EP0641344A1 - Xanthines 8-substituees utilisees en tant qu'inhibiteurs de la phosphodiesterase - Google Patents

Xanthines 8-substituees utilisees en tant qu'inhibiteurs de la phosphodiesterase

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Publication number
EP0641344A1
EP0641344A1 EP93910221A EP93910221A EP0641344A1 EP 0641344 A1 EP0641344 A1 EP 0641344A1 EP 93910221 A EP93910221 A EP 93910221A EP 93910221 A EP93910221 A EP 93910221A EP 0641344 A1 EP0641344 A1 EP 0641344A1
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European Patent Office
Prior art keywords
formula
compound
pharmaceutically acceptable
group
substituted
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EP93910221A
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German (de)
English (en)
Inventor
Ashley Edward Smithkline Beecham Pharm. Fenwick
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SmithKline Beecham Ltd
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SmithKline Beecham Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention relates to certain novel compounds having pharmacological activity, to a process for the preparation of such compounds, to pharmaceutical compositions containing such compounds and to the use of such compounds and compositions in medicine.
  • European Patent Application, Publication Number 0369744 also discloses certain 1,3- or 1,3,7- 8-H cycloalkylalkylene xanthines, for use inter alia as bronchodilators in the treatment of asthma.
  • These compounds are also indicated to have bronchodilator activity and thus to be of potential use in the treatment of disorders of the respiratory tract, such as reversible airways obstruction and asthma. These compounds also have a protective effect against the consequences of cerebral metabolic inhibition.
  • the said compounds improve data acquisition or retrieval following transient forebrain ischaemia and are therefore useful in the treatment of cerebral vascular and neuronal degenerative disorders associated with learning, memory and cognitive dysfunctions including cerebral senility, multi-infarct dementia, senile dementia of the Alzheimer type, age associated memory impairment and certain disorders associated with Parkinson's disease.
  • These compounds are also indicated to have neuroprotectant activity. They are therefore useful in the prophylaxis of disorders associated with neuronal degeneration resulting from ischaemic events, including cerebral ischaemia due to cardiac arrest, stroke and also after cerebral ischaemic events such as those resulting from surgery and/or during childbirth.
  • treatment with the compound is indicated to be of benefit for the treatment of functional disorders resulting from disturbed brain function following ischaemia.
  • the present compounds are also considered to be inhibitors of the in vivo production of Tumor Necrosis Factor (TNF) and hence they have potential for the treatment of diseases associated with excessive or unregulated TNF production including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resoiption diseases, reperfusion injury, graft vs.
  • TNF Tumor Necrosis Factor
  • the present compounds are also useful in the treatment of viral infections that produce TNF as a result of infection, or those which are sensitive to inhibition, such as by decreased replication, directly or indirectly, by the present compounds.
  • viruses include for example HIV-1, HIV-2 and HIV-3, Cytomegalovirus (CMV), Influenza, adenovirus and the Herpes group of viruses, such as but not limited to, Herpes Zoster and Herpes Simplex.
  • TNF mediated viral infections including, for example, feline immunodeficiency virus (FIV) or other retroviral infections such as equine infectious anaemia virus, caprine arthritis virus, visna virus, maedi virus and other lentiviruses.
  • FMV feline immunodeficiency virus
  • retroviral infections such as equine infectious anaemia virus, caprine arthritis virus, visna virus, maedi virus and other lentiviruses.
  • R- and R ⁇ each independently represent a moiety of formula (a):
  • m represents zero or an integer 1, 2 or 3 and A represents a substituted or unsubstituted cyclic hydrocarbon radical;
  • R represents hydrogen, substituted or unsubstituted alkyl or an aralkyl group substituted or unsubstituted in the aryl moiety; and R represents hydrogen, alkyl or alkylcarbonyl.
  • A is unsubstituted.
  • A represents a substituted or unsubstituted C3 ⁇ g cycloalkyl group, especially a C3-6 cycloalkyl group.
  • A represents a substituted or, preferably, unsubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group.
  • A represents a cyclopropyl group or a cyclobutyl group.
  • A represents a cyclopropyl group.
  • R3 represents unsubstituted alkyl
  • suitable examples include methyl.
  • R3 represents substituted alkyl
  • suitable examples include alkoxy methyl such as methoxymethyl.
  • R ⁇ represents an aralkyl group, for example a benzyl group, substituted or unsubstitued. in the aryl moiety.
