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WO1995033713A9 - Composes anti-inflammatoires - Google Patents

Composes anti-inflammatoires

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Publication number
WO1995033713A9
WO1995033713A9 PCT/US1995/007041 US9507041W WO9533713A9 WO 1995033713 A9 WO1995033713 A9 WO 1995033713A9 US 9507041 W US9507041 W US 9507041W WO 9533713 A9 WO9533713 A9 WO 9533713A9
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Prior art keywords
alkyl
inflammatory
compound according
hydrogen
acid
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PCT/US1995/007041
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English (en)
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WO1995033713A1 (fr
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  • This invention relates to pharmaceutical compositions and their use as anti- inflammatory agents in mammals.
  • lipid mediators are among the most potent and important products which are generated during inflammatory reactions.
  • the synthesis of most lipid mediators is initiated by the specific cleavage of complex phospholipid molecules which contain arachidonate at their sn-2 position.
  • Arachidonic acid is predominantly found in the sn-2 position of phospholipids after redistribution by transacylases and its release by sn-2 acylhydrolases from phospholipids represents the rate- limiting step in the formation of eicosanoids (leukotrienes, prostaglandins and thromboxanes) and other hydroxylated fatty acids.
  • arachidonic acid As arachidonic acid is released, it is then converted to oxygenated derivatives by at least two enzymatic systems (lipoxygenase and/or cyclooxygenase). Concomitant with arachidonate release, lysophospholipids are formed. One of these lyso phospholipids, l-alkyl-2-lyso-sn-glycero-3-phosphocholine, is then acetylated to form platelet-activating factor (PAF). Each of the cell types involved in the inflammatory response produce and secrete a unique subset of lipid mediators. The quantities and nature of the metabolites depend on which enzymes and precursor phospholipid pools are available to inflammatory cells.
  • PAF platelet-activating factor
  • lipid mediators such as PAF and eicosanoids are formed by the aforementioned pathways, they induce signs and symptoms observed in the pathogenesis of various inflammatory disorders. Indeed, the pathophysiological activity of arachidonic acid (and its metabolites) is well known to those skilled in the art. For example, these mediators have been implicated as having an important role in allergy, asthma, anaphylaxis, adult respiratory distress syndrome, reperfusion injury, inflammatory bowel disease, rheumatoid arthritis, endotoxic shock, and cardiovascular disease. Aalmon et al., Br. Med. Bull (1978) 43:285-296; Piper et al., Ann. NY Acad. Sci. (1991) 629:112-119; Holtzman, Am.
  • PAF is a potent proinflammatory mediator produced by a variety of cells.
  • PAF stimulates the movement and aggregation of neutrophils and the release therefrom of tissue-damaging enzymes and oxygen radicals.
  • PAF has also been implicated in activation of leukocytes, monocytes, and macrophages. These activities contribute to the actions of PAF as having (pathological) physiological activity in inflammatory and allergic responses.
  • PAF has also been implicated in smooth muscle contraction, pain, edema, hypotensive action, increases in vascular permeability, cardiovascular disorders, asthma, lung edema, endotoxin shock, and adult respiratory distress syndrome. PAF elicits these responses either directly through its own cellular receptor(s) or indirectly by inducing the synthesis of other mediators.
  • Phospholipase A2's are responsible for the liberation of arachidonic acid from the sn-2 position of phospholipid. They are thought to play an important role in the pathogenesis of inflammation and possibly in immunological dysfunction, both as a cell associated enzyme as well as an extracellular soluble enzyme.
  • Low molecular weight, mammalian Type ⁇ 14 kDa PLA2 has been well characterized and is known to exist in both an extracellular form in inflammatory fluids (Kramer et al., J.
  • PLA2 is important in the liberation of arachidoninc acid from phospholipid and may also play a role in the generation of PAF via lysophospholipid formation, inhibition of such an enzyme would be useful for the treatment of disease states caused thereby.
  • phospholipase A2's There are many novel forms of phospholipase A2's which have recently been discovered.
  • members of the sn-2 acylhydrolase family of PLA2's are divided into low and high molecular weight enzymes be it from mammalian, or non- mammalian sources.
  • Low molecular weight PLA2's will generally have a molecular weight in the range of 12,000 to 15,000.
  • High molecular weight will be in the range of 30,000 or 56,000 kDa to 110,000 by SDS electrophoresis analysis.
  • a high molecular weight, cytosolic 85 kDa PLA2 has been isolated and cloned from the human moncytic cell line, U937 (Clark et al., Proc. Natl. Acad. Sci., 87:7708-7712, 1990).
  • the cell-associated Type II- 14 kDa-PLA 2 in cell lipid metabolism was thought to be the key rate limiting enzyme in lipid mediator formation, until the recent identification of this cell-associated but structurally distinct 85 kDa sn-2 acylhydrolase, (Clark, et al., supra); and Kramer, et al., (1991) J. Biol. Chem. 266, 5268-5272.
  • this enzyme is active at neutral pH and Ca2+-dependent, but in contrast exhibits a preference for AA in the sn-2 position of phospholipid substrate and migrates from the cytosol to the membrane in a Ca2+-dependent manner and is regulated by phosphorylation (Kramer et al., J. Biol. Chem., 266:5268-5272 (1991).
