WO2009050159A1 - Combination of fluticasone furoate with 4- [ (4-chl0r0phenyl)methyl] -2- ({ (2r)-i- [4-(4-{ [3-(hexahydr0-lh-azepin-l-yl) propyl ] oxy} phenyl) butyl] -2-pyrrolidinyl}methyl) -1 (2h) - phthalaz inone - Google Patents

Abstract

The present invention provides aqueous pharmaceutical compositions which comprise a compound which is 4-[(4-chlorophenyl)methyl]-2-({(2/R)-1-[4-(4-{[3-(hexahydro-1H-azepin- 1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone (I) or a pharmaceutically acceptable salt thereof, and to their use in the treatment of various inflammatory and/or allergic diseases such as allergic rhinitis.

Description

PHARMACEUTICAL COMPOSITIONS

The present invention relates to pharmaceutical compositions comprising an antagonist of the histamine H1 and histamine H3 receptor or a pharmaceutically acceptable salt thereof, to processes for their preparation, and to their use in the treatment of various inflammatory and/or allergic diseases, such as allergic rhinitis.

Antagonists of the histamine H1 receptor are known and widely used for the treatment of inflammatory and/or allergic diseases that are associated with the release of histamine from cells. For example, antagonists of the H1 receptor (often referred to as antihistamines) are known and widely used for the treatment of allergic rhinitis.

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. Oral antihistamine compounds (such as chlorphenyramine, cetirizine, desloratidine and fexofenadine) and intranasal antihistamines (such as azelastine and levocabastine) which are selective H1 receptor antagonists 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 often associated with allergic rhinitis. These drugs are thought to produce a decongestant action by activating α-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 et al., 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., Br. J. Pharmacol., 1-8, (2005)], although the H3 mechanism in this regard would appear to be novel and unprecedented and may ultimately prove to be clinically silent.

Other compounds that may be effective in the treatment of inflammatory and/or allergic diseases, such as allergic rhinitis include glucocorticoid receptor agonists (referred to herein as glucocorticoids). Such glucocorticoids with proven anti-inflammatory properties and which are marketed for the treatment of allergic rhinitis, include but are not limited to, beclomethasone dipropionate which is marketed under the trademark Beconase™, fluticasone furoate which is marketed under the trademark Veramyst™ and fluticasone propionate which is marketed under the trademark Flixonase™.

Allergic rhinitis, pulmonary inflammation and congestion are medical conditions that are often associated with other conditions such as asthma and chronic obstructive pulmonary disease (COPD). 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, which includes '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.

A pharmaceutical composition that is effective in treating a broad spectrum of inflammatory and/or allergic symptoms, which offers the potential for a convenient dosing regimen and which has an improved side effect profile would therefore be desirable.

Accordingly, there is provided, in one embodiment, an aqueous pharmaceutical composition which comprises a compound which is 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1- [4-(4-{[3-(hexahydro-1H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone

or a pharmaceutically acceptable salt thereof.

4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone and pharmaceutically acceptable salts thereof are disclosed in International Patent Application No. PCT/EP2007/053773, published 1^st November 2007 as WO 2007/122156 (Glaxo Group Ltd). This compound is disclosed as a dual antagonist of the histamine H1 receptor and the histamine H3 receptor, and as such may be useful in the treatment of various inflammatory and/or allergic diseases associated with the release of histamine from cells, for example, allergic rhinitis. Further, this compound is believed to possess a prolonged duration of action and exhibit relatively low CNS penetration.

Aqueous pharmaceutical compositions comprising 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1- [4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone or a pharmaceutically acceptable salt thereof may be particularly suitable for intranasal delivery, and/or capable of once daily administration and/or may have an improved side effect profile compared with other existing therapies. 4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone may be used in the form of its free base or as a pharmaceutically acceptable salt, for example the 1 ,5-naphthalene disulfonate monohydrate salt or the dihydrochloride salt.

Compositions that comprise an anti-histamine and a glucocorticoid may be particularly effective in the treatment of inflammatory and/or allergic diseases.

Thus, in another embodiment, there is provided an aqueous pharmaceutical composition which comprises:

(i) a compound which is 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-

1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2/-/)-phthalazinone

or a pharmaceutically acceptable salt thereof; and

(ii) a compound which is 6α, 9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-1 1β-hydroxy-16α- methyl-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester

or a solvate thereof.

6α, 9α-Difluoro-17α-[(2-furanylcarbonyl)oxy]-1 1 β-hydroxy-16α-methyl-3-oxo-androsta-1 ,4 diene-17β-carbothioic acid S-fluoromethyl ester (hereinafter referred to as fluticasone furoate) and solvates thereof are disclosed in International Patent Application WO02/12265 and International Patent Application WO03/066024. Fluticasone furoate is sold in Europe as Veramyst™ for the treatment of seasonal allergy symptoms.

Fluticasone furoate may be used in the form of a solvate, or unsolvated. Thus, in one embodiment, aqueous pharmaceutical compositions of the invention comprise fluticasone furoate, in unsolvated form. In a further embodiment, compositions comprise unsolvated fluticasone furoate as polymorphic Form 1 , as defined in WO02/12265.

It will be appreciated that the aqueous pharmaceutical compositions of the invention comprising 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro1 Hazepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2/-/)-phthalazinone or a pharmaceutically acceptable salt thereof and fluticasone furoate or a solvate thereof may be delivered such that each compound may be delivered sequentially in separate pharmaceutical compositions, or may be delivered simultaneously in a combined pharmaceutical composition.

The aqueous pharmaceutical compositions of the invention are in the form of an aqueous suspension and/or an aqueous solution. Partial suspensions and/or partial solutions are encompassed within the scope of the present invention. Compositions comprising one compound which is in solution and the other compound which is in suspension are also included within the scope of the present invention.

In one embodiment, the compound(s) in the aqueous pharmaceutical compositions of the invention are in suspension.

The aqueous component of the compositions of the invention is typically a high grade quality of water, such as purified water (e.g. MilliQ™ water).

The compound(s) for use in the compositions of the invention will typically have a mass mean diameter (MMD) of less than 20 μm, such as from 0.5 to 10 μm, for example from 1 to 10 μm as measured by laser diffraction, for example. Particle size reduction, if necessary, may be achieved by techniques well known in the art such as micronisation, milling and/or microfluidisation. The aqueous pharmaceutical compositions of the invention may be suitable for topical administration, which includes intranasal, inhaled and ocular administration. Compositions suitable for intranasal administration are of particular interest.

The dose of 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 - yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone or a pharmaceutically acceptable salt thereof used will vary in the usual way with the seriousness of the diseases to be treated and other factors, for example the weight of the sufferer. However, as a general guide, suitable unit doses may be from about 0.05 to about 1000 mg, such as from about 0.05 to about 200 mg, for example from about 0.05 to about 2 mg, or from about 0.05 to about 1 mg and such unit doses may be administered once a day, or more than once a day, for example two or three times a day or as desired. Such therapy may extend for a number of weeks or months.

As used herein "% (w/w)" means the weight of a substance relative to the weight of the total composition expressed as a percentage, e.g. 50 % (w/w) = 0.5 g substance per 1 g total composition.

The proportion of 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 - yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone or a pharmaceutically acceptable salt thereof, in the aqueous pharmaceutical compositions of the invention will depend on the composition to be prepared and the particular route of administration, but will generally be within the range of from about 0.005 to about 2% (w/w), such as from about 0.01 to about 1% (w/w), based on the total weight of the composition. Particular concentrations of 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3- (hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone as the free base are 0.01% (w/w), 0.025% (w/w), 0.05% (w/w), 0.1% (w/w), 0.25% (w/w) and 0.5 % (w/w), based on the total weight of the composition. In one embodiment, the pharmaceutically acceptable salt is a dihydrochloride salt.

It will be appreciated that when 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro- 1 H-azepin-1 -yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone is used in the form of a pharmaceutically acceptable salt, the concentration will depend on the salt chosen but will be such as to provide the desired concentration of compound as the free base. Thus, particular concentrations of 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3- (hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone 1 ,5-naphthalene disulfonate monohydrate salt are in the range of from about 0.01 to about 0.75% (w/w), for example from about 0.01478 to about 0.739% (w/w), based on the total weight of the composition, to provide from about 0.01 to about 0.5% (w/w) of the free base

The proportion of fluticasone furoate or a solvate thereof in the aqueous pharmaceutical compositions of the invention will depend on the composition to be prepared and the particular route of administration, but will generally be within the range of from about 0.01 to about 1 % (w/w), such as from about 0.01 to about 0.5% (w/w) e.g. about 0.05% (w/w) based on the total weight of the composition. Typically, 50 μl_ of composition will deliver about 27.5 μg of fluticasone furoate, or a solvate thereof, giving a daily dose of about 55 to 110 μg of fluticasone furoate.

In one embodiment, the aqueous pharmaceutical compositions of the invention are suitable for intranasal administration.

Intranasal compositions may permit the compound(s) to be delivered to all areas of the nasal cavities (the target tissue) and further, may permit the compound(s) to remain in contact with the target tissue for longer periods of time. A suitable dosing regime for intranasal compositions would be for the patient to inhale slowly through the nose subsequent to the nasal cavity being cleared. During inhalation the composition would be administered to one nostril while the other is manually compressed. This procedure would then be repeated for the other nostril. Typically, one or two sprays per nostril would be administered by the above procedure up to two or three times each day, ideally once daily. Of particular interest are intranasal compositions suitable for once daily administration.

Aqueous pharmaceutical compositions of the invention, such as intranasal compositions, may optionally contain one or more suspending agents, one or more preservatives, one or more wetting agents and/or one or more isotonicity adjusting agents as desired. Further, the compositions of the invention, for example suitable for intranasal administration, may optionally further contain other excipients, such as antioxidants (for example sodium metabisulphite), taste-masking agents (such as menthol) and sweetening agents (for example dextrose, glycerol, saccharin and/or sorbitol). Compositions of the invention may also further contain one or more co-solvents, as desired.

The skilled person would readily appreciate that some excipients may perform more than one function, depending on the nature and number of excipients used in that composition and the particular properties of the compound(s) contained therein.

In one embodiment, there is provided an aqueous pharamaceutical composition of the invention comprising a suspending agent.

The suspending agent(s), if included, will typically be present in an amount of from about 0.1 to about 5% (w/w), such as from about 1.5% to about 2.4% (w/w), particularly about 2.4% (w/w) based on the total weight of the composition. Examples of pharmaceutically acceptable suspending agents include, but are not limited to, Avicel® (microcrystalline cellulose and carboxymethylcellulose sodium), carboxymethylcellulose sodium, veegum, tragacanth, bentonite, methylcellulose and polyethylene glycols. Further examples include xanthan gum and carbopol. In one embodiment, the suspending agents are microcrystalline cellulose and carboxy methylcellulose sodium.

In one embodiment, there is provided an aqueous pharmaceutical composition of the invention comprising a preservative.

For stability purposes, compositions of the invention (for example intranasal compositions) may be protected from microbial or fungal contamination and growth by inclusion of one or more preservatives. Examples of pharmaceutically acceptable anti-microbial agents or preservatives may include, but are not limited to, quaternary ammonium compounds (e.g. benzalkonium chloride, benzethonium chloride, cetrimide and cetylpyridinium chloride.

Other examples are myristyl picolinium chloride and lauralkonium chloride), mercurial agents (e.g. phenylmercuric nitrate, phenylmercuric acetate and thimerosal), alcoholic agents (e.g. chlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterial esters

(e.g. esters of para-hydroxybenzoic acid), chelating agents such as disodium ethylenediaminetetraacetate (EDTA) and other anti-microbial agents such as chlorhexidine, chlorocresol, sorbic acid and its salts (such as potassium sorbate) and polymyxin. Examples of pharmaceutically acceptable anti-fungal agents or preservatives may include, but are not limited to, sodium benzoate. Other anti-fungal agents include, sorbic acid, sodium propionate, methyl paraben, ethyl paraben, propyl paraben and butyl paraben. The preservative(s), if included, may be present in an amount of from about 0.001 to about 1% (w/w), such as from about 0.015% to about 0.5% (w/w), for example from about 0.015% to about 0.3% (w/w) based on the total weight of the composition. In a further embodiment, the preservative(s) are selected from benzalkonium chloride, EDTA and/or potassium sorbate. In a further embodiment, the preservative(s) are EDTA and/or potassium sorbate. In a further embodiment the preservatives are EDTA in a concentration of about 0.015% (w/w) and potassium sorbate in a concentration of about 0.3% (w/w), based on the total weight of the composition.

In a further embodiment, there is provided an aqueous pharmaceutical composition of the invention which is preservative free.

In one embodiment, there is provided an aqueous pharmaceutical composition of the invention comprising a wetting agent.

Compositions (for example wherein the compound is in suspension) may include one or more wetting agents which function(s) to wet the particles of medicament to facilitate dispersion thereof in the aqueous phase of the composition. Typically, the amount of wetting agent used will not cause foaming of the dispersion during mixing. Examples of pharmaceutically acceptable wetting agents include, but are not limited to, fatty alcohols, esters and ethers, such as polyoxyethylene (20) sorbitan monooleate (Polysorbate 80), macrogol ethers and poloxamers. The wetting agent may be present in an amount from about 0.005 to about 0.05, for example from about 0.01 to about 0.05% (w/w), for example about 0.025% (w/w), or from about 1.5 to 2.5%, such as about 2.0% (w/w), based on the total weight of the composition. In one embodiment, the wetting agent is polyoxyethylene (20) sorbitan monooleate (Polysorbate 80).

