WO2011012897A1

WO2011012897A1 - New combinations for the treatment of asthma

Info

Publication number: WO2011012897A1
Application number: PCT/GB2010/051243
Authority: WO
Inventors: Lilian Alcaraz; Nicholas Kindon; Andrew Lister
Original assignee: Astrazeneca Ab; Pulmagen Therapeutics (Syngery) Limited
Priority date: 2009-07-31
Filing date: 2010-07-29
Publication date: 2011-02-03
Also published as: GB0913345D0

Abstract

The invention provides a pharmaceutical product, kit or composition comprising a first active ingredient which is (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-1-hydroxyethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one or a salt thereof, and a second active ingredient selected from: a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist; an antioxidant; a CCR1 antagonist; a chemokine antagonist (not CCR1); a corticosteroid; a CRTh2 antagonist; a DP1 antagonist; an Histone Deacetylase activator; an IKK2 kinase inhibitor; a COX inhibitor; a lipoxygenase inhibitor; a leukotriene receptor antagonist; an MPO inhibitor; a PDE4 inhibitor; a PPARγ agonist; a protease inhibitor; a p38 inhibitor, a Statin; a thromboxane antagonist; a vasodilator; or, an ENAC blocker (Epithelial Sodium-channel blocker); and its use in the treatment of respiratory disease.

Description

NEW COMBINATIONS FOR THE TREATMENT OF ASTHMA

The present invention relates to a combination of two or more pharmaceutically active substances for use in the treatment of respiratory diseases (for example chronic obstructive pulmonary disease (COPD) or 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

corticosteroids. Corticosteroids (also known as glucocorticosteroids or glucocorticoids) are potent anti-inflammatory agents. Whilst their exact mechanism of action is not clear, the end result of corticosteroid treatment is a decrease in the number, activity and movement of inflammatory cells into the bronchial submucosa, leading to decreased airway responsiveness. Corticosteroids may also cause reduced shedding of bronchial epithelial lining, vascular permeability, and mucus secretion. Whilst corticosteroid treatment can yield important benefits, the efficacy of these agents is often far from satisfactory, particularly in COPD. Moreover, whilst the use of steroids may lead to therapeutic effects, it is desirable to be able to use steroids in low doses to minimise the occurrence and severity of undesirable side effects that may be associated with regular administration. Recent studies have also highlighted the problem of the acquisition of steroid resistance amongst patients suffering from respiratory diseases. For example, cigarette smokers with asthma have been found to be insensitive to short term inhaled corticosteroid therapy, but the disparity of the response between smokers and non-smokers appears to be reduced with high dose inhaled corticosteroid (Tomlinson et al., Thorax 2005; 60:282-287).

A further class of therapeutic agent used in the treatment of respiratory diseases are bronchodilators. Bronchodilators 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. Types of bronchodilators in clinical use include β₂ adrenoceptor agonists, muscarinic receptor antagonists and methylxanthines. Bronchodilators are prescribed mainly for symptomatic relief and they are not considered to alter the natural history of respiratory diseases.

(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one is a dual β₂ adrenoceptor agonist/M₃ receptor antagonist (MABA) compound.

Combination products comprising a β₂ adrenoceptor agonist and a corticosteroid are available. One such product is a combination of budesonide and formoterol fumarate

(marketed by AstraZeneca under the tradename Symbicort ®), which has proven to be effective in controlling asthma and COPD, and improving quality of life in many patients.

In view of the complexity of respiratory diseases such as asthma and COPD, it is unlikely that any one mediator can satisfactorily treat a respiratory disease alone.

Moreover, whilst combination treatments using a β₂ adrenoceptor agonist and a corticosteroid deliver significant patient benefits, there remains a medical need for new therapies against respiratory diseases such as asthma and COPD, in particular for therapies with disease modifying potential.

Accordingly, the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient which is Λ/-cyclopropyl-3-fluoro-4-methyl-5-[3-

[[ 1 -[2-[2-(methylamino)ethoxy]phenyl]cyclopropyl]amino]-2-oxo- 1 (2H)-pyrazinyl]- benzamide or a salt thereof, and a second active ingredient selected from:

an Adenosine A2A receptor antagonist;

an anti-infective;

a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist;

an antioxidant;

a CCRl antagonist;

a chemokine antagonist (not CCRl);

a corticosteroid;

a CRTh2 antagonist;

a DPI antagonist;

a formyl peptide receptor antagonist;

a Histone Deacetylase activator;

a chloride channel hCLCAl blocker an Epithelial sodium channel blocker (ENAC blocker) ).

an Inter-cellular adhesion molecule 1 blocker (ICAM blocker);

an IKK2 kinase inhibitor;

a JNK kinase inhibitor;

a cyclooxygenase inhibitor (COX inhibitor);

a lipoxygenase inhibitor;

a leukotriene receptor antagonist;

a MEK-I kinase inhibitor;

a myeloperoxidase inhibitor (MPO inhibitor);

a phosphodiesterase PDE4 inhibitor;

a phosphatidylinositol 3 (PB)-kinase γ inhibitor (PI 3 kinase γ inhibitor)

a peroxisome proliferator activated receptor agonist (PP ARγ agonist);

a protease inhibitor;

a p38 inhibitor

a retinoic acid receptor modulator (RAR γ modulator)

a Statin;

a thromboxane antagonist; or

a vasodilator.

The first active ingredient, which is (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole- 4-carbonyl)-l-oxa-4,9-diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l- hydroxyethyl)-4-hydroxybenzo[d]thiazol-2(3H)-one or a salt thereof, may be in the form of a solvate (such as a hydrate).

A suitable salt of (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one is, for example, a hydrochloride, hydrobromide, trifluoroacetate, sulphate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulphonate, /?-toluenesulphonate, bisulphate, benzenesulphonate, ethanesulphonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate, L-tartrate, D-tartrate, stearate, 2-furoate, 3-furoate, napadisylate (naphthalene- 1, 5 -disulfonate or naphthalene- 1 -(sulfonic acid)-5-sulfonate), edisylate (ethane- 1,2 -disulfonate or ethane- 1-

(sulfonic acid)-2-sulfonate), isethionate (2-hydroxyethylsulfonate), 2- mesitylenesulphonate, 2-naphthalenesulphonate, 2,5-dichlorobenzenesulphonate, D- mandelate, L-mandelate, cinnamate, benzoate, adipate, esylate, malonate, mesitylate (2- mesitylenesulphonate), napsylate (2-naphthalenesulfonate), camsylate (camphor- 10- sulphonate, for example (lS)-(+)-10-Camphorsulfonic acid salt), formate, glutamate, glutarate, glycolate, hippurate (2-(benzoylamino)acetate), orotate, xylate (p-xylene-2- sulphonate), pamoic (2,2'-dihydroxy-l,r-dinaphthylmethane-3,3'-dicarboxylate), palmitate or furoate. It is to be understood for the avoidance of confusion that salts may exist in varying stoichiometries, for example, but not limited to, hemi-, mono-, and di-, and that the invention encompasses all such forms.

In one aspect the present invention provides a pharmaceutical product wherein the first active ingredient is (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa- 4,9-diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l-hydroxyethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one.

The first and second active ingredients can be administered simultaneously (either in a single pharmaceutical preparation {that is, the active ingredients are in admixture} or via separate preparations), or sequentially or separately via separate pharmaceutical preparations.

An Adenosine A2A receptor antagonist is, for example, a compound such as UK- 432097.

An antiinfective is, for example, an antibiotic such as Amoxicillin, Doxycycline,

Trimethoprim sulpha, or a Cephalosporin.

A non-steroidal glucocorticoid receptor (GR) agonist is, for example, a compound disclosed in WO2008/076040, for example 2,2,2-trifluoro-N-[(lR,2S)-l-[l-(4- fluorophenyl)indazol-5-yl]oxy-l-(3-methoxyphenyl)propan-2-yl]acetamide; N-[(1R,2S)-1- [l-(4-fluorophenyl)indazol-5-yl]oxy-l-(4-methylsulfonylphenyl)propan-2-yl]-2-hydroxy- acetamide; N-[(lR*,2S*)-l-[l-(4-fluorophenyl)indazol-5-yl]oxy-l-(6-methoxypyridin-3- yl)propan-2-yl]cyclopropanecarboxamide; (25)-Λ/-[(li?,25)-l-[l-(4-fluorophenyl)indazol- 5-yl]oxy-l-phenyl-propan-2-yl]-2-hydroxy-propanamide; 2,2,2-trifluoro-N-[(2S*,3S*)-3- [1 -(4-fluorophenyl)indazol-5-yl]oxy-4-phenoxy-butan-2-yl]acetamide; Λ/"-[(li?,25)- 1 -[ 1 -(4- fluorophenyl)indazol-5-yl]oxy-l-(3-methoxyphenyl)propan-2-yl]-N-propan-2-yl-oxamide, or a pharmaceutically acceptable salt thereof. An antioxidant is, for example, Allopurinol, Erdosteine, Mannitol, N-acetyl cysteine choline ester, N-acetyl cysteine ethyl ester, N-Acetylcysteine, N- Acetylcysteine amide or Niacin.

