Tricyclic quinoxaline derivatives as protein tyrosine kinase inhibitors

The present invention relates to tricyclic quinoxaline compounds and physiologically acceptable salts and prodrugs thereof which modulate the activity of protein tyrosine kinases and therefore should be useful in the prevention and treatment of protein tyrosine kinase related cellular disorders such as cancer.

INTRODUCTION
 The present invention relates generally to organic chemistry, biochemistry,
 pharmacology and medicine. More particularly, it relates to tricyclic
 quinoxaline compounds, and their physiologically acceptable salts and
 prodrugs, which modulate the activity of protein tyrosine kinases ("PTKs")
 and, therefore, are expected to exhibit a salutary effect against
 disorders related to abnormal PTK activity.
 BACKGROUND OF THE INVENTION
 The following is offered as background information only and is not admitted
 to be prior art to the present invention.
 Protein kinases, PKs, are enzymes that catalyze the phosphorylation of
 hydroxy groups on tyrosine, serine and threonine residues of proteins. The
 consequences of this seemingly simple activity are staggering; cell
 growth, differentiation and proliferation; i.e., virtually all aspects of
 cell life in one way or another depend on PK activity. Furthermore,
 abnormal PK activity has been related to a host of disorders, ranging from
 relatively non-life threatening diseases such as psoriasis to extremely
 virulent diseases such as glioblastoma (brain cancer).
 The PKs can conveniently be broken down into two classes, the protein
 tyrosine kinases (PTKs) and the serine-threonine kinases (STKs).
 One of the prime aspects of PK activity is involvement with growth factor
 receptors. Growth factor receptors are cell-surface proteins. When bound
 by a growth factor ligand, growth factor receptors are converted to an
 active form which interacts with proteins on the inner surface of a cell
 membrane. This leads to phosphorylation on tyrosine residues of the
 receptor and other proteins and to the formation inside the cell of
 complexes with a variety of cytoplasmic signaling molecules that, in turn,
 affect numerous cellular responses such as cell division (proliferation),
 cell differentiation, cell growth, expression of metabolic effects on the
 extracellular microenvironment, etc. For a more complete discussion, see
 Schlessinger and Ullrich, Neuron, 9:303-391 (1992) which is incorporated
 by reference, including any drawings, as if fully set forth herein.
 Growth factor receptors with PK activity are known as receptor tyrosine
 kinases ("RTKs"). They comprise a large family of transmembrane receptors
 with diverse biological activity. At present, at least nineteen (19)
 distinct subfamilies of RTKs have been identified. An example of these is
 the subfamily designated the "HER" RTKs, which include EGFR (epithelial
 growth factor receptor), HER2, HER3 and HER4. These RTKs consist of an
 extracellular glycosylated ligand binding domain, a transmembrane domain
 and an intracellular cytoplasmic catalytic domain that can phosphorylate
 tyrosine residues on proteins.
 Another RTK subfamily consists of insulin receptor (IR), insulin-like
 growth factor I receptor (IGF-1R) and the insulin receptor related
 receptor (IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to
 form a heterotetramer of two entirely extracellular glycosylated .alpha.
 subunits and two .beta. subunits which cross the cell membrane and which
 contain the tyrosine kinase domain.
 A third RTK subfamily is referred to as the platelet derived growth factor
 receptor ("PDGFR") group, which includes PDGFR.alpha., PDGFR.beta., CSFIR,
 c-kit and c-fms. These receptors consist of glycosylated extracellular
 domains composed of variable numbers of immunoglobin-like loops and an
 intracellular domain wherein the tyrosine kinase domain is interrupted by
 unrelated amino acid sequences.
 Another group which, because of its similarity to the PDGFR subfamily, is
 sometimes subsumed in the latter group is the fetus liver kinase ("flk")
 receptor subfamily. This group is believed to be composed of kinase insert
 domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4 and
 fms-like tyrosine kinase 1 (flt-1).
 One further member of the tyrosine kinase growth factor receptor family is
 the fibroblast growth factor ("FGF")receptor group. This group consists of
 four receptors, FGFR1-4, and seven ligands, FGF1-7. While not yet well
 characterized, it appears that the receptors consist of a glycosylated
 extracellular domain containing a variable number of immunoglobin-like
 loops and an intracellular domain in which the PTK sequence is interrupted
 by regions of unrelated amino acid sequences.
 A more complete listing of the known RTK subfamilies is described in
 Plowman et al., DN&P, 7(6):334-339 (1994) which is incorporated by
 reference, including any drawings, as if fully set forth herein.
 In addition to the RTKs, there also exists a family of entirely
 intracellular PTKs called "non-receptor tyrosine kinases" or "cellular
 tyrosine kinases." This latter designation, abbreviated "CTK", will be
 used herein. CTKs do not contain extracellular and transmembrane domains.
 At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk, Abl,
 Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Src subfamily
 appear so far to be the largest group of CTKs and includes Src, Yes, Fyn,
 Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detailed discussion of CTKs,
 see Bolen, Oncogene, 8:2025-2031 (1993), which is incorporated by
 reference, including any drawings, as if fully set forth herein.
 The serine-threonine kinases or STKs, like the CTKs, are predominantly
 intracellular although there are a few receptor STKs. STKs are the most
 common of the cytosolic kinases; i.e., kinases which perform their
 function in that part of the cytoplasm other than the cytoplasmic
 organelles and cytoskelton. The cytosol is the region within the cell
 where much of the cell's intermediary metabolic and biosynthetic activity
 occurs; e.g., it is in the cytosol that proteins are synthesized on
 ribosomes.
 RTKs, CTKs and STKs have all been implicated in a host of pathogenic
 conditions including, significantly, cancer. Others pathogenic conditions
 which have been associated with PTKs include, without limitation,
 psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis,
 restenosis, ocular diseases, rheumatoid arthritis and other inflammatory
 disorders, autoimmune disease and a variety of renal disorders.
 With regard to cancer, two of the major hypotheses advanced to explain the
 excessive cellular proliferation that drives tumor development relate to
 functions known to be PK regulated. That is, it has been suggested that
 malignant cell growth results from a breakdown in the mechanisms that
 control cell division and/or differentiation. It has been shown that the
 protein products of a number of proto-oncogenes are involved in the signal
 transduction pathways that regulate cell growth and differentiation. These
 protein products of proto-oncogenes include the extracellular growth
 factors, transmembrane growth factor PTK receptors (RTKs), cytoplasmic
 PTKs (CTKs) and cytosolic STKs, discussed above.
 In view of the apparent link between PK-related cellular activities and a
 number of human disorders, it is no surprise that a great deal of effort
 is being spent in an attempt to identify ways to modulate PK activity.
 Some of these efforts have involved biomimetic approaches using large
 molecules patterned after those involved in the actual cellular processes
 (e.g., mutant ligands (U.S. application Ser. No. 4,966,849); soluble
 receptors and antibodies (App. No. WO 94/10202, Kendall and Thomas, Proc.
 Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature, 362:841-844
 (1993)); RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56); Takano,
 et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp. Cell Res.
 199:56-62 (1992); Wright, et al., J. Cellular Phys., 152:448-57) and
 tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO
 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., Proc. Am. Assoc.
 Cancer Res., 35:2268 (1994)).
 More recently, attempts have been made to identify small molecules which
 act as PK inhibitors. For example, bis-monocylic, bicyclic and
 heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole
 derivatives (PCT WO 94/14808) and 1-cyclopropyl-4-pyridylquinolones (U.S.
 Pat. No. 5,330,992) have been described as PTK inhibitors. Styryl
 compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds
 (U.S. Pat. No. 5,302,606), quinazoline derivatives (EP App. No. 0 566 266
 A1), selenaindoles and selenides (PCT WO 94/03427), tricyclic
 polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid
 compounds (PCT WO 91/15495) have all been described as PTK inhibitors
 useful in the treatment of cancer.
 SUMMARY OF THE INVENTION
 Our own efforts to identify small organic molecules which modulate PK
 activity and which, therefore, would be expected to be useful in the
 treatment or prevention of disorders driven by abnormal PK activity, has
 led us to the discovery of a family of tricyclic quinoxaline compounds
 which exhibit such PK-modulating activity and which are the subject of
 this invention.
 Thus, the present invention relates generally to tricyclic quinoxaline
 derivatives which modulate the activity of both receptor (RTK) and
 non-receptor (CTK) protein tyrosine kinases (PTKs). In addition, the
 present invention relates to the preparation and use of pharmacological
 compositions of the disclosed compounds and their physiologically
 acceptable salts and prodrugs in the treatment or prevention of PTK driven
 disorders such as, by way of example and not limitation, cancer, diabetes,
 hepatic cirrhosis, atherosclerosis, angiogenesis and renal disease.
 As used herein a "tricyclic quinoxaline derivative" refers to a chemical
 compound having the general structure shown in Formula 1.
 A "pharmacological composition" refers to a mixture of one or more of the
 compounds described herein, or physiologically acceptable salts or
 prodrugs thereof, with other chemical components such as physiologically
 acceptable carriers and excipients. The purpose of a pharmacological
 composition is to facilitate administration of a compound to an organism.
 A "prodrug" refers to an agent which is converted into the parent drug in
 vivo. Prodrugs are often useful because, in some situations, they may be
 easier to administer than the parent drug. They may, for instance, be
 bioavailable by oral administration whereas the parent drug is not. The
 prodrug may also have improved solubility compared to the parent drug in
 pharmacological compositions. An example, without limitation, of a prodrug
 would be a compound of the present invention which is administered as an
 ester (the "prodrug") to facilitate transmittal across a cell membrane
 where water solubility is not beneficial, but which then is metabolically
 hydrolyzed to the carboxylic acid once inside the cell where water
 solubility is beneficial.
 As used herein, an "ester" is a carboxy group, as defined herein, wherein
 R" is any of the listed groups other than hydrogen.
 As used herein, a "physiologically acceptable carrier" refers to a carrier
 or diluent that does not cause significant irritation to an organism and
 does not abrogate the biological activity and properties of the
 administered compound.
 An "excipient" refers to an inert substance added to a pharmacological
 composition to further facilitate administration of a compound. Examples,
 without limitation, of excipients include calcium carbonate, calcium
 phosphate, various sugars and types of starch, cellulose derivatives,
 gelatin, vegetable oils and polyethylene glycols.
 1. THE COMPOUNDS
 A. General Structural Features.
 In one aspect, the present invention relates to compounds having the
 structure shown in Formula 1:
 ##STR1##
 The dotted line in the five-member ring containing A, B and D means that
 either the A--B bond or the B--D bond is a double bond.
 A, B, and D are independently selected from the group consisting of carbon,
 nitrogen, oxygen and sulfur, such that the resulting fused 5-member
 ring/6-member ring is one known in the chemical arts. It is understood
 that when A, B or D is oxygen or sulfur, R.sup.1, R.sup.2 and R.sup.3,
 respectively, do not exist. Furthermore, when A, B or D is nitrogen and
 that nitrogen is participating in a double bond, R.sup.1, R.sup.2 or
 R.sup.3, respectively, does not exist.
 When A, B or D is nitrogen and that nitrogen is not participating in a
 double bond, R.sup.1, R.sup.2 or R.sup.3 is selected from the group
 consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
 hydroxy, alkoxy, C-carboxy, O-carboxy, carbonyl, thiocarbonyl, C-amido,
 guanyl, sulfonyl and trihalomethylsulfonyl.
 R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are independently selected from the
 group consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,
 alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,
 thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, N-sulfonamido,
 S-sulfonamido, trihalomethanesulfonamido, carbonyl, thiocarbonyl,
 C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl,
 N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, ureido, guanyl, guanidino,
 amino, and --NR.sup.10 R.sup.11, with the proviso that, when one of
 R.sup.5 or R.sup.6 is hydrogen, methyl or phenyl, the other is not any of
 hydrogen, methyl or phenyl.
 R.sup.10 and R.sup.11 are independently selected from the group consisting
 of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, sulfonyl and, combined, a
 five- or six-member heteroalicyclic ring.
 Physiologically acceptable salts and prodrugs of the claimed compounds are
 also within the scope of this invention.
 Examples of a "fused 5-member ring/6-member ring known in the chemical
 arts" include, but are not limited to:
 ##STR2##
 As used herein, to "participate in a double bond" means to be one of two
 atoms which are double-bonded to one another.
 As used herein, the term "alkyl" refers to a saturated aliphatic
 hydrocarbon including straight chain and branched chain groups.
 Preferably, the alkyl group has 1 to 20 carbon atoms (whenever a numerical
 range; e.g. "1-20", is stated herein, it means that the group, in this
 case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon
 atoms, etc. up to and including 20 carbon atoms). More preferably, it is a
 medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a
 lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted
 or unsubstituted. When substituted, the substituent group is preferably
 one or more independently selected from cycloalkyl, aryl, heteroaryl,
 heteroalicyclic, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
 thioaryloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl,
 O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,
 nitro, sulfonamido, trihalomethanesulfonamido, silyl, guanyl, guanidino,
 ureido, amino and NR.sup.10 R.sup.11, wherein R.sup.10 and R.sup.11 are
 independently selected from the group consisting of hydrogen, alkyl,
 cycloalkyl, aryl, carbonyl, sulfonyl, trihalomethysulfonyl and, combined,
 a five- or six-member heteroalicyclic ring.
 A "cycloalkyl" group refers to an all-carbon monocyclic or fused ring
 (i.e., rings which share an adjacent pair of carbon atoms) group wherein
 one of more of the rings does not have a completely conjugated pi-electron
 system. Examples, without limitation, of cycloalkyl groups are
 cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,
 cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. A
 cycloalkyl group may be substituted or unsubstituted. When substituted,
 the substituent group(s) is preferably one or more indepedently selected
 from alkyl, aryl, heteroaryl, heteroalycyclic, hydroxy, alkoxy, aryloxy,
 thiohydroxy, thioalkoxy, thioaryloxy, cyano, halo, carbonyl, thiocarbonyl,
 C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro,
 amino and NR.sup.10 R.sup.11, with R.sup.10 and R.sup.11 being as defined
 above.
 An "alkenyl" group refers to an alkyl group which consists of at least two
 carbon atoms and at least one carbon--carbon double bond.
 An "alkynyl" group refers to an alkyl group which consists of at least two
 carbon atoms and at least one carbon-carbon triple bond.
 An "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic
 (i.e., rings which share adjacent pairs of carbon atoms) groups having a
 completely conjugated pi-electron system. Examples, without limitation, of
 aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may
 be substituted or unsubstituted. When substituted, the substituent group
 is preferably one or more selected from halo, trihalomethyl, alkyl,
 hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano,
 nitro, azido, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl,
 N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl,
 sulfonyl, amino and NR.sup.10 R.sup.11 wherein R.sup.10 and R.sup.11 are
 previously defined herein.
 As used herein, a "heteroaryl" group refers to a monocyclic or fused ring
 (i.e., rings which share an adjacent pair of atoms) group having in the
 ring(s) one or more atoms selected from the group consisting of nitrogen,
 oxygen and sulfur and, in addition, having a completely conjugated
 pi-electron system. Examples, without limitation, of heteroaryl groups are
 pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole,
 pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl
 group may be substituted or unsubstituted. When substituted, the
 substituted group is preferably one or more selected from alkyl,
 cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, thiohydroxy,
 thioalkoxy, thioaryloxy, cyano, azido, nitro, carbonyl, thiocarbonyl,
 sulfonamido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O-carbamyl,
 N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and
 NR.sup.10 R.sup.11 where R.sup.10 and R.sup.11 are previously defined
 herein.
