WO2010015792A1

WO2010015792A1 - Nitrogen containing heterocyclic compounds useful as bifunctional modulators of m3 receptors and beta-2 receptors

Info

Publication number: WO2010015792A1
Application number: PCT/GB2008/002692
Authority: WO
Inventors: Jonathan Mark Sutton; Fabien Roussel; Marco Van Den Heuvel; Nicholas Charles Ray; Lilian Alcaraz
Original assignee: Argenta Discovery Limited; Astrazeneca Ab
Priority date: 2008-08-06
Filing date: 2008-08-06
Publication date: 2010-02-11

Abstract

The present invention relates to named compounds of the formula (I) having both M3 receptor antagonist and β2 agonist activity; compositions comprising such compounds; the use of such compounds in therapy (such as respiratory tract disorders); and a method of treating a patient with such compounds.

Description

NITROGEN CONTAINING HETEROCYCLIC COMPOUNDS USEFUL AS BIFUNCTIONAL MODULATORS OF M3 RECEPTORS AND BETA-2

RECEPTORS

Field of the Invention

This invention relates to heterocycles, pharmaceutical compositions, methods for their preparation and use in the treatment of diseases where compounds possessing both muscarinic receptor antagonist and β2-agonist activity present in the same molecule (bifunctional molecules) are useful (such as in the treatment of asthma or COPD).

Background to the invention

Compounds possessing both muscarinic receptor antagonist and β2-agonist activity present in the same molecule are known in the art. Such bifunctional molecules provide bronchodilation through two separate modes of action whilst possessing single molecule pharmacokinetics. Such a molecule might be easier to formulate for therapeutic use as compared to two separate compounds and might be more easily co-formulated with another active ingredient, for example a steroid. Such bifunctional molecules are described in, for example, WO04/074246, WO04/089892, WO05/111004, WO06/023457 and WO06/023460, all of which use different linker radicals for covalently linking an M3 antagonist to a β2-agonist.

Anti-cholinergic agents prevent the passage of, or effects resulting from the passage of, impulses through the parasympathetic nerves. This is a consequence of the ability of such compounds to inhibit the action of acetylcholine (Ach) by blocking its binding to the muscarinic cholinergic receptors.

There are five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1-M5, and each is the product of a distinct gene and each displays unique pharmacological properties. mAChRs are widely distributed in vertebrate organs, and these receptors can mediate both inhibitory and excitatory actions. For example, in smooth muscle found in the airways, bladder and gastrointestinal tract, M3 mAChRs mediate contractile responses (reviewed by Caulfield, 1993, Pharmac. Then, 58, 319 - 379).

In the lungs, muscarinic receptors M1 , M2 and M3 have been demonstrated to be important and are localized to the trachea, the bronchi, submucosal glands and parasympathetic ganglia (reviewed in Fryer and Jacoby, 1998, Am J Resp Crit Care Med., 158 (5 part 3) S 154 - 160). M3 receptors on airway smooth muscle mediate contraction and therefore bronchoconstriction. Stimulation of M3 receptors localised to submucosal glands results in mucus secretion.

Increased signalling through muscarinic acetylcholine receptors has been noted in a variety of different pathophysiological states including asthma and COPD. In COPD, vagal tone may either be increased (Gross et al. 1989, Chest; 96:984-987) and/or may provoke a higher degree of obstruction for geometric reasons if applied on top of oedematous or mucus-laden airway walls (Gross et al. 1984, Am Rev Respir Dis; 129:856-870). In addition, inflammatory conditions can lead to a loss of inhibitory M2 receptor activity which results in increased levels of acetylcholine release following vagal nerve stimulation (Fryer et al, 1999, Life Sci., 64, (6-7) 449-455). The resultant increased activation of M3 receptors leads to enhanced airway obstruction. Thus the identification of potent muscarinic receptor antagonists would be useful for the therapeutic treatment of those disease states where enhanced M3 receptor activity is implicated. Indeed, contemporary treatment strategies currently support regular use of M3 antagonist bronchodilators as first-line therapy for COPD patients (Pauwels et al. 2001 , Am Rev Respir Crit Care Med; 163:1256-1276)

Chem. Pharm. Bull. 27 (12) 3149-3152 (1979) and J. Pharm. Sci 69 (5) 534- 537 (1980) describe furyl derivatives as possessing atropine-like activities. Med. Chem. Res 10 (9), 615-633 (2001) describes isoxazoles and Δ²- isoxazolines as muscarinic antagonists. WO97/30994 describes oxadiazoles and thiadiazoles as muscarinic receptor antagonists.

EP0323864 describes oxadiazoles linked to a mono- or bicyclic ring as muscarinic receptor modulators.

The class of β2 adrenergic receptor agonists is well known. Many known β2- agonists, in particular, long-acting β2-agonists such as salmeterol and formoterol, have a role in the treatment of asthma and COPD. These compounds are also generally administered by inhalation. Compounds currently under evaluation as once-daily β2 agonists are described in Expert Opin. Investig. Drugs 14 (7), 775-783 (2005). A well known β2-agonist pharmacophore is the moiety:

Also known in the art are pharmaceutical compositions that contain both a muscarinic antagonist and a β2-agonist for use in the treatment of respiratory disorders. For example, US2005/0025718 describes a β2-agonist in combination with tiotropium, oxotropium, ipratropium or other muscarinic antagonist; WO02/060532 describes a combination of ipratropium with a β2- agonist; and, WO02/060533 describes a combination of oxotropium with a β2- agonist. Other M3 antagonist / β2-agonist combinations are described in WO04/105759 and WO03/087097.

Our co-pending application PCT/GB2008/000407 relates to compounds of formula I:

wherein

(i) R¹ is H or C_rC₆-alkyl; and R³ is lone pair or CrC₆-alkyl; or (ii) R¹ and R³ together with the nitrogen to which they are attached form a heterocycloalkyl ring;

R⁴ and R⁵ are independently selected from the group consisting of aryl, aryl- fused-heterocycloalkyl, heteroaryl, CrC₆-alkyl, cycloalkyl; R⁶ is -OH, CrC₆-alkyl, CrC₆-alkoxy, hydroxy-CrC₆-alkyl, nitrile, a group CON(R¹²)₂ or a hydrogen atom; one of W, V and A is N or NR¹¹; another of W, V and A is N, O, S or CR⁸; and the last one of W, V and A is N or CR⁸

X is an CrCβalkylene, C₂-C₆alkenylene or C₂-C₆alkynylene group; R⁸, R¹¹ and R¹² are, independently, hydrogen atom or CrC₆-alkyl group; L^a is a divalent linker radical of formula (Ia);

wherein L represents a linker comprising a hydrocarbyl chain of up to 14 carbon atoms, wherein up to three carbon atoms of the chain are replaced by groups independently selected from O, NR⁴⁵, S, S(O), S(O)₂, C(O)O, OC(O),

NR⁴⁶C(O), C(O)NR⁴⁷, NR⁴⁸S(O)_2, S(O)₂NR⁴⁹, NR⁵⁰C(O)NR⁵¹, NR⁵²S(O)₂NR⁵³, OC(O)NR⁵⁴, NR⁵⁵C(O)O, provided that any heteroatoms in the chain are separated by at least 2 carbon atoms; and/or up to four carbon atoms of the chain form part of a mono- or bicyclic aliphatic, heteroaliphatic, aromatic or heteroaromatic ring having up to four heteroatoms independently selected from N, O or S, said ring comprising up to 10 ring atoms, and wherein the ring is optionally substituted by up to three substituents independently selected from halogen, S(O)_0-2R⁵⁶, NR⁵⁷R⁵⁸, S(O)₂NR⁵⁹R⁶⁰, C(O)NR⁶¹R⁶², C(O)OR⁶³, NR⁶⁴S(O)₂R⁶⁵, NR⁶⁶C(O)R⁶⁷, NR⁶⁸C(O)OR⁶⁹, NR⁷⁰C(O)NR⁷¹ R⁷², OR⁷³, Ci_-6 alkyl and C_3-6 cycloalkyl, and wherein

Ci-₆ alkyl and C_3-6 cycloalkyl may be optionally substituted by up to three substituents independently selected from halogen, hydroxyl and Ci_-6 alkoxy; and the chain may comprise up to three of such rings each selected independently, and; wherein R⁵⁶, R⁶⁵ and R⁶⁹ each independently represent C_1-6 alkyl or C_3-6 cycloalkyl, wherein C_1-6 alkyl and C_3-6 cycloalkyl may be optionally substituted by up to three substituents independently selected from halogen, hydroxyl, Ci_-6 alkoxy; and wherein R⁴⁵, R⁴⁶, R⁴⁷, R⁴⁸, R⁴⁹, R⁵⁰, R⁵¹, R⁵², R⁵³, R⁵⁴, R⁵⁵, R⁵⁷, R⁵⁸, R⁵⁹, R⁶⁰, R⁶¹, R⁶², R⁶³, R⁶⁴, R⁶⁶, R⁶⁷, R⁶⁸ , R⁷⁰, R⁷¹, R⁷² and R⁷³ each independently represent hydrogen, d-₆ alkyl or C_3-6 cycloalkyl, wherein Ci_-6 alkyl and C_3-6 cycloalkyl may be optionally substituted by up to three substituents independently selected from halogen, hydroxyl, C₁-₆ alkoxy; or any of R⁵⁷ and R⁵⁸, R⁵⁹ and R⁶⁰, R⁶¹ and R⁶² or R⁷¹ and R⁷², together with the nitrogen atom to which they are both attached, may form a 4 to 8 membered aliphatic heterocyclic ring, wherein the aliphatic heterocyclic ring may comprise up to three heteroatoms independently selected from N, O and S, wherein the ring may be optionally substituted by up to three substituents independently selected from halogen, hydroxyl and Ci-₆ alkyl or C_3-6 cycloalkyl, wherein C_1-6 alkyl and C_3-6 cycloalkyl may be optionally substituted by one or more substituents independently selected from halogen and hydroxyl; and wherein the chain may additionally comprise up to three carbon-carbon double bonds; and, wherein the chain may additionally comprise up to three carbon-carbon triple bonds; L¹ and L² each independently represent hydrogen, C₁-₆ alkyl or C-3-6 cycloalkyl;

L³ and L⁴ each independently represent hydrogen, C_1-6 alkyl or C_3-6 cycloalkyl, wherein C_1-6 alkyl and C_3-6 cycloalkyl may be optionally substituted by one or more substituents independently selected from halogen and hydroxyl; and in addition L¹ and/or L³ may be linked to a carbon atom of the hydrocarbyl chain in linker L to form an aliphatic ring of up to 6 ring atoms, wherein the ring may comprise up to three heteroatoms independently selected from N, O and S; and wherein ^* denotes the point of attachement of the group of formula (Ia) to the non-aromatic nitrogen bearing R¹ and R³, and ^** denotes the point of attachment to the group Z¹;

Z¹ is a moiety having β2- adrenoreceptor binding activity; wherein, unless otherwise specified, each occurrence of alkyl, heterocycloalkyl, aryl, aryl-fused-heterocycloalkyl, heteroaryl, cycloalkyl, alkoxy, alkylene, alkenylene, alkynylene or aryl-fused-cycloalkyl may be optionally substituted; and wherein each alkenylene chain contains, where possible, up to 2 carbon- carbon double bonds and each alkynylene chain contains, where possible, up to 2 carbon-carbon triple bonds or a pharmaceutically acceptable salt thereof.

Summary of the Invention

The present invention provides compounds falling within the scope of, but not specifically disclosed in, our co-pending application PCT/GB2008/000407 referred to above.

Thus, the present invention provides a compound selected from the group consisting of: 8-[2-(2-{4-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-methyl]-phenyl}-ethylamino)-1-hydroxy-ethyl]-5-hydroxy-4H- benzo[1 ,4]oxazin-3-one;

3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-methyl- amino)-Λ/-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1 ,2-dihydro-quinolin-5-yl)- ethylamino]-methyl}-phenyl)-propionamide;

3-{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}-Λ/-(4-

{[(R)-2-hydroxy-2-(4-hydroxy-2-oxo-2,3-dihydro-benzothiazol-7-yl)- ethylamino]-methyl}-phenyl)-propionamide; 3-{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}-Λ/-(4-

{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1 ,2-dihydro-quinolin-5-yl)-ethylamino]- methyl}-phenyl)-propionamide;

3-{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}-N-(4-

{[2-hydroxy-2-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[1 ,4]oxazin-8-yl)- ethylamino]-methyl}-phenyl)-propionamide;

5-[(R)-2-(2-{3-[3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-propoxy]-phenyl}-ethylamino)-1-hydroxy-ethyl]-8- hydroxy-1 H-quinolin-2-one;

8-Hydroxy-5-((R)-1 -hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol- 5-ylmethyl]-methyl-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-1 /-/-quinolin-2- one;

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazoi-

5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-1 /-/-quinolin-2-one;

5-Hydroxy-8-(1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol-5- ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-4H-benzo[1 ,4]oxazin-3- one;

Λ/-[2-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-phenyl]- formamide; 4-((R)-1-Hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol-5- ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-2-hydroxymethyl-phenol; 7-[(R)-2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy-ethyl]-4- hydroxy-3H-benzothiazol-2-one;

5-[(R)-2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy-ethyl]-8- hydroxy-1 /-/-quinolin-2-one;

8-[2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy-ethyl]-5-hydroxy-

4H-benzo[1 ,4]oxazin-3-one; 5-((R)-2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-8-hydroxy-

1 /-/-quinolin-2-one;

8-(2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-5-hydroxy- 4/-/-benzo[1 ,4]oxazin-3-one;

/V-[5-((R)-2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-2-hydroxy- phenylj-formamide;

7-((R)-2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-4-hydroxy-

3/-/-benzothiazol-2-one;

5-((R)-2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-8-hydroxy-

1 /-/-quinolin-2-one; 8-(2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}- methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-5-hydroxy-4H- benzo[1 ,4]oxazin-3-one;

Λ/-[5-((R)-2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-2-hydroxy- phenyl]-formamide; 4-Hydroxy-7-((R)-1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5- yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-3/-/-benzothiazol-2- one;

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5- yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-1 /-/-quinolin-2-one;

5-Hydroxy-8-(1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-4H-benzo[1 ,4]oxazin-3- one;

Λ/-[2-Hydroxy-5-((R)-1 -hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)- oxazol-5-yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-phenyl]- formamide;

8-(2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-3-yl]-ethylamino}-1-hydroxy-ethyl)-5-hydroxy-

4H-benzo[1 ,4]oxazin-3-one; 8-Hydroxy-5-((R)-1-hydroxy-2-{2-[5-({[2-(hydroxy-diphenyl-methyl)-oxazol-5- ylmethyl]-amino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-1 /-/-quinolin-2-one;

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[2-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5- yl]-ethylamino}-methyl)-thiazol-5-yl]-ethylamino}-ethyl)-1 /-/-quinolin-2-one; and

4-((R)-1 -Hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-2-hydroxymethyl- phenol; and pharmaceutically acceptable salts thereof

In another aspect the present invention provides a prodrug of a compound as listed above, or a pharmaceutically acceptable salt thereof.

In yet another aspect the present invention provides an N-oxide of a compound as listed above, or a prodrug or pharmaceutically acceptable salt thereof.

In a further aspect the present invention provides a solvate (such as a hydrate) of a compound as listed above or an N-oxide, prodrug or pharmaceutically acceptable salt thereof.

