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WO1995033715A1 - Composes anti-inflammatoires - Google Patents

Composes anti-inflammatoires Download PDF

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Publication number
WO1995033715A1
WO1995033715A1 PCT/US1995/007019 US9507019W WO9533715A1 WO 1995033715 A1 WO1995033715 A1 WO 1995033715A1 US 9507019 W US9507019 W US 9507019W WO 9533715 A1 WO9533715 A1 WO 9533715A1
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Prior art keywords
hydrogen
alkyl
optionauy
halogen
compound according
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PCT/US1995/007019
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English (en)
Inventor
Drake S. Eggleston
Jerry Leroy Adams
Sherin Salaheldin Abdel-Meguid
Ralph Floyd Hall
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Smithkline Beecham Corporation
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Publication of WO1995033715A1 publication Critical patent/WO1995033715A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/62—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/78—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
    • C07C217/80—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings
    • C07C217/82—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings of the same non-condensed six-membered aromatic ring
    • C07C217/90—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings of the same non-condensed six-membered aromatic ring the oxygen atom of at least one of the etherified hydroxy groups being further bound to a carbon atom of a six-membered aromatic ring, e.g. amino-diphenylethers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/78—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
    • C07C217/80—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings
    • C07C217/82—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings of the same non-condensed six-membered aromatic ring
    • C07C217/92—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton having amino groups and etherified hydroxy groups bound to carbon atoms of non-condensed six-membered aromatic rings of the same non-condensed six-membered aromatic ring the nitrogen atom of at least one of the amino groups being further bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/40—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/42—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino groups bound to carbon atoms of at least one six-membered aromatic ring and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton with carboxyl groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by saturated carbon chains
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/52—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton
    • C07C229/54—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C229/64—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the same carbon skeleton with amino and carboxyl groups bound to carbon atoms of the same non-condensed six-membered aromatic ring the carbon skeleton being further substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/41—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton
    • C07C309/42—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton having the sulfo groups bound to carbon atoms of non-condensed six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • 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.
  • PAF is a potent proinflammatory mediator produced by a variety of cells. In vitro, 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.
  • PAF neuropeptide kinase kinase kinase kinase
  • PAF kinase kinase kinase
  • 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 II 14 kDa PLA2 has been well characterized and is known to exist in both an extracellular form in inflammatory fluids (Kramer et al., J. Biol.
  • 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.
  • 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 monocytic cell line, U937 (Clark et al., Proc. Natl. Acad. Sci., 87:7708-7712, 1990).
  • the cell-associated Type 11-14 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
  • 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 TSA 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 Ca 2 +-independent PLA 2 (Bomalaski and Clark, Arthritis and Rheumat. 36:190-198 (1993)) in that Ca 2 + is not required for catalysis and DTNB inhibition is observed.
  • 85 kDa-PLA 2 is not inhibited by the suicide inactivator bromoenol lactone, suggesting that the enzyme is distinct from the myocardial enzyme as well.
  • arachidonate-containing phospholipids are the key precursors for a broad range of lipid mediators it would not be surprising that inflammatory cells would treat these phospholipids differently than other fatty acid-containing phospholipids.
  • enzymes which control the amount of arachidonate in different phospholipid pools and these enzymes are tightly regulated to maintain arachidonate homeostasis.
  • the movement of arachidonate into and from all phospholipids was originally thought to be exclusively by Coenzyme A-dependent acyl transferase activities. Holub et al.. Adv. Lipid
  • CoA-IT Coenzyme A-independent transcylase
  • CoA-IT has a specificity for certain phosphoUpids 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 phosphoUpid 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 lysophosphoUpids with no selectivity for arachidonate.
  • CoA-IT is involved in arachidonic acid and phosphoUpid metaboUsm
  • inhibition of such an enzyme would be useful for the treatment of inflammatory, aUergic and hypersecretory conditions or disease states caused thereby. Therefore, a method by which CoA-IT is inhibited will consequently and preferentiaUy decrease the arachidonate content of 1-alkyl- and 1-alkenyl-linked phosphoUpids 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 pharmaceutical compositions of Formula (I) and Formula (II) comprising a compound of Formula (I) and/or (II), 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) or (II).
  • This invention also relates to a method of treating disease or disorders mediated by Upid inflammatory mediators, free arachidonic acid, its metaboUtes and/or PAF by adminis- tering to a patient in need thereof, an effective amount of a compound of Formula (I) or (II).
  • This invention also relates to a method of treating disease or disorders mediated by phospholipase A2 and Coenzyme A independent transacylase by administering to a patient in need thereof, an effective amount of a compound or composition of Formula (I) or (II).
  • the present invention also provides for a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceuticaUy acceptable carrier or diluent and a compound, or pharmaceuticaUy acceptable salt thereof, of Formula (I) or (II).
