EP1363993A2 - Methodes de traitement de maladies inflammatoires et immunitaires a l'aide d'inhibiteurs d'i kappa b kinase (ikk) - Google Patents

Methodes de traitement de maladies inflammatoires et immunitaires a l'aide d'inhibiteurs d'i kappa b kinase (ikk)

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Publication number
EP1363993A2
EP1363993A2 EP02714815A EP02714815A EP1363993A2 EP 1363993 A2 EP1363993 A2 EP 1363993A2 EP 02714815 A EP02714815 A EP 02714815A EP 02714815 A EP02714815 A EP 02714815A EP 1363993 A2 EP1363993 A2 EP 1363993A2
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Prior art keywords
ikk
inhibitor
activity
disease
inflammatory
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EP02714815A
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German (de)
English (en)
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EP1363993A4 (fr
Inventor
James R. Burke
Steven Nadler
Yuping Qiu
Robert M. Townsend
Fred Christopher Zusi
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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Priority claimed from US09/965,977 external-priority patent/US6960585B2/en
Application filed by Bristol Myers Squibb Co filed Critical Bristol Myers Squibb Co
Publication of EP1363993A2 publication Critical patent/EP1363993A2/fr
Publication of EP1363993A4 publication Critical patent/EP1363993A4/fr
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    • A61K31/47—Quinolines; Isoquinolines
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Definitions

  • the present invention relates to methods of using inhibitors of l ⁇ B kinase (IKK) in the treatment of inflammatory and immune diseases, to inhibitors of IKK, and to pharmaceutical compositions comprising such inhibitors, together with a pharmaceutically or physiologically-acceptable vehicle or excipient.
  • IKK inhibitors of l ⁇ B kinase
  • Tumor necrosis factor is a potent cytokine having pro- inflammatory properties that is released by many cell types when stimulated.
  • TNF- ⁇ Tumor necrosis factor
  • Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Creutzfeldt-Jacob disease also are reportedly associated with enhanced TNF- ⁇ levels.
  • NF-KB nuclear factor- ⁇ B
  • IMM-1 interleukin-1
  • VCAM-1 vascular cellular adhesion molecule-1
  • NF- ⁇ B and/or its activation pathway provides a means for treating a wide range of diseases including autoimmune diseases, Alzheimer's disease, atherosclerosis, oncogenesis, and so forth.
  • diseases including autoimmune diseases, Alzheimer's disease, atherosclerosis, oncogenesis, and so forth.
  • NF- ⁇ B is a transcriptional activator, which plays a central role in regulating the transcription of a number of genes including those, which encode proteins, involved in inflammatory and immune responses.
  • genes controlled by NF- ⁇ B include the cytokines tumor necrosis factor (TNF- ⁇ ), IL-1 ⁇ , IL-6, and IL-8; the adhesion molecules E-selectin and vascular cell adhesion molecule (VCAM)-1 ; and the enzyme nitric oxide (NO)-synthase (for reviews, see Siebenlist et al. Annu. Rev. Cell Biol. 10: 405-455, 1994; Bauerle and Baltimore, Cell 87:13- 20, 1997).
  • NF- ⁇ B has been shown to be inducible by several stimuli, in addition to mediators of immune function, such as UV irradiation, growth factors, and viral infection.
  • NF- ⁇ B transcription factor normally resides in the cytoplasm in unstimulated cells as an inactive complex with a member of the inhibitor KB (IKB) inhibitory protein family.
  • IKB inhibitor KB
  • the I ⁇ B class of proteins includes l ⁇ B- ⁇ , l ⁇ B- ⁇ , and l ⁇ B- ⁇ all of which contain ankyrin repeats for complexing with NF- ⁇ B (for review, see Whiteside et al., EMBO J. 16:1413-1426,1997).
  • I ⁇ B- ⁇ the most carefully studied member of this class, stimulation of cells with agents which activate NF- ⁇ B-dependent gene transcription results in the phosphorylation of l ⁇ B- ⁇ at serine-32 and serine-36 (Brown et al.
  • IKB is a cytoplasmic protein that controls NF- ⁇ B activity by retaining NF- ⁇ B in the cytoplasm.
  • IKB is phosphorylated by the IKB kinase (IKK), which has two isoforms, IKK-1 (or IKB kinase ⁇ , IKK ⁇ ) and IKK-2 (or IKB kinase ⁇ , IKK ⁇ ) .
  • NF- ⁇ B Upon phosphorylation of IKB by IKK, NF- ⁇ B is rapidly released into the cell and translocates to the nucleus where it binds to the promoters of many genes and up-regulates the transcription of pro-inflammatory genes.
  • Inhibitors of IKK can block the phosphorylation of IKB and further downstream effects, specifically those associated with NF- ⁇ B transcription factors.
  • Glucocorticoids reportedly inhibit NF- ⁇ B activity by two mechanisms, i.e., upregulating IKB protein levels and inhibiting NF- ⁇ B subunits.
  • Nitric oxide also reportedly inhibits NF- KB through upregulation of IKB. However, these mechanisms of interaction are complex; for example, production of nitric oxide in lymphocytes reportedly enhances NF- ⁇ B activity.
  • NF- ⁇ B- ⁇ Phosphorylation of l ⁇ B- ⁇ is critical for its subsequent ubiquitination and proteolysis, upon which NF- ⁇ B is released from complexing with IKB. NF- ⁇ B can then translocate into the nucleus and ultimately, activate gene transcription (Finco et al., Proc. Natl. Acad. Sci. USA 91 :11884-11888, 1994; Baldi et al. J. Biol. Chem. 271 :376-379, 1996; Roff et al. J. Biol.
  • NF- ⁇ B is released from the l ⁇ B/NF- ⁇ B complex and allowed to translocate from the cytoplasm to the nucleus and activate a number of genes, particularly those involved in inflammatory and immune responses. Since NF- ⁇ B is of significant importance in inflammation and immune responses, inhibition of the signal-inducible phosphorylation of IKB can be an important target for novel anti-inflammatory and immune-related agents in the treatment of inflammatory and immune system-related diseases and disorders, as described herein.
  • IKB kinase is a high molecular weight (500-900 kD) multisubunit enzyme which phosphorylates l ⁇ B- ⁇ at positions serine-32 and serine-36 and has been isolated from HeLa cells (Chen et al. Cell 84:853-862, 1996; Lee et al. Cell 88:213-222, 1997; DiDonato et al. Nature 388:548-554, 1997).
  • Two catalytic subunits termed IKK-1 and IKK-2 of IKK have been identified, cloned, and demonstrated to be widely expressed in human tissues (DiDonato et al. Nature 388:548-554, 1997; Zandi et al.
  • IKK-1 and IKK-2 catalytic subunits of IKK are highly homologous, having 50% sequence identity and more than 70% sequence similarity.
  • IKK- 1 and IKK-2 are 85- and 87-kDa proteins, respectively. Both kinases contain a catalytic domain followed by a leucine zipper domain and a helix- loop-helix (HLH) domain (Mercurio et al. Science, 278:860-866, 1997). When one subunit is recombinantly expressed without the other subunit, either one is still able to catalyze the phosphorylation of IKB (Li et al. J. Biol. Chem., 273:30736-30741 , 1998). Thus, IKK, either IKK-1 or IKK-2, can play an important role in signaling IKB for ubiquination and further degradation.
  • kinases which can phosphorylate IKB and which have been implicated in the activation of NF- ⁇ B.
  • two kinases pp90rsk and IKK- ⁇
  • IKK-1 and/ or IKK-2 have been demonstrated to phosphorylate l ⁇ B- ⁇ at serine-32 and/ or serine-36.
  • the overexpression of these kinases, and the use of dominant negative mutants to these kinases, have indicated a role for them in the phosphorylation of IKB in cells (Ghoda et al., J. Biol. Chem. 272:21281-21288, 1997; Peters et al., Mol. Cell. 5:513-522, 2000).
  • the existence of multiple IKB kinases is indicative of redundant signaling pathways. Therefore, it is possible that an inhibitor of IKK-1 and/ or IKK-2 can not necessarily show anti-inflammatory or immunosuppressive effects due to redundant signaling pathways in at least some cells.
  • IKK-1 and IKK-2 have been performed to further investigate IKK and its properties.
  • the in vitro types of cell biology studies for example, overexpression of either IKK-1 or IKK-2, or of dominant negative versions of these kinases (DiDonato et al. Nature 388:548-554, 1997; Mercurio et al. Science 278:860-866, 1997; Woronicz et al. Science 278:866-869, 1997; Regnier et al. Cell 90:373-383; Zandi et al. Cell 91 :243- 252)) that appear to implicate IKK-1 and IKK-2 in NF- ⁇ B activation are sometimes artifactual.
  • a particular example of such an artifactual study involves a kinase known as NF- ⁇ B-inducing kinase, NIK.
  • NIK is suggested to be essential for the activation of IKK and, subsequently, NF- ⁇ B.
  • aspirin acetylsalicyclic acid
  • salicylate have been demonstrated to be inhibitors of IKK-1 and IKK-2, but only at high, non- physiological concentrations which are much higher than those required to block prostaglandin synthesis through the inhibition of cyclooxygenase (Yin et al., Nature 396:77-80, 1998). Therefore, these agents are inappropriate for use in treatments or for testing the role of IKK in disease.
  • Sulindac is a cyclooxygenase inhibitor that has been demonstrated to also inhibit IKK-2 (Yamamoto et al., J. Biol. Chem. 274:27307-27314, 1999). However, in studies similar to those using aspirin, the concentrations of sulindac necessary for inhibiting IKK were much greater than those needed to inhibit cyclooxygenase. Therefore, this agent is also inappropriate for testing the role of IKK in disease.
  • IKK-2 cyclopentenone prostaglandins
  • 15dPGJ2 Rossi et al., Nature 403:103-108, 2000
  • 15dPGJ2 can also activate peroxisome proliferator-activated receptor-gamma (PPAR- ⁇ ), a nuclear receptor that interferes with NF- ⁇ B transcriptional activity. Therefore, any anti- inflammatory effect could be explained by PPAR- ⁇ activity rather than IKK inhibition.
  • PPAR- ⁇ peroxisome proliferator-activated receptor-gamma
  • this inhibitor is not specific for inhibiting the downstream effects of NF- ⁇ B-induced inflammation via IKK.
  • Arsenite another IKK inhibitor, is a reactive environmental toxin that has been shown to inhibit both IKK-1 and IKK-2 (Kapahi et al., J. Biol. Chem., 275:36062-36066, 2000). Due to its toxic effects, the therapeutic benefits or in vivo treatment utility of arsenite would therefore preclude its use in patients.