Particular compounds of the invention are:

3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-N-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1 ,2-dihydro-quinolin- 5-yl)-ethylamino]-methyl}-phenyl)-propionamide; 8-Hydroxy-5-((R)-1 -hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-methyl-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-1 H- quinolin-2-one;

5-Hydroxy-8-(1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-4H- benzo[1 ,4]oxazin-3-one;

N-[2-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl- methyl)-isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)- phenyl]-formamide;

7-[(R)-2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5- yl]-ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy-ethyl]-4- hydroxy-3H-benzothiazol-2-one;

5-[(R)-2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5- yl]-ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy-ethyl]-8- hydroxy-1 H-quinolin-2-one; 8-(2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1 -hydroxy-ethyl)-5-hydroxy- 4H-benzo[1 ,4]oxazin-3-one;

7-((R)-2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1 -hydroxy-ethyl)-4-hydroxy- 3H-benzothiazol-2-one;

5-((R)-2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-8-hydroxy- 1 H-quinolin-2-one;

4-Hydroxy-7-((R)-1 -hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)- oxazol-5-yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-3H- benzothiazol-2-one; 8-Hydroxy-5-((R)-1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)- oxazol-5-yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-1 H-quinolin- 2-one;

5-((R)-2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5- yl]-ethylamino}-methyl)-thiophen-3-yl]-ethylamino}-1 -hydroxy-ethyl)-8-hydroxy- 1 H-quinolin-2-one; and

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[5-({[2-(hydroxy-diphenyl-methyl)- oxazol-5-ylmethyl]-amino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-1 H- quinolin-2-one.

Each of the compounds identified above, taken alone or with any combination of the other identified compounds represents an independent aspect of the invention.

Certain compounds listed above also give rise to enantiomers. In such cases, both enantiomers of the invention generally exhibit affinity at the M₃ receptor, although one enantiomer is generally favoured on criteria of potency at the M₃ receptor and/or selectivity against the M₂ receptor. In some embodiments of the invention, the absolute stereochemistry of the favoured enantiomer is known.

Compounds of the invention may be useful in the treatment or prevention of diseases in which activation of muscarinic receptors are implicated, for example the present compounds are useful for treating a variety of indications, including but not limited to respiratory-tract disorders such as chronic obstructive lung disease (also known as chronic obstructive pulmonary disease or COPD), chronic bronchitis of all types (including dyspnoea associated therewith), asthma (allergic and non-allergic; 'wheezy-infant syndrome'), adult/acute respiratory distress syndrome (ARDS), chronic respiratory obstruction, bronchial hyperactivity, pulmonary fibrosis, pulmonary emphysema, and allergic rhinitis, exacerbation of airway hyperreactivity consequent to other drug therapy, particularly other inhaled drug therapy, pneumoconiosis (for example aluminosis, anthracosis, asbestosis, chalicosis, ptilosis, siderosis, silicosis, tabacosis and byssinosis); gastrointestinal-tract disorders such as irritable bowel syndrome, spasmodic colitis, gastroduodenal ulcers, gastrointestinal convulsions or hyperanakinesia, diverticulitis, pain accompanying spasms of gastrointestinal smooth musculature; urinary-tract disorders accompanying micturition disorders including neurogenic pollakisuria, neurogenic bladder, nocturnal enuresis, psychosomatic bladder, incontinence associated with bladder spasms or chronic cystitis, urinary urgency or pollakiuria; motion sickness; and cardiovascular disorders such as vagally induced sinus bradycardia.

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

For treatment of respiratory conditions, administration by inhalation will often be preferred. In many cases, the duration of action of quaternary ammonium salts of the invention administered by inhalation is may be more than 12, or more than 24 hours for a typical dose. For treatment of gastrointestinal-tract disorders and cardiovascular disorders, administration by the parenteral route, usually the oral route may be preferred.

Another aspect of the invention is a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier, diluent or excipient.

Another aspect of the invention is the use of a compound of the invention for the manufacture of a medicament for the treatment or prevention of a disease or condition in which muscarinic M3 receptor activity and β2-adrenergic activity are implicated. Diseases or conditions in which muscarinic M3 receptor activity and β2-adrenergic activity are implicated include respiratory- tract disorders, gastrointestinal-tract disorders and cardiovascular disorders. Specific examples of such diseases and conditions include those listed above.

Another aspect of the invention provides a compound of the invention for the treatment or prevention of a disease or condition in which muscarinic M3 receptor activity and β2-adrenergic activity are implicated. Diseases or conditions in which muscarinic M3 receptor activity and β2-adrenergic activity are implicated include respiratory-tract disorders, gastrointestinal-tract disorders and cardiovascular disorders. Specific examples of such diseases and conditions include those listed above.

Another aspect of the invention provides a method of treatment of a disease or condition in which M3 muscarinic receptor activity and β2-adrenergic activity are implicated comprising administration to a subject in need thereof a therapeutically effective amount of a compound of the invention. Diseases or conditions in which muscarinic M3 receptor activity and β2-adrenergic activity are implicated include respiratory-tract disorders, gastrointestinal-tract disorders and cardiovascular disorders. Specific examples of such diseases and conditions include those listed above. Another aspect of the invention provides a compound of the invention for use in therapy.

"Pharmaceutically acceptable salt" means a physiologically or toxicologically tolerable salt and includes, when appropriate, pharmaceutically acceptable base addition salts, pharmaceutically acceptable acid addition salts, and pharmaceutically acceptable quaternary ammonium salts. For example (i) where a compound of the invention contains one or more acidic groups, for example carboxy groups, pharmaceutically acceptable base addition salts that may be formed include sodium, potassium, calcium, magnesium and ammonium salts, or salts with organic amines, such as, diethylamine, Λ/-methyl-glucamine, diethanolamine or amino acids (e.g. lysine) and the like; (ii) where a compound of the invention contains a basic group, such as an amino group, pharmaceutically acceptable acid addition salts that may be formed include hydrochlorides, hydrobromides, sulfates, phosphates, acetates, citrates, lactates, tartrates, mesylates, napadisylate (naphthalene- 1 ,5-disulfonate or naphthalene-1 -(sulfonic acid)-5-sulfonate), edisylate (ethane-1 ,2-disulfonate or ethane-1 -(sulfonic acid)-2-sulfonate), maleates, fumarates, succinates, isethionates (2-hydroxyethyl-1 -sulfonates) and the like.

The present invention covers all permissible ratios of cationic ammonium species to counter-ion, for example hemi-napadisylate and napadisylate.

It will be understood that, as used herein, references to the compounds of the invention are meant to also include the pharmaceutically acceptable salts.

"Prodrug" refers to a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis, reduction or oxidation) to a compound of the invention. Suitable groups for forming pro-drugs are described in 'The

Practice of Medicinal Chemistry, 2^nd Ed. pp561-585 (2003) and in F. J.

Leinweber, Drug Metab. Res., , 18, 379. (1987) It will be understood that, as used in herein, references to the compounds of the invention are meant to also include the prodrug forms. Compounds of the invention may exist in one or more geometrical, optical, enantiomeric, diastereomeric and tautomeric forms, including but not limited to cis- and frans-forms, E- and Z-forms, R-, S- and meso-forms, keto-, and enol-forms. Unless otherwise stated a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Where appropriate such isomers can be separated from their mixtures by the application or adaptation of known methods (e.g. chromatographic techniques and recrystallisation techniques). Where appropriate such isomers may be prepared by the application of adaptation of known methods (e.g. asymmetric synthesis).

Compounds of the invention are β2-adrenergic binding compounds. Such compounds may be antagonists, partial agonists or full agonists. Compounds that are antagonists are useful tools, for example, for the generation of structure-activity relationships and as radioligands. Compounds that are partial or full agonists may be useful as pharmacological compounds for the treatment of the diseases described above.

The present invention is also concerned with pharmaceutical formulations comprising, as an active ingredient, a compound of the invention. Other compounds may be combined with compounds of this invention for the prevention and treatment of inflammatory diseases of the lung. Thus the present invention is also concerned with pharmaceutical compositions for preventing and treating respiratory-tract disorders such as chronic obstructive lung disease, chronic bronchitis, asthma, chronic respiratory obstruction, pulmonary fibrosis, pulmonary emphysema, and allergic rhinitis comprising a therapeutically effective amount of a compound of the invention and one or more other therapeutic agents. Other compounds may be combined with compounds of this invention for the prevention and treatment of inflammatory diseases of the lung. Accordingly the invention includes a combination of an agent of the invention as hereinbefore described with one or more anti-inflammatory, bronchodilator, antihistamine, decongestant or anti-tussive agents, said agents of the invention hereinbefore described and said combination agents existing in the same or different pharmaceutical compositions, administered separately or simultaneously. Preferred combinations would have two or three different pharmaceutical compositions. Suitable therapeutic agents for a combination therapy with compounds of the invention include:

One or more other bronchodilators such as PDE3 inhibitors;

Methyl xanthines such as theophylline;

A corticosteroid, for example fluticasone propionate, ciclesonide, mometasone furoate or budesonide, or steroids described in WO02/88167, WO02/12266, WO02/100879, WO02/00679, WO03/35668, WO03/48181 , WO03/62259, WO03/64445, WO03/72592, WO04/39827 and WO04/66920; A non-steroidal glucocorticoid receptor agonist;

A leukotriene modulator, for example montelukast, zafirlukast or pranlukast; protease inhibitors, such as inhibitors of matrix metalloprotease for example MMP12 and TACE inhibitors such as marimastat, DPC-333, GW-3333;

Human neutrophil elastase inhibitors, such as sivelestat and those described in WO04/043942, WO05/021509, WO05/021512, W 005/026123, WO05/026124, WO04/024700, WO04/024701 , WO04/020410, WO04/020412, WO05/080372, WO05/082863, WO05/082864, WO03/053930;

Phosphodiesterase-4 (PDE4) inhibitors, for example roflumilast, arofylline, cilomilast, ONO-6126 or IC-485;

Phosphodiesterase-7 inhibitors;

An antitussive agent, such as codeine or dextramorphan; Kinase inhibitors, particularly P38 MAPKinase inhibitors; P2X7 anatgonists; iNOS inhibitors; A non-steroidal anti-inflammatory agent (NSAID), for example ibuprofen or ketoprofen;

A dopamine receptor antagonist;

TNF-α inhibitors, for example anti-TNF monoclonal antibodies, such as Remicade and CDP-870 and TNF receptor immunoglobulin molecules, such as Enbrel;

A2a agonists such as those described in EP1052264 and EP1241176;

A2b antagonists such as those described in WO2002/42298;

Modulators of chemokine receptor function, for example antagonists of CCR 1 , CCR2, CCR3, CXCR2, CXCR3, CX3CR1 and CCR8, such as SB-332235,

SB-656933, SB-265610, SB-225002, MCP-1 (9-76), RS-504393, MLN-1202,

INCB-3284;

Compounds which modulate the action of prostanoid receptors, for example a

PGD₂ (DP1 or CRTH2), or a thromboxane A₂ antagonist eg ramatrobant; Compounds which modulate Th1 or Th2 function, for example, PPAR agonists; lnterleukin 1 receptor antagonists, such as Kineret; lnterleukin 10 agonists, such as llodecakin;

HMG-CoA reductase inhibitors (statins); for example rosuvastatin, mevastatin, lovastatin, simvastatin, pravastatin and fluvastatin;

Mucus regulators such as INS-37217, diquafosol, sibenadet, CS-003, talnetant, DNK-333, MSI-1956, gefitinib;

Antiinfective agents (antibiotic or antiviral), and antiallergic drugs including, but not limited to, anti-histamines.

In a further aspect the present invention provides a combination comprising a compound of the invention and an inhaled corticosteroid (for example fluticasone propionate, ciclesonide, mometasone furoate or budesonide), or an inhaled PDE₄ inhibitor (for example roflumilast, cilomilast, Tofimilast). The weight ratio of the first and second active ingredients may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used.

Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a compound of the present invention. In therapeutic use, the active compound may be administered by any convenient, suitable or effective route. Suitable routes of administration are known to those skilled in the art, and include oral, intravenous, rectal, parenteral, topical, ocular, nasal, buccal and pulmonary.

The magnitude of prophylactic or therapeutic dose of a compound of the invention will, of course, vary depending upon a range of factors, including the activity of the specific compound that is used, the age, body weight, diet, general health and sex of the patient, time of administration, the route of administration, the rate of excretion, the use of any other drugs, and the severity of the disease undergoing treatment. In general, the daily dose range for inhalation will lie within the range of from about 0.1 μg to about 10 mg per kg body weight of a human, preferably 0.1 μg to about 0.5 mg per kg, and more preferably 0.1 μg to 50μg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases. Compositions suitable for administration by inhalation are known, and may include carriers and/or diluents that are known for use in such compositions. The composition may contain 0.01-99% by weight of active compound. Preferably, a unit dose comprises the active compound in an amount of 1 μg to 10 mg. For oral administration suitable doses are 10μg per kg to 100mg per kg, preferably 40μg per kg to 4 mg per kg.

Another aspect of the present invention provides pharmaceutical compositions which comprise a compound of the invention and a pharmaceutically acceptable carrier. The term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the invention, additional active ingredient(s), and pharmaceutically acceptable excipients.

The pharmaceutical compositions of the present invention comprise a compound of the invention as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids, and salts of quaternary ammonium compounds with pharmaceutically acceptable counter-ions.

For delivery by inhalation, the active compound is preferably in the form of microparticles. They may be prepared by a variety of techniques, including spray-drying, freeze-drying and micronisation.

By way of example, a composition of the invention may be prepared as a suspension for delivery from a nebuliser or as an aerosol in a liquid propellant, for example for use in a pressurised metered dose inhaler (PMDI). Propellants suitable for use in a PMDI are known to the skilled person, and include CFC- 12, HFA-134a, HFA-227, HCFC-22 (CCI₂F₂) and HFA-152 (C₂H₄F₂) and isobutane.

In a preferred embodiment of the invention, a composition of the invention is in dry powder form, for delivery using a dry powder inhaler (DPI). Many types of DPI are known. Microparticles for delivery by administration may be formulated with excipients that aid delivery and release. For example, in a dry powder formulation, microparticles may be formulated with large carrier particles that aid flow from the DPI into the lung. Suitable carrier particles are known, and include lactose particles; they may have a mass median aerodynamic diameter of greater than 90 μm.

In the case of an aerosol-based formulation, an example is: Compound of the invention 24 mg / canister

Lecithin, NF Liq. Cone. 1.2 mg / canister Trichlorofluoromethane, NF 4.025 g / canister

Dichlorodif luoromethane, NF 12.15 g / canister.

The active compounds may be dosed as described depending on the inhaler system used. In addition to the active compounds, the administration forms may additionally contain excipients, such as, for example, propellants (e.g. Frigen in the case of metered aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, fillers (e.g. lactose in the case of powder inhalers) or, if appropriate, further active compounds.

For the purposes of inhalation, a large number of systems are available with which aerosols of optimum particle size can be generated and administered, using an inhalation technique which is appropriate for the patient. In addition to the use of adaptors (spacers, expanders) and pear-shaped containers (e.g. Nebulator®, Volumatic®), and automatic devices emitting a puffer spray (Autohaler®), for metered aerosols, in particular in the case of powder inhalers, a number of technical solutions are available (e.g. Diskhaler®, Rotadisk®, Turbohaler® or the inhalers for example as described EP-A- 0505321 ). Additionally, compounds of the invention may be delivered in multi- chamber devices thus allowing for delivery of combination agents. Methods of Synthesis

The compounds of the invention of the present invention can be prepared according to the procedures of the following examples, using appropriate materials, and are further exemplified by the following specific examples. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds.

The compounds of the invention may be isolated in the form of their pharmaceutically acceptable salts, such as those described previously herein above. It may be necessary to protect reactive functional groups (e.g. hydroxy, amino, thio or carboxy) in intermediates used in the preparation of compounds of the invention to avoid their unwanted participation in a reaction leading to the formation of the compounds. Conventional protecting groups, for example those described by T. W. Greene and P. G. M. Wuts in "Protective groups in organic chemistry" John Wiley and Sons, 1999, may be used.

