WO2000021934A1 - Compounds - Google Patents
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- WO2000021934A1 WO2000021934A1 PCT/SE1999/001829 SE9901829W WO0021934A1 WO 2000021934 A1 WO2000021934 A1 WO 2000021934A1 SE 9901829 W SE9901829 W SE 9901829W WO 0021934 A1 WO0021934 A1 WO 0021934A1
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- WIPO (PCT)
- Prior art keywords
- formula
- compound
- pharmaceutically acceptable
- acceptable salt
- methyl
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen 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
- C07D213/72—Nitrogen atoms
- C07D213/73—Unsubstituted amino or imino radicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
Definitions
- the present invention relates to novel 2-aminopyridine 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 particularly implicated in the pathogenesis of inflammatory diseases. Regulation of these enzymes should therefore offer considerable potential in the treatment of a wide variety of disease states (J. E. Macdonald, Ann. Rep. Med. Chem., 1996, 31, 221 - 230).
- the compounds of the present invention are clearly distinguished from those of the prior art by virtue of the nature of the particular substituents attached to the 2-aminopyridine ring.
- R represents hydrogen or one or more substituents selected independently from Cl to 6
- Y represents hydrogen or Cl to 4 alkyl
- Y is joined to the ortho position of ring A and represents ⁇ [CH_] ⁇ -;
- Z represents a bond or -CH -
- Q represents hydrogen, Cl to 6 alkyl, Cl to 6 alkoxy, Cl to 6 alkylthio, halogen, cyano, trifluoromethyl, trifluoromethoxy, hydroxy, nitro, methanesulphonyl, sulphamoyl, benzyloxy, -NR 8 R 9 , -CO 2 R 10 or -CONR H R 12 ;
- R , R and R independently represent hydrogen or Cl to 4 alkyl
- A represents phenyl, a five membered aromatic heterocyclic ring containing one or two heteroatoms selected independently from O, S or N, or a six membered aromatic azacyclic ring containing one or two nitrogen atoms;
- n an integer 0 to 5;
- n an integer 0 to 3;
- p represents an integer 0 to 4
- r represents an integer 0 to 3
- the compounds of formula (I) and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers have the advantage that they are inhibitors of the enzyme nitric oxide synthase (NOS).
- NOS nitric oxide synthase
- the compounds of formula (I) and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers have the advantage that they are inhibitors of the inducible isoform of the enzyme nitric oxide synthase (iNOS).
- the invention further provides a process for the preparation of compounds of formula (I) or a pharmaceutically acceptable salt, enantiomer, racemate or tautomer thereof.
- Another aspect of the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, racemate or tautomer thereof, in the manufacture of a medicament, for the treatment or prophylaxis of diseases or conditions in which inhibition of nitric oxide synthase activity is beneficial.
- a more particular aspect of the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, racemate or tautomer thereof, in the manufacture of a medicament, for the treatment or prophylaxis of inflammatory disease.
- a method of treating, or reducing the risk of, diseases or conditions in which inhibition of nitric oxide synthase activity is beneficial which comprises administering to a person suffering from or at risk of, said disease or condition, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, racemate or tautomer thereof. More particularly, there is also provided a method of treating, or reducing the risk of, inflammatory disease in a person suffering from or at risk of, said disease, wherein the method comprises administering to the person a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, racemate or tautomer thereof.
- the compounds of the present invention may also be used advantageously in combination with a second pharmaceutically active substance, particularly in combination with a selective inhibitor of the inducible isoform of cyclooxygenase (COX-2).
- COX-2 cyclooxygenase
- a method of treating, or reducing the risk of, inflammation, inflammatory disease and inflammatory related disorders in a person suffering from or at risk of, said disease or condition comprises administering to the person a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, racemate or tautomer thereof in combination with a COX-2 inhibitor.
- a in formula (I) represents a phenyl or pyridyl ring.
- Q in formula (I) represents hydrogen, halogen or cyano.
- R in formula (I) represents Cl to 6 alkyl or Cl to 6 alkoxy. More preferably, R in formula (I) represents methyl or methoxy.
