WO2002020511A1 - Derives d'amidine qui sont inhibiteurs de la synthase d'oxyde nitrique - Google Patents

Derives d'amidine qui sont inhibiteurs de la synthase d'oxyde nitrique Download PDF

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Publication number
WO2002020511A1
WO2002020511A1 PCT/SE2001/001868 SE0101868W WO0220511A1 WO 2002020511 A1 WO2002020511 A1 WO 2002020511A1 SE 0101868 W SE0101868 W SE 0101868W WO 0220511 A1 WO0220511 A1 WO 0220511A1
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Prior art keywords
phenyl
thiophenecarboximidamide
formula
methyl
methylsulfanyl
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PCT/SE2001/001868
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English (en)
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Deborah Chen
James Empfield
Kenneth Mattes
Robert Murray
Eifion Phillips
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Astrazeneca Ab
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Priority claimed from GB0021705A external-priority patent/GB0021705D0/en
Priority claimed from GB0021706A external-priority patent/GB0021706D0/en
Priority claimed from SE0102156A external-priority patent/SE0102156D0/xx
Application filed by Astrazeneca Ab filed Critical Astrazeneca Ab
Priority to AU2001282829A priority Critical patent/AU2001282829A1/en
Publication of WO2002020511A1 publication Critical patent/WO2002020511A1/fr

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    • 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
    • C07D333/26—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 hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • This invention relates to new amidine derivatives, processes for their preparation, compositions containing them and their use in therapy.
  • Nitric oxide is produced in mammalian cells from L-arginine by the action of specific nitric oxide synthases (NOSs). These enzymes fall into two distinct classes - constitutive NOS (cNOS) and inducible NOS (INOS). At the present time, two constitutive NOSs and one inducible NOS have been identified. Of the constitutive NOSs, an endothelial enzyme (ecNOS) is involved with smooth muscle relaxation and the regulation of blood pressure and blood flow, whereas the neuronal enzyme (ncNOS) serves as a neurotransmitter and appears to be involved in the regulation of various biological functions such as cerebral ischaemia. Inducible NOS has been implicated in the pathogenesis of inflammatory diseases. Specific regulation of these enzymes should therefore offer considerable potential in the treatment of a wide variety of disease states.
  • NOSs nitric oxide synthases
  • WO 95/05363 discloses compounds of generic structure
  • D represents an aromatic ring
  • R represents hydrogen, alkyl Cl to 6 or halogen
  • the compounds have nitric oxide synthase inhibitory activity.
  • Z represents a furan or thiophene ring, optionally substituted by one or more substituents selected from halogen, trifluoromethyl, Cl to 6 alkyl, Cl to 6 alkoxy, hydroxy, amino and S(O) a R 4 ;
  • X represents Cl to 6 alkyl or ⁇ CO ⁇
  • Y represents O, S(O) b or NR ;
  • W represents S(O) c ; a, b and c independently represent an integer 0, 1 or 2; R or phenyl; said phenyl being optionally substituted by one or more substituents selected from halogen, trifluoromethyl, Cl to 6 alkyl, Cl to 6 alkoxy, hydroxy and amino;
  • R and R independently represent hydrogen, Cl to 6 alkyl, C2 to 7 alkanoyl or
  • NR R represents azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl; or piperazinyl optionally 4-substituted by Cl to 6 alkyl; each of said azacyclic rings being
  • R , R , R , R , R , R and R independently represent hydrogen or Cl to 6 alkyl; or the groups NR R and NR R independently represent azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl; or piperazinyl optionally 4-substituted by Cl to 6 alkyl; n represents an integer 1 to 6; and optical isomers, racemates and tautomers thereof and pharmaceutically acceptable salts thereof.
  • the substituent -W-R in formula (I) is in the ortho or para position relative to the amidine group. More preferably the substituent -W-R in formula (I) is in the para position relative to the amidine group.
  • substituent -X-Y-R in formula (I) is in the meta position relative to the amidine group.
  • X represents CH2.
  • Y represents NR .
  • c 0.
  • R represents Cl to 6 alkyl. More preferably, c represents 0 and R represents
  • -W-R represents methylthio
  • Z represents unsubstituted thiophene
  • R represents methyl
  • X represents CH2 and Y represents NR .