  • R ⁇ is a benzyl group
  • examples include unsubstituted benzyl or benzyl substituted in the phenyl moiety by methoxy groups, particular examples include 4-methoxy benzyl.
  • R ⁇ represents hydrogen
  • R ⁇ represents alkylcarbonyl
  • alkycarbonyl groups are C ⁇ _4 alkylcarbonyl groups for example acetyl.
  • Suitable pharmaceutically acceptable salts are pharmaceutically acceptable base salts and pharmaceutically acceptable acid addition salts.
  • Suitable pharmaceutically acceptable base salts of the compounds of formula (I) include 7-N base salts including metal salts, such as alkali metal salts for example sodium salts, or organic amine salts such as that provided with ethylenediamine.
  • Suitable acid addition salts of the compounds of formula (I) are the acid addition salts including pharmaceutically acceptable inorganic salts such as the sulphate, nitrate, phosphate, borate, hydrochloride and hydrobromide and pharmaceutically acceptable organic acid addition salts such as acetate, tartrate, maleate, citrate, succinate, benzoate, ascorbate, methanesulphonate, ⁇ -keto glutarate, ⁇ -glycerophosphate and glucose- 1- phosphate.
  • the acid addition salt is a hydrochloride salt.
  • 'cyclic hydrocarbon radical' includes single ring and fused ring, alicyclic hydrocarbons comprising up to 8 carbon atoms in each ring, suitably up to 6 carbon atoms, for example 3, 4, 5 or 6 carbon atoms.
  • Suitable optional substituents for any cyclic hydrocarbon radical includes a C ⁇ - alkyl group or a halogen atom.
  • the term 'aryl' whether used alone or as part of another group includes phenyl and naphthyl optionally substituted with up to five, preferably up to three, groups selected from halogen, alkyl, phenyl, alkoxy, halo alkyl, hydroxy, amino, nitro, carboxy, alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, or alkylcarbonyl groups.
  • Optional substituents for any phenylene group include up to three of the substituents mentioned in relation to the aryl group.
  • Suitable optional substituents for the aryl moiety of any aralkyl group include those mentioned above in regard to the v aryl' group and in particular include alkoxy groups, for example methoxy groups.
  • alkyl' when used herein the term 'alkyl' whether used alone or when used as part of another group (for example as in an alkylcarbonyl group) includes straight and branched chain alkyl groups, containing from 1 to 12 carbon atoms, suitably 1 to 6 carbon atoms, for example methyl, ethyl, propyl or butyl. Suitable optional substituents for any alkyl group include up to five, preferably up to three of the substituents mentioned above in relation to the aryl group.
  • proliferative skin diseases means benign and malignant proliferative skin diseases which are characterized by accelerated cell division in the epidermis, dermis or appendages thereto, associated with incomplete tissue differentiation.
  • diseases include: psoriasis, atopic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, allergic contact dermatitis, basal and squamous cell carcinomas of the skin, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant sun induced keratosis, non-malignant keratosis, acne, and seborrheic dermatitis in humans and atopic dermatitis and mange in domesticated animals.
  • the compounds of formula (I) are preferably in pharmaceutically acceptable form.
  • pharmaceutically acceptable form is meant, inter alia, of a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels.
  • a pharmaceutically acceptable level of purity will generally be at least 50% excluding normal pharmaceutical additives, preferably 75%, more preferably 90% and still more preferably 95%.
  • the invention further provides a process for the preparation of a compound of formula (I), which process comprises reacting a compound of formula (II):
  • R- 3 - represents R- as defined in relation to formula (I) or a group convertible to R-
  • R ⁇ a represents R ⁇ as defined in relation to formula (I) or a group convertible thereto
  • R ⁇ a represents R ⁇ as defined in relation to formula (I) or a group convertible to R ⁇ >
  • R ⁇ represents a hydroxy protecting group and L represents a leaving group
  • a suitable leaving group L 1 is a halo atom, especially an iodine atom.
  • reaction between compounds of formulae (II) and (HI) may be carried out using conventional alkylation conditions, for example in an aprotic solvent such as dimethoxyethane, dimethylformamide or tetrahydrofuran, at any temperature providing a suitable rate of formation of the required product, such as in the range of from 0°C to 100°C, conveniently in the range of from 40°C to 80°C, for example 60°C; and preferably in an inert atmosphere such as nitrogen.
