WO2010007561A1 - Novel compounds active as muscarinic receptor antagonists - Google Patents

Abstract

The invention relates to compounds of formula (I), processes and intermediates for their preparation, their use as muscarinic antagonists and pharmaceutical compositions containing them.

Description

Novel compounds active as muscarinic receptor antagonists

This invention relates to compounds of general formula (I):

in which R¹ to R⁵ and X have the meanings indicated below, and to processes and intermediates for the preparation of, compositions containing and the uses of such derivatives.

Cholinergic muscarinic receptors are members of the G-protein coupled receptor super-family and are further divided into 5 subtypes, M₁ to M₅. Muscarinic receptor sub-types are widely and differentially expressed in the body. Genes have been cloned for all 5 sub-types and of these, M₁, M₂ and M₃ receptors have been extensively pharmacologically characterized in animal and human tissue. M₁ receptors are expressed in the brain (cortex and hippocampus), glands and in the ganglia of sympathetic and parasympathetic nerves. M₂ receptors are expressed in the heart, hindbrain, smooth muscle and in the synapses of the autonomic nervous system. M₃ receptors are expressed in the brain, glands and smooth muscle. In the airways, stimulation of M₃ receptors evokes contraction of airway smooth muscle leading to bronchoconstriction, while in the salivary gland M₃ receptor stimulation increases fluid and mucus secretion leading to increased salivation. M₂ receptors expressed on smooth muscle are understood to be pro-contractile while pre-synaptic M₂ receptors modulate acetylcholine release from parasympathetic nerves. Stimulation of M₂ receptors expressed in the heart produces bradycardia. Short and long-acting muscarinic antagonists are used in the management of asthma and COPD; these include the short acting agents Atrovent® (ipratropium bromide) and Oxivent® (oxitropium bromide) and the long acting agent Spiriva® (tiotropium bromide). These compounds produce bronchodilation following inhaled administration. In addition to improvements in spirometric values, anti-muscarinic use in chronic obstructive pulmonary disease (COPD) is associated with improvements in health status and quality of life scores.

As a consequence of the wide distribution of muscarinic receptors in the body, significant systemic exposure to muscarinic antagonists is associated with effects such as dry mouth, constipation, mydriasis, urinary retention (all predominantly mediated via blockade of M₃ receptors) and tachycardia (mediated by blockade of M₂ receptors). A commonly reported side-effect following inhaled administration of therapeutic dose of the current, clinically used non-selective muscarinic antagonists is dry-mouth and while this is reported as only mild in intensity it does limit the dose of inhaled agent given.

Accordingly, there is still a need for M₃ receptor antagonists that would have an appropriate pharmacological profile, for example in term of potency, pharmacokinetics or duration of action, in particular when administered by the inhalation route.

The invention thus relates to a compound of formula (I)

or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, wherein:

X is -CH₂-, -C(=O)CH₂- or -C(=O) -; one of R², R³, R⁴ and R⁵ is hydroxy, one of R², R³, R⁴ and R⁵ is H and two of R², R³, R⁴ and R⁵ are independently H or halo;

R¹ is H or methyl or alternatively when X is -CH₂-, R¹ can also represent a group of formula:

wherein one of R⁶, R⁷, R⁸ and R⁹ is hydroxy, one of R⁶, R⁷, R⁸ and R⁹ is H and two of R⁶, R⁷, R⁸ and R⁹ are independently H or halo.

In the here above general formula (I), halo denotes a halogen atom selected from the group consisting of fluoro, chloro, bromo and iodo. Preferred halo groups are fluoro and chloro.

In the above compounds of formula (I), the following definitions and combinations of such definitions are preferred :

Preferably R¹ is H or methyl.

Preferably, one of R², R³, R⁴ and R⁵ is hydroxy, one of R², R³, R⁴ and R⁵ is halo, one of R², R³, R⁴ and R⁵ is H, and one of R², R³, R⁴ and R⁵ is H or halo.

Preferably, one of R³ and R⁴ is hydroxy and the other is halo.

Preferably, one of R³ and R⁵ is hydroxy and the other is halo.

More preferably, R³ is hydroxy and R⁴ is halo.

Preferably halo is F or Cl.

In another preferred embodiment, one of R², R³, R⁴ and R⁵ is hydroxy and the others are H.

Preferred compounds according to the invention are: Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2,3-difluoro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3-chloro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-fluoro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin- 4-yl ester;

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[2-(3-fluoro-4-hydroxy-phenyl)-acetylamino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[2-(3-chloro-4-hydroxy-phenyl)-acetylamino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester; Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[2-(3-hydroxy-phenyl)-acetylamino]-ethyl}-phenyl)-ethyl]-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin- 4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,4-difluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(4-chloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-fluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-chloro-3-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin- 4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-fluoro-3-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-chloro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(5-chloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-fluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-4-yl ester; Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4,5-dichloro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester; Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-chloro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-chloro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3,5-dichloro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-fluoro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester; Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-5-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-difluoro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2,4-dichloro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-difluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(2-fluoro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-chloro-5-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[bis-(3-chloro-4-hydroxy-benzyl)-amino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester; and

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[bis-(2-fluoro-3-hydroxy-benzyl)-amino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester; or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts.

The compounds of formula (I) may be prepared in a variety of ways. The routes below illustrate ways of preparing these compounds but the skilled person will appreciate that other routes may be equally as practicable.

In particular, the compounds of formula (I) may be prepared according to the following procedures:

Scheme 1 wherein:

R¹⁰ is methyl or a suitable protecting group such as tert-butoxycarbonyl, R¹¹ is a suitable protecting group such as tert-butoxycarbonyl or alternatively R¹⁰ and R¹¹ may form together a suitable protecting group such as phthalimide;

R¹² is H or methyl; n = 0 or 1 ;

LG represents a suitable leaving group such as bromide or mesylate; and

R¹ to R⁹ and X are as defined for compounds of formula (I) unless otherwise stated.

The compounds of formula (II) may be prepared as described in US 2006205779. The compounds of formula (III) where R¹⁰ and R¹¹ together represent phthalimide may be prepared as described in Scheme 2:

Scheme 2

The compounds of formula (VIII) and (IX) are commercially available.

The compounds of formula (X) may be prepared from compounds of formula (VIII) and (IX) by Heck reaction (process step (vi)). Typical conditions comprise reaction of compound (VIII) with compound (IX), tri-o-tolylphosphine, palladium(ll)acetate and a suitable base such as diisopropylethylamine, in a suitable solvent such as acetonitrile, at 90⁰C for 21 hours.

The compounds of formula (Xl) may be prepared from compound of formula (X) by hydrogenation (process step (vii)). Typical_conditions comprise reaction of compound (X) with ammonium formate and 30% palladium hydroxide on carbon, in suitable solvents such as ethyl acetate and ethanol, at 8O⁰C for 18 hours. Alternative_conditions comprise reaction of compound (X) with hydrogen gas and rhodium tris(triphenylphosphine)chloride, in suitable solvents such as ethyl acetate and ethanol, at 20psi and room temperature for 24 hours.

The compounds of formula (III) where LG is bromide and R¹⁰ and R¹¹ together represent phthalimide may be prepared from compound of formula (Xl) by bromination (process step (viii)). Typical conditions comprise reaction of compound (Xl) with phosphorous tribromide, in a suitable solvent such as toluene, at reflux for 4 hours.

The compounds of formula (III) where LG is mesylate and R¹⁰ and R¹¹ together represent phthalimide may be prepared from compound of formula (Xl) by mesylation (process step (viii)). Typical conditions comprise reaction of compound (Xl) with methanesulfonyl chloride, in a suitable solvent such as methylethyl ketone, with a suitable base such as triethylamine, at O⁰C to room temperature for 1-4 hours.

Compounds of formula (III) where LG is mesylate, R¹⁰ is methyl and R¹¹ is tert-butoxycarbonyl may be prepared from a compound of formula (Xl) by a person skilled in the art, for example as described in Scheme 3:

(Xl)

(xiv)

Scheme 3 wherein PG represents a suitable protecting group such as benzyl or tert-butyl-dimethylsilyl.

The compounds of formula (XII) in Scheme 3 above have R¹⁰ and R¹¹ representing phthalimide and PG is tert-butyl dimethylsilyl or benzyl . They may be prepared from a compound of formula (Xl) where R¹⁰ and R¹¹ represent phthalimide by protection, using standard methodology as described in "Protecting Groups in Organic Synthesis" by T.W. Greene and P. Wutz. (process step (ix).

The compounds of formula (XIII) may be prepared from compounds of formula (XII) where R¹⁰ and R¹¹ represent phthalimide, by de-protection, using standard methodology as described in "Protecting Groups in Organic Synthesis" by T.W. Greene and P. Wutz (process step (x)).

The compounds of formula (XIV) in Scheme 3 above have have R¹¹ representing tert -butoxycarbonyl . They may be prepared from compounds of formula (XIII) by protection, using standard methodology as described in "Protecting Groups in Organic Synthesis" by T.W. Greene and P. Wutz (process step (xi)).

Compounds of formula (XIIa) in Scheme 3 above have R¹⁰ representing methyl and R¹¹ representing tert- butoxycarbonyl. They may be prepared from compounds of formula (XIV) by methylation (process step (xii)). Typical conditions comprise reaction of compound (XIV) with a suitable base such as sodium hydride, potassium tert-butoxide or lithium di-isopropylamide in a suitable solvent such as tetrahydrofuran or dimethylformamide, followed by addition of methyl iodide, at temperatures between -78⁰C to 25⁰C, for 1-4 hours.

The compound of formula (XIa) in Scheme 3 above have R¹⁰ representing methyl and R¹¹ representing tert-butoxycarbonyl. They may be prepared from compounds of formula (XIIa) where R¹⁰ is methyl and R¹¹ is tert-butoxycarbonyl, by de-protection, using standard methodology as described in "Protecting Groups in Organic Synthesis" by T.W. Greene and P. Wutz (process step (xiii)). The compound of formula (III) where R¹⁰ is methyl and R¹¹ is tert-butoxycarbonyl may be prepared from compound of formula (XIa) where R¹⁰ is methyl and R¹¹ is tert-butoxycarbonyl, by mesylation (process step (xiv)). Typical conditions comprise reaction of compound (XIa) with methane sulfonyl chloride and a suitable base such as triethylamine, in a suitable solvent such as dichloromethane, at 5⁰C to room temperature for 1 hour.

The compounds of formula (IV) may be prepared from compound of formula (II), and compound of formula (III) by alkylation (process step (i)). Typical conditions comprise reaction of compound (II) with compound (III) and a suitable base such as triethylamine, diisopropylethylamine, sodium carbonate or potassium carbonate, and optionally in the presence of a nucleophilic additive such as potassium iodide, in a suitable solvent such as dimethylformamide or acetonitrile, at temperatures between 60-100⁰C, for 18-48 hours.

The compounds of formula (V) may be prepared from compounds of formula (IV), by deprotection using standard methodology as described in "Protecting Groups in Organic Synthesis" by T.W. Greene and P. Wutz (process step (N)). When R¹⁰ and R¹¹ , or R¹¹ is tert-butoxycarbonyl; typical conditions comprise reaction of compound (IV) with hydrogen chloride in a suitable solvent such as dioxane, at room temperature, for 18 hours. When R¹⁰ and R¹¹ represent phthalimide; typical conditions comprise reaction of compound (IV) with hydrazine hydrate in a suitable solvent such as ethanol, at 9O⁰C for 3 hours.

The compounds of formula (Vl), (VII) and (Vila) are commercially available, known in the literature or they may be prepared by persons skilled in the art according to standard methodolgies.

The compounds of formula (I) where X is -C(=O)CH₂- or -C(=O)- may be prepared from the compounds of formula (V) and compounds of formula (Vl) by acylation (process step (iii)). Typical conditions comprise reaction of compound (V) and compound (Vl) with a suitable coupling agent such as (3-

(dimethylamino)propyl)ethyl carbodiimide hydrochloride or O-(1 H-benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate, optionally in the presence of an additive such as 1-hydroxy benzotriazole monohydrate or N,N-dimethylaminopyridine, with a suitable base such as triethylamine or N,N-diisopropylethylamine, in a suitable solvent such as dichloromethane, dimethylformamide, tetrahydrofuran, 1-methyl-2-pyrrolidinone or 2-methyltetrahydrofuran, at room temperature for 24 to 72 hours.

The compounds of formula (I) where X is CH₂ and R¹ is H or methyl may be prepared from the compounds of formula (V) and compounds of formula (VII) by reductive amination (process step (iv)).