  • Particular compounds of the invention include:
  • C 1 to 6 alkyl denotes a straight or branched chain alkyl group having from 1 to 6 carbon atoms and/or a cyclic alkyl group having from 3 to 6 carbon atoms.
  • examples of such groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, cyclopropyl, cyclopropylmethyl, cyclopentyl, methylcyclopentyl, cyclopentylmethyl and cyclohexyl.
  • C2 to 7 alkanoyl denotes a straight or branched chain alkyl group having from 1 to 6 carbon atoms or a cyclic alkyl group having from 3 to 6 carbon atoms bonded to a carbonyl (CO) group.
  • CO carbonyl
  • examples of such groups include acetyl, propionyl, iso-butyryl, valeryl, pivaloyl, cyclopentanoyl and cyclohexanoyl.
  • Cl to 6 alkoxy denotes an oxygen substituent bonded to a straight or branched chain alkyl group having from 1 to 6 carbon atoms and/or a cyclic alkyl group having from 3 to 6 carbon atoms.
  • groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, cyclopropyloxy, cyclopropylmethoxy, cyclopentyloxy, methylcyclopentyloxy, cyclopentylmethoxy and cyclohexyloxy.
  • halogen referred to herein denotes fluorine, chlorine, bromine and iodine.
  • the present invention includes compounds of formula (I) in the form of salts, in particular acid addition salts.
  • Suitable salts include those formed with both organic and inorganic acids.
  • Such acid addition salts will normally be pharmaceutically acceptable although salts of non-pharmaceuticaHy acceptable acids may be of utility in the preparation and purification of the compound in question.
  • preferred salts include those formed from hydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.
  • R , X, W and Z are as defined above and L is a leaving group, with a compound of formula (N) or a salt thereof
  • the reaction will take place on stirring a mixture of the reactants in a suitable solvent, for example a lower alkanol such as ethanol, 2-propanol or tert-butanol, at a temperature between room temperature and the reflux temperature of the solvent.
  • a suitable solvent for example a lower alkanol such as ethanol, 2-propanol or tert-butanol
  • the reaction may optionally be carried out under an atmosphere of an inert gas such as nitrogen or argon.
  • the reaction time will depend inter alia on the solvent and the nature of the leaving group, and may be up to 48 hours; however it will typically be from 1 to 5 hours.
  • Suitable leaving groups L include thioalkyl, sulfonate, trifluoromethylsulfonate, halide, alkoxide, aryloxide and tosylate groups; others are recited in "Advanced Organic
  • the displacement reaction is performed by reacting a compound of formula (IV) with a compound of formula (N) in an inert solvent.
  • Suitable leaving groups include sulfonate, trifluorosulfonate, tosylate, and halides selected from the group chloride, bromide or iodide.
  • the reaction is generally carried out in the presence of a base.
  • 3 base can be an additive to the reaction mixture, or in the case wherein Y represents ⁇ R , can alternatively be an excess of the amine nucleophile.
  • Potential basic additives are metal carbonate, especially alkali metal carbonates, metal oxides and hydroxides, and tertiary amine bases such as diisopropylethylamine.
  • Suitable organic solvents are those such as acetonitrile, dioxane, N,N-dimethylformamide, N-methyl-2-pyrrolidinone, tetrahydrofuran, dimethylsulfoxide, sulfolane and Cl to 4 alcohols.
  • the leaving group is chloride.
  • the reduction is generally performed using a suitable reducing agent in a inert solvent.
  • a suitable reducing agent may be diborane or lithium aluminium hydride
  • the solvent may be an ether such as diethyl ether or tetrahydrofuran.
  • Compounds of formula (I) wherein W represents -S(O) c - and c represents 1 or 2 may be prepared by oxidation of the corresponding compound of formula (I) wherein c represents 0 using a suitable selective oxidising agent. Such methods are generally well known in the art. For example, compounds wherein c represents 1 may be prepared using one equivalent of sodium periodate as oxidising agent. And compounds wherein c represents 2 may be prepared using two or more equivalents of oxone or of sodium periodate as the oxidising agent.
