WO2008096119A1

WO2008096119A1 - Salts 669

Info

Publication number: WO2008096119A1
Application number: PCT/GB2008/000394
Authority: WO
Inventors: Stephen Connolly
Original assignee: Astrazeneca Ab; Astrazeneca Uk Limited
Priority date: 2007-02-08
Filing date: 2008-02-06
Publication date: 2008-08-14
Also published as: GB0702459D0

Abstract

A pharmaceutically acceptable salt of N-[2-(Diethylamino)ethyl]-N-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-1,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]pro panamide provided it is not the ditrifluoroacetate salt; and the use of such a compound as a medicament (for example in the treatment of respiratory diseases (such as asthma or COPD).

Description

SALTS 669

The present invention concerns new salt forms of N-[2-(Diethylamino)ethyl]-7V-(2-{[2-(4- hydroxy-2-oxo-2,3-dihydro-l,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(3- chlorophenyl)ethoxy]propanamide, compositions comprising such new salt forms, processes for preparing such salt forms, and the use of such salt forms in the treatment of disease states (such as respiratory disease states, for example asthma or COPD).

TV- [2-(Diethy lamino)ethyl]-7V-(2- { [2-(4-hydroxy-2-oxo-2,3 -dihydro- 1 ,3 -benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide free base (in solution) and its ditrifluoroacetate salt are β2 adrenoceptor agonists and are disclosed in PCT/SE2006/000927 (now published as WO 2007/018461). These compounds show at least 10-fold selectivity for β2 adrenoceptor over adrenergic αlD, adrenergic βl and dopamine D2.

The present invention provides a pharmaceutically acceptable salt of /V- [2- (Diethylamino)ethyl]-N-(2- { [2-(4-hydroxy-2-oxo-2,3 -dihydro- 1 ,3 -benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide provided it is not the ditrifluoroacetate salt.

A pharmaceutically acceptable salt includes for example, a hydrochloride, hydrobromide (such as a dihydrobromide), sulphate, phosphate, acetate, fumarate, maleate, citrate, pyruvate, succinate, oxalate, methanesulphonate,p-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 or 2-naphthalenesulphonate. Further salts include D-mandelate or L-mandelate.

A salt of the invention can exist as a solvate (such as a hydrate), and the present invention covers all such solvents. In one particular aspect the present invention provides the dihydrobromide salt of N- [2- (Diethylamino)ethyl]-iV-(2- { [2-(4-hydroxy-2-oxo-2,3 -dihy dro- 1 ,3 -benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide.

In a further aspect the present invention provides a dihydrobromide salt of N- [2- (Diethylamino)ethyl]-iV-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-l,3-benzothiazol-7- yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide having an X-ray powder diffraction (XRPD) pattern containing specific peaks at: 5.1 (±0.1°), 17.5 (±0.1°), 18.5 (±0.1°), 20.6 (±0.1°), 21.0 (±0.1°) and 25.0 (±0.1°) 2Θ.

Alternative salts of N-[2-(Diethylamino)ethyl]-N-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro- l,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide can be prepared by methods known in the art. For example the dihydrobromide can be treated with a base to liberate iV-[2-(Diethylamino)ethyl]-iV-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro- l,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide, and then this can be reacted with an appropriate acid in a suitable solvent (such as an aliphatic alcohol, for example methanol) to produce the desired salt.

The salts of the present invention can be prepared by using or adapting: the methods presented above; the methods presented in the Preparations or Examples below; or, the methods described in the literature.

The salts of theinvention can be used in the treatment of: 1. respiratory tract: 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) or adenovirus; or eosinophilic esophagitis; 2. bone and joints: arthritides associated with or including osteoarthritis/osteoarthrosis, both primary and secondary to, for example, congenital hip dysplasia; cervical and lumbar spondylitis, and low back and neck pain; osteoarthritis; rheumatoid arthritis and Still's disease; seronegative spondyloarthropathies including ankylosing spondylitis, psoriatic arthritis, reactive arthritis and undifferentiated spondarthropathy; septic arthritis and other infection-related arthopathies and bone disorders such as tuberculosis, including Potts' disease and Poncet's syndrome; acute and chronic crystal-induced synovitis including urate gout, calcium pyrophosphate deposition disease, and calcium apatite related tendon, bursal and synovial inflammation; Behcet's disease; primary and secondary Sjogren's syndrome; systemic sclerosis and limited scleroderma; systemic lupus erythematosus, mixed connective tissue disease, and undifferentiated connective tissue disease; inflammatory myopathies including dermatomyositits and polymyositis; polymalgia rheumatica; juvenile arthritis including idiopathic inflammatory arthritides of whatever joint distribution and associated syndromes, and rheumatic fever and its systemic complications; vasculitides including giant cell arteritis, Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa, microscopic polyarteritis, and vasculitides associated with viral infection, hypersensitivity reactions, cryoglobulins, and paraproteins; low back pain; Familial Mediterranean fever, Muckle- Wells syndrome, and Familial Hibernian Fever, Kikuchi disease; drug-induced arthalgias, tendonititides, and myopathies; 3. pain and connective tissue remodelling of musculoskeletal disorders due to injury [for example sports injury] or disease: arthritides (for example rheumatoid arthritis, osteoarthritis, gout or crystal arthropathy), other joint disease (such as intervertebral disc degeneration or temporomandibular joint degeneration), bone remodelling disease (such as osteoporosis, Paget's disease or osteonecrosis), polychondritis, scleroderma, mixed connective tissue disorder, spondyloarthropathies or periodontal disease (such as periodontitis);

4. skin: psoriasis, atopic dermatitis, contact dermatitis or other eczematous dermatoses, and delayed-type hypersensitivity reactions; phyto- and photodermatitis; seborrhoeic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus et atrophica, pyoderma gangrenosum, skin sarcoid, discoid lupus erythematosus, pemphigus, pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitides, toxic erythemas, cutaneous eosinophilias, alopecia areata, male-pattern baldness, Sweet's syndrome, Weber-Christian syndrome, erythema multiforme; cellulitis, both infective and non-infective; panniculitis; cutaneous lymphomas, non-melanoma skin cancer and other dysplastic lesions; drug- induced disorders including fixed drug eruptions;