In one embodiment, there is provided an aqueous pharmaceutical composition of the invention comprising an isotonicity adjusting agent.

One or more isotonicity adjusting agent(s) may be included to achieve isotonicity with body fluids e.g. fluids of the nasal cavity or fluids of the eye, resulting in reduced levels of irritancy. Examples of pharmaceutically acceptable isotonicity adjusting agents include, but are not limited to, sodium chloride, dextrose, xylitol and calcium chloride. An isotonicity adjusting agent, if present, may be included in an amount of from about 0.1 to about 10% (w/w), such as from about 4.5 to about 5.5% (w/w), for example about 5.0% (w/w), or from about 0.5 to about 1 % (w/w), such as about 0.75% (w/w), based on the total weight of the composition. In a further embodiment, the isotonicity adjusting agents are dextrose (e.g. anhydrous dextrose) and/or xylitol. In a further embodiment the isotonicity adjusting agent is xylitol. In a further embodiment, the isotonicity adjusting agent is xylitol in a concentration of about 0.75% (w/w), based on the total weight of the composition. In a further embodiment, the composition does not contain an isotonicity adjusting agent.

In one embodiment, there is provided an aqueous pharmaceutical composition of the invention comprising a buffering agent.

The compositions of the invention may be buffered by the addition of suitable buffering agents such as sodium citrate, citric acid, phosphates such as disodium phosphate (for example the dodecahydrate, heptahydrate, dihydrate and anhydrous forms) or sodium phosphate and mixtures thereof. Another buffering agent is trometarol. In a further embodiment, the buffering agents are sodium citrate and/or citric acid. In a further embodiment, the buffering agents are sodium citrate in a concentration of about 1.48% (w/w) and citric acid (which may be anhydrous) in a concentration of about 0.96% (w/w), based on the total weight of the composition.

In one embodiment, there is provided an aqueous pharmaceutical composition of the invention comprising a co-solvent.

One or more co-solvent(s) may be included to aid solubility of the active compound(s) and/or other excipients. Examples of pharmaceutically acceptable co-solvents include, but are not limited to, propylene glycol, dipropylene glycol, ethylene glycol, glycerol, ethanol, polyethylene glycols (for example PEG300 or PEG400) and methanol. The cosolvent(s), if present, may be included in an amount of from about 0.05 to about 20% (w/w), such as from about 1.5 to about 17.5% (w/w), or from about 1.5 to about 7.5% (w/w), or from 0.05% to about 0.5% (w/w) based on the total weight of the composition.

In a further embodiment, there is provided an aqueous pharmaceutical composition of the invention further comprising: a) a suspending agent; b) a preservative; c) a wetting agent; and d) an isotonicity adjusting agent.

In a further embodiment, there is provided an aqueous pharmaceutical composition of the invention further comprising: a) a suspending agent; b) a preservative; c) a wetting agent; d) an isotonicity adjusting agent; and e) a co-solvent.

In a further embodiment, there is provided an aqueous pharmaceutical composition which comprises microcrystalline cellulose and carboxymethyl cellulose sodium (as suspending agents); benzalkonium chloride, EDTA and/or potassium sorbate (as preservatives); polyoxyethylene (20) sorbitan monooleate (supplied as polysorbate 80) (as a wetting agent); and dextrose and/or xylitol (as isononicity adjusting agents).

In a further embodiment, there is provided an aqueous pharmaceutical composition which comprises microcrystalline cellulose and carboxymethyl cellulose sodium (as suspending agents); benzalkonium chloride, EDTA and/or potassium sorbate (as preservatives); polyoxyethylene (20) sorbitan monooleate (supplied as polysorbate 80) (as a wetting agent); dextrose and/or xylitol (as isononicity adjusting agents); and propylene glycol (as a co-solvent).

In a further embodiment, there is provided an aqueous pharmaceutical composition which comprises microcrystalline cellulose and carboxymethyl cellulose sodium (as suspending agents); EDTA and/or potassium sorbate (as preservatives); polyoxyethylene (20) sorbitan monooleate (supplied as polysorbate 80) (as a wetting agent); xylitol (as isononicity adjusting agents); and propylene glycol (as a co-solvent).

Compositions for administration topically, to the nose or lung for example, for the treatment of rhinitis, include pressurised aqueous aerosol compositions and aqueous compositions delivered to the nasal cavities by pressurised pump. Aqueous compositions which are non-pressurised and adapted to be administered topically to the nasal cavity are of particular interest. Aqueous compositions may also be administered to the nose by nebulisation.

Accordingly, the aqueous pharmaceutical compositions of the invention are provided in a suitable container depending on the choice of route of administration. Further, for compositions comprising both compounds it will be appreciated that said container may be capable of delivering each compound sequentially in a separate pharmaceutical composition as well as simultaneously in a combined pharmaceutical composition.

A fluid dispenser typically used to deliver the aqueous pharmaceutical compositions of the invention to the nasal cavities may have a dispensing nozzle or dispensing orifice through which a metered dose of the fluid composition is dispensed upon the application of a user- applied force to a pump mechanism of the fluid dispenser. Such fluid dispensers are generally provided with a reservoir of multiple metered doses of the fluid composition, the doses being dispensable upon sequential pump actuations. The dispensing nozzle or orifice may be configured for insertion into the nostrils of the user for spray dispensing of the fluid composition into the nasal cavity. A fluid dispenser of the aforementioned type is described and illustrated in WO05/044354 the entire content of which is hereby incorporated herein by reference. The dispenser has a housing which houses a fluid discharge device having a compression pump mounted on a container for containing a fluid composition. The housing has at least one finger-operable side lever which is movable inwardly with respect to the housing to cam the container upwardly in the housing to cause the pump to compress and pump a metered dose of the composition out of a pump stem through a nasal nozzle of the housing. In one embodiment, the fluid dispenser is of the general type illustrated in Figures 30-40 of WO05/044354.

Further, the aqueous pharmaceutical compositions of the invention may be delivered by a pump as disclosed in WO2007/138084, for example as disclosed with reference to Figures 22-46 thereof, or as disclosed in GB0723418.0, for example as disclosed with reference to Figures 7-32 thereof, both of which are incorporated herein by reference in their entirety. The pump may be actuated by an actuator as disclosed in Figures 1-6 of GB0723418.0. Typically, fluid dispensers for use with the aqueous pharmaceutical compositions of the invention may be capable of holding 8 to 50 ml. (or less) of composition and each spray will typically deliver 50 to 100 μl_ (or less, for example 25 μl_) of composition. Typically therefore the fluid dispenser will be capable of providing at least 100 metered doses.

Accordingly, there is provided in a further aspect a container comprising an aqueous pharmaceutical composition of the invention.

In one embodiment, the container is suitable for delivering an aqueous pharmaceutical composition of the invention to the nasal cavities.

In a further embodiment, the container is suitable for delivering an aqueous pharmaceutical composition of the invention to the eye.

The compositions of the invention have potentially beneficial anti-inflammatory and/or antiallergic effects, particularly upon topical administration to the nose, demonstrated by, for example the ability of 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H- azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone, or a pharmaceutically acceptable salt thereof, to antagonise the H1 and H3 receptor, with long acting effect and the known activity of fluticasone furoate at the glucorcorticoid receptor.

Hence, the aqueous pharmaceutical compositions of the invention may be useful in the treatment of inflammatory and/or allergic diseases, particularly inflammatory and/or allergic diseases of the respiratory tract.

Examples of disease states in which the aqueous pharmaceutical compositions of the invention may have potentially beneficial anti-inflammatory and/or anti-allergic effects include inflammatory and/or allergic diseases of the respiratory tract, such as allergic rhinitis (seasonal and perennial) or other diseases such as bronchitis (including chronic bronchitis), asthma (including allergen-induced asthmatic reactions), chronic obstructive pulmonary disease (COPD) and sinusitis.

Furthermore, the aqueous pharmaceutical compositions of the invention may be of use in the treatment of nephritis, skin diseases such as psoriasis, eczema, allergic dermatitis and hypersensitivity reactions. Also, the aqueous pharmaceutical compositions of the invention may be useful in the treatment of insect bites and stings.

The aqueous pharmaceutical compositions of the invention may also be of use in the treatment of nasal polyposis, conjunctivitis (e.g. allergic conjunctivitis) or pruritis.

A disease of particular interest is allergic rhinitis.

Other diseases in which histamine may have may have a pathophysiological role include non-allergic rhinitis. Therefore, the aqueous pharmaceutical compositions of the invention may also be of use in the treatment of non-allergic rhinitis.

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

Accordingly, there is provided an aqueous pharmaceutical composition of the invention which comprises a compound which is 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3- (hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone or a pharmaceutically acceptable salt thereof and optionally a compound which is fluticasone furoate or a solvate thereof, for use in therapy.

In another embodiment, there is provided an aqueous pharmaceutical composition of the invention which comprises a compound which is 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4- (4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone or a pharmaceutically acceptable salt thereof and optionally a compound which is fluticasone furoate or a solvate thereof, for use in the treatment (or prophylaxis) of inflammatory and/or allergic diseases.

In another embodiment, there is provided an aqueous pharmaceutical composition of the invention which comprises a compound which is 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4- (4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone or a pharmaceutically acceptable salt thereof and optionally a compound which is fluticasone furoate or a solvate thereof, for use in the treatment (or prophylaxis) of inflammatory and/or allergic diseases of the respiratory tract, such as allergic rhinitis. In a further aspect, there is provided the use of an aqueous pharmaceutical composition of the invention which comprises a compound which is 4-[(4-chlorophenyl)methyl]-2- ({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone or a pharmaceutically acceptable salt thereof and optionally a compound which is fluticasone furoate or a solvate thereof, in the manufacture of a medicament for the treatment (or prophylaxis) of inflammatory and/or allergic diseases, such as inflammatory and/or allergic diseases of the respiratory tract, for example, allergic rhinitis.

In a further aspect, there is provided a method for the treatment (or prophylaxis) of any of the above mentioned diseases, which method comprises administering to a patient in need thereof a pharmaceutically effective amount of an aqueous pharmaceutical composition of the invention which comprises a compound which is 4-[(4- chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl) butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone or a pharmaceutically acceptable salt thereof and optionally a compound which is fluticasone furoate or a solvate thereof.

In one embodiment, there is provided a method for the treatment of allergic rhinitis.

The aqueous pharmaceutical compositions of the invention may be prepared by the methods described below or by similar methods. The following Examples illustrate the preparation of the aqueous pharmaceutical compositions but are not to be considered in limiting the the scope of the disclosure in any way.

Preparation of 4-r(4-chlorophenyl)methyll-2-({(2/?)-1 -r4-(4-{ r3-(hexahydro-1 H-azepin- 1-yl)propynoxy)phenyl)butvn-2-pyrrolidinyl)methyl)-1(2H)-phthalazinone

4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone and its salts may be prepared according to methods disclosed in International Patent Application WO 2007/122156 (Glaxo Group Ltd), published 1^st November 2007 (in particular, see Examples 24A, 24C and 24D).

Abbreviations BOC (Boc): te/f-butoxycarbonyl DMSO: Dimethylsulfoxide

HPLC: High Performance Liquid Chromotography

LCMS: Liquid Chromatography - Mass Spectroscopy mbar: millibar (pressure)

MDAP: Mass-Directed Autopreparative acetonitrile: Acetonitrile

NMR: Nuclear Magnetic Resonance

PTFE: Polytetrafluoroethylene

SiO₂: Silica

TLC: Thin Layer Chromotography h: Hours min: Minutes

RT: Retention Time

General Procedures

Flash silica gel refers to Merck Art No. 9385; silica gel refers to Merck Art No. 7734.

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.

LCMS was conducted on a Supelcosil LCABZ+PLUS column (3.3 cm x 4.6 mm ID) eluting with 0.1 % formic acid and 0.01 M ammonium acetate in water (solvent A) and 0.05% formic acid 5% water in acetonitrile (solvent B), using the following elution gradient 0.0 - 7 min 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 3 mlmin^"1. 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.

Mass directed autopreparative (MDAP) HPLC was conducted on a Waters FractionLynx system comprising of a Waters 600 pump with extended pump heads, Waters 2700 autosampler, Waters 996 diode array and Gilson 202 fraction collector on a 10 cm x 2.54 cm internal diameter ABZ+ column, eluting with 0.1 % formic acid in water (solvent A) and

0.1 % formic acid in acetonitrile (solvent B), using as appropriate elution gradient over 15 min at a flow rate of 20 mlmin^"1 and detecting at 200 - 320 nm at room temperature. Mass spectra were recorded on Micromass ZMD mass spectrometer using electrospray positive and negative mode, alternate scans. The software used was MassLynx 3.5 with

OpenLynx and FractionLynx options.

The ¹H NMR spectra were recorded on a Bruker AV400 operating at 400 MHz. Standard deuterated solvents were used. Typically, the NMR is taken with a deuterium lock for reference. Optionally, tetramethylsilane is used as internal standard.

Reactions are routinely monitored by methods well known to those skilled in the art, such as TLC, LCMS and/or HPLC. Such methods are used to assess whether a reaction has gone to completion, and reaction times may be varied accordingly.