  • Salts of compounds of formula (I) may be formed by reacting the free base or a salt, enantiomer, tautomer or protected derivative thereof, with one or more equivalents of the appropriate acid.
  • the reaction may be carried out in a solvent or medium in which the salt is insoluble, or in a solvent in which the salt is soluble followed by subsequent removal of the solvent in vacuo or by freeze drying.
  • Suitable solvents include, for example, water, dioxan, ethanol, 2-propanol, tetrahydrofuran or diethyl ether, or mixtures thereof.
  • the reaction may be a metathetical process or it may be carried out on an ion exchange resin.
  • R , R , X, Y and W are as defined above.
  • Z is as defined above; with an alkyliodide.
  • R , X and W are as defined above and Hal represents a halogen, with a nucleophile of formula (N);
  • X represents an alkyl group having one less CH2 group than X
  • R , X, W and Z are as defined above, using methods that are generally well known in the art.
  • Hal represents a halogen
  • M-SR metal thioalkoxide
  • R is as defined above and M represents a metal, particularly an alkali or alkaline earth metal such as sodium or potassium; followed by reduction of the nitro group and then amidination of the resulting amine.
  • Intermediate compounds may be prepared as such or in protected form.
  • amine and hydroxy groups may be protected.
  • Suitable protecting groups are described in the standard text "Protective Groups in Organic Synthesis", 3rd Edition (1999) by Greene and Wuts.
  • Amine protecting groups which may be mentioned include alkyloxycarbonyl such as t-butyloxycarbonyl, phenylalkyloxycarbonyl such as benzyloxycarbonyl, or trifluoroacetate. Deprotection will normally take place on treatment with aqueous base or aqueous acid.
  • the compounds of the invention and intermediates may be isolated from their reaction mixtures, and if necessary further purified, by using standard techniques.
  • the compounds of formula (I) may exist in tautomeric, enantiomeric or diastereoisomeric forms, all of which are included within the scope of the invention.
  • the various optical isomers may be isolated by separation of a racemic mixture of the compounds using conventional techniques, for example, fractional crystallisation or HPLC.
  • the individual enantiomers may be made by reaction of the appropriate optically active starting materials under reaction conditions that will not cause racemisation.
  • Intermediate compounds may also exist in enantiomeric forms and may be used as purified enantiomers, diastereomers, racemates or mixtures.
  • the compounds of formula (I), and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers, are useful because they possess pharmacological activity in animals.
  • the compounds are active as inhibitors of the enzyme nitric oxide synthase and as such are predicted to be useful in therapy. More particularly, they are in general selective inhibitors of the neuronal isoform of the enzyme nitric oxide synthase.
  • diseases or conditions include hypoxia, such as in cases of cardiac arrest, stroke and neonatal hypoxia, neurodegenerative conditions including nerve degeneration and/or nerve necrosis in disorders such as ischaemia, hypoxia, hypoglycemia, epilepsy, and in external wounds (such as spinal cord and head injury), hyperbaric oxygen convulsions and toxicity, dementia, for example, pre-senile dementia, Alzheimer's disease and AIDS-related dementia, Sydenham's chorea, Parkinson's disease, Huntington's disease, multiple sclerosis, Amyotrophic Lateral Sclerosis, Korsakoff s disease, imbecility relating to a cerebral vessel disorder, sleeping disorders, schizophrenia, -anxiety, depression, seasonal affective disorder, jet-lag, depression or other symptoms associated with Premenstrual Syndrome (PMS), anxiety and septic shock.
  • PMS Premenstrual Syndrome
  • the compounds of formula (I) are also useful in the treatment and alleviation of acute or persistent inflammatory or neuropathic pain, or pain of central origin.
  • the compounds of formula (I) may also be useful in the treatment or prophylaxis of inflammation.
