U.S. patent application number 10/395810 was filed with the patent office on 2004-04-15 for methods for treating inflammatory conditions or inhibiting jnk.
Invention is credited to Bennett, Brydon L., Bhagwat, Shripad S., Manning, Anthony M., Murray, Brion W., O'Leary, Eoin C., Satoh, Yoshitaka.
Application Number | 20040072888 10/395810 |
Document ID | / |
Family ID | 33096784 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072888 |
Kind Code |
A1 |
Bennett, Brydon L. ; et
al. |
April 15, 2004 |
Methods for treating inflammatory conditions or inhibiting JNK
Abstract
This invention generally relates to methods for treating or
preventing an inflammatory disease or disorder comprising
administering to a patient in need thereof an effective amount of a
Pyrazoloanthrone Derivative having the following structure: 1 or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 and
R.sub.2 are as defined herein.
Inventors: |
Bennett, Brydon L.; (San
Diego, CA) ; Bhagwat, Shripad S.; (San Diego, CA)
; Manning, Anthony M.; (San Diego, CA) ; Murray,
Brion W.; (San Diego, CA) ; O'Leary, Eoin C.;
(San Diego, CA) ; Satoh, Yoshitaka; (San Diego,
CA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST STREET
NEW YORK
NY
10017
US
|
Family ID: |
33096784 |
Appl. No.: |
10/395810 |
Filed: |
March 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10395810 |
Mar 24, 2003 |
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09642557 |
Aug 18, 2000 |
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60240928 |
Aug 19, 1999 |
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Current U.S.
Class: |
514/406 ;
548/358.5 |
Current CPC
Class: |
A61P 9/02 20180101; A61P
25/32 20180101; C07D 231/54 20130101; A61P 11/00 20180101; A61P
1/16 20180101; A61P 25/00 20180101; A61P 31/04 20180101; A61P 1/00
20180101; A61P 25/16 20180101; A61P 9/08 20180101; A61P 3/10
20180101; A61P 25/28 20180101; A61P 29/00 20180101; A61P 1/18
20180101; A61P 9/00 20180101; C07D 409/12 20130101; A61P 17/00
20180101; A61P 19/00 20180101; A61P 3/04 20180101; A61P 11/02
20180101; A61P 19/06 20180101; A61P 43/00 20180101; A61P 35/00
20180101; A61P 25/02 20180101; A61P 13/12 20180101; A61P 37/08
20180101; A61P 39/02 20180101; A61P 37/06 20180101; A61P 3/06
20180101; A61P 13/08 20180101; A61P 17/02 20180101; A61K 31/416
20130101; A61P 11/06 20180101; A61P 27/00 20180101; A61P 37/00
20180101; C07D 401/12 20130101; A61P 19/02 20180101; A61P 17/06
20180101; A61P 1/04 20180101; A61P 7/12 20180101; A61P 25/08
20180101; A61P 21/04 20180101; A61P 9/10 20180101; A61P 25/14
20180101; A61P 7/02 20180101 |
Class at
Publication: |
514/406 ;
548/358.5 |
International
Class: |
C07D 231/54; A61K
031/416 |
Claims
What is claimed is:
1. A method for the treating or preventing an inflammatory
condition, comprising administering to a patient in need thereof an
effective amount of a compound having the structure: 66or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 and
R.sub.2 are absent or present and independently represent alkyl
halogen, nitro, trifluoromethyl, sulfonyl, carboxyl,
alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy, arylalkyl,
cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy,
aminoalkoxy, mono- or di-alkylaminoalkoxy, or a group represented
by formula (a), (b), (c) or (d): 67R.sub.3 and R.sub.4 taken
together represent alkylidene or a heteroatom-containing
alkylidene, or R.sub.3 and R.sub.4 are the same or different and
independently represent hydrogen, alkyl, cycloalkyl, aryl,
arylalkyl, cycloalkylalkyl, aryloxyalkyl, alkoxyalkyl, alkoxyamono,
or alkoxy(mono- or di-alkylamino): and R.sub.5 represents hydrogen,
alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl, alkoxy, amino,
mono- or di-alkylamino, arylamino, arylalkylamino, cycloalkylamino,
or cycloalkylalkylamino.
2. The method of claim 1 wherein the treating or preventing
comprises inhibiting JNK in vivo.
3. The method of claim 2 wherein inhibiting JNK in vivo comprises
inhibiting TNF-.alpha. in vivo.
4. The method of claim 1 wherein the inflammatory condition is Type
II diabetes or obesity.
5. The method of claim 1 wherein the inflammatory condition is
rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gout,
asthma, bronchitis, allergic rhinitis, inflammatory bowel disease,
irritable bowel syndrome, mucous colitis, ulcerative colitis,
Crohn's disease, gastritis, esophagitis, transplant rejection,
endotoxin shock, psoriasis, eczema, dermatitis or multiple
sclerosis.
6. The method of claim 1 wherein the inflammatory condition is
hereditary obesity, dietary obesity, hormone related obesity or
obesity related to the administration of medication.
7. The method of claim 1 wherein the inflammatory condition is Type
I diabetes, diabetes insipidus, diabetes mellitus, maturity-onset
diabetes, juvenile diabetes, insulin-dependant diabetes,
non-insulin dependant diabetes, malnutrition-related diabetes,
ketosis-prone diabetes or ketosis-resistant diabetes.
8. The method of claim 1 wherein the inflammatory condition is
otitis externa, acute otitis media, hearing loss, systemic lupus
erythematosus, pancreatitis, peritonitis, sepsis, alcohol or
toxin-induced liver disease, sclerosis, steatosis,
ischemia-reperfusion injury, allergic rhinitis, an allergy,
nephropathy, acute respiratory distress syndrome, chronic
obstructive pulmonary disease, pulmonary interstitial fibrosis,
liver fibrosis, renal fibrosis, wound-healing or burn-healing.
9. The method of claim 1 wherein the compound has the following
structure: 68or a pharmaceutically acceptable salt thereof.
10. The method of claim 1 wherein the compound has one of the
following structures: 69or a pharmaceutically acceptable salt
thereof.
11. The method of claim 1 wherein the compound has one of the
following structures: 70or a pharmaceutically acceptable salt
thereof.
12. The method of claim 11 wherein in the compound or
pharmaceutically acceptable salt thereof, R.sub.1 and R.sub.2 are:
71
13. The method of claim 11 wherein in the compound or
pharmaceutically acceptable salt thereof, R.sub.1 and R.sub.2 are:
72
14. The method of claim 11 wherein in the compound or
pharmaceutically acceptable salt thereof, R.sub.1 and R.sub.2 are:
73
15. The method of claim 11 wherein in the compound or
pharmaceutically acceptable salt thereof, R.sub.1 and R.sub.2 are:
74
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/642,557, filed Aug. 18, 2000, which claims
the benefit of U.S. Provisional Application No. 60/240,928, filed
Aug. 19, 1999, each of which is incorporated by reference herein in
its entirety.
1. FIELD OF THE INVENTION
[0002] This invention is generally directed to Pyrazoloanthrones
and derivatives thereof which have utility over a wide range of
indications, including activity as Jun N-terminal kinase
inhibitors, and related compositions and methods.
2. BACKGROUND OF THE INVENTION
[0003] The Jun N-terminal kinase (JNK) pathway is activated by
exposure of cells to environmental stress or by treatment of cells
with pro-inflammatory cytokines. Targets of the JNK pathway include
the transcription factors c-jun and ATF2 (Whitmarsh A. J., and
Davis R. J. J. Mol. Med. 74:589-607, 1996). These transcription
factors are members of the basic leucine zipper (bZIP) group that
bind as homo- and hetero-dimeric complexes to AP-1 and AP-1-like
sites in the promoters of many genes (Karin M., Liu Z. G. and Zandi
E. Curr Opin Cell Biol 9:240-246, 1997). JNK binds to the
N-terminal region of c-jun and ATF-2 and phosphorylates two sites
within the activation domain of each transcription factor (Hibi M.
Lin A., Smeal T., Minden A., Karin M. Genes Dev. 7:2135-2148, 1993;
Mohit A. A., Martin M. H., and Miller C. A. Neuron 14:67-78, 1995].
Three JNK enzymes have been identified as products of distinct
genes (Hibi et al, supra; Mohit et al., supra). Ten different
isoforms of JNK have been identified. These represent alternatively
spliced forms of three different genes: JNK1, JNK2 and JNK3. JNK1
and 2 are ubiquitously expressed in human tissues, whereas JNK3 is
selectively expressed in the brain, heart and testis (Dong, C.,
Yang, D., Wysk. M., Whitmarsh, A., Davis, R., Flavell, R. Science
270:1-4, 1998). Gene transcripts are alternatively spliced to
produce four-JNK1 isoforms, four JNK2 isoforms and two-JNK3
isoforms. JNK1 and 2 are expressed widely in mammalian tissues,
whereas JNK3 is expressed almost exclusively in the brain.
Selectivity of JNK signaling is achieved via specific interactions
of JNK pathway components and by use of scaffold proteins that
selectively bind multiple components of the signaling cascade.
JIP-1 (JNK-interacting protein-1) selectively binds the MAPK
module, MLK.fwdarw.JNKK1.fwdarw.JNK.12,13 It has no binding
affinity for a variety of other MAPK cascade enzymes. Different
scaffold proteins are likely to exist for other MAPK signaling
cascades to preserve substrate specificity.
[0004] JNKs are activated by dual phosphorylation on Thr-183 and
Tyr-185. JNKK1 (also known as MKK 4) and JNKK2 (MKK7), two MAPKK
level enzymes, can mediate JNK activation in cells (Lin A., Minden
A., Martinetto H., Claret F.-Z., Lange-Carter C., Mercurio F.,
Johnson G. L., and Karin M. Science 268:286-289, 1995; Toumier C.,
Whitmarsh A. J., Cavanagh J., Barrett T., and Davis R. J. Proc.
Nat. Acad. Sci. USA 94:7337-7342, 1997). JNKK2 specifically
phosphorylates JNK, whereas JNKK1 can also phosphorylate and
activate p38. Both JNKK1 and JNKK2 are widely expressed in
mammalian tissues. JNKK1 and JNKK2 are activated by the MAPKKK
enzymes, MEKK1 and 2 (Lange-Carter C. A., Pleiman C. M., Gardner A.
M., Blumer K. J., and Johnson G. L. Science 260:315-319, 1993; Yan
M., Dai J. C., Deak J. C., Kyriakis J. M., Zon L. I., Woodgett J.
R., and Templeton D. J. Nature 372:798-781, 1994). Both MEKK1 and
MEKK2 are widely expressed in mammalian tissues.
[0005] Activation of the JNK pathway has been documented in a
number of disease settings, providing the rationale for targeting
this pathway for drug discovery. In addition, molecular genetic
approaches have validated the pathogenic role of this pathway in
several diseases. For example, autoimmune and inflammatory diseases
arise from the over activation of the immune system. Activated
immune cells express many genes encoding inflammatory molecules,
including cytokines, growth factors, cell surface receptors, cell
adhesion molecules and degradative enzymes. Many of these genes are
regulated by the JNK pathway, through activation of the
transcription factors AP-1 and ATF-2, including TNF-.alpha., IL-2,
E-selectin and matrix metalloproteinases such as collagenase-1
(Manning A. M. and Mercurio F. Exp Opin Invest Drugs 6: 555-567,
1997; International Publication No. WO 99/53927, published Oct. 28,
1999). Monocytes, tissue macrophages and tissue mast cells are key
sources of TNF-.alpha. production. The JNK pathway regulates
TNF-.alpha. production in bacterial lipopolysaccharide-stimulated
macrophages, and in mast cells stimulated through the FceRII
receptor (Swantek J. L., Cobb M. H., Geppert T. D. Mol. Cell. Biol.
17:6274-6282, 1997; Ishizuka, T., Tereda N., Gerwins, P., Hamelmann
E., Oshiba A., Fanger G. R., Johnson G. L., and Gelfland E. W.
Proc. Nat. Acad. Sci. USA 94:6358-6363, 1997). Inhibition of JNK
activation effectively modulates TNF-.alpha. secretion from these
cells. The JNK pathway therefore regulates production of this key
proinflammatory cytokine. Matrix metalloproteinases (MMPs) promote
cartilage and bone erosion in rheumatoid arthritis, and generalized
tissue destruction in other autoimmune diseases. Inducible
expression of MMPs, including MMP-3 and MMP-9, type II and IV
collagenases, are regulated via activation of the JNK pathway and
AP-1 (Gum, R., Wang, H., Lengyel, E., Juarez, J., and Boyd, D.
