WO2004035544A1

WO2004035544A1 - Benzo[d]azepine derivatives for the treatment of neurological and psychiatric disorders

Info

Publication number: WO2004035544A1
Application number: PCT/EP2003/011421
Authority: WO
Inventors: Thomas Daniel Heightman; David Matthew Wilson
Original assignee: Glaxo Group Limited
Priority date: 2002-10-16
Filing date: 2003-10-14
Publication date: 2004-04-29
Also published as: GB0224083D0; AU2003278084A1

Abstract

The present invention relates to novel benzazepine derivatives of formula (I) having pharmacological activity, processes for their preparation, to compositions containing them and to their use in the treatment of neurological and psychiatric disorders.

Description

BENZO ^ D I AZEPINE DERIVATIVES FOR THE TREATMENT OF NEUROLOGICAL AND PSYCHIATRIC DISORDERS

The present invention relates to novel benzazepine derivatives having pharmacological activity, processes for their preparation, to compositions containing them and to their use in the treatment of neurological and psychiatric disorders.

WO 02/76925 (Eli Lilly) describes a series of compounds which are claimed to be histamine H3 antagonists. WO 01/87834 (Takeda) describes a series of bicyclic heterocycles which are claimed to be melanin-concentrating hormone antagonists.

The histamine H3 receptor is predominantly expressed in the mammalian central nervous system (CNS), with minimal expression in peripheral tissues except on some sympathetic nerves (Leurs er a/., (1998), Trends Pharmacol. Sci. 19, 177-183). Activation of H3 receptors by selective agonists or histamine results in the inhibition of neurotransmitter release from a variety of different nerve populations, including histaminergic and cholinergic neurons (Schlicker ef al., (1994), Fundam. Clin. Pharmacol. 8, 128-137). Additionally, in vitro and in vivo studies have shown that H3 antagonists can facilitate neurotransmitter release in brain areas such as the cerebral cortex and hippocampus, relevant to cognition (Onodera et al., (1998), In: The Histamine H3 receptor, ed Leurs and Timmerman, pp255-267, Elsevier Science B.V.). Moreover, a number of reports in the literature have demonstrated the cognitive enhancing properties of H3 antagonists (e.g. thioperamide, clobenpropit, ciproxifan and GT-2331) in rodent models including the five choice task, object recognition, elevated plus maze, acquisition of novel task and passive avoidance (Giovanni et al., (1999), Behav. Brain Res. 104, 147-155). These data suggest that novel H3 antagonists and/or inverse agonists such as the current series could be useful for the treatment of cognitive impairments in neurological diseases such as Alzheimer's disease and related neurodegenerative disorders.

The present invention provides, in a first aspect, a compound of formula (I):

(I) wherein:

R¹ represents hydrogen, -Cι_e alkyl, -C₃.₈ cycloalkyl, aryl, heterocyclyl, heteroaryl, -C^ alkyl-aryl, -Cι_₆ alkyl-heteroaryl, -C^ alkyl-heterocyclyl, -aryl-aryl, -aryl-heterόaryl, -aryl- heterocyclyl,- heteroaryl-aryl, -fieteroaryl-heteroaryl, -heteroaryl-heterocyclyl, - heterocyclyl-aryl, -heterocyclyl-heteroaryl, -heterocyclyl-heterocyclyl, wherein R¹ may be optionally substituted by one or more substituents which may be the same or different, and which are selected from the group consisting of halogen, hydroxy, cyano, nitro, oxo, trifluoromethyl, trifluoromethoxy, fluoromethoxy, difluoromethoxy, Cι.₆ alkyl, pentafluoroethyl, Cι._e alkoxy, C₂.₆ alkenyl, arylCι.₆ alkoxy, d-₆ alkylthio, d-e alkoxyCι-6 alkyl, C₃.₇ cycloalkyld-6 alkoxy, d-6 alkanoyl, d-e alkoxycarbonyl, Cι_₆ alkylsulfonyl, d.₆ alkylsulfinyl, C₁-₆ alkylsulfonyloxy, d_6 alkylsulfonylC-ι.₆ alkyl, sulfonyl, arylsulfonyl, arylsulfonyloxy, arylsulfonylCvβ alkyl, aryloxy, C|.₆ alkylsulfonamido, d.₆ alkylamido, d.₆ alkylsulfonamidod-e alkyl, d_₆ alkylamidod-e alkyl, arylsulfonamido, arylcarboxamido, arylsulfonamidod.6 alkyl, arylcarboxamidod-e alkyl, aroyl, aroyld_₆ alkyl, aryld.₆ alkanoyl, or a group NR¹⁵R¹⁶, CONR¹⁵R¹⁶, NR¹⁵COR¹⁶, NR¹⁵R¹⁶SO₂ or SO₂NR¹⁵R¹⁶, wherein R¹⁵ and R¹⁶ independently represent hydrogen or d-e alkyl or together may be fused to form a 5- to 7- membered non-aromatic heterocyclic ring optionally interrupted by an O or S atom; X represents bond, d.₆ alkyl, CO, SO₂ or CONR⁴, such that when R¹ represents -C₃.₈ cycloalkyl, X represents d_₆ alkyl, CO, SO₂ or CONR⁴;

R² represents halogen, d_₆ alkyl, C₁-₆ alkoxy, cyano, amino or trifluoromethyl; n is 0, 1 or 2;

R⁴ represents hydrogen or d.₆ alkyl, or together with R¹ may form a heterocyclyl group;

R³ represents -(CH₂)_q-NR¹¹

wherein q is 2, 3 or 4;

R¹¹ and R¹² independently represent d.₆ alkyl or together with the nitrogen atom to which they are attached represent an N-linked nitrogen containing heterocyclic group optionally substituted by one or more R¹⁷ groups; R¹³ represents hydrogen, d_₆ alkyl, C₃.₈ cycloalkyl, -d.₆ alkyl-aryl or heterocyclyl;

R¹⁴ and R¹⁷ independently represent halogen, d.₆ alkyl, haloalkyl, OH, did.₆ alkylamino, d-e alkoxy or heterocyclyl; f and k independently represent 0, 1 or 2; g is 0, 1 or 2 and h is 0, 1 , 2 or 3, such that g and h cannot both be 0; or a pharmaceutically acceptable salt thereof.

Alkyl groups, whether alone or as part of another group, may be straight chain or branched and the groups alkoxy and alkanoyl shall be interpreted similarly. Alkyl moieties are more preferably C_M alkyl, eg. methyl or ethyl. The term 'halogen' is used herein to describe, unless otherwise stated, a group selected from fluorine, chlorine, bromine or iodine.

The term "aryl" includes phenyl and naphthyl. The term "heterocyclyl" is intended to mean a 4-7 membered monocyclic saturated or partially unsaturated aliphatic ring or a 4-7 membered saturated or partially unsaturated aliphatic ring fused to a benzene ring containing 1 to 3 heteroatoms selected from oxygen, nitrogen or sulphur. Suitable examples of such monocyclic rings include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, diazepanyl, azepanyl, azocanyl and dioxolanyl. Suitable examples of benzofused heterocyclic rings include indolinyl, isoindolinyl, benzodioxolyl and dihydroisoquinolinyl.

The term "nitrogen containing heterocyclyl" is intended to represent any heterocyclyl group as defined above which contains a nitrogen atom.