5. eyes: blepharitis; conjunctivitis, including perennial and vernal allergic conjunctivitis; iritis; anterior and posterior uveitis; choroiditis; autoimmune; degenerative or inflammatory disorders affecting the retina; ophthalmitis including sympathetic ophthalmitis; sarcoidosis; infections including viral, fungal, and bacterial;

6. gastrointestinal tract: glossitis, gingivitis, periodontitis; oesophagitis, including reflux; eosinophilic gastro-enteritis, mastocytosis, Crohn's disease, colitis including ulcerative colitis, proctitis, pruritis ani; coeliac disease, irritable bowel syndrome, and food-related allergies which may have effects remote from the gut (for example migraine, rhinitis or eczema);

7. abdominal: hepatitis, including autoimmune, alcoholic and viral; fibrosis and cirrhosis of the liver; cholecystitis; pancreatitis, both acute and chronic;

8. genitourinary: nephritis including interstitial and glomerulonephritis; nephrotic syndrome; cystitis including acute and chronic (interstitial) cystitis and Hunner's ulcer; acute and chronic urethritis, prostatitis, epididymitis, oophoritis and salpingitis; vulvovaginitis; Peyronie's disease; erectile dysfunction (both male and female);

9. allograft rejection: acute and chronic following, for example, transplantation of kidney, heart, liver, lung, bone marrow, skin or cornea or following blood transfusion; or chronic graft versus host disease;

10. CNS: Alzheimer's disease and other dementing disorders including CJD and nvCJD; amyloidosis; multiple sclerosis and other demyelinating syndromes; cerebral atherosclerosis and vasculitis; temporal arteritis; myasthenia gravis; acute and chronic pain (acute, intermittent or persistent, whether of central or peripheral origin) including visceral pain, headache, migraine, trigeminal neuralgia, atypical facial pain, joint and bone pain, pain arising from cancer and tumor invasion, neuropathic pain syndromes including diabetic, post-herpetic, and HIV-associated neuropathies; neurosarcoidosis; central and peripheral nervous system complications of malignant, infectious or autoimmune processes;

11. other auto-immune and allergic disorders including Hashimoto's thyroiditis, Graves' disease, Addison's disease, diabetes mellitus, idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-IgE syndrome, antiphospholipid syndrome;

12. other disorders with an inflammatory or immunological component; including acquired immune deficiency syndrome (AIDS), leprosy, Sezary syndrome, and paraneoplastic syndromes;

13. cardiovascular: atherosclerosis, affecting the coronary and peripheral circulation; pericarditis; myocarditis, inflammatory and auto-immune cardiomyopathies including myocardial sarcoid; ischaemic reperfusion injuries; endocarditis, valvulitis, and aortitis including infective (for example syphilitic); vasculitides; disorders of the proximal and peripheral veins including phlebitis and thrombosis, including deep vein thrombosis and complications of varicose veins; 14. oncology: treatment of common cancers including prostate, breast, lung, ovarian, pancreatic, bowel and colon, stomach, skin and brain tumors and malignancies affecting the bone marrow (including the leukaemias) and lymphoproliferative systems, such as Hodgkin's and non-Hodgkin's lymphoma; including the prevention and treatment of metastatic disease and tumour recurrences, and paraneoplastic syndromes; and, 15. gastrointestinal tract: Coeliac disease, proctitis, eosinopilic gastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis, microscopic colitis, indeterminant colitis, irritable bowel disorder, irritable bowel syndrome, non-inflammatory diarrhea, food- related allergies which have effects remote from the gut, e.g., migraine, rhinitis and eczema.

Thus, the present invention provides a salt as hereinbefore defined for use in therapy. In a further aspect, the present invention provides the use of a salt as hereinbefore defined in the manufacture of a medicament for use in therapy (for example a respiratory disease state).

In a still further aspect the present invention provides a salt as hereinbefore described for the treatment of a respiratory disease state.

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 disease or condition in question. Persons at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition.

The invention still further provides a method of treating, or reducing the risk of, an inflammatory disease or condition (including a reversible obstructive airways disease or condition) which comprises administering to a patient in need thereof a therapeutically effective amount of a salt as hereinbefore defined.

In particular, the compounds of this invention may be used in the treatment of adult respiratory distress syndrome (ARDS), pulmonary emphysema, bronchitis, bronchiectasis, chronic obstructive pulmonary disease (COPD), asthma and rhinitis.

For the above-mentioned therapeutic uses the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated. For example, the daily dosage of the compound of the invention, if inhaled, may be in the range from 0.05 micrograms per kilogram body weight (μg/kg) to 100 micrograms per kilogram body weight (μg/kg). Alternatively, if the compound is administered orally, then the daily dosage of the compound of the invention may be in the range from 0.01 micrograms per kilogram body weight (μg/kg) to 100 milligrams per kilogram body weight (mg/kg).

A salt of the invention may be used on its own but will generally be administered in the form of a pharmaceutical composition in which the salt (active ingredient) is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

Depending on the mode of administration, the pharmaceutical composition will for example comprise from 0.05 to 99 %w (per cent by weight), such as from 0.05 to 80 %w, for example from 0.10 to 70 %w, and such as from 0.10 to 50 %w, of active ingredient, all percentages by weight being based on total composition.

The present invention also provides a pharmaceutical composition comprising a salt as hereinbefore defined, in association with a pharmaceutically acceptable adjuvant, diluent or carrier.

The invention further provides a process for the preparation of a pharmaceutical composition of the invention which comprises mixing a salt as hereinbefore defined with a pharmaceutically acceptable adjuvant, diluent or carrier.

The pharmaceutical compositions may be administered topically (e.g. to the skin or to the lung and/or airways) in the form, e.g., of a cream, solution, suspension, heptafluoroalkane (HFA) aerosol or dry powder formulation, for example, a formulation in the inhaler device known as the Turbuhaler^®; or systemically, e.g. by oral administration in the form of a tablet, capsule, syrup, powder or granule; or by parenteral administration in the form of a solution or suspension; or by subcutaneous administration; or by rectal administration in the form of a suppository; or transdermally. A dry powder formulation or pressurized HFA aerosol of a salt of the invention may be administered by oral or nasal inhalation. For inhalation, the salt is desirably finely divided. The finely divided salt has, for example, 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 Cg- C₂₀ 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.