  • Ri is (CH2) n OH or (CH2) n CO2R8;
  • R8 is hydrogen or Ci-4 alkyl;
  • n is 0 or an integer having a value of 1;
  • X is oxygen or sulfur;
  • R2 is hydrogen, halogen, optionaUy substituted Ci-8 alkyl, or Ci-8 alkoxy;
  • m is an integer having a value of 1 or 2;
  • R3 is hydrogen, or optionally substituted Ci-8 alkyl, optionally substituted aryl, or
  • R4 is hydrogen, optionaUy substituted Ci- 8 alkyl, or optionally substituted aryl
  • R5 is hydrogen, halogen, CF 3 , CH 3 , (CH2)tC(O)2R7. or (CH2).OH
  • t is 0 or an integer having a value of 1 or 2
  • R($ is hydrogen or halogen
  • R7 is hydrogen or Ci-4 alkyl
  • R9 and Rio are independently hydrogen, optionaUy substituted C1-.4 alkyl, or optionaUy substituted aryl;
  • Rl 1 is an optionally substituted alkyl, optionaUy substituted C5-.7 cycloalkyl, or an optionaUy substituted aryl; or a pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention are the compounds represented by the structure having the formula:
  • Rl is SO3H or S(O) n Ci-4 alkyl; n is 0 or an integer having a value of 1 or 2;
  • X is oxygen or sulfur
  • R2 is hydrogen, halogen, optionaUy substituted Ci-8 alkyl, or Ci-8 alkoxy; m is an integer having a value of 1 to 2?
  • R3 is hydrogen, or optionally substituted Ci-8 alkyl, optionaUy substituted aryl, or (CR 9 RlO)p Rli; p is an integer having a value of 1 to 2;
  • R4 is hydrogen, optionaUy substituted Ci-8 alkyl, or optionaUy substituted aryl
  • R5 is hydrogen, halogen, CF 3 , CH3, (CH2)tC(O)2R7, or (CH2)tOH; t is 0 or an integer having a value of 1 or 2; R6 is hydrogen or halogen;
  • R7 is hydrogen or Ci-4 alkyl
  • R9 and Rio are independently hydrogen, optionaUy substituted Ci-4 alkyl, or optionaUy substituted aryl;
  • Rll is an optionally substituted C5-7 cycloalkyl, an optionally substituted aryl, or optionaUy substituted 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) or (II).
  • the compounds of Formula (I) and (II) may selectively inhibit the PLA2 enzyme, the CoA-IT enzyme or both. Inhibition of either or both enzymes wiU 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, coUagen 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) and (II) are preferential and selective inhibitors of the low molecular weight PLA2 enzyme.
  • Rl is suitably (CH2)nOH or (CH2)nCO2R8-
  • Rl is (CH2)nCO2R8 and n is preferably 0.
  • R8 is preferably hydrogen or methyl, more preferably hydrogen, or a pharmaceutically acceptable salt thereof.
  • R 2 is independently a substituent on the benzene ring from 1 to 2 times, and such substituent is selected from hydrogen, halogen, an optionally substituted Ci-8 alkyl, or Ci-8 alkoxy group.
  • R2 is halogen it is a chlorine or bromine.
  • R2 is an optionaUy substituted Ci-8 alkyl
  • the alkyl is substituted one to three times with halogen, such as fluorine, preferably the alkyl is a methyl substituted by fluorine to yield a trifluoromethyl group.
  • the optionaUy substituted Ci-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.
  • m is an integer having a value of 1 or 2.
  • R3 is hydrogen, optionally substituted Ci-8 alkyl, optionally substituted aryl, or (CR9R ⁇ o)p Rl 1. and p is an integer having a value of 1 to 2.
  • the ring is preferably phenyl.
  • R3 is hydrogen or (CR9RlO)p Rl l.
  • R9 and Rio are independently hydrogen, optionally substituted Ci-4 alkyl, or optionally substituted aryl.
  • Rl l is preferably an optionally substituted C5-7 cycloalkyl, an optionaUy substituted aryl, or an optionally substituted alkyl, more preferably an optionaUy substituted phenyl or cyclohexyl group.
  • p is 1, and at least one of R9 and R10 are preferably hydrogen.
  • one of R9 and Rio are hydrogen and the other an optionaUy substituted aryl, preferably a phenyl.
  • the aryl, aryl alkyl, or alkyl moieties of R3 are substituted independently, one to three times, by halogen, trifluoromethyl, optionaUy substituted aryl, optionally substituted aryloxy, methoxy, CH2OH, optionally substituted methyl, or C(O)2H.
  • the substituent groups are aryl, halogen, or trifluoromethyl.
  • the methyl group is preferably substituted by fluorine to yield a trifluoromethyl group.
  • the substituent halogen groups are preferably Cl, Br and fluorine.
