U.S. patent application number 09/854424 was filed with the patent office on 2002-02-07 for method and compositions for inhibition of adaptor protein/tyrosine kinase interactions.
Invention is credited to Harris, G. Davis, McMahon, Gerald, Tang, Peng Cho.
Application Number | 20020016353 09/854424 |
Document ID | / |
Family ID | 23890650 |
Filed Date | 2002-02-07 |
United States Patent
Application |
20020016353 |
Kind Code |
A1 |
Tang, Peng Cho ; et
al. |
February 7, 2002 |
Method and compositions for inhibition of adaptor protein/tyrosine
kinase interactions
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.
Inventors: |
Tang, Peng Cho; (Moraga,
CA) ; McMahon, Gerald; (Kenwood, CA) ; Harris,
G. Davis; (San Francisco, CA) |
Correspondence
Address: |
Beth A. Burrous
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
23890650 |
Appl. No.: |
09/854424 |
Filed: |
May 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09854424 |
May 14, 2001 |
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09565855 |
May 5, 2000 |
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6239161 |
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09565855 |
May 5, 2000 |
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09090737 |
Jun 4, 1998 |
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6090838 |
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09090737 |
Jun 4, 1998 |
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08658337 |
Jun 5, 1996 |
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5780496 |
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08658337 |
Jun 5, 1996 |
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08476136 |
Jun 7, 1995 |
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Current U.S.
Class: |
514/414 |
Current CPC
Class: |
C07D 209/14 20130101;
A61K 31/404 20130101; A61P 35/00 20180101; C07D 209/08
20130101 |
Class at
Publication: |
514/414 |
International
Class: |
A61K 031/404 |
Claims
What is claimed is:
1. A pharmaceutical composition suitable for administration to
humans which comprises the compound of the formula: 8or 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: 9or 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: 10
11
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: 12or 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: 13or 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: 14or 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: 15or 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: 16wherein 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: 17or 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: 18or 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-[2-(2-methylbut-2-en-4-yl)indo-
l-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-trifluoromethylphenylamin-
ocarbonyl)indol-3 -yl]1,4 -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-b-
utyl)indol-3-yl]1,4-quinone.
16. The compounds: (a)
2,5-Dihydroxy-3,6-di-[2-(methyl)indol-3-yl]-1,4-qui- none; (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-q-
uinone; (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-dih- ydroxy-1,4-quinone;
(n) 3,6-Di-[[5-amino-2-(n-propyl)]indol-3-yl]-2,5-dihy-
droxy-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)ind-
ol-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-me-
thylphenylsulfonylamino)-2-(n-propyl)]indol-3-yl]-1,4-quinone; (s)
2,5-Dihydroxy-3,6-di-[[2-(3-methyl-n-butyl)-5-(4-methylphenylsulfonylamin-
o)]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: 19or 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: 20or 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: 21or 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.
Description
1. INTRODUCTION
[0001] The present invention relates to methods and compositions
for 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 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
[0002] Cells rely, to a great extent, on extracellular molecules as
a means by which to receive stimuli from their immediate
environment. These extracellular signals are 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, act as ligands that bind specific cell surface
receptors. The binding of these ligands to their receptors triggers
a cascade of 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 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.
[0003] A central feature 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. G., 1990, 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 biological properties. Proteins are phosphorylated
by protein kinases and are dephosphorylated by protein
phosphatases. Protein kinases and phosphatases are classified
according to the amino acid residues they act on, with one class
being serine-threonine kinases and 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
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 defined as being receptors, i.e.,
the enzymes are an integral part of a transmembrane, ligand-binding
molecule, or as non-receptors, meaning 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 relative activities, at that instant, of the protein
kinases and phosphatases that catalyze these reactions.
[0004] 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 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 transformation is now well established (Cantley, L. C.
et al., 1991, Cell 64:281-302; Hunter T., 1991, Cell
64:249-270;
[0005] Nurse, 1990, Nature 344:503-508; Schlessinger, J. and
Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger,
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 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
[0006] 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-specif- ic protein kinases (Hanks et al., 1988,
Science 241:42-52).
[0007] Receptor-type protein tyrosine kinases having a
transmembrane topology 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 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., 1990, Cell
61:203-212).
[0008] The intracellular, cytoplasmic, non-receptor protein
tyrosine kinases, may be broadly defined as those protein tyrosine
kinases which do not contain a hydrophobic, transmembrane domain.
Within this broad classification, one can divide the known
cytoplasmic protein tyrosine kinases into eleven distinct
morphotypes, including the SRC family (Martinez, 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 43:393-404; 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), the ABL family (Shtivelman, E. et al., 1986, Cell
47:277-284; Kruh, G. D. et al., 1986, Science 234:1545-1548), the
Za.sub.p 70 family and the JAK family. While distinct in their
overall molecular structure, each of the members of these
morphotypic families of 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) domains and SH3 domains (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).
[0009] 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 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 (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 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
[0010] 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 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.
[0011] 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 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 domains also have independent sites for the
recognition of amino acid residues surrounding the phosphotyrosine
residue(s).
[0012] When a receptor protein tyrosine kinase binds an
extracellular ligand, receptor dimerization is induced, 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.
[0013] 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. 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 about the role of SH2. The current view is
that SH3 domains function, 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, J., 1993
Current Biology, 3:434-442).
2.4 G-Proteins and Signal Transduction
[0014] Guanine-nucleotide-binding proteins, (G-proteins; Simon, M.
I. et al., 1991, Science 252:802-808; Kaziro, Y. et al., 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 of mitogenic signals from
receptor tyrosine kinases. Taking Ras as an example, the activation
of receptor tyrosine kinases by ligand binding results in the
accumulation of the active GTP bound form 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. 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).
[0015] Ras activity is regulated by the opposing actions of the
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 of Ras function, while exchange factors act as Ras
activators.
[0016] Recently, a direct link between activated receptor tyrosine
kinases and Ras was established with the finding that the mammalian
GRB-2 protein, a 26 kilodalton protein comprised of a single SH2
and two SH3 domains (Lowenstein, E. J. et al., 1992, Cell
70:431-442), directly couples receptor tyrosine kinases to the Ras
exchange factor Sos in mammals 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 363:88-92; Rozakis-Adcock et al., 1993,
Nature 363:83-85; Chardin, P. et al., 1993, Science 260:1338-1343;
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 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).