A CCRl antagonist is, for example, a compound disclosed in WO2001/062728 or WO2001/098273, or a pharmaceutically acceptable salt thereof (such as a hydrochloride, trifluoroacetate, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt).

Also, a CCRl antagonist is, for example, N-{2-[((2S)-3-{[l-(4- chlorobenzyl)piperidin-4-yl] amino } -2-hydroxy-2-methylpropyl)oxy] -A- hydroxyphenyl}acetamide, also named as 4-({(25)-3-[2-(acetylamino)-5- hydroxyphenoxy]-2-hydroxy-2-methylpropyl} ammonio)- 1 -(4-chlorobenzyl)piperidine (see WO 2003/051839), or, 2-{2-Chloro-5-{[(2S)-3-(5-chloro-lΗ,3H-spiro[l-benzofuran-2,4'- piperidin]-r-yl)-2-hydroxypropyl]oxy}-4-[(methylamino)carbonyl]phenoxy}-2- methylpropanoic acid (see PCT publication no. WO 2008/010765), or a pharmaceutically acceptable salt thereof (for example a hydrochloride, sulphate, (hemi)fumarate, benzoate, furoate or succinate salt).

A chemokine antagonist (other than a CCRl antagonist), for example, 656933 (N- (2-bromophenyl)-N'-(4-cyano- IH-1 ,2,3 -benzotriazol-7-yl)urea), 766994 (4-( {[({ [(2R)-4- (3,4-dichlorobenzyl)morpholin-2-yl]methyl}amino)carbonyl]-amino}methyl)benzamide), CCX-282, CCX-915, Cyanovirin N, E-921, INCB-003284, INCB-9471, Maraviroc, MLN- 3701, MLN-3897, T-487 (N-{l-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3- d]pyrimidin-2-yl] ethyl} -N-(pyridin-3 -ylmethyl)-2- [4-(trifluoromethoxy)phenyl] acetamide) or Vicriviroc.

A chloride channel hCLCAl blocker is, for example, as disclosed in

WO2006/091112, WO2004/113286 and WO2001/038530.

A corticosteroid is, for example, Alclometasone dipropionate, Amelometasone,

Beclomethasone dipropionate, Budesonide, Butixocort propionate, Ciclesonide, Clobetasol propionate, Desisobutyrylciclesonide, Etiprednol dicloacetate, Fluocinolone acetonide, Fluticasone Furoate, Fluticasone propionate, Loteprednol etabonate (topical) or

Mometasone furoate.

A glucocorticosteroid compound is (lR,3aS,3bS,10aR,10bS,HS,12aS)l-

{ [(cyanomethyl)sulfanyl] carbonyl} -7-(4-fluorophenyl)- 11 -hydroxy- 1 Oa, 12a-dimethyl- l,2,3,3a,3b,4,5,7,10,10a,10b,l l,12,12a-tetradecahydrocyclopenta[5,6]naphtho[l,2- f]indazol-l-yl furan-2-carboxylate. The compound is disclosed in WO-2009/044200.

A CRTh2 antagonist is, for example, a compound from WO 2004/106302, WO2004/089885, WO2005/018529 or WO2007/039741.

A DPI antagonist is, for example, L888839 or MK0525.

An ENAC (Epithelial Sodium-channel blocker) is, for example, Amiloride, Benzamil, Triamterene, 552-02, PSA14984, PSA25569, PSA23682, AER002, Parion P- 522 or a compound from WO2008031048.

A formyl peptide receptor antagonist is, for example, a compound from WO2007/144198.

A histone deacetylase activator is, for example, ADC4022, Aminophylline, a

Methylxanthine or Theophylline.

An ICAM blocker is, for example, an anti-ICAM-1 monoclonal antibody (MAb) 1A6 from Antimicrobial Agents and Chemotherapy 2003, 47, 1503-1508.

An IKK2 inhibitor is, for example, 2-{[2-(2-Methylamino-pyrimidin-4-yl)-lH- indole-5 -carbonyl] -amino } -3 -(phenyl-pyridin-2-yl-amino)-propionic acid or a compound as disclosed in WO 01/58890, WO 03/010158, WO 03/010163, WO 04/063185,

WO 04/063186.

A JNK inhibitor is, for example, a compound from WO2005/003123 or

WO2003/051277.

A COX inhibitor is, for example, Celecoxib, Diclofenac sodium, Etodolac,

Ibuprofen, Indomethacin, Meloxicam, Nimesulide, OC1768, OC2125, OC2184, OC499, OCD9101, Parecoxib sodium, Piceatannol, Piroxicam, Rofecoxib or Valdecoxib.

A lipoxygenase inhibitor is, for example, Ajulemic acid, Darbufelone, Darbufelone mesilate, Dexibuprofen lysine (monohydrate), Etalocib sodium, Licofelone, Linazolast, Lonapalene, Masoprocol, MN-OOl , Tepoxalin, UCB-35440, Veliflapon, ZD-2138, ZD- 4007 or Zileuton ((±)-l-(l-Benzo[b]thien-2-ylethyl)-l -hydroxyurea)

A leukotriene receptor antagonist is, for example, Ablukast, Iralukast (CGP 45715A), Montelukast, Montelukast sodium, Ontazolast, Pranlukast, Pranlukast hydrate (mono Na salt), Verlukast (MK-679) or Zafirlukast.

A MEK-I inhibitor is, for example, a compound disclosed in WO2007123939,

WO2007025090 or WO2005051906. An MPO Inhibitor is, for example, a Hydroxamic acid derivative (N-(4-chloro-2- methyl-phenyl)-4-phenyl-4-[[(4-propan-2-ylphenyl)sulfonylamino]methyl]piperidine-l- carboxamide), Piceatannol or Resveratrol, or a compound disclosed within US7425560, WO2003/089430, WO2006/062465 and WO2007/120098.

p38 inhibitors are, for example, a compound from WO 2005/042502, 681323,

856553, AMG548 (2-[[(2S)-2-amino-3-phenylpropyl]amino]-3-methyl-5-(2-naphthalenyl)- 6-(4-pyridinyl)-4(3H)-pyrimidinone), Array-797, AZD6703, Doramapimod, KC-706, PH 797804, R1503, SC-80036, SCIO469, 6-chloro-5-[[(2^5i?)-4-[(4-fluorophenyl)methyl]- 2,5-domethyl- 1 -piperazinyl]carbonyl]-JV,Λf, 1 -trimethyl-α-oxo- lH-indole-3-acetamide, VX702 or VX745 (5-(2,6-dichlorophenyl)-2-(phenylthio)-6H-pyrimido[ 1 ,6-b]pyridazin-6- one). Λ/-cyclopropyl-3-fluoro-4-methyl-5-[3-[[ 1 -[2-[2-(methylamino)ethoxy]phenyl] cyclopropyl]amino]-2-oxo-l(2H)-pyrazinyl]-benzamide is exemplified in

WO2009/001132.

A PI 3 kinase γ inhibitor is, for example, a compound from WO2005/105801, WO2003/072557, and WO2007/082956.

A PPARγ agonist is, for example, Pioglitazone, Pioglitazone hydrochloride, Rosiglitazone Maleate, Rosiglitazone Maleate ((-)-enantiomer, free base), Rosiglitazone maleate/Metformin hydrochloride or Tesaglitizar.

A Protease Inhibitor is, for example, Alpha 1 -antitrypsin proteinase Inhibitor, EPI- HNE4, UT-77, ZD-0892 or a compound from WO 2006/004532, WO 2005/026123, WO 2002/0744767 or WO 22002/074751; or a TACE Inhibitor (for example DPC-333, Sch- 709156 or Doxycycline);. inhibitors of cathepsins for example inhibitors of cathepsin S (for example as disclosed in WO2002/14314), cathepsin L (for example as described within Bioorg. Med. Chem. 2004,12, 4081), cathepsin K (for example WO 2001/47886) , cathepsin B (for example tokaramide A and leupetin) and cathepsin C (dipeptidyl peptidase 1) (for example a compound from WO 2005/000800); inhibitors of neutrophil elastase, for example as disclosed in WO2005/026123 and WO2007/129963 (for example 6-[l-(4- cyanophenyl)-lH-pyrazol-5-yl]-N,5-dimethyl-3-oxo-4-[3-(trifluoromethyl)phenyl]-3,4- dihydropyrazine-2-carboxamide) and inhibitors of matrix metallo proteinases (for example ABT-518 or Ro-32-7315). A RAR γ modulator (Retinoic acid gamma receptor modulator) is, for example, palovarotene (R667), a compound disclosed in WO2008064136 (agonists) or

WO2006066978 (antagonists).

A Statin is, for example, Atorvastatin, Lovastatin, Pravastatin, Rosuvastatin or Simvastatin.

A Thromboxane Antagonist is, for example, Ramatroban or Seratrodast.