  • the 85 kDa- PLA 2 is also distinct from 14 kDa-PLA 2 s and Ca 2+ -independent PLA 2 as demonstrated by different biochemical characteristics such as stability of the 85 kDa-PLA 2 to DTT, instability to heat and the lack of inhibition by a phosphonate phospholipid TS A inhibitor of 14 kDa-PLA 2 .
  • 85 kDa-PLA 2 has been shown to possess a lysophospholipase Ai activity which is not observed with the 14 kDa-PLA 2 s .
  • the 85 kDa enzyme is similar to the myocardial Ca2+-independent PLA 2 (Bomalaski and Clark, Arthritis and Rheumat.
  • CoA-IT has a specificity for certain phospholipids as donor and acceptor molecules.
  • the fatty acid transferred is long chained and unsaturated, and almost exclusively arachidonate.
  • Other fatty acids such as the 16:0, 18: 1 or 18:2 are not moved into the sn-2 position of alkyl and 1-alkenyl phospholipid pools by CoA-IT.
  • the specificity of CoA-IT is in direct contrast to many other CoA-dependent acylation activities which acylate a wide variety of lysophospholipids with no selectivity for arachidonate.
  • CoA-IT is involved in arachidonic acid and phospholipid metabolism
  • inhibition of such an enzyme would be useful for the treatment of inflammatory, allergic and hypersecretory conditions or disease states caused thereby. Therefore, a method by which CoA-IT is inhibited will consequently and preferentially decrease the arachidonate content of 1-alkyl- and 1-alkenyl-linked phospholipids and will therefore decrease the production of pro-inflammatory mediators such as free arachidonic acid, prostaglandins, leukotriene and PAF during an inflammatory response.
  • This invention relates to the novel compounds of Formula (I) and compositions thereof which comprises a compound of Formula (I), or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.
  • This invention also relates to a method of treating or reducing inflammation in a mammal in need thereof, which comprises administering to said mammal an effective amount of a compound or composition of Formula (I).
  • This invention also relates to a method of treating disease or disorders mediated by lipid inflammatory mediators, free arachidonic acid, its metabolites and/or PAF by adminis- tering to a patient in need thereof, an effective amount of a compound of Formula (I).
  • This invention also relates to a method of treating disease or disorders mediated by phospholipase A2 (PLA2) and/or Coenzyme A independent transacylase (CoA-IT) by administering to a patient in need thereof, an effective amount of a compound or composition of Formula (I).
  • PHA2 phospholipase A2
  • CoA-IT Coenzyme A independent transacylase
  • One aspect of the present invention are compounds having the structure represented by the formula:
  • Rl is SO3H or S(O) n Ci-4 alkyl; n is 0 or an integer having a value 1 or 2; X is oxygen or sulfur;
  • R 2 is hydrogen, halogen, optionally substituted Ci -8 alkyl, or Ci-8 alkoxy; m is an integer having a value of 1 or 2; R3 is S(O)2R7; R4 is hydrogen or S(O)2R7 ;
  • R5 is hydrogen, halogen, CF3, CH3, (CH2)tC(O)2R8, or (CH2)tOH; t is 0 or an integer having a value of 1 or 2;
  • R ⁇ is hydrogen or halogen
  • R7 is optionally substituted aryl, optionally substituted arylCi-2 alkyl, or an optionally substituted C 1 -8 alkyl;
  • R8 is hydrogen or Ci-4 alkyl; or a pharmaceutically acceptable salt thereof.
  • the present invention is directed to a novel method of treating inflammatory disease in a mammal in need thereof by administering to said mammal an effective amount of a compound according to Formula (I).
  • the compounds of Formula (I) may selectively inhibit the PLA2 enzyme, the CoA-IT enzyme or both. Inhibition of either or both enzymes will result in the treatment of inflammatory occurrences in mammals. Inflammatory states in mammals may include, but are not limited to, allergic and asthmatic manifestations, dermatological diseases, inflammatory diseases, collagen diseases, reperfusion injury and stroke. Treatment of both acute and chronic diseases are possible. Preferred diseases for treatment are arthritis, asthma, allergic rhinitis, inflammatory bowel disease (IBD), psoriasis, reperfusion injury and stroke.
  • the compounds of Formula (I) are preferential and selective inhibitors of the low molecular weight PLA2 enzyme.
  • Ri is suitably SO3H or S(O)nCi-4 alkyl, and n is a number having a value of 0 to 2.
  • Ri is SO3H or a salt thereof.
  • R2 is independently a substitutent on the benzene ring from 1 to 2 times, and such substiutent is selected from hydrogen, halogen, an optionally substituted Ci-8 alkyl, or Ci-8 alkoxy group Suitably when R2 is halogen it is a chlorine or bromine.
  • R 2 is an optionally substituted Ci-8 alkyl, the alkyl is substituted one to three times with halogen, such as fluorine, preferably a trifluromethyl group.
  • the optionally substituted C 1-8 alkyl moiety if preferably a branched C5 chain, such as 1,1 -dimethyl propyl moiety or a C8 branched chain such as 1,1,3,3-tetramethyl butyl moiety.
  • X is oxygen or sulfur, preferably oxygen.
  • R3 is S(O)2R7; and R7 is an optionally substituted aryl, an optionally substituted arylCi-2 alkyl, or an optionally substituted Ci -8 alkyl group.
