Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/20—Hypnotics; Sedatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/22—Anxiolytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to imidazopyrimidylmethyl substituted piperidine derivatives and their use as pharmaceuticals.
• polypeptides and polynucleotides encoding polypeptides which are ligands for the orexin-1 receptor, e.g. orexin-A (Lig72A) are disclosed in EP849361.
• orexin receptor antagonist SB334867 potently reduced hedonic eating in rats (White et al (2005) Peptides 26 pp 2231 to 2238) and also attenuated high-fat pellet self- administration in rats (Nair et al (2008) British Journal of Pharmacology, published online 28 January 2008).
• the search for new therapies to treat obesity and other eating disorders is an important challenge.
• WHO definitions a mean of 35% of subjects in 39 studies were overweight and a further 22% clinically obese in westernised societies. It has been estimated that 5.7% of all healthcare costs in the USA are a consequence of obesity. About 85% of Type 2 diabetics are obese. Diet and exercise are of value in all diabetics.
• diabetes The incidence of diagnosed diabetes in westernised countries is typically 5% and there are estimated to be an equal number undiagnosed. The incidence of both diseases is rising, demonstrating the inadequacy of current treatments which maybe either ineffective or have toxicity risks including cardiovascular effects.
• Treatment of diabetes with sulfonylureas or insulin can cause hypoglycaemia, whilst metformin causes GI side-effects.
• No drug treatment for Type 2 diabetes has been shown to reduce the long-term complications of the disease. Insulin sensitisers will be useful for many diabetics, however they do not have an anti-obesity effect.
• Antagonists of the orexin receptors may therefore be useful in the treatment of sleep disorders including insomnia.
• WO01/96302 discloses cyclic amine derivatives.
• WO03/002561 discloses N-aroyl cyclic amine derivatives as orexin antagonists.
• Compounds disclosed in WO03/002561 include piperidine derivatives substituted at the 2- position with bicyclic heteroarylmethyl groups.
• piperidine derivatives substituted at the 2- position with an imidazo[l,2- ⁇ ]pyrimidin-2-ylmethyl group have beneficial properties including, for example, increased oral bioavailability and significantly increased solubility in physiologically relevant media compared to the prior art compounds.
• R 1 is (C 1 . 4 )alkyl, halo, halo(C 1 . 4 )alkyl, (C 1 . 4 )alkoxy, halo(C 1 . 4 )alkoxy, (C 1 . 4 )alkyl-O-( C 1 . 4 )alkyl, CN, NR 5 R 6 wherein R 5 is H or (C 1 . 4 )alkyl and R 6 is H or (C 1 . 4 )alkyl; R 2 is (C 1 . 4 )alkyl, halo, halo(C 1 . 4 )alkyl, (C 1 . 4 )alkoxy, halo(C 1 .
• R 3 is (C 1 . 4 )alkyl, halo, halo(C 1 . 4 )alkyl, (Ci. 4 )alkoxy, halo(C 1 . 4 )alkoxy, (C 1 . 4 )alkyl-O-( C 1 . 4 )alkyl, CN, NR 9 R 10 wherein R 9 is H or (C 1 . 4 )-alkyl and R 10 is H or (C 1 . 4 )-alkyl; n is O or 1; p is 0 or 1; and q is O or l; with the proviso that p and q are not both 0; or a pharmaceutically acceptable salt thereof.
• Ar is a group of formula (II).
• Ar is a group of formula (III). In one embodiment Ar is a group of formula (II) and n is 0.
• Ar is a group of formula (II) and n is 1.
• Ar is a group of formula (II), n is 1 and R 1 is halogen.
• Ar is a group of formula (II), n is 1 and R 1 is chlorine.
• Ar is a group of formula (II), n is 1, p is 1, q is 0, R 1 is chlorine and R 2 is methyl.
• Ar is a group of formula (II), n is 0, p is 1 , q is 0 and R 2 is methyl.
• Ar is a group of formula (II), n is 0, p is 1, q is 1 and one of R 2 and R 3 is halo and the other is (C 1 _ 4 )-alkyl. In one embodiment Ar is a group of formula (II), n is 0, p is 1, q is 1, R 2 is halo and R 3 is (C 1 _ 4 )-alkyl.
• Ar is a group of formula (II), n is 0, p is 1, q is 1, R 2 is chloro and R 3 is methyl. In one embodiment Ar is a group of formula (III) and n is 0.
• Ar is a group of formula (III) and n is 1.
• Ar is a group of formula (III), n is 1 and R 1 is halogen.
