Patent Abstract:
compounds with α7 nicotinic acetylcholine receptor agonistic activity , processes for their preparation , pharmaceutical compositions containing the same and the use thereof for the treatment of neurological and psychiatric diseases .

Detailed Description:
in a first aspect , the invention provides a compound of formula i z is ch 2 , n , o , s , s (═ o ), or s (═ o ) 2 ; t ′ represent , independently from one another when p is greater than 1 , hydroxy ; mercapto ; amino ; cyano ; nitro ; oxo ; linear , branched or cyclic ( c 1 - c 6 ) alkyl , trihaloalkyl , hydroxyalkyl , aminoalkyl , mercaptoalkyl , alkoxy , alkylthio , alkylcarbonyl , alkoxycarbonyl , alkylcarbonylamino ; ( c 5 - c 10 ) aryl - or heteroarylcarbonylamino ; mono - or di -, linear , branched or cyclic ( c 1 - c 6 ) alkylamino ; linear , branched or cyclic ( c 1 - c 6 ) alkoxy -( c 1 - c 6 ) alkyl , mono - or di -( c 1 - c 6 ) alkylamino -( c 1 - c 6 ) alkyl , or ( c 1 - c 6 ) alkylthio -( c 1 - c 6 ) alkyl ; ( c 5 - c 10 ) aryl - or heteroarylsulphonylamino ; ( c 1 - c 3 ) alkylsulphonylamino ; mono - or di -( c 5 - c 10 ) aryl - or heteroarylaminosulphonyl ; mono - or di -( c 1 - c 3 ) alkylaminosulphonyl ; sulphamoyl ; mono - or di -( c 5 - c 10 ) aryl - or heteroarylaminocarbonyl ; linear , branched or cyclic ( c 1 - c 6 ) alkylaminocarbonyl ; carbamoyl ; or , when p is 2 or 3 , two t ′ substituents , with the atoms they are attached to , may form a 5 - to 8 - membered ring with spiro or fused junction ; q and q ′ are , independently from one another , integers from 1 to 4 ; r represents a 5 to 10 - membered aromatic or heteroaromatic ring optionally substituted with one or more groups selected from : halogen ; hydroxy ; mercapto ; cyano ; nitro ; amino ; linear , branched or cyclic ( c 1 - c 6 ) alkyl , trihaloalkyl , alkoxy or alkylcarbonyl ; ( c 5 - c 10 ) aryl - or heteroaryl - carbonylamino ; linear , branched , or cyclic ( c 1 - c 6 ) alkylcarbonylamino , mono - or di -( c 5 - c 10 ) aryl - or heteroarylaminocarbonyl ; mono - or di , linear , branched , or cyclic ( c 1 - c 6 ) alkylamino or alkylaminocarbonyl ; carbamoyl ; ( c 5 - c 10 ) aryl - or heteroarylsulphonylamino ; linear , branched , or cyclic ( c 1 - c 6 ) alkylsulphonylamino ; ( c 5 - c 10 ) aryl - or heteroarylsulphonyl ; linear , branched , or cyclic ( c 1 - c 6 ) alkylsulphonyl ; mono - or di -( c 5 - c 10 ) aryl - or heteroarylsulphamoyl ; mono - or di - linear , branched , or cyclic ( c 1 - c 6 ) alkylsulphamoyl ; linear , branched or cyclic ( c 1 - c 6 ) alkoxy -( c 1 - c 6 ) alkyl , mono - or di -( c 1 - c 6 ) alkylamino -( c 1 - c 6 ) alkyl , ( c 1 - c 6 ) alkylthio -( c 1 - c 6 ) alkyl ; r ′ represent , independently from one another when j = 2 , halogen ; hydroxy ; mercapto ; cyano ; nitro ; trihalomethyl ; trihalomethoxy ; linear , branched or cyclic ( c 1 - c 6 ) alkyl , trihaloalkyl , alkoxy , hydroxyalkyl , mercaptoalkyl , alkoxycarbonyl , alkylcarbonyl , alkylsulphonyl ; linear , branched , or cyclic ( c 1 - c 6 ) alkylcarbonylamino ; mono - or di , linear , branched , or cyclic ( c 1 - c 6 ) alkylaminocarbonyl ; carbamoyl ; ( c 6 - c 10 ) aryl - or ( c 1 - c 6 ) alkylsulphonylamino ; ( c 6 - c 10 ) aryl - or linear , branched , or cyclic ( c 1 - c 6 ) alkylsulphamoyl ; linear , branched or cyclic ( c 1 - c 6 ) alkoxy -( c 1 - c 6 ) alkyl , mono - or di -( c 1 - c 6 ) alkylamino -( c 1 - c 6 ) alkyl , ( c 1 - c 6 ) alkylthio -( c 1 - c 6 ) alkyl . in a first preferred embodiment , the invention provides compounds of formula ( i ) wherein : t ′ represent , independently from one another when p is greater than 1 , linear , branched or