  • R7 is an aryl moiety it is phenyl or napthyl, preferably phenyl; when R7 is an aryl alkyl moiety it is preferably benzyl.
  • the aryl, aryl alkyl or alkyl moieties are substituted independently, one to three times, by halogen, trifluromethyl, aryloxy, methoxy, CH2OH, methyl, or C(O)2H.
  • the substituents are halogen, or trifluoromethyl.
  • the substituent halogen groups are preferably chlorine, bromine, or fluorine.
  • the substiutents are in the 3,5- position or the 4-position of the aryl ring. More preferably the aryl substituents are 3,5-bis-trifluoromethyl, 4-trifluoromethyl, 4-bromo, 4-chloro, or 4- fluoro.
  • R7 is an optionally substituted alkyl moiety
  • the alkyl group is preferably a methyl or a C8 unbranched chain.
  • the methyl moiety, if substituted, is preferably substituted by one or more fluorines, such as in a trifluromethyl group.
  • R4 is suitably hydrogen or S(O)2R7-
  • R4 is hydrogen.
  • R7 group is preferably the same as the R7 moiety the R3 group noted above, which will form a bis like structure.
  • R5 is hydrogen, halogen, CF3, CH3, CH2C(O)2R8, or CH2OH, wherein t is 1.
  • R5 is CH2C(O)2R8»
  • R8 is a Ci-4 alkyl, preferably t-butyl.
  • Prefered R5 groups are hydrogen, CF3, or halogen. More preferably R5 is hydrogen or CF3.
  • R6 is hydrogen or halogen; preferably hydrogen. If R6 is halogen it is preferably fluorine or chlorine.
  • 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.
  • Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.
  • halo all halogens, that is chloro, fluoro, bromo and iodo;
  • C ⁇ _8 alkyl or "alkyl” - both straight and branched chain radicals of 1 to 8 carbon atoms, unless the chain length is otherwise limited, including, but not limited to, methyl, ethyl, /i-propyl, wo-propyl, n-butyl, sec-butyl, tso-butyl, rf-butyl, and the like; • "aryl” - phenyl and naphthyl;
  • aralkyl is used herein to mean an aryl group connected to Ci-4 alkyl moiety wherein the alkyl group may be branched or straight as defined above, unless otherwise indicated.
  • the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds are included within the scope of the present invention.
  • Specifically exemplified compounds of Formula (I) are: 2-[2-[3,5-Bis(trifluoromethyl)sulfonamido-4-trifluoromethylphenoxy]-5-( 1,1- dimethylpropyl)benzenesulfonic acid; 2-[2-[3,5-Bis(trifluoromethyl)phenylsulfonamido]-4-trifluoromethylphenoxy]- benzenesulfonic acid;
  • Compounds of Formula (I) may be prepared by a process which comprises reacting a suitably protected compound of Formula (2), wherein Ri, R2, and m are as described in Formula (I) which are generally commercially available:
  • compounds of formula (2) may be prepared by sulfonating an appropriately substituted phenol with a suitable sulf onating reagent, such as fuming sulfuric acid.
  • compounds of Formula (4) where Ri is -S(O)3H may be prepared by sulfonation of a compound of Formula (4) where Ri is H with a suitable reagent such as fuming sulfuric acid.
  • Reaction of compound (5) with a sulfonyl halide in a suitable solvent such as pyridine provides a compound of Formula (6) wherein R4 is hydrogen.
  • Compounds wherein R4 is S(O)2R7 can be prepared by the reacion of compound (5) with excess sulfonyl halide.
  • the crude product was flash chromatographed (silica gel, methylene chloride/isopropanol/ammonium hydroxide) to yield the ammonium salt, an off-white solid.
  • the ammonium salt was mixed in methanol and water with sodium bicarbonate (361 mg, 0.0043 mol) to yield the title compound.
  • MS (FAB) m/e 448 [M+Na]+.
  • the compounds of Formula (I) or pharmaceutically acceptable salts thereof can be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of an inflammatory disease state in a mammal, preferably a human.
  • Inhibition of PLA2 and/or CoA-IT and the simultaneous reduction of PAF, free arachidonic acid and eicosanoid release from inflammatory cells according to this invention is of therapeutic benefit in a broad range of diseases or disorders.
  • the invention herein is therefore useful to treat such disease states both in humans and in other mammals.
  • Inhibition of CoA-IT and 14 kDa PLA2 by the compounds of Formula (I) is an effective means for simultaneously reducing PAF, free arachidonic acid and eicosanoids produced in inflammatory cells.
  • the therapeutic utility of blocking lipid mediator generation has been recognized for many years.
  • inhibitors of cyclooxygenase such as aspirin, indomethacin, acetaminophen and ibuprofen, have demonstrated broad therapeutic utilities.
  • CoA-IT inhibitors inhibit cyclooxygenase products.
  • Another class of inhibitors which are used in a broad range of inflammatory disorders are the corticosteroids. Corticosteroids act in a variety of ways, e.g.
  • Both 14 kDa PLA2 inhibitors and CoA-IT inhibitors block the release of free arachidonic acid.
  • Inhibitors of 5-lipoxygenase block the production of leukotrienes and leukotriene antagonists prevent the bioactions of leukotrienes. Recent studies indicate that both will have broad therapeutic utilities.