  • the Astrazeneca AB (WO 01/58890) publication discloses particular heteroaromatic carboxamide derivatives that are structurally different from the compounds of the present invention.
  • the compounds of the WO 01/58890 publication do not demonstrate in vivo efficacy of the IKK inhibitor compounds of the present invention in animal models of disease associated with inflammation and/or the immune system.
  • IKK knockout mice In animal models of disease (i.e. knock-out mice) designed to test inhibition of IKK, the deletion of IKK-1 or IKK-2 has been demonstrated to be embryonic lethal (Li et al. Science 284:321-325, 1999; Hu et al. Science 284:316-320, 1999). Therefore, the use of IKK knockout mice to demonstrate a role of IKK in disease is neither practical nor feasible. In contrast, the present invention demonstrates for the first time that inhibitors of the catalytic activity of IKK-1 and IKK-2 are effective in murine models of disease. Such models are believed to be predictive of similar effects in human patients.
  • Lactam-based tetracyclic compounds useful as antagonists of NMDA (N-methyl-D-aspartate) and AMPA ( ⁇ -3-hydroxy-5-methylisoxazole-4- propionate) receptors are disclosed in WO 94/07893, Preparation of 5H, 10H-imidazo[1 ,2-a]indeno[1 ,2-e]pyrazine-4-one AMPA/KA Receptor Antagonist, filed by Aloup et al ; and in articles by Mignani, Aloup, et al., "Synthesis and Pharmacological Properties of 5H, 10H-imidazo[1 ,2- a]indeno[1 ,2-e]pyrazine-4-one, a New Competitive AMPA/KA Receptor Antagonist," Drug.
  • the present invention relates to methods of preventing and treating inflammatory and immune-related diseases or disorders using IKB kinase (IKK) inhibitors, compounds of formula (I) as described herein. Also provided is the IKK inhibitor 4(2'-aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline used to prevent and treat inflammatory and immune-related diseases or disorders.
  • IKK IKB kinase
  • the methods comprise the use of IKK inhibitors for the prevention and treatment of inflammatory and immune- related diseases, non-limiting examples of which include, rheumatoid arthritis, transplant rejection, inflammatory bowel disease, osteoarthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, psoriasis, multiple sclerosis, stroke, systemic lupus erythematosus, Alzheimer's disease, brain ischemia, traumatic brain injury, Parkinson's disease, amyotrophic lateral sclerosis, subarachnoid hemorrhage or other diseases or disorders associated with excessive production of inflammatory mediators in the brain and central nervous system.
  • IKK inhibitors for the prevention and treatment of inflammatory and immune- related diseases, non-limiting examples of which include, rheumatoid arthritis, transplant rejection, inflammatory bowel disease, osteoarthritis, asthma, chronic obstructive pulmonary disease, atherosclerosis, psoriasis, multiple sclerosis, stroke, systemic lup
  • Yet another object of the present invention is to provide methods of inhibiting i) the catalytic activity of IKK; ii) the phosphorylation of IKB; and/or iii) NF- ⁇ B gene expression using IKK inhibitors for the prevention and treatment of inflammatory and immune-related diseases or disorders. .
  • a more particular object of the present invention is to provide an IKK inhibitor, 4(2'-aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline, also known as compound 6 herein, or a pharmaceutically-acceptable salt thereof, useful as a preventative and/or therapeutic agent and in methods of treating inflammatory and immune diseases.
  • compound 6 is shown by the below formula,
  • Yet another embodiment of the present invention relates to 4-amino substituted benzoquinoline, benzoquinoxaline, and benzoquinazoline compounds having five-membered heterocycles (e.g., pyrazolyl, imidazolyl, and thiazolyl rings) fused thereto.
  • the compounds are useful as anti- inflammatory agents and/or for treating conditions associated with TNF- ⁇ and NF- ⁇ B activity.
  • the compounds are useful as anti- inflammatory agents and/or for preventing and treating conditions associated with TNF- ⁇ and NF- ⁇ B.
  • One embodiment of the present invention is further directed to a compound of the formula (I), useful in treating inflammatory diseases or disorders:
  • R 2 is alkyl, substituted alkyl, alkenyl, alkynyl, alkoxy, alkylthio, aryl, heteroaryl, heterocyclo, cycloalkyl, or substituted cycloalkyl;
  • R 5 , ⁇ . R7, and R 8 are independently selected from hydrogen, alkyl, substituted alkyl, and phenyl, or when attached to the same nitrogen atom (as in NR 5 R 6 or NR 7 R 8 ) can join together to form a heterocycle or heteroaryl; and m, n and 1 are independently 0, 1 , or 2.
  • R 2 is an alkyl optionally substituted with OR' or NR'R", as defined in the specification.
  • the invention also relates to pharmaceutical compositions containing at least one compound of formula (I) and a pharmaceutically-acceptable carrier or diluent. Also included within the invention are methods of 1 preventing and treating inflammatory and immune-related diseases and disorders comprising administering to a mammal in need of such prevention and/or treatment an effective amount of at least one compound of formula
  • kits for treating inflammatory and immune-related diseases and disorders comprising one or more IKK inhibitors, compound 6, and/or compounds of formula (I) as described herein.
  • FIGURE 1 shows the effect of 4(2'-aminoethyl)amino- 1 ,8- dimethylimidazo(1 ,2-a) quinoxaline (compound 6) on disease incidence in the murine model of collagen-induced arthritis.
  • the compound was administered once daily in a preventative dosing mode. *p ⁇ 0.05, Fisher's Exact test.
  • FIGURE 2 shows the effect of 4(2'-aminoethyl)amino- 1 ,8- dimethylimidazo(1 ,2-a) quinoxaline (compound 6) on the average gross clinical score for all mice within each treatment in the murine model of collagen-induced arthritis based on clinical scores corresponding to the study day.
  • the test compound was administered once daily in the preventative dosing mode. * p ⁇ 0.05, Mann-Whitney U-test.
  • FIGURE 3 shows the effect of 4(2'-aminoethyl)amino- 1 ,8- dimethylimidazo(1 ,2-a) quinoxaline (compound 6) on disease incidence in the murine model of collagen-induced arthritis based on the clinical score corresponding to the days after disease onset.
  • the test compound was administered once daily in the established disease dosing mode. *p ⁇ 0.05, Mann-Whitney U-test.
  • the present invention describes methods of preventing and treating inflammatory and immune-related diseases or disorders using inhibitors of IKB kinase (IKK). Such methods are efficacious in vivo in inhibiting IKK activity in mammalian, including human and biological systems, e.g. animal models of disease.
  • the present invention relates to methods of inhibiting the catalytic activity of IKK using inhibitors.
  • the inhibitors of the present invention preferably inhibit the catalytic activity of the IKKs (see, Example 2).
  • inhibitors can include small molecule enzyme inhibitors which bind directly to the enzyme, preferably IKK, and more preferably IKK-2. Without wishing to be bound by theory, it is believed that the binding of the inhibitor to the active site of IKK also inhibits binding of substrate, such as, for example, IKB or ATP.
  • One embodiment of the present invention provides a novel IKK inhibitor, 4(2'-aminoethyl) amino-1 ,8-dimethylimidazo(1 ,2-a) quinoxaline (compound 6), wherein the compounds of the present invention can be used in methods for the prevention and treatment of inflammatory and immune- related diseases or disorders.
  • Another embodiment of the present invention provides 4-amino substituted benzoquinoline, benzoquinoxaline, and benzoquinazoline compounds having five-membered heterocycles (e.g., pyrazolyl, imidazolyl, and thiazolyl rings) fused thereto.
  • the compounds are useful as anti- inflammatory agents and/or for treating conditions associated with TNF- ⁇ and NF- ⁇ B.
  • compositions comprising IKK inhibitor compounds or salts thereof, and kits comprising IKK inhibitor compounds or salts thereof.
  • IKB is defined as Inhibitor KB.
  • the IKB family comprises l ⁇ B ⁇ , l ⁇ B ⁇ , and l ⁇ B ⁇ , all of which contain ankyrin repeats for complexing to NF- ⁇ B.
  • IKK refers to l ⁇ B-kinase (IKK). IKK comprises two catalytic subunits, IKK-1 and IKK-2, also known as IKK ⁇ and IKK ⁇ , respectively.
  • Activation of IKK means changing an inactive IKK protein into an active IKK protein that functions as an IKB kinase. Activated IKK phosphorylates serine-32 and serine-36 of IKB, which thus marks the IKB for ubiquit ⁇ nation and degradation.
  • IKK inhibitors refer to those compounds or molecules which prevent, block, abolish, antagonize, suppress, or reduce the activation of IKK as defined above.
  • IKK inhibitors inhibit the activity of IKK, preferably the in vivo activity of IKK, such that one or more of the following occurs: 1) inhibition of IKK catalytic activity; 2) inhibition of IKB phosphorylation, preferably catalyzed phosphorylation of IKB; and/or 3) inhibition of NF- ⁇ B-dependent gene expression activation.
  • An inhibitor is considered to be selective for one protein over another protein when there is preferably at least about a 5-fold or greater difference in inhibition of one protein compared with another protein. More preferably, there is at least about an 8-fold or greater difference in inhibition of one protein compared with another protein.
  • Nuclear factor- ⁇ B or "NF- ⁇ B” as used herein, is defined as a ubiquitously expressed family of eukaryotic transcription factors. This family comprises a homo- or hetero-dimer of DNA-binding proteins related to c-Rel, a proto-oncogene that controls the expression of many ⁇ B-dependent immune, inflammatory, and anti-apoptotic response genes.
  • NF- ⁇ B-dependent gene expression is defined as those immune-related and inflammatory genes that are under the regulatory control of the ⁇ B-enhancer.
  • IKB inhibitory proteins
  • Activation of NF- ⁇ B can be induced by cytokines, such as TNF ⁇ and IL-1 , where both TNF- ⁇ and IL-1 signaling results in sequential phosphorylation and activation of a cascade of proteins.
  • cytokines such as TNF ⁇ and IL-1
  • IKK activation of IKK or modification of the catalytic action of IKK results in phosphorylation, ubiquitination, and degradation of IKB.
  • treat refers to the prevention, reduction, or amelioration, partial or complete alleviation, or cure of a disease, disorder, or condition.
  • NF- ⁇ B plays an important role in inflammation and immune response; therefore, the inhibition of IKB phosphorylation can also be an important target for novel anti-inflammatory and immune-related agents.
  • NF- ⁇ B-associated condition refers to diseases that are characterized by release of NF- ⁇ B from the cytoplasm (e.g., upon phosphorylation of IKB).