cyclic ( c 1 - c 6 ) alkyl , trihaloalkyl , hydroxyalkyl , alkoxy , alkylcarbonyl , alkoxycarbonyl , alkylcarbonylamino ; linear , branched or cyclic ( c 1 - c 6 ) alkoxy -( c 1 - c 6 ) alkyl ; linear , branched or cyclic ( c 1 - c 6 ) alkylaminocarbonyl ; carbamoyl ; or , when p is 2 or 3 , two t ′ substituents form a 5 - to 8 - membered ring with spiro or fused junction ; q and q ′ are , independently from one another , integers from 1 to 4 ; r represents a 5 to 10 - membered aromatic or heteroaromatic ring optionally substituted with one or more groups selected from : halogen ; hydroxy ; mercapto ; cyano ; nitro ; amino ; linear , branched or cyclic ( c 1 - c 6 ) alkyl , trihaloalkyl , alkoxy or alkylcarbonyl ; linear , branched , or cyclic ( c 1 - c 6 ) alkylcarbonylamino ; mono - or di , linear , branched , or cyclic ( c 1 - c 6 ) alkylamino or alkylaminocarbonyl ; carbamoyl ; linear , branched or cyclic ( c 1 - c 6 ) alkoxy -( c 1 - c 6 ) alkyl ; r ′ represent , independently from one another when j = 2 , halogen ; hydroxy ; trihalomethyl ; trihalomethoxy ; linear , branched or cyclic ( c 1 - c 6 ) alkyl , trihaloalkyl , alkoxy , hydroxyalkyl . in this embodiment , particularly preferred are those compounds of formula i wherein : q and q ′ are , independently from one another , integers from 1 to 3 ; r represents a 5 to 10 - membered aromatic or heteroaromatic ring optionally substituted with one or more groups selected from : halogen ; linear , branched or cyclic ( c 1 - c 3 ) alkyl , alkoxy ; linear , branched , or cyclic ( c 1 - c 3 ) alkylcarbonylamino ; mono - or di , linear , branched , or cyclic ( c 1 - c 3 ) alkylaminocarbonyl ; carbamoyl ; yet more preferred are those compounds wherein both q and r are phenyl . the compounds of formula i can be prepared through a number of synthetic routes amongst which the ones illustrated in schemes 1 , 2 , 3 , and 4 below . according to scheme 1 , hereby exemplified by a = 2 and a = n , an amine 1 is reacted under reductive alkylation conditions , such as for example treatment with sodium triacetoxyborohydride , sodium cyanoborohydride or sodium borohydride , in the presence of a catalytic amount of acid , such as for example acetic acid or formic acid , in an organic solvent such as for example dichloromethane or tetrahydrofuran , with a cyclic ketone 2 containing a protected amine functionality , hereby exemplified by tert - butoxycarbonyl . other suitable protecting groups may be represented by benzyloxycarbonyl , fluorenylmethoxycarbonyl , and any other amine protecting group as described in greene , t . and wuts , p . g . m . protective groups in organic synthesis , john wiley and sons , 1999 . the thus obtained amine 3 is further modified by removal of the protecting group , for example in the case of the tert - butoxycarbonyl group by treatment with trifluoroacetic acid in dichloromethane or with hydrochloric acid in methanol or with any other suitable method as described in ref . 1 , to obtain amine 4 . amine 4 is then reacted with an isocyanate , hereby exemplified by a phenylisocyanate , in a suitable solvent such as for example dichloromethane , tetrahydrofuran , dimethylformamide or mixtures thereof , to yield the ureas ia . in the case of r being a halogen or a boronic acid ester , ia can be further processed — for example via a cross - coupling reaction , for example under the conditions described as suzuki coupling conditions ( suzuki , a . pure and appl . chem . 