A salt of the invention may also be administered by means of a dry powder inhaler. The inhaler may be a single or a multi dose inhaler, and may be a breath actuated dry powder inhaler.

One possibility is to mix the finely divided salt of the invention with a carrier substance, for example, a mono-, di- or polysaccharide, a sugar alcohol, or another polyol. A suitable carrier is, for example, a sugar, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol; or starch. Alternatively the finely divided salt 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.

For oral administration the salt of the invention may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If a coated tablet is required, the core, prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

For the preparation of a soft gelatine capsule, the salt of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. A hard gelatine capsule may contain granules of the salt using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the compound of the invention may be filled into hard gelatine capsules.

A liquid preparation for oral application may be in the form of a syrup or suspension, for example, a solution containing the salt of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such a liquid preparation may contain a colouring agent, flavouring agent, saccharine and/or carboxymethylcellulose as a thickening agent or other excipient known to those skilled in art.

A salt of the invention may also be administered in conjunction with another compound used for the treatment of one or more of the above conditions.

The invention therefore further relates to combination therapies wherein a salt of the invention or a pharmaceutical composition or formulation comprising a salt of the invention, is administered concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of one or more of the conditions listed.

In particular, for the treatment of the inflammatory diseases such as (but not restricted to) rheumatoid arthritis, osteoarthritis, asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), psoriasis, and inflammatory bowel disease, a salt of the invention may be combined with one of the following agents: non-steroidal anti-inflammatory agents (hereinafter NSAIDs) including non-selective cyclo-oxygenase COX-I / COX-2 inhibitors whether applied topically or systemically (such as piroxicam, diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such as mefenamic acid, indomethacin, sulindac, azapropazone, pyrazolones such as phenylbutazone, salicylates such as aspirin); selective COX-2 inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib, lumarocoxib, parecoxib and etoricoxib); cyclo-oxygenase inhibiting nitric oxide donors (CINODs); glucocorticosteroids (whether administered by topical, oral, intramuscular, intravenous, or intra-articular routes); methotrexate; leflunomide; hydroxychloroquine; d-penicillamine; auranofin or other parenteral or oral gold preparations; analgesics; diacerein; intra-articular therapies such as hyaluronic acid derivatives; and nutritional supplements such as glucosamine.

The present invention still further relates to the combination of a salt of the invention together with a cytokine or agonist or antagonist of cytokine function, (including agents which act on cytokine signalling pathways such as modulators of the SOCS system) including alpha-, beta-, and gamma-interferons; insulin-like growth factor type I (IGF-I); interleukins (IL) including ILl to 17, and interleukin antagonists or inhibitors such as anakinra; tumour necrosis factor alpha (TNF-α) inhibitors such as anti-TNF monoclonal antibodies (for example infliximab; adalimumab, and CDP-870) and TNF receptor antagonists including immunoglobulin molecules (such as etanercept) and low-molecular- weight agents such as pentoxyfylline.

In addition the invention relates to a combination of a salt of the invention with a monoclonal antibody targeting B-Lymphocytes (such as CD20 (rituximab), MRA-aIL16R and T-Lymphocytes, CTLA4-Ig, HuMax 11-15).

The present invention still further relates to the combination of a salt of the invention, with a modulator of chemokine receptor function such as an antagonist of CCRl , CCR2,

CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRlO and CCRI l (for the C-C family); CXCRl, CXCR2, CXCR3, CXCR4 and CXCR5 (for the C-X-C family) and CX₃CRl for the C-X₃-C family.

' The present invention further relates to the combination of a salt of the invention, with an inhibitor of matrix metalloprotease (MMPs), i.e., the stromelysins, the collagenases, and the gelatinases, as well as aggrecanase; especially collagenase-1 (MMP-I), collagenase-2 (MMP-8), collagenase-3 (MMP- 13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-IO), and stromelysin-3 (MMP-11) and MMP-9 and MMP- 12, including agents such as doxycycline.

The present invention still further relates to the combination of a salt of the invention, and a leukotriene biosynthesis inhibitor, 5 -lipoxygenase (5-LO) inhibitor or 5 -lipoxygenase activating protein (FLAP) antagonist such as; zileuton; ABT-761; fenleuton; tepoxalin; Abbott-79175; Abbott-85761; aiV-(5-substituted)-thiophene-2-alkylsulfonamide; 2,6-di- tert-butylphenolhydrazones; a methoxytetrahydropyrans such as Zeneca ZD-2138; the compound SB-210661; a pyridinyl-substituted 2-cyanonaphthalene compound such as L- 739,010; a 2-cyanoquinoline compound such as L-746,530; or an indole or quinoline compound such as MK-591, MK-886, and BAY x 1005.

The present invention further relates to the combination of a salt of the invention, and a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4 selected from the group consisting of the phenothiazin-3-ls such as L-651,392; amidino compounds such as CGS-25019c; benzoxalamines such as ontazolast; benzenecarboximidamides such as BIIL 284/260; and compounds such as zafirlukast, ablukast, montelukast, pranlukast, verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP 45715A), and BAY x 7195.

The present invention still further relates to the combination of a salt of the invention, and a phosphodiesterase (PDE) inhibitor such as a methylxanthanine including theophylline and aminophylline; a selective PDE isoenzyme inhibitor including a PDE4 inhibitor an inhibitor of the isoform PDE4D, or an inhibitor of PDE5.

The present invention further relates to the combination of a salt of the invention, and a histamine type 1 receptor antagonist such as cetirizine, loratadine, desloratadine, fexofenadine, acrivastine, terfenadine, astemizole, azelastine, levocabastine, chlorpheniramine, promethazine, cyclizine, or mizolastine; applied orally, topically or parenterally. The present invention still further relates to the combination of a salt of the invention, and a proton pump inhibitor (such as omeprazole) or a gastroprotective histamine type 2 receptor antagonist.

The present invention further relates to the combination of a salt of the invention, and an antagonist of the histamine type 4 receptor.

