WO2010071581A1 - Pharmaceutical product comprising a muscarinic receptor antagonist and a b2-adrenoceptor agonist - Google Patents

Abstract

The invention provides a pharmaceutical product, kit or composition comprising a first active ingredient which is a selected muscarinic receptor antagonist selected, and a second active ingredient which is a β_2- adrenoceptor agonist, of use in the treatment of respiratory diseases such as chronic obstructive pulmonary disease and asthma.

Description

PHARMACEUTICAL PRODUCT COMPRISING A MUSCARINIC RECEPTOR ANTAGONIST AND A β2- ADRENOCEPTOR AGONIST

The present invention relates to combinations of pharmaceutically active substances for use in the treatment of respiratory diseases, especially chronic obstructive pulmonary disease (COPD) and asthma.

The essential function of the lungs requires a fragile structure with enormous exposure to the environment, including pollutants, microbes, allergens, and carcinogens. Host factors, resulting from interactions of lifestyle choices and genetic composition, influence the response to this exposure. Damage or infection to the lungs can give rise to a wide range of diseases of the respiratory system (or respiratory diseases). A number of these diseases are of great public health importance. Respiratory diseases include Acute Lung Injury, Acute Respiratory Distress Syndrome (ARDS), occupational lung disease, lung cancer, tuberculosis, fibrosis, pneumoconiosis, pneumonia, emphysema, Chronic Obstructive Pulmonary Disease (COPD) and asthma.

Among the most common of the respiratory diseases is asthma. Asthma is generally defined as an inflammatory disorder of the airways with clinical symptoms arising from intermittent airflow obstruction. It is characterised clinically by paroxysms of wheezing, dyspnea and cough. It is a chronic disabling disorder that appears to be increasing in prevalence and severity. It is estimated that 15% of children and 5% of adults in the population of developed countries suffer from asthma. Therapy should therefore be aimed at controlling symptoms so that normal life is possible and at the same time provide basis for treating the underlying inflammation.

COPD is a term which refers to a large group of lung diseases which can interfere with normal breathing. Current clinical guidelines define COPD as a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases. The most important contributory source of such particles and gases, at least in the western world, is tobacco smoke. COPD patients have a variety of symptoms, including cough, shortness of breath, and excessive production of sputum; such symptoms arise from dysfunction of a number of cellular compartments, including neutrophils, macrophages, and epithelial cells. The two most important conditions covered by COPD are chronic bronchitis and emphysema.

Chronic bronchitis is a long-standing inflammation of the bronchi which causes increased production of mucous and other changes. The patients' symptoms are cough and expectoration of sputum. Chronic bronchitis can lead to more frequent and severe respiratory infections, narrowing and plugging of the bronchi, difficult breathing and disability.

Emphysema is a chronic lung disease which affects the alveoli and/or the ends of the smallest bronchi. The lung loses its elasticity and therefore these areas of the lungs become enlarged. These enlarged areas trap stale air and do not effectively exchange it with fresh air. This results in difficult breathing and may result in insufficient oxygen being delivered to the blood. The predominant symptom in patients with emphysema is shortness of breath.

Therapeutic agents used in the treatment of respiratory diseases include β₂-adrenoceptor agonists. These agents (also known as beta2 (β₂) - agonists) may be used to alleviate symptoms of respiratory diseases by relaxing the bronchial smooth muscles, reducing airway obstruction, reducing lung hyperinflation and decreasing shortness of breath. Compounds currently under evaluation as once-daily β2 agonists are described in Expert Opin. Investig. Drugs 14 (7), 775-783 (2005).

A further class of therapeutic agent used in the treatment of respiratory diseases are muscarinic antagonists. Muscarinic receptors are a G-protein coupled receptor (GPCR) family having five family members M₁, M₂, M₃, M₄ and M₅. Of the five muscarinic subtypes, three (M₁, M₂ and M₃) are known to exert physiological effects on human lung tissue. Parasympathetic nerves are the main pathway for reflex bronchoconstriction in human airways and mediate airway tone by releasing acetylcholine onto muscarinic receptors. Airway tone is increased in patients with respiratory disorders such as asthma and chronic obstructive pulmonary disease (COPD), and for this reason muscarinic receptor antagonists have been developed for use in treating airway diseases. Muscarinic receptor antagonsists, often called anticholinergics in clinical practice, have gained widespread acceptance as a first-line therapy for individuals with COPD, and their use has been extensively reviewed in the literature (e.g. Lee et al, Current Opinion in Pharmacology 2001 ,1 , 223-229).

Whilst treatment with a β₂-adrenoceptor agonist or a muscarinic antagonist can yield important benefits, the efficacy of these agents is often far from satisfactory. Moreover, in view of the complexity of respiratory diseases such as asthma and COPD, it is unlikely that any one mediator can satisfactorily treat the disease alone. Hence there is a pressing medical need for new therapies against respiratory diseases such as COPD and asthma, in particular for therapies with disease modifying potential.

The present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is a muscarinic antagonist selected from:

anf/-[(1S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl- ammonium X; a/tf/-(1 S, 2R) 2-(9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl- (3-phenoxy-propyl)-ammonium X; wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and a second active ingredient which is a β₂-adrenoceptor agonist.

A beneficial therapeutic effect may be observed in the treatment of respiratory diseases if a muscarinic antagonist according to the present invention is used in combination with a β₂-adrenoceptor agonist. The beneficial effect may be observed when the two active substances are administered simultaneously (either in a single pharmaceutical preparation or via separate preparations), or sequentially or separately via separate pharmaceutical preparations.

The pharmaceutical product of the present invention may, for example, be a pharmaceutical composition comprising the first and second active ingredients in admixture. Alternatively, the pharmaceutical product may, for example, be a kit comprising a preparation of the first active ingredient and a preparation of the second active ingredient and, optionally, instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof. The first active ingredient in the combination of the present invention is a muscarinic antagonist selected from: anf/-[(1S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl- ammonium X; a/tf/-(1 S, 2R) 2-(9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl- (3-phenoxy-propyl)-ammonium X; wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid.

The muscarinic antagonists of the invention are selected members of a novel class of compound described in co-pending application WO2007/017670 which display high potency to the M3 receptor. The names of the muscarinic antagonists are IUPAC names generated by the Autonom 2000 naming package , as supplied by MDL Information Systems Inc., based on the structures depicted in the examples, and stereochemistry assigned according to the Cahn-lngold-Prelog system. For example, the name anti- [(1 S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl- ammonium bromide, was generated from the structure:

The muscarinic antagonists 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. In an embodiment of the invention 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 toluenesulfonate (tosylate), edisylate (ethane-1 ,2-disulfonate), isethionate (2- hydroxyethylsulfonate),lactate, oleic, maleate ((Z)-3-carboxy-acrylate), succinate (3- carboxy-propionate), malate ((S)-3-carboxy -2-hydroxy-propionate), p- acetamidobenzoateacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, methanesulphonate, p-toluenesulphonate, benzenesulphonate, napadisylate (naphthalene-1 ,5-disulphonate) (e.g. a heminapadisylate), 2,5- dichlorobenzenesulphonate, (xinafoate) 1-hydroxy-2-naphthoate or 1- hydroxynaphthalene-2-sulphonate.

In an embodiment of the invention, the muscarinic receptor antagonist is in the form of a bromide salt.

In an embodiment of the invention, the muscarinic receptor antagonist is selected from

anf/-[(1S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl- ammonium bromide; and a/tf/-(1 S, 2R) 2-(9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl- (3-phenoxy-propyl)-ammonium bromide.

