Patent Publication Number: US-6906093-B2

Title: Indolinone combinatorial libraries and related products and methods for the treatment of disease

Description:
RELATED APPLICATIONS 
     This is a Continuation Application of application Ser. No. 08/702,232, filed Aug. 23, 1996 now abandoned, which is in turn a Continuation-in-Part of U.S. patent applications Ser. No. 08/655,255 now abandoned, Ser. No. 08/655,226 now U.S. Pat. No. 5,886,020, Ser. No. 08/655,223 U.S. Pat. No. 5,792,793, Ser. No. 08/655,224 U.S. Pat. No. 5,873,116, and Ser. No. 08/659,191 U.S. Pat. No. 5,813,113, all filed Jun. 5, 1996, and all of which are continuations-in-part of U.S. patent application Ser. No. 08/485,323 now U.S. Pat. No. 5,880,141, filed Jun. 7, 1995, all of which are incorporated herein by reference in their entirety, including any drawings. 
    
    
     1. INTRODUCTION 
     The present invention relates to novel compounds capable of modulating, regulating and/or inhibiting tyrosine kinase signal transduction. The present invention is also directed to methods of regulating, modulating or inhibiting tyrosine kinases, whether of the receptor or non-receptor class, for the prevention and/or treatment of disorders related to unregulated tyrosine kinase signal transduction, including cell proliferative and metabolic disorders. 
     2. BACKGROUND OF THE INVENTION 
     The following description of the background of the invention is provided merely to aid in understanding of the invention is not admitted to describe or constitute prior art to the invention. 
     Protein tyrosine kinases (PTKs) comprise a large and diverse class of proteins having enzymatic activity. The PTKs play an important role in the control of cell growth and differentiation (for review, see Schlessinger &amp; Ullrich, 1992 , Neuron  9:383-391). 
     For example, receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects to the extracellular microenvironment). See, Schlessinger and Ullrich, 1992 , Neuron  9:303-391. 
     With respect to receptor tyrosine kinases, it has been shown also that tyrosine phosphorylation sites function as high-affinity binding sites for SH2 (src homology) domains of 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 that associate with receptor tyrosine kinases (RTKs) have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993 , Cell  72:767-778. The specificity of the interactions between receptors or proteins and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993 , Cell  72:767-778. These observations suggest that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors. 
     Aberrant expression or mutations in the PTKs have been shown to lead to either uncontrolled cell proliferation (e.g. malignant tumor growth) or to defects in key developmental processes. Consequently, the biomedical community has expended significant resources to discover the specific biological role of members of the PTK family, their function in differentiation processes, their involvement in tumorigenesis and in other diseases, the biochemical mechanisms underlying their signal transduction pathways activated upon ligand stimulation and the development of novel drugs. 
     Tyrosine kinases can be of the receptor-type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular). Many of the tyrosine kinases, whether an RTK or non-receptor tyrosine kinase, have been found to be involved in cellular signaling pathways leading pathogenic conditions, including cancer, psoriasis and hyper immune response. 
     Development Of Compounds To Modulate The PTKs. In view of the surmised importance of PTKs to the control, regulation and modulation of cell proliferation the diseases and disorders associated with abnormal cell proliferation, many attempts have been made to identify receptor and non-receptor tyrosine kinase “inhibitors” using a variety of approaches, including the use of mutant ligands (U.S. Pat. No. 4,966,849), soluble receptors and antibodies (application Ser. No. WO 94/10202; Kendall &amp; Thomas, 1994 , Proc. Nat&#39;l Acad. Sci  90:10705-09; Kim, et al., 1993 , Nature  362:841-844), RNA ligands (Jellinek, et al.,  Biochemistry  33:10450-56); Takano, et al., 1993 , Mol. Bio. Cell  4:358A; Kinsella, et al., 1992 , Exp. Cell Res . 199:56-62; Wright, et al., 1992 , J. Cellular Phys . 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., 1994 , Proc. Am. Assoc. Cancer Res . 35:2268). 
     More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have been described generally as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer. 
     The identification of effective small compounds which specifically inhibit signal transduction by modulating the activity of receptor and non-receptor tyrosine kinases to regulate and modulate abnormal or inappropriate cell proliferation is therefore desirable and an object of this invention. 
     3. SUMMARY OF THE INVENTION 
     The present invention relates to organic molecules capable of modulating, regulating and/or inhibiting tyrosine kinase signal transduction. Such compounds are useful for the treatment of diseases related to unregulated TKS transduction, including cell proliferative diseases such as cancer, atherosclerosis, arthritis and restenosis and metabolic diseases such as diabetes. 
     In one aspect the invention features a combinatorial library of indolinone compounds. The library includes a series of at least ten (preferably at least 50-100, more preferably at least 100-500, and most preferably at least 500-5,000) indolinones that can be formed by reacting an oxindole compound with an aldehyde. In preferred embodiments the indolinones in the library can be formed by reacting a type A oxindole with a type B aldehyde. Type A oxindoles and type B aldehydes are shown in  FIGS. 1 and 2  respectively (and Tables 1 and 2 respectively), as explained in detail below. As can be seen, in the figures the oxindoles are labeled 01, 02, 03, . . . and the aldehydes are named A1, A2, A3, . . . . Thus, one can readily appreciate that the combinatorial library could include any and all combinations of oxindoles and aldehydes, including the indolinones resulting from 01 and A1, 01 and A2, 01 and A3, 02 and A1, 02 and A2, 02 and A3, 03 and A1, 03 and A2, 03 and A3 and so on. Similarly, the indolinones in the library can be formed by any combination of the oxindoles in Table 1 with any of the aldehydes listed in  FIG. 2  or Table 2. Finally, the indolinones may also, of course, come from any combination of aldehydes listed in Table 2 with any oxindoles from  FIG. 1  or Table 1. 
     The term “combinatorial library” refers to a series of compounds. In the present case, the combinatorial library contains a series of indolinone compounds that can be formed by reacting an oxindole and an aldehyde. A wide variety of oxindoles and aldehydes may be used to create the library of indolinones. 
     The term “indolinone” is used as that term is commonly understood in the art and includes substituted and unsubstituted indolinones, such as the compounds of structures I, II, III, IV, and V shown below. 
     The term “type A oxindole” is meant to include any and all of the oxindoles set forth in FIG.  1  and Table I. Oxindoles, as that term is used herein, typically have the structure set forth below: 
                 
 
wherein,
         (a) A 1 , A 2 , A 3 , and A 4  are independently carbon or nitrogen;   (b) R 1  is hydrogen or alkyl;   (c) R 2  is oxygen or sulfur;   (d) R 3  is hydrogen;   (e) R 4 , R 5 , R 6 , and R 7  (i) are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′ or (ii) any two adjacent R 4 , R 5 , R 6 , and R 7  taken together form a fused ring with the aryl portion of the oxindole-based portion of the indolinone.       

     It is to be understood that when A 1 , A 2 , A 3 , and A 4 , is nitrogen or sulfur that the corresponding R 4 , R 5 , R 6 , or R 7  is nothing and that the corresponding bond shown in structure VII does not exist. 
     Examples of oxindoles having such fused rings (as described in (e) (ii) above) are shown in  FIG. 1 , compounds 044, 045, 047, 048, 050, 051, 052, 053, 055, 056, 058, 059, 061, 062, 064, 066, 067, 069, 070, and 073. Other examples of suitable fused rings include the following: 
                 
 
     The six membered rings shown above also exemplify possible A rings in the structures V, VI, and VII. 
     The term “type B aldehyde” is meant to include any and all of the aldehydes set forth in FIG.  2  and Table 2. The term “aldehyde” is used as is commonly understood in the art to include substituted and unsubstituted aldehydes of the structure R d CHO where R d  can be a wide variety of substituted or unsubstituted groups such as alkyl, aryl, etc. 
     In yet another aspect, the invention provides a method of synthesizing an indolinone by reacting a type A oxindole with a type B aldehyde. The method of making the indolinones of the present invention may involve creating a combinatorial library of compounds as described above, testing each compound in biological assays such as those described herein, selecting one or more suitable compounds and synthesizing the selected compound or compounds. 
     Also featured is an indolinone compound having formula V or VI: 
                 
 
and pharmaceutically acceptable salts, esters, amides, prodrugs, isomers, and metabolites, thereof, wherein:
         (a) A 1 , A 2 , A 3 , and A 4  are independently carbon or nitrogen;   (b) R 1  is hydrogen or alkyl;   (c) R 2  is oxygen or sulfur;   (d) R 3  is hydrogen;   (e) R 4 , R 5 , R 6 , and R 7  (i) are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′ or (ii) any two adjacent R 4 , R 5 , R 6 , and R 7  taken together form a fused ring with the aryl ring of the oxindole-based portion of the indolinone;   (f) R 2 ′, R 3 ′, R 4 ′, R 5 ′, and R 6 ′ are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   (g) n is 0, 1, 2, or 3;   (h) R is hydrogen, alkyl or aryl;   (i) R′ is hydrogen, alkyl or aryl; and   (j) A is a five membered heteroaryl ring selected from the group consisting of thiophene, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, 2-sulfonylfuran, 4-alkylfuran, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3,4-oxatriazole, 1,2,3,5-oxatriazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3,4-thiatriazole, 1,2,3,5-thiatriazole, and tetrazole, optionally substituted at one or more positions with alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R or CONRR′.       

     It is to be understood that when A 1 , A 2 , A 3 , and A 4  is nitrogen or sulfur that the corresponding R 4 , R 5 , R 6 , or R 7  is nothing and that the corresponding bond shown in structure VII does not exist. 
     In preferred embodiments of structure VI, the A sustituent may be a five membered heterocycle of formula VII shown below: 
                 
 
wherein D, E, F, and G are nitrogen, carbon, or sulfur atoms. The specific juxtaposition of groups D-G is limited to examples of heterocyclic groups known in the chemistry arts. Specific examples of these heterocyclic groups include the following: 
                 
 
all of which may be optionally substituted as described above in paragraph (j).
 
     In preferred embodiments, the aryl ring (“the A ring”) of the oxindole-derived portion of the indolinone (i.e., the ring shown in structures V and VI with A 1 , A 2 , A 3 , and A 4 ) has a polar substituent, preferably selected from the group consisting of NH 2 , COOH, SO 3 H, Br, Cl, I, F, COCH 2 CH 2 COOH, COCH 2 Cl, piperazine, and CH 2 CH 2 NH 2  at the 4, 5, 6, and 7 carbon atom positions (identified by substituents R 4 , R 5 , R 6 , and R 7  respectively in structures V and VI), most preferably hydrophillic groups such as NH 2 , COOH, SO 3 , COCH 2 CH 2 COOH, piperazine and CH 2 CH 2 NH 2 , 
     One approach to choosing target inhibitors of the FGFR involves selecting target compounds with a substituent on the A ring that mimics the triphosphate of ATP and thereby increases the affinity of target compounds for the active-site of the FGFR. Hydrophillic groups may act to mimic the triphosphate at ATP, and also to improve the solubility of the final inhibitor. Without being bound to any theory, it appears that the trans form of the indolinones is generally a more favorable form for FGFR inhibitors. 
     Amine-based substituents at positions 4, 5, and 6 at the A ring of structures V and VI are a preferred class of substituents and an especially preferred class are amines of the structure: 
                 
 
wherein R a  is CO(CH 2 ) 2 COOH, aryl, alkyl, or contains COOH, OH, or NH 2 . These types of groups provide steric hindrance in order to force the isomer into a trans conformation which may be a favored property of FGFR inhibition and acts as a linker to a hydrophillic group.
 
     Another favored class of substituents on the aryl ring of structures V and VI includes piperazine type substituents of the structure: 
                 
 
wherein R b  is preferably a negatively charged group, such as a negatively charged alkyl or acyl.
 
     Yet another preferred class of substituents for the aryl ring of structures V and VI are C—COR groups of the formula: 
                 
 
wherein R c  is a hydrophilic or negatively charged group, preferably at the 5 and/or 6 positions of the A ring of structures V and VI, such as amide, ester, CH 2 CH 2 COOH, CH 2 Cl, or piperazine. R c  could also be linked to the aryl ring by a sp3 carbon or could be attached as R c O 3 S—.
 
     Yet another preferred set of substituents on the aryl ring are fused heterocyclic rings which can be synthesized by acylation of the arylamine followed by alkylation of the heterocyclic ring systems. Examples of several such compounds are set forth in  FIG. 1 , compounds 044, 045, 047, 048, 050, 051, 052, 053, 055, 056, 058, 059, 061, 062, 064, 066, 067, 069, 070, and 073. 
     Also featured is a pharmaceutical composition that has a pharmaceutically acceptable carrier or excipient and an indolinone compound as described above. 
     In another aspect the invention provides a method for treating diseases related to unregulated tyrosine kinase signal transduction, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of an indolinone compound described above. 
     A method for regulating tyrosine kinase signal transduction is also provided. The method involves administering to a subject a therapeutically effective amount of an indolinone compound described above. 
     In the embodiments set forth below, several preferred subclasses of compounds are set forth. In one embodiment, the invention provides compounds having the formula: 
                 
 
and the pharmaceutically acceptable salts thereof, wherein
         R 1  is hydrogen or alkyl;   R 2  is oxygen or sulfur;   R 3  is hydrogen;   R 4 , R 5 , R 6 , and R 7  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   R 3′ , R 5′ , and R 6′  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   n is 0-3;   X is Br, Cl, F or I;   R is hydrogen, alkyl or aryl; and   R′ is hydrogen, alkyl or aryl.       

     In a preferred embodiment of the compounds of formula I, R 3 ′ is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and R 5 ′ is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl. 
     A particularly preferred compound of formula I is 3-(2-chloro-4-hydroxybenzylidenyl)-2-indolinone (SU4932). 
     In another embodiment, the invention provides compounds having the formula: 
                 
 
and the pharmaceutically acceptable salts thereof, wherein
         R 1  is hydrogen or alkyl;   R 2  is oxygen or sulfer;   R 3  is hydrogen;   R 4 , R 5 , R 6 , and R 7  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   R 2′ , R 3′ , R 5′ , and R 6′  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   Ra and Rb are each independently selected from the group consisting of H, alkyl and C(O)R, or NRaRb taken together may be a heterocyclic ring of from 3 to 8 atoms optionally substituted at one or more positions with hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, s alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, or CONRR′;   n is 0-3;   X is Br, Cl, F or I; and   R is hydrogen, alkyl or aryl; and   R′ is hydrogen, alkyl or aryl.       

     In a preferred embodiment of the compounds of formula II, R 3 ′ is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and R 5 ′ is selected from the group consisting of hydrogen, methyl, -ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl. 
     A particularly preferred compound of formula II is 3-(4-Dimethylaminobenzylidenyl)-2-indolinone (SU4312). 
     In yet another embodiment, the invention provides compounds having the formula: 
                 
 
and the pharmaceutically acceptable salts thereof, wherein
         R 1  is hydrogen or alkyl;   R 2  is oxygen or sulfer;   R 3  is hydrogen;   R 4 , R 5 , R 6 , and R 7  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   R is hydrogen, alkyl or aryl; and   R′ is hydrogen, alkyl or aryl.       

     In a preferred embodiment of the invention, the compound of formula III is 3-[(2,3-Dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416). 
     In still another embodiment, the invention provides compounds having the formula: 
                 
 
and the pharmaceutically acceptable salts thereof, wherein:
         R 1  is hydrogen or alkyl;   R 2  is oxygen or sulfer;   R 3  is hydrogen;   R 4 , R 5 , R 6 , and R 7  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   R 3′ , R 4′ , R 5′ , and R 6′  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R, and CONRR′;   n is 0-3;   X is Br, Cl, F or I;   R is hydrogen, alkyl or aryl; and   R′ is hydrogen, alkyl or aryl.       

     In a preferred embodiment of the compound of formula IV, R 3 ′ is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and R 5 ′ is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl. 
     A particularly preferred compound of formula IV is 3-(2-Ethoxybenzylidenyl]-2-indolinone (SU5204). 
     In a final embodiment, the invention provides compounds having the formula: 
                 
 
and the pharmaceutically acceptable salts thereof, wherein:
         R 1  is hydrogen or alkyl;   R 2  is oxygen or sulfer;   R 3  is hydrogen;   R 4 , R 5 , R 6 , and R 7  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R and CONRR′;   R 2′ , R 3′ , R 5′ , and R 6′  are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, aryl, aryloxy, alkaryl, alkaryloxy, halogen, trihalomethyl, S(O)R, SO 2 NRR′, SO 3 R, SR, NO 2 , NRR′, OH, CN, C(O)R, OC(O)R, NHC(O)R, (CH 2 ) n CO 2 R and CONRR′;   n is 0-3;   Z is Br, Cl, F, I, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl;   R is hydrogen, alkyl or aryl; and   R′ is hydrogen, alkyl or aryl.       

     In a preferred embodiment of the compounds of formula V, R 3 ′ is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl; and R 5 ′ is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, halogen, aryl and OR, where R is H, alkyl or aryl. 
     A particularly preferred compound of formula V is 3-(4-Bromobenzylidenyl)-2-indolinone (SU4942). 
     The chemical formulae referred herein may exhibit the phenomena of tautomerism or structural isomerism. For example, the compounds described herein may be adopt a cis or trans conformation about the double bond connecting the indolinone 3-substituent to the indolinone ring, or may be mixtures of cis and trans isomers. As the formulae drawing within this specification can only represent one possible tautomeric or structural isomeric form, it should be understood that the invention encompasses any tautomeric or structural isomeric form, or mixtures thereof, which possesses the ability to regulate, inhibit and/or modulate tyrosine kinase signal transduction or cell proliferation and is not limited to any one tautomeric or structural isomeric form utilized within the formulae drawing. 
     In addition to the above-described compounds and their pharmaceutically acceptable salts, the invention is further directed, where applicable, to solvated as well as unsolvated forms of the compounds (e.g. hydrated forms) having the ability to regulate and/or modulate cell proliferation. 
     The compounds described herein may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Suitable processes are illustrated in the examples. Necessary starting materials may be obtained by standard procedures of organic chemistry. 
     An individual compound&#39;s relevant activity and efficacy as an agent to affect receptor tyrosine kinase mediated signal transduction may be determined using available techniques. Preferentially, a compound is subjected to a series of screens to determine the compound&#39;s ability to modulate, regulate and/or inhibit cell proliferation. These screens, in the order in which they are conducted, include biochemical assays, cell growth assays and in vivo experiments. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows illustrative type A oxindoles. 
         FIG. 2  shows illustrative type B aldehydes. 
     
    
    