The compounds of the invention have activity as pharmaceuticals, in particular as dual adrenergic β2 receptor agonists and anticholinergic agents including muscarinic receptor (M1 , M2, and M3) antagonists, in particular M3 antagonists. Diseases and conditions which may be treated with the compounds of the invention and their pharmaceutically acceptable salts include:

1. respiratory tract: obstructive diseases of the airways including: asthma, including bronchial, allergic, intrinsic, extrinsic, exercise-induced, drug-induced

(including aspirin and NSAID-induced) and dust-induced asthma, both intermittent and persistent and of all severities, and other causes of airway hyper-responsiveness; chronic obstructive pulmonary disease (COPD); bronchitis, including infectious and eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis; sarcoidosis; farmer's lung and related diseases; hypersensitivity pneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis, idiopathic interstitial pneumonias, 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; osteoporosis; 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: arthitides (for example rheumatoid arthritis, osteoarthritis, gout or crystal arthropathy), other joint disease (such as intervertebral disc degeneration or temporomandibular joint degeneration), bone remodelling disease (such as osteoporosis, Paget's disease or osteonecrosis), polychondritits, 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; vulvo-vaginitis; 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-lgE syndrome, antiphospholipid syndrome;

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

13. 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 gastroenteritis, 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 compound of the invention or a pharmaceutically-acceptable salt thereof as hereinbefore defined for use in therapy. In a further aspect, the present invention provides the use of a compound of the invention or a pharmaceutically acceptable salt thereof as hereinbefore defined in the manufacture of a medicament for use in therapy.

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 compound of the invention or a pharmaceutically acceptable salt thereof 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).

The compounds of the invention and pharmaceutically acceptable salts thereof may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the the invention compound/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 preferably comprise from 0.05 to 99 %w (per cent by weight), more preferably from 0.05 to 80 %w, still more preferably from 0.10 to 70 %w, and even more preferably 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 compound of the invention or a pharmaceutically acceptable salt thereof 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 compound of the invention or a pharmaceutically acceptable salt thereof 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 creams, solutions, suspensions, heptafluoroalkane (HFA) aerosols and dry powder formulations, for example, formulations in the inhaler device known as the Turbuhaler^®; or systemically, e.g. by oral administration in the form of tablets, capsules, syrups, powders or granules; or by parenteral administration in the form of solutions or suspensions; or by subcutaneous administration; or by rectal administration in the form of suppositories; or transdermally.

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

The compounds 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 compound of the invention with a carrier substance, for example, a mono-, di- or polysaccharide, a sugar alcohol, or another polyol. Suitable carriers are sugars, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol; and starch. Alternatively the finely divided compound may be coated by another substance. The powder mixture may also be dispensed into hard gelatine capsules, each containing the desired dose of the active compound. Another possibility is to process the finely divided powder into spheres which break up during the inhalation procedure. This spheronized powder may be filled into the drug reservoir of a multidose inhaler, for example, that known as the Turbuhaler^® in which a dosing unit meters the desired dose which is then inhaled by the patient. With this system the active ingredient, with or without a carrier substance, is delivered to the patient.

For oral administration the compound 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 coated tablets are required, the cores, 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 soft gelatine capsules, the compound of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound 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.

Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, saccharine and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art. The compounds of the invention may also be administered in conjunction with other compounds used for the treatment of the above conditions. The invention therefore further relates to combination therapies wherein a compound of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or formulation comprising a compound of the invention, is administered concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of one or more of the conditions listed.

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, the compounds of the invention may be combined with the following agents: non-steroidal anti-inflammatory agents (hereinafter NSAIDs) including non-selective cyclo-oxygenase COX-1 / COX-2 inhibitors whether applied topically or systemically (such as piroxicam, diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such as mefenamic acid, indomethacin, sulindac, azapropazone, pyrazolones such as phenylbutazone, salicylates such as aspirin); selective COX-2 inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib, lumarocoxib, parecoxib and etoricoxib); cyclo- oxygenase inhibiting nitric oxide donors (CINODs); glucocorticosteroids (whether administered by topical, oral, intramuscular, intravenous, or intraarticular 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 compound of the invention, or a pharmaceutically acceptable salt thereof, together with a cytokine or agonist or antagonist of cytokine function, (including agents which act on cytokine signalling pathways such as modulators of the SOCS system) including alpha-, beta-, and gamma- interferons; insulin-like growth factor type I (IGF-1); interleukins (IL) including IL1 to 17, and interleukin antagonists or inhibitors such as anakinra; tumour necrosis factor alpha (TNF-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 compound of the invention, or a pharmaceutically acceptable salt thereof, with a monoclonal antibody targeting B-Lymphocytes (such as CD20 (rituximab), MRA-alLI6R) or T-Lymphocytes (CTLA4-lg, HuMax 11-15).

The present invention still further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, with a modulator of chemokine receptor function such as an antagonist of CCR1 , CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C-C family); CXCR1 , CXCR2, CXCR3, CXCR4 and CXCR5 (for the C-X-C family) and CX₃CRI for the C-X₃-C family.

The present invention further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, with an inhibitor of matrix metalloprotease (MMPs), i.e., the stromelysins, the collagenases, and the gelatinases, as well as aggrecanase; especially collagenase-1 (MMP- 1 ), collagenase-2 (MMP-8), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11 ) and MMP-9 and MMP- 12, including agents such as doxycycline.

The present invention still further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, and a leukotriene biosynthesis inhibitor, 5-lipoxygenase (5-LO) inhibitor or 5- lipoxygenase activating protein (FLAP) antagonist such as; zileuton; ABT-761 ; fenleuton; tepoxalin; Abbott-79175; Abbott-85761 ; a N-(δ-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 compound of the invention, or a pharmaceutically acceptable salt thereof, and a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4. selected from the group consisting of the phenothiazin-3-1s such as L-651 ,392; amidino compounds such as CGS-25019c; benzoxalamines such as ontazolast; benzenecarboximidamides such as BIIL 284/260; and compounds such as zafirlukast, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, and an antagonist of the histamine type 4 receptor. The present invention still further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, and an alpha-1/alpha-2 adrenoceptor agonist vasoconstrictor sympathomimetic agent, such as propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, tramazoline hydrochloride or ethylnorepinephrine hydrochloride. The present invention further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, and a chromone, such as sodium cromoglycate or nedocromil sodium.

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

The present invention still further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, together with an immunoglobulin (Ig) or Ig preparation or an antagonist or antibody modulating Ig function such as anti-lgE (for example omalizumab).

The present invention further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, and another systemic or topically-applied anti-inflammatory agent, such as thalidomide or a derivative thereof, a retinoid, dithranol or calcipotriol. The present invention still further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, 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 antidepressant agent-s, paracetamol, or a non-steroidal anti-inflammatory agent. The present invention further relates to the combination of a compound of the invention, or a pharmaceutically acceptable salt thereof, together with a parenterally or topically-applied (including inhaled) local anaesthetic agent such as lignocaine or a derivative thereof.

A compound of the present invention, or a pharmaceutically acceptable salt thereof, 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 compound of the invention, or a pharmaceutically acceptable salt thereof, together with a: (i) tryptase inhibitor; (ii) platelet activating factor (PAF) antagonist; (iii) interleukin converting enzyme (ICE) inhibitor; (iv) IMPDH inhibitor; (v) adhesion molecule inhibitors including VLA-4 antagonist; (vi) cathepsin; (vii) kinase inhibitor such as an inhibitor of tyrosine kinase (such as Btk, Itk, Jak3 or MAP, for example Gefitinib or lmatinib mesylate), a serine / threonine kinase (such as an inhibitor of a MAP kinase such as p38, JNK, protein kinase A, B or C, or IKK), or a kinase involved in cell cycle regulation (such as a cylin dependent kinase); (viii) glucose-6 phosphate dehydrogenase inhibitor; (ix) kinin-B.subi . - 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.subL 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 (GR-receptor) agonist.

In a further aspect the present invention provides a combination (for example for the treatment of COPD, asthma or allergic rhinitis) of a compound of the invention and one or more agents selected from the list comprising: o a non-steroidal glucocorticoid receptor (GR-receptor) agonist; o a PDE4 inhibitor including an inhibitor of the isoform PDE4D; o a modulator of chemokine receptor function (such as a CCR 1 receptor antagonist); o a steroid (such as budesonide); and o an inhibitor of p38 kinase function. A compound of the invention, or a pharmaceutically acceptable salt thereof, can also be used in combination with an existing therapeutic agent for the treatment of cancer, for example suitable agents include: (i) an antiproliferative/antineoplastic drug or a combination thereof, as used in medical oncology, such as an alkylating agent (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan or a nitrosourea); an antimetabolite (for example an antifolate such as a fluoropyrimidine like 5-fluorouracil or tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, gemcitabine or paclitaxel); an antitumour antibiotic (for example an anthracycline such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin or mithramycin); an antimitotic agent (for example a vinca alkaloid such as vincristine, vinblastine, vindesine or vinorelbine, or a taxoid such as taxol or taxotere); or a topoisomerase inhibitor (for example an epipodophyllotoxin such as etoposide, teniposide, amsacrine, topotecan or a camptothecin); (ii) a cytostatic agent such as an antioestrogen (for example tamoxifen, toremifene, raloxifene, droloxifene or iodoxyfene), an oestrogen receptor down regulator (for example fulvestrant), an antiandrogen (for example bicalutamide, flutamide, nilutamide or cyproterone acetate), a LHRH antagonist or LHRH agonist (for example goserelin, leuprorelin or buserelin), a progestogen (for example megestrol acetate), an aromatase inhibitor (for example as anastrozole, letrozole, vorazole or exemestane) or an inhibitor of 5α-reductase such as finasteride;

(iii) an agent which inhibits cancer cell invasion (for example a metalloproteinase inhibitor like marimastat or an inhibitor of urokinase plasminogen activator receptor function);

(iv) an inhibitor of growth factor function, for example: a growth factor antibody (for example the anti-erbb2 antibody trastuzumab, or the anti-erbb1 antibody cetuximab [C225]), a farnesyl transferase inhibitor, a tyrosine kinase inhibitor or a serine/threonine kinase inhibitor, an inhibitor of the epidermal growth factor family (for example an EGFR family tyrosine kinase inhibitor such as N- (3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4- amine (gefitinib, AZD1839), N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) or 6-acrylamido-N-(3- chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine (Cl 1033)), an inhibitor of the platelet-derived growth factor family, or an inhibitor of the hepatocyte growth factor family; (v) an antiangiogenic agent such as one which inhibits the effects of vascular endothelial growth factor (for example the anti-vascular endothelial cell growth factor antibody bevacizumab, a compound disclosed in WO 97/22596, WO 97/30035, WO 97/32856 or WO 98/13354), or a compound that works by another mechanism (for example linomide, an inhibitor of integrin αvβ3 function or an angiostatin);

(vi) a vascular damaging agent such as combretastatin A4, or a compound disclosed in WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 or WO 02/08213; (vii) an agent used in antisense therapy, for example one directed to one of the targets listed above, such as ISIS 2503, an anti-ras antisense;

(viii) an agent used in a gene therapy approach, for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; or (ix) an agent used in an immunotherapeutic approach, for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.

The following detailed description and non-limiting Examples illustrate the invention.

GENERAL EXPERIMENTAL DETAILS:

Silica used for medium pressure column chromatography is 0.035 to 0.070 mm (220 to 440 mesh) (e.g. Fluka silica 60), and an applied pressure up to 10 psi accelerated column elution. Where thin layer chromatography (TLC) has been used, it refers to silica TLC using plates, typically 3 x 6 cm silica on aluminium foil plates with a fluorescent indicator (254 nm) (e.g. Fluka 60778). All solvents and commercial reagents were used as received. Purification by pre-packed SCX-2 cartridge refers to Isolute^® SCX-2, a strong cation exchange sorbent (Argonaut/IST).

All compounds containing a basic centre(s) and purified by reversed- phase HPLC were obtained as the TFA salt, unless stated otherwise.

Preparative reverse-phase HPLC conditions: HPLC System

Purification using a C18 Gemini column (250 x 21.20 mm with 5 μm particle size, Phenomenex), eluting using linear gradients of mixtures of solvent A (water with 0.1 % formic acid) and solvent B (MeCN with 0.1% formic acid) at a flow rate of 5-10 mL/min with UV detection set at 230 nm.

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

LC-MS Method 1 Micromass Platform LCT with a C18 reversed-phase column (30 x 4.6 mm i.d., Phenomenex Luna, 3 μm particle size), elution using linear gradients of mixtures of solvent A (water with 0.1% formic acid) and solvent B (MeCN with 0.1% formic acid) at a flow rate of 1 mL/min. Gradient:

Gradient - Time flow mL/min %A %B

0.00 2.0 95 5

0.50 2.0 95 5

4.50 2.0 5 95

5.50 2.0 5 95

6.00 2.0 95 5

Detection: MS, ELS, UV (100 μL split to MS with in-line UV detector). MS ionisation method: Electrospray (positive and negative ion).

LC-MS Method 2 Waters Micromass ZQ with a C18 reversed-phase column (30 x 4.6 mm i.d., Phenomenex Luna, 3 μm particle size), elution using linear gradients of mixtures of solvent A (water with 0.1 % formic acid) and solvent B (MeCN with 0.1% formic acid) at a flow rate of 1 ml_/min. Gradient:

GGrraaddiieenntt -- TTiimmee ffllooww mmll__//mmiinn %%AA %B

0.00 2.0 95 5

0.50 2.0 95 5

4.50 2.0 5 95

5.50 2.0 5 95

6.00 2.0 95 5

LC-MS Method 3

Waters ZMD with a C18 reversed-phase column (30 x 4.6 mm i.d., Phenomenex Luna, 3 μm particle size), elution using linear gradients of mixtures of solvent A (water with 0.1% formic acid) and solvent B (MeCN with 0.1 % formic acid) at a flow rate of 1 ml_/min. Gradient:

Gradient - Time flow mL/min %A %B 0.00 2.0 95 5

0.50 2.0 95 5

4.50 2.0 5 95

5.50 2.0 5 95

6.00 2.0 95 5

Detection: MS₁ ELS, UV (200μl_/min split to MS with in-line Waters 996 DAD detection).

MS ionisation method: Electrospray (positive and negative ion).

LC-MS Method 4

Waters Micromass ZQ with a C18 reversed-phase column (100 x 3.0 mm, Higgins Clipeus, 5 μm particle size), elution using linear gradients of mixtures of solvent A (water with 0.1 % formic acid) and solvent B (MeCN with 0.1 % formic acid) at a flow rate of 1 mL/min. Gradient:

Gradient - Time flow ml/min %A %B

0.00 1.0 95 5

1.00 1.0 95 5

15.00 1.0 5 95

20.00 1.0 5 95

22.00 1.0 95 5

25.00 1.0 95 5

Detection: MS, ELS, UV (100 μl split to MS with in-line UV detector at 254 nm) MS ionisation method: Electrospray (positive ion).

LCMS Method 5 Finnigan AQA series using a C18 reversed-phase column (30 x 4.6 mm, Luna, 2.0 μm particle size), eluting using linear gradients of mixtures of solvent A (water with 0.1 % Formic acid) and solvent B (MeCN with 0.1 % Formic acid) at a flow rate of 2.5 ml_/min. Gradient:

Gradient - Time flow mL/min %A %B

0.00 2.0 95 5

0.30 2.0 95 5

2.70 2.0 5 95

2.80 2.0 5 95

2.90 2.0 95 5

Detection: MS, ELS, UV (200 μL/min split to ESI source). MS ionisation method: Electrospray (positive ion).