Typical conditions comprise reaction of compound (V) with compound (VII) in a suitable solvent such as ethanol or dichloromethane, optionally in the presence of a suitable acid catalyst such as acetic acid, optionally in the presence of a drying agent such as sodium sulfate, and optionally in the presence of a suitable base such as triethylamine, at room temperature for up to 1 hour, followed by addition of a suitable reducing agent such as sodium triacetoxyborohydride or sodium cyanoborohydride at room temperature for 18 to 24 hours. The compounds of formula (I) where X is CH₂ and R¹ is of formula:

may be prepared from the compounds of formula (I) where X is CH₂ and R¹ is H, and compound of formula (Vila), by reductive amination (process step (v)). Typical conditions comprise reaction of compound (I) with compound (Vila) in a similar manner to that previously described for process step (iv).

The preparation of compound of formula (I) may require the protection of potential reactive functionality in addition to those methods already described. In such a case, examples of compatible protecting groups and their particular methods of protection and deprotection are described in "Protecting Groups in Organic Synthesis" by T. W. Greene and P. Wutz (Wiley-lnterscience Publication, 1981 ) or "Protecting groups" by P. J. Kocienski (Georg Thieme Verlag, 1994).

Compounds of formula (I) as well as intermediates for their preparation can be purified and isolated according to various well-known methods, for example crystallisation or chromatography.

Pharmaceutically acceptable salts of the compounds of formula (I) include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 1 ,5 -naphthalenedisulfonate, 2 -napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.

For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable salts of compounds of formula (I) may be prepared by one or more of three methods: (i) by reacting the compound of formula (I) with the desired acid or base; by removing an acid- or base-labile protecting group from a suitable precursor of the compound of formula (I) or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or by converting one salt of the compound of formula (I) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.

All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.

The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term 'amorphous' refers to a state in which the material lacks long range order at the molecular level and, depending upon temperature, may exhibit the physical properties of a solid or a liquid. Typically such materials do not give distinctive X-ray diffraction patterns and, while exhibiting the properties of a solid, are more formally described as a liquid. Upon heating, a change from solid to liquid properties occurs which is characterised by a change of state, typically second order ('glass transition'). The term 'crystalline' refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and gives a distinctive X-ray diffraction pattern with defined peaks. Such materials when heated sufficiently will also exhibit the properties of a liquid, but the change from solid to liquid is characterised by a phase change, typically first order ('melting point').

The compounds of the invention or salts thereof may also exist in unsolvated and solvated forms. The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

A currently accepted classification system for organic hydrates is one that defines isolated site, channel, or metal-ion coordinated hydrates - see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995). Isolated site hydrates are ones in which the water molecules are isolated from direct contact with each other by intervening organic molecules. In channel hydrates, the water molecules lie in lattice channels where they are next to other water molecules. In metal-ion coordinated hydrates, the water molecules are bonded to the metal ion.

When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will be dependent on humidity and drying conditions. In such cases, non-stoichiometry will be the norm.

Also included within the scope of the invention are multi-component complexes (other than salts and solvates) wherein the drug and at least one other component are present in stoichiometric or non- stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are typically defined as crystalline complexes of neutral molecular constituents which are bound together through non-covalent interactions, but could also be a complex of a neutral molecule with a salt. Co-crystals may be prepared by melt crystallisation, by recrystallisation from solvents, or by physically grinding the components together - see Chem Commun, X7_, 1889-1896, by O. Almarsson and M. J. Zaworotko (2004). For a general review of multi-component complexes, see J Pharm Sci, 64 (8), 1269-1288, by Haleblian (August 1975).

The compounds of the invention may also exist in a mesomorphic state (mesophase or liquid crystal) when subjected to suitable conditions. The mesomorphic state is intermediate between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism arising as the result of a change in temperature is described as 'thermotropic' and that resulting from the addition of a second component, such as water or another solvent, is described as 'lyotropic'. Compounds that have the potential to form lyotropic mesophases are described as 'amphiphilic' and consist of molecules which possess an ionic (such as -COO Na⁺, -COO K⁺, or -SO₃ Na⁺) or non-ionic (such as -N N⁺(CH₃)₃) polar head group. For more information, see Crystals and the Polarizing Microscope by N. H. Hartshorne and

A. Stuart, 4^th Edition (Edward Arnold, 1970).

Hereinafter all references to compounds of formula (I) include references to salts, solvates, multi- component complexes and liquid crystals thereof and to solvates, multi-component complexes and liquid crystals of salts thereof.

The compounds of the invention include compounds of formula (I) as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) as hereinafter defined and isotopically-labeled compounds of formula (I).

As indicated, so-called 'prodrugs' of the compounds of formula (I) are also within the scope of the invention. Thus certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as 'prodrugs'. Further information on the use of prodrugs may be found in Prodrugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (Ed. E. B. Roche, American Pharmaceutical Association).

Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985).

Some examples of prodrugs in accordance with the invention include

(i) where the compound of formula (I) contains a carboxylic acid functionality (-COOH), an ester thereof, for example, a compound wherein the hydrogen of the carboxylic acid functionality of the compound of formula (I) is replaced by (d-C₈)alkyl; where the compound of formula (I) contains an alcohol functionality (-OH), an ether thereof, for example, a compound wherein the hydrogen of the alcohol functionality of the compound of formula (I) is replaced by (Ci-C₆)alkanoyloxymethyl; and where the compound of formula (I) contains a primary or secondary amino functionality (-NH₂ or -NHR where R ≠ H), an amide thereof, for example, a compound wherein, as the case may be, one or both hydrogens of the amino functionality of the compound of formula (I) is/are replaced by (C-ι-C₁₀)alkanoyl.

Further examples of replacement groups in accordance with the foregoing examples and examples of other prodrug types may be found in the aforementioned references.

Moreover, certain compounds of formula (I) may themselves act as prodrugs of other compounds of formula

I.

Also included within the scope of the invention are metabolites of compounds of formula I, that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites in accordance with the invention include:

(i) where the compound of formula (I) contains a methyl group, an hydroxymethyl derivative thereof (-CH₃ -> -CH₂OH), and

(ii) where the compound of formula (I) contains a phenyl moiety, a phenol derivative thereof

(-Ph -> -PhOH).

The present invention includes all pharmaceutically acceptable isotopically-labelled compounds of formula (I) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as ²H and ³H, carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶CI, fluorine, such as ¹⁸F, iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, and oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O.

Certain isotopically-labelled compounds of formula I, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵ O and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D₂O, d₆-acetone, d₆-DMSO.

The compounds of formula (I) should be assessed for their biopharmaceutical properties, such as solubility and solution stability (across pH), permeability, etc. , in order to select the most appropriate dosage form and route of administration for treatment of the proposed indication.

Compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.

They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term 'excipient' is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

The compounds of the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.

Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids, or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules (made, for example, from gelatin or hydroxypropylmethylcellulose) and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet. The compounds of the invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, V\_ (6), 981-986, by Liang and Chen (2001 ).

For tablet dosage forms, depending on dose, the drug may make up from 1 weight % to 80 weight % of the dosage form, more typically from 5 weight % to 60 weight % of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disinteg rants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 weight % to 25 weight %, preferably from 5 weight % to 20 weight % of the dosage form.

Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may comprise from 0.2 weight % to 1 weight % of the tablet.

Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 weight % to 10 weight %, preferably from 0.5 weight % to 3 weight % of the tablet.

Other possible ingredients include anti-oxidants, colourants, flavouring agents, preservatives and taste- masking agents.

Exemplary tablets contain up to about 80% drug, from about 10 weight % to about 90 weight % binder, from about 0 weight % to about 85 weight % diluent, from about 2 weight % to about 10 weight % disintegrant, and from about 0.25 weight % to about 10 weight % lubricant.

Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of tablets is discussed in Pharmaceutical Dosage Forms: Tablets, Vol. 1 , by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

Consumable oral films for human or veterinary use are typically pliable water-soluble or water-swellable thin film dosage forms which may be rapidly dissolving or mucoadhesive and typically comprise a compound of formula I, a film-forming polymer, a binder, a solvent, a humectant, a plasticiser, a stabiliser or emulsifier, a viscosity-modifying agent and a solvent. Some components of the formulation may perform more than one function.

The compound of formula (I) may be water-soluble or insoluble. A water-soluble compound typically comprises from 1 weight % to 80 weight %, more typically from 20 weight % to 50 weight %, of the solutes. Less soluble compounds may comprise a greater proportion of the composition, typically up to 88 weight % of the solutes. Alternatively, the compound of formula (I) may be in the form of multiparticulate beads.

The film-forming polymer may be selected from natural polysaccharides, proteins, or synthetic hydrocolloids and is typically present in the range 0.01 to 99 weight %, more typically in the range 30 to 80 weight %.

Other possible ingredients include anti-oxidants, colorants, flavourings and flavour enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, surfactants and taste-masking agents.

Films in accordance with the invention are typically prepared by evaporative drying of thin aqueous films coated onto a peelable backing support or paper. This may be done in a drying oven or tunnel, typically a combined coater dryer, or by freeze-drying or vacuuming.

Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

Suitable modified release formulations for the purposes of the invention are described in US Patent No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in Pharmaceutical Technology On-line, 25(2), 1-14, by Verma et al (2001 ). The use of chewing gum to achieve controlled release is described in WO 00/35298.

The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of compounds of formula (I) used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility- enhancing agents.

Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the invention may be formulated as a suspension or as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and semi-solids and suspensions comprising drug-loaded poly(c//-lactic-coglycolic)acid (PGLA) microspheres.

The compounds of the invention may also be administered topically, (intra)dermally, or transdermally to the skin or mucosa. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated - see, for example, J Pharm Sci, 88 (10), 955-958, by Finnin and Morgan (October 1999).

Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.

Formulations for topical administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler, as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane, or as nasal drops. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin. The pressurised container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilising, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.

Capsules (made, for example, from gelatin or hydroxypropylmethylcellulose), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as /-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from 1 μg to 20mg of the compound of the invention per actuation and the actuation volume may vary from 1 μl to 10Oμl. A typical formulation may comprise a compound of formula I, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.

Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.

Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, for example, PGLA. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

In the case of dry powder inhalers and aerosols, the dosage unit is determined by a prefilled capsule, blister or pocket or by a system that utilises a gravimetrically fed dosing chamber. Units in accordance with the invention are typically arranged to administer a metered dose or "puff" containing from 1 to 5000 μg of (compound name here), or a salt thereof,. The overall daily dose will typically be in the range 1 μg to 20 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.

The compounds of formula (I) are particularly suitable for an administration by inhalation specifically using a dry powder inhaler. The compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

Formulations for rectal/vaginal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

The compounds of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, gels, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

Formulations for ocular/aural administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted, or programmed release.

The compounds of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol-containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.

Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma- cyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.

Inasmuch as it may desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound in accordance with the invention, may conveniently be combined in the form of a kit suitable for coadministration of the compositions.

Thus the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of formula (I) in accordance with the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.

The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically comprises directions for administration and may be provided with a so-called memory aid.

For administration to human patients, the total daily dose of the compounds of the invention is typically in the range 0.001 mg to 5000mg depending, of course, on the mode of administration. For example, oral administration may require a total daily dose of from 0.1 mg to 1000mg, while an intravenous dose may only require from 0.001 mg to 100mg. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein.

These dosages are based on an average human subject having a weight of about 60kg to 70kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.

For the avoidance of doubt, references herein to "treatment" include references to curative, palliative and prophylactic treatment.

The compounds of formula (I) have the ability to interact with muscarinic receptors and thereby have a wide range of therapeutic applications, as described further below, because of the essential role which muscarinic receptors play in the physiology of all mammals.

Thus the invention relates to the use of the compounds of formula (I), or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts, for the manufacture of a medicament for the treatment or the prevention of diseases, disorders, and conditions in which the muscarinic receptor is involved. The invention further relates to a method of treatment of a mammal, including a human being, with a muscarinic receptor antagonist including treating said mammal with an effective amount of a compound of the formula (I) or with a pharmaceutically acceptable salt, derived form or composition thereof.

