EP1019399A1 - Derives d'arylthiophene utilises comme inhibiteurs de la phosphodiesterase iv - Google Patents

Derives d'arylthiophene utilises comme inhibiteurs de la phosphodiesterase iv

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Publication number
EP1019399A1
EP1019399A1 EP98946190A EP98946190A EP1019399A1 EP 1019399 A1 EP1019399 A1 EP 1019399A1 EP 98946190 A EP98946190 A EP 98946190A EP 98946190 A EP98946190 A EP 98946190A EP 1019399 A1 EP1019399 A1 EP 1019399A1
Authority
EP
European Patent Office
Prior art keywords
optionally substituted
3alkyl
hydrogen
independently
alkyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98946190A
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German (de)
English (en)
Inventor
Yongxin Han
Dwight Macdonald
Andre Giroux
Robert N. Young
Helene Perrier
Carole Lepine
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Frosst Canada and Co
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Merck Frosst Canada and Co
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Filing date
Publication date
Priority claimed from GBGB9808109.4A external-priority patent/GB9808109D0/en
Application filed by Merck Frosst Canada and Co filed Critical Merck Frosst Canada and Co
Publication of EP1019399A1 publication Critical patent/EP1019399A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms
    • C07D333/24—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • This invention relates to compounds and pharmaceutical compositions for the treatment of diseases by raising the level of cyclic adenosine-3',5'-monophosphate (cAMP) through the inhibition of phosphodiesterase IV (PDE IV).
  • cAMP cyclic adenosine-3',5'-monophosphate
  • PDE IV phosphodiesterase IV
  • cAMP 3', ⁇ '-cyclic adenosine monophosphate
  • the cellular levels of cAMP are regulated by mechanisms which control synthesis and breakdown.
  • the synthesis of cAMP is controlled by adenyl cyclase which may be directly activated by agents such as forskolin or indirectly activated by the binding of specific agonists to cell surface receptors which are coupled to adenyl cyclase.
  • the breakdown of cAMP is controlled by a family of phosphodiesterase (PDE) isoenzymes, which also control the breakdown of guanosine 3',5'- cyclic monophosphate (cGMP).
  • PDE phosphodiesterase
  • PDE I-VII the distribution of which varies from tissue to tissue. This suggests that specific inhibitors of PDE isoenzymes could achieve differential elevation of cAMP in different tissues, [for reviews of PDE distribution, structure, function and regulation, see Beavo & Reifsnyder (1990) TIPS, 11: 150-155 and Nicholson et al (1991) TIPS, 19-27].
  • the availability of PDE isotype selective inhibitors has enabled the role of PDEs in a variety of cell types to be investigated. In particular it has been established that PDE IV controls the breakdown of cAMP in many inflammatory cells, for example, basophils (Peachell P.T. et al, (1992) J.
  • PDE IV inhibitors for the treatment of inflammatory diseases such as asthma, has met with limited success to date. Many of the PDE r inhibitors which have been synthesised have lacked potency and/or inhibit more than one type of PDE isoenzyme in a non-selective manner. PDE IV inhibitors that are relatively potent and selective for PDE IV, are reported to be emetic as well. Indeed this side effect has been so universal that experts have expressed their belief that the emesis experienced upon administration of a PDE IV inhibitor, may be mechanism based.
  • compounds according to the invention have good oral activity and at orally effective doses exhibit little or none of the side-effects associated with known PDE IV inhibitors, such as rolipram.
  • the compounds of the invention are therefore of use in medicine, especially in the prophylaxis and treatment of asthma and other inflammatory conditions.
  • the invention encompasses novel compounds of Formula I useful in the treatment of disease by inhibition of PDE IV, resulting in an elevation of cAMP.
  • the invention also encompasses certain pharmaceutical compositions and methods for treatment of diseases by inhibition of PDE IV, resulting in an elevation of cAMP, comprising the use of compounds of Formula I.
  • the invention encompasses the novel compound of Formula I useful in the treatment of disease by inhibition of PDE IV, resulting in an elevation of cAMP,
  • Ri is selected from: a) hydrogen, b) Cl-3alkyl, optionally substituted with -OH, or c) -X 1 -Y 1 -Ar 2 , wherein:
  • Xl is 1) -CH 2 -, or
  • Ar 2 is an aromatic ring selected from phenyl, naphthyl, pyrimidinyl, pyridinyl or thienyl, optionally substituted with up to two substituents chosen independently among:
  • R2 is selected from: a) hydrogen or b) Ci-3alkyl.
  • R3 is selected from phenyl, naphthyl, pyridinyl, furyl, thienyl, or ethinyl, optionally substituted with up to two substituents chosen independently among: a) Ci-3 alkyl, b) C ⁇ _3 fluoroalkyl, c) C ⁇ _6 alkoxy, d) Ci-3fluoroalkoxy, e) Ci-3alkylthio, f) halo, g) -OH, h) -N0 2 , i) -CH 2 OH, j) -NHCONR9R1°, k) -S(O) 2 NRllR 12 , 1) -SCH2(l,l-c-Pr)CH 2 CO 2 H, m) 1-piperazinyl, optionally substituted with Ci-3alkyl, n) 4-morpholinyl, or o)
  • Ar3 is phenyl, pyridinyl, pyrimidinyl or pyrazinyl, optionally substituted with up to two substituents chosen independently among:
  • Ci-3alkyl optionally substituted with -OH, or 2) -CH2CO2H.