 A "heteroalicyclic" group refers to a monocyclic or fused ring group having
 in the ring(s) one or more atoms selected from the group consisting of
 nitrogen, oxygen and sulfur. The rings may also have one or more double
 bonds. However, the rings do not have a completely conjugated pi-electron
 system. The heteroalicyclic ring may be substituted or unsubstituted. When
 substituted, the substituted group(s) is preferably one or more selected
 from alkyl, cycloaklyl, aryl, heteroaryl, halo, trihalomethyl, hydroxy,
 alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro,
 carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,
 O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido,
 amino and NR.sup.10 R.sup.11 where R.sup.10 and R.sup.11 are previously
 defined herein.
 A "hydroxy" group refers to an --OH group.
 An "azido" group refers to a --N.ident.N group.
 An "alkoxy" group refers to both an --O-alkyl and an --O-cycloalkyl group,
 as defined herein.
 An "aryloxy" group refers to both an --O-aryl and an --O-heteroaryl group,
 as defined herein.
 A "thiohydroxy" group refers to an --SH group.
 A "thioalkoxy" group refers to both an S-alkyl and an --S-cycloalkyl group,
 as defined herein.
 A "thioaryloxy" group refers to both an --S-aryl and an --S-heteroaryl
 group, as defined herein.
 A "carbonyl" group refers to a --C(.dbd.O)--R" group, where R" is selected
 from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl
 (bonded through a ring carbon) and heteroalicyclic (bonded through a ring
 carbon), as defined herein.
 An "aldehyde" group refers to a carbonyl group where R" is hydrogen.
 A "thiocarbonyl" group refers to a --C(.dbd.S)--R" group, with R" as
 defined herein.
 A "C-carboxy" group refers to a --C(.dbd.O)O--R" groups, with R" as defined
 herein.
 An "O-carboxy" group refers to a R"C(.dbd.O)O-- group, with R" as defined
 herein.
 A "carboxylic acid" group refers to a C-carboxyl group in which R" is
 hydrogen.
 A "halo" group refers to fluorine, chlorine, bromine or iodine.
 A "trihalomethyl" group refers to a --CX.sub.3 group wherein X is a halo
 group as defined herein.
 A "trihalomethanesulfonyl" group refers to a X.sub.3 CS(.dbd.O).sub.2 --
 groups with X as defined above.
 A "sulfinyl" group refers to a --S(.dbd.O)--R" group, with R" as defined
 herein.
 A "sulfonyl" group refers to a --S(.dbd.O).sub.2 R" group, with R" as
 defined herein.
 An "S-sulfonamido" group refers to a --S(.dbd.O).sub.2 NR.sup.10 R.sup.11
 group, with R.sup.10 and R.sup.11 as defined herein.
 An "N-sulfonamido" group refers to a R.sup.10 (.dbd.O).sub.2 NR.sup.11 --
 group, with R.sup.10 and R.sup.11 as defined herein.
 A "trihalomethanesulfonamido" group refers to a X.sub.3 CS(.dbd.O).sub.2
 NR.sup.10 -- group with R.sup.10 as defined herein.
 An "O-carbamyl" group refers to a --OC(.dbd.O)NR.sup.10 R.sup.11 group with
 R.sup.10 and R.sup.11 as defined herein.
 An "N-carbamyl" group refers to a R.sup.11 OC(.dbd.O)NR.sup.10 -- group,
 with R.sup.10 and R.sup.11 as defined herein.
 An "O-thiocarbamyl" group refers to a --OC(.dbd.S)NR.sup.10 R.sup.11 group
 with R.sup.10 and R.sup.11 as defined herein.
 An "N-thiocarbamyl" group refers to a R.sup.11 OC(.dbd.S)NR.sup.10 --
 group, with R.sup.10 and R.sup.11 as defined herein.
 An "amino" group refers to an --NH.sub.2 group.
 A "C-amido" group refers to a --C(.dbd.O)NR.sup.10 R.sup.11 group with
 R.sup.10 and R.sup.11 as defined herein.
 An "N-amido" group refers to a R.sup.11 C(.dbd.O)NR.sup.10 -- group, with
 R.sup.10 and R.sup.11 as defined herein.
 A "quaternary ammonium" group refers to a --.sup.+ NHR.sup.10 R.sup.11
 group wherein R.sup.10 and R.sup.11 are independently selected from the
 group consisting of alkyl, cycloalkyl, aryl, and heteroaryl.
 A "ureido" group refers to a --NR.sup.10 C(.dbd.O)NR.sup.11 R.sup.12 group,
 with R.sup.10 and R.sup.11 as defined herein and R.sup.12 defined the same
 as R.sup.10 and R.sup.11.
 A "guanidino" group refers to a --R.sup.10 NC(.dbd.N)NR.sup.11 R.sup.12
 group, with R.sup.10, R.sup.11 and R.sup.12 as defined herein.
 A "guanyl" group refers to a R.sup.10 R.sup.11 NC(.dbd.N)-- group, with
 R.sup.10 and R.sup.11 as defined herein.
 A "nitro" group refers to a --NO.sub.2 group.
 A "cyano" group refers to a --C.ident.N group.
 A "silyl" group refers to a --Si(R").sub.3, with R" as defined herein.
 B. Preferred Structural Features.
 Preferred structural features of this invention are those in which:
 A and D are nitrogen, B is carbon and either A or D is participating in a
 double bond.
 The "R" group bonded to whichever of A or D is not participating in a
 double bond; i.e., R.sup.1 or R.sup.3, is selected from the group
 consisting of hydrogen, alkyl, halo, cycloalkyl, aryl, carbonyl and
 C-carboxy.
 R.sup.2 is selected from the group consisting of hydrogen, alkyl,
 cycloalkyl, trihalomethyl, aryl, hydroxy, alkoxy, aryloxy, O-carboxy,
 amino and NR.sup.10 R.sup.11 where R.sup.10 and R.sup.11 are independently
 selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl,
 carbonyl, sulfonyl, trihalomethanesulfonyl and, combined, a five- or
 six-member heteroalicyclic ring.
 R.sup.4 and R.sup.7 are independently selected from the group consisting of
 hydrogen, alkyl, alkoxy, aryloxy, C-carboxy, cycloalkyl, hydroxy and halo.
 R.sup.5 and R.sup.6 are independently selected from the group consisting of
 hydrogen, alkyl, cycloalkyl, alkoxy, aryl, aryloxy, heteroaryl,
 heteroalicylic, hydroxy and halo with the proviso that, when one of
 R.sup.5 or R.sup.6 is hydrogen, methyl or phenyl, the other is not any of
 hydrogen, methyl or phenyl.
 Further preferred embodiments of this invention are those in which:
 A is nitrogen which is participating in a double bond.
 D is sulfur.
 R.sup.2, R.sup.4 and R.sup.7 are independently selected from the group
 consisting of hydrogen, alkyl and cycloalkyl.
 R.sup.5 and R.sup.6 are independently selected from the group consisting of
 hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy,
 alkoxy and aryloxy.
 Additional preferred embodiments of this invention are those in which:
 A is sulfur.
 B and D are carbon atoms which are participating in a double bond.
 R.sup.2 and R.sup.3 are independently selected from the group consisting of
 hydrogen, alkyl, cycloalkyl, aryl, C-carboxy and C-amido.
 R.sup.4 and R.sup.7 are independently selected from the group consisting of
 hydrogen, alkyl, cycloalkyl, hydroxy, alkoxy, aryloxy and carbonyl.
 R.sup.5 and R.sup.6 are independently selected from the group consisting of
 hydrogen, alkyl, aryl, heteroaryl and heteroalicyclic with the proviso
 that, when one of R.sup.5 or R.sup.6 is hydrogen, methyl or phenyl, the
 other is not any of hydrogen, methyl or phenyl.
 The compounds shown in Table 1, infra, comprise still further preferred
 embodiments of this invention. The substituent designations refer to
 Formula 1, supra.
 TABLE 1
 Compound
 No. A B D R.sup.1 R.sup.2 R.sup.3 R.sup.4 R.sup.5
 R.sup.6 R.sup.7
 1 N C N CH CH.sub.3 -- H p-CH.sub.3 Ph
 H H
 2 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 OPh
 H H
 3 N C N CH.sub.3 CH.sub.3 -- H p-HOPh
 H H
 4 N C N CH.sub.3 CH.sub.3 -- H p-(CH.sub.3).sub.2
 NPh H H
 5 N C N CH.sub.3 CH.sub.3 -- H p-N.sub.3 Ph
 H H
 6 N C N CH.sub.3 CH.sub.3 -- H furan
 H H
 7 N C N CH.sub.3 CH.sub.3 -- H H
 p-NO.sub.2 Ph H
 8 N C N CH.sub.3 CH.sub.3 -- H H
 p-CH.sub.3 Ph H
 9 N C N CH.sub.3 CH.sub.3 -- H H
 p-CH.sub.3 OPh H
 10 N C N CH.sub.3 CH.sub.3 -- H H
 p-HOPh H
 11 N C N CH.sub.3 CH.sub.3 -- H H
 p-(CH.sub.3).sub.2 NPh H
 12 N C N CH.sub.3 CH.sub.3 -- H H
 p-N.sub.3 Ph H
 13 N C N CH.sub.3 CH.sub.3 -- H H
 furan H
 14 N C N CH.sub.3 CH.sub.3 -- H p-BrPh
 p-BrPh H
 15 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 16 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 17 N C N CH.sub.3 CH.sub.3 -- H p-(CH.sub.3).sub.2
 NPh p-(CH.sub.3).sub.2 NPh H
 18 N C N CH.sub.3 CH.sub.3 -- H o-ClPh
 o-ClPh H
 19 N C N CH.sub.3 CH.sub.3 -- H m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 20 N C N CH.sub.3 CH.sub.3 -- H 3-Br-6-OHPh
 3-Br-6-OHPh H
 21 N C N CH.sub.3 CH.sub.3 -- H furan-2-yl
 furan-2-yl H
 22 N C N CH.sub.3 CH.sub.3 -- H thiophene-2-yl
 thiophene-2-yl H
 23 N C N CH.sub.3 CH.sub.3 -- H pyridine-2-yl
 pyridine-2-yl H
 24 N C N CH.sub.3 CH.sub.3 -- H 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 25 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 Ph
 H H
 26 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 Ph H
 27 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 Ph
 Ph H
 28 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-NO.sub.2 Ph
 H H
 29 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 Ph
 H H
 30 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 OPh
 H H
 31 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-HOPh
 H H
 32 N C N CH.sub.3 CH.sub.3 -- CH.sub.3
 p-(CH.sub.3).sub.2 NPh H H
 33 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-N.sub.3 Ph
 H H
 34 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 furan
 H H
 35 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-N.sub.2 Ph H
 36 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-CH.sub.3 Ph H
 37 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-CH.sub.3 OPh H
 38 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 o-OHPh H
 39 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-(CH.sub.3).sub.2 NPh H
 40 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-N.sub.3 Ph H
 41 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 furan H
 42 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-BrPh
 p-BrPh H
 43 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 44 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 45 N C N CH.sub.3 CH.sub.3 -- CH.sub.3
 p-(CH.sub.3).sub.2 NPh p-(CH.sub.3).sub.2 NPh H
 46 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 o-ClPh
 o-ClPh H
 47 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 48 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 3-Br-6-OHPh
 3-Br-6-OHPh H
 49 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 furan-2-yl
 furan-2-yl H
 50 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 thiophene-2-yl
 thiophene-2-yl H
 51 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 pyridine-2-yl
 pyridine-2-yl H
 52 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 53 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 CH.sub.3
 H H
 54 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 CH.sub.3 H
 55 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 CH.sub.3
 CH.sub.3 H
 56 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 Ph
 H H
 57 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 Ph H
 58 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 Ph
 Ph H
 59 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 CH.sub.3
 H H
 60 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 CH.sub.3 H
 61 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 CH.sub.3
 CH.sub.3 H
 62 N C N CH.sub.3 CH.sub.3 -- F Ph
 H H
 63 N C N CH.sub.3 CH.sub.3 -- F H
 Ph H
 64 N C N CH.sub.3 CH.sub.3 -- F Ph
 Ph H
 65 N C N CH.sub.3 CH.sub.3 -- F p-NO.sub.2 Ph
 H H
 66 N C N CH.sub.3 CH.sub.3 -- F p-CH.sub.3 Ph
 H H
 67 N C N CH.sub.3 CH.sub.3 -- F p-CH.sub.3 OPh
 H H
 68 N C N CH.sub.3 CH.sub.3 -- F p-HOPh
 H H
 69 N C N CH.sub.3 CH.sub.3 -- F p-(CH.sub.3).sub.2
 NPh H H
 70 N C N CH.sub.3 CH.sub.3 -- F p-N.sub.3 Ph
 H H
 71 N C N CH.sub.3 CH.sub.3 -- F furan
 H H
 72 N C N CH.sub.3 CH.sub.3 -- F H
 p-NO.sub.2 Ph H
 73 N C N CH.sub.3 CH.sub.3 -- F H
 p-CH.sub.3 Ph H
 74 N C N CH.sub.3 CH.sub.3 -- F H
 p-CH.sub.3 OPh H
 75 N C N CH.sub.3 CH.sub.3 -- F H
 p-HOPh H
 76 N C N CH.sub.3 CH.sub.3 -- F H
 p-(CH.sub.3).sub.2 NPh H
 77 N C N CH.sub.3 CH.sub.3 -- F H
 p-N.sub.3 Ph H
 78 N C N CH.sub.3 CH.sub.3 -- F H
 furan H
 79 N C N CH.sub.3 CH.sub.3 -- F p-BrPh
 p-BrPh H
 80 N C N CH.sub.3 CH.sub.3 -- F p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 81 N C N CH.sub.3 CH.sub.3 -- F p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 82 N C N CH.sub.3 CH.sub.3 -- F p-(CH.sub.3).sub.2
 NPh p-(CH.sub.3).sub.2 NPh H
 83 N C N CH.sub.3 CH.sub.3 -- F o-Clph
 o-ClPh H
 84 N C N CH.sub.3 CH.sub.3 -- F m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 85 N C N CH.sub.3 CH.sub.3 -- F 3-Br-6-OHPh
 3-Br-6-OHPh H
 86 N C N CH.sub.3 CH.sub.3 -- F furan-2-yl
 furan-2-yl H
 87 N C N CH.sub.3 CH.sub.3 -- F thiophene-2-yl
 thiophene-2-yl H
 88 N C N CH.sub.3 CH.sub.3 -- F pyridine-2-yl
 pyridine-2-yl H
 89 N C N CH.sub.3 CH.sub.3 -- F 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 90 N C N CH.sub.3 CH.sub.3 -- COOH Ph
 H H
 91 N C N CH.sub.3 CH.sub.3 -- COOH H
 Ph H
 92 N C N CH.sub.3 CH.sub.3 -- COOH Ph
 H H
 93 N C N CH.sub.3 CH.sub.3 -- COOH p-NO.sub.2 Ph
 H H
 94 N C N CH.sub.3 CH.sub.3 -- COOH p-CH.sub.3 Ph
 H H
 95 N C N CH.sub.3 CH.sub.3 -- COOH p-CH.sub.3 OPh
 H H
 96 N C N CH.sub.3 CH.sub.3 -- COOH p-HOPh
 H H
 97 N C N CH.sub.3 CH.sub.3 -- COOH p-(CH.sub.3).sub.2
 NPh H H
 98 N C N CH.sub.3 CH.sub.3 -- COOH p-N.sub.3 Ph
 H H
 99 N C N CH.sub.3 CH.sub.3 -- COOH furan
 H H
 100 N C N CH.sub.3 CH.sub.3 -- COOH H
 p-NO.sub.2 Ph H
 101 N C N CH.sub.3 CH.sub.3 -- COOH H
 p-CH.sub.3 Ph H
 102 N C N CH.sub.3 CH.sub.3 -- COOH H
 p-CH.sub.3 OPh H
 103 N C N CH.sub.3 CH.sub.3 -- COOH H
 p-HOPh H
 104 N C N CH.sub.3 CH.sub.3 -- COOH H
 p-(CH.sub.3).sub.2 NPh H
 105 N C N CH.sub.3 CH.sub.3 -- COOH H
 p-N.sub.3 Ph H
 106 N C N CH.sub.3 CH.sub.3 -- COOH H
 furan H
 107 N C N CH.sub.3 CH.sub.3 -- COOH p-BrPh
 p-BrPh H
 108 N C N CH.sub.3 CH.sub.3 -- COOH p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 109 N C N CH.sub.3 CH.sub.3 -- COOH p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 110 N C N CH.sub.3 CH.sub.3 -- COOH p-(CH.sub.3).sub.2
 NPh p-(CH.sub.3).sub.2 NPh H
 111 N C N CH.sub.3 CH.sub.3 -- COOH o-ClPh
 o-ClPh H
 112 N C N CH.sub.3 CH.sub.3 -- COOH m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 113 N C N CH.sub.3 CH.sub.3 -- COOH 3-Br-6-OHPh
 3-Br-6-OHPh H
 114 N C N CH.sub.3 CH.sub.3 -- COOH furan-2-yl
 furan-2-yl H
 115 N C N CH.sub.3 CH.sub.3 -- COOH thiophene-2-yl
 thiophene-2-yl H