[0017] Grb2 has been shown to be associated with CSF-1 receptor
(vanderGeer and Hunter, 1993, EMBO J. 12(13):5161-5172), PDGF
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
[0018] 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 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.
[0019] Taking as an example Philadelphia chromosome-positive 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-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-phosphorylated region of the BCR-ABL protein, and that
this interaction is required for the activation of the Ras
signaling pathway.
[0020] Thus, there are multiple events which occur along a 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 events which
contribute to the appearance of such disorders, by interfering with
one or more of these requisite events.
[0021] 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 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.
[0022] 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 expression of
signaling incompetent proteins in cells. For example, cells
expressing a mutant form of 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., supra). To date, however, no such
inhibitor of adaptor protein/protein tyrosine kinase interactions
has been identified.
3. SUMMARY OF THE INVENTION
[0023] 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 organic compounds and
methods utilizing such compounds.
[0024] "Protein tyrosine kinase" will, herein, be abbreviated
"PTK". It is to be understood that "PTK" may refer to either a
transmembrane, receptor-type protein tyrosine kinase or a
cytoplasmic protein tyrosine kinase, unless otherwise indicated.
The compounds of the invention inhibit PTK/adaptor protein
interactions, especially PTK/adaptor protein interactions wherein
the PTK is, for example, an epidermal growth factor receptor
(EGF-R) protein tyrosine 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-1R).
[0025] The compounds of the present invention are described by the
formula (I) below:
[0026] ID 2,5-bisindoly-3-yl-1,4-quinone 1
[0027] and pharmaceutically acceptable salts thereof, wherein:
[0028] R1 and R2 are independently H, acetate or aryl, alkylaryl
and higher alkyl acid ester;
[0029] 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.
[0030] Specific compounds within the scope of the present invention
are described by the formula (II) below. R1 and R2 of the formula
can be as listed in Table I following the formula. Illustrative
preparations or isolations of these compounds are found in the
working examples. 2
1TABLE 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 7. H 2-n-propyl 8. H
2-aminocarbonyl 9. acetyl 2-aminocarbonyl 10. benzoyl 2-allyl 11. H
2-cyano 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 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)
[0031] Specific compounds within the scope of the present invention
are also described by formula (III) below. R1-R12 of the formula
can be as listed in Table II following the formula. Illustrative
preparations or isolations of these compounds are found in the
working examples. 3
2TABLE II Ex. R1 = R2 R11 R12 R3-R10.sup.1 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) 33. 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) 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) .sup.1Unless
otherwise indicated, R3-R10 = hydrogen.
[0032] By the term "alkyl" as used herein is meant a straight 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 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 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
"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.
[0033] The term "substituted" as used herein means that the 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.
[0034] Preferred compounds of the present invention include the
following: 4
[0035] and pharmaceutically acceptable salts thereof.
[0036] In addition, the present invention encompasses a
pharmaceutical composition comprising a compound of the invention,
and methods for using a compound or pharmaceutical composition of
the invention in an animal, particularly a human, to ameliorate
symptoms of cell proliferative disorders involving protein tyrosine
kinase/adaptor protein interactions.
[0037] This invention is based, in part, on the discovery that the
disclosed compounds, while exhibiting no inhibitory effect 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, describes a method for the production of the
compounds of the present invention.
[0038] The present invention represents the first instance whereby
compounds have been discovered which directly inhibit the
interaction between adaptor proteins and protein tyrosine kinase
molecules.
4. DETAILED DESCRIPTION OF THE INVENTION
[0039] Described below are methods and compositions for the
inhibition of adaptor protein/protein tyrosine kinase protein
interactions, especially those interactions associated with a cell
proliferative disorder. Specifically, described below are
particular organic compounds, methods for the synthesis of such
compounds, and techniques utilizing such compounds.
4.1 Compounds
[0040] The compounds of the present invention are described by the
following formula (IV): 5
[0041] and pharmaceutically acceptable salts thereof, wherein:
[0042] R1 and R2 are independently H, acetate or aryl, alkylaryl
and higher alkyl acid ester;
[0043] R3 to R14 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 preceding Tables I and II. Illustrative preparations
or isolations of these compounds are found in the working
examples.
[0044] In one embodiment, compounds of the present invention are
described by the following formula (III): 6
[0045] and pharmaceutically acceptable salts thereof, wherein:
[0046] 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;
[0047] 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.
[0048] Groups R1-R12 may be substituted or unsubstituted where
appropriate.
[0049] In another embodiment, compounds of the present invention
are described by formula (III) above, and pharmaceutically
acceptable salts thereof, wherein:
[0050] R1 and R2 are both H;
[0051] 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
[0052] 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;
[0053] wherein at least one of R3 to R10 is other than H.
[0054] In another embodiment, compounds of the present invention
are described by formula (III) above, and pharmaceutically
acceptable salts thereof, wherein:
[0055] R1 and R2 are each independently aryl, alkylaryl and higher
alkyl acid ester; and
[0056] 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.
[0057] In another embodiment, compounds of the present invention
are described by formula (III) above, and pharmaceutically
acceptable salts thereof, wherein:
[0058] R1, R2, R11 and R12 are H; and
[0059] 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;
[0060] (a) when R4-R10 are each H, R3 may not be
2-methylbut-2-en-4-yl or 2-hydroxy-2-methylbut-4-yl;
[0061] (b) when R4-R6 and R8-R10 are each H, R3 and R7 may not
simultaneously be 2-methylbut-2-en-4-yl;
[0062] (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;
[0063] (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.
[0064] The present invention also encompasses compounds of formula
(III) above, and pharmaceutically acceptable salts thereof, wherein
R3-R5 and R7-R9 are H and either or both of R6 and R10 are
2-methylbut-2-en-4-yl.
[0065] In another embodiment, compounds of the present invention
are described by formula (III) above, and pharmaceutically
acceptable salts thereof, wherein:
[0066] at least one of R1 and R2 is acetyl;
[0067] R11 and R12 are H; and
[0068] 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:
[0069] (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;
[0070] (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;
[0071] (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.
[0072] In another embodiment, compounds of the present invention
are described by formula (III) above, and pharmaceutically
acceptable salts thereof, wherein:
[0073] at least one of R1 and R2 is lower alkyl;
[0074] R11 and R12 are H; and
[0075] 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:
[0076] (a) when both R1 and R2 are methyl, at least one of R3 to
R10 must be a group other than H;
[0077] (b) when both R1 and R2 are methyl, and R4-R10 are H, R3 may
not be 2-methylbut-2-en-4-yl;
[0078] (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;
[0079] (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.