The present invention still further relates to the combination of a salt of the invention, and an alpha- l/alpha-2 adrenoceptor agonist vasoconstrictor sympathomimetic agent, such as propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride or ethylnorepinephrine hydrochloride.

The present invention further relates to the combination of a salt of the invention, and an anticholinergic agents including muscarinic receptor (Ml, M2, and M3) antagonist such as atropine, hyoscine, glycopyrrrolate, ipratropium bromide, tiotropium bromide, oxitropium bromide, pirenzepine or telenzepine.

The present invention further relates to the combination of a salt of the invention, and a chromone, such as sodium cromoglycate or nedocromil sodium.

The present invention still further relates to the combination of a salt of the invention, with a glucocorticoid, such as flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide or mometasone furoate.

The present invention further relates to the combination of a salt of the invention, with an agent that modulates a nuclear hormone receptor such as PPARs.

The present invention still further relates to the combination of a salt of the invention, together with an immunoglobulin (Ig) or Ig preparation or an antagonist or antibody modulating Ig function such as anti-IgE (for example omalizumab). The present invention further relates to the combination of a salt of the invention, and another systemic or topically-applied anti-inflammatory agent, such as thalidomide or a derivative thereof, a retinoid, dithranol or calcipotriol.

The present invention still further relates to the combination of a salt of the invention, and combinations of aminosalicylates and sulfapyridine such as sulfasalazine, mesalazine, balsalazide, and olsalazine; and immunomodulatory agents such as the thiopurines, and corticosteroids such as budesonide.

The present invention further relates to the combination of a salt of the invention, together with an antibacterial agent such as a penicillin derivative, a tetracycline, a macrolide, a beta-lactam, a fluoroquinolone, metronidazole, an inhaled aminoglycoside; an antiviral agent including acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir, amantadine, rimantadine, ribavirin, zanamavir and oseltamavir; a protease inhibitor such as indinavir, nelfinavir, ritonavir, and saquinavir; a nucleoside reverse transcriptase inhibitor such as didanosine, lamivudine, stavudine, zalcitabine or zidovudine; or a non-nucleoside reverse transcriptase inhibitor such as nevirapine or efavirenz.

The present invention still further relates to the combination of a salt of the invention, and a cardiovascular agent such as a calcium channel blocker, a beta-adrenoceptor blocker, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin-2 receptor antagonist; a lipid lowering agent such as a statin or a fibrate; a modulator of blood cell morphology such as pentoxyfylline; thrombolytic, or an anticoagulant such as a platelet aggregation inhibitor.

The present invention further relates to the combination of a salt of the invention, and a CNS agent such as an antidepressant (such as sertraline), an anti-Parkinsonian drug (such as deprenyl, L-dopa, ropinirole, pramipexole, a MAOB inhibitor such as selegine and rasagiline, a comP inhibitor such as tasmar, an A-2 inhibitor, a dopamine reuptake inhibitor, an NMDA antagonist, a nicotine agonist, a dopamine agonist or an inhibitor of neuronal nitric oxide synthase), or an anti- Alzheimer's drug such as donepezil, rivastigmine, tacrine, a COX-2 inhibitor, propentofylline or metrifonate.

The present invention still further relates to the combination of a salt of the invention, and an agent for the treatment of acute or chronic pain, such as a centrally or peripherally- acting analgesic (for example an opioid or derivative thereof), carbamazepine, phenytoin, sodium valproate, amitryptiline or other anti-depressant agents, paracetamol, or a nonsteroidal anti-inflammatory agent.

The present invention further relates to the combination of a salt of the invention, together with a parenterally or topically-applied (including inhaled) local anaesthetic agent such as lignocaine or a derivative thereof.

A salt of the present invention, can also be used in combination with an anti-osteoporosis agent including a hormonal agent such as raloxifene, or a biphosphonate such as alendronate.

The present invention still further relates to the combination of a salt of the invention, together with a: (i) tryptase inhibitor; (ii) platelet activating factor (PAF) antagonist; (iii) interleukin converting enzyme (ICE) inhibitor; (iv) IMPDH inhibitor; (v) adhesion molecule inhibitors including VLA-4 antagonist; (vi) cathepsin; (vii) kinase inhibitor such as an inhibitor of tyrosine kinase (such as Btk, Itk, Jak3 or MAP, for example Gefitinib or Imatinib mesylate), a serine / threonine kinase (such as an inhibitor of a MAP kinase such as p38, JNK, protein kinase A, B or C, or IKK), or a kinase involved in cell cycle regulation (such as a cylin dependent kinase); (viii) glucose-6 phosphate dehydrogenase inhibitor; (ix) kinin-B.subl. - or B.sub2. -receptor antagonist; (x) anti-gout agent, for example colchicine; (xi) xanthine oxidase inhibitor, for example allopurinol; (xii) uricosuric agent, for example probenecid, sulfinpyrazone or benzbromarone; (xiii) growth hormone secretagogue; (xiv) transforming growth factor (TGFβ); (xv) platelet-derived growth factor (PDGF); (xvi) fibroblast growth factor for example basic fibroblast growth factor (bFGF); (xvii) granulocyte macrophage colony stimulating factor (GM-CSF); (xviii) capsaicin cream; (xix) tachykinin NK. sub 1. or NK.sub3. receptor antagonist such as NKP- 608C, SB-233412 (talnetant) or D-4418; (xx) elastase inhibitor such as UT-77 or ZD-0892; (xxi) TNF-alpha converting enzyme inhibitor (TACE); (xxii) induced nitric oxide synthase (iNOS) inhibitor; (xxiii) chemoattractant receptor-homologous molecule expressed on TH2 cells, (such as a CRTH2 antagonist); (xxiv) inhibitor of P38; (xxv) agent modulating the function of Toll-like receptors (TLR), (xxvi) agent modulating the activity of purinergic receptors such as P2X7; (xxvii) inhibitor of transcription factor activation such as NFkB, API, or STATS; or, (xxviii) a glucocorticoid receptor agonist.

