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WO1996006611A1 - Composes anti-inflammatoires - Google Patents

Composes anti-inflammatoires Download PDF

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Publication number
WO1996006611A1
WO1996006611A1 PCT/US1995/011044 US9511044W WO9606611A1 WO 1996006611 A1 WO1996006611 A1 WO 1996006611A1 US 9511044 W US9511044 W US 9511044W WO 9606611 A1 WO9606611 A1 WO 9606611A1
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WO
WIPO (PCT)
Prior art keywords
heptyl
pyridyl
hydroxy
alkyl
formula
Prior art date
Application number
PCT/US1995/011044
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English (en)
Inventor
Ruth Judik Mayer
Original Assignee
Smithkline Beecham Corporation
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Publication of WO1996006611A1 publication Critical patent/WO1996006611A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom

Definitions

  • This invenuon 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-phosphochol ⁇ ne, is then acetylated to form platelet- activating factor (PAP).
  • PAP platelet- activating factor
  • 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 mflammatory cells.
  • hpid mediators such as PAF and eicosanoids are formed by the aforementioned pathways, they induce signs and symptoms observed in the pathogenesis of various mflammatory 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 havmg 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.
  • 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 respirator)' 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 (PLA2, (EC 3.1.1.4) are responsible for the liberation of arachidonic acid (AA) from the sn-2 posiuon of phospholipid They are therefore thought to play an important role in the pathogenesis of inflammation and possibly in lmmunological dysfunction.
  • phospholipase A2's are now known for the purposes herein, 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 and include the non-pancreatic Type II 14 kDa PLA2
  • High molecular weight will be in the range of 30,000 or 56,000 Da to 1 10,000k ⁇ a by SDS electrophoresis analysis.
  • cytoso c 85 kDa PLA2 A high molecular weight, cytoso c 85 kDa PLA2 has been isolated and cloned from the human monocytic cell line, U937 (Clark et al., Proc Natl. Acad. Sci , 87 770S- 7712, 1990) This enzyme is active at neutral pH, preferentially releases AA over other fatty acids from the sn-2 position of phospholipids, is regulated by phosphorylation and 6/06611 PCMJS95/11044
  • the 85 kDa-PLA 2 is disunct from 14 kDa-PLA 2 s in biochemical characte ⁇ stics such as stability of the 85 kDa-PLA 2 to DTT, instability to heat and lack of inhibition by a phosphonate phosphohpid TSA inhibitor of 14 kDa-PLA 2 as well as in its mechamsm of regulation
  • 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 +- ⁇ ndependent PLA 2 (Hazen and Gross, Circ.
  • 85 kDa PLA2 The regulation of 85 kDa PLA2 by mechanisms related to signal transduction further imply a role for this enzyme in lipid mediator production in response to receptor mediated cell acuvation.
  • 85 kDa PLA2 is transcriptionally and translationally upregulated by inflammatory cytokines or growth factors (e.g. IL-1, TGFb, thrombin).
  • IL-1 IL-1
  • TGFb transforming growth factor
  • thrombin e.g. IL-1, TGFb, thrombin.
  • the enzyme becomes rapidly phosphorylated, enhanced activity is measured in in-vitro assays and translocation to the membrane is observed (Lin etal., L. Biol Chem. 267:23451-23454 (1992); Lin et al, Proc. Natl. Acad. Sci. U.S.A.
  • Phosphorylation is predominantly by the mitogen acuvated protein (MAP) kinase, known to be acuvated by a receptor mediated cascade of phosphorylation events Lin et al., Cell 12: 267-278 (1993) Collecuvely, the evidence supports a role for the 85 kDa-PLA2 in AA release in response to cell acuvauon
  • Addiuonal PLA2 may also be involved in the AA release, as noted above, most of the cellular lipid mediators found elevated in a va ⁇ ety of inflammatory fluids were formed in response to non-pancreatic PLA2 acuon, but not clearly attributable to one PLA2 lsoform.
  • Type 11- 14 kDa-PLA 2 in cell lipid metabolism was thought to be the key rate limiting enzyme in lipid mediator formauon, until the recent idenuficauon of the 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 Type II 14 kDa PLA2 has been found in a variety of cells and tissues or extracellularly when released in response to antigeruc activators or pro-inflammatory mediators such as Interleukin (IL)- 1 , IL-6 or tumor necrosis factor (TNF) Its presence in such inflammatory fluids, Ussue exudates or serum has therefore implicated Type II- 14 kDa-PLA 2 's role in inflammauon (Vadas, et al., (1985) Life Sci.