  • TNF- ⁇ -associated condition is a condition characterized by enhanced levels of TNF- ⁇ .
  • NF- ⁇ B-associated condition includes a TNF- ⁇ -associated condition but is not limited thereto as NF- ⁇ B is involved in the activity and upregulation of other pro-inflammatory proteins and genes.
  • inflammatory or immune diseases or disorders is used herein to encompass both IKK-associated conditions, NF- ⁇ B-associated conditions, TNF- ⁇ -associated conditions, e.g., any condition, disease, or disorder that is associated with release of NF- ⁇ B and/or enhanced levels of TNF- ⁇ , including each of the conditions specifically referenced below.
  • Bioavailability as referred to herein is the rate and extent to which an active ingredient or moiety is absorbed from a pharmaceutical composition and becomes available at the site of action. For drug products that are not intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements intended to reflect the rate and extent to which the active ingredient or active moiety becomes available at the site of action.
  • prodrug denotes a therapeutic agent that is prepared in an inactive form, which upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes by the action of endogenous enzymes or other chemicals and/or conditions, to yield the active compounds of the present invention, and/or salt and/or solvate thereof.
  • compounds containing a carboxy group can form physiologically hydrolyzable esters which serve as prodrugs by being hydrolyzed in the body to yield formula l-ll compounds per se.
  • alkyl refers to straight or branched chain hydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms. Lower alkyl groups, that is, alkyl groups of 1 to 4 carbon atoms, are most preferred. When numbers appear in subscript after the symbol "C", the subscript defines with more specificity the number of carbon atoms that a particular group can contain. For example, “C - 6 alkyl” refers to straight and branched chain alkyl groups with one to six carbon atoms, such as methyl, ethyl, n- propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and so forth.
  • substituted alkyl includes a polyfluoroalkyl, i.e., where two or more hydrogen atoms of an alkyl chain are replaced by a fluorine atom, such as with trifluoromethyl.
  • R' and R" can together form a heterocyclo or heteroaryi ring.
  • a substituted lower alkyl or substituted C ⁇ - alkyl refers to an alkyl group of 1- 4 carbon atoms having one to three substituents selected from those recited above for alkyl groups generally.
  • alkoxy refers to an alkyl group as defined above bonded through an oxygen atom (-O-)
  • alkylthio refers to an alkyl group as defined above bonded through a sulfur atom (-S-).
  • such groups include methoxy, methylthio, ethoxy, ethylthio, n-propoxy, n- propylthio, isopropoxy, isopropylthio, n-butoxy, n-butylthio, tert-butoxy, tert- butylthio, n-pentoxy, n-pentylthio, and so forth.
  • alkenyl refers to straight or branched chain hydrocarbon groups having 2 to 12 carbon atoms and at least one double bond. Alkenyl groups having 2 to 6 carbon atoms and one double bond are most preferred.
  • Exemplary alkenyl groups include ethenyl, 1-methyl-ethenyl, 1- or 2- propenyl, 1-methyl-1 -propenyl, 1-methyl-2-propenyl, 1 ,1-dimethyl-2- propenyl, 2-methyl-2-propenyl, 1- , 2- or 3-butenyl, 1-methyl-1-butenyl, 2- methyl-1-butenyl, 3-methyl-1-butenyl, 3,3-dimethyl-1-butenyl, 2,3-dimethyl- 1-butenyl, 1-methyl-2-butenyl, 1 ,1-dimethyl-2-butenyl, 2-methyl-2-butenyl, 3- methyl-2-butenyl, 1 ,3-butadienyl, 1 ,3-dimethyl-1 ,3-butadienyl, 1-, 2-, 3- or 4- pentenyl, and so forth.
  • alkynyl refers to straight or branched chain hydrocarbon groups having 2 to 12 carbon atoms and at least one triple bond. Alkynyl groups of 2 to 6 carbon atoms and having one triple bond are most preferred.
  • Exemplary alkynyl groups include ethynyl, 1- or 2-propynyl, 1- methyl-2-propynyl, 1 ,1-dimethyl-2-propynyl, 1-, 2- or 3-butynyl, 3-methyl-1- butynyl, 3, 3-dimethyl-1 -butynyl, 1-methyl-2-butynyl, 1 ,1-dimethyl-2-butynyl, 1-, 2- 3, or 4-pentynyl, and so forth.
  • amino when used alone refers to NH 2 .
  • amino refers to the group NR'R", wherein R' and R" are as defined above for alkyl.
  • halo or halogen refers to chloro, bromo, fluoro and iodo.
  • cycloalkyl refers to fully saturated and partially unsaturated hydrocarbon rings of 3 to 9, preferably 3 to 7, carbon atoms as well as such rings having a fused aryl ring such as indan or a bridge of three to four carbon atoms as in bicycloheptane.
  • aryl refers to phenyl, 1-naphthyl and 2-naphthyl, with phenyl being preferred.
  • heterocyclo refers to substituted and unsubstituted non-aromatic 3 to 7 membered monocyclic groups, 7 to 11 membered bicyclic groups, and 10 to 15 membered tricyclic groups which have at least one heteroatom (O, S, or N) in at least one of the rings.
  • heterocyclo is monocyclic, five- or six-membered rings are preferred; when bicyclic, fused 5,6- or 6,6-membered ring systems are preferred; and when tricyclic, ring systems having one five and two six membered rings, or three six membered rings, are preferred.
  • Each ring of the heterocyclo group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less, and further provided that the ring contains at least one carbon atom.
  • the fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated, or unsaturated.
  • the nitrogen and sulfur atoms can optionally be oxidized and the nitrogen atoms can optionally be quaternized.
  • the heterocyclo group can be attached at any available nitrogen or carbon atom.
  • Exemplary monocyclic groups include tetrahydrothienyl, tetrahydrofuryl, azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2- oxoazepinyl, azepinyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1 ,3- dioxolane and tetrahydro-1 ,1-dioxothienyl and
  • heteroaryl is monocyclic, five- or six membered rings are preferred; when bicyclic, fused 5,6- or 6,6- membered ring systems are preferred; and when tricyclic, ring systems having one five and two six membered rings, or three six-membered rings, are preferred.
  • Each ring of the heteroaryl group containing a heteroatom can contain one or two oxygen or sulfur atoms and/or from one to four nitrogen atoms provided that the total number of heteroatoms in each ring is four or less and each ring has at least one carbon atom.
  • the fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated, or unsaturated.
  • the nitrogen and sulfur atoms can optionally be oxidized and the nitrogen atoms can optionally be quaternized.
  • Heteroaryl groups which are bicyclic or tricyclic include at least one fully aromatic ring but the other fused ring or rings can be aromatic or non-aromatic.
  • the heteroaryl group can be attached at any available nitrogen or carbon atom of any ring.
  • Exemplary monocyclic heteroaryl groups include pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, and the like.
  • Exemplary bicyclic heteroaryl groups include indolyl, isoindolyl, benzothiazolyl, benzodioxolyl, benzoxaxolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, quinazolinyl, indazolyl, pyrrolopyridyl, furopyridinyl, dihydroisoindolyl, tetrahydroquinolinyl, napthyridinyl, pteridinyl, and the like.
  • Exemplary tricyclic heteroaryl groups include carbazolyl, benzidolyl, phenanthrollinyl, acridinyl, phenanthridinyl, xanthenyl and the like.
  • one emobidment of the present invention generally relates to the prevention and treatment of inflammatory and immune-related diseases, disorders, or conditions using IKK inhibitors.
  • embodiments of the present invention relate to: 1) methods of inhibiting IKK to prevent and/or treat inflammatory and immune-related diseases, disorders, or conditions; 2) methods of inhibiting the activation of NF- ⁇ B-dependent gene expression by inhibiting the IKK-dependent phosphorylation of IKB for preventing and treating inflammatory and/or immune-related diseases, disorders, or conditions; 3) inhibitors of IKK; 4) the use of inhibitors of IKK in the methods of the present invention; and 5) exemplary IKK inhibitor compounds which inhibit IKK.
  • an embodiment of the present invention demonstrates the in vivo employment of an IKK inhibitor for use in methods for treating and preventing inflammatory and immune-related diseases or disorders (Examples 4-7).
  • IKK inhibitors Prior to the present invention, there have been no demonstrations of the actual in vivo therapeutic effectiveness of IKK inhibitors in treating and preventing inflammatory or immune-related diseases, such as, for example, asthma, inflammatory bowel disease, tissue/organ transplant rejection, e.g., graft versus host rejection, and pulmonary diseases, for example, involving inflammatory cell infiltration in the lung.
  • an aspect of the present invention provides therapeutic and prophylactic methods of treating and preventing inflammatory or immune-related diseases, disorders, or conditions by administering an effective amount of one or more IKK inhibitors, or salt thereof, alone, or optionally, in combination with another IKK inhibitor, to a subject, preferably a mammal, in need thereof.
  • other therapeutic or biologically active agents can be used in combination with the IKK inhibitors, for example, drugs, hormones, or synthetic agents, such as those described below, for example.
  • such therapeutic or biologically active agents can be administered prior to, simultaneously with, or following the administration of one or more of the IKK inhibitors of the present invention.
  • IKK inhibitors can also be used in accordance with the described methods in conjunction with other drugs, such as cyclosporine A or CTLA4-lg, to alleviate transplant rejection.
  • Cyclosporine A is a potent immunosuppressive compound currently used therapeutically in humans to prevent graft rejection following solid organ transplantation.
  • Most current therapeutic protocols for transplant include cyclosporine A in combination with other potent immunosuppressive drugs such as steroids and antiproliferative agents such as mycophenolic acid.
  • the protocols When test compounds are examined for clinical efficacy in solid organ transplant, the protocols generally include treatment of the patients with test compounds in addition to standard therapeutics including cyclosporine A. Therefore, in order to progress with clinical development of new compounds, new compounds must work beneficially well with cyclosporine A.
  • CTLA4-lg is an immunoglobulin fusion protein that binds to the ligands for CD28 and CTLA4 (Linsley et al. J. Exp. Med., 174:561-569, 1991).
  • CTLA4-lg is a promising agent that is effective in the treatment of transplant rejection (Larsen et al., Nature, 381 :434-438, 1996). Since some form of CTLA4-lg could become part of the standard treatment paradigm for transplantation, new compounds developed for the prevention of graft rejection would be important, particularly in conjunction with CTLA4-lg.