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

According to one process, 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/- azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2/-/)-phthalazinone and its 1 ,5-naphthalene disulfonate salt may be prepared according to Scheme 1 , below. 4-[(4- Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/-azepin-1-yl)propyl]oxy}phenyl) butyl]-2-pyrrolidinyl}methyl)-1 (2/-/)-phthalazinone and its salts may also be prepared according to methods disclosed in International Patent Application WO 2007/122156 (Glaxo Group Ltd), published 1^st November 2007 (see Example 24).

Scheme 1 : Synthesis of 4-r(4-chlorophenyl)methyll-2-({(2f?)-1-r4-(4-{r3-(hexahvdro-1 /-/- azepin-1-yl)propyl1oxy)phenyl)butyl1-2-pyrrolidinyl) methyl)- 1 (2/-/)-phthalazinone, free base and 1 ,5-naphthalene disulfonate salt

Reagents and Conditions: Stage 1 ) suitable base e.g. sodium acetate, suitable solvent such as Λ/-methyl-2-pyrrolidinone, usually at an elevated temperature such as between 170 and 190 ⁰C; Stage 2) hydrazine, or hydrazine sulfate and sodium hydroxide, in a suitable solvent such as ethanol; Stage 3) appropriate base e.g. potassium carbonate; Stage 4a) appropriate activating agent such as mesyl chloride, suitable base e.g. triethylamine, appropriate solvent for example diethyl ether; Stage 4b) suitable base such as triethylamine and/or potassium carbonate, appropriate solvent e.g. methyl /so-butyl ketone, optionally with an activating agent such as potassium iodide, usually heated to an elevated temperature for example at reflux; Stage 5) deprotection using an appropriate agent such as boron tribromide or aqueous hydrogen bromide with acetic acid, in a suitable solvent such as dichloromethane, optionally cooled to a suitable lowered temperature e.g. approximately 0 ⁰C; Stage 6) suitable solvent such as 2-butanone, appropriate base e.g. potassium carbonate; Stage 7) suitable base such as potassium carbonate, in a suitable solvent e.g. 2-butanone, optionally with an activating agent such as potassium iodide; Stage 8) step 1 : suitable solvent e.g. methyl /so-butyl ketone and/or methanol, aqueous hydrogen chloride, 1 ,5-naphthalene disulfonic acid (commercially available, for example, from Aldrich); step 2 (optional): recrystallisation from Λ/./V- dimethylsulfoxide and aqueous tetrahydrofuran.

The compound of formula (I), phthalic anhydride, is commercially available, for example, from Sigma-Aldrich.

The compound of formula (II), 4-chlorophenylacetic acid, is commercially available, for example, from Sigma-Aldrich.

The compound of formula (III), 4-chlorobenzylidene phthalide, is commercially available, for example, from Honeywell.

The compound of formula (IV), 4-[(4-chlorophenyl)methyl]-1 (2H)-phthalazinone, is also disclosed in US patent 3,813,384, see Example 10, step 1.

The compound of formula (V), 1 ,1-dimethylethyl (2/?)-2-{[(methylsulfonyl)oxy]methyl}-1- pyrrolidinecarboxylate may be prepared by methods well known to those skilled in the art, for example by mesylation of the corresponding commercially available alcohol, 1 ,1- dimethylethyl (2/?)-2-(hydroxymethyl)-1-pyrrolidinecarboxylate, which is commercially available, for example, from Sigma-Aldrich. The activation reaction may typically be carried out using an appropriate activating agent, such as mesyl chloride, with a suitable base e.g. triethylamine in an appropriate solvent such as te/t-butyl methyl ether, usually at a lowered temperature, such as from 0 to 20 ⁰C (see Stage 3a, below).

The compound of formula (VII), 4-[4-(methyloxy)phenyl]-1-butanol, is commercially available from Sigma-Aldrich, for example.

The compound of formula (Xl), 1-bromo-3-chloropropane, is commercially available, for example, from Sigma-Aldrich.

The compound of formula (XIII), hexamethyleneimine, is commercially available, for example, from Aldrich.

Further, 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl] oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone as a pharmaceutically acceptable salt may be prepared by exchange of counterions, or precipitation of said salt from the free base.

It is to be further understood that references hereinafter to 4-[(4-chlorophenyl)methyl]-2- ({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone means that compound, as the free base, or as a pharmaceutically acceptable salt, or as a solvate.

4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone 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. ScL, 1977, 66, 1-19. Suitable pharmaceutically acceptable salts include acid addition salts. As used herein, the term "pharmaceutically acceptable salt", means any pharmaceutically acceptable salt or solvate of a compound of formula (I), which upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I), or an active metabolite or residue thereof.

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 4-[(4- chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl) butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone with a suitable inorganic or organic acid (such as hydrobromic, hydrochloric, sulphuric, nitric, phosphoric, succinc, maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic, benzoic, salicylic, glutamic, aspartic, p- toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, naphthalenesulfonic (e.g. 2-naphthalenesulfonic), naphthalene disulfonic or hexanoic 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. A pharmaceutically acceptable acid addition salt of a compound of formula (I) can comprise or be for example a hydrobromide, hydrochloride, sulfate, nitrate, phosphate, succinate, maleate, formate, acetate, propionate, fumarate, citrate, tartrate, lactate, benzoate, salicylate, glutamate, aspartate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate, naphthalenesulfonate (e.g. 2-naphthalenesulfonate), naphthalene disulfonate e.g. 1 ,5- naphthalene disulfonate salt or hexanoate salt.

It will be appreciated that 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H- azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone may form complexes with solvents in which it is reacted or from which it is precipitated or crystallised. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". It will be appreciated that 4-[(4-chlorophenyl)methyl]-2- ({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone or salts thereof may exist in solvated form, particularly as a hydrate. A particular solvated form is the 1 ,5-naphthalene disulfonate monohydrate salt.

Intermediate 1

1 , 1 -Dimethylethyl (2/?)-2-{[4-[(4-chlorophenyl)methyl]-1 -oxo-2(1 H)-phthalazinyl] methyl}-1 -pyrrolidinecarboxylate

To a solution of triphenylphosphine (1.86 g, 7.09 mmol) in dry tetrahydrofuran (6 ml) was added diisopropyl azodicarboxylate (1.12 ml, 5.69 mmol) at -15 ⁰C. The resulting pale yellow thick suspension was stirred at -15 ⁰C for 2 min. To aid stirring, more dry tetrahydrofuran (2 ml) was added. The reaction mixture was then treated with a suspension of 4-[(4-chlorophenyl)methyl]-1 (2H)-phthalazinone, (for example, as disclosed in US patent 3,813,384, Example 10, Step 1 ) (0.571 g, 2.11 mmol) and N-tert- butoxycarbonyl-D-prolinol (commercially available, for example, from Fluka), (0.650 g, 3.23 mmol) in dry tetrahydrofuran (10 ml) at -15 ⁰C. The reaction mixture was allowed to warm to room temperature and stirred at 20 ⁰C for 23 h. Methanol (20 ml) was then added and the solvents were removed in vacuo. The resultant residue was purified by Flashmaster Il chromatography (70 g silica cartridge) eluted with 0 - 50% ethylacetate- cyclohexane gradient over 40 min. The solvents were removed in vacuo to afford the title compound as a dark brown oil(1.05 g). LCMS RT = 3.71 min.

Intermediate 2

4-[(4-Chlorophenyl)methyl]-2-[(2/?)-2-pyrrolidinylmethyl]-1(2H)-phthalazinone

To a solution of 1 ,1 -dimethylethyl (2/?)-2-{[4-[(4-chlorophenyl)methyl]-1-oxo-2(1H)- phthalazinyl]methyl}-1 -pyrrolidinecarboxylate (for example, as prepared for Intermediate

1 ) (1.05 g, 2.31 mmol) in dry 1 ,4-dioxane (12 ml) was added a solution of hydrogen chloride in 1 ,4-dioxane (4.0 M, 6 ml). The solution was stirred at 20 ⁰C for 2 h. Trifluoroacetic acid (1 ml) was added to the mixture and stirred for 30 min, then more trifluoroacetic acid (3 x approximately 1 ml) was added at 10 minute intervals until deprotection was completed. The solvent was removed in vacuo and the residue applied onto an SCX cartridge (20 g), washed with methanol (x 2) and then eluted with 10% aqueous ammonia in methanol (2 x 50 ml). The solvents were removed in vacuo and the resultant residue purified by Flashmaster Il chromatography (50 g silica cartridge) eluted with 0 - 30% methanol + 1 % triethylamine - dichloromethane gradient over 40 min to afford the title compound as a dark brown foam (0.351 g). LCMS RT = 2.45 min.

Intermediate s

1-[(3-Chloropropyl)oxy]-4-iodobenzene

A mixture of 4-iodophenol (commercially available, for example, from Aldrich) (20 g), 1- bromo-3-chloropropane (commercially available, for example, from Aldrich) (17.91 g), and potassium carbonate (25.2 g) in 2-butanone (400 ml) was stirred at reflux under a nitrogen atmosphere for 18 h. The mixture was allowed to cool and was filtered. The filtrate was evaporated and the residue was dissolved in cyclohexane and purified by Flashmaster Il on a 100 g silica cartridge, eluting with cyclohexane and then 20% ethylacetate in cyclohexane. The solvent was evaporated from appropriate fractions giving the title compound as a colourless oil which crystallised on standing (15.928 g). LCMS RT = 3.70 min. The solvent was evaporated from a further set of fractions giving additional portion of the title compound as a pale yellow oil that solidified on standing (6.668 g). ¹H NMR (CDCI₃) δ 2.23 (2H, quint, J = 6 Hz), 3.74 (2H, t, J = 6 Hz), 4.09 (2H, t, J = 6 Hz), 6.70 (2H, m), 7.57 (2H, m).

Intermediate 4

1 -{3-[(4-lodophenyl)oxy]propyl}hexahydro-1 H-azepine

A mixture of 1-[(3-chloropropyl)oxy]-4-iodobenzene (for example, as prepared for Intermediate 3), (15.855 g), sodium iodide (8 g) and hexamethyleneimine (commercially available, for example, from Aldrich) (15.1 ml) in 2-butanone (200 ml) was stirred at 80 ⁰C under a nitrogen atmosphere for about 22 h. The reaction mixture was filtered and the filtrate was evaporated giving a beige residue. This material was triturated with diethyl ether and a pale solid was recovered by filtration. The solid was partitioned between dichloromethane (200 ml) and saturated sodium bicarbonate solution (200 ml). The organic layer was dried over anhydrous sodium sulfate and evaporated to give a residue that was purified by Flashmaster Il chromatography on a 50 g silica cartridge using a 0 - 30% methanol containing 1% triethylamine - dichloromethane gradient over 50 min. The solvent was evaporated from appropriate fractions to give the title compound as a yellow solid (1.2708 g). LCMS RT = 2.39 min, ES+ve m/z 360 (M+H)⁺. The filtrate from the trituration was evaporated yielding a brown residue that was purified by Flashmaster Il chromatography on 2 x 100 g silica cartridges using 0-30% methanol containing 1% triethylamine - dichloromethane gradient over 50 min. The solvent was evaporated from appropriate fractions giving the title compound as a yellow oil (15.9 g). LCMS RT = 2.40 min, ES+ve m/z 360 (M+H)⁺.

Intermediate s

4-(4-{[3-(Hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)-3-butyn-1 -ol

A mixture of 1-{3-[(4-iodophenyl)oxy]propyl}hexahydro-1H-azepine (for example, as prepared for Intermediate 4) (1.268 g), 3-butyn-1-ol (commercially available, for example, from Aldrich) (668 μl), triethylamine (2.5 ml), bis(triphenylphosphine)palladium(ll) chloride (124 mg) and copper (I) iodide (34 mg) in tetrahydrofuran (15 ml) was stirred at room temperature under a nitrogen atmosphere for about 5 h. The mixture was filtered (60 ml PTFE filter tube) and the filtrate was evaporated giving a residue. This material was purified by Flashmaster Il chromatography on a 100 g silica cartridge using 0 - 30% methanol containing 1% triethylamine - dichloromethane gradient over 60 min. Evaporation of the solvent from appropriate fractions gave a residue that was dissolved in methanol and loaded onto a SCX ion-exchange cartridge (50 g). The cartridge was washed with methanol and then eluted with 2 M ammonia in methanol. Evaporation of the solvent from the ammonia-containing fractions gave the title compound as a dark yellow residue (823 mg). LCMS RT = 2.10 min, ES+ve m/z 302 (M+H)⁺.

Intermediate 6

4-(4-{[3-(Hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)-1 -butanol

To a solution of 4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)-3-butyn-1-ol (for example, as prepared for Intermediate 5) (823 mg) in ethanol (20 ml) was added 1.25 M hydrogen chloride in methanol (3.25 ml) and the resulting mixture was hydrogenated over 10% w/w Palladium on carbon (350 mg) for about 4 h. The reaction mixture was filtered through celite and the solvent was evaporated. The residue was dissolved in dichloromethane (125 ml) and the resulting solution was washed with 2 N sodium hydroxide (100 ml) and brine (100 ml) and dried over anhydrous sodium sulfate. Evaporation of the solvent gave the title compound as a dark yellow residue (726 mg). LCMS RT = 2.18 min, ES+ve m/z 306 (M+H)⁺.

