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• • 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
  • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• • 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
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
  • A61P25/36—Opioid-abuse
• • 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
  • A61P3/04—Anorexiants; Antiobesity agents
• • 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
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • 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

• Thisinvention relatestoimidazopyridazinylmethylsubstitutedpiperidinederivativesandtheiruseaspharmaceuticals. ManymedicallysignificantbiologicalprocessesaremediatedbyproteinsparticipatinginsignaltransductionpathwaysthatinvolveG-proteinsand/orsecond messengergers. Polypeptidesandpolynucleotidesencodingthehuman7-transmembraneG-proteincoupledneuropeptidereceptor,orexin-1 (HFGAN72),havebeenidentifiedandaredisclosedinEP-A-875565,EP-A-875566andWO96/34877.
• Orexinreceptors arefoundinthemammalianhostandmayberesponsibleformanybiologicalfunctions,includingpathologiesincluding,butnotlimitedto,depression; anxiety;addictions;obsessivecompulsivedisorder;affectiveneurosis/disorder;depressiveneurosis/disorder;anxietyneurosis;dysthymicdisorder;behaviourdisorder;mooddisorder;sexualdysfunction;psychosexualdysfunction;sexdisorder;sexualdisorder;schizophrenia;manicdepression;delerium;dementia;severementalretardationanddyskinesiassuchasHuntington'sdiseaseandGillesdeIaTourett'ssyndrome;disturbedbiologicalandcircadianrhythms;feedingdisorders,suchasanorexia,bulimia,cachexia,andobesity;diabetes
• narcotics or withdrawal from narcotics sleep disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag syndrome; and neurodegenerative disorders, which includes nosological entities such as disinhibition-dementia-parkinsonism- amyotrophy complex; pallido-ponto-nigral degeneration, epilepsy, and seizure disorders.
• Rat sleep/EEG studies have also shown that central administration of orexin-A, an agonist of the orexin receptors, causes a dose-related increase in arousal, largely at the expense of a reduction in paradoxical sleep and slow wave sleep 2, when administered at the onset of the normal sleep period. Therefore antagonists of its receptor may be useful in the treatment of sleep disorders including insomnia.
• 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 imidazopyridazinylmethyl group have surprisingly beneficial properties including, for example, increased oral bioavailability and significantly increase solubility in physiologically relevant media compared to the prior art compounds.
• imidazopyridazinylmethyl substituted piperidine derivatives very attractive as potential pharmaceutical agents which may be useful in the prevention or treatment of obesity, including obesity observed in Type 2 (non-insulin-dependent) diabetes patients, sleep disorders, anxiety, depression, schizophrenia, drug dependency or compulsive behaviour. Additionally these compounds may be useful in the treatment of stroke, particularly ischemic or haemorrhagic stroke, and/or blocking the emetic response, i.e. useful in the treatment of nausea and vomiting.
• 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 4 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 .
• 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 1; with the proviso that p and q are not both 0; or a pharmaceutically acceptable salt thereof.
• Ar is a group of formula (II). In another embodiment Ar is a group of formula (III).
• Ar is a group of formula (II) and n is 0.
• 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.
• Ar is a group of formula (II), n is 0, p is 1, q is 1, R 2 is (C 1 _ 4 )- alkyl and R 3 is halo. In another embodiment Ar is a group of formula (II), n is 0, p is 1, q is 1, R 2 is methyl and R 3 is chloro.
• 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.
• Ar is a group of formula (III) and n is 0.
• Ar is a group of formula (III), n is 0, p is 1, q is 0 and R 2 is methyl. In one embodiment 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 (C 1 _ 4 )-alkyl.
• Ar is a group of formula (III), n is 0, p is 1, q is 1, R 2 is (C 1 _ 4 )- alkyl and R 3 is halo.
• Ar is a group of formula (III), n is 0, p is 1, q is 1, R 2 is methyl and R 3 is chloro.
• 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:
• halo(C 1 _ 4 )alkyl examples include trifluoromethyl (i.e. -CF 3 ).
• (C 1 _ 4 )alkoxy include methyloxy and ethyloxy.
• halo(C 1 _ 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.
• 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. 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.
• Particular compounds according to the invention include those mentioned in the examples and their pharmaceutically acceptable derivatives.
• pharmaceutically acceptable derivative includes any pharmaceutically acceptable salt, 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. 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, ppl-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. Also included within the scope of the invention are solvates and hydrates of compounds of formula (I).
• 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).
• the subject invention also includes isotopically-labeled compounds which are identical to those recited in formula (I) and following, 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.
• 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. According to a further aspect of the present invention there is provided a process for the preparation of compounds of formula (I) and derivatives thereof.
• the following scheme details an example of a synthetic route to compounds of the invention. In the following scheme 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.
• the compounds of formula (I) may be prepared singly or as compound libraries comprising at least 2, e.g. 5 to 1000, preferably 10 to 100 compounds of formula (I).
