CA2224103A1 - Method and compositions for inhibition of adaptor protein/tyrosine kinase interactions - Google Patents

Abstract

The present invention relates to methods and compositions for the inhibition of adaptor protein/protein tyrosine kinase protein interactions, especially wherein those interactions involving a protein tyrosine kinase capable of complexing with a member of the SH2-and/or SH3-containing family of adaptor proteins are associated with a cell proliferative disorder. Specifically, the present invention relates to particular compounds, especially quinazoline derivative compounds, and methods utilizing such compounds.

Description

CA 02224l03 l997-l2-08 h~.O~ AND COMPOSITIONS FOR INHIBITION OF
ADAPTOR PROTEIN/TYROSINE RINASE INTERACTIONS

1. INTRODUCTION
The present invention relates to methods and compositions ~or the inhibition of adaptor protein/phosphotyrosine interactions, especially wherein those interactions involve a protein tyrosine kinase capable of complexing with a member an the SH2 domain-containing 10 family of adaptor proteins associated with a cell proliferative disorder. Specifically, the present invention relates to particular organic compounds, and methods utilizing such compounds.

2. BACKGROUND OF THE INVENTION

2.1 PROTEIN PHOSPHORYLATION AND SIGNAL TRANSDUCTION

Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their ;mm~; ate environment. These extracellular signals are 20 essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell division, contact inhibition, and metabolism. The extracellular molecules, which can include, for example, hormones, growth factors, lymphokines, or neurotransmitters, 25 act as ligands that bind specific cell surface receptors.
The binding of these ligands to their receptors triggers a cascade o~ reactions that brings about both the amplification of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above. In addition to 30 normal cellular processes, receptors and their extracellular ligands may be involved in abnormal or potentially deleterious processes such as virus-receptor interaction, inflammation, and cellular transformation to a cancerous state.
A central ~eature of this process, referred to as signal transduction (for recent reviews, see Posada, J. and Cooper, J.A., 1992, Mol. Biol. Cell 3:583-592; Hardie, D~Go~ 1990, WO 96/40115 PCTrUS96/08741 Symp. Soc. Exp. Biol. 44:241-255), is the reversible phosphorylation of certain proteins. The phosphorylation or dephosphorylation of amino acid residues triggers conformational changes in regulated proteins that alter their r 5 biological properties. Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classi~ied according to the amino acid residues they act on, with one class being serine-threonine kinases and 10 phosphatases (reviewed in Scott, J.D. and Soderling, T.R., 1992, 2:289-295~, which act on serine and threonine residues, and the other class being the tyrosine kinases and phosphatases (reviewed in Fischer, E.H. et al., 1991 Science 253:401-406; Schlessinger, J. and Ullrich, A., 1992, Neuron 15 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212), which act on tyrosine residues. The protein kinases and phosphatases may be further de~ined as being receptors, i.e., the enzymes are an integral part of a transmembrane, ligand-binding molecule, or as non-receptors, 20 m~n;ng they respond to an extracellular molecule indirectly by being acted upon by a ligand-bound receptor.
Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the 25 relative activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions.
While the majority of protein phosphorylation occurs at serine and threonine amino acid residues, phosphorylation at tyrosine residues also occurs, and has begun to attract a 30 great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic 35 transformation is now well established (Cantley, L.C. et al., 1991, Cell 64:281-302i Hunter T., 1991, Cell 64:249-270;
Nurse, 1990, Nature 344:503-508; Schlessinger, J. and CA 02224l03 l997-l2-08 Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schles~inger, J., 1990, Cell 61:203-212). Subversion of normal growth control pathways leading to oncogenesis has been shown to be caused by activation or overexpression of 5 protein tyrosine kinases which constitute a large group of dominant oncogenic proteins (reviewed in Hunter, T., 1991, Cell 64:249-270).

2.2 PROTEIN TYROSINE KINASES
Protein tyrosine kinases comprise a large family of proteins, including many growth factor receptors and potential oncogenes, which share ancestry with, but nonetheless differ from, serine/threonine-specific protein kinases (Hanks et al., 1988, Science 241:42-52).
Receptor-type protein tyrosine kinases having a transmembrane topolosy have been studied extensively. The binding of a specific ligand to the extracellular domain of a receptor protein tyrosine kinase is thought to induce receptor dimerization and phosphorylation of their own 20 tyrosine residues. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signalling molecules, thereby activating various signal transduction pathways (Ullrich, A. and Schlessinger, J., 25 1990, Cell 61:203-212).
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases, may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane ~o~; n . Within this broad classification, one 30 can divide the known cytoplasmic protein tyrosine kinases into eleven distinct morphotypes, including the SRC family tMartinez~ R. et al., 1987, Science 237:411-414; Sukegawa, J.
et al., 1987, Mol. Cell. Biol., 7:41-47; Yamanishi, Y. et al., 1987, 7.237-243; Marth, J.D. et al., 1985, Cell 43:393-35 404; Dymecki, S.M. et al., 1990, Science 247:332-336), the FES family (Ruebroek, A.J.M. et al., 1985, EMBO J. 4:2897-2903; Hao, Q. et al., 1989, Mol. Cell. Biol. 9:1587-1593), W O9G/4011~ PCT/U~o/41 the ABL family (Shtivelman, E. et al., 1986, Cell 47:277-284;
Kruh, G.D~ et al., 1986, Science 234:1545-1548), the Zap 70 family and the JAK family. While distinct in their overall molecular structure, each of the members of these morphotypic 5 families o~ cytoplasmic protein tyrosine kinases share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domain are the SH2 (SRC
homology domain 2; Sadowski, I. et al., Mol. Cell. Biol. 6:
4396-4408; Koch, C.A. et al., 1991, Science 252:668-674) 10 domains and SH3 ~o~; n.C (Mayer, B.J. et al., 1988, Nature 332:269-272). Such non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson, T. and Gish, G., 1992, Cell 71:359-362).
While the metabolic roles of cytoplasmic protein tyrosine kinases are less well understood than that of the receptor-type protein tyrosine kinases, significant progress has been made in elucidating some of the processes in which this class of molecules is involved. For example, members of 20 the src family, lck and fyn, have been shown to interact with CD4/CD8 and the T cell receptor complex, and are thus implicated in T cell activation, (Veillette, A. and Davidson, D., 1992, TIG 8:61-66), certain cytoplasmic protein tyrosine kinases have been linked to certain phases of the cell cycle 25 (Morgan, D.O. et al., 1989, Cell 57: 775-786; Kipreos, E.T.
et al., 1990, Science 248: 217-220; Weaver et al., 1991, Mol.
Cell. Biol. 11:4415-4422), and cytoplasmic protein tyrosine kinases have been implicated in neuronal development (Maness, P., 1992, Dev. Neurosci 14:257-270). Deregulation of kinase 30 activity through mutation or overexpression is a well-established mechanism underlying cell transformation (Hunter et al., 1985, supra; Ullrich et al., supra).

2.3 ADAPTOR PROTEINS
Adaptor proteins are intracellular proteins having characteristic conserved peptide domains (SH2 and/or SH3 domains, as described below) which are critical to the signal transduction pathway. Such adaptor proteins serve to link protein tyrosine kinases, especially receptor-type protein tyrosine kinases to downstream intracellular signalling pathways such as the RAS signalling pathway. It is thought 5 that such adaptor proteins may be involved in targeting signal transduction proteins to the correct site in the plasma membrane or subcellular compartments, and may also be involved in the regulation of protein movement within the cell.
Such adaptor proteins are among the protein substrates of the receptor-type protein tyrosine kinases, and have in common one or two copies of an approximately 100 amino acid long motif. Because this motif was originally identified in c-Src-like cytoplasmic, non-receptor tyrosine kinases it is 15 referred to as a Src homology 2 (SH2) domain. SH2-containing polypeptides may otherwise, however, be structurally and functionally distinct from one another (Koch, C.A. et al., 1991, Science 252:668-674). SH2 domains directly recognize phosphorylated tyrosine amino acid residues. The peptide 20 domains also have independent sites for the recognition of amino acid residues surrounding the phosphotyrosine residue(s).
When a receptor protein tyrosine kinase binds an extracellular ligand, receptor dimerization is induced, 25 which, in turn, leads to intermolecular autophosphorylation of the dimerized kinases (Schlessinger, J. and Ullrich, A., 1992, Neuron 9: 383-391). Receptor phosphorylation, therefore, creates SH2-binding sites, to which an adaptor protein may bind.
In addition to SH2 peptide domains, many of the adaptor proteins involved in signal transduction contain a second conserved motif of 50-75 amino acids residues, the SH3 domain (Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391;
Pawson, T. and Gish, G.D., 1992, Cell 72:359-362; Mayer, B.J.
35 and Baltimore, D., 1993, Trends in Cell Biol. 3 8-13; Mayer, ~ B.J. et al., 1988, Nature 352:272-275). Much less is known about the biological role of the SH3 domain than is known W O 96/40115 PCT~US96/08741 about the role of SH2. The current view is that SH3 domains ~unction, in part, as protein-binding domains that act to link signals transmitted from the cell surface to downstream effector genes such as ras (Pawson, T. and Schlessinger, 5 1993 Current Biology, 3:434-442).

2.4 G-PROTEINS AND SIGNAL TRANSDUCTION
Guanine-nucleotide-binding proteins, (G-proteins; Simon, M.I. et al., 1991, Science 252:802-808; Kaziro, Y. et al., 10 1991, Ann. Rev. Biochem. 60:349-400) such as Ras (for review, see Lowy, D.R. and Willumsen, B.M., 1993, Ann Rev. Biochem.
62:851-891), play an essential role in the transmission o~
mitogenic signals ~rom receptor tyrosine kinases. Taking Ras as an example, the activation o~ receptor tyrosine kinases by 15 ligand binding results in the accumulation of the active GTP
bound ~orm of the Ras molecule (Gibbs, J.B. et al., 1990, J.
Biol. Chem. 265:20437-2044; Satoh, T. et al., 1990, Proc.
NaTl. Acad. Sci. USA 87:5993-5997; Li, B.-Q. et al., 1992, Science 256:1456-1459; Buday, L. and Downward, J., 1993, Mol.
20 Cell. Biol. 13:1903-1910, Medema, R.H. et al., 1993, Mol.
Cell. Biol. 13:155-162). Ras activation is also required for transformation by viral oncogenic tyrosine kinases (Smith, M.R. et al., 1986, Nature 320:540-43).
Ras activity is regulated by the opposing actions of the 25 GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, with GAPs stimulating the slow intrinsic rate of GTP hydrolysis on Ras and exchange factors stimulating the basal rate of exchange of GDP for GTP on Ras.
Thus, GAPs act as negative regulators o~ Ras function, while 30 ~x~h~nge ~actors act as Ras activators.
Recently, a direct link between activated receptor tyrosine kinases and Ras was established with the finding that the m~mm~l ian GRB-2 protein, a 26 kilodalton protein comprised of a single SH2 and two SH3 domains (Lowenstein, 35 E.J. et al., 1992, Cell 70-431-442), directly couples receptor tyrosine kinases to the Ras exchange factor Sos in m~mm~l S and Drosophila (Buday, L. and Downward, J., 1993, Cell 73:611-620; Egan, S.E. et al., 1993, Nature 363:45-51;
Li, N. et al., 1993, Nature 363:85-87; Gale, N.W. et al., 1993, Nature 363:88-92i Rozakis-Adcock et al., 1993, Nature 363:83-85; Chardin, P. et al., 1993, Science 260:1338-1343;
5 Oliver, J.P. et al., Cell 73:179-191; Simon, M.A. et al., 1993, Cell 73:169-177). The GRB-2 SH2 domain binds to specific tyrosine phosphorylated sequences in receptor tyrosine kinases while the GRB-2 SH3 domains bind to proline-rich sequences present in the Sos exchange factor. Binding 10 of GRB-2 to the receptor kinases, therefore, allows for the recruitment of Sos to the plasma membrane, where Ras is located (Schlessinger, J., 1993, TIBS 18:273-275).
Grb2 has been shown to be associated with CSF-1 receptor (vanderGeer and Hunter, 1993, EMBO J. 12(13):5161-5172), PDGF
15 receptor (Li et al., 1994, MCB 14(1):509-517), EGF-R (Matuoka et al., 1993, EMBO J. 12(9):3467-3475; Lowenstein et al., 1992, Cell 70:431-442) and Fak (Schlaepfer et al., 1994, Nature 372:786-791), amongst other proteins.

