyl, N-carbamyl, 0-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and -NR1; LR12, in the understanding, however, that at least one of R8, R9 or R10 is a group having the formula - (alk1) Z, wherein: Alk-L is selected from the group consisting of alkyl, alkenyl or alkynyl; and Z is a polar group. 2. The compound of claim 1, wherein R1, R2 and R7 are hydrogen.

3. The compound of claim 2, wherein one of R8, R9 or R10 is alk ^, wherein: alk-L is selected from the group consisting of unsubstituted lower alkyl, unsubstituted lower alkenyl, and unsubstituted lower alkynyl; and Z is a polar group selected from the group consisting of hydroxyl, alkoxy, C-carboxyl, carbonyl, nitro, cyano, amino, ammonium, -NR1: LR12, C-amido, S-sulfonamido, sulfinyl, sulfonyl, phosphonyl , ureido, amidino, guanidinyl, morpholino, piperidinyl and tetrazolo.

4. The compound of claim 1, wherein R3, R4, R5 and R6 are independently selected from the group consisting of: hydrogen; halogen; unsubstituted lower alkyl; lower alkyl substituted with one or more groups selected from the group consisting of: hydroxyl; halogen; C-carboxyl substituted with a group selected from the group consisting of: hydrogen; or, unsubstituted lower alkyl; Not me; or, -NR1: 1R12; lower alkyl-unsubstituted alkoxy; lower alkyl-alkoxy substituted with one or more halogen groups; unsubstituted aryloxy; aryloxy substituted with one or more groups independently selected from the group consisting of: unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; hydroxyl; lower alkyl-unsubstituted alkoxy; halogen; Not me; or, -NR1: LR12; S-sulfonamido, wherein R11 and R12 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl; unsubstituted aryl; aryl substituted with one or more groups independently selected from the group consisting of: halogen; unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; lower alkyl-unsubstituted alkoxy; Not me; or, -NR?: LR12; unsubstituted heteroaryl; heteroaryl substituted with one or more groups independently selected from the group consisting of: unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; lower alkyl-unsubstituted alkoxy; hydroxyl; halogen; Not me; or, -NR11R12; unsubstituted heteroalicyclic; heteroalicyclic substituted with one or more groups independently selected from the group consisting of: halogen; hydroxyl; unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; lower alkyl-unsubstituted alkoxy; Not me; or, R 11 R 12; unsubstituted lower alkyl-O-carboxyl; C-amido, wherein R11 and R12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, and unsubstituted aryl; and N-amido, wherein R11 and R12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, and unsubstituted aryl.

5. The compound of claim 3, wherein R3, R4, R5 and R6 are selected from the group consisting of: hydrogen; halogen; unsubstituted lower alkyl; lower alkyl substituted with one or more groups selected from the group consisting of: hydroxyl; halogen; C-carboxyl substituted with a group selected from the group consisting of: hydrogen; or, unsubstituted lower alkyl; Not me; or, -NR1: LR12; lower alkyl-unsubstituted alkoxy; lower alkyl-alkoxy substituted with one or more halogen groups; unsubstituted aryloxy; aryloxy substituted with one or more groups independently selected from the group consisting of: unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; hydroxyl; lower alkyl-unsubstituted alkoxy; halogen; Not me; or, -NR11R12; S-sulfonamido, wherein R11 and R12 are independently selected from the group consisting of hydrogen and unsubstituted lower alkyl; unsubstituted aryl; aryl substituted with one or more groups independently selected from the group consisting of: halogen; unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; lower alkyl-unsubstituted alkoxy; Not me; or, -NR ^ R12; unsubstituted heteroaryl; heteroaryl substituted with one or more groups independently selected from the group consisting of: unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; lower alkyl-unsubstituted alkoxy; hydroxyl; halogen; Not me; or, unsubstituted