Oncogene 14:1481-1493, 1997). In human rheumatoid synoviocytes
activated with TNF-.alpha., IL-1, or Fas ligand the JNK pathway is
activated (Han Z., Boyle D. L., Aupperle K. R., Bennett B., Manning
A. M., Firestein G. S. J. Pharm. Exp. Therap. 291:1-7, 1999;
Okamoto K., Fujisawa K., Hasunuma T., Kobata T., Sumida T., and
Nishioka K. Arth & Rheum 40: 919-92615, 1997). Inhibition of
JNK activation results in decreased AP-1 activation and
collagenase-1 expression (Han et al., supra). The JNK pathway
therefore regulates MMP expression in cells involved in rheumatoid
arthritis.
[0006] Inappropriate activation of T lymphocytes initiates and
perpetuates many autoimmune diseases, including asthma,
inflammatory bowel disease and multiple sclerosis. The JNK pathway
is activated in T cells by antigen stimulation and CD28 receptor
co-stimulation and regulates production of the growth factor IL-2
and cellular proliferation (Su B., Jacinto E., Hibi M., Kallunki
T., Karin M., Ben-Neriah Y. Cell 77:727-736, 1994; Faris M., Kokot
N., Lee L., and Nel A. E. J. Biol. Chem. 271:2736627373, 1996).
Peripheral T cells from mice genetically deficient in JNKK1 show
decreased proliferation and IL-2 production after CD28
co-stimulation and PMA/Ca2+ ionophore activation, providing
important validation for the role of the JNK pathway in these cells
(Nishina H., Bachmann M., Oliveria-dos-Santos A. J., et al. J. Exp.
Med. 186: 941-953, 1997). It is known that T cells activated by
antigen receptor stimulation in the absence of accessory
cell-derived co-stimulatory signals lose the capacity to synthesize
IL-2, a state called clonal anergy. This is an important process by
which auto-reactive T cell populations are eliminated from the
peripheral circulation. Of note, anergic T cells fail to activate
the JNK pathway in response to CD3- and CD28-receptor
co-stimulation, even though expression of the JNK enzymes is
unchanged (Li W., Whaley C. D., Mondino A., and Mueller D. L.
Science 271: 1272-1276, 1996). Recently, the examination of JNK
deficient mice revealed that the JNK pathway plays a key role in T
cell activation and differentiation to T helper 1 and 2 cell types.
JNK1 or JNK2 knockout mice develop normally and are phenotypically
unremarkable. Activated naive CD4+ T cells from these mice fail to
produce IL-2 and do not proliferate well (Sabapathy, K, Hu, Y,
Kallunki, T, Schreiber, M, David, J-P, Jochum, W, Wagner, E, Karin,
M. Curr Biol 9: 116-125, 1999). It is possible to induce T cell
differentiation in T cells from these mice, generating Th1 cells
(producers of IFN-g and TNF.beta. and Th2 effector cells (producers
of IL-4, IL-5, IL-6, IL-10 and IL-13) [22,23]. Deletion of either
JNK1 or JNK2 in mice resulted in a selective defect in the ability
of Th 1 effector cells to express IFNg. This suggests that JNK1 and
JNK2 do not have redundant functions in T cells and that they play
different roles in the, control of cell growth, differentiation and
death. The JNK pathway therefore, is an important point for
regulation of T cell responses to antigen.
[0007] Cardiovascular disease (CVD) accounts for nearly one quarter
of total annual deaths worldwide. Vascular disorders such as
atherosclerosis and restenosis result from dysregulated growth of
the vessel wall, restricting blood flow to vital organs. The JNK
pathway is activated by atherogenic stimuli and regulates local
cytokine and growth factor production in vascular cells (Yang, D D,
Conze, D, Whitmarsh, A J, et al, Immunity, 9:575, 1998). In
addition, alterations in blood flow, hemodynamic forces and blood
volume lead to JNK activation in vascular endothelium, leading to
AP-1 activation and pro-atherosclerotic gene expression (Aspenstrom
P., Lindberg U., and Hall A. Curr. Biol. 6:7077, 1996). Ischemia
and ischemia coupled with reperfusion in the heart, kidney or brain
results in cell death and scar formation, which can ultimately lead
to congestive heart failure, renal failure or cerebral dysfunction.
In organ transplantation, reperfusion of previously ischemic donor
organs results in acute leukocyte-mediated tissue injury and delay
of graft function. The JNK pathway is activated by ischemia and
reperfusion (Li Y., Shyy J., Li S., Lee J., Su B., Karin M., Chien
S Mol. Cell. Biol. 16:5947-5954, 1996), leading to the activation
of JNK-responsive genes and leukocyte-mediated tissue damage. In a
number of different settings JNK activation can be either pro- or
anti-apoptotic. JNK activation is correlated with enhanced
apoptosis in cardiac tissues following ischemia and reperfusion
(Pombo C M, Bonventre J V, Avruch J, Woodgett J R, Kyriakis J. M,
Force T. J. Biol. Chem. 269:26546-26551, 1994).
[0008] Cancer is characterized by uncontrolled growth,
proliferation and migration of cells. Cancer is the second leading
cause of death with 500,000 deaths and an estimated 1.3 million new
cases in the United States in 1996. The role of signal transduction
pathways contributing to cell transformation and cancer is a
generally accepted concept. The JNK pathway leading to AP-1 appears
to play a critical role in cancer. Expression of c-jun is altered
in early lung cancer and may mediate growth factor signaling in
non-small cell lung cancer (Yin T., Sandhu G., Wolfgang C. D.,
Burner A., Webb R. L., Rigel D. F. Hai T., and Whelan J. J. Biol.
Chem. 272:19943-19950, 1997). Indeed, over-expression of c-jun in
cells results in transformation, and blocking c-jun activity
inhibits MCF-7 colony formation (Szabo E., Riffe M., Steinberg S.
M., Birrer M. J., Linnoila R. I. Cancer Res. 56:305-315, 1996).
DNA-damaging agents, ionizing radiation and tumor necrosis factor
activate the JNK pathway. In addition to regulating c-jun
production and activity, JNK activation can regulate
phosphorylation of p53, and thus can modulate cell cycle
progression (Chen T. K., Smith L. M., Gebhardt D. K., Birrer M. J.,
Brown P. H. Mol. Carcinogenesis 15:215-226, 1996). The Oncogene
BCR-Abl, associated with t(9,22) Philadelphia chromosome
translocation of chronic myelogenous leukemia, activates JNK and
leads to transformation of hematopoietic cells (Milne D. M.,
Campbell L. E., Campbell D. G., Meek D. W. J. Biol. Chem.
270:5511-5518, 1995). Selective inhibition of JNK activation by a
naturally occurring JNK inhibitory protein, called JIP-1, blocks
cellular transformation caused by BCR-Abl expression (Raitano A.
B., Halpern J. R., Hambuch T. M., Sawyers C. L. Proc. Nat. Acad.
Sci USA 92:11746-11750, 1995). Thus, JNK inhibitors may block
transformation and tumor cell growth.
[0009] The involvement of JNK in insulin mediated diseases such as
Type II diabetes and obesity has also been confirmed (Hirosumi, J.
et al Nature 420:333-336, 2002; International Publication No. WO
02/085396). Without being limited by theory, it is thought that
phosphorylation at Ser 307 of insulin receptor substrate ("IRS-1")
is responsible for TNF-.alpha.-induced and FFA-induced insulin
resistance (Hotamisigil, G. H. Science 271:665-668, 1996). This was
demonstrated in a cellular model of insulin resistance in liver
cells where increased Ser 307 phosphorylation of IRS-1 was seen in
cells treated with TNF-.alpha. (Hirosumi, J. Id.). It was also
shown that the TNF-.alpha.-induced Ser 307 phosphorylation was
completely prevented by Compound 1 of the present invention (Id.).
Additional studies have demonstrated that inhibition of the JNK
pathway inhibits TNF-.alpha. lipolysis which has been implicated in
diseases characterized by insulin resistance (International
Publication No. WO 99/53927).
[0010] Accordingly, there is a need in the art for inhibitors of
JNK, as well as for methods for preparation thereof, pharmaceutical
compositions comprising such inhibitors, and methods of inhibiting
JNK's and treating diseases in mammals which are responsive to JNK
inhibition. The present invention fulfills these needs, and
provides further related advantages.
[0011] Citation of any reference in Section 2 of this application
is not an admission that the reference is prior art to the
application.
3. SUMMARY OF THE INVENTION
[0012] In brief, the present invention relates to methods for
treating or preventing a disease or disorder, comprising
administering to a patient in need thereof an effective amount of a
compound having the following formula (I): 2
[0013] and pharmaceutically acceptable salts thereof, wherein
R.sub.1 and R.sub.2 are as defined below.
[0014] A compound of formula (I), or a pharmaceutically acceptable
salt thereof, is hereinafter referred to as a "Pyrazoloanthrone
Derivative."
[0015] Pyrazoloanthrone Derivatives are useful for treating or
preventing an inflammatory condition including, but not limited to:
diabetes (such as Type II diabetes, Type I diabetes, diabetes
insipidus, diabetes mellitus, maturity-onset diabetes, juvenile
diabetes, insulin-dependant diabetes, non-insulin dependant
diabetes, malnutrition-related diabetes, ketosis-prone diabetes or
ketosis-resistant diabetes); nephropathy (such as
glomerulonephritis or acute/chronic kidney failure); obesity (such
as hereditary obesity, dietary obesity, hormone related obesity or
obesity related to the administration of medication); hearing loss
(such as that from otitis externa or acute otitis media); fibrosis
related diseases (such as pulmonary interstitial fibrosis, renal
fibrosis, cystic fibrosis, liver fibrosis, wound-healing or
burn-healing, wherein the burn is a first-, second- or third-degree
burn and/or a thermal, chemical or electrical burn); arthritis
(such as rheumatoid arthritis, rheumatoid spondylitis,
osteoarthritis or gout); an allergy; allergic rhinitis; acute
respiratory distress syndrome; asthma; bronchitis; an inflammatory
bowel disease (such as irritable bowel syndrome, mucous colitis,
ulcerative colitis, Crohn's disease, gastritis, esophagitis,
pancreatitis or peritonitis); or an autoimmune disease (such as
scleroderma, systemic lupus erythematosus, myasthenia gravis,
transplant rejection, endotoxin shock, sepsis, psoriasis, eczema,
dermatitis or multiple sclerosis).
[0016] Pyrazoloanthrone Derivatives are also useful for treating or
preventing a liver disease (such as hepatitis, alcohol-induced
liver disease, toxin-induced liver disease, steatosis or
sclerosis); a cardiovascular disease (such as atherosclerosis,
restenosis following angioplasty, left ventricular hypertrophy,
myocardial infarction, chronic obstructive pulmonary disease or
stroke); ischemic damage (such as to the heart, kidney, liver or
brain); ischemia-reperfusion injury (such as that caused by
transplant, surgical trauma, hypotension, thrombosis or trauma
injury); neurodegenerative disease (such as epilepsy, Alzheimer's
disease, Huntington's disease, Amyotrophic laterial sclerosis,
peripheral neuropathies, spinal cord damage or Parkinson's
disease); or cancer (such as cancer of the head, neck, eye, mouth,
throat, esophagus, chest, bone, lung, colon, rectum, stomach,
prostate, breast, ovaries, testicles or other reproductive organs,
skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, and
brain or central nervous system).
[0017] In one embodiment, the present methods for treating or
preventing further comprise the administration of an effective
amount of another therapeutic agent useful for treating or
preventing the diseases or disorders disclosed herein. In this
embodiment, the time in which the therapeutic effect of the other
therapeutic agent is exerted overlaps with the time in which the
therapeutic effect of the Pyrazoloanthrone Derivative is
exerted.
[0018] These and other aspects of this invention will be apparent
upon reference to the following detailed description. To that end,
certain patent and other documents are cited herein to more
specifically set forth various aspects of this invention. Each of
these documents are hereby incorporated by reference in their
entirety.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 illustrates the ability of a representative compound
of this invention to inhibit IL-2 in Jurkat T-Cell.
[0020] FIG. 2 illustrates the ability of a representative compound
of this invention to inhibit TNF-.alpha. in a mouse model of
endotoxin shock.
[0021] FIG. 3 illustrates the ability of a representative compound
of this invention to inhibit leukocyte recruitment in rat model for
inflamed lung.
[0022] FIG. 4 illustrates the ability of a representative compound
of this invention to inhibit paw swelling (FIG. 4A), joint
destruction (FIG. 4B), transcription factor AP-1 activation (FIG.