Intermediate 7 4-(4-{[3-(Hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)butyl methanesulfonate

To a solution of 4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy}phenyl)-1-butanol (for example, as prepared for Intermediate 6) (0.077 g, 0.25 mmol, in anhydrous dichloromethane (2 ml) was added Λ/,Λ/-diisopropylethylamine (0.053 ml, 0.30 mmol) and then methanesulfonyl chloride (commercially available, for example, from Aldrich) (0.023 ml, 0.30 mmol). The solution was stirred at 20 ⁰C for 2 h. The solution was diluted with dichloromethane (10 ml) and saturated sodium hydrogen carbonate (10 ml). The biphasic mixture was shaken and the phases separated using a hydrophobic frit. The organic phase was removed in vacuo to give the title compound (0.09 g). LCMS RT = 2.43 min, ES+ve m/z 384 (M+H)⁺.

Intermediate 8

4-[4-(Methyloxy)phenyl]butyl methanesulfonate

To a cooled, 0 ⁰C, solution of 4-[4-(methyloxy)phenyl]-1-butanol (commercially available, for example, from Aldrich) (0.36 g, 2 mmol) and triethylamine (1.39 ml, 10 mmol) in dry diethyl ether (10 ml) under nitrogen was added, dropwise, methanesulfonyl chloride (commercially available, for example, from Aldrich) (0.46 ml, 6 mmol). After stirring at room temperature for 4 h, the reaction mixture was separated between diethyl ether and water. The organic phase was washed with water, saturated sodium bicarbonate solution and dried over anhydrous magnesium sulfate. The solvent was removed in vacuo to afford the title compound as a colourless oil (0.52 g). ¹H NMR (400 MHz, CDCI₃) δ 7.10 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 4.24 (t, J = 6.3 Hz, 2H), 3.80 (s, 3H), 2.99 (s, 3H), 2.61 (t, J = 7 Hz, 2H), 1.82-1.68 (m, 4H).

Intermediate 9 4-[(4-Chlorophenyl)methyl]-2-[((2/?)-1-{4-[4-(methyloxy)phenyl]butyl}-2- pyrrolidinyl)methyl]-1(2H)-phthalazinone

To a solution of 4-[(4-chlorophenyl)methyl]-2-[(2R)-2-pyrrolidinylmethyl]-1 (2H)- phthalazinone (for example, as prepared for Intermediate 2) (35.35 g, 100 mmol) in 2- butanone (250 ml) under nitrogen was added 4-[4-(methyloxy)phenyl]butyl methanesulfonate (for example, as prepared for Intermediate 8) (31 g, 120 mmol) and potassium carbonate (27.6 g, 200 mmol). The reaction mixture was heated at reflux for 24 h. The solid was removed by filtration and washed with 2-butanone (3 x 100 ml). The combined filtrate and washings were evaporated in vacuo and the residue was dissolved in dichloromethane (70 ml). This was applied to a silica Biotage cartridge (800 g) and eluted with dichloromethane (5000 ml) and then 5% methanol in dichloromethane (5000 ml). The required fractions were evaporated in vacuo and dissolved in dichloromethane (70 ml). This was applied to aminopropyl cartridges (8 x 70 g) and eluted with a gradient of 0 - 100% dichloromethane in cyclohexane over 30 min. The required fractions were combined and evaporated in vacuo to afford the title compound as a pale brown oil (30.96 g). LCMS RT = 2.95 min, ES+ve m/z 516/518 [M+H] ⁺.

Intermediate 10

4-[(4-Chlorophenyl)methyl]-2-({(2/?)-1 -[4-(4-hydroxyphenyl)butyl]-2- pyrrolidinyl}methyl)-1(2H)-phthalazinone To a cooled -60 ⁰C solution of 4-[(4-chlorophenyl)methyl]-2-[((2R)-1-{4-[4- (methyloxy)phenyl]butyl}-2-pyrrolidinyl)methyl]-1 (2H)-phthalazinone (for example, as prepared for Intermediate 9) (24.35 g, 47 mmol) in dry dichloromethane (100 ml) under nitrogen was added, dropwise, a 1.0 M solution of boron tribromide in dichloromethane (52 ml, 52 mmol). The reaction mixture was allowed to warm to room temperature and stirred under nitrogen for 18 h. The reaction mixture was cooled in an ice/water bath and then quenched using 2 N hydrochloric acid (50 ml). The reaction mixture was basified using saturated sodium bicarbonate and extracted using dichloromethane (500 ml). The separated organic phase was dried over anhydrous magnesium sulfate and evaporated in vacuo to afford the title compound as an orange foam (22.04 g). LCMS RT = 2.80 min, ES+ve m/z 502/504 [M+H] ⁺.

Intermediate 11

4-[(4-Chlorophenyl)methyl]-2-{[C2/?;-1-(4-{4-[(3-chloropropyl)oxy]phenyl}butyl)-2- pyrrolidinyl]methyl}-1(2H)-phthalazinone To a solution of 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-hydroxyphenyl)butyl]-2- pyrrolidinyl}methyl)-1 (2H)-phthalazinone (for example, as prepared for Intermediate 10) (22.03 g, 44 mmol) in 2-butanone (220 ml) under nitrogen was added 1-bromo-3- chloropropane (commercially available, for example, from Aldrich) (5.2 ml, 53 mmol) and potassium carbonate (12.2 g, 88 mmol). The reaction mixture was heated at reflux under nitrogen for 18 h. The solid was removed by filtration and washed with 2-butanone (200 ml). The combined filtrate and washings were evaporated in vacuo and the residue was dissolved in dichloromethane (60 ml). This was applied to a silica cartridge (330 g) and eluted with a gradient of 0 - 25% methanol in dichloromethane over 12 column volumes. The required fractions were evaporated in vacuo. A portion of the residue (19.65 g) was dissolved in 2-butanone (200 ml) under nitrogen and to this was added 1-bromo-3- chloropropane (4.65 ml, 47 mmol) and potassium carbonate (10.8 g, 78.4 mmol). The reaction mixture was heated at reflux under nitrogen for 18 h. More 1-bromo-3- chloropropane (1 ml) was added and the reaction mixture was heated at reflux for a further 5 h. The solid was removed by filtration and washed with 2-butanone (3 x 100 ml). The combined filtrate and washings were evaporated in vacuo and the residue was dissolved in dichloromethane (50 ml). This was applied to a silica cartridge (330 g) and eluted with a gradient of 0 - 10% methanol in dichloromethane over 12 column volumes. The required fractions were evaporated in vacuo to afford the title compound as a brown oil (21.68 g). LCMS RT = 3.18 min, ES+ve m/z 578/580 [M+H] ⁺.

4-[(4-Chlorophenyl)methyl]-2-({(2/?)-1 -[4-(4-{[3-(hexahydro-1 H-azepin-1 - yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1(2H)-phthalazinone free base

Method 1 To a solution of 4-[(4-chlorophenyl)methyl]-2-{[(2R)-1-(4-{4-[(3- chloropropyl)oxy]phenyl}butyl)-2-pyrrolidinyl]methyl}-1 (2H)-phthalazinone (for example, as prepared for Intermediate 1 1 ) (20 g, 34.6 mmol) in 2-butanone (200 ml) under nitrogen was added potassium iodide (11.5 g, 69.2 mmol), potassium carbonate (9.6 g, 69.2 mmol) and hexamethylene imine (commercially available, for example, from Aldrich) (7.8 ml, 69.2 mmol). The reaction mixture was heated at reflux for 41 h. The solid was removed by filtration and washed with 2-butanone (2 x 100 ml). The combined filtrate and washings were evaporated in vacuo and the residue was dissolved in methanol-dimethylsulfoxide (30 ml, 1 :1 ). This was applied to a C18 reverse phase cartridge (2 x 330 g). This was eluted using a gradient of 0 - 50% acetonitrile (0.05% trifluoroacetic acid) in water (0.05% trifluoroacetic acid) over 12 column volumes. The required fractions were evaporated in vacuo and the residue was dissolved in methanol. This was applied to amino propyl cartridges (4 x 70 g) and eluted with methanol. The required fractions were evaporated in vacuo to afford the title compound as an orange gum (10.74 g). LCMS RT = 2.67 min, ES+ve m/z 641/643 [M+H]⁺. ¹H NMR (400 MHz, CDCI₃) δ 8.46 (m, 1 H), 7.74 - 7.62 (m, 3H), 7.26 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 4.42 (dd, J = 4,13 Hz, 1 H), 4.24 (s, 2H), 4.07 (dd, J = 8,13 Hz, 1 H), 3.98 (t, J = 6.3 Hz, 2H), 3.16 (m, 1 H), 2.97 (m, 1 H), 2.90 (m, 1 H), 2.65 (m, 6H), 2.55 (m, 2H), 2.37 (m, 1 H), 2.21 (m, 1 H), 1.93 (m, 2H), 1.89 - 1.52 (m, 16H).

Method 2

A mixture of 4-[(4-chlorophenyl)methyl]-2-[(2/?)-2-pyrrolidinylmethyl]-1 (2H)-phthalazinone (for example, as prepared for Intermediate 2) (1.017 g, 2.87 mmol), 4-(4-{[3-(hexahydro- 1 H-azepin-1-yl)propyl]oxy}phenyl)butyl methanesulfonate (for example, as prepared for Intermediate 7) (1.1 15 g, 2.91 mmol) and sodium bicarbonate (474 mg, 5.64 mmol) in dry acetonitrile (50 ml) was heated at 80 ⁰C with stirring for 5 days under a nitrogen atmosphere. The cooled reaction mixture was partitioned between water (70 ml) and ethyl acetate (70 ml). The aqueous layer was washed with further ethyl acetate (2 x 50 ml). The combined organic extracts were dried (magnesium sulfate), and concentrated in vacuo. The residue (1.35 g) was dissolved in Λ/,Λ/-dimethylformamide (10 ml), and divided into ten portions. Each was diluted with trifluoroacetic acid (0.5 ml). Each portion was purified by preparative HPLC, using a Kromasil C8 column (25 cm x 5 cm), eluting with a gradient of 5% to 45% of (0.25% trifluoroacetic acid in acetonitrile) in (0.25% trifluoroacetic acid in water) over 40 min, followed by holding the final concentration for a further 15 mins. The relevant fractions from each run were combined and concentrated in vacuo, to leave an aqueous solution. This was applied to an Amberchrom CG-161 M column (25 cm x 2.5 cm) to adsorb the compound. The column was washed with water to remove excess trifluoroacetic acid and eluted with acetonitrile, to afford the product as the trifluoroacetate salt. An SCX cartridge (20 g) was washed with methanol, then with acetonitrile. A portion of the above product (0.98 g) was dissolved in acetonitrile and applied to the SCX cartridge, eluting with acetonitrile, and then a solution of 10% aqueous ammonia in acetonitrile (200 ml). The appropriate fractions were concentrated in vacuo to give the title compound as an orange gum (651 mg). LCMS RT = 2.52 min, ES+ve m/z 641 [M+H]⁺ and 321/322 [M/2 +H]⁺. ¹H NMR (400 MHz, DMSOd₆) δ ppm 8.38 (dd, J = 7.7, 1.6 Hz, 1 H), 7.93 (m, 1 H), 7.86 (m, 1 H), 7.82 (m, 1 H), 7.30 (m, 4 H), 7.03 (d, J = 8.5 Hz, 2 H), 6.80 (d, J = 8.5 Hz, 2 H), 4.36 (m, 1 H), 4.33 (s, 2 H), 4.14 (dd, J = 13.1 , 8.0 Hz, 1 H), 3.98 (t, J = 6.1 Hz, 2 H), 3.14 (m, 1 H), 3.03 (dd, J = 7.8, 4.5 Hz, 1 H), 2.84 (m, 1 H), 2.75 (m, 6 H), 2.50 (t, J = 6.9 Hz, 2 H), 2.31 (m, 2 H), 1.97 (m, 2 H), 1.82 (m, 4 H), 1.68 (m, 8 H), 1.55 (m, 4 H).

4-[(4-Chlorophenyl)methyl]-2-({(2/?)-1 -[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl] oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1(2H)-phthalazinone, dihydrochloride salt 4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone (3.85 g, 6.0 mmol) was dissolved in MeOH (100 ml) and 2N hydrochloric acid (12 ml, 24 mmol). The solvent was removed in vacuo. The residue was dissolved in MeOH (50 ml) and then evaporated. This was repeated 3 times. The residue was dried in vacuo to give the title compound (4.3 g, 100%) as a crunchy foam. LCMS RT = 3.41 min, ES+ve m/z 641 (M+H)⁺.¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (1 H, br s), 10.49 (1 H, br s), 8.30 (1 H, dd, J = 7.5, 1.5 Hz), 7.96 (1 H, d, J = 7.5 Hz), 7.88-7.93 (1 H, m), 7.84-7.89 (1 H, m), 7.38 (2H, d, J = 8.5 Hz), 7.34 (2H, d, J = 8.5 Hz), 7.09 (2H, d, J = 8.5 Hz), 6.84 (2H, d, J = 8.5 Hz), 4.62 (1 H, dd, J = 14.0, 4.5 Hz), 4.55 (1 H, dd, J = 14.0, 7.0 Hz), 4.37 (1 H, d, J = 16.5 Hz), 4.33 (1 H, d, J = 16.5 Hz), 4.00 (2H, t, J = 6.0 Hz), 3.77-3.85 (1 H, m), 3.55-3.64 (1 H, m), 3.31-3.46 (3H, m), 3.15-3.22 (2H, m), 3.02-3.14 (4H, m), 2.47-2.53 (2H, m), 2.07-2.23 (4H, m), 1.49-1.99 (14H, m).