 116 N C N CH.sub.3 CH.sub.3 -- COOH pyridine-2-yl
 pyridine-2-yl H
 117 N C N CH.sub.3 CH.sub.3 -- COOH 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 118 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 Ph
 H H
 119 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 Ph H
 120 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 Ph
 Ph H
 121 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-NO.sub.2 Ph
 H H
 122 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-CH.sub.3 Ph
 H H
 123 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-CH.sub.3 OPh
 H H
 124 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-HOPh
 H H
 125 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3
 p-(CH.sub.3).sub.2 NPh H H
 126 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-N.sub.3 Ph
 H H
 127 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 furan
 H H
 128 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 p-NO.sub.2 Ph H
 129 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 p-CH.sub.3 Ph H
 130 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 p-CH.sub.3 OPh H
 131 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 p-HOPh H
 132 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 p-(CH.sub.3).sub.2 NPh H
 133 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 p-N.sub.3 Ph H
 134 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 H
 furan H
 135 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-BrPh
 p-BrPh H
 136 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 137 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 138 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3
 p-(CH.sub.3).sub.2 NPh p-(CH.sub.3).sub.2 NPh H
 139 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 o-Clph
 o-ClPh H
 140 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 141 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 3-Br-6-OHPh
 3-Br-6-OHPh H
 142 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 furan-2-yl
 furan-2-yl H
 143 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 thiophene-2-yl
 thiophene-2-yl H
 144 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 pyridine-2-yl
 pyridine-2-yl H
 145 N C N CH.sub.3 CH.sub.3 -- OCH.sub.3 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 146 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 Ph
 H H
 147 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 OPh
 H H
 148 N C N CH.sub.3 CH.sub.3 -- H p-HOPh
 H H
 149 N C N CH.sub.3 CH.sub.3 -- H p-(CH.sub.3).sub.2
 NPh H H
 150 N C N CH.sub.3 CH.sub.3 -- H p-N.sub.3 Ph
 H H
 151 N C N CH.sub.3 CH.sub.3 -- H furan
 H H
 152 N C N CH.sub.3 CH.sub.3 -- H H
 p-NO.sub.2 Ph H
 153 N C N CH.sub.3 CH.sub.3 -- H H
 p-CH.sub.3 Ph H
 154 N C N CH.sub.3 CH.sub.3 -- H H
 p-CH.sub.3 OPh H
 155 N C N CH.sub.3 CH.sub.3 -- H H
 p-HOPh H
 156 N C N CH.sub.3 CH.sub.3 -- H H
 p-(CH.sub.3).sub.2 NPh H
 157 N C N CH.sub.3 CH.sub.3 -- H H
 p-N.sub.3 Ph H
 158 N C N CH.sub.3 CH.sub.3 -- H H
 furan H
 159 N C N CH.sub.3 CH.sub.3 -- H p-BrPh
 p-BrPh H
 160 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 161 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 162 N C N CH.sub.3 CH.sub.3 -- H p-(CH.sub.3).sub.2
 NPh p-(CH.sub.3).sub.2 NPh H
 163 N C N CH.sub.3 CH.sub.3 -- H o-ClPh
 o-Clph H
 164 N C N CH.sub.3 CH.sub.3 -- H m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 165 N C N CH.sub.3 CH.sub.3 -- H 3-Br-6-OHPh
 3-Br-6-OHPh H
 166 N C N CH.sub.3 CH.sub.3 -- H furan-2-yl
 furan-2-yl H
 167 N C N CH.sub.3 CH.sub.3 -- H thiophene-2-yl
 thiophene-2-yl H
 168 N C N CH.sub.3 CH.sub.3 -- H pyridine-2-yl
 pyridine-2-yl H
 169 N C N CH.sub.3 CH.sub.3 -- H 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 170 N C N CH.sub.3 CH.sub.3 -- H Ph
 H H
 171 N C N CH.sub.3 CH.sub.3 -- H H
 Ph H
 172 N C N CH.sub.3 CH.sub.3 -- H Ph
 Ph H
 173 N C N CH.sub.3 CH.sub.3 -- H CH.sub.3
 H H
 174 N C N CH.sub.3 CH.sub.3 -- H H
 CH.sub.3 H
 175 N C N CH.sub.3 CH.sub.3 -- H CH.sub.3
 CH.sub.3 H
 176 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 Ph
 H H
 177 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 OPh
 H H
 178 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-HOPh
 H H
 179 N C N CH.sub.3 CH.sub.3 -- CH.sub.3
 p-(CH.sub.3).sub.2 NPh H H
 180 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-N.sub.3 Ph
 H H
 181 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 furan
 H H
 182 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-NO.sub.2 Ph H
 183 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-CH.sub.3 Ph H
 184 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-CH.sub.3 OPh H
 185 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-HOPh H
 186 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-(CH.sub.3).sub.2 NPh H
 187 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 p-N.sub.3 Ph H
 188 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 furan H
 189 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-BrPh
 p-BrPh H
 190 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 191 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 192 N C N CH.sub.3 CH.sub.3 -- CH.sub.3
 p-(CH.sub.3).sub.2 NPh p-(CH.sub.3).sub.2 NPh H
 193 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 o-Clph
 o-Clph H
 194 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 195 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 3-Br-6-OHPh
 3-Br-6-OHPh H
 196 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 furan-2-yl
 furan-2-yl H
 197 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 thiophene-2-yl
 thiophene-2-yl H
 198 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 pyridine-2-yl
 pyridine-2-yl H
 199 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 200 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 Ph
 H H
 201 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 H
 Ph H
 202 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 Ph
 Ph H
 203 N C N CH.sub.3 CH.sub.3 -- CH.sub.3 CH.sub.3
 CH.sub.3 H
 204 N C N CH.sub.3 CH.sub.3 -- H H
 p-NO.sub.2 Ph H
 205 N C N CH.sub.3 CH.sub.3 -- H H
 p-CH.sub.3 Ph H
 206 N C N CH.sub.3 CH.sub.3 -- H H
 p-CH.sub.3 OPh H
 207 N C N CH.sub.3 CH.sub.3 -- H H
 p-HOPh H
 208 N C N CH.sub.3 CH.sub.3 -- H H
 p-(CH.sub.3).sub.2 NPh H
 209 N C N CH.sub.3 CH.sub.3 -- H H
 p-N.sub.3 Ph H
 210 N C N CH.sub.3 CH.sub.3 -- H H
 furan H
 211 N C N CH.sub.3 CH.sub.3 -- H p-BrPh
 p-BrPh H
 212 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 Ph
 p-CH.sub.3 Ph H
 213 N C N CH.sub.3 CH.sub.3 -- H p-CH.sub.3 OPh
 p-CH.sub.3 OPh H
 214 N C N CH.sub.3 CH.sub.3 -- H p-(CH.sub.3).sub.2
 NPh p-(CH.sub.3).sub.2 NPh H
 215 N C N CH.sub.3 CH.sub.3 -- H o-ClPh
 o-ClPh H
 216 N C N CH.sub.3 CH.sub.3 -- H m-CH.sub.3 OPh
 m-CH.sub.3 OPh H
 217 N C N CH.sub.3 CH.sub.3 -- H 3-Br-6-OHPh
 3-Br-6-OHPh H
 218 N C N CH.sub.3 CH.sub.3 -- H furan-2-yl
 furan-2-yl H
 219 N C N CH.sub.3 CH.sub.3 -- H thiophene-2-yl
 thiophene-2-yl H
 220 N C N CH.sub.3 CH.sub.3 -- H pyridine-2-yl
 pyridine-2-yl H
 221 N C N CH.sub.3 CH.sub.3 -- H 6-CH.sub.3
 -pyridine-2-yl 6-CH.sub.3 -pyridine-2-yl H
 222 N C N CH.sub.3 CH.sub.3 -- H Ph
 H H
 223 N C N CH.sub.3 CH.sub.3 -- H H
 Ph H
 224 N C N CH.sub.3 CH.sub.3 -- H Ph
 Ph H
 225 N C N CH.sub.3 CH.sub.3 -- H CH.sub.3
 H H
 226 N C N CH.sub.3 CH.sub.3 -- H H
 CH.sub.3 H
 227 N C N CH.sub.3 CH.sub.3 -- H CH.sub.3
 CH.sub.3 H
 2. THE BIOCHEMISTRY
 In yet another embodiment, this invention relates to a method for the
 modulation of the catalytic activity of PTKs comprising administering a
 compound of this invention or a physiologically acceptable salt or a
 prodrug thereof to a PTK.
 By "PTK" is meant both RTKs and CTKs; i.e., the modulation of both RTK
 signal transduction and CTK signal transduction is contemplated by this
 invention.
 The term "method" refers to manners, means, techniques and procedures for
 accomplishing a given task including, but not limited to, those manners,
 means, techniques and procedures either known to, or readily developed
 from known manners, means, techniques and procedures by practitioners of
 the chemical, pharmacological, biological, biochemical and medical arts.
 As used herein, the term "modulation" or "modulating" refers to the
 alteration of the catalytic activity of RTKs and/or CTKs. In particular,
 modulating refers to the activation of the catalytic activity of RTKs
 and/or CTKs, more preferably to the activation or inhibition of the
 catalytic activity of RTKs and/or CTKs, depending on the concentration of
 the compound administered or, more preferably still, to the inhibition of
 the catalytic activity of RTKs and/or CTKs.
 The term "catalytic activity" as used herein refers to the rate of
 phosphorylation of tyrosine under the influence, direct or indirect, of
 RTKs and/or CTKs.
 The term "administering" as used herein refers to a method for bringing a
 compound of this invention and a target PK together in such a manner that
 the compound can affect the catalytic activity of the PK either directly;
 i.e., by interacting with the kinase itself, or indirectly; i.e., by
 interacting with another molecule on which the catalytic activity of the
 kinase is dependent. As used herein, administration can be accomplished
 either in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues
 of a living organism. Thus, the PTK mediated disorders which are the
 object of this invention can be studied, prevented or treated by the
 methods set forth herein whether the cells or tissues of the organism
 exist within the organism or outside the organism. Cells existing outside
 the organism can be maintained or grown in cell culture dishes. In this
 context, the ability of a particular compound to affect a PTK related
 disorder can be determined; i.e., the IC50 of the compound, defined below,
 can be ascertained before the use of the compounds in more complex living
 organisms is attempted. For cells outside the organism, multiple methods
 exist, and are well-known to those skilled in the art, to administer
 compounds including, but not limited to, direct cell microinjection and
 numerous transmembrane carrier techniques. For cells harbored within a
 living organism, myriad methods also exist, and are likewise well-known to
 those skilled in the art, to administer compounds including, but not
 limited to, oral, parenteral, dermal and aerosol applications.
 RTK 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 or metabolic responses
 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 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 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 RTK 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.
 PTK signal transduction results in, among other responses, cell
 proliferation, differentiation, growth 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 and hemangioma; psoriasis, arteriosclerosis,
 arthritis and diabetic retinopathy and other disorders related to
 uncontrolled angiogenesis and/or vasculogenesis.
 A precise understanding of the mechanism by which the compounds of this
 invention inhibit PTKs is not required in order to practice the present
 invention. However, while not being bound to any particular mechanism or
 theory, it is believed that the compounds interact with the amino acids of
 the catalytic region of PTKs. PTKs typically possess a bi-lobate structure
 wherein ATP appears to bind in the cleft between the two lobes in a region
 where the amino acids are conserved among PTKs. Inhibitors of PTKs are
 believed to bind by non-covalent interactions such as hydrogen bonding,
 van der Waals forces and ionic interactions in the same general region
 where the aforesaid ATP binds to the PTKs. More specifically, it is
 thought that the quinoxaline ring component of the compounds of this
 invention binds in the general space normally occupied by the adenine ring
 of ATP. Specificity of a particular quinoxaline for a particular PTK may
 arise as the result of additional interactions between the various
 substituents on the quinoxaline core and the amino acid domain specific to
 particular PTKs. That is, different quinoxaline substituents may
 contribute to preferential binding to particular PTKs. The ability to
 select compounds active at different ATP (or other nucleotide) binding
 sites makes the compounds of this invention useful for targeting any
 protein with such a site; i.e., not only PTKs but serine/threonine kinases
 and protein phosphatases as well. Thus, the compounds disclosed herein may
 be useful as in vitro assays for such proteins as well as in vivo
 therapeutic agents acting through such proteins.
 Thus, in another aspect, this invention relates to a method for treating or
 preventing a PTK related disorder by administering a therapeutically
 effective amount of a compound of this invention or a salt or a prodrug
 thereof to an organism.
 In addition to the compounds described above, the compounds shown in Table
 2, below, are expected to be useful in treating or preventing PTK related
 disorders in an organism.
 As used herein, "PTK related disorder," "PTK driven disorder," and
 "abnormal PTK activity" all refer to a disorder characterized by
 inappropriate PTK activity or over-activity of the PTK, which can be
 either an RTK or a CTK. Inappropriate activity refers to either: (1) PTK
 expression in cells which normally do not express PTKs; (2) increased PTK
 expression leading to unwanted cell proliferation, differentiation and/or
 growth; or, (3) decreased PTK expression leading to unwanted reductions in
 cell proliferation, differentiation and/or growth. Overactivity of PTKs
 refers to either amplification of the gene encoding a particular PTK or
 production of a level of PTK activity which can correlate with a cell
 proliferation, differentiation and/or growth disorder (that is, as the
 level of the PTK increases, the severity of one or more of the symptoms of
 the cellular disorder increases).
 As used herein, the terms "prevent", "preventing" and "prevention" refer to
 a method for barring an organism from acquiring a PTK mediated cellular
 disorder in the first place.