     4. DETAILED DESCRIPTION OF THE INVENTION 
     (a) Definitions 
     “Pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. 
     “Alkyl” refers to a straight-chain, branched or cyclic saturated aliphatic hydrocarbon. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like. The alkyl group may be optionally substituted with one or more substituents are selected from the group consisting of hydroxyl, cyano, alkoxy, ═O, ═S, NO 2 , halogen, N(CH 3 ) 2  amino, and SH. 
     “Alkenyl” refers to a straight-chain, branched or cyclic unsaturated hydrocarbon group containing at least one carbon-carbon double bond. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkoxy, ═O, ═S, NO 2 , halogen, N(CH 3 ) 2  amino, and SH. 
     “Alkynyl” refers to a straight-chain, branched or cyclic unsaturated hydrocarbon containing at least one carbon-carbon triple bond. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be optionally substituted with one or more substituents selected from the group consisting of hydroxyl, cyano, alkoxy, ═O, ═S, NO 2 , halogen, N(CH 3 ) 2  amino, and SH. 
     “Alkoxy” refers to an “-Oalkyl” group. 
     “Aryl” refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups. The aryl group may be optionally substituted with one or more substituents selected from the group consisting of halogen, trihalomethyl, hydroxyl, SH, OH, NO 2 , amine, thioether, cyano, alkoxy, alkyl, and amino. 
     “Alkaryl” refers to an alkyl that is covalently joined to an aryl group. Preferably, the alkyl is a lower alkyl. 
     “Carbocyclic aryl” refers to an aryl group wherein the ring atoms are carbon. 
     “Heterocyclic aryl” refers to an aryl group having from 1 to 3 heteroatoms as ring atoms, the remainder of the ring atoms being carbon. Heteroatoms include oxygen, sulfur, and nitrogen. Thus, heterocyclic aryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like. 
     “Amide” refers to —C(O)—NH—R, where R is alkyl, aryl, alkylaryl or hydrogen. 
     “Thioamide” refers to —C(S)—NH—R, where R is alkyl, aryl, alkylaryl or hydrogen. 
     “Amine” refers to a —N(R′)R″ group, where R′ and R″ are independently selected from the group consisting of alkyl, aryl, and alkylaryl. 
     “Thioether” refers to —S—R, where R is alkyl, aryl, or alkylaryl. 
     “Sulfonyl” refers to —S(O) 2 —R, where R is aryl, C(CN)═C-aryl, CH 2 CN, alkyaryl, sulfonamide, NH-alkyl, NH-alkylaryl, or NH-aryl. 
     The term “acyl” denotes groups _C(O)R, where R is alkyl as defined above, such as formyl, acetyl, propionyl, or butyryl. 
     The term “prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs may be easier to administer than the parent drug in some situations. For example, the prodrug may be bioavailable by oral administration but the parent is not, or the prodrug may improve solubility to allow for intravenous administration. 
     (b) The Invention 
     The present invention relates to compounds capable of regulating and/or modulating tyrosine kinase signal transduction and more particularly receptor and non-receptor tyrosine kinase signal transduction. 
     Receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, metabolic effects to the extracellular microenvironment). See, Schlessinger and Ullrich, 1992 , Neuron  9:303-391. 
     It has been shown that tyrosine phosphorylation sites in growth factor receptors function as high-affinity binding sites for SH2 (src homology) domains of 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 that associate with receptor tyrosine kinases have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and associate with catalytically active molecules. Songyang et al., 1993 , Cell  72:767-778. The specificity of the interactions between receptors and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles. Songyang et al., 1993 , Cell  72:767-778. These observations suggest that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors. 
     Tyrosine kinase signal transduction results in, among other responses, cell proliferation, differentiation and metabolism. Abnormal cell proliferation may result in a wide array of disorders and diseases, including the development of neoplasia such as carcinoma, sarcoma, leukemia, glioblastoma, hemangioma, psoriasis, arteriosclerosis, arthritis and diabetic retinopathy (or other disorders related to uncontrolled angiogenesis and/or vasculogenesis). 
     This invention is therefore directed to compounds which regulate, modulate and/or inhibit tyrosine kinase signal transduction by affecting the enzymatic activity of the RTKs and/or the non-receptor tyrosine kinases and interfering with the signal transduced such proteins. More particularly, the present invention is directed to compounds which regulate, modulate and/or inhibit the RTK and/or non-receptor tyrosine kinase mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors, including but not limited to carcinoma, sarcoma, leukemia, erythroblastoma, glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma. Indications may include, but are not limited to brain cancers, bladder cancers, ovarian cancers, gastric cancers, pancreas cancers, colon cancers, blood cancers, lung cancers and bone cancers. 
     (c) Indications 
     The compounds described herein are useful for treating disorders related to unregulated tyrosine kinase signal transduction, including cell proliferative disorders, fibrotic disorders and metabolic disorders. 
     Cell proliferative disorders which can be treated or further studied by the present invention include cancers, blood vessel proliferative disorders and mesangial cell proliferative disorders. 
     Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, play important roles in a variety of physiological processes such as embryonic development, corpus luteum formation, wound healing and organ regeneration. They also play a pivotal role in cancer development. Other examples of blood vessel proliferation disorders include arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. Conversely, disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, are also implicated. 
     Fibrotic disorders refer to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrohis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role ion hepatic cirrhosis. Other fibrotic disorders implicated include atherosclerosis (see, below). 
     Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies. The PDGF-R has been implicated in the maintenance of mesangial cell proliferation. Floege et al., 1993 , Kidney International  43:47S-54S. 
     PTKs have been associated with such cell proliferative disorders. For example, some members of the RTK family have been associated with the development of cancer. Some of these receptors, like the 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 the PDGF-R (Kumabe et al., 1992 , Oncogene  7:627-633) are overexpressed in many tumors and/or persistently activated by autocrine loops. In fact, in the most common and severe cancers these receptor overexpressions (Akbasak and Suner-Akbasak et al., 1992 , J. Neurol. Sci . 111:119-133; Dickson et al., 1992 , Cancer Treatment Res . 61:249-273; Korc et al., 1992 , J. Clin. Invest . 90:1352-1360) and autocrine loops (Lee and Donoghue, 1992 , J. Cell. Biol . 118:1057-1070; Korc et al., supra; Akbasak and Suner-Akbasak et al., supra) have been demonstrated. For example, the EGFR receptor has been associated with squamous cell carcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancer and bladder cancer. HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer. The PDGF-R has been associated with glioblastoma, lung, ovarian, melanoma and prostate. The RTK c-met has been generally associated with hepatocarcinogenesis and thus hepatocellular carcinoma. Additionally, c-met has been linked to malignant tumor formation. More specifically, the RTK c-met has been associated with, among other cancers, colorectal, thyroid, pancreatic and gastric carcinoma, leukemia and lymphoma. Additionally, over-expression of the c-met gene has been detected in patients with Hodgkins disease, Burkitts disease, and the lymphoma cell line. 
     The IGF-IR, in addition to being implicated in nutritional support and in type-II diabetes, has also been associated with several types of cancers. For example, IGF-I has been implicated as an autocrine growth stimulator for several tumor types, e.g. human breast cancer carcinoma cells (Arteaga et al., 1989 , J. Clin. Invest . 84:1418-1423) and small lung tumor cells (Macauley et al., 1990 , Cancer Res . 50:2511-2517). In addition, IGF-I, integrally involved in the normal growth and differentiation of the nervous system, appears to be an autocrine stimulator of human gliomas. Sandberg-Nordqvist et al., 1993 , Cancer Res . 53:2475-2478. The importance of the IGF-IR and its ligands in cell proliferation is further supported by the fact that many cell types in culture (fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes, myeloid cells, chondrocytes, osteoblasts, the stem cells of the bone marrow) are stimulated to grow by IGF-I. Goldring and Goldring, 1991 , Eukaryotic Gene Expression  1:301-326. In a series of recent publications, Baserga even suggests that IGF-I-R plays a central role in the mechanisms 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. 
     The association between abnormalities in RTKs and disease are not restricted to cancer, however. For example, RTKs have been associated with metabolic diseases like psoriasis, diabetes mellitus, wound healing, inflammation, and neurodegenerative diseases. For example, the EGF-R is indicated in corneal and dermal wound healing. Defects in the Insulin-R and the IGF-lR are indicated in type-II diabetes mellitus. A more complete correlation between specific RTKs and their therapeutic indications is set forth in Plowman et al., 1994 , DN &amp; P  7:334-339. 
     Not only receptor type tyrosine kinases, but also many cellular tyrosine kinases (CTKs) including src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr, yrk (reviewed by Bolen et al., 1992 , FASEB J . 6:3403-3409) are involved in the proliferative and metabolic signal transduction pathway and thus in indications of the present invention. For example, mutated src (v-src) has been demonstrated as an oncoprotein (pp60 v-src ) in chicken. Moreover, its cellular homolog, the proto-oncogene pp60 c-src  transmits oncogenic signals of many receptors. For example, overexpression of EGF-R or HER2/neu in tumors leads to the constitutive activation of pp60 c-src , which is characteristic for the malignant cell but absent from the normal cell. On the other hand, mice deficient for the expression of c-src exhibit an osteopetrotic phenotype, indicating a key participation of c-src in osteoclast function and a possible involvement in related disorders. Similarly, Zap 70 is implicated in T-cell signaling. 
     Furthermore, the identification of CTK modulating compounds to augment or even synergize with RTK aimed blockers is an aspect of the present invention. 
     Finally, both RTKs and non-receptor type kinases have been connected to hyperimmune disorders. 
     The KDR/FLK-1 Receptor and VEGF. Normal vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy. Folkman and Shing, 1992 , J. Biological Chem . 267:10931-34. However, many diseases are driven by persistent unregulated or inappropriate angiogenesis. For example, in arthritis, new capillary blood vessels invade the joint and destroy the cartilage. In diabetes, new capillaries in the retina invade the vitreous, bleed and cause blindness. Folkman, 1987, in:  Congress of Thrombosis and Haemostasis  (Verstraete, et. al, eds.), Leuven University Press, Leuven, pp.583-596. Ocular neovascularization is the most common cause of blindness and dominates approximately twenty (20) eye diseases. 
     Moreover, vasculogenesis and/or angiogenesis have been associated with the growth of malignant solid tumors and metastasis. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow. Furthermore, the new blood vessels embedded in a tumor provide a gateway for tumor cells to enter the circulation and to metastasize to distant sites in the body. Folkman, 1990 , J. Natl. Cancer Inst . 82:4-6; Klagsbrunn and Soker, 1993 , Current Biology  3:699-702; Folkman, 1991 , J. Natl., Cancer Inst . 82:4-6; Weidner et al., 1991 , New Engl. J. Med . 324:1-5. 
     Several polypeptides with in vitro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblastic growth factor (aFGF, bFGF), vascular endothelial growth factor (VEGF) and placental growth factor. Unlike aFGF and bFGF, VEGF has recently been reported to be an endothelial cell specific mitogen. Ferrara and Henzel, 1989 , Biochem. Biophys. Res. Comm . 161:851-858; Vaisman et al., 1990 , J. Biol. Chem . 265:19461-19566. 
     Thus, the identification of the specific receptors to which VEGF binds is an important advancement in the understanding of the regulation of endothelial cell proliferation. Two structurally closely related RTKs have been identified to bind VEGF with high affinity: the flt-1 receptor (Shibuya et al., 1990 , Oncogene  5:519-524; De Vries et al., 1992 , Science  255:989-991) and the KDR/FLK-1 receptor, discussed in the U.S. patent application Ser. No. 08/193,829. Consequently, it had been surmised that these RTKs may have a role in the modulation and regulation of endothelial cell proliferation. 
     Evidence, such as the disclosure set forth in copending U.S. application Ser. No. 08/193,829, strongly suggests that VEGF is not only responsible for endothelial cell proliferation, but also is a prime regulator of normal and pathological angiogenesis. See generally, Klagsburn and Soker, 1993 , Current Biology  3:699-702; Houck et al., 1992 , J. Biol. Chem . 267:26031-26037. Moreover, it has been shown that KDR/FLK-1 and flt-1 are abundantly expressed in the proliferating endothelial cells of a growing tumor, but not in the surrounding quiescent endothelial cells. Plate et al., 1992 , Nature  359:845-848; Shweiki et al., 1992 , Nature  359:843-845. 
     Identification Of Agonists And Antagonists To The KDR/PLK-1 Receptor. In view of the deduced importance of RTKs in the control, regulation and modulation of endothelial cell proliferation and potentially vasculogenesis and/or angiogenesis, many attempts have been made to identify RTK “inhibitors” using a variety of approaches. These include the use of mutant ligands (U.S. Pat. No. 4,966,849); soluble receptors and antibodies (Application No. WO 94/10202; Kendall and Thomas, 1994 , Proc. Natl. Acad. Sci. USA  90:10705-10709; Kim et al., 1993 , Nature  362:841-844); and RNA ligands (Jellinek et al., 1994 , Biochemistry  33:10450-10456). 
     Furthermore, tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani et al., 1994 , Proc. Am. Assoc. Cancer Res . 35:2268), and inhibitors acting on receptor tyrosine kinase signal transduction pathways, such as protein kinase C inhibitors have been identified (Schuchter et al., 1991 , Cancer Res . 51:682-687); Takano et al., 1993 , Mol. Bio. Cell  4:358A; Kinsella et al., 1992 , Exp. Cell Res . 199:56-62; Wright et al., 1992 , J. Cellular Phys . 152:448-57). 
     More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cycloproppyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have been described generally as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer. 
     Consequently, there is an unmet need for the identification and generation of effective small compounds which selectively inhibit the signal transduction of the KDR/FLK-1 receptor in order to effectively and specifically suppress vasculogenesis. 
     Some of the compounds of the present invention demonstrate excellent activity in biological assays and thus these compounds and related compounds are expected to be effective in treating Flk related disorders such as those driven by persistent unregulated or inappropriate angiogenesis. 
     (d) Pharmaceutical Formulations and Routes of Administration 
     The compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in “Remington&#39;s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. 
     (i) Routes of Administration. 
     Suitable routes of administration may, for example, is include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. 
     Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation. 
     Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor. 
     (ii) Composition/Formulation. 
     The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. 
     Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. 
     For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks&#39;s solution, Ringer&#39;s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. 
     For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. 
     Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. 
     Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. 
     For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. 
     For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. 
     The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. 
     Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. 
     Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. 
     The compounds may also be formulated in rectal compositions such as Suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. 
     In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. 
     A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. 
     Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed. 
     The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. 
     Many of the PTK modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. 
     (iii) Effective Dosage. 
     Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. 
     For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50  as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PTK activity). Such information can be used to more accurately determine useful doses in humans. 
     Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50  (the dose lethal to 50% of the population) and the ED 50  (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 50  and ED 50 . Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50  with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient&#39;s condition. (See e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1). 
     Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. 
     Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. 
     In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. 
     The amount of composition administered will, of course, be dependent on the subject being treated, on the subject&#39;s weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. 
     (iv) Packaging 
     The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like. 
     5. EXAMPLE 
     Compound Synthesis 
     The compounds of the present invention may be synthesized according to known techniques. The following represent preferred methods for synthesizing the compounds of the claimed invention. 
     (a) General Syntheses of 3-Substituted-2-Indolinone Analogs (SU4312 and SU4314 Analogs) 
     The following general methodologies were used to synthesize 3-substituted-2-indolinone compounds of the invention. 
     (i) Method A 
     A reaction mixture of the proper oxindole (2-indolinone) (1 equiv.), the appropriate aldehyde (1.2 equiv.), and piperidine (0.1 equiv.) in ethanol (1-2 mL/1 mmol oxindole) was stirred at 90° C. for 3-5 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield the target compound. 
     (ii) Method B 
     Preparation of The Proper Aldehydes via Vilsmeier Reaction. To a solution of N,N-dimethylformamide (1.2 equiv.) in 1,2-dichloroethane (2.0 mL/1.0 mmole of starting material) was added dropwise phosphorus oxychloride (1.2 equiv.) at 0° C. The ice-bath was removed and the reaction mixture was further stirred for 30 min. The proper starting material (1.0 equiv.) was added to the above solution portionwise and the reaction mixture was stirred at 50-70° C. for 5 h-2 days. The reaction mixture was poured into ice-cold 1N sodium hydroxide solution (pH=9 after mixing) and the resulting mixture was stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with brine until pH=7, dried over anhydrous sodium sulfate and evaporated. The residue was chromatographed on a silica gel column eluting with a solvent mixture of ethyl acetate and hexane to afford the title compound. 
     Synthesis for 3-Substituted-2-Indolinone Analogs. A reaction mixture of the proper oxindole (2-indolinone) (1 equiv.), the appropriate aldehyde (1.2 equiv.), and piperidine (0.1 equiv.) in ethanol (1-2 mL/1 mmol oxindole) was stirred at 90° C. for 3-5 h. After cooling, the precipitate was filtered, washed with cold ethanol and dried to yield the target compound. 
     (b) Synthesis of 3-Benzylidene-2-Indolinone (SU4928) 
     The preferred method for synthesizing 3-benzylidene-2-indolinone is as follows: Added 123.2 μl of benzaldehyde and 40 μl of piperidine to a solution of 137.0 mg of oxindole in 2.0 ml methanol. Reflux the reaction mixture for 3 hours and cool down the mixture in an ice-water bath. Filter the resulting precipitate, wash with cold methanol and dry in an oven at 40° C. overnight. Approximately 129.0 mg of the compound was obtained using such protocol. 
     (c) Synthesis of 3-[(Pyrid-4-yl)methylen]-2-indolinone (SU5212) 
     The preferred method for synthesizing 3-[(Pyrid-4-yl)methylene]-2-indolinone as follows: Add 117.0 μl of 4-pyridinecarboxaldehyde and 40 μl of piperidine to a solution of 138.0 mg of oxindole in 2.0 ml methanol. The reaction mixture was refluxed for 3 hours and cooled down in an ice-water bath. The resulting precipitate was filtered, washed with cold methanol and dried in an oven at 40° C. overnight to give 134.5 mg of the compound. 
     (d) Synthesis of 3-[4-(morpholin-4-yl)benzylidenyl]-2-indolinone (SU4991) (Method B): 
     4-(Morpholin-4-yl)benzaldehyde. To a solution of 15 mL of N,N-dimethylformamide in 50 mL of 1,2-dichloroethane was added dropwise 10 mL of phosphorus oxychloride at 0° C. The ice-bath was removed and the reaction mixture was further stirred for 30 min. 4-Phenylmorpholine (16.3 g) was added to the above solution portionwise and the reaction mixture was refluxed for 2 days. Triethylamine (2.5 mL) was added to the above reaction mixture and the reaction was refluxed for 2 day. The reaction mixture was poured into ice-cold 1N sodium hydroxide solution (pH=9 after mixing) and the resulting mixture was stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with 2×20 mL of dichloromethane. The combined organic layer was washed with brine until pH=7, dried over anhydrous sodium sulfate and evaporated. The residue was separated on a silica gel column eluting with a solvent mixture of ethyl acetate and hexane to afford 12.95 g (68%) of the title compound as a white solid. 
     3-[4-(Morpholin-4-yl)benzylidenyl]-2-indolinone (SU4981). A reaction mixture of 6.66 g of oxindole, 11.50 g of the 4-(morpholine-4-yl)benzaldehyde, and 5 mL of piperidine in 50 mL of ethanol was stirred at 90° C. for 5 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 15.0 g (98%) of the title compound as a yellow solid. 
     (e) Synthesis of 3-[4-(4-Formylpiperazin-1-yl)benzylidenyl]-2-indolinone (SU4984) (Method B): 
     4-(4-Formylpiperazin-1-yl)benzaldehyde. To a solution of 3.9 mL (30 mmoles) of N,N-dimethylformamide in 20 mL of 1,2-dichloroethane was added dropwise 3.0 mL (3.9 mmoles) of phosphorus oxychloride at 0° C. The ice-bath was removed and the reaction mixture was further stirred for 15 min. 1-Phenylpiperazine (16.0 g, 10 mmoles) was added to the above solution portionwise and the reaction mixture was stirred at 50° C. for 1 h. The reaction mixture was soured into ice-cold 1N sodium hydroxide solution and stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with 2×20 mL of ethyl acetate. The combined organic layer was washed with brine until pH=7, dried over anhydrous sodium sulfate and evaporated. The residue was separated on a silica gel column eluting with a mixture of ethyl acetate and hexane to afford 9.0 g (41%) of the title compound a light yellow solid. 
     3-[4-(4-Formylpiperazin-1-yl)benzylidenyl]-2-indolinone (SU4984). A reaction mixture of 133.15 mg of oxindole, 228.3 mg of 4-(piperazin-1yl)benzaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 5 h. After cooling, the precipitate was filtered, washed with cold ethanol and dried to yield 199.5 mg (65%) of the title compound a yellow solid. 
     (f) Synthesis of 3-[4-(Piperidin-1-yl)benzylidenyl]-2-indolinone (SU5450) (Method B). 
     4-(Piperidin-1-yl)benzaldehyde. To a solution of 2.3 mL (30 mmoles) of N,N-dimethylformamide in 10 mL of 1,2-dichloroethane was added dropwise 2.8 mL (30 mmoles) of phosphorus oxychloride at 0° C. The ice-bath was removed and the reaction mixture was stirred for 15 min. 1-Phenylpiperidine (3.2 mL, 20 mmoles) was added to the above solution portionwise and the reaction mixture was refluxed overnight. The reaction mixture was poured into ice-cold 2N sodium hydroxide solution and stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with 2×20 mL of ethyl acetate. The combined organic layer was washed with brine until pH=7, dried over anhydrous sodium sulfate and evaporated. The residue was separated on a silica gel column eluting with ethyl acetate and hexane to afford 1.5 g (40%) of the title compound as a white solid. 
     3-[4-(Piperidin-1-yl)benzylidenyl]-2-indolinone (SU5450). A reaction mixture of 134.0 mg of oxindole, 226.8 g of 4-(piperidine-1-yl)benzaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 5 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 268.5 mg (88%) of the title compound as a yellow solid. 
     (g) Synthesis of 3-[2-Chloro-4-methoxybenzylidenyl]-2-indolinone (SU5480). 
     2-Chloro-4-methoxybenzaldehyde. The reaction mixture 25 of 1.0 g (6.4 mmoles) of Z-chloro-4-hydroxybenzaldehyde, 4.4 g (32 mmoles) of potassium carbonate, and 1.4 g (9.6 mmoles) of methyl iodide in 10 mL of N,N-dimethylformamide was stirred at 70° C. for 2 h and poured into ice water. The precipitate was filtered, washed with water, and dried at 40° C. in vacuum oven overnight to yield 750 mg (68%) of the title compound as a light pink solid. 
     3-[2-Chloro-4-methoxybenzylidenyl]-2-indolinone (SU5480). The reaction mixture of 487.9 mg (3.7 mmoles) of oxindole, 750 mg (4.3 mmoles) of 2-chloro-4-methoxybenzaldehyde and 4 drops of piperidine in 5 mL of ethanol was heated to 90° C. for 2 h and cooled to room temperature. The yellow precipitate was filtered, washed with cold ethanol, and dried at 40° C. in a vacuum oven overnight to give 680.2 mg (62%) of the title compound. 
     (h) Synthesis of 3-[(4-Methylthien-2-yl)methylene]-2-indolinone (SU5401). 
     A reaction mixture of 133.0 mg of oxindole, 151.2 mg of the 4-methylthiophene-2-carboxaldehyde, and 3 drops of piperidine in 3 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 147.3 mg (61%) of the title compound as a yellow solid. 
     (i) Synthesis of 3-[(3-Methylpyrrol-2-yl)methylene]-2-indolinone (SU5404). 
     A reaction mixture of 133.0 mg of oxindole, 130.9 mg of the 3-methylpyrrole-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 150.9 mg (67%) of the title compound as a yellow solid. 
     (j) Synthesis of 3-[(3,4-Dimethylpyrrol-2-yl)methylene]-2-indolinone (SU5406) 
     3-[(3,4-Dimethylpyrrol-2-yl)methylene]-2-indolinone was synthesized as described in  J. Heterocyclic Chem . 13:1145-1147 (1976). 
     Ethyl 4-methylpyrrol-3-carboxylate. A solution of 11.86 g (0.1 moles) of ethyl crotonate and 19.50 g (0.1 moles) of p-toluenesulfonylmethylisocyanide in 500 mL of a 2:1 ether/dimethylsulfoxide was added dropwise into a suspension of 6.8 g of sodium hydride (60% mineral oil dispension, 0.17 moles) in ether at room temperature. Upon completion of addition the reaction mixture was stirred for 30 min and dilute with 400 mL of water. The aqueous layer was extracted with 3×100 mL of ether. The combined ether extracts were passed through a column of alumina eluting with dichloromethane. The organic solvent was evaporated and the resulting residue was solidified on standing. The solid was washed with hexane and dried at 40° C in vacuum oven overnight to yield 12.38 g (80%) of the title compound. 
     Preparation of 3,4-Dimethylpyrrole. To a solution of 23 g (80 mmoles) of sodium dihydrobis(2-methoxyehtoxy aluminate) was added dropwise of a solution of 5 g (34 mmoles) of ethyl 4-methylpyrrol-3-carboxylate in 50 mL of benzene at room temperature under nitrogen atmosphere. The reaction mixture was stirred for 18 h. Water (100 mL) was added to the reaction mixture. The organic layer was separated, washed with brine and dried over anhydrous sodium sulfate. The solvent was removed and the residue was distilled giving 1.2 g (44%) of the title compound. 
     Preparation of 3,4-Dimethylpyrrole-2-carboxaldehyde. To a solution of 0.92 mL (12 mmoles) of N,N-dimethylformamide in 10 mL of 1,2-dichloroethane was added dropwise 1.0 mL (12 mmoles) of phosphorus oxychloride at 0° C. The ice-bath was removed and the reaction mixture was further stirred for 30 min. 3,4-Dimethylpyrrole (960.0 mg, 10 mmoles) was added to the above solution portionwise and the reaction mixture was stirred at 50° C. for 5 h. The reaction mixture was poured into ice-cold 1N sodium hydroxide solution (pH=9 after mixing) and the resulting mixture was stirred at room temperature for 1 h. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with brine until pH=7, dried over anhydrous sodium sulfate and evaporated. The residue was chromatographed on a silica gel column eluting with a solvent mixture of ethyl acetate and hexane to afford 610 mg (50%) of the title compound. 
     3-[(3,4-Dimethylpyrrol-2-yl)methylene]-2-indolinone (SU5406). A reaction mixture of 67.0 mg (0.5 mmoles) of oxindole, 73.0 mg (0.6 mmoles) of the 3,4-dimethylpyrrole-2-carboxaldehyde, and 2 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 87.7 mg (37%) of the title compound as a yellow solid. 
     (k) Synthesis of 3-[(2,4-Dimethyl-3-ethoxycarbonylpyrrol-5-yl)methylene]-2-indolinone (SU5408) 
     A reaction mixture of 134.0 mg of oxindole, 234.3 mg of the 4-ethoxycarbonyl-3,5-dimethylpyrrole-2-carboxaldehyde, and 3 drops of piperidine in 3 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 244.6 mg (79%) of the title compound,as a yellow solid. 
     (l) Synthesis of 3-[(2,3-Dimetylpyrrol-5-yl)methylene]-2-indolinone (SU5416) 
     A reaction mixture of 134.0 mg of oxindole, 147.8 mg of the 3,5-dimethylpyrrole-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 136.7 mg (57%) of the title compound as a yellow solid. 
     (m) Synthesis of 3-[(2-Methylmercaptothien-5-yl)methylene]-2-indolinone (SU5419) 
     A reaction mixture of 134.0 mg of oxindole, 189.9 mg of the 5-methylmercaptothiophene-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 246.6 mg (90%) of the title compound as a orange solid. 
     (n) Synthesis of 3-[(2-Methylthien-5-yl)methylene]-2-indolinone (SU5424) 
     A reaction mixture of 134.0 mg of oxindole, 151.42 mg of the 5-methylthiophene-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 237.8 mg (99%) of the title compound as a yellow solid. 
     (o) Synthesis of 3-[(3-Methylthien-2-yl)methylene]-2-indolinone (SU5427) 
     A reaction mixture of 134.0 mg of oxindole, 151.4 mg of the 3-methylthiophene-2-carboxaldehyde, and 3 drops of piperidine in 2 mL of ethanol was stirred at 90° C. for 3 h. After cooling, the precipitate was filtered, washed with cold ethanol, and dried to yield 157.8 mg (65%) of the title compound as a yellow solid. 
     (p) Synthesis of 3-(2,5-Dimethoxybenzylidenyl)-2-indolinone (SU4793) 
     3-(2,5-Dimethoxybenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (q) Synthesis of 3-(2,3-dimethoxybenzylidenyl)-2-indolinone (SU4794) 
     3-(2,3-dimethoxybenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (r) Synthesis of 3-(3-bromo-6-methoxybenzylidenyl)-2-indolinone (SU4796) 
     3-(3-bromo-6-methoxybenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (s) Synthesis of 3-[4-(4-t-butylcarbonyl-piperazin-1-yl)benzylidenyl]-2-indolinone (SU5393) 
     3-[4-(4-t-butylcarbonyl-piperazin-1-yl)benzylidenyl]-2-indolinone is synthesized according to Method B. 
     (t) Synthesis of 3-[(furan-2-yl)methylene]-2-indolinone (SU4798) 
     3-[(furan-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (u) Synthesis of 3-(4-acetamidobenzylidenyl)-2-indolinone (SU4799) 
     3-(4-acetamidobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (v) Synthesis of 3-(2-chloro-4-hydroxybenzylidenyl)-2-indolinone (SU4932) 
     3-(2-chloro-4-hydroxybenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (w) Synthesis of 3-(4-Bromobenzylidenyl)-2-indolinone (SU4942) 
     3-(4-Bromobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (x) Synthesis of 3-(4-Acetylaminobenzylidenyl)-2-indolinone (SU4944) 
     3-(4-Acetylaminobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (y) Synthesis of 3-(2-Methoxybenzylidenyl)-2-indolinone (SU4949) 
     3-(2-Methoxybenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (z) Synthesis of 3-(4-Dimethylaminobenzylidenyl)-1-methyl-2-indolinone (SU4952) 
     3-(4-Dimethylaminobenzylidenyl)-1-methyl-2-indolinone is synthesized according to Method A. 
     (aa) Synthesis of 3-(4-Dimethylaminobenzylidenyl)-2-indolinone (SU4312) 
     3-(4-Dimethylaminobenzylidenyl)-2-indolinone is available from Maybridge Chemical Co. Ltd. 
     (bb) Synthesis of 3-(4-Bromobenzylidenyl)-1-methyl-2-indolinone (SU4956) 
     3-(4-Bromobenzylidenyl)-1-methyl-2-indolinone is synthesized according to Method A. 
     (cc) Synthesis of 5-Chloro-3-(4-dimethylaminobenzylidenyl)-2-indolinone (SU4967) 
     5-Chloro-3-(4-dimethylaminobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (dd) Synthesis of 3-(4-Bromobenzylidenyl)-5-chloro-2-indolinone (SU4972) 
     3-(4-Bromobenzylidenyl)-5-chloro-2-indolinone is synthesized according to Method A. 
     (ee) Synthesis of 3-(4-Diethylaminobenzylidenyl)-2-indolinone (SU4978) 
     3-(4-Diethylaminobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (ff) Synthesis of 3-(4-Di-n-butylaminobenzyldenyl)-2-indolinone (SU4979) 
     3-(4-Di-n-butylaminobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (gg) Synthesis of 1-Methyl-3-[4-(morpholin-4-yl)benzylidenyl]-2-indolinone (SU4982) 
     1-Methyl-3-[4-(morpholin-4-yl)benzylidenyl]-2-indolinone is synthesized according to Method B. 
     (hh) Synthesis of 5-Chloro-3-(4-(morpholine-4-yl)benzylidenyl)-2-indolinone (SU4983) 
     5-Chloro-3-(4-(morpholine-4-yl)benzylidenyl)-2-indolinone is synthesized according to Method B. 
     (ii) Synthesis of 3-(3,4-Dichlorobenzylidenyl)-2-indolinone (SU5201) 
     3-(3,4-Dichlorobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (jj) Synthesis of 3-(2-Ethoxybenzylidenyl]-2-indolinone (SU5204) 
     3-(2-Ethoxybenzylidenyl]-2-indolinone is synthesized according to Method A. 
     (kk) Synthesis of 3-(4-Fluorobenzylidenyl)-2-indolinone (SU5205) 
     3-(4-Fluorobenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (ll) Synthesis of 3-[(Thien-2-yl)methylene]-2-indolinone (SU5208) 
     3-[(Thien-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (mm) Synthesis of 3-(2-Methoxybenzylidenyl)-2-indolinone (SU5214) 
     3-(2-Methoxybenzylidenyl)-2-indolinone is synthesized according to Method A. 
     (nn) Synthesis of 3-[2-[3,5-Di-(trifluoromethyl)phenyl]furan-5-yl]methylene]-2-indolinone (SU5217) 
     3-[2-[3,5-Di-(trifluoromethyl)phenyl]furan-5-yl]methylene]-2-indolinone is synthesized according to Method A. 
     (oo) Synthesis of 2,6-Di-(dimethylamino)-3,5-di-[(indolin-2-one-3-ylidenyl)methyl]-phenylcyanide (SU5218) 
     2,6-Di-(dimethylamino)-35-di-[(indolin-2-one-3-ylidenyl)methyl]-phenylcyanide is synthesized according to Method A. 
     (pp) Synthesis of 3-[(3-(2-carboxyethyl)-4-methylpyrrol-5-yl)methylene]-2-indolinone SU5402) 
     3-[(3-(2-carboxyethyl)-4-methylpyrrol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (qq) Synthesis of 3-[(3,4-Dibromo-5-methylpyrrol-2-yl)methylene]-2-indolinone (SU5403) 
     3-[(3,4-Dibromo-5-methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method B. 
     (rr) Synthesis of 3-[(3,4-Dimethyl-2-formylpyrrole-5-yl)methylene)-2-indolinone (SU5405) 
     3-[(3,4-Dimethyl-2-formylpyrrole-5-yl)methylene)-2-indolinone is synthesized according to Method A. 
     (ss) Synthesis of 3-{[4-(2-methoxycarbonylethyl)-3-methylpyrrol-5-yl]methylene}-2-indolinone (SU5407) 
     3-{[4-(2-methoxycarbonylethyl)-3-methylpyrrol-5-yl]methylene}-2-indolinone is synthesized according to Method A. 
     (tt) Synthesis of 3-[2-Iodofuran-5-yl)methylene]-2-indolinone (SU5409) 
     3-[2-Iodofuran-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (uu) Synthesis of 3-[(3-Ethoxycarbonyl-2-methylfuran-5-yl)methylene]-2-indolinone (SU5410) 
     3-[(3-Ethoxycarbonyl-2-methylfuran-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (vv) Synthesis of 3-[(3-Bromothiene-2-yl)methylene]-2-indolinone (SU5418) 
     3-[(3-Bromothiene-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ww) Synthesis of 3-[(2-Chlorothiene-5-yl)methylene)-2-indolinone (SU5420) 
     3-[(2-Chlorothiene-5-yl)methylene)-2-indolinone is synthesized according to Method A. 
     (xx) Synthesis of 3-[(2,3-Dimethylfuran-5-yl)methylene]-2-indolinone (SU5421) 
     3-[(2,3-Dimethylfuran-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (yy) Synthesis of 3-[(5-Nitrothien-2-yl)methylene]-2-indolinone (SU5422) 
     3-[(5-Nitrothien-2-yl)methylene]-2-indolinone is synthesized according to method A. 
     (zz) synthesis of 3-[(2-Carboxythien-5-yl)methylene]-2-indolinone (SU5423) 
     3-[(2-Carboxythien-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (aaa) Synthesis of 3-[(2-Bromothiene-5-yl)methylene]-2-indolinone (SU5425) 
     3-[(2-Bromothiene-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (bbb) Synthesis of 3-[(4-Bromothiene-2-yl)methylene]-2-indolinone (SU5426) 
     3-[(4-Bromothiene-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ccc) Synthesis of 3-[(2-Sulphonylfuran-5-yl)methylene]-2-indolinone sodium salt (SU5428) 
     3-[(2-Sulphonylfuran-5-yl)methylene]-2-indolinone sodium salt is synthesized according to Method A. 
     (ddd) Synthesis of 3-[(Furan-2-yl)methylene]-2-indolinone (SU5429) 
     3-[(Furan-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (eee) Synthesis of 3-[(2-Methylfuran-5-yl)methylene]-2-indolinone (SU5430) 
     3-[(2-Methylfuran-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (fff) Synthesis of 3-[(2-Ethylfuran-5-yl)methylene-2-indolinone (SU5431) 
     3-[(2-Ethylfuran-5-yl)methylene-2-indolinone is synthesized according to Method A. 
     (ggg) Synthesis of 3-[(2-Nitrofuran-5-yl)methylene]-2-indolinone (SU5432) 
     3-[(2-Nitrofuran-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (hhh) Synthesis of 3-[(5-Bromofuran-2-yl)methylene]-2-indolinone (SU5438) 
     3-[(5-Bromofuran-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (iii) Synthesis of 3-[(2-Ethylthien-5-yl)methylene]-2-indolinone (SU5451) 
     3-[(2-Ethylthien-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (jjj) Synthesis of 3-[(4,5-Dimethyl-3-ethylpyrrol-2-yl)methylene]-2-indolinone (SU5453) 
     3-[(4,5-Dimethyl-3-ethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (kkk) Synthesis of 3-[(5-Ethoxycarbonyl-4-ethoxycarbonylethyl-3-ethoxycarbonylmethylpyrrol-2-yl)methylene]-2-indolinone (SU5454) 
     3-[(5-Ethoxycarbonyl-4-ethoxycarbonylethyl-3-ethoxycarbonylmethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (lll) Synthesis of 3-[(5-Carboxy-3-ethyl-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5455) 
     3-[(5-Carboxy-3-ethyl-4-methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (mmm) Synthesis of 3-[(3,5-Diiodo-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5456) 
     3-[(3,5-Diiodo-4-methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (nnn) Synthesis of 3-[(5-Chloro-3-methoxycarbonyl-4-methoxycarbonylmethylpyrrol-2-yl)methylene]-2-indolinone (SU5459) 
     3-[(5-Chloro-3-methoxycarbonyl-4-methoxycarbonylmethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ooo) Synthesis of 3-[(3-Acetyl-5-ethoxycarbonyl-4-methylpyrrol)-2-yl)methylene]-2-indolinone (SU5460) 
     3-[(3-Acetyl-5-ethoxycarbonyl-4-methylpyrrol)-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ppp) Synthesis of 3-{[1-(3,5-Dichlorophenyl)pyrrol-2-yl]methylene}-2-indolinone (SU5461) 
     3-{[1-(3,5-Dichlorophenyl)pyrrol-2-yl]methylene}-2-indolinone is synthesized according to Method A. 
     (qqq) Synthesis of 3-[1-(4-Chlorophenyl)pyrrol-2-yl)methylene]-2-indolinone (SU5462) 
     3-[1-(4-Chlorophenyl)pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (rrr) Synthesis of 3-[(4-Ethoxycarbonyl-3-methyl)pyrrol-2-yl)methylene]-2-indolinone (SU5463) 
     3-[(4-Ethoxycarbonyl-3-methyl)pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (sss) Synthesis of 3-[(1-Methylpyrrol-2-yl)methylene]-2-indolinone (SU5464) 
     3-[(-Methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ttt) Synthesis of 3-[(5-Ethoxycarbonyl-3-ethoxycarbonylethyl-4-ethoxylcarbonylmethylpyrrol-2-yl)methylene]-2-indolinone (SU5465) 
     3-[(5-Ethoxycarbonyl-3-ethoxycarbonylethyl-4-ethoxylcarbonylmethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (uuu) Synthesis of 3-[4-(Pyrrolidin-1-yl)benzylidenyl]-2-indolinone (SU5466) 
     3-[4-(Pyrrolidin-1-yl)benzylidenyl]-2-indolinone is synthesized according to Method A. 
       