• Ar is a group of formula (III), n is 1 and R 1 is chlorine.
• Ar is a group of formula (III), n is 1, p is 1, q is 0, R 1 is chlorine and R 2 is methyl.
• Ar is a group of formula (III), n is 0, p is 1 , q is 0 and R 2 is methyl.
• Ar is a group of formula (III), n is 0, p is 1, q is 1 and one of R 2 and R 3 is halo and the other is (Ci_ 4 )-alkyl. In one embodiment Ar is a group of formula (III), n is 0, p is 1 , q is 1 , R 2 is halo and
• R 3 is (C 1 _ 4 )-alkyl.
• Ar is a group of formula (III), n is 0, p is 1, q is 1, R 2 is chloro and R 3 is methyl.
• Examples of the compounds of the invention include: 7-methyl-2-( ⁇ (25)-l-[(2-methyl-5-phenyl-l,3-thiazol-4-yl)carbonyl]-2- piperidinyl ⁇ methyl)imidazo[l,2- ⁇ ]pyrimidine;
• the alkyl group maybe straight chain, branched or cyclic, or combinations thereof.
• (Ci_ 4 )alkyl are methyl or ethyl.
• An example Of(C 1 . 4 )alkoxy is methyloxy.
• Examples of halo(Ci_ 4 )alkyl include trifluoromethyl (i.e. -CF 3 ).
• Examples of (Ci_ 4 )alkoxy include methyloxy and ethyloxy.
• halo(Ci_ 4 )alkoxy examples include trifluoromethyloxy (i.e. - OCF 3 ).
• Halogen or "halo" when used, for example, in halo(C 1 _ 4 )alkyl means fluoro, chloro, bromo or iodo. It will be appreciated that for use in medicine the salts of the compounds of formula
• compositions of formula (I) should be pharmaceutically acceptable. Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art. Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse J.Pharm.Sci (1977) 66, pp 1-19. Such pharmaceutically acceptable salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid. Other salts e.g. oxalates or formates, may be used, for example in the isolation of compounds of formula (I) and are included within the scope of this invention.
• inorganic acids e.g. hydrochloric, hydrobromic, sulphuric, nitric
• Certain of the compounds of formula (I) may form acid addition salts with one or more equivalents of the acid.
• the present invention includes within its scope all possible stoichiometric and non-stoichiometric forms.
• the compounds of formula (I) may be prepared in crystalline or non-crystalline form and, if crystalline, may optionally be solvated, eg. as the hydrate.
• This invention includes within its scope stoichiometric solvates (eg. hydrates) as well as compounds containing variable amounts of solvent (eg. water). It will be understood that the invention includes pharmaceutically acceptable derivatives of compounds of formula (I) and that these are included within the scope of the invention.
• pharmaceutically acceptable derivative includes any pharmaceutically acceptable ester or salt of such ester 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.
• the compounds of formula (I) are S enantiomers. Where additional chiral centres are present in compounds of formula (I), the present invention includes within its scope all possible enantiomers and diastereoisomers, including mixtures thereof.
• the different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecif ⁇ c or asymmetric syntheses.
• the invention also extends to any tautomeric forms or mixtures thereof.
• the subject invention also includes isotopically-labeled compounds which are identical to those recited in formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.
• isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as 3 H, 11 C, 14 C, 18 F, 123 I or 125 I.
• Compounds of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention.
• Isotopically labeled compounds of the present invention for example those into which radioactive isotopes such as 3 H or 14 C have been incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, ie. 3 H, and carbon-14, ie. 14 C, isotopes are particularly preferred for their ease of preparation and detectability. 11 C and 18 F isotopes are particularly useful in PET (positron emission tomography).
• the compounds of formula (I) are intended for use in pharmaceutical compositions it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis).
• Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions.
• a process for the preparation of compounds of formula (I) and derivatives thereof The following schemes detail some synthetic routes to compounds of the invention. In the following schemes reactive groups can be protected with protecting groups and deprotected according to well established techniques.
• the starting materials for use in the scheme are commercially available, known in the literature or can be prepared by known methods.
• compositions may be prepared conventionally by reaction with the appropriate acid or acid derivative.
• the present invention provides compounds of formula (I) and their pharmaceutically acceptable derivatives for use in human or veterinary medicine.