  • R4 and R ⁇ are independently selected from: a) hydrogen, b) Ci_3alkyl, c) -S(0)2Ci-3alkyl, or d) -S(0)2phenyl, optionally mono-substituted with Ci-3alkyl,
  • Ci-3alkoxy, Ci-3alkylthio or halo are examples of compounds that are useful in the following abbreviations: Ci-3alkoxy, Ci-3alkylthio or halo.
  • R6 and R ⁇ are independently chosen from among: a) hydrogen, b ) C 1 _ 4 alkyl, c) -CO-C- ⁇ alkyl, or d) -CO-phenyl, optionally mono-substituted with C-, oalkyl,
  • Ci-3alkoxy, Ci-3alkylthio or halo R8 is chosen from among: a) hydrogen, or b) Cl- ⁇ alkyl;
  • R9 and ⁇ O are independently chosen from among: a)hydrogen, b) Ci-4alkyl, or c) phenyl;
  • RU and R ⁇ - 2 are independently chosen from among: a) hydrogen or b) Ci- ⁇ alkyl.
  • Ri is selected from : a) Cl-3alkyl, optionally substituted with -OH, or b) -X 1 -Y 1 -Ax ⁇ ;
  • R2 is hydrogen; and the remaining substituents are defined as in Formula I above.
  • Another preferred genus is that in which -Xl-Yl- is -CH2-S- and the remaining substituents are defined as in Formula I above.
  • Ar 2 is pyrimidinyl, optionally substituted with up to two substituents chosen independently among:
  • Arl is an aromatic ring selected from phenyl, quinolinyl, pyridinyl, furyl, thienyl or thiazolyl, optionally substituted with up to two substituents chosen independently from among: a) Cl-6alkyl, optionally substituted with -OH, -CO2H, CO2C1- 3alkyl, and CN, b) Cl-3alkoxy, c) Cl-3alkylthio, d) Cl-3alkylsulfinyl, e) Ci-3alkylsulfonyl, f) Ci-3_luoroalkyl, optionally substituted with -OH, g) halo, h) -OH, i) -CO2H, j) -C0 2 Ci- 3 alkyl,
  • Rl is selected from: b) Ci-3alkyl, optionally substituted with -OH, or c) -X 1 -Y 1 -Ar2, wherein: X s D -CH2-, or
  • Ar2 is pyrimidinyl optionally substituted with up to two substituents chosen independently among:
  • R 2 is selected from: a) hydrogen or R3 is selected from phenyl, naphthyl, pyridinyl, furyl, or thienyl, optionally substituted with up to two substituents chosen independently among: a) C 1-3 alkyl, b) Ci-3fluoroalkyl, c) C 1-6 alkoxy, d) Ci-3fluoroalkoxy, e) C i-3 alky lthio, f) halo, g) -OH, h) -N0 2 , i) -CH 2 OH, j) -N ⁇ CONRSR 10 , k) -S(0) 2 NRllRl2,
  • Ar3 is phenyl, pyridinyl, or pyrimidinyl optionally substituted with up to two substituents chosen independently among:
  • R4 and R ⁇ are independently selected from: a) hydrogen, b) Ci-3alkyl, c) -S(0)2Cl-3alkyl, or
  • R6 and R ⁇ are independently chosen from among: a) hydrogen, b) C ⁇ alkyl, c) -CO-C- j ⁇ alkyl, or
  • R ⁇ is chosen from among: a) hydrogen, or b) Ci-5alkyl;
  • R9 and R ⁇ -0 are independently chosen from among: a)hydrogen, b) Ci_4alkyl, and RU and R ⁇ 2 are independently chosen from among: a) hydrogen or b) Ci-5alkyl.
  • Arl is an aromatic ring selected from phenyl, quinolinyl, pyridinyl, furyl, thienyl or thiazolyl, optionally substituted with up to two substituents chosen independently from among: a) Ci-6alkyl, optionally substituted with -OH, -CO2H, CO2C1- 3alkyl, and CN, b) C ⁇ _3alkoxy, c) Ci-3alkylthio, d) Ci_3alkylsulfinyl, e) C 1-3 alkylsulfonyl, f) Ci_3fluoroalkyl, optionally substituted with -OH, g) halo, h) -OH, i) -CO2H, j) -C02Ci-3alkyl,
  • Ri is selected from: b) C _3alkyl, optionally substituted with -OH, or c) -Xl-Yl-Ar2, wherein:
  • Xl-Yl is CH 2 S;
  • Ar2 is pyrimidinyl optionally substituted with up to two substituents chosen independently among:
  • R 2 is hydrogen
  • R3 is selected from phenyl, pyridinyl, furyl, or thienyl, optionally substituted with up to two substituents chosen independently among: a) Ci-3 alkyl, b) Ci-3 fluoroalkyl, c) C 1-6 alkoxy, d) Ci-3fluoroalkoxy, e) Ci-3alkylthio, f) halo, g) -OH, h) -N02, i) -CH OH, j) -NHCONR 9 R 10 , k) -X2-Y2-Ar3, wherein, X2 is 1) -CH2-, or
  • a ⁇ 3 is phenyl, pyridinyl, or pyrimidinyl optionally substituted with up to two substituents chosen independently among:
  • R4 and R ⁇ are independently selected from: a) hydrogen, b) Ci-3alkyl,
  • R6 and R ⁇ are independently chosen from among: a) hydrogen, b) C- ⁇ alkyl,
  • R8, R11 nd R ⁇ 2 are chosen from among: a) hydrogen, or b) Ci-5alkyl;
  • R9 and R ⁇ -O are independently chosen from among: a)hydrogen, b) Cl-4alkyl.
  • halo is intended to include F, Cl, Br, and I.
  • alkyl is defined to include straight, branched, and cyclic structures of the indicated number of carbon atoms.