4-[(4-Chlorophenyl)methyl]-2-({(2/?)-1 -[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl] oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone, 1 ,5-naphthalene disulfonate monohydrate salt

Method 1

4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone free base (400 mg) was dissolved in methanol (4.44 ml). A solution of 1 ,5-naphthalene disulfonic acid (232 mg) in methanol (1 ml) was added and the resulting gummy solution was heated with an air gun. Small amounts of solid began to form and on cooling a solid precipitated. The slurry was stirred at room temperature for approximately 1 h. methanol (2 ml) was added to mobilise the slurry, which was heated and cooled again, and left to stir for a further hour. The solid was isolated by filtration and dried in vacuo at 40 ⁰C to give the title compound (464.5 mg, 73%).

Method 2 To 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone free base (3.82 g, 5.96 mmol) was added water (200 ml) and hydrochloric acid (2 N, 12 ml). The reaction mixture was heated to 90 ⁰C to obtain a clear solution. To this was added a solution of 1 ,5- naphthalenedisulfonic acid monohydrate (2.2 g, 6 mmol) in water (100 ml) over 20 min. The suspension was stirred at 90 ⁰C for 20 min and then allowed to cool to room temperature. The solid was collected by filtration and washed with water (100 ml). The solid was air-dried for 3 days and then in vacuo for 20 h to give the title compound (5.1 g, 92%) as a white solid. LCMS RT = 2.58 min, ES+ve m/z 641 (M+H)⁺.

Method 3 (Scheme 1 above)

For this method the following abbreviations are used: eq: equivalent (1 eq = 1 mole reagent per 1 mole of starting material) vol: volume (1 vol = 1 ml per gram starting material) vol/vol: volume/volume wt: weight (1 wt = 1 g reagent per 1 g starting material) wt/vol: weight/volume

Intermediate 12 (Stage 1)

(3E)-3-[(4-Chlorophenyl) methylidene]-2-benzofuran-1(3H)-one

4-Chlorophenylacetic acid (commercially available, for example, from Aldrich) (1.00 eq), phthalic anhydride (commercially available, for example, from Aldrich) (1.10 eq) and sodium acetate (0.04 eq) are mixed in Λ/-methyl pyrrolidinone (3 vol). The resulting suspension is heated to approximately 200 ⁰C and the resulting brown solution is stirred over 2 days. During the reaction Λ/-methyl pyrrolidinone/water (0.45 vol) is distilled off at ambient pressure. After checking complete conversion (99%, HPLC) the reaction mixture is cooled to approximately 70 ⁰C over 1 h and ethanol (4.5 vol) is added over 1 h at approximately 70 ⁰C. The resulting brown solution is cooled to approximately 50 ⁰C over 1.5 h during which it turns into a brown suspension. At approximately 50 ⁰C, ethanol (3.8 vol) is added over 1 h and the resulting brown suspension is cooled to approximately 2 ⁰C over 1 h and is stirred at approximately 0-5 ⁰C for 1 h. The brown solid is isolated by filtration through a suction strainer, washed with cold aqueous ethanol (ethanol/water = 1/1 , vol/vol, approximately 2 ⁰C, 3 x 1 vol) and dried on a suction strainer under nitrogen. The product is obtained as a pale brown and humid solid. The loss on drying is determined and the material is taken into the next stage. Yield (corrected for loss on drying and ¹H NMR assay): 80%.

¹H NMR (400MHz, CDCI₃), δ 6.37 (s, 1 H), 7.38 (d, 2H), 7.58 (t, 1 H), 7.77 (m, 4H), 7.95 (d, 1 H)

Intermediate 13 (Stage 2)

4-{[4-Chlorophenyl]methyl}-1(2H)-phthalazinone

(3£)-3-[(4-Chlorophenyl)methylidene]-2-benzofuran-1 (3H)-one (as prepared, for example, in stage 1 ) (1.0 eq, corrected for loss on drying) is suspended in ethanol (3.7 vol) and heated to approximately 85 ⁰C at slight reflux. A solution of hydrazine hydrate (commercially available, for example, from Aldrich) (1.2 eq) in ethanol (0.63 vol) is added through a dropping funnel over 1 h. At the end of the addition, ethanol (0.63 vol) is added through the dropping funnel into the reaction suspension in order to remove traces of hydrazine hydrate. The reaction suspension is heated at approximately 85 ⁰C at slight reflux for 14 h. It is cooled to approximately 20 ⁰C and a sample is taken to check the conversion (99% conversion, HPLC). Acetone (0.35 vol) is added to the reaction mixture over 30 min (exothermic reaction). The quenched suspension is stirred for at least 1 h and is then cooled to approximately 2 ⁰C over 30 min and stirred at approximately 2 ⁰C for 1 h. The product is isolated by filtration through a suction strainer and is washed with cold ethanol (approximately 0-5 ⁰C, 3 x 1.9 vol). The pale brown solid is completely dried on the suction strainer in vacuo under nitrogen. The title compound is obtained as a pale brown solid. Yield (corrected for ¹H NMR assay): 90-95%.

¹H NMR (400MHz, DMSOd₆), δ 4.30 (s, 2H), 7.35 (m, 4H), 7.88 (m, 3H), 8.26 (d, 1 H), 12.62 (s, 1 H)

Intermediate 14 (Stage 3a)

1,1 -Dimethylethyl 2-{[(methylsulfonyl)oxy]methyl}-1 -pyrrolidine carboxylate

A solution of Λ/-Boc-D-prolinol (commercially available, for example, from Aldrich) (1.0 eq) in methyl /so-butyl ketone (9.5 vol) is cooled to approximately 2 ⁰C and triethylamine (1.03 vol) is added. Methanesulfonyl chloride (1.2 eq) is added through a dropping funnel over 1 h and a white suspension forms. The dropping funnel is washed with additional methyl /so-butyl ketone (0.5 vol). The reaction mixture is warmed to approximately 22 ⁰C and stirred for 2 h. A sample is taken to check the conversion (complete conversion by TLC). Water (5.0 vol) is added. The phases are separated (good and quick phase separation). The organic phase is washed with saturated aqueous sodium hydrogen carbonate (5.0 vol) and finally with water (5.0 vol) (good and quick phase separations). The organic phase is dried by filtering over a suction strainer filled with magnesium sulfate (0.46 wt). The volume of the dried organic phase is determined (12.40 vol). The organic phase is concentrated to 43% wt/vol (based on Λ/-Boc-D-prolinol/solution) by distillation in vacuo at approximately 40 ⁰C to the final volume (2.20 vol). A loss on drying sample is taken and evaporated to dryness (approximately 40 ⁰C, <100 mbar); forming a yellow oil which is taken for analysis. The concentrated yellow organic phase (2.0 vol) is used immediately in the alkylation reaction. Yield (corrected for loss on drying and ¹H NMR assay): 100%.

Intermediate 2 (Stage 3b)

4-[(4-Chlorophenyl)methyl]-2-[(2/?)-2-pyrrolidinylmethyl]-1(2H)-phthalazinone, hydrochloride salt

A suspension of 4-{[4-chlorophenyl]methyl}-1 (2H)-phthalazinone (as prepared, for example, in stage 2) (1.0 eq) and cesium carbonate (2.5 eq) in methyl /so-butyl ketone (9.7 vol) is heated to approximately 100 ⁰C. A freshly prepared solution of 1 ,1- dimethylethyl 2-{[(methylsulfonyl)oxy]methyl}-1 -pyrrolidine carboxylate (as prepared, for example, in stage 3a) in methyl /so-butyl ketone (1.2 eq calculated for Λ/-Boc-D-prolinol) is added dropwise over 2 h at approximately 100 ⁰C. The dropping funnel is washed with methyl /so-butyl ketone (0.2 vol) which is added to the reaction mixture. The reaction mixture is stirred for 17 h at approximately 100 ⁰C. A brown suspension is formed. After cooling to approximately 50 ⁰C, a sample is taken to check the conversion (99% conversion, HPLC). The reaction mixture is cooled to approximately 22 ⁰C and water (16.7 vol) is added to the reaction mixture, followed by the addition of methyl /so-butyl ketone (16.7 vol). The phases are separated. The volume of the organic phase (18.8 vol) is determined and it is concentrated to 50% w/vol (4-{[4-chlorophenyl]methyl}-1 (2H)- phthalazinone/solution) by distillation in vacuo (approximately 45 ⁰C, < 100 mbar). Hydrogen chloride in /so-propyl alcohol (5-6 M, 3 eq, 2.0 vol) is added to the concentrated organic phase at approximately 22 ⁰C. The formation of gas is observed and a pale brown suspension forms over about 1 h. The reaction mixture is stirred at approximately 22 ⁰C for 14 h. A sample is taken to check the conversion (complete conversion, HPLC). The pale brown suspension is cooled to approximately 1 ⁰C over 2 h and the product is isolated by filtration through a fritted funnel, and is washed with cold methyl /so-butyl ketone (3 x 1 vol). A white solid results, which is dried on the suction strainer and subsequently in vacuo (45 ⁰C, < 20 mbar). The title compound (as hydrogen chloride salt) is obtained as a white solid. Yield (corrected for ¹H NMR assay): 86%. 1H NMR (400MHz, DMSOd₆), δ 1.76 (m, 1 H), 1.95 (m, 2H), 2.14 (m, 1 H), 3.15 (m, 1 H), 3.27 (m, 1 H), 3.91 (m, 1 H), 4.36 (d, 2H), 4.47 (m, 2H), 7.35 (d, 2H), 7.41 (d, 2H), 7.80- 8.00 (m, 3H), 8.31 (d, 1 H), 8.92 (bs, 1 H), 9.48 (bs, 1 H)

Intermediate 8 (Stage 4a) 4-[4-(Methyloxy)phenyl]butyl methanesulfonate

To a solution of 4-(4-methoxyphenyl)-1-butanol (commercially available, for example, from Aldrich) (1.0 eq) in methyl /so-butyl ketone (9.5 vol) is added triethylamine (1.5 eq, 1.16 vol) at approximately 21 ⁰C. The resulting solution is cooled to approximately 10-15 ⁰C and MsCI (1.2 eq, 0.52 vol) is added over 1 h, keeping the temperature at approximately 16 ⁰C. A white suspension forms immediately. At the end of the addition, the dropping funnel is washed with methyl /so-butyl ketone (0.5 vol) which is transferred into the reaction flask. The reaction mixture is warmed to approximately 22 ⁰C over 3 h and stirred at approximately 22 ⁰C for 15 h. A pale yellow suspension is present. A sample is taken to check the conversion (complete conversion, HPLC). The reaction mixture is cooled to approximately 10-15 ⁰C and water (5.6 vol) is added, keeping the temperature below approximately 18 ⁰C. The emulsion is stirred over 10 min at approximately 22°C. The phases are separated. The organic phase is washed with saturated aqueous NaHCO₃ (5.6 vol) and finally with water (5.6 vol). The organic phase is dried by filtering over a suction strainer filled with magnesium sulfate (0.5 wt) and the magnesium sulfate is washed with methyl /so-butyl ketone (2 x 0.2 vol). The volume of the dried organic phase is determined (12.40 vol). The organic phase is concentrated to 40% w/vol (4-(4- methoxyphenyl)-1-butanol /solution) by distillation in vacuo at approximately 45 ⁰C to 2.20 vol. A sample for loss on drying is taken and evaporated to dryness (approximately 40 ⁰C, < 100 mbar); a yellow oil remains which is taken for analysis. The product containing yellow organic phase (2.5 vol) is used in the subsequent alkylation reaction (stage 4b). Yield (corrected for loss on drying and ¹H NMR assay): 101%.

