WO2006125665A1 - Substituted piperidine antagonist of hi receptor to be used for the treatment of rhinitis - Google Patents

Abstract

The present invention relates to the compound, (I) and salts thereof, processes for its preparation, to compositions containing it and to its use in the treatment of various disorders, such as allergic rhinitis.

Description

SUBSTITUTED PIPERIDINE ANTAGONIST OF Hl RECEPTOR TO BE USED FOR THE TREATMENT OF RHINITIS

The present invention relates to a compound, processes for its preparation, compositions containing it and to its use in the treatment of various disorders, in particular inflammatory and/or allergic disorders of the respiratory tract.

Allergic rhinitis, pulmonary inflammation and congestion are medical conditions that are often associated with other conditions such as asthma, chronic obstructive pulmonary disease (COPD), seasonal allergic rhinitis and perennial allergic rhinitis. In general these conditions are mediated, at least in part, by inflammation associated with the release of histamine from various cells, in particular mast cells.

Allergic rhinitis, also known as 'hay fever' affects a large proportion of the population worldwide. There are two types of allergic rhinitis, seasonal and perennial. The clinical symptoms of seasonal allergic rhinitis, typically include nasal itching and irritation, sneezing and watery rhinorrhea which is often accompanied by nasal congestion. The clinical symptoms of perennial allergic rhinitis are similar except that nasal blockage may be more pronounced. Either type of allergic rhinitis may also cause other symptoms such as itching of the throat and/or eyes, epiphora and oedema around the eyes. The symptoms of allergic rhinitis may vary in intensity from the nuisance level to debilitating.

Allergic rhinitis and other allergic conditions are associated with the release of histamine from various cell types but particularly mast cells. The physiological effects of histamine are classically mediated by three receptor subtypes, termed H1 , H2 and H3. H1 receptors are widely distributed throughout the CNS and periphery, and are involved in wakefulness and acute inflammation. H2 receptors mediate gastric acid secretion in response to histamine. H3 receptors are present on the nerve endings in both the CNS and periphery and mediate inhibition of neurotransmitter release [Hill et al, Pharmacol. Rev. 49:253-278 (1997)]. Recently a fourth member of the histamine receptor family has been identified, termed the H4 receptor [Hough, MoI. Pharmacol. 59: 415-419, (2001)]. Whilst the distribution of the H4 receptor appears to be restricted to cells of the immune and inflammatory systems, a physiological role for this receptor remains to be identified.

The activation of H1 receptors in blood vessels and nerve endings are responsible for many of the symptoms of allergic rhinitis, which include itching, sneezing, and the production of watery rhinorrhea. Antihistamine compounds, i.e. drugs which are selective H1 receptor antagonists such as chlorphenyramine and cetirizine, are effective in treating the itching, sneezing and rhinorrhea associated with allergic rhinitis, but are not effective against the nasal congestion symptoms [Aaronson, Ann. Allergy, 67:541-547, (1991 )]. Thus, H1 receptor antagonists have been administered in combination with sympathomimetic agents such as pseudoephedrine or oxymetazoline to treat the nasal congestion symptoms of allergic rhinitis. These drugs are thought to produce a decongestant action by activating β-adrenergic receptors and increasing the vascular tone of blood vessels in the nasal mucosa. The use of sympathomimetic drugs for the treatment of nasal congestion is frequently limited by the CNS stimulant properties and their effects on blood pressure and heart rate. A treatment which decreases nasal congestion without having effects on the CNS and cardiovascular system may therefore offer advantages over existing therapies.

Histamine H3 receptors are expressed widely on both CNS and peripheral nerve endings and mediate the inhibition of neurotransmitter release. In vitro electrical stimulation of peripheral sympathetic nerves in isolated human saphenous vein results in an increase in noradrenaline release and smooth muscle contraction, which can be inhibited by histamine H3 receptor agonists [Molderings et al, Naunyn-Schmiedeberg's Arch. Pharmacol., 346: 46-50, (1992); Valentine et al,. Eur. J. Pharmacol., 366: 73-78, (1999)]. H3 receptor agonists also inhibit the effect of sympathetic nerve activation on vascular tone in porcine nasal mucosa [Varty & Hey. Eur. J. Pharmacol., 452:339-345, (2002)]. In vivo, H3 receptor agonists inhibit the decrease in nasal airway resistance produced by sympathetic nerve activation [Hey et al, Arzneim-Forsch Drug Res., 48:881-888 (1998)]. Activation of histamine H3 receptors in human nasal mucosa inhibits sympathetic vasoconstriction [Varty et al. Eur. J. Pharmacol., 484:83-89, (2004)]. Furthermore, H3 receptor antagonists in combination with histamine H1 receptor antagonists have been shown to reverse the effects of mast cell activation on nasal airway resistance and nasal cavity volume, an index of nasal congestion [Mcleod etal, AmJ. Rhinol., 13:391-399, (1999)], and further evidence for the contribution of H3 receptors to histamine-induced nasal blockage is provided by histamine nasal challenge studies performed on normal human subjects [Taylor-Clark et al. British J. Pharmacol., 1-8 (2005)].

The present invention relates to a compound (or salt thereof) that is a histamine H3 antagonist and/or inverse agonist. This compound (or salt thereof) may be useful in the treatment of various disorders in particular inflammatory and/or allergic disorders, such as inflammatory and/or allergic disorders of the respiratory tract, for example allergic rhinitis, that are associated with the release of histamine from cells such as mast cells. Further, the compound of the invention (or salt thereof) may show an improved profile over known

H3 antagonists/inverse agonists in that it may possess one or more of the following properties:

(i) potent H3 antagonist/inverse agonist activity with a pKi (pKb) of greater than about 9.5;

(ii) selective for the H3 receptor over the H1 receptor;

(iii) low CNS penetration;

(iv) improved bioavailability; and

(v) lower clearance and/or longer half-life in blood.

Compounds having such a profile may be orally effective, and/or capable of once daily administration and/or further may have an improved side effect profile compared with other existing therapies.

Thus, the present invention provides, in a first aspect, the compound 1-{[4-(1- azetidinylcarbonyl)phenyl]carbonyl}-4-(4-{[1-(1-methylethyl)-4- piperidinyl]oxy}phenyl)piperidine

(I)

or a salt thereof, such as a pharmaceutically acceptable salt.

It is to be understood that the present invention covers the compound of formula (I) as the free base and as a salt thereof e.g. a pharmaceutically acceptable salt.

It is to be further understood that references hereinafter to the compound of formula (I) or compound of the invention means a compound of formula (I) as the free base, or as a salt, or as a solvate. Thus, included within the scope of the invention are all salts, solvates, hydrates, complexes and polymorphic forms of the compound for formula (I).

The compound of the present invention may be in the form of and/or may be administered as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include acid addition salts. For a review on suitable salts see Berge et al., J. Pharm. ScL, 1977, 66, 1-19.

A pharmaceutically acceptable acid addition salt may be readily prepared by using a desired acid as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. Typically a pharmaceutically acceptable acid addition salt can be formed by reaction of the compound of formula (I) with a suitable inorganic or organic acid (such as hydrobromic, hydrochloric, formic, sulfuric, nitric, phosphoric, succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid), optionally in a suitable solvent such as an organic solvent, to give the salt which is usually isolated for example by crystallisation and filtration. Thus, a pharmaceutically acceptable acid addition salt of the compound of formula (I) can be for example a hydrobromide, hydrochloride, formate, sulfate, nitrate, phosphate, succinate, maleate, acetate, fumarate, citrate, tartrate, benzoate, p-toluenesulfonate, methanesulfonate or naphthalenesulfonate salt.