  • IL Interleukin
  • TNF tumor necrosis factor
  • PLA2 is important in the hberauon of arachidoninc acid from phosphohpid and may also play a role in the generation of PAF via lysophosphohpid formauon, inhibiuon of such an enzyme would be useful for the treatment of disease states caused thereby
  • This invenuon relates to the pharmaceuucal composiuons of Formula (I) comp ⁇ smg a compound of Formula (I), or pharmaceuucally acceptable salt thereof and a pharmaceuucally acceptable earner or diluent
  • This invenuon also relates to a method of treaung or reducing inflammation in a mammal in need thereof, which comprises administenng to said mammal an effecuve amount of a compound or composition of Formula (I)
  • This invenuon also relates to a method of treaung disease or disorders mediated b> PLA2, free arachidomc acid, its metabohtes and/or PAF by admmistermg to a pauent in need thereof, an effective amount of a compound of Formula (I)
  • One aspect of the present invention are the compounds represented by the structure corresponding to the formula:
  • Py is a 2-, 3- or 4- pyridyl
  • Rl is a Cj to Cjo alkyl
  • R2 is a Ci to Cio alkyl; provided that one of R ⁇ and R2 contains at least 6 carbon atoms; 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). Inhibition of the 85 kDa PLA2 enzyme will result in the treatment of inflammatory occurrences in mammals.
  • the present invenuon is also directed to a novel method of inhibiting this enzyme in a mammal in need thereof, which method comprises administering to said mammal an effective amount of a compound of Formula (I) sufficient to inhibit the 85 kDa PLA2 enzyme.
  • the treatment of mflammatory occurrences in a mammal includes, but is not limited to, allergic and asthmatic manifestations, dermatological diseases, collagen diseases, reperfusion injury, stroke and other well known inflammatory diseases. Treatment of both acute and chronic diseases are possible.
  • Preferred diseases for treatment herein are arthrius, asthma, allergic rhinitis, inflammatory bowel disease (IBD), psoriasis, reperfusion injury and stroke.
  • the compounds of Formula (I) are preferential and selective inhibitors of the high molecular weight PLA2 enzyme.
  • R ⁇ is a C ⁇ to C J O branched or unbranched alkyl chain, preferably a C6 to C ⁇ Q containing branched or unbranched alkyl chain More preferably it is a heptyl, a 2-methyl heptyl or a 2,3-d ⁇ meuhyl heptyl chain.
  • R2 is a C i to C JO containing branched or unbranched alkyl chain. More preferably it is a methyl, ethyl, iso-propyl, or hexyl chain.
  • C i. i ⁇ alkyl or "alkyl” as used herein refers to both straight and branched chain radicals of 1 to 10 carbon atoms, unless the chain length is otherwise hmited, including, but not limited to, methyl, ethyl, n-propyl, wo-propyl, ⁇ -butyl, sec- butyl, w ⁇ -butyl, rr-butyl, /i-pentyl, hexyl, 2-methyl-heptyl and the like.
  • the compounds of Formula (I) may form pharmaceuucally acceptable acid addition salts which may be obtained in known manner, for example by treatment thereof with an appropriate amount of acid in the presence of a suitable solvent P ⁇ ma ⁇ ly, the salt forms of interest are salts of the py ⁇ dyl moieties, and may specifically be referred to as py ⁇ dinium salts, or quaternary salts which are ionic or covalently bonded in character
  • Such salt forms mclude, but are not limited to the basic salts of inorganic and organic acids, such as hydrochlo ⁇ c acid, hydrobromic acid, sulphu ⁇ c acid, phospho ⁇ c acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, mabc acid, tarta ⁇ c acid, citric acid, lacuc acid, oxalic acid, succiruc acid, fuma ⁇ c acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandebc
  • Quaternary salts for use herein are those salts which may be obtained in a known manner with any suitable alkylating agent, for instance the alkyl, allyl or benzyl hahdes, such as methiodide, methbromide, ethyl iodide or ethyl bromide; suitably the alkyl group is not a branched alkyl, for instance, methyl, ethyl, n-propyl, n-butyl.
  • alkylating agents are the allyl or benzyl tnfliates It is possibly to exhange the counter anion (I- or Br-) after the salt form is made, for instance, by use of an ion exchange column
  • Specifically exemplified compounds of Formula (I) are: 7-Heptyl-3,4,-d ⁇ hydro-5-hydroxy-4-ethyl-4-(4-py ⁇ dyl)couma ⁇ n; 2-Methyl-7-Heptyl-3,4,-dihydro-5-hydroxy-4-e ⁇ yl-4-(2-py ⁇ dyl)couma ⁇ n ; and
  • methiodide and hydrochloride salt forms of die above noted compounds are preferred
  • Alternauve nomenclature for these compounds when a salt is present is as follows' 4-(4-Ethyl-7-heptyl-3,4-d ⁇ hydro-5-hydroxy-2-oxo-2H- 1 -benzopyran-4-yl)- 1-methylpynd ⁇ n ⁇ um iodide
