Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/04—Indoles; Hydrogenated indoles
  • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
  • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Definitions

• This invention relates to a novel group of indole containing compounds, processes for the preparation thereof, the use thereof in treating cytokine mediated diseases and pharmaceutical compositions for use in such therapy.
• Interleukin- 1 and Tumor Necrosis Factor (TNF) are biological substances produced by a variety of cells, such as monocytes or macrophages.
• IL- 1 has been demonstrated to mediate a variety of biological activities thought to be important in immunoregulation and other physiological conditions such as inflammation [See, e.g., Dinarello et al., Rev. Infect. Disease, 6, 51 (1984)].
• the myriad of known biological activities of IL- 1 include the activation of T helper cells, induction of fever, stimulation of prostaglandin or collagenase production, neutrophil chemotaxis, induction of acute phase proteins and the suppression of plasma iron levels.
• allograft rejections fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia, secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.
• AIDS cachexia secondary to infection or malignancy
• cachexia secondary to acquired immune deficiency syndrome
• AIDS AIDS
• ARC AIDS related complex
• keloid formation scar tissue formation
• Crohn's disease Crohn's disease
• ulcerative colitis or pyresis.
• HIV Human Immunodeficiency Virus
• HIV-1 HIV-1
• HIV-2 HIV-2
• HIV-3 HIV-3
• HIV entry into the T lymphocyte requires T lymphocyte activation.
• Other viruses, such as HIV-1, HIV-2 infect T lymphocytes after T Cell activation and such virus protein expression and/or replication is mediated or maintained by such T cell activation.
• Monokines are implicated in activated T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T lymphocyte activation. Therefore, interference with monokine activity such as by inhibition of monokine production, notably TNF, in an HIV-infected individual aids in limiting the maintenance of T cell activation, thereby reducing the progression of HIV infectivity to previously uninfected cells which results in a slowing or elimination of the progression of immune dysfunction caused by HIV infection.
• Monocytes, macrophages, and related cells such as kupffer and glial cells, have also been implicated in maintenance of the HIV infection. These cells, like T-cells, are targets for viral replication and the level of viral replication is dependent upon the activation state of the cells.
• TNF has also been implicated in various roles with other viral infections, such as the cytomegalia virus (CMV), influenza virus, and the herpes virus for similar reasons as those noted.
• CMV cytomegalia virus
• influenza virus influenza virus
• herpes virus herpes virus
• Interleukin-8 is a chemotactic factor first identified and characterized in 1987.
• IL-8 is produced by several cell types including mononuclear cells, fibroblasts, endothelial cells, and keratinocytes. Its production from endothelial cells is induced by IL- 1 , TNF, or lipopolysachharide (LPS).
• IL- 1 IL- 1 , TNF, or lipopolysachharide
• Human IL-8 has been shown to act on Mouse, Guinea Pig, Rat, and Rabbit Neutrophils. Many different names have been applied to IL-8, such as neutrophil attractant/activation protein- 1 (NAP-1), monocyte derived neutrophil chemotactic factor (MDNCF), neutrophil activating factor (NAF), and T-cell lymphocyte chemotactic factor.
• NAP-1 neutrophil attractant/activation protein- 1
• MDNCF monocyte derived neutrophil chemotactic factor
• NAF neutrophil activating
• IL-8 stimulates a number of functions in vitro. It has been shown to have che oattractant properties for neutrophils, T-lymphocytes, and basophils. In addition it induces histamine release from basophils from both normal and atopic individuals as well as lysozomal enzyme release and respiratory burst from neutrophils. IL-8 has also been shown to increase the surface expression of Mac- 1 (CD 11 b/CD 18) on neutrophils without de novo protein synthesis, this may contribute to increased adhesion of the neutrophils to vascular endothelial cells. Many diseases are characterized by massive neutrophil infiltration.
• IL-8 Conditions associated with an increased in IL-8 production (which is responsible for chemotaxis of neutrophil into the inflammatory site) would benefit by compounds which are suppressive of IL-8 production.
• IL- 1 and TNF affect a wide variety of cells and tissues and these cytokines as well as other leukocyte derived cytokines are important and critical inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these cytokines is of benefit in controlling, reducing and alleviating many of these disease states.
• compounds which are cytokine suppressive anti-inflammatory drugs i.e. compounds which are capable of inhibiting cytokines, such as IL-1 , IL-6, IL-8 and TNF.