Other non-pharmaceutically acceptable salts, eg. oxalates or trifluoroacetates, may be used, for example in the isolation of the compound of the invention, and are included within the scope of this invention. The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compound of formula (I).

It will be appreciated that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compound of the invention are within the scope of the invention.

The compound of formula (I) may be in crystalline or amorphous form. Furthermore, some of the crystalline forms of the compound of formula (I) may exist as polymorphs, which are included within the scope of the present invention. The most thermodynamically stable polymorphic form of the compound of formula (I) is of particular interest.

Polymorphic forms of the compound of formula (I) may be characterized and differentiated using a number of conventional analytical techniques, including, but not limited to, X-ray powder diffraction (XRPD) patterns, infrared (IR) spectra, Raman spectra, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and solid state nuclear magnetic resonance (NMR).

The compound of formula (I) may exist in one of several tautomeric forms. It will be understood that the present invention encompasses all tautomers of the compound of formula (I) whether as individual tautomers or as mixtures thereof.

The present invention also provides processes for the preparation of the compound of formula (I) or a salt thereof.

Thus, according to a first process A, the compound of formula (I) may be prepared by reacting 4-(azetidinyl-1-ylcarbonyl)benzoic acid shown in formula (II)

with a compound of formula (III)

or a salt thereof, for example an acid addition salt.

The reaction may be carried out in the presence of a suitable base such as diisopropylethylamine and a suitable coupling agent such as O-(benzotriazol-i-yl)- Λ/,Λ/,Λ/,Λ/-tetramethyluronium tetrafluoroborate (TBTU), in an appropriate solvent such as dichloromethane.

Compound of formula (III) may be prepared in accordance with the following or similar reaction scheme:

(VIl)

(Vl)

Reagents and Conditions: a) n-butyl lithium in hexane, phenylmethyl 4-oxo-1- piperidinecarboxylate, tetrahydrofuran; b) trifluoroacetic acid, triethylsilane, dichloromethane, -78⁰C; c) acetone, sodium triacetoxyborohydride; d) H₂, 10% palladium on carbon, ethanol. A compound of formula (Vl) is known (see Description 5 in WO0489373). A compound of formula (VII) is known (see Example 22(a) in WO0069819).

Examples of protecting groups that may be employed in the synthetic routes described and the means for their removal can be found in T. W. Greene 'Protective Groups in Organic Synthesis' (3rd edition, J. Wiley and Sons, 1999). Suitable amine protecting groups include sulphonyl (e.g. tosyl), acyl (e.g. acetyl, 2¹,2',2'-trichloroethoxycarbonyl₁ benzyloxycarbonyl or t-butoxycarbonyl) and arylalkyl (e.g. benzyl), which may be removed by hydrolysis (e.g. using an acid such as hydrogen chloride in dioxan or trifluoroacetic acid in dichloromethane) or reductively (e.g. hydrogenolysis of a benzyl group or reductive removal of a 2',2',2'-trichloroethoxycarbonyl group using zinc in acetic acid) as appropriate. Other suitable amine protecting groups include trifluoroacetyl (-COCF₃) which may be removed by base catalysed hydrolysis or a solid phase resin bound benzyl group, such as a Merrifield resin bound 2,6-dimethoxybenzyl group (Ellman linker), which may be removed by acid catalysed hydrolysis, for example with trifluoroacetic acid.

According to second process B, a salt of the compound of formula (I) may be prepared by exchange of counterions, or precipitation of the desired salt from the free base.

It will be appreciated that all novel intermediates used to prepare the compound of the invention form yet a further aspect of the present invention.

Examples of disease states in which the compound of formula (I), or a pharmaceutically acceptable salt thereof may have potentially beneficial anti-inflammatory and/or antiallergic effects include diseases of the respiratory tract such as bronchitis (including chronic bronchitis), asthma (including allergen-induced asthmatic reactions), chronic obstructive pulmonary disease (COPD), cystic fibrosis, sinusitis and allergic rhinitis (seasonal and perennial). Other disease states include diseases of the gastrointestinal tract such as intestinal inflammatory diseases including inflammatory bowel disease (e.g. Crohn's disease or ulcerative colitis) and intestinal inflammatory diseases secondary to radiation exposure or allergen exposure.

Furthermore, the compound of the invention may be used to treat nephritis, skin diseases such as psoriasis, eczema, allergic dermatitis and hypersensitivity reactions. The compound of the invention may also be of use in the treatment of nasal polyposis, conjunctivitis or pruritis.

Further diseases include inflammatory diseases of the gastrointestinal tract such as inflammatory bowel disease.

A disease of particular interest is allergic rhinitis.

Compounds that are antagonists and/or inverse agonists of the H3 receptor may also be of use in other diseases in which activation of the H3 receptor may be implicated. Such diseases may include non-allergic rhinitis.

It will be appreciated by those skilled in the art that references herein to treatment or therapy extend to prophylaxis as well as the treatment of established conditions.

As mentioned above, the compound of formula (I) may be useful as a therapeutic agent. There is thus provided, as a further aspect of the invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in therapy.

According to another aspect of the invention, there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of any of the above diseases.

In a further aspect there is provided a method for the treatment of any of the above diseases, in a human or animal subject in need thereof, which method comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof such as a pharmaceutically acceptable salt or solvate.

When used in therapy, the compound of formula (I) is usually formulated in a suitable composition. Such compositions can be prepared using standard procedures.

Thus, the present invention further provides a composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof optionally with one or more pharmaceutically acceptable carriers and/or excipients. A composition of the invention, which may be prepared by admixture, suitably at ambient temperature and atmospheric pressure, is usually adapted for oral, parenteral or rectal administration and, as such, may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable or infusible solutions or suspensions or suppositories. Orally administrable compositions are of particular interest.

Tablets and capsules for oral administration may be in unit dose form, and may contain conventional excipients, such as binding agents, fillers, tabletting lubricants, disintegrants and acceptable wetting agents. The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and, if desired, conventional flavourings or colorants.

For parenteral administration, fluid unit dosage forms are prepared utilising a compound of the invention or pharmaceutically acceptable salt thereof and a sterile vehicle. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, the compound can be dissolved for injection and filter sterilised before filling into a suitable vial or ampoule and sealing. Adjuvants such as a local anaesthetic, preservatives and buffering agents are dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilisation cannot be accomplished by filtration. The compound can be sterilised by exposure to ethylene oxide before suspension in a sterile vehicle. A surfactant or wetting agent may be included in the composition to facilitate uniform distribution of the compound.

The composition may contain from about 0.1 % to 99% by weight, such as from about 10 to 60% by weight, of the active material, depending on the method of administration. The dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses may be about 0.05 to 1000 mg, more suitably about 1.0 to 200 mg, and such unit doses may be administered more than once a day, for example two or three a day. Such therapy may extend for a number of weeks or months. In one embodiment compound and compositions according to the invention are suitable for oral administration and/or are capable of once daily administration.