• Compound libraries may be prepared by a combinatorial 'split and mix' approach or by multiple parallel synthesis using either solution phase or solid phase chemistry, by procedures known to those skilled in the art.
• a compound library comprising at least 2 compounds of formula (I), or pharmaceutically acceptable derivatives thereof.
• 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) and their pharmaceutically acceptable derivatives are useful for the treatment of diseases or disorders where an antagonist of a human Orexin receptor is required such as obesity, including obesity observed in Type 2 (non-insulin- dependent) diabetes patients, schizophrenia, anxiety, depression, obsessive compulsive disorder, drug dependency and/or 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); 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 condition; and substance- induced sleep disorder including the subtypes insomnia type, hypersomnia type
• the compounds of formula (I) and pharmaceutically acceptable derivatives are useful for the treatment of stroke, particularly ischemic or haemorrhagic and/or in blocking an emetic response i.e. nausea and vomiting.
• the invention also provides a method of treating or preventing diseases or disorders 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 derivative thereof.
• the invention also provides a compound of formula (I), or a pharmaceutically acceptable derivative thereof, for use in the treatment or prophylaxis of diseases or disorders 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 derivative thereof, in the manufacture of a medicament for the treatment or prophylaxis of diseases or disorders 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 derivative thereof, in the manufacture of a medicament for the treatment or prophylaxis of diseases or disorders where an antagonist of a human Orexin receptor is required such as obesity, including obesity observed in Type 2 (non-insulin- dependent) diabetes patients, schizophrenia, anxiety, depression, obsessive compulsive disorder, drug dependency and/or 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); 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 condition
• 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 derivative thereof, and a pharmaceutically acceptable carrier.
• the compounds of formula (I) and their pharmaceutically acceptable derivatives 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) and their pharmaceutically acceptable derivatives 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 nonaqueous 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.
• 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.
• the dose of the compound of formula (I), or a pharmaceutically acceptable derivative thereof, used in the treatment or prophylaxis of the abovementioned disorders or diseases will vary in the usual way with the particular disorder or disease being treated, the weight of the subject and other similar factors.
• suitable unit doses may be 0.05 to 1000 mg, more suitably 0.05 to 500 mg.
• Unit doses may be administered more than once a day for example two or three times a day, so that the total daily dosage is in the range of about 0.01 to 100 mg/kg; and such therapy may extend for a number of weeks or months.
• pharmaceutically acceptable derivatives the above figures are calculated as the parent compound of formula (I). No toxico logical effects are indicated/expected when a compound of formula (I) is administered in the above mentioned dosage range.
• Human Orexin-A has the amino acid sequence: pyroGlu Pro Leu Pro Asp Cys Cys Arg Gln Lys Thr Cys Ser Cys Arg Leu 1 5 10 15
• Leu-NH 2 Orexin-A can be employed in screening procedures for compounds which inhibit the ligand's activation of the orexin-1 receptor.
• screening procedures involve providing appropriate cells which express the orexin-1 receptor on their surface.
• Such cells include cells from mammals, yeast, Drosophila or E. coli.
• a polynucleotide encoding the orexin-1 receptor is used to transfect cells to express the receptor.
• the expressed receptor is then contacted with a test compound and an orexin-1 receptor ligand to observe inhibition of a functional response.
• One such screening procedure involves the use of melanophores which are transfected to express the orexin-1 receptor, as described in WO 92/01810.
• Another screening procedure involves introducing RNA encoding the orexin-1 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 receptor ligand to cells which have the receptor on their surface.
• This method involves transfecting a eukaryotic cell with DNA encoding the orexin-1 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 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 receptor ligand with the orexin-1 receptor.
• the invention is illustrated by the Examples described below. In the procedures that follow, after each starting material, reference to a description is typically provided. This is provided merely for assistance to the skilled chemist.
• the starting material may not necessarily have been prepared from the batch referred to.
• Mass spectra were taken on a 4 II triple quadrupole Mass Spectrometer (Micromass UK) or on a Agilent MSD 1100 Mass Spectrometer, operating in ES (+) and
• Flash silica gel chromatography was carried out on silica gel 230-400 mesh (supplied by Merck AG Darmstadt, Germany) or over Varian Mega Be-Si pre-packed cartridges or over pre-packed Biotage silica cartridges.
• SPE-SCX cartridges are ion exchange solid phase extraction columns supplied by Varian.
• the eluent used with SPE-SCX cartridges is methanol followed by 2N ammonia solution in methanol.
• purification was performed using either Biotage manual flash chromatography (Flash+) or automatic flash chromatography (Horizon) systems. All these instruments work with Biotage Silica cartridges.
• 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 °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 3 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).
• 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 8.8 to 9.1 at the human cloned orexin-1 receptor (having the amino acid residue alanine at position 280 and not glycine) and from 7.7 to 8.4 at the human cloned orexin-2 receptor.

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