heteroalicyclic; heteroalicyclic substituted with one or more groups independently selected from the group consisting of: halogen; hydroxyl; unsubstituted lower alkyl; lower alkyl substituted with one or more halogen groups; lower alkyl-unsubstituted alkoxy; Not me; or, R ^ R12; unsubstituted lower alkyl-O-carboxyl; C-amido, wherein R11 and R12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, and unsubstituted aryl; and, N-amido, wherein R11 and R12 are independently selected from the group consisting of hydroquin, unsubstituted lower alkyl, and unsubstituted aryl: The compound of claim 1, wherein: R1, R2, R3, R4 , R5, R6 and R7 are hydrogen; R8 and R10 are methyl; and R9 is - (CH2) (CH2) C (= 0) 0H. 7. A pharmaceutical composition, which comprises: the compound of claim 6; and a physiologically acceptable carrier or excipient. The compound of claim 1, wherein: R1, R2 and R7 are hydrogen; R3, R4, R5 and R6 are independently selected from the group consisting of: hydrogen; hydroxyl; halogen; unsubstituted lower alkyl; lower alkyl substituted with a carboxylic acid; unsubstituted lower alkoxy; carboxylic acid; unsubstituted aryl; aryl substituted with one or more of unsubstituted lower alkyl-alkoxy; or, morpholino; R8 is selected from the group consisting of hydrogen and unsubstituted lower alkyl; R9 is - (CH2) (CH2) C (= 0) OH; and, R10 is unsubstituted lower alkyl. 9. The compound of claim 2, wherein R7 is selected from the group consisting of: hydrogen; unsubstituted lower alkyl; and, lower alkyl substituted with a group selected from the group consisting of: unsubstituted cycloalkyl; unsubstituted aryl, and, aryl substituted with a group selected from hydroxyl, lower alkyl-unsubstituted alkoxy, and halogen. 10. The compound of claim 2, wherein Z is selected from the group consisting of: -C (= 0) NR13R14, wherein R13 and R14 are independently selected from the group consisting of: hydrogen, lower alkyl unsubstituted, lower alkyl substituted with a group selected from the group consisting of amino and -NR1: LR12, unsubstituted aryl, aryl substituted with one or more groups selected from the group consisting of halogen, hydroxyl, lower alkyl-unsubstituted alkoxy , and trihalomethyl, unsubstituted heteroaryl, unsubstituted heteroalicyclic, and, combined, a five-membered or six-membered unsubstituted heterocyclic, and, -NR1: LR12, wherein: R11 and R2 are independently selected from the group consisting of alkyl lower unsubstituted, and combined, a five-membered or six-membered unsubstituted heteroalicyclic ring. 11. The compound of claim 1, wherein: R7 is selected from the group consisting of unsubstituted lower alkyl; lower alkyl substituted with one or more groups selected from the group consisting of: unsubstituted cycloalkyl, unsubstituted aryl, aryl substituted with one or more groups independently selected from the group consisting of halogen, and unsubstituted lower alkyl-alkoxy, and alkyl unsubstituted lower carboxylalkyl, and, Z is selected from the group consisting of unsubstituted C-carboxyl, and unsubstituted C-lower alkylcarbonyl. The compound of claim 1, wherein: R3, R4, R5 and R6 are independently selected from the group consisting of: hydrogen, halogen, unsubstituted lower alkyl, lower alkyl substituted with one or more hydroxyl groups, lower alkoxy unsubstituted, unsubstituted aryl, aryl substituted with one or more unsubstituted lower alkoxy groups, and, S (0) 2NR1: LR12, R5 is hydrogen, R6 is -NR11R12, and, R11 and R12 are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, and in combination, a five-membered or six-membered unsubstituted heteroalicyclic ring. 13. A method for modulating the catalytic activity of a protein kinase, which comprises contacting this protein kinase with a compound, salt or prodrug of claim 1. 14. The method of claim 13, wherein the protein kinase is selected from the group consisting of a receptor tyrosine kinase, a non-receptor tyrosine kinase, and a serine-threonine kinase. 15. A pharmaceutical composition, which comprises: a compound, salt or prodrug of claim 1; and, a physiologically acceptable carrier or excipient. 1

