U.S. patent application number 10/243663 was filed with the patent office on 2003-03-06 for 1-(pyrrolidin-1-ylmethyl)-3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives.
This patent application is currently assigned to Pharmacia & Upjohn Company. Invention is credited to Gao, Ping, Moon, Malcolm Wilson, Morozowich, Walter.
Application Number | 20030045565 10/243663 |
Document ID | / |
Family ID | 26901875 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030045565 |
Kind Code |
A1 |
Moon, Malcolm Wilson ; et
al. |
March 6, 2003 |
1-(pyrrolidin-1-ylmethyl)-3-(pyrrol-2-ylmethylidene)-2-indolinone
derivatives
Abstract
The present invention is directed to
1-pyrrolidin-1-ylmethyl-3-(pyrrol-2-y- lmethylidene)-2-indolinone
derivatives that modulate the activity of protein kinases ("PKs").
Pharmaceutical compositions comprising these compounds, methods of
treating diseases related to abnormal PK activity utilizing
pharmaceutical compositions comprising these compounds and methods
of preparing them are also disclosed.
Inventors: |
Moon, Malcolm Wilson;
(Kalamazoo, MI) ; Morozowich, Walter; (Kalamazoo,
MI) ; Gao, Ping; (Portage, MI) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Pharmacia & Upjohn
Company
|
Family ID: |
26901875 |
Appl. No.: |
10/243663 |
Filed: |
September 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10243663 |
Sep 16, 2002 |
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09863905 |
May 24, 2001 |
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6451838 |
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60225045 |
Aug 11, 2000 |
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60207000 |
May 24, 2000 |
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Current U.S.
Class: |
514/411 ;
514/414; 548/430; 548/466; 548/467 |
Current CPC
Class: |
A61P 1/00 20180101; A61P
9/10 20180101; A61P 31/00 20180101; A61P 43/00 20180101; A61P 37/00
20180101; C07F 9/65583 20130101; A61P 3/00 20180101; A61P 3/10
20180101; C07D 403/06 20130101; A61P 9/00 20180101; A61P 11/00
20180101; A61P 19/04 20180101; A61P 13/12 20180101; C07F 9/5728
20130101; A61P 31/04 20180101; A61P 37/06 20180101; A61P 35/00
20180101; C07D 403/14 20130101; A61P 7/02 20180101; A61P 17/06
20180101; A61P 17/00 20180101; A61P 25/00 20180101; A61P 19/02
20180101; A61P 9/08 20180101; A61P 25/28 20180101; A61P 29/00
20180101; A61P 1/16 20180101; A61P 17/02 20180101 |
Class at
Publication: |
514/411 ;
514/414; 548/430; 548/466; 548/467 |
International
Class: |
A61K 031/405; A61K
031/407 |
Claims
What is claimed is:
1. A compound of the Formula (I): 10wherein: R.sup.3, R.sup.4,
R.sup.5 and R.sup.6 are independently selected from the group
consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,
alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,
aryloxy, mercapto, alkylthio, arylthio, sulfinyl, sulfonyl,
S-sulfonamido, N-sulfonamido, trihalomethane-sulfonamido, carbonyl,
C-carboxy, O-carboxy, C-amido, N-amido, cyano, nitro, halo,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, amino and
--NR.sup.11R.sup.12 where R.sup.11 and R.sup.12 are independently
selected from the group consisting of hydrogen, alkyl, cycloalkyl,
aryl, carbonyl, acetyl, sulfonyl, and trifluoromethanesulfony- l,
or R.sup.11 and R.sup.12, together with the nitrogen atom to which
they are attached, combine to form a five- or six-member
heteroalicyclic ring provided that at least two of R.sup.3,
R.sup.4, R.sup.5 and R.sup.6 are hydrogen; or R.sup.3 and R.sup.4,
R.sup.4 and R.sup.5, or R.sup.5 and R.sup.6 combine to form a
six-member aryl ring, a methylenedioxy or an ethylenedioxy group;
R.sup.7 is selected from the group consisting of hydrogen, alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic,
hydroxy, alkoxy, aryloxy, carbonyl, acetyl, C-amido, C-thioamido,
amidino, C-carboxy, O-carboxy, sulfonyl, and
trihalomethane-sulfonyl; R.sup.8, R.sup.9 and R.sup.10 are
independently selected from the group consisting of hydrogen,
alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,
heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,
alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido,
N-sulfonamido, carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, amino and --NR.sup.11R.sup.12, wherein R.sup.11 and
R.sup.12 are as defined above; or a pharmaceutically acceptable
salt thereof.
2. The compound of claim 1, wherein R.sup.7 is hydrogen.
3. The compound of claim 1 wherein R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, and R.sup.9 are hydrogen, R.sup.8 and R.sup.10
are unsubstituted lower alkyl.
4. The compound of claim 1, wherein R.sup.8 and R.sup.10 are
methyl.
5. The compound of claim 1, wherein the compound is 11
6. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier or excipient and a compound of claim 1.
7. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier or excipient and a compound of claim 5.
8. The pharmaceutical composition of claim 6, wherein said
composition is administered parenterally.
9. The pharmaceutical composition of claim 5, wherein said
composition is administered parenterally.
10. A method of treating a human having a disease capable of
treatment by administration of a protein kinase inhibitor,
comprising administration to that human of a therapeutically
effective amount of a compound of claim 1.
11. A method of treating a human having a disease capable of
treatment by administration of a protein kinase inhibitor,
comprising administration to that human of a therapeutically
effective amount of a compound of claim 5.
12. The method of claim 11, wherein said disease is selected from
the group cell proliferative disorders, metabolic diseases and
infectious diseases.
13. The method of claim 12, wherein the cancer is selected from the
group consisting of colorectal cancer, Kaposi's sarcoma and lung
cancer.
14. The method of claim 12, wherein the blood vessel proliferative
disorder is selected from the group consisting of arthritis and
restenosis.
15. The method of claim 12, wherein the fibrotic disorder is
selected from the group consisting of hepatic cirrhosis and
atherosclerosis.
16. The method of claim 12, wherein the mesangial cell
proliferative disorder is selected from the group consisting of
glomerulonephritis, diabetic nephropathy, malignant
nephrosclerosis, thrombotic microangiopathy syndromes, transplant
rejection and glomerulopathies.
17. The method of claim 12, wherein the metabolic disease is
selected from the group consisting of psoriasis, diabetes mellitus,
wound healing, inflammation and neurodegenerative diseases.
18. A process of preparing a compound of Formula (I) comprising
reacting a compound of Formula (II) 12with formaldehyde and
pyrrolidine.
19. The process of claim 18, wherein R.sup.3-R.sup.7 and R.sup.9
are hydrogen and R.sup.8 and R.sup.10 are methyl.
20. The process of claim 18, further comprising modifying any of
the R.sup.3-R.sup.10 groups.
21. The process of claim 18, further comprising preparing an acid
addition salt.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119(e) to
U.S. Provisional applications Serial Nos. 60/207,000 filed on May
24, 2000, and 60/225,045, filed on Aug. 11, 2000, the disclosures
of which are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] The present invention is directed to
1-(pyrrolidin-1-ylmethyl)-3-(p- yrrol-2-ylmethylidene)-2-indolinone
derivatives that modulate the activity of protein kinases ("PKs").
Pharmaceutical compositions comprising these compounds, methods of
treating diseases related to abnormal PK activity utilizing
pharmaceutical compositions comprising these compounds and methods
of preparing them are also disclosed.
[0004] 2. State of the Art
[0005] Protein kinases ("PKs") are enzymes that catalyze the
phosphorylation of hydroxy groups on tyrosine, serine and threonine
residues of proteins. PKs can be conveniently broken down into two
classes, the protein tyrosine kinases (PTKs) and the
serine-threonine kinases (STKs). One of the prime aspects of PTK
activity is their involvement with growth factor receptors. Growth
factor receptors are cell-surface proteins. When bound by a growth
factor ligand, growth factor receptors are converted to an active
form which interacts with proteins on the inner surface of a cell
membrane. This leads to phosphorylation on tyrosine residues of the
receptor and other proteins and to the formation inside the cell of
complexes with a variety of cytoplasmic signaling molecules that,
in turn, effect numerous cellular responses such as cell division
(proliferation), cell differentiation, cell growth, expression of
metabolic effects to the extracellular microenvironment, etc (See,
Schlessinger and Ullrich (1992) Neuron 9:303-391).
[0006] Growth factor receptors with PTK activity are known as
receptor tyrosine kinases ("RTKs"). They comprise a large family of
transmembrane receptors with diverse biological activity. At
present, at least nineteen (19) distinct subfamilies of RTKs have
been identified. An example of these is the subfamily designated
the "HER" RTKs, which include EGFR (epithelial growth factor
receptor), HER2, HER3 and HER4.
[0007] Another RTK subfamily consists of insulin receptor (IR),
insulin-like growth factor I receptor (IGF-1R) and insulin receptor
related receptor (IRR). IR and IGF-1R interact with insulin, IGF-I
and IGF-II to form a heterotetramer of two entirely extracellular
glycosylated .alpha. subunits and two .beta. subunits which cross
the cell membrane and which contain the tyrosine kinase domain.
[0008] A third RTK subfamily is referred to as the platelet derived
growth factor receptor ("PDGFR") group, which includes
PDGFR.alpha., PDGFR.beta., CSFIR, c-kit and c-fms. Another group is
the fetus liver kinase ("flk") receptor subfamily. This group is
believed to be made of up of kinase insert domain-receptor fetal
liver kinase-1 (KDR/FLK-1), flk-1R, flk-4 and fms-like tyrosine
kinase 1 (flt-1).
[0009] A further member of the tyrosine kinase growth factor
receptor family is the fibroblast growth factor ("FGF") receptor
subgroup. This group consists of four receptors, FGFR1-4, and seven
ligands, FGF-1-7. While not yet well defined, it appears that the
receptors consist of a glycosylated extracellular domain containing
a variable number of immunoglobin-like loops and an intracellular
domain in which the tyrosine kinase sequence is interrupted by
regions of unrelated amino acid sequences.
[0010] Still another member of the tyrosine kinase growth factor
receptor family is the vascular endothelial growth factor ("VEGF")
receptor subgroup. VEGF is a dimeric glycoprotein similar to PDGF
but has different biological functions and target cell specificity
in vivo. In particular, VEGF is presently thought to play an
essential role is vasculogenesis and angiogenesis.
[0011] A more complete listing of the known RTK subfamilies is
described in Plowman et al., DN&P, 7(6):334-339 (1994) which is
incorporated by reference, including any drawings, as if fully set
forth herein.
[0012] In addition to the RTKs, there also exists a family of
entirely intracellular PTKs called "non-receptor tyrosine kinases"
or "cellular tyrosine kinases." This latter designation,
abbreviated "CTK," will be used herein. CTKs do not contain
extracellular and transmembrane domains. At present, over 24 CTKs
in 11 subfamilies (Src, Frk, Btk, Csk, Abl, Zap7O, Fes, Fps, Fak,
Jak and Ack) have been identified. The Src subfamily appear so far
to be the largest group of CTKs and includes Src, Yes, Fyn, Lyn,
Lck, Blk, Hck, Fgr and Yrk. For a more detailed discussion of CTKs,
see Bolen, Oncogene, 8:2025-2031 (1993), which is incorporated by
reference, including any drawings, as if fully set forth
herein.
[0013] The serine/threonine kinases, STKs, like the CTKs, are
predominantly intracellular although there are a few receptor
kinases of the STK type. STKs are the most common of the cytosolic
kinases; i.e., kinases that perform their function in that part of
the cytoplasm other than the cytoplasmic organelles and
cytoskelton. The cytosol is the region within the cell where much
of the cell's intermediary metabolic and biosynthetic activity
occurs; e.g., it is in the cytosol that proteins are synthesized on
ribosomes.
[0014] RTKs, CTKs and STKs have all been implicated in a host of
pathogenic conditions including, significantly, cancer. Other
pathogenic conditions which have been associated with PTKs include,
without limitation, psoriasis, hepatic cirrhosis, diabetes,
angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and
other inflammatory disorders, immunological disorders such as
autoimmune disease, cardiovascular disease such as atherosclerosis
and a variety of renal disorders.
[0015] With regard to cancer, two of the major hypotheses advanced
to explain the excessive cellular proliferation that drives tumor
development relate to functions known to be PK regulated. That is,
it has been suggested that malignant cell growth results from a
breakdown in the mechanisms that control cell division and/or
differentiation. It has been shown that the protein products of a
number of proto-oncogenes are involved in the signal transduction
pathways that regulate cell growth and differentiation. These
protein products of proto-oncogenes include the extracellular
growth factors, transmembrane growth factor PTK receptors (RTKs),
cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.
[0016] In view of the apparent link between PK-related cellular
activities and wide variety of human disorders, it is no surprise
that a great deal of effort is being expended in an attempt to
identify ways to modulate PK activity. For example, attempts have
been made to identify small molecules which act as PK inhibitors.
For example, bis-monocylic, bicyclic and heterocyclic aryl
compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO
94/14808) and 1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No.
5,330,992) have been described as tyrosine kinase inhibitors.
Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted
pyridyl compounds (U.S. Pat. No. 5,302,606), quinazoline
derivatives (EP Application No. 0 566 266 A1), selenaindoles and
selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds
(PCT WO 92/21660), and benzylphosphonic acid compounds (PCT WO
91/15495). Additionally, a family of novel pyrrole-substituted
2-indolinone compounds have been discovered which exhibit PK
modulating ability and have a salutary effect against disorders
related to abnormal PK activity (U.S. Pat. No. 5,792,783 and PCT
Application Publication No. WO 99/61422). Administration of various
species of pyrrole-substituted 2-indolinone compounds has been
shown to be an effective therapeutic approach to cure many kinds of
solid tumors. For example,
3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one,
a highly active selective inhibitor of the vascular endothelial
growth factor receptor (Flk-1/KDR), inhibits tyrosine kinase
catalysis, tumor vascularization, and growth of multiple tumor
types (Fong et al. (1999) Cancer Res. 59:99-106). These compounds,
however, have high lipophilicity and low solubility in water and
most common vehicles at physiological pH limit their
adminstration.
[0017] Accordingly, there is a need for PK inhibitors that do not
exhibit such drawbacks. The present invention fulfills this and
related needs.
SUMMARY OF THE INVENTION
[0018] In one aspect, this invention is directed to a compound of
Formula (I): 1
[0019] wherein:
[0020] R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are independently
selected from the group consisting of hydrogen, alkyl,
trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,
arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,
trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy,
C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, amino and --NR.sup.11R.sup.12 where
R.sup.11 and R.sup.12 are independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl,
sulfonyl, and trifluoromethanesulfonyl, or R.sup.11 and R.sup.12,
together with the nitrogen atom to which they are attached, combine
to form a five- or six-member heteroalicyclic ring provided that at
least two of R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are hydrogen;
or
[0021] R.sup.3 and R.sup.4, R.sup.4 and R.sup.5, or R.sup.5 and
R.sup.6 combine to form a six-member aryl ring, a methylenedioxy or
an ethylenedioxy group;
[0022] R.sup.7 is selected from the group consisting of hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, hydroxy, alkoxy, aryloxy, carbonyl, acetyl,
C-amido, C-thioamido, amidino, C-carboxy, O-carboxy, sulfonyl, and
trihalomethane-sulfonyl;
[0023] R.sup.8, R.sup.9 and R.sup.10 are independently selected
from the group consisting of hydrogen, alkyl, trihaloalkyl,
cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic,
hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, sulfinyl,
sulfonyl, S-sulfonamido, N-sulfonamido, carbonyl, C-carboxy,
O-carboxy, cyano, nitro, halo, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and
--NR.sup.11R.sup.12, wherein R.sup.11 and R.sup.12 are as defined
above; or
[0024] a pharmaceutically acceptable salt thereof.
[0025] Preferably, R.sup.3, R.sup.5 and R.sup.6 are independently
selected from the group consisting of hydrogen, alkyl,
trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,
arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido,
trihalomethane-sulfonamido, carbonyl, C-carboxy, O-carboxy,
C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, amino and --NR.sup.11R.sup.12 where
R.sup.11 and R.sup.12 are independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl,
sulfonyl, trifluoromethanesulfonyl and, together with the nitrogen
to which they are attached form, a five- or six-member
heteroalicyclic ring; especially R.sup.3, R.sup.5, and R.sup.6 are
hydrogen;
[0026] R.sup.7 is hydrogen;
[0027] R.sup.4 is hydrogen or halo, especially hydrogen, fluoro, or
chloro, particularly hydrogen or fluoro;
[0028] R.sup.8 and R.sup.10 are independently unsubstituted lower
alkyl, especially methyl; and
[0029] R.sup.9 is hydrogen, lower alkyl substituted with C-carboxy
or --C(.dbd.O)NHR.sup.12 wherein R.sup.12 is lower alkyl
substituted with amino or heteroalicyclic and optionally
substituted with hydroxy; R.sup.9 is preferably hydrogen,
3-carboxypropyl, (2-diethylaminoethyl)-aminocarbo- nyl,
(2-ethylaminoethyl)aminocarbonyl,
3-(morpholin-4-yl)propyl-aminocarbo- nyl,
3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl; R.sup.9 is most
preferably hydrogen, 3-carboxypropyl,
(2-diethylaminoethyl)aminocarbonyl, or
(2-ethylaminoethyl)-aminocarbonyl.
[0030] A number of different preferences have been given above, and
following any one of these preferences results in a compound of
this invention that is more presently preferred than a compound in
which that particular preference is not followed. However, these
preferences are generally independent [although some (alternative)
preferences are mutually exclusive], and additive; and following
more than one of these preferences may result in a more presently
preferred compound than one in which fewer of the preferences are
followed.
[0031] Presently preferred classes of compounds of this invention
include those where:
[0032] (a) R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.9
are hydrogen and R.sup.8 and R.sup.10 are unsubstituted lower
alkyl, especially methyl.
[0033] (b) R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are
hydrogen; R.sup.8 and R.sup.10 are unsubstituted lower alkyl,
especially methyl; and R.sup.9 is lower alkyl substituted with
C-carboxy, especially 3-carboxypropyl.
[0034] Presently preferred compounds of this invention include:
[0035]
(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidiny-
lmethyl)-1,3-dihydro-2H-indol-2-one; and
[0036]
(3Z)-3-{[3,5-dimethyl-4-(3-carboxypropyl)-1H-pyrrol-2-yl]-methylide-
ne}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one.
[0037] The compounds of the present invention convert in vivo to
compounds of Formula (II): 2
[0038] that exhibit PK modulating ability, in particular PK
inhibiting ability, and are therefore useful in treating disorders
related to abnormal PK activity. The active compounds (II) formed
from the compounds of the present invention are described in U.S.
Pat. No. 5,792,783, PCT Application Publication No. WO 99/61422,
and U.S. patent application Ser. No. 09/783,264, filed on Feb. 15,
2001, and titled "PYRROLE SUBSTITUTED 2-INDOLINONE AS PROTEIN
KINASE INHIBITORS", the disclosures of which are hereby
incorporated by reference.
[0039] The prodrug compounds of the present invention have
advantages over compounds of Formula (II) by virtue of improved
aqueous solubility and formulability. For example, Applicants have
discovered that the N-pyrrolidin-1-ylmethyl prodrug of compound
(II) where R.sup.3-R.sup.7 and R.sup.9 are hydrogen and R.sup.8 and
R.sup.10 are methyl provides unexpected increased aqueous
solubility over the parent compound thus making it particularly
suitable for intravenous (IV) formulations. It is contemplated that
similar enhanced solubility will be observed for other compounds of
Formula (I) carrying the pyrrolidin-1-ylmethyl moiety at the
nitrogen of the indolinone ring. A general description of the
advantages and uses of prodrugs as pharmaceutically useful
compounds is given in an article by Waller and George in Br. J.
Clin. Pharmac., Vol. 28, pp. 497-507, 1989.
[0040] In a second aspect this invention is directed to a
pharmaceutical composition comprising one or more compound(s) of
Formula (I) or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable excipient.
[0041] In a third aspect, this invention is directed to a method of
treating diseases mediated by abnormal protein kinase activity, in
particular, receptor tyrosine kinases (RTKs), non-receptor protein
tyrosine kinases (CTKs) and serine/threonine protein kinases
(STKs), in an organism, in particular humans, which method
comprises administering to said organism a pharmaceutical
composition comprising a compound of Formula (I) or a
pharmaceutically acceptable salt thereof and a pharmaceutically
acceptable excipient. Such diseases include by way of example and
not limitation, cancer, diabetes, hepatic cirrhosis, cardiovascular
disease such as atherosclerosis, angiogenesis, immunological
disease such as autoimmune disease (e.g., AIDS and lupus) and renal
disease. Specifically, the diseases mediated by EGF, HER2, HER3,
HER4, IR, IGF-1R, IRR, PDGFR.alpha., PDGFR.beta., CSFIR, C-Kit,
C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R,
FGFR-4R, Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak, Jak, Ack,
Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, Yrk, CDK2 and Raf.
