WO1996006611A1

WO1996006611A1 - Anti-inflammatory compounds

Info

Publication number: WO1996006611A1
Application number: PCT/US1995/011044
Authority: WO
Inventors: Ruth Judik Mayer
Original assignee: SmithKline Beecham Corp
Current assignee: SmithKline Beecham Corp
Priority date: 1994-08-31
Filing date: 1995-08-29
Publication date: 1996-03-07
Anticipated expiration: 1997-02-28

Abstract

This invention relates to the novel pharmaceutical compositions of Formula (I) which comprises a compound of Formula (I) 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).

Description

ANTI-INFLAMMATORY COMPOUNDS

FIELD OF THE INVENTION

This invenuon relates to pharmaceutical compositions and their use as anti- inflammatory agents in mammals.

BACKGROUND OF THE INVENTION

An early event in the response of most inflammatory cells to immunologic activation and other stimuli is the release of newly formed products (mediators) which alter the function and biochemistry of surrounding cells and tissues. The ensuing biological responses, as well as much of the pathogenesis which is attributed to inflammation and allergy, are thought to be dependent on the effects that these newly- formed mediators have on adjacent cells within the inflammatory region.

In the last 20 years, it has become apparent that 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. 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-phosphocholιne, is then acetylated to form platelet- activating factor (PAP). 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.

Once 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. ( 1991) 629: 1 12- 1 19, Holtzman, Am. Rev. Respir. Dis. ( 1991 ) 143: 188-203; Snyder, Am J. Physiol Cell Physiol (1990) 259:C697-C708; Prescott et al., J Biol Chem (1990) 265: 17381- 17384

Similar to arachidonate products, 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 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.

Accordingly, a method which antagonizes the production of free arachidonic acid, its metabolites or PAF will have clinical utility in the treatment of a variety of allergic, inflammatory and hypersecretory conditions such as asthma, arthritis, rhinitis, bronchitis and urticaria, as well as reperfusion injury and other disease involving lipid mediators of inflammation. Many published patent applications or issued US patents exist which describe various compounds having utility as PAF or eicosanoid antagonists Such patents include U.S. Pat. No. 4,788,205, 4,801,598, 4,981 ,860, 4,992,455, 4,983,592, 5,01 1 ,847, 5,019,581 and 5,002,941.

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. Several forms of 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 10,000kϋa by SDS electrophoresis analysis.

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

-3-

migrates from the cytosol to the membrane in a Ca²⁺-dependent manner but does not require Ca²⁺ for catalysis (Kramer et al., J. Biol. Chem., 266:5268-5272 ( 1991). The 85 kDa-PLA₂ is disunct from 14 kDa-PLA₂s in biochemical characteπstics such as stability of the 85 kDa-PLA₂ to DTT, instability to heat and lack of inhibition by a phosphonate phosphohpid TSA inhibitor of 14 kDa-PLA₂ as well as in its mechamsm of regulation In addition, 85 kDa-PLA₂ has been shown to possess a lysophospholipase Ai activity which is not observed with the 14 kDa-PLA₂s The 85 kDa enzyme is similar to the myocardial Ca²+-ιndependent PLA₂ (Hazen and Gross, Circ. Res. 70:486-495 (1992)) in that Ca2+ is not required for catalysis and DTNB inhibition is observed. However, 85 kDa-PLA₂ is not inhibited by the suicide lnactivator bromoenol lactone, suggesung that the enzyme is disunct from the myocardial enzyme as well. These characteristics make the 85 kDa-PLA₂ a candidate for participation in the liberauon of AA from phospholipid stores for subsequent metabolism to lipid mediators. Both this cytosolic 85 kDa PLA2 and a cell associated Type II 14 kDa PLA2 have been found in the human immune cells such as the monocyte, neutrophil and platelet (Marshall and Roshak, Biochem Cell Biol. 71 :331-339

(1993)).

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). In response to cell acuvauon and subsequent Ca²⁺ increase, 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. 89: 6147-6151 (1992). 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 vaπety of inflammatory fluids were formed in response to non-pancreatic PLA2 acuon, but not clearly attributable to one PLA2 lsoform.