Abbreviations used in the experimental section: AIBN = (2,2'-azobis(2-methylproprionitrile) Atm = atmospheres

(R)-(+)-CBS = (R)-(+)-methyl-CBS-oxazaborolidine Companion = Teledyne ISCO CombiFlash Companion automated purification

DCM = dichloromethane DIPEA = diisopropylethylamine DMF = Λ/,/V-dimethylformamide DMSO = dimethylsulfoxide e.e. = enantiomeric excess EtOAc = ethyl acetate EtOH = ethanol equiv. = equivalents h = hour(s)

HATU = (>(7-azabenzotriazol-1-yl)-N,Λ/,Λ/'Λ/-tetramethyluronium- hexafluorophosphate HPLC = high performance liquid chromatography

LC-MS = liquid chromatography-mass spectrometry

MeCN = acetonitrile

MeOH = methanol min = minute(s)

NBS = Λ/-bromosuccinimide

NMP = Λ/-methyl-pyrrolidone quant. = quantitative

RT = room temperature Rf = retention factor (TLC)

Rt = retention time (LC-MS or HPLC)

SCX-2 = strong cation exchange resin

TBDMS = tert-butyldimethylsilyl

TBDPS = te/t-butyldiphenylsilyl TFA = trifluoroacetic acid

THF = tetrahydrofuran

TLC = thin layer chromatography

Intermediates

Intermediate 1 (5-Bromomethyl-oxazol-2-yl)-diphenyl-methanol

The title compound was prepared from (5-methyl-oxazol-2-yl)-diphenyl- methanol by a similar method to that disclosed in WO2007/017669. LC-MS (Method 1 ): Rt 3.53 min, m/z 344, 346 [MH]+. Intermediate 2 (RHδ-Bromomethyl-oxazol^-ylJ-cyclohexyl-phenyl-methanol

To a DCM (10 mL) solution of cyanogen bromide (3 M in DCM; 0.32 ml_, 0.96 mmol) was added, portion wise, a solution of (R)-cyclohexyl-(5- dimethylaminomethyl-oxazol-2-yl)-phenyl-methanol (150 mg, 0.48 mmol) (prepared according to WO2007/017669) in DCM (2 mL). After 45 min the reaction mixture was applied to a silica gel cartridge and purified by gradient elution from petrol ether (40-60 ⁰C) to DCM, and afforded the title compound as a clear oil that solidified on standing.

Yield: 168 mg (65%).

LC-MS (Method 3): Rt 4.15 min, m/z 350, 352 [MH]+. Rf = 0.24 (10% EtOAc/cyclohexane).

Intermediate 3 (R)-[5-(2-Amino-ethyl)-oxazol-2-yl]-cyclohexyl-phenyl-methanol

The title compound was prepared from (R)-(5-bromomethyl-oxazol-2- yl)-cyclohexyl-phenyl-methanol according to WO2007/017669. LC-MS (Method 3): Rt 2.31 min, m/z 301 [MH]+.

Intermediate 4 (RHδ^-Benzylamino-ethyO-oxazol^-yπ-cyclohexyl-phenyl-methanol

Benzaldehyde (2.90 g, 27.3 mmol) was added to a mixture of (R)-[5-(2- amino-ethyl)-oxazol-2-yl]-cyclohexyl-phenyl-methanol (4.26 g, 14.2 mmol) and 3A molecular sieves in dry methanol (200 mL) and the reaction mixture stirred at RT for 18 h. Sodium borohydride (2.19 g, 57.9 mmol) was added to the resulting cooled (0 ⁰C) brown suspension and stirred at this temperature for 3 h. The reaction mixture was treated with saturated NaHCO3 (aq) and vigorously stirred for 1 h. Water and DCM were added and the mixture was filtered over Celite, washed with DCM, and the phases separated. The aqueous layer was extracted with DCM and the combined organic extracts were washed with brine, dried (Na₂SO₄), and then concentrated in vacuo Xo afford the title compound. The compound was used without further purification.

LC-MS (Method 3): Rt 2.61 min, m/z 391 [MH]+.

Intermediate 5 ((RHδ-^Benzyl-methyl-aminoJ-ethylJ-oxazol^-ylj-cyclohexyl-phenyl- methanol

To a stirred solution of (R)-[5-(2-benzylamino-ethyl)-oxazol-2-yl]- cyclohexyl-phenyl-methanol (10.9 g, 28 mmol) in DCM (150 ml_) was added formaldehyde (37 wt% (aq), 10.60 mL, 142 mmol), followed by sodium triacetoxyborohydride (9.06 g, 42.7 mmol). The reaction mixture was stirred vigorously at RT for 1 h and was then treated with saturated NaHCO₃ (aq) (75 mL) and vigorous stirred for a further for 15 min, then left unstirred overnight. The phases were separated and the aqueous layer was extracted with DCM. The combined organic extracts were washed with brine, dried (Na₂SO₄), and concentrated to dryness to afford a brown gum that was purified over silica gel (330 g, Companion) eluting with 0-50% EtOAc/DCM to give the desired product as a yellow/brown viscous oil.

Yield: 8.66 g (78%, 2 steps).

LC-MS (Method 3): Rt 2.60 min, m/z 405 [MH]+.

Intermediate 6

(R)-Cyclohexyl-[5-(2-methylamino-ethyl)-oxazol-2-yl]-phenyl-methanol

A mixture of ((R)-{5-[2-(benzyl-methyl-amino)-ethyl]-oxazol-2-yl}- cyclohexyl-phenyl-methanol (3.25 g, 7.23 mmol) and palladium(ll) hydroxide on carbon (505 mg) in ethanol (75 mL) was stirred under a hydrogen atmosphere at RT for 18 h. The reaction mixture was filtered through Celite, the Celite pad washed with ethanol, and the filtrate concentrated in vacuo to afford a light yellow/brown gum. Analytical chiral HPLC (Chiralpak IA, 5%

EtOH/heptane + 0.1 % DEA) indicated an enantiomeric excess of >99% (other enantiomer not observed - racemic sample was used as a reference). Yield: 2.58 g (quant.). LC-MS (Method 3): Rt 2.42 min, m/z 315 [MH]+. Intermediate 7 [5-(2-Amino-ethyl)-oxazol-2-yl]-diphenyl-methanol

The title compound was prepared from (5-bromomethyl-oxazol-2-yl)-diphenyl- methanol (Intermediate 1 ) using a similar procedure to that disclosed in WO2007/017669. LC-MS (Method 2): Rt 1.82 min, m/z 295 [MH]+.

Intermediate 8 (5-Methyl-isoxazol-3-yl)-diphenyl-methanol

To a solution of 5-methyl-isoxazole-3-carboxylic acid methyl ester (Maybridge) (45 g, 0.32 mol) in anhydrous in THF (400 ml) under an atmosphere of nitrogen at 0 ⁰C was added drop-wise a 1 M solution of phenylmagnesium bromide in THF (418 ml, 0.42 mol). As soon as the addition was complete, the solution was allowed to warm to RT. After stirring for 18 h at RT, the solution was again cooled to 0 ⁰C and further phenyl magnesium bromide solution (260 ml_, 0.26 mol) was added. After stirring to RT overnight, the reaction mixture was quenched via addition of saturated NH₄CI (aq) (600 ml_) and extracted into EtOAc (3 x 300 mL). The organic layer was separated and washed successively with saturated NaHCO₃ (aq), water and brine, prior to drying (MgSO₄) and concentration in vacuo. The residue was purified by column chromatography (Companion, 300 g), eluting with 0-50% EtOAc/cyclohexane to afford the title compound. Yield: 41 g (48%).

LC-MS (Method 2): Rt 3.42 min, m/z 265 [MH]+.

Intermediate 9 (5-Bromomethyl-isoxazol-3-yl)-diphenyl-methanol

The title compound was prepared from (5-methyl-isoxazol-3-yl)- diphenyl-methanol by a similar method to that described for Intermediate 1. Product contained -50% un-reacted (5-methyl-isoxazol-3-yl)-diphenyl- methanol and was used without further purification.

LC-MS (Method 2): Rt 3.66 min, m/z 344, 346 [MH]+.

Intermediate 10 (5-Methylaminomethyl-isoxazol-3-yl)-diphenyl-methanol

A solution of (5-bromomethyl-isoxazol-3-yl)-diphenyl-methanol (45 mol%, 700 mg, 0.94 mmol) in THF (10 mL) was added by syringe pump over 2 h to a stirred solution of methylamine in ethanol (10 mL), and the reaction mixture was then stirred at RT overnight. The reaction mixture was concentrated in vacuo and taken up in MeCN/DCM (white solids formed) and passed through an SCX-2 cartridge which was rinsed with MeCN, then eluted with 2 M NH₃ in MeOH to afford the desired product.

Yield: 277 mg (quant.).

LC-MS (Method 3): Rt 2.28 min, m/z 295 [MH]+.

Intermediate 11 (5-Aminomethyl-isoxazol-3-yl)-diphenyl-methanol

Aqueous ammonia (10 mL, 33%) was added to a solution of (5- bromomethyl-isoxazol-3-yl)-diphenyl-methanol (770 mg, 2.23 mmol) in methanol (10 mL). The reaction mixture was stirred at RT overnight to afford a brown solution with some fine solids. The reaction mixture was concentrated in vacuo and partitioned between DCM and saturated NaHCOβ (aq). The aqueous layer was separated and back-extracted with DCM and the combined organic extracts were dried (Na₂SO₄) and concentrated to afford a brown viscous oil. This was purified over silica (12 g, Companion) eluting with 0-10% MeOH/DCM to afford the title compound as a light brown foam Yield: 190 mg (31%).

LC-MS (Method 3): Rt 2.10 min, m/z 281 [MH]+.

Intermediate 12

2-{4-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-methyl]-phenyl}-ethanol

A solution of 2-(4-(bromomethyl)phenyl)-ethanol (322 mg, 1.05 mmol), (prepared according to Tetrahedron Letters 1987, 28(13), 1401-4), (R)- cyclohexyl-[5-(2-methylamino-ethyl)-oxazol-2-yl]-phenyl-methanol (300 mg, 0.95 mmol) (Intermediate 6) and triethylamine (159 μl_, 1.14 mmol) in MeCN (7.8 ml_) was stirred at RT for 2 h. The solvent was concentrated in vacuo and the residue was partitioned between EtOAc (100 ml_) and water (10 ml_), the organic phase was separated and further washed with water, brine, then dried (Na₂SO₄). , Concentration in vacuo followed by purification by flash silica chromatography (Companion, 40 g cartridge), gradient eluting with 0- 10%MeOH/DCM, afforded the title compound as a foam. Yield: 232 mg (54%).

LC-MS (Method 3): Rt 2.57 min, m/z 449 [MH]+.

Intermediate 13

{4-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-methyl]-phenyl}-acetaldehyde

Dess-Martin periodinane (154 mg, 0.36 mmol) was added to a solution of 2-{4-[({2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-methyl]-phenyl}-ethanol (65 mg, 0.14 mmol) and TFA (11.2 μl_, 0.14 mmol) in DCM (5.2 ml_). After stirring for 30 min further Dess-Martin periodinane (62 mg, 0.14 mmol) was added and after another 40 min the reaction was quenched by addition of saturated NaHCO₃ (aq) (5.9 mL) and saturated aqueous sodium thiosulfate (5.9 mL). After stirring for 10 min the reaction mixture was extracted with EtOAc (50 mL) and the organic phase was washed with saturated NaHCO₃ (aq), water and then brine prior to drying (Na₂SO₄), filtering and concentrating in vacuo. The compound was used without further purification.

LC-MS (Method 3): Rt 2.56, 2.61 min, m/z 447 [MH]+.

Intermediate 14

8-Acetyl-5-benzyloxy-4Af-benzo[1 ,4]oxazin-3-one

Chloroacetyl chloride (1.25 mL, 15.5 mmol) was added to a stirred mixture of 1-(3-amino-4-benzyloxy-2-hydroxy-phenyl)-ethanone (US2005/0277632) (3.63 g, 14.1 mmol) and NaHCO₃ (2.69 g, 32.0 mmol) in dry DMF (20 mL). The reaction mixture was stirred at RT for 2 h. Caesium carbonate (5.53 g, 17.0 mmol) was added and the mixture was heated at 100 ⁰C, protected from moisture, for 2.5 h. The reaction mixture was cooled to RT and poured into water (-300 mL) to afford a turbid, milky solution from which a solid precipitated. The solid was removed by vacuum filtration and taken up in EtOAc by sonication and the aqueous layer was extracted with EtOAc. The combined organic layers were further washed with water, brine, dried (MgSO₄), and concentrated in vacuo Xo afford a dark brown solid. Yield: 3.60 g (86%). LC-MS (Method 3): Rt 3.24 min, m/z 298 [M+H]⁺.

Intermediate 15 5-Benzyioxy-8-(2-chloro-acetyl)-4f/-benzo[1 ,4]oxazin-3-one

Benzyltrimethylammonium dichloroiodate (9.27 g, 26.6 mmol) was added to a stirred solution of 8-acetyl-5-benzyloxy-4/-/-benzo[1 ,4]oxazin-3-one (3.60 g, 12.1 mmol) in a mixture of DCM (65 ml_), acetic acid (22 ml_), and water (3.6 mL) that was heated at 65 ⁰C and protected from light, for 18 h. The reaction mixture was cooled to RT and a solution of sodium bisulphite (3.78 g) in water (65 mL) was added and mixed vigorously to form a suspension. After stirring for 0.5 h diethyl ether (150 mL) was added and the solids collected by filtration, washed with water, then ether and dried at 40 ⁰C in vacuo to afford a light brown crystalline solid. Yield: 3.56 g (89%). LC-MS (Method 3): Rt 3.50 min, m/z 332 [M+H]⁺.

intermediate 16 8-(2-Azido-acetyl)-5-benzyloxy-4H-benzo[1,4]oxazin-3-one

Sodium azide (1.18 g, 18.09 mmol) was added to a suspension of 5- benzyloxy-8-(2-chloro-acetyl)-4/-/-benzo[1 ,4]oxazin-3-one (4.8 g, 14.47 mmol) in DMF (50 mL) and stirred for 2 h. The mixture was poured onto ice/water and the resulting solid filtered off, washed with water and dried in vacuo at 40

⁰C to afford the title compound as a light brown solid. Yield: 4.6 g (90%).

¹H NMR (300 MHz₁ d₆-DMSO): δ 10.42 (s, 1 H), 7.55 (m, 2H), 7.48 (m,

1 H), 7.43 - 7.29 (m, 3H), 6.97 (m, 1 H), 5.31 (s, 2H), 4.69 (s, 2H), 4.63

(s, 2H) ppm.

Intermediate 17 8-(2-Azido-1-hydroxy-ethyl)-5-benzyloxy-4H-benzo[1,4]oxazin-3-one

A suspension of 8-(2-azido-acetyl)-5-benzyloxy-4H-benzo[1 ,4]oxazin-3- one (2 g, 5.91 mmol) in EtOH (80 mL) was treated with sodium borohydride (224 mg, 5.91 mmol) and the resultant mixture stirred at 20 ⁰C for 1.5 h. The mixture was partitioned between EtOAc and brine, the organic layer was dried, filtered and the solvent removed in vacuo. The crude solid was triturated with acetone (20 mL) to yield the title compound as a white solid. Yield: 1.60 g (80%).

The enantiomers of the title compound were separated on a Chiralpak AS column 250 x 50 mm (20 μm), eluting with EtOH: From a total of 2.5 g of racemic alcohol was isolated 0.67 g (Rt = 8 min) of the first eluting enantiomer and 0.72 g (Rt = 12 min) of the second eluting enantiomer. Re-analysis of first and second eluting enantiomers (Chiralpak IS, EtOH) showed first enantiomer to be >99% e.e and second eluting enantiomer to be >96% e.e.

Intermediate 18 8-(2-Amino-1-hydroxy-ethyl)-5-hydroxy-4H-benzo[1 ,4]oxazin-3-one

A mixture of 8-(2-amino-1-hydroxy-ethyl)-5-hydroxy-4/-/- benzo[1 ,4]oxazin-3-one (Intermediate 17, first eluting enantiomer) (0.67 g, 1.97 mmol) in EtOH (30 ml_) with 10% Pd/C (210 mg, 1.97 mmol) was stirred vigorously under 4 atm pressure of hydrogen for 18 h. The catalyst was filtered off and the solvent removed in vacuo, and the residue was re- dissolved in acetic acid (15 ml_) and EtOH (15 ml_) and the mixture stirred with 10% Pd/C (210 mg, 1.97 mmol) under 4 atm pressure of hydrogen for a further 18 h. This procedure was repeated twice until the reaction was complete. The mixture was then filtered, the solvent removed in vacuo, and the residue triturated with MeCN (30 ml_) to yield the title compound as a grey solid.