  • R2 is a Ci to Cio alkyl, provided that one of Ri and R2 is at least a C6 to C7 alkyl containing chain; and further provided that when R2 is methyl, ethyl, or iso-propyl, then R 1 is not an un-branched heptyl chain; or a pharmaceuucally acceptable salt thereof
  • the compounds of Formula (I) and (la) can be used in the manufacture of a medicament for the prophylacuc or therapeuuc treatment of an mflammatory disease m a mammal, preferably a human Inhibition of PLA2, free arachidomc acid and eicosanoid release from mflammatory cells according to this invenuon is of therapeuuc benefit in a broad range of diseases or disorders
  • the invention herein is therefore useful to treat such disease states both in humans and m other mammals
  • InhibiUon of 85 kDa PLA2 by the compounds of Formula (I) is an effective means for reducmg free arachidonic acid and eicosanoids produced in inflammatory cells
  • the therapeutic utility of blocking hpid mediator generation has been recognized for many years
  • inhibitors of cyclooxygenase such as aspmn, indomethacin, • acetaminophen and lbuprofen, have demonstrated broad therapeutic utibues
  • Another class of inhibitors which are used in a broad range of inflammatory disorders are the corticosteroids
  • Corucosteroids act in a va ⁇ ety of ways, e.g to induce mflammatory cells to produce proteins which inhibit free arachidonic acid release or to down regulate PLA2 mRNA formauon
  • Inhibitors of 5-l ⁇ poxygenase block the producuon of leukot ⁇ enes and leukot ⁇ ene antagonists prevent the bioactions of leukot ⁇ enes
  • hpid mediator producuon diseases which could benefit from the inhibition of hpid mediator producuon include, but are not limited to, adult respiratory distress syndrome, asthma, arth ⁇ tis, reperfusion injury, endotoxic shock, mflammatory bowel disease, allergic rhinius and va ⁇ ous inflammatory skin disorders Each of these disorders is mediated in some part by bpid mediators of inflammation.
  • lipid mflammatory mediators i.e , arachidonate, eicosanoids and PAF
  • cardiovascular disorders such as but not limited to, myocardial infarction, stroke, circulatory shock, or hypotension, ischemia, reperfusion injury, inflammatory diseases such as, but not bmited to, arth ⁇ us, inflammatory bowel disease, Crohn's disease, or ulcerative cobus, respiratory disease such as but not limited to, asthma, or adult respiratory distress syndrome, anaphylaxis, shock such as but not limited to endotoxic shock, topical diseases, such as but not bmited to actinic keratosis, pso ⁇ asis, or contact dermatitis, or pyresis
  • a compound of formula (I), or a pharmaceuucally acceptable salt thereof, in therapy it wdl normally be formulated into a pharmaceutical composi on in accordance with standard pharmaceutical practice
  • This mvenuon therefore, also relates to a pharmaceuucal composiUon comprising an effecuve, non-toxic amount of a compound of formula (I) or (la) and a pharmaceutically acceptable earner or diluent
  • Compounds of formula (I), pharmaceuucally acceptable salts thereof and pharmaceutical compositions incorporating such may conveniently be administered by any of the routes convenuonally used for drug admmistrauon, 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 earners according to conventional procedures
  • Such pharmaceutically acceptable earners 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 bquid carriers are syrup, peanut oil, obve oil, water and the like.
  • the carrier or diluent may include time delay material well known to the art, such as glyceryl raono-stearate or glyceryl distearate alone or with a wax.
  • sobd carrier a sobd 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 sobd carrier will vary widely but preferably will be from about 25mg. to about lg.
  • a bquid carrier the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous bquid suspension.
  • Compounds of formula (I) may be administered topically, that is by non-systemic administration. This includes the appbcation of a compound of formula (I) externally to the epidermis or the buccal cavity and the instiUation of such a compound into die 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 bquid or semi-hquid 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 hniments for appbcation to the skin may also include an agent to hasten drying and to cool die 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 appbcation. 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 bquid paraffin, glycerol, beeswax, a metalhc 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 propyiene 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, ceUulose derivatives or inorganic materials such as silicaceous sibcas, and other ingredients such as lanobn, may also be included.