General Preparative Methods

¹H NMR spectra were recorded on a Varian Inova 400 MHz or a Varian Mercury-VX 300 MHz instrument. The central peaks of chloroform-fif (5_H 7.27 ppm), dimethylsulfoxide-flk (5_H 2.50 ppm), acetonitrile-^ (5_H 1.95 ppm) or methanol-^ (δπ 3.31 ppm) were used as internal references. Column chromatography was carried out using silica gel (0.040-0.063 mm, Merck). Unless stated otherwise, starting materials were commercially available. All solvents and commercial reagents were of laboratory grade and were used as received.

The following method was used for LC/MS analysis:

Instrument Agilent 1100; Column Waters Symmetry 2.1 x 30 mm; Mass APCI; Flow rate 0.7 ml/min; Wavelength 254 nm; Solvent A: water + 0.1% TFA; Solvent B: acetonitrile + 0.1% TFA ; Gradient 15-95%/B 8 min, 95% B 1 min.

Analytical chromatography was run on a Symmetry C]₈-column, 2.1 x 30 mm with 3.5 μm particle size, with acetonitrile/water/0.1% trifluoroacetic acid as mobile phase in a gradient from 5% to 95% acetonitrile over 8 minutes at a flow of 0.7 ml/min.

INSTRUMENT DETAILS:

o XRPD (X-ray powder diffraction) - Philips X-Pert MPD machine in θ - θ configuration over the scan range 2° to 40° 2Θ with 100-second exposure per 0.03° increment. The X-rays were generated by a copper long-fine focus tube operated at

45kV and 4OmA. The wavelengths of the copper X-rays were 1.5405A (K_αi) and 1.5444 A (K_α2). 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. XRPD data are presented in the tables below, and reflection angle (°2Θ) and D-spacing (A) data (bracketed) are provided.

o DSC (Differential Scanning Calorimetry) thermograms were measured using a TA QlOOO machine, with aluminium pans and pierced lids. The sample weights varied between 1 to 5mg. The procedure was carried out under a flow of nitrogen gas

(50ml/min) and the temperature studied from 25 to 300°C at a constant rate of temperature increase of 10°C per minute.

o GVS profiles were measured using a Dynamic Vapour Sorption DVS-I instrument. The solid sample ca. 4-10mg was placed into a glass vessel and the weight of the sample was recorded during a dual cycle step method (40 to 90 to 0 to 90 to 0% relative humidity (RH)₅ in steps of 10% RH).

o Ion-Stoichiometry — was measured using a KOH gradient and a Dionex AS 11 column with electrochemical detection and a Dionex IC3000 instrument.

o Solution ¹H NMR spectra were recorded using a Varian Unity Inova spectrometer at a proton frequency of 400 MHz.

The abbreviations or terms used in the examples have the following meanings: SCX: Solid phase extraction with a sulfonic acid sorbent

HPLC: High performance liquid chromatography DMF: ΛζiV-Dimethylformamide

The β₂-adrenoceptor agonists and the intermediates used in their preparation are herein named, based upon the structures depicted, using the IUPAC NAME, ACD Labs Version 8 naming package. The following Example illustrates the invention.

Example 1 N-[2-(Diethylamino)ethyI]-iV-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-l,3-benzothiazol-7- yl)ethyl]amino}ethyI)-3-[2-(3-chlorophenyl)ethoxy]propanamide dihydrobromide

a) tert-Butyl 3-[2-(3-chlorophenyl)ethoxy]propanoate

2-(3-chlorophenyl)ethanol (20 g) was treated with benzyltrimethylammonium hydroxide (Triton B^®) (2.67 mL) and the resultant mixture was stirred in vacuo for 30 minutes. The mixture was then cooled to O⁰C and treated with t-butyl acrylate (17.40 g). The reaction was warmed to room temperature and stirred for 16 hours. The mixture was filtered through aluminium oxide (15 g) eluting with ether (75 mL). The collected filtrate was concentrated to give the sub-titled compound (34.40 g) as an oil. 1H NMR (CDCl₃) δ 7.26-7.07 (m, 4H), 3.69-3.59 (m, 4H), 2.86-2.81 (t, 2H), 2.50-2.45 (t, 2H), 1.43 (s, 9H)

b) 3-[2-(3-chlorophenyl)ethoxy]propanoic acid tert-Butyl 3-[2-(3-chlorophenyl)ethoxy]propanoate (example Ia), 34.40 g) was dissolved in dichloromethane (150 mL) and treated with trifluoroacetic acid (50 mL). The mixture was stirred at room temperature for 3 hours, then concentrated in vacuo and azeotroped with dichloromethane (2 x 10 mL). The residue was taken up in dichlormethane (300 mL) and extracted with saturated sodium hydrogen carbonate (200 mL). The basic layer was washed with dichloromethane (20 mL) then acidified with 2M hydrochloric acid. The acidic layer was extracted with dichloromethane (2 x 200 mL). The organic layers were combined, washed with brine, dried over anhydrous magnesium sulphate, filtered and concentrated to yield the sub-titled compound (24.50 g) as an oil. m/e 227 [M-H] c) iV-[2-(DiethyIamino)ethyI]-A^(2,2-dimethoxyethyl)-3-[2-(3- chlorophenyl)ethoxy]propanamide

Oxalyl chloride (9.50 mL) was added dropwise over 45 minutes to a solution of 3-[2-(3- chlrophenyl)ethoxy]propanoic acid (22.50 g) (example Ib) in dichloromethane (120ml) and DMF (0.5 mL). The reaction mixture was stirred for a further 16 hours. The mixture was subsequently concentrated, redissolved in DCM (1.7 L) and added dropwise over 1.75 hours at O⁰C to a solution of ^-(l^-dimethoxyethy^-ΛζN-diethylethane-l^-diamine (20.20 g)(example 16a) and isopropyldiethylamine (34.43 mL) in DCM (200 mL). The resulting mixture was stirred at room temperature for 16 hours, washed with aqueous saturated sodium bicarbonate solution (3x1 L), water (1.5 L) and dried over sodium sulphate and concentrated to give 39.50 g of the sub-titled compound. m/e 415 (M+H⁺, 83%)

d) 7V-[2-(DiethyIamino)ethyl]-3-[2-(3-chIorophenyϊ)ethoxy]-N-(2- oxoethyl)proρanamide