• This invention relates to the novel compounds of Formula (I) or (II) and pharmaceutical compositions comprising a compound of Formula (I) or (II) and a pharmaceutically acceptable diluent or carrier.
• This invention also relates to a method of inhibiting cytokines and the treatment of a cytokine mediated disease, in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound of Formula (I) or (II).
• the present invention relates to a method of treating a CSBP/RK/p38 kinase mediated disease, in a mammal in need thereof.
• This invention more specifically relates to a method of inhibiting the production of IL- 1 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or (II).
• This invention more specifically relates to a method of inhibiting the production of IL-8 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or (II).
• This invention more specifically relates to a method of inhibiting the production of TNF in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or (II).
• R] is hydrogen, alkyl. aryl, heteroaryl, aryloxy, heteroaryloxy, nitro, amino, cyano, carboxy, carboxyalkoxy, carboxamido, or halogen, wherein the aryl, heteroaryl, heteroaryloxy, or aryloxy moieties may be optionally substituted; and
• R2 is aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkyl, cycloalkyl, or cycloalkyl alkyl, and wherein all of these moieties may be optionally substituted; or a pharmaceutically acceptable salt thereof.
• R j is suitably hydrogen, alkyl, aryl, heteroaryl, aryloxy, heteroaryloxy, nitro, amino, cyano, carboxy, carboxyalkoxy, carboxamido, or halogen, wherein the aryl, heteroaryl, heteroaryloxy, or aryloxy moieties may be optionally substituted one to three times independently by halogen; haloalkyl (such as CF3); alkyl; cyano; carboxy, carboxyalkoxy, carboxamido, nitro; alkoxy; hydroxy; amino; mono- or di-substituted alkyl amino; or S(O)m alkyl, wherein m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl.
• the Rj moiety is in the 5-position of the indole ring.
• the aryl, heteroaryl, aryloxy, heteroaryloxy moiety may be optionally substituted one or more times as defined herein.
• Rj is aryl it is preferably phenyl; when Rj is aryloxy it is preferably benzyloxy.
• R2 is suitably aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkyl, cycloalkyl, or a cycloalkyl alkyl group.
• Each of these moieties may be optionally substituted one or more times independently by halogen; haloalkyl (such as CF3); alkyl; cyano; carboxy, carboxyalkoxy, carboxamido, nitro; alkoxy; hydroxy; amino; mono- or di- substituted alkyl amino; or S(O)m alkyl, wherein is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl.
• R2 is aryl it is a phenyl moiety
• R2 is an arylalkyl group it is preferably benzyl, phenethyl or napthylmethyl.
• R2 is a heteroaryl or heteroarylalkyl, it is a pyridyl, or pyrimidine group.
• the alkyl moiety in the aryl alkyl group may also be optionally substituted, such as by halogen, hydroxy, alkoxy, alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, groups.
• halogen such as fluorine, chlorine, bromine or iodine
• hydroxy hydroxy substituted Ci- ioalkyl
• Ci-io alkoxy such as methoxy or ethoxy
• cyano carboxy, carboxyalkoxy, carboxamido, S(O)m alkyl, wherein is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl
• amino, mono & di-alkyl substituted amino Cj-io alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc.
• halosubstituted Cj-io alkyl such CF3; an optionally substituted heteroaryl, aryl, such as phenyl, or an optionally substituted heteroarylalkyl, arylalkyl, such as benzyl or phenethyl, wherein these aryl moieties may also be substituted one or more times by halogen; hydroxy; hydroxy substituted alkyl; Ci-io alkoxy; cyano; carboxy, carboxyalkoxy, carboxamido, S(O) m alkyl; amino, mono & di-alkyl substituted amino; alkyl, or halo substituted alkyl, such as CF3.
• Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic acid.
• pharmaceutically acceptable salts of compounds of Formula (I) may also be formed with a pharmaceutically acceptable cation, for instance, if a substituent group comprises a carboxy moiety.
• Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.
• halo or halogens
• halogens include the halogens: chloro, fluoro, bromo and iodo.
• cycloalkyl is used herein to mean cyclic radicals, preferably of 3 to 8 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like.
• heteroaryl (on its own or in any combination, such as “heteroaryloxy”, or “heteroaryl alkyl”) - a 5-10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole, thiadiazole, triazole, imidazole, or benzimidazole.
• heterocyclic (on its own or in any combination, such as
• heterocyclylalkyl a saturated or partially unsaturated 4-10 membered ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O, or S; such as, but not limited to, pyrrolidine, piperidine, piperazine, morpholine, tetrahydro pyran, or imidazolidine.