  • the substituents are in the 3,5- position or the 4- position of the phenyl ring. More preferably when the substituent group is an aryl group or an arylalkyl group, it is phenyl or benzyl and the aryl ring is substituted in the 3,5 or 4- positions by halogen, methoxy, CH2OH, hydroxy, optionaUy substituted methyl, or C(O)2H accordingly, such as but not limited to 3,5-bis-trifluoromethyl, 4-trifluoromethyl,
  • R4 is hydrogen, optionaUy substituted Ci-8 alkyl, or optionally substituted aryl.
  • R4 is hydrogen, methyl or phenyl, more preferably hydrogen.
  • R5 is hydrogen, halogen, CF 3 , CH 3 , (CH2)tC(O)2R7, or (CH2)tOH, and t is 0 or an integer having a value or 1 or 2;
  • R7 is suitably hydrogen or a Ci-4 alkyl group.
  • R5 is CH2C(O)2R7
  • R7 is suitably hydrogen or a Ci-4 alkyl, preferably hydrogen or t-butyl.
  • Preferred R5 groups are hydrogen, CF3, or halogen, such as Cl, or Br. More preferably R5 is hydrogen, chlorine, or CF3.
  • R 6 is hydrogen or halogen, preferably hydrogen or chlorine.
  • Specifically exemplified compounds of Formula (I) are: 2-[2-[(Diphenylmethyl)amino]-4-trifluoromethylphenoxy]benzoic acid; 2-[2-[(Phenylmethyl)amino-4-(trifluoromethyl)phenoxy]benzoic acid; 2-[2-[[(4-Hydroxyphenyl)methyl]amino]-4-(trifluoromethyl)phenoxy]benzoic acid; 2-[2-[Cyclohexyl_memyl)_unino]-4-(trifluoromethyl)phenoxy]benzoic acid
  • compounds of Formula (I) also have the following proviso's: a) when X is sulfur, and Rl is (CH2)nOH , R3 and R4 are both hydrogen, and R5 is hydrogen, halogen, CF3 or CH3; than n is 0; and b) when X is sulfur, Ri is (CH2)nCO2R8, R8 is Ci-4 alkyl, R3 and R4 are both hydrogen, and R5 is hydrogen, halogen, CF3 or alkyl, then n is 1; c) when X is oxygen, Ri is (CH2)nOH, n is 0, R4 is hydrogen, and R5 is hydrogen, halogen, CF3 or CH3, then R3 is other than an unsubstituted phenyl.
  • 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
  • R2, R5 and R6 are preferably the same as for compounds of Formula (I).
  • R3 and R4 suitably are the same as for compounds of Formula (I) above are also preferably both hydrogen.
  • Ri is SO3H
  • R2 is an optionaUy substituted C ⁇ _8 alkyl, preferably methyl or is 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
  • R3 and R4 are preferably hydrogen
  • R6 is hydrogen
  • R5 is CF3.
  • Suitable pharmaceuticaUy acceptable salts are well known to those slrilled 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, saUcyUc acid, phenylacetic acid and mandeUc acid.
  • 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, be
  • pharmaceutically acceptable salts of compounds of formula (I) may also be formed with a pharmaceuticaUy acceptable cation, for instance, if a substituent Ri comprises a carboxy group.
  • Suitable pharmaceutically acceptable cations are weU known to those skiUed in the art and include alkaUne, alkaline earth, ammonium and quaternary ammonium cations.
  • halo all halogens, that is chloro, fluoro, bromo and iodo;
  • C ⁇ _ 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, ⁇ -propyl, iro-propyl, n-butyl, sec-butyl, wo-butyl, . -butyl, and the like;
  • aryl (on its own or in any combination, such as “aryloxy”, or “arylalkyl”) means a phenyl and naphthyl ring;
  • 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 opticaUy active forms. All of these compounds are included within the scope of the present invention.
  • Compounds of Formula (I) and (II) may be prepared by the process indicated below which comprises reacting a suitably protected compound of Formula (2), which are generaUy commercially available, and Ri, R2, m are as defined in Formula (I) and/or Formula (II).
  • compounds of Formula (2) are not commerciaUy avaUable, such as where Ri is S(O)3H
  • compounds of formula (2) may be prepared by sulfonating an appropriately substituted phenol with a suitable sulfonating reagent, such as fuming sulfuric acid.
  • Reduction of compound (4) with a suitable reducing agent such as iron in a solvent such as acetic acid/ethanol/water, or titanium trichloride in acetic acid water, or H 2 in the presence of a catalyst such as paUadium on carbon in a solvent such as ethyl acetate or methanol, provides a compound of Formula (5), which may also after deprotection yield a compound of Formula (I) wherein R3 and R4 are hydrogen.
  • 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.
  • the alkylating agents are not commercially available, they can be prepared by one skilled in the art, for example from the corresponding carboxyUc acids or alcohols.