- n in formula (I) represents 0 orl.
- Particular compounds of the invention include:
- Cl to 6 alkyl 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. Examples of such groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, cyclopentyl and cyclohexyl.
- Cl to 6 alkoxy denotes a straight or branched chain alkoxy group having from 1 to 6 carbon atoms. Examples of such groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, s-butoxy and t-butoxy.
- Examples of a five membered aromatic heterocyclic ring containing one or two heteroatoms selected independently from O, S or N, or a six membered aromatic azacyclic ring containing one or two nitrogen atoms include furan, thiophene, pyrrole, thiazole, oxazole, imidazole, pyridine, pyrimidine, pyrazine and pyridazine.
- R 1 , R 2 , R 3 , R 4 , R 5 , X, Y and Z are as defined above with an acyl derivative of formula (HI)
- Q and A are as defined above and L represents a leaving group
- the reaction process will take place on stirring a mixture of the reactants in a suitable organic solvent at a suitable temperature, generally between 0 °C and the boiling point of the solvent.
- the reaction time will depend inter alia on the solvent used, the reaction temperature and the nature of the group L.
- the reaction may be catalysed by the addition of a base; bases that may be used include organic amines (for example, triethylamine or pyridine) and alkali metal hydroxides, alkoxides, carbonates or hydrides.
- Suitable leaving groups, L include halogen (especially chlorine) and hydroxyl.
- the reaction between compounds of formulae (II) and (HI) may also be achieved using a suitable coupling agent such as CDI (lJ'-carbonyldiimidazole) , DCC (1,3-dicyclohexylcarbodiimide) or HOBt ( 1 -hydroxybenzotriazole).
- CDI lJ'-carbonyldiimidazole
- DCC 1,3-dicyclohexylcarbodiimide
- HOBt 1 -hydroxybenzotriazole
- 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-pharmaceutically 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.
- Salts of compounds of formula (I) may be formed by reacting the free base, or a salt, enantiomer, racemate or tautomer 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, for example, water, dioxane, ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents, which may be removed in vacuo or by freeze drying.
- the reaction may also be a metathetical process or it may be carried out on an ion exchange resin.
- compounds of formula (H) may be prepared by metallation of a compound of formula (V)
- a suitable base such as butyl lithium or lithium diisopropylamide
- L is a suitable leaving group such as halide.
- the compounds of the invention and intermediates thereto may be isolated from their reaction mixtures and, if necessary further purified, by using standard techniques.
- the compounds of formula I may exist in enantiomeric forms. Therefore, all enantiomers, diastereomers, racemates and mixtures thereof 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.
- Intermediate compounds may also exist in enantiomeric forms and may be used as purified enantiomers, diastereomers, racemates or mixtures.
- the compounds of formula (I) may exist in alternative tautomeric forms.
- Compounds of formula (I) are provided in another tautomeric form or as a mixture thereof.
- 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. More particularly, they are inhibitors of the inducible isoform of the enzyme nitric oxide synthase and as such are predicted to be useful in therapy, for example, as anti-inflammatory agents. They may also have utility as inhibitors of the neuronal isoform of the enzyme nitric oxide synthase.
- the compounds and their pharmaceutically acceptable salts, enantiomers, racemates and tautomers are indicated for use in the treatment or prophylaxis of diseases or conditions in which synthesis or oversynthesis of nitric oxide synthase forms a contributory part.
- the compounds are indicated for use in the treatment of inflammatory conditions in mammals including man.
- Conditions that may be specifically mentioned are: 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, meningitis and pancreatitis; conditions of the gastrointestinal tract including inflammatory bowel disease, Crohn's disease, atrophic gastritis, gastritis varialoforme, ulcerative colitis, coeliac disease, regional ileit
- the compounds will also be useful in the treatment and alleviation of acute pain or persistent inflammatory pain or neuropathic pain or pain of a central origin.
- 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, glaucoma, 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 control of onset of diabetes, in the maintenance of pancreatic function in diabetes, in the treatment of vascular complications associated with diabetes and in cotherapy with cytokines, for example TNF or interleukins.