  • Embodiments of the present invention further embrace methods of inhibiting the IKK enzyme, comprising administering to an individual suffering from an inflammatory or immune-related disease or disorder, an IKK inhibitor in an amount effective to inhibit the catalytic activity of IKK and the IKK-dependent phosphorylation of IKB.
  • an IKK inhibitor in an amount effective to inhibit the catalytic activity of IKK and the IKK-dependent phosphorylation of IKB.
  • the mechanism of inhibition of IKK involves inhibiting the catalytic activity of the IKKs, either IKK-1 or IKK-2, preferably IKK-2, through binding to the active site of the enzyme and inhibiting the binding and/or phosphorylation of substrates such as IKB and ATP (see Example 2).
  • One embodiment of the present invention encompasses a method of inhibiting the phosphorylation of IKB inhibitor protein which comprises the administration of IKK inhibitors in an amount effective to inhibit the phosphorylation of the IKB protein.
  • inhibition of l ⁇ B- ⁇ phosphorylation is preferred, and the inhibition of phosphorylation of l ⁇ B- ⁇ at serine-32 and serine-36 is more preferred.
  • compounds which inhibit IKK can be used in the methods for preventing and treating inflammatory and immune system-related conditions, diseases, or disorders.
  • diseases, disorders, and conditions include arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, acute pancreatitis, chronic pancreatitis, psoriasis, glomerulonephritis, serum sickness, lupus (systematic lupus erythematosis), urticaria, scleraclerma, contact dermatitis, dermatomyositis, alopecia, atopic eczemas, ichthyosisrhinitis, inflammatory bowel disease (Crohn's and ulcerative colitis); Alzheimer's disease, brain ischemia, traumatic brain injury, Parkinson's Disease, Creutzfeldt-Jacob diseases, HIV encephalitis, cerebral malaria, and meningitis, at
  • IKK inhibitors of the present invention can be used to prevent, treat or ameliorate diseases and conditions, particularly inflammatory or immune- related diseases or conditions.
  • the IKK inhibitors are employed in preventing and treating diseases, disorders, or conditions characterized by NF- ⁇ B activation via phosphorylation of IKB by IKK.
  • the embodiments of the present invention further relate to IKK inhibitors that inhibit the catalytic activity of the IKK enzyme, thereby blocking phosphorylation of IKB and downstream transcriptional activation of KB gene expression, such as NF- ⁇ B.
  • an IKK inhibitor has an IC 50 against IKK activity ranging from about 0.01 ⁇ M to about 50 ⁇ M.
  • the IKK inhibitor has an IC 50 against IKK activity ranging from about 0.01 ⁇ M to about 10 ⁇ M, more preferably from about 0.01 ⁇ M to about 5 ⁇ M, and most preferably from about 0.01 ⁇ M to about 1 ⁇ M.
  • a selective IKK inhibitor has at least about a 5-fold or greater selectivity for IKK-2 over IKK-1. More preferably, a selective IKK inhibitor has at least about an 8-fold or greater selectivity for IKK-2 over IKK-1.
  • inhibition of IKK-2 activity is about 5- to 500-fold, or about 8- to 100-fold, relative to IKK-1 activity, preferably about 10- to 100-fold, and more preferably, about 10 to 50-fold.
  • selective inhibitors affect about 50% inhibition of IKK activity at an IC50 against IKK activity ranging from about 0.01 ⁇ M to about 50 ⁇ M.
  • selective inhibitors have an IC50 against IKK activity ranging from about 0.01 ⁇ M to about 10 ⁇ M, and preferably, from about 0.01 ⁇ M to about 5 ⁇ M.
  • selective inhibitors have an 1C50 ranging from about 0.01 ⁇ M to about 1 ⁇ M.
  • a preferred embodiment of the present invention comprises IKK inhibitors selective for IKK-2.
  • the selective IKK-2 inhibitor has an IC50 against IKK-2 activity, ranging from about 0.01 ⁇ M to about 5 ⁇ M, and more preferably, an IC50 against IKK-2 activity, ranging from about 0.01 ⁇ M to about 1 ⁇ M.
  • bioavailability of the inhibitor compounds was assessed.
  • the pharmacokinetics of compound 6 in mice is described in Example 3.
  • an effective oral bioavailability for the IKK inhibitor of the present invention ranges from about 10% to about 100%, preferably from about 50% to about 100%, and more preferably from about 80% to about 100%.
  • bioavailability data for a given compound estimate the relative fraction of the orally administered dose that is absorbed into the systemic circulation when compared to the bioavailability data for a solution, suspension, or intravenous dosage form.
  • the present invention further relates to methods of testing the inhibitory potential of candidate IKK inhibitors.
  • Example 2 analyzes test compounds and their capacity to inhibit IKK activity by measuring the concentration of phosphorylated substrate, specifically IKB, and the concentration of test IKK inhibitor compound necessary to achieve 50% inhibition of IKK activity.
  • One embodiment of the present invention provides beneficial and advantageous in vivo animal studies using IKK inhibitors that demonstrate compound efficacy using methods of the present invention.
  • 4 (2'-aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline, compound 6 herein, or a salt thereof, newly discovered by the present inventors inhibits the catalytic activity of IKB kinase by blocking the active site of IKK which is responsible for phosphorylating the IKB protein.
  • the examples described herein demonstrate that IKK inhibitors are particularly useful in methods of treating inflammatory and immune-related diseases or disorders in several different animal models of disease that are considered to be predictive of similar employment and effects in human patients.
  • the compounds and compositions of this invention are also useful in treating conditions that are characterized by release of NF- ⁇ B and/or enhanced levels of TNF- ⁇ . Inhibition or suppression of NF- ⁇ B and/or TNF- ⁇ may occur locally, for example, within certain tissues of the subject, or more extensively throughout the subject being treated for such a disease. Inhibition or suppression of NF- ⁇ B and/or TNF- ⁇ may occur by one or more mechanisms, e.g., by inhibiting or suppressing any step of the pathway(s), preferably inhibition of IKK.
  • the present invention thus provides methods for treating such conditions, comprising administering to a subject in need thereof an effective amount of at least one compound of formula (I), compound 6, or salt thereof.
  • therapeutic agents such as those described below may be employed in combination with the IKK inhibitors, compound 6, and/or compounds of formula (I). In the methods of the present invention, such other therapeutic agent(s) may be administered prior to, simultaneously with, or following the administration of the inventive compound.
  • Compound 6 is structurally represented by the following formula as shown below:
  • compounds of formula (I) are useful in treating inflammatory diseases or disorders:
  • R 2 is alkyl, substituted alkyl, alkenyl, alkynyl, alkoxy, alkylthio, aryl, heteroaryl, heterocyclo, cycloalkyl, or substituted cycloalkyl;
  • R5, Re. R7, and R 8 are independently selected from hydrogen, alkyl, substituted alkyl, and phenyl, or when attached to the same nitrogen atom (as in NR 5 R ⁇ or NR 7 R 8 ) can join together to form a heterocycle or heteroaryl; and m, n and q are independently 0, 1 , or 2.
  • Yet another embodiment of the present invention encompasses the compounds having the formula (If):
  • Yi and Y 2 are nitrogen or carbon, provided that a) when X is CR-i, one of Yi and Y 2 is nitrogen, and b) when one of Yi and Y 2 is carbon, either the other of Yi and Y 2 is nitrogen or X is NRi or S, so that ring A defines a five-membered heteroaryl ring having two heteroatoms;
  • R 1 is hydrogen, halogen, lower alkyl, or substituted lower alkyl;
  • R 2 is alkyl or substituted alkyl.
  • R 1 is hydrogen, halogen, lower alkyl, or trifluoromethyl
  • R is C 1 - 2 alkyl optionally substituted with ORg or NR 10 R 11
  • R 9 is hydrogen or lower alkyl
  • Most preferred are the compounds of formula (If), above, and/or pharmaceutically acceptable salts thereof, wherein ring A is selected from:
  • Ri does not include hydroxy or an alkyl substituted with hydroxy.
  • salts which are also within the scope of this invention.
  • reference to an inventive compound is understood to include reference to salts thereof.
  • the term “salt(s)” denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases.
  • the term “salt(s) can include zwitterions (inner salts), e.g., when a compound of formula (I) contains both a basic moiety, such as an amine or a pyridine or imidazole ring, and an acidic moiety, such as a carboxylic acid.
  • Salts of the compounds of the formula (I) can be formed, for example, by reacting a compound of the formula (1) with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
  • Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 2- hydroxyethanesulfonates, lactates, maleates (formed with maleic acid), methanesulfonates (formed with methanesul
  • Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; barium, zinc, and aluminum salts; salts with organic bases (for example, organic amines) such as trialkylamines such as TEA, procaine, dibenzylamine, N-benzyl- ⁇ -phenethylamine, 1- ephenamine, N,N'-dibenzylethylene-diamine, dehydroabietylamine, N- ethylpiperidine, benzylamine, dicyclohexylamine or similar pharmaceutically acceptable amines and salts with amino acids such as arginine, lysine and the like.
  • organic bases for example, organic amines
  • organic bases for example, organic amines
  • trialkylamines such as TEA, procaine, dibenzylamine, N-benzyl- ⁇ -phenethylamine, 1- e
  • Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.
  • Sodium or potassium salts are preferred.
  • inventive compounds can exist in their tautomeric form, in which hydrogen atoms are transposed to other parts of the molecules and the chemical bonds between the atoms of the molecules are consequently rearranged. It should be understood that the all tautomeric forms, insofar as they can exist, are included within the invention. Additionally, inventive compounds can have trans and cis (E and Z) isomers and can contain one or more chiral centers, therefore existing in enantiomeric and diastereomeric forms. The invention includes all such isomers, as well as mixtures of cis and trans isomers, mixtures of diastereomers and racemic mixtures of enantiomers (optical isomers). When no specific mention is made of the configuration (cis, trans or R or S) of a compound (or of an asymmetric carbon), then any one of the isomers or a mixture of more than one isomer is intended.
  • Another embodiment of the present invention relates to pharmaceutical or physiological compositions having at least one IKK inhibitor, or salt thereof, and preferably containing a pharmaceutically or physiologically acceptable vehicle, such as a carrier, diluent, or excipient.
  • these pharmaceutical or physiological compositions comprise one or more IKK inhibitors, or salt thereof, either alone or in combination with a biologically active agent, such as but not limited to drugs, steroids, or synthetic compounds, particularly for use in the methods according to the present invention.
  • the pharmaceutical compositions can preferably comprise any one of the IKK inhibitors of the present invention, for example, 4(2'-aminoethyl)amino-1 ,8-dimethylimidazo(1 ,2-a)quinoxaline (compound 6) or any one of the compounds of formula (I).
