WO2006090142A9 - l-{4- [ (l-CYCLOBUTYL-4-PIPERIDINYL) OXY] PHENYL] -4-{ [4- (METHYLSULFONYL) PHENYL]CARBONYL PIPERAZINE AS HISTAMINE H3 ANTAGONIST - Google Patents

Abstract

The present invention relates to the compound, of Formula (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

1-{4- [(1-CYCLOBUTYL^-PIPERIDINYL)OXY]PHENYL] -4- {[4- (METHYLSULFONYL) PHENYL] CARBONYL} PIPERAZINE AS HISTAMINE H3 ANTAGONIST

The present invention relates to a compound, processes for its preparation, pharmaceutical 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)].

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 a/., Naunyn-Schmiedeberg's Arch. Pharmacol., 346:46-50, (1992); Valentine et a/., 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 a/., 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 ef a/., Am. J. 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 receptor antagonist and/or inverse agonist. This compound may be useful in the treatment of various diseases in particular inflammatory and/or allergic diseases, such as inflammatory and/or allergic diseases 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 (or salt thereof) of the invention 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 of greater than about 9.5; (ii) approximately 10,000 fold 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-cyclobutyl-4- piperidinyl)oxy]phenyl}-4-{[4-(methylsulfonyl)phenyl]carbonyl}piperazine

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, for example a pharmaceutically acceptable salt .

It is to be further understood that references herinafter to a compound of the invention or to a compound of formula (I) means a compound of formula (I), as the free base or a compound of formula (I) as a salt.

As used herein, the term "pharmaceutically acceptable salt", means any pharmaceutically acceptable salt or solvate of a compound of the invention, which upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. 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 the present invention may be in the form of and/or may be administered as a pharmaceutically acceptable salt. For a review on suitable salts see Berge et al., J. Pharm. ScL₁ 66:1-19, (1977).

Typically, a pharmaceutically acceptable 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.

A pharmaceutically acceptable acid addition salt can be formed by reaction of a 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 a 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, e.g. 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).

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 a 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).

It will be appreciated from the foregoing that included within the scope of the invention are all solvates, hydrates, complexes and polymorphic forms of the compound of the invention and salts thereof.

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

Thus a compound of formula (I) may be prepared by reacting a compound of formula (II)

(H)

or salt thereof, such as an acid addition salt, with 4-(methylsulfonyl)benzoic acid, optionally in the presence of a suitable base such as triethylamine and a suitable coupling agent such as O-(benzotriazol-1-yl)-Λ/,Λ/,Λ/^',Λ/-tetramethyluronium tetrafluoroborate (TBTU), in an appropriate solvent such as dichloromethane.

Alternatively, acylation can be achieved using 4-(methylsulfonyl)benzoyl chloride optionally in the presence of a suitable base such as triethylamine, in an appropriate solvent such as dichloromethane.

A compound of formula (II) may be prepared in accordance with the following reaction scheme:

Compound of formula (II)

(III)

a) di-tert-butyl dicarbonate, dimethylformamide; b) di-terf-butyl azodicarboxylate, triphenyl phosphine, benzyl 4-hydroxy-1-piperidinecarboxylate, dichloromethane; c) H₂, 10% palladium on carbon, ethanol; d) sodium triacetoxyborohydride, cyclobutanone, dichloromethane; e) 4M HCI/dioxane, dichloromethane.

A compound of formula (VII) [56621-48-8] is available from Acros, Avocado and Lancaster and 4-(methylsulfonyl)-benzoic acid [4052-30-6] is available from Aldrich and Acros. A compound of formula (I) or salt thereof may also be synthesised by the methods described in WO 2004/035556 (see Example 292, step 4 thereof, which is a compound of formula (II).)

Examples of protecting groups that may be employed in the synthetic routes descrbied 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, 2^I,2',2'-trichloroethoxycarbonyl, benzyloxycarbonyl or f-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.

Examples of disease states in which a compound of formula (I), or a pharmaceutically acceptable salt thereof may have potentially beneficial anti-inflammatory and/or anti- allergic 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 of use in the treatment of 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 or alternative 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.

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, but particularly oral 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.