The term "heteroaryl" is intended to mean a 5-7 membered monocyclic aromatic or a fused 8-11 membered bicyclic aromatic ring containing 1 to 3 heteroatoms selected from oxygen, nitrogen and sulphur. Suitable examples of such monocyclic aromatic rings include thienyl, furyl, pyrrolyl, triazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, thiadiazolyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl and pyridyl. Suitable examples of such fused aromatic rings include benzofused aromatic rings such as quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, indolyl, indazolyl, pyrrolopyridinyl, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzoxadiazolyl, benzothiadiazolyl and the like.

Preferably, R¹ represents hydrogen, d-₆ alkyl (eg. methyl, i-propyl, t-butyl), C₃.₈ cycloalkyl (eg. cyclopropyl or cyclohexyl), aryl, heteroaryl (eg. thienyl) or heterocyclyl (eg. benzodioxolyl or tetrahydrofuranyl), optionally substituted by one or more d.₆ alkoxy groups (eg. methoxy). More preferably, R¹ represents d-e alkyl (eg. methyl, i-propyl, t- butyl) or C₃.₈ cycloalkyl (eg. cyclopropyl or cyclohexyl).

Preferably, X represents a bond, d.₆ alkyl (eg. CH₂) or CO, more preferably bond or d.₆ alkyl (eg. CH₂).

More preferably, R -X- represents hydrogen-;

Ci-₆ alkyl- (eg. methyl, i-butyl-, propyl- or ethyl-) optionally substituted by one or more halogen (eg. fluorine), cyano, d.₆ alkoxy groups (eg. methoxy), CONR¹⁵R¹⁶ (eg. - CON(ethyl)₂) or arylsulfonyl (eg. -SO₂-phenyl) groups;

C₃.₈ cycloalkyl-CH₂- (eg. cyclopropyl-CH₂- or cyclohexyl-CH₂-); aryl- (eg. phenyl) optionally substituted by one or more halogen (eg. chlorine) or C₂-₆alkenyl (eg. propenyl) groups; aryl-CH₂- (eg. phenyl-CH₂- or naphthyl-CH₂-) optionally substituted by one or more halogen (eg. chlorine), cyano, NR¹⁵R¹⁶ (eg. -N(Me)₂), NR¹⁵COR¹⁶ (eg. NHCOMe) or .₆ alkoxy groups (eg. methoxy); heterocyclyl-CH₂- (eg. tetrahydrofuranyl-CH₂- or dioxolanyl-CH₂-); d.₆ alkyl-CO- (eg. methyl-CO- or t-butyl-CO-); aryl-CO- (eg. phenyl-CO- or naphthyl-CO-) optionally substituted by one or more halogen (eg. chlorine) or d.₆ alkoxy groups (eg. methoxy); heteroaryl-CO- (eg. thienyl-CO- or benzofuranyl-CO-); heterocyclyl-CO- (eg. benzodioxolyl-CO-); d.₆alkyl-NR⁴CO- (eg. isopropyl-NH-CO- ); aryl-NR⁴CO- (eg. phenyl-NH-CO-) optionally substituted by one or more halogen (eg. chlorine) atoms;

Cι.₆alkyl-SO₂- (eg. butyl-SO₂-); or aryl-SO₂- (eg. phenyl-SO₂-) optionally substituted by one or more halogen (eg. chlorine) atoms.

Yet more preferably, R¹-X- represents hydrogen-; d-e alkyl- (eg. methyl, i-butyl- or ethyl-) optionally substituted by one or more halogen (eg. fluorine), cyano, d.₆ alkoxy groups (eg. methoxy), CONR¹⁵R¹⁶ (eg. -

CON(ethyl)₂) or arylsulfonyl (eg. -SO₂-pfιenyl) groups;

C₃_₈ cycloalkyl-CH₂- (eg. cyclopropyl-CH₂- or cyclohexyl-CH₂-); aryl- (eg. phenyl) optionally substituted by one or more C₂.₆ alkenyl (eg. propenyl) groups; aryl-CH₂- (eg. phenyl-CH₂- or naphthyl-CH₂-) optionally substituted by one or more halogen (eg. chlorine), cyano, NR¹⁵R¹⁶ (eg. -N(Me)₂), NR¹⁵COR¹⁶ (eg. NHCOMe) or d-₆ alkoxy groups (eg. methoxy); or heterocyclyl-CH₂- (eg. tetrahydrofuranyl-CH₂-).

Preferably, n represents 0 or 1 , more preferably 0.

When n represents 1 , R² is preferably an amino group.

Preferably, R³ represents ~(CH₂)_q-NR¹¹R¹².

Preferably, q represents 2 or 3, more preferably 3. Preferably, R¹¹ and R¹² independently represent d-e alkyl (eg. methyl or ethyl).

Alternatively and preferably, NR¹¹R¹² represents piperidinyl, pyrrolidinyl, morpholinyl, azepanyl or diazepanyl optionally substituted by one or more d.₆ alkyl (eg. methyl) or heterocyclic groups (eg. piperidine).

More preferably, NR¹¹R¹² represents unsubstituted piperidinyl, pyrrolidinyl or azepanyl. When R³ represents a group of formula (i), preferably f represents 0 or 1 , h represents 1 , g represents 2, k represents 0 and R¹³ represents d.₆ alkyl (eg. isopropyl). When R³ represents a group of formula (i), more preferably f represents 0, h represents 1 , g represents 2, k represents 0 and R¹³ represents d.₆ alkyl (eg. isopropyl).

Preferably, -O-R³ is present at the 7-position of the benzazepine group.

Preferred compounds according to the invention include examples E1-E54 as shown below, or a pharmaceutically acceptable salt thereof. Compounds of formula (I) may form acid addition salts with acids, such as conventional pharmaceutically acceptable acids, for example maleic, hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, sulphate, citric, lactic, mandelic, tartaric and methanesulphonic. Salts, solvates and hydrates of compounds of formula (I) therefore form an aspect of the invention.

Certain compounds of formula (I) are capable of existing in stereoisomeric forms. It will be understood that the invention encompasses all geometric and optical isomers of these compounds and the mixtures thereof including racemates. Tautomers also form an aspect of the invention.

The present invention also provides a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof, which process comprises:

(a) reacting a compound of formula (II)

(II) wherein R² and n are as defined above and P¹ represents R¹-X or a suitable protecting group such as t-butoxycarbonyl, wherein R¹ and X are as defined above, with a compound of formula R^3'-L\ wherein R^3' is as defined above for R³ or a group convertible thereto and L¹ represents a suitable leaving group such as a halogen atom (eg. bromine or chlorine) or an optionally activated hydroxyl group, followed as necessary by deprotection under acidic conditions and conversion of R^3' to R³; or

(b) deprotecting a compound of formula (I) which is protected; and optionally thereafter

(c) interconversion to other compounds of formula (I).

When R³ represents -(CH₂)_q-NR¹¹R¹², process (a) typically comprises the use of a suitable base, such as potassium carbonate in an appropriate solvent such as 2- butanone optionally in the presence of a transfer reagent such as potassium iodide at an appropriate temperature such as reflux.