4C), and expression of MMP-13 (FIG. 4D) in a rat model for adjuvant
arthritis.
[0023] FIG. 5 illustrates the ability of a representative compound
of this invention to reduce kainic acid-induced seizure
response.
5. DETAILED DESCRIPTION OF THE INVENTION
[0024] 5.1. Definitions
[0025] As used herein, the terms used above having following
meaning.
[0026] "Alkyl" means a straight chain or branched, saturated or
unsaturated alkyl, cyclic or non-cyclic hydrocarbon having from 1
to 10 carbon atoms. Representative saturated straight chain alkyls
include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and
the like; while saturated branched alkyls include isopropyl,
sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
Unsaturated alkyls contain at least one double or triple bond
between adjacent carbon atoms (also referred to as an "alkenyl" or
"alkynyl", respectively). Representative straight chain and
branched alkenyls include ethylenyl, propylenyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like; while representative straight chain and branched alkynyls
include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,
2-pentynyl, 3-methyl-1 butynyl, and the like. Representative
saturated cyclic alkyls include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic
alkyls include cyclopentenyl and cyclohexenyl, and the like.
Cycloalkyls are also referred to herein as "carbocyclic" rings
systems, and include bi- and tri-cyclic ring systems having from 8
to 14 carbon atoms such as a cycloalkyl (such as cyclopentane or
cyclohexane) fused to one or more aromatic (such as phenyl) or
non-aromatic (such as cyclohexane) carbocyclic rings.
[0027] "Halogen" means fluorine, chlorine, bromine or iodine.
[0028] "Trifluoromethyl" means --CF.sub.3.
[0029] "Sulfonyl" means --SO.sub.3H. "Carboxyl" means --COOH.
[0030] "Alkoxy" means --O-(alkyl), such as methoxy, ethoxy,
n-propyloxy, isopropyloxy, n-butyloxy, iso-butyloxy, and the
like.
[0031] "Alkoxyalkoxy" means --O-(alkyl)-O-(alkyl), such as
--OCH.sub.2CH.sub.2OCH.sub.3, and the like.
[0032] "Alkoxycarbonyl" means --C(.dbd.O)O-(alkyl), such as
--C(.dbd.O)OCH.sub.3, --C(.dbd.O)OCH.sub.2CH.sub.3, and the
like.
[0033] "Alkoxyalkyl" means -(alkyl)-O-(alkyl), such as
--CH.sub.2OCH.sub.3, --CH.sub.2OCH.sub.2CH.sub.3, and the like.
[0034] "Aryl" means a carbocyclic or heterocyclic aromatic group
containing from 5 to 10 ring atoms. The ring atoms of a carbocyclic
aryl group are all carbon atoms, and includes phenyl and naphthyl.
The ring atoms of a heterocyclic aryl group contains at least one
heteroatom selected from nitrogen, oxygen and sulfur, and include
pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl,
pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, and
indolyl.
[0035] "Aryloxy" means --O-(aryl), such as --O-phenyl,
--O-pyridinyl, and the like.
[0036] "Arylalkyl" means -(alkyl)-(aryl), such as benzyl (i.e.,
--CH.sub.2phenyl), --CH.sub.2-pyrindinyl, and the like.
[0037] "Arylalkyloxy" means --O-(alkyl)-(aryl), such as --O-benzyl,
--O--CH.sub.2-pyridinyl, and the like.
[0038] "Cycloalkyl" means a cyclic alkyl having from 3 to 7 carbon
atoms, such as cyclopropyl, cyclopentyl, cyclohexyl, and the
like.
[0039] "Cycloalkyloxy" means --O-(cycloalkyl), such as
--O-cyclohexyl, and the like.
[0040] "Cycloalkylalkyloxy" means --O-(alkyl)-(cycloalkyl), such as
--OCH.sub.2cyclohexyl, and the like.
[0041] "Alkylidene" means the divalent radical --CH.sub.2n-,
wherein n is an integer from 1 to 8, such as --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2--CH.sub.2--CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and the like.
[0042] "Heteroatom-containing alkylidene" means an alkylidene
wherein at least one carbon atom is replaced by a heteroatom
selected from nitrogen, oxygen or sulfur, such as
--CH.sub.2CH.sub.2OCH.sub.2CH2--, and the like.
[0043] "Aminoalkoxy" means --O-(alkyl)-NH.sub.2, such as
--OCH.sub.2NH.sub.2, --OCH.sub.2CH.sub.2NH.sub.2, and the like.
[0044] "Mono- or di-alkylamino" means --NH(alkyl) or
--N(alkyl)(alkyl), respectively, such as --NCH.sub.3,
--N(CH.sub.3).sub.2, and the like.
[0045] "Mono- or di-alkylaminoalkoxy" means --O-(alkyl)-NH(alkyl)
or --O-(alkyl)N(alkyl)(alkyl), respectively, such as
--OCH.sub.2NHCH.sub.3, --OCH.sub.2CH.sub.2N(CH.sub.3).sub.2, and
the like.
[0046] "Arylamino" means --NH(aryl), such as --NH-phenyl,
--NH-pyridinyl, and the like.
[0047] "Arylalkylamino" means --NH-(alkyl)-(aryl), such as
--NH-benzyl, --NHCH.sub.2pyridinyl, and the like.
[0048] "Alkylamino" means --NH(alkyl), such as --NHCH.sub.3,
--NHCH.sub.2CH.sub.3, and the like.
[0049] "Cycloalkylamino" means --NH-(cycloalkyl), such as
--NH-cyclohexyl, and the like.
[0050] "Cycloalkylalkylamino"-NH-(alkyl)-(cycloalkyl), such as
--NHCH.sub.2-cyclohexyl, and the like.
[0051] An "effective amount" when used in connection with a
Pyrazoloanthrone Derivative is an amount effective for treating or
preventing an inflammatory condition, a liver disease, a
cardiovascular disease, a neurodegenerative disease or cancer.
[0052] A "patient" includes an animal (e.g., cow, horse, sheep,
pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea
pig), in one embodiment a mammal such as a non-primate and a
primate (e.g., monkey and human), and in another embodiment a
human. In certain embodiments, the patient is an infant, child,
adolescent or adult.
[0053] 5.2. Compounds of the Invention
[0054] As mentioned above, the present invention is related to
methods for treating or preventing an inflammatory condition, a
liver disease, a cardiovascular disease, a neurodegenerative
disease or cancer, comprising administering to a patient in need
thereof an effective amount of a Pyrazoloanthrone Derivative of
formula (I): 3
[0055] wherein:
[0056] R.sub.1 and R.sub.2 are optional substituents that are the
same or different and independently represent alkyl, halogen,
nitro, trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy,
aryl, aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy,
cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono- or
di-alkylaminoalkoxy, or a group represented by formula (a), (b),
(c) or (d): 4
[0057] R.sub.3 and R.sub.4 taken together represent alkylidene or a
heteroatom-containing alkylidene, or R.sub.3 and R.sub.4 are the
same or different and independently represent hydrogen, alkyl,
cycloalkyl, aryl, arylalkyl, cycloalkylalkyl, aryloxyalkyl,
alkoxyalkyl, alkoxyamino, or alkoxy(mono- or di-alkylamino);
and
[0058] R.sub.5 represents hydrogen, alkyl, cycloalkyl, aryl,
arylalkyl, cycloalkylalkyl, alkoxy, amino, mono- or di-alkylamino,
arylamino, arylalkylamino, cycloalkylamino, or
cycloalkylalkylamino.
[0059] In the embodiment wherein R.sub.1 and R.sub.2 are not
present, Pyrazoloanthrone Derivatives have the following structure
(II) (also referred to herein as "Compound I"): 5
[0060] This compound is commercially available from Pfaltz-Bauer
(Conn., U.S.).
[0061] In the embodiment wherein only one of R.sub.1 and R.sub.2 is
present, Pyrazoloanthrone Derivatives have one of the following
structures (III) or (IV): 6
[0062] In the embodiment wherein both R.sub.1 and R.sub.2 are
present, Pyrazoloanthrone Derivatives have one of the following
structures (V), (VI) or (VII): 7
[0063] A Pyrazoloanthrone Derivative can be in the form of a
pharmaceutically acceptable salt or a free base. Pharmaceutically
acceptable salts of the Pyrazoloanthrone Derivatives can be formed
from organic and inorganic acids. Suitable non-toxic acids include,
but are not limited to, inorganic and organic acids such as acetic,
alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic,
citric, ethenesulfonic, formic, fumaric, furoic, galacturonic,
gluconic, glucuronic, glutamic, glycolic, hydrobromic,
hydrochloric, isethionic, lactic, maleic, malic, mandelic,
methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic,
phosphoric, propionic, salicylic, stearic, succinic, sulfanilic,
sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific
non-toxic acids include hydrochloric, hydrobromic, phosphoric,
sulfuric, and methanesulfonic acids. The compounds may also be used
in the form of base addition salts. Suitable pharmaceutically
acceptable base addition salts for the compound of the present
invention include, but are not limited to metallic salts made from
aluminum, calcium, lithium, magnesium, potassium, sodium and zinc
or organic salts made from lysine, N,N'-dibenzylethylenediamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine
(N-methylglucamine) and procaine. Examples of specific salts thus
include hydrochloride and mesylate salts. Others are well-known in
the art, see for example, Remington's Pharmaceutical Sciences, 18th
eds., Mack Publishing, Easton Pa. (1990) or Remington: The Science
and Practice of Pharmacy, 19th eds., Mack Publishing, Easton Pa.
(1995). Thus, the term "pharmaceutically acceptable salt" of a
compound of formula (I) is intended to encompass any and all
acceptable salt forms.
[0064] 5.3. Preparation of Compounds of the Invention
[0065] The Pyrazoloanthrone Derivatives can be made using organic
synthesis techniques known to those skilled in the art, as well as
by the following general techniques and by the procedures set forth
in the Examples. To that end, the Pyrazoloanthrone Derivatives can
be made according to the following Reaction Schemes 1 through 7.
8
[0066] In Reaction Scheme 1, Pyrazoloanthrone Derivatives can be
prepared by condensation of appropriate anthraquinones having a
leaving group at the 1-position (such as fluoro, chloro, bromo,
iodo, nitro, methanesulfonyloxy, tosyloxy or phenoxy) with
hydrazine in a suitable solvent (such as pyridine,
dimethylformamide, methylene chloride, chloroform, or dioxane). The
reaction is carried out at temperatures ranging 0.degree. C. to
200.degree. C. for 1 to 16 hours. Suitable anthraquinone starting
materials are commercially available from a variety of sources with
the R.sub.1 and/or R.sub.2 groups at various positions on the
anthraquinone ring. For purpose of illustration, the following
reaction schemes depict synthesis of 5-and/or 7-substituted
Pyrazoloanthrone Derivatives. One skilled in the art will recognize
that Pyrazoloanthrone Derivatives substituted at other positions
may be made in a similar manner from the appropriately substituted
pyrazoloanthrone starting material. 9
[0067] In Reaction Scheme 2, Pyrazoloanthrone Derivatives with
5-amino substituents can be prepared by condensation of
5-chloropyrazoloanthrone with mono- or disubstituted amines at 0 to
250.degree. C. for 1 to 16 hours, either in the absence or the
presence of a solvent. Typically solvents are pyridine,
dimethylformamide, dimethylsulfoxide, dichloroethane, chloroform,
tetrahydrofuran, dioxane, diglyme, or triglyme in the presence of
excess amount of the amine, or in the presence of an acid quenching
agent such as triethylamine, diisopropylethylamine, sodium
bicarbonate, potassium carbonate, or sodium hydroxide. 10
[0068] In Reaction Scheme 3, Pyrazoloanthrone Derivatives with
7-amino substituents can be prepared by condensation of
7-chloropyrazoloanthrone with mono- or disubstituted amines at 0 to
250.degree. C. for 1 to 16 hours either in the absence or the
presence of a solvent. Typically solvents are pyridine,
dimethylformamide, dimethylsulfoxide, dichloroethane, chloroform,
tetrahydrofuran, dioxane, diglyme, or triglyme in the presence of
excess amount of the amine, or in the presence of an acid quenching
agent such as triethylamine, diisopropylethylamine, sodium
bicarbonate, potassium carbonate, or sodium hydroxide. 11
[0069] In Reaction Scheme 4, Pyrazoloanthrone Derivatives with
5-acyl- or sulfonylamino substituents can be prepared by
condensation of 5-amino-2-(2-methoxyethoxymethyl) pyrazoloanthrone
with acid chlorides and sulfonyl chlorides followed by
deprotection. Condensation of 5-amino-2-(2-methoxyethoxymethyl)
pyrazoloanthrone with appropriate acid chlorides R.sub.6COCl or
sulfonyl chlorides R.sub.6SO.sub.2Cl is carried out in the presence
of an acid quenching agent such as triethylamine,
diisopropylethylamine, sodium bicarbonate, potassium carbonate, or
sodium hydroxide at -20 to 50.degree. C. for 0.5 to 16 hours in
solvents such as methylene chloride, chloroform, tetrahydrofuran,
dioxane, dimethylformamide, and ethyl acetate. The deprotection
step may be performed by the treatment of the product mentioned
above with an acid such as trifluoroacetic acid, aqueous
hydrochloric acid, aqueous hydrobromic acid, or aqueous sulfuric
acid.