The compound and compositions according to the invention may be used in combination with or include one or more other therapeutic agents, for example selected from antiinflammatory agents, anticholinergic agents (particularly an M₁ZM₂ZM₃ receptor antagonist), β₂-adrenoreceptor agonists, antiinfective agents (e.g. antibiotics, antivirals), or antihistamines. The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with one or more other therapeutically active agents, for example selected from an antiinflammatory agent (for example another corticosteroid or an NSAID), an anticholinergic agent, a β₂-adrenoreceptor agonist, an antiinfective agent (e.g. an antibiotic or an antiviral), or an antihistamine. Combinations comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with a β₂-adrenoreceptor agonist, andZor an anticholinergic, andZor a PDE-4 inhibitor form yet another aspect of the invention. The combinations of the invention may comprise one or two other therapeutic agents, and may optionally include one or more pharmaceutically acceptable carriers andZor excipients as desired.

It will be clear to a person skilled in the art that, where appropriate, the other therapeutic ingredient(s) may be used in the form of salts, (e.g. as alkali metal or amine salts or as acid addition salts), or prodrugs, or as esters (e.g. lower alkyl esters), or as solvates (e.g. hydrates) to optimise the activity andZor stability andZor physical characteristics (e.g. solubility) of the therapeutic ingredient. It will be clear also that where appropriate, the therapeutic ingredients may be used in optically pure form.

Examples of β₂-adrenoreceptor agonists include salmeterol (e.g. as racemate or a single enantiomer such as the R-enantiomer or the S-enantiomer), salbutamol (e.g. as racemate or a single enantiomer such as the ft-enantiomer), formoterol (e.g. as racemate or a single enantiomer such as the /?-enantiomer), salmefamol, fenoterol carmoterol, etanterol, naminterol, clenbuterol, pirbuterol, flerbuterol, reproterol, bambuterol, indacaterol, terbutaline and salts thereof, for example the xinafoate (1-hydroxy-2- naphthalenecarboxylate) salt of salmeterol, the sulphate salt or free base of salbutamol or the fumarate salt of formoterol. Combinations comprising a compound of the invention together with a longer-acting β₂-adrenoreceptor agonist, which provides effective bronchodilation for about 12 hours or longer, may be of particular interest.

Other β₂-adrenoreceptor agonists include those described in WO 02/066422, WO 02/070490, WO 02/076933, WO 03/024439, WO 03/072539, WO 03/091204, WO 04/016578, WO 2004/022547, WO 2004/037807, WO 2004/037773, WO 2004/037768, WO 2004/039762, WO 2004/039766, WO01/42193 and WO03/042160.

Exemplary β₂-adrenoreceptor agonists include: 3-(4-{[6-({(2f?)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino) hexyl] oxy} butyl) benzenesulfonamide;

3-(3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-hydroxymethyl) phenyl] ethylj-amino) heptyl] oxy} propyl) benzenesulfonamide;

4-{(1R)-2-[(6-{2-[(2, 6-dichlorobenzyl) oxy] ethoxy} hexyl) amino]-1-hydroxyethyl}-2-

(hydroxymethyl) phenol; 4-{(1R)-2-[(6-{4-[3-(cyclopentylsulfonyl)phenyl]butoxy}hexyl)amino]-1-hydroxyethyl}-2-

(hydroxymethyl)phenol;

Λ/-[2-hydroxyl-5-[(1 K)- 1 -hydroxy-2-[[2-4-[[(2f?)-2-hydroxy-2- phenylethyl]amino]phenyl]ethyl]amino]ethyl]phenyl]formamide;

Λ/-2{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-2-hydroxy-2-(8-hydroxy-2(1 H)- quinolinon-5-yl)ethylamine; and

5-[(f?)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1- hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one.

Anti-inflammatory agents include corticosteroids. Corticosteroids which may be used in combination with the compound of the invention are those oral and inhaled corticosteroids and their pro-drugs which have anti-inflammatory activity. Examples include methyl prednisolone, prednisolone, dexamethasone, fluticasone propionate, 6α,9α-difluoro-1 1 β- hydroxy-16α-methyl-17α-[(4-methyl-1 ,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1 ,4- diene-17β-carbothioic acid S-fluoromethyl ester, 6α,9α-difluoro-17α-[(2- furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester, 6α,9α-difluoro-1 1 β-hydroxy-16α-methyl-3-oxo-17α- propionyloxy- androsta-1 ,4-diene-17β-carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester, 6α,9α-difluoro-11 β-hydroxy-16α-methyl-3-oxo-17α-(2,2,3,3- tetramethycyclopropylcarbpnyOoxy-androsta-i ,4-diene-17β-carbothioic acid S- cyanomethyl ester, 6α,9α-difluoro-11 β-hydroxy-16α-methyl-17α-(1- methycyclopropylcarbonyl)oxy-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S- fluoromethyl ester, beclomethasone esters (eg. the 17-propionate ester or the 17,21- dipropionate ester), budesonide, flunisolide, mometasone esters (eg. the furoate ester), triamcinolone acetonide, rofleponide, ciclesonide (16α,17-[[(R)- cyclohexylmethylene]bis(oxy)]-11 β,21-dihydroxy-pregna-1 ,4-diene-3,20-dione), butixocort propionate, RPR-106541 , and ST-126. Corticosteroids that may be of interest include fluticasone propionate, 6α,9α-difluoro-11 β-hydroxy-16α-methyl-17α-[(4-methyl-1 ,3- thiazole-5-carbonyl)oxy]-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester and 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11 β-hydroxy-16α-methyl-3-oxo- androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester, 6α,9α-difluoro-11 β-hydroxy- 16α-methyl-3-oxo-17α-(2,2,3,3- tetramethycyclopropylcarbonyOoxy-androsta-i ,4-diene- 17β-carbothioic acid S-cyanomethyl ester and 6α,9α-difluoro-11 β-hydroxy-16α-methyl- 17α-(1 -methycyclopropylcarbonylJoxy-S-oxo-androsta-i ,4-diene-17β-carbothioic acid S- fluoromethyl ester, especially 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11 β-hydroxy- 16α-methyl-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester.

Non-steroidal compounds having glucocorticoid agonism that may possess selectivity for transrepression over transactivation and that may be useful in combination therapy include those covered in the following patents: WO03/082827, WO01/10143, WO98/54159, WO04/005229, WO04/009016, WO04/009017, WO04/018429, WO03/104195, WO03/082787, WO03/082280, WO03/059899, WO03/101932, WO02/02565, WO01/16128, WO00/66590, WO03/086294, WO04/026248, WO03/061651 , WO03/08277.

Anti-inflammatory agents include non-steroidal anti-inflammatory drugs (NSAID's). NSAID's include sodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors), leukotriene antagonists, inhibitors of leukotriene synthesis (eg. montelukast), iNOS inhibitors, tryptase and elastase inhibitors, beta-2 integrin antagonists and adenosine receptor agonists or antagonists (e.g. adenosine 2a agonists), cytokine antagonists (e.g. chemokine antagonists, such as a CCR3 antagonist) or inhibitors of cytokine synthesis, or 5- lipoxygenase inhibitors. iNOS inhibitors include those disclosed in WO93/13055, WO98/30537, WO02/50021 , WO95/34534 and WO99/62875. CCR3 inhibitors include those disclosed in WO02/26722. Adenosine 2a agonists include those disclosed in WO2005/1 16037.