• aralkyl or “heteroarylalkyl” or “heterocyclicalkyl” is used herein to mean Cj-4 alkyl as defined above attached to an aryl, heteroaryl or heterocyclic moiety as also defined herein unless otherwise indicate.
• Exemplified compounds of Formula (I) include:
• the compounds of Formula (I) or (II) may be obtained by applying synthetic procedures, some of which are illustrated in Schemes I, etc., herein.
• the synthesis provided for in these Schemes is applicable for the producing compounds of Formula (I) or (II) having a variety of different Rj and R2 groups which are reacted, employing optional substituents which are suitably protected, to achieve compatibility with the reactions outlined herein. Subsequent deprotection, in those cases, then affords compounds of the nature generally disclosed.
• further compounds of Formula (I) or (II) may be prepared by applying standard techniques for functional group interconversion, well known in the art.
• Piperazines which were not commercially available were prepared from 4-BOC-piperazine by reaction of the secondary amine with electrophiles and subsequent removal of the BOC protecting group with TFA (Scheme 3).
• Suitable protecting groups for use with hydroxyl, etc. are well known in the art and described in many references, for instance, Protecting Groups in Organic Synthesis, Greene T W, Wiley-Interscience, New York, 1981.
• Suitable examples of hydroxyl protecting groups include silyl ethers, such as t- butyldimethyl or t-butyldiphenyl, and alkyl ethers, such as methyl connected by an alkyl chain of variable link, (CRi()R20)n-
• compositions of Formula (I) or (II) may be obtained in known manner, for example by treatment thereof with an appropriate amount of acid in the presence of a suitable solvent.
• the compounds of Formula (I) or (II), or a pharmaceutically acceptable salt thereof can be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of any disease state in a human, or other mammal, which is exacerbated or caused by excessive or unregulated cytokine production by such mammal's cell, such as but not limited to monocytes and/or macrophages.
• Compounds of Formula (I) or (II) are capable of inhibiting proinflammatory cytokines, such as IL-1, IL-6, IL-8 and TNF and are therefore of use in therapy.
• IL- 1 , IL-6, IL-8 and TNF affect a wide variety of cells and tissues and these cytokines, as well as other leukocyte-derived cytokines, are important and critical inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these pro-inflammatory cytokines is of benefit in controlling, reducing and alleviating many of these disease states.
• the present invention provides a method of treating a cytokine-mediated disease which comprises administering an effective cytokine-interfering amount of a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof.
• a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof for purposes herein, the compounds of Formula (I) and (II) all have the same dosages, and formulations as that of Formula (I) are used interchangeably herewith.
• Compounds of Formula (I) are capable of inhibiting inducible proinflammatory proteins, such as COX-2, also referred to by many other names such as prostaglandin endoperoxide synthase-2 (PGHS-2) and are therefore of use in therapy.
• COX-2 proinflammatory lipid mediators of the cyclooxygenase (CO) pathway
• COX-2 which is responsible for the these products derived from arachido c acid, such as prostaglandins affect a wide variety of cells and tissues are important and critical inflammatory mediators of a wide variety of disease states and conditions
• Expression of COX- 1 is not effected by compounds of Formula (I)
• This selective inhibition of COX-2 may alleviate or spare ulcerogenic liability associated with inhibition of COX- 1 thereby inhibiting prostoglandms essential for cytoprotective effects
• inhibition of these pro-inflammatory mediators is of benefit in controlling, reducing and alleviating many of these disease states
• Most notably these inflammatory mediators, in particular prostaglandins have
• the present invention provides a method of inhibiting the synthesis of COX-2 which comp ⁇ ses administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• the present invention also provides for a method of prophylaxis treatment in a human, or other mammal, by inhibition of the synthesis of the COX-2 enzyme
• a new member of the MAP kinase family, alternatively termed CSBP, p38, or RK has been identified independently by several laboratories recently.
• Activation of this novel protein kinase via dual phosphorylation has been observed in different cell systems upon stimulation by a wide spectrum of stimuli, such as physicochemical stress and treatment with lipopolysaccharide or proinflammatory cytokines such as interleukin- 1 and tumor necrosis factor.
• the cytokine biosynthesis inhibitors, of the present invention, compounds of Formula (I) have been determined to be potent and selective inhibitors of CSBP/p38/RK kinase activity. These inhibitors are of aid in determining the signaling pathways involvement in inflammatory responses.