[0042] In a fourth aspect, this invention is directed to a method
of modulating the catalytic activity (e.g., inhibiting the
catalytic activity) of PKs, in particular receptor tyrosine kinases
(RTKs), non-receptor protein tyrosine kinases (CTKs) and
serine/threonine protein kinases (STKs), using a compound of this
invention or a pharmaceutical composition comprising a compound of
this invention and a pharmaceutically acceptable excipient. The
method may be carried out in vitro or in vivo. In particular, the
receptor protein kinase whose catalytic activity is modulated by a
compound of this invention is selected from the group consisting of
EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFR.alpha., PDGFR.beta.,
CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R,
FGFR-2R, FGFR-3R and FGFR-4R. The cellular tyrosine kinase whose
catalytic activity is modulated by a compound of this invention is
selected from the group consisting of Src, Frk, Btk, Csk, Abl,
ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr
and Yrk. The serine-threonine protein kinase whose catalytic
activity is modulated by a compound of this invention is selected
from the group consisting of CDK2 and Raf.
[0043] In a fifth aspect, this invention is directed to the use of
a compound of Formula (I) in the preparation of a medicament useful
in the treatment of a disease mediated by abnormal PK activity.
[0044] In a sixth aspect, this invention is directed to a method of
preparing a compound of Formula (I) which method comprises reacting
a compound of Formula (II) 3
[0045] with an pyrrolidine in the presence of formaldehyde;
[0046] optionally modifying any of the R.sup.3--R.sup.10 groups;
and
[0047] optionally preparing an acid addition salt thereof.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0048] Unless otherwise stated the following terms used in the
specification and claims have the meanings discussed below:
[0049] "Alkyl" refers to a saturated aliphatic hydrocarbon
including straight chain, or branched chain groups. Preferably, the
alkyl group has 1 to 20 carbon atoms (whenever a numerical range;
e.g., "1-20", is stated herein, it means that the group, in this
case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3
carbon atoms, etc. up to and including 20 carbon atoms). More
preferably, it is a medium size alkyl having 1 to 10 carbon atoms.
Most preferably, it is a lower alkyl having 1 to 4 carbon atoms
e.g., methyl, ethyl, n-propyl, isopropyl, butyl, iso-butyl,
tert-butyl and the like. The alkyl group may be substituted or
unsubstituted. When substituted, the substituent group(s) is
preferably one or more, more preferably one or two groups,
individually selected from the group consisting of cycloalkyl,
aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,
mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, C-carboxy, O-carboxy, nitro, silyl, amino, ammonium and
--NR.sup.13R.sup.14 where R.sup.13 and R.sup.14 are independently
selected from the group consisting of hydrogen, unsubstituted
alkyl, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, amino,
and trifluoromethanesulfonyl, or R.sup.13 and R.sup.14, together
with the nitrogen atom to which they are attached, combine to form
a five- or six-member heteroalicyclic ring. More preferably, the
substituent is hydroxy, amino, or --NR.sup.13R.sup.14 where
R.sup.13 and R.sup.14 are independently selected from the group
consisting of unsubstituted lower alkyl, lower alkyl substituted
with amino or hydroxy, or R.sup.13 and-R.sup.14, together with the
nitrogen atom to which they are attached, combine to form
pyrrolidine, morpholine, or piperazine.
[0050] A "cycloalkyl" group refers to an all-carbon monocyclic ring
(i.e., rings which share an adjacent pair of carbon atoms) of 3 to
6 ring atoms wherein one of more of the rings does not have a
completely conjugated pi-electron system e.g, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclobutenyl, cyclopentenyl,
cyclohexenyl, and the like. Examples, without limitation, of
cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane,
cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane
and, cycloheptatriene. A cycloalkyl group may be substituted or
unsubstituted. When substituted, the substituent group(s) is
preferably one or more, more preferably one or two groups,
individually selected from unsubstituted alkyl, alkyl, aryl,
heteroaryl, unsubstituted heteroalicyclic, heteroalicyclic,
hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano,
halo, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl,
N-carbamyl, C-amido, N-amido, nitro, amino and --NR.sup.13R.sup.14,
with R.sup.13 and R.sup.14 as defined above.
[0051] An "alkenyl" group refers to an alkyl group, as defined
herein, consisting of at least two carbon atoms and at least one
carbon-carbon double bond e.g., ethenyl, propenyl, butenyl or
pentenyl and their structural isomeric forms such as 1- or
2-propenyl, 1-, 2-, or 3-butenyl and the like.
[0052] An "alkynyl" group refers to an alkyl group, as defined
herein, consisting of at least two carbon atoms and at least one
carbon-carbon triple bond e.g., acetylene, ethnyl, propynyl,
butynyl, or pentnyl and their structural isomeric forms as
described above.
[0053] An "aryl" group refers to an all-carbon monocyclic or
fused-ring polycyclic (i.e., rings which share adjacent pairs of
carbon atoms) groups of 6 to 12 ring atoms and having a completely
conjugated pi-electron system. Examples, without limitation, of
aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl
group may be substituted or unsubstituted. When substituted, the
substituted group(s) is preferably one or more, more preferably
one, two, or three substituents, independently selected from the
group consisting of halo, trihalomethyl, alkyl, hydroxy, alkoxy,
aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl,
thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl,
sulfonyl, amino and --NR.sup.13R.sup.14, with R.sup.13 and R.sup.14
as defined above. Preferably the substituent(s) is/are
independently selected from chloro, fluoro, bromo, methyl, ethyl,
propyl including all its isomeric forms, butyl including all its
isomeric forms, hydroxy, methoxy, phenoxy, thio, methylthio,
phenylthio, cyano, nitro, carboxy, methoxycarbonyl, or amino.
[0054] A "heteroaryl" group refers to a monocyclic or fused
aromatic ring (i.e., rings which share an adjacent pair of atoms)
of 5 to 9 ring atoms in which one, two, three or four ring atoms
are selected from the group consisting of nitrogen, oxygen and
sulfur and the rest being carbon. Examples, without limitation, of
heteroaryl groups are pyrrole, furan, thiophene, imidazole,
oxazole, thiazole, pyrazole, tetrazole, pyridine, pyrimidine,
quinoline, isoquinoline, purine and carbazole. The heteroaryl group
may be substituted or unsubstituted. When substituted, the
substituted group(s) is preferably one or more, more preferably one
or two substituents, independently selected from the group
consisting of alkyl, cycloalkyl, halo, trihalomethyl, hydroxy,
alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro,
carbonyl, thiocarbonyl, sulfonamido, C-carboxy, O-carboxy,
sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,
N-thiocarbamyl, C-amido, N-amido, amino and --NR.sup.13R.sup.14,
with R.sup.13 and R.sup.14 as defined above. Preferably the
substituent(s) is/are independently selected from chloro, fluoro,
bromo, methyl, ethyl, propyl including all its isomeric forms,
butyl including all its isomeric forms, hydroxy, methoxy, phenoxy,
thio, methylthio, phenylthio, cyano, nitro, carboxy,
methoxycarbonyl, or amino.
[0055] A "heteroalicyclic" group refers to a monocyclic or fused
ring of 4 to 9 ring atoms containing one, two, or three heteroatoms
in the ring which are selected from the group consisting of
nitrogen, oxygen and --S(O).sub.n where n is 0-2, the remaining
ring atoms being carbon. The rings may also have one or more double
bonds. However, the rings do not have a completely conjugated
pi-electron system. Examples, without limitation, of
heteroalicyclic groups are pyrrolidine, piperidine, piperazine,
morpholine, imidazolidine, tetrahydropyridazine, tetrahydrofuran,
thiomorpholine, tetrahydropyridine, and the like. The
heteroalicyclic ring may be substituted or unsubstituted. When
substituted, the substituted group(s) is preferably one or more,
more preferably one, two, or three substituents, independently
selected from the group consisting of alkyl, cycloalkyl, halo,
trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,
arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy,
O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,
sulfinyl, sulfonyl, C-amido, N-amido, amino and
--NR.sup.13R.sup.14, with R.sup.13 and R.sup.14 as defined above.
Preferably the substituent(s) is/are independently selected from
chloro, fluoro, bromo, methyl, ethyl, propyl including all its
isomeric forms, butyl including all its isomeric forms, hydroxy,
methoxy, phenoxy, thio, methylthio, phenylthio, cyano, nitro,
carboxy, methoxycarbonyl, or amino.
[0056] A "hydroxy" group refers to an --OH group.
[0057] An "alkoxy" group refers to an --O-unsubstituted alkyl,
--O-substituted alkyl and an --O-unsubstitutedcycloalkyl group, as
defined herein. Examples include and are not limited to methoxy,
ethoxy, propoxy, butoxy, cyclopropyloxy, and the like, preferably
methoxy.
[0058] An "aryloxy" group refers to both an --O-aryl and an
--O-heteroaryl group, as defined herein. Examples include and are
not limited to phenoxy, napthyloxy, pyridyloxy, furanyloxy, and the
like.
[0059] A "mercapto" group refers to an --SH group.
[0060] A "alkylthio" group refers to both an S-alkyl and an
--S-cycloalkyl group, as defined herein. Examples include and are
not limited to methylthio, ethylthio, and the like. A "arylthio"
group refers to both an --S-aryl and an --S-heteroaryl group, as
defined herein. Examples include and are not limited to phenylthio,
napthylthio, pyridylthio, furanylthio, and the like.
[0061] A "sulfinyl" group refers to a --S(.dbd.O)--R" group
wherein, in addition to being as defined below, R" may also be a
hydroxy group, e.g., methylsulfinyl, phenylsulfinyl, and the
like.
[0062] A "sulfonyl" group refers to a --S(.dbd.O).sub.2R" group
wherein, in addition to being as defined below, R" may also be a
hydroxy group e.g., methylsulfonyl, ethylsulfonyl, phenylsulfonyl,
and the like.
[0063] A "trihalomethyl" group refers to a --CX.sub.3 group wherein
X is a halo group as defined herein e.g., trifluoromethyl,
trichloromethyl, tribromomethyl, dichlorofluoromethyl, and the
like.
[0064] A "trihalomethanesulfonyl" group refers to a
X.sub.3CS(.dbd.O).sub.2-- groups with X as defined above, e.g.,
trifluoromethylsulfonyl, trichloromethylsulfonyl,
tribromomethylsulfonyl, and the like.
[0065] A "trihalomethanesulfonylamido" group refers to a
--NH--S(.dbd.O).sub.2R groups wherein R is trihalomethyl as defined
above.
[0066] "Carbonyl" and "acyl" are used interchangeably herein to
refer to a --C(.dbd.O)--R" group, where R" is selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl
(bonded through a ring carbon) and heteroalicyclic (bonded through
a ring carbon), as defined herein. Representative examples include
and are not limited to acetyl, propionyl, benzoyl, formyl,
cyclopropylcarbonyl, pyridinylcarbonyl, pyrrolidin-1-ylcarbonyl,
and the like
[0067] An "aldehyde" group refers to a carbonyl group where R" is
hydrogen.
[0068] A "thiocarbonyl" group refers to a --C(.dbd.S)--R" group,
with R" as defined herein.
[0069] A "C-carboxy" group refers to a --C(.dbd.O)O--R" group, with
R" as defined herein e.g., --COOH, methoxycarbonyl, ethoxycarbonyl,
benzyloxycarbonyl, and the like.
[0070] An "O-carboxy" group refers to a --OC(.dbd.O)R" group, with
R" as defined herein e.g., methylcarbonyloxy, phenylcarbonyloxy,
benzylcarbonyloxy, and the like.
[0071] An "ester" group refers to a --C(.dbd.O)O--R" group with R"
as defined herein except that R" cannot be hydrogen e.g.,
methoxycarbonyl, benzyloxycarbonyl, and the like.
[0072] An "acetyl" group refers to a --C(.dbd.O)CH.sub.3 group.
[0073] A "carboxylic acid" group refers to a C-carboxy group in
which R" is hydrogen.
[0074] A "halo" group refers to fluorine, chlorine, bromine or
iodine.
[0075] A "cyano" group refers to a --C.ident.N group.
[0076] A "nitro" group refers to a --NO.sub.2 group.
[0077] A "methylenedioxy" group refers to --OCH.sub.2O-- group
where the two oxygen atoms are bonded to adjacent carbon atoms.
[0078] An "ethylenedioxy" group refers to --OCH.sub.2CH.sub.2O--
where the two oxygen atoms are bonded to adjacent carbon atoms.
[0079] An "S-sulfonamido" group refers to a
--S(.dbd.O).sub.2NR.sup.13R.su- p.14 group, with R.sup.13 and
R.sup.14 as defined herein. Representative examples include and are
not limited to dimethylaminosulfonyl, aminosulfonyl,
phenylmethylaminosulfonyl, phenylaminosulfonyl, and the like.
[0080] An "N-sulfonamido" group refers to a
--NR.sup.13S(.dbd.O).sub.2R.su- p.14 group, with R.sup.13 and
R.sup.14 as defined herein e.g., methylsulfonylamino,
ethylsulfonylamino, phenylsulfonylamino, benzylsulfonylamino, and
the like.
[0081] An "O-carbamyl" group refers to a
--OC(.dbd.O)NR.sup.13R.sup.14 group with R.sup.13 and R.sup.14 as
defined herein.
[0082] An "N-carbamyl" group refers to a
R.sup.14OC(.dbd.O)NR.sup.13-- group, with R.sup.13 and R.sup.14 as
defined herein.
[0083] An "O-thiocarbamyl" group refers to a
--OC(.dbd.S)NR.sup.13R.sup.14 group with R.sup.13 and R.sup.14 as
defined herein.
[0084] An "N-thiocarbamyl" group refers to a
R.sup.14OC(.dbd.S)NR.sup.13-- group, with R.sup.13 and R.sup.14 as
defined herein.
[0085] An "amino" group refers to an --NR.sup.13R.sup.14 group,
wherein R.sup.13 and R.sup.14 are independently hydrogen or
unsubstituted lower alkyl e.g, --NH.sub.2, dimethylamino,
diethylamino, ethylamino, methylamino, and the like.
[0086] A "C-amido" group refers to a
--C(.dbd.O)NR.sup.13R.sup.14group with R.sup.13 and R.sup.14 as
defined herein. Preferably R.sup.13 is hydrogen or unsubstituted
lower alkyl and R.sup.14 is hydrogen, lower alkyl optionally
substituted with heteroalicyclic, hydroxy, or amino. For example,
--C(.dbd.O)NR.sup.13R.sup.14 may be aminocarbonyl,
dimethylaminocarbonyl, diethylaminocarbonyl,
diethylaminoethylaminocarbon- yl, ethylaminoethylaminocarbonyl,
2-morpholinoethylaminocarbonyl, 3-morpholinopropylaminocarbonyl,
3-morpholino-2-hydroxypropylaminocarbony- l, and the like.
[0087] An "N-amido" group refers to a R.sup.14C(.dbd.O)NR.sup.13--
group, with R.sup.13 and R.sup.14 as defined herein e.g.,
acetylamino, and the like.
[0088] A "ammonium" group refers to a
--.sup.+NR.sup.15R.sup.16R.sup.17 group, wherein R.sup.15 and
R.sup.16 are independently selected from the group consisting of
alkyl, cycloalkyl, aryl, and heteroaryl, and R.sup.17 is selected
from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, and
heteroaryl.
[0089] A "amidino" group refers to a
R.sup.15R.sup.16NC(.dbd.NR.sup.17)-- group, with R.sup.15, R.sup.16
and R.sup.17 as defined herein.
[0090] A "morpholino" group refers to a group having the chemical
structure 4
[0091] A "piperazinyl" group refers to a group having the chemical
structure: 5
[0092] The terms "indolinone", "2-indolinone" and "indolin-2-one"
are used interchangeably herein to refer to a molecule having the
chemical structure: 6
[0093] "Pyrrole" refers to a molecule having the chemical
structure: 7
[0094] "Pyrrole-substituted 2-indolinone" and
"3-pyrrol-1-yl-2-indolinone" are used interchangeably herein to
refer to a chemical compound having the general structure shown in
Formula II. 8
[0095] A "prodrug" refers to an agent which is converted into the
parent drug in vivo. Prodrugs are often useful because, in some
situations, they may be easier to administer than the parent drug.
They may, for instance, be bioavailable by oral administration
whereas the parent drug is not. The prodrug may also have improved
solubility in pharmaceutical compositions over the parent drug. A
prodrug may be converted into the parent drug by various
mechanisms, including enzymatic processes and metabolic hydrolysis.
See Harper, "Drug Latentiation" in Jucker, ed. Progress in Drug
Research 4:221-294 (1962); Morozowich et al., "Application of
Physical Organic Principles to Prodrug Design" in E. B. Roche ed.
Design of Biopharmaceutical Properties through Prodrugs and
Analogs, APHA Acad. Pharm. Sci. (1977); Bioreversible Carriers in
Drug in Drug Design, Theory and Application, E. B. Roche, ed., APHA
Acad. Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard,
Elsevier (1985); Wang et al. "Prodrug approaches to the improved
delivery of peptide drug" in Curr. Pharm. Design. 5(4):265-287
(1999); Pauletti et al. (1997) Improvement in peptide
bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug.
Delivery Rev. 27:235-256; Mizen et al. (1998) "The Use of Esters as
Prodrugs for Oral Delivery of .beta.-Lactam antibiotics," Pharm.
Biotech. 11,:345-365; Gaignault et al. (1996) "Designing Prodrugs
and Bioprecursors I. Carrier Prodrugs," Pract. Med. Chem. 671-696;
Asgharnejad, "Improving Oral Drug Transport", in Transport
Processes in Pharmaceutical Systems, G. L. Amidon, P. I. Lee and E.
M. Topp, Eds., Marcell Dekker, p. 185-218 (2000); Balant et al.,
"Prodrugs for the improvement of drug absorption via different
routes of administration", Eur. J. Drug Metab. Pharmacokinet.,
15(2): 143-53 (1990); Balimane and Sinko, "Involvement of multiple
transporters in the oral absorption of nucleoside analogues", Adv.
Drug Delivery Rev., 39(1-3): 183-209 (1999); Browne, "Fosphenytoin
(Cerebyx)", Clin. Neuropharmacol. 20(1): 1-12 (1997); Bundgaard,
"Bioreversible derivatization of drugs--principle and applicability
to improve the therapeutic effects of drugs", Arch. Pharm. Chemi
86(1): 1-39 (1979); Bundgaard H. "Improved drug delivery by the
prodrug approach", Controlled Drug Delivery 17: 179-96 (1987);
Bundgaard H. "Prodrugs as a means to improve the delivery of
peptide drugs", Adv. Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher
et al. "Improved oral drug delivery: solubility limitations
overcome by the use of prodrugs", Adv. Drug Delivery Rev. 19(2):
115-130 (1996); Fleisher et al. "Design of prodrugs for improved
gastrointestinal absorption by intestinal enzyme targeting",
Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A): 360-81,
(1985); Farquhar D, et al., "Biologically Reversible
Phosphate-Protective Groups", J. Pharm. Sci., 72(3): 324-325
(1983); Freeman S, et al., "Bioreversible Protection for the
Phospho Group: Chemical Stability and Bioactivation of
Di(4-acetoxy-benzyl) Methylphosphonate with Carboxyesterase," J.
Chem. Soc., Chem. Commun., 875-877 (1991); Friis and Bundgaard,
"Prodrugs of phosphates and phosphonates: Novel lipophilic
alpha-acyloxyalkyl ester derivatives of phosphate- or phosphonate
containing drugs masking the negative charges of these groups",
Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al., "Pro-drug,
molecular structure and percutaneous delivery", Des. Biopharm.
Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21. (1977);
Nathwani and Wood, "Penicillins: a current review of their clinical
pharmacology and therapeutic use", Drugs 45(6): 866-94 (1993);
Sinhababu and Thakker, "Prodrugs of anticancer agents", Adv. Drug
Delivery Rev. 19(2): 241-273 (1996); Stella et al., "Prodrugs. Do
they have advantages in clinical practice?", Drugs 29(5): 455-73
(1985); Tan et al. "Development and optimization of anti-HIV
nucleoside analogs and prodrugs: A review of their cellular
pharmacology, structure-activity relationships and
pharmacokinetics",Adv. Drug Delivery Rev. 39(1-3): 117-151 (1999);
Taylor, "Improved passive oral drug delivery via prodrugs", Adv.
Drug Delivery Rev., 19(2): 131-148 (1996); Valentino and Borchardt,
"Prodrug strategies to enhance the intestinal absorption of
peptides", Drug Discovery Today 2(4): 148-155 (1997); Wiebe and
Knaus, "Concepts for the design of anti-HIV nucleoside prodrugs for
treating cephalic HIV infection", Adv. Drug Delivery Rev.:
39(1-3):63-80 (1999); Waller et al., "Prodrugs", Br. J. Clin.
Pharmac. 28: 497-507 (1989).