When a group R^3' convertible to R³ represents, for example, L²-(CH₂)_q-, process (a) typically comprises an alkylation reaction using analogous conditions to those described above. When R³ represents a group of formula (i) and L¹ represents an optionally activated hydroxyl group, process (a) typically comprises the use of a phosphine such as triphenylphosphine in a suitable solvent such as tetrahydrofuran, followed by addition of an azadicarboxylate such as diethylazaodicarboxylate at a suitable temperature such as room temperature.

In process (b), examples of protecting groups and the means for their removal can be found in T. W. Greene 'Protective Groups in Organic Synthesis' (J. Wiley and Sons, 1991). Suitable amine protecting groups include sulphonyl (e.g. tosyl), acyl (e.g. acetyl, 2',2',2^,-trichloroethoxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl) and arylalkyl (e.g. benzyl), which may be removed by hydrolysis (e.g. using an acid such as hydrochloric acid in dioxan or trifluoroacetic acid in dichloromethane) or reductively (e.g. hydrogenolysis of a benzyl group or reductive removal of a 2',2',2'- trichloroethoxycarbonyl group using zinc in acetic acid) as appropriate. Other suitable amine protecting groups include trifluoroacetyl (-COCF₃) which may be removed by base catalysed hydrolysis or a solid phase resin bound benzyl group, such as a Merrifield resin bound 2,6-dimethoxybenzyl group (Ellman linker), which may be removed by acid catalysed hydrolysis, for example with trifluoroacetic acid.

Process (c) may be performed using conventional interconversion procedures such as epimerisation, oxidation, reduction, alkylation, nucleophilic or electrophilic aromatic substitution, ester hydrolysis or amide bond formation. For example, compounds of formula (I) wherein X is other than a bond and R¹ is other than hydrogen may be prepared by the above interconversion procedures.

More specifically, compounds of formula (I) wherein -X-R¹ represents C₁-₆ alkyl may be prepared by interconverting a compound of formula (I) wherein -X-R¹ represents hydrogen in the presence of an appropriate carboxaldehyde or ketone. Such interconversion may occur in the presence of a suitable reducing agent such as a borohydride in the presence of a suitable solvent. Reaction with an appropriate carboxaldehyde may also be performed in the presence of sodium triacetoxyborohydride in the presence of a suitable solvent such as dichloromethane at a suitable temperature (eg. room temperature). Compounds of formula (I) wherein -X-R¹ represents d-e alkyl may also be prepared by interconverting a compound of formula (I) wherein -X-R¹ represents hydrogen in the presence of an appropriate d__β alkyl group containing a suitable leaving group (such as chlorine, bromine, iodine, methanesuifonyloxy and trifluoromethanesulfonyloxy). Such interconversion may occur in the presence of a suitable base (such as potassium carbonate) and a suitable solvent (such as butan-2-one) optionally in presence of heat. Compounds of formula (I) wherein X represents CO may be prepared by interconverting a compound of formula (I) wherein -X-R¹ represents hydrogen in the presence of an acid halide. Such interconversion may occur in the presence of a suitable base (such as triethylamine) and a suitable solvent (such as dichloromethane) at a suitable temperature (such as room temperature).

Compounds of formula (I) wherein X represents CO may also be prepared by interconverting a compound of formula (I) wherein -X-R¹ represents hydrogen in the presence of an appropriate carboxylic acid. Such interconversion may occur in the presence of a suitable coupling reagent (such as dicyclohexylcarbodiimide) in the presence of a suitable solvent (such as dichloromethane) at a suitable temperature

(such as room temperature).

Compounds of formula (I) wherein X represents SO₂ may be prepared by interconverting a compound of formula (I) wherein -X-R¹ represents hydrogen in the presence of a sulfonyl chloride. Such interconversion may occur in the presence of a suitable solvent

(such as 2-butanone).

Compounds of formula (I) wherein X represents CONR⁴ may be prepared by interconverting a compound of formula (I) wherein -X-R¹ represents hydrogen in the presence of a compound of formula R¹-N=C=O, wherein R¹ is as defined above. Such interconversion may occur in the presence of a suitable solvent (such as 2-butanone).

Compounds of formula (I) wherein X represents CONR⁴ may also be interconverted by reacting a compound of formula (I) wherein -X-R¹ represents hydrogen sequentially with phosgene in a solvent such as toluene followed by a compound of formula R⁴R¹-NH, in a solvent such as dichloromethane and a suitable base, such as triethylamine, wherein R¹ and R⁴ are as defined above.

Compounds of formula (II) may be prepared in an analogous manner to those described in Description 3 of WO 02/40471.

Compounds of formula (I) and their pharmaceutically acceptable salts have affinity for and are antagonists and/or inverse agonists of the histamine H3 receptor and are believed to be of potential use in the treatment of neurological diseases including Alzheimer's disease, dementia, age-related memory dysfunction, mild cognitive impairment, cognitive dysfunction, epilepsy, neuropathic pain, inflammatory pain, migraine, Parkinson's disease, multiple sclerosis, stroke and sleep disorders including narcolepsy; psychiatric disorders including schizophrenia, attention deficit hypereactivity disorder, depression and addiction; and other diseases including obesity, asthma, allergic rhinitis, nasal congestion, chronic obstructive pulmonary disease and gastro- intestinal disorders.

Thus the invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, for use as a therapeutic substance in the treatment or prophylaxis of the above disorders, in particular cognitive impairments in diseases such as Alzheimer's disease and related neurodegenerative disorders. The invention further provides a method of treatment or prophylaxis of the above disorders, in mammals including humans, which comprises administering to the sufferer a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of the above disorders.

When used in therapy, compounds of formula (I) are usually formulated in a standard pharmaceutical composition. Such compositions can be prepared using standard procedures.

Thus, the present invention further provides a pharmaceutical composition for use in the treatment of the above disorders which comprises the compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The present invention further provides a pharmaceutical composition which comprises the compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

A pharmaceutical composition of the invention, which may be prepared by admixture, suitably at ambient temperature and atmospheric pressure, is usually adapted for oral, parenteral or rectal administration and, as such, may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable or infusible solutions or suspensions or suppositories. Orally administrable compositions are generally preferred.

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

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and, if desired, conventional flavourings or colorants. For parenteral administration, fluid unit dosage forms are prepared utilising a compound of the invention or pharmaceutically acceptable salt thereof and a sterile vehicle. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions, the compound can be dissolved for injection and filter sterilised before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilisation cannot be accomplished by filtration. The compound can be sterilised by exposure to ethylene oxide before suspension in a sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The composition may contain from 0.1 % to 99% by weight, preferably from 10 to 60% by weight, of the active material, depending on the method of administration. The dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses may be 0.05 to 000 mg, more suitably 1.0 to 200 mg, and such unit doses may be administered more than once a day, for example two or three a day. Such therapy may extend for a number of weeks or months.

The following Descriptions and Examples illustrate the preparation of compounds of the invention.