A Vasodilator is, for example, A-306552, Ambrisentan, Avosentan, BMS-248360, BMS-346567, BMS-465149, BMS-509701, Bosentan, BSF-302146 (Ambrisentan), Calcitonin Gene-related Peptide, Daglutril, Darusentan, Fandosentan potassium, Fasudil, Iloprost, KC- 12615 (Daglutril), KC- 12792 2AB (Daglutril), Liposomal treprostinil, PS- 433540, Sitaxsentan sodium, Sodium Ferulate, TBC-11241 (Sitaxsentan), TBC-3214 (N- (2-acetyl-4,6-dimethylphenyl)-3-[[(4-chloro-3-methyl-5-isoxazolyl)amino]sulfonyl]-2- thiophenecarboxamide), TBC-3711, Trapidil, Treprostinil diethanolamine or Treprostinil sodium.

A PDE4 inhibitor is, for example, 6-fluoro-N-((ls,4s)-4-(6-fluoro-2,4-dioxo-l-(4'-

(piperazin- 1 -ylmethyl)-biphenyl-3-yl)- 1 ,2-dihydropyrido[2,3-d]pyrimidin-3(4H)- yl)cyclohexyl)imidazo[l,2-a]pyridine-2-carboxamide (as disclosed in. WO2008084223), or a salt thereof (for example a (lS)-(+)-10-Camphorsulfonic acid or trihydrochloride salt).

All the above second et seq active ingredients may be in the form of solvates, for example hydrates.

In one particular aspect the present invention provides a pharmaceutical product 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 and the second active ingredient of the

pharmaceutical product of the present invention may be administered simultaneously, sequentially or separately to treat respiratory diseases. By simultaneously is meant that the active ingredients are in admixture, or they could be in separate chambers of the same inhaler. By sequential it is meant that the active ingredients are administered, in any order, one immediately after the other. They still have the desired effect if they are administered separately, but when administered in this manner they are generally administered less than 4 hours apart, conveniently less than two hours apart, more conveniently less than 30 minutes apart and most conveniently less than 10 minutes apart, for example less than 10 minutes but not one immediately after the other.

The active ingredients may be delivered to the lung and/or airways via oral administration in the form of a solution, suspension, aerosol or dry powder formulation. These dosage forms will usually include one or more pharmaceutically acceptable ingredients which may be selected, for example, from an adjuvant, carrier, binder, lubricant, diluent, stabilising agent, buffering agent, emulsifying agent, viscosity- regulating agent, surfactant, preservative, flavouring or colorant. The active ingredients of the present invention may also 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. 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 another embodiment the first and second active ingredients are administered via a single pharmaceutical composition (that is, the first and second active ingredients are in admixture). Therefore, the present invention further provides a pharmaceutical

composition comprising, in admixture, a first active ingredient which is (R)-7-(2-(2-fluoro- 5-((4-(2-isopropylthiazole-4-carbonyl)- 1 -oxa-4,9-diazaspiro[5.5]undecan-9- yl)methyl)phenethylamino)-l-hydroxyethyl)-4-hydroxybenzo[d]thiazol-2(3H)-one or a salt thereof (such as the hydrochloride or L-tartaric acid salt) , and a second active ingredient as defined above. The pharmaceutical composition optionally further comprises a

pharmaceutically acceptable adjuvant, diluent or carrier.

The pharmaceutical compositions of the present invention can be prepared by mixing the first active ingredient with the 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 the first and second active ingredients 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 first active ingredient is administered via inhalation. When administered via inhalation the dose of the first active ingredient (that is(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one in: salt form, solvate form, or, solvate of salt form) 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 second active ingredient is administered by inhalation. When administered via inhalation the dose of the second active ingredient 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 another embodiment the present invention provides a pharmaceutical product wherein the molar ratio of first active ingredient to second active ingredient is from 1 : 1000 to 1000:1, such as from 1 :100 to 100:1, for example from 1 :50 to 50:1, for example 1 :20 to 20:1. In one embodiment, the present invention provides a pharmaceutical product comprising, in combination, a first active ingredient as defined above, and a second active ingredient as defined above, wherein each active ingredient is formulated for inhaled administration. In a further aspect of this embodiment, the pharmaceutical product is in the form of a pharmaceutical composition comprising the first and second active ingredients in admixture, and which composition is formulated for inhaled administration.

The active ingredients of the present invention are conveniently delivered via oral administration by inhalation to the lung and/or airways in the form of a solution, suspension, aerosol or dry powder (such as an agglomerated or ordered mixture) formulation. For example a metered dose inhaler device may be used to administer the active ingredients, dispersed in a suitable propellant and with or without an additional excipient such as ethanol, a surfactant, lubricant or stabilising agent. A suitable propellant includes a hydrocarbon, chlorofluorocarbon or a hydrofluoroalkane (e.g.

heptafluoroalkane) propellant, or a mixture of any such propellants, for example in a pressurised metered dose inhaler (pMDI). Preferred propellants are P 134a and P227, each of which may be used alone or in combination with other another propellant and/or surfactant and/or other excipient. A nebulised aqueous suspension or, preferably, solution may also be employed, with or without a suitable pH and/or tonicity adjustment, either as a unit-dose or multi-dose formulation. A suitable device for delivering a dry powder is Turbuhaler®.

The pharmaceutical product of the present invention can, for example, be administered: via an inhaler having the first and second active ingredients in separate chambers of the inhaler such that on administration the active ingredients mix in either the mouthpiece of the inhaler or the mouth of a patient or both (for simultaneous use); or, where the first and second active ingredients are in separate inhalers, via separate inhalers (for separate or sequential use); or the first and second active ingredients are in admixture in an inhaler when the inhaler is supplied to a patient (for simultaneous use).

A dry powder inhaler may be used to administer the active ingredients, alone or in combination with a pharmaceutically acceptable carrier (such as lactose), 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 is available.

The combination of the present invention may be used to treat diseases of the respiratory tract such as obstructive diseases of the airways including: asthma, including bronchial, allergic, intrinsic, extrinsic, exercise-induced, drug-induced (including aspirin and NS AID-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, fibrosis complicating anti-neoplastic therapy and chronic infection, including tuberculosis and aspergillosis and other fungal infections; complications of lung transplantation; vasculitic and thrombotic disorders of the lung vasculature, and pulmonary hypertension; antitussive activity including treatment of chronic cough associated with inflammatory and secretory conditions of the airways, and iatrogenic cough; acute and chronic rhinitis including rhinitis medicamentosa, and vasomotor rhinitis; perennial and seasonal allergic rhinitis including rhinitis nervosa (hay fever); nasal polyposis; acute viral infection including the common cold, and infection due to respiratory syncytial virus, influenza, coronavirus (including SARS) and adenovirus.

Accordingly, the present invention further provides a pharmaceutical product according to the invention for simultaneous, sequential or separate use in therapy.

The present invention further provides the use of a pharmaceutical product according to the invention in the manufacture of a medicament for the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).

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 as defined above; and, (b) a therapeutically effective dose of a second active ingredient as defined above;

to a patient in need thereof. In a further aspect the present invention provides the use of a pharmaceutical product, kit or composition as hereinbefore described for the treatment of a respiratory disease, in particular chronic obstructive pulmonary disease, asthma, rhinitis, emphysema or bronchitis (such as chronic obstructive pulmonary disease or asthma; for example chronic obstructive pulmonary disease).

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.

Example 1

Preparation of:

(i?)-7-(2-(2-Fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one ditrifluoroacetate

a) 2-(5-(Bromomethyl)-2-fluorophenyl)ethanol

Dibenzoyl peroxide (0.14 g) was added to a solution of NBS (1.53 g) and 2-(2- fluoro-5-methylphenyl)acetic acid (1.45 g) in DCM (50 mL) and the resulting mixture was heated at reflux for 12 h. The solvent was evaporated and the white solid partitioned between ethyl acetate (100 mL) and 10% sodium chloride solution (50 mL). The layers were separated and the organic phase washed with 10% sodium chloride solution (50 mL), dried over sodium sulphate, filtered and evaporated. The white solid obtained was redissolved in tetrahydrofuran (25 mL) and cooled in an ice bath. A solution of borane dimethyl sulfide complex (2M in THF, 13 mL) was added cautiously and the mixture was then allowed to warm to RT and stirred for 2 h. The reaction was cooled in an ice bath and cautiously quenched with methanol. Once bubbling had ceased the solvent was evaporated and the residue purified by silica gel chromatography eluting with 9:1 to 4: 1 ethyl acetate :isohexane gradient to give the subtitled compound as a clear oil. Yield 1.35 g. ¹H NMR (300 MHz, CDCl₃) δ 7.32 - 7.21 (m, 2H), 7.04 - 6.97 (m, IH), 4.46 (s, 2H), 3.87 (t, J = 6.5 Hz, 2H), 2.93 - 2.87 (m, 2H). One exchangeable proton not observed.

b) (9-(4-Fluoro-3-(2-hydroxyethyl)benzyl)-l-oxa-4,9-diazaspiro[5.5]undecan-4-yl)(2- isopropylthiazol-4-yl)methanone

2-(5-(Bromomethyl)-2-fluorophenyl)ethanol (example 47, step a) (0.16 g) was added to a solution of (2-isopropylthiazol-4-yl)(l-oxa-4,9-diazaspiro[5.5]undecan-4-yl)methanone trifluoroacetate (example 22, step b) (0.3 g) and triethylamine (0.3 mL) in acetonitrile (10 mL). The resulting mixture was stirred overnight, diluted with acetonitrile (20 mL) and applied to a SCX cartridge (10 g Varian, pre-wetted with acetonitrile (50 mL)). The cartridge was washed with acetonitrile (50 mL) and eluted with 10% '880' aqueous ammonia in acetonitrile solution (50 mL). The eluent was evaporated, azeotroped with toluene and purifed by silica gel chromatography eluting with 77.5:17.5:5 isohexane: ethyl acetate :triethylamine to 95:5 ethyl acetate :triethylamine gradient. The fractions containing product were combined and evaporated to give to the subtitled compound as a yellow gum.