[0096] The compounds of this invention may possess one or more
chiral centers, and can therefore be produced as individual
stereoisomers or as mixtures of stereoisomers, depending on whether
individual stereoisomers or mixtures of stereoisomers of the
starting materials are used. Unless indicated otherwise, the
description or naming of a compound or group of compounds is
intended to include both the individual stereoisomers or mixtures
(racemic or otherwise) of stereoisomers. Methods for the
determination of stereochemistry and the separation of
stereoisomers are well known to a person of ordinary skill in the
art [see the discussion in Chapter 4 of March J: Advanced Organic
Chemistry, 4th ed. John Wiley and Sons, New York, N.Y., 1992].
[0097] The chemical formulae referred to herein may exhibit the
phenomena of tautomerism and structural isomerism. For example, the
compounds described herein may adopt an E or a Z configuration
about the double bond connecting the 2-indolinone moiety to the
pyrrole moiety or they may be a mixture of E and Z. This invention
encompasses any tautomeric or structural isomeric form and mixtures
thereof.
[0098] The term "method" refers to manners, means, techniques and
procedures for accomplishing a given task including, but not
limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means,
techniques and procedures by, practitioners of the chemical,
pharmaceutical, biological, biochemical and medical arts.
[0099] As used herein, the term "modulation" or "modulating" refers
to the alteration of the catalytic activity of RTKs, CTKs and STKs.
In particular, modulating refers to the activation of the catalytic
activity of RTKs, CTKs and STKs, preferably the activation or
inhibition of the catalytic activity of RTKs, CTKs and STKs,
depending on the concentration of the compound or salt to which the
RTK, CTK or STK is exposed or, more preferably, the inhibition of
the catalytic activity of RTKs, CTKs and STKs.
[0100] The term "catalytic activity" as used herein refers to the
rate of phosphorylation of tyrosine under the influence, direct or
indirect, of RTKs and/or CTKs or the phosphorylation of serine and
threonine under the influence, direct or indirect, of STKs.
[0101] The term "contacting" as used herein refers to bringing a
compound of this invention and a target PK together in such a
manner that the compound can affect the catalytic activity of the
PK, either directly, i.e., by interacting with the kinase itself,
or indirectly, i.e., by interacting with another molecule on which
the catalytic activity of the kinase is dependent. Such
"contacting" can be accomplished in vitro, i.e., in a test tube, a
petri dish or the like. In a test tube, contacting may involve only
a compound and a PK of interest or it may involve whole cells.
Cells may also be maintained or grown in cell culture dishes and
contacted with a compound in that environment. In this context, the
ability of a particular compound to affect a PK related disorder,
i.e., the IC.sub.50 of the compound, defined below, can be
determined before use of the compounds in vivo with more complex
living organisms is attempted. For cells outside the organism,
multiple methods exist, and are well-known to those skilled in the
art, to get the PKs in contact with the compounds including, but
not limited to, direct cell microinjection and numerous
transmembrane carrier techniques.
[0102] "In vitro" refers to procedures performed in an artificial
environment such as, e.g., without limitation, in a test tube or
culture medium. The skilled artisan will understand that, for
example, an isolated PK may be contacted with a modulator in an in
vitro environment. Alternatively, an isolated cell may be contacted
with a modulator in an in vitro environment.
[0103] As used herein, "in vivo" refers to procedures performed
within a living organism such as, without limitation, a mouse, rat,
rabbit, ungulate, bovine, equine, porcine, canine, feline, primate,
or human.
[0104] As used herein, "PK related disorder," "PK driven disorder,"
and "abnormal PK activity" all refer to a condition characterized
by inappropriate, i.e., under or, more commonly, over PK catalytic
activity, where the particular PK can be an RTK, a CTK or an STK.
Inappropriate catalytic activity can arise as the result of either:
(1) PK expression in cells which normally do not express PKs, (2)
increased PK expression leading to unwanted cell proliferation,
differentiation and/or growth, or, (3) decreased PK expression
leading to unwanted reductions in cell proliferation,
differentiation and/or growth. Over-activity of a PK refers to
either amplification of the gene encoding a particular PK or
production of a level of PK activity which can correlate with a
cell proliferation, differentiation and/or growth disorder (that
is, as the level of the PK increases, the severity of one or more
of the symptoms of the cellular disorder increases). Under-activity
is, of course, the converse, wherein the severity of one or more
symptoms of a cellular disorder increase as the level of the PK
activity decreases.
[0105] The term "organism" refers to any living entity comprised of
at least one cell. A living organism can be as simple as, for
example, a single eukaryotic cell or as complex as a mammal,
including a human being.
[0106] The term "therapeutically effective amount" as used herein
refers to that amount of the compound being administered which will
relieve to some extent one or more of the symptoms of the disorder
being treated. In reference to the treatment of cancer, a
therapeutically effective amount refers to that amount which has
the effect of (1) reducing the size of the tumor, (2) inhibiting
(that is, slowing to some extent, preferably stopping) tumor
metastasis, (3) inhibiting to some extent (that is, slowing to some
extent, preferably stopping) tumor growth, and/or, (4) relieving to
some extent (or, preferably, eliminating) one or more symptoms
associated with the cancer.
[0107] "Pharmaceutically acceptable salt" refers to those salts
which retain the biological effectiveness and properties of the
free bases and which are obtained by reaction with inorganic or
organic acids such as hydrochloric acid, hydrobromic acid, sulfuric
acid, nitric acid, phosphoric acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic
acid, citric acid, maleic acid, succinic acid, tartaric acid, and
the like.
[0108] A "pharmaceutical composition" refers to a mixture of one or
more of the compounds described herein, or a pharmaceutically
acceptable salts thereof, with other chemical components, such as
physiologically acceptable carriers and excipients. The purpose of
a pharmaceutical composition is to facilitate administration of a
compound to an organism.
[0109] As used herein, a "pharmaceutically acceptable carrier"
refers to a carrier or diluent that does not cause significant
irritation to an organism and does not abrogate the biological
activity and properties of the administered compound.
[0110] An "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of
a compound. Examples, without limitation, of excipients include
calcium carbonate, calcium phosphate, various sugars and types of
starch, cellulose derivatives, gelatin, vegetable oils and
polyethylene glycols.
[0111] "Treating" or "treatment" of a disease includes preventing
the disease from occurring in an animal that may be predisposed to
the disease but does not yet experience or exhibit symptoms of the
disease (prophylactic treatment), inhibiting the disease (slowing
or arresting its development), providing relief from the symptoms
or side-effects of the disease (including palliative treatment),
and relieving the disease (causing regression of the disease). With
regard to cancer, these terms simply mean that the life expectancy
of an individual affected with a cancer will be increased or that
one or more of the symptoms of the disease will be reduced.
[0112] By "monitoring" is meant observing or detecting the effect
of contacting a compound with a cell expressing a particular PK.
The observed or detected effect can be a change in cell phenotype,
in the catalytic activity of a PK or a change in the interaction of
a PK with a natural binding partner. Techniques for observing or
detecting such effects are well-known in the art. For example, the
catalytic activity of a PK may be observed by determining the rate
or amount of phosphorylation of a target molecule. The
above-referenced effect is selected from a change or an absence of
change in a cell phenotype, a change or absence of change in the
catalytic activity of said protein kinase or a change or absence of
change in the interaction of said protein kinase with a natural
binding partner in a final aspect of this invention.
[0113] "Cell phenotype" refers to the outward appearance of a cell
or tissue or the biological function of the cell or tissue.
Examples, without limitation, of a cell phenotype are cell size,
cell growth, cell proliferation, cell differentiation, cell
survival, apoptosis, and nutrient uptake and use. Such phenotypic
characteristics are measurable by techniques well-known in the
art.
[0114] A "natural binding partner" refers to a polypeptide that
binds to a particular PK in a cell. Natural binding partners can
play a role in propagating a signal in a PK-mediated signal
transduction process. A change in the interaction of the natural
binding partner with the PK can manifest itself as an increased or
decreased concentration of the PK/natural binding partner complex
and, as a result, in an observable change in the ability of the PK
to mediate signal transduction.
General Synthetic Scheme
[0115] The starting materials and reagents used in preparing these
compounds are either available from commercial suppliers such as
Aldrich Chemical Co., (Milwaukee, Wis.), Bachem (Torrance, Calif.),
or Sigma (St. Louis, Mo.) or are prepared by methods known to those
skilled in the art following procedures set forth in references
such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes
1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon
Compounds, Volumes 1-5 and Supplementals (Elsevier Science
Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and
Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and
Sons, 4th Edition) and Larock's Comprehensive Organic
Transformations (VCH Publishers Inc., 1989). These schemes are
merely illustrative of some methods by which the compounds of this
invention can be synthesized, and various modifications to these
schemes can be made and will be suggested to one skilled in the art
having referred to this disclosure. The starting materials and the
intermediates of the reaction may be isolated and purified if
desired using conventional techniques, including but not limited to
filtration, distillation, crystallization, chromatography and the
like. Such materials may be characterized using conventional means,
including physical constants and spectral data. Unless specified to
the contrary, the reactions described herein take place at
atmospheric pressure over a temperature range from about
-78.degree. C. to about 150.degree. C., more preferably from about
0.degree. C. to about 125.degree. C. and most preferably at about
room (or ambient) temperature, e.g., about 20.degree. C.
[0116] Compounds of Formula (I) may be prepared as illustrated and
described below: 9
[0117] A compound of Formula (I) where R.sup.3-R.sup.10 are as
described in the Summary of the Invention can be prepared by
reacting a compound of formula (II) with formaldehyde, acetaldehyde
and pyrrolidine.
[0118] The solvent in which the reaction is carried out may be a
protic or an aprotic solvent, preferably it is a protic solvent
such as an alcohol e.g., methanol or ethanol, or an aqueous
alcohol. The reaction may be carried out at temperatures greater
than room temperature. The temperature is generally from about
20.degree. C. to about 100.degree. C., preferably about 40.degree.
C. to about 80.degree. C. By "about" is meant that the temperature
range is preferably within 10 degrees Celsius of the indicated
temperature, more preferably within 5.degree. C. of the indicated
temperature and, most preferably, within 2 degrees Celsius of the
indicated temperature. Thus, for example, by "about 60.degree. C."
is meant 60.degree. C..+-.10.degree. C., preferably 60.degree.
C..+-.5.degree. C. and most preferably, 60.degree. C..+-.2.degree.
C.
[0119] Compounds of Formula (II) can be prepared by methods well
known in the art. For example, compound (II) where R.sup.3-R.sup.6,
R.sup.7, and R.sup.9 are hydrogen and R.sup.8 and R.sup.10 are
methyl can be prepared by following the procedure described in U.S.
Pat. No. 5,792,783, at column 22, lines 60-67, the disclosure of
which is incorporated herein by reference. Other compounds of
Formula (II) can be prepared as described in U.S. Pat. No.
5,792,783, PCT Application Publication No. WO 99/61422, and U.S.
patent application Ser. No. 09/783,264, filed on Feb. 15, 2001, and
titled "PYRROLE SUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE
INHIBITORS", the disclosures of which are hereby incorporated by
reference.
[0120] The preparation of compounds of Formula (I) may further
include the step of removing a protecting group. "Protecting group"
refers to a group used to render a reactive moiety inert until
removal of the group. Reactive moieties are well known to the
skilled artisan; preferred reactive moieties include reactive
nitrogen, oxygen, sulfur, carboxyl and carbonyl groups. Exemplary
nitrogen protecting groups include, but are not limited to, benzyl,
benzyloxycarbonyl, tert-butoxycarbonyl, silyl groups (e.g.,
tert-butyldimethylsilyl), 9-fluorenylmethoxycarbonyl,
9-phenyl-9-fluorenyl and arylsulfonyl groups (e.g.,
toluenesulfonyl). Exemplary oxygen protecting groups include, but
are not limited to, allyloxycarbonyl, benzoyl, benzyl, tert-butyl,
silyl groups (e.g., tert-butyldimethylsilyl), 2-ethoxyethyl,
p-methoxybenzyl, methoxymethyl, pivaloyl, tetrahydropyran-2-yl and
trityl. Exemplary carboxyl protecting groups include, without
limitation, methyl, allyl, benzyl, silyl groups (e.g.,
tert-butyldimethylsilyl) and p-nitrobenzyl. Exemplary carbonyl
protecting groups include, but are not limited to, acetyl groups
(e.g., O,O-acetals).
[0121] Protecting groups may be removed using methods known in the
literature. For example, for the removal of nitrogen protecting
groups see Greene et al. (1991) Protecting Groups in Organic
Synthesis, 2.sup.nd ed., John Wiley & Sons, New York, pp.
309-405 and Kocienski (1994) Protecting Groups, Thieme, New York,
pp. 185-243. Methods for the removal of particular protecting
groups are exemplified herein.
Utility
[0122] The PKs whose catalytic activity is modulated by the
compounds of this invention include protein tyrosine kinases of
which there are two types, receptor tyrosine kinases (RTKs) and
cellular tyrosine kinases (CTKs), and serine-threonine kinases
(STKs). RTK mediated signal transduction, is initiated by
extracellular interaction with a specific growth factor (ligand),
followed by receptor dimerization, transient stimulation of the
intrinsic protein tyrosine kinase activity and phosphorylation.
Binding sites are thereby created for intracellular signal
transduction molecules and lead to the formation of complexes with
a spectrum of cytoplasmic signaling molecules that facilitate the
appropriate cellular response (e.g., cell division, metabolic
effects on the extracellular microenvironment, etc.). See,
Schlessinger and Ullrich, 1992, Neuron 9:303-391.
[0123] It has been shown that tyrosine phosphorylation sites on
growth factor receptors function as high-affinity binding sites for
SH2 (src homology) domains of signaling molecules. Fantl et al.,
1992, Cell 69:413-423, Songyang et al., 1994, Mol. Cell. Biol.
14:2777-2785), Songyang et al., 1993, Cell 72:767-778, and Koch et
al., 1991, Science 252:668-678. Several intracellular substrate
proteins that associate with RTKs have been identified. They may be
divided into two principal groups: (1) substrates that have a
catalytic domain, and (2) substrates which lack such domain but
which serve as adapters and associate with catalytically active
molecules. Songyang et al., 1993, Cell 72:767-778. The specificity
of the interactions between receptors and SH2 domains of their
substrates is determined by the amino acid residues immediately
surrounding the phosphorylated tyrosine residue. Differences in the
binding affinities between SH2 domains and the amino acid sequences
surrounding the phosphotyrosine residues on particular receptors
are consistent with the observed differences in their substrate
phosphorylation profiles. Songyang et al., 1993, Cell 72:767-778.
These observations suggest that the function of each RTK is
determined not only by its pattern of expression and ligand
availability but also by the array of downstream signal
transduction pathways that are activated by a particular receptor.
Thus, phosphorylation provides an important regulatory step which
determines the selectivity of signaling pathways recruited by
specific growth factor receptors, as well as differentiation factor
receptors.
[0124] STKs, being primarily cytosolic, affect the internal
biochemistry of the cell, often as a down-line response to a PTK
event. STKs have been implicated in the signaling process which
initiates DNA synthesis and subsequent mitosis leading to cell
proliferation.
[0125] Thus, PK signal transduction results in, among other
responses, cell proliferation, differentiation, growth and
metabolism. Abnormal cell proliferation may result in a wide array
of disorders and diseases, including the development of neoplasia
such as carcinoma, sarcoma, glioblastoma and hemangioma, disorders
such as leukemia, psoriasis, arteriosclerosis, arthritis and
diabetic retinopathy and other disorders related to uncontrolled
angiogenesis and/or vasculogenesis.
[0126] In another aspect, the protein kinase, the catalytic
activity of which is modulated by contact with a compound of this
invention, is a protein tyrosine kinase, more particularly, a
receptor protein tyrosine kinase. Among the receptor protein
tyrosine kinases whose catalytic activity can be modulated with a
compound of this invention, or salt thereof, are, without
limitation, EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFR.alpha.,
PDGFR.beta., CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1,
FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
[0127] The protein tyrosine kinase whose catalytic activity is
modulated by contact with a compound of this invention, or a salt
thereof, can also be a non-receptor or cellular protein tyrosine
kinase (CTK). Thus, the catalytic activity of CTKs such as, without
limitation, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak,
Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may be modulated by
contact with a compound or salt of this invention.
[0128] Still another group of PKs which may have their catalytic
activity modulated by contact with a compound of this invention are
the serine-threonine protein kinases such as, without limitation,
CDK2 and Raf.
[0129] In another aspect, this invention relates to a method for
treating or preventing a PK related disorder by administering a
therapeutically effective amount of a compound of this invention,
or a salt thereof, to an organism.
[0130] It is also an aspect of this invention that a pharmaceutical
composition containing a compound of this invention, or a salt
thereof, is administered to an organism for the purpose of
preventing or treating a PK related disorder.
[0131] This invention is therefore directed to compounds that
modulate PK signal transduction by affecting the enzymatic activity
of RTKs, CTKs and/or STKs, thereby interfering with the signals
transduced by such proteins. More particularly, the present
invention is directed to compounds which modulate RTK, CTK and/or
STK mediated signal transduction pathways as a therapeutic approach
to cure many kinds of solid tumors, including but not limited to
carcinomas, sarcomas including Kaposi's sarcoma, erythroblastoma,
glioblastoma, meningioma, astrocytoma, melanoma and myoblastoma.
Treatment or prevention of non-solid tumor cancers such as leukemia
are also contemplated by this invention. Indications may include,
but are not limited to brain cancers, bladder cancers, ovarian
cancers, gastric cancers, pancreatic cancers, colon cancers, blood
cancers, lung cancers and bone cancers.
[0132] Further examples, without limitation, of the types of
disorders related to inappropriate PK activity that the compounds
described herein may be useful in preventing, treating and
studying, are cell proliferative disorders, fibrotic disorders,
metabolic disorders and infectious diseases.
[0133] Cell proliferative disorders, which may be prevented,
treated or further studied by the present invention include cancer,
blood vessel proliferative disorders and mesangial cell
proliferative disorders.
[0134] Blood vessel proliferative disorders refer to disorders
related to abnormal vasculogenesis (blood vessel formation) and
angiogenesis (spreading of blood vessels). While vasculogenesis and
angiogenesis play important roles in a variety of normal
physiological processes such as embryonic development, corpus
luteum formation, wound healing and organ regeneration, they also
play a pivotal role in cancer development where they result in the
formation of new capillaries needed to keep a tumor alive. Other
examples of blood vessel proliferation disorders include arthritis,
where new capillary blood vessels invade the joint and destroy
cartilage, and ocular diseases, like diabetic retinopathy, where
new capillaries in the retina invade the vitreous, bleed and cause
blindness.
[0135] Two structurally related RTKs have been identified to bind
VEGF with high affinity: the fms-like tyrosine 1 (fit-1) receptor
(Shibuya et al., 1990, Oncogene, 5:519-524; De Vries et al., 1992,
Science, 255:989-991) and the KDR/FLK-1 receptor, also known as
VEGF-R2. Vascular endothelial growth factor (VEGF) has been
reported to be an endothelial cell specific mitogen with in vitro
endothelial cell growth promoting activity. Ferrara & Henzel,
1989, Biochem. Biophys. Res. Comm., 161:851-858; Vaisman et al.,
1990, J. Biol. Chem., 265:19461-19566. Information set forth in
U.S. application Ser. Nos. 08/193,829, 08/038,596 and 07/975,750,
strongly suggest that VEGF is not only responsible for endothelial
cell proliferation, but also is the prime regulator of normal and
pathological angiogenesis. See generally, Klagsburn & Soker,
1993, Current Biology, 3(10)699-702; Houck, et al., 1992, J. Biol.
Chem., 267:26031-26037.
[0136] Normal vasculogenesis and angiogenesis play important roles
in a variety of physiological processes such as embryonic
development, wound healing, organ regeneration and female
reproductive processes such as follicle development in the corpus
luteum during ovulation and placental growth after pregnancy.
Folkman & Shing, 1992, J. Biological Chem., 267(16):10931-34.
Uncontrolled vasculogenesis and/or angiogenesis has been associated
with diseases such as diabetes as well as with malignant solid
tumors that rely on vascularization for growth. Klagsburn &
Soker, 1993, Current Biology, 3(10):699-702; Folkham, 1991, J.
Natl. Cancer Inst., 82:4-6; Weidner, et al., 1991, New Engl. J.
Med., 324:1-5.
[0137] As presently understood, the role of VEGF in endothelial
cell proliferation and migration during angiogenesis and
vasculogenesis indicates an important role for the KDR/FLK-1
receptor in these processes. Diseases such as diabetes mellitus
(Folkman, 198, in XIth Congress of Thrombosis and Haemostasis
(Verstraeta, et al., eds.), pp. 583-596, Leuven University Press,
Leuven) and arthritis, as well as malignant tumor growth may result
from uncontrolled angiogenesis. See e.g., Folkman, 1971, N. Engl.