  • an aprotic solvent such as dimethoxyethane, dimethylformamide or tetrahydrofuran
  • the 8-amino group of compound (II) is in an activated form, favourably in an ionic form such as a salted form, for example an alkali metal salted form provided by treating the compound of formula (II) with an alkali metal base, for example potassium t-butoxide.
  • an ionic form such as a salted form, for example an alkali metal salted form provided by treating the compound of formula (II) with an alkali metal base, for example potassium t-butoxide.
  • a compound of formula (II) may be prepared using methods described in European Patent Application, Publication No. 0389282.
  • the compounds of formula (III) are known compounds or they may be prepared according to methods used to prepare known compounds, for example those discussed in Tetrahedron (1990), 46, 6903.
  • Conversions of one compound of formula (I) into another compound of formula (I) includes converting one group R ⁇ into another group R ⁇ , for example hydrolysing compounds wherein R ⁇ represents an alkylcarbonyl group into a compound of formula (I) wherein R ⁇ represents a hydrogen atom.
  • Suitable values for R* a and R 2a include R* and R 2 respectively or nitrogen protecting groups such as benzyl, nitrobenzyl or trimethoxybenzyl groups.
  • Suitable values for R ⁇ a include R ⁇ .
  • R* a and R 2a represent nitrogen protecting groups which can be inserted and removed without affecting Rj, for example trimethylsilyl groups.
  • R ⁇ is substituted or unsubstituted aralkyl
  • R* a is R ⁇ and R 2a is R 2 .
  • Rl a , R a or R ⁇ a represents other than R-, R 2 or R ⁇ repectively
  • the abovementioned conversions of R ⁇ a into R-, R 2a to R 2 and R ⁇ a into R-* may be carried out using the appropriate conventional procedure.
  • R a (or R a ) represents a nitrogen protecting group, such as a benzyl group
  • the protecting group may be removed using the appropriate conventional procedure, such as catalytic hydrogenation, and the resulting product reacted with a compound of formula (IV): X-(CH 2 ) m -A (IV)
  • a and m are as defined in relation to formula (IA) and X represents a leaving group, such as halide, for example bromide or iodide.
  • any reactive group or atom such as the xanthine nitrogen atom may be carried out at any appropriate stage in the aforementioned process.
  • Suitable protecting groups include those used conventionally in the art for the particular group or atom being protected, for example suitable protecting groups for the xanthine nitrogen atoms are alkylsilyl groups, especially trimethylsilyl or I-butyldimethylsilyl groups.
  • alkylsilyl protecting groups may be prepared and removed using the appropriate conventional procedure: For example, alkylsilyl protecting groups may be prepared by treating the compound of formula (II) with an appropriate alkylsilyl halide, for example trimethylsilyl chloride for trimethylsilyl groups and i- butyldimethylsilyl chloride for t-butyldimethylsilyl groups.
  • the silyl protecting groups may be removed by treatment with t-butylammonium fluoride in a suitable solvent, such as tetrahydrofuran conveniently at an ambient temperature.
  • the present invention accordingly provides a compound of formula (I) or where appropriate a pharmaceutically acceptable salt thereof and/or a pharmaceutically acceptable solvate thereof, for use as an active therapeutic substance.
  • the present invention provides a compound of formula (I) or where appropriate a pharmaceutically acceptable salt thereof and/or a pharmaceutically acceptable solvate thereof, for use in the treatment of and/or prophylaxis of disorders associated with increased numbers of eosinophils, such as asthma, and allergic disorders associated with atopy, such as urticaria, eczema and rhinitis.
  • the present invention also provides a compound of formula (I) or where appropriate a pharmaceutically acceptable salt thereof and/or a Opharmaceutically acceptable solvate thereof, for use as a phosphodiesterase inhibitor.
  • the present invention provides a compound of formula (I) or where appropriate a pharmaceutically acceptable salt thereof and or a pharmaceutically acceptable solvate thereof, for use in the treatment of disorders of the respiratory tract, such as reversible airways obstruction and asthma.
  • the present invention provides a compound of formula (I) or where appropriate a pharmaceutically acceptable salt thereof and/or a pharmaceutically acceptable solvate thereof, for use in the treatments mentioned hereinbefore, such as cerebral vascular and neuronal denerative disorders associated with learning, memory and cognitive dysfunctions, peripheral vascular disease or proliferate skin disease or for the prophylaxis of disorders associated with neuronal degeneration resulting from ischaemic events.