- cytokines for example TNF or interleukins.
- the compounds of formula (I) may also be useful in the treatment of hypoxia, for example in cases of cardiac arrest and stroke, neurodegenerative disorders including nerve degeneration and/or nerve necrosis in disorders such as ischaemia, hypoxia, hypoglycaemia, 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, Tourette's Syndrome, Huntington's disease, Amyotrophic Lateral Sclerosis, Multiple Sclerosis, Korsakoffs disease, imbecility relating to a cerebral vessel disorder, sleeping disorders, schizophrenia, depression, pain, autism, seasonal affective disorder, jet-lag, depression or other symptoms associated with Premenstrual Syndrome (PMS), anxiety and septic shock.
- PMS Premenstrual Syndrome
- Compounds of formula (I) may also be expected to show activity in the prevention and reversal of tolerance to opiates and diazepines, treatment of drug addiction, treatment of migraine and other vascular headaches, neurogenic inflammation, in the treatment of gastrointestinal motility disorders, cancer and in the induction of labour.
- 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.
- 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 at a dosage of the solid form of between 1 mg and 2000 mg per day.
- the compounds of formula (I), and pharmaceutically acceptable derivatives thereof may be used on their own, or in the form of appropriate pharmaceutical compositions in which the compound or derivative is in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
- Administration may be by, but is not limited to, enteral (including oral, sublingual or rectal), intranasal, intravenous, topical or other parenteral routes.
- enteral including oral, sublingual or rectal
- intranasal intranasal
- intravenous 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.
- the pharmaceutical composition preferably comprises less than 80% and more preferably less than 50% of a compound of formula (I), or a pharmaceutically acceptable salt, enantiomer, racemate or tautomer thereof.
- 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.
- 6-(4-Aminobutyl)-4-methyl-2-pyridinamine dihydrochloride (100 mg) was stirred in acetonitrile (20 ml) and treated with 4-cyanobenzoyl chloride (73 mg) followed by triethylamine (0.17 ml).
- the reaction mixture was stirred for 24 h, diluted with water, extracted with ethyl acetate, the combined extracts washed with sodium bicarbonate solution and brine, dried over magnesium sulphate and evaporated.
- the residue was dissolved in ethanol and treated with IN hydrochloric acid in ether, then evaporated.
- the residue was dissolved in water, washed with dichloromethane and basified with 40% sodium hydroxide solution.
- 6-(2-Aminoethyl)-4-methyl-2-pyridinarnine (111 mg, 0.738 mmol) prepared as in Example 1(c) and methyl 4-bromo-2-bromomethylbenzoate (227 mg, 0.738 mmol) in acetonitrile (5 ml) was treated with triethylamine (0.35 ml, 2.48 mmol) and the mixture heated under reflux for 2h. The reaction mixture was cooled, diluted with water (50 ml) and ethyl acetate (50 ml), the organic layer was separated washed with water (50 ml) then brine and dried (MgSO 4 ).
- the activity of compounds of formula (I), or a pharmaceutically acceptable salt, enantiomer or tautomer thereof, may be screened for nitric oxide synthetase inhibiting activity by a procedure based on that of Forstermann et al., Eur. J. Pharm., 1992, 225, 161-165.
- Nitric oxide synthase converts 3 H-L-arginine into 3 H-L-citrulline which can be separated by cation exchange chromatography and quantified by liquid scintillation counting.
- Enzyme is prepared, after induction, from the cultured murine macrophage cell line J774A-1 (obtained from the laboratories of the Imperial Cancer Research Fund). J774A-1 cells are cultured in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% foetal bovine serum, 4 mM L-glutamine and antibiotics (100 units/ml penicillin G, 100 mg ml streptomycin & 0J5 mg/ml amphotericin B). Cells are routinely grown in 225 cm 3 flasks containing 35 ml medium kept at 37 °C and in a humidified atmosphere containing 5% CO .