J. Med., 285:1182-1186. The receptors to which VEGF specifically
binds are an important and powerful therapeutic target for the
regulation and modulation of vasculogenesis and/or angiogenesis and
a variety of severe diseases which involve abnormal cellular growth
caused by such processes. Plowman, et al., 1994, DN&P,
7(6):334-339. More particularly, the KDR/FLK-1 receptor's highly
specific role in neovascularization make it a choice target for
therapeutic approaches to the treatment of cancer and other
diseases which involve the uncontrolled formation of blood
vessels.
[0138] Thus, one aspect of the present invention relates to
compounds capable of regulating and/or modulating tyrosine kinase
signal transduction including KDR/FLK-1 receptor signal
transduction in order to inhibit or promote angiogenesis and/or
vasculogenesis, that is, compounds that inhibit, prevent, or
interfere with the signal transduced by KDR/FLK-1 when activated by
ligands such as VEGF. Although it is believed that the compounds of
the present invention act on a receptor or other component along
the tyrosine kinase signal transduction pathway, they may also act
directly on the tumor cells that result from uncontrolled
angiogenesis.
[0139] Although the nomenclature of the human and murine
counterparts of the generic "flk-I" receptor differ, they are, in
many respects, interchangeable. The murine receptor, Flk-1, and its
human counterpart, KDR, share a sequence homology of 93.4% within
the intracellular domain. Likewise, murine FLK-I binds human VEGF
with the same affinity as mouse VEGF, and accordingly, is activated
by the ligand derived from either species. Millauer et al., 1993,
Cell, 72:835-846; Quinn et al., 1993, Proc. Natl. Acad. Sci. USA,
90:7533-7537. FLK-1 also associates with and subsequently tyrosine
phosphorylates human RTK substrates (e.g., PLC-.gamma. or p85) when
co-expressed in 293 cells (human embryonal kidney fibroblasts).
[0140] Models which rely upon the FLK-1 receptor therefore are
directly applicable to understanding the KDR receptor. For example,
use of the murine FLK-1 receptor in methods which identify
compounds that regulate the murine signal transduction pathway are
directly applicable to the identification of compounds which may be
used to regulate the human signal transduction pathway, that is,
which regulate activity related to the KDR receptor. Thus, chemical
compounds identified as inhibitors of KDR/FLK-1 in vitro, can be
confirmed in suitable in vivo models. Both in vivo mouse and rat
animal models have been demonstrated to be of excellent value for
the examination of the clinical potential of agents acting on the
KDR/FLK-1 induced signal transduction pathway.
[0141] Thus, in one aspect, this invention is directed to compounds
that regulate, modulate and/or inhibit vasculogenesis and/or
angiogenesis by affecting the enzymatic activity of the KDR/FLK-1
receptor and interfering with the signal transduced by KDR/FLK-1.
In another aspect, the present invention is directed to compounds
which regulate, modulate and/or inhibit the KDR/FLK-1 mediated
signal transduction pathway as a therapeutic approach to the
treatment of many kinds of solid tumors including, but not limited
to, glioblastoma, melanoma and Kaposi's sarcoma, and ovarian, lung,
mammary, prostate, pancreatic, colon and epidermoid carcinoma. In
addition, data suggest the administration of compounds which
inhibit the KDR/Flk-1 mediated signal transduction pathway may also
be used in the treatment of hemangioma, restenois and diabetic
retinopathy.
[0142] A further aspect of this invention relates to the inhibition
of vasculogenesis and angiogenesis by other receptor-mediated
pathways, including the pathway comprising the flt-1 receptor.
[0143] Receptor tyrosine kinase mediated signal transduction is
initiated by extracellular interaction with a specific growth
factor (ligand), followed by receptor dimerization, transient
stimulation of the intrinsic protein tyrosine kinase activity and
autophosphorylation. Binding sites are thereby created for
intracellular signal transduction molecules which leads to the
formation of complexes with a spectrum of cytoplasmic signaling
molecules that facilitate the appropriate cellular response, e.g.,
cell division and metabolic effects to the extracellular
microenvironment. See, Schlessinger and Ullrich, 1992, Neuron,
9:1-20.
[0144] The close homology of the intracellular regions of KDR/FLK-1
with that of the PDGF-.beta. receptor (50.3% homology) and/or the
related flt-1 receptor indicates the induction of overlapping
signal transduction pathways. For example, for the PDGF-.beta.
receptor, members of the src family (Twamley et al., 1993, Proc.
Natl. Acad. Sci. USA, 90:7696-7700), phosphatidylinositol-3'-kinase
(Hu et al., 1992, Mol. Cell. Biol., 12:981-990), phospholipase
c.gamma. (Kashishian & Cooper, 1993, Mol. Cell. Biol.,
4:49-51), ras-GTPase-activating protein, (Kashishian et al., 1992,
EMBO J., 11:1373-1382), PTP-ID/syp (Kazlauskas et al., 1993, Proc.
Natl. Acad. Sci. USA, 10 90:6939-6943), Grb2 (Arvidsson et al.,
1994, Mol. Cell. Biol., 14:6715-6726), and the adapter molecules
Shc and Nck (Nishimura et al., 1993, Mol. Cell. Biol.,
13:6889-6896), have been shown to bind to regions involving
different autophosphorylation sites. See generally, Claesson-Welsh,
1994, Prog. Growth Factor Res., 5:37-54. Thus, it is likely that
signal transduction pathways activated by KDR/FLK-1 include the ras
pathway (Rozakis et al., 1992, Nature, 360:689-692), the
PI-3'-kinase, the src-mediated and the plc.gamma.-mediated
pathways. Each of these pathways may play a critical role in the
angiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelial
cells. Consequently, a still further aspect of this invention
relates to the use of the organic compounds described herein to
modulate angiogenesis and vasculogenesis as such processes are
controlled by these pathways.
[0145] Conversely, disorders related to the shrinkage, contraction
or closing of blood vessels, such as restenosis, are also
implicated and may be treated or prevented by the methods of this
invention.
[0146] Fibrotic disorders refer to the abnormal formation of
extracellular matrices. Examples of fibrotic disorders include
hepatic cirrhosis and mesangial cell proliferative disorders.
Hepatic cirrhosis is characterized by the increase in extracellular
matrix constituents resulting in the formation of a hepatic scar.
An increased extracellular matrix resulting in a hepatic scar can
also be caused by a viral infection such as hepatitis. Lipocytes
appear to play a major role in hepatic cirrhosis. Other fibrotic
disorders implicated include atherosclerosis.
[0147] Mesangial cell proliferative disorders refer to disorders
brought about by abnormal proliferation of mesangial cells.
Mesangial proliferative disorders include various human renal
diseases such as glomerulonephritis, diabetic nephropathy and
malignant nephrosclerosis as well as such disorders as thrombotic
microangiopathy syndromes, transplant rejection, and
glomerulopathies. The RTK PDGFR has been implicated in the
maintenance of mesangial cell proliferation. Floege et al., 1993,
Kidney International 43:47S-54S.
[0148] Many cancers are cell proliferative disorders and, as noted
previously, PKs have been associated with cell proliferative
disorders. Thus, it is not surprising that PKs such as, for
example, members of the RTK family have been associated with the
development of cancer. Some of these receptors, like EGFR (Tuzi et
al., 1991, Br. J. Cancer 63:227-233, Torp et al, 1992, APMIS
100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712)
and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) are
over-expressed in many tumors and/or persistently activated by
autocrine loops. In fact, in the most common and severe cancers
these receptor over-expressions (Akbasak and Suner-Akbasak et al.,
1992, J. Neurol. Sci., 111: 119-133, Dickson et al., 1992, Cancer
Treatment Res. 61:249-273, Korc et al., 1992, J. Clin. Invest.
90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, J. Cell.
Biol., 118:1057-1070, Korc et al., supra, Akbasak and Suner-Akbasak
et al., supra) have been demonstrated. For example, EGFR has been
associated with squamous cell carcinoma, astrocytoma, glioblastoma,
head and neck cancer, lung cancer and bladder cancer. HER2 has been
associated with breast, ovarian, gastric, lung, pancreatic and
bladder cancer. PDGFR has been associated with glioblastoma and
melanoma as well as lung, ovarian and prostate cancer. The RTK
c-met has also been associated with malignant tumor formation. For
example, c-met has been associated with, among other cancers,
colorectal, thyroid, pancreatic, gastric and hepatocellular
carcinomas and lymphomas. Additionally c-met has been linked to
leukemia. Over-expression of the c-met gene has also been detected
in patients with Hodgkins disease and Burkitts disease.
[0149] IGF-IR, in addition to being implicated in nutritional
support and in type-II diabetes, has also been associated with
several types of cancers. For example, IGF-I has been implicated as
an autocrine growth stimulator for several tumor types, e.g. human
breast cancer carcinoma cells (Arteaga et al., 1989, J. Clin.
Invest. 84:1418-1423) and small lung tumor cells (Macauley et al.,
1990, Cancer Res., 50:2511-2517). In addition, IGF-I, while
integrally involved in the normal growth and differentiation of the
nervous system, also appears to be an autocrine stimulator of human
gliomas. Sandberg-Nordqvist et al., 1993, Cancer Res. 53:2475-2478.
The importance of IGF-IR and its ligands in cell proliferation is
further supported by the fact that many cell types in culture
(fibroblasts, epithelial cells, smooth muscle cells, T-lymphocytes,
myeloid cells, chondrocytes and osteoblasts (the stem cells of the
bone marrow)) are stimulated to grow by IGF-I. Goldring and
Goldring, 1991, Eukaryotic Gene Expression, 1:301-326. In a series
of recent publications, Baserga suggests that IGF-IR plays a
central role in the mechanism of transformation and, as such, could
be a preferred target for therapeutic interventions for a broad
spectrum of human malignancies. Baserga, 1995, Cancer Res.,
55:249-252, Baserga, 1994, Cell 79:927-930, Coppola et al., 1994,
Mol. Cell. Biol., 14:4588-4595.
[0150] STKs have been implicated in many types of cancer including,
notably, breast cancer (Cance, et al., Int. J. Cancer, 54:571-77
(1993)).
[0151] The association between abnormal PK activity and disease is
not restricted to cancer. For example, RTKs have been associated
with diseases such as psoriasis, diabetes mellitus, endometriosis,
angiogenesis, atheromatous plaque development, Alzheimer's disease,
von Hippel-Lindau disease, epidermal hyperproliferation,
neurodegenerative diseases, age-related macular degeneration and
hemangiomas. For example, EGFR has been indicated in corneal and
dermal wound healing. Defects in Insulin-R and IGF-1R are indicated
in type-II diabetes mellitus. A more complete correlation between
specific RTKs and their therapeutic indications is set forth in
Plowman et al., 1994, DN&P 7:334-339.
[0152] As noted previously, CTKs including, but not limited to,
src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed
by Bolen et al., 1992, FASEB J., 6:3403-3409), are also involved in
the proliferative and metabolic signal transduction pathway and
thus could be expected, and have been shown, to be involved in many
PTK-mediated disorders to which the present invention is directed.
For example, mutated src (v-src) has been shown to be an
oncoprotein (pp60.sup.v-src) in chicken. Moreover, its cellular
homolog, the proto-oncogene pp60.sup.c-src transmits oncogenic
signals of many receptors. Over-expression of EGFR or HER2/neu in
tumors leads to the constitutive activation of pp60.sup.c-src,
which is characteristic of malignant cells but absent in normal
cells. On the other hand, mice deficient in the expression of c-src
exhibit an osteopetrotic phenotype, indicating a key participation
of c-src in osteoclast function and a possible involvement in
related disorders.
[0153] Similarly, Zap70 has been implicated in T-cell signaling
which may relate to autoimmune disorders.
[0154] STKs have been associated with inflammation, autoimmune
disease, immunoresponses, and hyperproliferation disorders such as
restenosis, fibrosis, psoriasis, osteoarthritis and rheumatoid
arthritis.
[0155] PKs have also been implicated in embryo implantation. Thus,
the compounds of this invention may provide an effective method of
preventing such embryo implantation and thereby be useful as birth
control agents.
[0156] In yet another aspect, the compounds of the instant
invention can also be used as anti-infective agents. For example,
indolinone compounds are known to exhibit antibacterial and
antifungal activities. See, e.g., Singh and Jha (1989) "Indolinone
derivatives as potential antimicrobial agents," ZentralbL
Mikrobiol. 144(2):105-109. In addition, indolinone compounds have
been reported to exhibit significant antiviral activity. See, e.g.,
Maass et al. (1993) "Viral resistance to the
thiazolo-iso-indolinones, a new class of normucleoside inhibitors
of human immunodeficiency virus type 1 reverse transcriptase,"
Antimicrob. Agents Chemother. 37(12):2612-2617.
[0157] Finally, both RTKs and CTKs are currently suspected as being
involved in hyperimmune disorders.
[0158] A method for identifying a chemical compound that modulates
the catalytic activity of one or more of the above discussed
protein kinases is another aspect of this invention. The method
involves contacting cells expressing the desired protein kinase
with a compound of this invention (or its salt) and monitoring the
cells for any effect that the compound has on them. The effect may
be any observable, either to the naked eye or through the use of
instrumentation, change or absence of change in a cell phenotype.
The change or absence of change in the cell phenotype monitored may
be, for example, without limitation, a change or absence of change
in the catalytic activity of the protein kinase in the cells or a
change or absence of change in the interaction of the protein
kinase with a natural binding partner.
Pharmaceutical Compositions and Administration
[0159] A compound of the present invention or a physiologically
acceptable salt thereof, can be administered as such to a human
patient or can be administered in pharmaceutical compositions in
which the foregoing materials are mixed with suitable carriers or
excipient(s). Techniques for formulation and administration of
drugs may be found in "Remington's Pharmacological Sciences," Mack
Publishing Co., Easton, Pa., latest edition.
[0160] Routes of Administration
[0161] As used herein, "administer" or "administration" refers to
the delivery of a compound or salt of the present invention or of a
pharmaceutical composition containing a compound or salt of this
invention to an organism for the purpose of prevention or treatment
of a PK-related disorder.
[0162] Suitable routes of administration may include, without
limitation, oral, rectal, transmucosal or intestinal administration
or intramuscular, subcutaneous, intramedullary, intrathecal, direct
intraventricular, intravenous, intravitreal, intraperitoneal,
intranasal, or intraocular injections. The preferred routes of
administration are oral and parenteral.
[0163] Alternatively, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a solid tumor, often in a depot or sustained
release formulation.
[0164] Furthermore, one may administer the drug in a targeted drug
delivery system, for example, in a liposome coated with
tumor-specific antibody. The liposomes will be targeted to and
taken up selectively by the tumor.
[0165] Composition/Formulation
[0166] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, lyophilizing
processes or spray drying.
[0167] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0168] For injection, the compounds of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such buffers with or without a low concentration
of surfactant or cosolvent, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0169] For oral administration, the compounds can be formulated by
combining the active compounds with pharmaceutically acceptable
carriers well known in the art. Such carriers enable the compounds
of the invention to be formulated as tablets, pills, lozenges,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient. Pharmaceutical
preparations for oral use can be made using a solid excipient,
optionally grinding the resulting mixture, and processing the
mixture of granules, after adding other suitable auxiliaries if
desired, to obtain tablets or dragee cores. Useful excipients are,
in particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol, cellulose preparations such as, for example,
maize starch, wheat starch, rice starch and potato starch and other
materials such as gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinyl-pyrrolidone (PVP). If desired, disintegrating
agents may be added, such as cross-linked polyvinyl pyrrolidone,
agar, or alginic acid. A salt such as sodium alginate may also be
used.
[0170] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0171] Pharmaceutical compositions which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with a filler such as lactose, a binder such as starch,
and/or a lubricant such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, liquid polyethylene glycols, cremophor, capmul,
medium or long chain mono- di- or triglycerides. Stabilizers may be
added in these formulations, also.
[0172] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray using a pressurized pack or a
nebulizer and a suitable propellant, e.g., without limitation,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetra-fluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be controlled by providing
a valve to deliver a metered amount. Capsules and cartridges of,
for example, 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.
[0173] The compounds may also be formulated for parenteral
administration, e.g., by bolus injection or continuous infusion.
Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or in multi-dose containers, with an added
preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulating materials such as suspending, stabilizing and/or
dispersing agents.
[0174] Pharmaceutical compositions for parenteral administration
include aqueous solutions of a water soluble form, such as, without
limitation, a salt, of the active compound. Additionally,
suspensions of the active compounds may be prepared in a lipophilic
vehicle. Suitable lipophilic vehicles include fatty oils such as
sesame oil, synthetic fatty acid esters such as ethyl oleate and
triglycerides, or materials such as liposomes. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain suitable
stabilizers and/or agents that increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions.
[0175] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water with or without aditional sufactants or
cosolvents such as polysorbate 80, Cremophor, cyclodextrin
sulfobutyl ether, propylene glycol or polyethylene glycol such as
PEG-300 or PEG-400, before use.
[0176] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, using, e.g.,
conventional suppository bases such as cocoa butter or other
glycerides.
[0177] In addition to the formulations described previously, the
compounds may also be formulated as depot preparations. Such long
acting formulations may be administered by implantation (for
example, subcutaneously or intramuscularly) or by intramuscular
injection. A compound of this invention may be formulated for this
route of administration with suitable polymeric or hydrophobic
materials (for instance, in an emulsion with a pharmacologically
acceptable oil), with ion exchange resins, or as a sparingly
soluble derivative such as, without limitation, a sparingly soluble
salt.
[0178] A non-limiting example of a pharmaceutical carrier for the
hydrophobic compounds of the invention is a cosolvent system
comprising benzyl alcohol, a nonpolar surfactant, a water-miscible
organic polymer and an aqueous phase such as the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant Polysorbate 80.TM., and 65% w/v polyethylene
glycol 300, made up to volume in absolute ethanol. The VPD
co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a 5%
dextrose in water solution. This co-solvent system dissolves
hydrophobic compounds well, and itself produces low toxicity upon
systemic administration. Naturally, the proportions of such a
co-solvent system may be varied considerably without destroying its
solubility and toxicity characteristics. Furthermore, the identity
of the co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
Polysorbate 80.TM., the fraction size of polyethylene glycol may be
varied, other biocompatible polymers may replace polyethylene
glycol, e.g., polyvinyl pyrrolidone, and other sugars or
polysaccharides may substitute for dextrose.
[0179] Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds may be employed. Liposomes and emulsions
are well known examples of delivery vehicles or carriers for
hydrophobic drugs. In addition, certain organic solvents such as
dimethylsulfoxide also may be employed, although often at the cost
of greater toxicity.
[0180] Additionally, the compounds may be delivered using a
sustained-release system, such as semipermeable matrices of solid
hydrophobic polymers containing the therapeutic agent. Various
sustained-release materials have been established and are well
known by those skilled in the art. Sustained-release capsules may,
depending on their chemical nature, release the compounds for a few
weeks up to over 100 days. Depending on the chemical nature and the
biological stability of the therapeutic reagent, additional
strategies for protein stabilization may be employed.
[0181] The pharmaceutical compositions herein also may comprise
suitable solid or gel phase carriers or excipients. Examples of
such carriers or excipients include, but are not limited to,
calcium carbonate, calcium phosphate, various sugars, starches,
cellulose derivatives, gelatin, and polymers such as polyethylene
glycols.
[0182] Many of the PK modulating compounds of the invention may be
provided as physiologically acceptable salts wherein the claimed
compound may form the negatively or the positively charged species.
Examples of salts in which the compound forms the positively
charged moiety include, without limitation, quaternary ammonium
(defined elsewhere herein), salts such as the hydrochloride,
sulfate, citrate, mesylate, lactate, tartrate, maleate, succinate
wherein the nitrogen atom of the quaternary ammonium group is a
nitrogen of the selected compound of this invention which has
reacted with the appropriate acid. Salts in which a compound of
this invention forms the negatively charged species include,
without limitation, the sodium, potassium, calcium and magnesium
salts formed by the reaction of a carboxylic acid group in the
compound with an appropriate base (e.g. sodium hydroxide (NaOH),
potassium hydroxide (KOH), Calcium hydroxide (Ca(OH).sub.2),
etc.).
[0183] Dosage
[0184] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an amount sufficient to achieve the intended purpose,
i.e., the modulation of PK activity or the treatment or prevention
of a PK-related disorder.
[0185] More specifically, a therapeutically effective amount means
an amount of compound effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being
treated. Therapeutically effective amounts of compounds of Formula
I may range from approximately 10 mg/m.sup.2 to 400 mg/m.sup.2,
preferably 50 mg/m.sup.2 to 300 mg/m.sup.2, more preferably 100
mg/m.sup.2 to 220 mg/m.sup.2, even more preferably 195
mg/m.sup.2.
[0186] For any compound used in the methods of the invention, the
therapeutically effective amount or dose can be estimated initially
from cell culture assays. Then, the dosage can be formulated for
use in animal models so as to achieve a circulating concentration
range that includes the IC.sub.50 as determined in cell culture
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of the PK activity). Such information can
then be used to more accurately determine useful doses in
humans.