The PDE4-specific inhibitor useful in combinations of the invention may include any compound that is known to inhibit the PDE4 enzyme or which is discovered to act as a PDE4 inhibitor, and which are only PDE4 inhibitors, not compounds which inhibit other members of the PDE family, such as PDE3 and PDE5, as well as PDE4.

PDE4 inhibitors include c/s-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1- carboxylic acid, 2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4- difluoromethoxyphenyl)cyclohexan-1 -one and c/s-[4-cyano-4-(3-cyclopropylmethoxy-4- difluoromethoxyphenyljcyclohexan-i-ol]. Also, c/s-4-cyano-4-[3-(cyclopentyloxy)-4- methoxyphenyl]cyclohexane-1-carboxylic acid (also known as cilomilast) and its salts, esters, pro-drugs or physical forms, which is described in U.S. patent 5,552,438 issued 03 September, 1996.

Other PDE4 inhibitors include AWD-12-281 from Elbion (Hofgen, N. et al. 15th EFMC lnt Symp Med Chem (Sept 6-10, Edinburgh) 1998, Abst P.98; CAS reference No. 247584020-9); a 9-benzyladenine derivative nominated NCS-613 (INSERM); D-4418 from Chiroscience and Schering-Plough; a benzodiazepine PDE4 inhibitor identified as CI-1018 (PD-168787) and attributed to Pfizer; a benzodioxole derivative disclosed by Kyowa Hakko in WO99/16766; K-34 from Kyowa Hakko; V-1 1294A from Napp (Landells, LJ. et al., Eur. Resp. J. [Ann. Cong. Eur. Resp. Soc. (Sept 19-23, Geneva) 1998] 1998, 12 (Suppl. 28): Abst P2393); roflumilast (CAS reference No 162401-32-3) and a pthalazinone (WO99/47505) from Byk-Gulden; Pumafentrine, (-)-p-[(4aR^*,10/)S^*)-9-ethoxy- 1 , 2,3,4,4a, 10b-hexahydro-8-methoxy-2-methylbenzo[c][1 ,6]naphthyridin-6-yl]-N, N- diisopropylbenzamide which is a mixed PDE3/PDE4 inhibitor which has been prepared and published on by Byk-Gulden, now Altana; arofylline under development by Almirall- Prodesfarma; VM554/UM565 from Vemalis; or T-440 (Tanabe Seiyaku; Fuji, K. et al., J. Pharmacol. Exp. Then, 284(1 ): 162, 1998 ), and T2585. Further compounds of interest are disclosed in the published international patent application WO04/024728 (Glaxo Group Ltd), PCT/EP2003/014867 (Glaxo Group Ltd) and PCT/EP2004/005494 (Glaxo Group Ltd).

Anticholinergic agents are those compounds that act as antagonists at the muscarinic receptors, in particular those compounds which are antagonists of the M₁ or M₃ receptors, dual antagonists of the M₁ZM₃ or M₂/M₃, receptors or pan-antagonists of the M₁ZM₂ZM₃ receptors. Exemplary compounds for administration via inhalation include ipratropium (e.g. as the bromide, CAS 22254-24-6, sold under the name Atrovent), oxitropium (e.g. as the bromide, CAS 30286-75-0) and tiotropium (e.g. as the bromide, CAS 136310-93-5, sold under the name Spiriva). Also of interest are revatropate (e.g. as the hydrobromide, CAS 262586-79-8) and LAS-34273 which is disclosed in WO01Z04118. Exemplary compounds for oral administration include pirenzepine (CAS 28797-61-7), darifenacin (CAS 133099-04-4, or CAS 133099-07-7 for the hydrobromide sold under the name Enablex), oxybutynin (CAS 5633-20-5, sold under the name Ditropan), terodiline (CAS 15793-40-5), tolterodine (CAS 124937-51-5, or CAS 124937-52-6 for the tartrate, sold under the name Detrol), otilonium (e.g. as the bromide, CAS 26095-59-0, sold under the name Spasmomen), trospium chloride (CAS 10405-02-4) and solifenacin (CAS 242478- 37-1 , or CAS 242478-38-2 for the succinate also known as YM-905 and sold under the name Vesicare).

Other anticholinergic agents include compounds of formula (XXI), which are disclosed in US patent application 60Z487981 :

in which the orientation of the alkyl chain attached to the tropane ring may be endo; R³¹ and R³² are, independently, selected from the group consisting of straight or branched chain lower alkyl groups having, for example, from 1 to 6 carbon atoms, cycloalkyl groups having from 5 to 6 carbon atoms, cycloalkyl-alkyl having 6 to 10 carbon atoms, 2-thienyl, 2-pyridyl, phenyl, phenyl substituted with an alkyl group having not in excess of 4 carbon atoms and phenyl substituted with an alkoxy group having not in excess of 4 carbon atoms;

X^' represents an anion associated with the positive charge of the N atom. X^" may be but is not limited to chloride, bromide, iodide, sulfate, benzene sulfonate, and toluene sulfonate, including, for example:

(3-enc/o)-3-(2,2-di-2-thienylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide;

(3-enc/o)-3-(2,2-diphenylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1 Joctane bromide;

(3-e/icyo)-3-(2,2-diphenylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane 4- methylbenzenesulfonate;

(3-eA?c(o)-8,8-dimethyl-3-[2-phenyl-2-(2-thienyl)ethenyl]-8-azoniabicyclo[3.2.1]octane bromide; and/or

(3-enofo)-8,8-dimethyl-3-[2-phenyl-2-(2-pyridinyl)ethenyl]-8-azoniabicyclo[3.2.1]octane bromide.

Further anticholinergic agents include compounds of formula (XXII) or (XXIII), which are disclosed in US patent application 60/511009:

(XXII) (XXIII)

wherein: the H atom indicated is in the exo position;

R⁴¹ represents an anion associated with the positive charge of the N atom. R⁴¹ may be but is not limited to chloride, bromide, iodide, sulfate, benzene sulfonate and toluene sulfonate;

R and R are independently selected from the group consisting of straight or branched chain lower alkyl groups (having, for example, from 1 to 6 carbon atoms), cycloalkyl groups (having from 5 to 6 carbon atoms), cycloalkyl-alkyl (having 6 to 10 carbon atoms), heterocycloalkyl (having 5 to 6 carbon atoms) and N or O as the heteroatom, heterocycloalkyl-alkyl (having 6 to10 carbon atoms) and N or O as the heteroatom, aryl, optionally substituted aryl, heteroaryl, and optionally substituted heteroaryl; R⁴⁴ is sleeted from the group consisting of (d-C₆)alkyl, (C₃-C₁₂)cycloalkyl, (C₃-

C₇)heterocycloalkyl,

(Ci-C₆)alkyl(C₃-C₇)heterocycloalkyl, aryl, heteroaryl, (Ci-Cβ)alkyl-aryl, (Ci-Cβ)alkyl-heteroaryl, -OR⁴⁵, -CH₂OR⁴⁵, -CH₂OH, -CN,

-CF₃, -CH₂O(CO)R⁴⁶, -CO₂R⁴⁷, -CH₂NH₂, -CH₂N(R⁴⁷)SO₂R⁴⁵, -SO₂N(R⁴⁷)(R⁴⁸), - CON(R⁴⁷)(R⁴⁸), -CH₂N(R⁴⁸)CO(R⁴⁶), -CH₂N(R⁴⁸)SO₂(R⁴⁶), -CH₂N(R⁴⁸)CO₂(R⁴⁵), -