  • Conditions that may be specifically mentioned include osteoarthritis, rheumatoid arthritis, rheumatoid spondylitis, gouty arthritis and other arthritic conditions, inflamed joints; eczema, psoriasis, dermatitis or other inflammatory skin conditions such as sunburn; inflammatory eye conditions including uveitis and conjunctivitis; lung disorders in which inflammation is involved, for example, asthma, bronchitis, chronic obstructive pulmonary disease, pigeon fancier's disease, farmer's lung, acute respiratory distress syndrome; bacteraemia, endotoxaemia (septic shock), aphthous ulcers, gingivitis, pyresis, pain and pancreatitis; conditions of the gastrointestinal tract including inflammatory bowel disease, Crohn's disease, atrophic gastritis, gastritis vari
  • the compounds of formula (I) and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers may also be useful in the treatment or prophylaxis of diseases or conditions in addition to those mentioned above.
  • the compounds may be useful in the treatment of atherosclerosis, cystic fibrosis, hypotension associated with septic and/or toxic shock, in the treatment of dysfunction of the immune system, as an adjuvant to short- term immunosuppression in organ transplant therapy, in the treatment of vascular complications associated with diabetes and in cotherapy with cytokines, for example TNF or interleukins.
  • Compounds of formula (I) are also predicted to show activity in the prevention and reversal of tolerance to opiates and diazepines, treatment of drug addiction and treatment of migraine and other vascular headaches.
  • the compounds of the present invention may also show useful immunosuppressive activity, and be useful in the treatment of gastrointestinal motility disorders, and in the induction of labour.
  • the compounds may also be useful in the treatment of cancers that express nitric oxide synthase.
  • Compounds of formula (I) are predicted to be particularly useful in the treatment or prophylaxis of hypoxia or stroke or ischaemia or neurodegenerative conditions or schizophrenia or migraine or for the treatment of pain and especially in the treatment or prophylaxis of hypoxia or stroke or ischaemia or neurodegenerative disorders or schizophrenia or pain.
  • We are particularly interested in the conditions selected from the group consisting of hypoxia, ischaemia, stroke, pain, anxiety, schizophrenia, Parkinson's disease, Huntington's disease and migraine and other vascular headaches.
  • the compounds of formula (I) are expected to be particularly useful either alone, or in combination with other agents such as L-Dopa.
  • Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the disease or condition in question.
  • Persons at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition.
  • a compound of formula (I) or an optical isomer or racemate thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prophylaxis of the aforementioned diseases or conditions; and a method of treatment or prophylaxis of one of the aforementioned diseases or conditions which comprises administering a therapeutically effective amount of a compound of formula (I), or an optical isomer or racemate thereof or a pharmaceutically acceptable salt thereof, to a person suffering from or susceptible to such a disease or condition.
  • the dosage administered will, of course, vary with the compound employed, the mode of administration and the treatment desired. However, in general, satisfactory results are obtained when the compounds are administered to a human at a daily dosage of between 0.5 mg and 2000 mg (measured as the active ingredient) per day, particularly at a daily dosage of between 2 mg and 500 mg.
  • the compounds of formula (I), and optical isomers and racemates thereof and pharmaceutically acceptable salts thereof may be used on their own, or in the form of appropriate medicinal formulations. Administration may be by, but is not limited to, enteral (including oral, sublingual or rectal), intranasal, or topical or other parenteral routes. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.
  • a pharmaceutical formulation comprising preferably less than 95% by weight and more preferably less than 50% by weight of a compound of formula (I), or an optical isomer or racemate thereof or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable diluent or carrier.
  • the formulation may optionally also contain a second pharmacologically active ingredient such as L-Dopa.
  • the compounds of formula (I), and pharmaceutically acceptable derivatives thereof, may also be advantageously used in combination with a COX-2 inhibitor.
  • COX-2 inhibitors are Celecoxib and MK-966.
  • the NOS inhibitor and the COX-2 inhibitor may either be formulated together within the same pharmaceutical composition for administration in a single dosage unit, or each component may be individually formulated such that separate dosages may be administered either simultaneously or sequentially.
  • diluents and carriers are: for tablets and dragees: lactose, starch, talc, stearic acid; for capsules: tartaric acid or lactose; for injectable solutions: water, alcohols, glycerin, vegetable oils; for suppositories: natural or hardened oils or waxes.
  • compositions in a form suitable for oral, that is oesophageal, administration include: tablets, capsules and dragees; sustained release compositions include those in which the active ingredient is bound to an ion exchange resin which is optionally coated with a diffusion barrier to modify the release properties of the resin.