2.5 CELL PROLIFERATIVE DISORDERS
Growth factors and their receptors are crucial for normal development but can also act as oncogenes leading to cell transformation, oncogenesis, and cell proliferative disorders, including cancer. Activation of the oncogenic 25 potential of normal cellular proteins such as protein tyrosine kinases may, for example, occur by alteration of the proteins~ corresponding enzymatic activities, their inappropriate binding to other cellular components, or both.
Taking as an example Philadelphia chromosome-positive 30 human leukemias, it is known that the BCR-ABL oncoprotein is involved in the pathenogenesis of such leukemias. BCR-ABL
exhibits deregulated tyrosine kinase activity. It has recently been demonstrated (Pendergast, A.M. et al., 1993, Cell 75:175-185) that BCR-ABL binds the SH2/SH3 domain-35 containing GRB-2 adaptor protein. Further, it has been demonstrated that BCR-ABL/GRB-2 binding is mediated by the direct interaction the GRB-2 SH2 domain and a tyrosine-W O 96/40115 PCT~US96/08741 phosphorylated region of the BCR-ABL protein, and that this interaction is required for the activation of the Ras signaling pathway.
Thus, there are multiple events which occur along a 5 signal transduction pathway which appear to be required for the ultimate appearance of a cell proliferative disorder such as the form of leukemia described above. One approach to the treatment of oncogenenic, cell proliferative disorders would be to attempt to "short circuit" abnormal signal transduction 10 events which contribute to the appearance of such disorders, by interfering with one or more of these requisite events.
The amelioration of an abnormal kinase activity may be interfered with by targeting and directly inhibiting the enzymatic activity of the kinase involved in the cell 15 proliferative disorder. It has been proposed that certain compounds may have such anti-tyrosine kinase activity. See, for example, Levitzki and Gazit, 1995, Science 267:1782-1788, wherein certain quinazoline derivatives are proposed to directly inhibit receptor tyrosine kinase enzymatic activity.
In instances wherein the signal transduction event of interest involves an adaptor protein/protein tyrosine kinase interaction, the inhibition of such interactions may lead to the amelioration of cell proliferative disorder symptoms.
The utility of this approach has been demonstrated using 25 expression of signaling incompetent proteins in cells. For example, cells expressing a mutant form o~ Bcr-Abl which lacks the tyrosine residue necessary for binding of the GrB2 SH2 domain and is thus signaling incompetent no longer exhibits a transformed phenotype (RER) (Pendergast et al., 30 supra). To date, however, no such inhibitor of adaptor protein/protein tyrosine kinase interactions has been identified.

3. SUMMARY OF THE lNv~;NlIoN
The present invention relates to methods and compositions for the inhibition o~ adaptor protein/protein tyrosine kinase protein interactions, especially wherein CA 02224l03 l997-l2-08 W O 96/40115 PCTnUS96/08741 those interactions involving a protein tyrosine kinase capable of complexing with a member of the SH2-and/or S~3-containing family of adaptor proteins are associated with a cell proliferative disorder. Specifically, the present 5 invention relates to particular organic compounds and methods ~ utilizing such compounds.
"Protein tyrosine kinase" will, herein, be abbreviated "PTK". It is to be understood that "PTK" may re~er to either a transmembrane, receptor-type protein tyrosine kinase or a 10 cytoplasmic protein tyrosine kinase, unless otherwise indicated. The compounds of the invention ; nh; hit PTK/adaptor protein interactions, especially PTK/adaptor protein interactions wherein the PTK is, for example, an epidermal growth factor receptor (EGF-R) protein tyrosine 15 kinase molecule, a platelet derived growth factor receptor (PDGF-R) protein tyrosine kinase molecule, or an insulin growth factor-like receptor tyrosine kinase molecule (IGF-lR).
The compounds of the present invention are described by 20 the formula (I) below:
ID 2,5-bisindoly-3-yl-1,4-quinone RgtO R14 (I) and pharmaceutically acceptable salts thereof, wherein:
Rl and R2 are independently H, acetate or aryl, alkylaryl and higher alkyl acid ester;

R3 to R14 are independently H, alkyl, alkenyl, alkynyl, OH, hydroxyalkyl, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, and mercapto which can be substituted or substituted where appropriate.
Specific compounds within the scope of the present invention are described by the formula (II) below. Rl and R2 of the formula can be as listed in Table I following the 10 formula. Illustrative preparations or isolations of these compounds are found in the working examples.

0~

(II) TABLE I
Example R1 R2 1. H 2-(2-methylbut-2-en-4-yl) 2. acetyl 2-(2-methylbut-2-en-4-yl) 3. acetyl 2-(3-methyl-n-butyl) 4. H 2-(3-methyl-n-butyl) 5. H 5-bromo 6. H 2-allyl 10 7- H 2-n-propyl 8. H 2-aminocarbonyl 9. acetyl 2-aminocarbonyl 10. benzoyl 2-allyl 11. H 2-cyano 15 12. H 4-methoxycarbonyl 13. H 5,7-dimethoxy 14. H 4,7-dimethoxy 15. H 5-nitro 16. H 4-(4-chlorobenzoylamino) 17. H 4-(4-chlorophenyl) 18. H 2-(4-fluorophenyl) 19. H 4,6-dimethoxy 20. H 5-hydroxy-6-methoxy 25 21, H 4-cyano 22. H 5-(4-trifluoromethylphenyl-aminocarbonyl) 23. H 2-(4-trifluoromethylphenyl-aminocarbonyl) 24. H 2-ethyl 25. H 5-bromo-6-nitro 26. OMe 2-(2-methylbut-2-en-4-yl) 27. OMe 2-(3-methyl-n-butyl) SUBSTITUTE SHEET (RULE 26) W O 96/40115 PCT~US96/08741 Specific compounds within the scope of the present invention are also described by ~ormula (III) below. R1-R12 of the formula can be as listed in Table II following the formula. Illustrative preparations or isolations of these 5 compounds are ~ound in the working examples.

R

(III) W O 96/40115 PCT~US96/08741 TA~3LE II

Ex. R1=R2 R11 R12 R3-R10 28. H 2-(3-methyl- 2-(3-methyl-n-butyl) n-butyl) 29. H 2-methyl 2-methyl 30. H 2-ethyl 2-ethyl 31. H 2-butyl 2-butyl 32. H 2-(but-1-en- 2-(but-1-en-4-yl) 4-yl) 330 H 2-(4-methyl- 2-(4-methyl-n-pentyl) n-pentyl) 34. H 2-phenylethyl 2-phenylethyl 35. H H 2-(3-methyl-n-butyl) 36. H 2-ethyl 2-ethyl R5=R9=carboxy 37. H 2-(n-propyl) 2-(n-propyl) R5=R9=carboxy 38. H 2-(3-methyl- 2-(3-methyl- R5=R9=carboxy n-butyl) n-butyl) 39 H 2-(4-carboxy- 2-(4-carboxy-n-butyl) n-butyl) 40. H H 2-(3-methyl- R5=carboxy n-butyl) 41. H 2-ethyl 2-ethyl R5=R9=amino 42. H 2-(n-propyl) 2-(n-propyl) R5=R9=amino 43. H 2-(3-methyl- 2-(3-methyl- R5=R9=amino n-butyl) n-butyl) 44. acetyl 2-(3-methyl- 2-(3-methyl-n-butyl) n-butyl) 45. H 2-ethyl 2-ethyl R5=R9 = (4-methylphenyl-sulfonylamino) 46. H 2-(n-propyl) 2-(n-propyl) R5=R9 = (4-methylphenyl-sulfonylamino) Unless otherwise indicated, R3-R10 = hydrogen.

CA 02224l03 l997-l2-08 47. H 2-(3-methyl- 2-(3-methyl- R5=R9 = (4-n-butyl) n-butyl) methylphenyl-sulfonylamino) 48. H 2-(2- 2-(2-methylbut-1- methylbut-1-en-4-yl) en-4-yl) 49. H 2-(2- 2-(2-methylpent-2- methylpent-2-en-5-yl) en-5-yl) By the term "alkyl" as used herein is meant a straight 10 or branched chain saturated hydrocarbon group having from 1 to 20 carbons such as methyl, ethyl, isopropyl, n-butyl, s-butyl, t-butyl, n-amyl, isoamyl, n-hexyl, n-octyl and n-decyl; "alkenyl" and "alkynyl" are used to mean straight or branched chain hydrocarbon groups having from 2 to 10 carbons 15 and unsaturated by a double or triple bond respectively, such as vinyl, allyl, propargyl, 1-methylvinyl, but-1-enyl, but-2-enyl, but-2-ynyl, 1 methylbut-2-enyl, pent-1-enyl, pent-3-enyl, 3-methylbut-1-ynyl, 1,1-dimethylallyl, hex-2-enyl and 1-methyl-1-ethylallyl; "alkylaryl" means the aforementioned 20 alkyl groups substituted by a phenyl group such as benzyl, phenethyl, phenopropyl, 1-benzylethyl, phenobutyl and 2-benzylpropyl; "aryl" as used herein includes a monocyclic or bicyclic rings, wherein at least one ring is aromatic including aromatic or hetero-aromatic hydrocarbons; the term 25 "hydroxy-alkyl~ means the aforementioned alkyl groups substituted by a single hydroxyl group such as 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl, 1-hydroxybutyl and 6-hydroxyhexyl.
The term "substituted" as used herein means that the 30 group in question may bear one or more substituents including but not limited to halogen, hydroxy, cyano, alkyl, aryl, alkenyl, alkynyl, amino, nitro, mercapto, carboxy and other substituents known to those skilled in the art.

W O96/4011' PCT~US96/08741 Preferred compounds of the present invention include the following:

~~'? ~

~ ~ OH

Compound 1 H

20 O~

25~ OH

30Compound 2 and pharmaceutically acceptable salts thereof.
In addition, the present invention encompasses a ~ pharmaceutical composition comprising a compound of the 35 invention, and methods for using a compound or pharmaceutical composition of the invention in an ~n ~ m~ l, particularly a human, to ameliorate symptoms of cell proli~erative disorders W O 96/40115 PCT~US96/08741 involving protein tyrosine kinase/adaptor protein interactions.
This invention is based, in part, on the discovery that the disclosed compounds, while exhibiting no inhibitory 5 e~fect on protein tyrosine kinase enzymatic activity, act to inhibit the binding of an SH2-containing peptide to a tyrosine phosphorylated EGF receptor. The data representing this discovery is presented in the Examples in Sections 6, 7 and 8, below. The Example presented in Section 5, below, 10 describes a method for the production of the compounds of the present invention.
The present invention represents the first instance whereby compounds have been discovered which directly inhibit the interaction between adaptor proteins and protein tyrosine 15 kinase molecules.

4. DETAILED DESCRIPTION OF THE lNv~NlION
Described below are methods and compositions for the inhibition of adaptor protein/protein tyrosine kinase protein 20 interactions, especially those interactions associated with a cell proli~erative disorder. Specifically, described below are particular organic compounds, methods for the synthesis of such compounds, and techniques utilizing such compounds.

4.1 COMPOUNDS
The compounds o~ the present invention are described by the following ~ormula (IV):

o ~ ~< ~~
RgtO R14 (IV) and pharmaceutically acceptable salts thereof, wherein:
Rl and R2 are independently H, acetate or aryl, alkylaryl and higher alkyl acid ester;

R3 to Rl4 are independently H, alkyl, alkenyl, alkynyl, hydroxyalkyl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, and mercapto which can be substituted or substituted where appropriate. For example, alkyl groups of the compounds of the present invention may be substituted where appropriate with one or more carboxy or aryl groups. Alkenyl groups of compounds of the present invention may be substituted where appropriate with one or more carboxy groups. Specific compounds within the scope of the present invention are found in the pr~r~;ng Tables I and II. Illustrative preparations or isolations of these compounds are found in the working examples.

CA 02224l03 l997-l2-08 WO 96/40115 PCTfUS96/08741 In one embodiment, compounds of the present invention are described by the following formula (III):

R~

(III) and pharmaceutically acceptable salts thereof, wherein:

R1 and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, and wherein at least one of Rl and R2 is other than hydrogen;

R3 to R12 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, mercapto or 2-methylbut-2-en-4-yl; and wherein at least one of R11 and R12 is 2-methylbut-2-en-4-yl.
Groups R1-R12 may be substituted or unsubstituted where appropriate.

In another embodiment, compounds of the present invention are described by formula (III) above, and pharmaceutically acceptable salts thereo~, wherein:

Rl and R2 are both H;

R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, mercapto or 2-methylbut-2-en-4-yl; and R11 and R12 are each independently H or 2-methylbut-2-en-4-yl, wherein at least one of R11 and R12 is 2-methylbut-2-en-4-yl;

wherein at least one of R3 to R10 is other than H.