[0080] The present invention also includes compounds of formula
(III) above, and pharmaceutically acceptable salts thereof,
[0081] wherein R4 is 2-methylbut-2-en-4-yl and R3 and R5-R10 are
H;
[0082] or R5 is 2-methylbut-2-en-4-yl and R3-R4 and R6-R10 are
H;
[0083] or R6 is 2-methylbut-2-en-4-yl, and R3-R5 and R7-R10 are
H.
[0084] In another embodiment, compounds of the present invention
are described by formula (III) above, and pharmaceutically
acceptable salts thereof, wherein:
[0085] R1 and R2 are each independently hydrogen, lower alkyl,
acetyl, aryl, alkylaryl or higher alkyl acid ester,
[0086] 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
[0087] 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.
[0088] In yet another embodiment, compounds of the present
invention are described by formula (III) above, and
pharmaceutically acceptable salts thereof, wherein:
[0089] R1 and R2 are each independently hydrogen, lower alkyl,
acetyl, aryl, alkylaryl or higher alkyl acid ester,
[0090] 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
[0091] R11 and R12 are both 3-methyl-n-butyl.
[0092] In still another embodiment, compounds of the present
invention area described by formula (III) above, and
pharmaceutically acceptable salts thereof, wherein:
[0093] R1 and R2 are each independently hydrogen, lower alkyl,
acetyl, aryl, alkylaryl or higher alkyl acid ester,
[0094] 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
[0095] R11 and R12 are each independently hydrogen or
3-methyl-n-butyl. 7
[0096] The invention encompasses the compounds described above as
well as pharmaceutically acceptable salts thereof. The compounds of
the present invention can either be synthesized or isolated as
described herein.
[0097] The compounds of the present invention can be synthesized in
accordance with standard organic chemistry techniques using readily
available starting materials. Alternatively, the compounds can be
isolated as described in Section 5.2, below. Chemical synthesis and
isolation methods are provided herein solely for 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
[0098] The present Example employed a fungus culture (PenLabs Inc.
#592), and the following fermentation conditions: medium yeast malt
extract plus trace elements at 22.degree. 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 H.sub.2O to 1 liter. The pH of the seed
medium was adjusted to 7.0 before autoclaving. 30 mL seed medium
were dispensed per 250 ml flask (6 days 28.degree. C.), which was
then inoculated with 1 ml of spore/mycelium homogenate suspension
(2 days). Stock cultures were maintained frozen at -80.degree. C.
in spore storage solutions.
[0099] The fermentation mixture (mycelium and broth) was
homogenized and filtered through cheesecloth by suction filtration.
The filtrate was extracted three times with 0.5 v/v of 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 acetate layers were
combined and the solvent removed by rotary evaporation. The oily
residues both containing the asterriguinones were combined and
dried on a vacuum pump overnight.
[0100] The crude extract obtained above underwent CPC fractionation
on a PC Inc. high speed countercurrent chromatograph (HSCC)
containing a "tripple" coil column. A 1:3:3:3 v/v/v/v of 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. The upper layer was used as the mobile phase.
After two hours, the lower and upper layer were switched. The HSCC
run was completed after four hours. The crude metabolites eluted
from 8 to 12 minutes. The active fractions were pooled and
evaporated under reduced pressure to dryness.
[0101] The pooled HSCC fraction (8-12) was subjected to
semi-preparative HPLC (Water HPLC system with a Water 996
photodioarray detector using Millennium software) fractionations
using the following conditions:
[0102] Two semi-preparative C.sub.18-cartridges (25.times.100 mm
each, Nova Pak, 6.mu.); Flow rate: 10 mL/min.; 120 mg of the pooled
HSCC fraction 8-12 dissolved in 6 mL of DMSO; 250 .mu.L aliquots
per injection; PDA monitored at 270 nm; linear gradient of 70%
H.sub.2O/30% CH.sub.3CN to 100% CH.sub.3CN over 30 minutes; then
isocratic at 100% CH.sub.3CN for 6 minutes; the active material
eluted at 19 and 24 minutes. The active fractions from the 10 runs
were combined and evaporated under reduced pressure to dryness to
yield 17 mg of Asterriquinone C-3 (Compound I) and 3 mg of
Preasterriquinone C-3 (Compound II).
[0103] 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 compound I gave a
molecular ion of 507 (M+H).sup.+ (molecular weight:. 506). The
molecular formula C.sub.32H.sub.31N.sub.2O.sub.4(M.sup.++H) :
507.2289; found 507.2291). .sup.1H NMR spectra of compound I were
recorded in CDCl.sub.3 at 500 MHz on a Brucker DRX-500. Chemical
shifts are given in ppm relative to TMS at zero ppm using the
solvent peak at 7.26 ppm (CDCl.sub.3) 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). .sup.13C NMR
spectra of compound I were recorded in DMSO-d.sub.6 at 125 MHz on a
Brucker DRX-500. Chemical shifts are given in ppm relative to TMS
at zero using the solvent peak at 39.5 ppm (DMSO-d.sub.6) as an
internal standard. 138.8, 136.6, 136.3, 128.8, 128.2, 127.3, 122.3,
121.8, 121.0, 120.3, 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 of
150-154.degree. C.
[0104] Mass spectral analysis for compound II gave a molecular ion
of 439 (M+H).sup.+ (molecular weight: 438). .sup.1H NMR spectra of
compound II were recorded in DMSO-d.sub.6 at 500 MHz on a Brucker
DRX-500. Chemical shifts are given in ppm relative to TMS at zero
ppm using the solvent peak at 2.49 ppm (DMSO-d.sub.6) as an
internal standard. 11.35 (s, 1H), 10.96 (s, 1H), 10.62 (s, 1H),
7.48 (dJ=1 Hz, 1H), 7.39 (d, J=10.0 Hz, 1H), 7.29 (d, J=10.0Hz,
1H), 7.14 (d, J=10 Hz, 1H), 7.07 (t, J=10.0 Hz, 1H), 6.99 (t,
J=10.0 Hz, 1H), 6.93 (t, J=10.0 Hz, 1H), 6.88 (t, J=10.0 Hz, 1H),
5.26 (m, 1H), 3.30 (m, 2H) 1.64 (bs, 3H) and 1.61 ppm (bs, 3H).