[0187] Toxicity and therapeutic efficacy of the compounds described
herein can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., by determining the
IC.sub.50 and the LD.sub.50 (both of which are discussed elsewhere
herein) for a subject compound. The data obtained from these cell
culture assays and animal studies can be used in formulating a
range of dosage for use in humans. The dosage may vary depending
upon the dosage form employed and the route of administration
utilized. The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological
Basis of Therapeutics", Ch. 1 p. 1).
[0188] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active species which are sufficient to
maintain the kinase modulating effects. These plasma levels are
referred to as minimal effective concentrations (MECs). The MEC
will vary for each compound but can be estimated from in vitro
data, e.g., the concentration necessary to achieve 50-90%
inhibition of a kinase may be ascertained using the assays
described herein. Preferably, the Dosages necessary to achieve the
MEC will depend on individual characteristics and route of
administration. HPLC assays or bioassays can be used to determine
plasma concentrations.
[0189] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen that maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration and other procedures known in the art may be employed
to determine the correct dosage amount and interval.
[0190] The amount of a composition administered will, of course, be
dependent on the subject being treated, the severity of the
affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0191] Packaging
[0192] The compositions may, if desired, be presented in a pack or
dispenser device, such as an FDA approved kit, which may contain
one or more unit dosage forms containing the active ingredient. The
pack may for example comprise metal or plastic foil, such as a
blister pack. The pack or dispenser device may be accompanied by
instructions for administration. The pack or dispenser may also be
accompanied by a notice associated with the container in a form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals, which notice is reflective of approval
by the agency of the form of the compositions or of human or
veterinary administration. Such notice, for example, may be of the
labeling approved by the U.S. Food and Drug Administration for
prescription drugs or of an approved product insert. Compositions
comprising a compound of the invention formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated
condition. Suitable conditions indicated on the label may include
treatment of a tumor, inhibition of angiogenesis, treatment of
fibrosis, diabetes, and the like.
[0193] It is also an aspect of this invention that a compound
described herein, or its salt, might be combined with other
chemotherapeutic agents for the treatment of the diseases and
disorders discussed above. For instance, a compound or salt of this
invention might be combined with alkylating agents such as
fluorouracil (5-FU) alone or in further combination with
leukovorin; or other alkylating agents such as, without limitation,
other pyrimidine analogs such as UFT, capecitabine, gemcitabine and
cytarabine, the alkyl sulfonates, e.g., busulfan (used in the
treatment of chronic granulocytic leukemia), improsulfan and
piposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa and
uredepa; ethyleneimines and methylmelamines, e.g., altretamine,
triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphorami- de and trimethylolmelamine; and the
nitrogen mustards, e.g., chlorambucil (used in the treatment of
chronic lymphocytic leukemia, primary macroglobulinemia and
non-Hodgkin's lymphoma), cyclophosphamide (used in the treatment of
Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer,
ovarian cancer, lung cancer, Wilm's tumor and rhabdomyosarcoma),
estramustine, ifosfamide, novembrichin, prednimustine and uracil
mustard (used in the treatment of primary thrombocytosis,
non-Hodgkin's lymphoma, Hodgkin's disease and ovarian cancer); and
triazines, e.g., dacarbazine (used in the treatment of soft tissue
sarcoma).
[0194] Likewise a compound or salt of this invention might be
expected to have a beneficial effect in combination with other
antimetabolite chemotherapeutic agents such as, without limitation,
folic acid analogs, e.g. methotrexate (used in the treatment of
acute lymphocytic leukemia, choriocarcinoma, mycosis fungiodes
breast cancer, head and neck cancer and osteogenic sarcoma) and
pteropterin; and the purine analogs such as mercaptopurine and
thioguanine which find use in the treatment of acute granulocytic,
acute lymphocytic and chronic granulocytic leukemias.
[0195] A compound or salt of this invention might also be expected
to prove efficacious in combination with natural product based
chemotherapeutic agents such as, without limitation, the vinca
alkaloids, e.g., vinblastin (used in the treatment of breast and
testicular cancer), vincristine and vindesine; the
epipodophylotoxins, e.g., etoposide and teniposide, both of which
are useful in the treatment of testicular cancer and Kaposi's
sarcoma; the antibiotic chemotherapeutic agents, e.g.,
daunorubicin, doxorubicin, epirubicin, mitomycin (used to treat
stomach, cervix, colon, breast, bladder and pancreatic cancer),
dactinomycin, temozolomide, plicamycin, bleomycin (used in the
treatment of skin, esophagus and genitourinary tract cancer); and
the enzymatic chemotherapeutic agents such as L-asparaginase.
[0196] In addition to the above, a compound or salt of this
invention might be expected to have a beneficial effect used in
combination with the platinum coordination complexes (cisplatin,
etc.); substituted ureas such as hydroxyurea; methylhydrazine
derivatives, e.g., procarbazine; adrenocortical suppressants, e.g.,
mitotane, aminoglutethimide; and hormone and hormone antagonists
such as the adrenocorticosteriods (e.g., prednisone), progestins
(e.g., hydroxyprogesterone caproate); estrogens (e.g.,
diethylstilbesterol); antiestrogens such as tamoxifen; androgens,
e.g., testosterone propionate; and aromatase inhibitors (such as
anastrozole).
[0197] Finally, the combination of a compound of this invention
might be expected to be particularly effective in combination with
CAMPTOSAR.TM., GLEEVEC.TM., HERCEPTIN.TM., ENDOSTATIN.TM., Cox-2
inhibitors, MITOXANTRONE.TM. or PACLITAXEL.TM. for the treatment of
solid tumor cancers or leukemias such as, without limitation, acute
myelogenous (non-lymphocytic) leukemia.
EXAMPLES
[0198] The following preparations and examples are given to enable
those skilled in the art to more clearly understand and to practice
the present invention. They should not be considered as limiting
the scope of the invention, but merely as being illustrative and
representative thereof.
[0199] In general HPLC data was obtained with a Zorbax SB C18
column (4.6 mm ID.times.7.5 cm), a Perkin Elmer series 200 pump
programmed to run from 10% acetonitrile/water 0.1% TFA (solvent A)
to 90% acetonitrile/water (solvent B) with a flow rate of 1.5
mL/min. After 0.1 min on solvent A, a 5 min linear program to
solvent B was run, followed by 3 min on solvent B, before recycling
to solvent A (2 min). Detection was with a Perkin Elmer diode array
detector recording at 215 and 280 nM). NMR spectra were recorded on
a Bruker instrument at 300 MHz.
Synthetic Examples
Example 1
(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl-
)-1,3-dihydro-2H-indol-2-one
[0200] Pyrrolidine (450 mg, 6.3 mmol) was added to a stirred
solution of aqueous formaldehyde (500 mg of 38% solution, 6.0 mmol)
and
3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one
(900 mg, 3.8 mmol) in methanol (50 mL). After 15 min., the solution
was cooled to 0.degree. C. and the precipitate was filtered off,
washed with water, and dried to give 1.08 g of the title compound,
mp 129-132.degree. C. HPLC Rt 4.87 min. .sup.1H NMR
[(CD.sub.3).sub.2SO] .delta. 1.65 (m, 4H), 2.32 9s, 3H), 2.34 (s,
3H), 2.62 (m, 4H), 4.72 (s, 2H) 6.07 (d, 1H), 7.00 (m, 1H), 7.15
(m, 2H), 7.61 (s, 1H), 7.76 (d, 2H) and 13.1 (br s, 1H). Anal.
Calcd for C.sub.20H.sub.23N.sub.3O: C, 74.74; H, 7.21; N, 13.07.
Found: C, 74.61; H, 7.25; N, 13.03.
Biological Evaluation
[0201] It will be appreciated that, in any given series of
compounds, a range of biological activities will be observed. In
its presently preferred embodiments, this invention relates to
novel 1-substituted-3-pyrrolidinyl-2-indolinones capable of
generating in vivo 3-pyrrolidinyl -2-indolinones capable of
modulating, regulating and/or inhibiting protein kinase activity.
The following assays may be employed to select those compounds
demonstrating the optimal degree of the desired activity.
[0202] Assay Procedures
[0203] The following in vitro assays may be used to determine the
level of activity and effect of the different compounds of the
present invention on one or more of the PKs. Similar assays can be
designed along the same lines for any PK using techniques well
known in the art.
[0204] Several of the assays described herein are performed in an
ELISA (Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller,
et al., 1980, "Enzyme-Linked Immunosorbent Assay," Manual of
Clinical Immunology, 2d ed., Rose and Friedman, Am. Soc. Of
Microbiology, Washington, D.C., pp. 359-371). The general procedure
is as follows: a compound is introduced to cells expressing the
test kinase, either naturally or recombinantly, for a selected
period of time after which, if the test kinase is a receptor, a
ligand known to activate the receptor is added. The cells are lysed
and the lysate is transferred to the wells of an ELISA plate
previously coated with a specific antibody recognizing the
substrate of the enzymatic phosphorylation reaction. Non-substrate
components of the cell lysate are washed away and the amount of
phosphorylation on the substrate is detected with an antibody
specifically recognizing phosphotyrosine compared with control
cells that were not contacted with a test compound.
[0205] The presently preferred protocols for conducting the ELISA
experiments for specific PKs is provided below. However, adaptation
of these protocols for determining the activity of compounds
against other RTKs, as well as for CTKs and STKs, is well within
the scope of knowledge of those skilled in the art. Other assays
described herein measure the amount of DNA made in response to
activation of a test kinase, which is a general measure of a
proliferative response. The general procedure for this assay is as
follows: a compound is introduced to cells expressing the test
kinase, either naturally or recombinantly, for a selected period of
time after which, if the test kinase is a receptor, a ligand known
to activate the receptor is added. After incubation at least
overnight, a DNA labeling reagent such as 5-bromodeoxyuridine
(BrdU) or H.sup.3-thymidine is added. The amount of labeled DNA is
detected with either an anti-BrdU antibody or by measuring
radioactivity and is compared to control cells not contacted with a
test compound.
[0206] GST-Flk-1 Bioassay
[0207] This assay analyzes the tyrosine kinase activity of GST-Flk1
on poly(glu-tyr) peptides.
[0208] Materials and Reagents:
[0209] 1. Corning 96-well ELISA plates (Corning Catalog No.
25805-96).
[0210] 2. poly(glu-tyr) 4:1, lyophilizate (Sigma Catalog No.
P0275), 1 mg/ml in sterile PBS.
[0211] 3. PBS Buffer: for 1 L, mix 0.2 g KH.sub.2PO.sub.4, 1.15 g
NA.sub.2HPO.sub.4, 0.2 g KCl and 8 g NaCl in approx. 900 ml
dH.sub.2O. When all reagents have dissolved, adjust the pH to 7.2
with HCl. Bring total volume to 1 L with dH.sub.2O.
[0212] 4. PBST Buffer: to 1 L of PBS Buffer, add 1.0 ml
Tween-20.
[0213] 5. TBB-Blocking Buffer: for 1 L, mix 1.21 g TRIS, 8.77 g
NaCl, 1 ml TWEEN-20 in approximately 900 ml dH.sub.2O. Adjust pH to
7.2 with HCl. Add 10 g BSA, stir to dissolve. Bring total volume to
1 L with dH.sub.2O. Filter to remove particulate matter.
[0214] 6. 1% BSA in PBS: add 10 g BSA to approx. 990 ml PBS buffer,
stir to dissolve. Adjust total volume to 1 L with PBS buffer,
filter to remove particulate matter.
[0215] 7. 50 mM Hepes pH 7.5.
[0216] 8. GST-Flk1cd purified from sf9 recombinant baculovirus
transformation (SUGEN, Inc.).
[0217] 9. 4% DMSO in dH.sub.2O.
[0218] 10. 10 mM ATP in dH.sub.2O.
[0219] 11. 40 mM MnCl.sub.2
[0220] 12. Kinase Dilution Buffer (KDB): mix 10 ml Hepes (pH 7.5),
1 ml 5M NaCl, 40 .mu.L 100 mM sodium orthovanadate and 0.4 ml of 5%
BSA in dH.sub.2O with 88.56 ml dH.sub.2O.
[0221] 13. NUNC 96-well V bottom polypropylene plates, Applied
Scientific Catalog #AS-72092
[0222] 14. EDTA: mix 14.12 g ethylenediaminetetraacetic acid (EDTA)
with approx. 70 ml dH.sub.2O. Add 10 N NaOH until EDTA dissolves.
Adjust pH to 8.0. Adjust total volume to 100 ml with dH.sub.2O.
[0223] 15. 1.degree. and 20.degree. Antibody Dilution Buffer: mix
10 ml of 5% BSA in PBS buffer with 89.5 ml TBST.
[0224] 16. Anti-phosphotyrosine rabbit polyclonal antisera (SUGEN,
Inc.)
[0225] 17. Goat anti-rabbit HRP conjugate.
[0226] 18. ABST solution: To approx. 900 ml dH.sub.2O add 19.21 g
citric acid and 35.49 g Na.sub.2HPO.sub.4. Adjust pH to 4.0 with
phosphoric acid. Add
2,2'-Azinobis(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS, Sigma,
Cat. No. A-1888, hold for approx. 1/2 hour, filter.
[0227] 19. 30% Hydrogen Peroxide.
[0228] 20. ABST/H.sub.2O.sub.2: add 3 .mu.l of H.sub.2O.sub.2 to 15
ml of ABST solution.
[0229] 21. 0.2 M HCl.
[0230] Procedure:
[0231] 1. Coat Corning 96-well ELISA plates with 2 .mu.g of polyEY
in 100 .mu.l PBS/well, hold at room temperature for 2 hours or at
4.degree. C. overnight. Cover plates to prevent evaporation.
[0232] 2. Remove unbound liquid from wells by inverting plate. Wash
once with TBST. Pat the plate on a paper towel to remove excess
liquid.
[0233] 3. Add 100 .mu.l of 1% BSA in PBS to each well. Incubate,
with shaking, for 1 hr. at room temperature.
[0234] 4. Repeat step 2.
[0235] 5. Soak wells with 50 mM HEPES (pH 7.5, 150 .mu.l/well).
[0236] 6. Dilute test compound with dH.sub.2O/4% DMSO to 4 times
the desired final assay concentration in 96-well polypropylene
plates.
[0237] 7. Add 25 .mu.l diluted test compound to each well of ELISA
plate. In control wells, place 25 .mu.l of dH.sub.2O/4% DMSO.
[0238] 8. Dilute GST-Flk1 0.005 .mu.g (5 ng)/well in KDB.
[0239] 9. Add 50 .mu.l of diluted enzyme to each well.
[0240] 10. Add 25 .mu.l 0.5 M EDTA to negative control wells.
[0241] 11. Add 25 .mu.l of 40 mM MnCl.sub.2 with 4.times. ATP (2
.mu.M) to all wells (100 .mu.l final volume, 0.5 .mu.M ATP final
concentration in each well).
[0242] 12. Incubate, with shaking, for 15 minutes at room
temperature.
[0243] 13. Stop reaction by adding 25 .mu.l of 500 mM EDTA to each
well.
[0244] 14. Wash 3.times. with TBST and pat plate on paper towel to
remove excess liquid.
[0245] 15. Add 100 .mu.l per well anti-phosphotyrosine antisera,
1:10,000 dilution in antibody dilution buffer. Incubate, with
shaking, for 90 min. at room temperature.
[0246] 16. Wash as in step 14.
[0247] 17. Add 100 .mu.l/well of goat anti-rabbit HRP conjugate
(1:6,000 in antibody dilution buffer). Incubate, with shaking, for
90 minutes are room temperature.
[0248] 18. Wash as in Step 14.
[0249] 19. Add 100 .mu.l room temperature ABST/H.sub.2O.sub.2
solution to each well.
[0250] 20. Incubate, with shaking for 15 to 30 minutes at room
temperature.
[0251] 21. If necessary, stop reaction by adding 100 .mu.l of 0.2 M
HCl to each well.
[0252] 22. Read results on Dynatech MR7000 ELISA reader with test
filter at 410 nM and reference filter at 630 nM.
[0253] Pyk2 Bioassay
[0254] This assay is used to measure the in vitro kinase activity
of HA epitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA
assay.
[0255] Materials and Reagents:
[0256] 1. Corning 96-well ELISA plates.
[0257] 2. 12CA5 monoclonal anti-HA antibody (SUGEN, Inc.)
[0258] 3. PBS (Dulbecco's Phosphate-Buffered Saline (Gibco Catalog
#450-1300EB)
[0259] 4. TBST Buffer: for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and
1 ml of 0.1% Triton X-100 in approx. 900 ml dH.sub.2O. Adjust pH to
7.2, bring volume to 1 L.
[0260] 5. Blocking Buffer: for 1 L, mix 100 g 10% BSA, 12.1 g 100
mM TRIS, 58.44 g 1M NaCl and 10 mL of 1% TWEEN-20.
[0261] 6. FL.pyk2-HA from sf9 cell lysates (SUGEN, Inc.).
[0262] 7. 4% DMSO in MilliQue H.sub.2O.
[0263] 8. 10 nM ATP in dH.sub.2O.
[0264] 9. 1M MnCl.sub.2.
[0265] 10. 1M MgCl.sub.2.
[0266] 11. 1M Dithiothreitol (DTT).
[0267] 12. 10.times. Kinase buffer phosphorylation: mix 5.0 ml 1M
Hepes (pH 7.5), 0.2 ml 1M MnCl.sub.2, 1.0 ml 1 M MgCl.sub.2, 1.0 ml
10% Triton X-100 in 2.8 ml dH.sub.2O. Just prior to use, add 0.1 ml
1M DTT.
[0268] 13. NUNC 96-well V bottom polypropylene plates.
[0269] 14. 500 mM EDTA in dH.sub.2O.
[0270] 15. Antibody dilution buffer: for 100 mL, 1 mL 5% BSA/PBS
and 1 mL 10% Tween-20 in 88 mL TBS.
[0271] 16. HRP-conjugated anti-Ptyr (PY99, Santa Cruz Biotech Cat.
No. SC-7020).
[0272] 17. ABTS, Moss, Cat. No. ABST-2000.
[0273] 18. 10% SDS.
[0274] Procedure:
[0275] 1. Coat Corning 96 well ELISA plates with 0.5 .mu.g per well
12CA5 anti-HA antibody in 100 .mu.l PBS. Store overnight at
4.degree. C.
[0276] 2. Remove unbound HA antibody from wells by inverting plate.
Wash plate with dH.sub.2O. Pat the plate on a paper towel to remove
excess liquid.
[0277] 3. Add 150 .mu.l Blocking Buffer to each well. Incubate,
with shaking, for 30 min at room temperature.
[0278] 4. Wash plate 4.times. with TBS-T.
[0279] 5. Dilute lysate in PBS (1.5 .mu.g lysate/100 .mu.l
PBS).
[0280] 6. Add 100 .mu.l of diluted lysate to each well. Shake at
room temperature for 1 hr.
[0281] 7. Wash as in step 4.
[0282] 8. Add 50 .mu.l of 2.times. kinase Buffer to ELISA plate
containing captured pyk2-HA.
[0283] 9. Add 25 .mu.L of 400 .mu.M test compound in 4% DMSO to
each well. For control wells use 4% DMSO alone.
[0284] 10. Add 25 .mu.L of 0.5 M EDTA to negative control
wells.
[0285] 11. Add 25 .mu.l of 20 .mu.M ATP to all wells. Incubate,
with shaking, for 10 minutes.
[0286] 12. Stop reaction by adding 25 .mu.l 500 mM EDTA (pH 8.0) to
all wells.
[0287] 13. Wash as in step 4.
[0288] 14. Add 100 .mu.L HRP conjugated anti-Ptyr diluted 1:6000 in
Antibody Dilution Buffer to each well. Incubate, with shaking, for
1 hr. at room temperature.
[0289] 15. Wash plate 3.times. with TBST and 1.times. with PBS.
[0290] 16. Add 100 .mu.L of ABST solution to each well.
[0291] 17. If necessary, stop the development reaction by adding 20
.mu.L 10% SDS to each well.
[0292] 18. Read plate on ELISA reader with test filter at 410 nM
and reference filter at 630 nM.
[0293] FGFR1 Bioassay
[0294] This assay is used to measure the in vitro kinase activity
of FGF1-R in an ELISA assay.
[0295] Materials and Reagents:
[0296] 1. Costar 96-well ELISA plates (Coming Catalog #3369).
[0297] 2. Poly(Glu-Tyr) (Sigma Catalog #PO.sub.275).
[0298] 3. PBS (Gibco Catalog #450-1300EB)
[0299] 4. 50 mM Hepes Buffer Solution.
[0300] 5. Blocking Buffer (5% BSA/PBS).
[0301] 6. Purified GST-FGFR1 (SUGEN, Inc.)
[0302] 7. Kinase Dilution Buffer. Mix 500 .mu.l 1M Hepes (GIBCO),
20 .mu.l 5% BSA/PBS, 10 .mu.l 100 mM sodium orthovanadate and 50
.mu.l 5M NaCl.