  • a pharmaceutical or physiological composition can be administered to any individual in need of such therapy, including, for example, mammals such as monkeys, dogs, cats, cows, horses, rabbits, and most preferably, humans, for any of the above-described therapeutic or preventative uses and effects.
  • the present invention also provides pharmaceutical compositions capable of treating conditions that are related to the activity of NF- ⁇ B, TNF- ⁇ , and/or enzymes modulating NF- ⁇ B and/or TNF- ⁇ levels such as IKK.
  • Such compositions may contain other therapeutic agents and may be formulated, as described herein.
  • IKK inhibitors in the method embodiments of the present invention, can be administered alone or in combination with at least one other biologically active agent, which can be introduced in any sterile, biologically compatible pharmaceutical or physiologically acceptable carrier, excipient, or diluent, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a patient alone, or optionally in combination with other biologically active agents, drugs, or hormones.
  • the IKK inhibitors, or pharmaceutical or physiological compositions thereof can be administered by any means suitable for the condition to be treated, which can depend on the need for site-specific treatment or quantity of drug to be delivered, by any number of routes including, but not limited to, oral, nasal, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, and sublingual means.
  • IKK inhibitors are administered orally, nasally, topically, or by inhalation.
  • Administration of IKK inhibitor compositions of the invention can also include local or systemic administration, including injection, oral administration, particle gun, or catheterized administration, and topical administration.
  • IKK inhibitor compositions can be used directly to a specific site in the body.
  • topical delivery generally for skin-related diseases, transdermal patches and/or permeation enhancers can be used, a variety of which are commonly known in the art.
  • Systematic treatment is preferred for cancerous or pre-cancerous conditions, although other modes of delivery are contemplated.
  • Both the dose of an IKK inhibitor, or composition thereof, and the means of administration can be determined based on the specific qualities of the IKK inhibitor or therapeutic composition thereof; the condition, age, and weight of the patient; the progression of the disease; and other relevant factors.
  • an IKK inhibitor, or therapeutic composition thereof, according to the invention increases or decreases gene expression of NF- KB.
  • Methods well known in the art can be used to determine the effectiveness of the mechanism chosen to alter expression of the NF- ⁇ B gene, such as hybridization of nucleotide probes to mRNA of the NF- ⁇ B gene, quantitative RT-PCR, or detection of an NF- ⁇ B protein using specific antibodies (e.g. Santa Cruz Biotechnology; Santa Cruz, CA).
  • the pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers, diluents, or excipients comprising auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration are provided in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co.; Easton, Pa.).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • the compounds of the present invention can be delivered orally, such as in the form of tablets, capsules, granules, powders, or liquid formulations including syrups; topically, such as in the form of solutions, suspensions, gels, creams, or ointments; nasally such as by inhalation spray; parenterally, such as by subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); rectally such as in the form of suppositories; sublingually; bucally; or liposomally.
  • topically such as in the form of solutions, suspensions, gels, creams, or ointments
  • nasally such as by inhalation spray
  • parenterally such as by subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); rectally
  • compositions for oral use can be obtained by the combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropyl- methylcellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth, and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a physiologically acceptable salt thereof, such as sodium alginate.
  • compositions which can be orally administered in the methods according to the present invention, include push-fit capsules made of gelatin, as well as soft, scaled capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art.
  • the inventive compounds can also be orally delivered by sublingual and/or buccal administration, e.g., with molded, compressed, or freeze- dried tablets.
  • compositions can include fast-dissolving diluents such as mannitol, lactose, sucrose, and/or cyclodextrins.
  • fast-dissolving diluents such as mannitol, lactose, sucrose, and/or cyclodextrins.
  • high molecular weight excipients such as celluloses (AVICEL®) or polyethylene glycols (PEG); an excipient to aid mucosal adhesion such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC), and/or maleic anhydride copolymer (e.g., GANTREZ®); and agents to control release such as polyacrylic copolymer (e.g., CARBOPOL 934®).
  • Lubricants, glidants, flavors, coloring agents and stabilizers can also be added for ease of fabrication and use.
  • compositions for rectal administration include suppositories which can contain, for example, suitable non-irritating excipients, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures but liquefy and/or dissolve in the rectal cavity to release the drug.
  • suitable non-irritating excipients such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures but liquefy and/or dissolve in the rectal cavity to release the drug.
  • Dragee cores can be used in conjunction with physiologically suitable coatings, such as concentrated sugar solutions, which can also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification, or to characterize the quantity of active compound, i.e., dosage.
  • compositions suitable for parenteral administration in the methods of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyloleate or triglycerides, or liposomes.
  • the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • suitable compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • compositions for topical administration include a topical carrier such as PLASTIBASE® (mineral oil gelled with polyethylene).
  • exemplary compositions for nasal aerosol or inhalation administration include solutions which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance absorption and/or bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents can be administered.
  • the compounds can be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved with suitable pharmaceutical compositions or, particularly in the case of extended release, with devices such as subcutaneous implants or osmotic pumps.
  • the IKK inhibitor, or pharmaceutical composition thereof can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts tend to be more soluble in aqueous solvents, or other protonic solvents, than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1 %-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, combined with a buffer prior to use.
  • the pharmaceutical compositions After the pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of IKK inhibitors, such labeling would include amount, frequency, and method of administration.
  • compositions comprising one or more IKK inhibitors suitable for use in the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the intended purpose.
  • the determination of an effective dose or amount is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., using neoplastic cells, or in animal models, usually mice, rats, rabbits, dogs, or pigs.
  • the animal model can also be used to determine the appropriate dosage, concentration range and route of administration. Such information can then be used and extrapolated to determine useful doses, concentration ranges, and routes for administration in humans.
  • an effective amount of IKK inhibitor optimally results in a level of inhibition of IKK activity ranging from about 20-100%, preferably, about 50-100%, more preferably about 80-100%, and most preferably about 90-100%, for the treatment of inflammatory and/ or immune-related diseases.
  • a therapeutically effective dose refers to that amount of active ingredient or compound, such as an IKK inhibitor, which ameliorates, reduces, or eliminates the symptoms, condition, disease, or disorder.
  • Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio of therapeutic to toxic effects is the therapeutic index, which can be expressed as the ratio, ED50/LD50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • Preferred dosage of a pharmaceutical composition is within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors; the exact dosage will be determined by the practitioner, who will consider the factors related to the individual requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the IKK inhibitor active moiety or to maintain the desired effect. Factors which are taken into account include the severity of the individual's disease state, general health of the patient, age, weight, and gender of the patient, diet, and mode, time and frequency of administration, rate of excretion, drug combination(s), reaction sensitivities, and tolerance/response to therapy. As a general guide, long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks, depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from about 0.1 to about 100,000 micrograms ( ⁇ g), up to a total dose of about 1 gram (g), depending upon the route of administration. Preferably, dosages range from about 1 to about 100 ⁇ g.
  • Guidance as to particular dosages and methods of delivery is provided in the literature, via empirical determination, and is generally available to, and routinely able to be determined by practitioners in the art. Single or multiple doses can be administered on, for example, a daily, every other day, or weekly schedule.
  • the effective amount of a compound of the present invention can be determined by one of ordinary skill in the art, and includes exemplary dosage amounts for a mammal of from about 0.05 to 100 mg/kg of body weight of active compound per day, which can be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day.
  • Preferred subjects for treatment include animals, most preferably mammalian species such as humans, and domestic animals such as dogs, cats, horses, and the like.
  • the compounds of the invention, and pharmaceutically acceptable salts thereof also embrace prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Prodrugs and solvates of the inventive compounds are also contemplated.
  • prodrugs are suitable for use in the methods according to the present invention. Such prodrugs are preferably administered orally since hydrolysis in many instances occurs principally under the influence of the digestive enzymes. Parenteral administration can be used where the ester per se is active, or in those instances where hydrolysis occurs in the blood.
  • physiologically hydrolyzable esters of compounds of formula (I) include C ⁇ alkylbenzyl, 4-methoxybenzyl, indanyl, phthalyl, methoxymethyl, C ⁇ - 6 alkanoyloxy-C ⁇ - 6 alkyl, e.g.
  • esters used, for example, in the penicillin and cephalosporin arts. Such esters can be prepared by conventional techniques known in the art.
  • inventive compounds and compositions can be employed alone or in combination with each other and/or other suitable therapeutic agents useful in treating NF- ⁇ B and TNF- ⁇ associated conditions.
  • suitable therapeutic agents include corticosteroids, rolipram, calphostin, CSAIDs, 4-substituted imidazo [1 ,2-A]quinoxalines as disclosed in US Pat. No.
  • lnterleukin-10 glucocorticoids, salicylates, nitric oxide, and other immunosuppressants
  • nuclear translocation inhibitors such as deoxyspergualin (DSG); non-steroidal antiinflammatory drugs (NSAIDs) such as ibuprofen, celecoxib and rofecoxib; steroids such as prednisone or dexamethasone
  • NSAIDs non-steroidal antiinflammatory drugs
  • antiviral agents such as abacavir
  • antiproliferative agents such as methotrexate, leflunomide, FK506 (tacrolimus, Prograf)
  • cytotoxic drugs such as azathiprine and cyclophosphamide
  • TNF- ⁇ inhibitors such as tenidap, anti- TNF antibodies or soluble TNF receptor, and rapamycin (sirolimus or Rapamune) or derivatives thereof
  • other cancer drugs and treatments including radiation treatments and daunorubicin.
  • the IKK inhibitory compound 4(2'-aminoethyl)amino- 1 ,8- dimethylimidazo(1 ,2-a) quinoxaline, or compound 6 newly described herein, can be prepared by a method as described in the following scheme. Starting materials are commercially available or can be readily prepared by one of ordinary skill in the art, and/ or modifications can be made to the method of the scheme by one skilled in the art, using known methods and practices.
  • Compound 6 or 4(2'-aminoethyl)amino- 1,8-dimethylimidazo(1 ,2-a) quinoxaline can be prepared as shown in the above scheme: 3-fluoro-4- nitrotoluene (compound 1) is reacted with ethyl 5-methylimidazole-4- caboxylate (compound 2) according to known procedures to produce, after several steps, the imidazoquinoxalone-2-carboxylic acid (compound 3). Upon decarboxylation of said acid (compound 3) in a high-boiling solvent, such as diphenyl ether (Ph 2 O), 1 ,8-dimethylimidazoquinoxalone (compound 4) is produced.