¹H NMR (DMSOd₆) δ 1.53-1.71 (m, 4H), 2.52-2.57 (m, 2H), 3.11-3.20 (s, 3H), 3.68-3-76 (s, 3H), 4.15-4.26 (m, 2H), 6.81-6.87 (m, 2H), 7.08-7.15 (m, 2H)

Intermediate 9 (Stage 4b)

^[(^ChlorophenylJmethyll^-I^PJ-i^-^-tmethyloxyJphenyllbuty^^-pyrrolidinyl) methyl]-1 (2H)-phthalazinone

4-[(4-Chlorophenyl)methyl]-2-[(2/?)-2-pyrrolidinylmethyl]-1 (2H)-phthalazinone, hydrogen chloride salt (as prepared for example, in stage 3b) (1.0 eq) and potassium carbonate (5.0 eq, 1.77 wt) are mixed in methyl /so-butyl ketone (16.5 vol) and the resulting light brown suspension is heated to approximately 135 ⁰C at reflux. A clear orange solution of 4-[4- (methyloxy)phenyl]butyl methanesulfonate (as prepared for example in stage 4a) (2.4 eq, 1.59 wt) in methyl /so-butyl ketone (4.9 vol) is added at reflux over 1 h. The resulting yellow-brown suspension is stirred at reflux for 20 h. A sample is taken to check the conversion (88.5% conversion, HPLC). Water (24.7 vol) is added at approximately 19 ⁰C over 5 min (slightly exothermic). The turbid orange brown mixture thus formed is stirred for 15 min at approximately 20 ⁰C. The phases are separated. The organic phase is dried by filtration through a suction strainer filled with magnesium sulfate (0.92 wt); the magnesium sulfate is washed with methyl /so-butyl ketone (2 x 4.1 vol). The solvent of the resulting organic phase is completely removed in vacuo (approximately 40-45 ⁰C, 600 mbar to full suction). The obtained crude product (2.45 wt, dark brown oil, HPLC purity: 73.50% area/area) is combined with a crude product obtained in the same manner (2.41 wt) and purified by plug filtration (SiO₂). Therefore, the combined crude material (4.86 wt) is dissolved in dichloromethane and put on a suction strainer filled with SiO₂ (45.7 wt, height: 24.5 cm, diameter: 30 cm) to wash out impurities with dichloromethane (823 vol). The eluent is gradually changed from dichloromethane only to dichloromethane:methanol = 10:1 in order to elute the title compound. The product is obtained in solution (494 vol). Removal of the solvents by distillation in vacuo (45 ⁰C, 600 mbar to full suction) results in the title compound (2.46 wt) as a pale brown oil. Yield (corrected for ¹H NMR assay): 86% ¹H NMR (DMSO-de) δ 1.32-1.53 (m, 4H), 1.61-1.79 (m, 4H), 2.08-2.18 (m, 1 H), 2.20-2.27 (m, 1 H), 2.37-2.45 (m, 2H), 2.66-2.76 (m, 1 H), 2.84-2.93 (m, 1 H), 2.96-3.04 (m, 1 H), 3.69- 3.71 (m, 3H), 3.89-3.98 (m, 1 H), 4.18-4.26 (m, 1 H), 4.28-4.36 (m, 2H), 6.77-6.83 (m, 2H), 6.98-7.04 (m, 2H), 7.33-7.39 (m, 4H), 7.79-7.90 (m, 2H), 7.91-7.97 (m, 1 H), 8.26-8.31 (m, 1 H).

Intermediate 10 (Stage 5) ^[(^ChlorophenylJmethyll^-^PJ-i-μ^-hydroxyphenyObutyll^-pyrrolidinyl} methyl)-1 (2H)-phthalazinone

A solution of 4-[(4-chlorophenyl)methyl]-2-[((2R)-1-{4-[4-(methyloxy)phenyl]butyl}-2- pyrrolidinyl)methyl]-1 (2/-/)-phthalazinone (as prepared, for example, in stage 4b) (1.0 eq) in dichloromethane (4.2 vol) is cooled to approximately 0 ⁰C. A solution of boron tribromide (1.8 eq, 0.33 vol) in dichloromethane (3.4 vol) is added over 20 min keeping the temperature below approximately 2 ⁰C. The reaction mixture is stirred overnight at approximately 20 ⁰C. A sample is taken to check the conversion (90% conversion, HPLC). Additional boron tribromide (0.2 eq, 0.05 vol) is added at approximately -1 ⁰C over 10 min. The reaction mixture is warmed to approximately 20 ⁰C. After approximately 5 h, another sample is taken to check the conversion (96% conversion, HPLC). Additional boron tribromide (0.2 eq, 0.05 vol) is added and the reaction is stirred at approximately 25 ⁰C overnight and another sample is taken to check the conversion (> 99% conversion, HPLC). The reaction mixture is cooled to approximately 15 ⁰C and aqueous hydrogen chloride (2 N, 2.4 vol) is added dropwise over 15 min keeping the temperature below about 19 ⁰C. After approximately 2/3 of the addition of hydrogen chloride, exothermic reaction behaviour is observed. After complete addition, a brown suspension is formed which contains some brown, oily material. Aqueous saturated sodium hydrogen carbonate (5.1 vol) is slowly added over 20 min at approximately 11 ⁰C, keeping the temperature below about 13 ⁰C. A dark, slightly turbid emulsion is formed. The reaction mixture is warmed to approximately 20 ⁰C over 15 min and the phases are separated. The aqueous phase is back extracted with dichloromethane (4.28 vol). The combined organic phases are dried by filtration over a suction strainer filled with magnesium sulfate (0.69 wt), then the magnesium sulfate is washed with dichloromethane (3 x 1.7 vol). The dried organic phase is dark and clear. During removal of the solvent in vacuo (600 mbar - full suction, 35-40 ⁰C), a brown foam forms. The obtained brown solid (HPLC purity: 73.22% area/area) contains residual dichloromethane and is dried again over weekend (35 ⁰C, <20 mbar). The redried material displays decreased HPLC purity (63.98% area/area). The crude is divided into 2 equal portions (2 x 0.50 wt) which are purified by column chromatography (2 columns; SiO₂ (2 x 2.74 wt); height = 20.5 cm, diameter = 14 cm; dichloromethane:methanol = 20:1 ). The fractions containing product are combined and concentrated in vacuo (approximately 35 ⁰C, 600 mbar to full suction). The title compound is obtained as a light brown foamy solid. Yield (corrected for ¹H NMR assay): 65%. 1H NMR (DMSOd₆) δ 1.39-1.72 (m, 4H), 1.82-2.05 (m, 3H), 2.12-2.24 (m, 1 H), 2.35-2.48 (m, 2H), 3.00-3.25 (m, 2H,), 3.26-3.54 (m, 1 H), 3.56-3.71 (m, 1 H), 3.80-3.97 (m, 1 H), 4.25-4.43 (m, 2H), 4.49-4.62 (m, 2H), 6.58-6.74 (m, 2H), 6.91-7.03 (m, 2H), 7.27-7.48 (m, 4H), 7.79-8.04 (m, 3H), 8.23-8.39 (m, 1 H), 9.06-9.37 b, 2H)

Intermediate 11 (Stage 6) 4-[(4-Chlorophenyl)methyl]-2-{[(2/?)-1 -(4-{4-[(3-chloropropyl) oxy]phenyl}butyl)-2- pyrrolidinyl]methyl}-1(2H)-phthalazinone

4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-hydroxyphenyl)butyl]-2-pyrrolidinyl}methyl)- 1 (2H)-phthalazinone (as prepared, for example, in stage 5) (1.0 eq) and potassium carbonate (4.0 eq, 1.1 wt) are mixed in 2-butanone (7.1 vol) at approximately 20 ⁰C. To the resulting brown suspension, is added a solution of 1-bromo-3-chloropropane (2.0 eq, 0.40 vol) in 2-butanone (2.9 vol). The brown mixture is heated to reflux for 25 h. A sample is taken to check the conversion (98% conversion, HPLC). At approximately 20 ⁰C, water (14.8 vol) is added over 5 min (slightly exothermic). Methyl /so-butyl ketone (14.8 vol) is added and the orange mixture is stirred for 25 min; the phases are separated. The organic phase is dried by filtration over a suction strainer filled with sodium sulfate (1.90 wt) and the sodium sulfate is washed with methyl /so-butyl ketone (2 x 2.4 vol). Evaporation of the solvents in vacuo (40 ⁰C, 600 mbar - full suction) results in the title compound (as a mixture of chloro- and bromo-derivative) as a brown oil. Yield (corrected for HPLC purity): 91 %.

¹H NMR (CDCI₃) δ 1.47-1.91 (m, 6H), 2.16-2.27 (m, 3H), 2.33-2.43 (m, 1 H), 2.51-2.60 (m, 2H), 2.80-3.04 (m, 2H), 3.08-3.23 (m, 1 H), 3.47-3.86 (m, 3H), 4.01-4.14 (m, 3H), 4.18- 4.30 (m, 2H), 4.36-4.50 (m, 1 H), 6.74-6.88 (m, 2H), 7.01-7.14 (m, 2H), 7.15-7.34 (m, 4H), 7.57-7.77 (m, 3H), 8.37-8.52 (m, 1 H). Stage 7

4-[(4-Chlorophenyl)methyl]-2-({(2/?)-1 -[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl] oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1(2H)-phthalazinone, free base

4-[(4-Chlorophenyl)methyl]-2-{[(2R)-1-(4-{4-[(3-chloropropyl)oxy]phenyl}butyl)-2- pyrrolidinyl]methyl}-1 (2H)-phthalazinone (as prepared, for example, in stage 6) (1.0 eq), potassium iodide (3.0 eq, 0.86 wt), potassium carbonate (3.0 eq, 0.72 wt) and hexamethyleneimine (commercially available, for example, from Aldrich) (3.0 eq, 0.59 vol) are mixed in methyl /so-butyl ketone (10.9 vol) and the resulting brown suspension is heated at reflux for 18 h. A sample is taken to check the conversion (complete conversion, HPLC). The light brown suspension is cooled to approximately 30 ⁰C and water (6.9 vol) is added over 5 min. After stirring for 20 min the phases are separated. The aqueous phase is back extracted with methyl /so-butyl ketone (3.96 vol). Removal of the solvents in vacuo (40-50 ⁰C, 600 mbar - full suction) results in the title compound as a brown oil. Yield (corrected for ¹H NMR assay): 83%.

¹H NMR (CDCI₃) δ 1.41-2.05 (m, 14H), 2.13-2.27 (m, 1 H), 2.29-2.44 (m, 1 H), 2.47-2.72 (m, 6H), 2.81-3.02 (m, 2H), 3.08-3.22 (m, 1 H), 3.92-4.1 1 (m, 3H), 4.20-4.28 (m, 2H), 4.34- 4-49 (m, 1 H), 6.72-6.87 (m, 2H), 6.96-7.12 (m, 2H), 7.14-7.31 (m, 4H), 7.59-7.77 (m, 3H), 8.38-8.52 (m, 1 H).

Stage 8

4-[(4-Chlorophenyl)methyl]-2-({(2/?)-1 -[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl] oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1(2H)-phthalazinone, 1,5-naphthalene disulfonate monohydrate salt

Preparation 1 :

An orange solution of 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H- azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone (as prepared, for example, in stage 7) (1.0 eq) in methanol (41.41 vol) is cooled to approximately 15 ⁰C. Aqueous hydrogen chloride (2 N, 41.4 vol) is added over 20 min while keeping the temperature below about 18 ⁰C. The solvents are distilled off (approximately 80 ⁰C, 600 mbar to full suction) and an orange-brown oil remains, which is dissolved in water (32.1 vol). The resulting orange-brown, slightly turbid solution is heated to approximately 100 ⁰C (reflux) and methanol (61.1 vol) is added. To the resulting yellow solution, a solution of 1 ,5-naphthalenedisulfonic acid tetrahydrate (1.0 eq, 0.57 wt) in water (6.0 vol) is added over 2 min. The solution remains yellow and is cooled from approximately 58 ⁰C to approximately 20 ⁰C over 90 min. A white suspension forms, which is filtered through a suction strainer at approximately 20 ⁰C. The solid is washed with aqueous methanol (methanokwater = 1 :1 , 2 x 10.4 vol) and the recovered pale brown material is dried in vacuo (approximately 50 ⁰C, full suction). The title compound is obtained as a pale brown solid. Yield (corrected for HPLC purity): 69%.

Preparation 2:

A brown solution of 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1H-azepin- 1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2/-/)-phthalazinone (as prepared, for example, in stage 7) (1.0 eq) in methanol (39.9 vol) is cooled to approximately 15 ⁰C. Aqueous hydrogen chloride (2 N, 42.6 wt) is added over 20 min keeping the temperature below about 18 ⁰C. The solvents are distilled off (approximately 80 ⁰C, 600 mbar to full suction) and an orange-brown oil remains which is dissolved in water (32.9 vol). The resulting orange-brown, slightly turbid solution is washed with ethyl acetate (1 x 41.2 vol, 1 x 39.5 vol) and a white emulsion forms, which separates into two phases. The inorganic phase is evaporated to dryness and an orange-brown oil remains. The oil is dissolved in methanol (79.9 vol) and the resulting orange-brown solution is heated at approximately 90 ⁰C at reflux. To the solution is added a solution of 1 ,5-naphthalenedisulfonic acid tetrahydrate (1.0 eq, 0.56 wt) in water (4.8 vol) over 2 min. The solution remains clear and is cooled to approximately 2 ⁰C over 100 min. A white solid suspension results, which is stirred for 30 min at approximately 2 ⁰C. The solid is isolated by filtration (good) and is washed with cold aqueous methanol (3 x 1 1.0 vol). The brown solid is dried in vacuo (50 ⁰C, 10 mbar, 18 h). The pale brown material contains grains and is crushed mechanically. The title compound is obtained as a pale brown solid. Total recovery (corrected for HPLC purity): 74%.

¹H NMR (400MHz, DMSOd₆), δ 1.30-2.28 (m, 18H), 3.13 (m, 4H), 3.20 (m, 2H), 3.27-3.53 (m, 9H), 3.62 (m, 1 H), 3.85 (m, 1 H), 3.94 (m, 2H), 4.31 (m, 2H), 4.55 (d, 2H), 6.82 (m, 2H), 7.05 (d, 2H), 7.29-7.51 (m, 6H), 7.79-8.05 (m, 5H), 8.31 (m, 1 H), 8.86 (d, 2H), 9.15 (m, 2H)

Biological Data

4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone and various salts have been tested for in vitro and/or in vivo 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 is cloned using known procedures described in the literature [Biochem. Biophys. Res. Commun., 201 (2):894 (1994)]. Chinese hamster ovary (CHO) cells stably expressing the human H1 receptor are generated according to known procedures described in the literature [Br. J. Pharmacol., 1 17(6):1071 (1996)].