.sup.13C NMR spectra of compound II were recorded in CDCl.sub.3 at
125 MHz on a Brucker DRX-500. Chemical shifts are given in ppm
relative to TMS at zero ppm using the solvent peak at 77.0 ppm
(CDCl.sub.3) 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]1,4-quinone
[0105] 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-ditrimethylsilox- y-1,4-quinone,
3,6-dibromo-2,5-di-(t-butyldimethylsiloxy-1,4-quinone,
2,5-dibenzoxy-3,6-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-1,4-quinone and 180 mg of
3-[2-(2-methylbut-2-en-4-yl)indole, prepared by the Fisher indole
synthesis, in 10 ml of anhydrous dimethylforamide, or pyridine or
dimethylsulfoxide, with powdered potassium carbonate, was heated at
100.degree. C. for 24 hours. The cooled mixture was partitioned
between ethyl acetate and water. The ethyl 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 a solvent mixture of
dichloromethane and methanol to provide 25 mg of
2,5-diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]l,4-quinone.
2,5-Diacetoxy-3,6-di-(2(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone
was then hydrolysed with 1 N aqueous sodium hydroxide solution in
methanol. Acidification of the above mixture produced the crude
product after filtration. Further crystallization in ethanol and
water produced the title compound. Other aforementioned protecting
groups, they can be removed by 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).
[0106] 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.degree. C. to produce
the substituted 2,5-dibromo-3,6-(3-indolyl)-1,4-quinone which can
react with base such as sodium hydroxide to give the a substituted
2,5-dihydroxy-3,6-(3-indolyl)-- 1,4-quinone (Hoerher, J.;
Schwenner, E.; Franck, B., Liebigs Ann. Chem. 1986, 10:
1765-1771).
EXAMPLE 2
2,5-Diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quinone
[0107]
2,5-Diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)indol-3-yl]1,4-quino-
ne was prepared in Example 1.
EXAMPLE 3
2,5-Diacetoxy-3,6-di-[2
(3-methyl-n-butyl)indol-3-yl]1,4-quinone
[0108] Hydrogenation of
2,5-diacetoxy-3,6-di-[2-(2-methylbut-2-en-4-yl)ind- ol-3-yl],
1,4-quinone in methanol with 5% palladium on carbon under 1 atm of
hydrogen produced the title compound.
EXAMPLE 4
2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)indol-3-yl]1,4-quinone
[0109] Base hydrolysis of
2,5-diacetoxy-3,6-di-[2-(3-methyl-n-butyl)indol-- 3-yl],1,4-quinone
as described in Example 1 produced the title compound.
[0110] Under similar conditions as those described in Examples 1 to
4, the following 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
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
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
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
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
EXAMPLE 18
2,5-Dihydroxy-3,6-di-[2-(4-fluorophenyl)indol-3-yl]1,4-quinone
EXAMPLE 19
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
EXAMPLE 22
2,5-Dihydroxy-3,6-di-[5-(4-trifluoromethylphenylaminocarbonyl)indol-3-yl]1-
,4-quinone
EXAMPLE 23
2,5-Dihydroxy-3,6-di-[2-(4-trifluoromethylphenylaminocarbonyl)indol-3-yl]1-
,4-quinone
EXAMPLE 24
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
[0111] Methylation of Example 1 with methyl iodide and potassium
carbonate in dimethylforamide followed by purification produced the
title compound. This compound could also be prepared by heating
2,5-dibromo-3,6-di[2-(2-m- ethylbut-2-en-4-yl)indol-3-y]1,4-quinone
in methanol in the presence of powdered potassium carbonate.
EXAMPLE 27
2,5-Dimethoxy-3,6-di-[2(3-methyl-n-butyl)indol-3-yl]1,4-quinone
[0112] Hydrogenation of Example 26 under-conditions as those in
Example 3 produced the title compound.
EXAMPLE 28
Preparation of 2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)
indol-3-yl]-1,4-quinone
[0113] To a glass tube containing 2-(3-methyl-n-butyl)indole (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 ml). The organic layer was washed with
brine (100 ml) and dried with sodium sulfate. 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 pressure, and
the residue was purified by flash chromatography (150 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.
[0114] To a stirred solution of
2,5-dibromo-3,6-di-[2-(3-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 24 h, followed by dilution with water (50 ml). The product was
extracted with ethyl acetate (100 ml), washed with brine (50 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.
[0115] To a stirred solution of
2,5-dimethoxy-3,6-di-[2-(3-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.degree. 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
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.
[0116] 28a) Preparation of 2-(2-methyl-1-buten-4-yl)indole
[0117] To a stirred solution of 2-methylindole (1 g) in
diethylether (76 ml) under nitrogen was added a 1.6 M solution of
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 for 50 min., the mixture was cooled to -78.degree. 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.degree. C. for 2 h, 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 sulfate. The
solvent was removed under reduced pressure, and the crude residue
was purified by flash chromatography (4% ethyl acetate/hexane) to
afford 2-(2-methyl-1-butene-4-yl) indole (664 mg. 47%) as a waxy
yellow solid.
[0118] 28b) Preparation of 2-(3-methyl-n-butyl)indole
[0119] Into a 3-necked round bottom flask under a blanket of
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 flask, which was evacuated and charged
with hydrogen twice. The mixture was stirred vigorously under
hydrogen (1 atm) for 2 h, followed by filtration through a pad of
Celite. The solvent was removed under reduced pressure and the
crude residue was purified by flash chromatography (3% ethyl
acetate/hexane) to give 2-(3-methyl-n-butyl) indole (400 mg, 59%)
as a yellow crystalline solid.
EXAMPLE 29
Preparation of
2,5-Dihydroxy-3,6-di-[2-(methyl)indol-3-yl]-1,4-quinone
[0120] Refer to Example 28 using 2-methylindole as the starting
indole.
EXAMPLE 30
Preparation of
3,6-Di-(2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quinone
[0121] Refer to Example 28 using 2-ethylindole as the starting
indole.
[0122] 30a) Preparation of 2-ethylindole
[0123] Refer to 28a) using methyl iodide as the alkylating
agent.
EXAMPLE 31
Preparation of 3,6-Di-(2-butylindol-3-yl)
2,5-dihydroxy-1,4-quinone
[0124] Refer to Example 28 using 2-butylindole as the starting
indole.
[0125] 31a) Preparation of 2-(but-1-en-4-yl)indole
[0126] Refer to 28a) using allyl bromide as the alkylating
agent.
