Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/08—Bridged systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
  • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
  • C07D271/06—1,2,4-Oxadiazoles; Hydrogenated 1,2,4-oxadiazoles
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
  • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
  • C07D271/10—1,3,4-Oxadiazoles; Hydrogenated 1,3,4-oxadiazoles

Definitions

• This invention relates to heterocycles, pharmaceutical compositions, methods for their preparation and use in the treatment of diseases where enhanced M3 receptor activation is implicated.
• Anti-cholinergic agents prevent the passage of, or effects resulting from the passage of, impulses through the parasympathetic nerves. This is a consequence of the ability of such compounds to inhibit the action of acetylcholine (Ach) by blocking its binding to the muscarinic cholinergic receptors.
• mAChRs muscarinic acetylcholine receptors
• M1-M5 muscarinic acetylcholine receptors
• M3 mAChRs mediate contractile responses (reviewed by Caulfield, 1993, Pharmac. Ther., 58, 319 - 379).
• muscarinic receptors M1 , M2 and M3 have been demonstrated to be important and are localized to the trachea, the bronchi, submucosal glands and parasympathetic ganglia (reviewed in Fryer and Jacoby, 1998, Am J Resp Crit Care Med., 158 (5 part 3) S 154 - 160).
• M3 receptors on airway smooth muscle mediate contraction and therefore bronchoconstriction. Stimulation of M3 receptors localised to submucosal glands results in mucus secretion.
• vagal tone may either be increased (Gross et al. 1989, Chest; 96:984-987) and/or may provoke a higher degree of obstruction for geometric reasons if applied on top of oedematous or mucus-laden airway walls (Gross et al. 1984, Am Rev
• M3 mAChR antagonists may be useful as therapeutics in these mAChR-mediated diseases.
• Tiotropium (Spiriva TM) is a long-acting muscarinic antagonist currently marketed for the treatment of chronic obstructive pulmonary disease, administered by the inhaled route.
• ipratropium is a muscarinic antagonist marketed for the treatment of COPD.
• WO97/30994 describes oxadiazoles and thiadiazoles as muscarinic receptor antagonists.
• EP0323864 describes oxadiazoles linked to a mono- or bicyclic ring as muscarinic receptor modulators.
• R 2 is a group H, -(Z) p -R 7 , -Z-Y-R 7 or -Y-R 7 ; p is 0 or 1 ;
• R 4 and R 5 are independently selected from the group consisting of aryl, aryl-fused- heterocycloalkyl, heteroaryl, d-C 6 -alkyl, and cycloalkyl;
• R 6 is -OH, CrCe-alkyl, C r C 6 -alkoxy, hydroxy-CrC 6 -alkyl, nitrile, a group CONR 1 R 9 or a hydrogen atom; one of W, V and A is N or NR 11 ; another of W, V and A is N, O, S or CR 8 ; and the last one of W, V and A is N or CR 8 ;
• X is an C ⁇ C ⁇ alkylene, C 2 -C 4 -alkenylene or C 2 -C 4 -alkynylene group;
• R 7 is an CrC 6 -alkyl, C 2 -C 6 -alkenyl, aryl, aryl-fused-cycloalkyl, aryl-fused- heterocycloalkyl, heteroaryl, aryl(C r C 8 -alkyl)-, heteroaryl(CrC 8 -alkyl)-, heterocycloalky
• Z is a CrC 4 -alkylene, C 2 -C 4 -alkenylene or C 2 -C 4 -alkynylene group;
• Y is an oxygen atom, a group -OC(O)-, a group -N(H)C(O)- or a group -S(O) n ;
• n is O, 1 or 2;
• R 1 , R 8 , R 9 and R 11 are, independently, a hydrogen atom or Ci-C 6 -alkyl group
• D " is a pharmaceutically acceptable counter-ion
• each occurrence of alkyl, alkenyl, heterocycloalkyl, aryl, aryl-fused-heterocycloalkyl, heteroaryl, cycloalkyl, alkoxy, alkylene, alkenylene, alkynylene or aryl-fused-cycloalkyl may be optionally substituted; and wherein each alkenylene chain contains, where possible, up to 2 carbon-carbon double bonds and each alkynylene chain contains, where possible, up to 2 carbon- carbon triple bonds.
• the present invention provides compounds falling within the scope of, but not specifically disclosed in, our co-pending application PCT/GB2008/000519 referred to above.
• the present invention provides a compound which has a quaternary ammonium species selected from the group consisting of:
• X represents a pharmaceutically acceptable anion of a mono or polyvalent acid.
• the present invention provides a compound which has a quaternary ammonium species selected from the group consisting of:
• X represents a pharmaceutically acceptable anion of a mono or polyvalent acid.
• the present invention provides (R)-3-(3-Fluoro-4-methyl- phenoxy)-1-[3-(hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]-1-azonia- bicyclo[2.2.2]octane bromide
• the present invention provides (R)-3-(3-Fluoro-phenylsulfanyl)-1-
• the compounds of formula (I), referred to above, and those of the present invention comprise an anion X associated with the positive charge on the quaternary nitrogen atom.
• the anion X may be any pharmaceutically acceptable anion of a mono or polyvalent (e.g. bivalent) acid.
• X may be an anion of a mineral acid, for example chloride, bromide, iodide, sulfate, nitrate or phosphate; or an anion of a suitable organic acid, for example acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, methanesulphonate, p-toluenesulphonate, benzenesulphonate, napadisylate (naphthalene-1 ,5-disulfonate) (e.g. a heminapadisylate), 2,5-dichlorobenzenesulphonate, 1-hydroxynaphthalene-2- sulphonate or xinafoate (1-hydroxy-2-naphthoate).
• a mineral acid for example chloride, bromide, iodide, sulfate, nitrate or phosphate
• a suitable organic acid for example acetate, maleate, fum
• the present invention also comprises the following novel intermediate compounds which have utility in the synthesis of some of the claimed muscarinic antagonists of the present invention: (R)-(5-Bromomethyl-[1 ,3,4]oxadiazol-2-yl)-cyclohexyl-phenyl-methanol; (R)-(5-Chloromethyl-[1 ,3,4]oxadiazol-2-yl)-cyclohexyl-phenyl-methanol; and (RH ⁇ -Chloromethyl-fi ⁇ Joxadiazol-S-yO-cyclohexyl-phenyl-methanol.