Histamine H1 functional antagonist assay: Determination of functional pKi values

The histamine H1 cell line is seeded into non-coated black-walled clear bottom 384-well tissue culture plates in alpha minimum essential medium (Gibco/lnvitrogen, 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 is maintained overnight at 5% CO₂, 37 ⁰C.

Excess medium is removed from each well to leave 10 μl. 30 μl loading dye (250 μM Brilliant Black, 2 μM Fluo-4 diluted in Tyrodes buffer + probenecid (145 mM NaCI, 2.5 mM KCI, 10 mM HEPES, 10 mM D-glucose, 1.2 mM MgCI₂, 1.5 mM CaCI₂, 2.5 mM probenecid, pH adjusted to 7.40 with NaOH 1.0 M)) is added to each well and the plates are incubated for 60 min at 5% CO_2, 37 ⁰C. 10 μl of test compound, diluted to the required concentration in Tyrodes buffer + probenecid (or 10 μl Tyrodes buffer + probenecid as a control) is added to each well and the plate is incubated for 30 min at 37 ⁰C, 5% CO₂. The plates are then placed into a FLI PR™ (Molecular Devices, UK) to monitor cell fluorescence (λ_ex = 488 nm, A_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 10 μl 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 FLI PR™ system (Molecular Devices). By means of concentration effect curves, functional affinities are determined using standard pharmacological mathematical analysis.

Histamine H1 functional antagonist assay: Determination of antagonist pA2 and duration

The histamine H1 receptor expressing CHO cells are seeded into non-coated black-walled clear bottom 96-well tissue culture plates as described above.

Following overnight culture, growth medium is removed from each well, washed with 200 μl phosphate buffered saline (PBS) and is replaced with 50 μl loading dye (250 μM Brilliant Black, 1 μM Fluo-4 diluted in Tyrodes buffer + probenecid (145 mM NaCI, 2.5 mM KCI, 1OmM HEPES, 1 OmM D-glucose, 1.2 mM MgCI₂, 1.5 mM CaCI₂, 2.5 mM probenecid, pH adjusted to 7.40 with NaOH 1.0 M)). Cells are incubated for 45 min at 37 ⁰C. The loading buffer is removed and the cells are washed as above, and 90 μl of Tyrodes buffer + probenecid is added to each well. 10 μl of test compound, diluted to the required concentration in Tyrodes buffer + probenecid (or 10 μl Tyrodes buffer + probenecid as a control) is added to each well and the plate is incubated for 30 min at 37 ⁰C, 5% CO₂.

The plates are then placed into a FLIPR™ (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 50 μl histamine over a concentration range of 1 mM - 0.1 nM. The resultant concentration response curves are analysed by non-linear regression using a standard four parameter logistic equation to determine the histamine EC₅₀, the concentration of histamine required to produce a response of 50% of the maximum response to histamine. The antagonist pA2 is calculated using the following standard equation: pA2 = log(DR-1 )-log[B] where DR = dose ratio, defined as ECsoantagonist-treated/ECsoControl and [B] = concentration of antagonist.

To determine the antagonist duration, cells are cultured overnight in non-coated black- walled clear bottom 96-well tissue culture plates, are washed with PBS and are incubated with a concentration of antagonist chosen to give an approximate DR in the range 30 - 300. Following the 30 min antagonist incubation period, the cells are washed two or three times with 200 μl of PBS and then 100 μl Tyrodes buffer is added to each well to initiate antagonist dissociation. Following incubation for predetermined times, typically 30 - 270 min at 37 ⁰C, the cells are then washed again with 200 μl PBS and are incubated with 100 μl Tyrodes buffer containing Brilliant Black, probenecid and Fluo-4 for 45 min at 37 ⁰C, as described above. After this period, the cells are challenged with histamine in the FLIPR™ as described above. The dose ratio at each time point is used to determine the fractional H1 receptor occupancy by the following equation: fractional receptor occupancy = (DR- 1 )/DR. The decrease in receptor occupancy over time approximates to a straight line and is analysed by linear regression. The slope of this straight line fit is used as an index of the dissociation rate of the antagonist. The dose ratios for antagonist treated cells and for antagonist treated and washed cells at each time point are used to calculate a relative dose ratio (rel DR) which is also used as an index of antagonist duration. Antagonists with long duration of action produce rel DR values close to 1 , and antagonists with short duration of action produce rel DR values that approaches the dose ratio value obtained for antagonist treatment alone.

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

Generation of histamine H3 cell line

The histamine H3 cDNA is isolated from its holding vector, pCDNA3.1 TOPO (InVitrogen), by restriction digestion of plasmid DNA with the enzymes BamH1 and Not-1 and is 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) is performed as described in US Patents: 5,364,791 ; 5,874,534; and 5,935,934. Ligated DNA is transformed into competent DH5α E. coli host bacterial cells and is 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 50 μgml^"1. Colonies containing the re-ligated plasmid are identified by restriction analysis. DNA for transfection into mammalian cells is prepared from 250 ml cultures of the host bacterium containing the pGeneH3 plasmid and is 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) are seeded at 2x10⁶ 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 (100 μgml^"1), 24 h prior to use. Plasmid DNA is transfected into the cells using Lipofectamine plus according to the manufacturer's guidelines (InVitrogen). 48 h post transfection, cells are placed into complete medium supplemented with 500 μgml^"1 Zeocin™.

10-14 days post selection, 10 nM Mifepristone (InVitrogen) is added to the culture medium to induce the expression of the receptor. 18 h post induction, cells are detached from the flask using ethylenediamine tetra-acetic acid (EDTA; 1 :5000; InVitrogen), following several washes with PBS, pH 7.4 and are resuspended in Sorting Medium containing Minimum Essential Medium (MEM), without phenol red, and are supplemented with Earles salts and 3% Foetal Clone Il (Hyclone). Approximately 1 x10⁷ cells are examined for receptor expression by staining with a rabbit polyclonal antibody, 4a, raised against the Λ/-terminal domain of the histamine H3 receptor, are incubated on ice for 60 min, followed by two washes in sorting medium. Receptor bound antibody is detected by incubation of the cells for 60 min on ice with a goat anti rabbit antibody, conjugated with Alexa 488 fluorescence marker (Molecular Probes). Following two further washes with Sorting Medium, cells are filtered through a 50 μm Filcon™ (BD Biosciences) and then are analysed on a FACS Vantage SE Flow Cytometer fitted with an Automatic Cell Deposition Unit. Control cells are non-induced cells treated in a similar manner. Positively stained cells are sorted as single cells into 96-well plates, containing Complete Medium containing 500 μgml^"1 Zeocin™ and are allowed to expand before reanalysis for receptor expression via antibody and ligand binding studies. One clone, 3H3, is selected for membrane preparation. Membrane preparation from cultured cells

All steps of the protocol are carried out at 4 ⁰C and with pre-cooled reagents. The cell pellet is resuspended in 10 volumes of homogenisation buffer (50 mM Λ/-2- hydroxyethylpiperazine-/V-2-ethanesulfonic acid (HEPES), 1 mM ethylenediamine tetra- acetic acid (EDTA), pH 7.4 with KOH, supplemented with 10^"6 M leupeptin (acetyl-leucyl- leucyl-arginal; Sigma L2884), 25 μgml^"1 bacitracin (Sigma B0125), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2χ10^"6 M 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 500 g for 20 min. The supernatant is then spun at 48,000 g for 30 min. The pellet is resuspended in homogenisation buffer (4χ the volume of the original cell pellet) by vortexing for 5 sec, followed by homogenisation in a Dounce homogeniser (10- 15 strokes). At this point the preparation is aliquoted into polypropylene tubes and stored at -80 ⁰C.

Histamine H3 functional antagonist assay

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

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

DMSO as a control); (b) 30 μl bead/membrane/GDP mix which is 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 (20 mM /V-2-hydroxyethylpiperazine-/V-2-ethanesulfonic acid (HEPES) + 100 mM NaCI + 10 mM MgCI₂, pH 7.4 NaOH) to give a final volume of 30 μl which contains 5 μg protein, 0.25 mg bead per well and 10 μM final assay concentration of guanosine 5' diphosphate (GDP) (Sigma, diluted in assay buffer) incubating at room temperature for 60 min on a roller;

(c) 15 μl 0.38 nM [³⁵S]-GTPyS (Amersham; Radioactivity concentration = 37 MBqml^"1; Specific activity = 1160 Cimmol^"1), histamine (at a concentration that results in the final assay concentration of histamine being EC₈o).

After 2-6 h, the plate is centrifuged for 5 min at 1500 rpm and counted on a Viewlux counter using a 613/55 filter for 5 minplate^"1. Data is analysed using a 4-parameter logistic equation. Basal activity is used as minimum, i.e. histamine not added to well. Intranasal Challenge Method: Whole Body Plethysmography (a)Sensitisation

Female Dunkin-Hartley guinea pigs 150-250 g are sensitised twice daily for 5 days (week 1 ) with ovalbumin (OVA) and aluminium hydroxide (AI(OH)₃ or Alum) in physiological saline, 25 μl/nostril. Solution is made up at 20 μg/ml OVA, 180 mg/ml Alum. During weeks 2 and 3, animals receive 25 μl/nostril of OVA (5 mg/ml) once daily. During Week 4 guinea pigs will be entered into study but are continually sensitized as per weeks 2 and 3 until the day before dosing with compound or vehicle.

(b) Compound/Vehicle Pretreatment

Pretreatment with test compound is performed at various times prior to histamine challenge. Efficacy dose-response curves are determined 1 h after dosing whereas duration of action is studied up to 7 days post dose. Test compounds are formulated as solutions in 0.9% sterile saline or suspensions in 0.9% sterile saline/tweenδO.

Guinea pigs were anaesthetised with isoflurane (5%, 2-3 l/min O₂), placed in a supine position, and 25 μl of test compound or vehicle dosed into each nostril using a Gilson pipette. After dosing, animals remain supine for at least 30 seconds during recovery from anaesthesia.

(c) Histamine Challenge Protocol

At 30 min before the time of histamine challenge, guinea pigs are dosed with atropine sulphate (Sigma A0257, dissolved in saline), 1 mg/kg i.p. Animals are then placed into whole body plethysmograph systems (Buxco® Electronics) where the parameter PenH area under curve (AUC) is recorded as outlined in Hamelmann E., Schwarze, J., Takeda, K., Oshiba, A., Larsen, L., Irvin, CG. and Gelfand, E.W., Am. J. Respir. Crit. Care Med. 156:766-775 (1997). A 10 min baseline AUC is recorded and if this value is over 1000, the animals are excluded.

After the stipulated pre-dose time has been reached, guinea pigs are re-anaesthetised with isoflurane and dosed with either 15 mM histamine or phosphate-buffered saline (PBS), (25 μl per nostril). On recovery from anaesthesia, animals are returned to the individual plethysmograph chambers and 4x10 min consecutive PenH AUC recordings are made. These recordings are summed to give a cumulative AUC over 40 min post histamine challenge for each animal. Data are analysed using ANOVA with post-hoc Fishers LSD test (general linear models, Statistica®) and finally Hochberg adjustment. Inhibition of histamine-induced congestion is determined by statistically significant differences between the mean responses of compound pre-treated groups compared to the vehicle pre-treated, histamine-challenged group.

CNS penetration

(i) CNS penetration by bolus administration Compounds are dosed intravenously at a nominal dose level of 1 mg/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 min 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.

(ii) CNS penetration following intravenous infusion at steady state

A loading dose of the compounds is given to male CD Sprague Dawley rats at a nominal dose level of 0.4 mg/kg. The compounds are then infused intravenously for 4 h at a nominal dose level of 0.1 mg/kg/h. Compounds are formulated in 2% DMSO/30% PEG200/68% water. Serial or terminal blood samples are taken at 0.5, 1.5, 2.5, 3, 3.5 and 4 h post dose. The final blood sample is collected under terminal anaesthesia with isoflurane 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.

Results

The following data were obtained: i) an average pK, (pK_b) at H1 of greater than 7.5 and an average pA2 value at H1 of greater than 8.5. ii) an average pK, (pK_b) at H3 of greater than 9. iii) at one or more time points, a significantly longer duration of action than azelastine in the histamine H1 functional antagonist assay. iv) a statistically significant inhibition of nasal congestion at 24 hours after dosing compared to azelastine in the Guinea Pig whole body plethysmography model, (see Figure 1 ). v) lower CNS penetration than azelastine.

Preparation of 6α, 9α-Difluoro-17α-r(2-furanylcarbonyl)oxyl-11 β-hvdroxy-16α- methyl-3-oxo-androsta-1.4-diene-17β-carbothioic acid S-fluoromethyl ester

The preparation of 6α, 9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-1 1 β-hydroxy-16α-methyl- 3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester (fluticasone furoate), solvates and polymorphs thereof including polymorphic Form 1 , and biological activity thereof, are disclosed in International Patent Application WO02/12265 and International Patent Application WO03/066024 incorporated fully herein by reference.

Example Compositions

The aqueous pharmaceutical compositions of the invention may be prepared according to the following general method:

Where appropriate the isotonicity adjusting agent(s) is charged into a suitable mixing vessel containing purified water and dissolved with stirring.