Description 1

7-(3-Chloro-propoxy)-1 ,2,4,5-tetrahydro-benzo[d]azepine-3-carboxylic acid tert- butyl ester (D1) 7-Hydroxy-1 ,2,4,5-tetrahydro-benzo[o(]azepine-3-carboxylic acid terf-butyl ester (Description 3 in PCT publication number WO 02/40471) (13.15g, 50mmol) and potassium carbonate (20.7g, 150mmol) were suspended in 2-butanone (75ml). 1-Bromo- 3-chloropropane (7.4ml, 75mmol) was added dropwise to the mixture and heated at reflux for 48 hours. The solids were filtered and the filtrate concentrated in vacuo. The residue was purified by column chromatography, eluting with a mixture of ethyl acetate and hexane (1 :5) to afford the title compound (D1) (15.36g, 91%). ¹H NMR (CDCI₃) 7.03 (1H, d, J 8.1Hz), 6.62 (2H, m), 4.08 (2H, m), 3.73 (2H, m), 3.54 (4H, m), 2.84 (4H, m), 2.22 (2H, m), 1.48 (9H, s).

Description 2

7-(3-Piperidin-1 -yl-propoxy)-1 ,2,4,5-tetrahydro-benzo[ ]azepine-3-carboxylic acid fert-butyl ester (D2) 7-(3-Chloro-propoxy)-1 ,2,4,5-tetrahydro-benzo[o]azepine-3-carboxylic acid terf-butyl ester (D1) (6.80g, 20 mmol) and potassium carbonate (8.34g, 60mmol) were suspended in 2-butanone (50 ml). Piperidine (3.96ml, 40mmol) was added and the mixture heated at reflux for 24 hours. The solids were filtered and the filtrate concentrated in vacuo. The residue purified by column chromatography, eluting with a mixture of 0.880 ammonia:methanol:dichloromethane (1:9:90) to afford the title compound (D2) (6.45g, 83%). MS (ES+) m/e 390 [M+H]⁺.

Description 3 7-(2-Piperidin-1 -yl-ethoxy)-1 ,2,4,5-tetrahydro-benzo[d]azepine-3-carboxylic acid terf-butyl ester (D3)

A mixture of 7-hydroxy-1 ,2,4,5-tetrahydro-benzo[c|azepine-3-carboxylic acid tert-butyl ester (Description 3 in PCT publication number WO 02/40471) (2.63g, 10mmol), 1-(2- chloroethyl)piperidine hydrochloride (3.68g, 20mmol) and potassium carbonate (4.14g, 30mmol) in N,N-dimethyl formamide (30ml) was heated at 80°C for 24 hours. After cooling the mixture was diluted with water and ethyl acetate. The organic phase was separated washed with water, brine, dried and evaporated in vacuo. The residue was purified by column chromatography eluting with a mixture of 0.880 ammonia:methanol:dichloromethane (1:9:90) to afford the title compound (D3) (643mg, 17%); MS (ES+), m/e 375 [M+H]⁺.

Description 4

1,1-Dimethylethyl 7-({1-[(2-propen-1-yloxy)carbonyl]-4-piperidinyl}oxy)-1 ,2,4,5- tetrahydro-3r/-3-benzazepine-3-carboxylate (D4) 2-Propen-1-yl 4-hydroxy-1-piperidinecarboxylate (J.Med. Chem.,1998, 41(25), 4983- 4994) (6.64g, 36 mmol), and triphenylphosphine (9.39g, 36 mmol) were dissolved in tetrahydrofuran (100 ml) at 0°C. 7-Hydroxy-1 ,2,4,5-tetrahydro-benzo[ |azepine-3- carboxylic acid tert-butyl ester (Description 3 in PCT publication number WO 02/40471) (7.55g, 29 mmol) was added followed by dropwise addition of diethyl azodicarboxylate (5.65 ml, 36 mmol) and the solution stirred at room temperature for 72 hours. The reaction was concentrated in vacuo and the residue partitioned between 1 M sodium hydroxide solution and ethyl acetate. The organic layer was washed successively with 1M sodium hydroxide solution, water, brine, dried over anhydrous sodium sulfate and concentrated in vacuo to a crude oil which was purified by column chromatography, eluting with a mixture hexane:ethylacetate (1 :10) then ethylacetate to afford the title compound (7.01g, 56%) MS (ES+) m/e 431 [M+H]⁺.

Description 5

1,1-Dimethylethyl 7-(4-piperidinyloxy)-1,2,4,5-tetrahydro-3 -3-benzazepine-3- carboxylate (D5)

1 ,1-Dimethylethyl 7-({1-[(2-propen-1-yloxy)carbonyl]-4-piperidinyl}oxy)-1 ,2,4,5- tetrahydro-3H-3-benzazepine-3-carboxylate (D4) (4.30g, 10 mmol) and dimedone (14.02 g, 100 mmol) were dissolved in THF (300 ml) at 0°C.

Tetrakistriphenyphosphinepalladium(O) (2.30g, 2mmol) was added and the solution stirred at room temperature for 24 hours. Solvents were removed in vacuo and the residue was partitioned between 3M sodium hydroxide solution and diethyl ether. The organic phase was washed with 3M soidium hydroxide solution (x3), water (x3), brine, dried over anhydrous sodium sulfate and concentrated in vacuo to a crude solid. The solid was purified by column chromatography eluting with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1 :9:90) to afford the title compound (D9) (1.70g, 49%) MS (ES+) m/e 347 [M+H]⁺.

Description 6

1 ,1 -Dimethylethyl 7-{[1 -(1 -methylethyl)-4-piperidinyl]oxy}-1 ,2,4,5-tetrahydro-3H-3- benzazepine-3-carboxylate (D6)

1 ,1 -Dimethylethyl 7-(4-piperidinyloxy)-1 ,2,4,5-tetrahydro-3H-3-benzazepine-3- carboxylate (D5) (346 mg, 1mmol) and acetone (734 μL, 10 mmol) were stirred in a mixture of 5% acetic acid in dichloromethane for 1 hour. The solution was treated with sodium triacetoxyborohydride (2.11g, 10mmol) and the suspension stirred at room temperature for 16 hours. The mixture was partitioned between saturated sodium carbonate and dichloromethane. The organic layer was washed with water, brine, dried over anhydrous sodium sulfate and concentrated in vacuo to a crude solid. The solid was purified by column chromatography eluting with dichloromethane then a mixture of.880 ammonia:ethanol:dichloromethane (1 :9:90) to afford the title compound (211mg, 54%); MS (ES+) m/e 389 [M+H]⁺.

Description 7

1 ,1 -Dimethylethyl 7-[({1 -[(2-propen-1 -yloxy)carbonyl]-4-piperidinyl}methyl)oxy]- 1 ,2,4,5-tetrahydro-3H-3-benzazepine-3-carboxylate (D7)

The title compound was prepared in an analogous manner to that used for D4 using 2- propen-1-yl 4-(hydroxymethyl)-1-piperidinecarboxylate (EP 394991) and 7-hydroxy- 1 ,2,4,5-tetrahydro-benzo[o]azepine-3-carboxylic acid tetf-butyl ester (Description 3 in PCT publication number WO 02/40471) to afford the title compound (3.24g, 73%); MS (ES+) m/e 445 [M+H]\

Description 8 1,1-Dimethylethyl 7-[(4-piperidinylmethyl)oxy]-1,2,4,5-tetrahydro-3H-3- benzazepine-3-carboxylate (D8)

The title compound was prepared from 1,1 -dimethylethyl 7-[({1-[(2-propen-1- yloxy)carbonyl]-4-piperidinyl}methyl)oxy]-1 ,2,4,5-tetrahydro-3H-3-benzazepine-3- carboxylate (D7) in an analogous manner to that used in D5 to afford the title compound (1.06g, 59%); MS (ES+) m/e 361 [M+H]⁺.