  • nitric oxide synthase has a number of isoforms and compounds of formula (I), and optical isomers and racemates thereof and pharmaceutically acceptable salts thereof, may be screened for nitric oxide synthase inhibiting activity by following procedures based on those of Bredt and Snyder in Proc. Natl. Acad. Sci., 1990, 87, 682-685.
  • Nitric oxide synthase converts 3 H-L-arginine into 3 H-L-citrulline which can be separated by cation exchange chromatography and quantified by scintillation counting.
  • the enzyme is isolated from rat hippocampus or cerebellum.
  • the cerebellum or hippocampus of a male Sprague-Dawley rat (250-275g) is removed following CO 2 anaesthesia of the animal and decapitation.
  • Cerebellar or hippocampal supernatant is prepared by homogenisation in 50 mM Tris-HCl with 1 mM EDTA buffer (pH 7.2 at 25 °C) and centifugation for 15 minutes at 20,000 g. Residual L-arginine is removed from the supernatant by chromatography through Dowex AG-50W-X8 sodium form and hydrogen form columns successively, and further centrifugation at 1000 g for 30 seconds.
  • 25 ⁇ l of the final supernatant is added to each of 96 wells (of a 96 well filter plate) containing either 25 ⁇ l of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , pH 7.4) or 25 ⁇ l of test compound in the buffer at 22 °C and 25 ⁇ l of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , 1 mM DTT, 100 ⁇ M NADPH, 10 ⁇ g/ml calmodulin, pH 7.4).
  • an assay buffer 50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , pH 7.4
  • 25 ⁇ l of test compound in the buffer at 22 °C and 25 ⁇ l of complete assay buffer 50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , 1 mM DTT, 100 ⁇
  • an L-arginine solution (of concentration 18 ⁇ M 1 H-L-arginine, 96 nM H-L-arginine) is added to each well to initiate the reaction.
  • the reaction is stopped after 10 minutes by addition of 200 ⁇ l of a slurry of termination buffer (20 mM HEPES, 2 mM EDTA, pH 5.5) and Dowex AG-50W-X8 200-400.mesh.
  • Labelled L-citrulline is separated from labelled L-arginine by filtering each filter plate and 75 ⁇ l of each terminated reaction is added to 3 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting.
  • basal activity is increased by 20,000 dpm/ml of sample above a reagent blank that has an activity of 7,000 dpm/ml.
  • a reference standard, N-nifro-L-arginine which gives 80% inhibition of nitric oxide synthase at a concentration of 1 ⁇ M, is tested in the assay to verify the procedure.
  • Enzyme was isolated from human hippocampus, cortex or cerebellum. Cerebellar, cortical or hippocampal supernatant is prepared by homogenisation of frozen human tissue (1 to 5 g) in 50 mM Tris-HCl with 1 mM EDTA buffer (pH 7.2 at 25 °C) and centrifugation for 15 minutes at 20,000 g. Residual L-arginine is removed from the supernatant by chromatography through Dowex AG-50W-X8 sodium form and hydrogen form columns successively and further centrifugation at 1000 g for 30 seconds. Subsequently, the supernatant is passed tlirough 2'-5 ' ADP Sepharose and the human nNOS eluted with NADPH.
  • 25 ⁇ l of the final supernatant is added to each of 96 wells (of a 96 well filter plate) containing either 25 ⁇ l of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , pH 7.4) or 25 ⁇ l of test compound in the buffer at 22 °C and 25 ⁇ l of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , 1 mM DTT, 100 ⁇ M
  • Labelled L-citrulline is separated from labelled L-arginine by filtering each filter plate and 75 ⁇ l of each terminated reaction is added to 3 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting. In a typical experiment using the cerebellar supernatant, basal activity is increased by 20,000 dpm/ml of sample above a reagent blank that has an activity of 7,000 dpm/ml. A reference standard, N-nitro-L-arginine, which gives 80% inhibition of nitric oxide synthase at a concentration of 1 ⁇ M, is tested in the assay to verify the procedure.