• the compounds of the present invention display beneficial pharmaceutical properties.
• the compounds of the invention display activity as antagonists of muscarinic receptors, particularly muscarinic M 3 receptors.
• the compounds also display desirable plasma protein binding properties. Plasma protein binding may be an advantageous property for compounds administered via inhalation as it can lessen the impact of any systemic effect the compound may have.
• the compounds of the invention have activity as pharmaceuticals, in particular as anticholinergic agents including muscarinic receptor (M1 , M2, and M3) antagonists, in particular M3 antagonists.
• Diseases and conditions which may be treated with the compounds include: 1 . respiratory tract obstructive diseases of the airways including: asthma, including bronchial, allergic, intrinsic, extrinsic, exercise-induced, drug-induced (including aspirin and NSAID-induced) and dust-induced asthma, both intermittent and persistent and of all severities, and other causes of airway hyper-responsiveness; chronic obstructive pulmonary disease (COPD); bronchitis, including infectious and eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis; sarcoidosis; farmer's lung and related diseases; hypersensitivity pneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis, idiopathic interstitial pneumonias
• osteoarthritides associated with or including osteoarthritis/osteoarthrosis both primary and secondary to, for example, congenital hip dysplasia; cervical and lumbar spondylitis, and low back and neck pain; rheumatoid arthritis and Still's disease; seronegative spondyloarthropathies including ankylosing spondylitis, psoriatic arthritis, reactive arthritis and undifferentiated spondarthropathy; septic arthritis and other infection-related arthopathies and bone disorders such as tuberculosis, including Potts' disease and Poncet's syndrome; acute and chronic crystal-induced synovitis including urate gout, calcium pyrophosphate deposition disease, and calcium apatite related tendon, bursal and synovial inflammation; Behcet's disease; primary and secondary Sjogren's syndrome; systemic sclerosis and limited scleroderma; systemic lupus erythematosus, mixed connective tissue
• arthitides for example rheumatoid arthritis, osteoarthritis, gout or crystal arthropathy
• other joint disease such as intervertebral disc degeneration or temporomandibular joint degeneration
• bone remodelling disease such as osteoporosis, Paget's disease or osteonecrosis
• polychondritits such as osteoporosis, Paget's
• skin psoriasis, atopic dermatitis, contact dermatitis or other eczematous dermatoses, and delayed-type hypersensitivity reactions; phyto- and photodermatitis; seborrhoeic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus et atrophica, pyoderma gangrenosum, skin sarcoid, discoid lupus erythematosus, pemphigus, pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitides, toxic erythemas, cutaneous eosinophilias, alopecia areata, male-pattern baldness, Sweet's syndrome, Weber-Christian syndrome, erythema multiforme; cellulitis, both infective and non-infective; panniculitis;cutaneous lymphomas, non-melanoma
• eyes blepharitis; conjunctivitis, including perennial and vernal allergic conjunctivitis; ulceris; anterior and posterior uveitis; choroiditis; autoimmune; degenerative or inflammatory disorders affecting the retina; ophthalmitis including sympathetic ophthalmitis; sarcoidosis; infections including viral , fungal, and bacterial;
• abdominal hepatitis, including autoimmune, alcoholic and viral; fibrosis and cirrhosis of the liver; cholecystitis; pancreatitis, both acute and chronic;
• genitourinary, nephritis including interstitial and glomerulonephritis; nephrotic syndrome; cystitis including acute and chronic (interstitial) cystitis and Hunner's ulcer; acute and chronic urethritis, prostatitis, epididymitis, oophoritis and salpingitis; vulvovaginitis; Peyronie's disease; erectile dysfunction (both male and female);
• allograft rejection acute and chronic following, for example, transplantation of kidney, heart, liver, lung, bone marrow, skin or cornea or following blood transfusion; or chronic graft versus host disease;
• CNS Alzheimer's disease and other dementing disorders including CJD and nvCJD; amyloidosis; multiple sclerosis and other demyelinating syndromes; cerebral atherosclerosis and vasculitis; temporal arteritis; myasthenia gravis; acute and chronic pain (acute, intermittent or persistent, whether of central or peripheral origin) including visceral pain, headache, migraine, trigeminal neuralgia, atypical facial pain, joint and bone pain, pain arising from cancer and tumor invasion, neuropathic pain syndromes including diabetic, post-herpetic, and HIV-associated neuropathies; neurosarcoidosis; central and peripheral nervous system complications of malignant, infectious or autoimmune processes; 11. other auto-immune and allergic disorders including Hashimoto's thyroiditis,
• Graves' disease Addison's disease, diabetes mellitus, idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-lgE syndrome, antiphospholipid syndrome; 12. other disorders with an inflammatory or immunological component; including acquired immune deficiency syndrome (AIDS), leprosy, Sezary syndrome, and paraneoplastic syndromes; 13.
• AIDS acquired immune deficiency syndrome
• cardiovascular atherosclerosis, affecting the coronary and peripheral circulation
• pericarditis myocarditis , inflammatory and auto-immune cardiomyopathies including myocardial sarcoid; ischaemic reperfusion injuries; endocarditis, valvulitis, and aortitis including infective (for example syphilitic); vasculitides; disorders of the proximal and peripheral veins including phlebitis and thrombosis, including deep vein thrombosis and complications of varicose veins;
• oncology treatment of common cancers including prostate, breast, lung, ovarian, pancreatic, bowel and colon, stomach, skin and brain tumors and malignancies affecting the bone marrow (including the leukaemias) and lymphoproliferative systems, such as Hodgkin's and non-Hodgkin's lymphoma; including the prevention and treatment of metastatic disease and tumour recurrences, and paraneoplastic syndromes; and,
• gastrointestinal tract Coeliac disease, proctitis, eosinopilic gastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis, microscopic colitis, indeterminant colitis, irritable bowel disorder, irritable bowel syndrome, non-inflammatory diarrhea, food-related allergies which have effects remote from the gut, e.g., migraine, rhinitis and eczema.
• the present invention further provides a compound of the present invention, as hereinbefore defined, for use in therapy.
• the invention provides the use of a compound of the present invention, as hereinbefore defined, in the manufacture of a medicament for use in therapy.
• the term “therapy” also includes “prophylaxis” unless there are specific indications to the contrary.
• the terms “therapeutic” and “therapeutically” should be construed accordingly.