The suspending agent(s) is then charged into the mixing vessel and dispersed throughout the solution. The resulting suspending vehicle is allowed to hydrate for an appropriate period of time to ensure cross-linkage and gelation, which may take 60 minutes or longer.

Preservative(s) is pre-dissolved in purified water in a separate vessel, optionally with heating, for example to 50-60 ⁰C depending on the preservative chosen, to aid dissolution, and then added to the isotonicity adjusting agent(s) with continuous stirring. Buffering agents, if included, are dissolved in a minimum amount of purified water, optionally heated, for example to about 50-60 ⁰C as appropriate depending on the buffering agents chosen, and stirred to dissolve in separate containers. The separate solutions are combined, mixed well and then added to the bulk solution with continuous stirring.

In a separate mixing vessel, the wetting agent(s) is mixed with purified water which optionally may be heated, for example to about 50-60 ⁰C as appropriate depending on the wetting agent(s) chosen, and stirred to dissolve. A slurry or solution of active compound(s) is then prepared by adding the resultant wetting agent(s) solution to the active compound(s), which may be particle size reduced for example micronised, and mixed prior to homogenising/refining.

Additionally, in a separate mixing vessel, additional preservative(s), if needed, may be mixed with purified water and stirred to dissolve.

Following the dispersion and refining of the slurry of active compound(s) it is added to the mixing vessel containing the suspending agent and dispersed with stirring. Following the addition of the slurry of active compound(s), any additional preservative may be added to the bulk suspension and dispersed with continuous stirring. Finally, the suspension is made to its final mass by adding water and stirred.

Co-solvent(s), if included, may be added before or after the addition of the buffering agents. Alternatively, the co-solvent(s) may be added during the formation of the drug slurry or solution.

Preservative(s), if included, may be added before or after the addition of the suspending agent(s).

Fluticasone furoate is used in its unsolvated form as polymorphic Form 1.

4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of its free base, or as the 1 ,5-naphthalene disulfonate monohydrate salt, or as the dihydrochloride salt. Example 1

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

The composition components are weighed. The dextrose is charged into the mixing vessel containing purified water and dissolved with stirring. EDTA is pre-dissolved in purified water, heated to 50-60 ⁰C in a separate vessel, to aid dissolution, and then added to the dextrose solution with continuous stirring. The microcrystalline cellulose and carboxymethylcellulose sodium is then charged into the mixing vessel and dispersed throughout the solution. The resulting suspending vehicle is then allowed to build or hydrate for not less than 60 minutes to ensure complete cross-linkage and gelation.

In a separate mixing vessel, the polysorbate 80 is mixed with purified water which has been heated to 50-60 ⁰C and stirred to dissolve. A slurry of compound is then prepared by adding the resultant polysorbate 80 solution to the micronised compound and mixed with shaking or stirring prior to homogenising/refining with a high shear mixer. Additionally, in a separate mixing vessel, the benzalkonium chloride solution (50% w/v) is mixed and diluted with purified water and stirred to mix. Following the hydration period the slurry of compound is added to the mixing vessel containing the suspending agent and dispersed with stirring. Following the addition of the slurry of compound, the pre-diluted benzalkonium chloride solution is slowly added to the bulk suspension and dispersed with continuous stirring. Finally, the suspension is made to its final mass by adding water and stirred.

Example 2

Example 2 may be prepared by a generally similar method to the methods described above.

* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 3 Example 3 may be prepared by a generally similar method to the methods described above.

Component % (w/w)

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 4

Example 4 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 5

Example 5 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 6

Example 6 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 7

Example 7 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 8

Example 8 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 9

Example 9 may be prepared by a generally similar method to the methods described above.

Component % (w/w)

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of either the 1 ,5-naphthalene disulfonate monohydrate salt or the dihydrochloride salt

Example 10

Example 10 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of either the 1 ,5-naphthalene disulfonate monohydrate salt or the dihydrochloride salt

Example 11

Example 11 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt Example 12

Example 12 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 13 Example 13 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the dihydrochloride salt

Example 14

Example 14 may be prepared by a generally similar method to the methods described above.

Purified Water to 100%

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone is used in the form of the dihydrochloride salt

Example 15

Example 15 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 16

Example 16 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 17

Example 17 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 18

Example 18 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone is used in the form of the 1 ,5-naphthalene disulfonate monohydrate salt

Example 19

Example 19 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 /-/-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone is used in the form of either the 1 ,5-naphthalene disulfonate monohydrate salt or the dihydrchloride salt

Example 20 Example 20 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of either the 1 ,5-naphthalene disulfonate monohydrate salt or the dihydrchloride salt

Example 21

Example 21 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the dihydrochloride salt

Example 22

Example 22 may be prepared by a generally similar method to the methods described above.

^* micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the dihydrochloride salt

Example 23

Example 23 may be prepared by a generally similar method to the methods described above.

4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the free base Example 24

Example 24 may be prepared by a generally similar method to the methods described above.

4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the free base

Example 25

Example 25 may be prepared by a generally similar method to the methods described above.

4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1 -yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the free base

Example 26

Example 26 may be prepared by a generally similar method to the methods described above.

4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone is used in the form of the free base

In Examples 1 to 26 the concentation of micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1- [4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone is given as the free base, which concentrations are 0.01 % (w/w), 0.025% (w/w), 0.05% (w/w), 0.1% (w/w), 0.25% (w/w) and 0.5% (w/w), based on the total weight of the composition.

It will be appreciated that 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H- azepin-1-yl) propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2H)-phthalazinone may be used in the form of a pharmaceutically acceptable salt at an appropriate concentration, depending on the salt chosen, such as to provide the desired concentration of free base.

Thus, in Examples 1 to 12 and 15 to 20 concentrations of 4-[(4-chlorophenyl)methyl]-2- ({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone, 1 ,5-naphthalene disulfonate monohydrate (micronised) are 0.01478% (w/w), 0.03695% (w/w), 0.0739% (w/w), 0.1478% (w/w), 0.3695% (w/w) and 0.739 (w/w) based on the total weight of the composition, which will provide 0.01 to 0.5% (w/w) of free base.

In Examples 9, 10, 13, 14, 19, 20, 21 and 22 concentrations of 4-[(4-chlorophenyl)methyl]- 2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl} methyl)-1 (2/-/)-phthalazinone, dihydrochloride salt are such as to provide 0.01 to 0.5% (w/w) of free base.

In Examples 23 to 26 concentrations of micronised 4-[(4-chlorophenyl)methyl]-2-({(2R)-1- [4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone are given as the free base, which concentrations are 0.01% (w/w), 0.025% (w/w), 0.05% (w/w), 0.1% (w/w), 0.25% (w/w) and 0.5% (w/w), based on the total weight of the composition.

Example compositions may be filled into suitable containers depending on the chosen route of administration. For intransal administration, suitable containers are described hereinabove and typically are made of plastics and dispense 50 to 100 μl_ of composition per actuation.

Claims

1. An aqueous pharmaceutical composition which comprises a compound which is 4- [(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl] oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone,

or a pharmaceutically acceptable salt thereof.

An aqueous pharmaceutical composition which comprises

(i) a compound which is 4-[(4-chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-

(hexahydro-1 H azepin-1 -yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)- phthalazinone,

or a pharmaceutically acceptable salt thereof; and

(ii) a compound which is 6α, 9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-1 1 β-hydroxy- 16α-methyl-3-oxo-androsta-1 ,4-diene-17β-carbothioic acid S-fluoromethyl ester (fluticasone furoate)

or a solvate thereof.

3. An aqueous pharmaceutical composition according to claim 1 or claim 2 which comprises 0.005% to 2% (w/w) based on the total weight of the composition of 4-[(4- chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone, or a pharmaceutically acceptable salt thereof.

An aqueous pharmaceutical composition according to claim 3 which comprises 0.01% to 1% (w/w) based on the total weight of the composition of 4-[(4- chlorophenyl)methyl]-2-({(2/?)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1-yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone, or a pharmaceutically acceptable salt thereof.

5. An aqueous pharmaceutical composition according to any of claims 2 to 4, which comprises 0.01 to 1% (w/w) based on the total weight of the composition of fluticasone furoate, or a solvate thereof.

6. An aqueous pharmaceutical composition according to claim 5, which comprises about 0.05% (w/w) based on the total weight of the composition of fluticasone furoate, or a solvate thereof.

7. An aqueous pharmaceutical composition according to any of claims 1 to 6 further comprising a) a suspending agent; b) a preservative; c) a wetting agent; and d) an isotonicity adjusting agent.

8. An aqueous pharmaceutical composition according to claim 7 which further comprises a co-solvent.

9. An aqueous pharmaceutical composition according to claim 7 or claim 8 which comprises 1.5% to 2.4% (w/w) based on the total weight of the composition of a suspending agent.

10. An aqueous pharmaceutical composition according to any of claims 7 to 9 wherein the suspending agent comprises microcrystalline cellulose and/or carboxy methylcellulose sodium.

11. An aqueous pharmaceutical composition according to any of claims 7 to 10, which comprises 0.001 to 1% (w/w) based on the total weight of the composition of a preservative.

12. An aqueous pharmaceutical composition according to claim 1 1 , which comprises 0.015% to 0.3% (w/w) based on the total weight of the composition of a preservative.

13. An aqueous pharmaceutical composition according to any of claims 7 to 12, wherein the preservative comprises disodium ethylenediamine-tetraacetate (EDTA) and/or potassium sorbate.

14. An aqueous pharmaceutical composition according to any of claims 7 to 13 wherein the preservative comprises about 0.015% (w/w) disodium ethylenediamine- tetraacetate (EDTA) and about 0.3% (w/w), based on the total weight of the composition of potassium sorbate.

15. An aqueous pharmaceutical composition according to any of claims 7 to 14, which comprises 0.01 to 0.05% (w/w) based on the total weight of the composition of a wetting agent.

16. An aqueous pharmaceutical composition according to claim 15 which comprises about 0.025% (w/w) based on the total weight of the composition of a wetting agent.

17. An aqueous pharmaceutical composition according to any of claims 7 to 16 wherein the wetting agent comprises polyoxyethylene (20) sorbitan monooleate (Polysorbate

80).

18. An aqueous pharmaceutical composition according to any of claims 7 to 17, which comprises 0.1 to 10% (w/w) based on the total weight of the composition of an isotonicity adjusting agent.

19. An aqueous pharmaceutical composition according to claim 18, which comprises 4.5 to 5.5% (w/w) based on the total weight of the composition of an isotonicity adjusting agent.

20. An aqueous pharmaceutical composition according to any of claims 7 to 19, wherein the isotonicity adjusting agent comprises dextrose and/or xylitol.

21. An aqueous pharmaceutical composition according to any of claims 8 to 20, which comprises 1.5 to 17.5% (w/w) based on the total weight of the composition of a co- solvent.

22. An aqueous pharmaceutical composition according to claim 21 which comprises about 1.5, about 2.5, about 7.5 or about 17.5% (w/w) based on the total weight of the composition of a co-solvent.

23. An aqueous pharmaceutical composition according to any of claims 8 to 22, wherein the co-solvent is propylene glycol.

24. An aqueous pharmaceutical composition according to any of claims 7 to 23, further comprising a buffering agent.

25. An aqueous pharmaceutical composition according to any of claims 7 to 24, which is isotonic with fluids of the nasal cavity.

26. An aqueous pharmaceutical composition according to any of claims 7 to 24, which is isotonic with fluids of the eye.

27. An aqueous pharmaceutical composition according to any of claims 1 to 26, which is an aqueous suspension.

28. An aqueous pharmaceutical composition according to any of claims 1 to 26 which is an aqueous solution.

29. An aqueous pharmaceutical composition according to any of claims 1 to 28 comprising 4-[(4-chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1 H-azepin-1- yl)propyl]oxy} phenyl)butyl]-2-pyrrolidinyl}methyl)-1 (2H)-phthalazinone, 1 ,5-naphthalene disulfonate monohydrate salt.

30. An aqueous pharmaceutical composition according to any of claims 2 to 28 comprising fluticasone furoate in unsolvated form.

31. An aqueous pharmaceutical composition according to claim 30 comprising fluticasone furoate, polymorphic Form 1.

32. An aqueous pharmaceutical composition which is a composition as described in any of Examples 1 to 26.

33. A container comprising an aqueous pharmaceutical composition according to any of claims 1 to 32 suitable for delivering said composition to the nasal cavities.

34. A container comprising an aqueous pharmaceutical composition according to any of claims 1 to 32 suitable for delivering said composition to the eye.

35. An aqueous pharmaceutical composition according to any of claims 1 to 32 for use in the treatment of inflammatory and/or allergic diseases.

36. An aqueous pharmaceutical composition according to claim 35 for use in the treatment of allergic rhinitis.

37. An aqueous pharmaceutical composition according to claim 35 or claim 36 for use in treatment, once per day.

38. The use of an aqueous pharmaceutical composition according to any of claims 1 to 32 in the manufacture of a medicament for the treatment of inflammatory and/or allergic diseases.

39. The use according to claim 38, for treatment once per day.

40. The use according to claim 38 or claim 39, for the treatment of allergic rhinitis.

41. A method for the treatment (or prophylaxis) of inflammatory and/or allergic diseases which method comprises administering to a patient in need thereof a pharmaceutically effective amount of an aqueous pharmaceutical composition as defined in any of claims 1 to 32.