  • Ci-6alkyl includes methyl, ethyl, propyl, i-propyl, s- and t-butyl, pentyl, hexyl, 1,1-dimethylethyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • alkoxy, alkylthio, alkylsulfinyl, and alkylsulfonyl mean the corresponding groups of the indicated number of carbon atoms of a straight, branched, or cyclic configuration. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyl oxy, and the like.
  • alkylthio groups include methylthio, propylthio, isopropylthio, cycloheptylthio, etc.
  • the propylthio group signifies -SCH2CH2CH3.
  • Fluoroalkyl means an alkyl group of the indicated number of carbon atoms, of straight, branched or cyclic structure, in which one or more hydrogen atoms have been replaced by fluorine atoms; fluoroalkoxy, fluoroalkylthio, fluoroalkylsulfinyl, and fluoroalkylsulfonyl have the analogous meanings.
  • Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers.
  • the present invention is meant to comprehend such possible diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
  • Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
  • the invention encompasses pharmaceutical compositions for treatment of disease by inhibition of PDE IV, as disclosed herein comprising a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of compound of Formula I as described above.
  • compositions for treatment of disease by inhibition of PDE IV, resulting in an elevation of cAMP comprising a pharmaceutically acceptable carrier and a non-toxic therapeutically effective amount of compound of Formula I as described above.
  • a compound is said to selectively inhibit PDE IV in preference to other PDE's if the ratio of the IC50 concentration for all other PDE inhibition to PDE IV inhibition is 100 or greater.
  • compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt, thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N_-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. It will be understood that in the discussion of methods of treatment
  • Compounds according to the invention are selective and potent inhibitors of PDE IV.
  • the ability of the compounds to act in this way may be simply determined by the tests described in the Examples hereinafter.
  • the compounds according to the invention are thus of particular use in the prophylaxis and treatment of human diseases where an unwanted inflammatory response or muscular spasm (for example bladder or alimentary smooth muscle spasm) is present and where the elevation of cAMP levels may be expected to prevent or alleviate the inflammation and relax muscle.
  • an unwanted inflammatory response or muscular spasm for example bladder or alimentary smooth muscle spasm
  • the elevation of cAMP levels may be expected to prevent or alleviate the inflammation and relax muscle.
  • Particular uses to which the compounds of the invention may be put include the prophylaxis and treatment of asthma, especially inflamed lung associated with asthma, cystic fibrosis, or in the treatment of inflammatory airway disease, chronic bronchitis, eosinophilic granuloma, psoriasis and other benign and malignant proliferative skin diseases, endotoxic shock, septic shock, ulcerative colitis, Crohn's disease, reperfusion injury of the myocardium and brain, inflammatory arthritis, chronic glomerulonephritis, atopic dermatitis, urticaria, adult respiratory distress syndrome, diabetes Alzheimer's disease, allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis, arterial restenosis and artherosclerosis.
  • Compounds of the invention also suppress neurogenic inflammation through elevation of cAMP in sensory neurones. They are, therefore, analgesic, anti-tussive and anti-hyperalgesic in inflammatory diseases associated with irritation and pain.
  • Compounds according to the invention may also elevate cAMP in lymphocytes and thereby suppress unwanted lymphocyte activation in immune-based diseases such as rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, transplant rejection and graft versus host disease.
  • Compounds of the invention suppress cytokine synthesis by inflammatory cells in response to immune or infectious stimulation. They are, therefore, useful in the treatment of bacterial, fungal or viral induced sepsis and septic shock in which cytokines such as tumour necrosis factor (TNF) are key mediators. Also compounds of the invention suppress inflammation and pyrexia due to cytokines and are, therefore, useful in the treatment of inflammation and cytokine- mediated chronic tissue degeneration which occurs in diseases such as rheumatoid or osteo-arthritis.
  • cytokines such as tumour necrosis factor (TNF) are key mediators.
  • TNF tumour necrosis factor
  • cytokines such as TNF in bacterial, fungal or viral infections or in diseases such as cancer, leads to cachexia and muscle wasting.
  • Compounds of the invention ameliorate these symptoms with a consequent enhancement of quality of life.
  • Compounds of the invention also elevate cAMP in certain areas of the brain and thereby counteract depression and memory impairment.
  • Compounds of the invention suppress cell proliferation in certain tumour cells and can be used, therefore, to prevent tumour growth and invasion of normal tissues.
  • the compounds according to the invention may be administered as pharmaceutical compositions, and according to a further aspect of the invention we provide a pharmaceutical composition which comprises a compound of Formula I together with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • compounds of Formula I may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non- toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • the compound of the invention is effective in the treatment of humans.
  • compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • an oil medium for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxy- propylmethycellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene- oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n- propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
  • preservatives for example ethyl, or n- propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n- propyl, p-hydroxybenzoate
  • coloring agents for example ethyl, or n- propyl, p-hydroxybenzoate
  • flavoring agents such as sucrose, saccharin or aspartame.
  • sweetening agents such as sucrose, saccharin or aspartame.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
  • the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavouring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • compositions can be prepared by mixing the drug with a suitable non- irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non- irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula I are employed.
  • topical application shall include mouth washes and gargles.
  • Dosage levels of the order of from about 0.01 mg to about 140 mg/kg of body weight per day are useful in the treatment of the above- indicated conditions, or alternatively about 0.5 mg to about 7 g per patient per day.
  • inflammation may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively about 0.5 mg to about 3.5 g per patient per day.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition.
  • Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.
  • the compounds of the present invention can be prepared by the methods described below. It will be apparent to one skilled in the art that similar methodology could be used to prepare the enantiomers or the racemates of the illustrated compounds.