  • TNF Tumor Necrosis Factor
  • TNF production Diseases associated with excessive or unregulated TNF production include rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resorption diseases, reperfusion injury, graft vs.
  • allograft rejections fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia, secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.
  • AIDS cachexia secondary to infection or malignancy
  • cachexia secondary to acquired immune deficiency syndrome
  • AIDS AIDS
  • ARC AIDS related complex
  • keloid formation scar tissue formation
  • Crohn's disease Crohn's disease
  • ulcerative colitis or pyresis.
  • viruses include for example HIV-1 , HIV-2 and HIV-3, Cytomegalovirus (CMV), Influenza, adenovirus and the Herpes group of viruses, such as but not limited to, Herpes Zoster and Herpes Simplex.
  • a compound of formula (I) or where appropriate a pharmaceutically acceptable salt thereof and/or a pharmaceutically acceptable solvate thereof, may be administered per j ⁇ e or, preferably, as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) or where appropriate a pharmaceutically acceptable salt thereof and/or a pharmaceutically acceptable solvate thereof, and a pharmaceutically acceptable carrier.
  • the active compound may be formulated for administration by any suitable route, the preferred route depending upon the disorder for which treatment is required, and is preferably in unit dosage form or in a form that a human patient may administer to himself in a single dosage.
  • the composition is suitable for oral, rectal, topical, parenteral, intravenous or intramuscular administration or through the respiratory tract. Preparations may be designed to give slow release of the active ingredient.
  • compositions of the invention may be in the form of tablets, capsules, sachets, vials, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid preparations such as oral or sterile parenteral solutions or suspensions. Topical formulations are also envisaged where appropriate.
  • a composition of the invention is in the form of a unit dose.
  • Unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol orglycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrtolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone
  • fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol orglycine
  • tabletting lubricants for example magnesium stearate
  • disintegrants for example starch
  • the solid oral compositions may be prepared by conventional methods of blending, filling, tabletting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
  • Oral liquid preparations may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired conventional flavouring or colouring agents.
  • suspending agents for example sorbitol, syrup, methyl cellulose,
  • compositions may also suitably be presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.
  • the particles of active compound suitably have diameters of less than 50 microns, such as from 0J to 50 microns, preferably less than 10 microns, for example from 1 to 10 microns, 1 to 5 microns or from 2 to 5 microns.
  • small amounts of other anti-asthmatics and bronchodilators for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine; corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included.
  • sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine
  • corticosteroids such as prednisolone
  • adrenal stimulants such as ACTH
  • fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, and, depending on the concentration used, can be either suspended or dissolved in the vehicle.
  • the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing.
  • adjuvants such as a local anaesthetic, a preservative and buffering agents can be dissolved in the vehicle.
  • the composition can be frozen after filling into the vial and the water removed under vacuum.
  • Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration.
  • the compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
  • compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by weight, of the active material, depending on the method of administration.
  • Topical formulations may be presented as, for instance, ointments, creams or lotions, impregnated dressings, gels, gel sticks, spray and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.
  • the formulations may contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions.
  • Suitable cream, lotion, gel, stick, ointment, spray or aerosol formulations that may be used for compounds of formula (I) or if appropriate a pharmaceutically acceptable salt thereof, are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics and cosmetics, such as Harry's Cosmeticology published by Leonard Hill Books, Remington's Pharmaceutical Sciences, and the British and US Pharmacopoeias.
  • the compound of formula (I), or if appropriate a pharmaceutically acceptable salt thereof will comprise from about 0.5 to 20% by weight of the formulation, favourably from about 1 to 10%, for example 2 to 5%.
  • suitable unit doses may be 0J to lOOOmg, such as 0.5 to 200, 0.5 to 100 or 0.5 to 10 mg, for example 0.5, 1, 2, 3, 4 or 5 mg; and such unit doses may be administered more than once a day, for example 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total daily dosage for a 70kg adult is in the range of about 0.1 to 1000 mg, that is in the range of about 0.001 to 20 mg kg day, such as 0.007 to 3, 0.007 to 1.4, 0.007 to 0J4 or 0.01 to 0.5 mg kg day, for example 0.01, 0.02, 0.04, 0.05, 0.06, 0.08, 0J or 0.2 mg/kg day; and such therapy may extend for a number of weeks or months.
  • the term 'pharmaceutically acceptable encompasses materials suitable for both human and veterinary use. No toxicological effects have been established for the compounds of formula (I) in the abovementioned dosage ranges.