Yield: 330 mg (59%). 1 H NMR (300 MHz, d₆-DMSO): δ 6.85 (d, J = 8.5 Hz, 1 H)₁ 6.50 (d, J =

8.5 Hz, 1 H), 4.74 - 4.69 (m, 1 H), 4.52 - 4.42 (m, 2H), 2.74 - 2.50 (m,

2H), 1.83 (s, 3H) ppm.

Intermediate 19 Λ/-(4-Hydroxymethyl-phenyl)-acrylamide

To a solution of 4-aminobenzyl alcohol (2.5 g, 20.0 mmol) and triethylamine (5.6 mL, 41.0 mmol) in DCM (40 ml_) at 0 ⁰C was added drop- wise acryloyl chloride (1.60 mL, 20.0 mmol). An exotherm was observed and the mixture was then stirred at 0 ⁰C for 2 days. The reaction was quenched by careful addition of MeOH and concentrated in vacuo. The residue was partitioned between 2 M HCI (aq) and EtOAc and the aqueous phase was re- extracted with EtOAc. The combined organic extracts were washed with water and brine, dried (Na₂SO₄), then filtered and concentrated in vacuo. The residue was purified by column chromatography eluting from with 5-100% EtOAc/pentane to afford the desired product as a yellow solid .

Yield: 1.13 g (32%).

LC-MS (Method 1): Rt 2.20 min, m/z 176 [MH]+.

Intermediate 20 3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-/V-(4-hydroxymethyl-phenyl)-propionamide

To a solution of Λ/-(4-hydroxymethyl-phenyl)-acrylamide (68 mg, 0.38 mmol) in NMP (1 ml_) was added (R)-cyclohexyl-[5-(2-methylamino-ethyl)- oxazol-2-yl]-phenyl-methanol (100 mg, 1 mmol) (Intermediate 6), and the resulting mixture stirred at 70 °C for 3 days. The reaction mixture was cooled, diluted with MeOH and applied to a pre-equilibrated (MeOH) SCX-2 cartridge (5 g, Isolute). The cartridge was washed with MeOH (10OmL) and then eluted with 2 M NH₃ in MeOH solution. After concentrating in vacuo the residue was further purified by column chromatography (Companion, 4 g), eluting 0-10% (1 % triethylamine/MeOH)/DCM to afford the required product as a colourless glass. Yield: 63 mg (34%).

LC-MS (Method 5): Rt 2.54 min, m/z 492 [MH]+.

Intermediate 21 3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-/V-(4-formyl-phenyl)-propionamide

Manganese(IV) oxide (250 mg, 2.70 mmol) was added to a stirred solution of 3-({2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-Λ/-(4-hydroxymethyl-phenyl)-propionamide (137 mg, 0.27 mmol) in DCM (3 mL) and the resulting mixture heated to reflux for 1 h. The mixture was filtered through a pad of Celite, the pad washed with DCM and the filtrate concentrated in vacuo to afford the crude aldehyde, used without further purification.

Yield: 136 mg (quant.).

LC-MS (Method 5): Rt 2.64 min, m/z 490 [MH]+.

Intermediate 22

/V-(4-{[(R)-2-(tert-Butyl-dimethyl-silanyloxy)-2-(8-hydroxy-2-oxo-1 ,2- dihydro-quinolin-5-yl)-ethylamino]-methyl}-phenyl)-3-({2-[2-((R)- cyclohexyl-hydroxy-phenyl-methylj-oxazol-δ-yπ-ethyll-methyl-amino)- propionamide

A solution of 3-({2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5- yl]-ethyl}-methyl-amino)-Λ/-(4-formyl-phenyl)-propionamide (136 mg, 0.28 mmol) and 5-[(/7)-2-amino-1 -(te/?-butyl-dimethyl-silanyloxy)-ethyl]-8-(4- methoxy-benzyloxy)-1 /-/-quinolin-2-one (0.15 g, 0.33 mmol) (prepared according to WO2007/102771) in MeOH (2 ml_) was stirred over 3A molecular sieves at RT for 24 h. The solution was treated portion-wise with sodium triacetoxyborohydride (127 mg, 0.60 mmol) and allowed to stir at RT for 3 h. The reaction mixture was quenched by the addition of saturated NaHCO₃ (aq) and then extracted with DCM, and the organic layer dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was purified by column chromatography (Companion, 12 g), eluting with 0-20% (1% triethylamine/MeOH)/DCM to afford the required product as a yellow solid.

Yield: 36 mg (30%).

LC-MS (Method 5): Rt 2.66 min, m/z 808 [MH]+.

Intermediate 23

3-(Benzyl-{2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-amino)-propionic acid

A solution of (R)-[5-(2-benzylamino-ethyl)-oxazol-2-yl]-cyclohexyl- phenyl-methanol (300 mg, 0.77 mmol) (Intermediate 4) and acrylic acid (83 mg, 1.15 mmol) in DCM (5 mL) was heated in sealed tube at 45 ⁰C for 60 h. After cooling and concentration in vacuo the title compound was obtained as a beige solid and was used without further purification.

LC-MS (Method 2): Rt 2.87 min, m/z 463 [MH]+. Intermediate 24

3-(Benzyl-{2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-amino)-/V-(4-hydroxymethyl-phenyl)-propionamide

HATU (439 mg, 0.77 mmol) was added to a stirred solution of 3- (benzyl-{2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- amino)-propionic acid (356 mg, 0.77 mmol), (4-amino-phenyl)-methanol (142 mg, 1.15 mmol) and DIPEA (370 μL, 2.11 mmol) in DCM (25 ml_). After 20 h the reaction mixture was filtered and then concentrated in vacuo. The crude product was purified by flash silica chromatography (Companion, 12 g), gradient eluting with 0-5% (0.1 %triethylamine/MeOH)/DCM to afford the title compound as a yellow oil. Yield: 119 mg (27%).

LC-MS (Method 3): Rt 2.72 min, m/z 568 [MH]+.

Intermediate 25

{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-[2-(4- hydroxymethyl-phenylcarbamoyl)-ethyl]-carbamic acid terf-butyl ester

A solution of 3-(benzyl-{2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)- oxazol-5-yl]-ethyl}τamino)-Λ/-(4-hydroxymethyl-phenyl)-propionamide (66 mg, 0.12 mmol ), 10% Pd/C (~ 10 mg) and di-tert-butyl dicarbonate (25 mg, 0.15 mmol) in EtOAc (2.5 mL) was hydrogenated at RT overnight (H₂ filled balloon). The mixture was filtered through a pad of Celite eluting with DCM and the solution evaporated in vacuo to afford the title product as a white solid that was used without further purification.

Yield: 57 mg (82%).

LC-MS (Method 3): Rt 3.93 min, m/z 578 [MH]+.

Intermediate 26

{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-[2-(4- formyl-phenylcarbamoyl)-ethyl]-carbamic acid ferf-butyl ester

The title compound was prepared from {2-[2-((R)-cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-[2-(4-hydroxymethyl-phenylcarbamoyl)- ethyl]-carbamic acid te/t-butyl ester by similar methods to those employed for Intermediate 21.

LC-MS (Method 5): Rt 4.50 min, m/z 576 [MH]+.

Intermediate 27

{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-[2-(4- {[(R)-2-hydroxy-2-(4-hydroxy-2-oxo-2,3-dihydro-benzothiazol-7-yl)- ethylamino]-methyl}-phenylcarbamoyl)-ethyl]-carbamic acid terf-butyl ester

The title compound was prepared from {2-[2-((R)-cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-[2-(4-formyl-phenylcarbamoyl)-ethyl]- carbamic acid tert-butyl ester and 7-((R)-2-amino-1-hydroxy-ethyl)-4-hydroxy- 3H-benzothiazol-2-one hydrochloride (prepared according to WO2007/027133) by a similar method to that employed for Intermediate 22.

LC-MS (Method 5): Rt 3.05 min, m/z 786 [MH]+.

Intermediate 28

[2-(4-{[(R)-2-(tert-Butyl-dimethyl-silanyloxy)-2-(8-hydroxy-2-oxo-1,2- dihydro-quinolin-5-yl)-ethylamino]-methyl}-phenylcarbamoyl)-ethyl]-{2- [2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-carbamic acid terf-butyl ester

The title compound was prepared from {2-[2-((R)-cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-[2-(4-formyl-phenylcarbamoyl)-ethyl]- carbamic acid te/t-butyl ester and 5-[(f?)-2-amino-1-(te/t-butyl-dimethyl- silanyloxy)-ethyl]-8-(4-methoxy-benzyloxy)-1 /-/-quinolin-2-one by a similar method to that employed for Intermediate 22.

LC-MS (Method 3): Rt 3.22 min, m/z 894 [MH]+.

Intermediate 29

5-Benzyloxy-8-(2-chloro-1-hydroxy-ethyl)-4H-benzo[1,4]oxazin-3-one

To a suspension of 5-benzyloxy-8-(2-chloro-acetyl)-4H- benzo[1 ,4]oxazin-3-one (8.38 g, 25.3 mmol) in dry THF (150 ml_) under nitrogen was added (R)-(+)-CBS (5.0 mL, 5.0 mmol, 1 M in toluene) and the mixture was cooled to -15 ⁰C. Borane-THF complex (28 mL, 28 mmol, 1 M in THF) was slowly added via syringe pump over 2 h, after which the reaction mixture had become a clear solution with some solids present. The reaction was quenched by addition of MeOH (12 mL) (gas evolved) and was then allowed to warm to RT. After this time a beige crystalline material precipitated, which was removed by filtration. This was found to be unreacted starting material. The filtrate was partitioned between water and EtOAc, and the water layer was further extracted with EtOAc. The combined organic layers were washed with brine, dried (MgSO₄), and concentrated in vacuo to afford a beige, sticky gum. This material was dissolved in DCM and some insoluble white solids were removed by vacuum filtration, which were found to be CBS by-products. The filtrate was concentrated and purified by chromatography over silica (Companion, 120 g cartridge) eluting with 0-20% EtOAc/DCM to afford the title product as an off-white solid. Analysis by chiral HPLC (Chiralpak IA, 15% IPA/heptane, 1 ml_/min) revealed 90% e.e. of the first eluting enantiomer (Rt = 21.0 min) (Second enantiomer: Rt = 24.5 min). A single crystallisation from refluxing EtOAc (35 mL)/heptane (20 mL) afforded the title compound (first eluting enantiomer) as small colourless crystals in 97% e.e. as determined by chiral HPLC analysis.

Yield: 4.76 g (59%).

LC-MS (Method 3): Rt 3.16 min, m/z 332 [M-H]^".

Intermediate 30 δ-Benzyloxy-β-fi-ttert-butyl-dimethyl-silanyloxyJ^-chloro-ethylJ^W- benzo[1 ,4]oxazin-3-one

2,6-Lutidine (3.4 mL, 29 mmol) was added to a solution of 5-benzyloxy-8-(2- chloro-1-hydroxy-ethyl)-4/-/-benzo[1 ,4]oxazin-3-one (first eluting enantiomer) (4.76 g, 14.3 mmol) in dry DMF (30 mL) at 0 ⁰C under a nitrogen atmosphere, followed by slow addition of TBDMS trifluoromethanesulfonate (6.4 mL, 28 mmol). Fumes were formed and the reaction mixture was stirred below 10 °C for 5 min, after which the reaction mixture was allowed to warm to RT. After 45 min MeOH (3.0 mL) was added cautiously and the reaction mixture was poured into water to afford a white suspension. This was extracted with EtOAc (2x) and the combined organic layers were washed with water, brine, dried (MgSO₄), and concentrated in vacuo Xo afford the title compound as a white solid, which was used without further purification. Yield: 6.34 g (99%). LC-MS (Method 3): Rt 5.14 min, molecular ion no observed.

Intermediate 31

8-[2-Azido-1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-5-benzyloxy-4H- benzo[1 ,4]oxazin-3-one

A mixture of 5-benzyloxy-8-[1-(tert-butyl-dimethyl-silanyloxy)-2-chloro- ethyl]-4/-/-benzo[1 ,4]oxazin-3-one (3.00 g, 5.7 mmol), sodium azide (2.28 g, 35.1 mmol), and potassium iodide (5.85 g, 35.2 mmol) in dry DMF (24 ml_) was heated at 120 °C under a nitrogen atmosphere. After heating for 72 h the reaction mixture was cooled to RT and poured into water. The solids were collected by filtration and washed with water. This material was then taken up in EtOAc, dried (MgSO₄), filtered, and concentrated in vacuo. The crude product was purified over a short silica column to remove baseline material, eluting with 20-25% EtOAc/cyclohexane, and afforded the title compound as a white solid.

Yield: 1.2O g (46%). 1H NMR (400 MHz, CDCI₃): δ 7.77 (br. s, 1 H), 7.45 - 7.35 (m, 5H), 7.14

(d, J = 8.8 Hz, 1 H), 6.69 (d, J = 8.8 Hz, 1 H), 5.13 (m, 1 H), 5.08 (m, 2H), 4.48 (m, 2H), 3.25 (m, 2H), 0.92 (s, 9H), -0.04 (s, 6H) ppm.

Intermediate 32 8-[2-Amino-1-(ferf-butyl-dimethyl-silanyloxy)-ethyl]-5-hydroxy-4H- benzo[1 ,4]oxazin-3-one

A solution of 8-[2-azido-1 -(te/f-butyl-dimethyl-silanyloxy)-ethyl]-5- benzyloxy-4H-benzo[1 ,4]oxazin-3-one (1.20 g, 2.64 mmol) in EtOAc/EtOH (15 mL, 1 :1) in the presence of a catalytic amount of Pd/C was stirred under a hydrogen atmosphere at RT for 48 h. The reaction mixture was filtered through Celite, the pad was washed with EtOH/EtOAc, and the filtrate was concentrated in vacuo Xo afford the title compound as a light brown foam, which was used without further purification.

Yield: 892 mg (quant.). LC-MS (Method 3): Rt 2.33 min, m/z 339 [MH]⁺.

Intermediate 33

[2-(4-{[2-(fe/t-Butyl-dimethyl-silanyloxy)-2-(5-hydroxy-3-oxo-3,4-dihydro- 2H-benzo[1,4]oxa2in-8-yl)-ethylamino]-methyl}-phenylcarbamoyl)-ethyl]- {2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- carbamic acid te/t-butyl ester

The title compound was prepared from 8-[2-amino-1-(tert-butyl- dimethyl-silanyloxy)-ethyl]-5-hydroxy-4/-/-benzo[1 ,4]oxazin-3-one and {2-[2- ((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-[2-(4-formyl- phenylcarbamoyl)-ethyl]-carbamic acid tert-butyl ester (Intemediate 26) using a similar method to that employed for Intermediate 22. LC-MS (Method 3): Rt 3.34 min, m/z 898 [MH]+. Intermediate 34 2-[3-(3-Bromo-propoxy)-phenyl]-ethanol

A solution of 3-(2-hydroxy-ethyl)-phenol (10 g, 72.46 mmol), 1 ,3- dibromopropane (22 ml_, 0.218 mol) and K₂CO₃ (16 g, 0.115 mol) in acetone (80 ml_) was warmed to 60 ⁰C overnight. The reaction mixture was cooled, filtered, and then concentrated in vacuo. The residue was purified by silica chromatography, eluting with 25-50% EtOAc/pentane to afford the title compound as a colourless oil.

Yield: 16.69 g (89%).

TLC: Rf = 0.33 (25% EtOAc/pentane).