  • Both 14 kDa PLA2 inhibitors and CoA-IT inhibitors block the production of leukotrienes.
  • Inhibitors of phospholipase A2 block the release of free arachidonic acid and the formation of lyso PAF (the immediate precursor of PAF).
  • PLA2 inhibitors are recognized to have broad therapeutic utilities. It does not , however, follow that the disease states noted above must in fact be caused by altered CoA-IT or PLA2 activity. Thus, the disease state itself may not be directly mediated by CoA-IT or PLA2 activity. It only follows that CoA-IT or PLA2 activity is required for the continued expression of symptoms of the disease state and that CoA-IT or PLA2 inhibitors will be beneficial against the symptoms of these disease states.
  • PAF Intravenous infusion of PAF at doses of 20-200 pmol kg ⁇ - 1 > min ⁇ - 1 > into rats has been reported to result in the formation of extensive haemorrhagic erosions in the gastric mucosa.
  • Psoriasis is an inflammatory and proliferative disease characterised by skin lesions.
  • PAF is pro-inflammatory and has been isolated from lesioned scale of psoriatic patients indicating PAF has a role is the disease of psoriasis.
  • increasing evidence supports a potential patho-physiological role for PAF in cardiovascular disease.
  • a PLA2 inhibitor can be distinguished from the activity of a CoA-IT inhibitor based on their specific actions on their respective enzymes and by their different effects in cellular assays. For example only CoA-IT inhibitors have the ability to interfere with the mobilization of radiolabelled arachidonic acid to move from the alkyl-PC pool to the alkenyl PE pool. Selective inhibitors of 14 kDa PLA2 are without an effect in this assay
  • CoA-IT inhibitors will inhibit both LTC4 and PGE2 release from activated monocytes while selective PLA2 inhibitors inhibit LTC4 release but spare prostanoid formation or production (assay F).
  • lipid mediators of inflammation include, but are not limited to, adult respiratory distress syndrome, asthma, arthritis, reperfusion injury, endotoxic shock, inflammatory bowel disease, allergic rhinitis and various inflammatory skin disorders. Each of these disorders is mediated in some part by lipid mediators of inflammation. Compounds which inhibit CoA-IT, by virtue of their ability to block the generation of lipid mediators of inflammation, are of value in the treatment of any of these conditions. Similarly compounds which inhibit PLA2, by virtue of their ability to block the generation of lipid mediators of inflammation stemming from activation and/or release of this enzyme are of value in the treatment of these conditions.
  • an inhibitor of CoATT would offer an advantage over the classical NSAIDs which affect only prostanoid production (and not PAF biosynthesis) thereby inhibiting both the acute and cell-mediated "chronic" inflammatory processes.
  • an advantage of the PLA2 inhibitor would be their affect on human monocyte leukotrienes and PAF formation, while immunosuppressive prostanoids, such as PGE2, are spared.
  • selective inhibition of COX-2 is useful for the treatment of diseases mediated thereby, such as arthritis, and IBD, for relief of pain, and inflammation, without the gastric and renal side effects associated with COX-1 inhibition.
  • Treatment of disease states caused by these lipid inflammatory mediators i.e., arachidonate, eicosanoids and PAF include certain cardiovascular disorders such as but not limited to, myocardial infarction, stroke, circulatory shock, or hypotension, ischemia, reperfusion injury; inflammatory diseases such as, but not limited to, arthritis, inflammatory bowel disease, Crohn's disease, or ulcerative colitis; respiratory diseases such as but not limited to, asthma, or adult respiratory distress syndrome; analphylaxis, shock, such as but not limited to endotoxic shock; topical disesases, such as but not limited to actinic keratosis, psoriasis, or contact dermatitis; or pyresis.
  • cardiovascular disorders such as but not limited to, myocardial infarction, stroke, circulatory shock, or hypotension, ischemia, reperfusion injury
  • inflammatory diseases such as, but not limited to, arthritis, inflammatory bowel disease, Crohn's disease, or ulcerative colitis
  • 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.
  • Such pharmaceutically acceptable carriers or diluents and methods of making are well known to those of skill in the art, and reference can be found in such texts as Remington's
  • the compounds of formula (I) may also be administered in conventional dosages in combination with known second therapeutically active compounds, such as steroids or NSAID's for instance. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier 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.
  • Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • Exemplary of 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 steric 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 esteror 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.
  • Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of a compound of the structure (I) or a pharmaceutically acceptable salt thereof calculated as the free base.
  • the pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen. For an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered from 1 to 4 times per day.
  • BIOLOGICAL METHODS To determine activity of the compounds of Formula (I) various cellular assays can be used to determine in vitro activity. Additionally, various classical in vivo acute inflammatory models which have some aspect of their etilogy to elevated eicosanoid levels can be employed, such as the paw edema model, mouse zymosan peritonitis, reverse Arthus pleurisy or various skin inflammation assays which are described in Lewis et al., Experimental Models of Inflammation, in the Handbook of Inflammation. Vol. 5, Bonta Ed., Elsevier Science Publishers, NY (1985) whose disclosure is herein incorporated by reference.
  • TPA induced ear edema model as well as the carrageenan paw edema model in the rat are described herein as well.