 TABLE 2
 Compound
 No. A B D R.sup.1 R.sup.2 R.sup.3
 R.sup.4 R.sup.5 R.sup.6
 R.sup.7
 228 N C N H OH --
 H Ph Ph
 H
 229 N C N CI CF.sub.3 --
 H C1 CI
 H
 230 N C N H CH.sub.3 --
 H Ph Ph
 H
 231 N C N CH.sub.3 CH.sub.3 --
 H Ph Ph
 H
 232 N C N CH.sub.3 C(.dbd.O)-- CH.sub.3 --
 H CH.sub.3 CH.sub.3
 H
 233 N C N CH.sub.3 CH.sub.3 --
 H H H
 H
 234 N C N CH.sub.3 CH.sub.3 --
 H CH.sub.3 CH.sub.3
 H
 235 N C N CH.sub.3 NH.sub.2 --
 H H CH.sub.3
 CH.sub.3
 236 N C N CH.sub.3 CH.sub.3 --
 H Ph H
 H
 237 N C N CH.sub.3 CH.sub.3 --
 H H Ph
 H
 238 N C N H CH.sub.3 --
 H H Ph
 H
 239 N C N CH.sub.3 CH.sub.3 --
 H H CH.sub.3
 CH.sub.3
 240 O N C -- p-CH.sub.3 Ph-- --OCH.sub.3
 p-CH.sub.3 Ph-- p-CH.sub.3 Ph-- --OCH.sub.3
 H
 241 N N C CH.sub.3 H --OCH.sub.3
 p-CH.sub.3 Ph-- p-CH.sub.3 Ph-- --OCH.sub.3
 H
 242 N C C H H H
 H Ph Ph
 H
 243 S C N -- CH.sub.3 --
 H OH CH.sub.3
 H
 244 S C N -- CH.sub.3 --
 H OH OH
 H
 245 S C N -- H --
 H CH.sub.3 CH.sub.3
 H
 246 S C N -- CH.sub.3 --
 H Ph H
 H
 247 S C N -- CH.sub.3 --
 H H Ph
 H
 248 S C N -- CH.sub.3 --
 H CH.sub.3 CH.sub.3
 H
 249 S C N -- CH.sub.3 --
 H Ph Ph
 H
 250 S C N -- CH.sub.3 --
 H H H
 H
 251 S C N -- CH.sub.3 --
 H CH.sub.3 OH
 H
 252 S C N -- CH.sub.3 OC(.dbd.O)-- CH.sub.3
 --OCH.sub.3 Et.sub.2 NCH.sub.2 Ph-- Et.sub.2 NCH.sub.2 Ph--
 H
 253 S C N -- CH.sub.3 OC(.dbd.O)-- CH.sub.3
 --OCH.sub.3 Me.sub.2 NCH.sub.2 Ph-- Me.sub.2 NCH.sub.2 Ph--
 H
 254 S C N -- H.sub.2 NC(.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR3## ##STR4##
 H
 255 S C N -- CH.sub.3 OC(.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR5## ##STR6##
 H
 256 S C N -- HOC(--O)-- CH.sub.3
 --OCH.sub.3 ##STR7## ##STR8##
 H
 257 S C N -- ##STR9## H.sub.3
 --OCH.sub.3 ##STR10## ##STR11##
 H
 258 S C N -- CH.sub.3 OC(.dbd.O)-- H.sub.3
 --OCH.sub.3 ##STR12## ##STR13##
 H
 259 S C N -- Et.sub.2 NC(.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR14## ##STR15##
 H
 260 S C N -- CH.sub.3 OC(.dbd.O)-- CH.sub.3
 --OCH.sub.3 nPr.sub.2 NCH.sub.2 Ph-- nPr.sub.2 NCH.sub.2
 Ph-- H
 261 S C N -- CH.sub.3 O(C.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR16## ##STR17##
 H
 262 S C N -- CH.sub.3 O(C.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR18## ##STR19##
 H
 263 S C N -- CH.sub.3 O(C.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR20## ##STR21##
 H
 264 S C N -- ##STR22## CH.sub.3
 --OCH.sub.3 ##STR23## ##STR24##
 H
 265 S C N -- CH.sub.3 O(C.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR25## ##STR26##
 H
 266 S C N -- HO(C.dbd.O)-- CH.sub.3
 --OCH.sub.3 ##STR27## ##STR28##
 H
 267 S C N -- CH.sub.3 OC(.dbd.O)-- CH.sub.3
 --OCH.sub.3 p-CH3Ph-- p-CH3Ph--
 H
 268 S C N -- ##STR29## CH.sub.3
 --OCH.sub.3 ##STR30## ##STR31##
 H
 269 S C N -- CH.sub.3 O(C.dbd.O)-- CH.sub.3
 --OCH.sub.3 p-BrCH.sub.2 Ph-- p-BrCH.sub.2 Ph--
 H
 270 N C C CH.sub.3 H H
 H H H
 H
 As used herein, the terms "treat", "treating" and "treatment" refer to a
 method of alleviating or abrogating the PTK mediated cellular disorder
 and/or its attendant symptoms. With regard particularly to cancer, these
 terms simply mean that the life expectancy of an individual affected with
 a cancer will be increased or that one or more of the symptoms of the
 disease will be reduced.
 As used herein, the term "cancer" refers to various types of malignant
 neoplasms, most of which can invade surrounding tissues, and may
 metastasize to different sites, as defined by Stedman's Medical Dictionary
 25th edition (Hensyl ed. 1990). Examples of cancers which may be treated
 by the present invention include, but are not limited to, brain, ovarian,
 colon, prostate, kidney, bladder, breast, lung, oral and skin cancers
 which exhibit inappropriate PTK activity. These cancers can be further
 broken down. For example, brain cancers include glioblastoma multiforme,
 anaplastic astrocytoma, astrocytoma, ependymoma, oligodendroglioma,
 medulloblastoma, meningioma, sarcoma, hemangioblastoma, and pineal
 parenchymal. Likewise, skin cancers include melanoma and Kaposi's sarcoma.
 The term "organism" refers to any living entity comprised of at least one
 cell. A living organism can be as simple as, for example, a single
 eukariotic cell or as complex as a mammal, including a human being.
 The term "therapeutically effective amount" as used herein refers to that
 amount of the compound being administered which will relieve to some
 extent one or more of the symptoms of the disorder being treated. In
 reference to the treatment of cancer, a therapeutically effective amount
 refers to that amount which has the effect of (1) reducing the size of the
 tumor; (2) inhibiting (that is, slowing to some extent, preferably
 stopping) tumor metastasis; (3) inhibiting to some extent (that is,
 slowing to some extent, preferably stopping) tumor growth; and/or, (4)
 relieving to some extent (or preferably eliminating) one or more symptoms
 associated with the cancer.
 This invention is therefore directed to compounds which modulate PTK signal
 transduction by affecting the enzymatic activity of the RTKs and/or CTKs
 and thereby interfering with the signal transduced by such proteins. More
 particularly, the present invention is directed to compounds which
 modulate the RTK and/or CTK mediated signal transduction pathways as a
 therapeutic approach to cure many kinds of solid tumors, including but not
 limited to carcinoma, sarcoma, 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,
 bone cancers and leukemias.
 Further examples, without limitation, of the types of disorders related to
 unregulated PTK activity that the compounds described herein may be useful
 in preventing, treating and studying are cell proliferative disorders,
 fibrotic disorders and metabolic disorders.
 Cell proliferative disorders which may be prevented, treated or further
 studied by the present invention include cancer, 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
 matrices. Examples of fibrotic disorders include hepatic cirrhosis and
 mesangial cell proliferative disorders. Hepatic cirrhosis is characterized
 by the increase in extracellular matrix constituents resulting in the
 formation of a hepatic scar. Hepatic cirrhosis can lead to 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 in hepatic cirrhosis. Other fibrotic
 disorders implicated include atherosclerosis.
 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. In this regard, PDGFR has been implicated in the
 maintenance of mesangial cell proliferation. Floege et al., 1993, Kidney
 International 43:47S-54S.
 As noted previously, PTKs have been associated with the development of
 cancer. Some of these receptors, like 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 PDGFR (Kumabe et al., 1992,
 Oncogene, 7:627-633) are over-expressed in many tumors and/or are
 persistently activated by autocrine loops. In fact, in the most common and
 severe cancers, such receptor over-expression and autocrine loops have
 been demonstrated (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); (Lee and Donoghue,
 1992, J. Cell. Biol., 118:1057-1070; Korc et al., supra; Akbasak and
 Suner-Akbasak et al., supra). 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. PDGFR
 has been associated with glioblastoma, melanoma and lung, ovarian, and
 prostate cancer. 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,
 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 and Burkitts disease.
 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 including 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, while being 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 and 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-IR 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 abnormal RTK activity and disease are not
 restricted to cancer. For example, RTKs have been associated with
 metabolic diseases like psoriasis, diabetes mellitus, wound healing,
 inflammation, and neurodegenerative diseases. For example, EGFR has been
 indicated in corneal and dermal wound healing. Defects in the Insulin-R
 and IGF-1R 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&P 7:334-339.
 As noted previously, not only RTKs but CTKs as well including, but not
 limited to, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk
 (reviewed by Bolen et al., 1992, FASEB J., 6:3403-3409) are involved in
 the proliferative and metabolic signal transduction pathway and thus were
 expected, and have been shown, to be involved in many PTK-mediated
 disorders to which the present invention is directed. For example, mutated
 src (v-src) has been demonstrated as an oncoprotein (pp60.sup.v-src) in
 chicken. Moreover, its cellular homolog, the proto-oncogene pp60.sup.c-src
 transmits oncogenic signals of many receptors. Over-expression of EGFR or
 HER2/neu in tumors leads to the constitutive activation of pp60.sup.c-src,
 which is characteristic for the malignant cell but absent from the normal
 cell. On the other hand, mice deficient in 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, Zap70 is implicated in T-cell signaling.
 Finally, both RTKs and CTKs are currently suspected as being involved in
 hyperimmune disorders.
 3. PHARMACOLOGICAL COMPOSITIONS AND THERAPEUTIC APPLICATIONS
 A compound of the present invention, or its physiologically acceptable salt
 or prodrug, can be administered to a human patient per se, or in
 pharmacological compositions where it is mixed with suitable carriers or
 excipient(s). Techniques for formulation and administration of drugs may
 be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co.,
 Easton, Pa., latest edition.
 A. Routes Of Administration.
 Suitable routes of administration may, for example, include oral, rectal,
 transmucosal, intestinal or parenteral delivery, including intramuscular,
 subcutaneous and 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 a tumor-specific antibody.
 The liposomes will be targeted to and taken up selectively by the tumor.
 B. Composition/Formulation.
 Another aspect of this invention relates to pharmaceutical compositions of
 the compounds described herein and the physiologically acceptable salts
 and prodrugs thereof. Pharmacological compositions of the present
 invention may be manufactured by processes well known in the art; e.g., by
 means of conventional mixing, dissolving, granulating, dragee-making,
 levigating, emulsifying, encapsulating, entrapping or lyophilizing
 processes.
 Pharmacological 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 compounds of the invention may be formulated in aqueous
 solutions, preferably in physiologically compatible buffers such as Hanks'
 solution, Ringer's solution, or physiological saline buffer. For
 transmucosal administration, penetrants appropriate to the barrier to be
 permeated are used in the formulation. Such penetrants are generally known
 in the art.
 For oral administration, the compounds can be formulated readily by
 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. Pharmacological preparations for oral use can be made using a
 solid excipient, optionally grinding the resulting mixture, and processing
 the mixture of granules, after adding suitable 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
 physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If
 desired, disintegrating agents may be added, such as 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,
 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.
 Pharmacological compositions 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 may
 contain the active ingredients in admixture with filler such as lactose,
 binders such as starches, 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 the chosen route of 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 a pressurized pack or a nebulizer with the use of
 a suitable propellant, e.g., dichlorodifluoromethane,
 trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. 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 described herein may be formulated for parenteral
 administration, 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, optionally, an added
 preservative. The compositions may be suspensions, solutions or emulsions
 in oily or aqueous vehicles, and may contain formulatory agents such as
 suspending, stabilizing and/or dispersing agents.
 Pharmacological compositions for parenteral administration include aqueous
 solutions of the active compounds in water soluble form. Additionally,
 suspensions of the active compounds may be prepared as appropriate oily
 injection suspensions. Suitable lipophilic solvents or vehicles include
 fatty oils such as sesame oil, or synthetic fatty acid esters such as
 ethyl oleate, 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 of this invention may also be formulated in rectal
 compositions such as suppositories or retention enemas, using, e.g.,
 conventional suppository bases such as cocoa butter or other glycerides.
 In addition to the formulations described previously, a compound of this
 invention 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 such as
 sparingly soluble salts.
 The pharmacological compositions herein 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
 physiologically acceptable salts wherein the claimed compound may form the
 negatively or the positively charged species. Examples of salts in which
 the compound forms the positively charged moiety include, without
 limitation, quaternary ammonium (defined elsewhere herein), salts such as
 the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate,
 succinate, etc. wherein the nitrogen of the quaternary ammonium group is a
 nitrogen of a compound of this invention which reacts with an appropriate
 acid. Salts in which the compound forms the negatively charged species
 include, without limitation, the sodium, potassium, calcium and magnesium
 salts formed by the reaction of a carboxylic acid group in the molecule
 with the appropriate base (e.g. sodium hydroxide (NaOH), potassium
 hydroxide (KOH), Calcium hydroxide (Ca(OH).sub.2), etc.).
 C. Dosage.
 Pharmacological compositions suitable for use in the present invention
 include compositions wherein the active ingredients are contained in an
 amount effective to achieve the 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 amount or 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.sub.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., by determining the IC.sub.590 and the
 LD.sub.50 (both of which are discussed further, infra) for a subject
 compound. 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 may vary 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'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, termed the 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 a kinase
 may be ascertained using the assays described herein. Dosages necessary to
 achieve the MEC will depend on individual characteristics and route of
 administration. HPLC assays or bioassays can be used to determine plasma
 concentrations.
 Dosage intervals can also be determined using the 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%.
 It is noted that, in the case of local administration or selective uptake,
 the effective local concentration of the drug may not be related to plasma
 concentration. In such cases, other procedures known in the art can be
 employed to determine the effective local concentration.
 The amount of a composition to be administered will, of course, be
 dependent on the subject being treated, the severity of the affliction,
 the manner of administration, the judgment of the prescribing physician,
 etc.
 D. Packaging.
 Compositions of the present invention may, if desired, be presented in a
 pack or dispenser device, such as an FDA approved kit, which may contain
 one or more unit dosage forms containing the active ingredient. The pack
 may, for example, comprise metal or plastic foil, such as a blister pack.
 The pack or dispenser device may be accompanied by instructions for
 administration. The pack or dispenser may also be accompanied by a notice
 associated with the container in a form prescribed by a governmental
 agency regulating the manufacture, use or sale of pharmaceuticals, which
 notice is reflective of approval by the agency of the form of the
 compositions or human or veterinary administration. Such notice, for
 example, may be of the labeling approved by the U.S. Food and Drug
 Administration for prescription drugs or of an approved product insert.
 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.
 4. BRIEF DESCRIPTION OF THE TABLES
 Table 1 shows the chemical structure of specific compounds of this
 invention. The compounds shown are not to be construed as limiting the
 scope of this invention in any manner whatsoever.
 Table 2 shows the chemical structures of compounds which are not claimed
 herein as composition of matter but, rather, are claimed for their
 expected utility as modulators of PTK activity and for the treatment and
 prevention of PTK-related disorders.
 Table 3 shows the results of biological assays of some compounds of this
 invention. These results are provided as examples only and are not to be
 construed as limiting the scope of this invention in any manner including
 compound structure, the PTK against which the demonstrative compounds show
 activity or the level of activity (IC50) shown. PDGFR, FLK-1R and EGFR
 Kinase are defined and discussed elsewhere herein. IC50 refers to that
 amount of the tested compound needed to effect a 50% change in the
 activity of the PTK in comparison with a control in which no compound of
 this invention is present. With regard to the tests in the table, the 50%
 change being evaluated is a 50% inhibition of PTK activity compared to
 that in a control.