  • (CR9R ⁇ o)pRll may be prepared by reacting a compound of Formula (5) with an appropriate aldehyde or ketone under dehydrating conditions to form an inline which may be reduced with a suitable reducing agent such as sodium borohydride, sodium cyanoborohydride, or diborane. Deprotection (if required) of a compound of Formula (6) and/or conversion to suitable salt forms provides a final compound of Formula (1).
  • Methyl 2-[2-amino-4-(trifluoromethyl)phenoxy]benzoate 500 mg, 0.0016 mol
  • benzaldehyde 163 mL, 0.0016 mol
  • the solvent was evaporated and the residue flash chromatographed (siUca gel, ethyl acetate/hexane) to yield the title compound.
  • Methyl 2-[2-amino-4-(trifluoromethyl)phenoxy]benzoate (300 mg, 0.00096 mol) and cyclohexane carboxaldehyde (177 ⁇ l, 0.00096 mol) were dissolved in methanol (pH -4.0) and stirred under argon, at room temperature.
  • Sodium cyanoborohydride (30 mg) was added and mixing continued for several hours. The pH was taken to 2.0 and the methanol was evaporated. The residue was mixed in water and the pH adjusted to 10.0. This mixture was extracted with ethyl acetate (3x) and dried in vacuo.
  • Example 5 Preparation of 2-12-rr(4-Hvdroxyphenyl)memyllaminol-4-(trifluoromethviy phenoxylbenzoic acid a) 2-[2-[[(4-Hydroxyphenyl)methyl]amino]-4-(trifluoromethyl)phenoxy]benzoic acid Following the procedure of Example l(a-e) except substituting 4-hydroxy- benzaldehyde for benzaldehyde, gave the title compound; mp 136°C.
  • Methyl 2-(2-amino-4-trifluoromethylphenoxy)benzoate (0.2 g, 0.66 mmol) was dissolved in triethylamine (2 mL) and stirred under argon. Chlorodiphenylmethane (0.25 mL, 1.4 mmol) was added and the mixture was heated at reflux for 48 h. The solvent was evaporated and the residue was purified by flash chromatography (sUica gel, ethyl acetate/hexane)to give the title compound.
  • the crude product was flash chromatographed (sUica gel, methylene chloride/isopropanol/ammonium hydroxide) to yield the ammonium salt, an off-white soUd.
  • 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)/(II), or pharmaceuticaUy 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 ceUs 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) and/or Formula (II) is an effective means for simultaneously reducing PAF, free arachidonic acid and eicosanoids produced in inflammatory ceUs.
  • the therapeutic utility of blocking Upid mediator generation has been recognized for many years.
  • inhibitors of cyclooxygenase such as aspirin, indomethacin, acetaminophen and ibuprofen, have demonstrated broad therapeutic utiUties.
  • 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. to induce inflammatory cells to produce proteins which inhibit free arachidonic acid release or to down regulate PLA2 mRNA formation.
  • 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 utiUties. 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 foUows 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 wiU 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 proUferative disease characterized 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 abUity to interfere with the mobiUzation 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 (assay E ). Alternatively, CoA-IT inhibitors wiU inhibit both LTC4 and PGE2 release from activated monocytes whUe selective PLA2 inhibitors inhibit LTC4 release but spare prostanoid formation or production (assay F).
  • Upid mediators of inflammation include, but are not limited to, adult respiratory distress syndrome, asthma, arthritis, reperfusion injury, endotoxic shock, inflammatory bowel disease, aUergic rhinitis and various inflammatory skin disorders. Each of these disorders is mediated in some part by Upid mediators of inflammation. Compounds which inhibit CoA-IT, by virtue of their abUity to block the generation of Upid mediators of inflammation, are of value in the treatment of any of these conditions . Similarly compounds which inhibit PLA2, by virtue of their abUity to block the generation of Upid mediators of inflammation stemming from activation and/or release of this enzyme are of value in the treatment of these conditions.
  • an inhibitor of CoA-IT 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
  • Treatment of disease states caused by these Upid 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 Umited 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: anaphylaxis, shock, such as but not Umited to endotoxic shock; topical diseases, 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 Umited to, arthritis, inflammatory bowel disease, Crohn's disease, or ulcerative
  • a compound of formula (I)/(II) 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 pharmaceuticaUy acceptable carrier or diluent.
  • Compounds of formula (I) or (II), 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)/(_3) may be administered in conventional dosage forms prepared by combining a compound of formula (I) or (II) 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 Pharmaceutical Sciences, 18th Ed., Alfonso R. Genarao, Ed., 1990, Mack Publishing Co. and the Handbook of Pharmaceutical Excipients, APhA Publications, 1986.
  • the compounds of formula (I) or (II) may also be administered in conventional dosages in combination with known second therapeuticaUy 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 wiU be appreciated that the form and character of the pharmaceuticaUy acceptable carrier or dUuent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other weU-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.
  • soUd carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • liquid carriers are syrup, peanut oU, oUve oU, water and the like.