• a further aspect of the invention provides a method of treating a disease state in a mammal suffering from, or at risk of, said disease, which comprises administering to a mammal in need of such treatment a therapeutically effective amount of a compound of the present invention, as hereinbefore defined.
• the present invention also provides a compound of the present invention, as hereinbefore defined, for use in treating chronic obstructive pulmonary disease (COPD) (such as irreversible COPD).
• COPD chronic obstructive pulmonary disease
• the present invention also provides a compound of the present invention, as hereinbefore defined, for use in treating asthma.
• the present invention also provides the use of a compound of the present invention, as hereinbefore defined, in the manufacture of a medicament for use in the treatment of chronic obstructive pulmonary disease (COPD) (such as irreversible COPD).
• COPD chronic obstructive pulmonary disease
• the present invention also provides the use of a compound of the present invention, as hereinbefore defined, in the manufacture of a medicament for use in the treatment of asthma.
• the present invention further provides a method of treating chronic obstructive pulmonary disease (COPD) (such as irreversible COPD) 1 in a warm-blooded animal, such as man, which comprises administering to a mammal in need of such treatment an effective amount of a compound of the present invention, as hereinbefore defined.
• COPD chronic obstructive pulmonary disease
• the present invention further provides a method of treating asthma in a warmblooded animal, such as man, which comprises administering to a mammal in need of such treatment an effective amount of a compound of the present invention, as hereinbefore defined.
• a compound of the invention for the therapeutic treatment of a warmblooded animal, such as man, said ingredient is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
• the present invention provides a pharmaceutical composition that comprises a compound of the invention as hereinbefore defined and a pharmaceutically acceptable adjuvant, diluent or carrier.
• a process for the preparation of said composition which comprises mixing active ingredient with a pharmaceutically acceptable adjuvant, diluent or carrier.
• the pharmaceutical composition will, for example, comprise from 0.05 to 99%w (per cent by weight), such as from 0.05 to 80%w, for example from 0.10 to 70%w, such as from 0.10 to 50%w, of active ingredient, all percentages by weight being based on total composition.
• compositions of this invention may be administered in standard manner for the disease condition that it is desired to treat, for example by topical (such as to the lung and/or airways or to the skin), oral, rectal or parenteral administration.
• topical such as to the lung and/or airways or to the skin
• the compounds of this invention may be formulated by means known in the art into the form of, for example, aerosols, dry powder formulations, tablets, capsules, syrups, powders, granules, aqueous or oily solutions or suspensions, (lipid) emulsions, dispersible powders, suppositories, ointments, creams, drops and sterile injectable aqueous or oily solutions or suspensions.
• a suitable pharmaceutical composition of this invention is one suitable for oral administration in unit dosage form, for example a tablet or capsule, which contains between 0.1 mg and 1 g of active ingredient.
• a pharmaceutical composition of the invention is one suitable for intravenous, subcutaneous or intramuscular injection.
• Each patient may receive, for example, an intravenous, subcutaneous or intramuscular dose of 0.01 mgkg '1 to l OOmgkg "1 of the compound, for example in the range of 0.1 mgkg "1 to 20mgkg "1 of this invention, the composition being administered 1 to 4 times per day.
• the intravenous, subcutaneous and intramuscular dose may be given by means of a bolus injection.
• the intravenous dose may be given by continuous infusion over a period of time.
• each patient will receive a daily oral dose, which is approximately equivalent to the daily parenteral dose, the composition being administered 1 to 4 times per day
• Another suitable pharmaceutical composition of this invention is one suitable for inhaled administration, inhalation being a particularly useful method for administering the compounds of the invention when treating respiratory diseases such as chronic obstructive pulmonary disease (COPD) or asthma.
• COPD chronic obstructive pulmonary disease
• the compounds of the present invention may be used effectively at doses in the ⁇ g range, for example 0.1 to 500 ⁇ g, 0.1 to 50 ⁇ g, 0.1 to 40 ⁇ g, 0.1 to 30 ⁇ g, 0.1 to 20 ⁇ g, 0.1 to 10 ⁇ g, 5 to 10 ⁇ g, 5 to 50 ⁇ g, 5 to 40 ⁇ g, 5 to 30 ⁇ g, 5 to 20 ⁇ g, 5 to 10 ⁇ g, 10 to 50 ⁇ g, 10 to 40 ⁇ g 10 to 30 ⁇ g, or 10 to 20 ⁇ g of active ingredient.
• a pharmaceutical composition comprising a compound of the invention as hereinbefore defined, in association with a pharmaceutically acceptable adjuvant, diluent or carrier, which
• metered dose inhaler devices When administered by inhalation, metered dose inhaler devices may be used to administer the active ingredient, dispersed in a suitable propeliant and with or without additional excipients such as ethanol, surfactants, lubricants or stabilising agents.
• suitable propellants include hydrocarbon, chlorofluorocarbon and hydrofluoroalkane (e.g. heptafluoroalkane) propellants, or mixtures of any such propellants.
• Preferred propellants are P134a and P227, each of which may be used alone or in combination with other propellants and/or surfactant and/or other excipients.
• Nebulised aqueous suspensions or, preferably, solutions may also be employed, with or without a suitable pH and/or tonicity adjustment, either as a unit-dose or multi-dose formulations.
• Dry powder inhalers may be used to administer the active ingredient, alone or in combination with a pharmaceutically acceptable carrier, in the later case either as a finely divided powder or as an ordered mixture.
• the dry powder inhaler may be single dose or multi-dose and may utilise a dry powder or a powder-containing capsule.
• Metered dose inhaler, nebuliser and dry powder inhaler devices are well known and a variety of such devices are available.
• the invention further relates to combination therapies wherein a compound of the invention or a pharmaceutical composition or formulation comprising a compound of the invention, is administered concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of one or more of the conditions listed.
• the compounds of the invention may be combined with agents listed below.