Therefore, a further aspect of the present invention relates to the compounds of formula (I), or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts, for use in the treatment of diseases, disorders, and conditions in which muscarinic receptors are involved. Examples of such diseases, disorders, and conditions are Inflammatory Bowel Disease, Irritable

Bowel Disease, diverticular disease, motion sickness, gastric ulcers, radiological examination of the bowel, symptomatic treatment of BPH (benign prostatic hyperplasia), NSAID induced gastric ulceration, urinary Incontinence (including urgency, frequency, urge incontinence, overactive bladder, nocturia and

Lower urinary tract symptoms), cycloplegia, mydriatics, parkinsons disease. More specifically, the present invention also concerns the compounds of formula (I), or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts, for use in the treatment of diseases, disorders, and conditions selected from the group consisting of: • chronic or acute bronchoconstriction, chronic bronchitis, small airways obstruction, and emphysema;

• obstructive or inflammatory airways diseases of whatever type, etiology, or pathogenesis, in particular an obstructive or inflammatory airways disease that is a member selected from the group consisting of chronic eosinophilic pneumonia, chronic obstructive pulmonary disease (COPD), COPD that includes chronic bronchitis, pulmonary emphysema or dyspnea associated or not associated with COPD, COPD that is characterized by irreversible, progressive airways obstruction, adult respiratory distress syndrome (ARDS), exacerbation of airways hyper-reactivity consequent to other drug therapy and airways disease that is associated with pulmonary hypertension;

• bronchitis of whatever type, etiology, or pathogenesis, in particular bronchitis that is a member selected from the group consisting of acute bronchitis, acute laryngotracheal bronchitis, arachidic bronchitis, catarrhal bronchitis, croupus bronchitis, dry bronchitis, infectious asthmatic bronchitis, productive bronchitis, staphylococcus or streptococcal bronchitis and vesicular bronchitis;

• asthma of whatever type, etiology, or pathogenesis, in particular asthma that is a member selected from the group consisting of atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, intrinsic asthma caused by pathophysiologic disturbances, extrinsic asthma caused by environmental factors, essential asthma of unknown or inapparent cause, non-atopic asthma, bronchitic asthma, emphysematous asthma, exercise- induced asthma, allergen induced asthma, cold air induced asthma, occupational asthma, infective asthma caused by bacterial, fungal, protozoal, or viral infection, non-allergic asthma, incipient asthma, wheezy infant syndrome and bronchiolytis;

• acute lung injury; and

• bronchiectasis of whatever type, etiology, or pathogenesis, in particular bronchiectasis that is a member selected from the group consisting of cylindric bronchiectasis, sacculated bronchiectasis, fusiform bronchiectasis, capillary bronchiectasis, cystic bronchiectasis, dry bronchiectasis and follicular bronchiectasis.

More specifically, the present invention also concerns the compounds of formula (I), or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts, for use in the treatment of COPD or asthma.

Suitable examples of other therapeutic agents which may be used in combination with the compound(s) of formula (I), or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts, include, but are by no means limited to : (a) 5-Lipoxygenase (5-LO) inhibitors or 5-lipoxygenase activating protein (FLAP) antagonists; (b) Leukotriene antagonists (LTRAs) including antagonists of LTB₄, LTC₄, LTD₄, and LTE₄;

(c) Histamine receptor antagonists including H1 and H3 antagonists;

(d) oti- and α₂-adrenoceptor agonist vasoconstrictor sympathomimetic agents for decongestant use; (e) PDE inhibitors, e.g. PDE3, PDE4 and PDE5 inhibitors;

(f) Beta 2 receptor agonists;

(g) Theophylline;

(h) Sodium cromoglycate; (i) COX inhibitors both non-selective and selective COX-1 or COX-2 inhibitors (NSAIDs);

(j) Prostaglandin receptor antagonists and inhibitors of prostaglandin synthase;

(k) Oral and inhaled glucocorticosteroids;

(I) Dissociated agonists of the corticoid receptor (DAGR);

(m) Monoclonal antibodies active against endogenous inflammatory entities; (n) Anti-tumor necrosis factor (anti-TNF-α) agents;

(o) Adhesion molecule inhibitors including VLA-4 antagonists;

(p) KJnJn-B₁ - and B₂ -receptor antagonists;

(q) Immunosuppressive agents, including inhibitors of the IgE pathway and cyclosporine;

(r) Inhibitors of matrix metalloproteases (MMPs); (s) Tachykinin NK₁, NK₂ and NK₃ receptor antagonists;

(t) Protease inhibitors such as elastase inhibitors;

(u) Adenosine A2a receptor agonists and A2b antagonists;

(v) Inhibitors of urokinase;

(w) Compounds that act on dopamine receptors such as D2 agonists; (x) Modulators of the NFiφ pathway such as IKK inhibitors;

(y) Modulators of cytokine signalling pathyways such as p38 MAP kinase, PI3 kinase, JAK kinase, syk kinase, EGFR or MK-2;

(z) Agents that can be classed as mucolytics or anti-tussive;

(aa) Agents which enhance responses to inhaled corticosteroids; (bb) Antibiotics and antiviral agents effective against micro-organisms which can colonize the respiratory tract;

(cc) HDAC inhibitors;

(dd) CXCR2 antagonists;

(ee) lntegrin antagonists; (ff) Chemokines;

(gg) Epithelial sodium channel (ENaC) blockers or Epithelial sodium channel (ENaC) inhibitors;

(hh) P2Y2 Agonists and other Nucleotide receptor agonists;

(ii) Inhibitors of thromboxane;

(jj) Inhibitors of PGD₂ synthesis and PGD₂ receptors (DP1 and DP2/CRTH2); (kk) Niacin; and

(II) Adhesion factors including VLAM, ICAM, and ELAM.

According to the present invention, the combination of the compounds of formula (I), or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts, with H3 antagonists, β₂ agonists, PDE4 inhibitors, steroids (especially glucocorticosteroids),

Adenosine A2a receptor agonists, Modulators of cytokine signalling pathyways such as p38 MAP kinase or syk kinase, and/or with Leukotriene antagonists (LTRAs) including antagonists of LTB₄, LTC₄, LTD₄, and LTE₄, are preferred .

According to the present invention, the combination of the compounds of formula (I), or the pharmaceutically acceptable salts thereof or the pharmaceutically acceptable solvates of said compounds or salts, with:

- glucocorticosteroids, in particular inhaled glucocorticosteroids with reduced systemic side effects, including prednisone, prednisolone, flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide, and mometasone furoate; or

- β2 agonists including in particular salbutamol, terbutaline, bambuterol, fenoterol, salmeterol, formoterol, tulobuterol and their salts, are further preferred.

The following examples illustrate the preparation of the compounds of the formula (I):

PREPARATIONS

Preparation 1 2-{2-[4-(2-Hvdroxy-ethvD-phenyl1-vinyl)-isoindole-1 ,3-dione

To a solution of 2-(4-bromophenyl)ethanol (48.3g) in acetonitrile (480 ml) were added diisopropylethylamine (46.6g), N-vinylphthalimide (43.7g) and tri-o-tolylphosphine (7.31g) and the mixture was purged with nitrogen gas three times. Palladium acetate (2.7g) was added and the mixture was stirred at 9O⁰C for 21 hours under nitrogen. The reaction was cooled and the precipitated product collected by filtration. The resulting solid was re-dissolved in dichloromethane and ethyl acetate and filtered through silica gel. The filtrate was concentrated in vacuo to give the title compound, 24g. 1H NMR (400MHz, CDCI₃) δ = 2.81 -2.84 (t, 2H), 3.82-3.90 (t, 2H), 7.23-7.26 (d, 2H), 7.32-7.36 (d, 1 H), 7.40-7.43 (d, 2H), 7.61-7.64 (d, 1 H), 7.66-7.78 (d, 2H), 7.86-7.88 (d, 2H) ppm.

Preparation 2

2-{2-[4-(2-Hvdroxy-ethyl)-phenyl1-ethyl)-isoindole-1 ,3-dione

To a stirred solution of 2-{2-[4-(2-Hydroxy-ethyl)-phenyl]-vinyl}-isoindole-1 ,3-dione (Preparation 1 , 1Og) in ethanol and ethyl acetate (350ml each) was added 30% palladium hydroxide on carbon (1.44g), followed by ammonium formate (21.5g) and the reaction heated at 8O⁰C for 4 hours. Additional palladium hydroxide on carbon (1.44g) and ammonium formate (21.5g) were added and the reaction was stirred at 8O⁰C for a further 18 hours. The reaction was cooled, filtered through a pad of Arbocel™ and rinsed with methanol. The solvent was removed under reduced pressure and the resulting white solid partitioned between dichloromethane (200ml) and water (100ml). The aqueous layer was separated and extracted with further dichloromethane (2 x 50ml). The combined organic layers were dried (sodium sulphate) and the solvent removed in vacuo to give the title compound as an off-white solid, 9.11g. 1H NMR (400MHz, CDCI₃) δ = 2.80 -2.83 (t, 2H), 2.92-3.00 (t, 2H), 3.82-3.86 (t, 2H), 3.87-3.96 (t, 2H), 7.14-7.22 (2xd, 4H), 7.70-7.72 (dd, 2H), 7.82-7.84 (dd, 2H) ppm.

Alternatively, the title compound may be prepared according to the following procedure: 2-{2-[4-(2-hydroxy-ethyl)phenyl]-vinyl}-isoindole-1 ,3-dione (Preparation 1 , 62.Og, 211.37mmol) was dissolved in ethyl acetate (1200ml). To this was added rhodium tris(triphenylphosphine) chloride, (12.7g, 13.7mmol) and the mixture hydrogenated at 20psi, room temperature for 24 hours. The reaction was filtered and concentrated in vacuo. The residue was dissolved in ethyl acetate (1000ml) and passed through a pad of silica gel, washing with ethyl acetate. The solvent was removed in vacuo to yield a light brown solid which was recrystallised from ethyl acetate: heptane (4:1 , by volume) to yield the title compound as an off-white crystalline solid, in 85% yield, 53g,

¹H NMR (400MHz, CDCI₃) δ = 2.80-2.83 (t, 2H), 2.92-3.00 (t, 2H), 3.82-3.86 (t, 2H), 3.87-3.96 (t, 2H), 7.14-7.22 (2x d, 4H), 7.70-7.72 (dd, 2H), 7.82-7.84 (dd, 2H) ppm .

Preparation 3 2-{2-[4-(2-Bromo-ethyl)-phenyl1-ethyl)-isoindole-1 ,3-dione

A solution of 2-{2-[4-(2-Hydroxy-ethyl)-phenyl]-ethyl}-isoindole-1 ,3-dione (Preparation 2, 22.37g) and phosphorus tribromide (8.2Og) in toluene (500ml) was refluxed for 4 hours. The mixture was allowed to cool to room temperature, diluted with ethyl acetate (300ml) and carefully quenched with sodium bisulphite/sodium bicarbonate (1 :1 ) in water (100ml). The organic layer was separated and washed with further sodium bisulphite/sodium bicarbonate (1 :1 ) in water (100ml), dried over sodium sulphate and concentrated in vacuo. The resulting solid (24.26g) was triturated with heptane:tert-butyl methyl ether (100ml, 9:1 by volume) to give the title compound as a pale green solid, 17.94g. 1H NMR (400MHz, CDCI₃) δ = 2.93 -3.00 (t, 2H), 3.07-3.18 (t, 2H), 3.53-3.59 (t, 2H), 3.88-3.94 (t, 2H), 7.13-7.23 (2xd, 4H), 7.70-7.73 (dd, 2H), 7.83-7.85 (dd, 2H) ppm.

Preparation 4

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(1 ,3-dioxo-1 ,3-dihydro-isoindol-2-yl)-ethyl1-phenyl)-ethyl)-piperidin-

4-yl ester

To a stirred solution of 4-piperidinyl N-(2-biphenylyl)carbamate hydrochloride (US2006/205779, 21.9g) in dimethyl formamide (450 ml) was added sodium carbonate (21.2g) portion wise. The solution was stirred for 20 minutes at room temperature, then 2-{2-[4-(2-Bromo-ethyl)-phenyl]-ethyl}-isoindole-1 ,3-dione (Preparation 3, 23.85g) was added and the reaction heated to 7O⁰C for 20 hours. The flask was cooled to room temperature and the reaction mixture slowly poured into 1 L of water. The resulting solid was collected by filtration, ground to a fine powder and triturated with methanol. This solid was filtered and dried to give the title compound as an off-white solid, 36.2g.

¹H NMR (400MHz, CDCI₃) δ = 1.60-1.76 (m, 2H), 1.88-1.98 (m, 2H), 2.23-2.28 (m, 2H), 2.51-2.60 (m, 2H), 2.64-2.78 (m, 4H), 2.86-2.97 (t, 2H), 3.84-3.92 (t, 2H), 4.65-4.75 (m, 1 H), 6.61 (s, 1 H), 7.11-7.52 (m, 12H), 7.70-7.72 (dd, 2H), 7.83-7.85 (dd, 2H), 8.10-8.12 (bd, 1 H) ppm.