  • Drops according to die 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 steribzed by filtration and transferred to die 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 propyiene 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 pharmaceuticaby acceptable compounds of the invention will normaby be administered to a subject in a daily 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) 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 admmistrauon, as well as effective dosages will vary depending, inter aba, on the condition being treated The choice of mode of admimstrauon and dosage is within die skill of the 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
  • Va ⁇ ous cellular assays can be used to determine the acuvity of the compounds of Formula (I)
  • Described herein are in -vitro assays for PLA2 and 5-LO enzyme activities. These assays employ purified recombinant enzyme or a broken cebs (assays a, b and c, respectively) Alternatively, evaluauon of inhibitors can occur in intact cells, such as described assay (d) below Assay (a): Phospholipase A assav:
  • Phospholipase A2 activity of 85 kDa PLA2 semi-purified from U937 cebs or from Baculovirus infected Sf21 cells expressing 85 kDa PLA2 (Diez et al., J. Biol. Chem.,267,
  • [ 3 H]AA-E coli had up to 95% of the label incorporated into phospholipid which was locabzed almost exclusively in the sn-2 position, as demonstrated by punfied 14kDa PLA2 or low molecular weight PLA2 acylhydrolysis and separation of products by thin layer chromatography (TLC) (data not shown).
  • the reaction mixture (50 or 100 ul total volume) contained 25 mM HEPES, pH 7 4, 150 mM NaCl, 5 mM CaCb and [ 3 H]-AA-E coli (5-8 nmol PL Pi per assay). Assays were mcubated for a ume predetermined to be on the linear portion of a time versus hydrolysis plot (10 m ).
  • the following assay was used. 125 ul of 1 mg/ml dimyristoylphosphatidymethanol in chloroform and 8 ul of l-palmitoyl-[14C]-2-arachidonoyl-GPC (1.2 X105 dpm/nmol) were dried under Ar in a glass tube and then resuspended in 1 ml of water. This preparation was sonicated in a water bath high-power sonicator for 1.5 min to provide a mixed vesicle substrate.
  • PLA2 activity 25ul of this substrate, 10 ul of buffer (lOOmM HEPES, pH 8.0 and 10 mM Ca2+), and 10 uL of PLA2 diluted to give not more than 10 % hydrolysis in 7 min were incubated at 37° in a total volume of 50 ul. The reaction was terminated and the released radiolabeled fatty acid was determined as described in assay (a).
  • Representative compounds of Formula (I) which demonstrated a positive response in this assay include:
  • the assay for assessing inhibition of the 5-LO activity was a continuous assay which monitored the consumption of oxygen (O2).
  • the cell extract (100 ug) was preincubated
  • SUBSTITUTE SHEET (RULE 25) with the hibitor or its vehicle in 25 mM BisT ⁇ s buffer (pH 7.0) that contained 1 mM EDTA, 1 mM ATP, 150 mM NaCl and 5% etiiylene glycol for 2 minutes at 20°C (total volume 2.99 ml).
  • Arachidonic acid (10 uM) and CaCl2 (2 M) were added to start the reaction, and the decrease in O2 concentrauon followed with time using a Clark-type electrode and the Yellow Sp ⁇ ng O2 monitor (type 53) (Yebow Springs, OH). The optimum velocity was calculated from the progress curves. All compounds were dissolved in ethanol witii the final concentration of ethanol being 1% in the assay.
  • Leukocyte-rich leukopaks obtained from Biological Specialues (Lansdale, PA) were cobected from male volunteers who were not taking anti- inflammatory drugs. Leukopaks were centrifuged (90 x g for 15 min) twice to remove the platelet- ⁇ ch plasma. The cell pebet was washed by centrifugation and resuspended in HBSS widiout Ca 2 + or Mg 2 +. Histopaque 1077 was layered under the ceU suspension and cenuifuged at 400 x g for 30 mm to obtain the buffy coat The lnterfacial buffy coal, containing monocytes and lymphocytes, was removed and saved. The buffy coat was washed twice witii HBSS without Ca 2+ or Mg 2+ by centrifugauon. The ceU peUet (4-6 x
  • a molecule mat contains tnuum atoms, a radioactive isotope, A23187, a compound that abows free entry of calcium into a ceU;
  • AA arachidomc acid, arachidonate, arachidomc acid contained witiun a phospholipid; free arachidomc acid, arachidomc acid that is not contained wntun a phospholipid, 1-alkyl, l-O>alkyl; 1-alkenyl, l-O-alk-l '-enyl, BSA, bovine serum albumm; DTT, didiiothreitol; EGTA, [eti ⁇ yleneb ⁇ s(oxyethylenen ⁇ tnlo)]tetra acetic acid, a calcium chelator; GPC, sn-glycero-3-phosphochobne, EDTA, a metal ion chel