[0127] 31b) Preparation of 2-butylindole
[0128] Refer to 28b) using 2-(but-1-en-4-yl)indole as the starting
material.
EXAMPLE 32
Preparation of
3,6-Di-[2-(but-1-en-4-yl)indol-3-yl]2,5-dihydroxy-1,4-quino- ne
[0129] Refer to Example 28 using 2-(but-1-en-4-yl)indole as the
starting indole.
EXAMPLE 33
Preparation of 2,5-Dihydroxy-3,6-di-[2-(4-methyl-n-pentyl)
indol-3-yl)-1,4-quinone
[0130] Refer to Example 28 using 2-(4-methyl-n-pentyl)indole as the
starting indole.
[0131] 33a) Preparation of 2-(2-methyl-2-penten-5-yl) indole
[0132] Refer to 28a) using 4-bromo-2-methyl-2-butene as the
alkylating reagent.
[0133] 33b) Preparation of 2-(4-methyl-n-pentyl)indole
[0134] 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-quin-
one
[0135] Refer to Example 28 using 2-(2-phenylethyl)indole as the
starting indole.
[0136] 34a) Preparation of 2-(2-phenylethyl)indole
[0137] Refer to 28a) using benzyl bromide as the alkylating
agent.
EXAMPLE 35
Preparation of 2,5-Dihydroxy-6-(indol-3-yl)-3-[2-(3-methyl-n-butyl)
indol-3-yl]-1,4-quinone
[0138] This synthesis could be achieved by treating
2-(3-methyl-n-butyl) indole with 2 equivalents of bromanil in the
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
3,6-Di-(5-carboxy-2-ethylindol-3-yl)-2,5-dihydroxy-1,4-quin-
one
[0139] Refer to Example 28 using 5-carboxy-2-ethylindole as the
starting indole.
[0140] 36a) Preparation of 5-carboxy-2-ethylindole
[0141] 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 finish the synthesis.
EXAMPLE 37
Preparation of
3,6-Di-[5-carboxy-2-(n-propyl)indol-3-yl]-2,5-dihydroxy-1,4-
-quinone
[0142] Refer to Example 28 using 5-carboxy-2-propylindole as the
starting indole.
[0143] 37a) Preparation of 5-carboxy-2-propylindole
[0144] Refer to 36a) using ethyl iodide as the alkylating
agent.
EXAMPLE 38
Preparation of
3,6-Di-[5-carboxy-2-(3-methyl-n-butyl)indol-3-yl]-2,5-dihyd-
roxy-1,4-quinone
[0145] Refer to Example 28 using 5-carboxy-2-(3-methyl-n-butyl)
indole as the starting indole.
[0146] 38a) Preparation of
5-carboxy-2-(2-methyl-1-buten-4-yl)indole
[0147] Refer to 36a) using 3-bromo-2-methylpropene as the
alkylating agent.
[0148] 25 38b) Preparation of 5-carboxy-2-(3-methyl-n-butyl)
indole
[0149] Refer to 28b) using 5-carboxy-2-(2-methyl-1-buten-4-yl)
indole as the starting material.
EXAMPLE 39
Preparation of
3,6-Di-[2-(4-carboxy-n-butyl)indol-3-yl]-2,5-dihydroxy-1,4--
quinone
[0150] Refer to Example 28 using 2-(4-carboxy-n-butyl)indole as the
starting indole.
[0151] 35 39a) Preparation of 2-(4-carboxy-3-buten-1-yl) indole
[0152] Refer to 28a) using 4-bromo-2-butenoic acid as the
alkylating agent.
[0153] 39b) Preparation of 2-(4-carboxy-n-butyl)indole
[0154] Refer to 28b) using 2-(4-carboxy-3-buten-1-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
[0155] 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-dihydroxy-1,4-quinon- e
[0156] Refer to Example 28 using 5-amino-2-ethylindole as the
starting indole.
[0157] 41a) Preparation of 5-amino-2-ethylindole
[0158] 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; 1125-1128. 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-q-
uinone
[0159] Refer to Example 28 using 5-amino-2-(n-propyl)indole as the
starting indole.
[0160] 42a) Preparation of 5-amino-2-(n-propyl)indole
[0161] Refer to 41a) using 2-n-propylindole.
EXAMPLE 43
Preparation of
3,6-Di-(5-amino-2-(3-methyl-n-butyl)indol-3-yl]2,5-dihydrox-
y-1,4-quinone
[0162] Refer to Example 28 using 5-amino-2-(3-methyl-n-butyl)
indole as the starting indole.
[0163] 43a) Preparation of 5-amino-2-(3-methyl-n-butyl) indole
[0164] 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
[0165] 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
Preparation of
3,6-Di-[2-ethyl-5-(4-methylphenylsulfonylamino)indol-3-yl]--
2,5-dihydroxy-1,4-quinone
[0166] Refer to Example 28 using
2-ethyl-5-(4-methylphenylsulfonylamino) indole as the starting
indole.
[0167] 45a) Preparation of
2-ethyl-5-(4-methylphenylsulfonylamino)indole
[0168] 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
[0169] Refer to Example 28 using
5-(4-methylphenylsulfonylamino)-2-(n-prop- yl)indole as the
starting indole.
[0170] 46a) Preparation of
5-(4-methylphenylsulfonylamino)-2-(n-propyl)ind- ole
[0171] Refer to 45a) using 5-amino-2-propylindole.
EXAMPLE 47
Preparation of
2,5-Dihydroxy-3,6-di-[2-(3-methyl-n-butyl)-5-(4-methylpheny-
lsulfonylamino)indol-3-yl]-1,4-guinone
[0172] Refer to Example 28 using
2-(3-methyl-n-butyl)-5-(4-methylphenylsul- fonylamino) indole as
the starting indole.
[0173] 47a) Preparation of
2-(3-methyl-n-butyli-5-(4-methylphenylsulfonyla- mino)indole
[0174] 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
[0175] Refer to Example 28 using 2-(2-methylbut-1-en-4-yl) indole
as the starting indole.
4.3 Protein Tyrosine Kinase/Adaptor Protein Complexes
[0176] The PTK/adaptor protein complexes which may be disrupted by
the methods and compositions of the invention comprise at least one
member of the PTK family of proteins and at least one member of the
adaptor family of 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 PTK/adaptor protein complex components.
Preferably, 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 factor receptor (PDGF-R) protein tyrosine
kinase molecule or an insulin growth factor-like receptor tyrosine
kinase molecule (IGF-1R).