Description 9 1 ,1 -Dimethylethyl 7-({[1 -(1 -methylethyl)-4-piperidinyl]methyl}oxy)-1 ,2,4,5- tetrahydro-3H-3-benzazepine-3-carboxylate (D9)

The title compound was prepared from 1,1 -dimethylethyl 7-[(4-piperidinylmethyl)oxy]- 1 ,2,4,5-tetrahydro-3H-3-benzazepine-3-carboxylate (D8) in an analogous manner to that used in D6 to afford the title compound (283 mg, 70%); MS (ES+) m/e 403 [M+Η]⁺.

Description 10

7-Amino-8-(3-piperidin-1-yI-propoxy)-1,2,4,5-tetrahydro-benzo[d]azepine-3- carboxylic acid fe/τ-butyl ester (D10) Step 1 : 7-Nitro-8-(3-piperidin-1-yl-propoxy)-1,2,4,5-tetrahydro-benzo[d]azepine-3- carboxylic acid ferf-butyl ester

The title compound was prepared from 7-Hydroxy-8-nitro-1 ,2,4,5-tetrahydro- benzo[d]azepine-3-carboxylic acid ferf-butyl ester (WO 0368752) and 1-(2- chloroethyl)piperidine hydrochloride in an analogous manner to that used in D3 except methyl ethyl ketone was used instead of dimethylformamide; MS (ES+), m/e 434 [M+H]⁺.

Step 2: 7-Amino-8-(3-piperidin-1 -yl-propoxy)-1 ,2,4,5-tetrahydro-benzo[d]azepine-3- carboxylic acid ferf-butyl ester

The product of Step 1 (1.54g, 3.55mmol) was dissolved in ethanol (50ml), treated with

10% palladium on charcoal and shaken under a pressure of hydrogen (50psi) for 2 hours. The reaction mixture was then filtered and the filtrate reduced in vacuo to furnish the title compound; MS (ES+), m/e 404 [M+H]⁺.

Example 1

7-(3-Piperidin-1 -yl-propoxy)-1 ,2,4,5-tetrahydro-1 H-benzo[ ]azepine (E1)

7-(3-Piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-benzo[c/Iazepine-3-carboxylic acid tert- butyl ester (D2) (6.18g, 16mmol) was dissolved in dichloromethane (30ml) at 0°C and treated with trifluoroacetic acid (30ml). The solution was stirred at room temperature for one hour and solvents removed in vacuo. The residue was partitioned between ethyl acetate and 2N sodium hydroxide solution and the organic layer separated. The aqueous phase was extracted with ethyl acetate (x5). The combined organic extracts were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude residue was purified by column chromatography eluting initially with dichloromethane then a mixture of 0.880 ammonia:ethanol:dichloromethane (1 :9:90) to afford the title compound (E1) MS (ES+) m/e 289 [M+H]⁺.

Example 2 3-Cyclopropylmethyl-7-(3-piperidin-1 -yl-propoxy)-1 ,2,4,5-tetrahydro-1 H- benzo[d]azepine (E2)

The crude product of Example 1 (200 mg, 0.7 mmol) and cyclopropanecarboxaldehyde (79 μL, 1.1 mmol) were stirred at room temperature for 2 hours in 5% acetic acid in dichloromethane. The solution was then treated with sodium triacetoxyborohydride (297mg, 1.4mmol) and the suspension stirred at room temperature for 16 hours. The resulting solution was applied directly to a SCX cartridge (Varian bond-elute, 10g) and eluted with methanol then a mixture of .880 ammonia:methanol (1 :9). The basic fractions were then reduced in vacuo and the residue purified by column chromatography, eluting with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1 :9:90) to afford the title compound; (189mg, 79%), MS(ES+) m/e 343 [M+H]⁺.

Examples 3-11 Examples 3-11 (E3-E11) were prepared using an analogous method to that described in Example 2 (E2) by substituting cyclopropanecarboxaldehyde for the appropriate aldehyde indicated in the table.

Example 12

1 -[7-(3-Piperidin-1 -yl-propoxy)-1 ,2,4,5-tetrahydro-benzo[d]azepin-3-yl]-ethanone

7-(3-Piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-1H-benzo[o]azepine (E1) (200mg, 0.7mmol) and triethylamine (117μL, 0.84mmol) were dissolved in dichloromethane (3ml). The solution was treated with acetyl chloride (57μL, 0.77mmol) and stirred at room temperature for 16 hours. The solvent was removed in vacuo, and the residue purified by column chromatography eluting dichloromethane and then .880 ammonia:ethanol:dichloromethane (1 :9:90) to afford the title compound (E12) (178 mg, 63%), MS(ES+) m/e 331 [M+Hf .

Examples 13-17 Examples 13-17 (E13-E17) were prepared using an analogous method to that described in Example 12 (E12) by substituting acetyl chloride for the appropriate acid chloride indicated in the table.

Example 18

3-lsobutyl-7-(3-piperidin-1 -yl-propoxy)-1 ,2,4,5-tetrahydro-1 H-benzo[d]azepine (E18)

7-(3-Piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-1H-benzo[ ]azepine (E1) (361 mg, 1mmol) and potassium carbonate (417mg, 3 mmol) was suspended in 2-butanone (20 ml). The mixture was treated with isobutyliodide (127μL, 1.1 mmol) and heated at reflux for 16 hours. The solids were filtered and the filtrate concentrated in vacuo. The residue was purified by column chromatography eluting with a mixture of 0.880 ammonia:ethanol:dichloromethane (1:9:90) to afford the title compound (E18) (184mg, 53%), MS(ES+) m/e 345 [M+Hf .

Examples 19-26

Examples 19-26 (E19-E26) were prepared from Description 1 (D1) in a two step procedure using an analogous method to that described in Description 2 (D2) followed by an analogous method described in Example 1 (E1) by substituting piperidine for the appropriate amine indicated in the table.

Example 27

7-(4-Piperidin-1 -yl-butoxy)-2,3,4,5-tetrahydro-1 H-benzo[d]azepine (E27)

The title compound (E27) was prepared from 7-hydroxy-1 ,2,4,5-tetrahydro- benzo[ ]azepine-3-carboxylic acid tert-butyl ester (Description 3 in WO 02/40471) and 1 ,4-dibromobutane using an analogous method to that described in Description 1 (D1) followed by Description 2 (D2) and Example 1 (E1) MS(ES+) m/e 303 [M+Hf.

Example 28

7-(2-Piperidin-1 -yl-ethoxy)-2,3,4,5-tetrahydro-1 H-benzo[d]azepine (E28)

The title compound (E28) was prepared from 7-(2-piperidin-1-yl-ethoxy)-1 ,2,4,5- tetrahydro-benzo[o]azepine-3-carboxylic acid tert-butyl ester (D3) using the method described in Example 1 (E1) MS(ES+) m/e 274 [M+Hf.