A solution of N-[2-(Diethylamino)ethyl]-iV^r-(2,2-dimethoxyethyl)-3-[2-(3- chlorophenyl)ethoxy]propanamide (example Ic) (20 g) in DCM (500 mL) was treated dropwise at O⁰C with trifluoroacetic acid (50 mL) over 30 minutes. After the addition the reaction mixture was allowed to warm to room temperature and stirred for a further 1 hour. The reaction mixture was concentrated and the residue poured into aqueous saturated sodium bicarbonate solution (1800 mL, caution). The aqueous mixture was extracted with DCM (3x400 mL) and the combined extracts were dried over magnesium sulphate and concentrated. The residue was used directly in the following reaction. e) N-[2-(Diethylamino)ethyl]-N-(2-{[2-(4-hydroxy-2-oxo-2,3-dihydro-l,3-benzothiazol- 7-yl)ethyl]amino}ethyl)-3-[2-(3-chlorophenyl)ethoxy]propanamide dihydrobromide

A suspension of 7-(2-amino-ethyl)-4-hydiOxy-3H-benzothiazol-2-one hydrochloride (11.77 g) in dry NMP (50 mL) was heated to 65⁰C and treated in one portion with a solution of NaOH (1.83 g) in methanol (23 mL). The bright orange suspension was cooled to room temperature and treated dropwise with a solution of JV-[2-(diethylamino)ethyl]-3- [2-(3-chlorophenyl)ethoxy]-iV-(2-oxoethyl)propanamide (example Id) in dichloromethane (50 mL) over 30 minutes. The reaction was left to stir for 30 minutes. Sodium triacetoxyborohydride (20.33 g) was then added in portions over 20 minutes and the mixture stiffed for a further 16 hours. The reaction mixture was poured into water (1.8 L), basifϊed to pH8 by the addition of solid potassium carbonate and extracted with dichloromethane (2x500 mL); the combined organic extracts were dried over magnesium sulphate and concentrated to give a dark oil. The residue was purified by chromatography on silica with 10% (0.1% aqNH₃/MeOH)/DCM as eluent to give the sub-title compound as a brown oil. Yield (6.58 g). This was dissolved in ethanol (150 mL) and 48% aqueous hydrobromic acid (10 mL) was added. The solution was aged for 30 minutes then evaporated to dryness. The residue was triturated with ethanol (100 mL); the resultant solid was collected by filtration and dried in vacuo at 50. This material was recrystallised from ethanol/water (6:1, 500 mL); after standing overnight the resultant solid was collected by filtration and washed with ice-cold ethanol (75 mL). Drying in vacuo at 5O⁰C for 24hr afforded 4.96 g of the title compound. MS: APCI (+ve): 563 (M+l) 99.3% purity (T9505M). 1H NMR (DMSO, 90⁰C), δ 11.75-11.73 (m, IH), 10.08-10.06 (d, IH), 8.65 (bs, IH), 7.33- 7.19 (m, 4H), 6.89-6.84 (t, IH), 6.77-6.74 (m, IH), 3.68-3.58 (m, 8H), 3.17-3.16 (m, 10H), 2.86-2.80 (m, 4H), 2.67-2.62 (m, 2H), 1.23-1.19 (t, 6H). Elemental Analysis CHNS C:46.54%(46.39);H:5.75%(5.70);N:7.94%(7.73);S:4.46%(4.42)

Figure 1: XRPD of Polymorph A of Example 1 product Biological Assays

Adrenergic β2 mediated cAMP production

Cell preparation

H292 cells were grown in 225cm2 flasks incubator at 37°C, 5% CO₂ in RPMI medium containing, 10% (v/v) FBS (foetal bovine serum) and 2 mM L-glutamine.

Experimental Method Adherent H292 cells were removed from tissue culture flasks by treatment with

Accutase™ cell detachment solution for 15 minutes. Flasks were incubated for 15 minutes in a humidified incubator at 37⁰C, 5% CO₂. Detached cells were re-suspended in RPMI media (containing 10% (v/v) FBS and 2 mM L-glutamine) at 0.05 x 10⁶ cells per mL. 5000 cells in 100 μL were added to each well of a tissue-culture-treated 96-well plate and the cells incubated overnight in a humidified incubator at 37⁰C, 5% CO₂. The culture media was removed and cells were washed twice with 100 μL assay buffer and replaced with 50 μL assay buffer (HBSS solution containing 1OmM HEPES pH7.4 and 5 mM glucose). Cells were rested at room temperature for 20 minutes after which time 25 μL of rolipram (1.2 mM made up in assay buffer containing 2.4% (v/v) dimethylsulphoxide) was added. Cells were incubated with rolipram for 10 minutes after which time test compounds were added and the cells were incubated for 60 minutes at room temperature. The final rolipram concentration in the assay was 300 μM and final vehicle concentration was 1.6% (v/v) dimethylsulphoxide. The reaction was stopped by removing supernatants, washing once with 100 μL assay buffer and replacing with 50 μL lysis buffer. The cell monolayer was frozen at -8O⁰C for 30 minutes (or overnight).

AlphaScreen™ cAMP detection

The concentration of cAMP (cyclic adenosine monophosphate) in the cell lysate was determined using AlphaScreen™ methodology. The frozen cell plate was thawed for 20 minutes on a plate shaker then 10 μL of the cell lysate was transferred to a 96-well white plate. 40 μL of mixed AlphaScreen™ detection beads pre-incubated with biotinylated cAMP, was added to each well and the plate incubated at room temperature for 10 hours in the dark. The AlphaScreen™ signal was measured using an EnVision spectrophotometer (Perkin-Elmer Inc.) with the recommended manufacturer's settings. cAMP concentrations were determined by reference to a calibration curve determined in the same experiment using standard cAMP concentrations. Concentration response curves for agonists were constructed and data was fitted to a four parameter logistic equation to determine both the pEC₅₀ and Intrinsic Activity. Intrinsic Activity was expressed as a fraction relative to the maximum activity determined for formoterol in each experiment.

Selectivity Assays

Adrenergic αlD

Membrane Preparation Membranes were prepared from human embryonic kidney 293 (HEK293) cells expressing recombinant human αlo receptor. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.