6. A method for the treatment or prevention of a disorder related to protein kinase in an organism, which comprises administering a therapeutically effective amount of a compound, salt or prodrug of claim 1, to said organism. The method of claim 16, which comprises administering a therapeutically effective amount of 3- [2, 4-dimethyl-5- (2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrole- 3-yl] -propionic to this organism. 18. The method of claim 16, wherein the protein kinase related disorder is selected from the group consisting of a tyrosine receptor kinase related disorder, a non-receptor tyrosine kinase related disorder, and a related disorder with serine-threonine kinase. The method of claim 16, wherein the protein kinase related disorder is selected from the group consisting of an EGFR-related disorder, a disorder related to PDGFR, a disorder related to IGFR, and a disorder related to flk. The method of claim 16, wherein the protein kinase-related disorder is a cancer selected from the group consisting of squamous cell carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, lung cancer, bladder cancer , cancer of the head and neck, melanoma, ovarian cancer, prostate cancer, breast cancer, small cell lung cancer, glioma, colorectal cancer, genitourinary cancer and gastrointestinal cancer. The method of claim 16, wherein the protein kinase related disorder is selected from the group consisting of diabetes, an autoimmune disorder, a hyperproliferation disorder, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, angiogenesis , an inflammatory disorder, an immune disorder and a cardiovascular disorder. 22. The method of claim 16, wherein the organism is a human being. 23. A compound from the group consisting of: 3- [5- (5-chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, 3- [5- (6-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, 3- [5-] (5-chloro-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid, 3- [4-methy1-5- (2- oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- [2, 4-dimethyl-5- (2-oxo-l, 2-dihydroindol-3-ylidenemethyl] ) -lH-pyrrol-3-yl] -propionic acid, 3- [5- (5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] ] -propionic acid, 3- [5- (5-iodo-2-oxo-1,2-dihydroindol-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] -propionic acid, 3- [4] -methyl-5- (4-methyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- [4-methyl-5- (5-methyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- [5- (5,6-d) imethoxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, 3- [5- (6-chloro-2-oxo-1, 2 -dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, 3- [2-carboxyethyl] -3-methyl-lH-pyrrol-2-ylmethylene] -2-OXOmethyl ester -2, 3-dihydro-lH-indole-5-carboxylic acid, 3- [4- (2-carboxyethyl) -3-methyl-lH-pyrrol-2-ylmethylene] -2-OXO-2, 3-dihydro- lH-Indole-5-carboxylic acid, 3- [4-methyl-5- (2-oxo-5-sulfamoyl-l, 2-dihydroindol-3-ylidene-methyl) -lH-pyrrol-3-yl] -propionic acid , 3- [4-methy1-5- (5-methylsulfamoyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- acid. { 3- [4-2-carboxyethyl) -3-methyl-lH-pyrrol-2-ylmethylene] -2-oxo-2,3-dihydro-lH-indol-5-yl} -propionic, 3- [5- (5-eti1-2-oxo-1,2-dihydro-indol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, 3- [3- 5- (5-methoxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, 3- [5- (5-bromo-2- oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid, 3- [5- (5-iodo-2-oxo-l, 2-dihydroindole -3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid, 3- [2,4-dimethyl-5- (4-methyl-2-oxo-l, 2-dihydroindol- 3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- [2,4-dimethyl-5- (5-methyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH- pyrrol-3-yl] -propionic acid, 3- [5- (6-hydroxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] - propionic acid, 3- [5- (6-methoxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid, 3- [5] - (6-hydroxy-2-oxo-1,2-dihydroindol-3-ylidemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid 3,5-dimethoxybenzyl ester 