[0303] 8. 10 mM ATP
[0304] 9. ATP/MnCl.sub.2 phosphorylation mix: mix 20 .mu.L ATP, 400
.mu.L 1M MnCl.sub.2 and 9.56 ml dH.sub.2O.
[0305] 10. NUNC 96-well V bottom polypropylene plates (Applied
Scientific Catalog #AS-72092).
[0306] 11. 0.5M EDTA.
[0307] 12. 0.05% TBST Add 500 .mu.L TWEEN to 1 liter TBS.
[0308] 13. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN,
Inc.).
[0309] 14. Goat anti-rabbit IgG peroxidase conjugate (Biosource,
Catalog #AL10404).
[0310] 15. ABTS Solution.
[0311] 16. ABTS/H.sub.2O.sub.2 solution.
[0312] Procedure:
[0313] 1. Coat Costar 96 well ELISA plates with 1 .mu.g per well
Poly(Glu-Tyr) in 100 .mu.l PBS. Store overnight at 4.degree. C.
[0314] 2. Wash coated plates once with PBS.
[0315] 3. Add 150 .mu.L of 5% BSA/PBS Blocking Buffer to each well.
Incubate, with shaking, for 1 hr at room temperature.
[0316] 4. Wash plate 2.times. with PBS, then once with 50 mM Hepes.
Pat plates on a paper towel to remove excess liquid and
bubbles.
[0317] 5. Add 25 .mu.L of 0.4 mM test compound in 4% DMSO or 4%
DMSO alone (controls) to plate.
[0318] 6. Dilute purified GST-FGFR1 in Kinase Dilution Buffer (5 ng
kinase/50 .mu.l KDB/well).
[0319] 7. Add 50 .mu.L of diluted kinase to each well.
[0320] 8. Start kinase reaction by adding 25 .mu.l/well of freshly
prepared ATP/Mn++ (0.4 ml 1M MnCl.sub.2, 40 .mu.L 10 mM ATP, 9.56
ml dH.sub.2O), freshly prepared).
[0321] 9. Stop reaction with 25 .mu.L of 0.5M EDTA.
[0322] 10. Wash plate 4.times. with fresh TBST.
[0323] 11. Make up Antibody Dilution Buffer: For 50 ml, mix 5 ml of
5% BSA, 250 .mu.l of 5% milk and 50 .mu.l of 100 mM sodium
vanadate, bring to final volume with 0.05% TBST.
[0324] 12. Add 100 .mu.l per well of anti-phosphotyrosine (1:10000
dilution in ADB). Incubate, with shaking for 1 hr. at room
temperature.
[0325] 13. Wash as in step 10.
[0326] 14. Add 100 .mu.l per well of Biosource Goat anti-rabbit IgG
peroxidase conjugate (1:6000 dilution in ADB). Incubate, with
shaking for 1 hr. at room temperature.
[0327] 15. Wash as in step 10 and then with PBS to remove bubbles
and excess TWEEN.
[0328] 16. Add 100 .mu.l of ABTS/H.sub.2O.sub.2 solution to each
well.
[0329] 17. Incubate, with shaking, for 10 to 20 minutes. Remove any
bubbles.
[0330] 18. Read assay on Dynatech MR7000 ELISA reader: test filter
at 410 nM, reference filter at 630 nM.
[0331] EGFR Bioassay
[0332] This assay is used to the in vitro kinase activity of EGFR
in an ELISA assay.
[0333] Materials and Reagents:
[0334] 1. Corning 96-well ELISA plates.
[0335] 2. SUMO1 monoclonal anti-EGFR antibody (SUGEN, Inc.).
[0336] 3. PBS.
[0337] 4. TBST Buffer.
[0338] 5. Blocking Buffer: for 100 ml, mix 5.0 g Carnation.RTM.
Instant Non-fat Milk with 100 ml of PBS.
[0339] 6. A431 cell lysate (SUGEN, Inc.).
[0340] 7. TBS Buffer.
[0341] 8. TBS+10% DMSO: for 1L, mix 1.514 g TRIS, 2.192 g NaCl and
25 ml DMSO; bring to 1 liter total volume with dH.sub.2O.
[0342] 9. ATP (Adenosine-5'-triphosphate, from Equine muscle, Sigma
Cat. No. A-5394), 1.0 mM solution in dH.sub.2O. This reagent should
be made up immediately prior to use and kept on ice.
[0343] 10. 1.0 mM MnCl.sub.2.
[0344] 11. ATP/MnCl.sub.2 phosphorylation mix: for 10 ml, mix 300
.mu.l of 1 mM ATP, 500 .mu.l MnCl.sub.2 and 9.2 ml dH.sub.2O.
Prepare just prior to use, keep on ice.
[0345] 12. NUNC 96-well V bottom polypropylene plates.
[0346] 13. EDTA.
[0347] 14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN,
Inc.).
[0348] 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource
Cat. No. ALI0404).
[0349] 16. ABTS.
[0350] 17. 30% Hydrogen peroxide.
[0351] 18. ABTS/H.sub.2O.sub.2
[0352] 19. 0.2 M HCl.
[0353] Procedure:
[0354] 1. Coat Corning 96 well ELISA plates with 0.5 .mu.g SUMO1 in
100 .mu.l PBS per well, hold overnight at 4.degree. C.
[0355] 2. Remove unbound SUMO1 from wells by inverting plate to
remove liquid. Wash 1.times. with dH.sub.2O. Pat the plate on a
paper towel to remove excess liquid.
[0356] 3. Add 150 .mu.l of Blocking Buffer to each well. Incubate,
with shaking, for 30 min. at room temperature.
[0357] 4. Wash plate 3.times. with deionized water, then once with
TBST. Pat plate on a paper towel to remove excess liquid and
bubbles.
[0358] 5. Dilute lysate in PBS (7 .mu.g lysate/100 .mu.l PBS).
[0359] 6. Add 100 .mu.l of diluted lysate to each well. Shake at
room temperature for 1 hr.
[0360] 7. Wash plates as in 4, above.
[0361] 8. Add 120 .mu.l TBS to ELISA plate containing captured
EGFR.
[0362] 9. Dilute test compound 1:10 in TBS, place in well
[0363] 10. Add 13.5 .mu.l diluted test compound to ELISA plate. To
control wells, add 13.5 .mu.l TBS in 10% DMSO.
[0364] 11. Incubate, with shaking, for 30 minutes at room
temperature.
[0365] 12. Add 15 .mu.l phosphorylation mix to all wells except
negative control well. Final well volume should be approximately
150 .mu.l with 3 .mu.M ATP/5 mM MnCl.sub.2 final concentration in
each well. Incubate with shaking for 5 minutes.
[0366] 13. Stop reaction by adding 16.5 .mu.l of EDTA solution
while shaking. Shake for additional 1 min.
[0367] 14. Wash 4.times. with deionized water, 2.times. with
TBST.
[0368] 15. Add 100 .mu.l anti-phosphotyrosine (1:3000 dilution in
TBST) per well. Incubate, with shaking, for 30-45 min. at room
temperature.
[0369] 16. Wash as in 4, above.
[0370] 17. Add 100 .mu.l Biosource Goat anti-rabbit IgG peroxidase
conjugate (1:2000 dilution in TBST) to each well. Incubate with
shaking for 30 min. at room temperature.
[0371] 18. Wash as in 4, above.
[0372] 19. Add 100 .mu.l of ABTS/H.sub.2O.sub.2 solution to each
well.
[0373] 20. Incubate 5 to 10 minutes with shaking. Remove any
bubbles.
[0374] 21. If necessary, stop reaction by adding 100 .mu.l 0.2 M
HCl per well.
[0375] 22. Read assay on Dynatech MR7000 ELISA reader: test filter
at 410 nM, reference filter at 630 nM.
[0376] PDGFR Bioassay
[0377] This assay is used to the in vitro kinase activity of PDGFR
in an ELISA assay.
[0378] Materials and Reagents:
[0379] 1. Corning 96-well ELISA plates
[0380] 2. 28D4C10 monoclonal anti-PDGFR antibody (SUGEN, Inc.).
[0381] 3. PBS.
[0382] 4. TBST Buffer.
[0383] 5. Blocking Buffer (same as for EGFR bioassay).
[0384] 6. PDGFR-.beta. expressing NIH 3T3 cell lysate (SUGEN,
Inc.).
[0385] 7. TBS Buffer.
[0386] 8. TBS+10% DMSO.
[0387] 9. ATP.
[0388] 10. MnCl.sub.2.
[0389] 11. Kinase buffer phosphorylation mix: for 10 ml, mix 250
.mu.l 1M TRIS, 200 .mu.l 5M NaCl, 100 .mu.l IM MnCl.sub.2 and 50
.mu.l 100 mM Triton X-100 in enough dH.sub.2O to make 10 ml.
[0390] 12. NUNC 96-well V bottom polypropylene plates.
[0391] 13. EDTA.
[0392] 14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN,
Inc.).
[0393] 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource
Cat. No. ALI0404).
[0394] 16. ABTS.
[0395] 17. Hydrogen peroxide, 30% solution.
[0396] 18. ABTS/H.sub.2O.sub.2.
[0397] 19. 0.2 M HCl.
[0398] Procedure:
[0399] 1. Coat Coming 96 well ELISA plates with 0.5 .mu.g 28D4C10
in 100 .mu.l PBS per well, hold overnight at 4.degree. C.
[0400] 2. Remove unbound 28D4C10 from wells by inverting plate to
remove liquid. Wash 1.times. with dH.sub.2O. Pat the plate on a
paper towel to remove excess liquid.
[0401] 3. Add 150 .mu.l of Blocking Buffer to each well. Incubate
for 30 min. at room temperature with shaking.
[0402] 4. Wash plate 3.times. with deionized water, then once with
TBST. Pat plate on a paper towel to remove excess liquid and
bubbles.
[0403] 5. Dilute lysate in HNTG (10 .mu.g lysate/100 .mu.l
HNTG).
[0404] 6. Add 100 .mu.l of diluted lysate to each well. Shake at
room temperature for 60 min.
[0405] 7. Wash plates as described in Step 4.
[0406] 8. Add 80 l working kinase buffer mix to ELISA plate
containing captured PDGFR.
[0407] 9. Dilute test compound 1:10 in TBS in 96-well polypropylene
plates.
[0408] 10. Add 10 .mu.l diluted test compound to ELISA plate. To
control wells, add 10 .mu.l TBS+10% DMSO. Incubate with shaking for
30 minutes at room temperature.
[0409] 11. Add 10 .mu.l ATP directly to all wells except negative
control well (final well volume should be approximately 100 .mu.l
with 20 .mu.M ATP in each well.) Incubate 30 minutes with
shaking.
[0410] 12. Stop reaction by adding 10 .mu.l of EDTA solution to
each well.
[0411] 13. Wash 4.times. with deionized water, twice with TBST.
[0412] 14. Add 100 .mu.l anti-phosphotyrosine (1:3000 dilution in
TBST) per well. Incubate with shaking for 30-45 min. at room
temperature.
[0413] 15. Wash as in Step 4.
[0414] 16. Add 100 .mu.l Biosource Goat anti-rabbit IgG peroxidase
conjugate (1:2000 dilution in TBST) to each well. Incubate with
shaking for 30 min. at room temperature.
[0415] 17. Wash as in Step 4.
[0416] 18. Add 100 .mu.l of ABTS/H.sub.2O.sub.2 solution to each
well.
[0417] 19. Incubate 10 to 30 minutes with shaking. Remove any
bubbles.
[0418] 20. If necessary stop reaction with the addition of 100
.mu.l 0.2 M HCl per well.
[0419] 21. Read assay on Dynatech MR7000 ELISA reader with test
filter at 410 nM and reference filter at 630 nM.
[0420] Cellular HER-2 Kinase Assay
[0421] This assay is used to measure HER-2 kinase activity in whole
cells in an ELISA format.
[0422] Materials and Reagents:
[0423] 1. DMEM (GIBCO Catalog #11965-092).
[0424] 2. Fetal Bovine Serum (FBS, GIBCO Catalog #16000-044), heat
inactivated in a water bath for 30 min. at 56.degree. C.
[0425] 3. Trypsin (GIBCO Catalog #25200-056).
[0426] 4. L-Glutamine (GIBCO Catalog #25030-081).
[0427] 5. HEPES (GIBCO Catalog #15630-080).
[0428] 6. Growth Media: Mix 500 ml DMEM, 55 ml heat inactivated
FBS, 10 ml HEPES and 5.5 ml L-Glutamine.
[0429] 7. Starve Media: Mix 500 ml DMEM, 2.5 ml heat inactivated
FBS, 10 ml HEPES and 5.5 ml L-Glutamine.
[0430] 8. PBS.
[0431] 9. Flat Bottom 96-well Tissue Culture Micro Titer Plates
(Corning Catalog #25860).
[0432] 10. 15 cm Tissue Culture Dishes (Coming Catalog
#08757148).
[0433] 11. Corning 96-well ELISA Plates.
[0434] 12. NUNC 96-well V bottom polypropylene plates.
[0435] 13. Costar Transfer Cartridges for the Transtar 96 (Costar
Catalog #7610).
[0436] 14. SUMO 1: monoclonal anti-EGFR antibody (SUGEN, Inc.).
[0437] 15. TBST Buffer.
[0438] 16. Blocking Buffer: 5% Carnation Instant Milk.RTM. in
PBS.
[0439] 17. EGF Ligand: EGF-201, Shinko American, Japan. Suspend
powder in 100 .mu.L of 10 mM HCl. Add 100 uL 10 mM NaOH. Add 800
.mu.L PBS and transfer to an Eppendorf tube, store at -20.degree.
C. until ready to use.
[0440] 18. HNTG Lysis Buffer: For Stock 5.times. HNTG, mix 23.83 g
Hepes, 43.83 g NaCl, 500 ml glycerol and 100 ml Triton X-100 and
enough dH.sub.2O to make 1 L of total solution. For 1.times. HNTG*,
mix 2 ml 5.times. HNTG, 100 .mu.L 0.1M Na.sub.3VO.sub.4, 250 .mu.L
0.2M Na.sub.4P.sub.2O.sub.7 and 100 .mu.L EDTA.
[0441] 19. EDTA.
[0442] 20. Na.sub.3VO.sub.4: To make stock solution, mix 1.84 g
Na.sub.3VO.sub.4 with 90 ml dH.sub.2O. Adjust pH to 10. Boil in
microwave for one minute (solution becomes clear). Cool to room
temperature. Adjust pH to 10. Repeat heating/cooling cycle until pH
remains at 10.
[0443] 21. 200 mM Na.sub.4P.sub.2O.sub.7.
[0444] 22. Rabbit polyclonal antiserum specific for phosphotyrosine
(anti-Ptyr antibody, SUGEN, Inc.).
[0445] 23. Affinity purified antiserum, goat anti-rabbit IgG
antibody, peroxidase conjugate (Biosource Cat #AL10404).
[0446] 24. ABTS Solution.
[0447] 25. 30% Hydrogen peroxide solution.
[0448] 26. ABTS/H.sub.2O.sub.2.
[0449] 27. 0.2 M HCl.
[0450] Procedure:
[0451] 1. Coat Corning 96 well ELISA plates with SUMO1 at 1.0 .mu.g
per well in PBS, 100 .mu.l final volume/well. Store overnight at
4.degree. C.
[0452] 2. On day of use, remove coating buffer and wash plate 3
times with dH.sub.2O and once with TBST buffer. All washes in this
assay should be done in this manner, unless otherwise
specified.
[0453] 3. Add 100 .mu.L of Blocking Buffer to each well. Incubate
plate, with shaking, for 30 min. at room temperature. Just prior to
use, wash plate.
[0454] 4. Use EGFr/HER-2 chimera/3T3-C7 cell line for this
assay.
[0455] 5. Choose dishes having 80-90% confluence. Collect cells by
trypsinization and centrifuge at 1000 rpm at room temperature for 5
min.
[0456] 6. Resuspend cells in starve medium and count with trypan
blue. Viability above 90% is required. Seed cells in starve medium
at a density of 2,500 cells per well, 90 .mu.L per well, in a 96
well microtiter plate. Incubate seeded cells overnight at
37.degree. under 5% CO.sub.2.
[0457] 25. 7. Start the assay two days after seeding.
[0458] 8. Test compounds are dissolved in 4% DMSO. Samples are then
further diluted directly on plates with starve-DMEM. Typically,
this dilution will be 1:10 or greater. All wells are then
transferred to the cell plate at a further 1:10 dilution (10 .mu.l
sample and media into 90 .mu.l of starve media). The final DMSO
concentration should be 1% or lower. A standard serial dilution may
also be used.
[0459] 9. Incubate under 5% CO.sub.2 at 37.degree. C. for 2
hours.
[0460] 10. Prepare EGF ligand by diluting stock EGF (16.5 .mu.M) in
warm DMEM to 150 nM.
[0461] 11. Prepare fresh HNTG* sufficient for 100 .mu.L per well;
place on ice.
[0462] 12. After 2 hour incubation with test compound, add prepared
EGF ligand to cells, 50 .mu.L per well, for a final concentration
of 50 nM. Positive control wells receive the same amount of EGF.
Negative controls do not receive EGF. Incubate at 37.degree. C. for
10 min.
[0463] 13. Remove test compound, EGF, and DMEM. Wash cells once
with PBS.
[0464] 14. Transfer HNTG* to cells, 100 .mu.L per well. Place on
ice for 5 minutes. Meanwhile, remove blocking buffer from ELISA
plate and wash.
[0465] 15. Scrape cells from plate with a micropipettor and
homogenize cell material by repeatedly aspirating and dispensing
the HNTG* lysis buffer. Transfer lysate to a coated, blocked,
washed ELISA plate.
[0466] 16. Incubate, with shaking, at room temperature for 1
hr.
[0467] 17. Remove lysate, wash. Transfer freshly diluted anti-Ptyr
antibody (1:3000 in TBST) to ELISA plate, 100 .mu.L per well.
[0468] 18. Incubate, with shaking, at room temperature, for 30
min.
[0469] 19. Remove anti-Ptyr antibody, wash. Transfer freshly
diluted BIOSOURCE antibody to ELISA plate(1:8000 in TBST, 100 .mu.L
per well).
[0470] 20. Incubate, with shaking, at room temperature for 30
min.
[0471] 21. Remove BIOSOURCE antibody, wash. Transfer freshly
prepared ABTS/H.sub.2O.sub.2 solution to ELISA plate, 100 .mu.L per
well.
[0472] 22. Incubate, with shaking, for 5-10 minutes. Remove any
bubbles.
[0473] 23. Stop reaction by adding 100 .mu.L of 0.2M HCl per
well.
[0474] 24. Read assay on Dynatech MR7000 ELISA reader with test
filter set at 410 nM and reference filter at 630 nM.
[0475] Cdk2/Cyclin A Assay
[0476] This assay is used to measure the in vitro serine/threonine
kinase activity of human cdk2/cyclin A in a Scintillation Proximity
Assay (SPA).
[0477] Materials and Reagents.
[0478] 1. Wallac 96-well polyethylene terephthalate (flexi) plates
(Wallac Catalog #1450-401).
[0479] 2. Amersham Redivue [.gamma..sup.33P] ATP (Amersham catalog
#AH 9968).
[0480] 3. Amersham streptavidin coated polyvinyltoluene SPA beads
(Amersham catalog #RPNQ0007). The beads should be reconstituted in
PBS without magnesium or calcium, at 20 mg/ml.
[0481] 4. Activated cdk2/cyclin A enzyme complex purified from Sf9
cells (SUGEN, Inc.).
[0482] 5. Biotinylated peptide substrate (Debtide). Peptide
biotin-X-PKTPKKAKKL is dissolved in dH.sub.2O at a concentration of
5 mg/ml.
[0483] 6. 20% DMSO in dH.sub.2O.
[0484] 7. Kinase buffer: for 10 ml, mix 9.1 ml dH.sub.2O, 0.5 ml
TRIS(pH 7.4), 0.2 ml 1M MgCl.sub.2, 0.2 ml 10% NP40 and 0.02 ml 1M
DTT, added fresh just prior to use.
[0485] 8. 10 mM ATP in dH.sub.2O.
[0486] 9. 1M Tris, pH adjusted to 7.4 with HCl.
[0487] 10. 1M MgCl.sub.2.
[0488] 11. 1M DTT.
[0489] 12. PBS (Gibco Catalog #14190-144).
[0490] 13. 0.5M EDTA.
[0491] 14. Stop solution: For 10 ml, mix 9.25 ml PBS, 0.05 ml 10 mM
ATP, 0.1 ml 0.5 M EDTA, 0.1 ml 10% Triton X-100 and 1.5 ml of 50
mg/ml SPA beads.
[0492] Procedure:
[0493] 1. Prepare solutions of test compounds at 4.times. the
desired final concentration in 5% DMSO. Add 10 .mu.L to each well.