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. Advantageously, 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, the 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 anti- inflammatory 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 anti- inflammatory 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 f?-enantiomer or the S-enantiomer), salbutamol (e.g. as racemate or a single enantiomer such as the /^-enantiomer), formoterol (e.g. as racemate or a single enantiomer such as the f?-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-({(2fi)-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] ethyl}-amino) heptyl] oxy} propyl) benzenesulfonamide;

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

(hydroxymethyl) phenol; 4-{(1 R^-Kδ^-tS-CcyclopentylsulfonyOphenylJbutoxylhexyOaminoJ-i -hydroxyethyl}-2-

(hydroxymethyl)phenol;

N-[2-hydroxyl-5-[(1 ft)-1 -hydroxy-2-[[2-4-[[(2R)-2-hydroxy-2- phenylethyl]amino]phenyl]ethyl]amino]ethyl]phenyl]formamide;

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

5-[(R)-2-(2-{4-[4-(2-amino-2-methyl-propoxy)-phenylamino]-phenyl}-ethylamino)-1- hydroxy-ethyl]-8-hydroxy-1 H-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-11β- 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]-11 β-hydroxy-16α-methyl-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester, 6α,9α-difluoro-11β-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- tetramethycyclopropylcarbonyl)oxy-androsta-1 ,4-diene-17β-carbothioic acid S- cyanomethyl ester, 6α,9α-difluoro-11β-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-11β-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]-11β-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- tetramethycyclopropylcarbonyl)oxy-androsta-1 ,4-diene- 17β-carbothioic acid S-cyanomethyl ester and 6α,9α-difluoro-11 β-hydroxy-16α-methyl- 17α-(1-methycyclopropylcarbonyl)oxy-3-oxo-androsta~1 ,4-diene-17β-carbothioic acid S- fluoromethyl ester, especially 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-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.

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- difluoromethoxypheny))cyclohexan-1 -one and c/s-[4-cyano-4-(3-cyclopropylmethoxy-4- difluoromethoxyphenyl)cyclohexan-1 -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. ef a/. 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-11294A from Napp (Landells, L.J. ef a/., 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<bS*)-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 Vernalis; or T-440 (Tanabe Seiyaku; Fuji, K. et a/., J. Pharmacol. Exp. Ther., 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-e/7αto)-3-(2,2-di-2-thienylethenyl)-8,8-dimethyl-8-a2oniabicyclo[3.2.1]octane bromide;

(3-endo)-3-(2,2-diphenylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide;

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

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

(3-enαfo)-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; R⁴² and R⁴³ 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 (C_rC₆)alkyl, (C₃-C₁₂)cycloalkyl, (C₃-

C₇)heterocycloalkyl, (C_rC₆)alkyl(C₃-C₁₂)cycloalkyl, (C_rC₆)alkyl(C₃-C₇)heterocycloalkyl, aryl, heteroaryl, (C_rC₆)alkyl-aryl, (C_rC₆)alkyl-heteroaryl, -OR⁴⁵, -CH₂OR⁴⁵, -CH₂OH, -CN, -CF₃, -CH₂O(CO)R⁴⁶, -CO₂R⁴⁷, -CH₂NH₂, -CH₂N(R⁴⁷)SO₂R⁴⁵, -SO₂N(R⁴⁷)(R⁴⁸), -

CON(R⁴⁷XR⁴⁸), -CH₂N(R⁴⁸)CO(R⁴⁶), -CH₂N(R⁴⁸)SO₂(R⁴⁶), -CH₂N(R⁴⁸)CO₂(R⁴⁵), -

CH₂N(R⁴⁸)CONH(R⁴⁷);

R⁴⁵ is selected from the group consisting of (C_rC₆)alkyl, (Ci-C₆)alkyl(C₃-C₁₂)cycloalkyl,

(C_rC₆)alkyl(C₃-C₇)heterocycloalkyl, (C_rC₆)alkyl-aryl, (CrC_e)alkyl-heteroaryl; R⁴⁶ is selected from the group consisting of .

(C₃-Ci₂)cycloalkyl, (C₃-

C₇)heterocycloalkyl, (C_rC₆)alkyl(C₃-C₁₂)cycloalkyl, (C₁-C₆)alkyl(C₃-C₇)heterocycloalkyl, aryl, heteroaryl, (C_rC₆)alkyl-aryl, (C₁-C₆)alkyl-heteroaryl;

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

Ci₂)cycloalkyl, (C₃-C₇)heterocycloalkyl, (C_rC₆)alkyl(C₃-C₁₂)cycloalkyl, (C_rC₆)alkyl(C₃- C₇)heterocycloalkyl, (Ci-C₆)alkyl-aryl, and (CrC₆)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-cliphenyl-propyl]-acetamicie;

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;