  • iNOS inducible nitric oxide synthase inhibiting activity
  • Partially purified iNOS was prepared from cultured and lysed human DLDl cells which had been activated with TNF-alpha, interferon gamma, and LPS. Centrifugation at lOOOg removed cellular debris and residual L-arginine was removed from the supernatant by chromatography through Dowex AG-50W-X8 sodium form and hydrogen form columns successively.
  • 25 ⁇ l of the final supernatant is added to each of 96 wells (of a.96 well filter plate) containing either 25 ⁇ l of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , pH 7.4) or 25 ⁇ l of test compound in the buffer at 22 °C and 25 ⁇ l of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , 1 mM DTT, 100 ⁇ M NADPH, 10 ⁇ g/ml calmodulin, pH 7.4).
  • an assay buffer 50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , pH 7.4
  • 25 ⁇ l of test compound in the buffer at 22 °C and 25 ⁇ l of complete assay buffer 50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , 1 mM DTT, 100 ⁇
  • L-arginine solution (of concentration 12 ⁇ M 1 H-L-arginine, 96 nM 3 H-L-arginine) is added to each test tube to initiate the reaction.
  • the reaction is stopped after 30 minutes by addition of 200 ⁇ l of a slurry of termination buffer (20 mM HEPES, 2 mM EDTA, pH 5.5) and Dowex AG-50W-X8 200-400 mesh.
  • Labelled L-citrulline is separated from labelled L-arginine by filtering each filter plate and 75 ⁇ l of each terminated reaction is added to 3 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting.
  • basal activity is increased by 10,000 dpm/ml of sample above a reagent blank that has an activity of 5,000 dpm/ml.
  • HUNECs human umbilical vein endothelial cells
  • cells When cells reach confluency, they are resuspended in Dulbecco's phosphate buffered saline, centrifuged at 800 m for 10 minutes, and the cell pellet is then homogenised in ice-cold 50 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulphonylfluoride, 2 ⁇ M leupeptin at pH 4.2. Following centrifugation at 34,000 ⁇ m for 60 minutes, the pellet is solubilised in the homogenisation buffer which also contains 20 mM CHAPS. After a 30 minute incubation on ice, the suspension is centrifuged at 34,000 ⁇ m for 30 minutes.
  • the resulting supernatant is stored at -80 °C until use.
  • 25 ⁇ l of the final supernatant is added to each of 12 'test tubes containing 25 ⁇ l L-arginine solution (of concentration 12 ⁇ M ⁇ -L-arginine, 64 nM 3 H-L-arginine) and either 25 ⁇ l of an assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , pH 7.4) or 25 ⁇ l of test compound in the buffer at 22 °C.
  • an assay buffer 50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , pH 7.4
  • each test tube was added 25 ⁇ l of complete assay buffer (50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , 1 mM DTT, 100 ⁇ M NADPH, 10 ⁇ g/ml calmodulin, 12 ⁇ M tetrahydrobiopterin, pH 7.4) to initiate the reaction and the reaction is stopped after 10 minutes by addition of 2 ml of a termination buffer (20 mM HEPES, 2 mM EDTA, pH 5.5).
  • complete assay buffer 50 mM HEPES, 1 mM EDTA, 1.5 mM CaCl 2 , 1 mM DTT, 100 ⁇ M NADPH, 10 ⁇ g/ml calmodulin, 12 ⁇ M tetrahydrobiopterin, pH 7.4
  • Labelled L-citrulline is separated from labelled L-arginine by chromatography over a Dowex AG-50W-X8 200-400 mesh column. A 1 ml portion of each terminated reaction mixture is added to an individual 1 ml column and the eluant combined with that from two 1 ml distilled water washes and 16 ml of scintillation cocktail. The L-citrulline is then quantified by scintillation counting.
  • basal activity is increased by 5,000 dpm/ml of sample above a reagent blank that has an activity of 1500 dpm/ml.
  • IC 50 the concentration of drug substance which gives 50% enzyme inhibition in the assay.
  • IC 50 values for test compounds were initially estimated from the inhibiting activity of 1, 10 and 100 ⁇ M solutions of the compounds. Compounds that inhibited the enzyme by at least 50% at 10 ⁇ M were re-tested using more appropriate concentrations so that an IC 50 could be determined.