  • These classical models of inflammation will reflect the drug's ability to alter an inflammatory response but cannot address the specificity of drug action.
  • These models have been traditionally designed as non steriod antiinflammatory drug sensitive pharmacological screens and it is important to utilize models which can differentiate PLA2 and CoA-IT inhibitors from NSATDS.
  • evaluation of inhibitors can occur in intact cells such as described in the assay, assay (c and d) below.
  • CoA-IT activity can exclusively be measured, and differentiated from PLA2 inhibition, in intact cells by following the movement of a pulse of [ 3 H] arachidonate as it moves into the 1-alkyl and 1 - alkenyl phospholipids in inflammatory cells (assay e).
  • assays c, d, & f can both be used for PL A2 and CoA-IT inhibition determination.
  • Inflammatory responses are induced in the mouse ear by the topical application of a pro-inflammatory agent, such as 12-0- tetradecanoylphorbol 13-acetate (assay g). This produces an edematous response, as measured by increases in ear thickness, as well as increased inflammatory cellular infiltrate, as measured by increases in myeloperoxidase activity (as described in the methods).
  • a pro-inflammatory agent such as 12-0- tetradecanoylphorbol 13-acetate (assay g).
  • assay g 12-0- tetradecanoylphorbol 13-acetate
  • inflammation induced by the direct adminstration of arachidonic acid can be used. In this case compounds altering arachidonic acid mobilization or liberation should be with our effect
  • Phospholipase A2 activity of rh Type II- 14 kDa PLA2 or PLA2 semi-purified from human synovial joint fluid was measured by the acylhydrolysis of high specific activity (NEN)[ 3 H]-AA-£. coli (0.5 mCi 5nmol PL Pi) as previously described in Marshall et al., J. Rheumatology, 18:1, pp59-65 (1991).
  • Assays were incubated for a time predetermined to be on the linear portion of a time versus hydrolysis plot. Experiments were conducted with final % hydrolysis values ranging from 2% (400-1000 dpm) to 10% (2000-5000 dpm) acylhydrolysis after blank correction. Reactions were terminated by the addition of 1.0 L tetrahydrofuran (THF). The whole sample was placed over aminopropyl solid phase silica columns and eluted with THF:acetic acid (49:1) exclusively separating free fatty acids with greater than 95% recovery. Radiolabel in this eluate was quantitated by liquid scintillation counting.
  • THF tetrahydrofuran
  • Results were expressed as % of fatty acid hydrolyzed ([sample dpms - non-specific (blank) dpms/total dpms] x 100) or specific activity which was calculated from hydrolysis values found in the linear portion of time versus % hydrolysis plots (pmol free fatty acid hydrolyzed/mg/min). Non-specific activity was always less than 1% of the total counts added.
  • the following is a method to measure CoA-IT activity and the effects of compounds on CoA-IT activity.
  • the assay is based upon mixing cellular material containing CoA-IT activity with a stable lyso phospholipid such as l-alkyl-2-acyl-GPC and measuring the production of phospholipid product such as l-alkyl-2-acyl-GPC occurring in the absence of added CoA or CoA-fatty acids.
  • Any inflammatory cell that contains high levels of CoA-IT activity can be used, such as neutrophils, macrophages or cell lines such as U937 cells.
  • U937 cells were obtained from American Type Culture Collection and grown in RPMI-1640 media (Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) at 37°C, 5%C02- Cells were grown without differentiation (basal state) by any agent, such as dimethyl sulfoxide.
  • inflammatory cells include, but are not limited to neutrophils, macrophages, monocytes, lymphocytes, eosinophils, basophils, and mast cells.
  • Microsomes were prepared using standard techniques. In this case, cells were washed with a buffer of 250 mM sucrose, 10 mM Tris, 1 mM EGTA, 1 mM MgCl2, pH 7.4 and ruptured by N2 cavitation (750 psi, 10 minutes). The ruptured cells were centrifuged 1000 X g, 5 minutes. The resulting supernatant was centrifuged at 20,000 X g,-20 minutes. Microsomes were prepared from this supernatant by centrifugation at 100,000 x g, 60 minutes.
  • the resulting pellet was washed once with assay buffer (150 mM NaCl, 10 mM Na2KPO4, 1 mM EGTA, pH 7.4), recentrifuged and the pellet resuspended in assay buffer (4-20 mg protein/ml) and was stored at -80°C until assayed.
  • assay buffer 150 mM NaCl, 10 mM Na2KPO4, 1 mM EGTA, pH 7.4
  • CoA-IT activity was measured in 1.5 ml centrifuge tubes in a total volume of 100 ul. Microsomes were diluted in assay buffer to the desired protein concentration (6-20 ug/tube). The reaction was initiated by addition of [3H ] l-alkyl-2-lyso-sn-glycero-3- phosphocholine (GPC) ( ⁇ 0.1 uCi/tube) and 1 ⁇ M final cold l-alkyl-2-lyso-GPC in assay buffer with 0.25 mg/ml fatty acid-poor bovine serumalbumin (BSA) (Calbiochem, La Jolla, CA).
  • GPC l-alkyl-2-lyso-sn-glycero-3- phosphocholine
  • BSA bovine serumalbumin
  • Microsomes were pretreated with desired agents for the desired time (10 minutes) before the addition of [3H]l-alkyl-2-lyso-GPC.