In another embodiment, compounds of the present invention are described by formula (III) above, and pharmaceutically acceptable salts thereof, wherein:

R1 and R2 are each independently aryl, alkylaryl and higher alkyl acid ester; and R3 to R12 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, fluoro, chloro, iodo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sul~onamide, amino, or mercapto.

In another embodiment, compounds of the present invention are described by ~ormula (III) above, and 30 pharmaceutically acceptable salts thereof, wherein:

R1, R2, R11 and R12 are H; and R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, alkoxy, hydroxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sul~onamide, amino, or mercapto, wherein at least one o~ R3 to R10 is other than H;
(a) when R4-R10 are each H, R3 may not be 2-methylbut-2-en-4-yl or 2-hydroxy-2-methylbut-4-yl;
(b~ when R4-R6 and R8-R10 are each H, R3 and R7 may not simultaneously be 2-methylbut-2-en-4-yl;
(c) when R3-R4, R6-R8 and R10 are H, R5 and R9 may not simultaneously be 2-methylbut-2-en-4-yl or 3-methyl-n-butyl;
(d) when R3, R5-R7, R9-R10 are H, R4 and R8 may not both be 2-methylbut-2-en-4-yl or 2-methylbut-1,4-dien-4-yl, and R4 and R8 may not be 2-methylbut-2-en-4-yl and 2-methylbut-1,4-dien-4-yl.

The present invention also encompasses compounds o~
~ormula (III) above, and pharmaceutically acceptable salts 20 thereo~, wherein R3-R5 and R7-R9 are H and either or both o~
R6 and R10 are 2-methylbut-2-en-4-yl.

In another embodiment, compounds of the present invention are described by formula (III) above, and 25 pharmaceutically acceptable salts thereo~, wherein:

at least one o~ R1 and R2 is acetyl;

R11 and R12 are H; and R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sul~onyl, sul~onamide, amino, and mercapto, wherein:
(a) when both R1 and R2 are acetyl; or when one o~
R1 and R2 is acetyl and R3-R4, R6-R8 and R10-W O 96/40115 PCTAJS96/0~741 R12 are H; R5 and R9 may not simultaneously be 2-methylbut-2-en-4-yl;
(b) when both R1 and R2 are acetyl and when R4-R6 t and R8-R10 are H, R3 and R7 may not simultaneously be 2-methylbut-2-en-4-yl;
J ( C ) when both R1 and R2 are acetyl and when R3, R5-R7, and R9-R10 are H, R4 and R8 may not simultaneously be 2-methylbut-2-en-4-yl.

In another embodiment, compounds of the present invention are described by formula (III) above, and pharmaceutically acceptable salts thereof, wherein:

at least one of R1 and R2 is lower alkyl;
R11 and R12 are H; and R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, and mercapto, wherein:
(a) when both R1 and R2 are methyl, at lea~t one of R3 to R10 must be a group other than H;
(b) when both R1 and R2 are methyl, and R4-R10 are H, R3 may not be 2-methylbut-2-en-4-yl;
(c) when both R1 and R2 are methyl, and R4-R6 and R8-R10 are H, R3 and R7 may not simultaneously be 2-methylbut-2-en-4-yl;
(d) when both R1 and R2 are methyl, and R3-R4, R6-R8 and R10 are H, R5 and R9 may not simultaneously be 2-methylbut-2-en-4-yl.

The present invention also includes compounds of formula (III) above, and pharmaceutically acceptable salts thereof~
wherein R4 is 2-methylbut-2-en-4-yl and R3 and R5-R10 are H;

W O 96/40115 PCT~US96/08741 or R5 is 2-methylbut-2-en-4-yl and R3-R4 and R6-R10 are H;

or R6 is 2-methylbut-2-en-4-yl, and R3-R5 and R7-R10 are H.

In another embodiment, compounds of the present invention are described by formula (III) above, and pharmaceutically acceptable salts thereof, wherein:
R1 and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, R3 to R10 are each independently H, alkyl, alkylcarboxy, aryl, alkylaryl, alkenyl, alkenylcarboxy, OH, alkoxy, nitro, halo, trihalomethyl ! amide, carboxyamide, carboxy, sulfonyl, sulfonamide, amino, mercapto, 4-methylphenylsulfonylamino, or 2-methylbut-2-en-4-yl; and R11 and R12 are selected from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, aryl, alkylaryl, alkylcarboxy, alkenylcarboxy, but-1-en-4-yl, 2-methylbut-l-en-4-yl, 4-methyl-n-pentyl, 2-phenylethyl, 2-methylpent-2-en-4-yl, and 4-carboxy-n-butyl, wherein at least one of R11 and R12 is other than hydrogen.

In yet another embodiment, compounds of the present invention are described by formula (III) above, and pharmaceutically acceptable salts thereof, wherein:
Rl and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, R3 to R10 are each independently H, alkyl, alkylcarboxy, aryl, alkylaryl, alkenyl, alkenylcarboxy, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxyamide, CA 02224l03 l997-l2-08 WO 96/40115 PCT~US96/08741 carboxy, sul~onyl, sulfonamide, amino, mercapto, 4-methylphenylsul~onylamino, or 2-methylbut-2-en-4-yl; and R11 and R12 are both 3-methyl-n-butyl.

In still another embodiment, compounds o~ the present invention area described by formula (III) above, and pharmaceutically acceptable salts thereo~, wherein:

R1 and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, R3 to R10 are each independently H, alkyl, alkylcarboxy, aryl, alkylaryl, alkenyl, alkenylcarboxy, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxyamide, carboxy, sulfonyl, sul~onamide, amino, mercapto, 4-methylphenylsul~onylamino, or 2-methylbut-2-en-4-yl and wherein at least one o~ R3 to R10 is other than hydrogen; and R11 and R12 are each independently hydrogen or 3-methyl-n-butyl W O 96/40115 PCT~US96/08741 ~~

~ ~H
H

Compound 1 H

¦OH

Compound 2 The invention encompasses the compounds described above as well as pharmaceutically acceptable salts thereof. The compounds o~ the present invention can either be synthesized or isolated as described herein.
The compounds of the present invention can be 35 synthesized in accordance with standard organic chemistry techniques using readily available starting materials.

W O 96/40115 PCT~US96/08741 Alternatively, the compounds can be isolated as described in Section 5.2, below. Chemical synthesis and isolation methods are provided herein solely ~or illustration. Variation of these methods will be apparent to those skilled in the art.

4.2 PRODUCTION OF THE COMPOUNDS
4.2.1 ISOLATION OF NATURAL PRODUCTS
The present Example employed a fungus culture (PenLabs Inc. #592), and the ~ollowing ~ermentation conditions:
10 medium yeast malt extract plus trace elements at 22~C. The seed medium consisted of mannitol 60.0 g; soybean meal 12.5 g, citric acid 2.5 g, yeast extract 0.5 g, and H2O to 1 liter.
The pH o~ the seed medium was adjusted to 7.0 be~ore autoclaving. 30mL seed medium were dispensed per 250 ml 15 flask (6 days 28~C), which was then inoculated with 1 ml of spore/mycelium homogenate suspension (2 days). Stock cultures were maintained ~rozen at -80~C in spore storage solutions.
The ~ermentation mixture (mycelium and broth) was 20 homogenized and ~iltered through cheesecloth by suction ~iltration. The ~iltrate was extracted three times with 0.5 v/v o~ ethyl acetate. The ethyl acetate layers were combined and the solvent removed by rotary evaporation. The mycelium was extracted twice with 0.4 v/v of ethyl acetate. The ethyl 25 acetate layers were combined and the solvent removed by rotary evaporationO The oily residues both cont~;n~ng the asterriquinones were combined and dried on a vacuum pump overnight.
The crude extract obtained above underwent CPC
30 ~ractionation on a PC Inc. high speed countercurrent chromatograph (HSCC) containing a "tripple" coil column. A
1:3:3:3 v/v/v/v o~ n-hexane, ethyl acetate, methanol and water was mixed and allowed to settle overnight. The lower layer was pumped into HSCC column as the stationary phase.
35 The upper layer was used as the mobile phase. After two hours, the lower and upper layer were switched. The HSCC run was completed a~ter ~our hours. The crude metabolites eluted WO 96/4011~ PCT~US96/08741 from 8 to 12 minutes. The active fractions were pooled and evaporated under reduced pressure to dryness.
The pooled HSCC fraction (8-12) was subjected to semi-preparative HPLC (Water HPLC system with a Water 996 5 photodioarray detector using Millennium software) fractionations using the Eollowing conditions:
Two semi-preparative C18-cartridges (25 x 100 mm each, Nova Pak, 6 ~); Flow rate: 10 mL/min.; 120 mg oi~ the pooled HSCC fraction 8-12 dissolved in 6 mL oE DMSO; 250 IlL aliquots 10 per injection; PDA monitored at 270 nm; linear gradient o~
70~ H2O/ 30~ CH3CN to 100~ CH3CN over 30 minutes; then isocratic at 100% CH3CN Eor 6 minutes; the active material eluted at 19 and 24 minutes. The active fractions ~rom the 10 runs were combined and evaporated under reduced pressure 15 to dryness to yield 17 mg oE Asterriquinone C-3 (Compound I) and 3 mg of Preasterri~l~ non~ C-3 (Compound II).
Mass spectra were recorded on PE Sciex LC-MS model API
III (Ion Spray), exact mass measurements were performed at high resolution (HR-FAB). Mass spectral analysis for 20 compound I gave a molecular ion of 507 (M+H)+ (molecular weight: 506). The molecular Eormula C32H31N2O4(M~+H): 507.2289;
found 507.2291). lH NMR spectra o~ compound I were recorded in CDCl3 at 500 MHz on a Brucker DRX-500. Chemical shi~ts are given in ppm relative to TMS at zero ppm using the solvent 25 peak at 7.26 ppm (CDCl3) as an internal standard. Compound I:
8.18 (s, 2H), 8.05 (s, 2H), 7.35-7.10 (m, 8H), 5.40 (m, 2H), 3.45 (m, 4H), 1.81 (s, 6H~ and 1.75 ppm (s, 6H). 13C NMR
spectra of compound I were recorded in DMSO-d6 at 125 MHz on a Brucker DRX-500. Chemical shifts are given in ppm relative 30 to TMS at zero using the solvent peak at 39.5 ppm (DMSO-d6) as an internal standard. 138.8~ 136.6~ 136.3, 128.8, 128.2, 127.3, 122.3, 121.8, 121.0, 12003, 119.5, 119.3, 112.3, 111.8, 111.6, 105.2, 102.2, 27.3, 26.4 and 18.5 ppm.
Compound I gave a melting point o~ 150-154~C.
Mass spectral analysis for compound II gave a molecular ion of 439 (M+H)t (molecular weight: 438). lH NMR spectra of compound II were recorded in DMSO-d6 at 500 MHz on a Brucker W O 96/40115 ~CTAJS96/08741 DRX-500. Chemical shifts are given in ppm relative to TMS at zero ppm using the solvent peak at 2.49 ppm (DMSO-d6) as an internal standard. 11.35 (s, lH), 10.96 (s, lH), 10.62 (s, lH), 7.48 (d~ = 1 Hz, lH), 7.39 (d, J = 10.0 Hz, lH), 7.29 5 (d, J = 10.OHz, lH), 7.14 (d, J = 10 Hz, lH), 7.07 (t, J =
10.0 Hz, lH), 6.99 (t, J = 10.0 Hz, lH), 6.93 (t, J = 10.0 Hz, lH), 6.88 (t, J = 10.0 Hz, lH), 5.26 (m, lH), 3.30 (m, 2H) 1.64 (bs, 3H) and 1.61 ppm (bs, 3H). 13C NMR spectra of compound II were recorded in CDCl3 at 125 MHz on a Brucker 10 DRX-500. Chemical shifts are given in ppm relative to TMS at zero ppm using the solvent peak at 77.0 ppm (CDCl3) as an internal standard~ 138.4, 138.3, 135.7, 135.2, 127.7, 121.6, 120.0, 119.8, 119.6, 110.7, 110.6, 100.5, 26.8, 25.8 and 18.0 ppm.
4.2.2 COMPOUND SYNTHESIS
Example 1 2,5-Dihydroxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]l~4-quinone A mixture of 100 mg. of 2,5-diacetoxy-3,6-dibromo-1,4-quinone, or other suitably protected quinone such as 3,6-dibromo-2,5-ditrimethylsiloxy-1,4-quinone, 3,6-dibromo-2,5-di-(t-butyldimethylsiloxy-1,4-quinone, 2,5-dibenzoxy-3,6-25 dibromo-1,4-quinone, 3,6-dibromo-2,5-diisobutryoxy-1,4-quinone, 2,5-dibenzyloxy-3,6-dibromo-1,4-quinone or 2,5-diallyoxycarbonyloxy-3,6-dibromo-1,4-quinone which can be prepared from commercially available 2,4-dibromo-3,6-dihydroxy-1,~-quinone and 180 mg of 3-[2-(2-methylbut-2-en-4-30 yl)indole, prepared by the Fisher indole synthesis, in 10 mlof anhydrous dimethylforamide, or pyridine or dimethylsulfoxide, with powdered potassium carbonate, was heated at 100~C for 24 hours. The cooled mixture was partitioned between ethyl acetate and water. The ethyl 35 acetate layer was then washed with brine, dried over sodium sulfate, filtered and concentrated. The crude was then purified on a medium pressure liquid chromatography column in W O 96/40115 PCT~US96/08741 a solvent mixture of dichloromethane and methanol to provide 25 mg o~ 2,5-diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone. 2,5-Diacetoxy-3,6-di-[2(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone was then 5 hydrolysed with 1 N aqueous sodium hydroxide solution in methanol. Acidification of the above mixture produced the crude product after ~iltration. Further crystallization in ethanol and water produced the title compound. Other aforementioned protecting groups, they can be removed by 10 conventional deprotection methods such as diluted acid, potassium fluoride or palladium (0) complex or palladium on carbon with hydrogen or by methods described by Greene and Wuts (Protective groups in organic synthesis, John Wiley and Son, 1991).
Alternatively, under the similar conditions, 2,3,5,6-tetrabromo-1,4-quinone reacts with excess indole in the presence of potassium carbonate and aluminum oxide in dimethylformamide or dimethylsulfoxide at 100~C to produce the substituted 2,5-dibromo-3,6-(3-indolyl)-1,4-quinone which 20 can react with base such as sodium hydroxide to give the substituted 2,5-dihydroxy-3,6-(3-indolyl)-1,4-quinone (Hoerher, J.; Schwenner, E.; Franck, B., Liebigs Ann. Chem.
1986, 10: 1765-1771).