Yield 0.22 g.

m/z 462 (M+H)⁺ (APCI)

¹H NMR (400 MHz, D₆-DMSO, 9O⁰C) δ 7.91 (s, IH), 7.22 - 7.14 (m, IH), 7.14 - 7.06 (m,

IH), 7.00 (dd, J = 10.0, 8.3 Hz, IH), 4.40 (t, J = 5.3 Hz, IH), 3.72 - 3.52 (m, 8H), 3.43 -

3.23 (m, 3H), 2.74 (t, J = 7.0 Hz, 2H), 2.42 - 2.23 (m, 4H), 1.75 - 1.65 (m, 2H), 1.59 - 1.47

(m, 2H), 1.36 (d, J = 6.9 Hz, 6H).

c) (R)-7-(2-(2-Fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l-hydroxyethyl)-4- hydroxybenzo [d] thiazol-2(3H)-one ditrifluoroacetate

Trifluoroacetic acid (0.033 mL) was added to a solution of (9-(4-fluoro-3-(2- hydroxyethyl)benzyl)-l-oxa-4,9-diazaspiro[5.5]undecan-4-yl)(2-isopropylthiazol-4- yl)methanone (example 47, step b) (0.2 g) in DCM (5 mL) at O⁰C. The mixture was stirred for 5 min then Dess-Martin periodinane (0.28 g) was added. The resulting yellow solution was allowed to warm to RT and stirred for 1 h. A mixture of saturated sodium

thiosulphate solution (1 mL), saturated sodium bicarbonate solution (1 mL) and ethyl acetate (5 mL) was then added and the resulting mixture stirred vigorously for 10 min. The aqueous phase was separated and extracted with ethyl acetate (5 mL). The combined organic solutions were washed with brine (5 mL), acidified wth a few drops of acetic acid, dried over sodium sulphate, filtered and evaporated in vacuo. The residue was dissolved in methanol (2 mL), acetic acid (0.025 mL) and (7?)-7-(2-amino-l -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one hydrochloride (WO2007027134, Example 1, step d) (0.17 g) were then added and the mixture stirred for 5 min before cooling in an ice bath. Sodium cyanoborohydride (0.04 g) was then added, the mixture allowed to warm to RT and stirred overnight. The solvent was evaporated in vacuo. Purification was by silica gel chromatography eluting with 94.5:5:0.5 to 89:10:1 DCM:Methanol:'880' aqueous ammonia gradient. The fractions containing product were combined and evaporated in vacuo. Further purification was by preparative HPLC (Sunfϊre™, Gradient: 5-30% acetonitrile in 0.2% aqueous TFA). The fractions containing product were combined, evaporated in vacuo and the residue triturated with diethylether to give the titled compound as a white solid. Yield 0.14 g.

m/z 670 (M+H)⁺ (APCI)

¹H NMR (400 MHz, D₆-DMSO, 9O⁰C) δ 11.27 (s, IH), 7.94 (s, IH), 7.52 - 7.42 (m, 2H), 7.25 (t, J = 9.2 Hz, IH), 6.94 (d, J = 8.2 Hz, IH), 6.78 (d, J = 8.2 Hz, IH), 4.93 (dd, J = 7.9, 4.6 Hz, IH), 4.29 (s, 2H), 3.80 - 3.58 (m, 6H), 3.34 - 2.95 (m, HH), 2.11 - 1.95 (m, 2H), 1.87 - 1.64 (m, 2H), 1.34 (d, J = 6.7 Hz, 6H). Five exchangeable protons not observed.

Example IA

(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l-hydroxyethyl)-4- hydroxybenzo [d] thiazol-2(3H)-one

a) 2-(5-(Bromomethyl)-2-fluorophenyl)ethanol

Dibenzoyl peroxide (1 g) was added to a solution of NBS (10.6 g) and 2-(2-fluoro-5- methylphenyl)acetic acid (10 g) in DCM (250 mL) and the resulting mixture was heated under reflux for 12 h. The solvent was evaporated and the white solid partitioned between ethyl acetate (250 mL) and 10% sodium chloride solution (500 mL). The layers were separated and the organic phase washed with 10% sodium chloride solution (500 mL), dried over magnesium sulphate, filtered and evaporated. The white solid obtained was redissolved in tetrahydrofuran (150 niL) and cooled in an ice bath. A solution of borane dimethyl sulfide complex (2M in THF, 89 mL) was added cautiously and the mixture was then allowed to warm to RT and stirred for overnight. The reaction was cooled in an ice bath and cautiously quenched with methanol. Once bubbling had ceased the solvent was evaporated and the residue was triturated with a 4: 1 mixture of ώo-hexanes and ether. Purification was by silica gel chromatography eluting with 9:1 to 4:1 ethyl

acetate :isohexane gradient to give the subtitled compound as a clear oil. Yield 6.5 g. ¹H NMR (300 MHz, CDCl₃) δ 7.32 - 7.21 (m, 2H), 7.04 - 6.97 (m, IH), 4.46 (s, 2H), 3.87 (t, J = 6.5 Hz, 2H), 2.93 - 2.87 (m, 2H). One exchangeable proton not observed.

2-(5-(Bromomethyl)-2-fluorophenyl)ethanol (Example IA, step a) (5.2 g) was added to a suspension of (2-isopropylthiazol-4-yl)(l -oxa-4,9-diazaspiro[5.5]undecan-4-yl)methanone trifluoroacetate (example 22, step b) (9.4 g) and potassium carbonate (6.8 g) in ethanol (75 mL). The resulting mixture was stirred overnight and filtered. The filter cake was washed with ethanol (50 mL) and the combined filtrate and washings were evaporated. The residue was partioned between water (100 mL) and ethyl acetate (250 mL), The layers were separated and the organic washed with brine (100 mL), dried over sodium sulphate, filtered and evaporated. Purification was by silica gel chromatography eluting with 95:5 ethyl acetate :triethylamine gradient. The fractions containing product were combined and evaporated to give to the subtitled compound as a yellow gum. Yield 7.9 g.

m/z 462 (M+H)⁺ (APCI)

¹H NMR (400 MHz, D₆-DMSO, 9O⁰C) δ 7.91 (s, IH), 7.22 - 7.14 (m, IH), 7.14 - 7.06 (m, IH), 7.00 (dd, J = 10.0, 8.3 Hz, IH), 4.40 (t, J = 5.3 Hz, IH), 3.72 - 3.52 (m, 8H), 3.43 - 3.23 (m, 3H), 2.74 (t, J = 7.0 Hz, 2H), 2.42 - 2.23 (m, 4H), 1.75 - 1.65 (m, 2H), 1.59 - 1.47 (m, 2H), 1.36 (d, J = 6.9 Hz, 6H). c) (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one.

Trifluoroacetic acid (1.32 mL) was added to a solution of (9-(4-fluoro-3-(2- hydroxyethyl)benzyl)-l-oxa-4,9-diazaspiro[5.5]undecan-4-yl)(2-isopropylthiazol-4- yl)methanone (example 47 A, step b) (7.9 g) in DCM (200 mL) at O⁰C. The mixture was stirred for 5 min then Dess-Martin periodinane (12.3 g) was added. The resulting yellow solution was allowed to warm to RT and stirred for 1 h. A mixture of saturated sodium thiosulphate solution (100 mL), saturated sodium bicarbonate solution (100 mL) and ethyl acetate (500 mL) was then added and the resulting mixture stirred vigorously for 10 min. The aqueous phase was separated and extracted with ethyl acetate (10OmL). The combined organic solutions were washed with brine (100 mL), acidified wth acetic acid (2 mL), dried over sodium sulphate, filtered and evaporated in vacuo. The residue was dissolved in methanol (140 mL) and (i?)-7-(2-amino-l -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one hydrochloride (WO2007027134, Example 1 , step d) (4.5 g) was then added and the mixture stirred for 5 min before cooling in an ice bath. Sodium cyanoborohydride (1.6 g) was then added, the mixture allowed to warm to RT and stirred overnight. The reaction mixture was concentrated in vacuo and partioned between THF (10OmL) and a mixture of brine and saturated sodium bicarbonate solution (10:1, 10OmL). The layers were separated and the organic layer was dried over sodium sulphate, filtered, evaporated and the residue azeotroped with acetonitrile. Purification was by silica gel chromatography eluting with 94.5:5:0.5 to 89:10:1 DCM:Methanol:'880' aqueous ammonia gradient. The fractions containing the product were combined and evaporated in vacuo to give the titled coumpound as a white solid. Yield 4.1 g

m/z 670 (M+H)⁺ (APCI)