Example 29

3-Cyclopropylmethyl-7-(4-piperidin-1-yl-butoxy)-2,3,4,5-tetrahydro-1H- benzo[d]azepine (E29)

A mixture of 7-(4-piperidin-1-yl-but (E27) (150mg, O.δmmol), cyclopropanecarboxaldehyde (53mg, 0.75mmol), acetic acid (0.1ml) and (polystyrylmethyl)trimethylammonium cyanoborohydride (250mg, 4mmol/g loading, 1 mmol) in methanol (5ml) was stirred at room temperature for 18 hours. The reaction mixture was applied to an SCX column (Varian bond-elute, 10g) eluting with methanol followed by a mixture of 0.880 ammonia/methanol (1 :9). The basic fractions were combined and concentrated in vacuo giving the title compound (E29) MS(ES+) m/e 357 [M+Hf.

Example 30

3-Cyclopropylmethyl-7-(2-piperidin-1-yl-ethoxy)-2,3,4,5-tetrahydro-1H- benzo[d]azepine (E30)

The title compound (E30) was prepared from 7-(2-piperidin-1-yl-ethoxy)-2,3,4,5- tetrahydro-1 H-benzofαflazepine (E28) using an analogous method to that described in Example 29 (E29). MS(ES+) m/e 329 [M+Hf.

Example 31

7-{[1 -(1 -Methylethyl)-4-piperidinyl]oxy}-2,3,4,5-tetrahydro-1 W-3-benzazepine (E31)

1 ,1 -Dimethylethyl 7-{[1-(1-methylethyl)-4-piperidinyl]oxy}-1 ,2,4,5-tetrahydro-3H-3- benzazepine-3-carboxylate (D6) (200 mg, 0.69 mmol) was stirred at room temperature in a 1 :1 solution of dichloromethane:trifluoroacetic acid (10ml) for 1 hour. The solution was concentrated in vacuo and the residue co-evaporated with dichloromethane three times. The residue was passed through a SCX cartridge (Varian, 10g), washing with methanol and eluting products with 10% .880 ammonia in methanol. The ammonia solution was concentrated in vacuo and the residue purified by column chromatography, eluting with a dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1:9:190), to afford the title compound (160mg, 65%); MS (ES+) m/e 289 [M+Hf.

Example 32 7-{[1 -(1 -Methylethyl)-4-piperidinyl]oxy}-3-(phenylmethyl)-2,3,4,5-tetrahydro-1 H-3- benzazepine (E32)

The title compound was prepared from 7-{[1-(1-methylethyl)-4-piperidinyl]oxy}-2,3,4,5- tetrahydro-1H-3-benzazepine (E31) and benzaldyhyde using the general method described in Example 29 to afford the title compound (7.01 g, 56%); MS (ES+) m/e 431 [M+Hf.

Examples 33-34

The following examples were prepared according to the general method in Example 32, substituting benzaldehyde with the appropriate Aldehyde.

Example 35

7-({[1-(1-Methylethyl)-4-piperidinyl]methyl}oxy)-2,3,4,5-tetrahydro-1H-3- benzazepine (E35)

The title compound was prepared from 1 ,1 -dimethylethyl 7-({[1-(1-methylethyl)-4- piperidinyl]methyl}oxy)-1 ,2,4,5-tetrahydro-3H-3-benzazepine-3-carboxylate (D9) in an analogous manner to that used in E31 to afford the title compound (E35) (178 mg, 80%); MS (ES+) m/e 303 [M+Hf.

Example 36

3-Phenyl-7-{[3-(1-piperidinyl)propylloxy}-2,3,4,5-tetrahydro-1H-3-benzazepine (E36)

7-(3-Piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-1H-benzo[c |azepine (E1) (216mg, 0.75 mmol), iodobenzene (100 μL, 0.9 mmol), rac-2,2-bis(diphenylphosphino)-1,1-binapthyl (35 mg, 0.06 mmol) and sodium tert-butoxide (80 mg, 0.83 mmol) were suspended in toluene (10 ml) and treated with tris(dibenzylideneacetone) dipalladium(O) (35 mg, 0.04 mmol). The mixture was then heated at reflux for 24 hours, cooled, and then applied directly to a SCX ion exchange cartridge (Varian, 10g), eluting with methanol and then a mixture of .880 ammonia:methanol (1:9). The ammonia fractions were concentrated in vacuo and the residue purified by column chromatography, eluting with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1 :9:90) to afford the title compound (90mg, 33%); MS (ES+) m/e 365 [M+Hf.

Examples 37-38 The following examples were prepared according to the general method in E36, substituting iodobenzene with the appropriate aryl halide.

Example 39

W-(1 -Methylethyl)-7-{[3-(1 -piperidinyl)propyl]oxy}-1 ,2,4,5-tetrahydro-3H-3- benzazepine-3-carboxamide (E39)

The dihydrochloride salt of 7-(3-piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-1H- benzo[c/]azepine (E1) (361 mg, 1 mmol), and triethylamine (418 μl, 3 mmol), were dissolved in dichloromethane (5 ml). The solution was treated with isopropyl isocyanate (102 mg, 1.2 mmol) and stirred at room temperature for 16 hours. The solution was concentrated in vacuo to a crude solid that was purified by column chromatography eluting with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1 :9:90), to afford the title compound (270mg, 74%); MS (ES+) m/e 374 [M+H]⁺.

Examples 40-41

The following examples were prepared according to the general method described for E39 substituting phenyl isocyanate for the appropriate isocyanate.

Example 42

3-(Butylsulfonyl)-7-{[3-(1-piperidinyl)propyl]oxy}-2,3,4,5-tetrahydro-1W-3- benzazepine (E42)

The dihydrochloride salt of 7-(3-piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-1H- benzo[cQazepine (E1) (361 mg, 1 mmol), and triethylamine (418 μl, 3 mmol), were dissolved in dichloromethane (5 ml). The solution was treated with 1 -butane sulfonyl chloride (188 mg, 1.2 mmol) and stirred at room temperature for 16 hours. The solution was concentrated in vacuo to a crude solid that was purified by column chromatography eluting with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1 :9:90), to afford the title compound (176mg, 43%); MS (ES+) m/e 374 [M+Hf.

Examples 43-44

The following examples were prepared according to the general method general method described for E42 substituting 1-butane sulfonyl chloride for the appropriate sulphonyl chloride.

Example 45

3-[(3,4-Dichlorophenyl)carbonyl]-7-{[3-(1-piperidinyl)propyl]oxy}-2,3,4,5- tetrahydro-1 H-3-benzazepine (E45)

3,4-Dichlorobenzoic acid (172 mg, 0.9mmol), N-hydroxybenzotriazole (120 mg, 0.9 mmol) and N-cyclohexylcarbodiimide-N'-methyl polystyrene (1.8 mmol/g) (833 mg, 1.5 mmol) were stirred at room temperature in dichloromethane (5ml). After 15 minutes the product of Example 1 (E1) (216 mg, 0.75 mmol) was added and the slurry stirred for a further 16 hours. The mixture was then applied directly to a SCX ion exchange cartridge (Varian, 10g), eluting with methanol and then a mixture of .880 ammonia:methanol (1 :9). The ammonia fractions were concentrated in vacuo and the residue purified by column chromatography, eluting with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1:9:90), to afford the title compound (240mg, 69%); MS (ES+) m/e 461/462/463/464 [M+H]⁺.

Examples 46-47

The following examples were prepared according to the general method described for E45 substituting 3,4-dichlorobenzoic acid for the appropriate carboxylic acid.