  • the reaction was run for the desired time (10 minutes) at 37 °C.
  • the reaction was stopped and the lipids extracted by addition of 100 ul of chloroform :methanol (1:2, v/v) followed by 100 ul of chloroform and 100 ul of 1 M KCI.
  • the samples were vortexed and centrifuged at high speed in a microfuge for 2-3 minutes.
  • Protein concentration were assessed using the protein assay reagents from Bio-Rad (Richmond, California).
  • 5-LO 5-lipoxygenase
  • CO cyclooxygenase
  • the anti-oxidant BHT also has no effect at concentrations up to 100 ⁇ M.
  • Compounds which complex with phospholipids and inhibit PLA2 activity, such as quinacrine and aristolochic acid have no effect on CoA-IT activity at concentrations up to 500 ⁇ M.
  • Doxepine a compound reported to inhibit PAF release did not inhibit CoA-IT at concentrations up to 100 ⁇ M.
  • Sodium diclofenac reported to decrease leukotriene production by altering arachidonic acid metabolism, had no effect on CoA-IT activity at concentrations up to 500 ⁇ M.
  • Representative compounds of Formula (I) which inhibit CoA-IT activity in the microsomal CoA-IT assay (assay (a) above) [generally at 50 ⁇ M or less] are the compounds: 2-[2-[3,5-Bis(trifluoromethyl)sulf onamido-4-trifluoromethylphenoxy]-5-( 1 , 1 - dimethylpropyl)benzenesulfonic acid; 2-[2-[3,5-Bis(trifluoromethyl)phenylsulfonamido]-4-trifluoromethylphenoxy]- benzenesulfonic acid; 2-[3,5-Bis(trifluoromethyl)phenylsulfonamido]-l-(2-methylthiophenoxy)-4- trifluoromethylbenzene; 2-[3,5-B (trifluoromethyl)phenylsulfonamido]-l-[2-methylsulfonylphenoxy]-4- trifluoromethylbenzene; and
  • Human neutrophils are obtained in the laboratory using three different methods. One method uses leukophoresis packs from normal humans and neutrophils are isolated using the histopaque-1077 technique. The blood is centrifuged at 300 x g for 10 minutes. The cell pellets are resuspended in PBS composed of 137 mM NaCI, 8.8 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCI (Dulbecco's Gibco Laboratories, Long Island, New
  • the pellets are collected after centrifugation (300 x g for 30 minutes) and washed once in PBS.
  • the cell pellets are exposed briefly to deionized water to lyse any erythrocytes.
  • the remaining cells are collected by centrifugation, suspended in PBS, counted and identified after cytospinning and staining.
  • the final leukocyte preparation will be of greater than 95% purity and viability.
  • the second method isolates human neutrophils from fresh heparinized normal blood using the Histopaque-1077 technique.
  • the blood is layered over Histopaque-1077 (Sigma, St. Louis Missouri) and centrifuged at 400 x g for 30 minutes.
  • the cell pellets are resuspended in 35 ml of PBS and 12 ml of 6% Dextran, followed by Dextran sedimentation at room temperature for 45 minutes.
  • the upper layer is collected and further centrifugated for 10 minutes at 1000 rpm.
  • the cell pellets are exposed briefly to deionized water to lyse erythrocytes.
  • the remaining cells are collected by centrifugation, suspended in PBS, counted and identified after cytospinning and staining.
  • the final leukocyte preparation will be of greater than 95% purity and viability.
  • the third method isolates human neutrophils from freshly drawn heparinized normal blood using the Percoll technique.
  • the blood is first treated with 6% Dextran at room temperature for a 1 hour sedmination.
  • the upper layers of plasma are collected and centrifuged at 400 x g for 10 minutes.
  • the cell pellets are resuspended in Percoll 1.070 g/ml supplemented with 5% fetal bovine serumand layered on discontinuous gradients (1.080, 1.085, 1.090,1.095 g/ml) followed by centrifugation at 400 x g for 45 minutes.
  • the neutrophils are collected from interfaces of 1 ;080 and 1.085 and the 1.085 and 1.090 Percoll densities, followed by a centrifugation at 400 x g for 45 minutes.
  • the neutrophils are suspended in PBS, counted and identified after cytospinning and staining.
  • the final leukocyte preparation will be of greater than 95% purity and viability.
  • Neutrophils are suspended in PBS with 1 mM Ca 2+ and 1.1 mM Mg2+ at concentrations of 5 to 20 x 106 cells per ml. Cells are added to test tubes and treated with the desired compounds for 5 to 10 minutes, then challenged with calcium ionophere
  • the chloroform extract for each sample was evaporated to dryness and the material resuspended in hexane.
  • the hexane was passed through a Silica solid phase column (500 mg), washed 2x with hexane and a fatty acid enriched fraction eluted with hexane:ethyl ether (1:1, v/v).
  • Solvents were removed from the samples under a stream of nitrogen then the samples were converted to pentafluoro benzyl esters using pentafluorobenzyl bromide and d ⁇ sopropylethylamine in acetronitrile. Solvents were removed and samples were suspended in hexane.
  • GC/MS analysis is performed on a suitable instrument, such as a Finnigan MAT TSQ 700 GC/MS/MS/DS (San Jose, California) operated as a single stage quadruple system or a Hewlett-Packard 5890 with a 5989A M5 system.