¹H NMR (400 MHz, D₆-DMSO, 9O⁰C) δ 11.27 (s, IH), 7.94 (s, IH), 7.52 - 7.42 (m, 2H), 7.25 (t, J = 9.2 Hz, IH), 6.94 (d, J = 8.2 Hz, IH), 6.78 (d, J = 8.2 Hz, IH), 4.93 (dd, J = 7.9, 4.6 Hz, IH), 4.29 (s, 2H), 3.80 - 3.58 (m, 6H), 3.34 - 2.95 (m, HH), 2.11 - 1.95 (m, 2H), 1.87 - 1.64 (m, 2H), 1.34 (d, J = 6.7 Hz, 6H). Five exchangeable protons not observed. Example IB

(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l-hydroxyethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one di(lS)-(+)-10-camphorsulfonic acid salt

lS-(+)-Camphorsulphonic acid (41mg) was added to a solution of (R)-7-(2-(2-fluoro-5-((4- (2-isopropylthiazole-4-carbonyl)- 1 -oxa-4,9-diazaspiro[5.5]undecan-9- yl)methyl)phenethylamino)-l-hydroxyethyl)-4-hydroxybenzo[d]thiazol-2(3H)-one

(example 47A) (59mg) in ethanol (5mL) and the resulting clear solution evaporated to dryness to give (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one dicamphorsulfonic acid salt as an amorphous white solid. Yield 0. Ig

/so-propanol (ImL) was added to (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4- carbonyl)- 1 -oxa-4,9-diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)- 1 - hydroxyethyl)-4-hydroxybenzo[d]thiazol-2(3H)-one dicamphorsulfonic acid salt (20 mg) and the resulting clear solution was stirred 2 days. A white solid was formed and the suspension was stirred for a further 5 days. The solid was isolated by centrifugal filtration and dried under high vacuum to give (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4- carbonyl)- 1 -oxa-4,9-diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)- 1 - hydroxyethyl)-4-hydroxybenzo[d]thiazol-2(3H)-one dicamphorsulfonic acid salt as a white crystalline solid. Yield 5mg.

A mixture of (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l -oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one (4.3 g, 6.42 mmol) (example 47A), (lS)-(+)-10- camphorsulfonic acid (2.98 g, 12.84 mmol) and ώo-propanol (300 mL) was heated at 50⁰C until a clear solution formed, seeded, allowed to cool to RT and stirred for 4 days. The solid was isolated by filtration, washed with ώo-propanol (10OmL), ether (2x200mL) and sucked dry to give the titled compound as a white crystalline solid. Yield 5. Ig

The enantiomeric excess of the title compound is higher than the compound obtained in example 47A. (example 47A = 86% ee, title compound >96% ee) Analytical Chiral Method: Chiralcel OJ-H 4.6x250 mm, 80:20 isohexane:ethanol + 0.1% ethylenediamine, 1 ml/min, 35C, 225 +-10 nm over 30 min.

Retention time for the R enantiomer = 15.91 min

Retention time for the S enantiomer = 22.85min

¹H NMR (300 MHz, DMSO, 9O⁰C) δ 11.42 (s, IH), 9.95 (S, IH), 8.89 (S, 2H), 7.95 (S, IH), 7.61 - 7.54 (m, IH), 7.53 - 7.46 (m, IH), 7.27 (t, J = 9.2 Hz, IH), 6.94 (d, J = 8.5 Hz, IH), 6.77 (d, J= 8.3 Hz, IH), 4.97 - 4.90 (m, IH), 4.30 (s, 2H), 3.74 - 3.59 (m, 6H), 3.35 - 3.02 (m, 9H), 2.93 (d, J= 14.6 Hz, 2H), 2.76 - 2.61 (m, 2H), 2.44 (d, J= 14.6 Hz, 2H), 2.29 - 2.23 (m, 2H), 2.22 - 2.16 (m, 2H), 2.13 - 2.00 (m, 2H), 1.96 - 1.68 (m, 6H), 1.34 (d, J= 6.9 Hz, 6H), 1.32 - 1.20 (m, 4H), 1.06 (s, 6H), 0.76 (s, 6H) two exchangeable protons not observed.

An XRPD pattern of di(lS)-(+)-10-camphorsulfonic acid salt modification A is presented in Figure 1.

Some characteristic peaks for modification A

Example 1C

(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one fumarate Modification A

A solution of (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one (13 mg) (example 47D, step a) and fumaric acid (2 mg) in methanol (0.75 mL) was stirred at RT for 7 days. The resulting white solid formed was isolated by filtration and dried under high vacuum to give the titled compound as a white solid. Yield 5mg.

m/z 670 (M+H)⁺ (APCI) An XRPD pattern of fumarate salt modification A is presented in Figure 2.

Some characteristic peaks for the fumarate salt modification A

Example ID

(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l-hydroxyethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one mono fumarate Modification B

a) (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l-hydroxyethyl)-4- hydroxybenzo [d] thiazol-2(3H)-one

(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one di(lS)-(+)-10-camphorsulfonic acid salt (Example IB) (4.Ig) was partioned between freshly distilled 2-methyl-THF (100 mL) and saturated sodium bicarbonate solution (100 mL). The layers were separated and the organic layer washed with saturated sodium bicarbonate solution (100 mL), brine (10OmL), dried over sodium sulphate, filtered and evaporated in vacuo. The resulting glassy solid was triturated thrice with ether to give the subtitle compound as a white solid. Yield 2.6g. b) (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro[5.5]undecan-9-yl)methyl)phenethylamino)-l-hydroxyethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one mono fumarate Modification B

(R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one (2.4 g) was dissolved in ethanol (240 mL) at 50 ⁰C. Fumaric acid (0.416g) was added and the mixture heated under reflux until a clear solution formed. The resulting solution was allowed to cool to 60⁰C and seeded with form A (example 47C) (50 mg). The resulting mixture was stirred overnight at 50⁰C and then allowed to cool to RT over 3hrs. The resulting white solid was isolated by filtration, washed with ethanol (50 mL), ether (2x200 mL) and sucked dry to give the titled compound as a white solid. Yield 2.3g.

m/z 670 (M+H)⁺ (APCI)

¹H NMR (400 MHz, DMSO, 9O⁰C) δ 7.89 (s, IH), 7.19 - 7.09 (m, 2H), 7.01 (dd, J= 9.9,

8.3 Hz, IH), 6.86 (d, J= 8.2 Hz, IH), 6.69 (d, J= 8.5 Hz, IH), 6.59 (d, J= 1.0 Hz, 2H),

4.65 - 4.59 (m, IH), 3.73 - 3.56 (m, 6H), 3.41 (s, 2H), 3.30 (septet, J= 3.3 Hz, IH), 2.90

- 2.71 (m, 6H), 2.42 - 2.26 (m, 4H), 1.75 - 1.64 (m, 2H), 1.59 - 1.48 (m, 2H), 1.35 (dd, J

= 6.8, 1.2 Hz, 6H) + 6 exchangables not observed

An XRPD pattern of fumarate salt modification B is presented in Figure 3.

Some characteristic peaks for the fumarate salt modification B

XRPD - PANalvtical CubiX PRO

XRPD data was collected with a PANalytical CubiX PRO machine in θ- 2 <9 configuration over the scan range 2° to 40° 2 θ 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 A . 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. Example IE

a) tert-Butyl 4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9-diazaspiro[5.5]undecane-9- carboxylate

A mixture of tert-butyl l-oxa-4,9-diazaspiro[5.5]undecane-9-carboxylate hydrochloride (limiting reagent) and 2-isopropylthiazole-4-carboxylic acid (1.1 molar equivalents) was suspended in 2-MeTHF (10 volumes) and cooled to 10-15 ⁰C.

Triethylamine (7.2 molar equivalents) was added in portions at 10-15 ⁰C. The thick suspension was cooled to 5-10 ⁰C and T3P (1.3 molar equivalents of a 1.57M solution in THF) was added dropwise at 5-10 ⁰C over 0.5hr. The reaction mixture was allowed to warm to ambient temperature (35min) and stirred for 2.5hr. The mixture was diluted with water (10 volumes, 5°C exotherm) and the mixture vigourously stirred ; the aqueous (pHlO) was separated and extracted with 2-MeTHF (2x2 volumes) . The combined organics were washed with saturated aq sodium bicarbonate soln (2 volumes) and water (2x2 volumes). The organic phase was evaporated and azeotroped with MeCN (2x2 volumes) to give a brown gum, which was dried at 35°C in vacuo for 24hr. Yield: 89% of theoretical.