• a definitive signal transduction pathway can be prescribed to the action of lipopolysaccharide in cytokine production in macrophages.
• the cytokine inhibitors were subsequently tested in a number of animal models for anti-inflammatory activity. Model systems were chosen that were relatively insensitive to cyclooxygenase inhibitors in order to reveal the unique activities of cytokine suppressive agents. The inhibitors exhibited significant activity in many such in vivo studies. Most notable are its effectiveness in the collagen-induced arthritis model and inhibition of TNF production in the endotoxic shock model. In the latter study, the reduction in plasma level of TNF correlated with survival and protection from endotoxic shock related mortality. Also of great importance are the compounds effectiveness in inhibiting bone resorption in a rat fetal long bone organ culture system. Griswold et al., (1988) Arthritis Rheum.
• Another aspect of the present invention is the treatment of a CSBP/RK/p38 kinase mediated disease, in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound of Formula (I).
• Suitable diseases include those mentioned herein for IL-1, IL-6, IL-8 and TNF and more specifically those disease which are CSBP RK/p38 kinase mediated diseases.
• rheumatoid arthritis rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions
• sepsis septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, asthma, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injuries, such as neurotrauma and ischemia, including both open and closed head injuries
• psoriasis restenosis, such as occurs following coronary angioplasty, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcososis, bone resorption diseases, osteoporosis, , graft vs. host reaction, allograft rejections, Crohn's disease, ulcerative colitis or any other anti-inflammatory bowel disease (IBD), or pyresis.
• IBD anti-inflammatory bowel disease
• CNS injuries as defined herein include both open or penetrating head trauma, such as by surgery, or a closed head trauma injury, such as by an injury to the head region. Also included within this definition is ischemic stroke, particularly to the brain area.
• Ischemic stroke may be defined as a focal neurologic disorder that results from insufficient blood supply to a particular brain area, usually as a consequence of an embolus, thrombi, or local atheromatous closure of the blood vessel.
• the role of inflammatory cytokines in this are has been emerging and the present invention provides a mean for the potential treatment of these injuries. Relatively little treatment, for an acute injury such as these has been available.
• TNF- ⁇ is a cytokine with proinflammatory actions, including endothelial leukocyte adhesion molecule expression.
• Leukocytes infiltrate into ischemic brain lesions and hence compounds which inhibit or decrease levels of TNF would be useful for treatment of ischemic brain injury.
• Models of closed head injuries and treatment with mixed 5-LO/CO agents is discussed in Shohami et al., J. of Vaisc & Clinical Physiology and Pharmacology, Vol. 3, No. 2, pp. 99-107 ( 1992) whose disclosure is incorporated herein by reference. Treatment which reduced edema formation was found to improve functional outcome in those animals treated.
• compounds of Formula (I) or a pharmaceutically acceptable salt thereof are of use in the prophylaxis or therapy of any disease state in a human, or other mammal, which is exacerbated by or caused by excessive or unregulated IL-1 , IL-8 or TNF production by such mammal's cell, such as, but not limited to, monocytes and/or macrophages.
• this invention relates to a method of inhibiting the production of IL-1 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• a method of inhibiting the production of IL-1 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• this invention relates to a method of inhibiting the production of TNF in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• TNF production has been implicated in mediating or exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, such as osteoporosis, reperfusion injury, graft vs.
• diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcois
• allograft rejections fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis and pyresis.
• AIDS cachexia secondary to infection or malignancy
• AIDS cachexia secondary to acquired immune deficiency syndrome
• AIDS AIDS
• ARC AIDS related complex
• keloid formation scar tissue formation
• Crohn's disease Crohn's disease
• ulcerative colitis ulcerative colitis
• viruses of Formula (I) are also useful in the treatment of viral infections, where such viruses are sensitive to upregulation by TNF or will elicit TNF production in vivo.
• the viruses contemplated for treatment herein are those that produce TNF as a result of infection, or those which are sensitive to inhibition, such as by decreased replication, directly or indirectly, by the TNF inhibiting-compounds of Formula (1).
• viruses include, but are not limited to HIV-1, HIV-2 and HIV-3, Cytomegalovirus (CMV), Influenza, adenovirus and the Herpes group of viruses, such as but not limited to, Herpes Zoster and Herpes Simplex.
• this invention relates to a method of treating a mammal afflicted with a human immunodeficiency virus (HIV) which comprises administering to such mammal an effective TNF inhibiting amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• HAV human immunodeficiency virus
• TNF mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular viral infections.