[0070] The starting material may be prepared in two steps. The
2-position of 5-nitropyrazoloanthrone may be protected by a
protective group such as methoxymethyl (MOM), methoxyethoxymethyl
(MEM), 2-trimethylsilylethoxymet- hyl (SEM), or 4-methoxybenzyl
(PMB) with an aid of a base such as triethylamine,
diisopropylethylamine, pyridine, sodium hexamethyldisilazide,
potassium hexamethyldisilazide, or lithium diisopropylamide.
4-(N,N-Dimethylamino)pyridine (DM AP) may be used as a catalyst
when a tertiary amine is used as a base. The reaction is typically
carried out at -40 to 60.degree. C. for 1 to 16 hours in a solvent
such as methylene chloride, chloroform, tetrahydrofuran, dioxane,
or dimethoxyethane. As the nitrogen protective group, MEM group is
preferred.
[0071] N-Protected 5-nitropyrazoloanthorone is then reduced to its
5-amino derivative by a variety of reducing agents such as Sn or Fe
metal in acidic media such as acetic acid or aqueous hydrochloric
acid. The reaction is typically run at 20 to 160.degree. C. for 1
to 16 hours. The same transformation can be carried out by
hydrogenation in the presence of a transition-metal catalyst such
as palladium, platinum, rhodium, or iridium with or without a
support such as charcoal in a solvent such as ethanol, ethyl
acetate, tetrahydrofuran, dioxane, or dimethoxyethane at 1 to 20
atmospheres of hydrogen at 20 to 60.degree. C. for 1 to 16 hours.
The procedure using hydrogenation with palladium on charcoal as the
catalyst is preferred. 12
[0072] In Reaction Scheme 5, Pyrazoloanthrone Derivatives with
7-acyl- or sulfonylamino substituents can be prepared by
condensation of 7-amino-2-[(2-methoxyethoxymethyl) pyrazoloanthrone
with acid chlorides and sulfonyl chlorides followed by the
deprotection. Condensation of
7-amino-2-(2-methoxyethoxymethyl)pyrazoloanthrone with appropriate
acid chlorides R.sub.6COCl or sulfonyl chlorides R.sub.6SO.sub.2Cl
is carried out in the presence of an acid quenching agent such as
triethylamine, diisopropylethylamine, sodium bicarbonate, potassium
carbonate, or sodium hydroxide at -20 to 50.degree. C. for 0.5 to
16 hours in solvents such as methylene chloride, chloroform,
tetrahydrofuran, dioxane, dimethylformamide, or ethyl acetate. The
deprotection step may be performed by the treatment of the product
mentioned above with an acid such as trifluoroacetic acid, aqueous
hydrochloric acid, aqueous hydrobromic acid or aqueous sulfuric
acid.
[0073] The starting material is prepared in two steps. The
2-position of 7-nitropyrazoloanthrone is protected by a protective
group such as methoxymethyl (MOM) methoxyethoxymethyl (MEM),
2-trimethylsilylethoxymeth- yl (SEM), or 4-methoxybenzyl (PMB) with
an aid of a base such as triethylamine, diisopropylethylamine,
pyridine, sodium hexamethyldisilazide, potassium
hexamethyldisilazide, or lithium diisopropylamide. 4-(N,
N-dimethylamino)pyridine (DMAP) can be used as a catalyst when a
tertiary amine is use as a base. The reaction is typically carried
out at -40 to 60.degree. C. for 1 to 16 hours in a solvent such as
methylene chloride, chloroform, tetrahydrofuran, dioxane, or
dimethoxyethane. As the nitrogen protective group, MEM group is
preferred.
[0074] N-Protected 7-nitropyrazoloanthorone is then reduced to its
7-amine derivative by a variety of reducing agents such as Sn or Fe
metal in acidic media such a acetic acid or aqueous hydrochloric
acid. The reaction is typically run at 20 to 160.degree. C. for 5
to 16 hours. The same transformation can be carried out by
hydrogenation in the presence of a transition-metal catalyst such
as palladium, platinum, rhodium, or iridium with or without a
support such as charcoal in a solvent such as ethanol, ethyl
acetate, tetrahydrofuran, dioxane, or dimethoxyethane at 1 to 20
atmospheres of hydrogen at 20 to 60.degree. C. for 1 to 16 hours.
The procedure using hydrogenation with palladium on charcoal as the
catalyst is preferred. 13
[0075] In Reaction Scheme 6, Pyrazoloanthrone Derivatives with
5-alkoxy substituents can be prepared by condensation of
5-hydroxy-2-(2-methoxyeth- oxymethyl)-pyrazoloanthrone with alkyl
halides and sulfonates R.sub.7--X followed by deprotection. As the
leaving group X, chloride, bromide, iodide, methanesulfonate,
tosylate, benzenesulfonate, or triflate can be used. Condensation
of 5-hydroxy-2-(2-methoxyethoxymethyl) pyrazoloanthrone with
appropriate alkyl halides and sulfonates is carried out in the
presence of an acid quenching agent such as triethylamine,
diisopropylethylamine, sodium bicarbonate, potassium carbonate, or
sodium hydroxide at -20 to 50.degree. C. for 0.5 to 16 hours in
solvents such as methylene chloride, chloroform, tetrahydrofuran,
dioxane, dimethylformamide, or ethyl acetate. The deprotection step
is performed by the treatment of the product mentioned above with
an acid such as trifluoroacetic acid, aqueous hydrochloric acid,
aqueous hydrobromic acid, or aqueous sulfuric acid.
[0076] The starting material is prepared in two steps. The
2-position of 5-benzyloxypyrazoloanthrone is protected by a
protective group such as methoxymethyl (MOM), methoxyethoxymethyl
(MEM), 2-trimethylsilylethoxymet- hyl (SEM), or 4-methoxybenzyl
(PMB) with an aid of a base such as triethylamine,
diisopropylethylamine, pyridine, sodium hexamethyldisilazide,
potassium hexamethyldisilazide, or lithium diisopropylamide.
4-(N,N-dimethylamino)pyridine (DMAP) can be used as a catalyst when
a tertiary amine is used as a base. The reaction is typically
carried out at -40 to 60.degree. C. for 1 to 16 hours in a solvent
such as methylene chloride, chloroform, tetrahydrofuran, dioxane,
or dimethoxyethane. As the nitrogen protective group, MEM group is
preferred.
[0077] N-Protected 5-benzyloxypyrazoloanthorone is then reduced to
its 5-hydroxy derivative by hydrogenation in the presence of a
transition-metal catalyst, such as palladium platinum, rhodium, or
iridium with or without a support such as charcoal in a solvent
such as ethanol, ethyl acetate, tetrahydrofuran, dioxane, or
dimethoxyethane at 1 to 20 atmospheres of hydrogen at 20 to
60.degree. C. for 1 to 16 hours. The procedure using hydrogenation
with palladium on charcoal as the catalyst is preferred. 14
[0078] In Reaction Scheme 7, Pyrazoloanthrone Derivatives with
5-alkoxy substituents can be prepared by condensation of
7-hydroxy-2-(2-methoxyeth- oxymethyl)-pyrazoloanthrone with alkyl
halides and sulfonates R.sub.7--X followed by deprotection. As the
leaving group X, chloride, bromide, iodide, methanesulfonate,
tosylate, benzenesulfonate, or triflate can be used. Condensation
of 7-hydroxy-2-(2-methoxyethoxymethyl) pyrazoloanthrone with
appropriate alkyl halides and sulfonates is carried out in the
presence of an acid quenching agent such as triethylamine,
diisopropylethylamine, sodium bicarbonate, potassium carbonate, or
sodium hydroxide at -20 to 50.degree. C. for 0.5 to 16 hours in
solvents such as methylene chloride, chloroform, tetrahydrofuran,
dioxane, dimethylformamide, or ethyl acetate. The deprotection step
is performed by the treatment of the product mentioned above with
an acid such as trifluoroacetic acid, aqueous hydrochloric acid,
aqueous hydrobromic acid, or aqueous sulfuric acid.
[0079] The starting material is prepared in two steps. The
2-position of 7-benzyloxypyrazoloanthrone is protected by a
protective group such as methoxymethyl (MOM), methoxyethoxymethyl
(MEM), 2-trimethylsilylethoxymet- hyl (SEM), or 4-methoxybenzyl
(PMB) with an aid of a base such as triethylamine,
diisopropylethylamine, pyridine sodium hexamethyldisilazide,
potassium hexamethyldisilazide, or lithium diisopropylamide.
4-(N,N-dimethylamino)pyridine (DMAP) can be used as a catalyst when
a tertiary amine is used as a base. The reaction is typically
carried out at -40 to 60.degree. C. for 1 to 16 hours in a solvent
such as methylene chloride, chloroform, tetrahydrofuran, dioxane,
or dimethoxyethane. As the nitrogen protective group, MEM group is
preferred.
[0080] N-Protected 7-benzyloxypyrazoloanthorone is then reduced to
its 7-hydroxy derivative by hydrogenation in the presence of a
transition-metal catalyst, such as palladium platinum, rhodium, or
iridium with or without a support such as charcoal in a solvent
such as ethanol, ethyl acetate, tetrahydrofuran, dioxane, or
dimethoxyethane at 1 to 20 atmospheres of hydrogen at 20 to
60.degree. C. for 1 to 16 hours. The procedure using hydrogenation
with palladium on charcoal as the catalyst is preferred.
[0081] Pyrazoloanthrone Derivatives of structures (V), (VI) and
(VII) can be made by the same procedures as outlined above by
utilizing starting materials having multiple reactive sites at the
corresponding positions to the desired product.
[0082] 5.4. Methods of Use
[0083] The present invention provides methods for treating or
preventing a variety of conditions comprising administering an
effective amount of a Pyrazoloanthrone Derivative to a patient in
need thereof. Conditions that may be treated by the administration
of the Pyrazoloanthrone Derivatives, include any condition which is
responsive to JNK inhibition, and thereby benefits from
administration of a JNK inhibitor. Representative conditions
treatable or preventable by administering an effective amount of a
Pyrazoloanthrone Derivatives are useful for treating or preventing
an inflammatory condition including, but not limited to: diabetes
(such as Type II diabetes, Type I diabetes, diabetes insipidus,
diabetes mellitus, maturity-onset diabetes, juvenile diabetes,
insulin-dependant diabetes, non-insulin dependant diabetes,
malnutrition-related diabetes, ketosis-prone diabetes or
ketosis-resistant diabetes); nephropathy (such as
glomerulonephritis or acute/chronic kidney failure); obesity (such
as hereditary obesity, dietary obesity, hormone related obesity or
obesity related to the administration of medication); hearing loss
(such as that from otitis externa or acute otitis media); fibrosis
related diseases (such as pulmonary interstitial fibrosis, renal
fibrosis, cystic fibrosis, liver fibrosis, wound-healing or
burn-healing, wherein the burn is a first-, second- or third-degree
burn and/or a thermal, chemical or electrical burn); arthritis
(such as rheumatoid arthritis, rheumatoid spondylitis,
osteoarthritis or gout); an allergy; allergic rhinitis; acute
respiratory distress syndrome; asthma; bronchitis; an inflammatory
bowel disease (such as irritable bowel syndrome, mucous colitis,
ulcerative colitis, Crohn's disease, gastritis, esophagitis,
pancreatitis or peritonitis); or an autoimmune disease (such as
scleroderma, systemic lupus erythematosus, myasthenia gravis,
transplant rejection, endotoxin shock, sepsis, psoriasis, eczema,
dermatitis or multiple sclerosis).