Example 48

3-[2-(Methyloxy)ethyl]-7-{[3-(1-piperidinyI)propyl]oxy}-2,3,4,5-tetrahydro-1W-3- benzazepine (E48)

7-(3-Piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-1H-benzo[c/]azepine (E1) (216 mg, 0.75 mmol), 2-bromoethyl methyl ether (78μl, 0.83mmol) and potassium carbonate (311 mg, 2.25 mmol) were suspended in 2-butanone (10 ml) and heated at reflux for 24 hours. The solids were filtered, washed with acetone and concentrated in vacuo to a crude oil. The oil was purified by column chromatography eluting with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1:9:90) to afford the title compound (205mg, 59%); MS (ES+) m/e 347 [M+Hf.

Examples 49-52

The following examples were prepared according to the general method described for E50 substituting 2-bromoethyl methyl ether for the appropriate alkyl halide.

Example 53

N,N-diethyl-2-(7-{[3-(1-piperidinyl)propyl]oxy}-1,2,4,5-tetrahydro-3H-3-benzazepin- 3-yl)acetamide (E53)

7-(3-Piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-1H-benzo[c |azepine (E1) (361 mg, 1 mmol) and triethylamine (432 μL, 3.1 mmol) were dissolved in methanol (2ml) and treated with chloroacetic acid diethylamine (150 μL, 1 mmol) in acetone (8ml). The solution was stirred at room temperature for 16 hours and concentrated in vacuo. The residue was applied to a SCX ion exchange cartridge (Varian, 10g), eluting with methanol and then a mixture of .880 ammonia:methanol (1:9). The ammonia fractions were concentrated in vacuo and the residue purified by column chromatography, eluting with a with dichloromethane then a mixture of .880 ammonia:ethanol:dichloromethane (1 :9:90) to afford the title compound (353 mg, 88%); MS (ES+) m/e 402 [M+H]⁺.

Example 54 8-(3-Piperidin-1-yl-propoxy)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-ylamine (E54)

A solution of 7-amino-8-(3-piperidin-1-yl-propoxy)-1 ,2,4,5-tetrahydro-benzo[c]azepine-3- carboxylic acid tert-butyl ester (D10) (0,1g, 0.25mmol) in methanol (5ml) was treated with a 4M solution of HCl in dioxan (5ml) and stirred at room temperature for 2 hours. The reaction was then reduced in vacuo to to afford the title compound (0.085g); MS (ES+) m/e 304 [M+Hf.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

Biological Data A membrane preparation containing histamine H3 receptors may be prepared in accordance with the following procedures:

(i) Generation of histamine H3 cell line DNA encoding the human histamine H3 gene (Huvar, A. et al. (1999) Mol. Pharmacol. 55(6), 1101-1107) was cloned into a holding vector, pCDNA3.1 TOPO (InVitrogen) and its cDNA was isolated from this vector by restriction digestion of plasmid DNA with the enzymes BamH1 and Not-1 and ligated into the inducible expression vector pGene (InVitrogen) digested with the same enzymes. The GeneSwitch™ system (a system where in transgene expression is switched off in the absence of an inducer and switched on in the presence of an inducer) was performed as described in US Patent nos: 5,364,791; 5,874,534; and 5,935,934. Ligated DNA was transformed into competent DH5α E. coli host bacterial cells and plated onto Luria Broth (LB) agar containing Zeocin™ (an antibiotic which allows the selection of cells expressing the sh ble gene which is present on pGene and pSwitch) at 50μg ml^"1. Colonies containing the re-ligated plasmid were identified by restriction analysis. DNA for transfection into mammalian cells was prepared from 250ml cultures of the host bacterium containing the pGeneH3 plasmid and isolated using a DNA preparation kit (Qiagen Midi-Prep) as per manufacturers guidelines (Qiagen).

CHO K1 cells previously transfected with the pSwitch regulatory plasmid (InVitrogen) were seeded at 2x10e6 cells per T75 flask in Complete Medium, containing Hams F12 (GIBCOBRL, Life Technologies) medium supplemented with 10% v/v dialysed foetal bovine serum, L-glutamine, and hygromycin (100μg ml^"1), 24 hours prior to use. Plasmid DNA was transfected into the cells using Lipofectamine plus according to the manufacturers guidelines (InVitrogen). 48 hours post transfection cells were placed into complete medium supplemented with 500μg ml^"1 Zeocin™.

10-14 days post selection 10nM Mifepristone (InVitrogen), was added to the culture medium to induce the expression of the receptor. 18 hours post induction cells were detached from the flask using ethylenediamine tetra-acetic acid (EDTA; 1 :5000; InVitrogen), following several washes with phosphate buffered saline pH 7.4 and resuspended in Sorting Medium containing Minimum Essential Medium (MEM), without phenol red, and supplemented with Earles salts and 3% Foetal Clone II (Hyclone). Approximately 1x 10e7 cells were examined for receptor expression by staining with a rabbit polyclonal antibody, 4a, raised against the N-terminal domain of the histamine H3 receptor, incubated on ice for 60 minutes, followed by two washes in sorting medium. Receptor bound antibody was detected by incubation of the cells for 60 minutes on ice with a goat anti rabbit antibody, conjugated with Alexa 488 fluorescence marker (Molecular Probes). Following two further washes with Sorting Medium, cells were filtered through a 50μm Filcon™ (BD Biosciences) and then analysed on a FACS

Vantage SE Flow Cytometer fitted with an Automatic Cell Deposition Unit. Control cells were non-induced cells treated in a similar manner. Positively stained cells were sorted as single cells into 96-well plates, containing Complete Medium containing 500μg ml^"1 Zeocin™ and allowed to expand before reanalysis for receptor expression via antibody and ligand binding studies. One clone, 3H3, was selected for membrane preparation.

(ii) Membrane preparation from cultured cells All steps of the protocol are carried out at 4°C and with pre-cooled reagents. The cell pellet is resuspended in 10 volumes of buffer A2 containing 50mM N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.40) supplemented with 10e-4M leupeptin (acetyl-leucyl-leucyl-arginal; Sigma L2884), 25μg/ml bacitracin (Sigma B0125), 1mM ethylenediamine tetra-acetic acid (EDTA), 1mM phenylmethylsulfonyl fluoride (PMSF) and 2x10e-6M pepstain A (Sigma). The cells are then homogenised by 2 x 15 second bursts in a 1 litre glass Waring blender, followed by centrifugation at 500g for 20 minutes. The supernatant is then spun at 48,000g for 30 minutes. The pellet is resuspended in 4 volumes of buffer A2 by vortexing for 5 seconds, followed by homogenisation in a Dounce homogeniser (10-15 strokes). At this point the preparation is aliquoted into polypropylene tubes and stored at -70°C.