CH₂N(R⁴⁸)CONH(R⁴⁷);

R⁴⁵ is selected from the group consisting of (CrC₆)alkyl, (C₁-C₆)alkyl(C₃-C₁₂)cycloalkyl,

(d-C₆)alkyl(C₃-C₇)heterocycloalkyl, (d-C₆)alkyl-aryl, (C_rC₆)alkyl-heteroaryl;

R⁴⁶ is selected from the group consisting of (d-C₆)alkyl, (C₃-C₁₂)cycloalkyl, (C₃- C₇)heterocycloalkyl, (d-C₆)alkyl(C₃-C₁₂)cycloalkyl, (CrC₆)alkyl(C₃-C₇)heterocycloalkyl, aryl, heteroaryl, (d-C₆)alkyl-aryl, (d-CeJalkyl-heteroaryl;

R⁴⁷ and R⁴⁸ are, independently, selected from the group consisting of H, (d-C₆)alkyl, (C₃-

C₁₂)cycloalkyl, (C₃-C₇)heterocycloalkyl, (C₁-C₆)alkyl(C₃-Ci₂)cycloalkyl, (d-C₆)alkyl(C₃-

C₇)heterocycloalkyl, (d-C₆)alkyl-aryl, and (d-C₆)alkyl-heteroaryl, including, for example: (Endo)-3-(2-methoxy-2,2-di-thiophen-2-yl-ethyl)-8,8-dimethyl-8-azonia- bicyclo[3.2.1]octane iodide;

3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propionitrile;

(Endo)-8-methyl-3-(2,2,2-triphenyl-ethyl)-8-aza-bicyclo[3.2.1]octane;

3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propionamide; 3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propionic acid;

(Endo)-3-(2-cyano-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane iodide;

(Endo)-3-(2-cyano-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane bromide;

3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-diphenyl-propan-1 -ol; Λ/-Benzyl-3-((endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-diphenyl-propionamide;

(Endo)-3-(2-carbamoyl-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane iodide;

1-Benzyl-3-[3-((endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propyl]-urea;

1-Ethyl-3-[3-((endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propyl]-urea; Λ/-[3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propyl]-acetamide;

Λ/-[3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propyl]-benzamide;

3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-di-thiophen-2-yl-propionitrile;

(Endo)-3-(2-cyano-2,2-di-thiophen-2-yl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane iodide; /^-^-((EndoJ-δ-methyl-δ-aza-bicycloIS^.IJoct-S-yO^^-diphenyl-propyl]- benzenesulfonamide; [3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propyl]-urea;

Λ/-[3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-2,2-diphenyl-propyl]- methanesulfonamide; and/or

(Endo)-3-{2,2-diphenyl-3-[(1-phenyl-methanoyl)-amino]-propyl}-8,8-dimethyl-8-azonia- bicyclo[3.2.1]octane bromide.

Compounds of particular interest that may be useful in combinations of the invention include:

(Endo)-3-(2-methoxy-2,2-di-thiophen-2-yl-ethyl)-8,8-dimethyl-8-azonia- bicyclo[3.2.1]octane iodide; (Endo)-3-(2-cyano-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane iodide;

(Endo)-3-(2-cyano-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane bromide;

(Endo)-3-(2-carbamoyl-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane iodide; (Endo)-3-(2-cyano-2,2-di-thiophen-2-yl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane iodide; and/or

(EndoJ-S^^-diphenyl-S-ICI-phenyl-methanoyO-aminol-propylJ-δ.δ-dimethyl-δ-azonia- bicyclo[3.2.1]octane bromide.

Of particular interest is a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with an H1 antagonist. Suitable H1 antagonists include, without limitation, astemizole, azatadine, azelastine, acrivastine, brompheniramine, cetirizine, levocetirizine, efletirizine, chlorpheniramine, clemastine, cyclizine, carebastine, cyproheptadine, carbinoxamine, descarboethoxyloratadine, doxylamine, dimethindene, ebastine, epinastine, efletirizine, fexofenadine, hydroxyzine, ketotifen, loratadine, levocabastine, mizolastine, mequitazine, mianserin, noberastine, meclizine, norastemizole, olopatidine, picumast, pyrilamine, promethazine, terfenadine, tripelennamine, temelastine, trimeprazine and triprolidine, particularly cetirizine, levocetirizine, efletirizine and fexofenadine. Other histamine receptor antagonists which may be used alone, or in combination with an H3 receptor antagonist include antagonists (and/or inverse agonists) of the H4 receptor, for example, the compounds disclosed in Jablonowski et al J.Med Chem. 46:3957-3960 (2003).

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with a PDE4 inhibitor. The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with a β₂- adrenoreceptor agonist.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with an anticholinergic.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with a H1 receptor antagonist.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with a corticosteroid.

The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, together with a A2a receptor agonist.

The combinations referred to above may conveniently be presented for use in the form of a composition and thus compositions comprising a combination as defined above, optionally together with a pharmaceutically acceptable diluent or carrier represent a further aspect of the invention.

The individual compounds of such combinations may be administered either sequentially or simultaneously in separate or combined compositions. Suitably, the individual compounds will be administered simultaneously in a combined composition. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art.

The compound of the invention may be prepared by the methods described below or by similar methods. Thus , the following Descriptions and Examples illustrate the preparation of compounds of the invention. The Examples are not to be considered as limiting the scope of the invention in any way.

General Experimental

Throughout the examples, the following abbreviations are used: LCMS: Liquid Chromatography Mass Spectrometry RT: retention time DMF: Λ/,Λ/-dimethylformamide h: hour(s) min: minute(s) TBTU: O-(benzotriazol-1-yl)-Λ/,Λ/,Λ/,Λ/'-tetramethyluronium tetrafluoroborate PS-DIEA: polymer supported diisopropylethylamine

SCX cartridges are Ion Exchange SPE columns where the stationary phase is polymeric benzene sulfonic acid. These are used to isolate amines. SCX2 cartridges are Ion Exchange SPE columns where the stationary phase is polymeric propylsulfonic acid. These are used to isolate amines.

Organic solutions were dried either over magnesium or sodium sulfate.

LCMS was conducted on a Supelcosil LCABZ+PLUS column (3.3 cm x 4.6 mm ID) eluting with 0.1% HCO₂H and 0.01 M ammonium acetate in water (solvent A) and 0.05% HCO₂H 5% water in acetonitrile (solvent B), using the following elution gradient 0.0-7min 0%B, 0.7-4.2 min 100%B, 4.2-5.3 min 0%B, 5.3-5.5min 0%B at a flow rate of 3ml/min. The mass spectra were recorded on a Fisons VG Platform spectrometer using electrospray positive and negative mode (ES+ve and ES-ve).

The Flashmaster Il is an automated multi-user flash chromatography system, available from Argonaut Technologies Ltd, which utilises disposable, normal phase, SPE cartridges (2 g to 100 g). It provides quaternary on-line solvent mixing to enable gradient methods to be run. Samples are queued using the multi-functional open access software, which manages solvents, flow-rates, gradient profile and collection conditions. The system is equipped with a Knauer variable wavelength uv-detector and two Gilson FC204 fraction- collectors enabling automated peak cutting, collection and tracking.