  • a high-boiling solvent such as diphenyl ether (Ph 2 O)
  • Compound 4 is then chlorinated using standard reagents, such as phosphorus oxychloride (POCI 3 ) and N,N-diethylaniline (PhNEt 2 ) to give 4-chloro-1 ,8-dimethylimidazoquinoxaline (compound 5).
  • Compound 5 is then condensed with 1 ,2-diaminoethane or ethylenediamine to produce the final compound 4(2'-aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline (compound 6).
  • Compound 1 is commercially available from Aldrich Chemical Company (Milwaukee, Wl).
  • test compounds for measuring the inhibitory potential of test compounds, specifically compound 6, (4(2'-aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline), against IKK activity employed 3 P-labeled ATP and a recombinant l ⁇ B- ⁇ as substrates.
  • test compound was added to a solution of 0.5 mM l ⁇ B- ⁇ in 40 mM Tris-HCl, pH 8, containing 4 mM MgCI 2 , 1 mM dithiothreitol, and 2 mM 33 P-labeled ATP.
  • IKK enzyme (either the multisubunit complex from HeLa cells, recombinantly expressed IKK-1 , or recombinantly expressed IKK-2) was then added to initiate the reaction.
  • the multisubunit complex was isolated according to the procedure of Lee et al. [(1997) Cell 88:213-222] or Mercurio et al. [(1997) 278:860-866].
  • the recombinant IKK-1 and IKK-2 were expressed using the procedure of Burke et al. [(1999) J. Biol. Chem. 274:36146-36152]. After incubating for 10 minutes at 30°C, the reaction was quenched by the addition of EDTA to a final concentration of 30 mM.
  • the results presented in Table 1 show the inhibition of IKK activity using 33 P-labeled ATP and l ⁇ B- ⁇ as substrates for recombinantly expressed IKK-1 and IKK-2, wherein a test compound concentration of 5 ⁇ M and 0.5 ⁇ M, respectively, was shown to achieve inhibition of IKK activity by 50%.
  • the IKK inhibitor, compound 6, inhibits IKK-2 at an IC 5 o which is 10 fold less than that necessary to inhibit IKK-1 by 50%.
  • compound 6 inhibits IKK-2 with greater effectiveness than IKK-1 , thus demonstrating a selectivity of this compound for IKK-2 inhibition over IKK-1.
  • PHARMACOKINETICS OF COMPOUND 6 IN MICE Compound 6 was administered to BALB/c mice either per orally (p.o.) or intravenously (i.v.). Blood was drawn at various times (0.5 to 4 hours) after dosing and the plasma drug levels measured by liquid chromatography-mass spectral analysis. An oral bioavailability of 100% for compound 6 was calculated from the area under the curve (AUC) derived from a plot of plasma levels versus time after the p.o. dose, and then dividing by the AUC from the i.v. dose. An i.v. half-life of 7.9 hours was calculated from the apparent terminal elimination phase after the i.v. dose. Generally, to be considered, oral bioavailability ranges from about 10% to about 100%, more preferably from about 50% to about 100%, and most preferably from 80% to about 100%.
  • Neonatal hearts were obtained from newborn C57BL/6 mice not more than 48 hours after birth.
  • the hearts were transplanted into recipient BALB/c mice, which were prepared under anesthesia by making a small incision at the base of the ear pinna and forming a pocket by gently lifting the skin away from the ear. The incision permitted the insertion of the donor heart subcutaneously into the pocket.
  • the cardiac tissue typically does not begin to beat until 3 to 5 days after transplantation until vascular connections are established.
  • the pulsations of the heart were measured by contractile activity with an appropriate electrocardiogram (ECG) monitoring device.
  • ECG electrocardiogram
  • the contractile activity of the transplanted graft was monitored daily.
  • the time of graft rejection was defined as the day after transplantation on which contractile activity ceased.
  • the host mounts an immune response to the transplanted heart (i.e., graft rejection). This immune response causes the transplanted heart to cease beating typically 10-14 days post transplantation.
  • Test compounds were administered in daily doses either alone or in combination with other therapies, such as Cyclosporine A (15 mg/ kg, orally, "p.o.") or CTLA4-lg (200 ⁇ g in 200 ⁇ l PBS given intraperitoneally on only days 0, 2, and 4).
  • Table 2 presents the effects of test compounds on median survival time of transplanted hearts, either alone or in combination with Cyclosporine A (CsA).
  • Table 3 presents the effects of test compounds on median survival time of transplanted hearts, either alone or in combination with CTLA4-lg.
  • the murine model of collagen-induced arthritis is widely used to study disease mechanisms and potential therapies for rheumatoid arthritis in humans (Staines et al., Br. J. Rheumatol. 33:798-807, 1994; Feldmann et al., Annu. Rev. Immunol. 14:397-440, 1996).
  • test compounds 4(2'-aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline (compound 6) at a concentration of 100 mg/ kg, resulted in a significant difference in incidence of disease compared with vehicle alone.
  • the administration of test compounds was initiated only after paw inflammation of an animal had achieved a score of 2 (as defined below), at which time, the animal was randomly assigned to a treatment group. Treatment continued daily for all four paws as described below.
  • mice were regularly monitored for the development and severity of paw inflammation. Each paw was visually scored by the following clinical scoring scheme as presented in Table 4:
  • Clinical paw scores for all four paws were summed for each mouse and the mean ⁇ standard deviation (SD) was calculated for each treatment group.
  • the incidence of disease is defined as the percent of animals within a treatment group which showed any sign of disease regardless of severity.
  • Histological scoring involved evaluating histologically and grading semi-quantitatively tibiotarsal joints of sacrificed mice, specifically examining the severity of inflammation, synovial hyperplasia, bone resorption, and cartilage erosion. Cumulative arthritis injury scores for each group were compared by the nonparametric Kruskal-Wallis analysis of variance.
  • Figure 2 shows the average gross clinical score for all mice within each treatment in the collagen-induced arthritis murine model as determined on each study day.
  • the test compound was administered once daily in the preventative dosing mode.
  • Table 5 presents the histological evaluation of the effect of 4(2'-aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline (compound 6) on the cumulative arthritis injury scores + standard error mean (sem) upon sacrificing the animals at the end of the study in the murine model of collagen-induced arthritis.
  • Figure 3 shows the effects of 4(2'-aminoethyl)amino- 1 ,8- dimethylimidazo(1 ,2-a) quinoxaline (compound 6) administered in the established disease mode (i.e., after disease onset) on the average gross clinical score for all mice within each treatment in the murine model of collagen-induced arthritis as determined on the days following disease onset.
  • Table 6 presents the histological evaluation of the effect of 4(2'- aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline (compound 6) on the cumulative arthritis injury scores in the established disease mode of murine model of collagen-induced arthritis two weeks after disease onset.
  • Established Mode 4(2'- aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline
  • the murine model of collagen-induced arthritis shares many of the same biochemical and pathological mechanisms as does rheumatoid arthritis in humans, and was used for preventing and treating arthritis upon the administration of 4(2'-aminoethyl)amino-1 ,8-dimethylimidazo(1 ,2-a) quinoxaline (compound 6).
  • Figures 1 and 2 and Table 5 show that daily administration of 30 mg/kg (p.o.) of compound 6 inhibited disease onset, and that 100 mg/kg (p.o.) of compound 6 prevented any disease onset when dosed in a preventative mode.
  • Figure 3 and Table 6 show that treatment with 30 mg/kg (p.o.) of compound 6 significantly inhibited disease progression when compared with vehicle treated animals, while a dose of 100 mg/kg (p.o.) of compound 6 actually led to significant disease resolution.
  • Dextran sulfate sodium (DSS)-induced colitis in mice shows several of the same pathological mechanisms of inflammatory bowel disease (IBD) as it presents in humans, and has been used to study various treatments (Okayasu et al., Gastroenterology 98:694-702, 1990; Axelsson et al., Aliment. Pharmacol. Ther. 12:925-934, 1998).
  • the mouse models are used as predictors of IBD in humans and to develop effective treatments for human use.
  • Swiss-Webster mice (8 weeks old, 5 animals per treatment group) were given 6% DSS in their drinking water for 7 consecutive days to induce intestinal inflammation. Test compounds were administered daily throughout the study. On day 9, animals were sacrificed and the colons were removed for clinical and histological evaluation. Clinical scoring was determined by the gross clinical evaluation of the injury on a scale from 0 (normal) to 3 (severe) as detailed in Table 7.
  • colon sections were graded as to the severity of crypt injury and the degree of inflammation.
  • the crypt injury was scored as detailed in Table 8, wherein the crypt is part of the glandular make-up of the colon.
  • Tissue involvement was scored as detailed in Table 9. Any signs of crypt injury or inflammation, regardless of severity, constitute tissue involvement.
  • the injury histological score is defined as the product of the crypt injury grade and the tissue involvement grade.
  • the scoring of severity of inflammation is detailed in Table 10.
  • the inflammation histological score is the product of the severity of inflammation grade and the extent of tissue involvement grade. Crypt injury and inflammatory scoring were performed on each colon section and a mean score and standard error were determined for each section. Cumulative crypt injury and inflammatory scores for each group were determined. Table 11 presents the clinical scores of colons in mice with DSS-induced colitis. The results of Table 12 present the cumulative injury and inflammation scores of colons in mice with DSS-induced colitis.
  • results show that 4(2'-aminoethyl)amino- 1 ,8- dimethylimidazo(1 ,2-a) quinoxaline (compound 6) prevented and treated IBD in the well-established murine model of inflammatory bowel disease (IBD) in humans.
  • Tables 11 and 12 show that daily administration of the IKK inhibitor (compound 6) resulted in a statistically significant decrease in clinical and histological score when compared with untreated mice.
  • Such results support the advantageous outcome afforded by the present invention in which inhibition of the IKK enzyme prevents and/ or ameliorates inflammatory diseases and disorders such as IBD.
  • Pulmonary inflammation with infiltration of eosinophils and other inflammatory cells is a hallmark of asthma and other allergic respiratory disorders.
  • test compounds were administered in a murine model of pulmonary inflammation similar to that described by Kung et al. (Kung, T.T. et al., Int. Arch. Allergy Immunol. 105:83-90, 1994).
  • Kung et al. Kung, T.T. et al., Int. Arch. Allergy Immunol. 105:83-90, 1994.
  • BALB/c mice were sensitized by an intraperitoneal injection of 0.1 mL alum-precipitated antigen containing 40 ⁇ g ovalbumin adsorbed to aluminum hydroxide gel on days 0 and 10.