     (vvv) Synthesis of 3-[(5-Methylimidazol-2-yl)methylene]-2-indolinone (SU5468) 
     3-[(5-Methylimidazol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (www) Synthesis of 3-[(5-Methylthiazol-2-yl)methylene]-2-indolinone (SU5469) 
     3-[(5-Methylthiazol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (xxx) Synthesis of 3-[(3-Methylpyrazol-5-yl)methylene]-2-indolinone (SU5472) 
     3-[(3-Methylpyrazol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (yyy) Synthesis of 3-[(Imidazol-4-yl)methylene]-2-indolinone (SU5473) 
     3-[(Imidazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (zzz) Synthesis of 3-[( 4 -Chloropyrazol-3-yl)methylene]-2-indolinone (SU5474) 
     3-[(4-Chloropyrazol-3-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (aaaa) Synthesis of 3-[(4-Bromo-1-(4-chlorobenzyl)pyrazol-5-yl)methylene]-2-indolinone (SU5475) 
     3-[(4-Bromo-1-(4-chlorobenzyl)pyrazol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (bbbb) Synthesis of 3-[(4-Chloro-1-methylpyrazol-3-yl)methylene]-2-indolinone (SU5476) 
     3-[(4-Chloro-1-methylpyrazol-3-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (cccc) Synthesis of 3-[(4-Ethyl-3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone (SU5477) 
     3-[(4-Ethyl-3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method B. 
     (dddd) Synthesis of 3-[(5-Ethylpyrrol-2-yl)methylene]-2-indolinone (SU5478) 
     3-[(5-Ethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method B. 
     (eeee) Synthesis of 3-[3,5-Dimethyl-4-(propen-2-yl)pyrrol-2-yl)methylene]-2-indolinone (SU5479) 
     3-[3,5-Dimethyl-4-(propen-2-yl)pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method B. 
     (ffff) Synthesis of 5,6-Dimethoxyl-3-[2,3-dimethoxylbenzylidenyl]-2-indolinone (SU5495) 
     5,6-Dimethoxyl-3-[2,3-dimethoxylbenzylidenyl]-2-indolinone is synthesized according to Method A. 
     (gggg) Synthesis of 3-[2,4,6-Trimethoxybenzylidenyl]-2-indolinone (SU5607) 
     3-[2,4,6-Trimethoxybenzylidenyl]-2-indolinone is synthesized according to Method A. 
     (hhhh) Synthesis of 5-Chloro-3-[(pyrrol-2-yl)methylene]-2-indolinone (SU5612) 
     5-Chloro-3-[(pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (iiii) Synthesis of 5-Chloro-3-[(3-methylpyrrol-2-yl)methylene]-2-indolinone (SU5613) 
     5-Chloro-3-[(3-methylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (jjjj) Synthesis of 3-(4-isopropylbenzylidenyl)-2-indolinone (SU4313) 
     3-(4-isopropylbenzylidenyl)-2-indolinone is available from Maybridge Chemical Co. Ltd. 
     (kkkk) Synthesis of 5-Chloro-3-[(3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone (SU5614) 
     5-Chloro-3-[(3,5-dimethylpyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (llll) Synthesis of 3-[(pyrrol-2-yl)methylene]-2-indolinone (SU4314) 
     3-[(pyrrol-2-yl)methylene]-2-indolinone is available from Maybridge Chemical Co. Ltd. 
     (mmmm) Synthesis of 5-Chloro-3-[(indol-3-yl)methylene]-2-indolinone (SU5615) 
     5-Chloro-3-[(indol-3-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (nnnn) Synthesis of 5-Chloro-3-[(thien-2-yl)methylene]-2-indolinone (SU5616) 
     5-Chloro-3-[(thien-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (oooo) Synthesis of 5-Chloro-3-[(3-methylthien-2-yl)methylene]-2-indolinone (SU5617) 
     5-Chloro-3-[(3-methylthien-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (pppp) Synthesis of 5-Chloro-3-[(5-methylthien-2-yl)methylene]-2-indolinone (SU5618) 
     5-Chloro-3-[(5-methylthien-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (qqqq) Synthesis of 5-Chloro-3-[(5-ethylthien-2-yl)methylene]-2-indolinone (SU5619) 
     5-Chloro-3-[(5-ethylthien-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (rrrr) Synthesis of 5-Chloro-3-[(5-methylmercaptothien-2-yl)methylene]-2-indolinone (SU5620) 
     5-Chloro-3-[(5-methylmercaptothien-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ssss) Synthesis of 5-Chloro-3-[(imidazol-2-yl)methylene]-2-indolinone (SU5621) 
     5-Chloro-3-[(imidazol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (tttt) Synthesis of 3-[2,4-Dimethoxy-6-methylbenzylidenyl]-2-indolinone (SU5623) 
     3-[2,4-Dimethoxy-6-methylbenzylidenyl]-2-indolinone is synthesized according to Method A. 
     (uuuu) Synthesis of 5-Nitro-3-[(pyrrol-2-yl)methylene]-2-indolinone (SU5624) 
     5-Nitro-3-[(pyrrol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (vvvv) Synthesis of 3-[(3-Methylpyrrol-2-yl)methylene]-5-nitro-2-indolinone (SU5625) 
     3-[(3-Methylpyrrol-2-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (wwww) Synthesis of 3-[(3,5-Dimethylpyrrol-2-yl)methylene]-5-nitro-2-indolinone (SU5626) 
     3-[(3,5-Dimethylpyrrol-2-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (xxxx) Synthesis of 3-[(Indol-3-yl)methylene]-5-nitro-2-indolinone SU5627) 
     3-[(Indol-3-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (yyyy) Synthesis of 5-Nitro-3-[(thien-2-yl)methylene]-2-indolinone (SU5629) 
     5-Nitro-3-[(thien-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (zzzz) Synthesis of 3-[(3-Methylthien-2-yl)methylene]-5-nitro-2-indolinone (SU5629) 
     3-[(3-Methylthien-2-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (aaaaa) Synthesis of 3-[(5-Methylthien-2-yl)methylene]-5-nitro-2-indolinone (SU5630) 
     3-[(5-Methylthien-2-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (bbbbb) Synthesis of 3-[(5-Ethylthien-2-yl)methylene]-5-nitro-2-indolinone SU5631) 
     3-[(5-Ethylthien-2-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (ccccc) Synthesis of 3-[(5-Methylmercaptothien-2-yl)methylene]-5-nitro-2-indolinone (SU5632) 
     3-[(5-Methylmercaptothien-2-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (ddddd) Synthesis of 3-[(Imidazol-2-yl)methylene]-5-nitro-2-indolinone (SU5633) 
     3-[(Imidazol-2-yl)methylene]-5-nitro-2-indolinone is synthesized according to Method A. 
     (eeeee) Synthesis of 3-[(Oxazol-2-yl)methylene]-2-indolinone (CS7127) 
     3-[(Oxazol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (fffff) Synthesis of 3-[(Oxazol-4-yl)methylene]-2-indolinone (CS7128) 
     3-[(Oxazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ggggg) Synthesis of 3-[(Oxazol-5-yl)methylene]-2-indolinone (CS7129) 
     3-[(Oxazol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (hhhhh) Synthesis of 3-[(Thiazol-2-yl)methylene]-2-indolinone (CS7130) 
     3-[(Thiazol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (iiiii) Synthesis of 3-[(Thiazol-4-yl)methylene]-2-indolinone (CS7131) 
     3-[(Thiazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (jjjjj) Synthesis of 3-[(Thiazol-5-yl)methylene]-2-indolinone (CS7132) 
     3-[(Thiazol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (kkkkk) Synthesis of 3-[(Imdazol-2-yl)methylene]-2-indolinone (CS7133) 
     3-[(Imidazol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (lllll) Synthesis of 3-[(Pyrazol-3-yl)methylene]-2-indolinone (CS7135) 
     3-[(Pyrazol-3-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (mmmmm) Synthesis of 3-[(Pyrazol-4-yl)methylene]-2-indolinone (CS7136) 
     3-[(Pyrazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (nnnnn) Synthesis of 3-[(Isoxazol-3-yl)methylene]-2-indolinone (CS7137) 
     3-[(Isoxazol-3-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ooooo) Synthesis of 3-[(Isoxazol-4-yl)methylene]-2-indolinone (CS7138) 
     3-((Isoxazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ppppp) Synthesis of 3-[(Isaoxazol-5-yl)methylene]-2-indolinone (CS7139) 
     3-[(Isoxazol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (qqqqq) Synthesis of 3-[(Isothiazol-3-yl)methylene]-2-indolinone (CS7140) 
     3-[(Isothiazol-3-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (rrrrr) Synthesis of 3-[(Isothiazol-4-yl)methylene]-2-indolinone (CS7141) 
     3-[(Isothiazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (sssss) Synthesis of 3-[(Isothiazol-5-yl)methylene]-2-indolinone (CS7142) 
     3-[(isothiazol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (ttttt) Synthesis of 3-[(1,2,3-Triazol-4-yl)methylene]-2-indolinone (CS7143) 
     3-[(1,2,3-Triazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (uuuuu) Synthesis of 3-[(1,3,4-Thiadiazol-2-yl)methylene]-2-indolinone (CS7144) 
     3-[(1,3,4-Thiadiazol-2-yl)methylene]-2-indolinone is synthesized according to Method A. 
       