 5. SYNTHESIS
 The compounds of this invention may be readily synthesized using techniques
 well known in the chemical arts. Other synthetic pathways for forming the
 compounds of the invention will be apparent to those skilled in the art
 and are deemed to be within the scope and spirit of this invention.

A. EXAMPLES
 The following are examples of the synthesis of specific tricyclic
 quinoxaline compounds of this invention. These syntheses are provided by
 way of example only are not to be construed as limiting in any manner.
 Example 1
 1,2-Dimethyl-6,7-bis(4-bromo-phenyl))imidazo[4,5-g]quinoxaline
 ( ref: J. Gen. Chem. USSR (Enql. Transl.) EN, 1962, 32, 2829-2838).
 Step 1: 2-methyl-5,6-dinitrobenzimidazole: To a solution of
 2-methyl-5-nitrobenzimidazole in concentrated sulfuric acid was added
 fuming nitric acid at -5.degree. C. Upon addition of the fuming nitric
 acid, the reaction mixture was warmed to 0.degree. C. held for 1 h and
 poured into crushed ice. The precipitate which formed was filtered, washed
 with water, and dried overnight at 40.degree. C. to give the title
 compound.
 Step 2: 1,2-Dimethyl-5,6-dinitrobenzimidazole: To a solution of
 2-methyl-5,6-dinitro-benzimidazole in 5% NaOH was added methanol and
 dimethyl sulfate. The reaction mixture was heated for 30 min at
 90-95.degree. C. and then cooled to room temperature. The precipitate
 which formed was filtered, washed with 10% NaOH and then water, and dried
 overnight at 40.degree. C. to yield 1,2-dimethyl-5,6-dinitrobenzimidazole.
 Step 3: 1,2-Dimethyl-5,6-diaminobenzimidazole:
 1,2-Dimethyl-5,6-dinitrobenzimidazole was reduced with tin in hydrochloric
 acid. The solution obtained from the reduction was diluted with water,
 cooled to 10.degree. C., and treated with 40% NaOH. The precipitate which
 formed was filtered, washed with water and recrystallized from alcohol to
 yield 1,2 dimethly-5,6 diaminobenzimidazole.
 Step 4: 1,2-Dimethyl-6,7-bis(4-bromophenyl)imidazo[4,5-g]quinoxaline: To a
 solution of 1,2-dimethyl-5,6-diaminobenzoimidazole in methanol was added
 4,4'-dibromo-benzil. The reaction mixture was refluxed for 3 h and cooled
 to room temperature. The precipitate which formed was filtered and
 recrystallized from n-butyl alcohol to give the title quinoxaline.
 Example 2
 1,2,9-Trimethyl-6,7-bis(4-bromophenyl)-imidazol[4,5-g]quinoxaline
 (ref: Chem. Pharm. Bull. 1994, 42(2), 408-409.
 3-Fluoro-2-methylaniline was acetylated with acetic acid anhydride to give
 3-fluoro-2-methyl-N-acetylaniline. Displacing fluorine of the
 3-fluoro-2-methyl-N-acetylaniline with methylamine followed by nitration,
 deacetylation and reduction of the nitro group with palladium or charcoal
 gave 1,2-diamino-3-methyl-4-methylaminobenzene. Condensation of
 4,4'-dibromobenzil with the 1,2-diamino-3-methyl-4-methylaminobenzene in
 refluxing methanol gave
 2,3-bis(4-bromophenyl)-5-methyl-6-methylaminoquinoxaline which upon
 acetylation with acetic acid anhydride, nitration, reduction with
 palladium on charcoal and cyclization gave the title compound.
 Example 3
 2-Amino-6,7-bis(4-bromophenyl)-1,9-dimethylimidazol[4,5-g]quinoxaline
 2,3-bis(4-bromophenyl)-5-methyl-6-methylaminoquinoxaline was treated with
 trifluoroacetic anhydride to give
 2,3-bis-(4-bromophenyl)-5-methyl-6-(N-methyl-N-trifluoroacetylamino)quinox
 aline which was then nitrated with fuming nitric acid in trifluoroacetic
 anhydride followed by reduction with palladium on charcoal deprotection
 with potassium carbonate in methanol, and cyclization with cyanogen
 bromide to yield the title compound.
 Example 4
 6,7-Bis(4-bromophenyl)-2-Methylamino-1,9-dimethylimidazol-[4,5-g]quinoxalin
 e
 Methylation of
 2-amino-6,7-bis(4-bromophenyl)-1,9-dimethylimidazol[4,5-g]quinoxaline with
 methyl iodide in acetone in the presence of potassium carbonate gave the
 title compound.
 6. BIOLOGICAL EVALUATION
 It will be appreciated that, in any given series of compounds, a spectrum
 of biological activity will be afforded. In its most preferred
 embodiments, this invention relates to tricyclic quinoxaline compounds
 demonstrating the ability to modulate RTK and CTK activity. The following
 assays are employed to select those compounds demonstrating the optimal
 degree of the desired activity.
 As used herein, the phrase "optimal degree of the desired activity" refers
 to the lowest IC50, defined elsewhere herein, against a PTK related to a
 particular disorder so as to provide an organism, preferably a human, with
 a therapeutically effective amount of a compound of this invention at the
 lowest possible dosage.
 B. Assay Procedures.
 The following in vitro assays may be used to determine the level of
 activity and the effect of that activity produced by the compounds of the
 present invention with respect to specific PTKS. Similar assays can be
 designed along the same lines for any PK using techniques well known in
 the art.
 The cellular/catalytic assays described herein are performed in an ELISA
 format. The general procedure is as follows: a compound is introduced to
 cells expressing the test kinase, either naturally or recombinantly, for a
 selected period of time after which, if the test kinase is a receptor, a
 ligand known to activate the receptor's activity is added. The cells are
 lysed and the lysate is transferred to the wells of an ELISA plate
 previously coated with a specific antibody recognizing the substrate of
 the enzymatic phosphorylation reaction. Non-substrate components of the
 cell lysate are washed away and the amount of phosphorylation on the
 substrate is detected with an antibody specifically recognizing
 phosphotyrosine and is compared with that of control cells that were not
 contacted with the test compound.
 The cellular/biologic assays described herein measure the amount of DNA
 made in response to activation of a test kinase, which is a general
 measure of a proliferative response. The general procedure for this assay
 is as follows: a compound is introduced to cells expressing the test
 kinase, either naturally or recombinantly, for a selected period of time
 after which, if the test kinase is a receptor, a ligand known to activate
 the receptor's activity is added. After incubation at least overnight, a
 DNA labeling reagent such as Bromodeoxyuridine (BrdU) or 3H-thymidine is
 added. The amount of labeled DNA is detected with either an anti-BrdU
 antibody or by measuring radioactivity and is compared to control cells
 not contacted with a test compound.
 1. Cellular/Catalytic Assays
 Enzyme linked immunosorbent assays (ELISA) may be used to detect and
 measure the presence of PTK 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 PK. The preferred protocols for conducting the ELISA
 experiments for specific RTKs is provided below. Adaptation of these
 protocols for determining a compound's activity for other members of the
 RTK family, as well as for CTKs, is well within the scope of knowledge of
 those skilled in the art.
 a. FLK-1
 An ELISA assay is conducted to measure the kinase activity of the FLK-1
 receptor and more specifically, the inhibition or activation of TK
 activity on the FLK-1 receptor. Specifically, the following assay can be
 conducted to measure kinase activity of the FLK-1 receptor in cells
 genetically engineered to express Flk-1.
 MATERIALS AND METHODS
 Materials. The following reagents and supplies are 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.degree. 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.times.stock);
 h. Sodium orthovanadate (0.5 M as a 100.times.stock);
 i. Sodium pyrophosphate (0.2 M as a 100.times.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.times.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 .mu.g/100 .mu.l
 stock in Milli-Q dH.sub.2 O and stored at -20.degree. C.;
 p. Affinity purified anti-FLK-1 antiserum;
 q. UB40 monoclonal antibody specific for phosphotyrosine (see, Fendley, et
 al., 1990, Cancer Research 50:1550-1558);
 r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);
 s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution
 (100 mM citric acid (anhydrous), 250 mM Na.sub.2 HPO.sub.4 (pH 4.0), 0.5
 mg/ml ABTS (Sigma catalog no. A-1888)), solution should be stored in dark
 at 4.degree. C. until ready for use;
 t. H.sub.2 O.sub.2 (30% solution) (Fisher catalog no. H325);
 u. ABTS/H.sub.2 O.sub.2 (15 ml ABTS solution, 2 .mu.l H.sub.2 O.sub.2)
 prepared 5 minutes before use and left at room temperature;
 v. 0.2 M HCl stock in H.sub.2 O;
 w. dimethylsulfoxide (100%) (Sigma Catalog No. D-8418); and
 y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).
 Protocol. The following protocol can be used for conducting the assay:
 1. Coat Corning 96-well ELISA plates with 1.0 .mu.g per well Cappel
 Anti-rabbit IgG antibody in 0.1M Na.sub.2 CO.sub.3 pH 9.6. Bring final
 volume to 150 .mu.l per well. Coat plates overnight at 4.degree. C. Plates
 can be kept up to two weeks when stored at 4.degree. C.
 2. Grow cells in Growth media(DMEM, supplemented with 2.0 mM L-Glutamine,
 10% FBS) in suitable culture dishes until confluent at 37.degree. C., 5%
 CO.sub.2.
 3. Harvest cells by trypsinization and seed in Corning 25850 polystyrene
 96-well round bottom cell plates, 25.000 cells/well in 200 .mu.l of growth
 media.
 4. Grow cells at least one day at 37.degree. C., 5% CO.sub.2.
 5. Wash cells with D-PBS 1.times..
 6. Add 200 .mu.l/well of starvation media (DMEM, 2.0 mM 1-Glutamine, 0.1%
 FBS). Incubate overnight at 37.degree. C., 5% CO.sub.2.
 7. Dilute Compounds 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
 .mu.l of fresh starvation media to each well.
 9. Add 18 .mu.l of 1:20 diluted Compound 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.degree. C., 5%
 CO.sub.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 .mu.l 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 .mu.g/well affinity purified anti-FLU-1 polyclonal rabbit
 antiserum. Bring final volume to 150 .mu.l /well with TBSW+0.5%
 ethanolamine pH 7.0. Incubate plate for thirty minutes while shaking.
 14. Add 180 .mu.l starvation medium to the cells and stimulate cells with
 20 .mu.l /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.degree. C., 5% CO.sub.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 .mu.l/well PBS.
 16. Lyse cells in 150 .mu.l/well HNTG while shaking at room temperature for
 five minutes. HNTG formulation includes sodium ortho vanadate, sodium
 pyrophosphate 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 .mu.g/well UB40 in TBSW+05%
 ethanolamine. Bring final volume to 150 .mu.l/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 .mu.l/well. Incubate while shaking for thirty
 minutes.
 23. Wash plate as described in step 10.
 24. Add 100 .mu.l of ABTS/H.sub.2 O.sub.2 solution to well. Incubate ten
 minutes while shaking.
 25. Add 100 .mu.l 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.
 b. HER-2 ELISA
 Assay 1: EGF Receptor-HER2 Chimeric Receptor Assay in Whole Cells.
 HER2 kinase activity in whole EGFR-NIH3T3 cells are measured as described
 below:
 Materials and Reagents. The following materials and reagents can be used to
 conduct the assay:
 a. EGF: stock concentration: 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:

HNTG stock 2.0 ml
 milli-Q H.sub.2 O 7.3 ml
 EDTA, 100 mM, pH 7.0 0.5 ml
 Na.sub.3 VO.sub.4, 0.5 M 0.1 ml
 Na.sub.4 (P.sub.2 O.sub.7), 0.2 M 0.1 ml
 4. After 120 minutes incubation with drug, add prepared SGF ligand to
 cells, 10 .mu.l 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 .mu.l 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 .mu.l 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
 .mu.l 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.sub.2 O.sub.2 solution to ELISA plate, 100 .mu.l
 per well. Incubate shaking at room temperature for 20 minutes.
 (ABTS/H.sub.2 O.sub.2 solution: 1.0 .mu.l 30% H.sub.2 O.sub.2 in 10 ml
 ABTS stock).
 10. Stop reaction by adding 50 .mu.l 5N H.sub.2 SO.sub.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.
 c. PDGF-R ELISA
 All cell culture media, glutamine, and fetal bovine serum can be purchased
 from Gibco Life Technologies (Grand Island, N.Y.) unless otherwise
 specified. All cells are grown in a humid atmosphere of 90-95% air and
 5-10% CO.sub.2 at 37.degree. C. All cell lines are routinely subcultured
 twice a week and are negative for mycoplasma as determined by the Mycotect
 method (Gibco).
 For ELISA assays, cells (U1242, obtained from Joseph Schlessinger, NYU) are
 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 are changed to serum-free medium and
 treated with test compound for 2 hr in a 5% CO.sub.2, 37.degree. C.
 incubator. Cells are 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.sub.3 VO.sub.4, 0.2% Triton X-100, and 2 mM NaPyr). Cell lysates
 (0.5 mg/well in PBS) are 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 are incubated with shaking for 1 hour at
 room temperature. The plates are washed with TBST four times and then
 incubated with polyclonal anti-phosphotyrosine antibody at room
 temperature for 30 minutes. Excess anti-phosphotyrosine antibody is
 removed by rinsing the plate with TBST four times. Goat anti-rabbit IgG
 antibody is added to the ELISA plate for 30 min at room temperature
 followed by rinsing with TBST four more times. ABTS (100 mM citric acid,
 250 mM Na.sub.2 HPO.sub.4 and 0.5 mg/mL
 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) plus H.sub.2
 O.sub.2 (1.2 mL 30% H.sub.2 O.sub.2 to 10 ml ABTS) is added to the ELISA
 plates to start color development. Absorbance at 410 nm with a reference
 wavelength of 630 nm is recorded about 15 to 30 min after ABTS addition.
 d. IGF-I RECEPTOR 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 are used:
 a. The cell line used in this assay is 3T3/IGF-1R, a cell line genetically
 engineered to overexpresses IGF-1 receptor.
 b. NIH3T3/IGF-1R is grown in an incubator with 5% CO.sub.2 at 37.degree. C.
 The growth media is DMEM+10% FBS (heat inactivated)+2 mM L-glutamine.
 c. Affinity purified anti-IGF-1R antibody 17-69.
 d. D-PBS:

Citric acid 100 mM
 Na.sub.2 HPO.sub.4 250 mM (pH 4.0/1 N HCl)
 ABTS 0.5 mg/ml
 ABTS solution should be kept in dark and 4.degree. C. The solution should
 be discarded when it turns green.
 o. Hydrogen Peroxide: 30% solution is kept in the dark and at 4.degree. 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 .mu.l/well). Incubate for 1 day then replace medium to serum-free
 medium (90/.mu.l) and incubate in 5% CO.sub.2 and 37.degree. C. overnight.
 B. ELISA Plate Coating and Blocking:
 1. Coat the ELISA plate (Corning 25805-96) with Anti-IGF-1R Antibody at 0.5
 .mu.g/well in 100 .mu.l PBS at least 2 hours.
 2. Remove the coating solution, and replace with 100 .mu.l 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 .mu.l /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.sub.2 at 37.degree. C. for 2 hours.
 3. Prepare fresh cell lysis buffer (HNTG*)

HNTG 2 ml
 EDTA 0.1 ml
 Na.sub.3 VO.sub.4 0.1 ml
 Na.sub.4 (P.sub.2 O.sub.7) 0.1 ml
 H.sub.2 O 7.3 ml
 4. After drug incubation for two hours, transfer 10 .mu.l/well of 200 nM
 IGF-1 Ligand in PBS to the cells (Final Conc.=20 nM), and incubate at 5%
 CO.sub.2 at 37.degree. C. for 10 minutes.