25 Example 2 2,5-Diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone 2,5-Diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-30 yl]1,4-quinone was prepared in Example 1.

Example 3 2,5-Diacetoxy-3,6-di-[2t3-methyl-n-butyl)indol-3-yl]1,4-quinone Hydrogenation of 2,5-diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone in methanol with 5~ palladium on carbon under 1 atm o~ hydrogen produced the title compound.
i 5 Example 4 2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone Base hydrolysis of 2, 5-diacetoxy-3,6-di-[2-(3-methyl-n-10 butyl)indol-3-yl]1,4-quinone as described in Example 1 produced the title compound.

Under similar conditions as those described in Examples 1 to 4, the ~ollowing compounds are prepared using either 2,5-15 dibromo-3,6-dihydroxy-1,4-quinone or 2,3,5,6-tetrabromoquinone as starting materials:

Example 5 3,6-Di-[5-(bromo)indol-3-yl]-2,5-dihydroxy-1,4-quinone Example 6 3,6-Di-[2-(allyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone Example 7 25 2,5-Dihydroxy-3,6-di-[2-(n-propyl)indol-3-yl]1,4-quinone Example 8 3,6-Di-[2-(aminocarbonyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone Example 9 2,5-Diacetoxy-3,6-di-[2(aminocarbonyl)indol-3-yl]-1,4-quinone Example 10 35 3,6-Di-[2-allylindol-3-yl]-2,5-dibenzoyloxy-1,4-quinone Example 11 2,5-Dihydroxy-3,6-di-[2-(cyano)indol-3-yl]1,4-quinone Example 12 5 2,5-Dihydroxy-3,6-di-[4-(methoxycarbonyl)indol-3-yl]1,4-quinone Example 13 2,5-Dihydroxy-3,6-di-[5,7-(dimethoxy)indol-3-yl]1,4-quinone Example 14 2,5-Dihydroxy-3,6-di-[4,7-(dimethoxy)indol-3-yl]1,4-quinone Example 15 15 2,5-Dihydroxy-3,6-di-[5-(nitro)indol-3-yl]1,4-quinone Example 16 3,6-di-[4(4-chlorobenzoylamino)indol-3-yl]-2,5-dihydroxy-1,4-quinone Example 17 3,6-di-[2-(4-chlorophenyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone 25 Example 18 2,5-Dihydroxy-3,6-di-[2-(4-~luorophenyl)indol-3-yl]1,4-quinone Example 19 30 2,5-Dihydroxy-3,6-di-[4,6-~dimethoxy)indol-3-yl]1,4-quinone Example 20 2,5-Dihydroxy-3,6-di-[2-(5-hydroxy-6-methoxy)indol-3-yl]1,4-quinone Example 21 2,5-Dihydroxy-3,6-di-[4-(cyano)indol-3-yl]1,4-quinone WO 96/40115 PCT~US96/08741 Example 22 2,5-Dihydroxy-3,6-di-[5-(4-tri~luoromethylphenylaminocarbonyl)indol-3-yl]1,4-quinone 5 Example 23 2,5-Dihydroxy-3,6-di-[2-(4-tri~luoromethylphenylaminocarbonyl)indol-3-yl]1,4-quinone Example 24 10 2,5-Dihydroxy-3,6-di-[2-(ethyl)indol-3-yl]1,4-quinone Example 25 3,6-di-[2-(5-bromo-6-nitro)indol-3-yl]-2,5-dihydroxy-1,4-quinone Example 26 2,5-Dimethoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone 20 Methylation o~ Example 1 with methyl iodide and potassium carbonate in dimethyl~oramide ~ollowed by puri~ication produced the title compound. This compound could also be prepared by heating 2,5-dibromo-3,6-di[2-(2-methylbut-2-en-4-yl)indol-3-y]1,4-quinone in methanol in the pre~ence o~
25 powdered potassium carbonate.

Example 27 2,5-Dimethoxy-3,6-di-[2(3-methyl-n-butyl)indol-3-yl]1,4-quinone Hydrogenation o~ Example 26 under conditions as those in Example 3 produced the title compound.
.

W O 96/40115 PCT~US96/08741 Example 28 Preparation of 2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl) indol-3-yl]-1,4-quinone To a glass tube containing 2-(3-methyl-n-butyl) indole 5 (400 mg), bromanil (431 mg) and potassium carbonate (703 mg), equipped with a magnetic stir bar, was added dimethylformamide (10 ml). The mixture was stirred at room temperature for 40 h. Following dilution with 1 N HCl (100 ml), the crude mixture was extracted with ethyl acetate (200 10 ml)0 The organic layer was washed with brine (100 ml) and dried with sodium sul~ate. After removal of solvent under reduced pressure, the crude residue was filtered through a short plug of flash silica, eluting with 30~ ethyl acetate/hexane. The solvent was removed under reduced 15 pressure, and the residue was purified by flash chromatography (15~ ethyl acetate/hexane) to yield 2,5-dibromo-3,6-di-[2-(3-methyl-n-butyl) indol-3-yl]-1,4-quinone (40 mg, 7~) as a blue crystalline solid.
To a stirred solution of 2,5-dibromo-3,6-di-[2-(3-20 methyl-n-butyl) indol-3-yl]-1,4-quinone (40 mg) in methanol (1.5 ml) was added 2N methanolic sodium hydroxide (0.251 ml).
The solution was stirred at room temperature for 24h, followed by dilution with water (50 ml) The product was extracted with ethyl acetate (100 ml), washed with brine (50 25 ml) and dried with sodium sulfate. Removal of solvent under reduced pressure provided 2,5-methoxy-3,6-di-[2-(3-methyl-n-butyl) indol-3-yl]-1,4-quinone (30 mg, 90~) as a yellow crystalline solid.
To a stirred solution of 2,5-dimethoxy-3,6-di-[2-(3-30 methyl-n-butyl) indol-3-yl]-1,4-quinone (9 mg) in ethanol (2 ml) was added 1 N aqueous potassium hydroxide (1 ml). The mixture was heated at 85~C for 3.5 h, then diluted with 1 N
HCl (25 ml). The product was extracted with ethyl acetate (50 ml), washed with brine ~25 ml) and dried with sodium 35 sulfate. The solvent was removed under reduced pressure to afford 2,5-dihydroxy-3,6-di-[2-(3-methyl-n-butyl) indol-3-yl]-1,4-quinone (8 mg) as a reddish-brown crystalline solid.

W O 96/40115 PCT~US96/08741 28a) Preparation of 2-(2-methyl-1-buten-4-yl) indole To a stirred solution of 2-methylindole (lg) in diethylether (76 ml) under nitrogen was added a 1.6 M
solution o~ n-butyllithium in hexane (14.3 ml) slowly dropwise via syringe. Potassium tert-butoxide (1.711 g) was then added, producing a bright yellow mixture.
After stirring at room temperature under nitrogen ~or 50 min., the mixture was cooled to -78~C, whereupon 3-bromo-2-methylpropene (1.54 ml) was added dropwise via syringe, giving a red-orange solution. The reaction mixture was stirred at -78~C for 2h, then quenched with water (10 ml). After warming to room temperature, water (150 ml) and 1 N HCl (1 ml) was added to neutralize the reaction mixture. The mixture was extracted with ethyl acetate (250 ml), and the organic layer was washed with brine (100 ml) and dried with sodium sul~ate. The solvent was removed under reduced pressure, and the crude residue was puri~ied by ~lash chromatography (4~
ethyl acetate/hexane) to a~ford 2-(2-methyl-1-butene-4-yl) indole (664 mg. 47~) as a waxy yellow solid.

28b) Preparation of 2-(3-methyl-n-butyl) indole Into a 3-necked round bottom ~lask under a blanket o~
nitrogen was placed 5~ palladium catalyst on charcoal (771 mg!. A solution of 2-(2-methyl-1-buten-4-yl) indole (671 mg) in ethanol (36 ml) was added to the ~lask, which was evacuated and charged with hydrogen twice. The mixture was stirred vigorously under hydrogen (1 atm) ~or 2h, ~ollowed by ~iltration through a pad o~ Celite. The solvent was removed under reduced pressure and the crude residue was purified by ~lash chromatography (3~ ethyl acetate/hexane) to give 2-(3-methyl-n-butyl) indole (400 mg, 59~) as a yellow crystalline solid.

W O 96/40115 PCT~US96/08741 Example 29 Preparation o~ 2,5-Dihydroxy-3,6-di-[2-(methyl) indol-3-yl]-1,4-quinone Re~er to Example 28 using 2-methylindole as the starting 5 indole.

Example 30 Preparation o~ 3,6-Di-(2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quinone Re~er to Example 28 using 2-ethylindole as the starting indole.
3Oa) Preparation o~ 2-ethylindole Refer to 28a) using methyl iodide as the alkylating agent.
Example 31 Preparation o~ 3,6-Di-(2-butylindol-3-yl) 2,5-dihydroxy-1,4-quinone Re~er to Example 28 using 2-butylindole as the starting 20 indole.
31a) Preparation o~ 2-(but-1-en-4-yl) indole Refer to 28a) using allyl bromide as the alkylating agent.
31b) Preparation o~ 2-butylindole Re~er to 28b) using 2-(but-1-en-4-yl) indole as the starting material.

Example 32 Preparation o~ 3,6-Di-[2-(but-1-en-4-yl) indol-3-yl] 2,5-30 dihydroxy-1,4-quinone Re~er to Example 28 using 2-(but-1-en-4-yl) indole as the starting indole.

Example 33 35 Preparation o~ 2,5-Dihydroxy-3,6-di-[2-(4-methyl-n-pentyl) indol-3-yl]-1,4-quinone WO 96/4011~ PCT~US96/08741 Refer to Example 28 using 2-(4-methyl-n-pentyl) indole as the starting indole.
33a) Preparation of 2-(2-methyl-2-penten-5-yl) indole Refer to 28a) using 4-bromo-2-methyl-2-butene as the alkylating reagent.
33b) Preparation of 2-(4-methyl-n-pentyl) indole Refer to 28b) using 2-(2-methyl-2-penten-5-yl) indole as the starting material.
Example 34 Preparation of 2,5-Dihydroxy-3,6-di-[2-(2-phenylethyl) indol-3-yl]-1,4-quinone Refer to Example 28 using 2-(2-phenylethyl) indole as~5 the starting indole.
34a) Preparation of 2-(2-phenylethyl) indole Refer to 28a) using benzyl bromide as the alkylating agent.