  • the following pharmacological data and examples illustrate the invention.
  • the following preparations illustrate the preparation of intermediates to the novel compounds of formula (I).
  • 1,3-Di(cyclopropylmethyl)-8-(4-hydroxy-3- (hydroxymethyl)butylamino]-7-methylxanthine was prepared from 8-[4-acetoxy-3-(acetoxymethyl)butylamino]-1,3- di(cyclopropylmethyl)-7-methyl xanthine in 59% yield in an identical manner to Example 2, mp 173°C;
  • the Ca 2+ /calmodulin-stimulated PDE (PDE I, see Table 1 and Beavo and Reifsynder (1990) for nomenclature) was prepared from bovine cardiac ventricle. Following chromatography on a Mono Q column, the fractions showing stimulation of PDE activity by Ca 2+ and calmodulin were pooled and further purified on a calmodulin-affinity column.
  • cGMP-stimulated PDE (PDE II), cGMP-inhibited PDE (PDE III) and cAMP-specific PDE (PDE IV) were all isolated from guinea-pig cardiac ventricle.
  • This isoenzyme had high affinity for cAMP, the hydrolysis of which was not inhibited by cGMP. The activity of this isoenzyme was unaffected by the Ca2-calmodulin complex.
  • PDE V This isoenzyme had high affinity for cGMP. The activity of this isoenzyme was unaffected by the Ca 2+ -calmodulin complex.
  • PDE activity was assayed by the boronate column method as previously described (Reeves et al., 1987). The enzymes were assayed by incubation at 37°C for 4-30 min. in 50 mM Tris, 5 mM MgCl 2 , pH 7.5 with ⁇ H-labelled cyclic nucleotide (4 x 10 ⁇ disintegrations min ⁇ -) and l ⁇ C-labelled nucleotide 5'-monophosphate (3 x 10-* disintegrations min ⁇ l). The assay was stopped by boiling and the ⁇ H- labelled 5'-monophosphate product separated from substrate on boronate columns.
  • reaction mixture was diluted with 0.5 mL 100 mM HEPES [N-(2-hydroxyethyl)piperazine-Nl-2-ethanesulfonic acid], 100 mM NaCl, pH 8.5, and applied to the column.
  • the column was extensively washed with the same buffer, and the 5'-nucleotide eluted with 6 mL of 0.25 M acetic acid.
  • the recovery of product as judged by l ⁇ G-recovery was approximately 80%. All assays were linear with time of incubation and concentration of enzyme over the range used in these experiments.
  • IC50 values (the concentration of inhibitor required for 50% inhibition of activity) were obtained by incubation of the isoenzyme using 1 ⁇ M cGMP as a substrate for PDE I (in the absence of Ca + and calmodulin), PDE II and PDE V and with 1 ⁇ M cAMP as a substrate for PDE III and PDE IV.
  • Section I Assay set-up The effects of compounds of Formula (I) on the in vitro production of TNF by human monocytes was examined using the following protocol.
  • Human peripheral blood monocytes were isolated and purified from either blood bank buffy coats or plateletpheresis residues, according to the procedure of Colotta, R. et al., J. Immunol.. 132(2):936 (1984).
  • the monocytes were plated at a density of 1 X 10 ⁇ cells/ml medium/well in 24-well multi-dishes. The cells were allowed to adhere for 1 hour after which time the supernatant was aspirated and 1 ml fresh medium (RPMI-1640 (Whitaker Biomedical Products, Whitaker, CA) containing 1% fetal calf serum and penicillin and streptomycin at 10 units/ml was added.
  • the cells were incubated for 45 minutes in the presence or absence of test compounds at InM-lOuM dose ranges (compounds were solubilized in Dimethyl- sulfoxide/Ethanol such that the final solvent concentration in the culture medium was 0.5% Dimethyl sulfoxide/0.5% Ethanol).
  • Bacterial lipopolysaccharide E. coli 055:B5 [LPS] from Sigma Chemicals Co.
  • PBS Phosphate Buffered Saline
  • culture supematants were removed from the cells, centrifuged at 3000 revolutions per minute (rpm) to remove cell debris and .05 ml of the supernatant assayed for TNF activity using the radioimmunoassay described below.
  • the assay buffer consisted of 0.01M NaPO 0.15M NaCl, 0.025M EDTA and 0.1% sodium azide at pH 7.4.