 5. Remove media and add 100 .mu.l/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 repeated aspiration and dispensing. 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 .mu.l/well, and shake for 30 minutes.
 8. Remove anti-pTyr, wash the plate, transfer TAGO (1:3,000 with TBST) 100
 .mu.l/well, and shake for 30 minutes.
 9. Remove detection antibody , wash the plate, and transfer fresh
 ABTS/H.sub.2 O.sub.2 (1.2 .mu.l H.sub.2 O.sub.2 to 10 ml ABTS) 100
 .mu.l/well to the plate to start color development.
 10. Measure OD at 410 nm with a reference wavelength of 630 nm in Dynatec
 MR5000.
 e. EGF Receptor ELISA
 EGF Receptor kinase activity in cells genetically engineered to express
 human EGF-R can be measured as described below:
 Materials and Reagents. The following materials and reagents are used
 a. EGF Ligand: stock concentration=16.5 .mu.M; EGF 201, TOYOBO, Co., Ltd.
 Japan.
 b. 05-101 (UBI) (a monoclonal antibody recognizing an EGFR extracellular
 domain).
 c. Anti-phosphotyosine antibody (anti-Ptyr) (polyclonal).
 d. D etection antibody: Go at anti-rabbit lgG horse radish peroxidase
 conjugate, TAGO, Inc., Burlingame, Calif.
 e. TBST buffer:

EDTA 100 mM pH 7.0
 Na.sub.3 VO.sub.4 0.5 M
 Na.sub.4 (P.sub.2 O.sub.7) 0.2 M
 Procedure. The following protocol is used:
 A. Pre-coat ELISA Plate
 1. Coat ELISA plates (Corning, 96 well, Cat. #25805-96) with 05-101
 antibody at 0.5 .mu.g per well in PBS, 150 .mu.l final volume/well, and
 store overnight at 4.degree. C. Coated plates are good for up to 10 days
 when stored at 4.degree. 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.degree. C. to 25.degree. 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 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 .mu.l per well, in a 96 well microtiter plate. Incubate seeded cells
 in 5% CO.sub.2 at 37.degree. 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
 .mu.l 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.sub.2 at 37.degree. C. for one hour.
 2. Prepare EGF ligand: dilute stock EGF in DMEM so that upon transfer of 10
 .mu.l dilute EGF (1:12 dilution), 25 nM final concentration is attained.
 3. Prepare fresh 10 ml HNTG* sufficient for 100 .mu.l per well wherein
 HNTG* comprises: HNTG stock (2.0 ml), milli-Q H.sub.2 O (7.3 ml), EDTA,
 100 mM, pH 7.0 (0.5 ml), Na.sub.3 VO.sub.4 0.5 M (0.1 ml) and Na.sub.4
 (P.sub.2 O.sub.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 .mu.l 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 .mu.l 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 .mu.l 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
 .mu.l 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.sub.2 O.sub.2 solution to ELISA plate, 100 .mu.l per well.
 Incubate at room temperature for 20 minutes. ABTS/H.sub.2 O.sub.2
 solution: 1.2 .mu.l 30% H.sub.2 O.sub.2 in 10 ml ABTS stock.
 11. Stop reaction by adding 50 .mu.l 5N H.sub.2 SO.sub.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.
 f. Met Autophosphorylation Assay--ELISA
 This assay determines Met tyrosine kinase activity by analyzing Met protein
 tyrosine kinase levels on the Met receptor.
 1. Reagents
 a. HNTG (5.times.stock solution): Dissolve 23.83 g HEPES and 43.83 g NaCl
 in about 350 ml dH.sub.2 O. Adjust pH to 7.2 with HCl or NaOH, add 500 ml
 glycerol and 10 ml Triton X-100, mix, add dH.sub.2 O to 1 L total volume.
 To make 1 L of 1.times.working solution add 200 ml 5.times.stock solution
 to 800 ml dH.sub.2 O, check and adjust pH as necessary, store at 4.degree.
 C.
 b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco Cat. # 450-1300EB
 (1.times.solution).
 c. Blocking Buffer: in 500 ml dH.sub.2 O place 100 g BSA, 12.1 g
 Tris-pH7.5, 58.44 g NaCl and 10 ml Tween-20, dilute to 1 L total volume.
 d. Kinase Buffer: To 500 ml dH.sub.2 O add 12.1 g TRIS pH7.2, 58.4 g NaCl,
 40.7 g MgCl.sub.2 and 1.9 g EGTA; bring to 1 L total volume with dH.sub.2
 O.
 e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat. #P-7626, to 435.5 mg,
 add 100% ethanol to 25 ml total volume, vortex.
 f. ATP (Bacterial Source), Sigma Cat. # A-7699, store powder at -20.degree.
 C.; to make up solution for use, dissolve 3.31 mg in 1 ml dH.sub.2 O.
 g. RC-20H HRPO Conjugated Anti-Phosphotyrosine, Transduction Laboratories
 Cat. # E120H.
 h. Pierce 1-Step (TM) Turbo TMB-ELISA (3,3',5,5'-tetramethylbenzidine,
 Pierce Cat. # 34022.
 i. H.sub.2 SO.sub.4, add 1 ml conc. (18N) to 35 ml dH.sub.2 O.
 j. TRIS HCL, Fischer Cat. # BP152-5; to 121.14 g of material, add 600 ml
 MilliQ H.sub.2 O, adjust pH to 7.5 (or 7.2) with HCl , bring volume to 1 L
 with MilliQ H.sub.2 O.
 k. NaCl, Fischer Cat. # S271-10, make up 5M solution.
 l. Tween-20, Fischer Cat. # S337-500.
 m. Na.sub.3 VO.sub.4, Fischer Cat. # S454-50, to 1.8 g material add 80 ml
 MilliQ H.sub.2 O, adjust pH to 10.0 with HCl or NaOH, boil in microwave,
 cool, check pH, repeat procedure until pH stable at 10.0, add MilliQ
 H.sub.2 O to 100 ml total volume, make 1 ml aliquots and store at
 -80.degree. C.
 n. MgCl.sub.2, Fischer Cat. # M33-500, make up 1M solution.
 o. HEPES, Fischer Cat. # BP310-500, to 200 ml MilliQ H.sub.2 O, add 59.6 g
 material, adjust pH to 7.5, bring volume to 250 ml total, sterile filter.
 p. Albumin, Bovine (BSA), Sigma Cat. # A-4503, to 30 grams material add
 sterile distilled water to make total volume of 300 ml, store at 4.degree.
 C.
 q. TBST Buffer: to approx. 900 ml dH.sub.2 O in a 1 L graduated cylinder
 add 6.057 g TRIS and 8.766 g NaCl, when dissolved, adjust pH to 7.2 with
 HCl, add 1.0 ml Triton X-100 and bring to 1 L total volume with dH.sub.2
 O.
 r. Goat Affinity purified antibody Rabbit IgG (whole molecule), Cappel Cat.
 # 55641.
 s. Anti h-Met (C-28) rabbit polyclonal IgG antibody, Santa Cruz Chemical
 Cat. # SC-161.
 t. Transiently Transfected EGFR/Met chimeric cells (EMR) (Komada, et al.,
 Oncogene, 8:2381-2390 (1993).
 u. Sodium Carbonate Buffer, (Na.sub.2 CO.sub.4, Fischer Cat. # S495): to
 10.6 g material add 800 ml MilliQ H.sub.2 O, when dissolved adjust pH to
 9.6 with NaOH, bring up to 1 L total volume with MilliQ H.sub.2 O, filter,
 store at 4.degree. C.
 2. Procedure
 All of the following steps are conducted at room temperature unless it is
 specifically indicated otherwise. All ELISA plate washing is by rinsing
 4.times. with TBST.
 A. EMR Lysis
 This procedure can be performed the night before or immediately prior to
 the start of receptor capture.
 1. Quick thaw lysates in a 37.degree. C. waterbath with a swirling motion
 until the last crystals disappear.
 2. Lyse cell pellet with 1.times.HNTG containing 1 mM PMSF. Use 3 ml of
 HNTG per 15 cm dish of cells. Add 1/2 the calculated HNTG volume, vortex
 the tube for 1 min., add the remaining amount of HNTG, vortex for another
 min.
 3. Balance tubes, centrifuge at 10,000.times.g for 10 min at 4.degree. C.
 4. Pool supernatants, remove an aliquot for protein determination.
 5. Quick freeze pooled sample in dry ice/ethanol bath. This step is
 performed regardless of whether lysate will be stored overnight or used
 immediately following protein determination.
 6. Perform protein determination using standard bicinchoninic acid (BCA)
 method (BCA Assay Reagent Kit from Pierce Chemical Cat. # 23225).
 B. ELISA Procedure
 1. Coat Corning 96 well ELISA plates with 5 .mu.g per well Goat anti-Rabbit
 antibody in Carbonate Buffer for a total well volume of 50 .mu.l. Store
 overnight at 4.degree. C.
 2. Remove unbound Goat anti-rabbit antibody by inverting plate to remove
 liquid.
 3. Add 150 .mu.l of Blocking Buffer to each well. Incubate for 30 min. at
 room temperature with shaking.
 4. Wash 4.times. with TBST. Pat plate on a paper towel to remove excess
 liquid and bubbles.
 5. Add 1 .mu.g per well of Rabbit anti-Met antibody diluted in TBST for a
 total well volume of 100 .mu.l.
 6. Dilute lysate in HNTG (90 .mu.g lysate/100 .mu.l)
 7. Add 100 .mu.l of diluted lysate to each well. Shake at room temperature
 for 60 min.
 8. Wash 4.times. with TBST. Pat on paper towel to remove excess liquid and
 bubbles.
 9. Add 50 .mu.l of 1.times.lysate buffer per well.
 10. Dilute compounds/extracts 1:10 in 1.times.Kinase Buffer in a
 polypropylene 96 well plate.
 11. Transfer 5.5 .mu.l of diluted drug to ELISA plate wells. Incubate at
 room temperature with shaking for 20 min.
 12. Add 5.5 .mu.of 60 .mu.M ATP solution per well. Negative controls do not
 receive any ATP. Incubate at room temperature for 90 min., with shaking.
 13. Wash 4.times. with TEST. Pat plate on paper towel to remove excess
 liquid and bubbles.
 14. Add 100 .mu.l per well of RC20 (1:3000 dilution in Blocking Buffer).
 Incubate 30 min. at room temperature with shaking.
 15. Wash 4.times. with TBST. Pat plate on paper towel to remove excess
 liquid and bubbles.
 16. Add 100 .mu.l per well of Turbo-TMB. Incubate with shaking for 30-60
 min.
 17. Add 100 .mu.l per well of 1M H.sub.2 SO.sub.4 to stop reaction.
 18. Read assay on Dynatech MR7000 ELISA reader.
 Test Filter=450 nm, reference filter=410 nm.
 g. Biochemical Src Assay--ELISA
 This assay is used to determine src protein kinase activity measuring
 phosphorylation of a biotinylated peptide as the readout.
 1. Materials and Reagents:
 a. Yeast transformed with src from Courtneidge Laboratory (Sugen, Inc.,
 Redwood City, Calif.).
 b. Cell lysates: Yeast cells expressing src are pelleted, washed once with
 water, re-pelleted and stored at -80.degree. C. until use.
 c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is prepared by standard
 procedures well known to those skilled in the art.
 d. DMSO: Sigma, St. Louis, Mo.
 e. 96 Well ELISA Plate: Corning 96 Well Easy Wash, Modified flat Bottom
 Plate, Corning Cat. #25805-96.
 f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:
 Applied Scientific Cat. # A-72092.
 g. Vecastain ELITE ABC reagent: Vector, Burlingame, Calif.
 h. Anti-src (327) mab: Schizosaccharomyces Pombe is used to express
 recombinant Src (Superti-Furga, et al., EMBO J., 12:2625-2634;
 Superti-Furga, et al., Nature Biochem., 14:600-605). S. Pombe strain SP200
 (h-s leul.32 ura4 ade210) is grown as described and transformations are
 pRSP expression plasmids are done by the lithium acetate method
 (Superti-Furga, supra). Cells are grown in the presence of 1 .mu.M
 thiamine to repress expression from the nmtl promoter or in the absence of
 thiamine to induce expression.
 i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40 may be used instead).
 j. Turbo TMB-ELISA peroxidase substrate: Pierce Chemical.
 2. Buffer Solutions:
 a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO PBS, GIBCO Cat. #
 450-1300EB.
 b. Blocking Buffer: 5% Non-fat milk (Carnation) in PBS.
 c Carbonate Buffer: Na.sub.2 CO.sub.4 from Fischer, Cat. # S495, make up
 100 mM stock solution.
 d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl.sub.2 ; 0.2 ml (from
 a 1M stock solution) MnCl.sub.2 ; 0.2 ml (from a 1M stock solution) DTT;
 5.0 ml (from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml
 total volume with MilliQ H.sub.2 O.
 e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from 5M
 stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM
 stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml
 Na.sub.3 VO.sub.4 (from a 0.1 M stock solution); bring to 100 ml total
 volume with MilliQ H.sub.2 O.
 f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml).
 g TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H.sub.2 O add 121.14
 g material, adjust pH to 7.5 with HCl, bring to 1 L total volume with
 MilliQ H.sub.2 O.
 h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution with MilliQ
 H.sub.2 O.
 i. Na.sub.3 VO.sub.4 : Fischer Cat. # S454-50; to 80 ml MilliQ H.sub.2 O,
 add 1.8 g material; adjust pH to 10.0 with HCl or NaOH; boil in a
 microwave; cool; check pH, repeat pH adjustment until pH remains stable
 after heating/cooling cycle; bring to 100 ml total volume with MilliQ
 H.sub.2 O; make 1 ml aliquots and store at -80.degree. C.
 j. MgCl.sub.2 : Fischer Cat. # M33-500, make up 1M stock solution with
 MilliQ H.sub.2 O.
 k. HEPES: Fischer Cat. # BP 310-500; too 200 ml MilliQ H.sub.2 O, add 59.6
 g material, adjust pH to 7.5, bring to 250 ml total volume with MilliQ
 H.sub.2 O, sterile filter (1M stock solution).
 l. TBST Buffer: TBST Buffer: To 900 ml dH.sub.2 O add 6.057 g TRIS and
 8.766 g NaCl; adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to
 1 L total volume with dH.sub.2 O.
 m. MnCl.sub.2 : Fischer Cat. # M87-100, make up 1M stock solution with
 MilliQ H.sub.2 O.
 n. DTT: Fischer Cat. # BP172-5.
 o. TBS (TRIS Buffered Saline): to 900 ml MilliQ H.sub.2 O add 6.057 g TRIS
 and 8.777 g NaCl; bring to 1 L total volume with MilliQ H.sub.2 O.
 p. Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 ml
 Kinase Buffer, 200 .mu.g GST-.zeta., bring to final volume of 8.0 ml with
 MilliQ H.sub.2 O.
 q. Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide stock solution (1 mM,
 2.98 mg/ml) in water fresh just before use.
 r. Vectastain ELITE ABC reagent: To prepare 14 ml of working reagent, add 1
 drop of reagent A to 15 ml TBST and invert tube several times to mix. Then
 add 1 drop of reagent B. Put tube on orbital shaker at room temperature
 and mix for 30 minutes.
 3. Procedures:
 a. Preparation of Src Coated ELISA Plate.
 1. Coat ELISA plate with 0.5 .mu.g/well anti-src mab in 100 .mu.l of pH 9.6
 sodium carbonate buffer at 4.degree. C. overnight.