[0177] Intracellular, cytoplasmic PTK components of the PTK/adaptor
protein complexes may include, for example, members of the Src
family, such molecules as src, yes, fgr, fyn, lyn, hck, lck, and
blk; members of the Fes family, such as fes and fer; members of the
Abl family, such as abl and arg; and members of the Jak family,
such as jak1 and jak2. Transmembrane, receptor PTK components of
the PTK/adaptor protein complexes may include, for example, such
molecules as members of the FGF receptor, Sevenless/ROS, Insulin
receptor, PDGF receptor, and EGF receptor family of growth factor
receptors.
[0178] 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, 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.alpha., guanine triphosphatase activator protein
(GAP), and members of the GRB subfamily of proteins, such as GRB1,
GRB-2, GRB-3, GRB-4, GRB-7, and GRB-10.
4.4 Treatment of PTK/Adaptor Protein Complex-Related Cell
Proliferative Disorders
[0179] The compounds and/or pharmaceutical compositions (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 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-1R.
[0180] Among the oncogenic disorders which may be treated by 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 human prostate
cancers.
[0181] Assays for determining the effectiveness of a compound in
the disruption of a PTK/adaptor protein complex are 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.
[0182] "Disruption", 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 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 cell.
The compounds and pharmaceutical compositions of the invention do
not, however, directly interfere with (i.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
[0183] 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 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 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.
[0184] Additionally, in vivo complex formation may be assayed 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 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 standard immunoprecipitation techniques.
In cases where a complex is still formed, the immunoprecipitation
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.
[0185] The effect of a compound of the invention on the
transformation capability of the PTK/adaptor protein of interest
may be directly assayed. For example, one or more of the compounds
of the invention may be administered to a cell such as a fibroblast
or hematopoietic cell capable of 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 may
then be measured in vitro, by monitoring, for example, its ability
to form 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 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 mammary tumor cell lines established as SC xenografts can
be examined.
4.4.2 Pharmaceutical Compositions and Methods of Administration
[0186] 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 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.
[0187] Described, below, in Section 5.4.2.1, are methods for
determining the effective dosage of the compounds of the invention
for the treatment of cell proliferative disorders. Further,
described, below, in Section 5.4.2.2, are methods for formulations
and pharmaceutical compositions comprising the compounds of the
invention, and methods for the administration of such compounds,
formulations, and compositions.
4.4.2.1 Effective Dose
[0188] Toxicity and therapeutic efficacy of the compounds of the
invention can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining the
LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD.sub.50/ED.sub.50. Compounds which exhibit large therapeutic
indices are preferred. While compounds that exhibit toxic side
effects may be used, care should be 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.
[0189] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention; the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0190] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are 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 achieve the MEC will depend on individual
characteristics and route the administration. However, HPLC assays
or bioassays can be used to determine plasma concentrations.
[0191] Dosage intervals can also be determined using the MEC value.
Compounds should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%.
4.4.2.2 Formulations and Administrations
[0192] 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 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 Section.
[0193] Pharmaceutical compositions for use in accordance with the
compounds of the present invention may be formulated in
conventional manner using one or more physiologically acceptable
carriers or excipients.
[0194] Thus, the compounds and their physiologically acceptable
salts and solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral or rectal administration.
[0195] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., 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 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.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0196] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0197] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0198] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0199] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0200] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0201] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0202] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
5. EXAMPLE: THE COMPOUNDS INHIBIT EGF-RECEPTOR/GRB-2 SH2 DOMAIN
INTERACTION
[0203] 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
[0204] Adaptor-GST fusion protein: The adaptor-GST
(glutathione-S-transfer- ase) fusion 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
fusion proteins consisted of only the SH2 domain of 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 P-D-thiogalactopyranoside (IPTG) in order to express the
fusion protein.
[0205] After the 6 hour expression period, the cells are
precipitated, pelleted at 10,000.times.g for 10 minutes at
4.degree. C., washed, and resuspended in phosphate buffered saline
(PBS). Next, the cells are lysed by sonication (6 strokes, 5
seconds per stroke). Insoluble material is removed by
centrifugation at 10,000.times.g for 10 minutes at 4.degree. C.,
and the supernatant is passed over a Glutathion-Sepharose column.
Bound GRB-2-GST fusion protein is eluted off the column with 5 mM
reduced glutathion, then dialyzed against PBS.
[0206] Immobilized EGF-R tyrosine kinase molecule: Epidermal growth
factor 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 mM 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)
[0207] 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.
[0208] The EGF-R molecule was immobilized onto microtiter plates.
Microtiter plates were prepared by first coating the wells of the
plate, overnight at 4.degree. C., with an anti-EGF-R monoclonal
antibody directed against the extracellular domain of EGFR (UBI,
#05-101) at a concentration of 0.5 .mu.g (in PBS) per microtiter
well, at a final volume of 150 .mu.l per well.
[0209] After overnight coating, the coating solution was removed
from the microtiter wells, and replaced with blocking buffer (5%
dry 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, 50 mM Tris-HCl, pH 7.2, 0.1% Triton
X-100).
[0210] Cell lysate from EGF-R-expressing cells were added to each
well, in 150 .mu.l 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 ng of EGF-R
protein was bound per well.
[0211] 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, the phosphatases remove
phosphate groups from the EGF-R molecules, thus prohibiting
endogenous adaptor proteins, such as GRB proteins, 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 starved.
[0212] Preparation of autophophorylated EGF-R: The following in
vitro kinase reaction yielded autophosphorylated EGF-R. The kinase
reaction was initiated by the addition of 15 .mu.l of ATP/Mn.sup.2+
mix (in 50 mM MnCl.sub.2, final concentration of 10 .mu.M ATP, for
a total volume of 150 .mu.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.
[0213] Assay procedure: Either 30 ng GRB-2-GST fusion proteins
(i.e. a 1:1 ratio of EGF-R:GRB-2 proteins) or 5 ng GRB-2-GST fusion
proteins (i.e. a 4:1 ratio of EGF-R:GRB-2 proteins) were added to
the phosphorylated EGF-R coated microtiter wells in incubation
buffer (0.1 M potassium phosphate buffer, pH 6.5) for 30 minutes,
at room temperature, in the presence of Compound I. Control wells
were incubated with GRB-2-GST fusion proteins in the absence of
Compound I.
[0214] After incubation, wells were washed extensively with TBST.