¹H NMR (300 MHz, DMSO) δ 8.03 (s, IH), 3.76 - 3.44 (m, 8H), 3.37 - 3.25 (m, 3H), 3.16 - 3.00 (m, 2H), 1.76 - 1.66 (m, 2H), 1.39 (s, 9H), 1.33 (d, J = 6.9 Hz, 6H) b) (2-Isopropylthiazol-4-yl)(l-oxa-4,9-diazaspiro[5.5]undecan-4-yl)methanone, trifluoroacetate salt

To a solution of tert-butyl 4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro[5.5]undecane-9-carboxylate (limiting reagent) (step a) in DCM (5 volumes) was added trifluoroacetic acid (13.4 molar equivalents) in portions at 10-15 ⁰C. The solution was allowed to warm to ambient and stirred for 16hr. Solvent was evaporated and the residue was dissolved in diethyl ether (10 volumes). The solution was cooled in ice and scratched to give precipitation; the slurry was stirred at 0°-10°C for 0.5hr then filtered cold. The cake was washed with diethyl ether (5 volumes) and dried in vacuo at 35°C to give a beige coloured powder. Yield: 91% of theoretical.

¹H NMR (300 MHz, DMSO) δ 8.70 - 8.37 (m, 2H), 8.05 (s, IH), 3.82 - 3.52 (m, 6H), 3.42 - 3.25 (m, IH), 3.19 - 3.07 (m, 2H), 3.05 - 2.90 (m, 2H), 2.02 - 1.91 (m, 2H), 1.74 - 1.54 (m, 2H), 1.35 (d, J= 6.7 Hz, 6H)

c) 4-Bromo-l-fluoro-2-(2-methoxyvinyl)benzene (mixture of cis and trans isomers)

Potassiu m tert-butoxide (1.2 molar equivalents) was added portionwise, over 30 minutes, to a stirred suspension of (methoxymethyl)triphenylphosphonium chloride (1.3 molar equivalents) in THF (2.5 volumes) at -3⁰C (+ or - 2⁰C). A deep red colour developed.

The temperature was ramped to 18⁰C over 0.5 hour and the reaction stirred for a further 1.5 hours. 5-Bromo-2-fluorobenzaldehyde (limiting reagent) as a solution in THF (5 volumes) was then added over 90 minutes, such that the temperature of the reaction did not exceed 24⁰C. The reaction was then stirred for 2 hours at room temperature. The reaction mixture was poured into saturated aqueous ammonium chloride solution (10 volumes) and extracted with t-BME (2x 2.5 volumes). The combined extracts were washed with brine (3x 2 volumes), dried (Na2SC>4), filtered and evaporated in vacuo. The oily residue was extracted with ώo-hexane (8x 0.5 volumes). Combined ώo-hexane extracts were filtered, then washed with glacial acetic acid (2 volumes) and 50% glacial acetic acid in water (2 volumes). After drying (Na2SC>4) and filtering, the solvent was removed on a rotary evaporator to give a pale orange oil. Yield was 90% of theoretical. Cώ-isomer: ¹H NMR (300 MHz, D6-DMSO) δ 8.07 (d, J= 9.4 Hz, IH), 7.41 - 7.39 (m, IH), 7.13 (q, J= 1.8 Hz, IH), 6.54 (d, J= 7.1 Hz, IH), 5.31 (d, J= 7.1 Hz, IH), 3.83 (s, 3H)

Trans-isomer. ¹H NMR (300 MHz, D6-DMSO) δ 7.73 - 7.68 (m, IH), 7.44 (d, J= 12.9 Hz, IH), 7.41 - 7.39 (m, IH), 7.18 - 7.09 (m, IH), 5.79 (d, J= 12.9 Hz, IH), 3.67 (s, 3H) d) 4-Fluoro-3-(2-methoxyvinyl)benzaldehyde

4-Bromo-l-fluoro-2-(2-methoxyvinyl)benzene (limiting reagent) (step c) was dissolved in

2-methyltetrahydrofuran (5 volumes) and the solution cooled to -1O⁰C. Iso- propylmagnesium chloride (0.37 molar equivalents of a 2M solution in THF) was added dropwise, keeping the temperature at -7⁰C (+ or - 2⁰C), over about 30 minutes, followed by Butyllithium (0.74 molar equivalents of a 1.5M solution in hexane), controlling the temperature at -6⁰C (+ or - 2 ⁰C), over about 1 hour. Stirred for 45 minutes then added to a solution of 4-formylmorpholine (2 molar equivalents) in 2-methyltetrahydrofuran (7 volumes) at -5⁰C. This addition took about 90 minutes. The reaction was then stirred for

1 hour, during which time the internal temperature rose to 5⁰C. The reaction mixture was poured into saturated aq. ammonium chloride solution (10 volumes) and extracted with t- BME (2x 3 volumes). Combined extracts were washed with sat'd aq. ammonium chloride (until the pH of the washing was approx 6) (2x 3 volumes), water (5 volumes), dried (Na2SO4), filtered and evaporated to give a mobile orange oil.

The oil was chromatographed on silica using 5% ethyl acetate in ώo-hexane as eluent. Yield was 80% of theoretical.

Trans-isomer. ¹H NMR (400 MHz, CDCL₃) δ 9.93 (s, IH), 7.83 (d, J= 9.5 Hz,

IH), 7.66 - 7.61 (m, IH), 7.25 (d, J= 13.1 Hz, IH), 7.17 (d, J= 10.3 Hz, IH), 5.87 (d, J = 13.1 Hz, IH), 3.74 (s, 3H) Cώ-isomer: ¹H NMR (400 MHz, CDCL₃) δ 9.95 (s, IH), 8.57 (d, J= 9.5 Hz, IH), 7.70 - 7.66 (m, IH), 7.12 (d, J= 11.5 Hz, IH), 6.34 (d, J= 7.2 Hz, IH), 5.48 (d, J= 6.9 Hz, IH), 3.86 (s, 3H)

e) (9-(4-Fluoro-3-(2-methoxyvinyl)benzyl)- 1 -oxa-4,9-diazaspiro[5.5]undecan-4-yl)(2- isopropylthiazol-4-yl)methanone

To a suspension of 4-fluoro-3-(2-methoxyvinyl)benzaldehyde (limiting reagent) (step d) and (2-isopropylthiazol-4-yl)(l-oxa-4,9-diazaspiro[5.5]undecan-4-yl)methanone trifluoroacetate (1.05 molar equivalents) (step b) in 2-methyltetrahydrofuran (25 volumes) was added triethylamine (1.73 molar equivalents) in one portion (4°C exotherm). The mixture was stirred for 0.5hr; sodium triacetoxyborohydride (1.5 molar equivalents) was then added in one portion (no exotherm) and the resultant solution was stirred for 16hr. Saturated aq NaHCO_β (25 volumes) was added and the mixture vigourously stirred. The organic phase was separated and the aqueous (pH8) was extracted with 2-MeTHF

(10OmL); the combined organics were washed with water (2x50mL) and dried (Na2SC>4), filtered and evaporated to give a dark oil. Yield: 78% of theoretical.

¹H NMR (300 MHz, D6-DMSO) mixture of cis and trans isomers: δ 7.97 (s, IH), 7.86 - 7.79 (m, 0.5H), 7.34 - 7.20 (m, IH), 7.07 - 6.98 (m, 2H), 6.41 (d, J = 7.1 Hz, 0.5H), 5.82 (d, J = 12.9 Hz, 0.5H), 5.32 (d, J = 6.9 Hz, 0.5H), 3.77 (s, 0.5H), 3.66 (s,

2.5H), 3.42 - 3.19 (m, 7H), 2.41 - 2.26 (m, 4H), 1.76 - 1.63 (m, 3H), 1.59 - 1.45 (m, 3H), 1.35 (d, J = 6.7 Hz, 6H)

f) (R)-7-(2-(2-Fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one

A solution of (9-(4-fluoro-3-(2-methoxyvinyl)benzyl)-l-oxa-4,9- diazaspiro[5.5]undecan-4-yl)(2-isopropylthiazol-4-yl)methanone (limiting reagent) (step e) in THF (5 volumes) was treated with water (2.5 volumes) followed by cone, hydrochloric acid (6 molar equivalents) . The solution was heated at 55-60 ⁰C for 1.5hr; the solution was cooled to ambient and diluted with water (2.5 volumes). The THF was removed by evaporation and the aqueous residue was added to a stirred mixture of sodium bicarbonate (7 molar equivalents), water (5 volumes) and DCM (10 volumes).

The organic was separated and the aqueous (pH8) was extracted with DCM (5 volumes). The combined organic was washed with satd aq NaHCO₃ soln (5 volumes), water (2x5 volumes), dried (sodium sulphate) and filtered. The filtrate was diluted with MeOH (10 volumes) and the DCM evaporated at 35°C/405mbar. The methanol solution of aldehyde was treated with acetic acid (2 molar equivalents) and added to (R)-7-(2-amino- l-hydroxyethyl)-4-hydroxybenzo[d]thiazol-2(3H)-one hydrochloride (1 molar equivalent) in one portion; the mixture was cooled to 0-5⁰C and stirred for 5 min to give a solution. To this was added sodium cyanoborohydride (1.5 molar equivalents) in one portion and the mixture was stirred at 5°C for 0.5hr then warmed to ambient and stirred for 0.5hr. Solvent was evaporated and the residue was partitioned between 2-MeTHF (10 volumes) and satd NaHCO3 soln (5 volumes); the organics were washed with 20% brine soln (5 volumes), dried (Na₂SO₄), filtered and evaporated. Gave a yellow foam which was slurried in ethyl acetate (13 volumes) and the resultant solid was collected by filtration and dried in vacuo. Gave a pale yellow solid which was purified on silica (20 times weight of crude reaction mixture), eluent DCM/10-15% MeOH/1-1.5% ammonia.