 2. Wash wells once with PBS.
 3. Block plate with 0.15 ml 5% milk in PBS for 30 min. at room temperature.
 4. Wash plate 5.times. with PBS.
 5. Add 10 .mu.g/well of src transformed yeast lysates diluted in Lysis
 Buffer (0.1 ml total volume per well). (Amount of lysate may vary between
 batches.) Shake plate for 20 minutes at room temperature.
 b. Preparation of Phosphotyrosine Antibody-coated ELISA Plate.
 1. 4G10 plate: coat 0.5 .mu.g/well 4G10 in 100 .mu.l PBS overnight at
 4.degree. C. and block with 150 .mu.l of 5% milk in PBS for 30 minutes at
 room temperature.
 c. Kinase Assay Procedure.
 1. Remove unbound proteins from step 1-7, above, and wash plates 5.times.
 with PBS.
 2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 .mu.l of
 10.times.Kinase Buffer and 10 .mu.M (final concentration)
 biotin-EEEYEEYEEEYEEEYEEEY per well diluted in water.
 3. Add 10 .mu.l of compound diluted in water containing 10% DMSO and
 pre-incubate for 15 minutes at room temperature.
 4. Start kinase reaction by adding 10 .mu.l/well of 0.05 mM ATP in water (5
 .mu.M ATP final).
 5. Shake ELISA plate for 15 min. at room temperature.
 6. Stop kinase reaction by adding 10 .mu.l of 0.5 M EDTA per well.
 7. Transfer 90 .mu.l supernatant to a blocked 4G10 coated ELISA plate from
 section B, above.
 8. Incubate for 30 min. while shaking at room temperature.
 9. Wash plate 5.times. with TBST.
 10. Incubate with Vectastain ELITE ABC reagent (100 .mu.l/well) for 30 min.
 at room temperature.
 11. Wash the wells 5.times. with TBST.
 12. Develop with Turbo TMB.
 h. Biochemical Lck Assay--ELISA
 This assay is used to determine lck protein kinase activities measuring
 phosphorylation of GST-.zeta. as the readout.
 1. Materials and Reagents:
 a. Yeast transformed with lck. Schizosaccharomyces Pombe is used to express
 recombinant Lck (Superti-Furga, et al., EMBO J, 12:2625-2634;
 Superti-Furga, et al., Nature Biotech., 14:600-605). S. Pombe strain SP200
 (h-s leul.32 ura4 ade210) is grown as described and transformations with
 pRSP expression plasmids are done by the lithium acetate method
 (Superti-Furga, supra). Cells are grown in the presence of 1 .mu.M
 thiamine to induce expression.
 b. Cell lysates: Yeast cells expressing lck are pelleted, washed once in
 water, re-pelleted and stored frozen at -80.degree. C. until use.
 c. GST-.zeta.: DNA encoding for GST-.zeta. fusion protein for expression in
 bacteria obtained from Arthur Weiss of the Howard Hughes Medical Institute
 at the University of California, San Francisco. Transformed bacteria are
 grown overnight while shaking at 25.degree. C. GST-.zeta. is purified by
 glutathione affinity chromatography, Pharmacia, Alameda, Calif.
 d. DMSO: Sigma, St. Louis, Mo.
 e. 96-Well ELISA plate: Corning 96 Well Easy Wash, Modified Flat Bottom
 Plate, Corning Cat. #25805-96.
 f. NUNC 96-well V-bottom polypropylene plates for dilution of compounds:
 Applied Scientific Cat. # AS-72092.
 g. Purified Rabbit anti-GST antiserum: Amrad Corporation (Australia) Cat.
 #90001605.
 h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. # V010301.
 i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. # 5215-005-003.
 j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Cat # sc-433.
 k. Monoclonal anti-phosphotyrosine UBI 05-321 (UB40 may be used instead).
 2. Buffer Solutions:
 a. PBS (Dulbeccols Phosphate-Buffered Saline) 1.times.solution: GIBCO PBS,
 GIBCO Cat. # 450-1300EB.
 b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS-pH7.5, 58.44 g NaCl, 10 ml
 Tween-20, bring up to 1 L total volume with MilliQ H.sub.2 O.
 c. Carbonate Buffer: Na.sub.2 CO.sub.4 from Fischer, Cat. # S495; make up
 100 mM solution with MilliQ H.sub.2 O.
 d. Kinase Buffer: 1.0 ml (from 1M stock solution) MgCl.sub.2 ; 0.2 ml (from
 a 1M stock solution) MnCl.sub.2 ; 0.2 ml (from a 1M stock solution) DTT;
 5.0 ml (from a 1M stock solution) HEPES; 0.1 ml TX-100; bring to 10 ml
 total volume with MilliQ H.sub.2 O.
 e. Lysis Buffer: 5.0 HEPES (from 1M stock solution.); 2.74 ml NaCl (from 5M
 stock solution); 10 ml glycerol; 1.0 ml TX-100; 0.4 ml EDTA (from a 100 mM
 stock solution); 1.0 ml PMSF (from a 100 mM stock solution); 0.1 ml
 Na.sub.3 VO.sub.4 (from a 0.1 M stock solution); bring to 100 ml total
 volume with MilliQ H.sub.2 O.
 f. ATP: Sigma Cat. # A-7699, make up 10 mM stock solution (5.51 mg/ml).
 g TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml MilliQ H.sub.2 O add 121.14
 g material, adjust pH to 7.5 with HC1, bring to 1 L total volume with
 MilliQ H.sub.2 O.
 h. NaCl: Fischer Cat. # S271-10, Make up 5M stock solution with MilliQ
 H.sub.2 O.
 i Na.sub.3 VO.sub.4 : Fischer Cat. # S454-50; to 80 ml MilliQ H.sub.2 O,
 add 1.8 g material; adjust pH to 10.0 with HCl or NaOH; boil in a
 microwave; cool; check pH, repeat pH adjustment until pH remains stable
 after heating/cooling cycle; bring to 100 ml total volume with MilliQ
 H.sub.2 O; make 1 ml aliquots and store at -80.degree. C.
 j. MgCl.sub.2 : Fischer Cat. # M33-500, make up 1M stock solution with
 MilliQ H.sub.2 O.
 k. HEPES: Fischer Cat. # BP 310-500; to 200 ml MilliQ H.sub.2 O, add 59.6 g
 material, adjust pH to 7.5, bring to 250 ml total volume with MilliQ
 H.sub.2 O, sterile filter (1M stock solution).
 l. Albumin, Bovine (BSA), Sigma Cat. # A4503; to 150 ml MilliQ H.sub.2 O
 add 30 g material, bring 300 ml total volume with MilliQ H.sub.2 O, filter
 through 0.22 .mu.m filter, store at 4.degree. C.
 m. TBST Buffer: To 900 ml dH.sub.2 O add 6.057 g TRIS and 8.766 g NaCl;
 adjust pH to 7.2 with HCl, add 1.0 ml Triton-X100; bring to 1 L total
 volume with dH.sub.2 O.
 n. MnCl.sub.2 : Fischer Cat. # M87-100, make up 1M stock solution with
 MilliQ H.sub.2 O.
 o. DTT; Fischer Cat. # BP172-5.
 p. TBS (TRIS Buffered Saline): to 900 ml MilliQ H.sub.2 O add 6.057 g TRIS
 and 8.777 g NaCl; bring to 1 L total volume with MilliQ H.sub.2 O.
 q Kinase Reaction Mixture: Amount per assay plate (100 wells): 1.0 ml
 Kinase Buffer, 200 .mu.g GST-.zeta., bring to final volume of 8.0 ml with
 MilliQ H.sub.2 O.
 2. Procedures:
 a. Preparation of Lck Coated ELISA Plate.
 1. Coat 2.0 .mu.g/well Sheep anti-mouse IgG in 100 .mu.l of pH 9.6 sodium
 carbonate buffer at 4.degree. C. overnight.
 2. Wash well once with PBS.
 3. Block plate with 0.15 ml of blocking Buffer for 30 min. at room temp.
 4. Wash plate 5.times. with PBS.
 5. Add 0.5 .mu.g/well of anti-lck (mab 3A5) in 0.1 ml PBS at room
 temperature for 1-2 hours.
 6. Wash plate 5.times. with PBS.
 7. Add 20 .mu.g/well of lck transformed yeast lysates diluted in Lysis
 Buffer (0.1 ml total volume per well). (Amount of lysate may vary between
 batches) Shake plate at 4.degree. C. overnight to prevent loss of
 activity.
 b. Preparation of Phosphotyrosine Antibody-coated ELISA Plate.
 1. UB40 plate: 1.0 .mu.g/well UB40 in 100 .mu.l of PBS overnight at
 4.degree. C. and block with 150 .mu.l of Blocking Buffer for at least 1
 hour.
 c. Kinase Assay Procedure.
 1. Remove unbound proteins from step 1-7, above, and wash plates 5.times.
 with PBS.
 2. Add 0.08 ml Kinase Reaction Mixture per well (containing 10 .mu.l of
 10.times. Kinase Buffer and 2 .mu.g GST-.zeta. per well diluted with
 water).
 3. Add 10 .mu.l of compound diluted in water containing 10% DMSO and
 pre-incubate for 15 minutes at room temperature.
 4. Start kinase reaction by adding 10 .mu.l/well of 0.1 mM ATP in water (10
 .mu.M ATP final).
 5. Shake ELISA plate for 60 min. at room temperature.
 6. Stop kinase reaction by adding 10 .mu.l of 0.5 M EDTA per well.
 7. Transfer 90 .mu.l supernatant to a blocked 4G10 coated ELISA plate from
 section B, above.
 8. Incubate while shaking for 30 min. at room temperature.
 9. Wash plate 5.times. with TBST.
 10. Incubate with Rabbit anti-GST antibody at 1:5000 dilution in 100 .mu.l
 TBST for 30 min. at room temperature.
 11. Wash the wells 5.times. with TBST.
 12. Incubate with Goat anti-Rabbit-IgG-HRP at 1:20,000 dilution in 100
 .mu.l of TBST for 30 min. at room temperature.
 13. Wash the wells 5.times. with TBST.
 14. Develop with Turbo TMB.
 i. Assay Measuring Phosphorylating Function of Raf
 The following assay reports the amount of RAF-catalyzed phosphorylation of
 its target protein MEK as well as MEK's target MAPK. The RAF gene sequence
 is described in Bonner et al., 1985, Molec. Cell. Biol. 5: 1400-1407, and
 is readily accessible in multiple gene sequence data banks. Construction
 of the nucleic acid vector and cell lines utilized for this portion of the
 invention are fully described in Morrison et al., 1988, Proc. Natl. Acad.
 Sci. USA 85: 8855-8859.
 MATERIALS AND REAGENTS
 1. Sf9 (Spodoptera frugiperda) cells; GIBCO-BRL, Gaithersburg, Md.
 2. RIPA buffer: 20 mM Tris/HCl pH 7.4, 137 mM NaCl, 10% glycerol, 1 mM
 PMSF, 5 mg/L Aprotenin, 0.5% Triton X-100;
 3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK expression and
 purification by affinity chromatography are performed according to the
 manufacturer's procedures. Catalog# K 350-01 and R 350-40, Invitrogen
 Corp., San Diego, Calif.
 4. His-MAPK (ERK 2); His-tagged MAPK is expressed in XL1 Blue cells
 transformed with pUC18 vector encoding His-MAPK. His-MAPK is purified by
 Ni-affinity chromatography. Cat# 27-4949-01, Pharmacia, Alameda, Calif.,
 as described herein.
 5. Sheep anti mouse IgG: Jackson laboratories, West Grove, Pa. Catalog, #
 515-006-008, Lot# 28563
 6. RAF-1 protein kinase specific antibody: URP2653 from UBI.
 7. Coating buffer: PBS; phosphate buffered saline, GIBCO-BRL, Gaithersburg,
 Md.
 8. Wash buffer: TBST--50 mM Tris/HCL pH 7.2, 150 mM NaCl, 0.1% Triton X-100
 9. Block buffer: TBST, 0.1% ethanolamine pH 7.4
 10. DMSO, Sigma, St. Louis, Mo.
 11. Kinase buffer (KB): 20 mM HEPES/HCl pH 7.2, 150 mM NaCl, 0.1% Triton
 X-100, 1 mM PMSF, 5 mg/L Aprotenin, 75 mM sodium ortho vanadate, 0.5 MM
 DTT and 10 mM MgCl.sub.2.
 12. ATP mix: 100 mM MgCl.sub.2, 300 mM ATP, 10 mCi .sup.33 P ATP
 (Dupont-NEN)/mL.
 13 Stop solution: 1% phosphoric acid; Fisher, Pittsburgh, Pa.
 14. Wallac Cellulose Phosphate Filter mats; Wallac, Turku, Finnland.
 15. Filter wash solution: 1% phosphoric acid, Fisher, Pittsburgh, Pa.
 16. Tomtec plate harvester, Wallac, Turku, Finnland.
 17. Wallac beta plate reader # 1205, Wallac, Turku, Finnland.
 18. NUNC 96-well V bottom polypropylene plates for compounds Applied
 Scientific Catalog # AS-72092.
 PROCEDURE
 All of the following steps are conducted at room temperature unless
 specifically indicated.
 1. ELISA plate coating: ELISA wells are coated with 100 ml of Sheep anti
 mouse affinity purified antiserum (1 mg/100 mL coating buffer) over night
 at 4.degree. C. ELISA plates can be used for two weeks when stored at
 4.degree. C.
 2. Invert the plate and remove liquid. Add 100 mL of blocking solution and
 incubate for 30 min.
 3. Remove blocking solution and wash four times with wash buffer. Pat the
 plate on a paper towel to remove excess liquid.
 4. Add 1 mg of antibody specific for RAF-1 to each well and incubate for 1
 hour. Wash as described in step 3.
 5. Thaw lysates from RAS/RAF infected Sf9 cells and dilute with TBST to 10
 mg/100 mL. Add 10 mg of diluted lysate to the wells and incubate for 1
 hour. Shake the plate during incubation. Negative controls receive no
 lysate. Lysates from RAS/RAF infected Sf9 insect cells are prepared after
 cells are infected with recombinant baculoviruses at a MOI of 5 for each
 virus, and harvested 48 hours later. The cells are washed once with PBS
 and lysed in RIPA buffer. Insoluble material is removed by centrifugation
 (5 min at 10 000.times.g). Aliquots of lysates are frozen in dry
 ice/ethanol and stored at -80.degree. C. until use.
 6. Remove non-bound material and wash as outlined above (step 3).
 7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well and adjust the volume
 to 40 mL with kinase buffer. Methods for purifying T-MEK and MAPK from
 cell extracts are provided herein by example.
 8. Pre-dilute compounds (stock solution 10 mg/mL DMSO) or extracts 20 fold
 in TBST plus 1% DMSO. Add 5 mL of the pre-diluted compounds/extracts to
 the wells described in step 6. Incubate for 20 min. Controls receive no
 drug.
 9. Start the kinase reaction by addition of 5 mL ATP mix; Shake the plates
 on an ELISA plate shaker during incubation.
 10. Stop the kinase reaction after 60 min by addition of 30 mL stop
 solution to each well.
 11. Place the phosphocellulose mat and the ELISA plate in the Tomtec plate
 harvester. Harvest and wash the filter with the filter wash solution
 according to the manufacturers recommendation. Dry the filter mats. Seal
 the filter mats and place them in the holder. Insert the holder into
 radioactive detection apparatus and quantify the radioactive phosphorous
 on the filter mats.
 Alternatively, 40 mL aliquots from individual wells of the assay plate can
 be transferred to the corresponding positions on the phosphocellulose
 filter mat. After air drying the filters, put the filters in a tray.