The amount of GRB-2-GST fusion protein bound to the immobilized
EGF-R is then preferably determined by with a purified rabbit
antiserum against the GST-moiety of the fusion protein (AMRAD, New
Victoria, Australia; Catalog No. 00001605). Incubations were for 30
minutes at room temperature. After incubation, antibody was removed
and the wells are washed extensively with TBST. For visualization,
wells were next incubated with a TAGO goat-anti-rabbit peroxidase
antibody at room temperature for 30 minutes. After incubation, the
antibody was removed, the wells were washed with tap water, and
then with TBST. Substrate solution, ABTS
(2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid) /H.sub.2O.sub.2
(1.2 .mu.l H.sub.2O.sub.2 to 10 ml ABTS) was applied to the wells,
and incubated for 20 minutes at room temperature. The reaction was
stopped by addition of 5NH.sub.2SO.sub.4. The O.D. at 410 nm was
determined for each well. Utilizing this technique, it is normally
possible to detect as little as 2 ng GRB-2-GST over background.
[0215] 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 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.
[0216] 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 with 1
mM 1-chloro-2,4 dinitrobenzene (CDNB) and 1.54 mg/ml reduced
glutathion in incubation buffer. The OD 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 Mannervik and Danielson
(Mannervik, B. and Danielson, U. H., 1988, CRC Critical Reviews in
Biochemistry 23:238).
5.2 Results
[0217] 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 assays described,
above, in Section 5.1.
[0218] Compound I proves to be a potent inhibitor of GRB-2/SH2
binding, having an IC.sub.50 of 2.9 .mu.M. (IC.sub.50, as used
herein, return to the concentration of test compound required to
inhibit one-half of GRB-2/SH2 binding relative to the amount of
binding which occurs in the absence of test compound.)
6. COMPOUND I INHIBITS bcr/abl ACTIVITY
[0219] The Example presented herein demonstrates that compounds of
the invention inhibits cell survival in a bcr/abl-transformed cell
line.
6.1 Materials and Methods
[0220] (1) Cell lines used in this assay are:
[0221] 32D cl.3: murine lymphoblastoid cell, IL-3 dependent.
[0222] 32D cl.3 J2/leuk: 32D cl.3 expressing raf and myc, IL-3
independent.
[0223] 32D bcr/abl: 32D over expressing bcr/abl kinase, pooled,
IL-3 independent.
[0224] (2) All the above cell lines were grown in incubator with 5%
CO.sub.2 and 37.degree. C. Their growth media are:
[0225] 32D cl.3: RPMl+10% FBS+1 ng/ml IL-3+2 mM Glutamine.
[0226] 32D cl.3 J2/leuk: RPMl+10% FBS+2 mM Glutamine.
[0227] 32D bcr/abl: RPMl+10% FBS+2 mM Glutamine.
[0228] IL-3: Interleukin-3, mouse (UBI Cat. #01-374)
[0229] (3) PBS (Dulbecco's Phosphate Buffered Saline) Gibco Cat.
#450-1300EB
[0230] (4) MTT
(3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
Thiazolyl blue)
[0231] Sigma Cat. # M-2128
[0232] working solution: 5 mg/ ml PBS, store in dark @4.degree.
C.
[0233] (5) Solubilization Buffer
[0234] SDS Electrophoresis Grade, Fisher Cat. #BP 166.
[0235] N,N-Dimethyl-formamide (DMF), Fisher Cat. #BP1160.
[0236] Acetic Acid, Glacial, Fisher Cat. #A38.
[0237] working solution: Dissolve 200 g SDS in 250 ml warm H.sub.2O
and 500 ml DMF, stir in low heat. When SDS is almost solubilized,
add 25 ml 80% acetic acid and 25 ml 1N HCL to solution. Adjust
volume to 1000 ml.
6.2 Procedure
[0238] All of the following steps were conducted at room
temperature unless specifically indicated.
6.2.1 Cell Seeding
[0239] (1) The cells were grown in tissue culture dish (10 cm,
Corning 25020-100) to about 1.times.10.sup.6 cell/ml, subculture
every 2-3 days at 1:10 (1:20 for 32D bcr/abl line).
[0240] (2) Viable cells were counted with trypan blue according to
standard procedure.
[0241] (3) Cells were then resuspended in fresh medium at a density
of 2.times.10.sup.5 cells/ml, and transfer cells to 96-well tissue
culture plate (Corning, 25806-96) at 50 .mu.l per well to reach
about 2.times.10.sup.4 cells/well. Each cell line was plated with
its own positive and negative control: (negative control:medium
alone).
[0242] 32D cl.3 seeding medium should contain 2 ng/ml IL-3.
6.2.2 Assay Procedures
[0243] (1) Compound I drug stock (10 mM in DMSO) was diluted 1:50.
1:2 serial dilutions were performed for the remaining 8 wells in
each line of the tissue culture plate. 50 .mu.l were added to each
well. Control wells received medium alone. Cells were incubated
with drugs in 5% CO.sub.2 at 37.degree. for 15 hrs.
[0244] (2) 15 .mu.l MTT were added to each well. Plates were
incubated at 37.degree. C. for 4 hours.
[0245] (3) After 4 hours, 100 .mu.l solubilization solution was
added to each well.
[0246] (4) Plates were covered with Aluminum foil, and allowed to
sit on an ELISA plate shaker and shake overnight at room
temperature to completely solubilize formazan crystals.
[0247] (5) Absorbance was read at 570 nm wavelength with a
reference wavelength of 630 nm using a Dynatech ELISA plate reader,
Model MR 500.
6.3 Results
[0248] Compound I was tested herein for its ability to affect
bcr/abl activity, and was found to be an inhibitor of bcr/abl
function.
[0249] The effect of Compound I on bcr/abl function was tested
using the cell growth assay described, above, in Section 6.1.
Briefly, three cell lines were used in this assay. First, an IL-3
dependent cell line (32D cl.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 cl.3 J2/leuk, which
consists of the 32D cl.3 cell line transformed with raf and myc,
and 32D bcr/abl, which consists of the 32D cl.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 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 cl.3 bcr/abl cell line, cells will be unable to survive in
the absence of IL-3.
[0250] Compound I inhibits the ability of the 32D cl.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
[0251] The Example presented herein demonstrates that Compound I of
the invention is a potent inhibitor of cellular proliferation.