  • Human recombinant TNF (rhTNF) obtained using the procedure of Chen et al, Nature. 330:581-583 (1987) was iodinated by a modified Chloramine-T method described in Section III below.
  • samples 50 ⁇ l culture supematants
  • rhTNF standards a 1/9000 dilution of polyclonal rabbit anti-rhTNF (Genzyme, Boston, MA) and 8000 cpm of 125 I-TNF was added in a final volume of 400 ⁇ l buffer and incubated overnight (18 hours) at 4°C.
  • Normal rabbit serum and goat anti-rabbit IgG (Calbiochem) were titrated against each other for maximum precipitation of the anti-rhTNF.
  • the appropriate dilutions of carrier normal rabbit serum (1/200), goat anti-rabbit IgG (1/4) and 25 Units heparin (Calbiochem) were allowed to precipitate and 200 ⁇ l of this complex was added per assay tube and incubated overnight at 4°C. Tubes were centrifuged for 30 minutes at 2000 rpm, supematants were carefully aspirated, and radioactivity associated with the pellets measured in a Beckman Gamma 5500 counter. The logit-log linear transformation curve was used for the calculations.
  • the concentrations of TNF in the samples was read from a standard curve of rhTNF that was linear in the 157 to 20,000 pg/ml range.
  • Section III Radioiodination of rhTNF Iodination of rhTNF was performed using a modified chloramine-T method of Frolik et al., J. Biol. Chem..259:10995-11000 (1984). Briefly, 5 mg of rhTNF in 5 ml of 20MM Tris ph 7.5, was diluted with 15 ml of 0.5M KPO4 and 10 ml of carrier free l 2 5l(100mCi/ml;ICN). To initiate the reaction, a 5ml aliquot of a lOOmg ml (aqueous) chloramine-T solution was added.
  • ⁇ I-TNF Specific activity of ⁇ I-TNF was 80- 100 mCi/mg protein.
  • Biological activity of ⁇ odinated TNF was measured by the L929 cytotoxicity assay of Neale, M.L. et al., Eur. J. Can. Clin. Oncol..25(1): 133- 137 (1989) and was found to be 80% that of unlabeled TNF.
  • TNF TNF-binding protein
  • the ELISA employed a murine monoclonal anti-human TNF antibody, described below, as the capture antibody and a polyclonal rabbit anti- human TNF , described below, as the second antibody.
  • TNF levels in samples were calculated from a standard curve generated with recombinant human TNF produced in E. Coli (obtained from SmithKline Beecham Pharmaceuticals, King of Prussia, PA, USA).
  • Section V Production of anti-human TNF antibodies: Monoclonal antibodies to human TNF were prepared from spleens of
  • mice immunized with recombinant human TNF using a modification of the method of Kohler and Millstein, Nature 22 ⁇ :495 (1975), the entire disclosure of which is hereby incorporated by reference.
  • Polyclonal rabbit anti-human TNF antibodies were prepared by repeated immunization of New Zeland White (NZW) rabbits with recombinant human TNF emulsified in complete Freund's adjuvant (DIFCO, IL., USA). Results:
  • Plasma levels of TNF are measured using a modification of the basic sandwich ELISA method described in Winston et al., Current Protocols in Molecular Biology. Pg. 11.2.1, Ausubel et al, Ed. (1987) John Wiley and Sons, New York , USA.
  • the Elisa employed a hampster monoclonal anti-mouse TNF (Genzyme, Boston, MA, USA ) as the capture antibody and a polyclonal rabbit anti-murine TNF (Genzyme, Boston, MA, USA ) as the detecting antibody.
  • TNF levels in mouse samples are calculated from a standard curve generated with recombinant murine TNF (Genzyme, Boston, MA , USA).
  • TNF levels determined by ELISA correlated with levels detected by the L929 bioassay of Ruff et al., £. Immunol. 125:1671-1677 (1980), with 1 Unit of activity in the bioassay corresponding to 70 picograms (pg) of TNF in the ELISA.
  • the ELISA detected levels of TNF down to 25 pg ml.
  • Active compounds provide a positive in-vivo response in the above noted model and for example demonstrate an ED50 for reduction of serum TNF of about 2 50 mg kg orally.