Alternatively, the title compound may be prepared according to the following procedure: Methanesulfonic acid 2-{4-[2-(1 ,3-dioxo-1 ,3-dihydro-isoindol-2-yl)-ethyl]-phenyl}-ethyl ester (Preparation 6, 66.0Og, 176.74mmol) was dissolved in acetonitrile (500ml) and stirred at room temperature. Potassium iodide (147g, 884mmol) was added to the reaction vessel and heated at 7O⁰C for 30 minutes. In a separate vessel 4-piperidinyl N-(2-biphenylyl)carbamate hydrochloride (US2006205779, 58.8g, 176.74mmol) and diisopropylethylamine (77.0ml, 442mmol) were dissolved in acetonitrile (500ml) and stirred at room temperature for 30 minutes, then added to the vessel containing the preformed iodide. The resulting suspension was heated at 100⁰C for 18 hours. The reaction mixture was allowed to cool to room temperature and concentrated in vacuo to yield a brown solid that was partitioned between ethyl acetate (500ml) and saturated aqueous sodium hydrogen carbonate solution (500ml). The organic layer was washed with further saturated aqueous sodium hydrogen carbonate solution (500ml), water (2x500ml) and brine (500ml). The organic layer was then dried (magnesium sulphate) and concentrated in vacuo to yield a brown solid that was recrystallised from dichloromethane:heptane to yield the title compound as a light brown crystalline solid in 55% yield, 56.3g. LRMS: APCI ESI m/z 574 [M+H]⁺

¹H NMR (400 MHz, CDCI₃) δ = 1.68-1.76 (m, 2H), 1.92-2.02 (m, 2H), 2.24-2.35 (m, 2H), 2.54-2.58 (m, 2H), 2.73-2.77 (m, 4H), 2.93-2.97 (m, 2H), 3.87-3.91 (m, 2H), 4.70-4.78 (m, 1 H), 7.10-7.22 (m, 6H), 7.33- 7.52 (m, 6H), 7.69-7.71 (m, 2H), 7.81-7.85 (m, 2H), 8.09 (d, 1 H) ppm.

Preparation 5

Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl1-ethyl)-piperidin-4-vl ester

Hydrazine hydrate (0.947ml, 19.5mnnol) was added to a suspension of biphenyl-2-yl-carbamic acid 1-(2- {4-[2-(1 ,3-dioxo-1 ,3-dihydro-isoindol-2-yl)-ethyl]-phenyl}-ethyl)-piperidin-4-yl ester (Preparation 4, 5.6Og, 9.76mnnol) in ethanol (120ml) and the mixture heated to 90 C for 3 hours. The mixture was cooled to room temperature, the solid was collected by filtration and the filtrate concentrated in vacuo. The resulting residue was triturated with dichloromethane (50ml) and the insoluble material discarded. The solvent was removed in vacuo to give the title compound as a white solid, 3.34 g, 77%.

¹H NMR (400 MHz, CDCI₃) δ = 1.70-1.78 (m, 2H), 1.94-2.04 (m, 2H), 2.32-2.41 (m, 2H), 2.55-2.65 (m, 2H), 2.75-2.84 (m, 6H), 2.97-3.01 (m, 2H), 4.72-4.79 (m, 1 H), 6.61 (m, 1 H), 7.02-7.51 (m, 12H), 8.06-8.12 (m, 1 H) ppm .

Preparation 6

Methanesulfonic acid 2-{4-[2-(1 ,3-dioxo-1 ,3-dihvdro-isoindol-2-yl)-ethyl1-phenyl)-ethyl ester

A solution of 2-{2-[4-(2-hydroxy-ethyl)phenyl]-ethyl}-isoindole-1 ,3-dione (Preparation 2, 21.Og, 71.25mmol) in methyl ethyl ketone (315ml) was cooled to O⁰C. Triethylamine (14.9ml, 106.87mmol) was added followed by the drop wise addition of methane sulfonyl chloride (6.07ml, 78.37mmol). The reaction was stirred at 0-4⁰C for 1 hour and then allowed to warm to room temperature. An aqueous solution of potassium carbonate (1 M, 210ml) was added and the mixture stirred. The organic and aqueous layers were separated and the aqueous re-extracted with methyl ethyl ketone (50ml). The combined organic layers were concentrated to 40ml by distillation. Tert-butyl methyl ether (300ml) was slowly added to the hot solution causing crystallisation to occur. The mixture was allowed to cool to room temperature and then allowed to stir for 72 hours. The solid was removed by filtration and washed with tert-butyl methyl ether (3x 25ml). The solid was dried in vacuo at 45⁰C for 4 hours to yield the title compound as a light brown solid, in 92% yield, 24.6g.

¹H NMR (400MHz, CDCI₃) δ = 2.82 (s, 3H), 2.96-3.02 (m, 4H), 3.89-3.93 (t, 2H), 4.37-4.40 (t, 2H), 7.13- 7.23 (2xd, 4H), 7.70-7.72 (dd, 2H), 7.81-7.83 (dd, 2H) ppm.

Preparation 7

(2,6-Dichloro-phenoxy)-triethyl-silane

2,6-dichlorophenol (16.3g, lOOmmol) was dissolved in tetrahydrofuran (300ml). To this solution was added anhydrous pyridine (16.2ml, 200mmol) and chlorotriethylsilane (22.7ml, 135mmol). The resulting reaction mixture was stirred at room temperature for 18 hours, and then at 8O⁰C for 5 hours. The reaction was cooled to room temperature and poured onto saturated aqueous sodium hydrogen carbonate solution (150ml) and extracted with dichloromethane (3x 80ml). The combined organic layers were dried (sodium sulphate) and concentrated in vacuo to yield crude product. The residue was purified by column chromatography on silica gel eluting with heptane to furnish the title compound as a colourless oil, in 54% yield, 15g.

¹H NMR (400 MHz, CDCI₃) δ = 0.83-0.89 (q, 6H), 0.99-1.03 (t, 9H), 6.80-6.84 (m, 1 H), 7.23-7.26 (m, 2H) ppm.

Preparation 8 2,4-Dichloro-3-hvdroxy-benzaldehyde

Sec-butyllithium in cyclohexane (1.4M, 34.5ml, 48.3mmol) was added slowly to a solution of (2,6-dichloro- phenoxy)-triethyl-silane (Preparation 7, 12.17g, 43.89mmol) in tetrahydrofuran (100ml) at -72⁰C. When addition was complete, the reaction was stirred at -72⁰C for 1 hour. Anhydrous dimethylformamide (4.42ml, 57.1 mmol) was then added, keeping the temperature of the reaction below -65⁰C. Stirring was continued at -65⁰C for 10 minutes and then at -65⁰C to room temperature over 0.5 hours. The reaction was quenched by the addition of 2N hydrochloric acid solution saturated with sodium chloride (100ml) and the resulting mixture extracted with ethyl acetate (100ml). The organic layer was washed with brine (100ml), dried (sodium sulphate) and concentrated in vacuo to yield the crude product. Trituration in heptane:dichloromethane (10:1 , by volume; 220ml) yielded the title compound as a white solid, in 65% yield, 5.5g. LCMS: m/z 188 M- ¹H NMR (400 MHz, DMSO-c/₆ ) δ = 7.32 (d, 1 H), 7.49 (d, 1 H), 10.23 (s, 1 H) ppm.

Preparation 9

4-Chloro-5-fluoro-2-hvdroxy-benzaldehvde

Hexamethylenetetramine (21Og, 1.5 mol) was added to trifluoroacetic acid (3.6 L) in small portions and the resulting mixture was heated to reflux. A solution of 3-chloro-4-fluorophenol (21Og, 1.43 mol) in trifluoroacetic acid (1.4L) was then added drop wise and the mixture stirred for another 1 hour. The mixture was then cooled to room temperature and concentrated in vacuo. The residue was poured into ice-water (2L) and stirred overnight. The resulting solid was collected by filtration, dissolved in EtOAc (500ml), dried over magnesium sulphate and concentrated in vacuo. The crude residue was triturated in ethyl acetate/petroleum ether (10:1 , by volume) to give the title compound as a white solid, in 23% yield,

56.5g.

LCMS: m/z 172.9 M-

¹H NMR (400 MHz, CDCI₃) 5 = 7.00-7.01 (m, 1 H), 7.19-7.26 (m, 1 H), 9.75 (s, 1 H), 10.82 (s, 1 H) ppm.

EXAMPLES

Example 1

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2,3-difluoro-4-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

To a solution of biphenyl-2-yl-carbamic acid 1 -{2-[4-(2-amino-ethyl)-phenyl]-ethyl}-piperidin-4-yl ester (Preparation 5, 40.8mg, 92μmol) in dimethylformamide (1 ml), were added triethylamine (25.6μl, 184μmol), 2,3-difluoro-4-hydroxybenzoic acid (17.6mg, 101 μmol) and O-(1 H-benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluoro-phosphate (41.9mg, 110μmol) and the mixture was stirred at room temperature for 24 hours. Additional triethylamine (25.6μl, 184μmol) was added and stirring continued for a further 24 hours. The mixture was purified directly using an Isolute^® SCX -2 cartridge, eluting with methanol followed by 1 M ammonia in methanol. The basic fractions were evaporated under reduced pressure and the residue purified by HPLC method E to afford the title compound. LCMS Method E: RT 2.75 min (100%area) ES m/z 600.26 [M+H]⁺

Example 2

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-4-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40.8mg, 92μmol) and 3,5-dichloro-4-hydroxybenzoic acid (20.9mg, 101 μmol) using the same method as described in example 1. LCMS Method E: RT 2.62 min (100%area) ES m/z 632.2 [M+H]⁺

Example 3

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-4-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvπ-piperidin- 4-yl ester

(3-(Dimethylamino)propyl)ethylcarbodimide hydrochloride (135mg, 0.704mmol) was added to a solution of biphenyl-2-yl-carbamic acid 1 -{2-[4-(2-amino-ethyl)-phenyl}-ethyl}-piperidin-4-yl ester (Preparation 5, 223mg, 0.503mmol), 3-chloro-4-hydroxybenzoic acid hemihydrate (91.3mg, 0.50mmol) and 1- hydroxybenzotriazole monohydrate (81 mg, 0.528mmol) in a mixture of dichloromethane (2ml) and dimethylformamide (1 ml) and stirred for 3 days at room temperature under nitrogen. The solution was partitioned between dichloromethane (30ml) and saturated aqueous sodium hydrogen carbonate solution (20ml). The layers were separated and the aqueous layer extracted with further dichloromethane (30ml). The combined organic layers were dried (magnesium sulphate), the solvent removed in vacuo and the crude residue purified by HPLC method A to afford the title compound. LCMS Method A: RT 2.52 min (100%area) ES m/z 598 [M+H]⁺.

Example 4

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-4-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)-piperidin- 4-yl ester

Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl}-ethyl}-piperidin-4-yl ester (Preparation 5, 168mg, 0.379mmol), (3-(dimethylamino)propyl)ethylcarbodimide hydrochloride (87.2mg, 0.455mmol) and 3-fluoro-4-hydroxybenzoic acid (59.2 mg, 0.379 mmol) were added to the reaction flask. A mixture of 1- methyl-2-pyrrolidinone (2ml) and 2-methyltetrahydrofuran (4ml) was added, followed by N, N- diisopropylethylamine (0.165ml, 0.948mmol) and the reaction stirred for 24 hours at room temperature under nitrogen. 1-Hydroxybenzotriazole monohydrate (70mg, 0.455mmol) was added and stirring continued for a further 24 hours. The solvent was removed in vacuo an d the residue was partitioned between dichloromethane (50ml) and water (30ml). The layers were separated and the organic layer washed with further water (30ml), dried (magnesium sulphate) and the solvent removed in vacuo. The crude residue was purified by column chromatography on silica gel eluting with dichloromethane:methanol:880 ammonia, 99:1 :0.1 to 90:10:1 (by volume), to furnish the title compound as an off-white solid, 31 % yield, 67 mg.

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.57-1.69 (m, 2H), 1.81-1.90 (m, 2H), 2.29-2.39 (m, 2H), 2.48- 2.55 (m, 2H), 2.67-2.78 (m, 4H), 2.86 (t, J=7.22 Hz, 2H), 3.54 (t, J=7.22 Hz, 2H), 4.57-4.65 (m, 1 H), 6.91 (t, J=8.58 Hz, 1 H), 7.05 (t, J=8.78 Hz, 2H), 7.09 (s, 1 H), 7.18-7.57 (m, 12H) ppm. LCMS: APCI ESI m/z 582 [M+H]⁺

Example 5

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-fluoro-4-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)-piperidin- 4-yl ester

(3-(Dimethylamino)propyl)ethylcarbodimide hydrochloride (1.27g, 6.61 mmol) and N, N- diisopropylethylamine (2.40 ml, 13.8 mmol) were added to a solution of biphenyl-2-yl-carbamic acid 1-{2- [4-(2-amino-ethyl)-phenyl}-ethyl}-piperidin-4-yl ester (Preparation 5, 2.445g, 5.51 mmol), 2-fluoro-4- hydroxybenzoic acid (860mg, 5.51 mmol) and 1-hydroxybenzotriazole monohydrate (1.01 g, 6.61 mmol) in dimethylformamide (50ml) and stirred for 24 hours at room temperature under nitrogen. The solvent was removed in vacuo and the residue purified by column chromatography on silica gel eluting with dichloromethane:methanol:880 ammonia, 97:3:0.3 to 95:5:0.5 (by volume), to furnish the title compound as a white foam, 95% yield, 3.08 g.