- DMEM Dulbeccos Modified Eagles Medium
- Nitric oxide synthase is produced by cells in response to interferon-g (IFNg) and lipopolysaccharide (LPS).
- IFNg interferon-g
- LPS lipopolysaccharide
- the medium from confluent culture flasks is removed and replaced with 25 ml (per flask) of fresh medium containing 1 mg/ml LPS and 10 units/ml IFNg.
- harvesting of cells is accomplished by scraping the cell sheet from the flask surface into the culture medium.
- Cells are collected by centrifugation (1000 g for 10 minutes) and lysate prepared by adding to the cell pellet a solution containing 50 mM Tris-HCl (pH 7.5 at 20 °C), 10% (v/v) glycerol, 0.1% (v/v) Triton-X-100, 0.1 mM dithiothreitol and a cocktail of protease inhibitors comprising leupeptin (2 mg/ml), soya bean trypsin inhibitor (10 mg/ml), aprotinin (5 mg/ml) and phenylmethylsulphonyl fluoride (50 mg/ml).
- protease inhibitors comprising leupeptin (2 mg/ml), soya bean trypsin inhibitor (10 mg/ml), aprotinin (5 mg/ml) and phenylmethylsulphonyl fluoride (50 mg/ml).
- substrate cocktail 50 mM Tris-HCl (pH 7.5 at 20 °C), 400 ⁇ M NADPH, 20 ⁇ M flavin adenine dinucleotide, 20 ⁇ M flavin mononucleotide, 4 ⁇ M tetrahydrobiopterin, 12 ⁇ M L-arginine and 0.025 mCi L-[ 3 H] arginine
- substrate cocktail 50 mM Tris-HCl (pH 7.5 at 20 °C), 400 ⁇ M NADPH, 20 ⁇ M flavin adenine dinucleotide, 20 ⁇ M flavin mononucleotide, 4 ⁇ M tetrahydrobiopterin, 12 ⁇ M L-arginine and 0.025 mCi L-[ 3 H] arginine
- the reaction is started by adding 50 ⁇ l of cell lysate (prepared as above) and after incubation for 1 hour at room temperature is terminated by addition of 50 ⁇ l of an aqueous solution of 3 mM nitroarginine and 21 mM EDTA.
- Labelled L-citrulline is separated from labelled L-arginine using Dowex AG-50W.
- 150 ⁇ l of a 25% aqueous slurry of Dowex 50W (Na + form) is added to the assay after which the whole is filtered into 96 well plates.
- 75 ⁇ l of filtrate is sampled and added to wells of 96 well plates containing solid scintillant. After allowing the samples to dry the L-citrulline is quantified by scintillation counting.
- basal activity is 300 dpm per 75 ⁇ l sample which is increased to 1900 dpm in the reagent controls.
- Compound activity is expressed as IC 50 (the concentration of drug substance which gives 50% enzyme inhibition in the assay) and aminoguanidine, which gives an IC 50 (50% inhibitory concentration) of 10 ⁇ M, is tested as a standard to verify the procedure.
- Compounds are tested at a range of concentrations and from the inhibitions obtained, IC 50 values are calculated.
- Compounds that inhibit the enzyme by at least 25% at 100 ⁇ M are classed as being active and are subjected to at least one retest.
- Compounds also show activity against the human form of induced nitric oxide synthase as can be demonstrated in the following assay.
- Enzyme is prepared, after induction, from the cultured human colon adrenocarcinoma cell line DLDl (obtained from the European Collection of Animal Cell Culture - cell line number 90102540).
- DLDl cells are cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum, 4 mM L-glutamine and antibiotics (100 units/ml penicillin G, 100 ⁇ g/ml streptomycin and 0.25 ⁇ g/ml amphotericin B). Cells are routinely grown in 225 cm flasks containing 35 ml medium kept at 37 °C and in a humidified atmosphere containing 5% CO 2 .
- Nitric oxide synthase is produced by cells in response to interferon- ⁇ (IFN- ⁇ ) and interleukin-l ⁇ (IL-l ⁇ ).