For positive and negative controls, use 10 .mu.L 20% DMSO alone in
wells.
[0494] 2. Dilute the peptide substrate (deb-tide) 1:250 with
dH.sub.2O to give a final concentration of 0.02 mg/ml.
[0495] 3. Mix 24 .mu.L 0.1 mM ATP with 24 .mu.Ci .gamma..sup.33P
ATP and enough dH.sub.2O to make 600 .mu.L.
[0496] 4. Mix diluted peptide and ATP solutions 1:1 (600 .mu.L+600
.mu.L per plate). Add 10 .mu.L of this solution to each well.
[0497] 5. Dilute 5 .mu.L of cdk2/cyclin A solution into 2.1 ml
2.times. kinase buffer (per plate). Add 20 .mu.L enzyme per well.
For negative controls, add 20 .mu.L 2.times. kinase buffer without
enzyme.
[0498] 6. Mix briefly on a plate shaker; incubate for 60
minutes.
[0499] 7. Add 200 .mu.L stop solution per well.
[0500] 8. Let stand at least 10 min.
[0501] 9. Spin plate at approx. 2300 rpm for 10-15 min.
[0502] 10. Count plate on Trilux reader.
[0503] Met Transphosphorylation Assay
[0504] This assay is used to measure phosphotyrosine levels on a
poly(glutamic acid:tyrosine, 4:1) substrate as a means for
identifying agonists/antagonists of met transphosphorylation of the
substrate.
[0505] Materials and Reagents:
[0506] 1. Corning 96-well ELISA plates, Corning Catalog
#25805-96.
[0507] 2. Poly(glu-tyr), 4:1, Sigma, Cat. No; P 0275.
[0508] 3. PBS, Gibco Catalog #450-1300EB
[0509] 4. 50 mM HEPES
[0510] 5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin,
Sigma Cat. No A-7888, in 500 ml PBS, filter through a 4 .mu.m
filter.
[0511] 6. Purified GST fusion protein containing the Met kinase
domain, SUGEN, Inc.
[0512] 7. TBST Buffer.
[0513] 8. 10% aqueous (MilliQue H.sub.2O) DMSO.
[0514] 9. 10 mM aqueous (dH.sub.2O) Adenosine-5'-triphosphate,
Sigma Cat. No. A-5394.
[0515] 10. 2.times. Kinase Dilution Buffer: for 100 ml, mix 10 mL
1M HEPES at pH 7.5 with 0.4 mL 5% BSA/PBS, 0.2 mL 0.1 M sodium
orthovanadate and 1 mL 5M sodium chloride in 88.4 mL dH.sub.2O.
[0516] 11. 4.times. ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M
manganese chloride and 0.02 mL 0.1 M ATP in 9.56 mL dH.sub.2O.
[0517] 12. 4.times. Negative Controls Mixture: for 10 mL, mix 0.4
mL 1 M manganese chloride in 9.6 mL dH.sub.2O.
[0518] 13. NUNC 96-well V bottom polypropylene plates, Applied
Scientific Catalog #S-72092
[0519] 14. 500 mM EDTA.
[0520] 15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5%
BSA/PBS, 0.5 mL 5% Carnation.RTM. Instant Milk in PBS and 0.1 mL
0.1 M sodium orthovanadate in 88.4 mL TBST.
[0521] 16. Rabbit polyclonal antophosphotyrosine antibody, SUGEN,
Inc.
[0522] 17. Goat anti-rabbit horseradish peroxidase conjugated
antibody, Biosource, Inc.
[0523] 18. ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49 g
Na.sub.2HPO.sub.4 and 500 mg ABTS with sufficient dH.sub.2O to make
1 L.
[0524] 19. ABTS/H.sub.2O.sub.2: mix 15 mL ABST solution with 2
.mu.L H.sub.2O.sub.2 five minutes before use.
[0525] 20. 0.2 M HCl
[0526] Procedure:
[0527] 1. Coat ELISA plates with 2 .mu.g Poly(Glu-Tyr) in 100 .mu.L
PBS, hold overnight at 4.degree. C.
[0528] 2. Block plate with 150 .mu.L of 5% BSA/PBS for 60 min.
[0529] 3. Wash plate twice with PBS then once with 50 mM Hepes
buffer pH 7.4.
[0530] 4. Add 50 .mu.l of the diluted kinase to all wells.
(Purified kinase is diluted with Kinase Dilution Buffer. Final
concentration should be 10 ng/well.)
[0531] 5. Add 25 .mu.L of the test compound (in 4% DMSO) or DMSO
alone (4% in dH.sub.2O) for controls to plate.
[0532] 6. Incubate the kinase/compound mixture for 15 minutes.
[0533] 7. Add 25 .mu.L of 40 mM MnCl.sub.2 to the negative control
wells.
[0534] 8. Add 25 .mu.L ATP/MnCl.sub.2 mixture to the all other
wells (except the negative controls). Incubate for 5 min.
[0535] 9. Add 25 .mu.L 500 mM EDTA to stop reaction.
[0536] 10. Wash plate 3.times. with TBST.
[0537] 11. Add 100 .mu.L rabbit polyclonal anti-Ptyr diluted
1:10,000 in Antibody Dilution Buffer to each well. Incubate, with
shaking, at room temperature for one hour.
[0538] 12. Wash plate 3.times. with TBST.
[0539] 13. Dilute Biosource HRP conjugated anti-rabbit antibody
1:6,000 in Antibody Dilution buffer. Add 100 .mu.L per well and
incubate at room temperature, with shaking, for one hour.
[0540] 14. Wash plate 1X with PBS.
[0541] 15. Add 100 .mu.l of ABTS/H.sub.2O.sub.2 solution to each
well.
[0542] 16. If necessary, stop the development reaction with the
addition of 100 .mu.l of 0.2M HCl per well.
[0543] 17. Read plate on Dynatech MR7000 ELISA reader with the test
filter at 410 nM and the reference filter at 630 nM.
[0544] IGF-1 Transphosphorylation Assay
[0545] This assay is used to measure the phosphotyrosine level in
poly(glutamic acid:tyrosine, 4:1) for the identification of
agonists/antagonists of gst-IGF-1 transphosphorylation of a
substrate.
[0546] Materials and Reagents:
[0547] 1. Corning 96-well ELISA plates.
[0548] 2. Poly(Glu-Tyr),4: l, Sigma Cat. No. P 0275.
[0549] 3. PBS, Gibco Catalog #450-1300EB.
[0550] 4. 50 mM HEPES
[0551] 5. TBB Blocking Buffer: for 1 L, mix 100 g BSA, 12.1 gTRIS
(pH 7.5), 58.44 g sodium chloride and 10 mL 1% TWEEN-20.
[0552] 6. Purified GST fusion protein containing the IGF-1 kinase
domain (SUGEN, Inc.)
[0553] 7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodium
chloride and 0.5 ml TWEEN-20 with enough dH.sub.2O to make 1
liter.
[0554] 8. 4% DMSO in Milli-Q H.sub.2O.
[0555] 9. 10 mM ATP in dH.sub.2O.
[0556] 10. 2.times. Kinase Dilution Buffer: for 100 mL, mix 10 mL 1
M HEPES (pH 7.5), 0.4 mL 5% BSA in dH.sub.2O, 0.2 mL 0.1 M sodium
orthovanadate and 1 mL 5 M sodium chloride with enough dH.sub.2O to
make 100 mL.
[0557] 11. 4.times. ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M
MnCl.sub.2 and 0.008 mL 0.01 M ATP and 9.56 mL dH.sub.2O.
[0558] 12. 4.times. Negative Controls Mixture: mix 0.4 mL 1 M
MnCl.sub.2 in 9.60 mL dH.sub.2O.
[0559] 13. NUNC 96-well V bottom polypropylene plates.
[0560] 14. 500 mM EDTA in dH.sub.2O.
[0561] 15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA
in PBS, 0.5 mL 5% Carnation Instant Non-fat Milk in PBS and 0.1 mL
0.1 M sodium orthovanadate in 88.4 mL TBST.
[0562] 16. Rabbit Polyclonal antiphosphotyrosine antibody, SUGEN,
Inc.
[0563] 17. Goat anti-rabbit HRP conjugated antibody, Biosource.
[0564] 18. ABTS Solution.
[0565] 20. ABTS/H.sub.2O.sub.2: mix 15 mL ABTS with 2 .mu.L
H.sub.2O.sub.2 5 minutes before using.
[0566] 21. 0.2 M HCl in dH.sub.2O.
[0567] Procedure:
[0568] 1. Coat ELISA plate with 2.0 .mu.g/well Poly(Glu, Tyr), 4:1
(Sigma P0275) in 100 .mu.l PBS. Store plate overnight at 4.degree.
C.
[0569] 2. Wash plate once with PBS.
[0570] 3. Add 100 .mu.l of TBB Blocking Buffer to each well.
Incubate plate for 1 hour with shaking at room temperature.
[0571] 4. Wash plate once with PBS, then twice with 50 mM Hepes
buffer pH 7.5.
[0572] 5. Add 25 .mu.L of test compound in 4% DMSO (obtained by
diluting a stock solution of 10 mM test compound in 100% DMSO with
dH.sub.2O) to plate.
[0573] 6. Add 10.0 ng of gst-IGF-1 kinase in 50 .mu.l Kinase
Dilution Buffer to all wells.
[0574] 7. Start kinase reaction by adding 25 .mu.l 4.times. ATP
Reaction Mixture to all test wells and positive control wells. Add
25 .mu.l 4.times. Negative Controls Mixture to all negative control
wells. Incubates for 10 minutes, with shaking, at room
temperature.
[0575] 8. Add 25 .mu.l 0.5M EDTA (pH 8.0) to all wells.
[0576] 9. Wash plate 4.times. with TBST Buffer.
[0577] 10. Add rabbit polyclonal anti-phosphotyrosine antisera at a
dilution of 1:10,000 in 100 .mu.l Antibody Dilution Buffer to all
wells. Incubate, with shaking, at room temperature for 1 hour.
[0578] 11. Wash plate as in step 9.
[0579] 12. Add 100 .mu.L Biosource anti-rabbit HRP at a dilution of
1:10,000 in Antibody dilution buffer to all wells. Incubate, with
shaking, at room temperature for 1 hour.
[0580] 13. Wash plate as in step 9, follow with one wash with PBS
to remove bubbles and excess Tween-20.
[0581] 14. Develop by adding 100 .mu.l/well ABTS/H.sub.2O.sub.2 to
each well
[0582] 15. After about 5 minutes, read on ELISA reader with test
filter at 410 nm and referenced filter at 630 nm.
[0583] BrdU Incorporation Assays
[0584] The following assays use cells engineered to express a
selected receptor and then evaluate the effect of a compound of
interest on the activity of ligand-induced DNA synthesis by
determining BrdU incorporation into the DNA.
[0585] The following materials, reagents and procedure are general
to each of the following BrdU incorporation assays. Variances in
specific assays are noted.
[0586] General Materials and Reagents:
[0587] 1. The appropriate ligand.
[0588] 2. The appropriate engineered cells.
[0589] 3. BrdU Labeling Reagent: 10 mM, in PBS, pH 7.4(Roche
Molecular Biochemicals, Indianapolis, Ind.).
[0590] 4. FixDenat: fixation solution (Roche Molecular
Biochemicals, Indianapolis, Ind.).
[0591] 5. Anti-BrdU-POD: mouse monoclonal antibody conjugated with
peroxidase (Chemicon, Temecula, Calif.).
[0592] 6. TMB Substrate Solution: tetramethylbenzidine (TMB, ready
to use, Roche Molecular Biochemicals, Indianapolis, Ind.).
[0593] 7. PBS Washing Solution: 1.times. PBS, pH 7.4.
[0594] 8. Albumin, Bovine (BSA), fraction V powder (Sigma Chemical
Co., USA).
[0595] General Procedure:
[0596] 1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln
in DMEM, in a 96 well plate. Cells are incubated overnight at
37.degree. C. in 5% CO.sub.2.
[0597] 2. After 24 hours, the cells are washed with PBS, and then
are serum-starved in serum free medium (0% CS DMEM with 0.1% BSA)
for 24 hours.
[0598] 3. On day 3, the appropriate ligand and the test compound
are added to the cells simultaneously. The negative control wells
receive serum free DMEM with 0.1% BSA only; the positive control
cells receive the ligand but no test compound. Test compounds are
prepared in serum free DMEM with ligand in a 96 well plate, and
serially diluted for 7 test concentrations.
[0599] 4. After 18 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the cells
are incubated with BrdU (final concentration is 10 .mu.M) for 1.5
hours.
[0600] 5. After incubation with labeling reagent, the medium is
removed by decanting and tapping the inverted plate on a paper
towel. FixDenat solution is added (50 .mu.l/well) and the plates
are incubated at room temperature for 45 minutes on a plate
shaker.
[0601] 6. The FixDenat solution is removed by decanting and tapping
the inverted plate on a paper towel. Milk is added (5% dehydrated
milk in PBS, 200 .mu.l/well) as a blocking solution and the plate
is incubated for 30 minutes at room temperature on a plate
shaker.
[0602] 7. The blocking solution is removed by decanting and the
wells are washed once with PBS. Anti-BrdU-POD solution is added
(1:200 dilution in PBS, 1% BSA, 50 .mu.l/well) and the plate is
incubated for 90 minutes at room temperature on a plate shaker.
[0603] 8. The antibody conjugate is removed by decanting and
rinsing the wells 5 times with PBS, and the plate is dried by
inverting and tapping on a paper towel.
[0604] 9. TMB substrate solution is added (100 .mu.l/well) and
incubated for 20 minutes at room temperature on a plate shaker
until color development is sufficient for photometric
detection.
[0605] 10. The absorbance of the samples are measured at 410 nm (in
"dual wavelength" mode with a filter reading at 490 nm, as a
reference wavelength) on a Dynatech ELISA plate reader.
[0606] EGF-Induced BrdU Incorporation Assay
[0607] Materials and Reagents:
[0608] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).
[0609] 2. 3T3/EGFRc7.
[0610] Remaining Materials and Reagents and Procedure, as
above.
[0611] EGF-Induced Her-2-Driven BrdU Incorporation Assay
[0612] Materials and Reagents:
[0613] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).
[0614] 2. 3T3/EGFr/Her2/EGFr (EGFr with a Her-2 kinase domain).
[0615] Remaining Materials and Reagents and Procedure, as
above.
[0616] EGF-Induced Her-4-Driven BrdU Incorporation Assay
[0617] Materials and Reagents:
[0618] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).
[0619] 2. 3T3/EGFr/Her4/EGFr (EGFr with a Her-4 kinase domain).
[0620] Remaining Materials and Reagents and Procedure, as
above.
[0621] PDGF-Induced BrdU Incorporation Assay
[0622] Materials and Reagents:
[0623] 1. Human PDGF B/B (Boehringer Mannheim, Germany).
[0624] 2. 3T3/EGFRc7.
[0625] Remaining Materials and Reagents and Procedure, as
above.
[0626] FGF-Induced BrdU Incorporation Assay
[0627] Materials and Reagents:
[0628] 1. Human FGF2/bFGF (Gibco BRL, USA).
[0629] 2. 3T3c7/EGFr
[0630] Remaining Materials and Reagents and Procedure, as
above.
[0631] IGF1-Induced BrdU Incorporation Assay
[0632] Materials and Reagents:
[0633] 1. Human, recombinant (G511, Promega Corp., USA)
[0634] 2. 3T3/IGF1r.
[0635] Remaining Materials and Reagents and Procedure, as
above.
[0636] Insulin-Induced BrdU Incorporation Assay
[0637] Materials and Reagents:
[0638] 1. Insulin, crystalline, bovine, Zinc (13007, Gibco BRL,
USA).
[0639] 2. 3T3/H25.
[0640] Remaining Materials and Reagents and Procedure, as
above.
[0641] HGF-Induced BrdU Incorporation Assay
[0642] Materials and Reagents:
[0643] 1. Recombinant human HGF (Cat. No. 249-HG, R&D Systems,
Inc. USA).
[0644] 2. BxPC-3 cells (ATCC CRL-1687).
[0645] Remaining Materials and Reagents, as above.
[0646] Procedure:
[0647] 1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a
96 well plate. Cells are incubated overnight at 37.degree. C. in 5%
CO.sub.2.
[0648] 2. After 24 hours, the cells are washed with PBS, and then
are serum starved in 100 .mu.l serum-free medium (RPMI with 0.1%
BSA) for 24 hours.
[0649] 3. On day 3, 25 .mu.l containing ligand (prepared at 1
.mu.g/ml in RPMI with 0.1% BSA; final HGF conc. is 200 ng/ml) and
test compounds are added to the cells. The negative control wells
receive 25 .mu.l serum-free RPMI with 0.1% BSA only; the positive
control cells receive the ligand (HGF) but no test compound. Test
compounds are prepared at 5 times their final concentration in
serum-free RPMI with ligand in a 96 well plate, and serially
diluted to give 7 test concentrations. Typically, the highest final
concentration of test compound is 100 .mu.M, and 1:3 dilutions are
used (i.e. final test compound concentration range is 0.137-100
.mu.M).
[0650] 4. After 18 hours of ligand activation, 12.5 .mu.l of
diluted BrdU labeling reagent (1:100 in RPMI, 0.1% BSA) is added to
each well and the cells are incubated with BrdU (final
concentration is 10 .mu.M) for 1 hour.
[0651] 5. Same as General Procedure.
[0652] 6. Same as General Procedure.
[0653] 7. The blocking solution is removed by decanting and the
wells are washed once with PBS. Anti-BrdU-POD solution (1:100
dilution in PBS, 1% BSA) is added (100 .mu.l/well) and the plate is
incubated for 90 minutes at room temperature on a plate shaker.
[0654] 8. Same as General Procedure.
[0655] 9. Same as General Procedure.
[0656] 10. Same as General Procedure.
[0657] Exponential BrdU Incorporation Assay
[0658] This assay is used to measure the proliferation (DNA
synthesis) of exponentially growing A431 cells. The assay will
screen for compounds that inhibit cell cycle progression.
[0659] Materials and Reagents:
[0660] Healthy growing A431 cells. The remainder of the Materials
and Reagents are the same as listed above in the general protocol
section.
[0661] Procedure:
[0662] 1. A431 cells are seeded at 8000 cells/well in 10% FBS, 2 mM
Gln in DMEM, on a 96-well plate. Cells are incubated overnight at
37.degree. C. in 5% CO.sub.2.
[0663] 2. On day 2, test compounds are serially diluted to 7 test
concentrations in the same growth medium on a 96-well plate and
then are added to the cells on a 96-well tissue culture plate.
[0664] 3. After 20-24 hours of incubation, diluted BrdU labeling
reagent (1:100 in DMEM, 0.1% BSA) is added and the cells are
incubated with BrdU (final concentration is 10 .mu.M) for 2 hours.
Steps 5-10 of the General Procedure are used to complete the
assay.
[0665] ZenSrc Assay
[0666] This assay is used to screen for inhibitors of the tyrosine
kinase Src.
[0667] Materials and Reagents:
[0668] 1. Coating buffer: PBS containing sodium azide (0.2
mg/ml).
[0669] 2. 1% w/v BSA in PBS.
[0670] 3. Wash buffer: PBS containing 0.05% v/v Tween 20
(PBS-TWEEN)
[0671] 4. 500 mM HEPES pH 7.4.
[0672] 5. ATP (40 .mu.M)+MgCl.sub.2 (80 mM) in distilled water.
[0673] 6. MgCl.sub.2 (80 mM) in distilled water (for no ATP
blanks).
[0674] 7. Test compounds, 10 mM in DMSO.
[0675] 8. Assay Buffer: 100 mM HEPES, pH 7.4, containing 2 mM DTT,
0.2 mM sodium orthovanadate and 0.2 mgs/ml BSA.
[0676] 9. Partially purified recombinant human Src (UBI
(14-117)
[0677] 10. Anti-phosphotyrosine (SUGEN rabbit polyclonal
anti-PY).
[0678] 11. HRP-linked goat anti-rabbit Ig (Biosource International
#6430)
[0679] 12. HRP substrate ABTS or Pierce Peroxidase substrate.
[0680] 13. Corning ELISA plates.
[0681] Procedure:
[0682] 1. Coat plates with 100 .mu.l of 20 .mu.g/ml poly(Glu-Tyr)
(Sigma Cat. No. P0275) containing 0.01% sodium azide. Hold
overnight at 4.degree. C.
[0683] 2. Block with 1% BSA at 100 .mu.l/well for one hour at room
temperature.
[0684] 3. Plate test compounds (10 mM in DMSO) at 2 ul/well on a
Costar plate ready for dilution with dH.sub.2O and plating to
reaction plates.
[0685] 4. Dilute Src kinase 1:10,000 in Reaction Buffer, for 5
plates prepare 25 ml as follows: 2.5 mls 1M HEPES pH 7.4 (stored
sterile at 4.degree. C.), 21.85 ml distilled water, 0.1 ml 5% BSA,
0.5 ml 10 mM sodium orthovanadate (stored sterile at 4.degree. C.),
50 .mu.l 1.0M DTT (stored frozen at -20.degree. C.), and 2.5 .mu.l
Src Kinase (stored frozen at -80.degree. C.).