7.1 Materials and Methods
Sulforhodamine B (SRB) Growth Assays
[0252] 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 cells was incubated for about 24
hours at 37.degree. C. after which it received an equal volume of
compound dilution per well making the total volume per well 200
.mu.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 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.degree. C. in a
humidified 5% Co.sub.2 incubator. Four days following dosing of
compound, the media was discarded and 200 .mu.l/well of ice-cold
10% TCA (Trichloroacetic Acid) was added to fix cells. After 60
minutes at 4.degree. C., the TCA was discarded and the plate was
rinsed 5 times with water. The plate was then air-dried and 100
.mu.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 completely dried, 100
.mu.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.
[0253] Assay 2: PDGF-R/SRB Adherent Cells Growth Assay
[0254] Compounds were tested for inhibition of anchorage-dependent
tumor cell growth using the colorimetric assay described by Skehan
et al., 1990. J. Natl. Cancer Inst. 82:1107-1112. The assay
measures protein content of acid-fixed cells using the counterion
binding dye sulforhodamine B (SRB, Sigma). The 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
.mu.l) were added to 96-well plates containing attached cellular
monolayers (2000 cells/well in 100 .mu.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.degree. C.,
5% CO.sub.2) the monolayers were washed 3 times with PBS and fixed
with 200 .mu.l ice-cold 10% TCA (Fisher Scientific), and kept at
4.degree. 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
protein was stained for 10 min with 100 .mu.l 0.4% SRB dissolved in
1% acetic acid. After 5 washes with tap water, the dye was
solubilized in 10 mM Tris base (100 .mu.l per well) and absorbance
read at 570 nm on a Dynatech plate reader model MR5000. Growth
inhibition data were expressed as a 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. IC.sub.50 values were determined using a four parameter
curve fit function.
[0255] 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 solidified agarose base layer (0.8%
agarose). After a 2 to 3 week incubation at 37.degree. C., colonies
larger than 50 .mu.m were quantified using an Omnicon 3800 Tumor
Colony counter.
[0256] Assay 3: MCF-7/HER-2B Growth Assay. The protocol used herein
is essentially similar to that described above (for 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
for four days and developed as per standard techniques.
[0257] 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
[0258] 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.
[0259] As shown below, Compound I proved to be a potent inhibitor
of cells proliferation of each of the four cell lines tested.
3 Compound I Cell Line IC.sub.50 (MM) C6 8 A431 7.5 MCF7 10
MCF7-HER 2 6
[0260] IC.sub.50, 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).
[0261] 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 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, for example, protein tyrosine kinase
molecules). Given this Compound I activity, the compound may
represent an anti-cell proliferation agent.
8. EXAMPLE: 3T3 CELLULAR PROLIFERATION INHIBITION ASSAY
[0262] The following protocol describes the procedures used to
determine the ability of the compounds to inhibit cellular
proliferation in 3T3 engineered cell lines that over expressing
EGFr, IGF1r, or PDGFr.
8.1 Materials and Reagents
[0263] (1) EGF Ligand: stock concentration=16.5 .mu.M; EGF 201,
TOYOBO, Co., Ltd. Japan.
[0264] (2) IGF1 Ligand: human, recombinant; G511, Promega Corp,
USA.
[0265] (3) PDGF Ligand: human PDGF B/B; 1276-956, Boehringer
Mannheim, Germany.
[0266] (4) SRB: sulfohodamine B; S-9012, Sigma Chemical Co.,
USA.
[0267] SRB Dye Solution: 0.4% SRB in 1% acetic acid, glacial.
[0268] (5) Acetic Acid, Glacial: A38-212, Fisher Scientific,
USA.
[0269] (6) Albumin, Bovine: fraction V powder; A-8551, Sigma
Chemical Co., USA.
[0270] (7) TCA Buffer: 10% trichloroacetic acid (A32-500, Fisher
Scientific, USA).
[0271] (8) Tris Base Buffer: 10 mM tris base (BP152-5, Fisher
Scientific, USA).
8.2 Procedure
[0272] (1) N-1H 3T3 (ATCC#1658) engineered cell liens: 3T3-EGFr,
3T3-IGF1r, 3T3-PDGFr.
[0273] (2) Cells are seeded at 8000 cells/well in 10% FBS+2 mM GLN
DMEM, in a 96 well plate. Cells are incubated at 37.degree. C. 5%
CO.sub.2 for overnight to allow the cells attach plate.
[0274] (3) On day 2, the cells are quiesced in the serum free
medium (0% FBS DMEM) for 24 hours.
[0275] (4) On day 3, the cells are treated with the ligands (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 free DMEM
with 0.1% bovine albumin only; the positive control cells receive
the ligands (EGF, IGF1, or PDGF) but no drugs. The drugs are
prepared in the serum free DMEM in a 96 well plate, and a serial
dilution is taken the place. A total of 10 .mu.l/well medium of the
diluted drugs are added into the cells. The total volume of each
well is 200 .mu.L. Quadruplicates (wells) and 11 concentration
points are applied to each drug tested.
[0276] (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.
[0277] (6) On day 5, the cells were washed with PBS and fixed with
200 .mu.l/well ice cold 10% TCA for 1 hour under 0-5.degree. C.
condition.
[0278] (7) 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 .mu.L/well for 10 minutes.
[0279] (8) Pour off SRB and rinse plate 5 times with 1% acetic
acid. Dry plate completely.
[0280] (9) Solubilize the dye with 10 mM Tris-base at 100
.mu.L/well for 10 minutes on a shaker.
[0281] (10) Read the plate at dual wavelengths at 570 nm and 630 nm
on Dynatech Elsia plate reader.
8.3 Assay Procedures
[0282] (1) Dilute drug stock (10 mM in DMSO) 1:50 in RPMI medium in
first well, then do 1:2 dilution for 8-points in tissue culture
plate. Transfer 50 .mu.l/well of this solution to the cells.
Control wells receive medium alone. Incubate the cells with drugs
in 50% CO.sub.2 at 37.degree. for 15 hrs.
[0283] (2) Add 15 .mu.l MTT to each well. Incubate plate at
37.degree. C. for 4 hours.
[0284] (3) After 4 hours, add 100 .mu.l solubilization solution to
each well.
[0285] (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.
[0286] (5) Read absorbance at 570 nm wavelength with a reference
wavelength of 630 nm using a Dynatech ELISA plate reader, Model MR
500.
[0287] It is apparent that many modifications and variations of
this invention as set forth here may be made without 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 terms of the appended claims.
* * * * *