1994 66 213 - 222 ), with a boronic acid or an aryl or heteroaryl halide — to yield compounds iβ . a = ch , an amine 1 is reacted with an activated acid 5 ( lg = leaving group ) containing a protected amine functionality , hereby exemplified by tert - butoxycarbonyl , to afford an amide 6 in a solvent such as for example dichloromethane , tetrahydrofuran , dimethylformamide or mixtures thereof . suitable activation for acids may be represented by acid chloride , an acyl imidazolide as obtained by treatment of an acid with a stoichiometric amount of carbonyldiimidazole , an activated ester such as for example a benzotriazolyl ester or a pentafluorophenyl ester , a mixed anhydride such as for example the one obtained by reaction of an acid with a iso - butyl chloroformate in the presence of a tertiary amine . the thus obtained amide 6 is further modified by removal of the protecting group , for example in the case of the tert - butoxycarbonyl group by treatment with trifluoroacetic acid in dichloromethane or with hydrochloric acid in methanol or any other suitable method as mentioned above , to obtain amine 7 . amine 7 is then reacted with a reducing agent , such as lithium aluminium hydride or borane in a suitable solvent such as for example tetrahydrofuran to afford amine 8 , which is further reacted with an isocyanate , hereby exemplified by a phenylisocyanate , in a suitable solvent such as for example dichloromethane , tetrahydrofuran , dimethylformamide or mixtures thereof , to yield the ureas iα . in the case of r being a halogen or a boronic acid ester , iα can be further processed — for example via a cross - coupling reaction , for example under the suzuki coupling conditions , with a boronic acid or an aryl or heteroaryl halide — to yield compounds iβ . according to scheme 3 , hereby exemplified by n = 2 and a = ch , an activated acid 9 ( lg = leaving group ) is reacted with an aromatic or heteroaromatic amine , hereby exemplified an a substituted aniline , to afford an aromatic or heteroaromatic amide 10 . suitable activation for acids may be represented by acid chloride , an acyl imidazolide as obtained by treatment of an acid with a stoichiometric amount of carbonyldiimidazole , an activated ester such as for example a benzotriazolyl ester or a pentafluorophenyl ester , a mixed anhydride such as for example the one obtained by reaction of an acid with a iso - butyl chloroformate in the presence of a tertiary amine . the resulting amide is then reacted with an amine x under reductive alkylation conditions , such as for example treatment with sodium triacetoxyborohydride , sodium cyanoborohydride or sodium borohydride , in the presence of a catalytic amount of acid , such as for example acetic acid or formic acid , in an organic solvent such as for example dichloromethane or tetrahydrofuran . in the case of r being a halogen or a boronic acid ester , iα can be further processed — for example via a cross - coupling reaction , for example under the suzuki coupling conditions , with a boronic acid or an aryl or heteroaryl halide — to yield compounds iβ . according to scheme 4 , the hydroxy group of a suitably protected hydroxyalkylcyclamine , hereby exemplified , but not limited to , by 4 -( 2 - hydroxyethyl ) piperidine n - protected as its tert - butylcarbamate derivative , is activated by the transformation into a leaving group , hereby exemplified by , but not limited to , a para - toluenesulphonyl group and subsequently displaced in the reaction with a primary or secondary amine . following removal of the cyclamine protecting group , this is then reacted with an aryl or heteroaryl isocyanate in a suitable solvent , such as for example dichloromethane , or tetrahydrofurane to furnish product iα . in the case of x being a halogen or a boronic acid ester , iα can be further