3- [5- (6-hydroxy-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-1H-pyrrol-3-yl] -propionic acid, 3-acid. { 5- [6- (3-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-lH-pyrrol-3-yl} -propionic, 3- [5- (6-bromo-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, acid 3-. { 5- [6-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-lH-pyrrol-3-yl} -propionic, acid 3-. { 5- [3-ethoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-lH-pyrrol-3-yl} -propionic, acid 3-. { 5- [6- (3-Ethoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-lH-pyrrol-3-yl} -propionic, 3- [2,4-dimethyl-5- (2-oxo-6-phenyl-1, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- [3- 4-Methyl-5- (2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- acid. { 5- [6- (4-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-lH-pyrrole-3-yl} -propionic, acid 3-. { 5- [6- (4-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-lH-pyrrol-3-yl} -propionic, acid 3-. { 5- [6- (2-methoxyphenyl) -2-oxys-l, 2-dihydroindol-3-ylidenemethyl] -4-methyl-lH-pyrrol-3-yl} -propionic, acid 3-. { 5- [6- (2-methoxyphenyl) -2-oxo-l, 2-dihydroindol-3-ylidenemethyl] -2,4-dimethyl-lH-pyrrol-3-yl} -propionic, 3- [2,4-dimethyl-5- (6-morpholin-4-yl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, 3- [5- (5-Chloro-4-methyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -2,4-dimethyl-lH-pyrrol-3-yl] -propionic acid, 3- acid [5- (5-chloro-4-methyl-2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -4-methyl-lH-pyrrol-3-yl] -propionic acid, 3- [2, 4- dimethyl-5- (2-oxo-l, 2-dihydroindol-3-ylidenemethyl) -lH-pyrrol-3-yl] -propionic acid, sodium salt. 24. A compound selected from the group consisting of: 3- [3,5-dimethyl-4- (3-morpholin-4-ylpropyl) -lH-pyrrol-2-ylmethylene] -1,3-dihydroindole-2 -one, 5-bromo-3- [3, 5-dimethyl-4- (3-morpholin-4-ylpropyl) -1H-pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one, 3- [ 3,5-dimethyl-4- (3-morpholin-4-ylpropyl) -lH-pyrrol-2-ylmethylene] -6-phenyl-1,3-dihydroinds-2-one, 3- [3,5-dimethyl- 4- (3-morpholin-4-ylpropyl) -lH-pyrrol-2-ylmethylene] -6- (2-methoxyphenyl) -1,3-dihydroindol-2-one, 3- [3,5-dimethyl-4-] (3-morpholin-4-ylpropyl) -lH-pyrrol-2-ylmethylene] -6- (3-methoxyphenyl) -1,3-dihydroindol-2-one, 3- [3,5-dimethyl-4- (3 -morpholin-4-ylpropyl) -lH-pyrrol-2-ylmethylene] -6- (4-methoxyphenyl) -1,3-dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3,5- dimethyl-lH-pyrrol-2-yl-ethylene] -1,3-dihydroindol-2-one, 5-bromo-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene ] -1,3-dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6-phenyl-1,3-dihydroindole -2-one, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6- (2-methoxyphenyl) -1,3-dihydroindol-2-one, 3 - [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6- (3-methoxyphenyl) -1,3-dihydroindol-2-one, 3- [4- (3 -dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6- (4-methoxyphenyl) -1,3-dihydroindol-2-one, 5-chloro-3- [4- (3-dimethylaminopropyl) ) -3,5-dimethyl-1H-pyrrol-2-ylmethylene] -l, 3-dihydroindol-2-one, 6-chloro-3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-1H- pyrrol-2-ylmethylene] -1,3-dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -5-methoxy-3 -dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -6-methoxy-l, 3-dihydroindol-2-one, 3- [ 4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -5-methyl-1,3-dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3, 5-dimethyl-lH-pyrrol-2-ylmethylene] -4-methyl-l, 3-dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3,5-dime til-lH-pyrrol-2-ylmethylene] -4- (2-hydroxyethyl) -1,3-dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-amide -pyrrole-2-ylmethylene-2-oxo-2,3-dihydro-lH-indole-5-sulphonic acid, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrole-2-isopropylamide] -ethylmethylene] -2-oxo-2,3-dihydro-lH-indol-5-sulphonic acid, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -5- (morpholin-4-sulfonyl) -1,3-dihydroindol-2-one, 3- [4- (3-dimethylaminopropyl) -3,5-dimethyl-lH-pyrrol-2-ylmethylene] -2-OXO dimethylamide -2, 3-dihydro-lH-indole-5-sulfonic acid.