     (vvvvv) Synthesis of 3-[(5-Phenyl-1,2,4-oxadiazol-3-yl)methylene]-2-indolinone (CS7145) 
     3-[(5-Phenyl-1,2,4-oxadiazol-3-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (wwwww) Synthesis of 3-[(3-Phenyl-1,2,4-oxadiazol-5-yl)methylene]-2-indolinone (CS7146) 
     3-[(3-Phenyl-1,2,4-oxadiazol-5-yl)methylene]-2-indolinone is synthesized according to Method A. 
     (xxxxx) synthesis of 3-[(3-Phenyl-1,2,5-oxadiazol-4-yl)methylene]-2-indolinone (CS7147) 3-[(3-Phenyl-1,2,5-oxadiazol-4-yl)methylene]-2-indolinone is synthesized according to Method A. 
     6. EXAMPLES 
     In Vitro RTK Assays 
     The following in vitro assays may be used to determine the level of activity and effect of the different compounds of the present invention on one or more of the RTKs. Similar assays can be designed along the same lines for any tyrosine kinase using techniques well known in the art. 
     (a) Enzyme Linked Immunosorbent Assay (ELISA) 
     Enzyme linked immunosorbent assays (ELISA) may be used to detect and measure the presence of tyrosine kinase activity. The ELISA may be conducted according to known protocols which are described in, for example, Voller, et al., 1980, “Enzyme-Linked Immunosorbent Assay,” In:  Manual of Clinical Immunology , 2d ed., edited by Rose and Friedman, pp 359-371 Am. Soc. Of Microbiology, Washington, D.C. 
     The disclosed protocol may be adapted for determining activity with respect to a specific RTK. For example, the preferred protocols for conducting the ELISA experiments for specific RTKs is provided below. Adaptation of these protocols for determining a compound&#39;s activity for other members of the RTK family, as well as non-receptor tyrosine kinases, are within the scope of those in the art. 
     (i) FLK-1 ELISA 
     An ELISA assay was conducted to measure the kinase activity of the FLK-1 receptor and more specifically, the inhibition or activation of protein tyrosine kinase activity on the FLK-1 receptor. Specifically, the following assay was conducted to measure kinase activity of the FLK-1 receptor in FLK-1/NIH3T3 cells. 
     Materials and Methods. 
     Materials. The following reagents and supplies were used:
         a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);   b. Cappel goat anti-rabbit IgG (catalog no. 55641);.   c. PBS (Gibco Catalog No. 450-1300EB);   d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and 0.1% Tween-20);   e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4° C.);   f. HNTG buffer (20 mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% Triton X-100, and 10% glycerol);   g. EDTA (0.5 M (pH 7.0) as a 100× stock);   h. Sodium ortho vanadate (0.5 M as a 100× stock);   i. Sodium pyro phosphate (0.2M as a 100× stock);   j. NUNC 96 well V bottom polypropylene plates (Applied Scientific Catalog No. AS-72092);   k. NIH3T3 C7 #3 Cells (FLK-1 expressing cells);   l. DMEM with 1× high glucose L Glutamine (catalog No. 11965-050);   m. FBS, Gibco (catalog no. 16000-028);   n. L-glutamine, Gibco (catalog no. 25030-016);   o. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept as 1 μg/100 μl stock in Milli-Q dH 2 O and stored at −20° C.;   p. Affinity purified anti-FLK-1 antiserum, Enzymology Lab, Sugen, Inc.;   q. UB40 monoclonal antibody specific for phosphotyrosine, Enzymology Lab, Sugen, Inc. (see, Fendley, et al., 1990 , Cancer Research  50:1550-1558);   r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);   s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution (100 mM citric acid (anhydrous), 250 mM Na 2 HPO 4 , (pH 4.0), 0.5 mg/ml ABTS (Sigma catalog no. A-1888)), solution should be stored in dark at 4° C. until ready for use;   t. H 2 O 2  (30% solution) (Fisher catalog no. H325);   u. ABTS/H 2 O 2 , (15 ml ABTS solution, 2 μl H 2 O 2 ) prepared 5 minutes before use and left at room temperature;   v. 0.2 M HCl stock in H 2 O;       

     w. dimethylsulfoxide (100%) (Sigma Catalog No. D-8418); and
         y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).       

     Protocol. The following protocol was used for conducting the assay: 
     1. Coat Corning 96-well elisa plates with 1.0 μg per well Cappel Anti-rabbit IgG antibody in 0.1M Na 2 CO 3  pH 9.6. Bring final volume to 150 μl per well. Coat plates overnight at 4° C. Plates can be kept up to two weeks when stored at 4° C. 
     2. Grow cells in Growth media(DMEM, supplemental with 2.0 mM L-Glutamine, 10% FBS) in suitable culture dishes until confluent at 37° C., 5% CO 2 . 
     3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene 96-well roundbottom cell plates, 25.000 cells/well in 200 μl of growth media. 
     4. Grow cells at least one day at 37° C., 5% CO 2 . 
     5. Wash cells with D-PBS 1×. 
     6. Add 200 μl/well of starvation media (DMEM, 2.0 mM l-Glutamine, 0.1% PBS). Incubate overnight at 37° C., 5% CO 2 . 
     7. Dilute Compounds/Extracts 1:20 in polypropylene 96 well plates using starvation media. Dilute dimethylsulfoxide 1:20 for use in control wells. 
     8. Remove starvation media from 96 well cell culture plates and add 162 μl of fresh starvation media to each well. 
     9. Add 18 μl of 1:20 diluted Compound/Extract dilution (from step 7) to each well plus the 1:20 dimethylsulfoxide dilution to the control wells (+/−VEGF), for a final dilution of 1:200 after cell stimulation. Final dimethylsulfoxide is 0.5%. Incubate the plate at 37° C. 5% CO 2  for two hours. 
     10. Remove unbound antibody from ELISA plates by inverting plate to remove liquid. Wash 3 times with TBSW+0.5% ethanolamine, pH 7.0. Pat the plate on a paper towel to remove excess liquid and bubbles. 
     11. Block plates with TBSW+0.5% Ethanolamine, pH 7.0, 150 μl per well. Incubate plate thirty minutes while shaking on a microtiter plate shaker. 
     12. Wash plate 3 times as described in step 10. 
     13. Add 0.5 μg/well affinity purified anti-FLU-1 polyclonal rabbit antiserum. Bring final volume to 150 μl/well with TBSW+0.5% ethanolamine pH 7.0. Incubate plate for thirty minutes while shaking. 
     14. Add 180 μl starvation medium to the cells and stimulate cells with 20 μl/well 10.0 mM sodium ortho vanadate and 500 ng/ml VEGF (resulting in a final concentration of 1.0 mM sodium ortho vanadate and 50 ng/ml VEGF per well) for eight minutes at 37° C., 5% CO 2 . Negative control wells receive only starvation medium. 
     15. After eight minutes, media should be removed from the cells and washed one time with 200 μl/well PBS. 
     16. Lyse cells in 50 μl/well HNTG while shaking at room temperature for five minutes. HNTG formulation includes sodium ortho vanadate, sodium pyro phosphate and EDTA. 
     17. Wash ELISA plate three times as described in step 10. 
     18. Transfer cell lysates from the cell plate to elisa plate and incubate while shaking for two hours. To transfer cell lysate pipette up and down while scrapping the wells. 
     19. Wash plate three times as described in step 10. 
     20. Incubate ELISA plate with 0.02 μg/well UB40 in TBSW+05% ethanolamine. Bring final volume to 150 μl/well. Incubate while shaking for 30 minutes. 
     21. Wash plate three times as described in step 10. 
     22. Incubate ELISA plate with 1:10,000 diluted EIA grade goat anti-mouse IgG conjugated horseradish peroxidase in TBSW+0.5% ethanolamine, pH 7.0. Bring final volume to 150 μl/well. Incubate while shaking for thirty minutes. 
     23. Wash plate as described in step 10. 
     24. Add 100 μl of ABTS/H 2 O 2  solution to well. Incubate ten minutes while shaking. 
     25. Add 100 μl of 0.2 M HCl for 0.1 M HCl final to stop the color development reaction. Shake 1 minute at room temperature. Remove bubbles with slow stream of air and read the ELISA plate in an ELISA plate reader at 410 nm. 
     (ii) HER-2 ELISA 
     Assay 1: EGF Receptor-HER2 Chimeric Receptor Assay In Whole Cells. HER2 kinase activity in is whole EGFR-NIH3T3 cells was measured as described below: 
     Materials and Reagents. The following materials and reagents were used to conduct the assay:
         a. EGF: stock concentrations 16.5 ILM; EGF 201, TOYOBO, Co., Ltd. Japan.   b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain).   c. Anti-phosphotyrosine antibody (anti-Ptyr) (polyclonal) (see, Fendley, et al., supra).   d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, Calif.   e. TBST buffer:       

     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Tris-HCl, pH 7.2 
                  50 mM 
               
               
                   
                 NaCl 
                 150 mM 
               
               
                   
                 Triton X-100 
                 0.1 
               
               
                   
                   
               
            
           
         
       
         
         
           
             f. HNTG 5X stock: 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 HEPES 
                  0.1 M 
               
               
                   
                 NaCl 
                 0.75 M 
               
               
                   
                 Glycerol 
                 50% 
               
               
                   
                 Triton X-100 
                 1.0% 
               
               
                   
                   
               
            
           
         
       
         
         
           
             g. ABTS stock: 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Citric Acid 
                 100 mM 
               
               
                   
                 Na 2 HPO 4   
                 250 mM 
               
               
                   
                 HCl, conc. 
                  0.5 pM 
               
               
                   
                 ABTS* 
                  0.5 mg/ml 
               
               
                   
                   
               
               
                   
                 *(2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)). Keep solution in dark at 4° C. until use.  
               
            
           
         
       
         
         
           
             h. Stock reagents of:
           EDTA 100 mM pH 7.0   Na 3 VO 4  0.5 M   Na 4 (P 2 O 7 ) 0.2 M   
         
           
         
       
    
     Procedure. The following protocol was used: 
     A. Pre-coat ELISA Plate 
     1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 g per well in PBS, 100 μl final volume/well, and store overnight at 4° C. Coated plates are good for up to 10 days when stored at 4° C. 
     2. On day of use, remove coating buffer and replace with 100 μl blocking buffer (5% Carnation Instant Non-Fat Dry Milk in PBS). Incubate the plate, shaking, at room temperature (about 23° C. to 25° C.) for 30 minutes. Just prior to use, remove blocking buffer and wash plate 4 times with TBST buffer. 
     B. Seeding Cells 
     1. An NIH3T3 cell line overexpressing a chimeric receptor containing the EGFR extracellular domain and extracellular HER2 kinase domain can be used for this assay. 
     2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding 10% fetal bovine serum. Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1500 rpm, at room temperature for 5 minutes. 
     3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count the cells using trypan blue. Viability above 90% is acceptable. Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 μl per well, in a 96 well microtiter plate. Incubate seeded cells in 5% CO 2  at 37° C. for about 40 hours. 
     C. Assay Procedures 
     1. Check seeded cells for contamination using an inverted microscope. Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer 5 l to a TBST well for a final drug dilution of 1:200 and a final DMSO concentration of 1%. Control wells receive DMSO alone. Incubate in 5% CO 2  at 37° C. for two hours. 
     2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10 μl dilute EGF (1:12 dilution), 100 nM final concentration is attained. 
     3. Prepare fresh HNTG* sufficient for 100 μl per well; and place on ice. 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 HNTG stock 
                 2.0 ml 
               
               
                   
                 milli-Q H 2 O 
                 7.3 ml 
               
               
                   
                 EDTA, 100 mM, pH 7.0 
                 0.5 ml 
               
               
                   
                 Na 3 VO 4 , 0.5 M 
                 0.1 ml 
               
               
                   
                 Na 4 (P 2 O 7 ), 0.2 M 
                 0.1 ml 
               
               
                   
                   
               
            
           
         
       
     
     4. After 120 minutes incubation with drug, add prepared SGF ligand to cells, 10 μl per well, to a final concentration of 100 nM. Control wells receive DMEM alone. Incubate, shaking, at room temperature, for 5 minutes. 
     5. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG* to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above. 
     6. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate. Incubate shaking at room temperature for one hour. 
     7. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000 dilution in TBST). 
     8. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transfer the freshly diluted TAGO anti-rabbit IgG antibody to the ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST). 
     9. Remove TAGO detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS/H 2 O 2  solution to ELISA plate, 100 μl per well. Incubate shaking at room temperature for 20 minutes. (ABTS/H 2 O 2  solution: 1.0 μl 30% H 2 O 2  in 10 ml ABTS stock). 
     10. Stop reaction by adding 50 μl 5N H 2 SO 4  (optional), and determine O.D. at 410 nm. 
     11. The maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls. 
     Assay 2: HER-2-BT474 ELISA. A second assay may be conducted to measure whole cell HER2 activity. Such assay may be conducted as follows: 
     Materials And Reagents. The following materials and reagents were used:
         a. BT-474 (ATCC HBT20), a human breast tumor cell line which expresses high levels of HER2 kinase.   b. Growth media comprising RPMI+10% FBS+GMS-G (Gibco supplement)+glutamine for use in growing BT-474 in an incubator with 5% CO 2  at 37° C.   c. A monoclonal anti-HER2 antibody.   d. D-PBS:       

     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 KH 2 HPO 4   
                 0.20 g/l 10 (GIBCO, 310-4190AJ) 
               
               
                   
                 K 2 HPO 4   
                 2.16 g/l 
               
               
                   
                 KCl 
                 0.20 g/l 
               
               
                   
                 NaCl 
                 8.00 g/l (pH 7.2) 
               
               
                   
                   
               
            
           
         
       
         
         
           
             e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk). 
             f. TBST buffer: 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Tris-HCl 
                  50 mM 
               
               
                   
                 NaCl 
                 150 mM (pH 7.2, HCl 10 N) 
               
               
                   
                 Triton X-100 
                 0.1% 
               
               
                   
                   
               
            
           
         
       
         
         
           
              wherein stock solution of TES (10×) is prepared, and Triton X-100 is added to the buffer during dilution. 
             g. HNTG buffer (5×): 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 HEPES 
                  0.1 M 
               
               
                   
                 NaCl 
                 750 mM (pH 7.2 (HCl, 1 N) 
               
               
                   
                 Glycerol 
                 50% 
               
               
                   
                 Triton X-100 
                 1.0% 
               
               
                   
                   
               
            
           
         
       
         
         
           
              Stock solution (5×) is prepared and kept in 4° C. 
             h. EDTA-HCl: 0.5 M pH 7.0 (10 N HCl) as 500× stock. 
             i. Na 3 VO 4 : 0.5 M as 100× stock is kept at −80° C. as aliquots. 
             j. Na 4 (P 2 O 7 ): 0.2 M as 100× stock. 
             k. Polyclonal antiserum anti-phosphotyrosine. 
             l. Goat anti-rabbit IgG, horseradish peroxidase (POD) conjugate (detection antibody), Tago (Cat. No. 4520; Lot No. 1802): Tago, Inc., Burlingame, Calif. 
             m. ABTS solution: 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Citric acid 
                 100 mM 
               
               
                   
                 Na 2 HPO 4   
                 250 mM (pH 4.0, 1 N HCl) 
               
               
                   
                 ABTS 
                  0.5 mg/ml 
               
               
                   
                   
               
            
           
         
       
         
         
           
              wherein ABTS is 2.2′-azinobis(3-ethylbenzthiazoline sulfonic acid). For this assay, the ABTS solution should be kept in the dark at 4° C. The solution should be discarded when it turns green. 
             n. Hydrogen peroxide: 30% solution is kept in dark and 4° C. 
           
         
       
    
     Procedure. All the following steps are at room temperature and aseptically, unless stated otherwise. All ELISA plate washing is by rinsing with distilled water three times and once with TBST. 
     A. Cell Seeding 
     1. Grow BT474 cells in tissue culture dishes (Corning 25020-100) to 80-90% confluence and collect using. Trypsin-EDTA (0.25%, GIBCO). 
     2. Resuspend the cells in fresh medium and transfer to 96-well tissue culture plates (Corning, 25806-96) at about 25,000-50,000 cells/well (1.00 μl/well) Incubate the cells in 5% CO 2  at 37° C. overnight. 
     B. ELISA Plate Coating and Blocking 
     1. Coat the ELISA plate (Corning 25805-96) with anti HER2 antibody at 0.5 μg/well in 150 μl PBS overnight at 4° C., and seal with parafilm. The antibody coated plates can be used up to 2 weeks, when stored at 4° C. 
     2. On the day of use, remove the coating solution, replace with 200 μl of Blocking Buffer, shake the plate, and then remove the blocking buffer and wash the plate just before adding lysate. 
     C. Assay Procedures 
     1. TBST the drugs in serum-free condition. Before adding drugs, the old media is replaced with serum-free RPMI (90 μl/well) 
     2. Dilute drug stock (in 100% DMSO) 1:10 with RPMI, and transfer 10 μl/well of this solution to the cells to achieve a final drug DMSO concentration at 1%. Incubate the cells in 5% CO 2  at 37° C. 
     3. Prepare fresh cell lysis buffer (HNTG*) 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 5xHNTG 
                 2 
                 ml 
               
               
                   
                 EDTA 
                 0.2 
                 ml 
               
               
                   
                 Na 3 VO 4   
                 0.1 
                 ml 
               
               
                   
                 Na 4 P 2 O 7   
                 0.1 
                 ml 
               
               
                   
                 H 2 O 
                 7.3 
                 ml 
               
               
                   
                   
               
            
           
         
       
     
     4. After drug preincubation for two hours remove all the solution from the plate, transfer HNTG* (100 μl/well) to the cells, and shake for 10 minutes. 
     5. Use a 12-channel pipette to scrape the cells from the plate, and homogenize the lysate by repeat aspiration and dispensing. Transfer all the lysate to the ELISA plate and shake for 1 hour. 
     6. Remove the lysate, wash the plate, add anti-pTyr (1:3,000 with TBST) 100 μl/well, and shake for 30 minutes. 
     7. Remove anti-pTyr, wash the plate, add goat anti-rabbit IgG conjugated antibody (1:5,000 with TBST) 100 μl/well, and shake for 30 minutes. 
     8. Remove anti-rabbit IgG antibody, wash the plate, and add fresh ABTS/H 2 O 2  (1.2 μl H 2 O 2  to 10 ml ABTS) 100 l/well to the plate to start color development, which usually takes 20 minutes. 
     9. Measure OD 410 nM, Dynatec MR5000. 
     (iii) PDGF-R ELISA 
     All cell culture media, glutamine, and fetal bovine serum were purchased from Gibco Life Technologies (Grand Island, N.Y.) unless otherwise specified. All cells were grown in a humid atmosphere of 90-95% air and 5-10%; CO 2  at 37° C. All cell lines were routinely subcultured twice a week and were negative for mycoplasma as determined by the Mycotect method (Gibco). 
     For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU) were grown to 80-90% confluency in growth medium (MEM with 10% FBS, NEAA, 1 mM NaPyr and 2 mM GLN) and seeded in 96-well tissue culture plates in 0.5% serum at 25,000 to 30,000 cells per well. After overnight incubation in 0.5% serum-containing medium, cells were changed to serum-free medium and treated with test compound for 2 hr in a 5% CO 2 , 37° C. incubator. Cells were then stimulated with ligand for 5-10 minute followed by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM Na 3 VO 4 , 0.2% Triton X-100, and 2 mM NaPyr). Cell lysates (0.5 mg/well in PBS) were transferred to ELISA plates previously coated with receptor-specific antibody and which had been blocked with 5% milk in TBST (50 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.1% Triton X-100) at room temperature for 30 min. Lysates were incubated with shaking for 1 hour at room temperature. The plates were washed with TBST four times and then incubated with polyclonal anti-phosphotyrosine antibody at room temperature for 30 minutes. Excess anti-phosphotyrosine antibody was removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG antibody was added to the ELISA plate for 30 min at room temperature followed by rinsing with TBST four more times. ABTS (100 mM citric acid, 250 mM Na 2 HPO 4  and 0.5 mg/mL 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H 2 O 2  (1.2 mL 30% H 2 O 2  to 10 ml ABTS) was added to the ELISA plates to start color development. Absorbance at 410 nm with a reference wavelength of 630 nm was recorded about 15 to 30 min after ABTS addition. 
     (iv) IGF-I ELISA 
     The following protocol may be used to measure phosphotyrosine level on IGF-I receptor, which indicates IGF-I receptor tyrosine kinase activity. 
     Materials And Reagents. The following materials and reagents were used:
         a. The cell line used in this assay is 3T3/IGF-1R, a cell line which overexpresses IGF-1 receptor.   b. NIH3T3/IGF-1R is grown in an incubator with 5% CO 2  at. 37° C. The growth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.   c. Anti-IGF-1R antibody named 17-69 is used. Antibodies are purified by the Enzymology Lab, SUGEN, Inc.   d. D-PBS:       

     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 KH 2 PO 4   
                 0.20 g/l 
               
               
                   
                 K 2 HPO 4   
                 2.16 g/l 
               
               
                   
                 KCl 
                 0.20 g/l 
               
               
                   
                 NaCl 
                 8.00 g/l (pH 7.2) 
               
               
                   
                   
               
            
           
         
       
         
         
           
             e. Blocking Buffer: TBST plus 5% Milk (Carnation Instant Non-Fat Dry Milk). 
             f. TBST buffer: 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Tris-HCl 
                  50 mM 
               
               
                   
                 NaCl 
                 150 mM (pH 7.2/HCl 10 N) 
               
               
                   
                 Triton X-100 
                 0.1% 
               
               
                   
                   
               
            
           
         
       
         
         
           
              Stock solution of TBS (10×) is prepared, and Triton X-100 is added to the buffer during dilution. 
             g. HNTG buffer: 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 HEPES 
                  20 mM 
               
               
                   
                 NaCl 
                 150 mM (pH 7.2/HCl 1 N) 
               
               
                   
                 Glycerol 
                 10% 
               
               
                   
                 Triton X-100 
                 0.2% 
               
               
                   
                   
               
            
           
         
       
         
         
           
              Stock solution (5×) is prepared and kept at 4° C. 
             h. EDTA/HCl: 0.5 M pH 7.0 (NaOH) as 100× stock. 
             i. Na 3 VO 4 : 0.5 M as 100× stock and aliquots are kept in −80° C. 
             j. Na 4 P 2 O 7 : 0.2 M as 100× stock. 
             k. Insulin-like growth factor-1 from Promega (Cat# G5111). 
             l. Polyclonal antiserum anti-phosphotyrosine: rabbit sera generated by Enzymology Lab., SUGEN Inc. 
             m. Goat anti-rabbit IgG, POD conjugate (detection antibody), Tago (Cat. No. 4520, Lot No. 1802): Tago, Inc., Burlingame, Calif. 
             n. ABTS (2.2′-azinobis(3-ethylbenzthiazolinesulfonic acid)) solution: 
           
         
       
    
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Citric acid 
                 100 
                 mM 
               
               
                   
                 Na 2 HPO 4   
                 250 
                 mM (pH 4.0/1 N HCl) 
               
               
                   
                 ABTS 
                 0.5 
                 mg/ml 
               
               
                   
                   
               
            
           
         
       
         
         
           
              ABTS solution should be kept in dark and 4° C. The solution should be discarded when it turns green. 
           