processed — for example via a cross - coupling reaction , for example under the suzuki coupling conditions , with a boronic acid or an aryl or heteroaryl halide — to yield compounds iβ . the compounds of formula i , their optical isomers or diastereomers can be purified or separated according to well - known procedures , including but not limited to chromatography with a chiral matrix and fractional crystallisation . the pharmacological activity of a representative group of compounds of formula i was demonstrated in an in vitro assay utilising cells stably transfected with the alpha 7 nicotinic acetylcholine receptor and cells expressing the alpha 1 and alpha 3 nicotinic acetylcholine receptors and 5ht3 receptor as controls for selectivity . according to a further aspect , the invention is therefore directed to a method of treating neurological and psychiatric disorders , which comprises administering to a subject , preferably a human subject in need thereof , an effective amount of a compound of formula i . neurological and psychiatric disorders that may benefit from the treatment with the invention compounds include but are not limited to senile dementia , attention deficit disorders , alzheimer &# 39 ; s disease and schizophrenia . in general , the compounds of formula i can be used for treating any disease condition , disorder or dysfunction that may benefit from the activation of the alpha 7 nicotinic acetylcholine receptor , including but not limited to parkinson &# 39 ; s disease , huntington &# 39 ; s chorea , amyotrophic lateral sclerosis , multiple sclerosis , epilepsy , memory or learning deficit , panic disorders , cognitive disorders , depression , sepsis and arthritis . the dosage of the compounds for use in therapy may vary depending upon , for example , the administration route , the nature and severity of the disease . in general , an acceptable pharmacological effect in humans may be obtained with daily dosages ranging from 0 . 01 to 200 mg / kg . in yet a further aspect , the invention refers to a pharmaceutical composition containing one or more compounds of formula i , in association with pharmaceutically acceptable carriers and excipients . the pharmaceutical compositions can be in the form of solid , semi - solid or liquid preparations , preferably in form of solutions , suspensions , powders , granules , tablets , capsules , syrups , suppositories , aerosols or controlled delivery systems . the compositions can be administered by a variety of routes , including oral , transdermal , subcutaneous , intravenous , intramuscular , rectal and intranasal , and are preferably formulated in unit dosage form , each dosage containing from about 1 to about 1000 mg , preferably from 1 to 600 mg of the active ingredient . the compounds of the invention can be in the form of free bases or as acid addition salts , preferably salts with pharmaceutically acceptable acids . the invention also includes separated isomers and diastereomers of compounds i , or mixtures thereof ( e . g . racemic mixtures ). the principles and methods for the preparation of pharmaceutical compositions are described for example in remington &# 39 ; s pharmaceutical science , mack publishing company , easton ( pa ). unless otherwise specified all nuclear magnetic resonance spectra were recorded using a varian mercury plus 400 mhz spectrometer equipped with a pfg atb broadband probe . hplc - ms analyses were performed with a waters 2795 separation module equipped with a waters micromass zq ( es ionisation ) and waters pda 2996 , using a waters xterra ms c18 3 . 5 μm 2 . 