Experimental Method

Assays were performed in U-bottomed 96-well polypropylene plates. 10 μL [³H]-prazosin (0.3 nM final concentration) and 10 μL of test compound (10x final concentration) were added to each test well. For each assay plate 8 replicates were obtained for [³H]-prazosin binding in the presence of 10 μL vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or lOμL BMY7378 (10 μM final concentration; defining non-specific binding (NSB)). Membranes were then added to achieve a final volume of 100 μL. The plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 μL wash buffer (5OmM HEPES, ImM EDTA, pH 7.4) were performed at 4⁰C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 μL) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3 -minute counting protocol.

Total specific binding (B₀) was determined by subtracting the mean NSB from the mean maximum binding. NSB values were also subtracted from values from all other wells. These data were expressed as percent of B₀. Compound concentration-effect curves (inhibition of [³H]-prazosin binding) were determined using serial dilutions typically in the range 0.1 nM to 10 μM. Data was fitted to a four parameter logistic equation to determine the compound potency, which was expressed as pIC50 (negative log molar concentration inducing 50% inhibition of [³H]-prazosin binding).

Adrenergic βl

Membrane Preparation

Membranes containing recombinant human adrenergic beta 1 receptors were obtained from Euroscreen. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.

Experimental Method

Assays were performed in U-bottomed 96- well polypropylene plates. 10 μL [¹²⁵I]- Iodocyanopindolol (0.036 nM final concentration) and 10 μL of test compound (10x final concentration) were added to each test well. For each assay plate 8 replicates were obtained for [¹²³I]-Iodocyanopindolol binding in the presence of 10 μL vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or 10 μL Propranolol (10 μM final concentration; defining non-specific binding (NSB)). Membranes were then added to achieve a final volume of 100 μL. The plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 μL wash buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, pH 7.4) were performed at 4°C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 μL) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3-minute counting protocol.

Total specific binding (B₀) was determined by subtracting the mean NSB from the mean maximum binding. NSB values were also subtracted from values from all other wells. These data were expressed as percent of B₀. Compound concentration-effect curves (inhibition of [¹²⁵I] -Iodocyanopindolol binding) were determined using serial dilutions typically in the range 0.1 nM to 10 μM. Data was fitted to a four parameter logistic equation to determine the compound potency, which was expressed as pi C₅₀ (negative log molar concentration inducing 50% inhibition of [¹²⁵I]-Iodocyanopindolol binding).

Dopamine D2

Membrane Preparation

Membranes containing recombinant human Dopamine Subtype D2s receptors were obtained from Perkin Elmer. These were diluted in Assay Buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, 0.1% gelatin, pH 7.4) to provide a final concentration of membranes that gave a clear window between maximum and minimum specific binding.

Experimental Method

Assays were performed in U-bottomed 96-well polypropylene plates. 30 μL [³H]- spiperone (0.16 nM final concentration) and 30 μL of test compound (1Ox final concentration) were added to each test well. For each assay plate 8 replicates were obtained for [³H] -spiperone binding in the presence of 30 μL vehicle (10% (v/v) DMSO in Assay Buffer; defining maximum binding) or 30 μL Haloperidol (10 μM final concentration; defining non-specific binding (NSB)). Membranes were then added to achieve a final volume of 300 μL. The plates were incubated for 2 hours at room temperature and then filtered onto PEI coated GF/B filter plates, pre-soaked for 1 hour in Assay Buffer, using a 96-well plate Tomtec cell harvester. Five washes with 250 μL wash buffer (5OmM HEPES, ImM EDTA, 12OmM NaCl, pH 7.4) were performed at 4⁰C to remove unbound radioactivity. The plates were dried then sealed from underneath using Packard plate sealers and MicroScint-0 (50 μL) was added to each well. The plates were sealed (TopSeal A) and filter-bound radioactivity was measured with a scintillation counter (TopCount, Packard BioScience) using a 3-minute counting protocol.

Total specific binding (B₀) was determined by subtracting the mean NSB from the mean maximum binding. NSB values were also subtracted from values from all other wells. These data were expressed as percent of B₀. Compound concentration-effect curves (inhibition of [³H] -spiperone binding) were determined using serial dilutions typically in the range 0.1 nM to 10 μM. Data was fitted to a four parameter logistic equation to determine the compound potency, which was expressed as pICso (negative log molar concentration inducing 50% inhibition of [³H]-spiperone binding).

Table 1

Compound β2 pEC50 β2 Int Act αl bind pIC50 βl bind p IC50 D2 bind pIC50

Example 1 8.3 0.7 <6. 1 <5 5.6

Onset Assay

Dunkin-Hartley guinea-pigs (between 200 g and 300 g on delivery) were supplied by a designated breeding establishment. The guinea-pigs were killed by cervical dislocation and the trachea removed. The adherent connective tissue was removed and each trachea cut into four rings. The tissue rings were then attached to an isometric transducer. The tissues were washed and a force of 1 g was applied to each ring. In all experiments a paired curve design was used. A priming dose of 1 μM methacholine was applied to the tissues. The tissues were then washed (three times, one minute between washes), the resting tension of Ig was reapplied and the tissues were allowed to rest for 1 hour to equilibrate. Tissues were then contracted with 1 μM methacholine and once a steady response was obtained a cumulative concentration response curve to isoprenaline (10^"9M - 10^"5 M) was constructed. The tissues were then washed (three times, one minute between washes) and left to rest for an hour. At the end of the resting period the tissues were contracted with 1 μM methacholine and a p[A]₅₀ concentration of test compound added. Once the tissue had reached maximum relaxation, a 30 x p[A]₅₀ concentration of test compound was added. Once the tissue response had reached a plateau, 10 μM sotalol was added to the bath to confirm that the relaxation was β₂ mediated

Data were collected using the ADInstruments chart4forwindows software, which measured the maximum tension generated at each concentration of agonist.

For each concentration of the isoprenaline cumulative concentration curve, the response was calculated as % relaxation of the methacholine-induced contraction. A curve was plotted of log io [agonist] (M) versus percentage inhibition of the methacholine-induced contraction. These data were then fitted to a non-linear regression curve fit. For each experiment, E/[A] curve data were fitted using a 4-parameter logistic function of the form:

E and [A] are the pharmacological effect (% relaxation) and concentration of the agonist respectively; α, β, [A]₅₀ and m are the asymptote, baseline, location and slope parameters, respectively. The P[A]₅₀ and IA of each isoprenaline curve was determined from this fit, to determine if the tissue was viable for generating an onset time for the test compounds.