The second active ingredient in the combination of the present invention is a β₂- adrenoceptor agonist. The β₂-adrenoceptor agonist of the present invention may be any compound or substance capable of stimulating the β₂ -receptors and acting as a bronchodilator. In the context of the present specification, unless otherwise stated, any reference to a β₂-adrenoceptor agonist includes active salts, solvates or derivatives that may be formed from said β₂-adrenoceptor agonist and any enantiomers and mixtures thereof. Examples of possible salts or derivatives of β₂-adrenoceptor agonist are acid addition salts such as the salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, 1-hydroxy-2-naphthalenecarboxylic acid, maleic acid, and pharmaceutically acceptable esters (e.g. CrC₆ alkyl esters). The β₂ -agonists may also be in the form of solvates, e.g. hydrates.

Examples of a β₂-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 β₂-adrenoceptor agonist of this embodiment may be a long-acting β₂-agonist (i.e. a β₂-agonist with activity that persists for more than 24 hours), for example salmeterol (e.g. as xinafoate), formoterol (e.g. as fumarate), bambuterol (e.g. as hydrochloride), carmoterol (TA 2005, chemically identified as 2(1 H)-Quinolone, 8-hydroxy-5-[1-hydroxy-2- [[2-(4-methoxy-phenyl)-1 -methylethyl]-amino]ethyl]-monohydrochloride, [R-(R^*, R^*)] also identified by Chemical Abstract Service Registry Number 137888-1 1-0 and disclosed in U.S. Patent No 4,579,854), indacaterol (CAS no 312753-06-3; QAB-149), formanilide derivatives e.g. 3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2- hydroxyethyl}amino)hexyl]oxy}-butyl)-benzenesulfonamide as disclosed in WO 2002/76933, benzenesulfonamide derivatives e.g. 3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy- 3-(hydroxy-methyl)phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamide as disclosed in WO 2002/88167, aryl aniline receptor agonists as disclosed in WO 2003/042164 and WO 2005/025555, indole derivatives as disclosed in WO 2004/032921 , in US

2005/222144, compounds GSK 159797, GSK 159802, GSK 597901 , GSK 642444 and GSK 678007.

In an embodiment of the present invention, the β₂-adrenoceptor agonist is formoterol. The chemical name for formoterol is Λ/-[2-hydroxy-5-[(1 )-1-hydroxy-2-[[(1 )-2-(4- methoxyphenyl)-1-methylethyl]amino]ethyl]phenyl]-formamide. The preparation of formoterol is described, for example, in WO 92/05147. In one aspect of this embodiment, the β₂-adrenoceptor agonist is formoterol fumarate. It will be understood that the invention encompasses the use of all optical isomers of formoterol and mixtures thereof including racemates. Thus for example, the term formoterol encompasses Λ/-[2-hydroxy-5-[(1 R)-1- hydroxy-2-[[(1 R)-2-(4-methoxyphenyl)-1 -methylethyl]amino]ethyl]phenyl]-formamide, Λ/-[2- hydroxy-5-[(1 S)- 1 -hydroxy-2-[[(1 S)-2-(4-methoxyphenyl)-1 - methylethyl]amino]ethyl]phenyl]-formamide and a mixture of such enantiomers, including a racemate.

In an embodiment of the invention, the β₂-adrenoceptor agonist is selected from:

Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(1-naphthyl)ethoxy]propanamide,

Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide, and

7-[(1 f?)-2-({2-[(3-{[2-(2-Chlorophenyl)ethyl]amino}propyl)thio]ethyl}amino)-1-hydroxyethyl]-

4-hydroxy-1 ,3-benzothiazol-2(3H)-one, or a pharmaceutically acceptable salt thereof. The β₂-adrenoceptor agonists according to this embodiment may be prepared as described in the experimental preparation section of the present application. The names of the β₂-adrenoceptor agonists of this embodiment are IUPAC names generated by the IUPAC NAME, ACD Labs Version 8 naming package.

In a further embodiment of the invention, the β₂-adrenoceptor agonist is selected from: Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(1-naphthyl)ethoxy]propanamide dihydrobromide, Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide dihydrobromide, and 7-[(1 f?)-2-({2-[(3-{[2-(2-Chlorophenyl)ethyl]amino}propyl)thio]ethyl}amino)-1-hydroxyethyl]- 4-hydroxy-1 ,3-benzothiazol-2(3H)-one dihydrobromide.

In an embodiment of the invention, the muscarinic receptor antagonist is anf/-[(1S,2R)-2- (2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl-ammonium X, wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and the β₂-adrenoceptor agonist is formoterol (e.g. as fumarate). In one aspect of this embodiment, the muscarinic receptor antagonist is an//-[(1S,2R)-2-(2-Hydroxy-2,2-di- thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl-ammonium bromide.

In an embodiment of the invention, the muscarinic receptor antagonist is anf/-(1S, 2R) 2- (9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl-(3-phenoxy- propyl)-ammonium X, wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and the β₂-adrenoceptor agonist is formoterol (e.g. as fumarate). In one aspect of this embodiment, the muscarinic receptor antagonist is anf/-(1S, 2R) 2-(9- Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl-(3-phenoxy- propyl)-ammonium bromide.

In an embodiment of the invention, the muscarinic receptor antagonist is anf/-[(1S,2R)-2- (2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl-ammonium X, wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and the β₂-adrenoceptor agonist is Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo- 2,3-dihydro-1 ,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(1-naphthyl)ethoxy]propanamide or a pharmaceutically acceptable salt thereof (e.g. dihydrobromide). In one aspect of this embodiment, the muscarinic receptor antagonist is an//-[(1S,2R)-2-(2-Hydroxy-2,2-di- thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl-ammonium bromide. In an embodiment of the invention, the muscarinic receptor antagonist is anf/-(1S, 2R) 2- (9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl-(3-phenoxy- propyl)-ammonium X, wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and the β₂-adrenoceptor agonist is Λ/-[2-(Diethylamino)ethyl]-Λ/- (2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(1- naphthyl)ethoxy]propanamide or a pharmaceutically acceptable salt thereof (e.g. dihydrobromide). In one aspect of this embodiment, the muscarinic receptor antagonist is anf/-(1 S, 2R) 2-(9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl- (3-phenoxy-propyl)-ammonium bromide.

In an embodiment of the invention, the muscarinic receptor antagonist is anf/-[(1S,2R)-2- (2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl-ammonium X, wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and the β₂-adrenoceptor agonist is 7-[(1 fl)-2-({2-[(3-{[2-(2-

Chlorophenyl)ethyl]amino}propyl)thio]ethyl}amino)-1-hydroxyethyl]-4-hydroxy-1 ,3- benzothiazol-2(3A7)-one or a pharmaceutically acceptable salt thereof (e.g. dihydrobromide). In one aspect of this embodiment, the muscarinic receptor antagonist is anf/-[(1S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl- ammonium bromide.

In an embodiment of the invention, the muscarinic receptor antagonist is anf/-(1S, 2R) 2- (9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl-(3-phenoxy- propyl)-ammonium X, wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and the β₂-adrenoceptor agonist is 7-[(1 f?)-2-({2-[(3-{[2-(2- Chlorophenyl)ethyl]amino}propyl)thio]ethyl}amino)-1-hydroxyethyl]-4-hydroxy-1 ,3- benzothiazol-2(3H)-one or a pharmaceutically acceptable salt thereof (e.g. dihydrobromide). In one aspect of this embodiment, the muscarinic receptor antagonist is anf/-(1 S, 2R) 2-(9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl- (3-phenoxy-propyl)-ammonium bromide.

The combination of the present invention may provide a beneficial therapeutic effect in the treatment of respiratory diseases. Examples of such possible effects include improvements in one or more of the following parameters: reducing inflammatory cell influx into the lung, mild and severe exacerbations, FEV₁ (forced expiratory volume in one second), vital capacity (VC), peak expiratory flow (PEF), symptom scores and Quality of Life.