¹H NMR (400 MHz, CDCI₃) D 7.21 (m, 1 H), 6.84-6.73 (m, 3H), 4.08 (t, J = 6 Hz, 2H), 3.81 (m, 2H), 3.58 (t, J = 6.5 Hz, 2H), 2.81 (t, J = 6.5 Hz,

2H), 2.29 (m, 2H), 1.81 (br s, 1 H) ppm.

Intermediate 35

2-{3-[3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-am i no)-propoxy]-pheny l}-ethanol

A solution of 2-[3-(3-bromo-propoxy)-phenyl]-ethanol (284 mg, 0.95 mmol), (R)-cyclohexyl-[5-(2-methylamino-ethyl)-oxazol-2-yl]-phenyl-methanol (Intermediate 6) (300 mg, 0.95 mmol) and triethylamine (159 μL, 1.14 mmol) in MeCN (7.8 ml_) was stirred at RT overnight. The solvent was removed in vacuo and the residue was partitioned between EtOAc (100 ml_) and water (10 ml_). The organic phase was further washed with water and brine prior to drying (Na₂SO₄), filtering and concentrating in vacuo. Purification by flash silica chromatography (Companion, 12 g cartridge), gradient eluting with 0- 10% MeOH/DCM afforded the title compound as a colourless gum.

Yield: 281 mg (60%).

LC-MS (Method 3): Rt 2.57 min, m/z 493 [MH]+.

Intermediate 36 (R)-{5-[2-({3-[3-(2-Bromo-ethyl)-phenoxy]-propyl}-methyl-amino)-ethyl]- oxazol-2-yl}-cyclohexyl-phenyl-methanol

A cooled (0 ⁰C) solution of 2-{3-[3-({2-[2-((R)-cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-methyl-amino)-propoxy]-phenyl}-ethanol (130 mg, 0.26 mmol) and carbon tetrabromide (175 mg, 0.53 mmol) in DCM (5 ml_) was treated dropwise with a solution of triphenylphospine (104 mg, 0.40 mmol) in DCM (0.5 ml_). The reaction mixture was allowed to stir at this temperature for 1 h and then to warm to RT for 1 h. Water was added and the DCM layer separated and dried (Na₂SO₄), filtered and concentrated in vacuo. The crude product was purified on silica (Companion, 12 g cartridge), loading in DCM and gradient eluting with 0-50% triethylamine/cyclohexane to afford the desired compound. Yield: 56 mg (38%).

LC-MS (Method 3): Rt 2.95 min, m/z 555 & 557 [MH]+.

Intermediate 37 5-[(R)-1-(te/t-Butyl-dimethyl-silanyloxy)-2-(2-{3-[3-({2-[2-((R)-cyclohexyl- hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-methyl-amino)-propoxy]- phenyl}-ethylamino)-ethyl]-8-hydroxy-1 W-quinolin-2-one

A solution (R)-{5-[2-({3-[3-(2-bromo-ethyl)-phenoxy]-propyl}-methyl- amino)-ethyl]-oxazol-2-yl}-cyclohexyl-phenyl-methanol (66 mg, 0.12 mmol), 5- [(/?)-2-amino-1-(te/t-butyl-dimethyl-silanyloxy)-ethyl]-8-(4-methoxy-benzyloxy)- 1 H-quinolin-2-one (27 mg, 0.081 mmol) and DIPEA (21 μl_, 0.12 mmol) in

DMF (1 ml_) was stirred at 80 ⁰C for 20 h. Water (10 ml_) was added and ether contents extracted with EtOAc (2 x 20 mL), the organic phases separated, dried (Na₂SO₄), filtered, and then concentrated in vacuo. The crude product was purified by HPLC to give afford the desired product as a formic acid salt. Yield: 45 mg (46%).

LC-MS (Method 3): Rt 2.63 min, m/z 809 [MH]+.

Intermediate 38

{5-[({3-[4-(2-Bromo-ethyl)-phenoxy]-propyl}-methyl-amino)-methyl]- isoxazol-3-yl}-diphenyl-methanol

The title compound was prepared from (5-methylaminomethyl-isoxazol- 3-yl)-diphenyl-methanol (Intermediate 10) and 2-[4-(3-bromo-propoxy)- phenyl]-ethanol (prepared according to US 2004/0167167) using similar methods to those employed for Intermediates 35 and 36, respectively.

LC-MS (Method 3): Rt 2.95 min, m/z 535 & 537 [MH]+.

Intermediate 39

5-((R)-1-(fert-Butyl-dimethyl-silanyloxy)-2-{2-[4-(3-{[3-(hydroxy-diphenyl- methyl)-isoxazol-5-ylmethyl]-methyl-amino}-propoxy)-phenyl]- ethylamino}-ethyl)-8-hydroxy-1 W-quinolin-2-one

The title compound was prepared from {5-[({3-[4-(2-bromo-ethyl)- phenoxy]-propyl}-methyl-amino)-methyl]-isoxazol-3-yl}-diphenyl-methanol and 5-[(/?)-2-amino-1-(tøAt-butyl-dimethyl-silanyloxy)-ethyl]-8-(4-methoxy- benzyloxy)-1 H-quinolin-2-one using a similar method to that employed for 37.

LC-MS (Method 3): Rt 2.51 min, m/z 789 [MH]+.

Intermediate 40 [3-(Hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]-{3-[4-(2-hydroxy- ethyl)-phenoxy]-propyl}-carbamic acid terf-butyl ester

A solution of (5-methylaminomethyl-isoxazol-3-yl)-diphenyl-methanol (Intermediate 10) (540 mg, 1.93 mmol) and 2-[4-(3-bromo-propoxy)-phenyl]- ethanol (759 mg, 2.72 mmol) was converted into the intermediate secondary amine using a similar method to that employed for Intermediate 35. The crude reaction mixture was cooled (0 ⁰C) and treated with a solution in THF (2 ml_) of di-tert-butyl dicarbonate (505 mg, 2.31 mmol) in THF (2 mL) and left to stir at RT for 72 h. The reaction mixture was diluted with EtOAc, washed with diluted saturated NaHCO₃ (aq), 2x water, brine, dried (MgSO₄), and then concentrated in vacuo to afford a light yellow/brown viscous oil (1.51 g). This was purified over silica (40 g, Companion), gradient eluting with 0-50% EtOAc/cyclohexane to afford the title compound as colourless gum. Yield: 574 mg (53%).

LC-MS (Method 3): Rt 4.09 min, m/z 559 [MH]+.

Intermediate 41

{3-[4-(2-Bromo-ethyl)-phenoxy]-propyl}-[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-carbamic acid ferf-butyl ester

The title compound was prepared from [3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-{3-[4-(2-hydroxy-ethyl)-phenoxy]-propyl}-carbamic acid terf-butyl ester using a similar method to that employed for Intermediate 36. LC-MS (Method 3): Rt 4.84 min, m/z 621 & 623 [MH]+.

Intermediate 42 5-((R)-1-(fert-Butyl-dimethyl-silanyloxy)-2-{2-[4-(3-{[3-(hydroxy-diphenyl- methyl)-isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}- ethyl)-8-hydroxy-1 W-quinolin-2-one trifluoroacetate salt

A solution of {3-[4-(2-bromo-ethyl)-phenoxy]-propyl}-[3-(hydroxy- diphenyl-methyl)-isoxazol-5-ylmethyl]-carbamic acid tert-butyl ester (52 mg, 0.083 mmol) and 5-[(fl)-2-amino-1-(te/t-butyl-dimethyl-silanyloxy)-ethyl]-8-(4- methoxy-benzyloxy)-1 /-/-quinolin-2-one (43 mg, 0.13 mmol) was converted into the intermediate Boc/TBDMS compound using a similar method to that employed for Intermediate 37. After purification over silica, eluting with 0-7% MeOH/DCM, the intermediate was dissolved in DCM (8 ml_) and treated with TFA (1 ml_) and allowed to stir at RT for 45 min. Toluene was added and the reaction was concentrated in vacuo to remove excess TFA. This procedure was repeated to afford the title compound as a TFA salt. Yield: 30 mg (41%). LC-MS (Method 3): Rt 2.53 min, m/z 775 [MH]+.

Intermediate 43

[3-(4-{2-[(R)-2-(2,2-Dimethyl-4H-benzo[1,3]dioxin-6-yl)-2-hydroxy- ethylamino]-ethyl}-phenoxy)-propyl]-[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-carbamic acid terf-butyl ester

The title compound was prepared from {3-[4-(2-bromo-ethyl)-phenoxy]- propyl}-[3-(hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]-carbamic acid tert- butyl ester (Intermediate 41 ) and (R)-2-amino-1 -(2,2-dimethyl-4/-/- benzo[1 ,3]dioxin-6-yl)-ethanol (Prepared according to WO 2005/044787) using a similar method to that employed for Intermediate 37.

LC-MS (Method 3): Rt 3.04 min, m/z 764 [MH]+.

Intermediate 44

(R)-[5-(2-{[5-(2-Bromo-ethyl)-thiophen-2-ylmethyl]-methyl-amino}-ethyl)- oxazol-2-yl]-cyclohexyl-phenyl-methanol

The title compound was prepared from 5-(2-hydroxy-ethyl)-thiophene- 2-carbaldehyde (Prepared according to Phosphorus, Sulfur and Silicon and the Related Elements 1990, 53, 75-79) and (R)-cyclohexyl-[5-(2-methylamino- ethyl)-oxazol-2-yl]-phenyl-methanol (Intermediate 6) using similar methods to those employed for Intermediates 22 and 36, respectively.

LC-MS (Method 3): Rt 2.84 min, m/z 517 & 519 [MH]+.

Intermediate 45 {2-[2-(Hydroxy-diphenyl-methyl)-oxazol-5-yl]-ethyl}-[5-(2-hydroxy-ethyl)- thiophen-2-ylmethyl]-carbamic acid fe/t-butyl ester

A solution of 5-(2-hydroxy-ethyl)-thiophene-2-carbaldehyde (0.60 g, 3.84 mmol) and [5-(2-amino-ethyl)-oxazol-2-yl]-diphenyl-methanol (Intermediate 7) (1.13 g, 3.84 mmol) in dry MeOH (20 ml_) was allowed to stir at RT over 3A molecular sieves for 20 h. The reaction mixture was cooled in an ice bath, and then treated portion-wise with sodium triacetoxyborohydride. After warming to RT over 1.5 h, the reaction was quenched by addition of saturated NaHCO₃ (aq), and then extracted into EtOAc (2 x 50 ml_). The combined organics were washed with brine, dried (Na₂SO₄), filtered and concentrated in vacuo. The crude secondary amine (1.54 g, 3.55 mmol) was dissolved in THF (15 ml_) and treated with Et₃N (0.95 ml_, 7.1 mmol) and di- tert-butyl dicarbonate (0.93 g, 4.26 mmol). After 2 h the reaction was quenched with saturated NaHCO₃ (aq) and extracted into EtOAc (2 x 10 ml_). The combined organics were washed with water, then with brine, dried (Na₂SO₄), filtered and concentrated in vacuo. Purification by silica chromatography (Isolute cartridge; 25g, 70 ml_) eluting with DCM, then 0.5-1 % MeOH/DCM gave the title compound as an amber oil. Yield: 913 mg (44%). LC-MS (Method 3): Rt 3.80 min, m/z 535 [MH]+.

Intermediate 46

[5-(2-Bromo-ethyl)-thiophen-2-ylmethyl]-{2-[2-(hydroxy-diphenyl-methyl)- oxazol-5-yl]-ethyl}-carbamic acid fe/t-butyl ester

The title compound was prepared from {2-[2-(hydroxy-diphenyl-methyl)- oxazol-5-yl]-ethyl}-[5-(2-hydroxy-ethyl)-thiophen-2-ylmethyl]-carbamic acid terf-butyl ester using a similar method to that employed for Intermediate 36. LC-MS (Method 3): Rt 4.45 min, m/z 597 & 599 [MH]+.

Intermediate 47

[5-(2-Bromo-ethyl)-thiophen-2-ylmethyl]-{2-[2-((R)-cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-carbamic acid terf-butyl ester

The title compound was prepared from (R)-[5-(2-amino-ethyl)-oxazol-2- yl]-cyclohexyl-phenyl-methanol (Intermediate 3) and 5-(2-hydroxy-ethyl)- thiophene-2-carbaldehyde using similar methods to those employed for Intermediates 45 and 46, respectively.

LC-MS (Method 3): Rt 5.23 min, m/z 603 & 605 [MH]+.

Intermediate 48 Cyclobutyl-(5-methyl-oxazol-2-yl)-phenyl-methanol

A crystal of iodine was added to a stirred suspension of magnesium (2.35 g, 95.8 mmol) in dry THF (50 ml_) under a nitrogen atmosphere. After stirring at RT for 5 min the iodine colour was still present. Cyclobutyl bromide (12.10 g, 86.0 mmol) was added dropwise to the mixture and after about 2 ml_ was added the colour disappeared and heat was evolved. The addition was continued to maintain a steady exotherm. Dry THF (50 ml_) was added simultaneously. After the addition, the reaction mixture was heated at 65 ⁰C for 0.5 h (only a small portion of magnesium remaining in clear brown solution), allowed to cool to RT and then cooled on ice to afford a suspension. A solution of (5-methyl-oxazol-2-yl)-phenyl-methanone (Prepared according to WO 2008/017827) (16.5 g, 88.1 mmol) in dry THF (100 ml_) was added via a canula. Heat was evolved, but kept below 20 ⁰C by cooling on ice. The yellow/brown solution became a yellow/brown suspension. After the addition was complete the reaction was stirred at RT for 1 h and was then quenched by addition of saturated NH₄CI (aq) and the mixture was stirred vigorously for 0.5 h. EtOAc was added and the phases were separated. The water layer was extracted with 2x EtOAc and the combined organic extracts were washed with brine, dried (MgSO₄), and concentrated to afford a yellow/brown oil that crystallised on standing to afford the title compound. Yield: 17.4 g (Quant.).

¹H NMR (300 MHz, d₆-DMSO): δ 7.55 - 7.46 (m, 2H), 7.36 - 7.23 (m, 3H), 6.64 (s, 1 H), 3.56 (s, 1 H), 3.35 (m, 1 H), 2.27 (s, 3H), 2.18 - 2.00 (m, 2H), 1.97 - 1.56 (m, 4H) ppm.

Intermediate 49 (δ-Bromomethyl-oxazol^-yO-cyclobutyl-phenyl-methanol

AIBN (815 mg, 4.96 mmol) was added to a stirred solution of cyclobutyl-(5- methyl-oxazol-2-yl)-phenyl-methanol (11.73 g, 48.2 mmol) and NBS (9.54 g, 53.6 mmol) under a nitrogen atmosphere. The reaction mixture was heated at 80 ⁰C for 2 h, then allowed to cool to RT and washed with saturated NaHCO₃ (aq). The organic phase was separated and the aqueous layer was extracted with DCM and the combined organic layers were concentrated in vacuo to afford a brown viscous oil. This crude product was purified over silica (Companion, 330 g) eluting with 0-100% EtOAc/cyclohexane to afford the racemic product as a yellow/brown viscous oil.

Yield: 14.2 g (92%).

LC-MS (Method 3): Rt 3.77 min, m/z 304 & 306 [M-H₂O+H]+.

Separation of the enantiomers of (5-bromomethyl-oxazol-2-yl)- cyclobutyl-phenyl-methanol

Both enantiomers were separated on a preparative chiral HPLC (IA column) eluting with 30% THF/heptane:

Enantiomer 1 : Rt = 4.0 min, sharp peak (5.78 g), off-white solid; Enantiomer 2: Rt = 5.5 min, broad, tailing peak (5.67 g), off-white solid. Re-analysis of EnantiomeM and 2 (Chiralpak IA, 10% EtOAc/heptane) showed both enantiomers to be >99% e.e.

Intermediate 50 [5-(2-Amino-ethyl)-oxazol-2-yl]-cyclobutyl-phenyl-methanol

The title compound was prepared from [5-(2-amino-ethyl)-oxazol-2-yl]- cyclohexyl-phenyl-methanol (Intermediate 49, Enantiomer 1 ) using similar methods to those employed to prepare Intermediate 3.