• Non-steroidal anti-inflammatory agents including non-selective cyclo-oxygenase COX-1 / COX-2 inhibitors whether applied topically or systemically (such as piroxicam, diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such as mefenamic acid, indomethacin, sulindac, azapropazone, pyrazolones such as phenylbutazone, salicylates such as aspirin); selective COX-2 inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib, lumarocoxib, parecoxib and etoricoxib); cyclo- oxygenase inhibiting nitric oxide donors (CINODs); glucocorticosteroids (whether administered by topical, oral, intramuscular
• COX-2 inhibitors such
• the present invention still further relates to the combination of a compound of the invention together with a cytokine or agonist or antagonist of cytokine function, (including agents which act on cytokine signalling pathways such as modulators of the SOCS system) including alpha-, beta-, and gamma-interferons; insulin-like growth factor type I (IGF-1); interleukins (IL) including IL1 to 17, and interleukin antagonists or inhibitors such as anakinra; tumour necrosis factor alpha (TNF- ⁇ ) inhibitors such as anti-TNF monoclonal antibodies (for example infliximab; adalimumab, and CDP-870) and TNF receptor antagonists including immunoglobulin molecules (such as etanercept) and low-molecular-weight agents such as pentoxyfylline.
• a cytokine or agonist or antagonist of cytokine function including agents which act on cytokine signalling pathways such as modulators of the SOCS system
• the invention relates to a combination of a compound of the invention with a monoclonal antibody targeting B-Lymphocytes (such as CD20 (rituximab), MRA- alLl6R and T-Lymphocytes, CTLA4-lg, HuMax 11-15).
• B-Lymphocytes such as CD20 (rituximab), MRA- alLl6R and T-Lymphocytes, CTLA4-lg, HuMax 11-15.
• the present invention still further relates to the combination of a compound of the invention with a modulator of chemokine receptor function such as an antagonist of CCR1 , CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C-C family); CXCR1 , CXCR2, CXCR3, CXCR4 and CXCR5 (for the C-X-C family) and CX 3 CRI for the C-X 3 -C family.
• a modulator of chemokine receptor function such as an antagonist of CCR1 , CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C-C family); CXCR1 , CXCR2, CXCR3, CXCR4 and CXCR5 (for the C-X-C family) and CX 3 CRI for
• the present invention further relates to the combination of a compound of the invention with an inhibitor of matrix metalloprotease (MMPs), i.e., the stromelysins, the collagenases, and the gelatinases, as well as aggrecanase; especially collagenase-1 (MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-1 1 ) and MMP-9 and MMP-12, including agents such as doxycycline.
• MMPs matrix metalloprotease
• the present invention still further relates to the combination of a compound of the invention and a leukotriene biosynthesis inhibitor, 5- lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activating protein (FLAP) antagonist such as; zileuton; ABT-761 ; fenleuton; tepoxalin; Abbott-79175; Abbott-85761 ; a N-(5-substituted)-thiophene-2- alkylsulfonamide; 2,6-di-tert-butylphenolhydrazones; a methoxytetrahydropyrans such as Zeneca ZD-2138; the compound SB-210661 ; a pyridinyl-substituted 2- cyanonaphthalene compound such as L-739,010; a 2-cyanoquinoline compound such as L-746,530; or an indole or quinoline compound such as MK-591 , MK-886, and BAY x 100
• the present invention further relates to the combination of a compound of the invention and a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4 selected from the group consisting of the phenothiazin-3-1s such as L-651 ,392; amidino compounds such as CGS-25019c; benzoxalamines such as ontazolast; benzenecarboximidamides such as BIIL 284/260; and compounds such as zafirlukast, abiukast, montelukast, pranlukast, verlukast (MK-679), RG-12525, Ro- 245913, iralukast (CGP 45715A), and BAY x 7195.
• LT leukotrienes
• the present invention still further relates to the combination of a compound of the invention and a phosphodiesterase (PDE) inhibitor such as a methylxanthanine including theophylline and aminophylline; a selective PDE isoenzyme inhibitor including a PDE4 inhibitor an inhibitor of the isoform PDE4D, or an inhibitor of PDE5.
• PDE phosphodiesterase
• the present invention further relates to the combination of a compound of the invention and a histamine type 1 receptor antagonist such as cetirizine, loratadine, desloratadine, fexofenadine, acrivastine, terfenadine, astemizole, azelastine, levocabastine, chlorpheniramine, promethazine, cyclizine, or mizolastine; applied orally, topically or parenterally.
• a histamine type 1 receptor antagonist such as cetirizine, loratadine, desloratadine, fexofenadine, acrivastine, terfenadine, astemizole, azelastine, levocabastine, chlorpheniramine, promethazine, cyclizine, or mizolastine; applied orally, topically or parenterally.
• the present invention still further relates to the combination of a compound of the invention and a proton pump
• the present invention further relates to the combination of a compound of the invention and an antagonist of the histamine type 4 receptor.
• the present invention still further relates to the combination of a compound of the invention and an alpha-1/alpha-2 adrenoceptor agonist vasoconstrictor sympathomimetic agent, such as propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride or ethylnorepinephrine hydrochloride.
• an alpha-1/alpha-2 adrenoceptor agonist vasoconstrictor sympathomimetic agent such as propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline hydrochloride, oxymetazoline hydrochloride, t
• the present invention still further relates to the combination of a compound of the invention and a beta-adrenoceptor agonist (including beta receptor subtypes 1-4) such as isoprenaline, salbutamol, formoterol, salmeterol, terbutaline, orciprenaline, bitolterol mesylate, pirbuterol, or indacaterol or a chiral enantiomer thereof.
• a beta-adrenoceptor agonist including beta receptor subtypes 1-4
• the present invention further relates to the combination of a compound of the invention and a chromone, such as sodium cromoglycate or nedocromil sodium.
• a chromone such as sodium cromoglycate or nedocromil sodium.
• the present invention still further relates to the combination of a compound of the invention with a glucocorticoid, such as flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide or mometasone furoate.
• a glucocorticoid such as flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide or mometasone furoate.
• the present invention further relates to the combination of a compound of the invention with an agent that modulates a nuclear hormone receptor such as PPARs.
• the present invention still further relates to the combination of a compound of the invention together with an immunoglobulin (Ig) or Ig preparation or an antagonist or antibody modulating Ig function such as anti-lgE (for example omalizumab).
• the present invention further relates to the combination of a compound of the invention and another systemic or topically-applied anti-inflammatory agent, such as thalidomide or a derivative thereof, a retinoid, dithranol or calcipotriol.
• the present invention still further relates to the combination of a compound of the invention and combinations of aminosalicylates and sulfapyridine such as sulfasalazine, mesalazine, balsalazide, and olsalazine; and immunomodulatory agents such as the thiopurines, and corticosteroids such as budesonide.
• aminosalicylates and sulfapyridine such as sulfasalazine, mesalazine, balsalazide, and olsalazine
• immunomodulatory agents such as the thiopurines, and corticosteroids such as budesonide.