 Gently rock the tray, changing the wash solution at 15 min intervals for 1
 hour. Air-dry the filter mats. Seal the filter mats and place them in a
 holder suitable for measuring the radioactive phosphorous in the samples.
 Insert the holder into a detection device and quantify the radioactive
 phosphorous on the filter mats.
 j. CDK2/Cyclin A--Inhibition Assay
 This assay analyzes the protein kinase activity of CDK2 in exogenous
 substrate.
 REAGENTS
 A. Buffer A (80 mM Tris ( pH 7.2), 40 mM MgCl.sub.2): 4.84 G. Tris
 (F.W.=121.1 g/mol), 4.07 g. MgCl.sub.2 (F.W.=203.31 g/mol) dissolved in
 500 ml H.sub.2 O. Adjust pH to 7.2 with HCl.
 B. Histone H1 solution (0.45 mg/ml Histone H1 and 20 mM HEPES pH 7.2 (pH
 7.4 is OK): 5 mg Histone H1 (Boehinger Mannheim) in 11.111 ml 20 mM HEPES
 pH 7.2 (477 mg HEPES (F.W.=238.3 g/mol) dissolved in 100 ml ddH.sub.2 O,
 stored in 1 ml aliquots at -80.degree. C.
 C. ATP solution (60 .mu.M ATP, 300 .mu.g/ml BSA, 3 mM DTT): 120 .mu.l 10 mM
 ATP, 600 .mu.l 10 mg/ml BSA to 20 ml, stored in 1 ml aliquots at
 -80.degree. C.
 D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH 7.2, 25 mM NaCl, 0.5 mM
 DTT, 10% glycerol, stored in 9 .mu.l aliquots at -80.degree. C.
 DESCRIPTION OF ASSAY
 1. Prepare solutions of inhibitors at three times the desired final assay
 concentration in ddH.sub.2 O/15% DMSO by volume.
 2. Dispense 20 .mu.l of inhibitors to wells of polypropylene 96-well plates
 (or 20 .mu.l 15% DMSO for positive and negative controls).
 3. Thaw Histone H1 solution (1 ml/plate), ATP solution (1 ml/plate plus 1
 aliquot for negative control), and CDK2 solution (9 .mu.l/plate). Keep
 CDK2 on ice until use.
 Aliquot CDK2 solution appropriately to avoid repeated freeze-thaw cycles.
 4. Dilute 9 .mu.l CDK2 solution into 2.1 ml Buffer A (per plate). Mix.
 Dispense 20 .mu.l into each well.
 5. Mix 1 ml Histone H1 solution with 1 ml ATP solution (per plate) into a
 10 ml screw cap tube. Add .gamma..sup.33 P ATP to a concentration of 0.15
 .mu.Ci/20 .mu.l (0.15 .mu.Ci/well in assay). Mix carefully to avoid BSA
 frothing. Add 20 .mu.l to appropriate wells. Mix plates on plate shaker.
 For negative control, mix ATP solution with an equal amount of 20 mM HEPES
 pH 7.2 and add .gamma..sup.33 P ATP to a concentration of 0.15 .mu.Ci/20
 .mu.l solution. Add 20 .mu.l to appropriate wells.
 6. Let reactions proceed for 60 minutes.
 7. Add 35 .mu.l 10% TCA to each well. Mix plates on plate shaker.
 8. Spot 40 .mu.l of each sample onto P30 filter mat squares. Allow mats to
 dry (approx. 10-20 minutes).
 9 Wash filter mats 4.times.10 minutes with 250 ml 1% phosphoric acid (10 ml
 phosphoric acid per liter ddH.sub.2 O).
 10. Count filter mats with beta plate reader.
 2. Cellular/Biologic Assays
 Assay 1: PDGF-Induced BrdU Incorporation Assay
 MATERIALS AND REAGENTS
 (1) PDGF: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany.
 (2) BrdU Labeling Reagent: 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.times.PBS, pH 7.4, made in house (Sugen, Inc.,
 Redwood City, Calif.).
 (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
 USA.
 (8) 3T3 cell line genetically engineered to express human PDGF-R.
 PROTOCOL
 (1) 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.degree. C. in 5% CO.sub.2.
 (2) 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.
 (3) 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.
 (4) 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 .mu.M) for 1.5 hours.
 (5) 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 .mu.l /well) and the plates are incubated at room
 temperature for 45 minutes on a plate shaker.
 (6) 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 .mu.l/well) as a blocking solution and the plate is incubated for
 30 minutes at room temperature on a plate shaker.
 (7) The blocking solution is removed by decanting and the wells are washed
 once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
 added (100 .mu.l/well) and the plate is incubated for 90 minutes at room
 temperature on a plate shaker.
 (8) 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.
 (9) TMB substrate solution is added (100 .mu.l/well) and incubated for 20
 minutes at room temperature on a plate shaker until color development is
 sufficient for photometric detection.
 (10) 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.times.PBS, pH 7.4, made in house (Sugen, Inc.,
 Redwood City, Calif.).
 (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
 USA.
 (8) 3T3 cell line genetically engineered to express human EGF-R.
 PROTOCOL
 (1) 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.degree. C. in 5%
 CO.sub.2.
 (2) 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.
 (3) 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.
 (4) 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 .mu.M) for 1.5 hours.
 (5) 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 .mu.l/well) and the plates are incubated at room
 temperature for 45 minutes on a plate shaker.
 (6) 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 .mu.l /well) as a blocking solution and the plate is incubated
 for 30 minutes at room temperature on a plate shaker.
 (7) The blocking solution is removed by decanting and the wells are washed
 once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
 added (100 .mu.l/well) and the plate is incubated for 90 minutes at room
 temperature on a plate shaker.
 (8) 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.
 (9) TMB substrate solution is added (100 .mu.l/well) and incubated for 20
 minutes at room temperature on a plate shaker until color development is
 sufficient for photometric detection.
 (10) 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.times.PBS, pH 7.4, made in house.
 (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
 USA.
 (8) 3T3 cell line engineered to express a chimeric receptor having the
 extra-cellular domain of EGF-R and the intra-cellular domain of Her2.
 PROTOCOL
 (1) Cells are seeded at 8000 cells/well in DMEM, 10% CS, 2 mM Gln in a
 96-well plate. Cells are incubated overnight at 370 in 5% CO.sub.2.
 (2) 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.
 (3) 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.
 (4) 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 .mu.M) for 1.5 hours.
 (5) 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 .mu.l/well) and the plates are incubated at room
 temperature for 45 minutes on a plate shaker.
 (6) 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 .mu.l/well) as a blocking solution and the plate is incubated for
 30 minutes at room temperature on a plate shaker.
 (7) The blocking solution is removed by decanting and the wells are washed
 once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
 added (100 .mu.l/well) and the plate is incubated for 90 minutes at room
 temperature on a plate shaker.
 (8) 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.
 (9) TMB substrate solution is added (100 .mu.l/well) and incubated for 20
 minutes at room temperature on a plate shaker until color development is
 sufficient for photometric detection.
 (10) 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.times.PBS, pH 7.4, made in house (Sugen, Inc.,
 Redwood City, Calif.).
 (7) Albumin, Bovine (BSA): fraction V powder; A-8551, Sigma Chemical Co.,
 USA.
 (8) 3T3 cell line genetically engineered to express human IGF-1 receptor.
 PROTOCOL
 (1) 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.degree. C. in 5%
 CO.sub.2.
 (2) 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.
 (3) 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.
 (4) 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 .mu.M) for 1.5 hours.
 (5) 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 .mu.l/well) and the plates are incubated at room
 temperature for 45 minutes on a plate shaker.
 (6) 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 .mu.l/well) as a blocking solution and the plate is incubated for
 30 minutes at room temperature on a plate shaker.
 (7) The blocking solution is removed by decanting and the wells are washed
 once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1% BSA) is
 added (100 .mu.l/well) and the plate is incubated for 90 minutes at room
 temperature on a plate shaker.
 (8) 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.
 (9) TMB substrate solution is added (100 .mu.l/well) and incubated for 20
 minutes at room temperature on a plate shaker until color development is
 sufficient for photometric detection.
 (10) 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.
 g. HUV-EC-C Assay
 The following protocol may also be used to measure a compound's activity
 against PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally
 expressed by HUV-EC cells.
 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's phosphate-buffered saline (D-PBS; obtained from Gibco BRL;
 catalogue no. 14190-029) 2 times at about 1 ml/10 cm.sup.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 is 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.sup.2 of tissue culture flask for about 5 minutes
 at 37.degree. 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.sup.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, Coulter Electronics, Inc.) and add assay medium to the cells to
 obtain a concentration of 0.8-1.0.times.10.sup.5 cells/ml.
 3. Add cells to 96-well flat-bottom plates at 100 .mu.l/well or
 0.8-1.0.times.10.sup.4 cells/well; incubate .about.24 h at 37.degree. C.,
 5% CO.sub.2.
 DAY 1
 1. Make up two-fold drug titrations in separate 96-well plates, generally
 50 .mu.M on down to 0 .mu.M. Use the same assay medium as mentioned in day
 0, step 2 above. Titrations are made by adding 90 .mu.l/well of drug at
 200 .mu.M (4.times. 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 .mu.M 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 .mu.l/well. Take 60 .mu.l from
 the 120 .mu.l of 200 .mu.M drug dilution in the top well of the column and
 mix with the 60 .mu.l in the second well of the column. Take 60 .mu.l from
 this well and mix with the 60 .mu.l 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 .mu.l of the 120 .mu.l in this well and discard it.
 Leave the last well with 60 .mu.l 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); or, 3) human PDGF
 B/B (1276-956, Boehringer Mannheim, Germany) and assay media control. ECGF
 comes as a preparation with sodium heparin.
 2. Transfer 50 .mu.l/well of the drug dilutions to the 96-well assay plates
 containing the 0.8-1.0.times.10.sup.4 cells/100 .mu.l/well of the HUV-EC-C
 cells from day 0 and incubate .about.2 h at 37.degree. C., 5% CO.sub.2.
 3. In triplicate, add 50 .mu.l/well of 80 .mu.g/ml VEGF, 20 ng/ml ECGF, or
 media control to each drug condition. As with the drugs, the growth factor
 concentrations are 4.times. 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.degree. C., 5% CO.sub.2. Each well
 will have 50 .mu.l drug dilution, 50 .mu.l growth factor or media, and 100
 ul cells,=200 ul/well total. Thus the 4.times. concentrations of drugs and
 growth factors become 1.times. once everything has been added to the
 wells.
 DAY 2
 1. Add .sup.3 H-thymidine (Amersham; catalogue no. TRK-686) at 1
 .mu.Ci/well (10 .mu.l/well of 100 .mu.Ci/ml solution made up in RPMI
 media+10% heat-inactivated fetal bovine serum) and incubate .about.24 h at
 37.degree. C., 5% CO.sub.2. RPMI is obtained from Gibco BRL, catalogue no.
 11875-051.
 DAY 3
 1. Freeze plates overnight at -20.degree. C.
 DAY 4
 1. Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester
 96.sup.(R)) onto filter mats (Wallac; catalogue no. 1205-401); read counts
 on a Wallac Betaplate(.TM.) liquid scintillation counter.
 3. In Vivo Animal Models
 A. 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,
 gastro-intestinal, head and neck, glioblastoma, bone, and malignant
 melanomas) have been transplanted and successfully grown in nude mice. The
 following assays may be used to determine the level of activity,
 specificity and effect of the different compounds of the present
 invention. Three general types of assays are useful for evaluating
 compounds: cellular/catalytic, cellular/biological and in vivo. The object
 of the cellular/catalytic assays is to determine the effect of a compound
 on the ability of a TK to phosphorylate tyrosines on a known substrate in
 a cell. The object of the cellular/biological assays is to determine the
 effect of a compound on the biological response stimulated by a TK in a
 cell. The object of the in vivo assays is to determine the effect of a
 compound in an animal model of a particular disorder such as cancer.
 Suitable cell lines for subcutaneous xenograft experiments include C6 cells
 (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC # CRL 1619), A431
 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC #
 HTB 56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cells and NIH 3T3
 fibroblasts genetically engineered to overexpress EGFR, PDGFR, IGF-lR or
 any other test kinase. The following protocol can be used to perform
 xenograft experiments:
 Female athymic mice (BALB/c, nu/nu) are obtained from Simonsen Laboratories
 (Gilroy, Calif.). All animals are maintained under clean-room conditions
 in Micro-isolator cages with Alpha-dri bedding. They receive sterile
 rodent chow and water ad libitum.
 Cell lines are grown in appropriate medium (for example, MEM, DMEM, Ham's
 F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mM glutamine
 (GLN)). All cell culture media, glutamine, and fetal bovine serum are
 purchased from Gibco Life Technologies (Grand Island, N.Y.) unless
 otherwise specified. All cells are grown in a humid atmosphere of 90-95%
 air and 5-10% CO.sub.2 at 37.degree. C. All cell lines are routinely
 subcultured twice a week and are negative for mycoplasma as determined by
 the Mycotect method (Gibco).
 Cells are harvested at or near confluency with 0.05% Trypsin-EDTA and
 pelleted at 450.times.g for 10 min. Pellets are resuspended in sterile PBS
 or media (without FBS) to a particular concentration and the cells are
 implanted into the hindflank of the mice (8-10 mice per group,
 2-10.times.10.sup.6 cells/animal). Tumor growth is measured over 3 to 6
 weeks using venier calipers. Tumor volumes are calculated as a product of
 length.times.width.times.height unless otherwise indicated. P values are
 calculated using the Students t-test. Test compounds in 50-100 .mu.L
 excipient (DMSO, or VPD:D5W) can be delivered by IP injection at different
 concentrations generally starting at day one after implantation.
 B. 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.
 PROCEDURE
 8 week old nude mice (female) (Simonsen Inc.) are used as experimental
 animals. Implantation of tumor cells can be performed in a laminar flow
 hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and 5
 mg/kg Xylazine) are administered intraperitoneally. A midline incision is
 done to expose the abdominal cavity (approximately 1.5 cm in length) to
 inject 10.sup.7 tumor cells in a volume of 100 .mu.l 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 is closed by using wound clips. Animals are
 observed daily.
 ANALYSIS
 After 2-6 weeks, depending on gross observations of the animals, the mice
 are sacrificed, and the local tumor metastases in various organs (lung,
 liver, brain, stomach, spleen, heart, muscle) are excised and analyzed
 (measurement of tumor size, grade of invasion, immunochemistry, in situ
 hybridization, etc.).
 D. 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.sub.50 /LD.sub.50. IC.sub.50, the
 dose required to achieve 50% inhibition, can be measured using standard
 techniques such as those described herein. LD.sub.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.
 TABLE 3
 RESULTS OF ASSAYS OF SELECTED COMPOUNDS
 PDGFR
 KINASE FLK KINASE EGFR KINASE
 NAME IC50 (.mu.M) IC50 (.mu.M) IC50 (.mu.M)
 5,6,-dimethyl-2- 1.9 &gt;10 &gt;10
 phenylimidazo [4,5-
 e] quinoxaline
 6,7,-dimethyl-2- 4.3 88.4 &gt;100
 phenylimidazo [4,5-
 e] quinozaline
 CONCLUSION
 Thus, it will be appreciated that the compounds, methods and
 pharmacological compositions of the present invention are expected to
 modulate RTK and CTK activity and therefore to be effective as therapeutic
 agents against RTK- and CTK-related disorders.
 Although certain embodiments and examples have been used to describe the
 present invention, it will be apparent to those skilled in the art that
 changes to the embodiments and examples shown may be made without
 departing from the scope and spirit of the invention.
 Other embodiments are within the following claims.