Intermediates and compounds were named using ACD/Name PRO 6.02 chemical naming software Advanced Chemistry Developments Inc.; Toronto, Ontario, M5H2L3, Canada.

Description 1

1 ,1 -Dimethylethyl 4-[(4-bromophenyl)oxy]-1 -piperidinecarboxylate

To a stirring solution of 4-bromophenol (5.19 g), 1 ,1-dimethylethyl 4-hydroxy-1- piperidinecarboxylate (Aldrich) (5.032 g) and triphenylphosphine (7.869 g) in dichloromethane (125 ml) was added, portionwise, di-t-butyl azodicarboxylate (6.908 g). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for about 65 h. The solvent was evaporated from the mixture giving a viscous residue. A 3 g sample of this residue was purified by Flashmaster Il on a 100 g silica cartridge using a 0- 100% ethyl acetate in cyclohexane gradient over 80 minutes. The solvent was evaporated from appropriate fractions to give the title compound (604 mg). LCMS RT= 3.72 min, ES+ve m/z 341/343 (M+H)⁺.

The remaining crude material was purified by flash column chromatography on Merck Kieselgel 60 (9385) silica (40Og) using 10% ethyl acetate in cyclohexane as the eluent. Evaporation of the solvent from appropriate fractions gave a colourless oil that was further purified by Flashmaster Il on a 100g silica cartridge using a 0-50% ethyl acetate in cyclohexane gradient over 60 minutes. Evaporation of the solvent from approriate fractions gave the title compoundas a colourless oil that crystallised on standing (5.725g). LCMS RT= 3.72 mins, ES+ve m/z 341/343 (M+H)+.

Description 2

Phenylmethyl 4-{4-[(1 -{[(1 ,1 -dimethylethyl)oxy]carbonyl}-4-piperidinyl)oxy]phenyl}-

4-hydroxy-1-piperidinecarboxylate

n-Butyl lithium (13.9 ml of 1.6M solution in hexane) was added dropwise to a stirring solution of 1 ,1-dimethylethyl 4-[(4-bromophenyl)oxy]-1-piperidinecarboxylate (D1 ) (6.32 g) in anhydrous tetrahydrofuran (75 ml) such that the reaction mixture temperature did not exceed -7O⁰C. The resulting mixture was stirred at -75°C for 20 min. A solution of phenylmethyl 4-oxo-i-piperidinecarboxylate (Aldrich) (5.173 g) in anhydrous tetrahydrofuran (15 ml) was then added dropwise such that the reaction temperature was maintained below -7O⁰C. The reaction mixture was then allowed to warm slowly to room temperature overnight. The reaction mixture was partitioned between saturated aqueous ammonium chloride (200 ml) and dichloromethane (150 ml). The organic layer was washed with brine (100 ml), dried over anhydrous magnesium sulphate and evaporated to give a viscous residue. This material was purified by Flashmaster Il on a 100g silica cartridge eluted with a 0-50% ethyl acetate in cyclohexane gradient over 60 min. The solvent was evaporated from appropriate fractions to give the title compound (1.606 g). LCMS RT= 3.59 min, ES+ve m/z 528 (M+NH₄)⁺.

Additional fractions containing the desired material were combined and evaporated to give a residue that was repurified by Flashmaster Il using a 0-50% ethyl acetate in cyclohexane gradient. Evaporation of the solvent from approriate fractions gave the title compoundas a colourless gum (521 mg). LCMS RT= 3.59 mins, ES+ve m/z 528 (M+NH4)+.

Description 3

Phenylmethyl 4-[4-(4-piperidinyloxy)phenyl]-1-piperidinecarboxylate

A stirring solution of phenylmethyl 4-{4-[(1-{[(1 ,1-dimethylethyl)oxy]carbonyl}-4- piperidinyl)oxy]phenyl}-4-hydroxy-1-piperidinecarboxylate (D2) (1.62 g) and triethylsilane (2.54 g) in anhydrous dichloromethane at -78°C was treated, dropwise, with trifluoroacetic acid (1.18 ml). The resulting mixture was stirred at -78°C for 15 min and at room temperature overnight. Evaporation of the solvent gave a residue that was re-dissolved in toluene (10 ml) and evaporated once more to give a semi-solid. This material was dissolved in methanol and applied to two 2Og SCX ion-exchange cartridges. The cartridges were eluted with methanol, followed by 2N ammonia in methanol. Evaporation of the solvent from the ammonia-containing fractions gave the title compound (945mg). LCMS RT= 2.59 min, ES+ve m/z 395 (M+H)⁺.

Description 4

Phenylmethyl 4-(4-{[1 -(1 -methylethyl)-4-piperidinyl]oxy}phenyl)-1 ■ piperidinecarboxylate

A stirring solution of phenylmethyl 4-[4-(4-piperidinyloxy)phenyl]-1 -piperidinecarboxylate (D3) (614 mg), acetic acid (0.1 ml) and acetone (1 ml) in dichloromethane (10 ml) was treated with sodium triacetoxyborohydride (661 mg). The resulting reaction mixture was stirred at room temperature under a nitrogen atmosphere for approximately 20 h. Further quantities of acetone (1 ml) and sodium triacetoxyborohydride (500 mg) were added and the mixture stirred for an additional 4 h. The reaction mixture was partitioned between dichloromethane (50 ml) and saturated sodium bicarbonate solution (50 ml). The organic layer was washed with brine (50 ml), dried over anhydrous magnesium sulphate and evaporated to give the title compound (667 mg). LCMS RT= 2.70 min, ES+ve m/z 437 (M+H)⁺.

Description 5

1 -(1 -Methylethyl)-4-{[4-(4-piperidinyl)phenyl]oxy}piperidine

A solution of phenylmethyl 4-(4-{[1-(1-methylethyl)-4-piperidinyl]oxy}phenyl)-1- piperidinecarboxylate (D4) (667 mg) in ethanol (20 ml) was hydrogenated at atmospheric pressure in the presence of 10%wt palladium on carbon (300 mg). After 3 h the reaction mixture was filtered through celite and the filtrate evaporated. The residue was dissolved in methanol and applied to a 1Og SCX ion-exchange cartridge. The cartridge was eluted with methanol, followed by 2N ammonia in methanol. Evaporation of the solvent from the ammonia-containing fractions gave the title compound (416 mg). LCMS RT=1.5 min, ES+ve m/z 303 (M+H)⁺.

Description 6

Methyl 4-(azetidin-1 -ylcarbonyl)benzoate Methyl 4-chlorocarbonylbenzoate (2.Og) was stirred with triethylamine (2.0ml) in dichloromethane (20ml) at 20⁰C and azetidine (0.81 ml) was added. (It is to be noted that this reaction was vigorous, and thus cooling and slower addition of azetidine may be advisable.) After stirring at room temperature under nitrogen for 2h, the solution was diluted with dichloromethane and washed with water twice. The organic solution was then washed with dilute hydrochloric acid (2N) and water, each time back extracting with dichloromethane. The combined organic layers were dried with magnesium sulphate and evaporated to give the title compound (1.92g), LCMS RT= 2.33 min, ES+ve m/z 220 (M+H)⁺.