Low molecular weight, mammalian Type II 14 kDa PLA2 has been well charactenzed and is known to exist in both an extracellular form in inflammatory fluids (Kramer et al., J Biol. Chem., 264:5768-5775 (1989)) and in a cell associated form (Kanda et al . Biochemical and Biophysical Research Communica ons, 163 42-48

( 1989)) The cell -associated Type 11- 14 kDa-PLA₂ 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-PLA₂'s role in inflammauon (Vadas, et al., (1985) Life Sci. 36, 579-587, and Seilhamer, et al , (1989) J Biol Chem 264, 5335-5338). Recently, the elevated serum levels of PLA₂ activity dunng an mflammatory insult has been attributed to cytokine inducuon of acute phase protein release from liver, of which the 14 kDa-PLA₂ is suggested to be a part (Crowl, et al , (1991) J Biol Chem 266, 2647-2651) In addition, soluble PLA2 acuvity is markedly elevated in the serum and synovial fluid of pauents with rheumatoid arthritis (Stefanski et al , J Biochem. 100.1297-303 (1986). Furthermore, increasing serum PLA2 levels have been shown to posittvely correlate with clinical seventy (Bomalaski and Clark, Arthntis and Rheumat. 36: 190-198 (1993))

Various inhibitors of PLA2 have been descnbed in publicauons and in US Patents See for instance US Patents 4,959,357, 4,933,365, 5,208,223; 5,208244, Marshall et al , J Rheumatology 18:1 (1991); Marshall et al , Phosphobpase A2, Ed Pyu Wong, Plenum Press, NY (1990) pages 169-181; Wilkerson, et al , Eur J Med Chem , 26:667, 1991 and Wilkerson, Antunflammatory Phosphobpase A2 Inhibitors, Drugs of the Future,

Vol 15, No. 2 p 139-148(1990)

Accordingly, as 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

SUMMARY OF THE INVENTION

This invenuon relates to the pharmaceuucal composiuons of Formula (I) compπsmg 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:

wherein 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.

DETAILED DESCRIPTION OF THE INVENTION

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. There 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. For purposes herein, the compounds of Formula (I) are preferential and selective inhibitors of the high molecular weight PLA2 enzyme.

For compounds of Formula (I), 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-dιmeuhyl heptyl chain.

Preferably, 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.

The term "C i. iøalkyl" 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, π-butyl, sec- butyl, wσ-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 Pπmaπly, the salt forms of interest are salts of the pyπdyl moieties, and may specifically be referred to as pyπdinium 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 hydrochloπc acid, hydrobromic acid, sulphuπc acid, phosphoπc acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, mabc acid, tartaπc acid, citric acid, lacuc acid, oxalic acid, succiruc acid, fumaπc acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandebc acid. 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. Also useful as 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,-dιhydro-5-hydroxy-4-ethyl-4-(4-pyπdyl)coumaπn; 2-Methyl-7-Heptyl-3,4,-dihydro-5-hydroxy-4-eϋ yl-4-(2-pyπdyl)coumaπn ; and

7-Heptyl-3,4,-dιhydro-5-hydroxy-4-hexyl-4-(4-pyπdyl)coumaπn

Preferably the 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-dιhydro-5-hydroxy-2-oxo-2H- 1 -benzopyran-4-yl)- 1-methylpyndιnιum iodide

4-(7-Heptyl-4-hexyl-3,4-dιhydro-5-hydroxy-2-oxo-2H- l-benzopyran-4-yl)- 1-methylpyπdιnιum iodide

4-Ethyl-3,4-dιhydro-5-hydroxy-7-( l-methylheptyl)-4-(2-pyπdιnyl)- 2H-l-benzopyran-2-one hydrochloπde

Another aspect of trie present invention are the novel compounds of Formula (la). a subset of the compounds of Formula (I) herein Compounds of Formula (la) are represented by the structure

wherein Py is a 2-, 3- or 4- pyπdyl, Rl is a Ci to Cio alkyl;

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

Compounds of Formula (I) can be readily prepared by one skilled in the art in an analogous manner to the synthesis as described in Tickle et al , J. Chem. Soc., Perkin Trans 1 (1974) (5), pg 569-74 whose disclosure is incorporated by reference herein in its enurety.