• viruses include, but are not limited to, lentivirus infections such as, equine infectious anaemia virus, caprine arthritis virus, visna virus, or maedi virus or retrovirus infections, such as but not limited to feline immunodeficiency virus (FIV), bovine immunodeficiency virus, or canine immunodeficiency virus or other retroviral infections.
• the compounds of Formula (I) may also be used topically in the treatment or prophylaxis of topical disease states mediated by or exacerbated by excessive cytokine production, such as by IL-1 or TNF respectively, such as inflamed joints, eczema, psoriasis and other inflammatory skin conditions such as sunburn; inflammatory eye conditions including conjunctivitis; pyresis, pain and other conditions associated with inflammation.
• cytokine production such as by IL-1 or TNF respectively, such as inflamed joints, eczema, psoriasis and other inflammatory skin conditions such as sunburn; inflammatory eye conditions including conjunctivitis; pyresis, pain and other conditions associated with inflammation.
• this invention relates to a method of inhibiting the production of IL-8 in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
• IL-8 IL-8
• IL-8 has the unique property of promoting neutrophil chemotaxis and activation. Therefore, the inhibition of IL-8 production would lead to a direct reduction in the neutrophil infiltration.
• the compounds of Formula (I) are administered in an amount sufficient to inhibit cytokine, in particular IL-1, IL-6, IL-8 or TNF, production such that it is regulated down to normal levels, or in some case to subnormal levels, so as to ameliorate or prevent the disease state.
• cytokine in particular IL-1, IL-6, IL-8 or TNF
• Abnormal levels of IL-1 , IL-6, IL-8 or TNF constitute: (i) levels of free (not cell bound) IL-1, IL-6, IL-8 or TNF greater than or equal to 1 picogram per ml; (ii) any cell associated IL-1, IL-6, IL-8 or TNF; or (iii) the presence of IL- 1 , IL-6, IL-8 or TNF mRNA above basal levels in cells or tissues in which IL-1, IL-6, IL-8 or TNF, respectively, is produced.
• the compounds of Formula (I) are inhibitors of cytokines, specifically IL-1, IL-6, IL-8 and TNF is based upon the effects of the compounds of Formulas (I) on the production of the IL-1, IL-8 and TNF in in vitro assays which are described herein.
• TNF refers to: a) a decrease of excessive in vivo levels of the cytokine (IL- 1 , IL-6, IL- 8 or TNF) in a human to normal or sub-normal levels by inhibition of the in vivo release of the cytokine by all cells, including but not limited to monocytes or macrophages; b) a down regulation, at the genomic level, of excessive in vivo levels of the cytokine (IL- 1, IL-6, IL-8 or TNF) in a human to normal or sub-normal levels; c) a down regulation, by inhibition of the direct synthesis of the cytokine (IL- 1 , IL-6, IL-8 or TNF) as a postranslational event; or d) a down regulation, at the translational level, of excessive in vivo levels of the cytokine (IL- 1 , IL-6, IL-8 or TNF) in a human to normal or subnormal levels.
• TNF mediated disease or disease state refers to any and all disease states in which TNF plays a role, either by production of TNF itself, or by TNF causing another monokine to be released, such as but not limited to IL-1, IL-6 or IL-8.
• cytokine refers to any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response.
• a cytokine includes, but is not limited to, monokines and lymphokines, regardless of which cells produce them.
• a monokine is generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte.
• Lymphokines are generally referred to as being produced by lymphocyte cells.
• cytokines include, but are not limited to, Interleukin- 1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL- 8), Tumor Necrosis Factor-alpha (TNF-a) and Tumor Necrosis Factor beta (TNF- ⁇ ).
• cytokine interfering or "cytokine suppressive amount” refers to an effective amount of a compound of Formula (I) which will cause a decrease in the in vivo levels of the cytokine to normal or sub-normal levels, when given to a patient for the prophylaxis or treatment of a disease state which is exacerbated by, or caused by, excessive or unregulated cytokine production.
• the cytokine referred to in the phrase "inhibition of a cytokine, for use in the treatment of a HIV-infected human” is a cytokine which is implicated in (a) the initiation and/or maintenance of T cell activation and/or activated T cell-mediated HIV gene expression and/or replication and or (b) any cytokine-mediated disease associated problem such as cachexia or muscle degeneration.