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Abstract

L'invention se rapporte à un composé représenté par la formule (I), ou éventuellement à un sel dudit composé acceptable pharmaceutiquement. Dans ladite formule, R1 et R2 représentent indépendamment chacun une fraction de formule (a): -CH¿2?)m-A, dans laquelle m représente 0 ou un entier 1, 2 ou 3 et A représente un radical d'hydrocarbure cyclique substitué ou non substitué; R?3¿ représente hydrogène, alkyle substitué ou non substitué ou un groupe aralkyle substitué ou non substitué dans la fraction aryle; R4 représente hydrogène, alkyle ou alkylcarbonyle. On décrit un procédé de préparation dudit composé, une composition pharmaceutique contenant ledit composé et l'utilisation dudit composé dans un médicament.
EP93910221A 1992-05-21 1993-05-18 Xanthines 8-substituees utilisees en tant qu'inhibiteurs de la phosphodiesterase Withdrawn EP0641344A1 (fr)

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GB929210839A GB9210839D0 (en) 1992-05-21 1992-05-21 Novel compounds
GB9210839 1992-05-21
PCT/GB1993/001014 WO1993023401A1 (fr) 1992-05-21 1993-05-18 Xanthines 8-substituees utilisees en tant qu'inhibiteurs de la phosphodiesterase

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GB9703044D0 (en) * 1997-02-14 1997-04-02 Glaxo Group Ltd Phenyl xanthine esters and amides
CN1130363C (zh) 1997-11-12 2003-12-10 三菱化学株式会社 嘌呤衍生物以及含有其作为有效成分的药物
GB9817623D0 (en) 1998-08-13 1998-10-07 Glaxo Group Ltd Pharmaceutical compounds
ATE254616T1 (de) 1999-05-11 2003-12-15 Mitsubishi Chem Corp Dihydrat eines purinderivates, medikamente die dieses als aktiven wirkstoff enthalten und zwischenprodukte zu dessen herstellung
US6403567B1 (en) 1999-06-22 2002-06-11 Cv Therapeutics, Inc. N-pyrazole A2A adenosine receptor agonists
USRE47351E1 (en) 1999-06-22 2019-04-16 Gilead Sciences, Inc. 2-(N-pyrazolo)adenosines with application as adenosine A2A receptor agonists
MXPA02002262A (es) * 1999-08-31 2002-09-30 Univ Vanderbilt Antagonistas selectivos de los receptores adenosina a2b.
US6815446B1 (en) 1999-08-31 2004-11-09 Vanderbilt University Selective antagonists of A2B adenosine receptors
AU2001238665A1 (en) 2000-02-23 2001-09-03 Cv Therapeutics, Inc. Method of identifying partial agonists of the a2a receptor
US6821978B2 (en) 2000-09-19 2004-11-23 Schering Corporation Xanthine phosphodiesterase V inhibitors
ATE395349T1 (de) 2001-08-28 2008-05-15 Schering Corp Polycyclische guanin phosphodiesterase inhibitoren
US6943171B2 (en) 2001-11-09 2005-09-13 Schering Corporation Polycyclic guanine derivative phosphodiesterase V inhibitors
EP1719772A1 (fr) 2002-05-31 2006-11-08 Schering Corporation Procédé de préparation d'inhibiteurs de la phosphodiesterase v de la xanthine et leur précurseurs
WO2004009091A1 (fr) 2002-06-17 2004-01-29 Glaxo Group Limited Agonistes des recepteurs x du foie
US20050020915A1 (en) 2002-07-29 2005-01-27 Cv Therapeutics, Inc. Myocardial perfusion imaging methods and compositions
AU2003259264A1 (en) 2002-07-29 2004-02-16 Cv Therapeutics, Inc. Myocardial perfusion imaging using a2a receptor agonists
US8470801B2 (en) 2002-07-29 2013-06-25 Gilead Sciences, Inc. Myocardial perfusion imaging methods and compositions
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CN101076343A (zh) 2004-10-20 2007-11-21 Cv医药有限公司 A2a腺苷受体激动剂的应用
EP2581381A3 (fr) 2006-02-03 2013-10-30 Gilead Sciences, Inc. Procédé de préparation d'un agoniste du récepteur A2A-adénosine et ses polymorphes

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JPH07506591A (ja) 1995-07-20
AU4081493A (en) 1993-12-13
CN1094046A (zh) 1994-10-26
MX9302957A (es) 1994-05-31
WO1993023401A1 (fr) 1993-11-25
TW277062B (fr) 1996-06-01
CA2136196A1 (fr) 1993-11-25
GB9210839D0 (en) 1992-07-08

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