LC-MS (Method 3): Rt 1.98, 2.08 min, m/z 273 [MH]+.

Intermediate 51

[S^-Bromo-ethyO-thiophen-Z-ylmethyll^-^-^RJ-cyclobutyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-carbamic acid terf-butyl ester

The title compound was prepared from [5-(2-amino-ethyl)-oxazol-2-yl]- cyclobutyl-phenyl-methanol (intermediate 50) and 5-(2-hydroxy-ethyl)- thiophene-2-carbaldehyde using similar methods to those employed for Intermediates 45 and 36, respectively.

LC-MS (Method 3): Rt 4.86 min, m/z 575 & 577 [MH]+.

Intermediate 52 te/t-Butyl-diphenyl-(2-thiophen-3-yl-ethoxy)-silane

A solution of 2-thiophen-3-yl-ethanol (4.89 g, 38.14 mmol) and imidazole (7.70 g, 0.11 mol) in DMF (30 ml_) was cooled to 0⁰C. A solution of TBDPS-CI in DMF (5 ml_) was added dropwise over 5 min and the reaction mixture was allowed to warm to RT. After a total of 5 h stirring, the reaction was partitioned between EtOAc/cyclohexane [1 :1] (200 ml_) and water/brine [1 :1] (200 ml_), and the organic phase was washed with water/brine [1 :1] (200 ml_), dried (Na₂SO₄), filtered and concentrated in vacuo. Purification by flash chromatography (Companion, 330 g) eluting with 0-10% EtOAc/cyclohexane the title compound.

Yield: 11.3 g (80%).

¹H NMR (400 MHz, CDCI₃): δ 7.64 - 7.57 (m, 4H), 7.43 - 7.32 (m, 6H),

3.84 (t, J = 7.1 Hz, 2H), 2.88 (t, J = 7.1 Hz, 2H), 1.03 (s, 9H).

Intermediate 53 te/*-Butyl-diphenyl-(2-thiophen-3-yl-ethoxy)-silane

A cooled -78°C solution of ter/-butyl-diphenyl-(2-thiophen-3-yl-ethoxy)- silane (17.44 g, 47.6 mmol) in THF was treated dropwise with nBuLi (1.6 M in hexanes: 37.8 ml_, 60.44 mmol) over 10 min. After a further 10 min of stirring, the reaction mixture was allowed to warm to -5°C. After stirring at -5 °C for 1 h reaction mixture was cooled to -78°C and dry DMF (36 ml_, 47.6 mmol) was added drop-wise over 10 min. After another 45 min of stirring, the reaction was allowed to warm to RT. The reaction was quenched by addition of water (15 ml_) and extracted into EtOAc (200 ml_). The organic phase was further washed with water, brine, dried (Na₂SO₄) and concentrated in vacuo. Purification by flash chromatography (Companion, 330 g) eluting with 0-10% EtOAc/cyclohexane afforded the title compound, which was found to contain -10% of the undesired 2,3-isomer as confirmed by ¹H NMR, but was used without further purification.

Yield: 15 g (77%).

¹H NMR (400 MHz, CDCI₃): δ 9.83 (d, J = 1.3 Hz, 1 H), 7.61 - 7.54 (m,

5H), 7.43 - 7.32 (m, 7H), 3.86 (t, J = 6.3 Hz, 2H), 2.87 (t, J = 6.3 Hz,

2H), 1.03 (s, 9H) ppm.

Intermediate 54

[4-(2-Bromo-ethyl)-thiophen-2-ylmethyl]-{2-[2-((R)-cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-carbamic acid terf-butyl ester

The title compound was prepared from teA?-butyl-diphenyl-(2-thiophen- 3-yl-ethoxy)-silane (1.31 g, 3.33 mol) and (R)-[5-(2-amino-ethyl)-oxazol-2-yl]- cyclohexyl-phenyl-methanol (Intermediate 3) (1g, 3.33 mmol) using a similar method to that employed for Intermediate 45. Prior to conversion to the desired product, the intermediate TBDPS ether was converted to the alcohol: A solution of {4-[2-(te/t-butyl-diphenyl-silanyloxy)-ethyl]-thiophen-2-ylmethyl}- {2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}carbamic acid tert-butyl ester in THF (5 ml_) was treated with TBAF (1 M in THF; 1.49 ml_, 1.49 mmol) and allowed to stir at RT for 2 h. The reaction was concentrated in vacuo and then partitioned between water and EtOAc (2 x 25 ml_). The combined organic extracts were washed with brine, dried (Na₂SO₄), filtered and concentrated in vacuo. Purification by silica chromatography (Isolute cartridge; 10 g, 70 ml_), eluting with 0-2% MeOH/DCM afforded the intermediate alcohol as an amber oil. The title compound was then prepared using a similar method to that employed for Intermediate 36.

Yield: 327 mg (16%, 4 steps). LC-MS (Method 2): Rt 4.91 min, m/z 603 & 605 [MH]+.

Intermediate 55 (5-Aminomethyl-oxazol-2-yl)-diphenyl-methanol

The title compound was prepared from (5-bromomethyl-oxazol-2-yl)- diphenyl-methanol (Intermediate 1) using a similar method to that employed for Intermediate 11. LC-MS (Method 2): Rt 2.02 min, m/z 281 [MH]+.

Intermediate 56

[5-(2-Bromo-ethyl)-thiophen-2-ylmethyl]-[2-(hydroxy-diphenyl-methyl)- oxazol-5-ylmethyl]-carbamic acid tert-butyl ester

The title compound was prepared from (5-aminomethyl-oxazol-2-yl)- diphenyl-methanol using similar methods to those employed for Intermediates 45 and 36, respectively.

LC-MS (Method 2): Rt 4.45 min, m/z 597 & 599 [MH]+.

Intermediate 57 2-(2-Bromo-thiazol-5-yl)-ethanol

A solution of copper(ll) bromide (268 mg, 1.20 mmol) in MeCN (5 ml_) was treated with tert-butyl nitrite (178 μl_, 1.50 mmol). To this mixture was added drop-wise a solution of 2-(2-amino-thiazol-5-yl)-ethanol (prepared according to WO8911476) (144 mg, 1 mmol) in MeCN. The reaction mixture was allowed to stir at RT for 4 h, and then partitioned between water and EtOAc. The organic extract was washed with brine, dried (Na₂SO₄), filtered and concentrated in vacuo. Purification by silica chromatography, eluting with 0-100% EtOAc/DCM afforded the title compound as an amber oil.

Yield: 38 mg (15%).

LC-MS (Method 2): Rt 2.27 min, m/z 207 & 209 [MH]+.

Intermediate 58 5-(2-Hydroxy-ethyl)-thiazole-2-carbaldehyde

The title compound was prepared from 2-(2-bromo-thiazol-5-yl)-ethanol using a similar method to that employed for Intermediate 53. LC-MS (Method 2): Rt 1.91 min, m/z 158 [MH]+.

Intermediate 59

[5-(2-Bromo-ethyl)-thiazol-2-ylmethyl]-{2-[2-(hydroxy-diphenyl-methyl)- oxazol-5-yl]-ethyl}-carbamic acid tert-butyl ester

The title compound was prepared from [5-(2-amino-ethyl)-oxazol-2-yl]- diphenyl-methanol (Intermediate 7) and 5-(2-hydroxy-ethyl)-thiazole-2- carbaldehyde using similar methods to those employed for Intermediates 45 and 36, respectively.

LC-MS (Method 2): Rt 4.19 min, m/z 598 & 600 [MH]+.

The invention will now be illustrated by reference to the following Examples 1 30:

Example 1

8-[2-(2-{4-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-methyl]-phenyl}-ethylamino)-1-hydroxy-ethyl]-5- hydroxy-4H-benzo[1,4]oxazin-3-one diformate salt

{4-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-methyl]-phenyl}-acetaldehyde (Intermediate 13) (64 mg, 0.144 mmol) and 8-(2-amino-1-hydroxy-ethyl)-5-hydroxy-4/-/-benzo[1 ,4]oxazin-3-one (Intermediate 18) (48 mg, 0.144 mmol) were dissolved in dry MeOH (6.8 ml_) and then acetic acid (8 μL, 0.144 mmol) and 4 M HCI in dioxane (36 μl_, 0.144 mmol) were added. After stirring for 1 h the reaction mixture was cooled to 0 °C and sodium triacetoxyborohydride was added at 0°C. The reaction mixture then was allowed to warm to RT over 15 min. After stirring for 72 h the reaction was quenched by addition of saturated NaHCO₃ (aq) (3.0 ml_) and extracted with EtOAc, and the organic phase was further washed with water, brine, dried (Na₂SO₄), filtered and concentrated in vacuo. The crude product was purified by HPLC to afford the desired product as a bisformic acid salt.

Yield: 7 mg (7%).

LC-MS (Method 4): Rt 5.68 min, m/z 655 [MH]+.

Example 2

3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-Λ/-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1 ,2-dihydro- quinolin-5-yl)-ethylamino]-methyl}-phenyl)-propionamide diformate salt

A solution of Λ/-(4-{[(R)-2-(te/?-butyl-dimethyl-silanyloxy)-2-(8-hydroxy- 2-0X0-1 , 2-dihydro-quinolin-5-yl)-ethylamino]-methyl}-phenyl)-3-({2-[2-((R)- cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}-methyl-amino)- propionamide (Intermediate 22) (140 mg, 0.17 mmol) in THF (6 ml_) under nitrogen was treated with triethylamine trihydrofluoride (70 μl_, 0.43 mmol). After stirring at RT overnight, the reaction mixture was neutralised with saturated NaHCO₃ (aq) and extracted with DCM. The combined organic extracts were washed with brine, dried (Na₂SO₄), filtered, and concentrated to dryness. The crude product was purified by HPLC to afford the desired product as a bisformic acid salt. Yield: 36 mg (30%).

LC-MS (Method 4): Rt 5.65 min, m/z 694 [MH]+.

Example 3

S^-^-^RJ-Cyclohexyl-hydroxy-phenyl-methylJ-oxazol-δ-yll-ethylamino}- Λ/-(4-{[(R)-2-hydroxy-2-(4-hydroxy-2-oxo-2,3-dihydro-benzothiazol-7-yl)- ethylamino]-methyl}-phenyl)-propionamide diformate salt

A solution of {2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-[2-(4-{[(R)-2-hydroxy-2-(4-hydroxy-2-oxo-2,3-dihydro-benzothiazol-7-yl)- ethylaminoj-methylj-phenylcarbamoylj-ethylj-carbamic acid te/t-butyl ester (Intermediate 27) (18 mg, 0.017 mmol) in DCM (5 ml_) was treated with TFA (800 μl_) and allowed to stir at RT for 1 h. The reaction mixture was treated with toluene (5 ml_) and concentrated in vacuo to afford the title compound as a white solid. The crude product was purified by HPLC (System 5) to afford the desired product as a bisformic acid salt.

Yield: 3.1 mg (27%).

LC-MS (Method 4): Rt 5.78 min, m/z 686 [MH]+.

Example 4 3-{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}- /V-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)- ethylamino]-methyl}-phenyl)-propionamide formate salt

The title compound was prepared from [2-(4-{[(R)-2-(te/ϊ-butyl-dimethyl- silanyloxy)-2-(8-hydroxy-2-oxo-1 ,2-dihydro-quinolin-5-yl)-ethylamino]-methyl}- phenylcarbamoyl)-ethyl]-{2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol- 5-yl]-ethyl}-carbamic acid te/f-butyl ester (Intermediate 28) by similar methods to those employed for Examples 3 and 2, respectively. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.77 min, m/z 680 [MH]+.

Example 5 S^-^-^RJ-Cyclohexyl-hydroxy-phenyl-methylJ-oxazol-S-ylJ-ethylamino}- N-(4-{[2-hydroxy-2-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[1 ,4]oxazin-8- yl)-ethylamino]-methyl}-phenyl)-propionamide formate salt

The title compound was prepared from [2-(4-{[2-(te/?-butyl-dimethyl- silanyloxy)-2-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[1 ,4]oxazin-8-yl)- ethylamino]-methyl}-phenylcarbamoyl)-ethyl]-{2-[2-((R)-cyclohexyl-hydroxy- phenyl-methyl)-oxazol-5-yl]-ethyl}-carbamic acid tert-butyl (Intermediate 33) by similar methods to those employed for Examples 3 and 2, respectively. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.76 min, m/z 684 [MH]+.

Example 6 S-ftRJ^^-IS-tS-tia-^-URJ-Cyclohexyl-hydroxy-phenyl-methyO-oxazol-δ- yl]-ethyl}-methyl-amino)-propoxy]-phenyl}-ethylamino)-1-hydroxy-ethyl]- 8-hydroxy-1 W-quinolin-2-one diformate salt

The title compound was prepared from 5-[(R)-1 -(te/?-butyl-dimethyl- silanyloxy)-2-(2-{3-[3-({2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5- yl]-ethyl}-methyl-amino)-propoxy]-phenyl}-ethylamino)-ethyl]-8-hydroxy-1 H- quinolin-2-one (Intermediate 37) by a similar method to that employed for Example 2. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 6.07 min, m/z 695 [MH]+.

Example 7

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-methyl-amino}-propoxy)-phenyl]-ethylamino}-ethyl)- 1 H-quinolin-2-one formate salt

The title compound was prepared from 5-((R)-1-(tert-butyl-dimethyl- silanyloxy)-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]- methyl-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-8-hydroxy-1 H-quinolin-2- one (Intermediate 39) by a similar method to that employed for Example 2. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.57 min, m/z 675 [MH]+.

Example 8 8-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-1 Ay- quinolin-2-one formate salt

The title compound was prepared from 5-((R)-1-(terf-butyl-dimethyl- silanyloxy)-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]- amino}-propoxy)-phenyl]-ethylamino}-ethyl)-8-hydroxy-1 /-/-quinolin-2-one trifluoroacetate salt (Intermediate 42) by a similar method to that employed for Example 2. The crude product was purified by HPLC to the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.46 min, m/z 661 [MH]+.

Example 9

5-Hydroxy-8-(1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-4H- benzo[1,4]oxazin-3-one formate salt

The title compound was prepared from {3-[4-(2-bromo-ethyl)-phenoxy]- propyl}-[3-(hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]-carbamic acid tert- butyl ester (Intermediate 41) and 8-[2-amino-1-(terf-butyl-dimethyl-silanyloxy)- ethyl]-5-hydroxy-4/-/-benzo[1 ,4]oxazin-3-one (Intermediate 32) by similar methods to those employed for Intermediate 42 and Example 2. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.55 min, m/z 665 [MH]+.

Example 10 yV-[2-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)- phenyl]-formamide formate salt

The title compound was prepared from {3-[4-(2-bromo-ethyl)-phenoxy]- propyl}-[3-(hydroxy-diphenyl-methyl)-isoxazol-5-ylmethyl]-carbamic acid tert- butyl ester (Intermediate 41 ) and Λ/-{5-[(R)-2-amino-1 -(tert-butyl-dimethyl- silanyloxy)-ethyl]-2-hydroxy-phenyl}-formamide (Prepared according to US 2007/0249675) by similar methods to those employed for Intermediate 42 and Example 2. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.62 min, m/z 637 [MH]+.

Example 11

4-((R)-1-Hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol-5- ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-2-hydroxymethyl- phenol formate salt

A solution of [3-(4-{2-[(R)-2-(2,2-dimethyl-4H benzo[1 ,3]dioxin-6-yl)-2- hydroxy-ethylamino]-ethyl}-phenoxy)-propyl]-[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-carbamic acid tert-butyl ester (Intermediate 43) (144 mg, 0.64 mmol) in DCM (4 mL) was treated with TFA (2 mL) and allowed to stir at RT for 30 min. Water (0.5 mL) was added to the reaction mixture and the resulting mixture stirred ar RT for 2 h. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.54 min, m/z 624 [MH]+.