20 Example 35 Preparation of 2,5-Dihydroxy-6-(indol-3-yl)-3-[2-(3-methyl-n-butyl) indol-3-yl]-1,4-quinone This synthesis could be achieved by treating 2-(3-methyl-n-butyl) indole with 2 equivalents of bromanil in the 25 presence of potassium carbonate in dimethylformamide, followed by workup and purification similar to Example 28.
The resultant mono-indolyl adduct could then be treated with 2 equivalents of indole under the same conditions as above to provide the bis-indolyl product.
Example 36 Preparation of 3l6-Di-(5-carboxy-2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quinone Refer to Example 28 using 5-carboxy-2-ethylindole as the 35 starting indole.
36a) Preparation of 5-carboxy-2-ethylindole W O 96/40115 PCT~US96/08741 This synthesis could start with 5-chloro-2-methylindole~ which could be alkylated with methyl indole (see 28a). The product chloroindole could then be converted to its Grignard species and exposed to carbon dioxide to ~inish the synthesis.

Example 37 Preparation of 3,6-Di-[5-carboxy-2-(n-propyl) indol-3-yl]-2,5-dihydroxy-1,4-quinone Refer to Example 28 using 5-carboxy-2-propylindole as the starting indole.
37a) Preparation o~ 5-carboxy-2-propylindole Re~er to 36a) using ethyl iodide as the alkylating agent.
Example 38 Preparation o~ 3,6-Di-[5-carboxy-2-~3-methyl-n-butyl) indol-3-yl]-2,5-dihydroxy-1,4-ql~lnon~
Re~er to Example 28 using 5-carboxy-2-(3-methyl-n-butyl) 20 indole as the starting indole.
38a) Preparation o~ 5-carboxy-2-(2-methyl-1-buten-4-yl) indole Re~er to 36a) using 3-bromo-2-methylpropene as the alkylating agent.
38b) Preparation o~ 5-carboxy-2-(3-methyl-n-butyl) indole Re~er to 28b) using 5-carboxy-2-(2-methyl-1-buten-4-yl) indole as the starting material.

30 Example 39 Preparation o~ 3,6-Di-[2-(4-carboxy-n-butyl) indol-3-yl]-2,5-dihydroxy-1,4-quinone Re~er to Example 28 using 2-(4-carboxy-n-butyl) indole as the starting indole.
39a) Preparation o~ 2-(4-carboxy-3-buten-1-yl) indole WO 96/40115 PCT~US96/08741 Refer to 28a) using 4-bromo-2-butenoic acid as the alkylating agent.
39b) Preparation of 2-(4-carboxy-n-butyl) indole Refer to 28b) using 2-(4-carboxy-3-buten-l-yl) indole as the starting material.

Example 40 Preparation of 3-[5-Carboxy-2-(3-methyl-n-butyl) indol-3-yl]-2,5-dihydroxy-6-(indol-3-yl)-1,4-quinone Refer to Example 35 using 5-carboxy-2-(3-methyl-n-butyl) indole in the first step.

Example 41 Preparation of 3,6-Di-(5-amino-2-ethylindol-3-yl)-2,5-15 dihydroxy-1,4-quinone Refer to Example 28 using 5-amino-2-ethylindole as the starting indole.
41~) Preparation of 5-amino-2-ethylindole This synthesis could be achieved beginning with a standard nitration of 2-ethylindole using sodium nitrate and sulfuric acid similar to that cited in Yokoyama; Tanaka; Yamane; Kurita; Chem. Lett.; 7;
1991; 1125-1128O The resultant 5-nitro-2-ethylindole could be reduced to the desired amino compound using catalytic hydrogenation as in 28b).

Example 42 Preparation of 3,6-Di-[5-amino-2-(n-propyl) indol-3-yl]-2,5-dihydroxy-1,4-quinone Refer to Example 28 using 5-amino-2-(n-propyl) indole as the starting indole.
42a) Preparation of 5-amino-2-(n-propyl) indole Rerer to 41a) using 2-n-propylindole.

35 Example 43 Preparation of 3,6-Di-[5-amino-2-(3-methyl-n-butyl) indol-3-yl] 2,5-dihydroxy-1,4-quinone W O 96/40115 PCT~US96/08741 Refer to Example 28 using 5-amino-2-(3-methyl-n-butyl) indole as the starting indole.
43a) Preparation of 5-amino-2-(3-methyl-n-butyl) indole J
Refer to 41a) using 2-(3-methyl-n-butyl) indole.

Example 44 Preparation of 2,5-Diacetoxy-3,6-di-[2-(3-methyl-n-butyl) indol-3-yl]-1,4-quinone This synthesis could be accomplished by treating 2,5-hydroxy-3,6-di-[2-(3-methyl-n-butyl) indol-3-yl]-1,4-quinone with acetic anhydride in the presence of pyridine.

Example 45 15 Preparation o~ 3,6-Di-[2-ethyl-5-(4-methylphenylsulfonylamino) indol-3-yl]-2,5-dihydroxy-1,4-quinone Refer to Example 28 using 2-ethyl-5-(4-methylphenylsulfonylamino) indole as the starting indole.
45a) Preparation of 2-ethyl-5-(4-methylphenylsulfonylamino) indole The above compound could be synthesized by treating 5-amino-2-ethylindole with p-toluenesulfonyl chloride in the presence of triethylamine.
Example 46 Preparation of 2,5-Dihydroxy-3,6-di-[5-(4-methylphenylsulfonylamino)-2-(n-propyl) indol-3-yl]-1,4-quinone Refer to Example 28 using 5-(4-methylphenylsul~onylamino)-2-(n-propyl) indole as the starting indole.
46a) Preparation of 5-(4-methylphenylsul~onylamino)-2-(n-propyl) indole Refer to 45a) using 5-amino-2-propylindole.

W O 96/40115 PCT~US96/08741 Example 47 Preparation of 2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)-5-(4-methylphenylsulfonylamino) indol-3-yl]-1,4-quinone Re~er to Example 28 using 2-(3-methyl-n-butyl)-5-(4-5 methylphenylsulfonylamino) indole as the starting indole.
47a) Preparation of 2-(3-methyl-n-butyl)-5-(4-methylphenylsul~onylamino) indole Refer to 45a) using 5-amino-2-(3-methyl-n-butyl) indole.
Example 48 Preparation of 2,5-Dihydroxy-3,6-di-[2-(2-methylbut-1-en-4-yl) indol-3-yl]-1,4-quinone Re~er to Example 28 usinq 2-(2-methylbut-1-en-4-yl) 15 indole as the starting indole.

4.3 PROTEIN TYROSINE KINASE/ADAPTOR PROTEIN COMPLEXES
The PTK/adaptor protein complexes which may be disrupted by the methods and compositions of the invention comprise at 20 least one member o~ the PTK family of proteins and at least one member of the adaptor family o~ proteins, as described below. Under standard physiological conditions, the components of such complexes are capable of forming stable, non-covalent attachments with one or more of the other 25 PTK/adaptor protein complex components. Pre~erably, the compounds of he invention inhibit PTK/adaptor protein complexes wherein the PTK component is an epidermal growth factor receptor (EGF-R) protein tyrosine kinase molecule, a platelet derived growth ~actor receptor (PDGF-R) protein 30 tyrosine kinase molecule or an insulin growth factor-like receptor tyrosine kinase molecule (IGF-lR).
Intracellular, cytoplasmic PTK components o~ the PTK/adaptor protein complexes may include, ~or example, members of the Src family, such molecules as src, yes, fgr, 35 fyn, lyn, hck, lck, and blk; members of the Fes family, such as ~es and fer; members of the Abl family, such as abl and arg; and members o~ the Jak ~amily, such as jakl and jak2.

Transmembrane, receptor PTK components of the PTK/adaptor protein complexes may include, ~or example, such molecules as members of the FGF receptor, Sevenless/ROS, Insulin receptor, PDGF receptor, and EGF receptor family of growth factor r 5 receptors.
The adaptor protein components of the PTK/adaptor protein complexes comprise one or more SH2 and/or one or more SH3 non-catalytic domains. The SH2 and SH3 domains which may be a part of the adaptor proteins are as described, above, 10 for the PTK components. Adaptor proteins which may be components of the PTK/adaptor protein complexes may include, for example, p85, c-Crk, SHC, Nck, ISGF3~, guanine triphosphatase activator protein (GAP), and members of the GRB subfamily of proteins, such as GRBl, GRB-2, GRB-3, GRB-4, 15 GRB-7, and GRB-10.

4.4 TREATMENT OF PTK/ADAPTOR PROTEIN COMPLEX-R~LATED
CELL PROLIFERATIVE DISORDERS
The compounds and/or pharmaceutical compositions 20 (described in Section 4.4.2, below) of the invention may be used for the treatment of cell proliferative disorders, such as oncogenic disorders, involving a PTK capable of complexing with a member of the SH2- and/or SH3-containing family of adaptor proteins. The compounds of the invention may be 25 preferentially utilized in the treatment of cell proliferative disorders involving PTK/adaptor protein complexes wherein the PTK component is EGF-R, PDGF-R, MCT or IGF-lR.
Among the oncogenic disorders which may be treated by 30 the compounds of the invention are, for example, BCR-ABL-associated cancers (such as, for example, chronic myelogenous and acute lymphocytic leukemias), gliomas, glioblastomas, melanoma, human ovarian cancers, human breast cancers (especially HER-2/GRB-7-associated human breast cancers), and 35 human prostate cancers.
Assays for determining the effectiveness of a compound in the disruption of a PTK/adaptor protein complex are CA 02224l03 l997-l2-08 described, below, in Section 4.4.1. Methods for the administering the compounds and/or pharmaceutical compositions of the invention to patients are described, below, in Section 4.4.2.
"Disruption'l, as used here, is meant to refer not only to a physical separation of PTK/adaptor protein complex components, but is also meant to refer to a perturbation of the activity of the PTK/adaptor complexes, regardless of whether or not such complexes remain able, physically, to 10 form. "Activity", as used here, refers to the function the PTK/adaptor protein complex in the signal transduction cascade of the cell in which such a complex is formed, i.e., refers to the function of the complex in effecting or inhibiting the transduction of an extracellular signal into a 15 cell. The compounds and pharmaceutical compositions of the invention do not, however, directly interfere with (l.e., inhibit or enhance) the enzymatic activity of the protein tyrosine kinase of interest.

4.4.1 ASSAYS FOR THE DISRUPTION OF
PTK/ADAPTOR PROTEIN COMPLEXES
A variety of methods may be used to assay the ability that the compounds of the invention exhibit to disrupt PTK/adaptor protein complexes. For example, in vitro complex 25 formation may be assayed by, first, immobilizing one component, or a functional portion thereof, of the complex of interest to a solid support. Second, the immobilized complex component may be exposed to a compound such as one identified as above, and to the second component, or a functional 30 portion thereof, of the complex of interest. Third, it may be determined whether or not the second component is still capable of forming a complex with the immobilized component in the presence of the compound.
Additionally, in vivo complex formation may be assayed 35 by utilizing co-immunoprecipitation techniques well known to those of skill in the art. Briefly, a cell line capable of forming a PTK/adaptor complex of interest may be exposed to W O 96/40115 PCT~US96/08741 one or more of the compounds of the invention, and a cell lysate may be prepared from this exposed cell line. An antibody raised against one of the components of the complex of interest may be added to the cell lysate, and subjected to 5 standard lmmllnoprecipitation techniques. In cases where a complex is still formed, the lmml~noprecipitation will precipitate the complex, whereas in cases where the complex has been disrupted, only the complex component to which the antibody is raised will be precipitated.
The effect of a compound of the invention on the transformation capability of the PTK/adap~or protein of interest may be directly assayed. For example, one or more of the compounds o~ the invention may be administered to a cell such as a fibroblast or hematopoietic cell capable of 15 forming a PTK/adaptor complex which, in the absence of a compound of the invention, would lead to the cell's transformation (Muller, A.J. et al., 1991, Mol. Cell. Biol.
11:1785-1792; McLaughlin, J. et al., 1987, Proc. Natl. Acad.
Sci. USA 84:6558-6562). The transformation state of the cell 20 may then be measured in vitro, by monitoring, for example, its ability to ~orm colonies in soft agar (Lugo and Witte, 1989, Mol. Cell. Biol. 9:1263-1270; Gishizky, M.L. and Witte, O.N., 1992, Science 256:836-839). Alternatively, a cell's transformation state may be monitored in vivo by determining 25 its ability to form tumors in immunodeficient nude or severe combined immunodeficiency (SCID) mice (Sawyers, C.L. et al., 1992, Blood 79:2089-2098~. Further, the ability of the compounds of the present invention, to inhibit various tumor cell lines, such as for example, melanoma, prostate, lung and 30 m~mm~ry tumor cell lines established as SC xenografts can be ~mlned.