  • Both Ar a and R a can be further (Mitsunobo reaction) modified to give derivatives
  • TBAF tetra-n-butylammonium fluoride
  • Example 26 A mixture of Example 26 (48mg), 4-chloropyridine (31mg) and K 2 CO 3 (45mg) in DMAC was heated to reflux for 3h and cooled to rt. The mixture was diluted with water and extracted with ethyl acetate. The extract was washed with brine dried over MgS0 4 , filtered and concentrated. The residue was purified by flash chromatography. Eluting with 4:1 ethyl acetate in hexanes afforded the title compound (36mg, 64% yield) as a white solid. ⁇ NMR: see Table 1.
  • Step 1 Preparation of Intermediate 2: To a solution of ethyl 4- bromobenzoate (2.29g, lOmmol) in DME (40mL) was added boronic acid 1 (2.37g, l ⁇ mmol), Pd(PPh 3 ) 4 (346mg, 0.3mmol) and Na 2 C0 3 (2M, 7.5mL) and the mixture was deoxygenated under a stream of N 2 for 5 min and then heated to 80° C for 50 min. After cooling to rt, the mixture was diluted with H 2 0 and extracted with ethyl acetate (3 x 50mL).
  • Step 2 Bromination to Intermediate 3: To a solution of intermediate 2 (2.7g, lOmmol) in THF (50mL) at 0°C was added NBS (3.56g, 20mmol) and water (5mL) and the mixture was stirred at 0°C for 30 min and quenched with excess 5% Na 2 S 2 0 3 aqueous solution. The mixture was then extracted with ethyl acetate (3 x 50mL) and the extracts combined, washed with water, brine, dried over Mg 2 S0 4 and filtered.
  • Step 3 Preparation of Intermediate 4: To a solution of bromide 3 (610mg, 1.79mmol) in DME (8mL) was added 3,4-dimethoxyphenylboronic acid (391mg, 2.15mmol) (see: Yokoe, I. et al, (1989) Chem. Pharm. Bull. 37, 529), Pd(PPh 3 ) 4 (62mg, 0.054mmol) and Na 2 C0 3 (lmL, 2M) and the mixture was deoxygenated under a stream of nitrogen for 5 min, heated to reflux for 4h, and then cooled to rt and diluted with water.
  • 3,4-dimethoxyphenylboronic acid 391mg, 2.15mmol
  • Step 4 Preparation of Intermediate 5: To a solution of compound 4 (461mg, l.l ⁇ mmol) in CH 2 C1 2 (6mL) at 0°C was added Br 2 PPh 3 (586mg, 1.39mmol) and the solution was allowed to warm to rt and stirred for 30 min under N 2 . Then to the solution was added 4,6-dimethyl-2- mercaptopyrimidine (325mg, 2.32mmol) and diisopropylethylamine (0.8mL) and the resultant solution was stirred at rt for lh and concentrated.
  • Br 2 PPh 3 586mg, 1.39mmol
  • Step 5 A mixture of compound 5 (213mg, 0.41 mmol) and LiOH (1.23mL, IM) in dioxane (5mL) was heated to 80°C for 2h and cooled to rt, diluted with water and acetic acid and then extracted with ethyl acetate. The extract was washed with water, brine, dried over MgSO 4 , filtered and concentrated. The crude was crystallized from ethyl acetate and hexanes to give the title compound as a white powdery solid (189mg, 94% yield). MP 238.8°C .
  • Step 1 Preparation of Intermediate 6: To a solution of bromide 3 (lllmg, 0.36mmol) dissolved in CH 2 C1 2 (2 mL) was added Br 2 PPh 3 (227mg, 0.54mmol) at 0°C and the mixture was stirred at 0°C under N 2 for lh. To the solution was added a solution of 4,6-dimethyl-2-mercaptopyrimidine in DMF (IM, 0.54mL) and diisopropylethylamine (93uL) and the mixture stirred at rt for 30 min, diluted with water and extracted with ethyl acetate. The extract was washed with brine, dried over MgS0 4 , filtered and concentrated.
  • IM 4,6-dimethyl-2-mercaptopyrimidine
  • 93uL diisopropylethylamine
  • Step 2 Preparation of Intermediate 5: Bromide 6 from above was reacted with 3,4-dimethoxyphenylboronic acid in a similar manner to that used to prepare compound 4, to yield compound 5.
  • Examples 27, 32, 34, 39, 42, 44, 49, 52, 54, 58, 59, 63, 73, 84, and 101 were prepared using technology similar to that described above.
  • Examples 38, 74, and 76 were prepared in a similar manner.
  • Step 1 One pot preparation of 4-trimethylsilylphenylboronic acid (see: Kaufmann et al, (1987) Chem. Ber. 120, 901): To a solution of p- dibromobenzene (11.8g, 50mmol) in THF (250mL) at -78°C was added n- BuLi (20mL, 2.5M in hexanes) over 2 min and the mixture was stirred at -78°C for 2 min. TMSC1 (6.3mL, 50mmol) was added in one portion and the resultant mixture was stirred at -78° C for 10 min.
  • Step 2 Preparation of Intermediate 10.
  • the boronic acid (4.96g, 25.5mmol) from Step 1 was reacted with 2-bromo-3- hydroxymethylthiophene (4.7g, 24.3mmol) (prepared from 3- thiophenemethanol with NBS in THF) under the standard Suzuki coupling conditions to yield intermediate 10 in 73% yield.
  • ⁇ H NMR 400MHz, acetone-d 6 ): ⁇ 7.62 (d, 2H), 7.54 (d, 2H), 7.39 (d, IH), 7.21 (d, IH), 4.62 (d, 2H), 4.17 (t, IH, OH), 0.29 (s, 9H).