1 × 50 mm column . preparative hlpc was run using a waters 2767 system with a binary gradient module waters 2525 pump and coupled to a waters micromass zq ( es ) or waters 2487 dad , using a supelco discovery hs c18 5 . 0 μm 10 × 21 . 2 mm column . gradients were run using 0 . 1 % formic acid / water and 0 . 1 % formic acid / acetonitrile with gradient 5 / 95 to 95 / 5 in the run time indicated . all column chromatography was performed following the method of still , c . ; j . org chem 43 , 2923 ( 1978 ). all tlc analyses were performed on silica gel ( merck 60 f254 ) and spots revealed by uv visualisation at 254 nm and kmno4 or ninhydrin stain . when specified for array synthesis , heating was performed on a buchi syncore ® system . the secondary amine ( 1 . 0 eq ) was dissolved in dcm and n - boc - piperidone ( 1 . 0 eq ) was added . the reaction was stirred for 1 hour and then nabh ( oac ) 3 was added and reaction stirred for other 18 hours . the mixture was then extracted into a hcl solution at ph2 and non - basic impurities removed by washing with dcm . the aqueous phase was then brought to ph 12 with sodium hydroxide and extracted with dcm . the organic phase was concentrated under reduced pressure affording the reductive alkylation product pure enough for the following step . the product obtained from the above reductive alkylation step ( 1 eq ) was dissolved in dcm and an excess of tfa ( 80 eq ) was slowly added . the reaction was stirred for 1 h and then concentrated under reduced pressure . the mixture was neutralised with naoh 10 % and extracted with dcm , affording a piperidine product pure enough for the following step . to a suspension of n - boc - isonipecotic acid ( 1 eq ) and tbtu ( 1 eq ) in ch 3 cn , the appropriate amine ( 2 eq ) was added . the resulting solution was stirred at 85 ° c . for 6 hours . the reaction mixture was concentrated under reduced pressure and then dissolved in dcm and washed twice with sat . aqueous na 2 co 3 solution . solvent removal gave the 4 -( alkyl ) carbamoyl - piperidine - 1 - carboxylic acid tert - butyl ester product usually pure enough for the next step . the amide thus obtained was dissolved in 6n hcl solution at 0 ° c . after 10 minutes tlc analysis generally showed disappearance of starting material , and the mixture was basified to ph 12 with naoh ( pellets ) and extracted with acoet . the organic phase concentrated under reduced pressure gave the piperidine - 4 - carboxylic acid amide product that was used without further purification for the next step . the piperidine - amide was added ( at 0 ° c .) to a suspension of lialh 4 in anhydrous thf . after stirring for 30 minutes the reaction was heated at reflux for 1 hour and when tlc analysis generally showed complete conversion of the starting amide . the reaction was cooled to 0 ° c . and lialh 4 was quenched with h 2 o and naoh ( 10 % aqueous solution ). the inorganic salts were filtered and the solution was concentrated under reduced pressure , yielding the piperidin - 4 - yl methylamine product usually pure enough for the next step . to a solution of 4 -( 2 - hydroxy - ethyl )- piperidine - 1 - carboxylic acid tert - butyl ester in dcm ( 0 . 65 mmol / ml ) p - toluensulphonyl chloride ( 1 . 5 eq ) and dimethylaminopyridine ( 1 eq ) were added . the reaction was left at rt for 18 h and then tlc showed complete conversion of the starting material . the mixture was washed with naoh 2n and then with hcl 2n , the organic phase was dried over na 2 so 4 and then concentrated under reduced pressure . the oil obtained was purified with sio 2 column , eluting with dcm , giving the pure product in quantitative yield . 1 h - nmr ( 400 mhz , cdcl3 ): 1 . 