• the compounds of formula (I) or their pharmaceutically acceptable salts may be of use for the treatment or prophylaxis of a disease or disorder where an antagonist of a human orexin receptor is required such as sleep disorders selected from the group consisting of Dyssomnias such as Primary Insomnia (307.42), Primary Hypersomnia (307.44), Narcolepsy (347), Breathing-Related Sleep Disorders (780.59), Circadian Rhythm Sleep Disorder (307.45) and Dyssomnia Not Otherwise Specified (307.47); primary sleep disorders such as Parasomnias such as Nightmare Disorder (307.47), Sleep Terror Disorder (307.46), Sleepwalking Disorder (307.46) and Parasomnia Not Otherwise Specified (307.47); Sleep Disorders Related to Another Mental Disorder such as Insomnia Related to Another Mental Disorder (307.42) and Hypersomnia Related to Another Mental Disorder (307.44); Sleep Disorder Due to a General Medical
• the compounds of formula (I) or their pharmaceutically acceptable salts may be of use for the treatment or prophylaxis of a disease or disorder where an antagonist of a human orexin receptor is required such as depression and mood disorders including Major Depressive Episode, Manic Episode, Mixed Episode and Hypomanic Episode; Depressive Disorders including Major Depressive Disorder, Dysthymic Disorder (300.4), Depressive Disorder Not Otherwise Specified (311); Bipolar Disorders including Bipolar I Disorder, Bipolar II Disorder (Recurrent Major Depressive Episodes with Hypomanic Episodes) (296.89), Cyclothymic Disorder (301.13) and Bipolar Disorder Not Otherwise Specified (296.80); Other Mood Disorders including Mood Disorder Due to a General Medical Condition (293.83) which includes the subtypes With Depressive Features, With Major Depressive-like Episode, With Manic Features and With Mixed Features),
• the compounds of formula (I) or their pharmaceutically acceptable salts may be of use for the treatment or prophylaxis of a disease or disorder where an antagonist of a human orexin receptor is required such as anxiety disorders including Panic Attack; Panic Disorder including Panic Disorder without Agoraphobia (300.01) and Panic Disorder with Agoraphobia (300.21); Agoraphobia; Agoraphobia Without History of Panic Disorder (300.22), Specific Phobia (300.29, formerly Simple Phobia) including the subtypes Animal Type, Natural Environment Type, Blood-Injection-Injury Type, Situational Type and Other Type), Social Phobia (Social Anxiety Disorder, 300.23), Obsessive-Compulsive Disorder (300.3), Posttraumatic Stress Disorder (309.81), Acute Stress Disorder (308.3), Generalized Anxiety Disorder (300.02), Anxiety Disorder Due to a General Medical Condition (293.84), Substance-Induced Anxiety Disorder, Separation Anxiety Disorder (309.21), Adjustment Disorders with Anxiety (309.24
• Substance Use Disorders such as Substance Dependence, Substance Craving and Substance Abuse
• Substance-Induced Disorders such as Substance Intoxication, Substance Withdrawal, Substance-Induced Delirium, Substance-Induced Persisting Dementia, Substance-Induced Persisting Amnestic Disorder, Substance-Induced Psychotic Disorder, Substance-Induced Mood Disorder, Substance-Induced Anxiety Disorder, Substance- Induced sexual Dysfunction, Substance-Induced Sleep Disorder and Hallucinogen Persisting Perception Disorder (Flashbacks); Alcohol-Related Disorders such as Alcohol Dependence (303.90), Alcohol Abuse (305.00), Alcohol Intoxication (303.00), Alcohol Withdrawal (291.81), Alcohol Intoxication Delirium, Alcohol Withdrawal Delirium, Alcohol-Induced Persisting Dementia, Alcohol-Induced Persisting Amnestic Disorder, Alcohol-Induced Psychotic Disorder, Alcohol-Induced
• Cannabis-Related Disorders such as Cannabis Dependence (304.30), Cannabis Abuse (305.20), Cannabis Intoxication (292.89), Cannabis Intoxication Delirium, Cannabis- Induced Psychotic Disorder, Cannabis-Induced Anxiety Disorder and Cannabis-Related Disorder Not Otherwise Specified (292.9); Cocaine-Related Disorders such as Cocaine Dependence (304.20), Cocaine Abuse (305.60), Cocaine Intoxication (292.89), Cocaine Withdrawal (292.0), Cocaine Intoxication Delirium, Cocaine-Induced Psychotic Disorder, Cocaine-Induced Mood Disorder, Cocaine-Induced Anxiety Disorder, Cocaine-Induced Sexual Dysfunction, Cocaine-Induced Sleep Disorder and Cocaine-Related Disorder Not Otherwise Specified (292.9); Hallucinogen-Related Disorders such as Hallucinogen
• Phencyclidine (or Phencyclidine-Like)-Related Disorders such as Phencyclidine Dependence (304.60), Phencyclidine Abuse (305.90), Phencyclidine Intoxication (292.89), Phencyclidine Intoxication Delirium, Phencyclidine-Induced Psychotic Disorder, Phencyclidine-Induced Mood Disorder, Phencyclidine-Induced Anxiety Disorder and Phencyclidine-Related Disorder Not Otherwise Specified (292.9); Sedative-, Hypnotic-, or Anxiolytic-Related Disorders such as Sedative, Hypnotic, or Anxiolytic Dependence (304.10), Sedative, Hypnotic, or Anxiolytic Abuse (305.40), Sedative,
• the compounds of formula (I) or their pharmaceutically acceptable salts may be of use for the treatment or prophylaxis of a disease or disorder where an antagonist of a human orexin receptor is required such as feeding disorders such as bulimia nervosa, binge eating, obesity, including obesity observed in Type 2 (non-insulin-dependent) diabetes patients.