  • mice received intranasal challenge with 100 ⁇ g ovalbumin in 50 ⁇ l saline (PBS). Mice were injected intraperitoneally with antigen to sensitize the animals prior to intranasal challenge to induce lung inflammation. Administration of the test compound was given on days 14 to 17. On day 18, bronchoalveolar lavage fluid (BAL) was collected for measurements of total inflammatory cell infiltration (eosinophils, monocytes, lymphocytes, and neutrophils).
  • BAL bronchoalveolar lavage fluid
  • Table 13 presents the effect of 4(2'- aminoethyI)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline (compound 6) on ovalbumin-induced inflammatory cell infiltration into the lungs of sensitized mice after daily administration of compound.
  • Ovalbumin-induced inflammatory cell infiltration in murine lung provides a model system for pulmonary inflammation, specifically asthma and other allergic respiratory disorders in humans.
  • Compound 6, 4(2'- aminoethyl)amino- 1 ,8-dimethylimidazo(1 ,2-a) quinoxaline was administered in a murine model of pulmonary inflammation for the treatment of inflammation and immune-related conditions.
  • Table 13 show that administration of compound 6 after ovalbumin challenge to sensitized mice resulted in a statistically significant decrease in total inflammatory cells in the lungs, as determined by measurements from bronchoalveolar lavage fluid when compared with untreated mice.
  • THP-1 human monocytic cell line obtained from ATCC was cultured in RPMI-1640 supplemented with 10% FBS, sodium pyruvate, HEPES, 2- mercaptoethanol, Penicillin/Streptomycin.
  • RPMI-1640 was added 20 ⁇ L of the test compound in 10% DMSO.
  • test compound concentrations 0.1-100 ⁇ M were used in the assay.
  • compound 6 was determined to have an IC 50 of 4 ⁇ M.
  • Compounds of Examples 12-19 demonstrated IC 50 values of below 9 ⁇ M, with preferred and more preferred compounds having IC 50 values of below 2 ⁇ M and 1 ⁇ M, respectively, in this assay.
  • TNF ⁇ -STIMULATED DEGRADATION OF l ⁇ B ⁇ IN THP-1 CELLS TNF ⁇ stimulation of monocytic THP-1 cells leads to the proteolytic degradation of l ⁇ B ⁇ .
  • Both an IKK-dependent phosphorylation and ubiquitin ligase-dependent ubiquitination of l ⁇ B ⁇ are essential steps in the TNF ⁇ - stimulated pathway targeting l ⁇ B ⁇ for proteolytic degradation by the proteosome.
  • THP-1 cells were suspended in RPMI-1640 supplemented with 10% fetal bovine serum, preincubated for 60 mins with test agent, then stimulated for 15 mins with TNF ⁇ (100 ng/mL, R&D Systems).
  • TNF ⁇ 100 ng/mL, R&D Systems
  • Total cell lysates were fractionated by sodium dodecylsulfate-polyacrylamide electrophoresis followed by Western blot analysis.
  • l ⁇ B ⁇ was detected using a polyclonal antibody from Santa Cruz (catalog #sc-4094) and ECL reagents (Amersham). Films were scanned using a Kodak ID Image Analysis system to quantitate the amount of l ⁇ B ⁇ .
  • test compounds were administered to BALB/c mice subsequently challenged with an intravenous dose of E. coli LPS (1 ⁇ g in 100 ⁇ L phosphate-buffered saline).
  • E. coli LPS 1 ⁇ g in 100 ⁇ L phosphate-buffered saline.
  • EIA EIA
  • Compounds of formula (I) demonstrated inhibition of the LPS-induced serum TNF ⁇ levels in vivo in the range of about 30 to 65% at 10 mg/kg, p.o; in the range of about 70 to 90% at 30 mg/kg, p.o.; and in the range of above 90% at 100 mg/kg p.o.
  • Compound 6 demonstrated inhibition of the LPS-induced serum TNF levels in vivo in the range of about 27-63% at 10 mg/kg, p.o.; in the range of about 53-89% at 30 mg/kg, p.o.; and in the range of about 83-92% at 50 mg/kg, p.o. Accordingly, compounds of formula (I) have been tested and shown active as inhibitors of TNF- ⁇ and the NF- ⁇ B pathway.
  • inventive compounds of formula (I) can be prepared by methods such as those illustrated in the following Schemes I to V in this example.
  • chlorinating agents can include phosphorous oxychloride
  • catalytic agents can include metals such as Pd
  • solvents can be selected from 1,2- dichlorobenzene, methylene chloride, DMF, THF, alcohols, ethers, dioxane, acetonitrile, water, mixtures of ethers and water, and the like.
  • TFA trifluoroacetic acid
  • THF tetrahydrofuran
  • K 2 CO 3 potassium carbonate
  • Acid halide formation includes methods of converting a carboxylic acid to an acid halide. For example, this reaction can be performed with thionyl chloride, oxalyl chloride or bromide in the presence of DMF in DCM and phosphorus trichloride or tribromide.
  • Alkylation includes all alkylation procedures such as alkylation of desired alcohol or ketone groups by treatment with organic or inorganic base in an appropriate organic solvent, followed by addition of an alkylating agent such as an alkyl, allyl or benzyl halide, mesylate or tosylate to the generated enolate, phenolate or thiophenolate.
  • an alkylating agent such as an alkyl, allyl or benzyl halide, mesylate or tosylate to the generated enolate, phenolate or thiophenolate.
  • Aromatic substitution includes all aromatic substitution methods known in the field including nucleophilic substitutions of aromatic halides by water in presence of sulfuric acid or trifluoroacetic acid, or by alkoxides, aryloxides, thioalkoxides or thioaryloxides in an inert organic solvent. Copper salts can be used to promote the reaction of aryl halides with alkoxides, and Pd(0) salts can be used to promote the reaction with thioalkoxides.
  • Aromatic halogenation includes the addition of chlorine, bromine or iodine to an aromatic ring optionally with a catalyst, e.g. iron or a Lewis acid. It also includes the reaction of N-chloro and N-bromoamides catalyzed by the addition of acids.
  • iodine can be used with copper salts, silver trifluoromethanesulfonate, thallium(l) acetate, or with an oxidizing agent such as nitric acid, iodic acid, sulfur trioxide or hydrogen peroxide.
  • Iodine monochloride can also be used.
  • Cross-coupling includes all cross-coupling methods known by those skilled in the art. Such methods include the reaction of a vinyl or aromatic triflate, bromide or iodide with a tin (Stille-type), zinc, magnesium or boronate (Suzuki-type) derivative catalyzed by a palladium(O), palladium(ll), nickel(O) or nickel(ll) catalyst. Copper iodide, lithium chloride, zinc chloride or triphenylarsine, tris(2-furyl)phosphine or tris(2,4,6- trimethoxyphenyl)phosphine advantageously can also be added. When a boronic acid derivative is used, the reaction proceeds in the presence of an inorganic base such as potassium phosphate or carbonate or sodium carbonate. The cross-coupling reactions are performed in an inert organic solvent.
  • an inorganic base such as potassium phosphate or carbonate or sodium carbonate.
  • Grignard type reaction includes the addition of an organometallic compound to a carbonyl-containing compound. This includes addition of Grignard reagents, alkyl or aryllithiums, alkylzinc, alkylaluminum, organotitanium, organozirconium or organocerium compounds in an inert organic solvent such as ethyl ether, THF, DCM, benzene, toluene, or the like. Complexing of the ketone or the Grignard reagent with cerium halides, perchlorate salts or tetraalkylammonium halides can sometimes be advantageous to improve the addition reaction.
  • Grignard type reaction is also intended to include the addition of a Grignard reagent to an acid chloride that has been first reacted with tributylphosphine to form the corresponding phosphonium salt. The reaction is performed in an inert organic solvent.
  • “Hydrolysis” includes the hydrolysis of esters and carbonyl protecting groups.
  • methyl or ethyl esters can be removed with aqueous ' solutions of sodium or potassium alkoxides in THF or EtOH.
  • the hydrolysis of tert-butyl esters advantageously is carried out under acidic conditions such as 90% trifluoroacetic acid or 6N hydrochloric acid in solvents such as THF or DCM. Allyl esters can be removed with Pd(0) catalyst in an organic solvent.
  • Silyl esters such as trimethylsilylethyl esters can be cleaved with tetrabutylammonium fluoride in THF.
  • the hydrolysis of ketals and acetals can be carried out under acidic conditions such as 1 N hydrochloric acid, 80% acetic acid or p-toluenesulfonic acid in solvents such as THF or acetone.
  • ketone formation procedures are known in the field. For example, a ketone can be reacted with an amine in presence of an acid with or without a drying agent.
  • Various inorganic and organic acids can be used, such as zinc chloride, titanium chloride, hydrochloric acid, sulfuric acid, trifluoroacetic acid, acetic acid, p-toluenesulfonic acid and the like, in solvents such as DCM, EtOH, benzene, toluene, THF, DMF and the like.
  • Benzo-imidazo-quinoxalines of formula ]a (wherein Yi of formula (I) is N, X is CRi and Ri is hydrogen) can be prepared as above.
  • Substituted 2,3- diaminonaphthalenes are reacted with, for example, diethyl oxalate or oxalyl chloride to give annulated quinoxalinediones 2.
  • Diones 2 are reacted with a chlorinating agent to give dichlorides 3.
  • Dichlorides 3 are reacted with amine reagents comprising aminoacetaldehyde acetal and an appropriate primary amine (R 2 -NH 2 ), and cyclized, wherein the order of reactivity can be adjusted depending upon chlorine atom reactivity.
  • dichlorides 3 can first be reacted with an aminoacetaldehyde acetal to give amino-chloro derivatives 4a.
  • the amino- chloro derivatives 4a can be cyclized to give the chloro-benzo- imidazoquinoxaline 5a. which can, in turn, be reacted with various primary amines (R 2 -NH 2 ) to give compounds of formula (la).
  • the order of addition of the amine reagents can be reversed, as shown for structures 4b and 5b.
  • Dichloride 3 is reacted with primary amines (R2-NH 2 ) to give amino-chloro derivatives 4b, which are then reacted with an aminoacetaldhyde acetal to give diamino compounds 5b, which are then cyclized to give the compound of formula la.
  • Exemplary aminoacetaldehyde acetals comprise aminoacetaldehyde, dimethyl acetal, or aminoacetaldehyde, diethyl acetal. Cyclization is typically performed under acidic conditions.
  • the order of reactivity can be adjusted, wherein dichloro compounds 3 are first reacted with suitable primary amines to give derivatives 4b, as in Scheme I, and then reacted with propargyl amines to amino derivatives 6b, which can be cyclized as above to give compounds of formula lb.