Yield: 29% of theoretical.

m/z 670 (M+H)⁺ (APCI) ¹H NMR (300 MHz, D6-DMSO) δ 7.99 (s, IH), 7.23 - 7.02 (m, 3H), 6.84 (d, J= 8.3 Hz, IH), 6.69 (d, J= 8.3 Hz, IH), 4.58 (q, J= 4.1 Hz, IH), 3.75 - 3.24 (br m, 10H), 2.80 - 2.59 (m, 6H), 2.41 - 2.11 (m, 7H), 1.76 - 1.41 (m, 4H), 1.34 (d, J= 6.9 Hz, 6H)

The following abbreviations have been used:

DMF Λ/,Λ/-dimethylformamide

DMSO dimethylsulphoxide

THF tetrahydrofuran

DMA JV,iV-dimethylacetamide

DCM dichloromethane

TBME tert-butyl methyl ether

Example 2

Evaluation of compound activity on intra-alveolar neutrophil migration after aerosol challenge with lipopolysaccharride (LPS) in rats

LPS challenge in rats causes an influx of inflammatory cells into the lungs. Rats are challenged either with an aerosol of 0.9% w/v saline or 0.1-0.5 mg/mL LPS in 0.9% saline for 30 min or an intratracheal dose of 0.1-10μg/kg. This is repeated up to 8 times according to the experimental protocol. Rats are dosed with vehicle, standard compound or test compound by the appropriate route and frequency at various time points before and after challenge depending upon the experimental protocol. Test compound groups may either be the same compound at different doses or single doses of different compounds or a combination of the two. Test compounds are given by intraperitoneal, intravenous or subcutaneous injection or by inhalation or intratracheal administration.

The rats are euthanized at various time points after challenge depending upon the nature of the study, but typically 4hr after LPS challenge with ImL pentobarbitone sodium. A tracheotomy is performed and a cannula inserted. The airway is then lavaged using 3 mL sterile PBS at room temperature. The PBS is left in the airway for 10 seconds before being removed. The PBS containing cells is placed into a 15 mL centrifuge tube on ice. This process is repeated three times.

An aliquot of BAL fluid is removed and counted on Sysmex (Sysmex UK, Milton

Keynes). Cytospin slides are prepared by adding a 100 μl aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin3 operated at 700 rpm for 5 min. Slides are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells (mononuclear cells included monocytes, macrophages and lymphocytes) and are expressed as a percentage of the total count.

Example 3

Evaluation of lung function in anaesthetised guinea-pigs

Male Dunkin-Hartley guinea-pigs (300-60Og) are weighed and dosed with either vehicle or compound in an appropriate vehicle according to the experimental protocol via the intratracheal route under recoverable gaseous anaesthesia (5% halothane in oxygen).

Following dosing, the animals are administered supplemental oxygen and monitored until full recovery. Typically a dose volume of 0.5 mL/kg is used for the intratracheal route. In a dose response study, animals are dosed with compound or vehicle two hours prior to the administration of bronchoconstrictor agent (e.g. histamine or methacholine). Test compound groups could either be the same compound at different doses or single doses of different compounds or a combination of the two.

The guinea-pigs are anaesthetised with pentobarbitone (1 mL/kg of 60 mg/mL solution intraperitoneally) 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 bronchoconstrictor agent or maintenance anaesthetic (0.1 mL of pentobarbitone solution, 60 mg/mL, as required).

The animals are then 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 CmH₂O is applied. Respiratory resistance is measured using the Flexivent "snapshot" facility (1 second duration, 1 Hz frequency). Once stable baseline resistance value has been obtained the animals are given histamine

dihydrochloride or methacholine in ascending doses (histamine; 0.5, 1, 2, 3 and 5μg/kg, i.v., methacholine; 3, 10 and 30 μg/kg, i.v.) at approximately 4-minute intervals via the jugular catheter. After each administration of histamine or methacholine, 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 a compound is calculated at each dose of histamine or methacholine as follows:

% changeR_veh - % changeR_cmpd

% bronchoprotection =^■

% changeR_V •_ιeh

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

Example 4

Evaluation of Compounds on Antigen induced Eosinophilia in Ovalbumin Sensitised Brown Norway Rats

On day 0 of the study Brown Norway rats are given a subcutaneous injection of 500 μg ovalbumin adsorbed onto 100 mg aluminium hydroxide in 0.4 mL saline in two distinct sites, approximately 0.2 mL per site. Day 14 and 15 following sensitisation the rats are challenged with aerosolised ovalbumin for 15 minutes. The rats are placed in groups of 10 in an acrylic box (internal dimensions 320mm wide x 320mm deep x 195 mm high, 2OL volume). 8mL of 10 mg/mL ovalbumin in 0.9% saline, or 0.9% saline alone, is placed in each of two jet nebulizers (Sidestream®, Profile Respiratory Systems Ltd.). Compressed air at 6 L/min is passed through each nebulizer and the output of the nebulizers is passed into the box containing the rats.

Rats are dosed via the appropriate route with vehicle, standard compound or test compound at various time points before and after challenge depending upon the experimental protocol. Rats are euthanised with 0.5 mL pentobarbitone sodium (Euthatal) intraperitoneally at various times after challenge. A tracheotomy is performed and the trachea cannulated. The airway is then lavaged using 3 mL sterile PBS at room

temperature. The PBS is left in the airway for 10 seconds before being removed. The PBS containing cells is placed into a 15 mL centrifuge tube on ice. This process is repeated three times. The final volume recovered is recorded. An aliquot of BAL fluid is removed and counted using a Sysmex (Sysmex UK, Milton Keynes).

Cytospin slides are prepared by adding a 100 μl aliquot of BAL fluid into cytospin funnels in a Shandon Cytospin 3 operated at 700 rpm for 5 min. Slides are stained on the Hema-Tek-2000 automatic slide stainer, using Wright-Giemsa stain and typically, 200 cells are counted under a microscope. Cells are classified as eosinophils, neutrophils and mononuclear cells. Mononuclear cells included monocytes, macrophages and lymphocytes.

Example 5

Evaluation on the effect of compound on lung function and BAL-neutrophilia following acute smoke exposure in the mouse

BALB/c or C57BL6/J mice undergo whole body exposure to main stream smoke (50 min/12 cigarettes) and fresh air once or twice a day for 1-9 days. Mice are dosed via the appropriate route with vehicle, standard compound or test compound at various time points before and after challenge depending upon the experimental protocol. On the final day of the experiment, mice are either killed with euthatal 0.2 ml i.p. and broncho-aveolar lavage fluid obtained for analysis of white blood cell infiltration (as described above) or lung function is assessed using a Flexivent System (SCIREQ, Montreal, Canada). Alternatively lung mechanics are measured using a forced manoeuvres system (EMMS).

Mice are anaesthetised with pentobarbitone (1/lOdilution at a dose volume of

1 mL/kg intraperitoneally). The trachea is cannulated and the animal transferred to the Flexivent System where they are ventilated (quasi-sinusoidal ventilation pattern) at a rate of 150 breath/min and a tidal volume of 10 ml/kg in order to measure airways resistance. Respiratory resistance is measured using the Flexivent "snapshot" facility (1 second duration, 1 Hz frequency). Mice are euthanised with approximately 0.5mL pentobarbitone sodium (Euthatal) intravenously after the completion of the lung function measurements. Example 6

Evaluation of bronchodilator activity in the guinea-pig isolated tracheal ring preparation.

Guinea-pigs (300-60Og) are killed by cervical dislocation and the trachea removed. After clearing the adherent connective tissue, the trachea is cut into four ring segments (2-3 cartilage rings in width) and suspended in 10ml organ baths containing modified Krebs' solution (gassed with 5% CO₂, 95% O₂ at 37°C). The tracheal rings are attached to an isometric force transducer for the measurement of isometric tension. The tissues are washed and a force of Ig was applied to each tissue. Protocol A: The rings are

precontracted with methacholine (1 μM) and a cumulative (10^"9M - 10^"5M) isoprenaline concentration effect curve is constructed. Responses are expressed as a percentage relaxation of the methacholine induced contraction. The rings are washed and a second concentration of methacholine (lμM) is added. Once the contraction has reached a plateau isoprenaline or the compound under investigation is added until a maximum effective dose is reached. Data are collected using the ADInstruments chart4forwindows software, which measures the maximum tension generated at each concentration of agonist and the response expressed as percentage relaxation.