The title compound (1.63 g) was dissolved in the minimum volume of hot ethyl acetate and allowed to cool slowly to room temperature overnight. The resulting white crystals were collected by filtration and dried in vacuo to afford the title compound as a white crystalline solid, 1.05 g, 64% yield. 1H NMR (400 MHz, METHANOL-c/₄) δ = 1.59-1.70 (m, 2H), 1.82-1.92 (m, 2H), 2.34 -2.43 (m, 2H), 2.54- 2.61 (m, 2H), 2.70-2.80 (m, 4H), 2.86 (t, J=7.41 Hz, 2H), 3.56 (t, J=7.22 Hz, 2H), 4.58-4.65 (m, 1 H), 6.54 (d, J=2.34 Hz, 1 H), 6.64 (dd, J=8.58, 2.34 Hz, 1 H), 7.13-7.44 (m, 12H), 7.55 (d, J=7.80 Hz, 1 H), 7.61 (t, J=8.78 Hz, 1 H) ppm. LCMS: ESI m/z 582 [M+H]⁺ Example 6

Biphenyl-2-yl-carbannic acid 1-[2-(4-{2-[2-(3-fluoro-4-hvdroxy-phenvπ-acetylaπnino1-ethyl)-phenvπ-ethyl1- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 168mg, 0.379mmol) and S-fluoro^-hydroxyphenylacetic acid (64.5mg, 0.379mmol) using the same method as described in example 4, as an off-white solid, 24% yield, 55 mg. ¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.57-1.69 (m, 2H), 1.81-1.90 (m, 2H), 2.31-2.41 (m, 2H), 2.50- 2.58 (m, 2H), 2.68-2.78 (m, 6H), 3.31-3.33 (m, 2H), 3.38-3.41 (m, 2H), 4.57-4.65 (m, 1 H), 6.78-7.57 (m, 16H) ppm. LCMS: APCI ESI m/z 596 [M+H]⁺

Alternatively, the title compound may be prepared according to the following procedure; Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl}-ethyl}-piperidin-4-yl ester (Preparation 5, 1.95g, 4.40mmol) was dissolved in dichloromethane (20ml), to which were added 3-fluoro-4- hydroxyphenylacetic acid (785mg, 4.62mmol) and triethylamine (610μl_, 4.40mmol) and the reaction stirred to the point of full dissolution. To the reaction were then added a solution of N, N- dimethylaminopyridine (215mg, 1.76mmol) in dichloromethane (5ml), and (3- (dimethylamino)propyl)ethylcarbodimide hydrochloride (1.01g, 5.28mmol) as a suspension in dichloromethane (15ml) and the reaction stirred for 48 hours at room temperature. The reaction mixture was diluted with dichloromethane (20ml) and washed with saturated aqueous sodium bicarbonate solution (50ml), followed by brine (50ml), dried over magnesium sulphate and the solvent removed in vacuo. The residue was dissolved in methanol/water (40ml/5ml), treated with potassium carbonate (1.22g, 8.79mmol) and heated at 5O⁰C for 5 hours, then allowed to cool to room temperature overnight. The solvent was removed in vacuo and residue partitioned between ethyl acetate (70ml) and water (70ml). The aqueous layer was further extracted with ethyl acetate (70ml) and the combined organic layers washed with brine (70ml), dried over magnesium sulphate and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with ethyl acetate:methanol:880 ammonia (99:1 :0.1 to 95:5:0.5, by volume). Evaporation of the chromatography solvents to dryness resulted in isolation of the title compound as a white crystalline solid, in 55% yield, 1.45g. LCMS: APCI ESI m/z 596 [M+H]⁺

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.57-1.69 (m, 2H), 1.81-1.90 (m, 2H), 2.31-2.41 (m, 2H), 2.50- 2.58 (m, 2H), 2.68-2.78 (m, 6H), 3.31-3.33 (m, 2H), 3.38-3.41 (m, 2H), 4.57-4.65 (m, 1 H), 6.78-7.57 (m, 16H) ppm. Example 7

Biphenyl-2-yl-carbannic acid 1-[2-(4-{2-[2-(3-chloro-4-hvdroxy-phenvπ-acetylaπnino1-ethyl)-phenvπ-ethyl1- piperidin-4-yl ester

(3-(Dimethylamino)propyl)ethylcarbodimide hydrochloride (1.32g, 6.90mnnol) and N, N- diisopropylethylamine (2.50ml, 14.4mnnol) were added to a solution of biphenyl-2-yl-carbamic acid 1-{2- [4-(2-amino-ethyl)-phenyl}-ethyl}-piperidin-4-yl ester (Preparation 5, 2.55g, 5.75mnnol), 3-chloro-4- hydroxy-phenylacetic acid (1.07g, 5.75mnnol) and 1-hydroxybenzotriazole monohydrate (1.06g, 6.90mnnol) in 1-methyl-2-pyrrolidinone (50ml) and stirred for 24 hours at room temperature under nitrogen. The solvent was removed in vacuo and the crude residue purified by column chromatography on silica gel eluting with dichloromethane:methanol:880 ammonia, 98:2:0.2 to 96:4:0.4 (by volume), to furnish the title compound as a white foam, 47% yield, 1.66 g.

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.58-1.70 (m, 2H), 1.83-1.92 (m, 2H), 2.32-2.42 (m, 2H), 2.51- 2.58 (m, 2H), 2.69-2.77 (m, 6H), 3.29-3.34 (m, 2H), 3.40 (t, J=7.02 Hz, 2H), 4.58-4.66 (m, 1 H), 6.80-7.56 (m, 16H) ppm.

LCMS: ESI m/z 612 [M+H]⁺

Example 8

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[2-(3-hvdroxy-phenyl)-acetylamino1-ethyl)-phenyl)-ethyl1-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 1.00g, 2.3mmol) and 3-hydroxyphenyl acetic acid (343mg, 2.25mmol) using the same method as described in example 3, to give crude product as a yellow oil. This oil was dissolved in dichloromethane (1 ml) and acetonitrile (15ml), and the solution left to stand at room temperature for 1 hour. The resulting solid was collected by filtration and washed with acetonitrile to give a white solid, 900mg. This solid was dissolved in methanol (25ml) and water (5ml), and further purified by addition of potassium carbonate (800mg) and stirred at room temperature for 18 hours. The methanol was removed in vacuo and the residue partitioned between water (10ml) and ethyl acetate (30ml). The aqueous layer was separated and extracted with additional ethyl acetate (2 x 10ml). The combined organic layers were dried (magnesium sulfate) and concentrated in vacuo to give the title compound as a solid, 60% yield, 775mg. LCMS: APCI ESI m/z 578 [M+H]⁺

¹H NMR (400 MHz, CDCI₃) δ = 1.70-1.80 (m, 2H), 1.94-2.03 (m, 2H), 2.34-2.43 (m, 2H), 2.67-2.74 (m, 4H), 2.79-2.88 (m, 4H), 3.41 (s, 2H), 3.46-3.51 (m, 2H), 4.74-4.81 (m, 1 H), 5.16-5.22 (m, 1 H), 6.16 (s, 1 H), 6.60 (s, 1 H), 6.64-6.68 (t, 2H), 6.89 (d, 2H), 7.09-7.17 (m, 4H), 7.21-7.23 (m, 1 H), 7.34-7.53 (m, 6H), 8.09 (d, 1 H) ppm.

Alternatively, the title compound may be prepared according to the following procedure: Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}-piperidin-4-yl ester (Preparation 5, 2.38g, 5.365mmol) and 3-hydroxyphenyl acetic acid (980mg, 6.44mmol) were dissolved in tetrahydrofuran (30ml). To this mixture was added N,N-dimethylaminopyridine (262mg, 2.15mmol) and (3-(dimethylamino)propyl)ethyl-carbodimide hydrochloride (1.23g, 6.44mmol) and the reaction mixture allowed to stir at room temperature for 18 hours. Dimethylformamide (4ml) was added to form a homogeneous solution and stirring continued for a further 4 hours. The mixture was partitioned between ethyl acetate (50ml) and water (50ml). The organic layer was washed with saturated aqueous sodium bicarbonate (50ml) and brine (30ml), dried over magnesium sulphate and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with ethyl acetate:methanol:880 ammonia, (100:0:0 to 94:6:0.6, by volume), to furnish the title compound as a white solid, in 67% yield, 2.2g.

The title compound (100mg) was added to hot acetonitrile (3ml) and mixture heated to reflux for 1 hour. The temperature was reduced to 7O⁰C for 30 minutes, then to 6O⁰C for 30 minutes, then back up to reflux for 8 hours. The mixture was cooled to room temperature gradually over 18 hours and the resulting solid filtered from solution to yield the title compound as a crystalline white solid, 80mg. LCMS: APCI ESI m/z 578 [M+H]⁺

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.59-1.70 (m, 2H), 1.83-1.91 (m, 2H), 2.32-2.42 (m, 2H), 2.51- 2.58 (m, 4H), 2.68-2.78 (m, 4H), 3.34-3.39 (m, 4H), 4.58-4.66 (m, 1 H), 6.65-6.69 (m, 3H), 7.03-7.11 (m, 5H), 7.23-7.44 (m, 8H), 7.56 (d, 1 H) ppm .

Example 9

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}-piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) was dissolved in dimethylformannide (0.5ml) and added to a reaction vessel containing 3,5-dichloro-2-hydroxybenzoic acid (18.6mg, 90μmol). To the reaction mixture was then added a solution of 1 -hydroxybenzotriazole monohydrate (16.5mg, 108μmol) in dimethylformamide (0.2ml), a solution of (3-(dimethylamino)propyl)ethyl carbodimide hydrochloride (20.7mg, 108μmol) in dimethylformamide (0.4ml) and diisopropylethylamine (39.2μl, 225μmol). The reaction was stirred at room temperature for 48 hours. The solvent was removed in vacuo and the residue partitioned between dichloromethane (2ml) and water (2ml). The aqueous phase was separated and extracted with further dichloromethane (1ml). The combined organic layers was concentrated in vacuo and the crude residue purified by HPLC method G to afford the title compound. LCMS Method G: RT 2.77 min (100%area) ES m/z 632 [M+H]⁺.

Example 10

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 3-chloro-2-hydroxy benzoic acid (15.5mg, 90μmol) using the same method as described in example 9. LCMS Method G: RT 2.63 min (100%area) ES m/z 598 [M+H]⁺.

Example 11 Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,4-difluoro-2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

The title compound was prepared from bi phenyl -2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 3,4-difluoro-2-hydroxy benzoic acid (15.7mg, 90μmol) using the same method as described in example 9. LCMS Method G: RT 2.59 min (100%area) ES m/z 600 [M+H]⁺.

Example 12

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvO-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 3-fluoro-2-hydroxy benzoic acid (14.1 mg, 90μmol) using the same method as described in example 9. LCMS Method G: RT 2.60 min (100%area) ES m/z 582 [M+H]⁺.

Example 13

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-chloro-2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvπ-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 4-chloro-2-hydroxy benzoic acid (15.5mg, 90μmol) using the same method as described in example 9. LCMS Method G: RT 2.82 min (100%area) ES m/z 598 [M+H]⁺.

Example 14

Biphenyl-2-yl-carbamic acid 1-(2-{4-r2-(4-fluoro-2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvO-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 4-fluoro-2-hydroxy benzoic acid (14.1 mg, 90μmol) using the same method as described in example 9. The crude product was purified by HPLC method E to yield the title compound.

LCMS Method E: RT 2.69 min (100%area)

ES m/z 582 [M+H]⁺.

Example 15 Biphenyl-2-yl-carbamic acid 1-(2-(4-[2-(4-chloro-3-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvπ-piperidin- 4-yl ester

(3-(Dimethylamino)propyl)ethylcarbodimide hydrochloride (1.36g, 7.10mnnol) was added to a solution of biphenyl-2-yl-carbamic acid 1 -{2-[4-(2-amino-ethyl)-phenyl}-ethyl}-piperidin-4-yl ester (Preparation 5,

2.25g, 5.07mnnol), 4-chloro-3-hydroxybenzoic acid (963mg, 5.58mnnol), triethylamine (1.06ml, 7.61 mmol) and 4-dimethylanninopyridine (248mg, 2.03mnnol) in tetrahydrofuran (100ml). The reaction was stirred at room temperature for 15 minutes, then heated at 5O⁰C for 18h. The solvent was removed in vacuo and the residue partitioned between ethyl acetate (75ml) and water (75ml). The aqueous layer was further extracted with ethyl acetate (75ml). The combined organic layers were washed with brine (75ml), dried

(magnesium sulphate) and concentrated in vacuo to yield a crude oil. The residue was passed through a plug of silica gel eluting with dichloromethane:methanol:880 ammonia (80:20:2, by volume) to yield the crude product as a pale yellow oil. This residue was dissolved in methanol (50ml) and water (10ml), and further purified by addition of potassium carbonate (5.54g) with stirr ing at 5O⁰C for 2.5 hours. The methanol was removed in vacuo and the aqueous residue adjusted to pH8. The aqueous layer was diluted with water (50ml) and extracted with ethyl acetate (2x 100ml). The combined organic layers were dried (sodium sulphate) and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with dichloromethane:methanol:880 ammonia (98:2:0.2 to 90:10:1 , by volume), to furnish the title compound as a white solid, in 34% yield, 1.02 g. LCMS: APCI ESI m/z 598 [M+H]⁺ 1H NMR (400 MHz, METHANOL-c/₄) δ = 1.60-1.70 (m, 2H), 1.82-1.90 (m, 2H), 2.35-2.40 (m, 2H), 2.55- 2.59 (m, 2H), 2.70-2.78 (m, 4H), 2.84-2.88 (m, 2H), 3.52-3.56 (m, 2H), 4.58-4.65 (m, 1 H), 7.12-7.18 (m, 5H), 7.23-7.44 (m, 10H), 7.56 (d, 1 H) ppm.