  • the carrier or dUuent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
  • soUd carrier a soUd carrier
  • 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 soUd carrier wiU vary widely but preferably wUl be from about 25 mg. to about lg.
  • wUl be from about 25 mg. 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)/(_3) may be administered topically, that is by non- systemic administration. This includes the application of a compound of formula (I)/(II) 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 Uquid or semi-Uquid 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 simUar 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 appUcation. 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 metalUc soap; a mucilage; an oU 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 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 steriUzed by autoclaving or maintaining at 98-100 °C. for half an hour.
  • the solution may be steriUzed 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 pharmaceuticaUy acceptable compounds of the invention will normaUy be administered to a subject in a daUy dosage regimen.
  • 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)/(II) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered from 1 to 4 times per day.
  • the choice of form for administration, as well as effective dosages, wiU vary depending, inter aUa, on the condition being treated. The choice of mode of administration and dosage is within the skiU of the art.
  • evaluation of inhibitors can occur in intact ceUs 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 phosphoUpids in inflammatory ceUs (assay e).
  • assays c, d, & f can both be used for PLA2 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).
  • a pro-inflammatory agent such as 12-0- tetradecanoylphorbol 13-acetate (assay g).
  • Assay g 12-0- tetradecanoylphorbol 13-acetate
  • 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).
  • inflammation induced by the direct administration of arachidonic acid can be used. In this case compounds altering arachidonic acid mobilization or liberation should be with our effect.
  • PhosphoUpase 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-E. coli (0.5 mCi 5nmol PL Pi) as previously described in Marshall et al., J. Rheumatology, 18:1, p ⁇ 59-65 (1991). High specific activity [ 3 H]AA-E.
  • coli had up to 95% of the label incorporated into phospholipid which was locaUzed almost exclusively in the sn-2 position, as demonstrated by purified 14kDa PLA2 or low molecular weight PLA2 acylhydrolysis and separation of products by thin layer chromatography (TLC) (data not shown).
  • TLC thin layer chromatography
  • Predominately used herein was rh Type ⁇ 14 kDa PLA2, or alternatively bovine pancreatic PLA2 was also be used.
  • the reaction mixture (50 or 100 ml total volume) contained 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl 2 and [ 3 H]-AA-E. coli (low specific activity; 5-8 nmol PL Pi per assay).
  • Assays were incubated for a time predetermined to be on the Unear 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 mL 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 Uquid scintiUation 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.
  • AU protein concentrations were determined by Bradford protein analysis kits (Biorad, Richmond, CA).
  • Assay (h. : CoA-IT Activity
  • the foUowing is a method to measure CoA-IT activity and the effects of compounds on CoA-IT activity.
  • the assay is based upon mixing ceUular material containing CoA-IT activity with a stable lyso phosphoUpid 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 ceU that contains high levels of CoA-IT activity can be used, such as neutrophils, macrophages or ceU Unes such as U937 cells.
  • U937 ceUs were obtained from American Type Culture CoUection 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 ceUs” include, but are not Umited to neutrophUs, macrophages, monocytes, lymphocytes, eosinophils, basophils, and mast cells.
  • Microsomes are prepared using standard techniques. In this case, ceUs are 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 are centrifuged 1000 X g, 5 minutes. The resulting supernatant is centrifuged at 20,000 X g, ⁇ 20 minutes. Microsomes are prepared from this supernatant by centrifugation at 100,000 x g, 60 minutes.
  • the resulting pellet is 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 is stored at -80°C until assayed.
  • assay buffer 150 mM NaCl, 10 mM Na2KPO4, 1 mM EGTA, pH 7.4
  • CoA-IT activity is measured in 1.5 ml centrifuge tubes in a total volume of 100 ul. Microsomes are dUuted in assay buffer to the desired protein concentration (6-20 ug/tube). The reaction is initiated by addition of [3H ] l-alkyl-2-lyso-sn-glycero-3-phosphochoUne (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 JoUa, CA).
  • GPC l-alkyl-2-lyso-sn-glycero-3-phosphochoUne
  • BSA bovine serumalbumin
  • [3H]1- alkyl-2-lyso-GPC approximately 50 Ci/mmol, is from NEN-Dupont (Boston, Massachusetts) and cold l-alkyl-2-lyso-GPC is from Biomol (Plymouth Meeting, Pennsylvania).
  • Microsomes are pretreated with desired agents for the desired time (10 minutes) before the addition of [3H]l-alkyl-2-lyso-GPC.
  • the reaction is run for the desired time (10 minutes) at 37°C.
  • the reaction is stopped and the Upids 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.
  • 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.
  • Human neutrophUs are obtained in the laboratory using three different methods. One method uses leukophoresis packs from normal humans and neutrophUs are isolated using the histopaque-1077 technique. The blood is centrifuged at 300 x g for 10 minutes.
  • the ceU 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 York) and layered over histopaque-1077 (Sigma, St. Louis, Missouri).