[0686] 5. Add 48 .mu.l of distilled water to the 2 .mu.l of each
compound in the dilution plate then add 25 .mu.l/well of this to
the reaction plate.
[0687] 6. Add 50 .mu.l of HRP to each reaction buffer well and then
25 .mu.l ATP-MgCl.sub.2/well (MgCl.sub.2 only to no ATP blanks).
Incubate at room temperature for 15 minutes on plate shaker. Stop
reaction by adding 25 .mu.l of 0.5M EDTA to each well.
[0688] 7. Wash 4.times. with PBS-TWEEN.
[0689] 8. Add 100 .mu.l anti-phosphotyrosine (1:10,000 of anti-pTyr
serum or 1:3,000 of 10% glycerol diluted PA-affinity purified
antibody) in PBS-TWEEN containing 0.5% BSA, 0.025% Non-fat milk
powder and 100 .mu.M sodium orthovanadate. Incubate with continuous
shaking at room temperature for one hour.
[0690] 9. Wash plates 4.times. with PBS-TWEEN.
[0691] 10. Add 100 .mu.l HRP-linked Ig (1:5,000) in PBS-TWEEN
containing 0.5% BSA, 0.025% Non-fat milk powder, 100 .mu.M sodium
orthovanadate. Incubate with shaking at room temperature for one
hour.
[0692] 11. Wash plates 4.times. with PBS-TWEEN and then once with
PBS.
[0693] 12. Develop plate using ABTS or other peroxidase
substrate.
[0694] Cell Cycle Analysis:
[0695] A431 cells in standard growth medium are exposed to a
desired concentration of a test compound for 20-24 hours at
37.degree. C. The cells are then collected, suspended in PBS, fixed
with 70% ice-cold methanol and stained with propidium iodide. The
DNA content is then measured using a FACScan flow cytometer. Cell
cycle phase distribution can then be estimated using CellFIT
software (Becton-Dickinson).
[0696] HUV-EC-C Assay
[0697] This assay is used to measure a compound's activity against
PDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally
expressed by HUV-EC cells.
[0698] Day 0
[0699] 1. Wash and trypsinize HUV-EC-C cells (human umbilical vein
endothelial cells, (American Type Culture Collection, catalogue no.
1730 CRL). Wash with Dulbecco's phosphate-buffered saline (D-PBS,
obtained from Gibco BRL, catalogue no. 14190-029) 2 times at about
1 ml/10 cm.sup.2 of tissue culture flask. Trypsinize with 0.05%
trypsin-EDTA in non-enzymatic cell dissociation solution (Sigma
Chemical Company, catalogue no. C-1544). The 0.05% trypsin is made
by diluting 0.25% trypsin/1 mM EDTA (Gibco, catalogue no.
25200-049) in the cell dissociation solution. Trypsinize with about
1 ml/25-30 cm.sup.2 of tissue culture flask for about 5 minutes at
37.degree. C. After cells have detached from the flask, add an
equal volume of assay medium and transfer to a 50 ml sterile
centrifuge tube (Fisher Scientific, catalogue no. 05-539-6).
[0700] 2. Wash the cells with about 35 ml assay medium in the 50 ml
sterile centrifuge tube by adding the assay medium, centrifuge for
10 minutes at approximately 200.times. g, aspirate the supernatant,
and resuspend with 35 ml D-PBS. Repeat the wash two more times with
D-PBS, resuspend the cells in about 1 ml assay medium/15 cm.sup.2
of tissue culture flask. Assay medium consists of F12K medium
(Gibco BRL, catalogue no. 21127-014) and 0.5% heat-inactivated
fetal bovine serum. Count the cells with a Coulter Counter.RTM.
(Coulter Electronics, Inc.) and add assay medium to the cells to
obtain a concentration of 0.8-1.0.times.10.sup.5 cells/ml.
[0701] 3. Add cells to 96-well flat-bottom plates at 100 .mu.l/well
or 0.8-1.0.times.10.sup.4 cells/well, incubate .about.24 h at
37.degree. C., 5% CO.sub.2.
[0702] Day 1
[0703] 1. Make up two-fold test compound titrations in separate
96-well plates, generally 50 .mu.M on down to 0 .mu.M. Use the same
assay medium as mentioned in day 0, step 2 above. Titrations are
made by adding 90 .mu.l/well of test compound at 200 .mu.M
(4.times. the final well concentration) to the top well of a
particular plate column. Since the stock test compound is usually
20 mM in DMSO, the 200 .mu.M drug concentration contains 2%
DMSO.
[0704] A diluent made up to 2% DMSO in assay medium (F12K+0.5%
fetal bovine serum) is used as diluent for the test compound
titrations in order to dilute the test compound but keep the DMSO
concentration constant. Add this diluent to the remaining wells in
the column at 60 .mu.l/well. Take 60 .mu.l from the 120 .mu.l of
200 .mu.M test compound dilution in the top well of the column and
mix with the 60 .mu.l in the second well of the column. Take 60
.mu.l from this well and mix with the 60 .mu.l in the third well of
the column, and so on until two-fold titrations are completed. When
the next-to-the-last well is mixed, take 60 .mu.l of the 120 .mu.l
in this well and discard it. Leave the last well with 60 .mu.l of
DMSO/media diluent as a non-test compound-containing control. Make
9 columns of titrated test compound, enough for triplicate wells
each for: (1) VEGF (obtained from Pepro Tech Inc., catalogue no.
100-200, (2) endothelial cell growth factor (ECGF) (also known as
acidic fibroblast growth factor, or aFGF) (obtained from Boehringer
Mannheim Biochemica, catalogue no. 1439 600), or, (3) human PDGF
B/B (1276-956, Boehringer Mannheim, Germany) and assay media
control. ECGF comes as a preparation with sodium heparin.
[0705] 2. Transfer 50 .mu.l/well of the test compound dilutions to
the 96-well assay plates containing the 0.8-1.0.times.10.sup.4
cells/100 .mu.l/well of the HUV-EC-C cells from day 0 and incubate
.about.2 h at 37.degree. C., 5% CO.sub.2.
[0706] 3. In triplicate, add 50 .mu.l/well of 80 .mu.g/ml VEGF, 20
ng/ml ECGF, or media control to each test compound condition. As
with the test compounds, the growth factor concentrations are
4.times. the desired final concentration. Use the assay media from
day 0 step 2 to make the concentrations of growth factors. Incubate
approximately 24 hours at 37.degree. C., 5% CO.sub.2. Each well
will have 50 .mu.l test compound dilution, 50 .mu.l growth factor
or media, and 100 .mu.l cells, which calculates to 200 .mu.l/well
total. Thus the 4.times. concentrations of test compound and growth
factors become 1.times. once everything has been added to the
wells.
[0707] Day 2
[0708] 1. Add .sup.3H-thymidine (Amersham, catalogue no. TRK-686)
at 1 .mu.Ci/well (10 .mu.l/well of 100 .mu.Ci/ml solution made up
in RPMI media+10% heat-inactivated fetal bovine serum) and incubate
.about.24 h at 37.degree. C., 5% CO.sub.2. RPMI is obtained from
Gibco BRL, catalogue no. 11875-051.
[0709] Day 3
[0710] 1. Freeze plates overnight at -20.degree. C.
[0711] Day 4
[0712] Thaw plates and harvest with a 96-well plate harvester
(Tomtec Harvester 96.RTM.) onto filter mats (Wallac, catalogue no.
1205-401), read counts on a Wallac Betaplate.TM. liquid
scintillation counter.
[0713] Vascular Permeability Assay
[0714] Increased vascular permeability in tumor-dependent
angiogenesis is due to a loosening of gap junctions in response to
vascular endothelial growth factor (VEGF). The Miles assay for
vascular permeability (Miles and Miles, J. Physiol. 118: 228-257
(1952)) has been adapted to athymic mice in order to evaluate the
ability of the compounds of the present invention to inhibit
VEGF-induced vascular permeability in vivo.
[0715] General Procedure:
[0716] Test compound or vehicle is administered prior to (typically
it is 4 hours prior) to VEGF injection. 100 .mu.l of 0.5% Evan's
blue dye in PBS is injected intravenously via lateral tail vein
injections using a 27 gauge needle. Sixty minutes later, animals
are anesthetized using the inhalant Isofluorane. Following
anesthesia, VEGF (100 ng of VEGF in 20 .mu.l of PBS) is injected
intradermally in two spots and PBS (20 .mu.l) is injected in two
spots in a grid pattern in the back of each animal. At a designated
timepoint of up to 1 hour after VEGF injection, the animals are
euthanized by CO.sub.2 and the skin patches are dissected and
photographed. Based on a published report (Alicieri et al., Mol.
Cell 4: 915-914 (1999)) quantitative evaluation of the
VEGF-dependent dye leakage into mouse skin can be achieved
following elution of the dye from skin patches.
[0717] In vivo Animal Models
[0718] Xenograft Animal Models
[0719] The ability of human tumors to grow as xenografts in athymic
mice (e.g., Balb/c, nu/nu) provides a useful in vivo model for
studying the biological response to therapies for human tumors.
Since the first successful xenotransplantation of human tumors into
athymic mice, (Rygaard and Povlsen, 1969, Acta Pathol. Microbial.
Scand. 77:758-760), many different human tumor cell lines (e.g.,
mammary, lung, genitourinary, gastro-intestinal, head and neck,
glioblastoma, bone, and malignant melanomas) have been transplanted
and successfully grown in nude mice. The following assays may be
used to determine the level of activity, specificity and effect of
the different compounds of the present invention. Three general
types of assays are useful for evaluating compounds:
cellular/catalytic, cellular/biological and in vivo. The object of
the cellular/catalytic assays is to determine the effect of a
compound on the ability of a TK to phosphorylate tyrosines on a
known substrate in a cell. The object of the cellular/biological
assays is to determine the effect of a compound on the biological
response stimulated by a TK in a cell. The object of the in vivo
assays is to determine the effect of a compound in an animal model
of a particular disorder such as cancer.
[0720] Suitable cell lines for subcutaneous xenograft experiments
include C6 cells (glioma, ATCC #CCL 107), A375 cells (melanoma,
ATCC #CRL 1619), A431 cells (epidermoid carcinoma, ATCC #CRL 1555),
Calu 6 cells (lung, ATCC #HTB 56), PC3 cells (prostate, ATCC #CRL
1435), SKOV3TP5 cells and NIH 3T3 fibroblasts genetically
engineered to overexpress EGFR, PDGFR, IGF-1R or any other test
kinase. The following protocol can be used to perform xenograft
experiments:
[0721] Female athymic mice (BALB/c, nu/nu) are obtained from
Simonsen Laboratories (Gilroy, Calif.). All animals are maintained
under clean-room conditions in Micro-isolator cages with Alpha-dri
bedding. They receive sterile rodent chow and water ad libitum.
[0722] Cell lines are grown in appropriate medium (for example,
MEM, DMEM, Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum
(FBS) and 2 mM glutamine (GLN)). All cell culture media, glutamine,
and fetal bovine serum are purchased from Gibco Life Technologies
(Grand Island, N.Y.) unless otherwise specified. All cells are
grown in a humid atmosphere of 90-95% air and 5-10% CO.sub.2 at
37.degree. C. All cell lines are routinely subcultured twice a week
and are negative for mycoplasma as determined by the Mycotect
method (Gibco).
[0723] Cells are harvested at or near confluency with 0.05%
Trypsin-EDTA and pelleted at 450.times. g for 10 min. Pellets are
resuspended in sterile PBS or media (without FBS) to a particular
concentration and the cells are implanted into the hindflank of the
mice (8-10 mice per group, 2-10.times.10.sup.6 cells/animal). Tumor
growth is measured over 3 to 6 weeks using venier calipers. Tumor
volumes are calculated as a product of
length.times.width.times.height unless otherwise indicated. P
values are calculated using the Students t-test. Test compounds in
50-100 .mu.L excipient (DMSO, or VPD:D5W) can be delivered by IP
injection at different concentrations generally starting at day one
after implantation.
[0724] Tumor Invasion Model
[0725] The following tumor invasion model has been developed and
may be used for the evaluation of therapeutic value and efficacy of
the compounds identified to selectively inhibit KDR/FLK-1
receptor.
[0726] Procedure
[0727] 8 week old nude mice (female) (Simonsen Inc.) are used as
experimental animals. Implantation of tumor cells can be performed
in a laminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail
(100 mg/kg ketamine and 5 mg/kg Xylazine) are administered
intraperitoneally. A midline incision is done to expose the
abdominal cavity (approximately 1.5 cm in length) to inject
10.sup.7 tumor cells in a volume of 100 .mu.l medium. The cells are
injected either into the duodenal lobe of the pancreas or under the
serosa of the colon. The peritoneum and muscles are closed with a
6-0 silk continuous suture and the skin is closed by using wound
clips. Animals are observed daily.
[0728] Analysis
[0729] After 2-6 weeks, depending on gross observations of the
animals, the mice are sacrificed, and the local tumor metastases to
various organs (lung, liver, brain, stomach, spleen, heart, muscle)
are excised and analyzed (measurement of tumor size, grade of
invasion, immunochemistry, in situ hybridization determination,
etc.).
[0730] Additional Assays
[0731] Additional assays which may be used to evaluate the
compounds of this invention include, without limitation, a
bio-flk-1 assay, an EGF receptor-HER2 chimeric receptor assay in
whole cells, a bio-src assay, a bio-lck assay and an assay
measuring the phosphorylation function of raf. The protocols for
each of these assays may be found in U.S. application Ser. No.
09/099,842, which is incorporated by reference, including any
drawings, herein. Additionally, U.S. Pat. No. 5,792,783, filed Jun.
5, 1996 and U.S. application Ser. No. 09/322,297, filed May 28,
1999 are incorporated by reference as if fully set forth
herein.
[0732] Measurement of Cell Toxicity
[0733] Therapeutic compounds should be more potent in inhibiting
receptor tyrosine kinase activity than in exerting a cytotoxic
effect. A measure of the effectiveness and cell toxicity of a
compound can be obtained by determining the therapeutic index,
i.e., IC.sub.5/LD.sub.50. IC.sub.50, the dose required to achieve
50% inhibition, can be measured using standard techniques such as
those described herein. LD.sub.50, the dosage which results in 50%
toxicity, can also be measured by standard techniques as well
(Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring the
amount of LDH released (Korzeniewski and Callewaert, 1983, J.
Immunol. Methods, 64:313, Decker and Lohmann-Matthes, 1988, J.
Immunol. Methods, 115:61), or by measuring the lethal dose in
animal models. Compounds with a large therapeutic index are
preferred. The therapeutic index should be greater than 2,
preferably at least 10, more preferably at least 50.
[0734] Plasma Stability Test:
[0735] The prodrug
(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-
-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one was administered IV
at 2 mg/mL to dogs. Levels of both prodrug and drug
(3(Z)-3-[(3,5-dimethyl-1H--
pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one) were followed
by HPLC analysis of blood plasma for 4 hours following dosing. This
study showed that the half-life for conversion of the prodrug to
drug was 7.3 min. From a plot of drug concentration vs. time, the
area under the curve indicated that 80% of the prodrug was
converted into drug.
Formulation Examples
[0736] The formulations being evaluated are listed in Tables 1 and
2 and are described below:
[0737] [A] Solid Formulations to be Reconstituted to a Stable
Infusate (Table 1):
[0738] (1) Lyophilized Formulation:
[0739] (a) Captisol Based: This formulation uses Captisol and an
acidic agent to form an in situ salt at a pH of 1.5-2.0 to compound
and lyophilize solutions of drug at concentrations of 20.0-25.0
mg/mL. The lyophilized cake is reconstituted with an IV fluid to
provide a stable infusate at 2 mg/mL or higher at pH 3.
[0740] (b) Non-Captisol based: This formulation uses small amounts
of a surfactant such as Polysorbate-80 or Cremophor EL and an
acidic agent to form an in-situ salt at a pH of 1.5-2.0 to compound
and lyophilize solutions of drug at concentrations of 20.0-25.0
mg/mL. The lyophilized cake is reconstituted with
cosolvent-surfactant based aqueous diluent such as
PEG-300-Polysorbate 80 or PEG-300-Cremophor EL to provide a stable
infusate at 2 mg/mL or higher at pH 3.
[0741] (2) Sterile API Fill:
[0742] The drug is filled as a sterile powder fill in a container
and will be reconstituted with a specific co-solvent-surfactant
based aqueous diluent to provide a stable infusate at 2 mg/mL or
higher of drug at pH 3.
[0743] [B] Solution Concentrate to be Diluted to a Stable Infusate
(Table 2):
[0744] The drug is solubilized in a non-aqueous mixture of
co-solvents and surfactants at a high concentration such that it
can be diluted with aqueous diluents to a stable infusate. The
concentration of the drug in the infusate is at a concentration of
2 mg/mL or higher, at pH 3. The total levels of the co-solvent is
less than 15% and the levels of surfactant is than 0.5%.
1TABLE 1 Formulation (1) Solid formulations Attributes
Lyophilized-Captisol based Lyophilized Non-Captisol based Sterile
API Fill Dose/50 CC 200-300 200-300 300-400 vial (mgs) Sterile API
fill NA NA 300-400 mg in vial Composition- drug (mg) 200-300 drug
(mg) 200-300 NA Lyophilized Acid (M) 1.4 Acid (M) 1.4 Cake
Antioxidant (mg) 0-10 Antioxidant (mg) 0-10 Captisol (mg) 2000-3000
Filler (mg) 200-300 Polysorbate-80 (mg) 0-50 Composition- 0.9%
NaCl, D5W, PEG-300 (% w/v) 5-20 PEG-300 (% w/v) 5-20 Reconstitution
Polysorbate-80 (% w/v) 0-1.0 Polysorbate-80 (% w/v) 0-1.0 Fluid
Citrate Buffer pH 3.0 Citrate Buffer pH 3.0 0.1 M (% w/v) 30-40 0.1
M (% w/v) 30-40 Water (qs to volume) Water (qs to volume)
Composition- drug (mg/mL) 2-3 drug (mg/mL) 2-3 drug (mg/mL) 2-3
Resconstituted Acid (Molar) 1.4 Acid (Molar) 1.4 Acid (Molar) 1.4
Infusate Antioxidant (mg/mL) 0-1.0 Antioxidant (mgmL) 0-1.0
Antioxidant (mg/mL) 0-1.0 (Administered Captisol (mg/mL) 20-30
Filler (mg/mL) 2-3 Filler (mg/mL) 2-3 to Patient) IV fluid (qs to
volume) PEG-300 (mg/mL) 50-200 PEG-300 (mg/mL) 50-200 pH 3.0
Polysorbate-80 (mg/mL) 0-10 Polysorbate-80 (mg/mL) 0-10 Citrate
Buffer 0.3 Citrate Buffer 0.3 0.1 M, pH 3.0 (mL) 0.1 M, pH 3.0 (mL)
Water (qs to volume) Water (qs to volume) pH 3.0 pH 3.0
Composition- drug (mg/mL) 20-30 drug (mg/mL) 20-30 NA Prelyophilate
Acid (Molar) 1.4 Acid (Molar) 1.4 Antioxidant (mg/mL) 0-10
Antioxidant (mg/mL) 0-10 Captisol (mg/mL) 200-300 Filler (mg/mL)
20-30 Water for Injection qs to 1.0 mL Polysorbate-80 (mg) 0-50 pH
1.5-2.0 Water for Injection qs to 1.0 mL pH 1.5-2.0 Acids used:
Methane sulfonic acid, Tartaric acid, Citric acid, succinic acid is
used in a 1:1.4 molar ratio for in situ salt formation
Cosolvents-PEG-300, PEG-400 Surfactants: Polysorbate-80, Cremophor
EL Captisol .RTM.: Sulfobutylether Cyclodextrin Drug: Compound of
Example 2
[0745]
2TABLE 2 Composition of Solution Formulation Composition
Ingredients (% w/v) or mg/mL Drug 1.2-2.5 (12-25 mg/mL)
Cosolvent-(PEG-300, PEG-400) 70-90 Surfactant 0-10
Dimethylacetamide 0-2 Salt forming agent (Methane Equivalent to 1.4
M ratio of sulfonic acid, Tartaric acid, the API Citric Acid,
Succinic Acid) Alcohol Qs to Volume
[0746] Formulation to be diluted 10 fold with pharmaceutically
acceptable IV fluids.
[0747] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the inventions, and any clones, DNA or amino acid
sequences which are functionally equivalent are within the scope of
the invention. Indeed, various modifications of the invention in
addition to those described herein will become apparent to those
skilled in the art from the foregoing description and accompanying
drawings. Such modifications are intended to fall within the scope
of the appended claims.
[0748] All references cited herein are hereby incorporated by
reference in their entirety.
* * * * *