01 - 1 . 15 ( m , 2h ), 1 . 44 ( s , 9h ), 1 . 47 - 1 . 57 ( m , 2h ), 1 . 62 - 1 . 72 ( m , 3h ), 2 . 44 ( s , 3h ), 2 . 54 - 2 . 65 ( m , 2h ), 3 . 95 - 4 . 1 ( m , 4h ), 7 . 36 ( d , 2h , j = 7 . 3 ), 7 . 78 ( d , 2h , j = 7 . 3 ). to the chosen alkylamine ( 2 eq ) was added a solution of 4 -[ 2 -( toluene - 4 - sulfonyloxy )- ethyl ]- piperidine - 1 - carboxylic acid tert - butyl ester in ch 3 cn ( 0 . 65 mmol / ml , 1 eq ) and the reaction was heated at 80 ° c . for about 6 hours . upon complete conversion ( as monitored by thin layer chromatography ) the mixture was cooled down to room temperature and washed with saturated nacl water solution . the organic phase was dried over na 2 so 4 and then concentrated under reduced pressure . the oil obtained was purified by sio 2 column eluting with gradient starting from 100 % dcm to dcm - nh3 ( 2n meoh solution ) 9 : 1 . to a solution of hcl 6n ( 30 eq ) was added the 4 -( 2 -( n - alkylamino )- ethyl )- piperidine - 1 - carboxylic acid tert - butyl ester and the reaction was stirred at room temperature for 10 minutes . the mixture was basified to ph 12 and the product was extracted with dcm . the organic phase was dried over na 2 so 4 and then concentrated under reduced pressure affording the pure product . to a cooled solution of amine ( 1 eq ) in dichloromethane an equimolar amount of an aryl or heteroaryl isocyanate was added . in the case of the amine being in the form of a hydrochloride or bis - hydrochloride salt , equimolar amounts of tea were added to free - base the amine . the mixture was left stirring at 0 ° c . for 1 - 4 hours . the p - bromophenyl ureas generally precipitated out of solution as white solids , were recovered by filtration and if necessary purified further by washing with et 2 o or by flash chromatography . the m - bromophenylureas were isolated by solvent removal under reduced pressure and purified by crystallisation from an mixture of acoet : et 2 o . to a degassed solution of aryl or heteroaryl bromide prepared following the general procedure for urea synthesis described above ( 1 eq ), the appropriate boronic acid ( 1 . 3 eq ) was added dissolved in 40 volumes ( wt / vol ) of acetonitrile / 0 . 4n aqueous na 2 co 3 ( 1 / 1 ), pd [( pph 3 )] 4 ( 10 % mol ). the solution was refluxed overnight under nitrogen either in a round - bottom flask or in a glass test tube in a buchi syncore ® apparatus . the acetonitrile phase was separated and the desired products purified over a scx or silica column . fractions containing the desired product were combined and dried under reduced pressure . to a degassed solution of bromide prepared following the general procedure for urea synthesis ( 1 eq ), the appropriate boronic acid ( 1 eq ) and na 2 co 3 ( 3 eq ) in 20 volumes ( wt / vol ) of acetonitrile / water ( 1 / 1 ), pd [( pph 3 )] 4 ( 10 % mol ) were added . the solution was irradiated with microwave using following parameters : power 200 watt ; ramp time 1 min ; hold time 20 min ; temperature 90 ° c . ; pressure 200 psi . the acetonitrile phase was separated , the solvent was removed under reduced pressure and the crude material purified using scx column ( eluting with a gradient of dcm / meoh , meoh , nh 3 / meoh ). the fractions containing the desired product were combined and dried under reduced pressure . to a degassed solution of aryl / heteroaryl bromide prepared following the general procedure ( 1 eq ), in dme / h 2 o ( 1 . 8 / 0 . 3 ), the appropriate boronic acid . 1 . 