• the present invention further relates to the combination of a compound of the invention together with an antibacterial agent such as a penicillin derivative, a tetracycline, a macrolide, a beta-lactam, a fluoroquinolone, metronidazole, an inhaled aminoglycoside; an antiviral agent including acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir, amantadine, rimantadine, ribavirin, zanamavir and oseltamavir; a protease inhibitor such as indinavir, nelfinavir, ritonavir, and saquinavir; a nucleoside reverse transcriptase inhibitor such as didanosine, lamivudine, stavudine, zalcitabine or zidovudine; or a non-nucleoside reverse transcriptase inhibitor such as nevirapine
• the present invention still further relates to the combination of a compound of the invention and a cardiovascular agent such as a calcium channel blocker, a beta- adrenoceptor blocker, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin-2 receptor antagonist; a lipid lowering agent such as a statin or a fibrate; a modulator of blood cell morphology such as pentoxyfylline; thrombolytic, or an anticoagulant such as a platelet aggregation inhibitor.
• a cardiovascular agent such as a calcium channel blocker, a beta- adrenoceptor blocker, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin-2 receptor antagonist
• ACE angiotensin-converting enzyme
• angiotensin-2 receptor antagonist angiotensin-2 receptor antagonist
• a lipid lowering agent such as a statin or a fibrate
• a modulator of blood cell morphology such as pentoxyfylline
• the present invention further relates to the combination of a compound of the invention and a CNS agent such as an antidepressant (such as sertraline), an antiparkinsonian drug (such as deprenyl, L-dopa, ropinirole, pramipexole, a MAOB inhibitor such as selegine and rasagiline, a comP inhibitor such as tasmar, an A-2 inhibitor, a dopamine reuptake inhibitor, an NMDA antagonist, a nicotine agonist, a dopamine agonist or an inhibitor of neuronal nitric oxide synthase), or an anti- Alzheimer's drug such as donepezil, rivastigmine, tacrine, a COX-2 inhibitor, propentofylline or metrifonate.
• a CNS agent such as an antidepressant (such as sertraline), an antiparkinsonian drug (such as deprenyl, L-dopa, ropinirole, pramip
• the present invention still further relates to the combination of a compound of the invention and an agent for the treatment of acute or chronic pain, such as a centrally or peripherally-acting analgesic (for example an opioid or derivative thereof), carbamazepine, phenytoin, sodium valproate, amitryptiline or other anti-depressant agent-s, paracetamol, or a non-steroidal anti-inflammatory agent.
• analgesic for example an opioid or derivative thereof
• carbamazepine for example an opioid or derivative thereof
• phenytoin for example an opioid or derivative thereof
• sodium valproate for example an opioid or derivative thereof
• amitryptiline or other anti-depressant agent-s for example an opioid or derivative thereof
• paracetamol for example an opioid or derivative thereof
• non-steroidal anti-inflammatory agent for example an opioid or derivative thereof
• the present invention further relates to the combination of a compound of the invention together with a parenteral ⁇ or topically-applied (including inhaled) local anaesthetic agent such as lignocaine or a derivative thereof.
• a parenteral ⁇ or topically-applied (including inhaled) local anaesthetic agent such as lignocaine or a derivative thereof.
• a compound of the present invention can also be used in combination with an anti- osteoporosis agent including a hormonal agent such as raloxifene, or a biphosphonate such as alendronate.
• a hormonal agent such as raloxifene
• a biphosphonate such as alendronate.
• the present invention still further relates to the combination of a compound of the invention together with a: (i) tryptase inhibitor; (ii) platelet activating factor (PAF) antagonist; (iii) interleukin converting enzyme (ICE) inhibitor; (iv) IMPDH inhibitor; (v) adhesion molecule inhibitors including VLA-4 antagonist; (vi) cathepsin; (vii) kinase inhibitor such as an inhibitor of tyrosine kinase (such as Btk, Itk, Jak3 or MAP, for example Gefitinib or lmatinib mesylate), a serine / threonine kinase (such as an inhibitor of a MAP kinase such as p38, JNK, protein kinase A, B or C, or IKK), or a kinase involved in cell cycle regulation (such as a cylin dependent kinase); (viii) glucose-6 phosphate
• -receptor antagonist for example colchicine
• anti-gout agent for example colchicine
• xanthine oxidase inhibitor for example allopurinol
• uricosuric agent for example probenecid, sulfinpyrazone or benzbromarone
• growth hormone secretagogue for example transforming growth factor (TGF ⁇ );
• PDGF platelet-derived growth factor
• fibroblast growth factor for example basic fibroblast growth factor (bFGF);
• GM-CSF granulocyte macrophage colony stimulating factor
• capsaicin cream for example tachykinin NK1 or NK3 receptor antagonist such as NKP-608C, SB-233412
• elastase inhibitor such as UT-77 or 2D-0892
• TACE TNF- alpha converting enzyme inhibitor
• iNOS induced nitric oxide synthase
• inhibitor of P38 agent modulating the function of Toll-like receptors (TLR),
• agent modulating the activity of purinergic receptors such as P2X7; or
• inhibitor of transcription factor activation such as NFkB, API, or STATS.
• a compound of the invention can also be used in combination with an existing therapeutic agent for the treatment of cancer, for example suitable agents include: (i) an antiproliferative/antineoplastic drug or a combination thereof, as used in medical oncology, such as an alkylating agent (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan or a nitrosourea); an antimetabolite (for example an antifolate such as a fluoropyrimidine like 5-fluorouracil or tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, gemcitabine or paclitaxel); an antitumour antibiotic (for example an anthracycline such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dact
• a cytostatic agent such as an antioestrogen (for example tamoxifen, toremifene, raloxifene, droloxifene or iodoxyfene), an oestrogen receptor down regulator (for example fulvestrant), an antiandrogen (for example bicalutamide, flutamide, nilutamide or cyproterone acetate), a LHRH antagonist or LHRH agonist (for example goserelin, leuprorelin or buserelin), a progestogen (for example megestrol acetate), an aromatase inhibitor (for example as anastrozole, letrozole, vorazole or exemestane) or an inhibitor of 5 ⁇ -reductase such as finasteride; (iii) an agent which inhibits cancer cell invasion (for example a metalloproteinase inhibitor like marimastat or an inhibitor of urokinase plasm
• an inhibitor of growth factor function for example: a growth factor antibody (for example the anti-erbb2 antibody trastuzumab, or the anti-erbb1 antibody cetuximab [C225]), a farnesyl transferase inhibitor, a tyrosine kinase inhibitor or a serine/threonine kinase inhibitor, an inhibitor of the epidermal growth factor family (for example an EGFR family tyrosine kinase inhibitor such as N-(3-chloro-4- fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, AZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) or 6-acrylamido-N-(3-chloro-4-fluorophenyl
• an agent used in antisense therapy for example one directed to one of the targets listed above, such as ISIS 2503, an anti-ras antisense
• an agent used in a gene therapy approach for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy
• an agent used in an immunotherapeutic approach for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transf
• the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is a compound of the present invention, as hereinbefore described, and at least one further active ingredient selected from:-
• the pharmaceutical product according to this embodiment may, for example, be a pharmaceutical composition comprising the first and further active ingredients in admixture.