The muscarinic antagonist (first active ingredient) and β₂-adrenoceptor agonist (second active ingredient) of the present invention may be administered simultaneously, sequentially or separately to treat respiratory diseases. By sequential it is meant that the active ingredients are administered, in any order, one immediately after the other. They may still have the desired effect if they are administered separately, but when administered in this manner they will generally be administered less than 4 hours apart, more conveniently less than two hours apart, more conveniently less than 30 minutes apart and most conveniently less than 10 minutes apart.

The active ingredients of the present invention may be administered by oral or parenteral (e.g. intravenous, subcutaneous, intramuscular or intraarticular) administration using conventional systemic dosage forms, such as tablets, capsules, pills, powders, aqueous or oily solutions or suspensions, emulsions and sterile injectable aqueous or oily solutions or suspensions. The active ingredients may also be administered topically (to the lung and/or airways) in the form of solutions, suspensions, aerosols and dry powder . These dosage forms will usually include one or more pharmaceutically acceptable ingredients which may be selected, for example, from adjuvants, carriers, binders, lubricants, diluents, stabilising agents, buffering agents, emulsifying agents, viscosity-regulating agents, surfactants, preservatives, flavourings and colorants. As will be understood by those skilled in the art, the most appropriate method of administering the active ingredients is dependent on a number of factors.

In one embodiment of the present invention the active ingredients are administered via separate pharmaceutical preparations. Therefore, in one aspect, the present invention provides a kit comprising a preparation of a first active ingredient which is a muscarinic antagonist according to the present invention, and a preparation of a second active ingredient which is a β₂-adrenoceptor agonist, and optionally instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof. In another embodiment the active ingredients may be administered via a single pharmaceutical composition. Therefore, the present invention further provides a pharmaceutical composition comprising, in admixture, a first active ingredient, which is a muscarinic antagonist according to the present invention, and a second active ingredient, which is a β₂-adrenoceptor agonist.

The pharmaceutical compositions of the present invention may be prepared by mixing the muscarinic antagonist (first active ingredient) with a β₂-adrenoceptor agonist (second active ingredient) and a pharmaceutically acceptable adjuvant, diluent or carrier. Therefore, in a further aspect of the present invention there is provided a process for the preparation of a pharmaceutical composition, which comprises mixing a muscarinic antagonist according to the present invention with a β₂-adrenoceptor agonist and a pharmaceutically acceptable adjuvant, diluent or carrier.

It will be understood that the therapeutic dose of each active ingredient administered in accordance with the present invention will vary depending upon the particular active ingredient employed, the mode by which the active ingredient is to be administered, and the condition or disorder to be treated.

In one embodiment of the present invention, the muscarinic antagonist according to the present invention is administered via inhalation. When administered via inhalation the dose of the muscarinic antagonist according to the present invention will generally be in the range of from 0.1 microgram (μg) to 5000 μg, 0.1 to 1000 μg, 0.1 to 500 μg, 0.1 to 100 μg, 0.1 to 50 μg, 0.1 to 5 μg, 5 to 5000 μg, 5 to 1000 μg, 5 to 500 μg, 5 to 100 μg, 5 to 50 μg, 5 to 10 μg, 10 to 5000 μg, 10 to 1000 μg, 10 to 500 μg, 10 to 100 μg, 10 to 50 μg, 20 to 5000 μg, 20 to 1000 μg, 20 to 500 μg, 20 to 100 μg, 20 to 50 μg, 50 to 5000 μg, 50 to 1000 μg, 50 to 500 μg, 50 to 100 μg, 100 to 5000 μg, 100 to 1000 μg or 100 to 500 μg. The dose will generally be administered from 1 to 4 times a day, conveniently once or twice a day, and most conveniently once a day.

In one embodiment of the present invention the β₂-adrenoceptor agonist may conveniently be administered by inhalation. When administered via inhalation the dose of the β₂- adrenoceptor agonist will generally be in the range of from 0.1 to 50 μg, 0.1 to 40 μg, 0.1 to 30 μg, 0.1 to 20 μg, 0.1 to 10 μg, 5 to 10 μg, 5 to 50 μg, 5 to 40 μg, 5 to 30 μg, 5 to 20 μg, 5 to 10 μg, 10 to 50 μg, 10 to 40 μg 10 to 30 μg, or 10 to 20 μg. The dose will generally be administered from 1 to 4 times a day, conveniently once or twice a day, and most conveniently once a day.

In one embodiment, the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is a muscarinic antagonist, and a second active ingredient which is a β₂-adrenoceptor agonist, wherein each active ingredient is formulated for inhaled administration.

In an embodiment the pharmaceutical preparations of active ingredients may be administered simultaneously.

In an embodiment the different pharmaceutical preparations of active ingredients may be administered sequentially.

In an embodiment the different pharmaceutical preparations of active ingredients may be administered separately.

The active ingredients of the present invention are conveniently administered via inhalation (e.g. topically to the lung and/or airways) in the form of solutions, suspensions, aerosols and dry powder formulations. For example metered dose inhaler devices may be used to administer the active ingredients, dispersed in a suitable propellant 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.

Dry powders and pressurized HFA aerosols of the active ingredients may be administered by oral or nasal inhalation. For inhalation, the compound is desirably finely divided. The finely divided compound preferably has a mass median diameter of less than 10 μm, and may be suspended in a propellant mixture with the assistance of a dispersant, such as a C₈-C_2O fatty acid or salt thereof, (for example, oleic acid), a bile salt, a phospholipid, an alkyl saccharide, a perfluorinated or polyethoxylated surfactant, or other pharmaceutically acceptable dispersant.

One possibility is to mix the finely divided compound of the invention with a carrier substance, for example, a mono-, di- or polysaccharide, a sugar alcohol, or another polyol. Suitable carriers are sugars, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol; and starch. Alternatively the finely divided compound may be coated by another substance. The powder mixture may also be dispensed into hard gelatine capsules, each containing the desired dose of the active compound.

Another possibility is to process the finely divided powder into spheres which break up during the inhalation procedure. This spheronized powder may be filled into the drug reservoir of a multidose inhaler, for example, that known as the Turbuhaler^® in which a dosing unit meters the desired dose which is then inhaled by the patient. With this system the active ingredient, with or without a carrier substance, is delivered to the patient.

The combination of the present invention is useful in the treatment or prevention of respiratory-tract disorders such as chronic obstructive pulmonary disease (COPD), chronic bronchitis of all types (including dyspnoea associated therewith), asthma (allergic and non-allergic; 'wheezy-infant syndrome'), adult/acute respiratory distress syndrome (ARDS), chronic respiratory obstruction, bronchial hyperactivity, pulmonary fibrosis, pulmonary emphysema, and allergic rhinitis, exacerbation of airway hyperreactivity consequent to other drug therapy, particularly other inhaled drug therapy or pneumoconiosis (for example aluminosis, anthracosis, asbestosis, chalicosis, ptilosis, siderosis, silicosis, tabacosis and byssinosis).

Dry powder inhalers may be used to administer the active ingredients, 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 present invention further provides a pharmaceutical product, kit or pharmaceutical composition according to the invention for simultaneous, sequential or separate use in therapy.

The present invention further provides the use of a pharmaceutical product, kit or pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease or asthma.

The present invention further provides a pharmaceutical product, kit or pharmaceutical composition according to the invention for use in the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease or asthma.