  • the peUets are collected after centrifugation (300 x g for 30 minutes) and washed once in PBS.
  • the ceU 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 wiU be of greater than 95% purity and viability.
  • the second method isolates human neutrophUs from fresh heparinized normal blood using the Histopaque-1077 technique.
  • the blood is layered over Histopaque-1077 (Sigma,
  • 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 ceU peUets are exposed briefly to deionized water to lyse erythrocytes. The remaining cells are coUected by centrifugation, suspended in PBS, counted and identified after cytospinning and staining.
  • the final leukocyte preparation wiU be of greater than 95% purity and viabUity.
  • the third method isolates human neutrophUs from freshly drawn heparinized normal blood using the PercoU technique.
  • the blood is first treated with 6% Dextran at room temperature for a 1 hour sedmination.
  • the upper layers of plasma are coUected and centrifuged at 400 x g for 10 minutes.
  • the cell pellets are resuspended in PercoU 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 neutrophUs are coUected 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 neutrophUs are suspended in PBS, counted and identified after cytospinning and staining.
  • the final leukocyte preparation wUl be of greater than 95% purity and viabiUty.
  • NeutrophUs are suspended in PBS with 1 mM Ca ⁇ + and 1.1 mM Mg2+ at concentrations of 5 to 20 x 106 cells per ml. CeUs are added to test tubes and treated with the desired compounds for 5 to 10 minutes, then challenged with calcium ionophere A23187, 2 ⁇ M, or vehicle control, PBS containing 0.25-1 mg/ml BSA. After 5 to 20 minutes, the reactions are terminated by addition of an equal volume of chloroform:methanol (1:2, v/v) to the samples. acid (50, 100 or 200 ng) is added as an internal standard and the Upids ware extracted by addition of equal volumes of chloroform and distilled water. The samples are vortexed and centrifuged at high speed and the chloroform layer removed to a clean tube.
  • the chloroform extract for each sample is evaporated to dryness and the material resuspended in hexane.
  • the hexane is passed through a Silica soUd phase column (500 mg), washed 2x with hexane and a fatty acid enriched fraction eluted with hexane:ethyl ether (1:1, v/v).
  • Solvents are removed from the samples under a stream of nitrogen then the samples are converted to pentafluorobenzyl esters using pentafluorobenzyl bromide and diisopropylethylamine in acetronitrile. Solvents are removed and samples are 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.
  • Blood is obtained from normal humans and neutrophUs are isolated as described for the arachidonic acid release assay, above.
  • the final leukocyte preparation should be of greater than 95% purity and viabiUty.
  • NeutrophUs are suspended in PBS at concentrations of 5 to 20 x 10 ⁇ cells per ml. CeUs are 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
  • the chloroform from each tube is 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 SiUca TLC plate.
  • the plates are developed in chloroform methanol/ acetic acid/water (50:25:8:4, v/v) visuaUzed by radioscanning (Bioscan) and the product, [3H]PAF, is scraped and quantified by Uquid scintiUation spectroscopy.
  • the Rf value for a synthetic standard of PAF is approximately 0.33.
  • the phospholipids are converted into diradylglycerols by addition of phosphohpase C, 20 units-40 units of Bacillus cereus phospholipase C (Sigma Type XIII) in 100 mM Tris HCl buffer (pH 7.4) for 2.5-6 hr, then converted into l,2-diradyl-3-acetylglycerols by incubation with acetic anhydride and pyridine (Chilton, F. H. [Methods Enzymol. (1990)187, 157-166]).
  • the phospholipid subclasses are 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 is determined by zonal scraping and Uquid scintillation counting.
  • the following is the method for assessing the abiUty of a compound to alter arachidonate content of cellular phospholipids, which can be generaUzed for any desired ceU.
  • SpecificaUy mouse bone marrow-derived mast ceUs are removed from culture and provided with exogenous [ ] arachidonic acid for 30 minutes.
  • the labeled arachidonic acid which had not been incorporated into the ceUs is then removed by washing the ceUs 2 times with an albumin-containing buffer. At that point, the ceUs 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 BUgh and Dyer [Can. J. Biochem. Physiol. (1959) 37, 911-917] and phosphoUpids separated by normal phase HPLC by the method of
  • ChUton [Methods Enzymol. (1990)187, 157-166].
  • the radioactive and mole quantities of • arachidonate in complex Upids are determined.
  • cellular Upid extracts are treated with KOH (0.5 M) to remove fatty acids from complex Upids (phospholipids) and the quantities of arachidonate in these extracts can then be determined by various methods, including gas chromatography and mass spectrometry (Chilton [Methods Enzymol.
  • Leukopaks are centrifuged (90 x g for 15 min) twice to remove the platelet-rich plasma. The cell pellet is washed by centrifugation and are resuspended in HBSS without Ca 2+ or Mg 2+ . Histopaque 1077 is 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, is removed and saved. The buffy coat is washed twice with HBSS without Ca 2+ or Mg 2+ by centrifugation.