4.4.2 PHARMACEUTICAL COMPOSITIONS
AND METHODS OF ADMINISTRATION
The compounds of the invention, as described, above, in Section 5.1, may be administered to a patient at therapeutically effective doses to treat or ameliorate cell W O 96/40115 PCT~US96/08741 proliferative disorders involving PTK/adaptor protein interactions. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of a cell proliferative disorder.
Described, below, in Section 5.4.2.1, are methods for determining the effective dosage of the compounds of the invention fo~ the treatment of cell proliferative disorders.
Further, described, below, in Section 5.4.2.2, are methods for formulations and pharmaceutical compositions comprising 10 the compounds of the invention, and methods for the administration of such compounds, formulations, and compositions.

4.4.2.1 EFFECTIVE DOSE
Toxicity and therapeutic efficacy of the compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.q., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective 20 in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be 25 taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and ~n~m~l studies can be used in formulating a range of dosage 30 for use in humans. The dosage of ~uch compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within -his range depending upon the dosage form employed and the route of administration utilized. For any 35 compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell cu_ture assays. A dose may be formulated in ~n;m~l W O 96/40115 PCT~US96/087~1 models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information 5 can be used to more accurately determine useful doses in hllm~nc. Levels in plasma may be measured, ~or example, by high performance liquid chromatography.
Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are 10 sufficient to maintain inhibition of adaptor protein/protein tyrosine kinase interactions, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data, e.g., the interactions using the assays described herein. Dosages necessary to 15 achieve the MEC will depend on individual characteristics and route the administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using the MEC
valueO Compounds should be administered using a regimen 20 which maintains plasma levels above the MEC for 10-90~ of the time, preferably between 30-90~ and most preferably between 50-90~.

4.4.2.2 FORMULATIONS AND ADMINISTRATION
As discussed, above, adaptor proteins are intracellular proteins. Thus, PTK/adaptor protein interactions are intracellular~ regardless of whether the PTK
of interest is of the transmembrane or the intracellular type. Therefore, the compounds of the invention act 30 intracellularly to interfere with the formation and/or activity of the PTK/adaptor complexes. A variety of methods are known to those of skill in the art for administration of compounds which act intracellularly, as, for example, discussed in this SectionO
Pharmaceutical compositions for use in accordance with the compounds of the present invention may be formulated in WO 96/4011~ PCT~US96/08741 conventional m~nn~r using one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by 5 inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically 10 acceptable excipients such as binding agents (e.a., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.a., lactose, microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.q., magnesium stearate, talc or silica);
15 disintegrants (e.a., potato starch or sodium starch glycolate); or wetting agents (e.a., sodium lauryl sulphate).
The tablets may be coated by methods well known in the art.
Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they 20 may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.a., sorbitol syrup, cellulose derivatives or 25 hydrogenated edible fats); emulsifying agents (e.a., lecithin or acacia); non-aqueous vehicles (e.q., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.q., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer 30 salts, flavoring, coloring and sweetening agents as appropriateO
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration the compositions may take the 35 form of tablets or lozenges formulated in conventional manner.

For admini~tration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, 5 e.q., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.q. gelatin for use in 10 an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral a~m;n;.qtration by injection, e.a., by bolus injection or 15 continuous infusion. Formulations ~or injection may be presented in unit dosage form, e.q., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain 20 formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.q., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal 25 compositions such as suppositories or retention enemas, e.q., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be ~ormulated as a depot preparation.
30 Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, ~or example, the compounds may be formulated with suitable polymeric or hydrophobic materials (~or example as an emulsion in an 35 acceptable oil) or ion exchange resins, or as sparingly soluble deri-~atives, ~or example, as a sparingly soluble salt.

W O 96/40115 PCT~US96/08741 The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage ~orms containing the active ingredient. The pack may ~or example comprise metal or plastic foil, such as a blister 5 pack. The pack or dispenser device may be accompanied by instructions ~or administration.

5. EXAMPLE: THE COMPOUNDS INHIBIT EGF-RECEPTOR/GRB-2 In the Example presented in this Section, Compound I is demonstrated to effectively inhibit the binding of tyrosine phosphorylated EGF-receptor to a GRB-2 SH2 peptide domain.

5.1 MATERIALS AND METHODS
Ada~tor-GST fusion protein: The adaptor-GST
(glutathione-S-trans~erase) ~usion proteins used herein were GRB-2-GST fusion proteins prepared by expression in E. coli transformed with GRB-2/pGEX constructs. The GRB-2 portions of these ~usion proteins consisted of only the SH2 domain of 20 the GRB-2 protein. Transformed cells are grown in Luria broth (LB) supplemented with ampicillin. After reaching an optical density (OD) at 600 nm of 0.3, the cells are induced for 6 hours with isopropyl ~-D-thiogalactopyranoside (IPTG) in order to express the fusion protein.
A~ter the 6 hour expression period, the cells are precipitated, pelleted at 10,000 x g for 10 minutes at 4~C, washed, and -;esuspended in phosphate buffered saline (PBS).
Next, the cells are lysed by sonication (6 strokes, 5 seconds per stroke). Insoluble material is removed by centri~ugation 30 at 10,000 x g for 10 minutes at 4~C, and the supernatant is passed over a Glutathion-Sepharose column. Bound GRB-2-GST
~usion protein is eluted off the column with 5 mM reduced glutathion, then dialyzed against PBS.

Immobilized EGF-R tYrosine kinase molecule: Epidermal growth ~actor receptor tyrosine kinase (EGF-R). EGF-R was isolated from cells overexpressing EGF-R, specifically, the A431 (ATCC CRL 1551), cell line~ The cells are lysed in HNTG
buffer (20 m~ Hepes/HCl, pH 7.4, 150 mM NaCl, 1.0~ Triton X-100, 5~ glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/L aprotonin, 1 mg/L leupeptin, 10 mg/L benzamidine).
EGF-R protein was isolated from the cell lysates by immobilization onto microtiter plates, as described below.
EGF-R was subsequently phosphorylated in vitro, as explained below.
The EGF-R molecule was immobilized onto microtiter 10 plates. Microtiter plates were prepared by first coating the wells of the plate, overnight at 4~C, with an anti-EGF-R
monoclonal antibody directed against the extracellular domain of EGFR ~UBI, ~05-101) at a concentration of 0.5 ~g (in PBS) per microtiter well, at a final volume o~ 150 ~1 per well.
After overnight coating, the coating solution was removed from the microtiter wells, and replaced with blocking buffer (5~ d~y milk in PBS) for 30 minutes at room temperature, after which the blocking buffer is removed and the wells were washed 4 times with TBST buffer (150 mM NaCl, 20 50 mM Tris-HCl, pH 7.2, 0.1~ Triton X-100).
Cell lysate from EGF-R-expressing cells were added to each well, in 150 ~1 of PBS, incubated 30 minutes at room temperature, with shaking. Unbound EGF-R was removed by washing wells 5 times with TBST buffer. Approximately 50-100 25 ng of EGF-R protein was bound per well.
It was important to use an EGF-R overexpressing cell line which exhibits a high endogenous phosphatase activity, such as the A431 cell line used herein. This is because during lysis and incubation with the immobilized antibody, 30 the phosphatases remove phosphate groups from the EGF-R
molecules, thus prohibiting endogenous adaptor proteins, such as GRB prote;ns, to bind EGFR, which could potentially lead to artifactual results. Alternatively, cells may be starved before lysis, if the cell line utilized may be readily 35 starved.

WO 96/4011r PCT~US96/08741 .

Pre~aration o~ auto~hophorylated EGF-R: The following in vitro kinase reaction yielded autophosphorylated EGF-R.
The kinase reaction was initiated by the addition o~ 15 ~l o~
ATP/Mn2~ mix (in 50 mM MnCl2, ~inal concentration o~ 10 ~M
5 ATP, for a total volume of 150 ~l. The plate was incubated for 5 minutes at room temperature, shaking, the supernatant was aspirated, and the plates were then washed 5 times with TBST.

Assa~ procedure: Either 30 ng GRB-2-GST ~usion proteins (i.e. a 1:1 ratio o~ EGF-R:GRB-2 proteins) or 5 ng GRB-2-GST
~usion proteins (i.e. a 4:1 ratio o~ EGF-R:GRB-2 proteins) were added to the phosphorylated EGF-R coated microtiter wells in incubation bu~er (0.1 M potassium phosphate bu~er, 15 pH 6.5) ~or 30 minutes, at room temperature, in the presence o~ Compound I. Control wells were incubated with GRB-2-GST
~usion proteins in the absence of Compound I.
A~ter incubation, wells were washed extensively with TBST. The amount o~ GRB-2-GST fusion protein bound to the 20 immobilized EGF-R is then pre~erably determined by with a puri~ied rabbit antiserum against the GST-moiety of the ~usion protein (AMRAD, New Victoria, Australia; Catalog No.
00001605). Incubations were ~or 30 minutes at room temperature. A~ter incubation, antibody was removed and the 25 wells are washed extensively with TBST. For visualization, wells were next incubated with a TAGO goat-anti-rabbit peroxidase antibody at room temperature ~or 30 minutes.
A~ter incubation, the antibody was removed, the wells were washed with tap water, and then with TBST. Substrate 30 solution, ABTS (2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid)/H2O2 (1.2 ~l H2O2 to 10 ml ABTS) was applied to the wells, and incubated ~or 20 minutes at room temperature. The reaction was stopped by addition o~ 5NH2SO4. The O.D. at 410 nm was determined ~or each well. Utilizing this technique, 35 it is normally possible to detect as little as 2 ng GRB-2-GST
over background.

Alternatively, after incubation of the test substance and the GRB-2-GST fusion protein on the EGF-R wells, biotinylated monoclonal antibodies e.g., EL-6 or EL-12, may be utilized to assay fusion protein binding. The epitopes 5 recognized by such antibodies map on the SH2 domain of GRB-2, but do not interfere with GRB-2 binding to phosphorylated EGFR. Binding of these antibodies is then determined by using a streptavidin-biotinylated horseradish peroxidase reactant.
Additionally, after incubation of the test substance and the GRB-2-GST fusion protein on the EGF-R wells, binding of the fusion protein to the immobilized EGFR may be assayed by incubating w th 1 mM 1-chloro-2,4 dinitrobenzene (CDNB) and 1.54 mg/ml reduced glutathion in incubation buffer. The OD
15 is then measured at 340 nm. This reaction is linear up to OD
1.0, and can be stopped with competitive GST inhibitors, as described in M~nne~vik and Danielson (Mannervik, B. and Danielson, U.H., 1988, CRC Critical Reviews in Biochemistry 23:238).
5.2 RESULTS
Compound I was tested for its ability to inhibit the binding of tyrosine phosphorylated EGF-receptor to an SH2 peptide domain of the GRB-2 adaptor protein, according to the 25 assays described, above, in Section 5.1 Compound I proves to be a potent inhibitor of GRB-2/SH2 binding, having an IC50 of 2.9~M. tIC50, as used herein, return to the concentration of test compound required to inhibit one-half of GRB-2/SH2 binding relative to the amount 30 of binding which occurs in the absence of test compound.) 6. COMPOUND I INHIBITS bcr/abl A~llvllY
The Example presented herein demonstrates that compounds of the invention inhibits cell survival in a bcr/abl-35 transformed ceIl line.

W O 96/40115 PCTrUS96/08741 6.1 MATERIALS AND METHODS
(1) Cell lines used in this assay are:
32D c1.3: murine lymphoblastoid cell, IL-3 dependent.
32D c1.3 J2/leuk: 32D c1.3 expressing raf and myc, IL-3 independent.
32D bcr/ablo 32D over expressing bcr/abl kinase, pooled, IL-3 independent.
(2) All the above cell lines were grown in incubator with 5% CO2 and 37~C. Their growth media are:
32D c1.3: RPMl + 10% FBS + 1 ng/ml IL-3 + 2 mM
Glutamine.
32D c1.3 J2/leuk: RPMl + 10% FBS + 2 mM Glutamine.
32D bcr/abl: RPMl + 10% FBS + 2 mM Glutamine.
IL-3: Interleukin-3, mouse tUBI Cat. # 01-37~) (3) PBS (Dulbecco's Phosphate Buffered Saline) Gibco Cat. #450-130OEB
(4) MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) Sigma Cat. # M-2128 working solution: 5 mg/ ml PBS, store in dark 4~C.
(5) Solubilization Buffer SDS Electrophoresis Grade, Fisher Cat. #BP 166.
N,N-Dimethyl-formamide (DMF), Fisher Cat. #BP1160.
Acetic Acid, Glacial, Fisher Cat. #A38.
working solution: Dissolve 200 g SDS in 250 ml warm H2O and 500 ml DMF, stir in low heat. When SDS is almost solubilized, add 25 ml 80% acetic acid and 25 ml lN HCL to solution. Adjust volume to 1000 ml:

6.2 PROCEDURE
All of the following steps were conducted at room 35 temperature unless specifically indicated.

WO 96/40115 PCT~US96/08741 6.2.1 CELL SEEDING
(1) The cells were grown in tissue culture dish (10 cm, Corning 25020-100) to about 1x106 cell/ml, subculture every 2-3 days at 1:10 (1:20 for 32D bcr/abl line).
(2) Viable cells were counted with trypan blue according to standard procedure.
(3) Cells were then resuspended in fresh medium at a density of 2 x 105 cells/ml, and transfer cells to 96-well tissue culture plate (Corning, 25806-96) at 50 ~l per well to 10 reach about 2 x 10~ cells/well. Each cell line was plated with its own positive and negative control: (negative control:medium alone).
32D c1.3 seeding medium should contain 2 ng/ml IL-3.