• the pharmaceutical product may, for example, comprise the first and further active ingredients in separate pharmaceutical preparations suitable for simultaneous, sequential or separate administration to a patient in need thereof.
• the pharmaceutical product of this embodiment is of particular use in treating respiratory diseases such as asthma, COPD or rhinitis.
• Examples of a phosphodiesterase inhibitor that may be used in the pharmaceutical product according to this embodiment include a PDE4 inhibitor such as an inhibitor of the isoform PDE4D, a PDE3 inhibitor and a PDE5 inhibitor.
• PDE4 inhibitor such as an inhibitor of the isoform PDE4D
• PDE3 inhibitor a PDE3 inhibitor
• PDE5 inhibitor examples include the compounds (Z)-3-(3,5-dichloro-4-pyridyl)-2-[4-(2-indanyloxy-5-methoxy-2-pyridyl]propenenitrile,
• Examples of a ⁇ 2 -adrenoceptor agonist that may be used in the pharmaceutical product according to this embodiment include metaproterenol, isoproterenol, isoprenaline, albuterol, salbutamol (e.g. as sulphate), formoterol (e.g. as fumarate), salmeterol (e.g. as xinafoate), terbutaline, orciprenaline, bitolterol (e.g. as mesylate), pirbuterol or indacaterol.
• the ⁇ 2 -adrenoceptor agonist of this embodiment may be a long-acting ⁇ 2 -agonists, for example salmeterol (e.g.
• Examples of a modulator of chemokine receptor function that may be used in the pharmaceutical product according to this embodiment include a CCR1 receptor antagonist.
• Examples of an inhibitor of kinase function that may be used in the pharmaceutical product according to this embodiment include a p38 kinase inhibitor and an IKK inhibitor.
• protease inhibitor examples include an inhibitor of neutrophil elastase or an inhibitor of MMP12.
• Examples of a steroidal glucocorticoid receptor agonist that may be used in the pharmaceutical product according to this embodiment include budesonide, fluticasone (e.g. as propionate ester), mometasone (e.g. as furoate ester), beclomethasone (e.g. as 17-propionate or 17,21 -dipropionate esters), ciclesonide, loteprednol (as e.g. etabonate), etiprednol (as e.g. dicloacetate), triamcinolone (e.g.
• Examples of a modulator of a non-steroidal glucocorticoid receptor agonist that may be used in the pharmaceutical product according to this embodiment include those described in WO2006/046916.
• Figure 1 X-ray powder diffraction pattern of (R)-3-(3-Fluoro-4-methyl-phenoxy)-1 - [3-(hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]-1 -azonia-bicyclo[2.2.2]octane bromide (Example 42).
• Figure 2 DSC trace of (R)-3-(3-Fluoro-4-methy
• NMR spectra were obtained on a Varian Unity Inova 400 spectrometer with a 5 mm inverse detection triple resonance probe operating at 400 MHz or on a Bruker Avance DRX 400 spectrometer with a 5 mm inverse detection triple resonance TXI probe operating at 400 MHz or on a Bruker Avance DPX 300 spectrometer with a standard 5 mm dual frequency probe operating at 300 MHz. Shifts are given in ppm relative to tetramethylsilane.
• 'flash silica' refers to silica gel for chromatography, 0.035 to 0.070 mm (220 to 440 mesh) (e.g. Fluka silica gel 60), and an applied pressure of nitrogen up to 10 p.s.i accelerated column elution or use of the semi-automated CombiFlash ® Companion purification system or by manual elution of Biotage ® lsolute Flash Si Il cartridges under reduced pressure. All solvents and commercial reagents were used as received. SCX chromatography was performed on Biotage ® lsolute SCX or SCX-2 pre-packed cartridges.
• MS ionisation method Electrospray (positive and negative ion).
• X-Ray Powder Diffraction - PANalytical X'Pert machine in 20 - 0 configuration or a PANalytical Cubix machine in 0 - 0 configuration over the scan range 2° to 40° 20 with 100-second exposure per 0.02° increment.
• the X-rays were generated by a copper long-fine focus tube operated at 45kV and 4OmA.
• the wavelength of the copper X-rays was 1.5418 ⁇ .
• the Data was collected on zero background holders on which ⁇ 2mg of the compound was placed.
• the holder was made from a single crystal of silicon, which had been cut along a non-diffracting plane and then polished on an optically flat finish.
• the X-rays incident upon this surface were negated by Bragg extinction.
• DSC Differential Scanning Calorimetry
• Thermogravimetric Vapour Sorption (TGA) thermograms were measured using a TA Q500 Thermogravimetric Analyser, with platinum pans. The sample weights varied between 1 and 5mg. The procedure was carried out under a flow of nitrogen gas (60ml/min) and the temperature studied from Room Temperature to 300°C at a constant rate of temperature increase of 10°C per minute.
• Step 1 A solution of borane-THF (1.0 M in THF, 24.8 mL) was added dropwise to a solution of (R)-1-aza-bicyclo[2.2.2]octan-3-ol (3 g) in THF (20 mL) at 0 0 C. The reaction mixture was allowed to warm to room temperature, stirred for 24 h, and evaporated in vacuo. The resulting residue was diluted with chloroform, washed with water, brine, dried (MgSO 4 ), filtered and evaporated in vacuo. The resulting residue was dissolved in ether and treated with 0-50% DCM/petroleum spirit (bp 40-60 0 C).