  • the compounds of Examples 1 to 26 below showed IC50 values for inhibition of neuronal nitric oxide synthase of less than 10 ⁇ M and good selectivity compared to inhibition of the endothelial isoform of the enzyme, indicating that they are predicted to show particularly useful therapeutic activity.
  • N-(3- ( risopropylaminolmethyl) -4-(methylsulfanyl)phenvn-2 -thiophenecarboximidamide A mixture of [3-(chloromethyl)-4-(methylsulfanyl)phenyl]-2 -thiophenecarboximidamide hydrochloride (534 mg, 1.6 mmol), isopropylamine (0.77 ml, 9.0 mmol) and diisopropylethylamine (1.56 ml, 9.0 mmol) in N,N-dimethylformamide (10 ml) was stirred at room temperature for 16 h. The mixture was diluted with water. Potassium carbonate was added and a white precipitate was formed. The solid was collected by filtration and washed with water, then dried to give the title compound as a white solid (360 mg, 1.13 mmol, 70%).
  • N-r3-r(Methylamino methyll-4-( ' methyl ' sulfanyl)phenyll-2 -thiophenecarboximidamide Prepared by a method analogous to that described in Example 1 and using [3-(chloromethyl)-4-(methylsulfanyl)phenyl]-2 -thiophenecarboximidamide hydrochloride, methylamine (2M) and diisopropylethylamine in N,N-dimethylformamide.
  • ® was purified by reverse phase HPLC on a Waters Bondapak C ⁇ 8 column using a gradient of acetonitrile and 0.1% aqueous trifluoroacetic acid as the eluent.
  • the free base product was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extracts were dried (magnesium sulfate) and evaporated to give the title compound as a white solid (43%).
  • N- -T3 - [(Methylamino)methyll -4-f methylsulfonvDphenyll -2-thiophenecarboximidamide dihydrochloride To a solution of N-[3-[(methylamino)methyl]-4-(methylsulfanyl)phenyl]-2- thiophenecarboximidamide (460 mg, 1.58 mmol) in methanol (15 ml) was added oxone (1.89 g, 3.07 mmol). The residue was partitioned between water and dichloromethane. The organic layer was collected, then dried (magnesium sulfate), filtered, and evaporated. The
  • ® compound was purified by reverse phase HPLC on a Waters Bondapak C ⁇ column using a gradient of acetonitrile and 0.1% aqueous trifluoroacetic acid as the eluent.
  • the free base was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extract was dried (magnesium sulfate), filtered and evaporated. Dissolution of the residue in methanol, addition of excess hydrogen chloride solution (1M in diethyl ether) and evaporation gave the title compound as a yellow solid (95 mg, 24%).
  • N-r3- ⁇ r(2-Hvdroxyethyl aminolmethyll-4-(methylsulfanyl phenyll-2- thiophenecarboximidamide dihydrochloride A mixture of [3-(chloromethyl)-4-(methylsulfanyl)phenyl]-2-thiophenecarboximidamide hydrochloride (500 mg, 1.5 mmol), ethanolamine (513.1 mg, 8.4 mmol) and diisopropylethylamine (1.46 ml, 8.4 mmol) in N,N-dimethylformamide (10 ml) was stirred for 6 h at room temperature. The reaction was diluted with water. Excess solid potassium carbonate was added. The aqueous layer was extracted with dichloromethane. The organic layer was dried (magnesium sulfate), filtered and evaporated. The compound was purified
  • ® compound was purified by reverse phase HPLC on a Waters Bondapak C ⁇ 8 column using a gradient of acetonitrile and 0.1% aqueous trifluoroacetic acid as the eluent.
  • the free base was prepared by basification of the product-containing fractions with potassium carbonate and extraction with dichloromethane. The organic extract was dried (magnesium sulfate), filtered and evaporated. Dissolution of the residue in methanol, addition of excess hydrogen chloride solution (1M in diethyl ether) and evaporation gave the title compound as a white solid (398.3 mg, 1.13 mmol, 58%).