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

^-((EndoJ-δ-methyl-δ-aza-bicycloP^.iloct-S-yO^^-diphenyl-propyO-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 (Endo)-3-{2,2-diphenyl-3-[(1-phenyl-methanoyl)-amino]-propyl}-8,8-dimethyl-8-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, 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 the compound of the invention, and 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)

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)

Description 1

1 ,1 -Dimethylethyl 4-(4-hydroxyphenyl)-1 -piperazinecarboxylate (D1) A solution di-te/Y-butyl dicarbonate (257 g, 1.18 mol) in Λ/,Λ/-dimethylformamide (400 ml) was added dropwise to a stirred suspension of Λ/-(4-hydroxyphenyl)piperazine (200 g, 1.12 mol) in /V,Λ/-dimethylformamide (800 ml) at 5-10 ⁰C. The mixture was stirred in the cold bath for a further hour, then allowed to warm to ambient and then stirred for 2 days. The mixture was washed in a separating funnel with hexane (1200 ml) and the DMF phase was separated and evaporated in vacuo. Trituration of the residue with diethyl ether and the solid washed with diethylether to give the title compound (304 g). LCMS RT = 2.76 min, ES+ve m/z 279 [M+H]⁺.

Description 2

1,1-Dimethylethyl 4-{4-[(1-{[(phenylmethyl)oxy]carbonyl}-4-piperidinyl)oxy]phenyl}- 1-piperazinecarboxylate (D2)

A solution of di-terf-butyl azodicarboxylate (25 g, 108 mmol) in dry dichloromethane (150 ml) was added dropwise to a stirred mixture of 1 ,1-dimethylethyl 4-(4-hydroxyphenyl)-1- piperazinecarboxylate (D1 )(25.16g, 90.4 mmol), benzyl 4-hydroxy-1-piperidinecarboxylate (Aldrich)(25.56 g, 109 mmol), and triphenylphosphine (28.45 g, 108 mmol) in dry dichloromethane (300 ml) with cooling as necessary to keep the reaction temperature below 25°C. The mixture was then stirred at ambient temperature overnight and then for a further 24 hours. The solvent was evaporated and the residue purified on a silica column eluting with dichloromethane-ethyl acetate (9:1) to afford the title compound (17.9g). LCMS RT =3.79 min, ES+ve m/z 496 [M+H]⁺.

Description 3

1 ,1 -Dimethylethyl 4-[4-(4-piperidinyloxy)phenyl]-1 -piperazinecarboxylate (D3)

1 , 1 -Dimethylethyl 4-{4-[(1 -{[(phenylmethyl)oxy]carbonyl}-4-piperidinyl)oxy]phenyl}-1 - piperazinecarboxylate (D2) (5.1 g) was hydrogenated over 10% palladium on carbon (2 g) in ethanol (50 ml) over 1 hour at atmospheric pressure. The catalyst was removed by filtration over a Celite cartridge and washed with ethanol. The combined filtrate and washings were concentrated to give the title compound (2.023 g). LCMS RT= 2.30 min, ES+ve m/z 362 (M+H)⁺.

Description 4

1,1-Dimethylethyl 4-{4-[(1-cyclobutyl-4-piperidinyl)oxy]phenyl}-1- piperazinecarboxylate (D4)

To a solution of 1 ,1-dimethylethyl 4-[4-(4-piperidinyloxy)phenyl]-1-piperazinecarboxylate (D3) (2.023 g, 5.60 mmol) in dichloromethane (30 ml) were added acetic acid (0.32 ml, 5.60 mmol) and cyclobutanone (0.836 ml, 11.2 mmol). The resulting solution was stirred at room temperature for 5 minutes. Sodium triacetoxyborohydride (1.78 g, 8.40 mmol) was then added to the solution, which was stirred at room temperature overnight. The reaction mixture was then partitioned between dichloromethane and water. The organic layer was washed with brine, concentrated and purified by SCX-2 cartridge (20 g) eluting with methanol, followed by 2M ammonia solution in methanol, collecting the ammoniacal fractions to afford the title compound (1.136 g). LCMS RT= 2.42 min, ES+ve m/z 416 (MH-H)⁺.