- IFN- ⁇ interferon- ⁇
- IL-l ⁇ interleukin-l ⁇
- the medium from confluent flasks is removed and replaced with 25 ml (per flask) of fresh medium containing 250 units/ml EL-l ⁇ and 1000 units/ml IFN- ⁇ .
- harvesting of cells is accomplished by scraping the cell monolayer from the flask surface into the culture medium.
- Cells are collected by centrifugation (lOOOg for 10 minutes) and lysate prepared by adding to the cell pellet a solution containing 50 mM Tris-HCl (pH 7.5 at 20°C), 10% (v/v) glycerol, 0.1% (v/v) Triton-XlOO, 0J mM dithiothreitol and a cocktail of protease inhibitors including leupeptin (2 ⁇ g/ml), soya bean trypsin inhibitor (10 ⁇ g/ml), aprotonin (5 ⁇ g/ml) and phenylmethylsulphonyl fluoride (50 ⁇ g/ml).
- substrate cocktail 50 mM Tris-HCl (pH 7.5), 400 ⁇ M NADPH, 20 ⁇ M flavin adenine dinucleotide, 20 ⁇ M flavin mononucleotide and 4 ⁇ M tetrahydrobiopterin
- substrate cocktail 50 mM Tris-HCl (pH 7.5), 400 ⁇ M NADPH, 20 ⁇ M flavin adenine dinucleotide, 20 ⁇ M flavin mononucleotide and 4 ⁇ M tetrahydrobiopterin
- Test compounds are preincubated with enzyme by adding together with 40 ⁇ l of cell lysate (prepared as above) and incubating for 1 hour at 37 °C at the end of which period 10 ⁇ l of 30 ⁇ M L-arginine and 0.025 ⁇ Ci of L-[ 3 H]-arginine in 50 mM Tris-HCl is added to start the enzymatic reaction. Incubation is continued for a further 1 hour at 37 °C. The reaction is terminated by addition of 50 ⁇ l of an aqueous solution of 3 mM nitroarginine and 21 mM EDTA.
- Labelled L-citrulline is separated from labelled L-arginine using Dowex AG-50W. 120 ⁇ l of a 25% aqueous slurry of Dowex 50W is added to 96 well filter plates (0.45 ⁇ m pore size). To this is added 120 ⁇ l of terminated assay mix. 75 ⁇ l of filtrate is sampled and added to the wells of 96 well plates containing solid scintillant. After allowing the samples to dry the L-citrulline is quantified by scintillation counting.
- basal activity is 300 dpm per 75 ⁇ l sample of reagent controls, which is increased to 3000 dpm in the presence of enzyme.
- Compound activity is expressed as IC 50 (the concentration of drug substance which gives 50% enzyme inhibition in the assay) and L-NMMA, which gives an IC 50 of about 0.4 ⁇ M is tested as a standard to verify the procedure.
- Compounds are tested at a range of concentrations and from the inhibitions.obtained IC 5 o values are calculated.