  • Benzo-pyrazolo-quinazolines of formula Jc (wherein Y 2 of formula (I) is N, X is CHRi and Ri is as defined above) can be prepared as shown in Scheme III.
  • Reagents 8c and 9c are condensed in the presence of catalyst in a Stille-type reaction to give coupled product 10, which is hydrolyzed to give compound V
  • Treatment of 12 with a chlorinating agent gives chloro compound 13, which can be reacted with suitable primary amines (R 2 -NH 2 ) to give compounds of formula jc.
  • Reagents 8 and 9 for Scheme III above can be prepared as shown in Scheme Ilia.
  • Substituted 3-amino-2-naphthoic acids 8a can be converted to substituted 3-iodo-2-naphthoic acids 8b by, for example, diazotization followed by treatment with an iodide salt.
  • Esterification of compound 8b produces 3-iodo-2-napthoic acid esters 8.
  • 4-Substituted pyrazoles 9a can be treated with a p-toluenesulfonating agent, such as p-toluenesulfonyl chloride, to give p-toluenesulfonamide 9
  • a p-toluenesulfonating agent such as p-toluenesulfonyl chloride
  • a strong base such as t-butyl lithium
  • a stannylating agent such as trimethyl chloro stannane
  • NRi and Ri is methyl
  • Scheme IV Substituted 3-iodo-2-naphthoic acid esters 8 prepared as in Scheme Ilia are converted to boronate derivatives 14 using catalyst and a reagent such as bis(pinacolato)diboron. Condensation of compound 14 with 1-methyl-5- bromoimidazole and catalyst in a Suzuki-type reaction gives coupled ester 15. Ester 15 is hydrolyzed to give acid 16, which is converted via treatment with diphenylphosphoryl azide (through an intermediate isocyanate - see 11b) and t-butanol into protected amine 17.
  • Acid treatment converts 17 to free amine 18, which is heated in a high-boiling point inert solvent such as 1 ,2-dichlorobenzene with carbonyldiimidazole to give cyclized product 19.
  • Treatment with a chlorinating agent converts compound 19 to the chloro derivative 20, which can be converted by treatment with suitable primary amines (R 2 -NH 2 ) to give compounds of formula id.
  • Benzo-thiazolo-quinolines can be prepared as shown above in Scheme V.
  • Substituted 3-boronato-2-naphthoic acid ester 14 is condensed with 5-bromothiazole in a Suzuki-type reaction, catalyzed by a metal such as Pd, to give coupled product 21.
  • the ester 21 is hydrolyzed under standard conditions to give acid 22, which is then converted via treatment with diphenylphosphoryl azide (through an intermediate isocyanate similar to 11 b) and t-butanol into the protected amine 23.
  • Acid treatment can convert compound 23 to the free amine 24, which is heated in a high-boiling inert solvent such as 1 ,2-dichlorobenzene with carbonyldiimidazole to give cyclized product 25.
  • Compound 25 can be converted to the chloro derivative 26 by treatment with a chlorinating agent, and compound 26 is converted by treatment with suitable primary amines (R 2 -NH 2 ) to give the compound of formula le.
  • HPLC purity determinations were done using either an HP 1090 DR5 with a diode array detector and a Waters Nova-Pak C18 column (3.9 x 150 mm), or a Shimadzu LC-10AS with a SPD-10AV UV-Vis detector and one of the following columns: YMC Combiscreen ODS-A (4.6 x 50 mm); HP Zorbax
  • p-TsCI (9.81g,51.46 mmol) was added in batches over a few minutes to a CH 2 CI 2 (100 mL) solution of 4-methylpyrazole (4.00 g, 48.72 mmol) and pyridine (6.0 mL, 74.18 mmol).
  • the reaction mixture was stirred for 95 min, then diluted with CH 2 CI 2 (250 mL) and washed with NaHC ⁇ 3 solution [a mixture of sat'd NaHCO 3 solution (20 mL) and H 2 O (40 mL)], and water (60 mL).
  • the organic layer was dried (MgSO ), filtered and evaporated in vacuo.
  • PhNEt. 2 (1.50 mL) and POCI 3 (15.0 mL) were added to 1- methylbenzo(g)pyrazolo(1 ,5-c)quinazolin-5-one (Preparation 11 ) (184.7 mg, 0.741 mmol), and the resulting heterogeneous mixture was heated at reflux for 46 hr. During heating, the suspension (the starting material), gradually started to dissolve. The dark-green reaction mixture was filtered to remove non-consumed starting material (16.2 mg, 8.8%), and the filtrate was exposed to high vacuum to remove most of the POCI 3 . The residue was partitioned between water (30 mL) and EtOAc (70 mL).
  • Examples 12-19 provide compounds as described herein.
  • N-methyl-ethylenediamine 250 ⁇ L, 2.836 mmol was added rapidly to a THF (2.0 mL) semi-suspension of 4-methyl-1-(4-tolunesulfonyl)-5- trimethylstannylpyrazole (Preparation 8 in Example 11) (30.5 mg, 0.114 mmol).
  • the reaction mixture was stirred at RT for 1 hr and at 75°C for 2.3 hr. After the reaction mixture was cooled to RT, it was treated with NaHCO 3 (57 mg) and water (2 pipet-drops) and stirred for a few minutes. The volatile components, including the excess diamine, were removed in vacuo.

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Abstract

L'invention concerne des méthodes de prévention et de traitement de troubles ou de maladies d'origine immune et inflammatoires à l'aide d'inhibiteurs d'IλB kinase (IKK). L'invention concerne également des inhibiteurs IKK efficaces dans la prévention et le traitement de troubles ou de maladies d'origine immune et inflammatoires, comme démontré in vivo. D'autres modes de réalisation de l'invention concerne des inhibiteurs IKK spécifiques, 4(2'-aminoéthyl)amino- 1,8-diméthylimidazo(1,2-a) quinoxaline et des composés de formule (I), les sels de ces composés, ainsi que des compositions pharmaceutiques.
EP02714815A 2001-02-01 2002-02-01 Methodes de traitement de maladies inflammatoires et immunitaires a l'aide d'inhibiteurs d'i kappa b kinase (ikk) Withdrawn EP1363993A4 (fr)

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US26585301P 2001-02-01 2001-02-01
US265853P 2001-02-01
US09/965,977 US6960585B2 (en) 2000-10-03 2001-09-27 Amino-substituted tetracyclic compounds useful as anti-inflammatory agents and pharmaceutical compositions comprising same
US965977 2001-09-27
PCT/US2002/003060 WO2002060386A2 (fr) 2001-02-01 2002-02-01 Methodes de traitement de maladies inflammatoires et immunitaires a l'aide d'inhibiteurs d'i$g(k)b kinase (ikk)

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DE60117835T2 (de) * 2000-10-03 2006-10-19 Bristol-Myers Squibb Co. Amino- substituierte tetracyclische Verbindungen,die als entzündungshemmende Mittel nützlich sind, und diese enthaltende Arzneimittel
ES2305125T3 (es) 2000-10-26 2008-11-01 Amgen Inc. Agentes anti-inflamatorios.
AU2002365611A1 (en) 2001-12-05 2003-06-17 F. Hoffmann - La Roche Ag Inflammation modulators
DE10237722A1 (de) 2002-08-17 2004-08-19 Aventis Pharma Deutschland Gmbh Indol- oder Benzimidazolderivate zur Modulation der IKappaB-Kinase
JP2006512313A (ja) 2002-10-31 2006-04-13 アムジェン インコーポレイテッド 抗炎症剤
AR050253A1 (es) 2004-06-24 2006-10-11 Smithkline Beecham Corp Compuesto derivado de indazol carboxamida, composicion que lo comprende y su uso para la preparacion de un medicamento
PE20060748A1 (es) 2004-09-21 2006-10-01 Smithkline Beecham Corp Derivados de indolcarboxamida como inhibidores de quinasa ikk2
US8063071B2 (en) 2007-10-31 2011-11-22 GlaxoSmithKline, LLC Chemical compounds
CA2613068A1 (fr) 2005-06-30 2007-01-11 Smithkline Beecham Corporation Indole carboxamides en tant qu'inhibiteurs d'ikk2
EP2266561A3 (fr) * 2005-09-07 2011-05-18 Merck Serono S.A. Inhibiteurs de l'IKK pour le traitement de l'endométriose
US20090175852A1 (en) 2006-06-06 2009-07-09 Schering Corporation Imidazopyrazines as protein kinase inhibitors
EP2103613B1 (fr) * 2006-12-13 2016-02-17 ASKA Pharmaceutical Co., Ltd. Dérivé de quinoxaline
PE20081889A1 (es) 2007-03-23 2009-03-05 Smithkline Beecham Corp Indol carboxamidas como inhibidores de ikk2
CA2721927C (fr) 2008-04-21 2014-01-28 Otonomy, Inc. Preparations auriculaires de traitement de maladies et etats otiques
US11969501B2 (en) 2008-04-21 2024-04-30 Dompé Farmaceutici S.P.A. Auris formulations for treating otic diseases and conditions
WO2010000903A2 (fr) * 2008-06-30 2010-01-07 Centro De Investigaciones Energéticas, Medioambientales Y Tecnológicas (Ciemat) Composition permettant de moduler l'activité d'ikkalpha, destinée au traitement de maladies associées à l'acantholyse
JP5312466B2 (ja) 2008-10-02 2013-10-09 旭化成ファーマ株式会社 ８位置換イソキノリン誘導体及びその用途
JP2012520257A (ja) 2009-03-10 2012-09-06 グラクソ グループ リミテッド Ｉｋｋ２阻害剤としてのインドール誘導体
JP7033789B2 (ja) 2016-06-29 2022-03-11 オトノミー，インク． トリグリセリド耳用製剤とその使用
EP3494994A1 (fr) * 2017-12-07 2019-06-12 Universität zu Köln Combinaisons d'inhibiteurs de ripk1 et d'ikk pour la prévention ou le traitement de maladies immunitaires

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US6235740B1 (en) * 1997-08-25 2001-05-22 Bristol-Myers Squibb Co. Imidazoquinoxaline protein tyrosine kinase inhibitors
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GB0003154D0 (en) * 2000-02-12 2000-04-05 Astrazeneca Uk Ltd Novel compounds
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TSAI S -H ET AL: "Suppression of TNF[alpha]-mediated NF[kappa]B activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells" JOURNAL OF CELLULAR BIOCHEMISTRY 19990915 US, vol. 74, no. 4, 15 September 1999 (1999-09-15), pages 606-615, XP002497486 ISSN: 0730-2312 *

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