Description s

1 -{4-[(1 -Cyclobutyl-4-piperidinyl)oxy]phenyl}piperazine trihydrochloride(D5)

3 CIH

To a solution of 1 ,1-dimethylethyl 4-{4-[(1-cyclobutyl-4-piperidinyl)oxy]phenyl}-1- piperazinecarboxylate (D4) (1.136 g, 2.74 mmol) in dichloromethane (15 ml) was added 4M hydrogen chloride solution in dioxane (15 ml), and the resulting solution was stirred at room temperature for 1 h. The reaction mixture was then concentrated to afford the title compound (1.158 g). LCMS RT= 0.21 min and 0.28 min, ES+ve m/z 316 (M+H)⁺.

Example 1

1 -{4-[(1 -Cyclobutyl-4-piperidinyl)oxy]phenyl}-4-{[4- (methylsulfonyl)phenyl]carbonyl}piperazine

To a mixture of 4-(methylsulfonyl)benzoic acid (78 mg, 0.39 mmol) and O-(benzotriazol-1- yl)-Λ/,Λ/,Λ/^;,Λ/'-tetramethyluronium tetrafluoroborate (125 mg, 0.39 mmol) was added a solution of 1-{4-[(1-cyclobutyl-4-piperidinyl)oxy]phenyl}piperazine trihydrochloride (D5)(125 mg, 0.3 mmol) in dichloromethane (4 ml) and triethylamine (0.54 ml, 3.90 mmol). The resulting solution was stirred at room temperature overnight. The reaction mixture was then partitioned between dichloromethane and saturated aqueous NaHCO₃ solution. The organic layer was concentrated and purified by SCX-2 cartridge (5 g) eluting with methanol, followed by 2M ammonia solution in methanol, collecting the ammoniacal fractions to afford the title compound (118.7 mg). LCMS RT = 2.12 min, ES+ve m/z 498 (M+H)⁺; ¹H NMR δ (CD₃OD; 400 MHz) 8.06 (2H, m), 7.69 (2H, m), 6.95 (2H, m), 6.87 (2H, m), 4.32 (1 H, br m), 3.92 (2H, br m), 3.53 (2H, br m), 3.15 (5H, s and br m), 3.02 (2H, br m), 2.90 (1 H, br m), 2.70 (2H, br m), 2.32 (2H, br m), 2.15-1.68 (10H, m).

Biological Data

The 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, 37°C.

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, 1 OmM 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 ai, (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 4⁰C 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), , 1mM 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 EC₈₀).

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

Rat Pharmacokinetics

Compounds were 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 were formulated in 5%DMSO, 45%PEG200, 50%water. An intravenous profile was 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 was obtained by taking serial or terminal blood samples at 0.25, 0.5, 1 , 2, 4, 7 and 12 hours post dose. Blood samples were taken directly into heparinised tubes. Blood samples were prepared by protein precipitation and subjected to quantitative analysis by LC-MS/MS using compound-specific mass transitions. Drug concentration-time profiles were generated and non-compartmental PK analysis used to generate estimates of half-life, clearance, volume of distribution and oral bioavailability.

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

Results

In the above or similar assays/methods the compound of formula (I) had (i) an average pki (pKb) at H3 of approximately 9.5 (ii) an average pki (pKb) at H1 of approximately 5.6 (iii) low CNS penetration (less than 100 ng compound/g of brain tissue) (iv) oral bioavailability in the rat of greater than about 50% and confirmed in the dog (oral bioavailability of greater than 90%)

(v) a half-life in the rat of approximately 2 hours (IV route) and in the dog approximately 5 hours (IV route); and clearance in the rat of about 29 ml/min/kg and in dog of about 10 ml/mi n/kg.

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

Claims

1. 1-{4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl}-4-{[4- (methylsulfonyl)phenyl]carbonyl}piperazine

or a salt thereof.

2. Compound according to claim 1 or a pharmaceutically acceptable salt thereof.

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

(")

or salt thereof, with 4-(methylsulfonyl)-benzoic acid.

4. A compound of formula (I) or a pharmaceutically acceptable salt thereof for use in therapy.

5. Compound according to claim 4 for use in the treatment of inflammatory and/or allergic diseases.

6. Compound according to claim 4 for use in the treatment of rhinitis, particularly allergic rhinitis

7. 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.

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

9. A combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents.

10. A combination according to claim 9, wherein the other therapeutic agent is an H1 receptor antagonist.

11. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prophylaxis of inflammatory and/or allergic diseases.

12. The use according to claim 11 in which the disease is allergic rhinitis.

13. A method for the treatment or prophylaxis of inflammatory and/or allergic diseases which comprises administering to a patient in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

14. A method according to claim 13 wherein the disease is allergic rhinitis.