         
       
    
     o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4° C. 
     Procedure. All the following steps are conducted at room temperature unless it is specifically indicated. All ELISA plate washings are performed by rinsing the plate with tap water three times, followed by one TBST rinse. Pat plate dry with paper towels. 
     A. Cell Seeding: 
     1. The cells, grown in tissue culture dish (Corning 25020-100) to 80-90% confluence, are harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100, GIBCO). 
     2. Resuspend the cells in fresh DMEM+10% FBS+2 mM L-Glutamine, and transfer to 96-well tissue culture plate (Corning, 25806-96) at 20,000 cells/well (100 μl/well). Incubate for 1 day then replace medium to serum-free medium (90/μl) and incubate in 5% CO 2  and 37° C. overnight. 
     B. ELISA Plate Coating and Blocking: 
     1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1R Antibody at 0.5 μg/well in 100 μl PBS at least 2 hours. 
     2. Remove the coating solution, and replace with 100 μl Blocking Buffer, and shake for 30 minutes. Remove the blocking buffer and wash the plate just before adding lysate. 
     C. Assay Procedures: 
     1. The drugs are tested in serum-free condition. 
     2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-well poly-propylene plate, and transfer 10 μl/well of this solution to the cells to achieve final drug dilution 1:100, and final DMSO concentration of 1.0%. Incubate the cells in 5% CO 2  at 37° C. for 2 hours. 
     3. Prepare fresh cell lysis buffer (HNTG*) 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 HNTG 
                 2 
                 ml 
               
               
                   
                 EDTA 
                 0.1 
                 ml 
               
               
                   
                 Na 3 VO 4   
                 0.1 
                 ml 
               
               
                   
                 Na 4 (P 2 O 7 ) 
                 0.1 
                 ml 
               
               
                   
                 H 2 O 
                 7.3 
                 ml 
               
               
                   
                   
               
            
           
         
       
     
     4. After drug incubation for two hours, transfer 10 μl/well of 200 nM IGF-1 Ligand in PBS to the cells (Final Conc.=20 nM), and incubate at 5% CO 2  at 37° C. for 10 minutes. 
     5. Remove media and add 100 μl/well HNTG* and shake for 10 minutes. Look at cells under microscope to see if they are adequately lysed. 
     6. Use a 12-channel pipette to scrape the cells from the plate, and homogenize the lysate by repeat aspiration and dispense. Transfer all the lysate to the antibody coated ELISA plate, and shake for 1 hour. 
     7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 with TBST) 100 μl/well, and shake for 30 minutes. 
     8. Remove anti-pTyr, wash the plate, transfer Tago (1:3,000 with TBST) 100 μl/well, and shake for 30 minutes. 
     9. Remove detection antibody, wash the plate, and transfer fresh ABTS/H2O2 (1.2 μl H 2 O 2  to 10 ml ABTS) 100 μl/well to the plate to start color development. 
     10. Measure OD in Dynatec MR5000, which is connected to Ingres. 
     (v) EGF Receptor ELISA 
     EGF Receptor kinase activity (EGFR-NIH3T3 assay) in whole cells was measured as described below: 
     Materials and Reagents. The following materials and reagents were used
         a. EGF Ligand: stock concentration=16.5 μM; EGF 201, TOYOSO, Co., Ltd. Japan.   b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular domain).   c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).   d. Detection antibody: Goat anti-rabbit lgG horse radish peroxidase conjugate, TAGO, Inc., Burlingame, Calif.   e. TBST buffer:       

     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Tris-HCl, pH 7 
                 50 
                 mM 
               
               
                   
                 NaCl 
                 150 
                 mM 
               
               
                   
                 Triton X-100 
                 0.1 
               
               
                   
                   
               
            
           
         
       
         
         
           
             f. HNTG 5X stock: 
           
         
       
    
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 HEPES 
                  0.1 M 
               
               
                   
                 NaCl 
                  0.75 M 
               
               
                   
                 Glycerol 
                 50 
               
               
                   
                 Triton X-100 
                  1.0% 
               
               
                   
                   
               
            
           
         
       
         
         
           
             g. ABTS stock: 
           
         
       
    
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Citric Acid 
                 100 
                 mM 
               
               
                   
                 Na 2 HPO 4   
                 250 
                 mM 
               
               
                   
                 HCl, conc. 
                 4.0 
                 pH 
               
               
                   
                 ABTS* 
                 0.5 
                 mg/ml 
               
               
                   
                   
               
            
           
         
       
         
         
           
              Keep solution in dark at 4° C. until used. 
             h. Stock reagents of:
           EDTA 100 mM pH 7.0   Na 3 VO 4  0.5 M   Na 4 (P 2 O 7 ) 0.2 M   
         
           
         
       
    
     Procedure. The following protocol was used: 
     A. Pre-coat ELISA Plate 
     1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101 antibody at 0.5 μg per well in PBS, 150 μl final volume/well, and store overnight at 4° C. Coated plates are good for up to 10 days when stored at 4° C. 
     2. On day of use, remove coating buffer and replace with blocking buffer (5% Carnation Instant NonFat Dry Milk in PBS). Incubate the plate, shaking, at room temperature (about 23° C. to 25° C.) for 30 minutes. Just prior to use, remove blocking buffer and wash plate 4 times with TBST buffer. 
     B. Seeding Cells 
     1. NIH 3T3/C7 cell line (Honegger, et al., Cell 51:199-209, 1987) can be use for this assay. 
     2. Choose dishes having 80-90% confluence for the experiment. Trypsinize cells and stop reaction by adding. 10% CS DMEM medium. Suspend cells in DMEM medium (10% CS DMEM medium) and centrifuge once at 1000 rpm, and once at room temperature for 5 minutes. 
     3. Resuspend cells in seeding medium (DMEM, 0.5% bovine serum), and count the cells using trypan blue. Viability above 90% is acceptable. Seed cells in DMEM medium (0.5% bovine serum) at a density of 10,000 cells per well, 100 μl per well, in a 96 well microtiter plate. Incubate seeded cells in 5% CO 2  at 37° C. for about 40 hours. 
     C. Assay Procedures. 
     1. Check seeded cells for contamination using an inverted microscope. Dilute drug stock (10 mg/ml in DMSO) 1:10 in DMEM medium, then transfer 5 μl to a test well for a final drug dilution of 1:200 and a final DMSO concentration of 1%. Control wells receive DMSO alone. Incubate in 5% CO 2  at 37° C. for one hour. 
     2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10 μl dilute EGF (1:12 dilution), 25 nM final concentration is attained. 
     3. Prepare fresh 10 ml HNTG* sufficient for 100 μl per well wherein HNTG* comprises: HNTG stock (2.0 ml), milli-Q H 2 O (7.3 ml), EDTA, 100 mM, pH 7.0 (0.5 ml), Na 3 VO 4  0.5 M (0.1 ml) and Na 4  (P 2 O 7 ), 0.2 M (0.1 ml). 
     4. Place on ice. 
     5. After two hours incubation with drug, add prepared EGF ligand to cells, 10 μl per well, to yield a final concentration of 25 nM. Control wells receive. DMEM alone. Incubate, shaking, at room temperature, for 5 minutes. 
     6. Remove drug, EGF, and DMEM. Wash cells twice with PBS. Transfer HNTG* to cells, 100 μl per well. Place on ice for 5 minutes. Meanwhile, remove blocking buffer from other ELISA plate and wash with TBST as described above. 
     7. With a pipette tip securely fitted to a micropipettor, scrape cells from plate and homogenize cell material by repeatedly aspirating and dispensing the HNTG* lysis buffer. Transfer lysate to a coated, blocked, and washed ELISA plate. Incubate shaking at room temperature for one hour. 
     8. Remove lysate and wash 4 times with TBST. Transfer freshly diluted anti-Ptyr antibody to ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes in the presence of the anti-Ptyr antiserum (1:3000 dilution in TBST). 
     9. Remove the anti-Ptyr antibody and wash 4 times with TBST. Transfer the freshly diluted TAGO 30 anti-rabbit IgG antibody to the ELISA plate at 100 μl per well. Incubate shaking at room temperature for 30 minutes (anti-rabbit IgG antibody: 1:3000 dilution in TBST). 
     10. Remove detection antibody and wash 4 times with TBST. Transfer freshly prepared ABTS/H 2 O 2  solution to ELISA plate, 100 μl per well. Incubate at room temperature for 20 minutes. ABTS/H 2 O 2  solution: 1.2 μl 30% H 2 O 2 in 10 ml ABTS stock. 
     11. Stop reaction by adding 50 μl 5N H 2 SO 4  (optional), and determine O.D. at 410 nm. 
     12. The maximal phosphotyrosine signal is determined by subtracting the value of the negative controls from the positive controls. The percent inhibition of phosphotyrosine content for extract-containing wells is then calculated, after subtraction of the negative controls. 
     (vi) Cellular Insulin Receptor ELISA 
     The following protocol was used to determine whether the compounds of the present invention possessed insulin receptor tyrosine kinase activity. 
     Materials And Reagents. The following materials and reagents were used to measure phophotyrosine levels on the insulin receptor (indicating insulin receptor tyrosine kinase activity):
         1. The preferred cell line was an NIH3T3 cell line (ATCC No. 1658) which overexpresses Insulin Receptor (H25 cells);   2. H25 cells are grown in an incubator with 5% CO 2  at 37° C. The growth media is DMEM+10% FBS (heat inactivated)+2mm L-Glutamine;   3. For ELISA plate coating, the monoclonal anti-IR antibody named BBE is used. Said antibodies was purified by the Enzymology Lab, SUGEN, Inc.;       

     4. D-PBS, comprising: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 KH 2 PO 4   
                 0.20 
                 g/l (GIBCO, 310-4190AJ) 
               
               
                   
                 K 2 HPO 4   
                 2.16 
                 g/l 
               
               
                   
                 KCl 
                 0.20 
                 g/l 
               
               
                   
                 NaCl 
                 8.00 
                 g/l (pH 7.2); 
               
               
                   
                   
               
            
           
         
       
     
     5. Blocking Buffer: TBST plus 5% Milk (Carnation Instant N6n-Fat Dry Milk); 
     6. TBST buffer, comprising: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Tris-HCl 
                 50 
                 mM 
               
               
                   
                 NaCl 
                 150 
                 mM pH 7.2 (HCI, 1N) 
               
               
                   
                 Triton X-100 
                 0.1% 
               
               
                   
                   
               
            
           
         
       
         
         
           
              Note: Stock solution of TBS (10×) is prepared, and Triton X-100 is added to the buffer during dilution; 
           
         
       
    
     7. HNTG buffer, comprising: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 HEPES 
                 20 
                 mM 
               
               
                   
                 NaCl 
                 150 
                 mM pH 7.2 (HCI, 1N) 
               
               
                   
                 Glycerol 
                 10% 
               
               
                   
                 Triton X-100 
                 0.2% 
               
               
                   
                   
               
            
           
         
       
         
         
           
              Note: Stock solution (5×) is prepared and kept at 4° C.; 
             8. EDTA.HCI: 0.5 M pH 7.0 (NaOH) as 100× stock; 
             9. Na 3 VO 4 : 0.5 M as 100× stock and aliquots are kept in −80° C.; 
             10. Na 4 P 2 O 7 : 0.2 M as 100× stock; 
             11. Insulin from GIBCO BRL (Cat# 18125039); 
             12. Polyclonal antiserum Anti-phosphotyrosine: rabbit sera generated by Enzymology Lab., SUGEN Inc.; 
             13. Detection antibody, preferably goat anti-rabbit IgG, POD conjuate, Tago (Cat. No. 4520: Lot No. 1802): Tago, Inc., Burlingame, Calif.; 
           
         
       
    
     14. ABTS solution, comprising: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Citric acid 
                 100 
                 mM 
               
               
                   
                 Na 2 HPO 4   
                 250 
                 mM pH 4.0 (1N HCI) 
               
               
                   
                 ABTS 
                 0.5 
                 mg/ml 
               
               
                   
                   
               
            
           
         
       
         
         
           
              wherein ABTS is 2,2′-azinobis (3-ethylbenathiazoline sulfonic acid) and stored in the dark at 4° C. and discarded when it turns green; 
             15. Hydrogen Peroxide: 30% solution is kept in the dark and at 40° C. 
           
         
       
    
     Protocol. All the following steps are conducted at room temperature unless it is specifically indicated. All ELISA plate washings are performed by rinsing the plate with tap water three times, followed by one TBST rinse. All plates were tapped dry with paper towels prior to use. 
     A. Cell Seeding: 
     1. The cells were grown in tissue culture dish (10 cm, Corning 25020-100) to 80-90% confluence and harvested with Trypsin-EDTA (0.25%, 0.5 ml/D-100, GIBCO); 
     2. Resuspend the cells in fresh DMEM+10; FBS+2 mM L-Glutamine, and transfer to 96-well tissue culture plate (Corning, 25806-96) at 20,000 cells/well (100 μl/well). The cells are then incubated for 1 day. Following such incubation, 0.0% serum medium (90/μl) replaces the old media and the cells incubate in 5% CO 2  and 37° C. overnight. 
     B. Plate Coating and Blocking: 
     1. Coat the ELISA plate (Corning 25805-96) with Anti-IR Antibody at 0.5 μg/well in 100 μl PBS at least 2 hours. 
     2. Remove the coating solution, and replace with 100 u; blocking Buffer, and shake for 30 minutes. Remove the blocking buffer and wash the plate just before adding lysate. 
     C. Assay Procedures 
     1. The drugs are tested in serum-free condition. 
     2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM in 96-well poly-propylene plate, and transfer 10 μl/well of this solution to the cells to achieve final drug dilution 1:100, and final DMSO concentration of 1.0%. Incubate the cells in 5% CO 2  at 37° C. for 2 hours. 
     3. Prepare fresh cells lysis buffer (HNTG*) 
     
       
         
           
               
             
               
                   
               
               
                 Prepare fresh cells lysis buffer (HNTG*) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 HNTG (5x) 
                 2 
                 ml 
               
               
                   
                 EDTA 
                 0.1 
                 ml 
               
               
                   
                 Na 3 VO 4   
                 0.1 
                 ml 
               
               
                   
                 Na 4 P 2 O 7   
                 0.1 
                 ml 
               
               
                   
                 H 2 O 
                 7.3 
                 ml 
               
               
                   
                 HNTG* 
                 10 
                 ml 
               
               
                   
                   
               
            
           
         
       
     
     4. After drug incubation for two hours, transfer 10 μl/well of 1 μM insulin in PBS to the cells (Final concentration=100 nM), and incubate at 5% CO 2  at 37° C. for 10 minutes. 
     5. Remove media and add 100 μl/well HNTG* and shake for 10 minutes. Look at cells under microscope to see if they are adequately lysed. 
     6. Using a 12-channel pipette, scrape the cells from the plate, and homogenize the lysate by repeat aspiration and dispense. Transfer all the lysate to the antibody coated ELISA plate, and shake for 1 hour. 
     7. Remove the lysate, wash the plate, transfer anti-pTyr (1:3,000 with TBST) 100 μl/well, and shake for 30 minutes. 
     8. Remove anti-pTyr, wash the plate, transfer Tago (1:3,000 with TEST) 100 μl/well, and shake for 30 minutes. 
     9. Remove detection antibody, wash the plate, and transfer fresh ABTS/H 2 O 2  (1.2 μl H 2 O 2  to 10 ml ABTS) 100 μl/well to the plate to start color development. 
     10. Measure OD in Dynatec MR5000, which is connected to Ingres. All following steps should follow Ingres instruction. 
     (vii) Experimental Results from ELISA Assays 
     The experimental results for various compounds according to the invention using the above-described protocols are set forth at Table 3: 
                     TABLE 3                  ELISA Assay Results                                                     HER2           COM-   PDGFR   FLK-1   EGFR   Kinase   IGF-1R       POUND   IC50 (μM)   IC50 (μM)   IC50 (μM)   IC50 (μM)   IC50 (μM)                                             SU4312   19.4   0.8                   SU4313   14.5   18.8   11   16.9   8.0       SU4314   12   0.39       SU4793   87.4   4.2       SU4794       11.8       SU4798       28.8       SU4799       9       SU4932       2.2       SU4944       8.5       SU4952       22.6       SU4956               22.5       SU4967   7.9   11.2       SU4979       20.9       SU4981   33.1   2.1       SU4982       21.6       39.4       SU4983       4.1       SU4984   5.8   1.6       90.2       SU5204       4       51.5       SU5205       9.6       SU5208       4.7       SU5214       14.8   36.7       SU5218       6.4       SU5401       2.9       89.8       SU5402       0.4       SU5403       1.8       SU5404   17   0.24       SU5405       23.8       SU5406       0.17       SU5407   53.7   1.1       SU5408       0.07       SU5416   10.8   0.11       SU5418       15.4       SU5419       2.3       SU5421       4.6       SU5424       2.4       SU5425       51.4       SU5427       4.5       70.6       SU5428       8.6       SU5430       73.4       SU5431       41.2       SU5432       22.8       SU5450       4.5       92.6       SU5451       3.4   44       SU5453   65.5   0.14       SU5455       36.2       SU5463       0.18       SU5464       20.3       SU5466   86   1.6       SU5468   55.9   2.7       SU5472       8.7       SU5473   14.2   1.5       SU5474       7.4       SU5477       0.15       SU5480       5.3   39.6   30.4                    
(b) Cell Growth Assays
 
     The following assays may be conducted to measure the effect of the claimed compounds upon cell growth as a result of the compound&#39;s interaction with one or more RTKs. Unless otherwise specified, the following assays may be generally applied to measure the activity of a compound against any particular RTK. To the extent that an assay, set forth below, refers to a specific RTK, one skilled in the art would be able to adapt the disclosed protocol for use to measure the activity of a second RTK. 
     (i) Soft Agar Assay 
     The soft agar assay may be used to measure the effects of substances on cell growth. Unless otherwise stated the soft agar assays were carried out as follows: 
     Material And Reagents. The following materials and reagents were used:
         a. A water bath set at 39° C. and another water bath at 37° C.   b. 2× assay medium is comprised of 2× Dulbecco&#39;s 5 Modified Eagle&#39;s Medium (DMEM) (Gibco Cat. # CA400-4ANO3) supplemented by the following:
           20% Fetal Bovine Serum (FBS) 2 mM sodium pyruvate 4 mM glutamine amine; and   20 mM HEPES Non-essential Amino Acids (1:50 from 100× stock).   
           c. 1× assay medium made of 1×DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM glutamine, 10 mM HEPES, non-essential amino acid (1:100 from 100× stock).   d. 1.6% SeaPlaque Agarose in autoclave bottle.   e. Sterile 35 mm Corning plates (FMC Bioproducts Cat. #50102).   f. Sterile 5 ml glass pipets (individually wrapped).   g. Sterile 15 ml and 50 ml conical centrifuge tubes.   h. Pipets and sterile tips.   i. Sterile microcentrifuge tubes.   j. Cells in T75 flasks: SKOV-3 (ATCC HTB77).   k. 0.25% Trypsin solution (Gibco #25200-015).       

     Procedure. The following procedure was used to conduct the soft agar assay: 
     A. Procedure for Making the Base Layer 
     1. Have all the media warmed up in the 37° C. water bath. 
     2. To make 1× of assay medium+0.8% agar: make a 1:2 (vol:vol) dilution of melted agar (cooled to 39° C.), with 2× assay medium. 
     3. Keep all media with agar warm in the 39° C. water bath when not in use. 
     4. Dispense 1 ml of 1× assay medium+0.8% agar into dishes and gently swirl plate to form a uniform base layer. Bubbles should be avoided. 
     5. Refrigerate base layers to solidify (about 20 minutes). Base layers can be stored overnight in the refrigerator. 
     B. Procedure for Collecting Cells 
     1. Take out one flask per cell line from the incubator; aspirate off medium; wash once with PBS and aspirate off; add 3 ml of trypsin solution. 
     2. After all cells dissociate from the flask, add 3 ml of 1× assay media to inhibit trypsin activity. Pipet the cells up and down, then transfer the suspension into a 15 ml tube. 
     3. Determine the concentration of cells using a Coulter counter, and the viability by trypan blue exclusion. 
     4. Take out the appropriate volume needed to seed 3300 viable cells per plate and dilute it to 1.5 ml with 1× assay medium. 
     C. Procedure for Making the Upper 0.4% Agarose Layer: 
     1. Add TBST compounds at twice the desired final assay concentration;+1.5 ml of cell suspension in 1× assay medium 10% FBS;+1.5 ml of 1× assay medium+0.8% agarose*: Total=3.0 ml 1× media 10% FBS+0.4% agarose with 3300 viable cells/ml, with and without TBST compounds. 
     *(Made by 1:2 dilution of 2× media with 1.6% agar 30 for the base layer procedure above.)  
     2. Plate 1 ml of the Assay Mix onto the 1 ml base layer. The duplicates are plated from the 3 ml volume. 
     3. Incubate the dishes for 2-3 weeks in a 100% humidified, 10% CO 2  incubator. 
     4. Colonies that are 60 microns and larger are scored positive. 
     (ii) Sulforhodamine B (SRB) Growth Assays 
     The SRB assays may be used to measure the effects of substances or cell growth. The assays are carried out as follows: 
     Assay 1: 3T3/E/H+TGF-a(T) Cell Growth SRB Assay 
     Materials:
         96-well flat bottom sterile plates   96-well round bottom sterile plates   sterile 25 ml or 100 ml reservoir   pipets, multi-channel pipetman   sterile pipet tips   sterile 15 ml and 50 ml tubes       

     Reagents:
         0.4% SRB in 1% acetic acid   10 mM Tris base   10% TCA   1% acetic acid   sterile DMSO (Sigma)   compound in DMSO (100 mM or less stock solution)   25% Trypsin-EDTA in Cell Dissociation Solution (Sigma)       

     Cell Line and Growth Medium:
         3T3/E/H+TGF-a(T) (NIH 3T3 clone 7 cells expressing EGF-R/HER2 chimera and TGF-a, tumor-derived autocrine loop cells)   2% calf serum/DMEM+2 mM glutamine       