  • a suitable instrument such as a Finnigan MAT TSQ 700 GC/MS/MS/DS (San Jose, California) operated as a single stage quadruple system or a Hewlett-Packard 5890 with a 5989A M5 system.
  • the peaks corresponding to arachidonic acid and [ ⁇ HglArachidonic acid were identified and the areas of those peaks compared and the released arachidonic acid calculated as ng of arachidonic acid for each sample.
  • Protein concentrations are assessed using the protein assay reagents from Bio-Rad (Richmond, CA).
  • Blood is obtained from normal humans and neutrophils were isolated as described for the arachidonic acid release assay, above.
  • the final leukocyte preparation should be of greater than 95% purity and viability.
  • Neutrophils were suspended in PBS at concentrations of 5 to 20 x 10 ⁇ cells per ml. Cells were added to test tubes and treated with the desired compounds for 5 to 10 minutes, then challenged with calcium ionophore A23187, 2 ⁇ M and 20-30 ⁇ Ci of [3H]acetic acid (NEN-Dupont, Boston, Massachusetts), or the vehicle of PBS with 0.25- 1 mg/ml. After 5 to 20 minutes, the reactions were terminated by addition of an equal volume of chlorofomrmethanol (1:2, v/v) to the samples and the lipids were extracted by addition of equal volumes of chloroform and distilled water. The samples were vortexed and centrifuged at high speed and the chloroform layer removed to a clean tube.
  • the chloroform from each tube was evaporated to dryness and the material suspended in a small volume of chloroform or chloroform:methanol (25-100 ⁇ l) and the total material spotted on a Silica TLC plate.
  • the plates were developed in chlorof ⁇ - ⁇ n methanol/ acetic acid/water (50:25:8:4, v/v) visualized by radioscanning
  • a representative compounds of Formula (I) herein which demonstrated positive activity, i.e., inhibition of PAF production, in this assay is 2-[2-[3,5-Bis(trifluoromethyl)- sulfonamido-4-trifluoromethylphenoxy]-5-( 1 , 1 -dimethylpropyl)benzenesulfonic acid;
  • HBSS Hanks Balanced Salt Solution
  • [5,6,8,9,1 l,12,14,15- 3 H]-Arachidonic acid (100 Ci/mmol; New England Nuclear) complexed to 200 ⁇ l HBSS containing 0.25 mg/ml HSA was added to the cell suspension (1 ⁇ Ci/ml). The cells were incubated with gentle shaking at 37°C for 5 min. The reaction was terminated by the addition of 40 ml ice-cold HBSS containing HSA (0.25 mg/ml). The cells were then removed from the supernatant fluid by centrifugation (225 g, 8 min). Unincorporated [ 3 H] -arachidonic acid was completely removed by two more washes of HBSS containing 0.25 mg/ml HSA.
  • the neutrophils were resuspended in fresh buffer, exposed to various concentrations of a CoA-IT inhibitor or its vehicle and incubated without stimulation for 2 hrs. At that time, the tubes containing the cells and buffer were extracted (Bligh & Dyer [Can. J. Biochem. Physiol. (1959) 37, 911-917]) and the phospholipid classes separated and collected by normal phase HPLC, using a Ultrasphere
  • the phospholipid subclasses were separated by TLC in benzene/hexane/ethyl ether (50:45:4, v/v), located by image analysis (Bioscan) and the amount of radioactivity in each class was determined by zonal scraping and liquid scintillation counting.
  • mouse bone marrow-derived mast cells are removed from culture and provided with exogenous [3H]arachidonic acid for 30 minutes.
  • the labeled arachidonic acid which had not been incorporated into the cells is then removed by washing the cells 2 times with an albumm-containing buffer. At that point, the cells are treated with various concentrations of CoA-IT inhibitors and then placed back in culture for 24-48 hours.
  • the phospholipids are extracted by the method of Bligh and Dyer [Can. J. Biochem. Physiol.
  • Leukocyte-rich leukopaks obtained from Biological Specialties (Lansdale, PA) were collected from male volunteers who were not taking anti- inflammatory drugs. Leukopaks were centrifuged (90 x g for 15 min) twice to remove the platelet-rich plasma. The cell pellet was washed by centrifugation and resuspended in HBSS without Ca 2+ or Mg 2+ . Histopaque 1077 was layered under the cell suspension and centrifuged at 400 x g for 30 min to obtain the buffy coat The interfacial buffy coat, containing monocytes and lymphocytes, was removed and saved. The buffy coat was washed twice with HBSS without Ca 2+ or Mg 2+ by centrifugation. The cell pellet (4-6 x
  • Monocytes (5 x 106/ml) were incubated as a suspension in serum-free RPMI- 1640 medium containing the vehicle
  • DMSO DMSO ( ⁇ 1%) or drug for 30 min at 27°C after which vehicle or stimuli was added for the indicated time.
  • the stimulating agent is solubilized in DMSO and appropriate vehicle controls were included in all experiments.
  • the amount of stimuli was chosen from the linear portion of a concentration versus product curve usually representing 60-80% maximal stimulation over the indicated incubation time at 37°C (A23187, 1 ⁇ M,(15 min).