  • the ceU peUet (4-6 x 108 ceUs/30mls) is resuspended in iso-osmotic media (RPMI-1640, 10% heat inactivated fetal bovine serum (FBS), 0.2 mM L-glutamine, 2.5 mM HEPES) and layered over an equal volume of 46% Percol mixture (10 X PBS/ Percol; 9.25 / 0.75) and 54% iso-osmotic media and centrifuged for 30 min at 1000 x g (Marshall and Roshak, Biochem. Cell Biol. 71: 331- 339, 1993).
  • the monocyte population located at the interface of the PercoU gradient is removed and washed twice in HBSS without Ca 2+ or Mg 2+ . This resulted in a greater than 85-90 % pure monocyte population as assessed by differential staining.
  • Monocytes (5 x 106/ml) are incubated as a suspension in serum-free RPMI-1640 medium containing the vehicle DMSO
  • the stimulating agent is solubUized in DMSO and appropriate vehicle controls are included in all experiments.
  • the amount of stimuU is 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 is terminated by reduction of pH through addition of citric acid and centrifugation (10 min, 400 x g, 4°C). CeU viabUity is monitored before and after experiments using trypan blue exclusion.
  • the cell-free media is decanted and stored at -70° C untU analyzed.
  • Prostaglandin E2 and LTC4 are directly measured in ceU-free media using enzyme immunoassay (EIA) kits purchased from Caymen Chemical Co. (Ann Arbor, MI). Sample or standard dUutions are made with appropriate media and analyzed in tripUcate. Results are obtained by extrapolation from a standard curve prepared in the media and expressed as pg or ng/ml of sample.
  • EIA enzyme immunoassay
  • Assays (g and h) : Assay (Method) for TPA (assay g) or Arachidonic acid (assay h)-induced Inflammation
  • mice Male Balb/c inbred mice are obtained from Charle River Breeding Laboratories (Kingston, NY). Within a single experiment mice (22-25g) are age-matched. • These in vivo experiments typically involve the use of 5-6 animals/group.
  • TPA (12-0-tetradecanoylphorbol 13-acetate) (Sigma Chemical Company) in acetone (4 mg/20ml) is appUed to the inner and outer surfaces of the left ear of B ALB/c male mice.
  • the thickness of both ears is then measured with a dial micrometer (Mitutoyo, Japan) at both 2 and 4 hours after treatment, and the data expressed as the change in thickness (10" 3 cm) between treated and untreated ears.
  • the application of acetone does not cause an edematous response; therefore, the difference in ear thickness representes the response to the TPA.
  • the inflammed left ears are removed and stored at -70°C untU they were assayed for MPO (myeloperoxidase) activity where appropriate.
  • MPO myeloperoxidase
  • MPO Myeloperoxidase
  • MPO-dependent reaction of o-dianisidine (0.167 mg/ml; Sigma) and hydrogen peroxide (0.0005%; Sigma) is measured spectrophotometricaUy at 460 nm.
  • Supernatant MPO activity is quantified kineticaUy (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 is dissolved in acetone (lmg/ear) to the left ear of BALB/c male mice.
  • the thickness of both ears is 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 ceU 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 anti-inflammatory effect of various standard inhibitors topically administered in the AA and TPA induced mouse ear edema model were measured for dexamethasone, scalaradial and Wyeth's compound WY 50,295 at does of 0.2, 0.1 and 0.3 respectively.
  • 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).
  • AA administration to the ear overrides the need for PLA2 mediated Uberation of substrate for subsequent pro- inflammatory Upid mediator generation or AA mobUization by CoA-IT.
  • an inhibitor of an AA-metaboUzing enzyme should be effective while and inhibitor of PLA2 would be ineffective.
  • scalaradial and dexamethasone have Uttle or no effect in the AA ear model at concentrations which were effective in the TPA ear model.
  • [ 3 H] a molecule that contains tritium atoms, a radioactive isotope
  • A23187 a compound that allows free entry of calcium into a ceU
  • AA arachidonic acid
  • arachidonate arachidonic acid contained within a phospholipid
  • free arachidonic acid arachidonic acid that is not contained within a phospholipid
  • [ 2 Hs]arachidonic acid the form of arachidonic acid labeled with 8 deuterium atoms, a stable isotope
  • BSA bovine serum albumin
  • CoA coenzyme A
  • CoA-IT CoA-independent transacylase
  • DTT dithiothreitol
  • EGTA [ethylenebis(oxyethylenenitrilo)]tetra

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Abstract

Cette invention se rapporte à de nouveaux composés et de nouvelles compositions pharmaceutiques des formules (I) et (II), ainsi qu'à un procédé de traitement ou de réduction d'une inflammation chez un mammifère, ce procédé consistant à administrer à ce mammifère une dose efficace du composé ou de la composition des formules (I) ou (II).