Description 7

4-(Azetidin-1 -ylcarbonyl)benzoic acid

Methyl 4-(azetidin-1-ylcarbonyl)benzoate (D6) (1.91g) was heated at 70⁰C in MeOH (20ml) and 2M sodium hydroxide solution (10ml) with stirring for 1.5 h. After cooling, the mixture was acidified with 2M hydrochloric acid (10ml) to pH 5. The white solid, which separated, was collected by filtration and washed with water. The combined filtrate and washings were acidified by adding further 2M hydrochloric acid to pH 1 and the solution was extracted three times with ethylacetate. The combined organic extracts were washed with brine, dried with magnesium sulphate and evaporated to give the title compound (0.25g). LCMS RT= 2.04 min, ES+ve m/z 206 (M+H)⁺.

Example 1

1 -{[4-(1 -Azetidinylcarbonyl)phenyl]carbonyl}-4-(4-{[1 -(1 -methylethyl)-4- piperidinyl]oxy}phenyl)piperidine

A solution of 1-(1-methylethyl)-4-{[4-(4-piperidinyl)phenyl]oxy}piperidine (D5) (24.2 mg) in dichloromethane (1.5 ml) was added to a mixture of 4-(1-azetidinylcarbonyl)benzoic acid (D7) (17.4 mg), TBTU (28.9 mg) and PS-DIEA (100 mg of 3.88 mmol/g). The resulting mixture was agitated at room temperature in a sealed vial for about 65 h. The PS-DIEA was removed by filtration and washed with dichloromethane (3 ml). The combined filtrate and washings were evaporated under a stream of nitrogen giving a residue that was dissolved in methanol (3 ml) and applied to an SCX ion-exchange cartridge (1 g). The cartridge was eluted with methanol (3 x 3 ml) and then 2N ammonia in methanol (2 x 3 ml). Evaporation of the solvent from the ammonia-containing fractions gave the title compound (38.6 mg), LCMS RT= 2.24 min, ES+ve m/z 490 (M+H)⁺, 1 H NMR (400MHz, DMSO-D6) δ ppm 0.98 (d, J=6.5 Hz, 6H) 1.50-1.97 (m, 8H) 2.21-2.43 (m, 4H) 2.65-2.92 (m, 5H) 3.13 (br m, 1 H) 3.59 (br m, 1 H) 4.05 (m, 2H) 4.25-4.36 (m, 3H) 4.62 (br m, 1 H) 6.86 (d, J=8.5 Hz, 2H) 7.16 (d, J=8.5 Hz, 2H) 7.48 (d, J=8 Hz, 2H) 7.67 (d, J=8 Hz, 2H)

Biological Data

Compound of the invention may be tested for in vitro biological activity in accordance with the following or similar assays: H1 receptor cell line generation and FLIPR assay protocol

1. Generation of histamine H1 cell line The human H1 receptor was cloned using known procedures described in the literature [Biochem. Biophys. Res. Commun. 1994, 201 (2), 894]. Chinese hamster ovary cells stably expressing the human H1 receptor were generated according to known procedures described in the literature [Br. J. Pharmacol. 1996, 117(6), 1071].

Histamine H1 functional antagonist assay

The histamine H1 cell line was seeded into non-coated black-walled clear bottom 384-well tissue culture plates in alpha minimum essential medium (Gibco /Invitrogen, cat no. 22561-021), supplemented with 10% dialysed foetal calf serum (Gibco/lnvitrogen cat no. 12480-021 ) and 2 mM L-glutamine (Gibco/lnvitrogen cat no 25030-024) and maintained overnight at 5% CO₂, 37⁰C.

Excess medium was removed from each well to leave 10μl. 30μl loading dye (250μM Brilliant Black, 2μM Fluo-4 diluted in Tyrodes buffer + probenecid (145 mM NaCI, 2.5 mM KCI, 1OmM HEPES, 1OmM D-glucose, 1.2 mM MgCI₂, 1.5 mM CaCI₂, 2.5 mM probenecid, pH adjusted to 7.40 with NaOH 1.0 M)) was added to each well and the plates were incubated for 60 minutes at 5% CO₂, 37⁰C.

10μl of test compound, diluted to the required concentration in Tyrodes buffer + probenecid (or 10μl Tyrodes buffer + probenecid as a control) was added to each well and the plate incubated for 30 min at 37⁰C, 5% CO₂. The plates were then placed into a PLIPR^TM (Molecular Devices, UK) to monitor cell fluorescence (λ_ex= 488 nm, λ_EM= 540 nm) in the manner described in Sullivan et al. (In: Lambert DG (ed.), Calcium Signaling Protocols, New Jersey: Humana Press, 1999, 125-136) before and after the addition of 10μl histamine at a concentration that results in the final assay concentration of histamine being EC₈₀.

Functional antagonism is indicated by a suppression of histamine induced increase in fluorescence, as measured by the FLIPR™ system (Molecular Devices). By means of concentration effect curves, functional affinities are determined using standard pharmacological mathematical analysis.

2. H3 receptor cell line generation, membrane preparation and functional GTPYS assay protocols

Generation of histamine H3 cell line

The histamine H3 cDNA was isolated from its holding vector, pCDNA3.1 TOPO (InVitrogen), by restriction digestion of plasmid DNA with the enzymes BamH1 and Not-1 and ligated into the inducible expression vector pGene (InVitrogen) digested with the same enzymes. The GeneSwitch™ system (a system where in transgene expression is switched off in the absence of an inducer and switched on in the presence of an inducer) was performed as described in US Patent nos: 5,364,791 ; 5,874,534; and 5,935,934. Ligated DNA was transformed into competent DH5α E. coli host bacterial cells and plated onto Luria Broth (LB) agar containing Zeocin™ (an antibiotic which allows the selection of cells expressing the sh ble gene which is present on pGene and pSwitch) at 50μg ml^"1. Colonies containing the re-ligated plasmid were identified by restriction analysis. DNA for transfection into mammalian cells was prepared from 250ml cultures of the host bacterium containing the pGeneH3 plasmid and isolated using a DNA preparation kit (Qiagen Midi- Prep) as per manufacturers guidelines (Qiagen).

CHO K1 cells previously transfected with the pSwitch regulatory plasmid (InVitrogen) were seeded at 2x10e6 cells per T75 flask in Complete Medium, containing Hams F12

(GIBCOBRL, Life Technologies) medium supplemented with 10% v/v dialysed foetal bovine serum, L-glutamine, and hygromycin (100μg ml^"1), 24 hours prior to use. Plasmid

DNA was transfected into the cells using Lipofectamine plus according to the manufacturers guidelines (InVitrogen). 48 hours post transfection cells were placed into complete medium supplemented with 500μg ml^"1 Zeocin™.

10-14 days post selection 1OnM Mifepristone (InVitrogen), was added to the culture medium to induce the expression of the receptor. 18 hours post induction cells were detached from the flask using ethylenediamine tetra-acetic acid (EDTA; 1 :5000; InVitrogen), following several washes with phosphate buffered saline pH 7.4 and resuspended in Sorting Medium containing Minimum Essential Medium (MEM), without phenol red, and supplemented with Earles salts and 3% Foetal Clone Il (Hyclone). Approximately 1x 10e7 cells were examined for receptor expression by staining with a rabbit polyclonal antibody, 4a, raised against the N-terminal domain of the histamine H3 receptor, incubated on ice for 60 minutes, followed by two washes in sorting medium. Receptor bound antibody was detected by incubation of the cells for 60 minutes on ice with a goat anti rabbit antibody, conjugated with Alexa 488 fluorescence marker (Molecular Probes). Following two further washes with Sorting Medium, cells were filtered through a 50μm Filcon™ (BD Biosciences) and then analysed on a FACS Vantage SE Flow Cytometer fitted with an Automatic Cell Deposition Unit. Control cells were non- induced cells treated in a similar manner. Positively stained cells were sorted as single cells into 96-well plates, containing Complete Medium containing 500μg ml^"1 Zeocin™ and allowed to expand before reanalysis for receptor expression via antibody and ligand binding studies. One clone, 3H3, was selected for membrane preparation.