     Protocol: 
     Day 0: Cell Plating: 
     This part of assay is carried out in a laminar flow hood. 
     1. Trypsinize cells as usual. Transfer 100 μl of cell suspension to 10 ml of isotone. Count cells with the Coulter Counter. 
     2. Dilute cells in growth medium to 60,000 cells/ml. Transfer 100 μl of cells to each well in a 96-well flat bottom plate to give 6000 cells/well. 
     3. Use half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, a set of 4 wells for medium control and 4 wells for DMSO control. 
     4. Gently shake plates to allow for uniform attachment of the cells. 
     5. Incubate the plates at 37° C. in a 10% CO2 incubator. 
     Day 1: Addition of Compound: 
     This part of assay is carried out in a laminar flow hood. 
     1. In 96 well-round bottom plate, add 125 μl of growth medium to columns 3 to 11. This plate is used to titrate out the compound, 4 rows per compound. 
     2. In a sterile 15 ml tube, make a 2× solution of the highest concentration of compound by adding 8 μl of the compound to a total of 2 ml growth medium for a dilution of 1:250. At this dilution, the concentration of DMSO is 0.4% for a 2× solution or 0.2% for 1× solution on the cells. The starting concentration of the compound is usually 100 uM but this concentration may vary depending upon the solubility of the compound. 
     3. Transfer the 2× starting compound solution to quadruplicate wells in column 12 of the 96-well round bottom plate. Do 1:2 serial dilutions across the plate from right to left by transferring 125 μl from column 12 to column 11, column 11 to 10 and so on. Transfer 100 μl of compound dilutions onto 100 μl medium on cells in corresponding wells of 96-well flat bottom plate. Total volume per well should be 200 μl. 
     4. For vehicle control, prepare a 2× solution of DMSO at 0.4% DMSO in growth medium. Transfer 100 μl of the DMSO solution to the appropriate wells of cells. The final concentration of DMSO is 0.2%. 
     5. For the medium control wells, add 100 μl/well of growth medium to the appropriate wells of cells. 
     6. Return the plate to the incubator and incubate for 4 days. 
     Day 5: Development of Assay 
     This part of assay is carried out on the bench. 
     1. Aspirate or pour off medium. Add 200 μl cold 10% TCA to each well to fix cells. Incubate plate for at least 60 min. at 4° C. 
     2. Discard TCA and rinse wells 5 times with water. Dry plates upside down on paper towels. 
     3. Stain cells with 100 μl/well 0.4% SRB for 10 min. 
     4. Pour off SRB and rinse wells 5 times with 1% acetic acid. Dry plates completely upside down on paper towels. 
     5. Solubilize dye with 100 μl/well 10 mM Tris base for 5-10 min. on shaker. 
     6. Read plates on Dynatech ELISA Plate Reader at 570 nm with reference at 630 nm. 
     Assay 2: 3T3/EGF-R+TGF-a(T) Cell Growth SRB Assay 
     Materials and Reagents same as for Assay 1. 
     Cell line and growth medium:
         3T3/EGF-R+TGF-a(T) (NIH 3T3 clone 7 cells expressing EGF-R and TGF-a, tumor-derived autocrine loop cells)   2% calf serum/DMEM+2 mM glutamine       

     Protocol: 
     Day 0: Cell Plating: 
     This part of assay is carried out in a laminar flow hood. 
     1. Trypsinize cells as usual. Transfer 100 μl of cell suspension to 10 ml of isotone. Count cells with the Coulter Counter. 
     2. Dilute cells in growth medium to 60,000 cells/ml. Transfer 100 μl of cells to each well in a 96-well flat bottom plate to give 6000 cells/well. 
     3. Use half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, a set of 4 wells for medium control and 4 wells for DMSO control. 
     4. Gently shake plates to allow for uniform attachment of the cells. 
     5. Incubate the plates at 37° C. in a 10% CO2 incubator. 
     Day 1: Addition of Compound: same as for Assay 1. 
     Day 5: Development of Assay: same as for Assay 1. 
     Assay 3: 3T3/PDGF-βR/PDGF-BB(T) Cell Growth SRB Assay 
     Cell Line and Growth Medium: 3T3/PDGF-βR/PDGF-BB(T) (NIH 3T3 clone 7 cells expressing PDGF β-receptor and PDGF-BB, from tumors resected from athymic mice)
         2% calf serum/DMEM+2 mM glutamine       

     Protocol: 
     Day 0: Cell Plating: 
     This part of assay is carried out in a laminar flow hood. 
     1. Trypsinize cells as usual. Transfer 200 μl of cell suspension to 10 ml of isotone. Count cells on the Coulter Counter. 
     2. Dilute cells in growth medium to 60,000 cells/ml. Transfer 100 μl of cells to each well in a 96-well flat bottom plate to give 6000 cells/well. 
     3. Allow half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, a set of 4 wells for medium control and 4 wells for DMSO control. 
     4. Gently shake plates to allow for uniform attachment of the cells to the plate. 
     5. Incubate the plates at 37° C. in a 10% CO 2  incubator. 
     Day 1: Addition of Compound: same as for Assay 1. 
     Day 5: Development of Assay: same as for Assay 1. 
     Assay 4: Euman Smooth Muscle Cells (SMC) Growth SRB Assay 
     Materials and Reagents same as for Assay 1: 
     Cell Line and Growth Medium: 
     Human Aortic Smooth Muscle cells (Clonetics) 
     Clonetics&#39;s Bullet Kit: Smooth Muscle Basal Medium (SmBM) which is modified MCDB 131 containing fetal bovine serum (5%), hFGF (2 ng/ml), HEGF (0.1 ng/ml), insulin (5.0 ug/ml), gentamicin (50 ug/ml) and amphotericin B (50 ng/ml) 
     Protocol: 
     Day 0: Cell Plating: 
     This part of assay is carried out in a laminar flow hood. 
     1. Trypsinize cells as usual. Transfer 200 μl of cell suspension to 10 ml of isotone. Count cells on the Coulter Counter. 
     2. Dilute cells in growth medium to 20,000 cells/ml. Transfer 100 μl of cells to each well in a 96-well flat bottom plate to give 2000 cells/well. 
     3. Allow half of plate (4 rows) for each compound and quadruplicate wells for each compound concentration, a set of 4 wells for medium control and 4 wells for DMSO control. 
     4. Gently shake plates to allow for uniform attachment of the cells to the plate. 
     5. Incubate the plates at 37° C. in a 10% CO2 incubator. 
     Day 1: Addition of Compound: same as for Assay 1. 
     Day 5: Development of Assay: same as for Assay 1. 
     (iii) 3T3 Cell Growth Assay 
     Assay 1: PDGF-Induced BrdU Incorporation Assay 
     Materials and Reagents:
         (1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany   (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (6) PBS Washing Solution: 1×PBS, pH 7.4, made in house.   (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.       

     Protocol
         (1) 3T3 engineered cell line: 3T3/EGFRc7.   (2) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO 2 .   (3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.   (4) On day 3, ligand (PDGF=3.8 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (PDGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.   (5) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours.   (6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.   (7) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.   (8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.   (9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.   (10) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.   (11) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
 
Assay 2: EGF-Induced BrdU Incorporation Assay
       

     Materials and Reagents
         (1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan   (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (6) PBS Washing Solution: 1×PBS, pH 7.4, made in house.   (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.       

     Protocol
         (1) 3T3 engineered cell line: 3T3/EGFRc7   (2) Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, in a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO 2 .   (3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.   (4) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.   5) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours.   6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.   (7) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.   (8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.   (9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.   (10) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.   (11) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
 
Assay 3: EGF-Induced Her2-Driven BrdU Incorporation)
       

     Materials and Reagents:
         (1) EGF: mouse EGF, 201; Toyobo, Co., Ltd. Japan   (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (6) PBS Washing Solution: 1×PBS, pH 7.4, made in house.   (7) Albumin, Bovine (BSA): fraction V powder; A-8551, sigma Chemical Co., USA.       

     Protocol:
         (1) 3T3 engineered cell line: 3T3/EGFr/Her2/EGFr (EGFr with a Her2 kinase domain)   (2) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96-well plate. Cells are incubated overnight at 37° in 5% CO 2 .   (3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.   (4) On day 3, ligand (EGF=2 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (EGF) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.   5) After 20 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours.   (6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.   (7) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.   (8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.   (9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.   (10) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.   (11) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
 
Assay. 4: IGF1-Induced BrdU Incorporation Assay
       

     Materials and Reagents:
         (1) IGF1 Ligand: human, recombinant; G511, Promega Corp, USA.   (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (4) Anti-BrdU-POD; mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (6) PBS Washing Solution: 1×PBS, pH 7.4, made in house.   (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.       

     Protocol:
         (1) 3T3 engineered cell line: 3T3/IGF1r.   (2) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96-well plate. Cells are incubated overnight at 37° C. in 5% CO 2 .   (3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.   (4) On day 3, ligand (IGF1=3.3 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (IGF1) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.   5) After 16 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours.   (6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.   (7) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.   (8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.   (9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.   (10) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.   (11) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.
 
Assay 5: Insulin-Induced BrdU Incorporation Assay
       

     Materials and Reagents:
         (1) Insulin: crystalline, bovine, Zinc; 13007, Gibco BRL, USA.   (2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4), Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (3) FixDenat: fixation solution (ready to use), Cat. No. 1 647-229, Boehringer Mannheim, Germany.   (4) Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (5) TMB Substrate Solution: tetramethylbenzidine (TMB), ready to use, Cat. No. 1 647 229, Boehringer Mannheim, Germany.   (6) PBS Washing Solution: 1×PBS, pH 7.4, made in house.   (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co., USA.       

     Protocol:
         (1) 3T3 engineered cell line: H25   (2) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a 96 well plate. Cells are incubated overnight at 37° C. in 5% CO 2 .   (3) After 24 hours, the cells are washed with PBS, and then are serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.   (4) on day 3, ligand (Insulin=10 nM, prepared in DMEM with 0.1% BSA) and test compounds are added to the cells simultaneously. The negative control wells receive serum free DMEM with 0.1% BSA only; the positive control cells receive the ligand (Insulin) but no test compound. Test compounds are prepared in serum free DMEM with ligand in a 96 well plate, and serially diluted for 7 test concentrations.   (5) After 16 hours of ligand activation, diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (final concentration=10 μM) for 1.5 hours.   (6) After incubation with labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is added (50 μl/well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.   (7) The FixDenat solution is thoroughly removed by decanting and tapping the inverted plate on a paper towel. Milk is added (5% dehydrated milk in PBS, 200 μl/well) as a blocking solution and the plate is incubated for 30 minutes at room temperature on a plate shaker.   (8) The blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.   (9) The antibody conjugate is thoroughly removed by decanting and rinsing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.   (10) TMB substrate solution is added (100 μl/well) and incubated for 20 minutes at room temperature on a plate shaker until color development is sufficient for photometric detection.   (11) The absorbance of the samples are measured at 410 nm (in “dual wavelength” mode with a filter reading at 490 nm, as a reference wavelength) on a Dynatech ELISA plate reader.       

     (iv) HUV-EC-C Assay 
     The following protocol may also be used to measure a compound&#39;s activity: 
     DAY 0 
     1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelial cells, (American Type Culture Collection; catalogue no. 1730 CRL). Wash with Dulbecco&#39;s phosphate-buffered saline (D-PBS; obtained from Gibco BRL; catalogue no. 14190-029) 2 times at about 1 ml/10 cm 2  of tissue culture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic cell dissociation solution (Sigma Chemical Company; catalogue no. C-1544). The 0.05% trypsin was made by diluting 0.25% trypsin/1 mM EDTA (Gibco; catalogue no. 25200-049) in the cell dissociation solution. Trypsinize with about 1 ml/25-30 cm 2  of tissue culture flask for about 5 minutes at 37° C. After cells have detached from the flask, add an equal volume of assay medium and transfer to a 50 ml sterile centrifuge tube (Fisher Scientific; catalogue no. 05-539-6). 
     2. Wash the cells with about 35 ml assay medium in the 50 ml sterile centrifuge tube by adding the assay medium, centrifuge for 10 minutes at approximately 200×g, aspirate the supernatant, and resuspend with 35 ml D-PBS. Repeat the wash two more times with D-PBS, resuspend the cells in about 1 ml assay medium/15 cm 2  of tissue culture flask. Assay medium consists of F12K medium (Gibco BRL; catalogue no. 21127-014)+0.5% heat-inactivated fetal bovine serum. Count the cells with a Coulter Counter®v Coulter Electronics, Inc.) and add assay medium to the cells to obtain a concentration of 0.8-1.0×10 5  cells/ml. 
     3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10 4  cells/well; incubate ˜24 h at 37° C., 5% CO 2 . 
     1. Make up two-fold drug titrations in separate 96-well plates, generally 50 μM on down to 0 μM. Use the same assay medium as mentioned in day 0, step 2 above. Titrations are made by adding 90 μl/well of drug at 200 μM (4× the final well concentration) to the top well of a particular plate column. Since the stock drug concentration-is usually 20 mM in DMSO, the 200 μM drug concentration contains 2% DMSO. 
     Therefore, diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovine serum) is used as diluent for the drug titrations in order to dilute the drug but keep the DMSO concentration constant. Add this diluent to the remaining wells in the column at 60 μl/well. Take 60 μl from the 120 μl of 200 μM drug dilution in the top well of the column and mix with the 60 μl in the second well of the column. Take 60 μl from this well and mix with the 60 μl in the third well of the column, and so on until two-fold titrations-are completed. When the next-to-the-last well is mixed, take 60 μl of the 120 μl in this well and discard it. Leave the last well with 60 μl of DMSO/media diluent as a non-drug-containing control. Make. 9 columns of titrated drug, enough for triplicate wells each for 1) VEGF (obtained from Pepro Tech Inc., catalogue no. 100-200, 2) endothelial cell growth factor (ECGF) (also known as acidic fibroblast growth factor, or aFGF) (obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600), and assay media control. ECGF comes as a preparation with sodium heparin. 
     2. Transfer 50 μl/well of the drug dilutions to the 96-well assay plates containing the 0.8-1.0×10 4  cells/100 μl/well of the HUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO 2 . 
     3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, or media control to each drug condition. As with the drugs, the growth factor concentrations are 4× the desired final concentration. Use the assay media from day 0 step 2 to make the concentrations of growth factors. Incubate approximately 24 hours at 37° C., 5% CO 2 . Each well will have 50 μl drug dilution, 50 μl growth factor or media, and 100 ul cells, =200 ul/well total. Thus the 4× concentrations of drugs and growth factors become 1× once everything has been added to the wells. 
     DAY 2 
     1. Add  3 H-thymidine (Amersham; catalogue no. TRK-686) at 1 μCi/well (10 μl/well of 100 μCi/ml solution made up in RPMI media+10% heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5% CO 2 . Note:  3 H-thymidine is made up in RPMI media because all of the other applications for which we use the  3 H-thymidine involve experiments done in RPMI. The media difference at this step is probably not significant. RPMI was obtained from Gibco BRL, catalogue no. 11875-051. 
     DAY 3 
     1. Freeze plates overnight at −20° C. 
     DAY 4 
     1. Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester 96®) onto filter mats (Wallac; catalogue no. 1205-401); read counts on a Wallac Betaplate™ liquid scintillation counter. 
     (v) PDGF-R Cellular Assay 
     The PDGF cellular kinase assay was carried out as follows: cells are lysed in 0.2 M Hepes, 0.15 M NaCl, 10% V/V glycerol, 0.04% Triton X-100, 5 mM EDTA, 5 mM sodium vanadate and 2 mM Na+ pyrophosphate; cell lysates are then added to an ELISA plate coated with an anti-PDGF receptor antibody (Genzyme); ELISA plates are coated at 0.5 μg of antibody/well in 150 μl of PBS for 18 hours at 4° C. prior to the addition of the lysate; the lysate is incubated in the coated plates for 1 hour and then washed four times in TBST (35 mM Tris-HCl pH 7.0, 0.15 M NaCl, 0.1% Triton X100); anti-phosphotyrosine antibody (100 μl in PBS) is added and the mixture is incubated for 30 minutes at room temperature; the wells were then washed four times in TBST, a secondary antibody conjugated to POD (TAGO) is added to each well, and the treated well are incubated for 30 minutes at room temperature; the wells are then washed four times in TBST, ABTS/H 2 O 2  solution is added to each well and the wells are incubated for two minutes; absorbance is then measured at 410 nm. 
     (vi) Experimental Results of Cell Growth Assay 
     Results for various compounds obtained from the above-described assays are set forth in the Tables that follow: 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Mitogenesis in Endothelial Cells 
               
               
                 [3H] Thymidine Incorporation 
               
            
           
           
               
               
               
            
               
                   
                 HUV-EC Assay 
                   
               
            
           
           
               
               
               
            
               
                 COMPOUND 
                 VEGF (μM) 
                 a-FGF (μM) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 SU4312 
                 1.1 
                 153.8 
               
               
                 SU4314 
                 0.2 
                 6.0 
               
               
                 SU4793 
                 6.6 
                 3.4 
               
               
                 SU4794 
                 4.8 
                 35.7 
               
               
                 SU4796 
                 30.7 
                 35.8 
               
               
                 SU4798 
                 43.2 
               
               
                 SU4799 
                 19.9 
               
               
                 SU4932 
                 2.5 
                 45.2 
               
               
                 SU4942 
                 1.6 
                 4.6 
               
               
                 SU4944 
                 14.8 
               
               
                 SU4949 
                 3.4 
                 3.7 
               
               
                 SU4952 
                 25.6 
                 19.3 
               
               
                 SU4956 
                 8.0 
                 13.0 
               
               
                 SU4967 
                 34.3 
                 16.3 
               
               
                 SU4972 
                 1.0 
                 1.4 
               
               
                 SU4979 
                 4.4 
                 4.9 
               
               
                 SU4981 
                 0.6 
               
               
                 SU4982 
                 46.1 
                 27.3 
               
               
                 SU4984 
                 0.8 
                 25.8 
               
               
                 SU5201 
                 2.5 
                 2.3 
               
               
                 SU5204 
                 2.3 
                 0.7 
               
               
                 SU5205 
                 5.1 
                 11.8 
               
               
                 SU5208 
                 2.9 
                 130 
               
               
                 SU5217 
                 9.6 
                 10.5 
               
               
                 SU5218 
                 2.4 
                 2.7 
               
               
                 SU5401 
                 2.2 
               
               
                 SU5402 
                 &lt;0.8 
                 2.0 
               
               
                 SU5404 
                 &lt;0.8 
                 31.1 
               
               
                 SU5405 
                 0.9 
                 0.6 
               
               
                 SU5406 
                 &lt;0.8 
               
               
                 SU5407 
                 39.8 
                 35.5 
               
               
                 SU5408 
                 &lt;0.8 
                 22.7 
               
               
                 SU5409 
                 26.0 
               
               
                 SU5416 
                 &lt;0.8 
               
               
                 SU5418 
                 13.6 
                 40 
               
               
                 SU5419 
                 0.7 
               
               
                 SU5421 
                 11.4 
               
               
                 SU5424 
                 2.5 
               
               
                 SU5427 
                 5.7 
               
               
                 SU5429 
                 27.6 
               
               
                 SU5432 
                 0.16 
                 0.14 
               
               
                 SU5438 
                 39.8 
                 33.0 
               
               
                 SU5451 
                 1.2 
                 30.0 
               
               
                 SU5454 
                 3.8 
                 3.4 
               
               
                 SU5455 
                 20 
                 20 
               
               
                 SU5461 
                 &lt;0.07 
                 &lt;0.07 
               
               
                 SU5462 
                 0.5 
                 0.8 
               
               
                 SU5463 
                 0.14 
                 7.9 
               
               
                 SU5464 
                 3.8 
                 12.9 
               
               
                 SU5466 
                 1.3 
                 3.2 
               
               
                 SU5468 
                 0.54 
                 8.7 
               
               
                 SU5472 
                 2.0 
                 5.0 
               
               
                 SU5473 
                 1.2 
                 14.1 
               
               
                 SU5477 
                 0.05 
                 37.8 
               
               
                 SU5480 
                 1.2 
                 3.8 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Mitogenesis in 3T3/EGFR Cells 
               
               
                 BrdU Incorporation 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 PDGFR 
                 FGFR 
                 EGFR 
               
               
                   
                   
                 PDGF Ligand 
                 FGF Ligand 
                 EGF Ligand 
               
               
                   
                 COMPOUND 
                 IC50 (μM) 
                 IC50 (μM) 
                 IC50 (μM) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 SU4312 
                 75 
                   
                   
               
               
                   
                 SU4313 
                 6 
                 5.5 
                 5.5 
               
               
                   
                 SU4314 
                 2.5 
               
               
                   
                 SU4967 
                 9 
                 4.9 
                 60 
               
               
                   
                 SU4981 
                 3 
                 10 
                 20 
               
               
                   
                 SU5402 
                 50 
                 40 
               
               
                   
                 SU5404 
                 3 
                 25 
               
               
                   
                 SU5406 
                 5.2 
               
               
                   
                 S05407 
                 7.5 
                 70 
                 100 
               
               
                   
                 SU5416 
                 2.8 
                 70 
               
               
                   
                 SU5451 
                 30 
                 16 
               
               
                   
                 SU5463 
                   
                   
                 23 
               
               
                   
                 SU5464 
                 70 
                 60 
                 95 
               
               
                   
                 SU5465 
                 40 
                 25 
                 50 
               
               
                   
                 SU5466 
                 18 
                 15 
                 17 
               
               
                   
                 SU5468 
                 8 
               
               
                   
                 SU5469 
                 4 
                 15 
                 28 
               
               
                   
                 SU5473 
                 4 
                 50 
                 54 
               
               
                   
                 SU5475 
                 6.5 
                 9 
                 48 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Cell Growth Assay on Various Cell Lines 
               
               
                 SRB Readout 
               
            
           
           
               
               
               
               
               
            
               
                   
                 3T3/E/H + 
                 3T3/EGFR + 
                 3T3/PDGFR + 
                   
               
               
                 COM- 
                 TGF-a (T) 
                 TGF-a (T) 
                 PDGF (T) 
                 SMC 
               
               
                 POUND 
                 IC50 (μM) 
                 IC50 (μM) 
                 IC50 (μM) 
                 IC50 (μM) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 SU4312 
                 36 
                   
                   
                   
               
               
                 SU4313 
                 32 
                 10.7 
                   
                 8.8 
               
               
                 SU4314 
                 78 
                   
                 10 
               
               
                 SU4984 
                   
                   
                 22.2 
               
               
                   
               
               
                 3T3/E/H + TGF-α (T): NIH 3T3 cells expressing EGFR/HER2 chimera and TGF-α, tumor-derived  
               
               
                 3T3/EGFR + TGF-α (T): NIH 3T3 cells expressing EGFR and TGF-α, tumor-derived  
               
               
                 3T3/PDGFR + PDGF (T): NIH 3T3 cells expressing PDGF-βR and PDGF-ββ, tumor-derived  
               
               
                 SMC: human smooth muscle cells from Clonetics  
               
            
           
         
       
     
     6.3. Measurement of Cell Toxicity 
     Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect. A measure of the effectiveness and cell toxicity of a compound can be obtained by determining the therapeutic index: IC 50 /LD 50 . IC 50 , the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein. LD 50 , the dosage which results in 50% toxicity, can also be measured by standard techniques (Mossman, 1983 , J. Immunol. Methods , 65:55-63), by measuring the amount of LDH released (Korzeniewski and Callewaert, 1983 , J. Immunol. Methods  64:313; Decker and Lohmann-Matthes, 1988 , J. Immunol. Methods  115:61), or by measuring the lethal dose in animal models. Compounds with a large therapeutic index are preferred. The therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50. 
     (c) In Vivo Animal Models 
     (i) Xenograft Animal Models 
     The ability of human tumors to grow as xenografts in athymic mice (e.g., Balb/c, nu/nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice, (Rygaard and Povlsen, 1969 , Acta Pathol. Microbial. Scand . 77:758-760), many different human tumor cell lines (e.g., mammary, lung, genitourinary, gastrointestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice. Human mammary tumor cell lines, including MCF-7, ZR75-1, and MDA-MB-231, have been established as subcutaneous xenografts in nude mice (Warri et al., 1991 , Int. J. Cancer  49:616-623; Ozzello and Sordat, 1980 , Eur. J. Cancer  16:553-559; Osborne et al., 1985 , Cancer Res . 45:584-590; Seibert et al., 1983 , Cancer Res . 43:2223-2239). 
     Assay 1: HER2/Xenograft Animal Model 
     To study the effect of anti-tumor drug candidates on HER2 expressing tumors, the tumor cells should be able to grow in the absence of supplemental estrogen. Many mammary cell lines are dependent on estrogen for in vivo growth in nude mice (Osborne et al., supra), however, exogenous estrogen suppresses HER2 expression in nude mice (Warri et al., supra, Dati et al., 1990 , Oncogene  5:1001-1006). For example, in the presence of estrogen, MCF-7, ZR-75-1, and T47D cells grow well in vivo, but express very low levels of HER2 (Warri et al., supra, Dati et al., supra). 
     The following type of xenograft protocol can be used:
         1) implant tumor cells (subcutaneously) into the hindflank of five- to six-week-old female Balb/c nu/nu athymic mice;   2) administer the anti-tumor compound;   3) measure tumor growth by measuring tumor volume.       