The present invention still further provides a method of treating a respiratory disease which comprises simultaneously, sequentially or separately administering:

(a) a (therapeutically effective) dose of a first active ingredient which is a muscarinic antagonist according to the present invention; and

(b) a (therapeutically effective) dose of a second active which is a β₂-adrenoceptor agonist; to a patient in need thereof.

In the context of the present specification, the term "therapy" also includes "prophylaxis" unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be construed accordingly. 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 condition or disorder in question. Persons at risk of developing a particular condition or disorder generally include those having a family history of the condition or disorder, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition or disorder.

The term "disease, unless stated otherwise, has the same meaning as the terms "condition" and "disorder" and are used interchangeably throughout the description and claims. The term "agent" and "active ingredient" means the compounds comprised in the combination of the present invention, e.g. a muscarine antagonist or a β₂-adrenoceptor agonist.

The pharmaceutical product, kit or composition of the present invention may optionally comprise a third active ingredient which third active ingredient is a substance suitable for use in the treatment of respiratory diseases. Examples of third active ingredients that may be incorporated into the present invention include

• a phosphodiesterase inhibitor,

• a modulator of chemokine receptor function, • an inhibitor of kinase function,

• a protease inhibitor,

• a steroidal glucocorticoid receptor agonist, and

• a non-steroidal glucocorticoid receptor agonist.

Examples of a phosphodiesterase inhibitor that may be used as a third active ingredient according to this embodiment include a PDE4 inhibitor such as an inhibitor of the isoform PDE4D, a PDE3 inhibitor and a PDE5 inhibitor. Examples include the compounds (Z)-3-(3,5-dichloro-4-pyridyl)-2-[4-(2-indanyloxy-5-methoxy-2-pyridyl]propenenitrile, N-[9-amino-4-oxo-1-phenyl-3,4,6,7-tetrahydropyrrolo[3,2,1-jk][1 ,4]benzodiazepin-3(R)- yl]pyridine-3-carboxamide (CI-1044),

3-(benzyloxy)-1-(4-fluorobenzyl)-N-[3-(methylsulphonyl)phenyl]-1 H-indole-2-carboxamide, (1S-exo)-5-[3-(bicyclo[2.2.1]hept-2-yloxy)-4-methoxyphenyl]tetrahydro-2(1 H)-pyrimidinone (Atizoram), N-(3,5,dichloro-4-pyridinyl)-2-[1-(4-fluorobenzyl)-5-hydroxy-1 H-indol-3-yl]-2-oxoacetamide (AWD-12-281 ), β-[3-(cyclopentyloxy)-4-methoxyphenyl]-1 ,3-dihydro-1 ,3-dioxo-2H-isoindole-2- propanamide (CDC-801 ),

N-[9-methyl-4-oxo-1-phenyl-3,4,6,7-tetrahydropyrrolo[3,2,1-jk][1 ,4]benzodiazepin-3(R)- yl]pyridine-4-carboxamide (CI-1018), cis-[4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexane-1 -carboxylic acid (Cilomilast),

8-amino-1 ,3-bis(cyclopropylmethyl)xanthine (Cipamfylline), N-(2,5-dichloro-3-pyridinyl)-8-methoxy-5-quinolinecarboxamide (D-4418), 5-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2-iminothiazolidin-4-one (Darbufelone), 2-methyl-1-[2-(1-methylethyl)pyrazolo[1 ,5-a]pyridin-3-yl]-1-propanone (Ibudilast), 2-(2,4-dichlorophenylcarbonyl)-3-ureidobenzofuran-6-yl methanesulphonate (Lirimilast), (-)-(R)-5-(4-methoxy-3-propoxyphenyl)-5-methyloxazolidin-2-one (Mesopram), (-)-cis-9-ethoxy-8-methoxy-2-methyl-1 ,2,3,4,4a, 10b-hexahydro-6-(4- diisopropylaminocarbonylphenyl)-benzo[c][1 ,6]naphthyridine (Pumafentrine), 3-(cyclopropylmethoxy)-N-(3,5-dichloro-4-pyridyl)-4-(difluoromethoxy)benzamide (Roflumilast), the N-oxide of Roflumilast,

5,6-diethoxybenzo[b]thiophene-2-carboxylic acid (Tibenelast), 2,3,6,7-tetrahydro-2-(mesitylimino)-9, 10-dimethoxy-3-methyl-4H-pyrimido[6, 1 - a]isoquinolin-4-one (trequinsin), and

3-[[3-(cyclopentyloxy)-4-methoxyphenyl]-methyl]-N-ethyl-8-(1-methylethyl)-3H-purine-6- amine (V-11294A).

Examples of a modulator of chemokine receptor function that may be used as a third active ingredient according to this embodiment include a CCR3 receptor antagonist, a CCR4 receptor antagonist, a CCR5 receptor antagonist and a CCR8 receptor antagonist.

Examples of an inhibitor of kinase function that may be used as a third active ingredient according to this embodiment include a p38 kinase inhibitor and an IKK inhibitor.

Examples of a protease inhibitor that may be used as a third active ingredient according to this embodiment include an inhibitor of neutrophil elastase or an inhibitor of MMP12.

Examples of a steroidal glucocorticoid receptor agonist that may be used as a third active ingredient 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. as acetonide), flunisolide, zoticasone, flumoxonide, rofleponide, butixocort (e.g. as propionate ester), prednisolone, prednisone, tipredane, steroid esters e.g. 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11 β-hydroxy- 16α-methyl-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester, 6α,9α- difluoro-1 1 β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1 ,4-diene-17β- carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester and 6α,9α-difluoro-1 1 β-hydroxy- 16α-methyl-17α-[(4-methyl-1 ,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1 ,4-diene-17β- carbothioic acid S-fluoromethyl ester, steroid esters according to DE 4129535, steroids according to WO 2002/00679, WO 2005/041980, or steroids GSK 870086, GSK 685698 and GSK 799943.

Examples of a modulator of a non-steroidal glucocorticoid receptor agonist that may be used as a third active ingredient according to this embodiment include those described in WO2006/046916.

The invention is illustrated by the following Examples.

Preparation of Muscarinic Antagonists

Muscarinic antagonists according to the present invention may be prepared as follows. Alternative salts to those described herein may be prepared by conventional chemistry using methods analogous to those described.

General Experimental Details for Preparation of Muscarinic Antagonists

Unless otherwise stsated the following general conditions were used in the preparation of the Muscarinic Antagonists

All reactions were carried out under an atmosphere of nitrogen unless specified otherwise. 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.

Where products were purified by column chromatography, '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. Where thin layer chromatography (TLC) has been used, it refers to silica gel TLC using plates, typically 3 * 6 cm silica gel on aluminium foil plates with a fluorescent indicator (254 nm), (e.g. Fluka 60778). All solvents and commercial reagents were used as received. LC-MS Systems

The Liquid Chromatography Mass Spectroscopy (LC-MS) systems used:

LC-MS method 1 :

Waters ZMD with a C18-reverse-phase column (30 x 4.6 mm, Phenomenex Luna 3 μm particle size), elution with A: water + 0.1 % formic acid; B: acetonitrile + 0.1 % formic acid.

Gradient:

Gradient - Time flow mL/min %A %B

0.00 2.0 95 5

0.50 2.0 95 5

4.50 2.0 5 95

5.50 2.0 5 95

6.00 2.0 95 5

Detection: MS, ELS, UV (200μl/min split to MS with in-line Waters 996 DAD detection). MS ionisation method: Electrospray (positive and negative ion).