• TNF- ⁇ also known as lymphotoxin
• TNF-a also known as cachectin
• a compound of Formula (I) or a pharmaceutically acceptable salt thereof in therapy it will normally be Formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice.
• This invention also relates to a pharmaceutical composition comprising an effective, non-toxic amount of a compound of Formula (I) and a pharmaceutically acceptable carrier or diluent.
• Compounds of Formula (I), pharmaceutically acceptable salts thereof and pharmaceutical compositions incorporating such may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation.
• the compounds of Formula (I) may be administered in conventional dosage forms prepared by combining a compound of Formula (I) with standard pharmaceutical carriers according to conventional procedures.
• the compounds of Formula (I) may also be administered in conventional dosages in combination with a known, second therapeutically active compound. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
• the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
• the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the Formulation and not deleterious to the recipient thereof.
• the pharmaceutical carrier employed may be, for example, either a solid or liquid.
• solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
• liquid carriers are syrup, peanut oil, olive oil, water and the like.
• the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
• the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
• the amount of solid carrier will vary widely but preferably will be from about 25mg. to about lg.
• the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
• Compounds of Formula (I) may be administered topically, that is by non-systemic administration. This includes the application of a compound of Formula (I) externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream.
• systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
• Formulations suitable for topical administration include liquid or semi- liquid preparations suitable for penetration through the skin to the site of inflammation such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
• the active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, for instance from 1 % to 2% by weight of the Formulation. It may however comprise as much as 10% w/w but preferably will comprise less than 5% w/w, more preferably from 0.1% to 1% w/w of the Formulation.
• Lotions according to the present invention include those suitable for application to the skin or eye.
• An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.
• Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
• Creams, ointments or pastes according to the present invention are semi- solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base.
• the base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or a macrogel.
• the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof.
• Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
• Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent.
• the resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100° C. for half an hour.
• the solution may be sterilized by filtration and transferred to the container by an aseptic technique.
• bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01 %).
• Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
• Compounds of formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parental administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques.
• Compounds of Formula (I) may also be administered by inhalation, that is by intranasal and oral inhalation administration.
• Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
• the daily oral dosage regimen will preferably be from about 0.01 to about 80 mg/kg of total body weight, preferably from about 0.1 to 30 mg/kg, more preferably from about 0.2 mg to 15 mg.
• the daily parenteral dosage regimen about 0.01 to about 80 mg/kg of total body weight, preferably from about 0.1 to about 30 mg/kg, and more preferably from about 0.2 mg to 15mg/kg.
• the daily topical dosage regimen will preferably be from 0.1 mg to 150 mg, administered one to four, preferably two or three times daily.
• the daily inhalation dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day.
• the optimal quantity and spacing of individual dosages of a compound of Formula (I) or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound of Formula (I) or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
• novel compounds of Formula (I) may also be used in association with the veterinary treatment of mammals, other than humans, in need of inhibition of cytokine inhibition or production.
• cytokine mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted herein in the Methods of Treatment section, but in particular viral infections.
• viruses include, but are not limited to, lentivirus infections such as, equine infectious anaemia virus, caprine arthritis virus, visna virus, or maedi virus or retrovirus infections, such as but not limited to feline immunodeficiency virus (FIV), bovine immunodeficiency virus, or canine immunodeficiency virus or other retroviral infections.
• lentivirus infections such as, equine infectious anaemia virus, caprine arthritis virus, visna virus, or maedi virus or retrovirus infections, such as but not limited to feline immunodeficiency virus (FIV), bovine immunodeficiency virus, or canine immunodeficiency virus or
• Human peripheral blood monocytes are isolated and purified from either fresh blood preparations from volunteer donors, or from blood bank buffy coats, according to the procedure of Colotta et al, J Immunol, 132, 936 ( 1984). These monocytes ( 1x10") are plated in 24- well plates at a concentration of 1-2 million/ml per well. The cells are allowed to adhere for 2 hours, after which time non-adherent cells are removed by gentle washing. Test compounds are then added to the cells for Ih before the addition of lipopolysaccharide (50 ng/ml), and the cultures are incubated at 37°C for an additional 24h. At the end of this period, culture supernatants are removed and clarified of cells and all debris.
• Culture supernatants are then immediately assayed for IL-1 biological activity, either by the method of Simon et al., J. Immunol. Methods, 84, 85, (1985) (based on ability of IL-1 to stimulate a Interleukin 2 producing cell line (EL-4) to secrete IL-2, in concert with A23187 ionophore) or the method of Lee et ai, J. ImmunoTherapy, 6 ( 1), 1-12 (1990) (ELISA assay).