Compounds of the invention may be tested for in vitro biological activity in accordance with the following assays:

(I) Histamine H3 binding assay

For each compound being assayed, in a white walled clear bottom 96 well plate, is added:-

(a) 10μl of test compound (or 10μl of iodophenpropit (a known histamine H3 antagonist) at a final concentration of 10mM) diluted to the required concentration in 10% DMSO;

(b) 10μl ¹²⁵l 4-[3-(4-iodophenylmethoxy)propyl]-1H-imidazolium (iodoproxyfan) (Amersham; 1.85MBq/μl or 50μCi/ml; Specific Activity ~2000Ci/mmol) diluted to 200pM in assay buffer (50mM Tris(hydroxymethyl)aminomethane buffer (TRIS) pH 7.4, 0.5mM ethylenediamine tetra-acetic acid (EDTA)) to give 20pM final concentration; and

(c) 80μl bead/membrane mix prepared by suspending Scintillation Proximity Assay (SPA) bead type WGA-PVT at 100mg/ml in assay buffer followed by mixing with membrane (prepared in accordance with the methodology described above) and diluting in assay buffer to give a final volume of 80μl which contains 7.5μg protein and 0.25mg bead per well - mixture was pre-mixed at room temperature for 60 minutes on a roller. The plate is shaken for 5 minutes and then allowed to stand at room temperature for 3-4 hours prior to reading in a Wallac Microbeta counter on a 1 minute normalised tritium count protocol. Data was analysed using a 4-parameter logistic equation.

(II) Histamine H3 functional antagonist assay

(a) 10μl of test compound (or 10μl of guanosine 5'- triphosphate (GTP) (Sigma) as non-specific binding control) diluted to required concentration in assay buffer (20mM N- 2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) + 100mM NaCI + 10mM MgCI₂, pH7.4 NaOH); (b) 60μl bead/membrane/GDP mix prepared by suspending wheat germ agglutinin- polyvinyltoluene (WGA-PVT) scintillation proximity assay (SPA) beads at 100mg/ml in assay buffer followed by mixing with membrane (prepared in accordance with the methodology described above) and diluting in assay buffer to give a final volume of 60μl which contains 10μg protein and 0.5mg bead per well - mixture is pre-mixed at 4°C for 30 minutes on a roller and just prior to addition to the plate, 10μM final concentration of guanosine 5' diphosphate (GDP) (Sigma; diluted in assay buffer) is added; The plate is incubated at room temperature to equilibrate antagonist with receptor/beads by shaking for 30 minutes followed by addition of: (c) 10μl histamine (Tocris) at a final concentration of 0.3μM; and

(d) 20μl guanosine 5' [γ35-S] thiotriphosphate, triethylamine salt (Amersham; radioactivity concentration = 37kBq/μl or 1mCi/ml; Specific Activity 1160Ci/mmol) diluted to 1.9nM in assay buffer to give 0.38nM final. The plate is then incubated on a shaker at room temperature for 30 minutes followed by centrifugation for 5 minutes at 1500 rpm. The plate is read between 3 and 6 hours after completion of centrifuge run in a Wallac Microbeta counter on a 1 minute normalised tritium count protocol. Data is analysed using a 4-parameter logistic equation. Basal activity used as minimum i.e. histamine not added to well.

Results

The compounds of Examples E1-54 were tested in the histamine H3 functional antagonist assay and exhibited antagonism in the following range: 6.0.-10.0 pK_b. More particularly, the compounds of Examples E1-11 , E13-19, E26, E29-34, E37, E40, E42-44 and E46-54 exhibited antagonism in the following range: 8.0-10.0 pK_b. Yet more particularly, the compounds of Examples E2-11 , E18-19, E26, E29-34, E48 and E50-53 exhibited antagonism in the following range: 8.5-10.0 pK_b.

Claims

CLAIMS:

1. A compound of formula (I):

(I) wherein:

R¹ represents hydrogen, -Cι_₆ alkyl, -C₃.₈ cycloalkyl, aryl, heterocyclyl, heteroaryl, -d.₆ alkyl-aryl, -d„₆ alkyl-heteroaryl, -Cι_₆ alkyl-heterocyclyl, -aryl-aryl, -aryl-heteroaryl, -aryl- heterocyclyl,- heteroaryl-aryl, -heteroaryl-heteroaryl, -heteroaryl-heterocyclyl, - heterocyclyl-aryl, -heterocyclyl-heteroaryl, -heterocyclyl-heterocyclyl, wherein R¹ may be optionally substituted by one or more substituents which may be the same or different, and which are selected from the group consisting of halogen, hydroxy, cyano, nitro, oxo, trifluoromethyl, trifluoromethoxy, fluoromethoxy, difluoromethoxy, d.₆ alkyl, pentafluoroethyl, d-e alkoxy, C₂.₆ alkenyl, aryld.₆ alkoxy, d„₆ alkylthio, d-e alkoxyd-₆ alkyl, C₃.₇ cycloalkyld.₆ alkoxy, d.₆ alkanoyl, d-e alkoxycarbonyl, C|_₆ alkylsulfonyl, Cι.₆ alkylsulfinyl, d-₆ alkylsulfonyloxy, C₁-6 alkylsulfonylCι._e alkyl, sulfonyl, arylsulfonyl, arylsulfonyloxy, arylsulfonylC|-6 alkyl, aryloxy, d-e alkylsulfonamido, d-e alkylamido, d-e alkylsulfonamidoCι.₆ alkyl, d-e alkylamidod-e alkyl, arylsulfonamido, arylcarboxamido, arylsulfonamidoCι_₆ alkyl, arylcarboxamidoCι_₆ alkyl, aroyl, aroyld.₆ alkyl, arylCi.₆ alkanoyl, or a group NR¹⁵R¹⁶, CONR¹⁵R¹⁶, NR¹⁵COR¹⁶, NR¹⁵R¹⁶SO₂ or SO₂NR¹⁵R¹⁶, wherein R¹⁵ and R¹⁶ independently represent hydrogen or d-e alkyl or together may be fused to form a 5- to 7- membered non-aromatic heterocyclic ring optionally interrupted by an O or S atom;

X represents bond, d_₆ alkyl, CO, SO₂ or CONR⁴, such that when R¹ represents -C₃._B cycloalkyl, X represents C|.₆ alkyl, CO, SO₂ or CONR⁴; R² represents halogen, d„₆ alkyl, d.₆ alkoxy, cyano, amino or trifluoromethyl; n is 0, 1 or 2;

R⁴ represents hydrogen or d.₆ alkyl, or together with R¹ may form a heterocyclyl group; R³ represents -(CH₂)_q-NR¹¹

wherein q is 2, 3 or 4;

R¹¹ and R¹² independently represent Cι-₆ alkyl or together with the nitrogen atom to which they are attached represent an N-linked nitrogen containing heterocyclic group optionally substituted by one or more R¹⁷ groups;

R¹³ represents hydrogen, d.₆ alkyl, C₃.₈ cycloalkyl, -C₁-₆ alkyl-aryl or heterocyclyl; R¹⁴ and R¹⁷ independently represent halogen, d_₆ alkyl, haloalkyl, OH, did-e alkylamino, d„₆ alkoxy or heterocyclyl; f and k independently represent 0, 1 or 2; g is 0, 1 or 2 and h is 0, 1 , 2 or 3, such that g and h cannot both be 0; or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 which is a compound of formula E1-E54 or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition which comprises the compound of formula (I) as defined in claim 1 or claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

4. A compound as defined in claim 1 or claim 2 for use in therapy.

5. A compound as defined in claim 1 or claim 2 for use in the treatment of neurological diseases.

6. Use of a compound as defined in claim 1 or claim 2 in the manufacture of a medicament for the treatment of neurological diseases.

7. A method of treatment of neurological diseases which comprises administering to a host in need thereof an effective amount of a compound of formula (I) as defined in claim 1 or claim 2 or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition for use in the treatment of neurological diseases which comprises the compound of formula (I) as defined in claim 1 or claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.