  • the reaction was terminated by reduction of pH through addition of citric acid and centrifugation (10 min, 400 x g, 4°C). Cell viability was monitored before and after experiments using trypan blue exclusion. The cell-free media was decanted and stored at -70° C until analyzed.
  • Prostaglandin E2 and LTC4 were directly measured in cell-free media using enzyme immunoassay (EIA) kits purchased from Caymen Chemical Co. (Ann Arbor, MI). Sample or standard dilutions were made with appropriate media and analyzed in triplicate. Results were obtained by extrapolation from a standard curve prepared in the media and expressed as pg or ng/ml of sample.
  • EIA enzyme immunoassay
  • Representative compounds of Formula (I) herein which demonstrated positive activity in this assay is the compound 2-[2-[3,5-Bis(trifluoromethyl)sulfonamido-4- trifluoromethylphenoxy]-5-( 1 , l-dimethylpropyl)benzenesulfonic acid, for which the IC50 of PGE2 was greater than 10 and for LTC4 1.2.
  • Assays (g and h) : Assay (Method) for TPA (assay g ⁇ or Arachidonic acid (assay h)-induced Inflammation
  • mice Male Balb/c inbred mice were obtained from Charle River Breeding Laboratories (Kingston, NY). Within a single experiment mice (22-25g) were age-matched. These in vivo experiments typically involved use of 5-6 animals/group.
  • MPO1 Myeloperoxidase
  • MPO activity was quantified kinetically (change in absorbance measured over 3 min, sampled at 15-sec intervals) using a Beckman DU-7 spectrophotometer and a Kinetics Analysis package (Beckman Instruments, Inc.).
  • One unit of MPO activity is defined as that degrading one micromole of peroxide per minute at 25°C.
  • the ED50 are values which cause a 50% inhibition of the inflammatory response and are calculated by regression analysis of the dose response data.
  • Arachidonic acid induced ear inflammation assay Arachidonic acid is dissolved in acetone (lmg/ear) to the left ear of BALB/c male mice. The thickness of both ears was measured with a constant pressure thickness guage 1 hour after treatment and the data expressed as the change in thickness between treated and untreated ears. Test compounds or vehicle are given at the time of AA applciation. The inflammatory cell infiltration is measured by MPO activity as described above in the TPA ear edema assay. After the edema measurements are made, the inflamed ears are removed and assayed for MPO activity.
  • the TPA % change in edema was -50 (p ⁇ 0.001), -46 (p ⁇ 0.01) and -18 (ns) respectively; for AA the change was -10 (ns), -1 l(ns) and -50 (p ⁇ 0.001).
  • the change in MPO for TPA model was -54 (p ⁇ 0.001), -65 (p ⁇ 0.001) and -36 (p ⁇ 0.05) respectively; for AA it was 0 (ns), -33 (ns) and -90 (p ⁇ 0.001).
  • One hypothesis is that the AA administration to the ear overrides the need for PLA2 mediated liberation of substrate for subsequent pro- inflammatory lipid mediator generation or AA moblization by CoA-IT.
  • an inhibitor of an AA-metabolizing enzyme should be effective while and inhibitor of PLA2 would be ineffective.
  • scalaradial and dexamethasone have little or no effect in the AA ear model at concentrations which were effective in the TPA ear model. This can be contrasted to the activity of the selective 5-LO inhibitor WY 50,295 which strongly inhibits inflammation in response to AA.
  • the AA ear model therefore responds well to compounds that exhibit 5-LO inhibitory action and appears to be uneffected by putative PLA2 inhibitors. This model therefore provides a unique tool with which the contribution of the 5-LO inhibition to the in vivo anti-inflammatory activity of various compounds can be separated from LMW-PLA2 inhibition.
  • the positive activity of compounds of Formula (I) in this animal model demonstrate a clear utility in the treatment of topically administered diseases associated with inflammation as noted herein such as, but not limited to, inflammatory bowel disease, contact dermatoses, actinic keratosis, psoriasis, or conjunctivitis.
  • [ 3 H] a molecule that contains tritium atoms, a radioactive isotope
  • A23187 a compound that allows free entry of calcium into a cell
  • AA arachidonic acid
  • arachidonate arachidonic acid contained within a phospholipid
  • free arachidonic acid arachidonic acid that is not contained within a phospholipid
  • [ 2 Hg]arachidonic acid the form of arachidonic acid labeled with 8 deuterium atoms, a stable isotope
  • 1-alkyl, 1-Oj-alkyl 1-alkenyl, l- ⁇ alk-l'- enyl
  • BSA bovine serum albumin
  • Co A coenzyme A
  • CoA-IT CoA-independent transacylase
  • DTT dithiothreitol
  • EGTA [ethylenebis(oxyethylenenitrilo)]tetra acetic acid, a
  • TLC thin layer chromatography
  • U937 American Type Tissue Culture designated cell line similar to a monocyte.

Abstract

Cette invention se rapporte à de nouveaux composés et compositions pharmaceutiques représentés par la formule (I). Cette invention se rapporte également à un procédé pour traiter ou réduire l'inflammation chez un mammifère nécessitant un tel traitement, ce procédé consistant à administrer audit mammifère une quantité efficace d'un composé ou d'une composition de la formule (I).
PCT/US1995/007041 1994-06-02 1995-06-02 Composes anti-inflammatoires WO1995033713A1 (fr)

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