5 eq ) na 2 co 3 ( 2 eq ), pd ( oac ) 2 ( 10 % mol ) and tri - o - tolylphosphine ( 40 % mol ), were added . the solution was irradiated under microwave conditions for 20 minutes at power = 200 w . the organic phase was separated and the desired products purified using scx column and / or prep - hplc . fractions containing the desired product were combined and dried under reduced pressure . azepine ( 0 . 99 g , 10 mmol ) was dissolved in 20 ml of dcm and n - boc - piperidone ( 2 . 58 g , 13 mmol ) was added . the reaction was stirred for 1 hour and then nabh ( oac ) 3 ( 3 . 16 g , 15 mmol ) was added and the mixture stirred for further 18 hours . the mixture was extracted with hcl solution at ph2 and then the aqueous phase was basified to ph 12 and extracted with dcm . the organic phase was concentrated under reduced pressure affording the title product ( 1 . 7 g , 60 % yield ). 1 h - nmr ( 400 mhz , cdcl3 ) 1 . 29 - 1 . 52 ( 1h , m ); 1 . 53 - 1 . 67 ( 8h , m ); 1 . 67 - 1 . 81 ( 2h , m ); 2 . 48 - 2 . 80 ( 7h , m ); 3 . 98 - 4 . 28 ( 2h , m ). 4 - azepan - 1 - yl - piperidine - 1 - carboxylic acid tert - butyl ester ( 1 . 7 g , 6 . 1 mmol ) was dissolved in 10 ml of dcm and 10 ml of tfa was slowly added . the reaction was stirred for 1 hour and then concentrated under reduced pressure . the mixture was neutralised with naoh 10 % and extracted with dcm , affording the title compound ( 800 mg , 72 % yield ). 1 h - nmr ( 400 mhz , cdcl3 ) 1 . 30 - 1 . 46 ( 2h , m ), 1 . 49 - 1 . 67 ( 9h , m ), 1 . 70 - 1 . 82 ( 2h , m ), 2 . 44 - 2 . 61 ( 3h , m ), 2 . 63 - 2 . 67 ( 4h , m ), 3 . 04 - 3 . 17 ( 2h , m ). to a cooled solution of 1 - piperidin - 4 - yl - azepane ( 0 . 80 g , 4 . 4 mmol ) in dichloromethane ( 20 ml ), 4 - bromophenylisocyanate ( 0 . 88 g , 4 . 4 mmol ) was added . the mixture was stirred at 0 ° c . until precipitation of a white solid was observed after 2 hours . the white solid was filtered and washed with et 2 o to afford 1 . 49 g of the title product ( 90 % yield ). nmr ( 400 mhz , dmso ): 1 . 19 - 1 . 39 ( 2h , m ); 1 . 43 - 1 . 58 ( 8h , m ); 1 . 61 - 1 . 63 ( 2h , m ); 2 . 52 - 2 . 65 ( 4h , m ); 2 . 65 - 2 . 82 ( 2h , m ); 4 . 03 - 4 . 19 ( 2h , m ); 7 . 36 ( 2h , d , j = 8 hz ); 7 . 42 ( 2h , d , j = 8 hz ), 8 . 57 ( 1h , s ). 4 - azepan - 1 - yl - piperidine - 1 - carboxylic acid ( 4 - bromo - phenyl )- amide was weighed ( 0 . 1 g , 0 . 26 mmol ), placed in a glass test tube and dissolved in 4 ml of a degassed solution of acetonitrile / 0 . 4n aqueous na 2 co 3 ( 1 / 1 ). to this solution , 2 - chlorophenyl boronic acid ( 0 . 066 g , 0 . 42 mmol ) and pd [ p ( ph ) 3 ] 4 ( 10 % mol ) were added . the mixture was heated at 80 ° c . and shaken in a buchi syncore ® for 18 hours . the solution was diluted with acoet and the organic phase was separated , and dried under reduced pressure ; the crude was purified over a sio 2 column ( eluent : gradient from dcm to dcm / meoh 9 / 1 ). the fractions containing the product were collected and dried under reduced pressure ( 14 % yield ). [ 1 , 4 ′] bipiperidinyl - 1 ′- carboxylic acid ( 4 - bromo - phenyl )- amide was weighed ( 0 . 1 g , 0 . 28 mmol ), placed in a glass test tube and dissolved with 4 ml of a previously degassed solution of acetonitrile / 0 . 4n aqueous na 2 co 3 ( 1 / 1 ). to this solution , 2 - chlorophenyl boronic acid ( 0 . 066 g , 0 . 42 mmol ) and pd [ p ( ph ) 3 ] 4 ( 10 % mol equivalents ) were added . the mixture was heated at 80 ° c . and shaken in a buchi syncore ® for 18 hours . the solution was diluted with acoet and the organic phase was separated , and dried under reduced pressure ; the crude was purified over a sio 2 column ( eluent : gradient from dcm to dcm / meoh 9 / 1 ). the fractions containing the product were collected and dried under reduced pressure to give the title compound ( 13 % yield ). 