• Tumour Necrosis Factor Human peripheral blood monocytes are isolated and purified from either blood bank buffy coats or platelet pheresis residues, according to the procedure of Colotta, R. et al., J Immunol, 132(2), 936 ( 1984). The monocytes are plated at a density of 1x10-5 cells/ml medium/well in 24- well multi-dishes. The cells are allowed to adhere for 1 hour after which time the supernatant is aspirated and fresh medium (1ml, RPMI-1640, Whitaker Biomedical Products, Whitaker, CA) containing 1% fetal calf serum plus penicillin and streptomycin (10 units/ml) added.
• fresh medium (1ml, RPMI-1640, Whitaker Biomedical Products, Whitaker, CA
• the cells are incubated for 45 minutes in the presence or absence of a test compound at InM-lOmM dose ranges (compounds are solubilized in dimethyl sulfoxide/ethanol, such that the final solvent concentration in the culture medium is 0.5% dimethyl sulfoxide/0.5% ethanol).
• Bacterial lipopolysaccharide E. coli 055.B5 [LPS] from Sigma Chemicals Co.
• LPS Bacterial lipopolysaccharide
• culture supernatants are removed from the cells, centrifuged at 3000 rpm to remove cell debris. The supernatant is then assayed for TNF activity using either a radio-immuno or an ELISA assay, as described in WO 92/10190 and by Becker et al, J Immunol, 1991, 147, 4307.
• IL-1 and TNF inhibitory activity does not seem to correlate with the property of the compounds of Formula (I) in mediating arachidonic acid metabolism inhibition. Further the ability to inhibit production of prostaglandin and/or leukotriene synthesis, by nonsteroidal anti-inflammatory drugs with potent cyclooxygenase and/or lipoxygenase inhibitory activity does not mean that the compound will necessarily also inhibit TNF or IL- 1 production, at non- toxic doses.
• Interleukin -8 (IL-8 ):
• HUVEC Primary human umbilical cord endothelial cells
• CELL Systems Cell Systems, Kirland, Wa
• CS-HBGF aFGF and heparin.
• the cells are then diluted 20-fold before being plated (250 ⁇ l) into gelating coated 96-well plates. Prior to use, culture medium are replaced with fresh medium (200 ⁇ l).
• Buffer or test compound (25 ⁇ l, at concentrations between 1 and lO ⁇ M) is then added to each well in quadruplicate wells and the plates incubated for 6h in a humidified incubator at 37°C in an atmosphere of 5% CO2- At the end of the incubation period, supernatant is removed and assayed for IL-8 concentration using an IL-8 ELISA kit obtained from R&D Systems (Minneapolis, MN). All data is presented as mean value (ng/ml) of multiple samples based on the standard curve. IC50 S where appropriate are generated by non-linear regression analysis. Cytokine Specific Binding Protein Assay
• a radiocompetitive binding assay was developed to provide a highly reproducible primary screen for structure-activity studies. This assay provides many advantages over the conventional bioassays which utilize freshly isolated human monocytes as a source of cytokines and ELISA assays to quantify them. Besides being a much more facile assay, the binding assay has been extensively validated to highly correlate with the results of the bioassay.
• a specific and reproducible cytokine inhibitor binding assay was developed using soluble cystosolic fraction from THP. l cells and a radiolabeled compound.
• CSBP cytokine specific binding protein
• the binding protein may be in isolated form in solution, or in immobilized form, or may be genetically engineered to be expressed on the surface of recombinant host cells such as in phage display system or as fusion proteins.
• whole cells or cytosolic fractions comprising the CSBP may be employed in the screening protocol.
• a plurality of compounds are contacted with the binding protein under conditions sufficient to form a compound/ binding protein complex and compound capable of forming, enhancing or interfering with said complexes are detected.
• the following assay describes a method for determining the inhibitory effects of compounds of Formula (I) on human PGHS-2 protein expression in LPS stimulated human monocytes.
• the assay shown below is demonstrated with compounds other than that of Formula (I) herein:
• Human peripheral blood monocytes were isolated from buffy coats by centrifugation through Ficoll and Percoll gradients. Cells were seeded at 2 X lO well in 24 well plates and allowed to adhere for 1 hour in RPMI supplemented with 1% human AB serum, 20mM L-glutamine, Penicillin- Streptomycin and lOmM HEPES. Compounds were added at various concentrations and incubated at 37°C for 10 minutes. LPS was added at 50 ng/well (to induce enzyme expression) and incubated overnight at 37°C. The supernatant was removed and cells washed once in cold PBS.