Protocol B: A cumulative methacholine concentration effect curve was constructed and then the tissue was washed. Vehicle or test compound was added to the tissue and allowed to equilibrate. A second extended cumulative concentration response curve to

methacholine was constructed. Data are collected using the ADInstruments

chart4forwindows software, which measures the maximum tension generated at each concentration of agonist and the response expressed as percentage relaxation. Results were expressed as percentage of the maximum response measured in the first curve. Then, pA₅₀ values were calculated from the first (untreated) and second (compound treated) methacholine concentration response curves and a potency value, pA₂, was calculated.

Example 7

Inhibition of lipopolysaccharride (LPS)-induced TNFα production in human peripheral blood mononuclear cells.

Human isolated peripheral blood mononuclear cells (PBMCs) are pre-incubated with a range of concentrations of the test compound, alone or in the presence of a range of concentrations of a second compound with a distinct pharmacological activity for 18 hours at 37°C. After the pre-incubation period, the cells are then incubated with LPS (lμg/mL) for 4 hours at 37°C to induce TNFα production. The total assay volume is 200 μL. At the end of the incubation period, 25 μL of the culture supernatant is analysed to quantify the TNFα released using a Flourescence-linked immunosorbance assay (FLISA).

Fluorescence levels are read on an FMAT plate reader. Inhibition curves are fitted using a 4-parameter logistic equation in a non-linear curve fitting routine and activity is expressed as pIC₅o- Example 8

Inhibition of lipopolysaccharride (LPS)-induced TNFα production in human peripheral blood mononuclear cells. Human isolated peripheral blood mononuclear cells (PBMCs) were pre-incubated with a range of concentrations of the dual muscarinic antagonist beta₂ adrenoceptor agonist (compound A), alone or in the presence of a range of concentrations of a second compound for 30 min at 37°C. After the pre-incubation period, LPS (lμg/mL) was added to the cells, which were then incubated with for 18 hours at 37°C to induce TNFα production. The total assay volume was 100 μL. At the end of the incubation period, 25 μL of the culture supernatant was analysed to quantify the TNFα released using a Flourescence-linked immunosorbance assay (FLISA). Fluorescence levels were read on an FMAT plate reader. Data were expressed as a percentage inhibition of LPS-induced TNFα release

(mean±s.e.mean, n = 3-4 donors).

The percentage inhibition achieved for combinations of compound A with compound B (Figure 4) is shown in Table 1. The percentage inhibition achieved for combinations of compound A with compound C (Figure 5) is shown in Table 2. The percentage inhibition achieved for combinations of compound A with budesonide (Figure 6) is shown in Table 3.

Description of Figures

Figure 1 shows the XRPD pattern of di(lS)-(+)-10-camphorsulfonic acid salt modification A - Example IB

Figure 2 shows the XRPD pattern of fumarate salt modification A - Example 1C

Figure 3 shows the XRPD pattern of fumarate salt modification B - Example ID

Figure 4 shows the effect of the combination of compound A (InM) and compound B (0.1-10OnM) on LPS stimulated TNFα production from human PBMC. Data were expressed as a percentage inhibition of TNFα production (mean ± s.e.mean, n = 3-4).

Figure 5 shows the effect of the combination of compound A (InM) and compound C (0.1-10OnM) on LPS stimulated TNFα production from human PBMC. Data were expressed as a percentage inhibition of TNFα production (mean ± s.e.mean, n = 3-4).

Figure 6 shows the effect of the combination of compound A (InM) and budesonide (1- 10OnM) on LPS stimulated TNFα production from human PBMC. Data were expressed as a percentage inhibition of TNFα production (mean ± s.e.mean, n = 4). Table 1

Effect of the combination of compound A and compound B on LPS stimulated TNFα production from human PBMC compound A (nM) compound B (nM) % Inhibition

1 0 13

0 100 83

1 0.1 19

1 1 42

1 10 73

1 100 88

Table 2

Effect of the combination of compound A and compound C on LPS stimulated TNFα production from human PBMC compound A (nM) compound C (nM) % Inhibition

1 0 20

0 100 27

1 0.1 24

1 1 41

1 10 41

1 100 39 Table 3

Effect of the combination of compound A and budesonide on LPS stimulated TNFα production from human PBMC compound A (nM) budesonide (nM) % Inhibition

1 0 26

0 10 51

1 1 40

1 10 54

1 100 58

Claims

1. A pharmaceutical product comprising, in combination, a first active ingredient which is (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one or a salt thereof, and a second active ingredient selected from:

an Adenosine A2A receptor antagonist;

an anti-infective;

a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist;

an antioxidant;

a CCRl antagonist;

a chemokine antagonist (not CCRl);

a corticosteroid;

a CRTh2 antagonist;

a DPI antagonist;

a formyl peptide receptor antagonist;

a Histone Deacetylase activator;

a chloride channel hCLCAl blocker

an Epithelial sodium channel blocker (ENAC blocker) ).

an Inter-cellular adhesion molecule 1 blocker (ICAM blocker);

an IKK2 kinase inhibitor;

a JNK kinase inhibitor;

a cyclooxygenase inhibitor (COX inhibitor);

a lipoxygenase inhibitor;

a leukotriene receptor antagonist;

a MEK-I kinase inhibitor

a myeloperoxidase inhibitor (MPO inhibitor);

a phosphodiesterase PDE4 inhibitor;

a phosphatidylinositol 3 (PB)-kinase D inhibitor (PI 3 kinase D inhibitor) a peroxisome proliferator activated receptor agonist (PP ARγ agonist);

a protease inhibitor; a p38 inhibitor

a retinoic acid receptor modulator (RAR D modulator)

a Statin;

a thromboxane antagonist; or

a vasodilator.

2. A pharmaceutical product as claimed in claim 1 wherein the first active ingredient is in the form of a salt which is a hydrochloride, hydrobromide, trifluoroacetate, sulphate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulphonate, /?-toluenesulphonate, bisulphate, benzenesulphonate,

ethanesulphonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate, L-tartrate, D-tartrate,, stearate, 2- furoate, 3-furoate, napadisylate (naphthalene- 1, 5 -disulfonate or naphthalene- 1 -(sulfonic acid)-5-sulfonate), edisylate (ethane- 1 ,2-disulfonate or ethane- 1 -(sulfonic acid)-2- sulfonate), isethionate (2-hydroxyethylsulfonate), 2-mesitylenesulphonate, 2- naphthalenesulphonate, 2,5-dichlorobenzenesulphonate, D-mandelate, L-mandelate, cinnamate, benzoate, adipate, esylate, malonate, mesitylate (2-mesitylenesulphonate), napsylate (2-naphthalenesulfonate), camsylate (camphor- 10-sulphonate), formate, glutamate, glutarate, glycolate, hippurate (2-(benzoylamino)acetate), orotate, xylate (p- xylene-2-sulphonate), pamoic (2,2'-dihydroxy-l,r-dinaphthylmethane-3,3'-dicarboxylate), palmitate or furoate.

3. A pharmaceutical composition which comprises a compound of the formula (I), or a pharmaceutically acceptable salt thereof as claimed in claims 1-2, and a pharmaceutically acceptable adjuvant, diluent or carrier.

4. Use of a product according to any one of claims 1-3 in therapy.

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. Use according to claim 5, wherein the respiratory disease is asthma.

8. 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 as defined in claim 1 ;

(b) a (therapeutically effective) dose of a second active ingredient which is as defined in claim 1 ;

to a patient in need thereof.

9. A kit comprising a preparation of a first active ingredient, a preparation of a second active ingredient, both as defined in claim 1, and optionally instructions for the simultaneous, sequential or separate administration of the preparations to a patient in need thereof.

10. A pharmaceutical composition comprising, in admixture, a first active ingredient which is (R)-7-(2-(2-fluoro-5-((4-(2-isopropylthiazole-4-carbonyl)-l-oxa-4,9- diazaspiro [5.5 ]undecan-9-yl)methyl)phenethylamino)- 1 -hydroxy ethyl)-4- hydroxybenzo[d]thiazol-2(3H)-one or a salt thereof, and a second active ingredient which is selected from:

an Adenosine A2A receptor antagonist;

an anti-infective;

a non-steroidal Glucocorticoid Receptor (GR Receptor) Agonist;

an antioxidant;

a CCRl antagonist;

a chemokine antagonist (not CCRl);

a corticosteroid;

a CRTh2 antagonist;

a DPI antagonist; a formyl peptide receptor antagonist;

a Histone Deacetylase activator;

a chloride channel hCLCAl blocker

an Epithelial sodium channel blocker (ENAC blocker) ).

an Inter-cellular adhesion molecule 1 blocker (ICAM blocker);

an IKK2 kinase inhibitor;

a JNK kinase inhibitor;

a cyclooxygenase inhibitor (COX inhibitor);

a lipoxygenase inhibitor;

a leukotriene receptor antagonist;

a MEK-I kinase inhibitor

a myeloperoxidase inhibitor (MPO inhibitor);

a phosphodiesterase PDE4 inhibitor;

a phosphatidylinositol 3 (PB)-kinase D inhibitor (PI 3 kinase D inhibitor) a peroxisome proliferator activated receptor agonist (PP ARγ agonist); a protease inhibitor;

a p38 inhibitor

a retinoic acid receptor modulator (RAR D modulator)

a Statin;

a thromboxane antagonist; or

a vasodilator.