     The tumors can also be analyzed for the presence of a receptor, such as HER2, EGF or PDGF, by Western and immunohistochemical analyses. Using techniques known in the art, one skilled in the art can vary the above procedures, for example through the use of different treatment regimes. 
     Assay 2: FLK-1/Xenograft Model. 
     The ability of the compounds of the present invention to inhibit ovarian, melanoma, prostate, lung and mammary tumor cell lines established as SC xenografts was examined. These studies were conducted using doses ranging from 1 to 75 mg/kg/day. 
     Materials And Methods. The tumor cells were implanted subcutaneously into the indicated strains of mice. Treatment was initiated on day 1 post implantation unless otherwise indicated (e.g. treatment of the SCID mouse related to the A375 melanoma cell line began on Day 9). Eight (8) to sixteen (16) mice comprised each test group. 
     Specifically: 
     Animals. Female athymic mice (BALB/c, nu/nu), BALB/c mice, Wistar rats and Fisher 344 rats were obtained from Simonsen Laboratories (Gilroy, Calif.). Female A/I mice were obtained from Jackson Laboratory (Bar Harbor, Me.). DA rats were obtained from B&amp;K Universal, Inc. (Fremont, Calif.). Athymic R/Nu rats, DBA/2N mice, and BALB/c mice were obtained from Harlan Sprague Dawley (Indianapolis, Ind.). Female C57BL/6 mice were obtained from Taconic (Germantown, N.Y.). All animals were maintained under clean-room conditions in Micro-isolator cages with Alpha-dri bedding. They received sterile rodent chow and water ad libitum. 
     All procedures were conducted in accordance with the NIH Guide for the Care and Use Of Laboratory Animals. 
     Subcutaneous Xenograft Model. Cell lines were grown in appropriate medium as described (See Section 6). Cells were harvested at or near confluency with 0.05% Trypsin-EDTA and pelleted at 450×g for 10 min. Pellets were resuspended in sterile PBS or media (without FBS) to a suitable concentration indicated in the Figure legends and the cells were implanted into the hindflank of mice. Tumor growth was measured over 3 to 6 weeks using venier calipers tumor volumes were calculated as a product of length×width×height unless otherwise indicated. P values were calculated using the Students&#39; t-test. Compound in 50-100 μL excipient (dimethylsulfoxide, PETE, PBTE6C:D5W, or PBTE:D5W) was delivered by IP injection at concentrations indicated in the Figure legends. 
     Intracerebral Xenograft Model. For the mouse IC model, rat C6 glioma cells were harvested and suspended in sterile PBS at a concentration of 2.5×10 7  cells/ml and placed on ice. Cells were implanted into BALB/c, nu/nu mice in the following manner: the frontoparietal scalps of mice were shaved with animal clippers if necessary before swabbing with 70% ethanol. Animals were anesthetized with isofluorane and the needle was inserted through the skull into the left hemisphere of the brain. Cells were dispensed from Hamilton Gas-tight Syringes using 30 ga ½ inch needles fitted with sleeves that allowed only a 3 mm penetration. A repeater dispenser was used for accurate delivery of 4 μL of cell suspension. Animals were monitored daily for well-being and were sacrificed when they had a weight loss of about 40% and/or showed neurological symptoms. 
     For the rat IC model, rats (Wistar, Sprague Dawley, Fisher 344, or athymic R/Nu; approximately 200-400 g (some 3-400 g)) were anesthetized by an IP injection of 100 mg/kg Ketaset (ketamine hydrochloride; Aveco, Fort Dodge, Iowa) and 5 mg/kg Rompun (xylazine, 2% solution; Bayer, Germany). After onset of anesthesia, the scalp was shaved and the animal was oriented in a stereotaxic apparatus (Stoelting, Wood Dale, Ill.). The skin at the incision site was cleaned 3 times with alternating swabs of 70% ethanol and 10% Povidone-Iodine. A median 1.0-1.5 cm incision was made in the scalp using a sterile surgical blade. The skin was detached slightly and pulled to the sides to expose the sutures on the skull surface. A dental drill (Stoelting, Wood Dale, Ill.) was used to make a small (1-2 mm diameter) burrhole in the skull approximately 1 mm anterior and 2 mm lateral to the bregma. The cell suspension was drawn into a 50 μL Hamilton syringe fitted with a 23 or 25 g a standard bevel needle. The syringe was oriented in the burrhole at the level of the arachnoidea and lowered until the tip of the needle was 3 mm deep into the brain structure, where the cell suspension was slowly injected. After cells were injected, the needle was left in the burrhole for 1-2 minutes to allow for complete delivery of the cells. The skull was cleaned and the skin was closed with 2 to 3 sutures. Animals were observed for recovery from surgery and anesthesia. Throughout the experiment, animals were observed at least twice each day for development of symptoms associated with progression of intracerebral tumor. Animals displaying advanced symptoms (leaning, loss of balance, dehydration, loss of appetite, loss of coordination, cessation of grooming activities, and/or significant weight loss) were humanely sacrificed and the organs and tissues of interest were resected. 
     Intraperitoneal Model. Cell lines were grown in the appropriate media. Cells were harvested and washed in sterile PBS or medium without FBS, resuspended to a suitable concentration, and injected into the IP cavity of mice of the appropriate strain. Mice were observed daily for the occurrence of ascites formation. Individual animals were sacrificed when they presented with a weight gain of 40%, or when the IP tumor burden began to cause undue stress and pain to the animal. 
     (ii) In Vivo VEGF Pellet Model 
     In the following example, the Pellet Model was used to test a compound&#39;s activity against the FLK-1 receptor and against disorders associated with the formation of blood vessels. In this model, VEGF is packaged into a time-release pellet and implanted subcutaneously on the abdomen of nude mice to induce a ‘reddening’ response and subsequent swelling around the pellet. Potential FLK-1 inhibitors may then be implanted in methylcellulose near the VEGF pellet to determine whether such inhibitor may be used to inhibit the “reddening” response and subsequent swelling. 
     Materials And Methods. The following materials were used:
         1) VEGF-human recombinant lyophilized product is commercially may be obtained from Peprotech, Inc., Princeton Business Park, G2; P.O. box 275, Rocky Hill, N.J. 08553.   2) VEGF packaged into 21 day release pellets were obtained from Innovative Research of America (Innovative Research of America, 3361 Executive Parkway, P.O. Box 2746, Toledo, Ohio 43606), using patented matrix driven delivery system. Pellets were packaged at 0.20, 0.21, or 2.1 μg VEGF/pellet. These doses approximate 10 and 100 ng/day release of VEGF.   3) Methylcellulose   4) Water (sterile)   5) Methanol   6) Appropriate drugs/inhibitors   7) 10 cm culture plates   8) parafilm       

     The following protocol was then followed to conduct the VEGF pellet model:
         1) VEGF, purchased from Peprotech, was sent to Innovative Research for Custom Pellet preparation;   2) Methylcellulose prepared at 1.5% (w/v) in sterile water;   3) Drugs solubilized in methanol (usual concentration range=10 to 20 mg/ml);   4) Place sterile parafilm in sterile 10 cm plates;   5) 150 μl of drug in methanol added to 1.35 ml of 1.5% methylcellulose and mixed/vortexed thoroughly;   6) 25 μl aliquots of homogenate placed on parafilm and dried into discs;   7) Mice (6-10 wk. Balb/C athymic nu/nu, female) were anesthetized via isoflurane inhalation;   8) VEGF pellets and methylcellulose discs were implanted subcutaneously on the abdomen; and   9) Mice were scored at 24 hours and 48 hours for reddening and swelling response.       

     The specific experimental design used in this example was:
         N=4 animals/group   Controls: VEGF pellet+drug placebo
           VEGF placebo+drug pellet   
               

     Experimental Results. The compounds of the present invention are expected to demonstrate activity according to this assay. 
       
     (iii) Mammary Pat Pad Model 
     Because of the established role played by many of the RTKs, e.g., the HER2 receptor, in breast cancer, the mammary fat pad model is particularly useful for measuring the efficacy of compounds which inhibit such RTKs. By implanting tumor cells directly into the location of interest, in situ models more accurately reflect the biology of tumor development than do subcutaneous models. Human mammary cell lines, including MCF-7, have been grown in the mammary fat pad of athymic mice. Shafie and Grantham, 1981 , Natl. Cancer Instit . 67:51-56; Gottardis et al., 1988 , J. Steroid Biochem . 30:311-314. More specifically, the following procedure can be used to measure the inhibitory effect of a compound on the HER2 receptor:
         1) Implant, at various concentrations, MDA-MB-231 and MCF-7 cells transfected with HER-2 into the axillary mammary fat pads of female athymic mice;   2) Administer the compound; and   3) Measure the tumor growth at various time points.       

     The tumors can also be analyzed for the presence of a receptor such as HER2, by Western and immunohistochemical analyses. Using techniques known in the art, one skilled in the art can vary the above procedures, for example through the use of different treatment regimes. 
     (iv) Tumor Invasion Model 
     The following tumor invasion model has been developed and may be used for the evaluation of therapeutic value and efficacy of the compounds identified to selectively inhibit KDR/FLK-1 receptor. 
     (A) Procedure 
     8 week old nude mice (female) (Simonsen Inc.) were used as experimental animals. Implantation of tumor cells was performed in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5 mg/kg) are administered intraperitoneally. A midline incision is done to expose the abdominal cavity (approximately 1.5 cm in length) to inject 10 7  tumor cells in a volume of 100 μl medium. The cells are injected either into the duodenal lobe of the pancreas or under the serosa of the colon. The peritoneum and muscles are closed with a 6-0 silk continuous suture and the skin was closed by using would clips. Animals were observed daily. 
     (B) Analysis 
     After 2-6 weeks, depending on gross observations of the animals, the mice are sacrificed, and the local tumor metastases, to various organs (lung, liver, brain, stomach, spleen, heart, muscle) are excised and analyzed (measurements of tumor size, grade of invasion, immunochemistry, and in situ hybridization). 
     (v) RESULTS 
     Results for various compounds obtained from the above-described in vivo assays are set forth at Table 5, below: 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 In Vivo Data 
               
            
           
           
               
               
               
            
               
                   
                   
                 EpH4-VEGF 
               
               
                   
                 COMPOUND 
                 % inhibition @ mg/kg 
               
               
                   
                   
               
               
                   
                 SU4312 
                 56% @ 75 
               
               
                   
                   
                 ---------------------------------- 
               
               
                   
                   
                 50% @ 75 
               
               
                   
                   
                 63% @ 50 
               
               
                   
                 SU4932 
                 42% @ 75 
               
               
                   
                   
                 ---------------------------------- 
               
               
                   
                   
                 — 
               
               
                   
                   
                   42% @ 50/50 
               
               
                   
                 SU4942 
                 46% @ 50 
               
               
                   
                   
                 47% @ 25 
               
               
                   
                 SU5416 
                 50% @ 25 
               
               
                   
                   
                 ---------------------------------- 
               
               
                   
                   
                 — 
               
               
                   
                   
                     57% @ 37.5/37.5 
               
               
                   
                 SU5424 
                 45% @ 50 
               
               
                   
                   
                 ---------------------------------- 
               
               
                   
                   
                 — 
               
               
                   
                   
                 65% @ 50 
               
               
                   
                 SU5427 
                 47% @ 50 
               
               
                   
                   
                 ---------------------------------- 
               
               
                   
                   
                 — 
               
               
                   
                   
                 65% @ 50 
               
               
                   
                   
               
            
           
         
       
     
     The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. 
     All references cited herein are hereby incorporated by reference in their entirety. 
     Other embodiments are within the following claims. 
     
       
         
           
               
               
               
             
               
                 TABLE I 
               
               
                   
               
             
            
               
                 5-aminooxindole 
                 5-bromooxindole 
                 5-chlorooxindole 
               
               
                 oxindote-001 
                 oxindole-002 
                 oxindole-003 
               
               
                 4,6-dimethyloxindole 
                 5,6-dimethoxyoxindole 
                 oxindole 
               
               
                 oxindole-004 
                 oxindole-005 
                 oxindole-006 
               
               
                 4-methyloxindole 
                 5,7-dibromooxindole 
                 7-bromo-5-chloro- 
               
               
                 oxindole-007 
                 oxindole-008 
                 oxindole 
               
               
                   
                   
                 oxindole-009 
               
               
                 5-fluorooxindole 
                 5-nitrooxindole 
                 5-iodooxindole 
               
               
                 oxindole-010 
                 oxindole-011 
                 oxindole-012 
               
               
                 5-chlora-7- 
                 5-methyloxindole 
                 5-bromo-4-methyl- 
               
               
                 methyloxindole 
                 oxindole-014 
                 oxindole 
               
               
                 oxindole-013 
                   
                 oxindole-015 
               
               
                 7-fluorooxindole 
                 7-chlorooxindole 
                 4-fluorooxindole 
               
               
                 oxindole-016 
                 oxindole-017 
                 oxindole-018 
               
               
                 6-fluorooxindole 
                 4-chlorooxindole 
                 6-chlorooxindole 
               
               
                 oxindole-019 
                 oxindole-020 
                 oxindole-021 
               
               
                 5-bromo-7-methyl- 
                 7-chloro-5- 
                 4-bromooxindole 
               
               
                 oxindole 
                 cyanoxindole 
                 oxindole-024 
               
               
                 oxindole-022 
                 oxindole-023 
               
               
                 7-methoxyoxindole 
                 4-methyl-5-carboxy- 
                 4-methyl-5-carboxy- 
               
               
                 oxindole-025 
                 oxindole 
                 methyloxindole 
               
               
                   
                 oxindole-026 
                 oxindole-027 
               
               
                 4-methyl-5-carboxy- 
                 4-methyl-5- 
                 4-methyl-5-hydroxy- 
               
               
                 ethyloxindole 
                 (3-carboxy-n-propyl) 
                 methyloxindole 
               
               
                 oxindole-028 
                 oxindole-029 
                 oxindole-030 
               
               
                 4-methyl-5-methoxy- 
                 4-methyl-5-(2-hy- 
                 4-methyl-5-(2-meth- 
               
               
                 methyloxindole 
                 droxyethyl)oxindole 
                 oxyethyl)oxindole 
               
               
                 oxindole-031 
                 oxindole-032 
                 oxindole-033 
               
               
                 4-methyl-5-(3- 
                 4-methyl-5-(3- 
                 5-aminosulfon- 
               
               
                 hydroxy-n-propyl) 
                 methoxy-n-propyl) 
                 yloxindole 
               
               
                 oxindole 
                 oxindole 
                 oxindole-036 
               
               
                 oxindole-034 
                 oxindole-035 
               
               
                 5-methlamino- 
                 5-(4-trifluoro- 
                 5-(morpholin-1- 
               
               
                 sulfonyloxindole 
                 methylanilino- 
                 yl-sulfonyl) 
               
               
                 oxindole-037 
                 sulfonyl)oxindole 
                 oxindole 
               
               
                   
                 oxindole-038 
                 oxindole-039 
               
               
                 6-trifluorometh- 
                 5-(2-chloroeth- 
                 5-carboxymethyl- 
               
               
                 yloxindole 
                 yl)oxindole 
                 oxindole 
               
               
                 oxindole-040 
                 oxindole-041 
                 oxindole-042 
               
               
                 6-carboxymethyl- 
                 4-methoxycarbonyl- 
                 5-methoxycarbonyl- 
               
               
                 oxindole 
                 oxindole 
                 oxindole 
               
               
                 oxindole-043 
                 oxindole-044 
                 oxindole-045 
               
               
                 6-methoxycarbonyl- 
                 4-carboxyoxindole 
                 5-carboxyoxindole 
               
               
                 oxindole 
                 oxindole-047 
                 oxindole-048 
               
               
                 oxindole-046 
               
               
                 6-carboxyoxindole 
                 5-carboxyethyl- 
                 5-hydroxyethyl- 
               
               
                 oxtndole-049 
                 oxindole 
                 oxindole 
               
               
                   
                 oxindole-050 
                 oxindole-051 
               
               
                 4-methyl-5-amino- 
                 4-methyl-5-nitro- 
                 4-methyl-5-iodo- 
               
               
                 oxindole 
                 oxindole 
                 oxindole 
               
               
                 oxindole-052 
                 oxindole-053 
                 oxindole-054 
               
               
                 4-methyl-5-chloro- 
               
               
                 oxindole 
               
               
                 oxindole-055 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE II 
               
               
                   
               
             
            
               
                 2-ethoxybenzaldehyde 
                 2-thiophenecarboxal- 
                 1-methylpyrrol-2- 
               
               
                 CHO-001 
                 dehydehyde 
                 carboxaldehyde 
               
               
                   
                 CHO-002 
                 CHO-003 
               
               
                 4-fluorobenzaldehyde 
                 indole-3-carboxal- 
                 5-methylthiophene-2- 
               
               
                 CHO-004 
                 dehyde 
                 carboxaldehyde 
               
               
                   
                 CHO-005 
                 CHO-006 
               
               
                 4-bromobenzaldehyde 
                 pyrrole-2-carb- 
                 hyde 
               
               
                 CHO-007 
                 oxaldehyde 
                 2-Hydroxy-8-meth- 
               
               
                   
                 CHO-008 
                 oxybenzaldehyde 
               
               
                   
                   
                 CHO-009 
               
               
                 3-methyl-2-thiophene- 
                 3,4-Dibromo-5- 
                 Ethyl-2,4-Dimethyl- 
               
               
                 carboxaldehyde 
                 methyl-2-pyrrole- 
                 5-formyl-3-pyrrole- 
               
               
                 CHO-010 
                 carboxaldehyde 
                 carboxylate 
               
               
                   
                 CHO-011 
                 CHO-012 
               
               
                 3-Bromo-2-hydroxy- 
                 1-Hydroxy-2- 
                 Ethyl-2-(ethoxycar- 
               
               
                 5-methoxybenzal- 
                 napthaldehyde 
                 bonyl)-4-(ethoxy- 
               
               
                 dehyde 
                 CHO-014 
                 carbonylmethyl)- 
               
               
                 CHO-013 
                   
                 5-farmyl-3-pyrr- 
               
               
                   
                   
                 olepropionate 
               
               
                   
                   
                 CHO-015 
               
               
                 Ethyl-5-formyl- 
                 4-Formyl-3-methoy- 
                 2-Hydroxy-3-nitro- 
               
               
                 2-methyl-3-furan- 
                 carbonymethyl-5- 
                 benzaldehyde 
               
               
                 carboxylate 
                 me-1H-pyrrole-2- 
                 CHO-018 
               
               
                 CHO-016 
                 carboxylic acid 
               
               
                   
                 methyl ester 
               
               
                   
                 CHO-017 
               
               
                 2,4-Dihydroxy-3- 
                 Methyl-5-formyl-4- 
                 2-furaldehyde 
               
               
                 methylbenzaldehyde 
                 methyl-3-pyrrole- 
                 CHO-021 
               
               
                 CHO-019 
                 propionate 
               
               
                   
                 CHO-020 
               
               
                 5-Nitro-2-furaldehyde 
                 4-Ethoxy-3-methoxy- 
                 3,4-Dihyroxybenzal- 
               
               
                 CHO-022 
                 benzaldehyde 
                 dehyde 
               
               
                   
                 CHO-023 
                 CHO-024 
               
               
                 2,4-Dimethoxy- 
                 3,5-Dimethyl-4- 
                 2,4,6-trimethoxy- 
               
               
                 benzaldehyde 
                 ethyl-2-pyrrol- 
                 benzaldehyde 
               
               
                 CHO-025 
                 carboxaldehyde 
                 CHO-027 
               
               
                   
                 CHO-026 
               
               
                 4-Hydroxyben- 
                 4-(Dimethylamino)- 
                 2,4-Dimethyl-3- 
               
               
                 zaldehyde 
                 benzaldehyde 
                 carbethoxyopyrrole- 
               
               
                 CHO-028 
                 CHO-029 
                 5-carboxaldehyde 
               
               
                   
                   
                 CHO-030 
               
               
                 2-chloro-4-fluoro- 
                 3-Nitrobenzaldehyde 
                 4-Fluoro-2-(tri- 
               
               
                 benzaldehyde 
                 CHO-032 
                 fluoromethyl)ben- 
               
               
                 CHO-031 
                   
                 zaldehyde 
               
               
                   
                   
                 CHO-033 
               
               
                 2,4,6-Trifluoroben- 
                 4-Hydroxy-2-meth- 
                 3,4-Dimethoxyben- 
               
               
                 zaldehyde 
                 oxybenzaldehyde 
                 zaldehyde 
               
               
                 CHO-034 
                 CHO-035 
                 CHO-036 
               
               
                 Salicylaldehyde 
                 Benzaldehyde 
                 3,5-diethylpyrrole- 
               
               
                 CHO-037 
                 CHO-038 
                 2-carboxaldehyde 
               
               
                   