• the cells were lysed in 100ml of cold lysis buffer(50mM Tris/HCl pH 7.5, 150mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1 % SDS, 300ug/ml DNAse, 0.1 %
• PBS/0.1 % Tween 20 with 5% non-fat dry milk After washing 3 times in PBS/Tween buffer, the membrane was incubated with a 1:2000 dilution of a monospecific antiserum to PGHS-2 or a 1 : 1000 dilution of an antiserum to PGHs-1 in PBS/Tween with 1% BSA for one hour with continuous shaking. The membrane was washed 3X in PBS/Tween and then incubated with a 1:3000 dilution of horseradish peroxidase conjugated donkey antiserum to rabbit lg (Amersham) in PBS/Tween with 1% BSA for one hour with continuous shaking.
• the present assay provides for examination of the expression of tumor necrosis factor mRNA in specific brain regions which follow experimentally induced lateral fluid-percussion traumatic brain injury (TBI) in rats.
• TBI experimentally induced lateral fluid-percussion traumatic brain injury
• LC left (injured) parietal cortex
• RC contralateral right cortex
• LA cortex adjacent to injured parietal cortex
• RA right cortex
• RH right hippocampus
• TNF- ⁇ mRNA expression is observed in LH (104 ⁇ 17% of positive control, p ⁇ 0.05 compared with sham), LC (105 ⁇ 21%, p ⁇ 0.05) and LA (69 ⁇ 8%, p ⁇ 0.01) in the traumatized hemisphere 1 hr. following injury.
• An increased TNF- a mRNA expression is also observed in LH (46 ⁇ 8%, p ⁇ 0.05), LC (30 ⁇ 3%, p ⁇ 0.01) and LA (32 ⁇ 3%, p ⁇ 0.01) at 6 hr. which resolves by 24 hr. following injury.
• TNF- ⁇ mRNA In the contralateral hemisphere, expression of TNF- ⁇ mRNA is increased in RH (46 ⁇ 2%, p ⁇ 0.01), RC (4 ⁇ 3%) and RA (22 ⁇ 8%) at 1 hr. and in RH (28 ⁇ 1 1 %), RC (7 ⁇ 5%) and RA (26 ⁇ 6%, p ⁇ 0.05) at 6 hr. but not at 24 hr. following injury. In sham (surgery without injury) or naive animals, no consistent changes in expression of TNF- ⁇ mRNA is observed in any of the 6 brain areas in either hemisphere at any times.
• TNF- o mRNA is altered in specific brain regions, including those of the non- traumatized hemisphere. Since TNF- ⁇ is able to induce nerve growth factor (NGF) and stimulate the release of other cytokines from activated astrocytes, this post-traumatic alteration in gene expression of TNF- ⁇ plays an important role in both the acute and regenerative response to CNS trauma.
• NGF nerve growth factor
• CNS Injury model for IL- ⁇ mRNA This assay characterizes the regional expression of interleukin- l ⁇ (IL- l ⁇ ) mRNA in specific brain regions following experimental lateral fluid- percussion traumatic brain injury (TBI) in rats.
• TBI lateral fluid- percussion traumatic brain injury
• LC left (injured) parietal cortex
• RC contralateral right cortex
• LA cortex adjacent to injured parietal cortex
• RA right cortex
• RH right hippocampus
• Total RNA is isolated and Northern blot hybridization is performed and the quantity of brain tissue IL-l ⁇ mRNA is presented as percent relative radioactivity of IL-l ⁇ positive macrophage RNA which is loaded on same gel.
• Example 7 1 -( " 1 H-Indol-3-ylcarbonyl)-4-(4-trifluoromethylphenylmethy Ppiperazine a) - 1 -Tertbutoxycarbonyl-4-f4-trifluoromethylbenzyl) piperazine
• Example 1 1 1 -( lH-Indol-3-ylcarbonyl)-4-(cvclopropylmethyl)piperazine
• Example 15 1 -( 1 H-Indol-3-y lcarbony P-4-(2-phenylethyl)piperazine The title compound was prepared according to the general method of
• Example 24 ( 1 H-5-Phenylindol-3-ylcarbonvP-4-( benzyPpiperazine The product of example 17 (200 mg, 0.5 mmol), phenylboronic acid

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