[0084] Pyrazoloanthrone Derivatives are also useful for treating or
preventing a liver disease (such as hepatitis, alcohol-induced
liver disease, toxin-induced liver disease, steatosis or
sclerosis); a cardiovascular disease (such as atherosclerosis,
restenosis following angioplasty, left ventricular hypertrophy,
myocardial infarction, chronic obstructive pulmonary disease or
stroke); ischemic damage (such as to the heart, kidney, liver or
brain); ischemia-reperfusion injury (such as that caused by
transplant, surgical trauma, hypotension, thrombosis or trauma
injury); neurodegenerative disease (such as epilepsy, Alzheimer's
disease, Huntington's disease, Amyotrophic laterial sclerosis,
peripheral neuropathies, spinal cord damage or Parkinson's
disease); or cancer (cancer of the head, neck, eye, mouth, throat,
esophagus, chest, bone, lung, colon, rectum, stomach, prostate,
breast, ovaries, testicles or other reproductive organs, skin,
thyroid, blood, lymph nodes, kidney, liver, pancreas, and brain or
central nervous system).
[0085] In one embodiment, the present methods for treating or
preventing further comprise the administration of an effective
amount of another therapeutic agent useful for treating or
preventing the diseases or disorders disclosed herein. In this
embodiment, the time in which the therapeutic effect of the other
therapeutic agent is exerted overlaps with the time in which the
therapeutic effect of the Pyrazoloanthrone Derivative is
exerted.
[0086] In one embodiment, the other therapeutic agent is an
anti-inflammatory agent. Examples of anti-inflammatory agents
include, but are not limited to, steroids (e.g., cortisol,
cortisone, fludrocortisone, prednisone, 6.alpha.-methylprednisone,
triamcinolone, betamethasone or dexamethasone), nonsteroidal
antiinflammatory drugs (NSAIDS (e.g., aspirin, acetaminophen,
tolmetin, ibuprofen, mefenamic acid, piroxicam, nabumetone,
rofecoxib, celecoxib, etodolac or nimesulide). In another
embodiment, the other therapeutic agent is an antiobiotic (e.g.,
vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime,
ceftriaxone, cefixime, rifampinmetronidazole, doxycycline or
streptomycin). In another embodiment, the other therapeutic agent
is a PDE4 inhibitor (e.g., roflumilast or rolipram). In another
embodiment, the other therapeutic agent is an antihistamine (e.g.,
cyclizine, hydroxyzine, promethazine or diphenhydramine). In
another embodiment, the other therapeutic agent is an anti-malarial
(e.g., artemisinin, artemether, artsunate, chloroquine phosphate,
mefloquine hydrochloride, doxycycline hyclate, proguanil
hydrochloride, atovaquone or halofantrine). In one embodiment, the
other therapeutic agent is drotrecogin alfa.
[0087] In one embodiment, inhibiting JNK in vivo comprises
inhibiting TNF-.alpha. in vivo.
[0088] In one embodiment the JNK is JNK1. In another embodiment the
JNK is JNK2 In another embodiment the JNK is JNK3.
[0089] 5.5. Pharmaceutical Compositions
[0090] Pharmaceutical compositions can be used in the preparation
of individual, single unit dosage forms. Pharmaceutical
compositions and dosage forms of the invention comprise a
Pyrazoloanthrone Derivative and one or more excipients.
[0091] Single unit dosage forms of the invention are suitable for
oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or
rectal), or parenteral (e.g., subcutaneous, intravenous, bolus
injection, intramuscular, or intraarterial), transdermal or
transcutaneous administration to a patient. Examples of dosage
forms include, but are not limited to: tablets; caplets; capsules,
such as soft elastic gelatin capsules; cachets; troches; lozenges;
dispersions; suppositories; powders; aerosols (e.g., nasal sprays
or inhalers); gels, liquid dosage forms suitable for oral or
mucosal administration to a patient, including suspensions (e.g.,
aqueous or non-aqueous liquid suspensions, oil-in-water emulsions,
or a water-in-oil liquid emulsions), solutions, and elixirs; liquid
dosage forms suitable for parenteral administration to a patient;
and sterile solids (e.g., crystalline or amorphous solids) that can
be reconstituted to provide liquid dosage forms suitable for
parenteral administration to a patient.
[0092] The composition, shape, and type of dosage forms of the
invention will typically vary depending on their use. For example,
a dosage form used in the acute treatment of a disease may contain
larger amounts of one or more Pyrazoloanthrone Derivatives than a
dosage form used in the chronic treatment of the same disease.
Similarly, a parenteral dosage form may contain smaller amounts of
a Pyrazoloanthrone Derivative than an oral dosage form used to
treat the same disease. These and other ways in which specific
dosage forms encompassed by this invention will vary from one
another will be readily apparent to those skilled in the art. See,
e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing, Easton Pa. (1990).
[0093] Typical pharmaceutical compositions and dosage forms
comprise one or more excipients. Suitable excipients are well known
to those skilled in the art of pharmacy, and non-limiting examples
of suitable excipients are provided herein. Whether a particular
excipient is suitable for incorporation into a pharmaceutical
composition or dosage form depends on a variety of factors well
known in the art including, but not limited to, the way in which
the dosage form will be administered to a patient. For example,
oral dosage forms such as tablets may contain excipients not suited
for use in parenteral dosage forms. The suitability of a particular
excipient may also depend on the specific active agents in the
dosage form. For example, the decomposition of some active agents
may be accelerated by some excipients such as lactose, or when
exposed to water. Active agents that comprise primary or secondary
amines are particularly susceptible to such accelerated
decomposition. Consequently, this invention encompasses
pharmaceutical compositions and dosage forms that contain little,
if any, lactose other mono- or di-saccharides. As used herein, the
term "lactose-free" means that the amount of lactose present, if
any, is insufficient to substantially increase the degradation rate
of an active agent.
[0094] Lactose-free compositions of the invention can comprise
excipients that are well known in the art and are listed, for
example, in the U.S. Pharmacopeia (USP) 25-NF20 (2002). In general,
lactose-free compositions comprise active agents, a binder/filler,
and a lubricant in pharmaceutically compatible and pharmaceutically
acceptable amounts. Preferred lactose-free dosage forms comprise
active agents, microcrystalline cellulose, pre-gelatinized starch,
and magnesium stearate.
[0095] Anhydrous (less than 5% water) pharmaceutical compositions
and dosage forms of the invention can be prepared using anhydrous
or low moisture containing agents and low moisture or low humidity
conditions. Pharmaceutical compositions and dosage forms that
comprise lactose and at least one active agent that comprises a
primary or secondary amine are preferably anhydrous if substantial
contact with moisture and/or humidity during manufacturing,
packaging, and/or storage is expected.
[0096] An anhydrous pharmaceutical composition should be prepared
and stored such that its anhydrous nature is maintained.
Accordingly, anhydrous compositions are preferably packaged using
materials known to prevent exposure to water such that they can be
included in suitable formulary kits. Examples of suitable packaging
include, but are not limited to, hermetically sealed foils,
plastics, unit dose containers (e.g. vials), blister packs, and
strip packs.
[0097] The invention further encompasses pharmaceutical
compositions and dosage forms that comprise one or more compounds
that reduce the rate by which an active agent will decompose. Such
compounds, which are referred to herein as "stabilizers," include,
but are not limited to, antioxidants such as ascorbic acid, pH
buffers, or salt buffers.
[0098] The amount of a Pyrazoloanthrone Derivative in a dosage form
may differ depending on factors such as, but not limited to, the
route by which it is to be administered to patients. However,
typical dosage forms of the invention comprise a Pyrazoloanthrone
Derivative in an amount of from about 0.10 mg to about 3500 mg,
from about 1 mg to about 2500 mg, from about 10 mg to about 500 mg,
from about 25 mg to about 250 mg, from about 50 mg to about 100 mg.
Typical dosage forms comprise a Pyrazoloanthrone Derivative in an
amount of about 0.1, 1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50,
100, 150, 200, 250, 500, 750, 1000, 1500, 2000, 2500, 3000 or 3500
mg. In a particular embodiment, a dosage form comprises a
Pyrazoloanthrone Derivative in an amount of about 1, 2, 5, 10, 25,
50, 100, 250 or 500 mg. In a specific embodiment, a dosage form
comprises an amount of about 5, 10, 25 or 50 mg of a
Pyrazoloanthrone Derivative.
[0099] 5.5.1. Oral Dosage Forms
[0100] Pharmaceutical compositions of the invention that are
suitable for oral administration can be presented as discrete
dosage forms, such as, but are not limited to, tablets (e.g.,
chewable tablets), caplets, capsules, and liquids (e.g., flavored
syrups). Such dosage forms contain predetermined amounts of a
Pyrazoloanthrone Derivative, and may be prepared by methods of
pharmacy well known to those skilled in the art. See generally,
Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing,
Easton Pa. (1990).
[0101] Typical oral dosage forms of the invention are prepared by
combining a Pyrazoloanthrone Derivative in an intimate admixture
with at least one excipient according to conventional
pharmaceutical compounding techniques. Excipients can take a wide
variety of forms depending on the form of preparation desired for
administration. For example, excipients suitable for use in oral
liquid or aerosol dosage forms include, but are not limited to,
water, glycols, oils, alcohols, flavoring agents, preservatives,
and coloring agents. Examples of excipients suitable for use in
solid oral dosage forms (e.g., powders, tablets, capsules, and
caplets) include, but are not limited to, starches, sugars,
micro-crystalline cellulose, diluents, granulating agents,
lubricants, binders, and disintegrating agents.
[0102] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit forms, in
which case solid excipients are employed. If desired, tablets can
be coated by standard aqueous or nonaqueous techniques. Such dosage
forms can be prepared by any of the methods of pharmacy. In
general, pharmaceutical compositions and dosage forms are prepared
by uniformly and intimately admixing the Pyrazoloanthrone
Derivative with liquid carriers, finely divided solid carriers, or
both, and then shaping the product into the desired presentation if
necessary.
[0103] For example, a tablet can be prepared by compression or
molding. Compressed tablets can be prepared by compressing in a
suitable machine the active agents in a free-flowing form such as
powder or granules optionally mixed with an excipient. Molded
tablets can be made by molding in a suitable machine a mixture of
the powdered compound moistened with an inert liquid diluent.
[0104] Examples of excipients that can be used in oral dosage forms
of the invention include, but are not limited to, binders, fillers,
disintegrants, and lubricants. Binders suitable for use in
pharmaceutical compositions and dosage forms include, but are not
limited to, corn starch, potato starch, or other starches, gelatin,
natural and synthetic gums such as acacia, sodium alginate, alginic
acid, other alginates, powdered tragacanth, guar gum, cellulose and
its derivatives (e.g., ethyl cellulose, cellulose acetate,
carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),
polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch,
hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),
microcrystalline cellulose, and mixtures thereof.
[0105] Suitable forms of microcrystalline cellulose include, but
are not limited to, the materials sold as AVICEL-PH-101,
AVICEL-PH-103, AVICEL RC-581, AVICEL-PH-105 (available from FMC
Corporation, American Viscose Division, Avicel Sales, Marcus Hook,
Pa.), and mixtures thereof. An specific binder is a mixture of
microcrystalline cellulose and sodium carboxymethyl cellulose sold
as AVICEL RC-581. Suitable anhydrous or low moisture excipients or
additives include AVICEL-PH-103 and Starch 1500 LM.
[0106] Examples of fillers suitable for use in the pharmaceutical
compositions and dosage forms disclosed herein include, but are not
limited to, talc, calcium carbonate (e.g., granules or powder),
microcrystalline cellulose, powdered cellulose, dextrates, kaolin,
mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,
and mixtures thereof. The binder or filler in pharmaceutical
compositions of the invention is typically present in from about 50
to about 99 weight percent of the pharmaceutical composition or
dosage form.
[0107] Disintegrants are used in the compositions of the invention
to provide tablets that disintegrate when exposed to an aqueous
environment. Tablets that contain too much disintegrant may
disintegrate in storage, while those that contain too little may
not disintegrate at a desired rate or under the desired conditions.
Thus, a sufficient amount of disintegrant that is neither too much
nor too little to detrimentally alter the release of the active
agents should be used to form solid oral dosage forms of the
invention. The amount of disintegrant used varies based upon the
type of formulation, and is readily discernible to those of
ordinary skill in the art. Typical pharmaceutical compositions
comprise from about 0.5 to about 15 weight percent of disintegrant,
preferably from about 1 to about 5 weight percent of
disintegrant.
[0108] Disintegrants that can be used in pharmaceutical
compositions and dosage forms of the invention include, but are not
limited to, agar-agar, alginic acid, calcium carbonate,
microcrystalline cellulose, croscarmellose sodium, crospovidone,
polacrilin potassium, sodium starch glycolate, potato or tapioca
starch, other starches, pre-gelatinized starch, other starches,
clays, other algins, other celluloses, gums, and mixtures
thereof.