  • Step 3 Preparation of Intermediate 11 Compound 10 (4.6g, 17.5mmol) in THF (80mL) was treated with NBS (6.25g) at rt for 2h and quenched with 10% Na 2 S 2 0 3 and then extracted with ethyl acetate. The extract was washed with water, brine, dried over MgS0 4 , filtered and concentrated. The residue was purified by flash chromatography (eluting with 20% ethyl acetate in hexanes) to furnish 5.42g (90% yield) of bromide 11.
  • Step 4 Preparation of Intermediate 12: Bromide 11 (2g, 5.86mmol) was reacted with 3,4-dimethoxyphenylboronic acid (1.28g, 7mmol) under the standard Suzuki coupling reaction conditions to afford intermediate 12 (2.2g, 94% yield) as a white solid.
  • Step 5 Preparation of Intermediate 13: Compound 12 (1.41g, 3.54mmol) was treated with a solution of IC1 (8.85mL, IM in CH 2 C1 2 ) at rt for lh and quenched with 10% Na 2 S 2 0 3 . The mixture was extracted with CH 2 C1 2 and the extract was concentrated. The residue was purified by flash chromatography (40% ethyl acetate, 10% CH 2 C1 2 in hexanes) to give iodide 13 (1.3g, 81% yield) as a light brown solid.
  • Step 6 Preparation of Intermediate 14: To a solution of iodide 13 (lg, 2.21mmol) in CH 2 C1 2 was added Br 2 PPh 3 (1.12g, 2.65mmol) and the mixture stirred at rt for 30 min. 4,6-Dimethyl-2-mercaptopyrimidine (619mg, 4.42mmol) and diisopropylethylamine (1.54mL) were introduced and the mixture was stirred at rt for lh, concentrated and the residue purified by flash chromatography (40% ethyl acetate in hexanes) to give compound 14 (1.28g, 100% yield) as a light yellow solid.
  • Step 7 To a solution of iodide 14 (68mg) and cyclobutanone (20 ⁇ L) in ether (2mL) and THF (2mL) cooled to -100°C was added n-BuLi (O.llmL) and the mixture was allowed to warm to -70°C, quenched with water and extracted with ethyl acetate. The extract was washed with brine, dried over MgS0 4 , filtered, concentrated and the crude was purified by flash chromatography (2:1 hexanes/ethyl acetate) to yield the title compound (52mg, 85% yield) as a white solid. ⁇ NMR: see Table 1.
  • Example 21 5-(3.4-Dimethoxyphenyl)-3-(4.6-dimethylpyrimidin-2- yl,thiomethyl-2-(4-methylthiophenyl)thiophene Step 1.
  • Preparation of Intermediate 7 A mixture of thioanisolyl-4- boronic acid (2.2g, 13mmol) (see: Santucci et al, (1958) J. Am. Chem. Soc.
  • Step 2 Preparation of Intermediate 8: The product from Step 1 was dissolved in 50mL of THF/1N HCl (4/1) and heated to reflux for 3h and cooled to rt. To the mixture was then added NBS (4.53g) and the resultant solution stirred at rt for 2h and quenched with 5% Na 2 S 2 O 3 and then extracted with ethyl acetate (3 x 50mL). The extracts were combined, washed with water, brine, dried over MgS0 4 , filtered and concentrated. The crude was purified by flash chromatography (5% ethanol in ethyl acetate) to yield 1.8g of sulfoxide 8.
  • Step 3 Preparation of Intermediate 9: To a solution of sulfoxide 8 (1.8g, 6mmol) in CH 2 C1 2 (40mL) was added Br 2 PPh 3 (5.08g, 12mmol) at rt and the mixture stirred at rt for lh. To the mixture was then added 4,6- dimethyl-2-mercaptopyrimidine (2.52g, 18mmol) and diisopropylethylamine (5.2mL, 30mmol) and the mixture stirred at rt for lh and concentrated. The residue was purified by flash chromatography to yield compound 9 as a white solid (2.3g, 88% yield).
  • Step 4 A mixture of 9 (700mg, 1.6mmol), 3,4-dimethoxyphenylboronic acid (350mg, 1.92mmol), Pd(PPh 3 ) 4 (55mg, 0.048mmol) and Na 2 C0 3 (lmL, 2M) in DME (5mL) was heated to reflux for 2h and worked up as usual.
  • Examples 4, 7, 15, 24, and 28 were prepared similarly by reacting intermediate 9 with the appropriate boronic acids and further modifications.
  • Step 1 Preparation of Intermediate 15: To a solution of compound 2 (4g) in THF (80mL) was added MeMgBr (51mL, 3M in THF) in THF (80mL) at 0°C under N 2 and the resulting mixture was stirred at that temperature for 1 h, quenched with IN HCl and extracted with ethyl acetate. The crude was purified by flash chromatography (2:1 hexanes/ethyl acetate) to give 3g (79%) of product 15.
  • Step 2 Preparation of Intermediate 16: Compound 15 (3.0g) was dissolved in THF (lOOmL) and cooled to 0°C. To the solution was added NBS (4.3g) and H 2 0 (lmL) and the mixture stirred at 0°C for lh, quenched with Na 2 S 2 0 3 and NaHC0 3 and extracted with ethyl acetate. The crude product was purified by flash chromatography (2:1 hexanes/ethyl acetate) to yield bromide 16.