6.2.2 ASSAY PROCEDURES
(1) Compound I drug stock (10 mM in DMSO) was diluted 1:50. 1:2 serial dilutions were per~ormed for the r~m~;n;ng 8 wells in each line of the tissue culture plate. 50 ~l were added to each well. Control wells received medium alone.
20 Cells were incubated with drugs in 5~ CO2 at 37~ for 15 hrs.
(2) 15 ~l MTT were added to each well. Plates were incubated at 37~C for 4 hours.
(3) After 4 hours, 100 ~l solubilization solution was added to each well.
(4) Plates were covered with All7m;nl~m foil, and allowed to sit on an ELISA plate shaker and shake overnight at room temperature to completely solubilize formazan crystals.
(5) Absorbance was read at 570 nm wavelength with a reference wavelength of 630 nm using a Dynatech ELISA plate 30 reader, Model MR 500.

6.3 RESULTS
Compound I was tested herein for its ability to affect bcr/abl activity, and was found to be an inhibitor of bcr/abl 35 function.
The effect of Compound I on bcr/abl function was tested using the cell growth assay described, above, in Section 6.1.

W O 96/40115 PCT~US96/08741 Briefly, three cell lines were used in this assay~ First, an IL-3 dependent cell line (32D c1.3) was used, which requires the presence of the IL-3 cytokine for survival. Next, two IL-3 independent cell lines were used, including 32D c1.3 5 J2/leuk, which consists of the 32D c1.3 cell line transformed with raf and myc, and 32D bcr/abl, which consists of the 32D
c1.3 cell line transformed with bcr/abl. Because these latter cell lines are made IL-3 independent due to the activity of the products produced by the gene sequences they 10 have been transformed by, if these products become inactive and the cells are not exposed to IL-3, the cell will not survive. Thus, for example, if bcr/abl is inactivated in the 32D c1.3 bcr/abl cell line, cells will be unable to survive in the absence of IL-3.
Compound I inhibits the ability of the 32D c1.3 bcr/abl cell line to survive in the absence of IL-3. This result is significant as this cell line is quite robust.

7. EXAMPLE: COMPOUND I INHIBITS CELLULAR PROLIFERATION
The Example presented herein demonstrates that Compound I of the invention is a potent inhibitor of cellular proliferation.

7.î MATERIALS AND METHODS
Sulforhodamine B (SRB) Growth Assays Assay 1: MCF-7SRB Growth Assay. MCF-7 (ATCC~ HTB 22) cells (H+B22) were seeded at 2000 cells/well in a 96-well flat bottom plate in normal growth media, which was 10~
FBS/RPMI supplemented with 2 mM Glutamine. The plate of 30 cells was incubated for about 24 nours at 37~C after which it received an equal volume of compound dilution per well making the total volume per well 200 ~l. The compound was prepared at 2 times the desired highest final concentration and serially diluted in the normal growth media in a 96-well 35 round bottom plate and then transferred to plate of cells.
DMSO serves as the vector control up to 0.2~ as final concentration. The cells were then incubated at 37~C in a _ 5~ _ W O 9G/40115 PCT~US96/08741 humidified 5~ CO2incubator. Four days following dosing of compound, the media was discarded and 200 ~l/well of ice-cold 10~ TCA (Trichloroacetic Acid) was added to fix cells. After 60 minutes at 4~C, the TCA was discarded and the plate was 5 rinsed 5 times with water. The plate was then air-dried and 100 ~l/well of 0.4~ SRB (Sulforhodamine B from Sigma) 20 in 1% Acetic Acid was added to stain cells for 10 minutes at room temperature. The SRB was discarded and the plate was rinsed 5 times with 1% Acetic Acid. After the plate was 10 completely dried, 100 ~l/well of 10 mM Tris-base was added to solubilize the dye. After 5 to 10 minutes, the plate was read on a Dynatech ELISA Plate Reader at dual wavelengths at 570 nm and 630 nm.
Assav 2: PDGF-R/SRB Adherent Cells Growth Assay.
15 Compounds were tested for inhibition of anchorage-dependent tumor cell growth using the colorimetric assay described by Skehan et al ., 1990. ~. Natl . Ca~cer Inst. 82 :1107-1112. The assay measures protein content of acid-fixed cells using the counterion binding dye sulforhodamine B (SRB, Sigma). The 20 compounds were solubilized in DMSO (Sigma, cell culture grade) and diluted into appropriate growth medium at two-fold the desired final assay concentration. In assays using C6 cells (CCL 107), compounds (100 ~l) were added to 96-well plates containing at~ached cellular monolayers (2000 25 cells/well in 100 ~l). C6 (ATCC# CCL 107) cells were maintained in Ham's F10 supplemented with 5~ fetal bovine serum (FBS) and 2 mM glutamine (GLN). After 4 days (37~C, 5 CO2) the monolayers were washed 3 times with PBS and fixed with 200 ~l ice-cold 10~ TCA (Fisher Scientific)~ and kept at 30 4~C for 60 min. The TCA was removed and the fixed monolayers were washed 5 times with tap water and allowed to dry completely at room temperature on absorbent paper. The cellular proteln was stained for lQ min with 100 ~1 0.4~ SRB
dissolved in 1~ acetic acid. After 5 washes with tap water, 35 the dye was solubilized in 10 mM Tris base (100 ~l per well) and absorbance read at 570 nm on a Dynatech plate reader model MR5000 Growth inhibition data were expressed as a W O 96/40115 PCT~US96/08741 percentage of absorbance detected in control wells which were treated with 0.4~ DMSO alone. DMSO controls were not different from cells grown in regular growth medium. IC50 values were determined using a ~our parameter curve fit 5 function.
For the anchorage-independent tumor cell growth assay, cells (3000 to 5000 per dish) suspended in 0.4~ agarose in assay medium (DMEM containing 10~ FCS) with and without Compounds were plated into 35 mm dishes coated with a 10 solidified agarose base layer (0.8~ agarose). After a 2 to 3 week incubation at 37~C, colonies larger than 50 ~m were quantified using an Omnicon 3800 Tumor Colony counter.
Assav 3: MCF-7/HER-2B Growth Assay. The protocol used herein is essentially similar to that described above (for 15 the MCF-7 Growth Assay) except that immediately before Compound I was added, the normal growth media was removed and 0~5~ FBS/RPMI supplemented with 2 mM Glutamine is added onto the cells. The compound was also prepared in this 0.5~ serum media. The plate of cells was incubated ~or four days and 20 developed as per standard techniques.
Assay 4: A431/SRB Growth Assay. A431 (ATCC# CRL 1555) cells were tested essentially according to the protocol described, above, for the MCF-7~HER-2B growth assay.

7.2 RESULTS
A number of cell lines were contacted to Compound I to test Compound I's effects on cell proliferation, utilizing the SRB protocols described, above, in Section 7.1.
As shown below, Compound I proved to be a potent 30 inhibitor of ceIls proliferation of each of the four cell lines tested.
Compound I
Cell Line IC 50 (MM) W O96/40115 PCT~US96/08741 IC50, as used herein, refers to the concentration of test compound required to inhibit cell proliferation to 50~ of the level seen in the same cell line which has not been contacted to test compound (in this case, Compound I).
Thus, the results depicted in this Section demonstrate that Compound I acts to inhibit cell proliferation. These results, taken together with those shown in the Example presented in Section 5, above, which demonstrated that Compound I acts to inhibit adaptor protein binding to the SH2 10 domain of the protein tyrosine kinase receptor EGFR, indicate that Compound I acts as a cell growth inhibitor that acts by blocking adaptor protein interaction with its binding partners (such as, ~or example, protein tyrosine kinase molecules). Given this Compound I activity, the compound may 15 represent an anti-cell proliferation agent.

8. EXAMPLE: 3T3 CELLULAR PROLIFERATION INHIBITION ASSAY
The following protocol describes the procedures used to determine the ability of the compounds to inhibit cellular 20 proliferation in 3T3 engineered cell lines that over expressing EGFr, IGFlr, or PDGFr.

8.1 MATERIALS AND REAGENTS
(1) EGF Ligand: stock concentration = 16.5 ~M; EGF 201, 25 TOYOBO, Co., Ltd. Japan.
(2) IGF1 Ligand: human, recombinant; G511, Promega Corp, USA.
(3) PDGF Ligand: human PDGF B/B; 1276-956, Boehringer Mannheim, Germany.
(4) SRB: sulfohodamine B; S-9012, Sigma Chemical Co., USA.
SRB Dye Solution: 0.4~ SRB in 1~ acetic acid, glacial.
(5) Acetic Acid, Glacial: A38-212, Fisher Scientific, 35 USA.
(6) Albumin, Bovine: fraction V powder; A-8551, Sigma Chemical Co., USA.

CA 02224l03 l997-l2-08 (7) TCA Bu~er: 10~ trichloroacetic acid (A32-500, Fisher Scientific, USA).
(8) Tris Base Bu~er: 10 mM tris base (BP152-5, Fisher ~ Scienti~ic, USA).

8.2 PROCEDURE
(1) N-lH 3T3 (ATCC# 1658) engineered cell liens: 3T3-EGFrr 3T3-IGFlr, 3T3-PDGFr.
(2) Cells are seeded at 8000 cells/well in 10~ FBS+2mM
10 GLN DMEM, in a 96 well plate. Cells are inçubated at 37~C 5 CO2 ~or overnight to allow the cells attach plate.
(3) On day 2, the cells are quiesced in the serum ~ree medium (0~FBS DMEM) ~or 24 hours.
(4) On day 3, the cells are treated with the ligands 15 (EGF=5 nM, IGF1=20 nM, or PDGF=100 ng/ml) and drugs at the same time. The ligands are prepared in the serum free DMEM
with 0.1~ bovine albumin. The negative control cells receive the serum ~ree DMEM with 0.1~ bovine albumin only; the positive control cells receive the ligands (EGF, IGF1, or 20 PDGF) but no drugs. The drugs are prepared in the serum ~ree DMEM in a 96 well plate, and a serial dilution is taken the place. A total o~ 10~1/well medium o~ the diluted drugs are added into the cells. The total volume o~ each well is 200~L. Quadruplicates (wells) and 11 concentration points 25 are applied to each drug tested (5) On day 4, adding the ligands (EGF, IGF1, or PDGF) to the cells again, and to keep the final ligand concentration in the cells as same as previous.
(6) On day 5, the cells were washed with PBS and ~ixed 30 with 200 ~l/well ice cold 10~ TCA for 1 hour under 0-5~C
condition.
t7) Remove TCA and rinse wells 5 times with de-ionized water. Dry plates upside down with paper towels. Stain cells with 0 4~ SRB at 100 ~L/well ~or 10 minutes.
(8~ Pour of~ SRB and rinse plate 5 times with 1~ acetic acid. Dry plate completely.

W O 96/40115 PCTrUS96/08741 (9) Solubilize the dye with 10 mM Tris-base at 100 ~L/well ~or 10 minutes on a shaker.

(10) Read the plate at dual wavelengths at 570 nm and 630 nm on Dynatech Elsia plate reader.

8.3 ASSAY PROCEDURES
(1) Dilute drug stock (10 mM in DMSO) 1:50 in RPMI
medium in ~irst well, then do 1:2 dilution for 8-points in tissue culture plate. Transfer 50 ~l/well of this solution 10 to the cells. Control wells receive medium alone. Incubate the cells with drugs in 5~ CO2 at 37~ for 15 hrs.
(2) Add 15 ~l MTT to each well. Incubate plate at 37~C
for 4 hours.
(3) After 4 hours, add 100 ~l solubilization solution 15 to each well.
(4) Cover the plate with Aluminum foil, let plate sit on ELISA plate shaker and shake overnight at room temperature to completely solubilize formazan crystals.
(5) Read absorbance at 570 nm wavelength with a 20 reference wavelength of 630 nm using a Dynatech ELISA plate reader, Model MR 500.