LC-MS method 2: Micromass Platform LCT with a C18-reverse-phase column (100 x 3.0 mm Higgins Clipeus with 5 μm particle size), elution with A: water + 0.1% formic acid; B: acetonitrile + 0.1 % formic acid. Gradient:

Gradient - Time flow ml/min %A %B 00..0000 11..00 9955 5

1.00 1.0 95 5

15.00 1.0 5 95

20.00 1.0 5 95

22.00 1.0 95 5 2255..0000 11..00 9955 5

Detection - MS, ELS, UV (100 μl split to MS with in-line UV detector) MS ionisation method - Electrospray (positive ion)

Abbreviations used in the experimental section: AIBN = 2,2'-azobis(2-methyl-propionitrile) aq = aqueous bpt = boiling point

DCE = dichloroethane DCM = dichloromethane

Et₂O = diethyl ether

EtOH = ethanol equiv. = equivalents h = hour(s) IPA = propan-2-ol

MeCN = acetonitrile

MeOH = methanol min = minutes

O/N = over night ppt = precipitate

R_f = retention factor

RM = reaction mixture

Rt = retention time rt = room temperature sat. = saturated

SM = starting material

THF = tetrahydrofuran

Example 1 - anf/-[(1 S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)- bicyclo[2.2.1 ]hept-7-yl]-trimethyl-ammonium bromide

H

THF

DCM, rt, 0/N

2

3) Na₂CO₃ (aq)

CO₂Me Intermediate 6

NaOMe, Toluene,

110 ⁰C (external), 2 h Reduced pressure distillation int^oπncαiatc o

Scheme 1

Intermediate 1

A solution of (S)-(-)-α-methylbenzylamine (84 g, 0.69 mol) in dichloromethane (600 ml.) was treated with water (14 ml.) and cooled in an ice/water bath. Sulphur dioxide gas was bubbled through the stirred mixture at such a rate that the internal temperature was maintained at 20-25 ⁰C. A solid formed immediately to give a thick slurry but gas addition was continued until this disappeared and the mixture became homogeneous. The yellow solution was treated with bicyclo[3.2.0]hept-2-en-6-one (75 g, 0.69 mol) and the mixture stirred until a solid began to deposit. The mixture was then allowed to stand at 4 ⁰C overnight. The slurry was filtered and the solid washed with dichloromethane and dried to afford a white solid (102.7 g). This solid (102 g) was stirred with 1 M aqueous sodium carbonate solution (700 ml.) for ca. 5 mins and the mixture extracted three times with diethyl ether. The organic layers were combined and washed with water, 2 M hydrochloric acid, brine, dried (Na₂SO₄) and evaporated carefully to leave a straw-coloured liquid. Yield: 25.8 g, 34%.

[α]_D +29.9 (c = 5% in MeOH) R_f = 0.44 (15% Et₂O/pentane).

Intermediate 2 A solution of Intermediate 1 (14.14 g, 131 mmol) in toluene (160 mL) was cooled in an ice/acetone bath to give an internal temperature of 0 ⁰C. Bromine (6.7 mL, 131 mmol) was added dropwise over 30 mins such that the internal temperature remained < 5 ⁰C. After addition the mixture was allowed to warm to room temperature. Sodium thiosulphate (aq. 100 mL) was added and the mixture extracted with ethyl acetate. Combined organics were dried over Na₂SO₄, filtered and evaporated to dryness. Purification over silica using automated Biotage SP1 system (340 g column eluting with 0-20% Et₂O/cyclohexane gave the product as a yellow viscous oil which solidifies on extended refrigeration. Yield: 24.3 g (69%). R_f = 0.34 (20% Et₂O/pentane).

Intermediate 3

To a chilled (-10 ⁰C) DCM (20 mL) solution of Intermediate 2 (1.5 g, 5.6 mmol) was added a THF solution of dimethylamine (2 M, 8.23 mL, 16.5 mmol) dropwise. The reaction was allowed to slowly warm to ambient temperature over several hours. After 17 hours the reaction mixture was concentrated and the residue dissolved in Et₂O and filtered. The filtrate was absorbed onto diatomaceous earth and chromatographed on a column of silica gel eluting with 25 % Et₂O in pentane to give the product as an orange solid.

Yield: 0.88 g (58%).

1 H NMR (400 MHz, CDCI₃) 4.70-4.64 (1 H, m), 2.86-2.77 (3H, m), 2.62-2.60 (1 H, m), 2.49- 2.47 (1 H, m), 2.24-2.17 (7H, m), 1.71 (1 H, ddd, J = 14.4, 4.2, 1.1 ).

Intermediate 4

Toluene was degassed prior to the reaction by vigorously bubbling nitrogen gas through it for 30 minutes. To Intermediate 3 (200 mg, 0.75 mmol) and AIBN (12 mg, 0.07 mmol) in a nitrogen atmosphere was added the degassed toluene (8 mL). To this was added tributyltin hydride (221 μl_, 0.82 mmol) and the solution was stirred under a nitrogen atmosphere at 80 ⁰C for 1 hour. A toluene (300 μl_) solution of tributyltin hydride (30 μl_, 0.11 mmol) and a few flakes of AIBN were added and the reaction was heated at 80 ⁰C for 1.5 hours. The reaction mixture was evaporated to give 0.5 g of a yellow liquid that was chromatographed on a column of silica gel eluting with pentane, then 20 % Et₂O in pentane up to 100 % Et₂O to provide a colourless oil. Yield: 214 mg (>100%, tin residue contamination). Rf = 0.25 (50 % Et₂O in pentane)

1 H NMR (400 MHz, CDCI₃) (only peaks for the product reported) 2.55-2.52 (1 H, m), 2.50- 2.47 (1 H, m), 2.20 (1 H, s), 2.13 (6H, s), 2.10-1.87 (4H, m), 1.65-1.52 (2H, m).

Intermediate 5

To a chilled (-10 ⁰C) THF (4 ml.) solution of Intermediate 4 (214 mg, < 0.75 mmol) was added lithium tri-te/t-butoxyaluminohydride (380 mg, 1.49 mol.) The reaction mixture was stirred at 0 ⁰C for 1.5 hours and then another portion of lithium tri-te/t- butoxyaluminohydride (190 mg, 0.75 mol) was added and the reaction mixture was stirred for a further 1.5 hours. The reaction mixture was diluted with DCM and directly evaporated on to diatomaceous earth and chromatographed on a column of silica gel eluting with 5 % MeOH in DCM with a 2.5 % MeOH gradient up to 20 % MeOH in DCM to give the product as a white solid. Yield: 120 mg (100 %) Rf = 0.1 (75 % Et₂O in pentane)

1 H NMR (400 MHz, CDCI₃) 4.23-4.18 (1 H, m), 2.28-2.25 (1 H, m), 2.17 (6H, s), 2.12-2.09 (1 H, m), 2.07-1.98 (2H, m), 1.89-1.79 (2H, m), 1.68-1.53 (2H, brm), 1.30-1.25 (1 H, m), 0.94 (1 H, dd, J = 13.3, 3.7).

Intermediate 6

The preparation of Intermediate 6 is described in WO2008/008376.

Intermediate 7

A solution of norbornane alcohol (Intermediate 5, 1.21 g, 7.8 mmol) was dissolved in toluene (140 ml) and treated with sodium methoxide (505 mg, 9.4 mmol). A microdistillation head was attached to the reaction vessel, the pressure was reduced to 480 mbar and the reaction was heated to 100 ⁰C. A solution of Intermediate 6 (2.97 g, 11.7 mmol) in toluene (75 ml) was prepared. After 25 ml of distillate had been collected from the reaction mixture, one-third of the solution of Intermediate 6 was added to the reaction mixture. A further 25 ml of distillate was collected from the reaction mixture, which was replaced with a further 25 ml of the solution of Intermediate 6. This was repeated again, so that all of the solution of Intermediate 6 had been added to the reaction. A slurry of sodium methoxide (210 mg, 3.9 mmol) in toluene (60 ml) was formed and added to the reaction mixture. A volume of 50 ml of distillate was collected from the reaction mixture and replaced with toluene (50 ml). This process was repeated four times. The reaction mixture was allowed to cool to room temperature, diluted with ammonium chloride solution and transferred to a separating funnel. The organic layer was separated and the aqueous layer was extracted again with toluene. The combined organic layers were washed with brine, dried (MgSO₄), filtered and evaporated in vacuo. Purification by column chromatography (eluting with 0-20% methanol in DCM) gave Intermediate 7 (1.95 g, 66%) as a tan-coloured solid.