  • Step 3 Preparation of Intermediate 17: To a solution of bromide 16 (2.6g) in THF (lOmL) and CH 2 C1 2 (lOmL) at 0°C was added MsCl (0.54mL) and diisopropylethylamine (1.33mL) and the mixture was stirred at 0°C for 2h. 4,6-Dimethyl-2-mercaptopyrimidine (1.36g) was added followed by diisopropylethylamine (3.63mL) and the mixture was stirred for an additional 15 min at 0°C and then at rt for 20 min.
  • Example 26 A mixture of the bromide 17 (983mg), 3-allyloxy- phenylboronic acid (623mg, prepared by reacting 3-bromophenol allyl ether with n-BuLi and triisopropylborate), Pd(PPh 3 ) 4 (81mg) and Na 2 C0 3 (2.3mL, 2M) in DME was deoxygenated under nitrogen and then heated to reflux overnight. The mixture was cooled to rt and worked up as usual.
  • Step 1 Preparation of 3-hydroxymethylthiophene-2-boronic acid (1): To a solution of 2-(dihydroxyboranyl)thiophene-3-carboxaldehyde (prepared from thiophene-3-carboxaldehyde dimethyl acetal by a modified literature procedure, see: Gronowitz, S. et al, (1967) Ada Chem. Scand. 21, 2151) (3.34g, 0.214mol) in ethanol (20mL) at 0°C under nitrogen was added NaBH 4 (0.81g, 0.21mol) in portions in 10 min. After stirring for 30 min at 0°C, the mixture was quenched with water and NH 4 C1 (sat'd aq.).
  • 2-(dihydroxyboranyl)thiophene-3-carboxaldehyde prepared from thiophene-3-carboxaldehyde dimethyl acetal by a modified literature procedure, see: Gronowitz, S. et al, (1967) Ada Chem
  • Step 2 Preparation of Resin B: Reaction of 1 with polymer bound (Wang resin) 4-bromobenzoate (resin A): To a suspension of the resin (6.16g, 5.3 mmol, 0.86mmol g loading) in DME (30mL) was added boronic acid 1 (1.67g, 10.6mmol), Pd(PPh 3 ) 4 (184mg, O.l ⁇ mmol) and Na 2 C0 3 (2M solution, 5.3mL) and the mixture was deoxygenated under a stream of nitrogen for 5 min under gentle stirring and then heated to 85° C under nitrogen overnight. The mixture was filtered when hot and the resin washed sequentially with DMF (3x), DMF/H 2 0 (3x), DMF (2x), THF (2x) and then MeOH (3x) and dried under nitrogen flow for 48 h to yield resin B.
  • boronic acid 1 (1.67g, 10.6mmol
  • Pd(PPh 3 ) 4 184mg, O.l
  • Step 3 Preparation of Resin C: Resin B was suspended in 60mL of THF and cooled to 0°C. NBS (1.9g, 10.6mmol) was added followed by lmL H 2 O and the mixture was allowed to warm to rt for 11/2 h and filtered. The resin was then washed with THF (3x), DMF (3x), THF (2x) and MeOH (3x) and dried under nitrogen and then under vacuum to give resin C.
  • NBS 1.9g, 10.6mmol
  • Step 4 Preparation of 3-Cyclopentyloxy-4-methoxyphenylboronic acid: To a solution of 4-bromo-2-cyclopentyloxy-l-methoxybenzene (3.4g, 12.5mmol) (see: Meyer, A. I. et al, (1993) J. Org. Chem. 58, 36) in THF (60mL) at -78°C was added n-BuLi (5.2mL, 2.4M in hexanes) over 2 min and the resultant solution stirred at -78°C for 5 min.
  • Step 6 Preparation of Resin E: Converting CH 2 OH to CH 2 Br: To a suspension of resin D (2.9g) in dichloromethane (30mL) under N 2 at 0°C was added Br 2 PPh 3 (1.5g, 3.48mmol) and the mixture was allowed to warm to rt with gentle stirring and then filtered. The residual resin was washed with CH 2 C1 2 (3x), THF (3x), ethyl acetate (2x) and ether (3x), and dried under reduced pressure to give resin E.
  • Step 7 To a suspension of resin E (50mg) in DMF (0.5mL) in a 5-mL fritted polypropylene tube equipped with a TeflonTM stopcock was added a solution of 4,6-dimethyl-2-mercaptopyrimidine in DMF( 200 ⁇ L, IM) and diisopropylethylamine (50 ⁇ L) and the mixture was shaken for 1 h at rt. The solvent was drained and the residue washed with DMF (3x), THF (3x), MeOH (3x ) and CH 2 C1 2 (3x) and the resultant resin was then treated with lmL 20%TFA in CH 2 C1 2 (containing 5% dimethyl sulfide) for 30 min. The liquid was drained to a round bottom
  • Examples 27, 82, 83, 86, 94, 95, 96, 97, 100, 102, 103, 104, 108, 114, 115, and 116 were prepared similarly.
  • Examples 14, 40, 43, 48, 50, 78, 80, 81, 85, 87, 89, and 90 were synthesized from the corresponding polymer (Merrifield or Wang resins) bound bromides according to similar procedures as described for Example 35 except for the cleavage.
  • the Cleavage were carried out using MeMgBr (1.4M in THF/toluene, 20eq) in THF at room temperature for 2-12h and filtered through fritted polypropylene reservoirs. The filtrates were quenched with NH 4 C1 (saturated aq.) and extracted with ethyl acetate. The crude products were purified by preparative TLC.
  • Step 1 Preparation of boronic acid 18: To a solution of 3-trimethylsilyl- thiophene (1.56g) in THF (15mL) at -78°C was added a THF solution of LDA (pre-prepared from 1.68mL of diisopropylamine and 4.6mL 2.4M n- BuLi in THF) via a cannula and the resultant solution was stirred at - 78°C for 10 min, rt for 30 min and cooled to -78°C again.