It is apparent that many modifications and variations of this invention as set forth here may be made without 25 departing from the spirit and scope thereof. The specific embodiments described hereinabove are given by way of example only and the invention is limited only by the ~erms of the appended claims.

Claims (21)

WHAT IS CLAIMED IS:

1. A pharmaceutical composition suitable for administration to humans which comprises the compound of the formula:

or a pharmaceutically salt thereof; and a pharmaceutically acceptable carrier.

2. A pharmaceutical composition suitable for administration to humans which comprises: the compound of the formula:

or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

3. A method of ameliorating symptoms of a cell proliferative disorder wherein the cell proliferative disorder involves a protein tyrosine kinase polypeptide/adaptor polypeptide complex with an amount of a compound sufficient to disrupt protein tyrosine kinase polypeptide/adaptor polypeptide complexes of the cell so that symptoms of the cell proliferative disorder are ameliorated;
wherein said compound has either of the following formulas:

(1) (2)

4. The method of claim 3 wherein the cell proliferative disorder occurs in a mammal and the compound contacts the cell within a mammal so that the symptoms of the cell proliferative disorder in the mammal are ameliorated.

5. The method of Claim 3 wherein the cell proliferative disorder is a BCR-ABL-associated cancer, a glioma, a glioblastoma, a melanoma, an ovarian cancer, a breast cancer, or a prostate cancer.

6. A method of ameliorating symptoms of a cell proliferative disorder wherein the cell proliferative disorder involves a protein tyrosine kinase polypeptide/adaptor polypeptide complex, comprising:
contacting a cell capable of forming the protein tyrosine kinase polypeptide/adaptor polypeptide complex with an amount of the pharmaceutical composition of claim 1 or 2 sufficient to disrupt protein tyrosine kinase polypeptide/adaptor polypeptide complexes of the cell so that symptoms of the cell proliferative disorder are ameliorated.

7. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

R1 and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, and wherein at least one of R1 and R2 is other than hydrogen;

R3 to R12 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, mercapto or 2-methylbut-2-en-4-yl; and wherein at least one of R11 and R12 is 2-methylbut-2-en-4-yl.

8. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

R1 and R2 are both H;

R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, mercapto or 2-methylbut-2-en-4-yl; and R11 and R12 are each independently H or 2-methylbut-2-en-4-yl, wherein at least one of R11 and R12 is 2-methylbut-2-en-4-yl;

wherein at least one of R3 to R10 is other than H.

9. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

R1 and R2 are each independently aryl, alkylaryl and higher alkyl acid ester; and R3 to R12 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, fluoro, chloro, iodo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, or mercapto.

10. A compound of the formula or a pharmaceutically acceptable salt thereof, wherein:

R1, R2, R11 and R12 are H; and R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, alkoxy, hydroxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, or mercapto, wherein at least one of R3 to R10 is other than H;
(a) when R4-R10 are each H, R3 may not be 2-methylbut-2-en-4-yl or 2-hydroxy-2-methylbut-4-yl;
(b) when R4-R6 and R8-R10 are each H, R3 and R7 may not simultaneously be 2-methylbut-2-en-4-yl;
(c) when R3-R4, R6-R8 and R10 are H, R5 and R9 may not simultaneously be 2-methylbut-2-en-4-yl or 3-methyl-n-butyl;
(d) when R3, R5-R7, R9-R10 are H, R4 and R8 may not both be 2-methylbut-2-en-4-yl or 2-methylbut-1,3-dien-4-yl, and R4 and R8 may not be 2-methylbut-2-en-4-yl and 2-methylbut-1,3-dien-4-yl.

11. The compound of claim 10, wherein the compound is of the formula:
wherein R3-R5 and R7-R9 are H and either or both of R6 and R10 are 2-methylbut-2-en-4-yl.

12. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

at least one of R1 and R2 is acetyl;

R11 and R12 are H; and R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, and mercapto, wherein:
(a) when both R1 and R2 are acetyl; or when one of R1 and R2 is acetyl and R3-R4, R6-R8 and R10-R12 are H; R5 and R9 may not simultaneously be 2-methylbut-2-en-4-yl;
(b) when both R1 and R2 are acetyl and when R4-R6 and R8-R10 are H, R3 and R7 may not simultaneously be 2-methylbut-2-en-4-yl;
(c) when both R1 and R2 are acetyl and when R3, R5-R7, and R9-R10 are H, R4 and R8 may not simultaneously be 2-methylbut-2-en-4-yl.

13. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

at least one of R1 and R2 is lower alkyl;

R11 and R12 are H; and R3 to R10 are each independently H, alkyl, alkylcarboxy, alkenyl, alkenylcarboxy, aryl, alkylaryl, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxamide, carboxy, sulfonyl, sulfonamide, amino, and mercapto, wherein:
(a) when both R1 and R2 are methyl, at least one of R3 to R10 must be a group other than H;
(b) when both R1 and R2 are methyl, and R4-R10 are H, R3 may not be 2-methylbut-2-en-4-yl;
(c) when both R1 and R2 are methyl, and R4-R6 and R8-R10 are H, R3 and R7 may not simultaneously be 2-methylbut-2-en-4-yl;
(d) when both R1 and R2 are methyl, and R3-R4, R6-R8 and R10 are H, R5 and R9 may not simultaneously be 2-methylbut-2-en-4-yl.

14. The compound of claim 10 wherein R4 is 2-methylbut-2-en-4-yl and R3 and R5-R10 are H; or R5 is 2-methylbut-2-en-4-yl and R3-R4 and R6-R10 are H; or R6 is 2-methylbut-2-en-4-yl, and R3-R5 and R7-R10 are H.

15. The Compounds:
(a) 2,5-Diacetoxy-3,6-di-t2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone;
(b) 2,5-Diacetoxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone;
(c) 2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone;
(d) 3,6-Di-[5-(bromo)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(e) 3,6-Di-[2-(allyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(f) 2,5-Dihydroxy-3,6-di-[2-(n-propyl)indol-3-yl]1,4-quinone;
(g) 3,6,-Di-[2-(aminocarbonyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(h) 2,5-Diacetoxy-3,6-di-[2(aminocarbonyl)indol-3-yl]-1,4-quinone;

(i) 3,6-Di-[2-allylindol-3-yl]-2,5-dibenzoyloxy-1,4-quinone;
(j) 2,5-Dihydroxy-3,6-di-[2-(cyano)indol-3-yl]1,4-quinone;
(k) 2,5-Dihydroxy-3,6-di-[4-(methoxycarbonyl)indol-3-yl]1,4-quinone;
(l) 2,5-Dihydroxy-3,6-di-[5,7-(dimethoxy)indol-3-yl]1,4-quinone;
(m) 2,5-Dihydroxy-3,6-di-[4,7-(dimethoxy)indol-3-yl]1,4-quinone;
(n) 2,5-Dihydroxy-3,6-di-[5-(nitro)indol-3-yl]1,4-quinone;
(o) 3,6-Di-[4(4-chlorobenzoylamino)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(p) 3,6-di-[2-(4-chlorophenyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(q) 2,5-Dihydroxy-3,6-di-[2-(4-fluorophenyl)indol-3-yl]1,4-quinone;
(r) 2,5-Dihydroxy-3,6-di-[4,6-(dimethoxy)indol-3-yl]1,4-quinone;
(s) 2,5-Dihydroxy-3,6-di-[2-(5-hydroxy-6-methoxy)indol-3-yl]1,4-quinone;
(t) 2,5-Dihydroxy-3,6-di-[4-(cyano)indol-3-yl]1,4-quinone;
(u) 2,5-Dihydroxy-3,6-di-[5-(4-trifluoromethylphenylaminocarbonyl)indol-3-yl]1,4-quinone;
(v) 2,5-Dihydroxy-3,6-di-[2-(4-trifluoromethylphenylaminocarbonyl)indol-3-yl]l,4-quinone;
(w) 3,6-Di-[2-(5-bromo-6-nitro)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(x) 2,5-Dimethoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone;
(y) 2,5-Dimethoxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone.

16. The compounds:
(a) 2,5-Dihydroxy-3,6-di-[2-(methyl)indol-3-yl]-1,4-quinone;
(b) 3,6-Di-(2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quinone;
(c) 3,6-Di-(2-butylindol-3-yl)-2,5-dihydroxy-1,4-quinone;
(d) 3,6-Di-[2-(but-1-en-4-yl)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(e) 2,5-Dihydroxy-3,6-di-[2-(2-methylbut-1-en-4-yl)indol-3-yl]-1,4-quinone;
(f) 2,5-Dihydroxy-3,6-di-[2-(4-methyl-n-pentyl)indol-3-yl]-1,4-quinone;
(g) 2,5-Dihydroxy-3,6-di-[2-(2-phenylethyl)indol-3-yl]-1,4-quinone;
(h) 3,6-Di-[(5-carboxy-2-ethyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(i) 3,6-Di-[[5-carboxy-2-(n-propyl)]indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(j) 3,6-Di-[[5-carboxy-2-(3-methyl-n-butyl)]indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(k) 3,6-Di-[2-(4-carboxy-n-butyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(l) 3-[[5-Carboxy-2-(3-methyl-n-butyl)]indol-3-yl]-2,5-dihydroxy-6-(indol-3-yl)1,4-quinone;
(m) 3,6-Di-[(5-amino-2-ethyl)indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(n) 3,6-Di-[[5-amino-2-(n-propyl)]indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(o) 3,6-Di-[5-amino-2-(3-methyl-n-butyl)]indol-3-yl]-2,5-dihydroxy-1,4-quinone;
(p) 2,5-Diacetoxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]-1,4-quinone;
(q) 3,6-Di-[[2-ethyl-5-(4-methylphenylsulfonylamino)]
indol-3-yl]-2,5-dihydroxy-1,4-quinone;

(r) 2,5-Dihydroxy-3,6-di-[[5-(4-methylphenylsulfonylamino)-2-(n-propyl)]indol-3-yl]-1,4-quinone;
(s) 2,5-Dihydroxy-3,6-di-[[2-(3-methyl-n-butyl)-5-(4-methylphenylsulfonylamino)]indol-3-yl]-1,4-quinone;
(t) 2,5-Dihydroxy-3,6-di-[2-(2-methylpent-2-en-5-yl) indol-3-yl]-1,4-quinone.

17. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

R1 and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, R3 to R10 are each independently H, alkyl, alkylcarboxy, aryl, alkylaryl, alkenyl, alkenylcarboxy, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxyamide, carboxy, sulfonyl, sulfonamide, amino, mercapto, 4-methylphenylsulfonylamino, or 2-methylbut-2-en-4-yl; and R11 and R12 are selected from the group consisting of hydrogen, methyl, ethyl, propyl, butyl, aryl, alkylaryl, alkylcarboxy, alkenylcarboxy, but-1-en-4-yl, 2-methylbut-1-en-4-yl, 4-methyl-n-pentyl, 2-phenylethyl, 2-methylpent-2-en-4-yl, and 4-carboxy-n-butyl, wherein at least one of R11 and R12 is other than hydrogen.

18. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

R1 and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, R3 to R10 are each independently H, alkyl, alkylcarboxy, aryl, alkylaryl, alkenyl, alkenylcarboxy, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxyamide, carboxy, sulfonyl, sulfonamide, amino, mercapto, 4-methylphenylsulfonylamino, or 2-methylbut-2-en-4-yl; and R11 and R12 are both 3-methyl-n-butyl.

19. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein:

R1 and R2 are each independently hydrogen, lower alkyl, acetyl, aryl, alkylaryl or higher alkyl acid ester, R3 to R10 are each independently H, alkyl, alkylcarboxy aryl, alkylaryl, alkenyl, alkenylcarboxy, OH, alkoxy, nitro, halo, trihalomethyl, amide, carboxyamide, carboxy, sulfonyl, sulfonamide, amino, mercapto, 4-methylphenylsulfonylamino, or 2-methylbut-2-en-4-yl and wherein at least one of R3 to R10 is other than hydrogen; and R11 and R12 are each independently hydrogen or 3-methyl-n-butyl.

20. A pharmaceutical composition suitable for administration to humans comprising a compound of claims 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; and a pharmaceutically acceptable carrier.

21. A method of ameliorating symptoms of a cell proliferative disorder wherein the cell proliferative disorder involves a protein tyrosine kinase polypeptide/adaptor polypeptide complex, comprising:
contacting a cell capable of forming the protein tyrosine kinase polypeptide/adaptor polypeptide complex with an amount of the pharmaceutical composition of claim 20 sufficient to disrupt protein tyrosine kinase polypeptide/adaptor polypeptide complexes of the cell so that symptoms of the cell proliferative disorder are ameliorated.