Example 16

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-fluoro-3-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethyl)-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 4-fluoro-3-hydroxy benzoic acid (14.1 mg, 90μmol) using the same method as described in example 9. The crude product was purified by HPLC method E to yield the title compound.

LCMS Method E: RT 2.65 min (95%area)

ES m/z 582 [M+H]⁺.

Example 17

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-chloro-4-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvπ-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 3.04g, 6.85mmol) and 2-chloro-4-hydroxybenzoic acid (1.42g, 8.22mmol) using the same method as described in example 15. Following aqueous work-up, the crude residue was dissolved in methanol (100ml) and water (20ml), and purified by addition of potassium carbonate (11.0g) with stirr ing at 5O⁰C for 2.5 hours. The methanol was removed in vacuo and the aqueous residue acidified to pH8. The aqueous layer was diluted with water (50ml) and extracted with ethyl acetate (2x 100ml). The combined organic layers were dried (sodium sulphate) and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with ethyl acetate:methanol:880 ammonia (100:0:0 to 90:10:1 , by volume), to furnish the title compound as a yellow foam, in 52% yield, 2.15g. LCMS: APCI ESI m/z 598 [M+H]⁺

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.60-1.69 (m, 2H), 1.83-1.90 (m, 2H), 2.35-2.40 (m, 2H), 2.55- 2.59 (m, 2H), 2.71-2.79 (m, 4H), 2.84-2.89 (m, 2H), 3.53-3.56 (m, 2H), 4.59-4.65 (m, 1 H), 6.71 (d, 1 H), 6.81 (s, 1 H), 7.13-7.44 (m, 13H), 7.56 (d, 1 H).

Alternatively, following purification, the title compound may be isolated by the following crystallisation procedure:

A solution of the title compound (100mg) in acetonitrile (6ml) was heated to give a clear solution and then allowed to cool to room temperature. The resulting solid was filtered and dried in vacuo to afford the title compound as a crystalline white solid, 40mg.

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.60-1.68 (m, 2H), 1.83-1.90 (m, 2H), 2.33-2.41 (m, 2H), 2.54-

2.59 (m, 2H), 2.70-2.79 (m, 4H), 2.85-2.89 (m, 2H), 3.53-3.56 (m, 2H), 4.59-4.64 (m, 1 H), 6.71 (d, 1 H),

6.81 (s, 1 H), 7.13-7.44 (m, 13H), 7.55 (d, 1 H) ppm . Example 18

Biphenyl-2-yl-carbannic acid 1 -(2-{4-[2-(5-chloro-2-hvdroxy-benzoylaπnino)-ethyl1-phenyl)-ethvπ-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 5-chloro-2-hydroxy benzoic acid (15.5mg, 90μmol) using the same method as described in example 9. The crude product was purified by HPLC method E to yield the title compound.

LCMS Method E: RT 2.75 min (100%area)

ES m/z 598 [M+H]⁺.

Example 19

Biphenyl^-yl-carbamic acid 1-(2-(4-r2-(5-fluoro-2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvO-piperidin- 4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 5-fluoro-2-hydroxy benzoic acid (14.1 mg, 90μmol) using the same method as described in example 9. The crude product was purified by HPLC method E to yield the title compound.

LCMS Method E: RT 2.78 min (100%area)

ES m/z 582 [M+H]⁺.

Example 20

Biphenyl^-yl-carbamic acid 1-(2-(4-r2-(3-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvD-piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 3-hydroxy benzoic acid (12.4mg, 90μmol) using the same method as described in example 9. The crude product was purified by HPLC method E to yield the title compound.

LCMS Method E: RT 2.64 min (85%area) ES m/z 564 [M+H]⁺.

Example 21 Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(2-hvdroxy-benzoylamino)-ethyl1-phenyl)-ethvO-piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 2-hydroxy benzoic acid (12.4mg, 90μmol) using the same method as described in example 9. The crude product was purified by HPLC method E to yield the title compound.

LCMS Method E: RT 2.70 min (100%area)

ES m/z 564 [M+H]⁺.

Example 22 Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4,5-dichloro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethvD- piperidin-4-yl ester

Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}-piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) was dissolved in ethanol (0.5ml) and added to a reaction vessel containing 4,5-dichloro-2- hydroxybenzaldehyde (17.2mg, 90μmol). To the reaction mixture was then added acetic acid (5.1 μl, 90μmol) and sodium sulphate (drying agent). The resulting mixture was allowed to stir for 1 hour. Sodium tri(acetoxy)borohydride (38mg, 178μmol) was then added, and the reaction allowed to stir at room temperature for 18h. The reaction was quenched with water (20μl) and stirred for 5 minutes. The solvents were removed in vacuo and the residue partitioned between dichloromethane (2ml) and saturated aqueous sodium hydrogen carbonate solution (2ml). The aqueous phase was separated and extracted with further dichloromethane (1 ml). The combined organic layers were concentrated in vacuo and the crude residue purified using an Isolute^® SCX-2 cartridge, eluting with methanol, followed by 1 M ammonia in methanol. The basic fractions were evaporated under reduced pressure and the residue purified by HPLC method E to afford the title compound. LCMS Method E: RT 2.27 min (100%area) ES m/z 618 [M+H]⁺.

Example 23

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-chloro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4- yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 5-chloro-2-hydroxy benzaldehyde (14.1 mg,

90μmol) using the same method as described in example 22.

LCMS Method E: RT 2.25 min (97%area) ES m/z 584 [M+H]⁺.

Example 24

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-chloro-3-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4-

Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}-piperidin-4-yl ester (Preparation 5, 2.Og, 4.51 mmol) was dissolved in ethanol (15ml). To this was added 2-chloro-3-hydroxybenzaldehyde (1.06g, 6.76mnnol) and titanium tefra-isopropoxide (2.64ml, 9.02mmol) with stirring at room temperature for 18 hours. Sodium borohydride (512mg, 13.5mmol) was then added with stirring at room temperature for a further 2 hours. The reaction was quenched by drop wise addition of water (10ml) and allowed to stir at room temperature for 10 minutes. The mixture was concentrated in vacuo, and the residue purified by column chromatography on silica gel eluting with ethyl acetate:methanol:880 ammonia, (100:0:0 to 90:10:1 , by volume), to furnish the title compound as a white foam, in 60% yield, 1.57g. LCMS: ESI m/z 584 [M+H]⁺

¹H NMR (400 MHz, METHANOL-^) δ = 1.61 -1.68 (m, 2H), 1.83-1.90 (m, 2H), 2.33-2.39 (m, 2H), 2.54- 2.58 (m, 2H), 2.70-2.85 (m, 8H), 3.83 (s, 2H), 4.59-4.65 (m, 1 H), 6.81 -6.85 (m, 2H), 7.05-7.13 (m, 5H), 7.23-7.44 (m, 8H), 7.56 (d, 1 H) ppm .

Alternatively, following purification, the title compound may be isolated by the following crystallisation procedure:

A solution of the title compound (50mg) in methanol (0.25ml) was heated to give a clear solution and then allowed to cool to room temperature. The resulting solid was filtered and dried in vacuo to afford the title compound as a crystalline white solid, 37mg. 1H NMR (400 MHz, METHANOL-c/₄) δ = 1.60-1.68 (m, 2H), 1.83-1.90 (m, 2H), 2.33-2.41 (m, 2H), 2.54-

2.59 (m, 2H), 2.70-2.79 (m, 4H), 2.85-2.89 (m, 2H), 3.53-3.56 (m, 2H), 4.59-4.64 (m, 1 H), 6.71 (d, 1 H),

6.81 (s, 1 H), 7.13-7.44 (m, 13H), 7.55 (d, 1 H) ppm .

Example 25 Biphenyl^-yl-carbamic acid 1 -(2-{4-[2-(3,5-dichloro-4-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}-piperidin-4-yl ester (Preparation 5, 200mg, 0.451 mmol) was dissolved in dichloroethane (5ml). To this was added 3,5-dichloro-4- hydroxybenzaldehyde (82.1 mg, 0.430mnnol) with stirring at room temperature for 1 hour. Sodium tri(acetoxy)borohydride (127mg, 0.601 mmol) was then added with stirr ing at room temperature for a further 18 hours. The reaction was quenched by dropwise addition of water (1 ml) and solvent removed in vacuo. The residue was partitioned between dichloromethane (20ml) and saturated aqueous sodium hydrogen carbonate (20ml). The organic layer was dried (magnesium sulphate) and concentrated in vacuo and the residue purified by column chromatography on silica gel eluting with dichloromethane:methanol:880 ammonia (100:0:0 to 95:5:0.5, by volume), to furnish the title compound as an off white solid, in 24% yield, 67mg. LCMS: ESI m/z 618 [M+H]⁺

¹H NMR (400 MHz, CDCI₃) δ = 1.68-1.76 (m, 2H), 1.94-2.00 (m, 2H), 2.28-2.33 (m, 2H), 2.56-2.60 (m, 2H), 2.70-2.88 (m, 8H), 3.65 (s, 2H), 4.72-4.78 (m, 1 H), 7.1 1-7.15 (m, 7H), 7.22 (d, 1 H), 7.34-7.51 (m, 6H), 8.10 (d, 1 H) ppm.

Example 26

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 3,5-dichloro-2-hydroxy benzaldehyde (17.2mg, 90μmol) using the same method as described in example 22. The crude product was purified by HPLC method G to yield the title compound. LCMS Method G: RT 4.15 min (94%area) ES m/z 616 [M-H]^".

Example 27

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-fluoro-3-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4- yl ester

Biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}-piperidin-4-yl ester (Preparation 5, 150mg, 0.338mnnol) was dissolved in dichloromethane (3ml). To this was added 4-fluoro-3- hydroxybenzaldehyde (Bioorg. Med. Chem, 2001, 9, 677; 82.1 mg, 0.338mmol), acetic acid (19.4μl, 0.338mnnol) and sodium tri(acetoxy)borohydride (143mg, 0.676mmol) and the mixture stirred at room temperature for 18 hours. The reaction was quenched by dropwise addition of aqueous sodium carbonate solution (2M, 15ml) and then partitioned between ethyl acetate (20ml) and water (10ml). The organic layer was washed with brine (15ml), dried (sodium sulphate) and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with dichloromethane:methanol:880 ammonia (100:0:0 to 90:10:1 , by volume), to furnish the title compound as an off white solid, in 59% yield, 113mg. LCMS: ESI m/z 568 [M+H]⁺

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.60-1.68 (m, 2H), 1.83-1.89 (m, 2H), 2.33-2.38 (m, 2H), 2.54- 2.58 (m, 2H), 2.70-2.80 (m, 8H), 3.66 (s, 2H), 4.59-4.66 (m, 1 H), 6.67-6.70 (m, 1 H), 6.84 (d, 1 H), 6.95 (m, 1 H), 7.09-7.15 (m, 4H), 7.23-7.44 (m, 8H), 7.55 (d, 1 H) ppm.

Example 28

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-4-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4- yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 150mg, 0.338mmol) and 3-chloro-4-hydroxybenzaldehyde (79.4mg, 0.507mmol) using the same method as described in example 24, to furnish the title compound as a white foam, in 82% yield, 163mg. LCMS: ESI m/z 584 [M+H]⁺

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.59-1.68 (m, 2H), 1.82-1.90 (m, 2H), 2.33-2.38 (m, 2H), 2.54- 2.58 (m, 2H), 2.71-2.80 (m, 8H), 3.65 (s, 2H), 4.58-4.64 (m, 1 H), 6.83 (d, 1 H), 7.03 (d, 1 H), 7.10-7.14 (m, 4H), 7.22-7.44 (m, 9H), 7.55 (d, 1 H) ppm . Example 29

Biphenyl-2-yl-carbannic acid 1 -(2-{4-[2-(3-fluoro-4-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4- yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 150mg, 0.338mmol) and 3-fluoro-4-hydroxy benzaldehyde (71.0mg, 0.507mmol) using the same method as described in example 24, to furnish the title compound as a white foam, in 61 % yield, 117mg. LCMS: ESI m/z 568 [M+H]⁺

¹H NMR (400 MHz, METHANOL-^) δ = 1.60-1.68 (m, 2H), 1.82-1.89 (m, 2H), 2.33-2.38 (m, 2H), 2.54- 2.58 (m, 2H), 2.70-2.80 (m, 8H), 3.66 (s, 2H), 4.58-4.64 (m, 1 H), 6.81-6.90 (m, 2H), 6.98 (d, 1 H), 7.09- 7.14 (m, 4H), 7.23-7.44 (m, 8H), 7.55 (d, 1 H) ppm.