• the compounds of formula (I) or their pharmaceutically acceptable salts may be of use for the treatment or prophylaxis of a disease or disorder where an antagonist of a human orexin receptor is required such as stroke, particularly ischemic or haemorrhagic and/or in blocking an emetic response i.e. nausea and vomiting.
• the numbers in brackets after the listed diseases refer to the classification code in DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, published by the American Psychiatric Association.
• the various subtypes of the disorders mentioned herein are contemplated as part of the present invention.
• the invention also provides a method of treating or preventing a disease or disorder where an antagonist of a human orexin receptor is required, for example those diseases and disorders mentioned hereinabove, which comprises administering to a subject in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
• the invention also provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of a disease or disorder where an antagonist of a human orexin receptor is required, for example those diseases and disorders mentioned hereinabove.
• the invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of a disease or disorder where an antagonist of a human Orexin receptor is required, for example those diseases and disorders mentioned hereinabove.
• the compounds of the invention are usually administered as a pharmaceutical composition.
• the invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the compounds of formula (I) or their pharmaceutically acceptable salts may be administered by any convenient method, e.g. by oral, parenteral, buccal, sublingual, nasal, rectal or transdermal administration, and the pharmaceutical compositions adapted accordingly.
• the compounds of formula (I) or their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids or solids, e.g. as syrups, suspensions, emulsions, tablets, capsules or lozenges.
• a liquid formulation will generally consist of a suspension or solution of the active ingredient in a suitable liquid carrier(s) e.g. an aqueous solvent such as water, ethanol or glycerine, or a non-aqueous solvent, such as polyethylene glycol or an oil.
• a suitable liquid carrier(s) e.g. an aqueous solvent such as water, ethanol or glycerine, or a non-aqueous solvent, such as polyethylene glycol or an oil.
• the formulation may also contain a suspending agent, preservative, flavouring and/or colouring agent.
• a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations, such as magnesium stearate, starch, lactose, sucrose and cellulose.
• a composition in the form of a capsule can be prepared using routine encapsulation procedures, e.g. pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), e.g. aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
• suitable pharmaceutical carrier(s) e.g. aqueous gums, celluloses, silicates or oils
• Typical parenteral compositions consist of a solution or suspension of the active ingredient in a sterile aqueous carrier or parenterally acceptable oil, e.g. polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
• a sterile aqueous carrier or parenterally acceptable oil e.g. polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
• the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.
• compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders.
• Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a pharmaceutically acceptable aqueous or non- aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container which can take the form of a cartridge or refill for use with an atomising device.
• the sealed container may be a disposable dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve.
• the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas e.g. air, or an organic propellant such as a fluorochlorohydrocarbon or hydrofluorocarbon.
• Aerosol dosage forms can also take the form of pump-atomisers.
• Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles where the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.
• compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
• compositions suitable for transdermal administration include ointments, gels and patches.
• the composition is in unit dose form such as a tablet, capsule or ampoule.
• suitable unit doses may contain from 0.1% to 100% by weight, for example from 10 to 60% by weight, of the active material, depending on the method of administration.
• the composition may contain from 0% to 99% by weight, for example 40% to 90% by weight, of the carrier, depending on the method of administration.
• the composition may contain from 0.05mg to lOOOmg, for example from 1.Omg to 500mg, of the active material, depending on the method of administration.
• the composition may contain from 50 mg to 1000 mg, for example from lOOmg to 400mg of the carrier, 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.
• suitable unit doses may be 0.05 to 1000 mg, more suitably 1.0 to 500mg, and such unit doses maybe administered more than once a day, for example two or three a day. Such therapy may extend for a number of weeks or months.