1 H NMR (400 MHz, CDCI₃) 7.30-7.27 (2H, m), 7.21-7.19 (1 H, m), 7.18-7.16 (1 H, m), 7.00- 6.97 (2H, m), 5.11-5.05 (1 H, m), 4.76 (1 H, s), 2.53-2.49 (1 H, m), 2.15 (6H, s), 2.15-2.07 (2H, m), 2.04-2.02 (1 H, m), 1.87-1.77 (1 H, m), 1.66-1.58 (1 H, m), 1.52-1.45 (1 H, m), 1.18- 1.08 (2H, m). Rf Intermediate 7 = 0.27 (5%MeOH/DCM)

Target compound (an/H(1 S,2R)-2-(2-Hvdroxy-2,2-di-thiophen-2-yl-acetoxy)- bicvclor2.2.1lhept-7-yll-trimethyl-ammonium bromide) A solution of methyl bromide in MeCN was formed by bubbling methyl bromide gas through cooled MeCN (0 ⁰C) until the mass increase indicated an approx 50% w/w solution. Intermediate 7 (6.66 g, 17.6 mmol) was dissolved in this solution (50 ml) and heated in a sealed vessel at 50 ⁰C for 2 days. The mixture was then cooled to 0 ⁰C and the resulting white precipitate collected by filtration to give the product (6.68 g, 80%) as a crystalline solid.

1 H NMR (400 MHz, DMSO) 7.52-7.49 (2H, m), 7.38 (1 H, s), 7.11-7.09 (2H, m) 7.02-6.99 (2H, m), 5.00-4.94 (1 H, m), 3.54 (1 H, s), 3.13 (9H, s), 2.98-2.97 (1 H, m), 2.67-2.66 (1 H, m), 2.21-2.13 (1 H, m), 2.01-1.92 (1 H, m), 1.77-1.62 (1 H, m), 1.39-1.32 (1 H, m), 1.04 (1 H, dd, J = 13.3, 3.2). LC-MS (Method 2): R_t 6.18 min, m/z 392 [M-Br]⁺. Mpt: 229-231 ⁰C (decomp)

A sample of the foregoing compound (480 mg) was crystallized from boiling 2% aqueous- MeCN (10 ml) to give the crystalline monohydrate.

Example 2 - anf/-(1S, 2R) 2-(9-Hydroxy-9H-xanthene-9-carbonyloxy)- bicyclo[2.2.1]hept-7-yl]-dimethyl-(3-phenoxy-propyl)-ammonium bromide

3) Na₂CO₃ (aq)

"Bu₃SnH, AIBN (cat) LiAIH(CBu).

Toluene, 80°C, N₂

TH F, -15°C, N₂ 1 - 2 h Intermediate 5 2 h - O/N

NaOMe, Toluene,

110 °C (external), 2 h Reduced pressure distillation

.tfcrrr,fcdιa*e ^■>

Scheme 2 Intermediate 1

A solution of (S)-(-)-α-methylbenzylamine (84 g, 0.69 mol) in dichloromethane (600 mL) was treated with water (14 mL) and cooled in an ice/water bath. Sulphur dioxide gas was bubbled through the stirred mixture at such a rate that the internal temperature was maintained at 20-25 ⁰C. A solid formed immediately to give a thick slurry but gas addition was continued until this disappeared and the mixture became homogeneous. The yellow solution was treated with bicyclo[3.2.0]hept-2-en-6-one (75 g, 0.69 mol) and the mixture stirred until a solid began to deposit. The mixture was then allowed to stand at 4 ⁰C overnight. The slurry was filtered and the solid washed with dichloromethane and dried to afford a white solid (102.7 g). This solid (102 g) was stirred with 1 M aqueous sodium carbonate solution (700 mL) for ca. 5 mins and the mixture extracted three times with diethyl ether. The organic layers were combined and washed with water, 2 M hydrochloric acid, brine, dried (Na₂SO₄) and evaporated carefully to leave a straw-coloured liquid. Yield: 25.8 g, 34%.

[α]_D +29.9 (c = 5% in MeOH) R_f = 0.44 (15% Et₂O/pentane).

Intermediate 2 A solution of Intermediate 1 (14.14 g, 131 mmol) in toluene (160 mL) was cooled in an ice/acetone bath to give an internal temperature of 0 ⁰C. Bromine (6.7 mL, 131 mmol) was added dropwise over 30 mins such that the internal temperature remained < 5 ⁰C. After addition the mixture was allowed to warm to room temperature. Sodium thiosulphate (aq.

100 mL) was added and the mixture extracted with ethyl acetate. Combined organics were dried over Na₂SO₄, filtered and evaporated to dryness. Purification over silica using automated Biotage SP1 system (340 g column eluting with 0-20% Et₂O/cyclohexane gave the product as a yellow viscous oil which solidifies on extended refrigeration.

Yield: 24.3 g (69%).

R_f = 0.34 (20% Et₂O/pentane).

Intermediate 3

A solution of 3-phenoxy-1 -propyl bromide (25.25 g, 0.1 17 mol) in dry EtOH (100 ml.) was added over 20 min via a dropping funnel to a cooled (0 ⁰C) 33 wt. % solution of methylamine in absolute EtOH (250 ml_, 2.0 mol, 17 equiv.) and slowly allowed to warm to RT overnight. The RM was concentrated to dryness to afford a white solid that was partitioned between sat. sodium carbonate solution (aq.) and DCM with vigorous stirring. After separating the organic layer the water layer was extracted with DCM (2x). The combined organic layers were washed with brine, dried (Na₂SO₄), and concentrated to dryness to afford a light brown oil. Yield: 18.3 g (94%). LC-MS (Method 1 ): R₁ 1.86 min, m/z 166 [MH]⁺.

Intermediate 4

To a solution of Intermediate 2 (4.70 g, 17.5 mmol) in acetone (50 ml.) was added

Intermediate 3 (6.69 g, 40.5 mmol, 2.3 equiv) and stirred at rt for 18 h. The solids were removed by filtration and washed with acetone. The filtrate was concentrated to dryness, the dark brown residue taken up in EtOAc, washed with sat. sodium bicarbonate solution (aq.), brine, dried (Na₂SO₄), and absorbed onto diatomaceous earth. This was then purified by column chromatography over silica gel eluting with 20-30% Et₂O/pentane to afford the title product as a yellow/brown viscous oil. Yield: 3.39 g (55%).

R_f = 0.40 (25% EtOAc/cyclohexane).

Intermediate 5

To a solution of Intermediate 4 (3.20 g, 9.10 mmol) and tri-n-butyltin hydride (2.7 ml_, 10.0 mmol, 1.1 equiv.) in dry toluene (80 ml_, degassed prior to the reaction by vigorously bubbling nitrogen gas through it for 30 minutes) was added AIBN (149 mg, 0.91 mmol, 0.1 equiv.) and heated at 80 ⁰C on a preheated oil bath for 1 h. After cooling to rt the RM was extracted with 1 M HCI (aq., 3x). The combined water layers were washed with pentane, neutralised with Na₂CO₃ (s), and extracted with EtOAc (2x). The combined organic layers were washed with brine, dried (Na₂SO₄), and concentrated to dryness to afford a light brown liquid.