  • LDA pre-prepared from 1.68mL of diisopropylamine and 4.6mL 2.4M n- BuLi in THF
  • Triisopropylborate (1.37mL) was added and stirring was continued for lh at -78° C and rt for 30 min. The mixture was then quenched with H 2 0 and partitioned between hexanes and H 2 0. The aqueous phase was acidified with acetic acid to pH ⁇ 5 and extracted with ethyl acetate (3x). The extracts were concentrated and the crude was recrystallized from acetone/H 2 0 to yield 18 (1.41g) as a white powder. ⁇ NMR (400MHz, acetone-d 6 ): 57.79 (m, 2H), 7.25 (bs, 2H), 0.25 (s, 9H).
  • Step 3 Preparation of Intermediate 20: A solution of 19 (470mg), NBS (550mg) and AcOH (lmL) in THF was heated to reflux for 2h and cooled to rt. The mixture was treated with aqueous Na 2 S 2 O 3 and extracted with ethyl acetate. The extract was concentrated and the crude purified by flash chromatography to give compound 20. ⁇ NMR (CDC1 3 , 400MHz): 58.05 (d, 2H), 7.53 (d, 2H), 7.18 (s, IH), 4.36 (q, 2H), 1.40 (t, 3H).
  • Step 4 A mixture of 20 (303mg), 3-cyclopentyloxy-4- methoxyphenylboronic acid (373mg), Pd(PPh 3 ) 4 (27mg) and Na 2 CO 3 (0.8mL, 2M) in DME (7mL) was deoxygenated under N 2 and heated to reflux for 5h and worked up as usual. Purification of the crude by flash chromatography failed to give compound 21 in its pure form. As a result, the fraction containing 21 (180mg) was dissolved in dioxane (lmL) and H 2 0 (lmL) containing LiOH monohydrate (46mg) and the mixture was heated to 70° C for 2h and cooled to rt.
  • Example 110 3-(3-Cvclopentyloxy-4-methoxy)benzyl-2-r4-(l-hydroxy-l- methylethyl)phenyl.-5-(3-pyridin-l-yl)thiophene
  • Step 1 Preparation of Resin F: A suspension of resin C (4.3g), lithium pyridine-3-trimethylboronate (1.81g) (see: Fischer et al, (1974) Reel Tra ⁇ .
  • Step 2 Preparation of Resin G: To a suspension of resin F (4.3g) in CH 2 C1 2 (30mL) was added Br 2 PPh 3 (2.18g) and the suspension was stirred at rt for lh and filtered. The residue was washed with CH 2 C1 2 (3x), DMF (3x), THF(2x), ethyl acetate (2x) and ether (2x), and then dried under reduced pressure to afford resin G. Step 3.
  • CHO-Kl cells stably expressing the prostacyclin receptor and grown under G418 selection as described previously (Y. Boie, et al. J. Biol. Chem.: 269, 12173-12178, 1994) were plated at a density of 1.75 x 10 6 cells/175cm 2 in a T-175 flask (Gibco, Burlington, VT) containing alpha MEM media; 10% heat inactivated fetal bovine serum (FBS); 1% (v/v) penicillin/streptomycin; 25 mM Hepes, pH 7.4; and 500 ⁇ g/ml G418 (complete media). The cells were placed in an incubator for 24 hr at 37°C and 5% C0 .
  • the cells were then washed with warmed sterile phosphate buffered saline (PBS) and incubated with 2 ⁇ g/ml DNA, and 9 ⁇ g/ml lipofectamine reagent in Opti-MEM for 7 hr. At 37°C and 5% C0 2 .
  • the incubation solution was diluted 1:2 with Opti-MEM containing 20% FBS and incubated overnight. Following the overnight incubation, the media was replaced by complete media containing 500 ⁇ g/ml hygromycin B. Colonies were identified and grown in T-175 flasks for further characterization.
  • the flasks were maintained in an incubator at 37°C with 5.0% C0 2 for 72 hr.
  • the media was changed and the cells were allowed to grow overnight.
  • the cells were washed and dissociated from the plate with PBS containing 0.5 mM EDTA.
  • Cellular cAMP content was measured by centrifuging the cell suspension at 150 g x 10 min. And resuspending the cells in a Hanks buffered salt solution at a density of 0.2 x 10 cells/ml.
  • the cells were preincubated at room temperature for 15 min. and then incubated with 10 ⁇ M prostaglandin I 2 (PGI 2 ) and the indicated compound for an additional 10 min.
  • Basal cAMP levels were determined by incubating the cells in 0.1% DMSO. The incubations were terminated by the addition of HCl (0.1 N final) and the cells measured for cAMP as described below.
  • Determinations of whole-cell cAMP content were performed by incubating 100 ⁇ l reconstituted rabbit anti-succinyl cAMP serum with 100 ⁇ l of the whole-cell reaction or known cAMP standard and 30 pmol of 125 I-cAMP TME in a ScintiStripTM well (300 ⁇ l final volume) at room temperature for 18 h. Total cpm (B 0 ) was determined in the absence of sample of cAMP standard. The reaction mixture was then aspirated out of the well, and the individual wells were counted in a Beckman LS 6000SC with the window open from 10-999 for 1 min.
  • %B/B 0 [(standard or sample cpm - non-specific cpm) / (B 0 cpm - non-specific cpm)] x 100.
  • Non-specific cpm were determined by incubating only the 125 I-cAMP TME with assay buffer (50 nM acetate; pH 5.8) in the ScintiStrip well. All determinations were performed in triplicate.