[0109] Lubricants that can be used in pharmaceutical compositions
and dosage forms of the invention include, but are not limited to,
calcium stearate, magnesium stearate, mineral oil, light mineral
oil, glycerin, sorbitol, mannitol, polyethylene glycol, other
glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated
vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil,
sesame oil, olive oil, corn oil, and soybean oil), zinc stearate,
ethyl oleate, ethyl laureate, agar, and mixtures thereof.
Additional lubricants include, for example, a syloid silica gel
(AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, Md.), a
coagulated aerosol of synthetic silica (marketed by Degussa Co. of
Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold
by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at
all, lubricants are typically used in an amount of less than about
1 weight percent of the pharmaceutical compositions or dosage forms
into which they are incorporated.
[0110] A preferred solid oral dosage form of the invention
comprises a Pyrazoloanthrone Derivative, anhydrous lactose,
microcrystalline cellulose, polyvinylpyrrolidone, stearic acid,
colloidal anhydrous silica, and gelatin.
[0111] 5.5.2. Delayed Release Dosage Forms
[0112] A JNK Inhibitor can be administered by controlled release
means or by delivery devices that are well known to those of
ordinary skill in the art. Examples include, but are not limited
to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;
3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595,
5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and
5,733,566, each of which is incorporated herein by reference. Such
dosage forms can be used to provide slow or controlled-release of
one or more Pyrazoloanthrone Derivatives using, for example,
hydropropylmethyl cellulose, other polymer matrices, gels,
permeable membranes, osmotic systems, multilayer coatings,
microparticles, liposomes, microspheres, or a combination thereof
to provide the desired release profile in varying proportions.
Suitable controlled-release formulations known to those of ordinary
skill in the art, including those described herein, can be readily
selected for use with a Pyrazoloanthrone Derivative. The invention
thus encompasses single unit dosage forms suitable for oral
administration such as, but not limited to, tablets, capsules,
gelcaps, and caplets that are adapted for controlled-release.
[0113] All controlled-release pharmaceutical products have a common
goal-of improving drug therapy over that achieved by their
non-controlled counterparts. Ideally, the use of an optimally
designed controlled-release preparation in medical treatment is
characterized by a minimum of drug substance being employed to cure
or control the condition in a minimum amount of time. Advantages of
controlled-release formulations include extended activity of the
drug, reduced dosage frequency, and increased patient compliance.
In addition, controlled-release formulations can be used to affect
the time of onset of action or other characteristics, such as blood
levels of the drug, and can thus affect the occurrence of side
(e.g., adverse) effects.
[0114] Most controlled-release formulations are designed to
initially release an amount of drug (e.g., a Pyrazoloanthrone
Derivative) that promptly produces the desired therapeutic effect,
and gradually and continually release of other amounts of drug to
maintain this level of therapeutic or prophylactic effect over an
extended period of time. In order to maintain this constant level
of drug in the body, the drug must be released from the dosage form
at a rate that will replace the amount of drug being metabolized
and excreted from the body. Controlled-release of an active agent
can be stimulated by -various conditions including, but not limited
to, pH, temperature, enzymes, water, or other physiological
conditions or compounds.
[0115] 5.5.3. Parenteral Dosage Forms
[0116] Parenteral dosage forms can be administered to patients by
various routes including, but not limited to, subcutaneous,
intravenous (including bolus injection), intramuscular, and
intraarterial. Because their administration typically bypasses
patients' natural defenses against contaminants, parenteral dosage
forms are preferably sterile or capable of being sterilized prior
to administration to a patient. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
and emulsions.
[0117] Suitable vehicles that can be used to provide parenteral
dosage forms of the invention are well known to those skilled in
the art. Examples include, but are not limited to: Water for
Injection USP; aqueous vehicles such as, but not limited to, Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection,
Dextrose and Sodium Chloride Injection, and Lactaied Ringer's
Injection; water-miscible vehicles such as, but not limited to,
ethyl alcohol, polyethylene glycol, and polypropylene glycol; and
non-aqueous vehicles such as, but not limited to, corn oil,
cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl
myristate, and benzyl benzoate.
[0118] Compounds that increase the solubility of one or more of the
active agents disclosed herein can also be incorporated into the
parenteral dosage forms of the invention. For example, cyclodextrin
and its derivatives can be used to increase the solubility of a
Pyrazoloanthrone Derivative. See, e.g., U.S. Pat. No. 5,134,127,
which is incorporated herein by reference.
[0119] 5.5.4. Topical and Mucosal Dosage Forms
[0120] Topical and mucosal dosage forms of the invention include,
but are not limited to, sprays, aerosols, solutions, emulsions,
suspensions, or other forms known to one of skill in the art. See,
e.g., Remington's Pharmaceutical Sciences, 16.sup.th and 18.sup.th
eds., Mack Publishing, Easton Pa. (1980 & 1990); and
Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea &
Febiger, Philadelphia (1985). Dosage forms suitable for treating
mucosal tissues within the oral cavity can be formulated as
mouthwashes or as oral gels.
[0121] Suitable excipients (e.g., carriers and diltients) and other
materials that can be used to provide topical and mucosal dosage
forms encompassed by this invention are well known to those skilled
in the pharmaceutical arts, and depend on the particular tissue to
which a given pharmaceutical composition or dosage form will be
applied. With that fact in mind, typical excipients include, but
are not limited to, water, acetone, ethanol, ethylene glycol,
propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl
palmitate, mineral oil, and mixtures thereof to form solutions,
emulsions or gels, which are non-toxic and pharmaceutically
acceptable. Moisturizers or humectants can also be added to
pharmaceutical compositions and dosage forms if desired. Examples
of such additional agents are well known in the art. See, e.g.,
Remington's Pharmaceutical Sciences, 16.sup.th and 18.sup.th eds.,
Mack Publishing, Easton Pa. (1980 & 1990).
[0122] The pH of a pharmaceutical composition or dosage form may
also be adjusted to improve delivery of one or more active agents.
Similarly, the polarity of a solvent carrier, its ionic strength,
or tonicity can be adjusted to improve delivery. Compounds such as
stearates can also be added to pharmaceutical compositions or
dosage forms to advantageously alter the hydrophilicity or
lipophilicity of one or more active agents so as to improve
delivery. In this regard, stearates can serve as a lipid vehicle
for the formulation, as an emulsifying agent or surfactant, and as
a delivery-enhancing or penetration-enhancing agent. Different
salts, hydrates or solvates of the active agents can be used to
further adjust the properties of the resulting composition.
[0123] 5.5.5. Kits
[0124] Typically, active agents of the invention are preferably not
administered to a patient at the same time or by the same route of
administration. This invention therefore encompasses kits which,
when used by the medical practitioner, can simplify the
administration of appropriate amounts of active agents to a
patient.
[0125] A typical kit of the invention comprises a dosage form of a
Pyrazoloanthrone Derivative, or a pharmaceutically acceptable salt
salt, solvate, hydrate, stereoisomer, prodrug, or clathrate
thereof. Kits encompassed by this invention can further comprise
additional active agents. Examples of the additional active agents
include, but are not limited to, antidepressants, anticonvulsants,
antihypertensives, anxiolytics, calcium channel blockers, muscle
relaxants, non-narcotic analgesics, opioid analgesics,
non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, or
other therapeutics discussed herein (see, e.g., section 4.2).
[0126] Kits of the invention can further comprise devices that are
used to administer the Pyrazoloanthrone Derivative. Examples of
such devices include, but are not limited to, syringes, drip bags,
patches, and inhalers.
[0127] Kits of the invention can further comprise pharmaceutically
acceptable vehicles that can be used to administer one or more
Pyrazoloanthrone Derivatives. For example, if a Pyrazoloanthrone
Derivative is provided in a solid form that must be reconstituted
for parenteral administration, the kit can comprise a sealed
container of a suitable vehicle in which the a Pyrazoloanthrone
Derivative can be dissolved to form a particulate-free sterile
solution that is suitable for parenteral administration. Examples
of pharmaceutically acceptable vehicles include, but are not
limited to: Water for Injection USP; aqueous vehicles such as, but
not limited to, Sodium Chloride Injection, Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection, and
Lactated Ringer's Injection; water-miscible vehicles such as, but
not limited to, ethyl alcohol, polyethylene glycol, and
polypropylene glycol; and non-aqueous vehicles such as, but not
limited to, corn oil, cottonseed oil, peanut oil, sesame oil,
soybean oil, ethyl oleate, isopropyl myristate, and benzyl
benzoate.
[0128] The following examples are offered by way of illustration,
not limitation.
6.1. EXAMPLES
Example 1
Synthesis of Representative Compounds
[0129] 15
[0130] A. Anthra[1,9cd]pyrazol-6(2H)-one ("Compound 1") 16
[0131] Anhydrous hydrazine is added to a solution of
2-chloroanthraquinone (Aldrich) in 10 ml pyridine, and the mixture
heated at 100.degree. C. for 16 hours. The mixture is cooled and
the solvent is evaporated in vacuo. The residue is taken in hot 6N
HCl, and the solid is collected by filtration. Flash chromatography
of the crude material on silica gel affords
anthra[1,9cd]pyrazol-6(2H)-one ("Compound 1") as yellow solids.
[0132] Due to limited solubility of Compound 1, purification of the
same may be achieved by first derivatizing Compound 1 to a more
soluble intermediate, such as the corresponding acetate,
recrystallizing the intermediate, and then converting the
intermediate to yield purified Compound 1 in good yield. More
specifically, to solution of the pyrazoloanthrone (9.67 g, 43.9
mmol) in acetic, acid (700 mL)-is added acetic anhydride (12.4 mL,
132 mmol). The solution is heated to 80.degree. C. for 5 hours and
then cooled to room temperature. After 16 hours, the reaction is
cooled to 0.degree. C. for 2 hours. The reaction is then filtered
to give the N-acetylpyrazoloanthrone intermediate. This
intermediate is recrystallized in acetic acid to give the pure
intermediate (5.96 g, 52%). 1H NMR (CDCL.sub.3) .delta. 10.6 (br
s,1H), 8.46 (d,1H), 8.33 (d, 1H), 8.26 (d, 1H), 8.08 (d, 1H),
7.96-7.87 (m, 2H), 7.78 (t, 1H), 2.83 (s, 3H); ES-MS (m/z) 263
[M+1].sup.+. To a solution of the pure intermediate, (5.96 g, 23
mmol) in methanol (600 mL) is added ammonium hydroxide (60 mL). The
reaction is stirred at room temperature for 16 hours and then
filtered and dried in a vacuum oven. A second crop of crystals is
recovered to give a total of 4.8 g of Compound 1 at greater than
98% purity. ES-MS (m/z) 221 [M+1].sup.-.
[0133] B. 5-Chloroanthra[1,9cd]pyrazol-6(2H)-one 17
[0134] This compound may be made in the same manner from 1,4
dichloroanthraquinone (commercial product).
[0135] C. 7-Chloroanthra[1,9cd]pyrazol-6(2H)-one 18
[0136] This compound may be made in the same manner from
1,5-dichloroanthraquinone (commercial product).
[0137] D. 5-Nitroanthra[1,9cd]pyrazol-6(2H)-one 19
[0138] This compound may be made from 1,4-dinitroanthraquinone
(Krapcho, A. P.; Avery, K. L., Jr. J. Org. Chem. 55, 5562-4,
1990).
[0139] E. 7-Nitroanthra[1,9cd]pyrazol-6(2H)-one 20
[0140] This compound may be made in the same manner from
1-chloroanthraquinone (commercial product).
[0141] F. 5-Benzyloxyanthra[1,9cd]pyrazol-6(2H)-one 21
[0142] This compound may be made in the same manner from
1-nitro-4-benzyloxyanthraquinone. This starting material may be
prepared as follows. Benzyl bromide is added to
1-nitro-4-hydroxyanthraquinone (Aldrich) and potassium carbonate in
dimethylformamide, and the mixture is stirred for 16 hours. Water
is added and the mixture is extracted with ethyl acetate (x2). The
combined organic layer is washed sequentially with sodium
bicarbonate solution, water, 1N hydrochloric acid, and brine,
dried, and evaporated. The residue is chromatographed on silica gel
to afford 1-nitro-4-benzyloxyanthraquinone.
[0143] G. 7-Benzyloxyanthra[1,9cd]pyrazol-6(2H)-one 22
[0144] This compound may be made in the same manner from
1-nitro-5-benzyloxyanthraquinone, which starting material may
prepared as disclosed in German Patent No. DE 2254199 to Reubke,
Hohmann and Bien.