Yield: 2.44 g (98%).

LC-MS (Method 1 ): R₁ 1.91 min, m/z 274 [MH]⁺.

R_f = 0.35 (25% EtOAc/cyclohexane). Intermediate 6

To a solution of Intermediate 5 (2.43 g, 8.90 mmol) in dry THF (70 ml.) at -15 ⁰C was added lithium tri-te/t-butoxyaluminum hydride (3.39 g, 13.3 mmol, 1.5 equiv.). After 2 h a further 1.O g was added (only partial conversion by TLC) and the RM allowed to warm to rt overnight. The RM was quenched with sat. ammonium chloride solution (aq.), the solids removed by filtration, and washed with DCM. The filtrate was concentrated to dryness, partitioned between DCM and water. The organic layer was dried (Na₂SO₄) and concentrated to dryness to afford a light brown viscous oil.

Yield: 2.30 g (94%).

LC-MS (Method 1 ): R₁ 1.73 min, m/z 276 [MH]⁺.

R_f = 0.04-0.18 (30% EtOAc/cyclohexane).

Intermediate 7

i0u °C u,, 1 i--2z h π

A suspension of xanthene-9-carboxylic acid (50 g, 221 mmol) was formed in a 1.75 M HCI solution in MeOH (100 mL) and heated to reflux over night giving a pale yellow solution. The mixture was allowed to cool slowly to rt. The resulting crystalline solid was filtered, washed with cold MeOH and dried under vacuum to give methyl xanthene-9-carboxylate. Yield: 48 g (90%). R_f = 0.40 (20% EtOAc/cyclohexane).

A solution of methyl xanthene-9-carboxylate (25 g, 104 mmol) was formed in THF (600 mL) and cooled to -10 ⁰C. Potassium te/t-butoxide (1 1.7 g, 104 mmol) was added portionwise over 30 mins to give a bright yellow solution. After 5 mins, dry air (from compressed air supply) was gently bubbled through solution (maintained at -10 ⁰C) resulting in a gradual loss of yellow colour and the formation of a white ppt. After approx. 1 hour tic shows loss of SM. The reaction was quenched by the careful addition of 1 M HCI (aq) until pH ~3. Test for peroxides was negative. The reaction was partitioned between Et₂O and water. The organics were washed with sodium sulphite (aq) and brine then dried over Na₂SO₄, filtered and evaporated. Careful purification by column chromatography over silica using 0-20% EtOAc/cyclohexane gives Intermediate 7 as a white solid. Yield: 11 g (41 %) R_f = 0.23 (20% EtOAc/cyclohexane). Intermediate 8

A solution of Intermediate 6 (0.97 g, 3.5 mmol) was formed in toluene (60 ml_). DMF (2 ml.) was added followed by sodium hydride (350 mg, 60% in mineral oil, 8.75 mmol) in one portion (N. B. H₂ evolution). After 5 minutes Intermediate 7 (0.9 g, 3.5 mmol) was added portionwise (N. B. H₂ evolution). After complete addition the mixture was aged for 5 mins then rapidly heated in a pre-heated oil bath to 80 ⁰C for 2 hours. The mixture was allowed to cool to rt then quenched by careful addition of Na₂CO₃ (sat. aq.). The mixture was extracted with both EtOAc and then DCM. The combined organics were dried over MgSO₄, filtered and evaporated. Purification by silica cartridge on lsco Companion using 0-25% EtOAc/cyclohexane gave Intermediate 8 as a colourless gum. Yield: 0.72 g (41%) LC-MS (Method 2): R_t 8.09 min, m/z 500 [MH]⁺.

Alternatively, the following procedure may be employed:

A solution of norbornane alcohol (Intermediate 6, 90 g, 0.327 mol) was dissolved in toluene (2.5 L) and treated with a suspension of sodium methoxide (21.2 g, 0.392 mol) in toluene (0.5 L). Distillation apparatus was attached to the reaction vessel, the pressure was reduced to 570 mbar and the reaction was heated to 93 ⁰C. A solution of Intermediate 7 (125.6 g, 0.490 mol) in toluene (1.2 L) was prepared. After 300 ml of distillate had been collected from the reaction mixture, one-fourth of the solution of Intermediate 7 was added to the reaction mixture. A further 300 ml of distillate was collected from the reaction mixture, which was replaced with a further 300 ml of the solution of Intermediate 7. This was repeated again until all of the solution of Intermediate 7 had been added to the reaction. A suspension of sodium methoxide (8.9 g, 0.164 mol) in toluene (250 ml) was formed and added to the reaction mixture. A volume of 300 ml of distillate was collected. The reaction mixture was allowed to cool to room temperature using an ice bath, diluted with ammonium chloride solution (1 L) and transferred to a separating funnel using EtOAc (900 ml). The organic layer was separated and washed with brine (2 x 1 L), dried (MgSO₄), filtered and evaporated in vacuo. The crude product (205 g) was purified using a sinter funnel under suction using silica (2 kg) eluting firstly with 0-50% DCM in cyclohexane and then with 0-8% EtOAc in DCM to give Intermediate 8 (11 1.7 g, 68%) as a thick yellow oil.

Target compound (anf/-(1S, 2R) 2-(9-Hvdroxy-9H-xanthene-9-carbonyloxy)- bicvclor2.2.1lhept-7-yll-dimethyl-(3-phenoxy-propyl)-ammonium bromide)

A solution of methyl bromide in MeCN is formed by bubbling methyl bromide gas through cooled MeCN (0 ⁰C) until the mass increase indicates an approx 30% w/w solution. Intermediate 8 (0.72 g, 1.44 mmol) is dissolved in this solution (8 ml.) and heated in a sealed tube at 50 ⁰C for 2 days. The mixture is then allowed to cool to rt and concentrated. Purification by silica cartridge on lsco Companion using 0-15% MeOH/DCM gives the Target compound. Recrystallisation from MeCN gives crystalline material. Yield: 630 mg (76%) LC-MS (Method 2): R_t 8.18 min, m/z 514 [M-Br]⁺. Mpt: 203-205 ⁰C

Biological Activity of Muscarinic Antagonists

The inhibitory effects of the muscarinic antagonists can be determined using the muscarinic receptor radioligand binding assay described in WO2007/017670. In this assay, anti-[(1 S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]- trimethyl-ammonium bromide and anti-(1 S, 2R) 2-(9-Hydroxy-9H-xanthene-9- carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl-(3-phenoxy-propyl)-ammonium bromide displayed M3 binding potencies (Ki values) of 0.25 nM and 0.04 nM respectively.

Preparation of βg-adrenoceptor agonists The β₂-adrenoceptor agonists Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3- dihydro-1 ,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(1-naphthyl)ethoxy]propanamide, /V- [2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide and 7-[(1 f?)-2-({2-[(3-{[2- (2-Chlorophenyl)ethyl]amino}propyl)thio]ethyl}amino)-1-hydroxyethyl]-4-hydroxy-1 ,3- benzothiazol-2(3H)-one, which may be employed in the combination of the present invention, may be prepared according to the methods described in WO2008/096126.

Biological Activity of β?-Adrenoceptor Agonists

The biological activity of the β₂-adrenoceptor agonists Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2- (4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(1- naphthyl)ethoxy]propanamide (BA1 ), Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo- 2,3-dihydro-1 ,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(3- chlorophenyl)ethoxy]propanamide (BA2) and 7-[(1 fl)-2-({2-[(3-{[2-(2- Chlorophenyl)ethyl]amino}propyl)thio]ethyl}amino)-1-hydroxyethyl]-4-hydroxy-1 ,3- benzothiazol-2(3H)-one (BA3), can be determined using the "Adrenergic β2 mediated cAMP production", "Adrenergic α1 D", "Adrenergic β1" and "Dopamine D2" assays described in WO2008/096126. The data obtained from these assays are shown in Table 1 below.