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Abstract

L'invention concerne de nouvelles compositions pharmaceutiques représentées par la formule (I) comprenant un composé représenté par la formule (I) et un diluant ou un véhicule utilisés en pharmacie. Elle concerne également un procédé de traitement ou de limitation de l'inflammation chez un mammifère consistant à administrer audit mammifère une quantité efficace d'un composé ou de la composition représentés par la formule (I).
PCT/US1995/011044 1994-08-31 1995-08-29 Composes anti-inflammatoires WO1996006611A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003105842A1 (fr) * 2002-06-13 2003-12-24 Novuspharma S.P.A. Derives de chromen-2-one utilises comme inhibiteurs de la production des vegf dans les cellules mammaliennes
CN108349959A (zh) * 2015-09-24 2018-07-31 贵州百灵企业集团制药股份有限公司 4位取代的香豆素衍生物及其制备方法和用途

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US3947462A (en) * 1974-05-22 1976-03-30 Abbott Laboratories 2,4,7-substituted 5-hydroxy benzopyrans and esters

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US3947462A (en) * 1974-05-22 1976-03-30 Abbott Laboratories 2,4,7-substituted 5-hydroxy benzopyrans and esters

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CHEMICAL ABSTRACTS, Volume 81, Number 1, issued 08 July 1994, TICKLE et al., "Interaction of Grignard Reagents With Coumarins. 1. Novel 1,4-Addition", Abstract No. 3725; & J. CHEM. SOC., PERKIN TRANS. 1, (1974), (5), 569-574. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003105842A1 (fr) * 2002-06-13 2003-12-24 Novuspharma S.P.A. Derives de chromen-2-one utilises comme inhibiteurs de la production des vegf dans les cellules mammaliennes
CN108349959A (zh) * 2015-09-24 2018-07-31 贵州百灵企业集团制药股份有限公司 4位取代的香豆素衍生物及其制备方法和用途
CN108349959B (zh) * 2015-09-24 2021-11-16 成都赜灵生物医药科技有限公司 4位取代的香豆素衍生物及其制备方法和用途

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