  • N-[3-r(Benzylamino)methyn-4-(methylsulfanyl phenyl1-2-thiophenecarboximidamide A mixture ofN-[3-(aminomethyl)-4-(methylsulfanyl)phenyl]-2-thiophenecarboximidamide (0.185 g, 0.67 mmol), benzaldehyde (0.081 g, 0.76 mmol) and glacial acetic acid (38 ⁇ l, 0.67 mmol) in 1 ,2-dichloroethane (5 ml) was treated with sodium triacetoxyborohydri.de (0.222 g, 1.05 mmol).

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Abstract

L'invention concerne des composés de la formule (I) où R1, R2, X, Y, W et Z sont définis dans la description, et leurs isomères optiques, racémates et tautomères ainsi que leurs sels pharmaceutiquement acceptables. L'invention concerne également leurs procédés de préparation, les compositions les contenant et leur utilisation en thérapie. Ces composés sont inhibiteurs de l'enzyme synthase d'oxyde nitrique.
PCT/SE2001/001868 2000-09-05 2001-08-30 Derives d'amidine qui sont inhibiteurs de la synthase d'oxyde nitrique WO2002020511A1 (fr)

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AU2001282829A AU2001282829A1 (en) 2000-09-05 2001-08-30 Amidine derivatives which are inhibitors of nitric oxide synthase

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GB0021705A GB0021705D0 (en) 2000-09-05 2000-09-05 Novel compounds
GB0021705.9 2000-09-05
GB0021706.7 2000-09-05
GB0021706A GB0021706D0 (en) 2000-09-05 2000-09-05 Novel compounds
SE0102156-7 2001-06-14
SE0102156A SE0102156D0 (sv) 2001-06-14 2001-06-14 Novel compounds

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003053914A1 (fr) * 2001-12-12 2003-07-03 Schering Aktiengesellschaft Nouveaux derives amidine et utilisation dans des agents pharmaceutiques

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005363A1 (fr) * 1993-08-12 1995-02-23 Astra Aktiebolag Derives de l'amidine ayant des activites de synthetase de l'oxyde nitrique
WO1998042696A1 (fr) * 1997-03-24 1998-10-01 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Nouveaux derives du 2-(iminomethyl)amino-phenyle, leur preparation, leur application a titre de medicaments et les compositions pharmaceutiques les contenant
JPH10265450A (ja) * 1997-03-25 1998-10-06 Mitsui Chem Inc 一酸化窒素合成酵素阻害作用を有する新規なアミジン誘導体
WO1998058934A1 (fr) * 1997-06-20 1998-12-30 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Nouveaux derives du 2-(iminomethyl)amino-phenyle, leur preparation, leur application a titre de medicaments et les compositions pharmaceutiques les contenant
WO2001046171A1 (fr) * 1999-12-20 2001-06-28 Astrazeneca Ab Derives d'amidine inhibiteurs d'oxyde nitrique synthase
WO2001046170A1 (fr) * 1999-12-20 2001-06-28 Astrazeneca Ab Derives d'amidine inhibiteurs de l'oxyde nitrique synthase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005363A1 (fr) * 1993-08-12 1995-02-23 Astra Aktiebolag Derives de l'amidine ayant des activites de synthetase de l'oxyde nitrique
WO1998042696A1 (fr) * 1997-03-24 1998-10-01 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Nouveaux derives du 2-(iminomethyl)amino-phenyle, leur preparation, leur application a titre de medicaments et les compositions pharmaceutiques les contenant
JPH10265450A (ja) * 1997-03-25 1998-10-06 Mitsui Chem Inc 一酸化窒素合成酵素阻害作用を有する新規なアミジン誘導体
WO1998058934A1 (fr) * 1997-06-20 1998-12-30 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Nouveaux derives du 2-(iminomethyl)amino-phenyle, leur preparation, leur application a titre de medicaments et les compositions pharmaceutiques les contenant
WO2001046171A1 (fr) * 1999-12-20 2001-06-28 Astrazeneca Ab Derives d'amidine inhibiteurs d'oxyde nitrique synthase
WO2001046170A1 (fr) * 1999-12-20 2001-06-28 Astrazeneca Ab Derives d'amidine inhibiteurs de l'oxyde nitrique synthase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003053914A1 (fr) * 2001-12-12 2003-07-03 Schering Aktiengesellschaft Nouveaux derives amidine et utilisation dans des agents pharmaceutiques

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