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Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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AU14242/00A AU1424200A (en) | 1998-10-15 | 1999-10-11 | Compounds |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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SE9803518-1 | 1998-10-15 | ||
SE9803518A SE9803518D0 (en) | 1998-10-15 | 1998-10-15 | Novel compounds |
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WO2000021934A1 true WO2000021934A1 (en) | 2000-04-20 |
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PCT/SE1999/001829 WO2000021934A1 (en) | 1998-10-15 | 1999-10-11 | Compounds |
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AU (1) | AU1424200A (en) |
SE (1) | SE9803518D0 (en) |
WO (1) | WO2000021934A1 (en) |
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US7105544B2 (en) | 2001-07-05 | 2006-09-12 | Synaptic Pharmaceutical Corporation | Substituted alkyl amido piperidines |
US7199135B2 (en) | 2001-07-05 | 2007-04-03 | H. Lundbeck A/S | Substituted alkyl amido piperidines |
JP2008543958A (en) * | 2005-06-28 | 2008-12-04 | サノフィ−アベンティス | Heteroaryl-substituted amides containing saturated linker groups and their use as pharmaceuticals |
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US8993591B2 (en) | 2010-11-08 | 2015-03-31 | Janssen Pharmaceuticals, Inc. | 1,2,4-triazolo[4,3-a] pyridine derivatives and their use as positive allosteric modulators of MGLUR2 receptors |
US9012448B2 (en) | 2010-11-08 | 2015-04-21 | Janssen Pharmaceuticals, Inc. | 1,2,4-triazolo[4,3-a]pyridine derivatives and their use as positive allosteric modulators of MGLUR2 receptors |
US9067891B2 (en) | 2007-03-07 | 2015-06-30 | Janssen Pharmaceuticals, Inc. | 1,4-disubstituted 3-cyano-pyridone derivatives and their use as positive allosteric modulators of mGluR2-receptors |
US9085577B2 (en) | 2009-05-12 | 2015-07-21 | Janssen Pharmaceuticals, Inc. | 7-aryl-1,2,4-triazolo[4,3-A]pyridine derivatives and their use as positive allosteric modulators of mGluR2 receptors |
US9114138B2 (en) | 2007-09-14 | 2015-08-25 | Janssen Pharmaceuticals, Inc. | 1′,3′-disubstituted-4-phenyl-3,4,5,6-tetrahydro-2H,1′H-[1,4′] bipyridinyl-2′-ones |
US9271967B2 (en) | 2010-11-08 | 2016-03-01 | Janssen Pharmaceuticals, Inc. | 1,2,4-triazolo[4,3-a]pyridine derivatives and their use as positive allosteric modulators of mGluR2 receptors |
US9708315B2 (en) | 2013-09-06 | 2017-07-18 | Janssen Pharmaceutica Nv | 1,2,4-triazolo[4,3-a]pyridine compounds and their use as positive allosteric modulators of MGLUR2 receptors |
US10106542B2 (en) | 2013-06-04 | 2018-10-23 | Janssen Pharmaceutica Nv | Substituted 6,7-dihydropyrazolo[1,5-a]pyrazines as negative allosteric modulators of mGluR2 receptors |
US10537573B2 (en) | 2014-01-21 | 2020-01-21 | Janssen Pharmaceutica Nv | Combinations comprising positive allosteric modulators or orthosteric agonists of metabotropic glutamatergic receptor subtype 2 and their use |
US11369606B2 (en) | 2014-01-21 | 2022-06-28 | Janssen Pharmaceutica Nv | Combinations comprising positive allosteric modulators or orthosteric agonists of metabotropic glutamatergic receptor subtype 2 and their use |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997024119A1 (en) * | 1995-12-29 | 1997-07-10 | Smithkline Beecham Corporation | Vitronectin receptor antagonists |
WO1997024122A1 (en) * | 1995-12-29 | 1997-07-10 | Smithkline Beecham Corporation | Vitronectin receptor antagonists |
WO1997036871A1 (en) * | 1996-03-29 | 1997-10-09 | Pfizer Inc. | 6-phenylpyridyl-2-amine derivatives |
-
1998
- 1998-10-15 SE SE9803518A patent/SE9803518D0/en unknown
-
1999
- 1999-10-11 WO PCT/SE1999/001829 patent/WO2000021934A1/en active Application Filing
- 1999-10-11 AU AU14242/00A patent/AU1424200A/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997024119A1 (en) * | 1995-12-29 | 1997-07-10 | Smithkline Beecham Corporation | Vitronectin receptor antagonists |
WO1997024122A1 (en) * | 1995-12-29 | 1997-07-10 | Smithkline Beecham Corporation | Vitronectin receptor antagonists |
WO1997036871A1 (en) * | 1996-03-29 | 1997-10-09 | Pfizer Inc. | 6-phenylpyridyl-2-amine derivatives |
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US7199135B2 (en) | 2001-07-05 | 2007-04-03 | H. Lundbeck A/S | Substituted alkyl amido piperidines |
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Also Published As
Publication number | Publication date |
---|---|
SE9803518D0 (en) | 1998-10-15 |
AU1424200A (en) | 2000-05-01 |
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