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 For example, 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 vaπety 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-lιpoxygenase block the producuon of leukotπenes and leukotπene antagonists prevent the bioactions of leukotπenes Recent studies indicate thai both will have broad therapeutic utilities Inhibitors of phospholipase A2 block the release of free arachidonic acid PLA2 inhibitors are recognized to have broad therapeuuc uUhties It does not, however, follow that the disease states noted above must in fact be caused by altered PLA2 activity Thus, the disease state itself may not be directly mediated by PLA2 activity It only follows that PLA2 activity is required for the continued expression of symptoms of the disease state and that PLA2 inhibitors will be beneficial against the symptoms of these disease states

Disease states which could benefit from the inhibition of hpid mediator producuon include, but are not limited to, adult respiratory distress syndrome, asthma, arthπtis, reperfusion injury, endotoxic shock, mflammatory bowel disease, allergic rhinius and vaπous inflammatory skin disorders Each of these disorders is mediated in some part by bpid mediators of inflammation. Compounds which inhibit PLA2, by virtue of their ability to block the generauon of bpid mediators of inflammation stemming from acuvation and/or release of this enzyme are of value in the treatment of these condiuons Treatment of disease states caused by these lipid mflammatory 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 bmited to, arthπus, 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, psoπasis, or contact dermatitis, or pyresis

In order to use 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

Pharmaceuucal Sciences, 18th Ed , Alfonso R Genarao, Ed , 1990, Mack Publishing Co and the Handbook of Pharmaceutical Excipients, APhA Publications, 1986

SUBSTITUTE SHEET (RULE 25) 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. Similarly, 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.

A wide variety of pharmaceutical forms can be employed. Thus, if a sobd carrier is used, 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. When a bquid carrier is used, 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. In contrast, 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. Alternatively, the solution may be steribzed by filtration and transferred to die container by an aseptic technique. Examples of 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. For an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compound of the Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered from 1 to 4 times per day. 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

BIOLOGICAL METHODS:

Vaπous cellular assays can be used to determine the acuvity of the compounds of Formula (I)

Cell-free and Cellular Assessment of Inhibitors

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,

18342-18348 (1992), Amegadzie et al., Gene, 128: 307-308 (1993)) was measured by the acylhydrolysis of (NEN)[³H]-AA-£. coli (specific activity 100,000-200,000 dpm /5nmol PL Pi) as previously described m Marshall et al., J. Rheumatology, 18: 1, pp59- 65 (1991). Activity of 14 kDa PLA2 could also be measured by this method. [³H]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 [³H]-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 ). Experiments were conducted with final % hydrolysis values ranging from 2% (2000-4000 dpm) to 7% (7000-14000 dpm) acylhydrolysis after blank correction All assays contained 10% dimethylsulfoxide (DMSO) even when compounds were solubbzed in water to enhance the rate of hydrolysis approximately 2-fold over the rate without DMSO Reacuons were terminated by die addiuon of 1 0 mL tetrahydrofuran (THF) The whole sample was placed over aminopropyl solid phase sibca columns and eluted with THF:aceuc acid (49:1) exclusiveh separaung free fatty acids with greater than 95% recovery Radiolabel in this eluate was quantitated by bquid scmtiUaUon counting Results were expressed as % of fatty acid hydrolyzed ([sample dpms - non-specific (blank) dpms/total dpms] \ 100) Non-specific acuvity was always less than 1% of the total counts added Protein determination

Ab protein concentrations were determined by Bradford protein analysis kits (Biorad,

Richmond, CA). Results:

The foUowing representative compounds of Formula. (I) below demonstrated positive PLA2 inhibition in the above noted method. Compounds generally tested positive at 1-25 μm levels. Such compounds include:

7-Heptyl-3,4,-dihydro-5-hydroxy-4-eύ yl-4-(4-pyridyl)coumarin methiodide salt; 2-Methyl-7-Heptyl-3,4,-dihydro-5-hydroxy-4-ethyl-4-(2-pyridyl)coumarin; and 7-Heptyl-3,4,-dihydro-5-hydroxy-4-hexyl-4-(4-pyridyl)coumarin methiodide salt;

Assay (b) for measurement of PLA activity

To measure PLA2 activity using a vesicle substrate which is less susceptible to artifactual interference by, for example, high levels of protein or non-specific inhibitors, 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. To determine 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).

Results: Compounds tested positive in this assay at concentrations from l-25uM and no more than 2-fold different from the concentration in assay (a).