PCT/US1995/007019 1994-06-02 1995-06-02 Composes anti-inflammatoires WO1995033715A1 (fr)

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US25271294A 1994-06-02 1994-06-02
US08/252,712 1994-06-02

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006353A1 (fr) * 1997-08-04 1999-02-11 Taisho Pharmaceutical Co., Ltd. Derives aryloxyaniline
WO1999015493A1 (fr) * 1997-09-24 1999-04-01 Roche Diagnostics Gmbh Derives de 9,10-dihydro-9,10-ethanoanthracene utilises comme inhibiteurs de phohspholipase
US7579002B2 (en) * 2003-12-05 2009-08-25 Wisconsin Alumni Research Foundation Method for improving body weight uniformity and increasing carcass yield in animals

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DE1100647B (de) * 1956-05-02 1961-03-02 Albert Ag Chem Werke Verfahren zum UEberfuehren von Arylaethern in Phenole und aromatisch substituierte Amine
US3629477A (en) * 1966-08-08 1971-12-21 Geigy Chem Corp Halogenated diphenyether-containing compositions and control of pests therewith
US3646200A (en) * 1969-06-25 1972-02-29 Merck & Co Inc Treatment of inflammation with salicylic acids
US3755449A (en) * 1968-02-28 1973-08-28 Sumitomo Chemical Co Process for producing aminodiphenyl ether derivatives
US3927093A (en) * 1972-10-10 1975-12-16 Squibb & Sons Inc 2-(O-aminophenylthio)benzyl alcohols
US3997401A (en) * 1975-12-08 1976-12-14 G. D. Searle & Co. Microbial transformation of 8-chloro-10,11-dihydrodibenz[b,f][1,4]oxazepine
JPS572278A (en) * 1980-06-04 1982-01-07 Chugai Pharmaceut Co Ltd Dibenzoxazepin derivative
JPS58208278A (ja) * 1982-05-31 1983-12-03 Chugai Pharmaceut Co Ltd ジベンゾ〔ｂ，ｆ〕〔１，４〕オキサゼピン誘導体の製造方法
WO1992000285A1 (fr) * 1990-06-22 1992-01-09 Nippon Shinyaku Co., Ltd. Derive de tetrazole et medicament

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1100647B (de) * 1956-05-02 1961-03-02 Albert Ag Chem Werke Verfahren zum UEberfuehren von Arylaethern in Phenole und aromatisch substituierte Amine
US3629477A (en) * 1966-08-08 1971-12-21 Geigy Chem Corp Halogenated diphenyether-containing compositions and control of pests therewith
US3755449A (en) * 1968-02-28 1973-08-28 Sumitomo Chemical Co Process for producing aminodiphenyl ether derivatives
US3646200A (en) * 1969-06-25 1972-02-29 Merck & Co Inc Treatment of inflammation with salicylic acids
US3927093A (en) * 1972-10-10 1975-12-16 Squibb & Sons Inc 2-(O-aminophenylthio)benzyl alcohols
US3997401A (en) * 1975-12-08 1976-12-14 G. D. Searle & Co. Microbial transformation of 8-chloro-10,11-dihydrodibenz[b,f][1,4]oxazepine
JPS572278A (en) * 1980-06-04 1982-01-07 Chugai Pharmaceut Co Ltd Dibenzoxazepin derivative
JPS58208278A (ja) * 1982-05-31 1983-12-03 Chugai Pharmaceut Co Ltd ジベンゾ〔ｂ，ｆ〕〔１，４〕オキサゼピン誘導体の製造方法
WO1992000285A1 (fr) * 1990-06-22 1992-01-09 Nippon Shinyaku Co., Ltd. Derive de tetrazole et medicament

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Title
CHEMICAL ABSTRACTS, Volume 56, issued 1968, K.L. OLIVIER, "Conversion of Aryl Ethers Into Phenols and Substituted Aromatic Amines", Columns 1395-1396; & DE,B,1 100 647. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006353A1 (fr) * 1997-08-04 1999-02-11 Taisho Pharmaceutical Co., Ltd. Derives aryloxyaniline
US6333358B1 (en) 1997-08-04 2001-12-25 Taisho Pharmaceutical Co., Ltd. Aryloxyaniline derivatives
US6476056B2 (en) 1997-08-04 2002-11-05 Taisho Pharmaceutical Co., Ltd. Heterocycle substituted aryloxyaniline derivatives and their use as MDR ligands
WO1999015493A1 (fr) * 1997-09-24 1999-04-01 Roche Diagnostics Gmbh Derives de 9,10-dihydro-9,10-ethanoanthracene utilises comme inhibiteurs de phohspholipase
US7579002B2 (en) * 2003-12-05 2009-08-25 Wisconsin Alumni Research Foundation Method for improving body weight uniformity and increasing carcass yield in animals

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