Example 30

Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3-chloro-5-fluoro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 3-chloro-5-fluoro-2-hydroxy benzaldehyde (15.7mg, 90μmol) using the same method as described in example 22. The crude product was purified by HPLC method G to yield the title compound. LCMS Method G: RT 4.15 min (100%area) ES m/z 602 [M+H]⁺.

Example 31 Biphenyl-2-yl-carbannic acid 1-(2-{4-[2-(3,5-difluoro-4-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

H

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 200mg, 0.451 mmol) and 3,5-difluoro-4-hydroxy benzaldehyde (68.0mg, 0.430mmol) using the same method as described in example 25, to furnish the title compound as an off white solid, in 10% yield, 25mg. LCMS: ESI m/z 586 [M+H]⁺

¹H NMR (400 MHz, CDCI₃) δ = 1.70-1.79 (m, 2H), 1.95-2.01 (m, 2H), 2.31-2.36 (m, 2H), 2.57-2.65 (m, 2H), 2.75-2.97 (m, 8H), 3.61 (s, 2H), 4.73-4.79 (m, 1 H), 6.66 (d, 2H), 7.11-7.15 (m, 5H), 7.22 (d, 1 H), 7.34-7.43 (m, 4H), 7.47-7.51 (m, 2H), 8.10 (d, 1 H) ppm.

Example 32

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-fluoro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4- yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 5-fluoro-2-hydroxy benzaldehyde (12.6mg, 90μmol) using the same method as described in example 22. The crude product was purified by HPLC method G to yield the title compound. LCMS Method G: RT 4.01 min (100%area) ES m/z 568 [M+H]⁺.

Example 33 Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2,4-dichloro-3-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 1.5g, 3.381 mmol) and 2,4-dichloro-3-hydroxybenzaldehyde (Preparation 8, 969mg, 5.07mmol) using the same method as described in example 24, to furnish the title compound as a white solid, in 87% yield, 1.82g. LCMS: ESI m/z 618 [M+H]⁺

¹H NMR (400 MHz, DMSO-c/₆) δ = 1.37-1.46 (m, 2H), 1.67-1.75 (m, 2H), 2.13-2.18 (m, 2H), 2.42-2.46 (m, 2H), 2.62-2.72 (m, 8H), 3.73 (s, 2H), 4.40-4.47 (m, 1 H), 6.94 (d, 1 H), 7.08 (s, 4H), 7.24-4.42 (m, 10H) ppm.

Example 34

Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3,5-difluoro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 40mg, 90μmol) and 3,5-difluoro-2-hydroxy benzaldehyde (14.2mg, 90μmol) using the same method as described in example 22. The crude product was purified by HPLC method G and analysed by HPLC method E to yield the title compound. LCMS Method E: RT 2.34 min (100%area) ES m/z 586 [M+H]⁺.

Example 35

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-fluoro-3-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4- yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 150mg, 0.338mmol) and 2-fluoro-3-hydroxy benzaldehyde (Synthesis, (9), 710-12, 1988; 47.4mg, 0.338mmol) using the same method as described in example 27, to furnish the title compound as a white solid, in 53% yield, 102mg. LCMS: ESI m/z 568 [M+H]⁺

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.60-1.68 (m, 2H), 1.83-1.90 (m, 2H), 2.34-2.39 (m, 2H), 2.54- 2.58 (m, 2H), 2.71-2.84 (m, 8H), 3.80 (s, 2H), 4.58-4.65 (m, 1 H), 6.72-6.76 (m, 1 H), 6.80-6.85 (m, 1 H), 6.89-6.93 (m, 1 H), 7.09-7.14 (m, 4H), 7.23-7.44 (m, 8H), 7.55 (d, 1 H) ppm.

Example 36

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)-piperidin-4- yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 200mg, 0.451 mmol) and 3-fluoro-2-hydroxy benzaldehyde (68.0mg, 0.430mmol) using the same method as described in example 25, to furnish the title compound as an off white solid, in 10% yield, 25mg. LCMS: ESI m/z 568 [M+H]⁺

¹H NMR (400 MHz, CDCI₃) δ = 1.60-1.75 (m, 2H), 1.94-1.99 (m, 2H), 2.26-2.31 (m, 2H), 2.54-2.58 (m, 2H), 2.74-2.84 (m, 6H), 2.92-2.98 (m, 2H), 4.00 (s, 2H), 4.73-4.78 (m, 1 H), 6.64-6.76 (m, 2H), 6.94-6.99 (m, 1 H), 7.11-7.15 (m, 5H), 7.22 (d, 1 H), 7.34-7.43 (m, 4H), 7.47-7.52 (m, 2H), 8.10 (d, 1 H) ppm .

Example 37 Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-chloro-5-fluoro-2-hvdroxy-benzylamino)-ethyl1-phenyl)-ethyl)- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 200mg, 0.451 mmol) and 4-chloro-5-fluoro-2-hydroxy benzaldehyde (Preparation 9, 75.1 mg, 0.430mmol) using the same method as described in example 25, to furnish the title compound as a yellow oil, in 40% yield, 110mg. LCMS: ESI m/z 602 [M+H]⁺

¹H NMR (400 MHz, CDCI₃) δ = 1.67-1.76 (m, 2H), 1.94-2.00 (m, 2H), 2.26-2.32 (m, 2H), 2.55-2.59 (m, 2H), 2.74-2.82 (m, 6H), 2.89-2.92 (m, 2H), 3.90 (s, 2H), 4.72-4.78 (m, 1 H), 6.74 (d, 1 H), 6.82-6.83 (d, 1 H), 7.09-7.15 (m, 5H), 7.22 (d, 1 H), 7.34-7.44 (m, 4H), 7.47-7.51 (m, 2H), 8.10 (d, 1 H) ppm.

Example 38

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[bis-(3-chloro-4-hvdroxy-benzyl)-amino1-ethyl)-phenyl)-ethyl1- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 150mg, 0.338mmol) and 3-chloro-4-hydroxy benzaldehyde (52.9mg, 0.338mmol) using the same method as described in example 27, to furnish the title compound as a white solid, in 15% yield, 33mg. LCMS: ESI m/z 724 [M+H]⁺

¹H NMR (400 MHz, METHANOL-c/₄) δ = 1.60-1.70 (m, 2H), 1.84-1.91 (m, 2H), 2.37-2.44 (m, 2H), 2.59- 2.64 (m, 4H), 2.71-2.80 (m, 6H), 3.46 (s, 4H), 4.58-4.67 (m, 1 H), 6.80 (d, 2H), 6.98 (d, 4H), 7.08 (d, 2H), 7.14 (s, 2H), 7.23-7.44 (m, 8H), 7.55 (d, 1 H) ppm .

Example 39 Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[bis-(2-fluoro-3-hvdroxy-benzyl)-amino1-ethyl)-phenyl)-ethyl1- piperidin-4-yl ester

The title compound was prepared from biphenyl-2-yl-carbamic acid 1-{2-[4-(2-amino-ethyl)-phenyl]-ethyl}- piperidin-4-yl ester (Preparation 5, 150mg, 0.338mmol) and 2-fluoro-3-hydroxy benzaldehyde (Synthesis, (9), 710-12, 1988; 47.4mg, 0.338mmol) using the same method as described in example 27, to furnish the title compound as a white solid, in 14% yield, 32mg. LCMS: ESI m/z 692 [M+H]⁺

¹H NMR (400 MHz, METHANOL-^) δ = 1.60-1.69 (m, 2H), 1.83-1.90 (m, 2H), 2.35-2.41 (m, 2H), 2.54- 2.59 (m, 2H), 2.63-2.67 (m, 2H), 2.71-2.78 (m, 6H), 3.70 (s, 4H), 4.59-4.65 (m, 1 H), 6.76-6.89 (m, 6H), 7.00 (d,2H), 7.05 (d, 2H), 7.25-7.44 (m, 8H), 7.55 (d, 1 H) ppm.

HPLC methodology

Method A:

Method E:

Method G:

Cell based potency assessment at the human recombinant IVU muscarinic receptor Potency assay

M₃ potency was determined in CHO-K1 cells transfected with the NFAT-Betalactamase gene. CHO (Chinese Hamster Ovary) cells recombinantly expressing the human muscarinic M₃ receptor were transfected with the NFAT_β-Lac_Zeo plasmid. Cells were grown in DMEM with Glutamax-1 , supplemented with 25mM HEPES(Life Technologies 32430-027), containing 10% FCS (Foetal Calf Serum; Sigma F-7524), 1 nM Sodium pyruvate (Sigma S-8636), NEAA (non-Essential Amino Acids; Invitrogen 11140-035) and 200μg/ml Zeocin (Invitrogen R250-01 ).

hM₃ β-Lactamase Assay Protocol Cells were harvested for assay when they reached 80-90% confluency using enzyme free cell Dissociation Solution (Life technologies 13151 -014) incubated with the cells for 5 min at 37°C in an atmosphere containing 5% CO₂. Detached cells were collected in warmed growth media and centrifuged at 2000rpm for 10min, washed in PBS (Phosphate Buffered Saline; Life Technologies 14190-094) and centrifuged again as just described. The cells were re-suspended at 2x10⁵ cells/ml in growth medium (composition as described above). 20μl of this cell suspension was added to each well of a 384 well black clear bottomed plate (Greiner Bio One 781091-PFI). The assay buffer used was PBS supplemented with 0.05% Pluronic F-127 (Sigma 9003-11-6) and 2.5% DMSO. Muscarinic M₃ receptor signalling was stimulated using 8OnM carbamyl choline (Aldrich N240-9) incubated with the cells for 4h at 37°C /5% CO₂ and monitored at the end of the incubation period using a Tecan SpectraFluor÷ plate reader (λ - excitation 405nm, emission 450nm and 503nm). M₃ receptor antagonists under test were added to the assay at the beginning of the 4h incubation period and compound activity measured as the concentration dependent inhibition of the carbamyl choline induced signal. Inhibition curves were plotted and IC₅₀ values generated using a 4-parameter sigmoid fit and converted to Ki values using the Cheng- Prusoff correction and the K_D value for carbamyl choline in the assay.

Binding affinity assessment at the human recombinant M₃ muscarinic receptor

Membrane preparation

Cell Pellets from CHO (Chinese Hamster Ovary) cells recombinantly expressing the human muscarinic

M₃ receptor were homogenised in 2OmM HEPES (pH7.4) and centrifuged at 48000 x g for 20min at 4°C. The pellet was re-suspended in buffer and the homogenisation and centrifugation steps repeated. The resulting pellet was re-suspended in 1 ml buffer per 1 ml original packed cell volume and the homogenisation step repeated. Protein estimation was carried out on the suspension and 1 ml aliquots of ~1 mg/ml frozen at -8O⁰C.

hM₃ competition binding Assay Protocol

Membranes (5μg/well) were incubated with ³H-NMS (at a concentration 5 x K_D) plus/minus test compound for 24hr at RT (room temperature) in a 1 ml polystyrene 96-well deep well block. The final assay volume was 200μl, comprising of: 20μl plus/minus test compound; 20μl ³H-NMS (Perkin Elmer NEN 636) and 160μl membrane solution. Total Binding was defined with 0.1 % DMSO; Non-Specific Binding was defined with 1μM Atropine. Assay buffer was 2OmM Hepes (pH 7.4). Once all assay components were added, plates were covered and incubated at room temperature for 24 hrs with shaking. The assay was terminated by rapidly filtering through GF/B Unifilter plates pre-soaked with 0.5% polyethylenimine, using a Packard filtermate harvester, the filter plate was then washed with 3x1 ml 4⁰C assay buffer. The filter plates were dried at 45°C for 1 hour. The bottoms of the filter plates were sealed and 50μl/well of Microscint '0' added, the top of the plates were sealed with a Topseal. Following 90mins, the plates were read on an NXT Topcount (1 minute read time per well). The resulting data was expressed as a percentage of the specific binding (Specific binding = Total binding - Non-Specific Binding). % specific binding versus test compound concentration was plotted to determine an IC₅₀ from a sigmoid curve using an in-house data analysis programme. IC₅₀ values corrected to Ki values by applying the Cheng-Prussoff equation:

Cheng-Prussoff equation: ια '50

K =

1 + [L]/K_D where IC₅₀ is the concentration of unlabelled drug which inhibits by 50% the specific radioligand binding. [L] is the free radioligand concentrations and K_D and K, are the equilibrium dissociation constants of the radioligand and unlabelled drug respectively.