Representative compounds of Formula (I) which demonstrated a positive response in this assay include:

7-Heptyl-3,4,-dihydro-5-hydroxy-4-eUiyl-4-(4-pyridyl)coumarin meϋ iodide salt: and 2-Methyl-7-Heptyl-3,4,-dihydro-5-hydroxy-4-ethyl-4-(2-pyridyl)coumarin.

Assay (c) for measurement of 5-lipoxygenase activity The 5-lipoxygenase (5-LO) enzyme was isolated from extracts of RBL- 1 cells.

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 BisTπs 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 Spπng 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.

Drug-induced effects on enzyme acuviϋes are descπbed as the concentration of drug causing a 50% inhibiuon of oxygen consumpuon (IC50).

The following representauve compounds demonstrated positive 5-bpoxygenase inhibiuon in the above noted method at 10-25 μm levels.

7-Heptyl-3,4,-dihydro-5-hydroxy-4-ethyl-4-(4-pyridyl)coumarin methiodide salt; and 2-Methyl-7-Heptyl-3,4,-dιhydro-5-hydroxy-4-ethyl-4-(2-pyridyl)coumarin;

Assay (d) for measurement of stimulated eicosanoid release by human monocvtes.

Human Monocvte Isolation. 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-πch plasma. The cell pebet was washed by centrifugation and resuspended in HBSS widiout Ca²+ or Mg²+. 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 Ca²⁺ or Mg²⁺ by centrifugauon. The ceU peUet (4-6 x

108 cells/30mls) was resuspended in lso-osmotic media O^PMI-1640, 10% heat inactivated fetal bovine serum (FBS), 0.2 M L-glutamine, 2.5 mM HEPES) and layered over an equal volume of 46% Percol mixture (10X PBS/ Percol; 9.25 / 0.75) and 54% iso-osmouc media and centrifuged for 30 mm at 1000 x g (Marshall and Roshak, Biochem CeU Biol 71: 331-339, 1993) The monocyte populauon located at die interface of the Percob gradient was removed and washed twice in HBSS without Ca²+ or Mg²⁺ This resulted a greater than 85-90 % pure monocyte population as assessed by differenual staining

Measurement of Stimuli-Induced Eicosanoid Release Monocytes (5 x 106/ml) were incubated as a suspension m serum-free RPMI- 1640 medium containing the vehicle DMSO (< 1%) or drug for 30 min at 27°C after which vehicle or su ub was added for d e dicated time The stimulating agent is solubibzed in DMSO and appropπate vehicle controls were included in ab experiments The amount of stimuli was chosen from die linear portion of a concentration versus product curve usuaby represenung 60-80% maximal sumulation over die indicated incubation ume at 37°C (A23187, 1 μM,(15 min) The reaction was terminated by reducuon of pH through addition of citπc acid and centπfugauon (10 mm, 400 x g, 4°C) CeU viabdity was monitored before and after experiments usmg trypan blue exclusion The ceU-free media was decanted and stored at -70° C untd analyzed Prostaglandin E2 and LTC4 were directly measured n ceU-free media us g enzyme immunoassay (EIA) kits purchased from Caymen Chemical Co (Ann Arbor, MI) Sample or standard dduuons were made with appropπate media and analyzed in tπpbcate Results were obtained by extrapolauon from a standard curve prepared in the media and expressed as pg or ng/ml of sample

RepresentaUve compounds of Formula (I) which demonstrated positive acuvity in dns assay and were acuve at concentrations consistent with the activites m Assay (a) (0 1- lOμM) were.