Membrane preparation from cultured cells

All steps of the protocol are carried out at 4°C and with pre-cooled reagents. The cell pellet is resuspended in 10 volumes of homogenisation buffer (5OmM N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1mM ethylenediamine tetra- acetic acid (EDTA), pH 7.4 with KOH, supplemented with 10e-6M leupeptin (acetyl-leucyl- leucyl-arginal; Sigma L2884), 25μg/ml bacitracin (Sigma B0125), , 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2x10e-6M pepstain A (Sigma)). The cells are then homogenised by 2 x 15 second bursts in a 1 litre glass Waring blender, followed by centrifugation at 50Og for 20 minutes. The supernatant is then spun at 48,00Og for 30 minutes. The pellet is resuspended in homogenisation buffer (4X the volume of the original cell pellet) by vortexing for 5 seconds, followed by homogenisation in a Dounce homogeniser (10-15 strokes). At this point the preparation is aliquoted into polypropylene tubes and stored at -80⁰C.

Histamine H3 functional antagonist assay

For each compound being assayed, in a solid white 384 well plate, is added:-

(a) 0.5μl of test compound diluted to the required concentration in DMSO (or 0.5μl DMSO as a control);

(b) 30μl bead/membrane/GDP mix prepared by mixing Wheat Germ Agglutinin Polystyrene LeadSeeker® (WGA PS LS) scintillation proximity assay (SPA) beads with membrane (prepared in accordance with the methodology described above) and diluting in assay buffer (2OmM N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) + 10OmM NaCI + 1OmM MgCI₂, pH7.4 NaOH) to give a final volume of 30μl which contains 5μg protein and 0.25mg bead per well, incubating at room temperature for 60 minutes on a roller and, just prior to addition to the plate, adding 10μM final concentration of guanosine 5' diphosphate (GDP) (Sigma; diluted in assay buffer);

(c) 15μl 0.38nM [³⁵S]-GTPyS (Amersham; Radioactivity concentration=37MBq/ml; Specific activity=1160Ci/mmol), histamine (at a concentration that results in the final assay concentration of histamine being EC₈o). After 2-6 hours, the plate is centrifuged for 5 min at 1500 rpm and counted on a Viewlux counter using a 613/55 filter for 5 min/plate. Data is analysed using a 4-parameter logistical equation. Basal activity used as minimum i.e. histamine not added to well.

CNS Penetration

Compounds are dosed intravenously at a nominal dose level of 1mg/kg to male CD Sprague Dawley rats. Compounds are formulated in 5% DMSO/45% PEG200/50% water. Blood samples are taken under terminal anaesthesia with isoflurane at 5 minutes post- dose and the brains are also removed for assessment of brain penetration. Blood samples are taken directly into heparinised tubes. Blood samples are prepared for analysis using protein precipitation and brain samples are prepared using extraction of drug from brain by homogenisation and subsequent protein precipitation. The concentration of parent drug in blood and brain extracts is determined by quantitative LC- MS/MS analysis using compound-specific mass transitions.

Rat Pharmacokinetics

Compounds are dosed to male CD Sprague Dawley rats by single intravenous or oral administration at a nominal dose level of 1mg/kg and 3mg/kg respectively. Compounds are formulated in 5% DMSO/45% PEG200/50% water. An intravenous profile is obtained by taking serial or terminal blood samples at 0.083, 0.25, 0.5, 1 , 2, 4, and 7 hours post dose. An oral profile is obtained by taking serial or terminal blood samples at 0.25, 0.5, 1 , 2, 4, 7 and 12 hours post dose. Blood samples are taken directly into heparinised tubes. Blood samples are prepared by protein precipitation and subjected to quantitative analysis by LC-MS/MS using compound-specific mass transitions. Drug concentration-time profiles are generated and non-compartmental PK analysis used to generate estimates of half-life, clearance, volume of distribution and oral bioavailability.

Dog Pharmacokinetics

Compounds are dosed to male Beagle dogs by single intravenous or oal administration at a nominal dose level of 1mg/kg and 2mg/kg respectively. The study is carried out according to a crossover design such that the same dog is used for both dosing events and the dosing events occurred 1 week apart. Compounds are formulated in 5%DMSO/45%Peg200/50%water. An intravenous profile is obtained by taking serial blood samples at 0.083, 0.25, 0.5, 0.75, 1 , 2, 4, 6 & 12hr post dose. An oral profile is obtained by taking serial blood samples at 0.25, 0.5, 0.75, 1 , 2, 4, 6, 12 & 24hr post dose. Blood samples are taken directly into heparinised tubes. Blood samples are prepared by protein precipitation and subjected to quantitative analysis by LC-MS/MS using compound-specific mass transitions. Drug concentration-time profiles are generated and non-compartmental PK analysis used to generate estimates of half-life, clearance, volume of distribution and oral bioavailability.

Results

In the above assays or similar assays compound of formula (I) had (i) an average pki (pkb) at H3 of approximately 9.6 (ii) an average pki (pkb) at H1 of approximately 5.6 (iii) low CNS penetration (less than 100ng compound/g of brain tissue)

(iv) good oral bioavailability (in the rat about 22%F and in the dog about 87%F)

(v) half-life in the rat of greater than about 2hrs and in the dog greater than about 4 hours

The content of all documents including literature references, patents and patent applications referred to hereinabove are to be considered as incorporated in full herein.

Claims

1. 1-{[4-(1-Azetidinylcarbonyl)phenyl]carbonyl}-4-(4-{[1-(1-methylethyl)-4- piperidinyl]oxy}phenyl)piperidine

(I)

or a salt thereof.

2. Compound according to claim 1 wherein the salt is a pharmaceutically acceptable salt or solvate.

3. A process for the preparation of the compound of formula (I) or a salt thereof, the process comprising reacting a compound of formula (III)

or a salt thereof, with 4-(azetidin-1-ylcarbonyl) benzoic acid.

4. A compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2 for use in therapy.

5. A compound or a pharmaceutically acceptable salt thereof according to claim 4 for use in the treatment of inflammatory and/or allergic disorders.

6. Compound or a pharmaceutically acceptable salt thereof according to claim 4 for use in the treatment of rhinitis e.g. allergic rhinitis.

7. A composition which comprises a compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, optionally with one or more pharmaceutically acceptable carriers and/or excipients.

8. A composition according to claim 7 which further comprises an H1 receptor antagonist.

9. A combination comprising a compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and a H1 receptor antagonist.

10. The use of a compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the manufacture of a medicament for the treatment or prophylaxis of inflammatory and/or allergic disorders.

11. The use according to claim 10 in which the disorder is allergic rhinitis.

12. A method for the treatment or prophylaxis of inflammatory and/or allergic disorders which comprises administering to a patient in need thereof an effective amount of a compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2.