Table 1

In Vitro Combination Data

Evaluation of compound activity on isolated tracheal rings from guinea-pig preconstricted with methacholine.

Addition of β2-adrenoceptor agonists and/or muscarinic M3 receptor antagonists causes relaxation of isolated guinea-pig tracheal rings precontracted with the muscarinic agonist, methacholine. Male albino Dunkin Hartley guinea-pigs (300-350 g) are killed by cervical dislocation and the trachea excised. Adherent connective tissue is removed and the trachea cut into ring segments (2-3 mm wide). These are suspended in 1 OmL organ baths containing a modified Krebs solution composition (mM): NaCI 1 17.56, KCI 5.36, NaH₂PO₄ 1.15, MgSO₄ 1.18, glucose 11.10, NaHCO₃ 25.00 and CaCI₂ 2.55. This is maintained at 37°C and continually gassed with 5% CO₂ in O₂, lndomethacin (2.8 μM), corticosterone (10 μM), ascorbate (1 mM), CGP20712A (1 μM) and phentolamine (3 μM) are added to the Krebs solution: indomethacin to prevent development of smooth muscle tone due to the synthesis of cyclooxygenase products, corticosterone to inhibit the uptake 2 process, ascorbate to prevent catecholamine oxidation and CGP20712A and phentolamine to avoid any complicating effects of β1- and α-adrenoceptor activation respectively. The tracheal rings are suspended between two stainless steel hooks, one attached to an isometric force transducer and the other to a stationary support in the organ bath. Changes in isometric force are recorded. Acetyl-β-methylcholine chloride (Methacholine), Indomethacin, Corticosterone-21 -acetate, Phentolamine hydrochloride, Ascorbic acid, CGP20712A methanesulphate are obtained from the Sigma Chemical Company. Indomethacin is dissolved in 10% w/v Na₂CO₃, corticosterone 21-acetate in ethanol and other compounds in DMSO. The muscarinic antagonist and formoterol are diluted in Krebs prior to adding to tissues and the level of DMSO in the bath was < 0.1 %.

At the beginning of each experiment a force of 1.0 g.wt. is applied to the tissues and this is reinstated over a 30min equilibration period until it remained steady. Tissues are then exposed to 1 μM of the muscarinic agonist, methacholine, to assess tissue viability. Tissues are washed by exchanging the bathing Krebs solution three times. After 30 minutes the tissues are again precontracted with 1 μM methacholine. When the contraction has reached a plateau, 1 nM Formoterol, 1OnM of the muscarinic antagonist or a combination of both is added to the bathing media and left for 60 minutes.

Data are collected using the ADInstruments Chartδ for windows software, the tension generated is measured before addition of methacholine and after its response has reached plateau. The response to the mucorinic antagonist and/or formoterol is measured at 10 minute intervals following their addition. All responses are expressed as percentage inhibition of the methacholine-induced contraction.

In Vivo Combination Data

Evaluation of lung function in anaesthetised guinea pigs.

Male Dunkin-Hartley guinea pigs (300-60Og) are weighed and dosed with vehicle (0.05M phosphate, 0.1% Tween 80, 0.6% saline, pH 6) or compound via the intratracheal route under recoverable gaseous anaesthesia (5% halothane in oxygen). Animals are dosed with compound or vehicle two hours prior to the administration of methacholine. Guinea pigs are anaesthetised with pentobarbitone (1 mL/kg of 60 mg/ml_ solution Lp.) approximately 30 minutes prior to the first bronchoconstrictor administration. The trachea is cannulated and the animal ventilated using a constant volume respiratory pump

(Harvard Rodent Ventilator model 683) at a rate of 60 breath/min and a tidal volume of 5 mL/kg. A jugular vein is cannulated for the administration of methacholine or maintenance anaesthetic (0.1 ml. of pentobarbitone solution, 60 mg/ml_, as required).

The animals are transferred to a Flexivent System (SCIREQ, Montreal, Canada) in order to measure airway resistance. The animals are ventilated (quasi-sinusoidal ventilation pattern) at 60 breaths/min at a tidal volume of 5 mL/kg. A positive end expiratory pressure of 2-3 cm H₂O was applied. Respiratory resistance is measured using the Flexivent "snapshot" facility (1 second duration, 1 Hz frequency). Once a stable baseline resistance value has been obtained the animals are given methacholine in ascending doses (0.5, 1 , 2, 3 and 5μg/kg, Lv) at approximately 4-minute intervals via the jugular catheter. After each administration of bronchoconstrictor the peak resistance value is recorded. Guinea pigs are euthanised with approximately 1.OmL pentobarbitone sodium (Euthatal) intravenously after the completion of the lung function measurements. Percentage bronchoprotection produced by the compound is calculated at each dose of brochoconstrictor as follows:

. % chαngeR_veh - % chαngeR _d

% bronchoprotection =

% chαngeR_veh

Where % change R_VΘh is the mean of the maximum percentage change in airway resistance in the vehicle treated group.

Claims

1. A pharmaceutical product comprising, in combination, a first active ingredient which is a muscarinic antagonist selected from: anf/-[(1S,2R)-2-(2-Hydroxy-2,2-di-thiophen-2-yl-acetoxy)-bicyclo[2.2.1]hept-7-yl]-trimethyl- ammonium X; a/tf/-(1 S, 2R) 2-(9-Hydroxy-9H-xanthene-9-carbonyloxy)-bicyclo[2.2.1]hept-7-yl]-dimethyl- (3-phenoxy-propyl)-ammonium X; wherein X represents a pharmaceutically acceptable anion of a mono or polyvalent acid, and a second active ingredient which is a β₂-adrenoceptor agonist.

2. A product according to claim 1 wherein the first active ingredient is a muscarinic antagonist, which is a bromide salt.

3. A product according to claim 1 or claim 2, wherein the β₂-adrenoceptor agonist is formoterol.

4. A product according to claim 3 wherein wherein the β₂-adrenoceptor agonist is selected from: Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(1-naphthyl)ethoxy]propanamide,

Λ/-[2-(Diethylamino)ethyl]-Λ/-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1 ,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide, and 7-[(1 f?)-2-({2-[(3-{[2-(2-Chlorophenyl)ethyl]amino}propyl)thio]ethyl}amino)-1-hydroxyethyl]- 4-hydroxy-1 ,3-benzothiazol-2(3H)-one, or a pharmaceutically acceptable salt thereof.

5. Use of a product according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a respiratory disease.

6. Use according to claim 5, wherein the respiratory disease is chronic obstructive pulmonary disease.

7. A method of treating a respiratory disease, which method comprises simultaneously, sequentially or separately administering:

(a) a (therapeutically effective) dose of a first active ingredient which is a muscarinic receptor antagonist as defined in claim 1 or claim 2; and (b) a (therapeutically effective) dose of a second active ingredient which is a β₂- adrenoceptor agonist; to a patient in need thereof.

8. A kit comprising a preparation of a first active ingredient which is a muscarinic receptor antagonist as defined in claim 1 or claim 2, and a preparation of a second active ingredient which is a β₂-adrenoceptor agonist and optionally instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof.

9. A pharmaceutical composition comprising, in admixture, a first active ingredient which is a muscarinic receptor antagonist as defined in claim 1 or claim 2 and a second active ingredient which is a β₂-adrenoceptor agonist.