It has thus been found that compounds of formula (I) according to the present invention that have been tested in the above assays show hM₃ receptor antagonist activity as listed in the table below:

n.d. - not determined

Guinea Pig Trachea assay

Male, Dunkin-Hartley guinea-pigs weighing 350-45Og are culled in a rising concentration of CO₂, followed by exsanguinations of the vena cava. Tracheas are dissected from the larynx to the entry point into the chest cavity and then placed in fresh, oxygenated, modified Krebs buffer solution (Krebs containing 10μM propranolol, 10μM guanethidine and 3μM indomethacin) at room temperature. The tracheas are opened by cutting through the cartilage opposite the trachealis muscle. Strips approximately 3-5 cartilage rings wide are cut. A cotton thread is attached to the cartilage at one end of the strip for attachment to the force transducer and a cotton loop made at the other end to anchor the tissue in the organ bath. The strips are mounted in 5ml organ baths filled with warm (37⁰C) aerated modified Krebs. The pump flow rate is set to 1.0 ml/ min and the tissues washed continuously. Tissues are placed under an initial tension of 1000mg. Tissues are re-tensioned after 15 and 30 minutes, then allowed to equilibrate for a further 30-45 minutes. Tissues are subjected to electrical field stimulation (EFS) of the following parameters: 10s trains every 2 minutes, 0.1 ms pulse width, 10Hz and 10-30V. The voltage is raised 5V every 10min within the stated range until a maximum contractile response for each tissue is observed. This just maximum voltage for each tissue is then used throughout the remainder of the experiment. Following equilibration to EFS for 20min, the pump is stopped, and after 15min control readings are taken over a 8-10 min period (4-5 responses). Compound is then added to each tissue as a bolus dose at 3OxKi (determined at the human M₃ receptor expressed in CHO cells in a filtration binding assay), and left to incubate for 2h. Compound is then washed from tissues using a rapid wash with modified Krebs for 1 min and flow is restored to 1 ml/min for the remainder of the experiment. At the end of the experiment tissues are challenged with histamine (1μM) to determine viability. Readings taken during the experiment are automatically collected using Notocord ® software. The raw data are converted into percent response taking into account measurements of inhibition of the EFS response. After starting washout, the times taken for the tissue to recover by 25% from the inhibition induced are recorded and used as a measure of compound duration of action. Tissue viability limits the duration of the experiment to 16h post-compound washout. Compounds are typically tested at n=2 to 5 to estimate duration of action.

Alternatively the following Guinea Pig Trachea assay can also be used:

Trachea were removed from male Dunkin-Hartley guinea-pigs (wt 350-45Og) and following removal of adherent connective tissue, an incision was made through the cartilage opposite the trachealis muscle and tracheal strips 3-5 cartilage rings wide prepared. The tracheal strips were suspended between an isometric strain gauge and a fixed tissue hook with the muscle in the horizontal plane in 5ml tissue baths under an initial tension of 1g and bathed in warmed (37°C) aerated (95%0₂/5%C0₂) Krebs solution containing 3μM indomethacin and 10μM guanethidine. The tissues were positioned between parallel platinum wire electrodes (~1 cm gap). A constant 1 ml/min flow of fresh Krebs solution (of the above composition) was maintained through the tissue baths using peristaltic pumps. The tissues were allowed to equilibrate for an hour with re-tensioning to 1g at 15min and 30min from the start of the equilibration period. At the end of the equilibration, tissues were electrically field stimulated (EFS) using the following parameters: 10V, 10Hz 0.1 ms pulse width with 10sec trains every 2 min. In each tissue a voltage response curve was constructed over the range 10v - 30V (keeping all other stimulation parameters constant) to determine a just maximal stimulation. Using these stimulation parameters EFS responses were 100% nerve mediated and 100% cholinergic as confirmed by blockade by 1μM tetrodotoxin or 1 μM atropine. Tissues were then repeatedly stimulated at 2 min intervals until the responses were reproducible. The peristaltic pump was stopped 20 min prior to the addition of the study compound and the average twitch contraction over the last 10min recorded as the control response. The study compound was added to the tissue baths, with each tissue receiving a single concentration of compound and allowed to equilibrate for 2h. At 2h post addition the inhibition of the EFS response was recorded and IC₅₀ curves generated using a range of compound concentrations over tracheal strips from the same animal. The tissues were then rapidly washed and the 1 ml/min perfusion with Krebs solution reestablished. Tissues were stimulated for a further 16h and recovery of the EFS response recorded. At the end of the 16h, 10μM histamine was added to the baths to confirm tissue viability. The just max concentration (tested concentration giving a response > 70% inhibition but less than 100%) of antagonist was identified from the IC₅₀ curve and the time to 25% recovery of the induced inhibition (T₂₅) calculated in tissues receiving this concentration. Compounds are typically tested at n=2 to 5 to estimate duration of action.

Claims

Claims

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, wherein:

X is -CH₂-, -C(=O)CH₂- or -C(=O) -; one of R², R³, R⁴ and R⁵ is hydroxy, one of R², R³, R⁴ and R⁵ is H and two of R², R³, R⁴ and R⁵ are independently H or halo;

R¹ is H or methyl or alternatively when X is -CH₂-, R¹ can also represent a group of formula:

wherein one of R⁶, R⁷, R⁸ and R⁹ is hydroxy, one of R⁶, R⁷, R⁸ and R⁹ is H and two of R⁶, R⁷, R⁸ and R⁹ are independently H or halo.

2. A compound according to claim 1 , or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, where R¹ is H or methyl.

3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, wherein: one of R², R³, R⁴ and R⁵ is hydroxy, one of R², R³, R⁴ and R⁵ is halo, one of R², R³, R⁴ and R⁵ is H, and, one of R², R³, R⁴ and R⁵ is selected from H or halo.

4. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, where one of R³ and R⁴ is hydroxy and the other is halo.

5. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, where one of R³ and R⁵ is hydroxy and the other is halo.

6. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, where R³ is hydroxy and R⁴ is halo.

7. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, where one of R², R³, R⁴ and R⁵ is hydroxy and the others are H.

8. A compound according to claim 1 , or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, said compound being selected from: Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2,3-difluoro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin- 4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-fluoro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester; Biphenyl-2-yl-carbamic acid 1 -[2-(4-{2-[2-(3-fluoro-4-hydroxy-phenyl)-acetylamino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[2-(3-chloro-4-hydroxy-phenyl)-acetylamino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[2-(3-hydroxy-phenyl)-acetylamino]-ethyl}-phenyl)-ethyl]-piperidin- 4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3,4-difluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-chloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin- 4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-fluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-chloro-3-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(4-fluoro-3-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester; Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-chloro-4-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-chloro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(5-fluoro-2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-

4-yl ester;

Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(2-hydroxy-benzoylamino)-ethyl]-phenyl}-ethyl)-piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4,5-dichloro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-chloro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2-chloro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester; Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-dichloro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-fluoro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-chloro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(3-chloro-5-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-difluoro-4-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(5-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(2,4-dichloro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3,5-difluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester; Biphenyl-2-yl-carbamic acid 1 -(2-{4-[2-(2-fluoro-3-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(3-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)-piperidin-4- yl ester;

Biphenyl-2-yl-carbamic acid 1-(2-{4-[2-(4-chloro-5-fluoro-2-hydroxy-benzylamino)-ethyl]-phenyl}-ethyl)- piperidin-4-yl ester;

Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[bis-(3-chloro-4-hydroxy-benzyl)-amino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester; and Biphenyl-2-yl-carbamic acid 1-[2-(4-{2-[bis-(2-fluoro-3-hydroxy-benzyl)-amino]-ethyl}-phenyl)-ethyl]- piperidin-4-yl ester.

9. A pharmaceutical composition comprising at least an effective amount of a compound of the formula (I) as described in any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt.

10. A compound of the formula (I) as described in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, for use as a medicament.

11. A compound of the formula (I) as described in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, for use in the treatment of diseases, disorders, and conditions selected from the group consisting of: - chronic or acute bronchoconstriction, chronic bronchitis, small airways obstruction, and emphysema;

- obstructive or inflammatory airways diseases of whatever type, etiology, or pathogenesis, in particular an obstructive or inflammatory airways disease that is a member selected from the group consisting of chronic eosinophilic pneumonia, chronic obstructive pulmonary disease (COPD), COPD that includes chronic bronchitis, pulmonary emphysema or dyspnea associated or not associated with COPD, COPD that is characterized by irreversible, progressive airways obstruction, adult respiratory distress syndrome (ARDS), exacerbation of airways hyper-reactivity consequent to other drug therapy and airways disease that is associated with pulmonary hypertension,

- bronchitis of whatever type, etiology, or pathogenesis, in particular bronchitis that is a member selected from the group consisting of acute bronchitis, acute laryngotracheal bronchitis, arachidic bronchitis, catarrhal bronchitis, croupus bronchitis, dry bronchitis, infectious asthmatic bronchitis, productive bronchitis, staphylococcus or streptococcal bronchitis and vesicular bronchitis;

- asthma of whatever type, etiology, or pathogenesis, in particular asthma that is a member selected from the group consisting of atopic asthma, non-atopic asthma, allergic asthma, atopic bronchial IgE-mediated asthma, bronchial asthma, essential asthma, true asthma, intrinsic asthma caused by pathophysiologic disturbances, extrinsic asthma caused by environmental factors, essential asthma of unknown or inapparent cause, non-atopic asthma, bronchitic asthma, emphysematous asthma, exercise-induced asthma, allergen induced asthma, cold air induced asthma, occupational asthma, infective asthma caused by bacterial, fungal, protozoal, or viral infection, non-allergic asthma, incipient asthma, wheezy infant syndrome and bronchiolytis; - acute lung injury; and

- bronchiectasis of whatever type, etiology, or pathogenesis, in particular bronchiectasis that is a member selected from the group consisting of cylindric bronchiectasis, sacculated bronchiectasis, fusiform bronchiectasis, capillary bronchiectasis, cystic bronchiectasis, dry bronchiectasis and follicular bronchiectasis.

12. The use of a compound of the formula (I) as described in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, for the manufacture of a drug for the treatment of treatment of the diseases, disorders, and conditions as defined in claim 11.

13. Combination of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt or solvate thereof, with other therapeutic agent(s) selected from:

(a) 5-Lipoxygenase (5-LO) inhibitors or 5-lipoxygenase activating protein (FLAP) antagonists;

(b) Leukotriene antagonists (LTRAs) including antagonists of LTB₄, LTC₄, LTD₄, and LTE₄;

(c) Histamine receptor antagonists including H1 and H3 antagonists;

(d) OC₁- and oc₂-adrenoceptor agonist vasoconstrictor sympathomimetic agents for decongestant use; (e) PDE inhibitors, e.g. PDE3, PDE4 and PDE5 inhibitors;

(f) Beta 2 receptor agonists;

(g) Theophylline;

(h) Sodium cromoglycate;

(i) COX inhibitors both non-selective and selective COX-1 or COX-2 inhibitors (NSAIDs); (j) Prostaglandin receptor antagonists and inhibitors of prostaglandin synthase;

(k) Oral and inhaled glucocorticosteroids;

(I) Dissociated agonists of the corticoid receptor (DAGR);

(m) Monoclonal antibodies active against endogenous inflammatory entities;

(n) Anti-tumor necrosis factor (anti-TNF-α) agents; (o) Adhesion molecule inhibitors including VLA-4 antagonists;

(p) KJnJn-B₁ - and B₂ -receptor antagonists;

(q) Immunosuppressive agents, including inhibitors of the IgE pathway and cyclosporine;

(r) Inhibitors of matrix metalloproteases (MMPs);

(s) Tachykinin NK₁, NK₂ and NK₃ receptor antagonists; (t) Protease inhibitors such as elastase inhibitors;

(u) Adenosine A2a receptor agonists and A2b antagonists;

(v) Inhibitors of urokinase;

(w) Compounds that act on dopamine receptors, such as D2 agonists;

(x) Modulators of the NFiφ pathway, such as IKK inhibitors; (y) modulators of cytokine signalling pathyways such as p38 MAP kinase, PI3 kinase, JAK kinase, syk kinase, EGFR or MK-2;

(z) Agents that can be classed as mucolytics or anti-tussive;

(aa) Agents, which enhance responses to inhaled corticosteroids;

(bb) Antibiotics and antivral agents effective against micro-organisms which can colonize the respiratory tract;

(cc) HDAC inhibitors;

(dd) CXCR2 antagonists;

(ee) lntegrin antagonists;

(ff) Chemokines; (gg) Epithelial sodium channel (ENaC) blockers or Epithelial sodium channel (ENaC) inhibitors;

(hh) P2Y2 Agonists and other Nucleotide receptor agonists;

(ii) Inhibitors of thromboxane; (jj) Inhibitors of PGD₂ synthesis and PGD₂ receptors (DP1 and DP2/CRTH2);

(kk) Niacin; and

(II) Adhesion factors including VLAM, ICAM, and ELAM.