[0145] 5-(Acetylamino)anthra[1,9cd]pyrazol-6(2H)-one 23
[0146] This compound may be made in the same manner from
4-acetylamino-1-chloroanthraquinone. This starting material may be
prepared as follows. 4-Amino-1-chloroanthraquinone is taken in
pyridine and treated with acetic anhydride. The mixture is stirred
for 1 hour, and poured onto water. The solids are collected by
filtration, washed with water, and dried in vacuo to give
4-acetylamino-1-chloroanthraquinone as a colorless solid.
Example 2
Synthesis of Representative Compounds
[0147] 24
[0148] A. 5-(Dimethylamino)anthra[1,9cd]pyrazol-6(2H)-one 25
[0149] A mixture of 5-chloroanthra[1,9cd]pyrazol-6(2H)-one (Example
1-B) and dimethylamine in pyridine is heated at 100.degree. C. for
16 hours. The mixture is cooled and evaporated. The residue is
chromatographed on silica gel to give the desired compound as
yellow solids.
[0150] B. 5-(1-Piperidinyl)anthra[1,9cd]pyrazol-6(2H)-one 26
[0151] This compound may be made in the same manner using
piperidine as the amine.
[0152] C. 5-(1-Morpholinyl)anthra[1,9cd]pyrazol-6(2H)-one 27
[0153] This compound may be made in the same manner using
morpholine as the amine.
[0154] D. 5-(Benzylamino)anthra[1,9cd]pyrazol-6(2H)-one 28
[0155] This compound may be made in the same manner using
benzylamine as the amine.
[0156] E. 5-{(4-Pyridylmethyl)lamino}anthra[1,9cd]pyrazol-6(2H)-one
29
[0157] This compound may be made in the same manner using
4-pyridylmethylamine as the amine.
[0158] F.
5-{2-(1-Piperidinyl)ethylamino}anthra[1,9cd]pyrazol-6(2H)-one
30
[0159] This compound may be made in the same manner using
2-(1-piperidyl)ethylamine as the amine.
Example 3
Synthesis of Representative Compounds
[0160] 31
[0161] A. 7-(Dimethylamino)anthra[1,9cd]pyrazol-6(2H)-one 32
[0162] A mixture of 6-chloroanthra[1,9cd]pyrazol-6(2H)-one (Example
1-C) and dimethylamine in pyridine is heated at 100.degree. C. for
16 hours. The mixture is cooled and evaporated. The residue is
chromatographed on silica gel to give the desired compound as
yellow solids.
[0163] B. 5-(1-Piperidinyl)anthra[1,9cd]pyrazol-6(2H)-one 33
[0164] This compound may be made in the same manner using
piperidine as the amine.
[0165] C. 5-(1-Morpholinyl)anthra[1,9cd]pyrazol-6(2H)-one 34
[0166] This compound may be made in the same manner using
morpholine as the amine.
[0167] D. 5-(Benzylamino)anthra[1,9cd]pyrazol-6(2H)-one 35
[0168] This compound may be made in the same manner using
benzylamine as the amine.
[0169] E. 5-{(4-Pyridylmethyl)amino}anthra[1,9cd]pyrazol-6(2H)-one
36
[0170] This compound may be made in the same manner using
4-pyridylmethylamine as the amine.
[0171] F.
5-{2-(1-Piperidinyl)ethylamino)anthra[1,9cd]pyrazol-6(2H)-one
37
[0172] This compound may be made in the same manner using
2-(1-piperidyl)ethylamine as the amine.
Example 4
Synthesis of Representative Compounds
[0173] 38
[0174] A. 5-(Benzoylamino)anthra[1,9cd]pyrazol-6(2H)-one 39
[0175] Benzoyl chloride is added to a solution of
2-(methoxyethoxymethyl)-- 5-aminoanthra[1,9cd]pyrazol-6-(2H)one and
triethylamine in methylene chloride at 0.degree. C. The mixture is
stirred for 16 hours, quenched with water, and extracted with ethyl
acetate (x2). The combined organic layer is washed with sodium
bicarbonate solution, and brine, dried and evaporated. The crude
reaction mixture is then taken in aqueous 6N hydrochloric acid, and
heated at 80.degree. C. for 4 hours. After cooling, the mixture is
extracted with ethyl acetate (x2), washed with brine, dried, and
evaporated. The residue is chromatographed on silica gel to furnish
the desired amide as a yellow solid.
[0176] The starting material is prepared as follows. Sodium
hexamethyldisilazide is added to a cooled (0.degree. C.) solution
of 5-nitroanthra[1,9cd]pyrazol-6(2H)-one (Example 1-D) in
tetrahydrofuran, and the mixture is stirred for 30 minutes at
0.degree. C. MEM-chloride is added, and the mixture is stirred for
16 hours at room temperature. Water is added and the mixture is
extracted with ethyl acetate (x2). The combined organic layer is
washed with aqueous sodium bicarbonate solution, water, 1N
hydrochloric acid, and brine, dried and evaporated. The residue is
chromatographed on silica gel to give
2-MEM-5-nitroanthra[1,9cd]pyrazol-6(2H)-one as an oil.
[0177] Palladium(10%) on charcoal and
2-MEM-5-nitroanthra[1,9cd]pyrazol-6(- 2H)-one in ethanol is placed
under 1-atm of hydrogen, and the mixture was stirred for 6 hours.
The catalyst is filtered off over celite, and the filtrate is
evaporated to dryness to give 2-(methoxyethoxymethyl)-5-amino-
anthra[1,9cd]pyrazol-6-(2H)one, which is used without further
purification.
[0178] B. 5-(Isonicotinylamino)anthra[1,9cd]pyrazol-6(2H)-one
40
[0179] This compound may be made in the same manner using
isonicotinoyl chloride as the acid chloride.
[0180] C. 5-(Nicotinylamino)anthra[1,9cd]pyrazol-6(2H-one 41
[0181] This compound may be made in the same manner using
nicotinoyl chloride as the acid chloride.
[0182] D.
5-(2-Thiophenecarbonylamino)anthra[1,9cd]pyrazol-6(2H)-one 42
[0183] This compound may be made in the same manner using
2-thiophenecarboxylic acid as the acid chloride.
[0184] E. 5-(3-Methylbutyrylamino)anthra[1,9cd]pyrazol-6(2H)-one
43
[0185] This compound may be made in the same manner using
isopentanoyl chloride as the acid chloride.
[0186] F. 5-(3-Methanesulfonylamino)anthra[1,9cd]pyrazol-6(2H)-one
44
[0187] This compound may be made in the same manner using
methanesulfonyl chloride as the sulfonyl chloride.
[0188] G. 5-(3-Benzenesulfonylamino)anthra[1,9cd]pyrazol-6(2H)-one
45
[0189] This compound may be made in the same manner using
benzenesulfonyl chloride as the sulfonyl chloride.
Example 5
Synthesis of Representative Compounds
[0190] 46
[0191] A. 7-(Benzoylamino)anthra[1,9cd]pyrazol-6(2H)-one 47
[0192] Benzoyl chloride is added to a solution of
2-(methoxyethoxymethyl)-- 7-aminoanthra[1,9cd]pyrazol-6-(2H)one and
triethylamine in methylene chloride at 0.degree. C. The mixture is
stirred for 16 hours, quenched with water, and extracted with ethyl
acetate (x2). The combined organic layer is washed with sodium
bicarbonate solution, and brine, dried and evaporated. The crude
reaction mixture is then taken in aqueous 6N hydrochloric acid, and
heated at 80.degree. C. for 4 hours. After cooling, the mixture is
extracted with ethyl acetate (x2), washed with brine, dried, and
evaporated. The residue is chromatographed on silica gel to furnish
the desired amide as a yellow solid.
[0193] The starting material is prepared as follows. Sodium
hexamethyldisilazide is added to a cooled (0.degree. C.) solution
of 7-nitroanthra[1,9cd]pyrazol-6(2H)-one (Example 1-E) in
tetrahydrofuran, and the mixture is stirred for 30 minutes at
0.degree. C. MEM-chloride is added, and the mixture is stirred for
16 hours at room temperature. Water is added and the mixture is
extracted with ethyl acetate (x2). The combined organic layer is
washed with aqueous sodium bicarbonate solution, water, 1 N
hydrochloric acid, and brine, dried and evaporated. The residue is
chromatographed on silica gel to give
2-MEM-7-nitroanthra[1,9cd]pyrazol-6(2H)-one as an oil.
[0194] Palladium(10%) on charcoal and
2-MEM-5-nitroanthra[1,9cd]pyrazol-6(- 2H)-one in ethanol is placed
under 1-atm of hydrogen, and the mixture was stirred for 6 hours.
The catalyst is filtered off over celite, and the filtrate is
evaporated to dryness to give 2-(methoxyethoxymethyl)-7-amino-
anthra[1,9cd]pyrazol-6-(2H)one, which is used without further
purification.
[0195] B. 7-(Isonicotinylamino)anthra[1,9cd]pyrazol-6(2H)-one
48
[0196] This compound may be made in the same manner using
isonicotinoyl chloride as the acid chloride.
[0197] C. 7-(Nicotinylamino)anthra[1,9cd]pyrazol-6(2H)-one 49
[0198] This compound may be made in the same manner using
nicotinoyl chloride as the acid chloride.
[0199] D.
5-(2-Thiophenecarbonylamino)anthra[1,9cd]pyrazol-6(2H)-one 50
[0200] This compound may be made in the same manner using
2-thiophenecarboxylic acid chloride as the acid chloride.
[0201] E. 7-(3-Methylbutyrylamino)anthra[1,9cd]pyrazol-6(2H)-one
51
[0202] This compound may be made in the same manner using
isopentanoyl chloride as the acid chloride.
[0203] F. 7-(3-Methanesulfonylamino)anthra[1,9cd]pyrazol-6(2H)-one
52
[0204] This compound may be made in the same manner using
methanesulfonyl chloride as the sulfonyl chloride.
[0205] G. 7-(3-Benzenesulfonylamino)anthra[1,9cd]pyrazol-6(2H-one
53
[0206] This compound may be made in the same manner using
benzenesulfonyl chloride as the sulfonyl chloride.
Example 6
Synthesis of Representative Compounds
[0207] 54
[0208] A. 5-(3-Methylbutyloxy)anthra[1,9]cd]pyrazol-6(2H)-one
55
[0209] Isopentyl bromide is added to a mixture of
3-(2-methoxyethoxymethyl- )5-hydroxyanthra[1,9cd]pyrazol-6(2H)-one
and potassium carbonate in dimethylformamide at room temperature.
After stirring the mixture for sixteen hours, water is added, and
the mixture was extracted with ethyl acetate (x2). The combined
organic layer is washed with aqueous sodium bicarbonate, water, 1N
hydrochloric acid, and brine, dried and evaporated. The reside is
taken in 6N hydrochloric acid and heated at 80.degree. C. for 4
hours. After cooling, the mixture is extracted with ethyl acetate
(x2), and the combined organic layer is washed with brine, dried,
and evaporated. The residue is purified by column chromatography to
afford the title compound as yellow solid.
[0210] The starting material is prepared as follows. Sodium
hexamethyldisilazide is added to a cooled (0.degree. C.) solution
of 5-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one (Example 1-F) in
tetrahydrofuran, and the mixture is stirred for 30 minutes at
0.degree. C. MEM-chloride is added, and the mixture is stirred for
16 hours at room temperature. Water is added and the mixture is
extracted with ethyl acetate (x2). The combined organic layer is
washed with aqueous sodium bicarbonate solution, water, 1 N
hydrochloric acid, and brine, dried and evaporated. The residue is
chromatographed on silica gel to give
2-MEM-5-henzyloxyanthra[1,9cd]pyrazol-6(2H)-one as an oil.
[0211] Palladium(10%) on charcoal and
2-MEM-5-benzyloxyanthra[1,9cd]pyrazo- l-6(2H)-one in ethanol is
placed under 1-atm of hydrogen, and the mixture stirred for 6
hours. The catalyst is filtered off over celite, and the filtrate
is evaporated to dryness to give 2-(2-methoxyethoxymethyl)-5-hyd-
roxyanthra[1,9cd]pyrazol-6-(2H)one, which is used without further
purification.
[0212] B. 5-(4-Pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one 56
[0213] This compound may be made in the same manner using
chloromethyl-4-pyridine as the alkyl halide.
[0214] C. 5-(3-Pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one 57
[0215] This compound may be made in the same manner using
chloromethyl-3-pyridine as the alkyl halide.