• Step 1 Diisopropylamine (21.6 ml_) was added dropwise to a stirred suspension of ethyl 2-oximinooxamate (15 g) in dry DCM (300 mL) cooled to -10°C. After stirring for 10 minutes a solution of chloroacetyl chloride (9.96 mL) in dry DCM (30 mL) was added dropwise over 20 minutes to the cooled mixture. After stirring at room temperature overnight, the reaction was poured into ice/water mixture (1 L) to obtain two layers.
• Step 2 A thick suspension of the foregoing compound (11.43 g) in toluene (200 mL) was allowed to reflux in a Dean-Stark apparatus for 12 h. The reaction mixture was allowed to cool down to room temperature and dried with MgSO 4 . Filtration of the solid residue and evaporation of the solvent in vacuo gave 5-chioromethyl-
• Step 3 Phenylmagnesium chloride (2 M solution in THF; 91.2 mL) was added drop wise to a stirred solution of 5-chloromethyl-[1 ,2,4]oxadiazole-3-carboxylic acid ethyl ester (15.8 g) in anhydrous THF (300 mL) at -30°C .
• the reaction mixture was stirred at -30 0 C for 5 mins, warmed to 12°C, recooled to 0 0 C, quenched with ammonium chloride solution (90 mL) and warmed to room temperature. Water (60 mL) was added and the reaction mixture was extracted with ethyl acetate.
• Step 2 Dry 1 ,2-DCE (500 mL) was purged with argon for 15 mins.
• (5-Methyl- isoxazol-3-yl)-diphenyl-methanol (37.9 g) was added under nitrogen with stirring followed by NBS (28.0 g) and AIBN (4.7 g).
• NBS 28.0 g
• AIBN 4.7 g
• the reaction mixture was stirred at 80 0 C for 1 hour. Further NBS (28.Og) and AIBN (4.7 g) was added to the reaction mixture and stirring continued at 80 0 C for 3 hours.
• the reaction mixture was allowed to cool to RT, poured into 1 M HCI (500 mL) and extracted "with ether.
• the title compound may also be obtained from ethyl 2-chloro-2- (hydroxyimino)acetate as follows:
• Step B (5-Bromomethyl-isoxazol-3-yl)-diphenyl-methanol
• Phenylmagnesium bromide (3 M solution in diethyl ether; 3.8 mL) was added dropwise to a solution of 3-chloromethyl-[1 ,2,4]oxadiazole-5-carboxylic acid ethyl ester (0.969 g) (prepared as described in DE1915495) in anhydrous THF (85 mL) at -78°C under N 2 .
• the reaction mixture was stirred at -78°C for 45 mins, then was allowed to warm to -20 0 C and stirred for a further 30 mins. It was then allowed to warm to room temperature.
• Step 1 Dimethylamino-acetic acid N'-((R)-2-cyclohexyl-2-hydroxy-2-phenyl-acetyl)- hydrazide
• Step 2 A solution of the foregoing compound (5.13 g) in acetic anhydride (65 mL) was heated at 90°C for 1 h. The reaction mixture was cooled and poured into an ice- water-NaHCO 3 mixture. More NaHCO 3 solution was added until the mixture was basic. The mixture was extracted with DCM and the organic phase was washed with brine, dried (Na 2 SO 4 ), filtered and evaporated in vacuo to give a crude solid.
• Step 3 (R)-(5-Bromomethyl-[1 ,3,4]oxadiazol-2-yl)-cyclohexyl-phenyl-methanol
• DCM dimethyl methacrylate
• cyanogen bromide 3.0 M in DCM; 1.05 mL
• the reaction mixture was left to stand for four days at room temperature, evaporated under a stream of nitrogen and purified by column chromatography (silica, cyclohexane to 20% EtOAc in DCM) to give the desired compound (93.7 mg, 34%) as a thick syrup.
• Step 1 (R)-Cyclohexyl-hydroxy-phenyl-acetic acid hydrazide
• Step 2 Chloro-acetic acid N'-((R)-2-cyclohexyl-2-hydroxy-2-phenyl-acetyl)-hydrazide
• DCM diisopropylethylamine
• chloroacetyl chloride 0.39 ml
• Step 3 (R)-(5-Chloromethyl-[1 ,3,4]oxadiazol-2-yl)-cyclohexyl-phenyl-methanol
• Step 1 1 ,1 '-Carbonyl diimidazole (25.0 g, 154 mmol) was added to a stirred suspension of (R)-cyclohexyl-hydroxy-phenyl-acetic acid (30.0 g, 128 mmol) in dry THF (600 ml_). After stirring for 90 mins at room temperature, sodium borohydride (1 1.6 g, 307 mmol) was added portionwise over a period of 1 hour. The reaction mixture was then left to stir at room temperature overnight. The reaction was quenched by the addition of water (100 ml_) then extracted with DCM. The combined organic phases were dried (MgSO 4 ), filtered and evaporated in vacuo to give a crude solid.
• Step 2 A solution of oxalyl chloride (15.5 ml_, 201 mmol) in dry DCM (900 ml_) was cooled to -78 0 C under a nitrogen atmosphere. A solution of DMSO (28.5 mL, 401 mmol) in DCM (25 mL) was added drop wise then the mixture stirred at -78 0 C for 10 mins. A solution of (R)-1-cyclohexyl-1 -phenyl-ethane-1 ,2-diol (29.5 g, 134 mmol) in DCM (250 mL) was added dropwise over the course of 1 hour giving a thick slurry. The internal temperature was allowed to reach -45 0 C.
• Triethylamine (92.8 mL, 669 mmol) was added dropwise and after complete addition the mixture was allowed to warm to room temperature. The mixture was washed with 1 N hydrochloric acid (500 mL x 2), water (500 mL) and brine (500 mL) then dried (MgSO 4 ), filtered and evaporated to give an orange oil. This was dissolved in IMS (320 mL) and added portionwise to a preformed solution of hydroxylamine hydrochloride (14.0 g, 201 mmol) and sodium carbonate (21.3 g, 201 mmol) in water (210 mL). The resulting emulsion was stirred at room temperature overnight then partitioned between DCM and water.
• Step 3 A solution of (R)-cyclohexyl-hydroxy-phenyl-acetaldehyde oxime (8 g, 34 mmol) and 2,6-lutidine (10 ml_, 86 mmol) in DCM (150 ml_) was cooled in an ice-bath.