                   
                 CHO-038 
               
               
                 5-(Methylthio)thio- 
                 2,4-Dihydroxy-6- 
                 Methyl-5-formyl- 
               
               
                 phene-2-carboxal- 
                 methylbenzaldehyde 
                 4-methyl-3-pyrrole- 
               
               
                 dehyde 
                 CHO-040 
                 propionate 
               
               
                 CHO-039 
                   
                 CHO-041 
               
               
                 3-Ethoxy-4-hydroxy- 
                 2-Hydroxy-5-meth- 
                 2-Imidazolecar- 
               
               
                 benzaldehyde 
                 oxylbenzaldehyde 
                 boxaldehyde 
               
               
                 CHO-042 
                 CHO-043 
                 CHO-044 
               
               
                 1-Methyl-2-formyl- 
                 4-Chloro-1-methyl- 
                 2,3-dimethyl-5-for- 
               
               
                 benzimidazole 
                 pyrazole-3-car- 
                 mylthiophene 
               
               
                 CHO-045 
                 boxaldehyde 
                 CHO-047 
               
               
                   
                 CHO-046 
               
               
                 2-Formyl-4,5,6,7- 
                 3-Chloromethyl-5- 
                 1-(3,5-Dichlorophen- 
               
               
                 tetrahydroindole 
                 nitrosalicylaldehyde 
                 yl)pyrrole-2- 
               
               
                 CHO-048 
                 CHO-049 
                 carboxaldehyde 
               
               
                   
                   
                 CHO-050 
               
               
                 5-Chlorothiophene-2- 
                 3,5-dimethyl-5- 
                 3-t-Butyl-4-hydroxy- 
               
               
                 carboxaldehyde 
                 formylpyrrole 
                 benzaldehyde 
               
               
                 CHO-051 
                 CHO-052 
                 CHO-053 
               
               
                 3-t-Butyl-5-bromo- 
                 3,5-Di-tert-butyl-4- 
                 3-t-Butyl-4-hydroxy- 
               
               
                 4-hydroxybenzal- 
                 hydroxybenzaldehyde 
                 5-nitrobenzal- 
               
               
                 dehyde 
                 hemihydrate 
                 dehyde 
               
               
                 CHO-054 
                 CHO-055 
                 CHO-056 
               
               
                 2,4,6-Trihydroxy- 
                 2-formyl-5-nitro- 
                 4-Carboxybenzal- 
               
               
                 benzaldehyde 
                 thiophene 
                 dehyde 
               
               
                 CHO-057 
                 CHO-058 
                 CHO-059 
               
               
                 2,4-difluoroben- 
                 3,5-Dimethyl-4-hy- 
                 3-Chloro-4-hydroxy- 
               
               
                 zaldehyde 
                 droxybenzaldehyde 
                 5-t-butylbenzal- 
               
               
                 CHO-060 
                 CHO-061 
                 dehyde 
               
               
                   
                   
                 CHO-062 
               
               
                 4-Ethoxy-3-methoxy- 
                 2-Nitrothiophene-4- 
                 4-(Dibutylamino)ben- 
               
               
                 benzaldehyde 
                 carboxaldehyde 
                 zaldehyde 
               
               
                 CHO-063 
                 CHO-064 
                 CHO-065 
               
               
                 4-(Trifluorometh- 
                 4,6-Dimethoxy-sali- 
                 2,3,4-Trihydroxyben- 
               
               
                 yl)benzaldehyde 
                 cylaldehyde 
                 zaldehyde 
               
               
                 CHO-066 
                 CHO-O67 
                 CHO-068 
               
               
                 2-Hydroxy-3-meth- 
                 5-Bromo,3,4-dihy- 
                 3,4-Diacetoxyben- 
               
               
                 oxybenzaldehyde 
                 roxybenzaldehyde 
                 zaldehyde 
               
               
                 CHO-069 
                 CHO-070 
                 CHO-071 
               
               
                 4-Hydroxy-3-meth- 
                 2-Bromobenzaldehyde 
                 2,4-Dihydroxyben- 
               
               
                 ylbenzaldehyde 
                 CHO-073 
                 zaldehyde 
               
               
                 CHO-072 
                   
                 CHO-074 
               
               
                 2-Hydroxy-4-meth- 
                 3-Bromobenzaldehyde 
                 3,5-Di-tert-butyl- 
               
               
                 oxybenzaldehyde 
                 CHO-076 
                 2-hydroxybenzaldehyde 
               
               
                 CHO-075 
                   
                 CHO-077 
               
               
                 4-Carboxybenzal- 
                 4-Dimethylamino-1- 
                 4-Hydroxy-3-nitroben- 
               
               
                 dehyde 
                 naphthaldehyde 
                 zaldehyde 
               
               
                 CHO-078 
                 CHO-079 
                 CHO-080 
               
               
                 2-Hydroxy-4-methoxy- 
                 3-Hydroxy-4-nitro- 
                 4-Bromobenzaldehyde 
               
               
                 benzaldehyde 
                 benzaldehyde 
                 CHO-083 
               
               
                 CHO-081 
                 CHO-082 
               
               
                 2,3,6,7-Tetrahy- 
                 3,5-Diisopropyl-4- 
                 Benzo[b]furan-2- 
               
               
                 dro-8-hydroxy-1 
                 hydroxybenzaldehyde 
                 carboxaldehyde 
               
               
                 H, 5H-benzo[ij] 
                 CHO-085 
                 CHO-086 
               
               
                 quinolizine,9 
               
               
                 carboxaldehyde 
               
               
                 CHO-084 
               
               
                 3,5-Diiodo-4-meth- 
                 1-(4-chlorophen- 
                 5-Ethyl-2-furaldehyde 
               
               
                 yl-2-pyrrolcar- 
                 yl) pyrrole-2- 
                 CHO-089 
               
               
                 boxaldehyde 
                 carboxaldehyde 
               
               
                 CHO-087 
                 CHO-088 
               
               
                 3,4-Dimethylthi- 
                 3-Bromothiophene- 
                 6-Bromo-2-hydroxy- 
               
               
                 eno[b]thio- 
                 2-carboxaldehyde 
                 3-methoxybenzal- 
               
               
                 phene-2-carbox- 
                 CHO-091 
                 dehyde 
               
               
                 aldehyde 
                   
                 CHO-092 
               
               
                 CHO-090 
               
               
                 5-Methylfurfural 
                 3-Methyl-1H- 
                 5-Iodo-2-furaldehyde 
               
               
                 CHO-093 
                 Pyrazole-5- 
                 CHO-095 
               
               
                   
                 carboxaldehyde 
               
               
                   
                 CHO-094 
               
               
                 6-Methoxy-4-methyl- 
                 Ethyl 
                 4-Ethyl-5-formy-3- 
               
               
                 sailcylaldehyde 
                 2,4-Dimethyl-5- 
                 methyl-2-pyrrole- 
               
               
                 CHO-096 
                 formyl-3-pyrrole- 
                 carboxylic acid 
               
               
                   
                 carboxylate 
                 CHO-098 
               
               
                   
                 CHO-097 
               
               
                 Ethyl-5-formy-1,2,4- 
                 4-(4-Formylpiper- 
                 4-(4-Formylmorphon- 
               
               
                 trimethyl-3-pyrrole- 
                 azine-1-yl)-ben- 
                 ino-1-yl)-benzal- 
               
               
                 carboxylate 
                 zaldehyde 
                 dehyde 
               
               
                 CHO-099 
                 CHO-100 
                 CHO-101 
               
               
                 5-Chloro-3-methoxy- 
                 1-(4-chlorobenzyl)- 
                 Imidazole-4-carbox- 
               
               
                 carbonyl-4-methoxy- 
                 4-bromo-pyrazole- 
                 aldehyde 
               
               
                 carbonylmethyl- 
                 5-carboxaldehyde 
                 CHO-104 
               
               
                 pyrrole-2-carbox- 
                 CHO-103 
               
               
                 aldehyde 
               
               
                 CHO-102 
               
               
                 4-Chloro-pyrazole- 
                 5-Ethoxycarbonyl- 
                 5-t-Butyl-4-hydro- 
               
               
                 5-carboxaldehyde 
                 4-methyl-3-methyl- 
                 xy-3-iodobenzal- 
               
               
                 CHO-105 
                 carbonyl-pyrrole- 
                 dehyde 
               
               
                   
                 2-carboxaldehyde 
                 CHO-107 
               
               
                   
                 CHO-106 
               
               
                 5-Bromofuran-2- 
                 1,4-Dimethyl-3- 
                 1,4-Dihydroxy-2- 
               
               
                 carboxaldehyde 
                 formylcarbazole 
                 formyl-5,6,7,8,- 
               
               
                 CHO-108 
                 CHO-109 
                 tetrahydronaph- 
               
               
                   
                   
                 thalene 
               
               
                   
                   
                 CHO-110 
               
               
                 5-fluoroisatin 
                 3,4-dimethyl-2- 
                 isatin 
               
               
                 CHO-111 
                 formylpyrrole 
                 CHO-113 
               
               
                   
                 CHO-112 
               
               
                 5-ethyl-2-formyl- 
                 4-methoxybenzal- 
                 4-diethylamino- 
               
               
                 thiophene 
                 dehyde 
                 benzaldehyde 
               
               
                 CHO-114 
                 CHO-115 
                 CHO-116 
               
               
                 3,5-diethylpyrrole- 
                 5-Benzyloxyindole- 
                 3-Bromo-5-chloro- 
               
               
                 2-carboxaldehyde 
                 3-carboxaldehyde 
                 2-hydroxybenzaldehyde 
               
               
                 CHO-117 
                 CHO-118 
                 CHO-119 
               
               
                 2-(4-chlorophenyl- 
                 6-Chloropiperonal 
                 Chromone-3-carbox- 
               
               
                 (thio)benzaldehyde 
                 CHO-121 
                 aldehyde 
               
               
                 CHO-120 
                   
                 CHO-122 
               
               
                 3-Cyanobenzal- 
                 4-Cyanobenzal- 
                 6,8-Dichlorochromone- 
               
               
                 dehyde 
                 dehyde 
                 3-carboxaldehyde 
               
               
                 CHO-123 
                 CHO-124 
                 CHO-125 
               
               
                 2,5-dihydroxy- 
                 2,3-Dimethoxy- 
                 2,4-Dimethoxy- 
               
               
                 benzaldehyde 
                 benzaldehyde 
                 benzaldehyde 
               
               
                 CHO-126 
                 CHO-127 
                 CHO-128 
               
               
                 2,5-Dimethoxy- 
                 2,6-Dimethoxy- 
                 3,5-Dimethoxy- 
               
               
                 benzaldehyde 
                 benzaldehyde, 
                 benzaldehyde 
               
               
                 CHO-129 
                 CHO-130 
                 CHO-131 
               
               
                 4-Dimethylamino- 
                 3,4-Dimethyl- 
                 5,7-Dimethylchro- 
               
               
                 2-methoxyben- 
                 benzaldehyde 
                 mone-3-carboxal- 
               
               
                 zaldehyde 
                 CHO-133 
                 dehyde 
               
               
                 CHO-132 
                   
                 CHO-134 
               
               
                 5-Ethylfurfural 
                 Ferrocenecar- 
                 Fluorene-2-carbox- 
               
               
                 CHO-135 
                 boxaldehyde 
                 aldehyde 
               
               
                   
                 CHO-136 
                 CHO-137 
               
               
                 2-Fluoro-3-(tri- 
                 2-Fluoro-4-(tri- 
                 2-Fluoro-5-(tri- 
               
               
                 fluoromethyl)ben- 
                 fluoromethyl)ben- 
                 fluoromethyl)ben- 
               
               
                 zaldehyde 
                 zaldehyde 
                 zaldehyde 
               
               
                 CHO-138 
                 CHO-139 
                 CHO-140 
               
               
                 2-Fluoro-6-(tri- 
                 2-Formylphenoxy- 
                 3-Methoxy-5,-methyl- 
               
               
                 fluoromethyl)ben- 
                 acetic acid 
                 lenedioxybenzaldehyde 
               
               
                 zaldehyde 
                 CHO-142 
                 CHO-143 
               
               
                 CHO-141 
               
               
                 2-Methoxy-1-naph- 
                 4-Methoxy-1-naph- 
                 4-(Methoylthio)ben- 
               
               
                 thaldehyde 
                 thaldehyde 
                 zaldehyde 
               
               
                 CHO-144 
                 CHO-145 
                 CHO-146 
               
               
                 3-Methylthiophene- 
                 5-Methylthiophene- 
                 pentamethylbenzal- 
               
               
                 2-carboxaldehyde 
                 2-carboxaldehyde 
                 dehyde 
               
               
                 CHO-147 
                 CHO-148 
                 CHO-149 
               
               
                 3-Phenoxybenzal- 
                 Pyridine-2-carbox- 
                 Pyridine-3-carbox- 
               
               
                 dehyde 
                 aldehyde 
                 aldehyde 
               
               
                 CHO-150 
                 CHO-151 
                 CHO-152 
               
               
                 Pyridine-4-carbox- 
                 4-Pyrrolidino- 
                 1,2,3,6-Tetrahydro- 
               
               
                 aldehyde 
                 benzaldehyde, 
                 benzaldehyde 
               
               
                 CHO-153 
                 98+% 
                 CHO-155 
               
               
                   
                 CHO-154 
               
               
                 2,3,4-Trimethoxy- 
                 2,4,5-trimethoxy- 
                 2,4,6-Trimethoxy- 
               
               
                 benzaldehyde 
                 benzaldehyde 
                 benzaldehyde 
               
               
                 CHO-156 
                 CHO-157 
                 CHO-158 
               
               
                 3,4,5-Trimethoxy- 
                 1-Acetyl-3-indole- 
                 6-Chloro-3-formyl- 
               
               
                 benzaldehyde 
                 carboxaldehyde 
                 chromone 
               
               
                 CHO-159 
                 CHO-160 
                 CHO-161 
               
               
                 6-Chloro-3-formyl- 
                 5-(2-Chlorophenyl)- 
                 2-Chloro-3-quinoline- 
               
               
                 7-methylchlromone 
                 furfural 
                 carboxaldehyde 
               
               
                 CHO-162 
                 CHO-163 
                 CHO-164 
               
               
                 6,8-Dibromo-3- 
                 2,5-Dimethoxy-3- 
                 4,5-Dimethyl-2- 
               
               
                 formylchromone 
                 tetrahydrofura- 
                 furaldehyde 
               
               
                 CHO-165 
                 carboxaldehyde 
                 CHO-167 
               
               
                   
                 CHO-166 
               
               
                 9-Ethyl-3-carba- 
                 3-Formyl-6,7- 
                 3-formyl-6,9-di- 
               
               
                 zolecarboxaldehyde 
                 diemthylchromone 
                 methylchromone 
               
               
                 CHO-168 
                 CHO-169 
                 CHO-170 
               
               
                 3-formyl-6-isopro- 
                 3-formyl-6-methyl- 
                 3-formyl-6-nitro- 
               
               
                 pylchromone 
                 chromone 
                 chromone 
               
               
                 CHO-171 
                 CHO-172 
                 CHO-173 
               
               
                 5-Formyluracil 
                 5-Methyoxyindole- 
                 1-Methylisatin 
               
               
                 CHO-174 
                 3-carboxaldehyde 
                 CHO-176 
               
               
                   
                 CHO-175 
               
               
                 5-(2-Nitrophan- 
                 (S)-(-)-Perill- 
                 2-(Trifluoro- 
               
               
                 yl)furfural 
                 aldehyde 
                 acetyl)thiophene 
               
               
                 CHO-177 
                 CHO-178 
                 CHO-179 
               
               
                 3,5-diisopropyl- 
                 4-benzyloxy-3,5- 
                 3-t-butyl-4-meth- 
               
               
                 4-methoxybenzal- 
                 diisopropylbenz- 
                 oxybenzaldehyde 
               
               
                 dehyde 
                 aldehyde 
                 CHO-182 
               
               
                 CHO-180 
                 CHO-181 
               
               
                 4-benzyloxy-3-t- 
                 3-bromo-5-t-butyl-4- 
                 4-benzyloxy-3-bromo- 
               
               
                 butylbenzaldehyde 
                 methoxybenzaldehyde 
                 5-t-butylbenzaldehyde 
               
               
                 CHO-183 
                 CHO-184 
                 CHO-185 
               
               
                 3-t-butyl-5-chloro- 
                 4-benzyloxy-3-t- 
                 3-t-butyl-5-iodo-4- 
               
               
                 4-methoxybenzal- 
                 butyl-5-chloroben- 
                 methoxybenzaldehyde 
               
               
                 dehyde 
                 zaldehyde 
                 CHO-188 
               
               
                 CHO-186 
                 CHO-187 
               
               
                 4-benzyloxy-3-t- 
                 3-t-butyl-4-methoxy- 
                 4-benzyloxy-3-t-butyl- 
               
               
                 butyl-5-iodoben- 
                 5-nitrobenzal- 
                 5-nitrobenzaldehyde 
               
               
                 zaldehyde 
                 dehyde 
                 CHO-191 
               
               
                 CHO-189 
                 CHO-190 
               
               
                 3,5-di-t-butyl-4- 
                 4-benzyloxy-3,5- 
                 3,5-dimethyl-4-meth- 
               
               
                 methoxybenzaldehyde 
                 di-t-butylbenzal- 
                 oxybenzaldehyde 
               
               
                 CHO-192 
                 dehyde 
                 CHO-194 
               
               
                   
                 CHO-193 
               
               
                 4-benzyloxy-3,5- 
                 5-bromo-2-hydroxy- 
                 5-bromosalicyal- 
               
               
                 dimethylbenzal- 
                 3-methoxy-benzal- 
                 dehyde 201 
               
               
                 dehyde 
                 dehyde 
                 CHO-197 
               
               
                 CHO-195 
                 CHO-196 
               
               
                 2-hydroxy-5-ni- 
                 4-hydroxy-2-ni- 
                 3-ethoxysalicyal- 
               
               
                 trobenzaldehyde 
                 tro-3-methoxy- 
                 dehyde 
               
               
                 CHO-198 
                 benzaldehyde 
                 CHO-200 
               
               
                   
                 CHO-199 
               
               
                 3,5-dichlorosali- 
                 5-chlorosalicyal- 
                 4-(diethylamino)sali- 
               
               
                 cyaldehyde 
                 dehyde 
                 cyaldehyde 
               
               
                 CHO-201 
                 CHO-202 
                 CHO-203 
               
               
                 5-(trifluorometh- 
                 3,5-dibromoalicyal- 
                 3-fluorosalicyal- 
               
               
                 oxy)salicylaldehyde 
                 dehyde 
                 dehyde 
               
               
                 CHO-204 
                 CHO-205 
                 CHO-206 
               
               
                 3-bromo-4-hydroxy- 
                 5-chlorosalicyal- 
                 2,4-dimethyl-5- 
               
               
                 benzaldehyde 
                 dehyde 
                 formylpyrrole 
               
               
                 CHO-207 
                 CHO-208 
                 CHO-209 
               
               
                 3,5-diisopropyl- 
                 3,5-dimethylthio- 
                 3-methyl-5-ethyl- 
               
               
                 2-formylpyrrole 
                 phene-2-carboxal- 
                 thiophene-2-carbox- 
               
               
                 CHO-210 
                 dehyde 
                 aldehyde 
               
               
                   
                 CHO-211 
                 CHO-212 
               
               
                 3-methyl-5-isopro- 
                 3-methyl-5-cyclo- 
                 3-methyl-5-cyclopro- 
               
               
                 propylthiophene- 
                 pentylmethylthi- 
                 pylthiophene-2-car- 
               
               
                 2-carboxaldehyde 
                 ophene-2-carbox- 
                 boxaldehyde 
               
               
                 CHO-213 
                 aldehyde 
                 CHO-215 
               
               
                   
                 CHO-214 
               
               
                 4-methyl-5-ethyl- 
                 4-methyl-5-isopro- 
                 4-methyl-5-cyclo- 
               
               
                 thiophene-2-car- 
                 pylthiophene-2- 
                 pentylmethylthi- 
               
               
                 boxaldehyde 
                 carboxaldehyde 
                 ophene-2-carboxal- 
               
               
                 CHO-216 
                 CHO-217 
                 dehyde 
               
               
                   
                   
                 CHO-218 
               
               
                 4-methyl-5-cyclo- 
                 5-isopropylthio- 
                 5-phenylmehylthio- 
               
               
                 propylmethylthio- 
                 phene-2-carboxal- 
                 phene-2-carboxal- 
               
               
                 phene-2-carboxal- 
                 dehyde 
                 dehyde 
               
               
                 dehyde 
                 CHO-220 
                 CHO-221 
               
               
                 CHO-219 
               
               
                 5-cyclohexylmeth- 
                 5-cyclohexylthio- 
                 5-phenylthiophene-2- 
               
               
                 thiophene-2-car- 
                 phene-2-carbox- 
                 carboxaldehyde 
               
               
                 boxaldehyde 
                 aldehyde 
                 CHO-224 
               
               
                 CHO-222 
                 CHO-223 
               
               
                 3-methyl-5-propyl- 
                 3-methyl-5-cyclo- 
                 4-methyl-5-propyl- 
               
               
                 thiophene-2-carbox- 
                 hexylmethylthio- 
                 thiophene-2-carbox- 
               
               
                 aldehyde 
                 2-carboxaldehyde 
                 aldehyde 
               
               
                 CHO-225 
                 CHO-226 
                 CHO-227 
               
               
                 4-methyl-5-cyclo- 
                 5-n-butylthiophene- 
                 5-cyclopropylmethyl- 
               
               
                 hexylmethylthio- 
                 2-carboxaldehyde 
                 thiophene-2-carbox- 
               
               
                 phene-2-carboxal- 
                 CHO-229 
                 aldehyde 
               
               
                 dehyde 
                   
                 CHO-230 
               
               
                 CHO-228 
               
               
                 5-cyclopropylthio- 
                 3-methyl-5-phenyl- 
                 4-methyl-5-phenyl- 
               
               
                 phene-2-carboxal- 
                 methylthiophene- 
                 methylthiophene- 
               
               
                 dehyde 
                 2-carboxaldehyde 
                 2-carboxaldehyde 
               
               
                 CHO-231 
                 CHO-232 
                 CHO-233 
               
               
                 5-cyclopentylmeth- 
                 5-cyclophentylthio- 
                 4,5-dimethylthio- 
               
               
                 ylthiophene-2-car- 
                 phene-2-carboxal- 
                 phene-2-carboxal- 
               
               
                 boxaldehyde 
                 dehyde 
                 dehyde 
               
               
                 CHO-234 
                 CHO-235 
                 CHO-236 
               
               
                 5-n-propylthio- 
               
               
                 phene-2-carboxal- 
               
               
                 dehyde 
               
               
                 CHO-237