[0216] D. 5-(2-Methoxyethoxy)anthra[1,9cd]pyrazol-6(2H)-one 58
[0217] This compound may be made in the same manner using
2-methoxyethyl bromide as the alkyl halide.
[0218] E. 5-(2-Dimethylaminoethoxy)anthra[1,9cd]pyrazol-6(2H)-one
59
[0219] This compound may be made in the same manner using
2-dimethylaminoethyl chloride as the alkyl halide.
Example 7
Synthesis of Representative Compounds
[0220] 60
[0221] A. 7-(3-Methylbutyloxy)anthra[1,9cd]pyrazol-6(2H)-one 61
[0222] Isopentyl bromide is added to a mixture of
3-(2-methoxyethoxymethyl- )-7-hydroxyanthra[1,9cd]pyrazol-6(2H)-one
and potassium carbonate in dimethylformamide at room temperature.
After stirring the mixture for sixteen hours, water is added, and
the mixture was extracted with ethyl acetate (x2). The combined
organic layer is washed with aqueous sodium bicarbonate, water, 1 N
hydrochloric acid, and brine, dried and evaporated. The residue is
taken in 6N hydrochloric acid and heated at 80.degree. C. for 4
hours. After cooling, the mixture is extracted with ethyl acetate
(x2), and the combined organic layer is washed with brine, dried,
and evaporated. The residue is purified by column chromatography to
afford the title compound as yellow solid.
[0223] The starting material is prepared as follows. Sodium
hexamethyldisilazide is added to a cooled (0.degree. C.) solution
of 7-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one (Example 1-F) in
tetrahydrofuran, and the mixture is stirred for 30 minutes at
0.degree. C. MEM-chloride is added, and the mixture is stirred for
16 hours at room temperature. Water is added and the mixture is
extracted with ethyl acetate (x2). The combined organic layer is
washed with aqueous sodium bicarbonate solution, water, 1N
hydrochloric acid, and brine, dried and evaporated. The residue is
chromatographed on silica gel to give
2-MEM-7-benzyloxyanthra[1,9cd]pyrazol-6(2H)-one as an oil.
[0224] Palladium(10%) on charcoal and
2-MEM-7-benzyloxyanthra[1,9cd]pyrazo- l-6-(2H)-one in ethanol is
placed under 1-atm of hydrogen, and the mixture was stirred for 6
h. The catalyst is filtered off over celite, and the filtrate is
evaporated to dryness to give 2-(2-methoxyethoxymethyl)-7-hyd-
roxyanthra[1,9cd]pyrazol-6-(2H)one, which is used without further
purification.
[0225] B. 7-(4-Pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one 62
[0226] This compound may be made in the same manner using
chloromethyl-4-pyridine as the alkyl halide.
[0227] C. 7-(3-Pyridylmethoxy)anthra[1,9cd]pyrazol-6(2H)-one 63
[0228] This compound may be made in the same manner using
chloromethyl-3-pyridine as the alkyl halide.
[0229] D. 7-(2-Methoxyethoxy)anthra[1,9cd]pyrazol-6(2H)-one 64
[0230] This compound may be made in the same manner using
2-methoxyethyl bromide as the alkyl halide.
[0231] E. 7-(2-Dimethylaminoethoxy)anthra[1,9cd]pyrazol-6(2H)-one
65
[0232] This compound may be made in the same manner using
2-dimethylaminoethyl chloride as the alkyl halide.
Example 8
Activity of Representative Compound
[0233] The compounds of this invention may be assayed for their
activity accordingly to the following procedures.
[0234] JNK Assay
[0235] To 10 .mu.L of the test compound in 20% DMSO/80% dilution
buffer consisting of 20 mM HEPES (pH 7.6), 0.1 mM EDTA, 2.5 mM
magnesium chloride, 0.004% Triton x100, 2 .mu.g/mL leupeptin, 20 mM
.beta.-glycerolphosphate, 0.1 mM sodium vanadate, and 2 mM DTT in
water is added 30 .mu.L of 50-200 ng His6-JNK1, JNK2 or JNK3 in the
same dilution buffer. The mixture is preincubated for 30 minutes at
room temperature. Sixty microliter of 10 .mu.g GST-c-Jun(1-79) in
assay buffer consisting of 20 mM HEPES (pH 7.6), 50 mM sodium
chloride, 0.1 mM EDTA, 24 mM magnesium chloride, 1 mM DTT, 25 mM
PNPP, 0.05% Triton x100, 11 .mu.M ATP, and 0.5 .mu.Ci .gamma.-32P
ATP in water is added and the reaction is allowed to proceed for 1
hour at room temperature. The c-Jun phosphorylation is terminated
by addition of 150 .mu.L of 12.5% trichloroacetic acid. After 30
minutes, the precipitate is harvested onto a filter plate, diluted
with 50 .mu.L of the scintillation fluid and quantified by a
counter. The IC.sub.50 values are calculated as the concentration
of the test compound at which the c-Jun phosphorylation is reduced
to 50% of the control value. Preferred compounds of the present
invention have an IC.sub.50 value ranging 0.01-10 .mu.M in this
assay. To this end, a preferred compound of this invention is
Compound 1, which has an IC.sub.50 according to this assay of 0.11
.mu.M for JNK1 and JNK2, and 0.15 .mu.M for JNK3.
[0236] Selectivity For JNK
[0237] Compound 1 was also assayed for its inhibitory activity
against the following protein kinases by techniques known to those
skilled in this field (see, e.g., Protein Phosphorlation, Sefton
& Hunter, Eds., Academic Press, pp. 97-367, 1998):
1 Enzyme IC.sub.50 p38-2 >30,000 nM ERK 1 >30,000 nM MEKK 1
>30,000 nM IKK 1 >30,000 nM IKK 2 >30,000 nM PKA
>30,000 nM PKC >10,000 nM EGF-TK >10,000 nM
[0238] Jurkat T-Cell IL-2 Production Assay
[0239] Jurkat T cells (clone E6-1) are purchased from, the American
Tissue Culture Collection and maintained in growth media consisting
of RPMI 1640 medium containing 2 mM L-glutamine (Mediatech), with
10% fetal bovine serum (Hyclone) and penicillin/streptomycin. All
cells are cultured at 37.degree. C. in 95% air and 5% CO.sub.2.
Cells are plated at a density of 0.2.times.10.sup.6 cells per well
in 200 .mu.L of media. Compound stock (20 mM) is diluted in growth
media and added to each well as a 10.times. concentrated solution
in a volume of 25 .mu.L, mixed, and allowed to pre-incubate with
cells for 30 minutes. The compound vehicle (dimethylsulfoxide) is
maintained at a final concentration of 0.5% in all samples. After
30 minutes the cells are activated with PMA (phorbol myristate
acetate; final concentration 50 ng/mL) and PHA (phytohemagglutinin;
final concentration 2 .mu.g/mL). PMA and PHA are added as a
10.times. concentrated solution made up in growth media and added
in a volume of 25 .mu.L per well. Cell plates are cultured for 10
hours. Cells are pelleted by centrifugation and the media removed
and stored at -20.degree. C. Media aliquots are analyzed by
sandwich ELISA for the presence of IL-2 as per the manufacturers
instructions (Endogen). The IC.sub.50 values are calculated as the
concentration of the test compound at which the IL-2 production was
reduced to 50% of the control value. Preferred compounds of the
present invention have an IC.sub.50 value ranging 0.1-30 .mu.M in
this assay. FIG. 1 presents the dose dependent inhibition of IL-2
in Jarkat T-Cells by Compound 1 according to this procedure, with a
resulting IC.sub.50 of 5 .mu.M.
[0240] Mouse In Vivo LPS-Induced TNF-.alpha. Production Assay
[0241] Non-fasted mice are acclimatized for at least 7 days. Groups
of 4 to 6 female BALB/c or CD-1 mice (8-10 weeks of age from
Charles River laboratories) are pretreated with test compound,
either by intravenous injection or by oral gavage 15-180 minutes
prior to the injection of 0.5 mg/kg Bacto LPS from E. coli 055:B5
(Difco Labs). Ninety minutes after LPS challenge, a terminal bleed
is performed via abdominal vena cava and blood is allowed to clot
at room temperature for 30 minutes in Microtainer serum separator
tubes. After separation by centrifugation, the serum is stored
frozen at -80.degree. C. ELISA is performed on thawed, diluted
samples (1:10 to 1:20) using a Mouse TNF-alpha kit (Biosource
International). The ED.sub.50 values are calculated as the dose of
the test compound at which the TNF-.alpha. production is reduced to
50% of the control value. Preferred compounds of the present
invention have an ED.sub.50 value ranging 1-30 mg/kg in this assay.
FIG. 2 illustrates the results of this experiment utilizing
Compound 1 administered by intravenous injection (I.V.) at 15 and
30 mg/kg, as well as by per os (P.O.) at 7.5, 15 and 30 mg/kg.
Vehicle alone (PEG-400, propylene glycol, cremophor EL, and ethanol
in normal saline, "PPCES") and dexamethasone-21 acetate ("DEX") (1
mg/kg P.O.) were run as controls (n=6, *=p 0.01). Compound 1 was
administered 15 minutes pre-LPS challenge, and bleed occurred 90
minutes post LPS.
[0242] Inhibition of Leukocyte Recruitment in Rat Inflamed Lung
[0243] Aerosol administration of ovalbumun in Brown Norway Rats
previously sensitized by injection of ovalbumin (OA) results in an
allergic airway inflammation marked by the generation of an
eosinophil- and T-lymphocyte-rich leukocytic infiltration in the
lungs (see Richards et al., Am. J. Physiol. 271:2 Pt 1, L267-76,
1996). Compound 1 was administered by subcutaneous injection at a
dose of 30 mg/kg, b.i.d. for 3 days prior to ovalbumin challenge by
aerosol. Cell counts were obtained from samples of bronchoalveolar
lavage, the results of which are illustrated in FIG. 3 (V=PPCES
vehicle).
[0244] Rat In Vivo Adjuvant Arthritis
[0245] Male Lewis rats were immunized with complete Freund's
adjuvant on day 0 to induce an aggressive arthritis characterized
by joint destruction and paw swelling. Compound 1 was administered
subcutaneously once daily from day 8 to day 20. Paw swelling was
determined be water displacement plethysmometry (see FIG. 4A;
*=p<0.01). Radiographs were obtained of the right hind paw to
assess bone changes using a semi-quantitative scoring system:
demineralization (0-2+), calcaneal erosion (0-1+), and heteretropic
bone formation (0-1+), with a maximum possible score=6 (see FIG.
4B). Activation of AP-1 (see FIG. 4C) was determined by DNA binding
activity in an electrophoretic mobility shift assay (EMSA) (Ausubel
et al., Short Protocols in Molecular Biology, Second Edition, John
Wiley & Sons Publisher, New York, 1992). Matrix
metalloproteinase-13 expression (see FIG. 4D) was measured by
nothern blot analysis of MMP-13 mRNA (Ausebel et al., supra) (see
also Winter et al., Arthritis and Rheumatism 9(3):394-404, 1966;
Weichman et al., Pharmacological Methods in the Control of
Inflammation, Chang and Lewis Eds., AlaD R. Liss, Inc., Publ., New
York, 1989).
[0246] Kainic Acid-Induced Seizure Response
[0247] Compound 1 was administered to male CD rats at 10 mg/kg
intravenously through a tail vein catheter. This was followed
immediately by a 30 mg/kg subcutaneous injection. Vehicle controls
received the same injection volumes of the PPCES vehicle alone.
Thirty minutes later, animals were given a 1-mg/kg i.p. injection
of kainic acid in normal saline solution. This dose of kainic acid
has been previously reported to induce a seizure syndrome in rats
(Maj et al., Eur. J. Pharm. 359:27-32, 1992). Seizure behavior was
monitored for 4 hours following kainic acid injection. As presented
in FIG. 5, behaviors were assessed based on the following
cumulative scoring system: 1 pt.=arrest of motion; 2 pts.=myoclonic
jerks of the head and neck (moderate); 3 pts.=unilateral or
bilateral forelimb clonic activity; 4 pts.=whole body clonus; 5
pts.=clonic-tonic seizures; 6 pts.=status epilepticus (see also
Mathis and Ungerer, Exp. Brain Res. 88:277-282, 1992; Rong et al.,
Proc. Natl. Acad. Sci. USA 96:9897-9902, 1999; Yang et al., Nature
389:865-870, 1997).
[0248] It will be appreciated that, although specific embodiments
of the invention have been described herein for purposes of
illustration, various modifications may be made without departing
from the spirit and scope of the invention. Accordingly, the
invention is not limited except as by the appended claims.
* * * * *