For each p[A]₅o concentration of the test compound, the response was calculated as % relaxation of the methacholine-induced contraction. The results were plotted % relaxation against time and the time taken to reach a 90% relaxation value was calculated and recorded.

The addition of a 30 x p [A]₅₀ concentration enabled determination of the maximum compound effect within the individual tissue. Hence, the % of the maximum compound effect at the p[A]₅o concentration was calculated and recorded. Pharmacokinetics in the Rat

A dose solution of the test compound was prepared using a suitable dose vehicle. The concentration of the compound in the dose solution was assayed by diluting an aliquot to a nominal concentration of SOμg-ml^"1 and calibrating against duplicate injections of a standard solution and a QC standard at this concentration. Compounds were administered intravenously as a bolus into a caudal vein to groups of three 250-35Og rats (approximately 1 ml-kg^'1). For the oral dose, a separate group of 2 or 3 animals were dosed by oral gavage (3 ml-kg^"1). Delivered doses were estimated by weight loss. Food was not usually withdrawn from animals prior to dosing, although this effect was investigated if necessary.

Blood samples (0.25ml) were taken into ImI syringes from the caudal vein, transferred to EDTA tubes and plasma was prepared by centrifugation (5 min at 13000rprn) soon after sample collection, before storage at -2O⁰C. Typical sampling times were 2, 4, 8, 15, 30, 60, 120, 180, 240, 300 (min) or until the terminal tl/2 was accurately described.

The concentration of the analyte(s) were determined in plasma by quantitative mass spectrometry. Standard and quality control stock solutions were prepared at a concentration lmg/ml in methanol. A range of standard and QC stocks produced by serial dilution were added to control rat plasma (50μl). The range of concentrations covered the range of levels of analyte present in the rat samples. Standards, QCs and samples underwent liquid extraction using 50μl of organic solvent and lOOμl of organic solvent containing an internal standard, chosen to closely resemble the analyte. The samples were then mixed by repeated inversion, stored at -2O⁰C for at least 1 h, and centrifuged at 3500 rpm in a centrifuge for 20 minutes. Aliquots (120 μl) of each sample were transferred for analysis using LC-MSMS. Standard and quality control samples covering the range of concentrations found in the test samples were within 25 % of the nominal concentration.

Pharmacokinetic data analysis was achieved using WinNonlin. A standard non- compartmental analysis was used to estimate the parameters such as Tmax, Cmax, Lambda_z, tl/2_Lambda_z, AUCaIl₅ AUCINF(observed), Cl(observed), Vss(observed).

Claims

1. A pharmaceutically acceptable salt of N-[2-(Diethylamino)ethyl]-iV-(2-{[2-(4- hydroxy-2-oxo-2,3-dihydro-l,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(3-

5 chlorophenyl)ethoxy]propanamide provided it is not the ditrifluoroacetate salt.

2. A pharmaceutically acceptable salt as claimed in claim wherein the salt is a hydrochloride, hydrobromide, sulphate, phosphate, acetate, fumarate, maleate, citrate, pyruvate, succinate, oxalate, methanesulphonate, /?-toluenesulphonate,

I₀ 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- i₅ hydroxy ethylsulfonate), 2-mesitylenesulphonate, 2-naphthalenesulphonate, D- mandelate or L-mandelate.

3. A pharmaceutically acceptable salt of λf-[2-(Diethylamino)ethyi]-JV-(2-{[2-(4- hydroxy-2-oxo-2,3-dihydro-l,3-benzothiazol-7-yl)ethyl]amino}ethyl)-3-[2-(3-

20 chlorophenyl)ethoxy]propanamide which is the dihydrobromide salt.

4. A processes for preparing a salt as calimed in claim 1, the process comprising reacting a suitable acid with Λ⁷-[2-(Diethylamino)ethyl]-Λ^r-(2-{[2-(4-hydroxy-2-oxo- 2,3-dihydro-l,3-benzothiazol-7-yl)ethyl]aniino}ethyl)-3-[2-(3-

2₅ chlorophenyl)ethoxy]propanamide in a suitable solvent, optionally at elevated temperature.

5. A pharmaceutical composition comprising a pharmaceutically acceptable salt as claimed in claim 1 in association with a pharmaceutically acceptable adjuvant,

30 diluent or carrier.

6. A pharmaceutically acceptable salt as claimed in claim 1 for use in therapy.

7. Use of a pharmaceutically acceptable salt as claimed in claim 1 in the manufacture of a medicament for the treatment of human diseases or conditions in which modulation of β2 adrenoreceptor activity is beneficial.

8. Use of a pharmaceutically acceptable salt as claimed in claim 1 in the manufacture of a medicament for use in treating adult respiratory distress syndrome (ARDS), pulmonary emphysema, bronchitis, bronchiectasis, chronic obstructive pulmonary disease (COPD), asthma or rhinitis.

9. A pharmaceutically acceptable salt as claimed in claim 1 for the treatment of a respiratory disease state.

10. A pharmaceutically acceptable salt as claimed in claim 1 for the treatment of adult respiratory distress syndrome (ARDS), pulmonary emphysema, bronchitis, bronchiectasis, chronic obstructive pulmonary disease (COPD), asthma or rhinitis.

11. A method of treating, or reducing the risk of, a disease or condition in which modulation of β2 adrenoreceptor activity is beneficial which comprises administering to a patient in need thereof a therapeutically effective amount of a pharmaceutically acceptable salt as claimed in claim 1.

12. A method of treating, or reducing the risk of, an inflammatory disease or condition which comprises administering to a patient in need thereof a therapeutically effective amount of a pharmaceutically acceptable salt as claimed in claim 1.

13. A method according to claim 10 or claim 11, wherein the disease or condition is adult respiratory distress syndrome (ARDS), pulmonary emphysema, bronchitis, bronchiectasis, chronic obstructive pulmonary disease (COPD), asthma or rhinitis.