  • the most potent compounds according to the invention induced a concentration-dependent elevation of cAMP in neutrophils and/or eosinophils at concentrations of O.lnM to l ⁇ M.
  • Human Whole Blood Assay Fresh blood was collected in heparinized tubes by venipuncture from healthy volunteers. These subjects had no apparent inflammatory conditions and had not taken any NSAIDs for at least 4 days prior to blood collection. Five hundred ⁇ L aliquots of human blood were initially pre-incubated at 37°C with either 2 ⁇ L DMSO (vehicle) or 2 ⁇ L of a test compound at a final concentration of up to 100 ⁇ M.
  • LPS lipopolysaccharide
  • f-MLP n-formyl-Met-Leu-Phe
  • f-MLP n-formyl-Met-Leu-Phe
  • the blood was immediately centrifuged at 4°C for 10 min. at 3,300 rpms to obtain plasma.
  • a plasma aliquot was diluted in PBS and assayed for TNF- ⁇ using a commercial ELISA kit (Cistron).
  • An additional plasma aliquot was de-proteinized with methanol and the supernatant was assayed for LTB 4 using a commercial EIA kit (Cayman).
  • the instant compounds showed IC 50 values ranging from 1 nM to 5 ⁇ M.
  • Fresh blood was collected from healthy volunteers by venipuncture into tubes containing 0.13M sodium citrate as anticoagulant (final 10%v/v in blood).
  • the blood was diluted in equal parts with PBS and gently layered on top of one half volume histopaque (1.077 density) and was centrifuged at 1400 rpm for 35 min at room temperature. After centrifugation, a distinct layer of mononuclear cells (monocytes and lymphocytes) located between the blood and histopaque layers could be aspirated off using a transfer pipette.
  • the mononuclear cells were washed in calcium and magnesium free PBS.
  • the cell pellet was resuspended in RPMI 1640 (Gibco BRL) complete media (containing streptomycin/penicillin and HEPES buffer) at a cell density of 1 x 10 6 cells/ml.
  • RPMI 1640 Gibco BRL
  • DMSO fetal sulfate
  • test compound containing streptomycin/penicillin and HEPES buffer
  • DMSO fetal sulfate
  • test compound containing streptomycin/penicillin and HEPES buffer
  • the instant compounds showed IC 50 values ranging from 0.1 nM to 5 ⁇ M.
  • Guinea pigs 200 g are sensitized with a lOO ⁇ g/ml ovalbumin in an A1 2 0 3 suspension in physiological saline. Five hundred ⁇ l of this solution are injected mtraperitoneally and another 500 ⁇ l are injected in 6 ganglionic regions ( ⁇ 75 ⁇ l/ site). The animals are then housed for 4 to 6 weeks. Thirty minutes prior to the experimentation, the guinea pigs are treated with the test compound or vehicle and with mepyramine maleate, 1 mg/kg. The injection volume is 1 ml/kg of body weight.
  • test compound dissolved in 2 ul DMSO
  • substrate buffer containing [2,8- 3 H] adenosine 3',5'-cyclic phosphate (cAMP, 100 nM)
  • cAMP adenosine 3',5'-cyclic phosphate
  • EDTA 50 mM Tris, pH 7.5.
  • the reaction was initiated by the addition of 10 ⁇ l of human recombinant PDE-rV isozymes, either expressed and purified from sf9 cells, or from CHO-Kl cells (the amount was controlled so that ⁇ 10% product was formed in 10 min. at 30 °C).
  • the reaction was stopped after 10 min. by the addition of 1 mg of PDE-SPA beads (Amersham).
  • the product AMP generated was quantified on a Microbeta 96-well plate counter.
  • the signal in the absence of enzyme was defined as the background. 100% activity was defined as the signal detected in the presence of enzyme and DMSO with the background subtracted. Percentage of inhibition was calculated accordingly.
  • the IC50 value was approximated by the non-linear regression fitting of a ten point titration using the standard 4 parameter equation.
  • IC50 values were determined with 100 nM cAMP using the purified GST fusion protein of the human recombinant phosphodiesterase IVa (met-248) produced from a baculovirus/Sf-9 expression system. The instant compounds were shown to have IC 50 values of 0.01 to 1000 nM.

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Abstract

L'invention concerne un nouveau composé de formule (I) servant dans le traitement de maladies telles que l'asthme. Ce composé élève le taux de l'adénosine-3',5'-monosphophate cyclique (cAMP) par inhibition de la phosphodiestérase IV (PDE IV). L'invention concerne également l'utilisation des composés de formule (I) dans certaines compositions pharmaceutiques et dans des méthodes de traitement de maladies par inhibition de la PDE IV, et par conséquent élévation du taux de cAMP.
EP98946190A 1997-10-03 1998-10-01 Derives d'arylthiophene utilises comme inhibiteurs de la phosphodiesterase iv Withdrawn EP1019399A1 (fr)

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GBGB9808109.4A GB9808109D0 (en) 1998-04-16 1998-04-16 Aryl thiophene derivatives as pde iv inhibitors
PCT/CA1998/000931 WO1999018099A1 (fr) 1997-10-03 1998-10-01 Derives d'arylthiophene utilises comme inhibiteurs de la phosphodiesterase iv

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CA2305414A1 (fr) 1999-04-15
JP2001519347A (ja) 2001-10-23
JP3409029B2 (ja) 2003-05-19
AU9334798A (en) 1999-04-27
WO1999018099B1 (fr) 1999-05-27
AU732406B2 (en) 2001-04-26
WO1999018099A1 (fr) 1999-04-15

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