• Compounds of the invention may be identified and characterised using screening procedures and assays including, for example, activity assays and functional assays.
• Orexin-A (Sakurai, T. et al (1998) Cell, 92 pp 573-585)) can be employed in screening procedures for compounds which inhibit the ligand's activation of the orexin-1 or orexin-2 receptors.
• screening procedures involve providing appropriate cells which express the orexin-1 or orexin-2 receptor on their surface.
• Such cells include cells from mammals, yeast, Drosophila or E. coli.
• a polynucleotide encoding the orexin- 1 or orexin-2 receptor is used to transfect cells to express the receptor.
• the expressed receptor is then contacted with a test compound and an orexin-1 or orexin-2 receptor ligand, as appropriate, to observe inhibition of a functional response.
• One such screening procedure involves the use of melanophores which are transfected to express the orexin-1 or orexin-2 receptor, as described in WO 92/01810.
• Another screening procedure involves introducing RNA encoding the orexin-1 or orexin-2 receptor into Xenopus oocytes to transiently express the receptor.
• the receptor oocytes are then contacted with a receptor ligand and a test compound, followed by detection of inhibition of a signal in the case of screening for compounds which are thought to inhibit activation of the receptor by the ligand.
• Another method involves screening for compounds which inhibit activation of the receptor by determining inhibition of binding of a labelled orexin-1 or orexin-2 receptor ligand to cells which have the orexin-1 or orexin-2 receptor (as appropriate) on their surface.
• This method involves transfecting a eukaryotic cell with DNA encoding the orexin-1 or orexin-2 receptor such that the cell expresses the receptor on its surface and contacting the cell or cell membrane preparation with a compound in the presence of a labelled form of an orexin-1 or orexin-2 receptor ligand.
• the ligand may contain a radioactive label. The amount of labelled ligand bound to the receptors is measured, e.g. by measuring radioactivity.
• Yet another screening technique involves the use of FLIPR equipment for high throughput screening of test compounds that inhibit mobilisation of intracellular calcium ions, or other ions, by affecting the interaction of an orexin-1 or orexin-2 receptor ligand with the orexin-1 or orexin-2 receptor as appropriate.
• the NMR spectra were recorded at a temperature ranging from 25 to 9O 0 C. When more than one conformer was detected the chemical shifts for the most abundant one is reported.
• the eluent used with SPE-SCX cartridges is methanol followed by 2N ammonia solution in methanol.
• SPE-Si cartridges are silica solid phase extraction columns supplied by Varian.
• reaction mixture was charged into a dropping funnel and then added dropwise to a 2 L round-bottomed flask containing -400 ml of NaOH 1 M aqueous solution cooled at 0 0 C.
• the resulting grey suspension was diluted with EtOAc (250 ml) and allowed to stir overnight (mechanical stirring).
• the resulting yellow suspension was then filtered over a Gooch funnel (using Sterimat): salts were washed with EtOAc (-500 ml). Phases were then separated and the organic layer was washed with brine (2 x 500 ml). The combined organic phases were dried (Na 2 SO 4 ), filtered and concentrated to give a deep orange oil.
• Example 4 Determination of antagonist affinity at human Orexin-1 and 2 receptors using FLIPR
• Adherent Chinese Hamster Ovary (CHO) cells stably expressing the recombinant human Orexin-1 (hOXl) or human Orexin-2 receptors (hOX2), were maintained in culture in Alpha Minimum Essential Medium (Gibco/Invitrogen, cat. no.; 22571-020), supplemented with 10% decomplemented foetal bovine serum (Life Technologies, cat. no.
• CHO-hOXl or CHO-hOX2 cells were seeded into black clear-bottom 384-well plates at a density of 20,000 cells per well in culture medium as described above and maintained overnight (95%:5% air:CO 2 at 37°C).
• the plates were then incubated at room temperature for 60 minutes in the dark with 1 ⁇ M FLUO-4AM dye to allow cell uptake of the FLUO-4AM, which is then converted by intracellular esterases to FLUO-4, which is unable to leave the cells.
• DMSO dimethylsulfoxide
• hOrexinA human orexin A
• the loaded cells were then incubated for lOmin at 37° C with test compound.
• FLIPR fluometric imaging plate reader
• Compounds of the Examples tested according to this method had fpKi values in the range from 8.1 to 9.6 at the human cloned orexin-1 receptor (having the amino acid residue alanine at position 280 and not glycine) and from 6.3 to 7.9 at the human cloned orexin-2 receptor.

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