7-Heptyl-3,4,-dιhydro-5-hydroxy-4-ethyl-4-(4-pyπdyl)coumaπn methiodide salt; and

2-Memyl-7-Heptyl-3,4,-dihydro-5-hydroxy-4-edιyl-4-(2-pyπdyl)coumaπn

As used herein, various abbreviations and explanations are as foUows [³H], 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, [etiιylenebιs(oxyethylenenιtnlo)]tetra acetic acid, a calcium chelator; GPC, sn-glycero-3-phosphochobne, EDTA, a metal ion chelator, GPE, sn-glycero-3-phosphoethanolamme, 5HETE, 5(S)-hydroxyeιcosa- 6,8,1 1 ,14-tetraenoιc acid, 15HETE, 15(S)-hydroxyeιcosa-5,8, l l, 13-tetraenoιc acid, LTB4, leukotπene B4, LTC4, leukotnene C4; LTD₄, leukotnene D₄, lyso PAF, l-alkyl-2 lyso-GPC, lyso platelet-activating factor, PLA2, phospholipase A2, PBS, phosphate buffered saline, PAF, platelet activaung factor, l -alkyl-2-acetyl-GPC, PL, phosphohpid PC, phosphatidylchohne, PE, phosphatidylethanolamine PI, phospha dybnositol PMN polymo honuclear neutrophibc ceU, neutrophil, PS phosphaudylseπne, TLC. thin layer chromatography, TSA, transiuon state analog, U937, A eπcan Type Tissue Culture designated ceU line simdar to a monocyte The above description fully discloses die invention including preferred embodiments thereof. Modifications and improvements of the embodiments specificaUy disclosed herein are within the scope of the foUowing claims. Without further elaboration, it is believed tiiat one skilled in die art can, using the preceding description, utibze the present invention to its fuUest extent. Therefore, the Examples herein are to be construed as merely Ulustrative and not a bmitation of the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privϋege is claimed are defined as follows.

Claims

What is claimed is

1 A method for treaung an inflammatory disease or disorder in a mammal in need thereof which method compπses administering to said mammal an effective amount of a compound of Formula (I)

where Py is a 2-, 3- or 4- pyπdyl; Rl is a Ci to Cio alkyl;

R2 is a Ci to Cio alkyl; provided that one of Ri and R2 contains at least 6 carbon atoms, or pharmaceuucally acceptable salts thereof

2 The method according to Claim 1 wherein the inflammatory disease or disorder is allergic rhinius, ischemia, reperfusion injury, arthπus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, astiima, adult respiratory distress syndrome, anaphylaxis, actimc keratosis, psoπasis, contact dermatiUs, or pyresis

3 The method according to Claim 1 wherein the inflammatory disease or disorder is mediated by bpid inflammatory mediators, arachidonic acid, its metabohtes and/or platelet acuvaung factor (PAF)

4 The metiiod according to Claim 3 wherein the bpid inflammatory mediators are inhibited by an inhibitor of the enzyme phosphobpase A2 (PLA2)

5 The method according to Claim 1 wherein R] is a C to C$ alkvl containing chain

6 The method according to Claim 1 wherem R2 is ethyl or hexyl

7 The method according to Claim 1 wherem the compound is

7-Heptyl-3,4,-dιhydro-5-hydroxy-4-etiιyl-4-(4-pyπdyl)cournaπn, 2-Methyl-7-Heptyl-3,4,-dihydro-5-hydroxy-4-ethyl-4-(2-pyridyl)coumarin; and 7-Heptyl-3,4,-dihydro-5-hydroxy-4-hexyl-4-(4-pyridyl)coumarin.

8. A pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and a compound according to the formula

wherein

Py is a 2-, 3- or 4- pyridyl; Ri s a C i to Cio alkyl;

R2 is a Ci to Cio alkyl; provided tiiat one of Ri and R2 contains at least 6 carbon atoms; or pharmaceuticaby acceptable salts thereof.

9. The composition according to Claim 8 wherein R 1 is a Cβ to C8 alkyl.

10. The composition according to Claim 9 wherein R2 is a C > to Cs alkyl.

1 1. The composition according to Claim 9 wherein R2 is ethane or hexane.

12. The composition according to Claim 8 wherein the compound is:

7-Heptyl-3,4,-dihydro-5-hydroxy-4-edιyl-4-(4-pyridyl)coumarin; 2-Methyl-7-Heptyl-3,4,-dihydro-5-hydroxy-4-ethyl-4-(2-pyridyl)coumarin; and 7-Heptyl-3,4,-dihydro-5-hydroxy-4-hexyl-4-(4-pyridyl)coumarin.

13. A compound of the formula:

wherein

Py is a 2-, 3- or 4- pyridyl; Rl is a Ci to o alkyl;

R2 is a Cj to C JO alkyl; provided that one of Rj and R2 contains at least 6 carbon atoms; and further provided that when R2 is methyl, ethyl, or iso-propyl, then Ri is not a heptyl, or 2,3-dιmethyI heptyl; or p harm ace uucaby acceptable salts diereof.