• Trimethylsilyl trifluoromethanesulfonate (15.6 mL, 86 mmol) was added dropwise.
• Step 4 A solution of (R)-cyclohexyl-phenyl-trimethylsilanyloxy-acetaldehyde oxime (6 g, 19.6 mmol) was formed in dry DCM (400 mL) and cooled to -78 0 C. Under reduced lighting, a solution of tert-butylhypochlorite (4.3 g, 39.3 mmol) in DCM (10 mL) was added dropwise. After 2 hours at -78 °C a solution of triethylamine (4.1 mL, 29.4 mmol) in DCM (10 mL) was added dropwise. After a further 10 mins at -78 0 C the mixture was allowed to warm to 0 0 C.
• the melting temperature of this solid form was determined by DSC ( Figure 2) and was found to be 241.6 0 C (onset). GVS determination showed a mass increase of approximately 0.1% was measured at 80%RH.
• Recombinant human M3 receptor was expressed in CHO-K1 cells.
• Cell membranes were prepared and binding of [3H]-N-methyl scopolamine ([3H]-NMS) and compounds was assessed by a scintillation proximity assay (SPA).
• SPA scintillation proximity assay
• the incubation time was 16 hours at ambient temperature in the presence of 1% (v/v) DMSO.
• the assay was performed in white 96 well clear-bottomed NBS plates (Corning). Prior to the assay, the CHO cell membranes containing M3 receptor were coated onto SPA WGA (Wheat germ agglutinin) beads (GE Healthcare). Non specific binding was determined in the presence of 1 ⁇ M Atropine.
• Radioactivity was measured on a Microbeta scintillation counter (PerkinElmer) using a 3H protocol with a 2 minutes per well read time.
• Compound inhibition of [3H]-NMS binding was determined typically using concentrations in the range 0.03 nM to 1 ⁇ M and expressed as percent inhibition relative to the plate specific radioligand binding for the plate. Concentration dependent inhibition of [3H]-NMS binding by compounds was expressed as plC50. All compounds tested exhibited potencies (as Ki values) in the M3 binding assay of less than 1 nM. In particular, Example 24 exhibited a Ki value of 0.52nM, Example 42 exhibited a Ki value of 0.4OnM and Example 45 exhibited a Ki value of 0.31 nM in the M3 binding assay.
• the compounds of the invention may also be tested for appropriate pharmaceutical activity using assays know in the art, such as for example:
• Tissues are washed with Krebs solution and allowed to stabilize under stimulation prior to addition of test compound.
• Concentration response curves are obtained by a cumulative addition of test compound in half-log increments. Once the response to each addition had reached a plateau the next addition is made. Percentage inhibition of EFS- stimulated contraction is calculated for each concentration of each compound added and dose response curves constructed using Graphpad Prism software and the IC 50 calculated for each compound. By means of illustration, Example 42 and Example 45 both showed an IC 50 ⁇ 4nM in this assay.
• Onset time and duration of action studies may be performed by adding the previously determined EC 50 concentration of compound to EFS contracted tissues and the response allowed to plateau. The time taken to reach 50% of this response is determined to be the onset time. Tissues are then washed free of compound by flushing the tissue bath with fresh Krebs solution and the time taken for the contraction in response to EFS to return to 50% of the response in the presence of compound is measured. This is termed the duration of action.
• mice Male Guinea pigs (Dunkin Hartley), weighing 500-60Og housed in groups of 5 are individually identified. Animals are allowed to acclimatize to their local surroundings for at least 5 days. Throughout this time and study time animals are allowed access to water and food ad libitum.
• Guinea pigs are anaesthetized with the inhaled anaesthetic Halothane (5%).
• Test compound or vehicle (0.25 - 0.50 mLVkg) is administered intranasally. Animals are placed on a heated pad and allowed to recover before being returned to their home cages. Up to 72hrs post dosing guinea pigs are terminally anaesthetized with Urethane (250 ⁇ g/mL, 2mL7kg).
• the jugular vein is cannulated with a portex i.v. cannula filled with heparinised phosphate buffered saline (hPBS) (10U/mL) for i.v.
• hPBS heparinised phosphate buffered saline
• a pulmonary measurement system consisting of a flow pneumotach and a pressure transducer.
• the tracheal cannula is attached to a pneumotach and the oesophageal cannula attached to a pressure transducer.
• the oesophageal cannula is positioned to give a baseline resistance of between 0.1 and 0.2cm H20/mL/s.
• a 2 minute baseline reading is recorded before i.v. administration of methacholine (up to 30 ⁇ g/kg, 0.5mL_/kg).
• a 2 minute recording of the induced constriction is taken from the point of i.v. administration.
• the software calculates a peak resistance and a resistance area under the curve (AUC) during each 2 minute recording period which are used to analyse the bronchoprotective effects of test compounds.
• AUC resistance area under the curve
• Guinea pigs (450-55Og) supplied by Harlan UK or David Hall, Staffs UK are acclimatised to the in-house facilities for a minimum of three days before use. Guinea pigs are randomly assigned into treatment groups and weighed. Each animal is lightly anaesthetised (4% Halothane) and administered compound or vehicle intranasally (O. ⁇ mL ⁇ g) at up to 24 hours before challenge with pilocarpine. At the test time point, guinea pigs are terminally anaesthetised with urethane (25% solution in H 2 O, 1.5g/kg).
• each animal has an absorbent pad placed in the mouth for 5 minutes to dry residual saliva, this pad is removed and replaced with a new pre-weighed pad for 5 minutes to establish a reading of baseline saliva production. At the end of this 5 minute period the pad is removed and weighed. A new pre-weighed pad is inserted into the mouth before each animal receives s.c. pilocarpine administered under the skin at the back of the neck (0.6mg/kg @ 2ml_/kg). The pad is removed, weighed and replaced with a new pre-weighed pad every 5 minutes up to 15 minutes.
• Saliva production is calculated by subtracting the pre-weighed weight of the pad from each 5 minute period post weighed pad and these numbers added together to produce an accumulation of saliva over 15 minutes. Each 5 minute period could be analysed in addition to the whole 15 minute recording period. Baseline production of saliva is assumed to be constant and multiplied by three to produce a reading for baseline saliva production over 15 minutes.
• Example 42 showed no reduction in salivation when dosed at 10 ⁇ g/kg 4hrs prior to pilocarpine (0.6mg/kg s.c.) challenge.

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