Example 12

7-[(R)-2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy- ethyl]-4-hydroxy-3H-benzothiazol-2-one formate salt

The title compound was prepared from (R)-[5-(2-{[5-(2-bromo-ethyl)- thiophen^-ylmethylj-methyl-aminoJ-ethyO-oxazol^-ylj-cyclohexyl-phenyl- methanol (Intermediate 44) and 7-((R)-2-amino-1-hydroxy-ethyl)-4-hydroxy- 3H-benzothiazol-2-one hydrochloride (prepared according to WO2007/027133) by similar a method to that employed for Intermediate 37. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.74 min, m/z 663 [MH]+.

Example 13

5-[(R)-2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy- ethyl]-8-hydroxy-1 H-quinolin-2-one formate salt

The title compound was prepared from (R)-[5-(2-{[5-(2-bromo-ethyl)- thiophen-2-ylmethyl]-methyl-amino}-ethyl)-oxazol-2-yl]-cyclohexyl-phenyl- methanol (Intermediate 44) by similar methods to those employed for Intermediate 37 and Example 2, respectively. The crude product was purified by HPLC to afford the desired product as a formic acid salt. LC-MS (Method 4): Rt 5.59 min, m/z 657 [MH]+.

Example 14

8-[2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy- ethyl]-5-hydroxy-4H-benzo[1 ,4]oxazin-3-one formate salt

The title compound was prepared from (R)-[5-(2-{[5-(2-bromo-ethyl)- thiophen-2-ylmethyl]-methyl-amino}-ethyl)-oxazol-2-yl]-cyclohexyl-phenyl- methanol (Intermediate 44) and 8-[2-amino-1-(te/?-butyl-dimethyl-silanyloxy)- ethyl]-5-hydroxy-4/-/-benzo[1 ,4]oxazin-3-one (Intermediate 32) by similar methods to those employed for Intermediate 37 and Example 2, respectively. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.83 min, m/z 661 [MH]+.

Example 15

5-((R)-2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-8- hydroxy-1 H-quinolin-2-one formate salt

The title compound was prepared from [5-(2-bromo-ethyl)-thiophen-2- ylmethyl]-{2-[2-((R)-cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- carbamic acid terf-butyl ester (Intermediate 47) and 5-[(/?)-2-amino-1-(terf- butyl-dimethyl-silanyloxy)-ethyl]-8-(4-methoxy-benzyloxy)-1 /-/-quinolin-2-one by similar methods to those employed for Intermediate 42 and Example 2, respectively. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.61 min, m/z 643 [MH]+.

Examples 16-29, shown in the table below, with the exception of the benzothiazolone containing compounds, were prepared using similar methods to those described for Example 15. The benzothiazolone containing examples were prepared from 7-((R)-2-amino-1-hydroxy-ethyl)-4-hydroxy-3H- benzothiazol-2-one by similar methods to those employed for Intermediate 42. All compounds were purified by HPLC to afford the desired compounds as formic acid salts.

5-Hydroxy-8-(1 -hydroxy-2-{2-[5-

({2-[2-(hydroxy-diphenyl- 46 5.13 641 methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2- yl]-ethylamino}-ethyl)-4/-/- benzo[1 ,4]oxazin-3-one

Λ/-[2-Hydroxy-5-((R)-1 -hydroxy-

2-{2-[5-({2-[2-(hydroxy-diphenyl- 46 5.14 613 methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2- yl]-ethylamino}-ethyl)-phenyl]- formamide

5-((R)-2-{2-[5-({2-[2-((R)-

Cyclohexyl-hydroxy-phenyl- 54 5.53 643 methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-3- yl]-ethylamino}-1-hydroxy-ethyl)-

8-hydroxy-1 H-quinolin-2-one

Example 30 4-((R)-1-Hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-2-hydroxymethyl- phenol

The title compound was prepared from [5-(2-bromo-ethyl)-thiophen-2- ylmethyl]-{2-[2-(hydroxy-diphenyl-methyl)-oxazol-5-yl]-ethyl}-carbamic acid tert-butyl ester (Intermediate 46) and (R)-2-amino-1-(2,2-dimethyl-4H- benzo[1 ,3]dioxin-6-yl)-ethanol (Prepared according to WO 2005/044787) by similar methods to those employed for Intermediate 37 and Example 11 , respectively. The crude product was purified by HPLC to afford the desired product as a formic acid salt.

LC-MS (Method 4): Rt 5.00 min, m/z 600 [MH]+.

BIOLOGICAL ASSAYS

The inhibitory effects of compounds of the present invention at the M₃ muscarinic receptor and the β₂ adrenergic receptor may be determined by the following binding assays:

Assay for adrenergic β2 mediated cAMP production

CELL PREPARATION

H292 cells are grown in RPMI (Roswell Park Memorial Institute) medium containing, 10% (v/v) FBS (foetal bovine serum) and 2 mM L- glutamine. Cells are grown in 225cm2 flasks containing 25 mL media in a humidified incubator at 37°C, 5% CO₂. Cells are harvested from the flask and passaged at a 1 in 10 dilution once per week. EXPERIMENTAL METHOD

The media from flasks containing H292 cells is removed, rinsed with 10 mL PBS (phosphate buffered saline) and replaced with 10 ml_ Accutase™ cell detachment solution. Flasks are incubated for 15 minutes in a humidified incubator at 37°C, 5% CO₂. The cell suspension is counted and the cells 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 are added to each well of a tissue-culture-treated 96-well plate and the cells incubated overnight in a humidified incubator at 37°C, 5% CO₂. The culture media is removed, washed twice with 100 μL assay buffer and replaced with 50 μL assay buffer. Cells are 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) is added. Cells are incubated with rolipram for 10 minutes after which time test compounds (made up as x4 concentrated stocks in assay buffer containing 4% (v/v) dimethylsulphoxide) are added and the cells are incubated for 10 minutes at room temperature. Final rolipram concentration in the assay is 300 μM and final vehicle concentration is 1.6% (v/v) dimethylsulphoxide. The reaction is stopped by removing supematants, washing once with 100 μL assay buffer and replacing with 50 μL lysis buffer. The cell monolayer is frozen at -80 ⁰C for 30 minutes (or overnight).

ALPHASCREEN™ CAMP DETECTION

The concentration of cAMP (cyclic adenosine monophosphate) in the cell lysate is determined using the AlphaScreen™ methodology. The frozen cell plate is thawed for 20 minutes on a plate shaker then 10 μL of the cell lysate is transferred to a 96-well white plate. 40 μL of mixed AlphaScreen™ detection beads (containing equal volumes of donor beads (pre-incubated with biotinylated cAMP in the dark for 30 minutes) and acceptor beads), is added to each well and the plate incubated at room temperature for 10 hours in the dark. The AlphaScreen™ signal is measured using an EnVision spectrophotometer (Perkin-Elmer Inc.) with the recommended manufacturer's settings. cAMP concentrations are determined by reference to a calibration curve determined in the same experiment using standard cAMP concentrations (made up in lysis buffer in a 96-well tissure-culture-treated plate and frozen/thawed alongside the test samples) and detected using the same protocol. Concentration response curves for agonists are constructed to determine both the pEC₅₀ and Intrinsic Activity. Intrinsic Activity is expressed as a fraction relative to the maximum activity determined for formoterol in each experiment.

Alternative assay for Adrenergic β2 mediated cAMP production Cell preparation

H292 cells are grown in 225cm2 flasks incubator at 37°C, 5% CO₂ in RPMI medium containing10% (v/v) FBS (foetal bovine serum) and 2 mM L- glutamine. Experimental Method Adherent H292 cells re removed from tissue culture flasks by treatment with Accutase™ cell detachment solution for 15 minutes. Flasks are incubated for 15 minutes in a humidified incubator at 37°C, 5% CO₂. Detached cells are 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_ are added to each well of a tissue-culture-treated 96-well plate and the cells incubated overnight in a humidified incubator at 37°C, 5% CO₂. The culture media is removed and cells are 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 are 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) is added. Cells are incubated with rolipram for 10 minutes after which time test compounds are added and the cells are incubated for 60 minutes at room temperature. The final rolipram concentration in the assay is 300 μM and final vehicle concentration is 1.6% (v/v) dimethylsulphoxide. The reaction is stopped by removing supematants, washing once with 100 μL assay buffer and replacing with 50 μL lysis buffer. The cell monolayer is frozen at -80 °C for 30 minutes (or overnight). AlphaScreen™ cAMP detection

The concentration of cAMP (cyclic adenosine monophosphate) in the cell lysate is determined using AlphaScreen™ methodology. The frozen cell plate is thawed for 20 minutes on a plate shaker then 10 μl_ of the cell lysate is transferred to a 96-well white plate. 40 μL of mixed AlphaScreen™ detection beads pre-incubated with biotinylated cAMP, is added to each well and the plate incubated at room temperature for 10 hours in the dark. The AlphaScreen™ signal is measured using an EnVision spectrophotometer (Perkin-Elmer Inc.) or a Fusion αHT (Packard) with the recommended manufacturer's settings. cAMP concentrations are determined by reference to a calibration curve determined in the same experiment using standard cAMP concentrations. Concentration response curves for agonists are constructed and data is fitted to a four parameter logistic equation to determine both the pEC-50 and Intrinsic Activity. Intrinsic Activity is expressed as a fraction relative to the maximum activity determined for formoterol in each experiment. Compounds of the invention tested in this assay had a pEC₅₀ of < 1 μM and an intrinsic activity of > 0.6.

Muscarinic M3 receptor binding assay

The affinity (plC₅₀) of compounds to the M₃ receptor is determined by competition binding of [³H]N-methyl scopolamine (NMS) to CHO-K1 (Chinese Hamster Ovary) cell membranes expressing the human muscarinic acetylcholine M₃ receptor (M₃-ACh) in a scintillation proximity assay (SPA) format.

SPA beads are precoated with membranes and then incubated at 2mg of beads per well with serial dilutions of compounds of the invention, [³H]NMS at 0.2nM, half Kd (experimentally determined dissociation constant) and assay buffer (20 mM HEPES pH 7.4 containing 5 mM MgCI₂). The assay is conducted in a final volume of 200 μL, in the presence of 1 % (v/v) dimethyl sulphoxide (DMSO). Total binding of [³H]NMS is determined in the absence of competing compound and non-specific binding of [³H]NMS is determined in the presence of 1 μM atropine. The plates are incubated for 16 hours at room temperature and then read on Wallac Microbeta^™ using a normalised ³H protocol. The plC₅₀, defined as the negative logarithm of the concentration of compound required for 50% reduction in specific [³H]-NMS binding, is determined.

The compounds of Examples 1-30 were tested in the above M3 receptor binding and β2 cAMP functional assays and the following results obtained:

^*lntrinsic activity relative to Formoterol is shown in parenthesis. NT = Not Tested

Claims

1. A compound selected from the group consisting of

8-[2-(2-{4-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethyl}- methyl-amino)-methyl]-phenyl}-ethylamino)-1-hydroxy-ethyl]-5-hydroxy-4H- benzo[1 ,4]oxazin-3-one;

3-{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}-Λ/-(4- {[(R)-2-hydroxy-2-(4-hydroxy-2-oxo-2,3-dihydro-benzothiazol-7-yl)- ethylamino]-methyl}-phenyl)-propionamide; 3-{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}-Λ/-(4- {[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1 ,2-dihydro-quinolin-5-yl)-ethylamino]- methyl}-phenyl)-propionamide;

3-{2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}-N-(4- {[2-hydroxy-2-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[1 ,4]oxazin-8-yl)- ethylamino]-methyl}-phenyl)-propionamide; 5-[(R)-2-(2-{3-[3-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-propoxy]-phenyl}-ethylamino)-1 -hydroxy-ethyl]-8- hydroxy-1 H-quinolin-2-one;

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol- 5-ylmethyl]-methyl-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-1 /-/-quinolin-2- one;

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol- 5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-1 /-/-quinolin-2-one; 5-Hydroxy-8-(1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol-5- ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-4H-benzo[1 ,4]oxazin-3- one; Λ/-[2-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)- isoxazol-5-ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-phenyl]- formamide;

4-((R)-1-Hydroxy-2-{2-[4-(3-{[3-(hydroxy-diphenyl-methyl)-isoxazol-5- ylmethyl]-amino}-propoxy)-phenyl]-ethylamino}-ethyl)-2-hydroxymethyl-phenol;

7-[(R)-2-(2-{5-[({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyl}-methyl-amino)-methyl]-thiophen-2-yl}-ethylamino)-1-hydroxy-ethyl]-4- hydroxy-3H-benzothiazol-2-one;

1 /-/-quinolin-2-one;

8-(2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-5-hydroxy- 4H-benzo[1 ,4]oxazin-3-one;

/V-[5-((R)-2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethyiamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-2-hydroxy- phenyl]-formamide;

7-((R)-2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1 -hydroxy-ethyl)-4-hydroxy-

3/-/-benzothiazol-2-one;

1 /-/-quinolin-2-one; 8-(2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]-ethylamino}- methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-5-hydroxy-4/-/- benzo[1 ,4]oxazin-3-one; Λ/-[5-((R)-2-{2-[5-({2-[2-(Cyclobutyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-1-hydroxy-ethyl)-2-hydroxy- phenyl]-formamide;

4-Hydroxy-7-((R)-1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5- yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-3/-/-benzothiazol-2- one;

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5- yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-1 H-quinolin-2-one;

5-Hydroxy-8-(1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-4/-/-benzo[1 ,4]oxazin-3- one;

Λ/-[2-Hydroxy-5-((R)-1-hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)- oxazol-5-yl]-ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-phenyl]- formamide; 8-(2-{2-[5-({2-[2-((R)-Cyclohexyl-hydroxy-phenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-3-yl]-ethylamino}-1-hydroxy-ethyl)-5-hydroxy-

4H-benzo[1 ,4]oxazin-3-one;

8-Hydroxy-5-((R)-1-hydroxy-2-{2-[5-({[2-(hydroxy-diphenyl-methyl)-oxazol-5- ylmethyl]-amino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-1 /-/-quinolin-2-one; 8-Hydroxy-5-((R)-1 -hydroxy-2-{2-[2-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5- yl]-ethylamino}-methyl)-thiazol-5-yl]-ethylamino}-ethyl)-1 /-/-quinolin-2-one; and

4-((R)-1-Hydroxy-2-{2-[5-({2-[2-(hydroxy-diphenyl-methyl)-oxazol-5-yl]- ethylamino}-methyl)-thiophen-2-yl]-ethylamino}-ethyl)-2-hydroxymethyl- phenol; and pharmaceutically acceptable salts thereof.

2. A pharmaceutical composition comprising a compound as claimed in claim 1 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

3. A pharmaceutical composition as claimed in claim 2 in a form suitable for inhalation.

4. A compound as claimed in claim 1 , or a pharmaceutically acceptable salt thereof, for use in therapy.

5. A compound as claimed in claim 1 , or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the treatment of prevention of a disease or condition in which M3 muscarinic receptor activity and β-adrenergic activity is implicated.

6. A method of treatment of a disease or condition in which M3 muscarinic receptor activity is implicated comprising administration to a subject in need thereof of an effective amount of a compound, or a pharmaceutically acceptable salt thereof, as claimed in claim 1.

7. Use as claimed in claim 4 or 5 or a method of treatment as claimed in claim 6, wherein the disease or condition is a respiratory-tract disorder.

8. Use as claimed in claim 4 or 5 or a method of treatment as claimed in claim 6, wherein the disease or condition is chronic obstructive lung disease, chronic bronchitis, asthma, chronic respiratory obstruction, bronchial hyperactivity, pulmonary fibrosis, pulmonary emphysema, or allergic rhinitis.

9. A combination comprising a compound as claimed in claim 1 , or a pharmaceutically acceptable salt thereof, and an inhaled corticosteroid, or an inhaled PDE₄ inhibitor.