4 - pyrrolidin - 1 - yl - piperidine - 1 - carboxylic acid ( 4 - bromo - phenyl )- amide was weighed ( 0 . 1 g , 0 . 30 mmol ), placed in a glass test tube and dissolved in 4 ml of a degassed solution of acetonitrile / 0 . 4n aqueous na 2 co 3 ( 1 / 1 ). to this solution , 3 - benzamide - phenyl boronic acid ( 0 . 069 g , 0 . 42 mmol ) and pd [ p ( ph ) 3 ] 4 ( 10 % mol ) were added . the mixture was heated at 80 ° c . and shaken in a buchi syncore ® for 18 hours . the solution was diluted with acoet and the organic phase was separated , and dried under reduced pressure ; the crude was purified over a sio 2 column ( eluent : gradient from dcm to dcm / meoh 9 / 1 ). the fractions containing the product were collected and dried under reduced pressure ( 28 % yield ). 1 h - nmr ( cd3od ): 1 . 30 - 1 . 48 ( 2h , m ), 1 . 65 - 1 . 79 ( 4h , m ), 1 . 85 - 2 . 01 ( 1h , m ), 2 . 49 - 2 . 66 ( 4h , m ), 2 . 76 - 2 . 92 ( 2h , m ), 4 . 06 - 4 . 21 ( 2h , m ), 7 . 36 - 7 . 47 ( 3h , m ), 7 . 49 - 7 . 56 ( 2h , m ), 7 . 68 - 7 . 75 ( 1h , m ), 8 . 03 ( 1h , s ). to a suspension of n - boc - isonipecotic acid ( 3 . 0 g , 13 . 1 mmol ) and tbtu ( 4 . 2 g , 13 . 1 mmol ) in 60 ml of ch 3 cn , piperidine ( 1 . 67 g , 19 . 6 mmol ) was added . the resulting solution was stirred at 85 ° c . for 6 hours . the reaction mixture was concentrated under reduced pressure and then dissolved in dcm and washed twice with saturated aqueous na 2 co 3 solution . solvent removal gave 3 . 2 g of title compound that was used without further purification in the next step . 3 . 2 g ( 10 . 8 mmol ) of 4 -( piperidine - 1 - carbonyl )- piperidine - 1 - carboxylic acid tert - butyl ester were dissolved in 25 ml of 6n hcl solution at 0 ° c . after 10 minutes tlc showed complete conversion of the starting material , the mixture was cooled at 0 ° c ., basified to ph 10 with naoh ( pellets ) and extracted with acoet . the organic phase concentrated under reduced pressure gave 1 . 9 g of title compound ( 91 % yield ). nmr ( 400 mhz , dmso ): 1 . 43 - 1 . 59 ( 4h , m ), 1 . 60 - 1 . 75 ( 8h , m ), 2 . 53 - 2 . 71 ( 3h , m ), 3 . 06 - 3 . 20 ( 2h , m ), 3 . 36 - 3 . 47 ( 2h , m ), 3 . 50 - 3 . 61 ( 2h , m ). 1 . 9 g ( 9 . 8 mmol ) of piperidin - 4 - yl - piperidin - 1 - yl - methanone were added ( at 0 ° c .) to a suspension of 0 . 74 g ( 17 . 7 mmol ) of lialh 4 in anhydrous thf . after stirring for 30 minutes the reaction was heated at reflux for 1 hour when tlc analysis showed complete conversion of the starting amide . the reaction was cooled to 0 ° c . and lialh 4 was quenched with 0 . 7 ml of h 2 o and 2 . 8 ml of naoh ( 10 % aqueous solution ). the inorganic salts were filtered and the solution was concentrated under reduced pressure , affording 1 . 2 g of the title compound , with nmr purity about 80 %, that was used for the following step . nmr ( 400 mhz , cdcl3 ): 0 . 99 - 1 . 23 ( 2h , m ), 1 . 30 - 1 . 45 ( 2h , m ), 1 . 46 - 1 . 67 ( 4h , m ), 1 . 67 - 1 . 77 ( 2h , m ), 2 . 15 - 2 . 37 ( 4h , m ), 2 . 52 - 2 . 63 ( 1h , m ), 3 . 02 - 3 . 11 ( 1h , m ). to a cooled solution of 4 - piperidin - 1 - ylmethyl - piperidine ( 1 . 2 g , 80 % purity , 6 . 3 mmol ) in dichloromethane ( 20 ml ) p - bromophenylisocyanate ( 1 . 24 g , 6 . 3 mmol ) was added and the mixture stirred at 0 ° c . until a white solid precipitated out of solution after 2 hours . the white solid was filtered off and washed with et 2 o to give 1 . 2 g of pure title compound ( 50 % yield ). nmr ( 400 mhz , dmso ): 0 . 81 - 1 . 07 ( 2h , m ), 1 . 29 - 1 . 39 ( 2h , m ), 1 . 40 - 1 . 50 ( 4h , m ), 1 . 57 - 1 . 73 ( 3h , m ), 1 . 95 - 2 . 08 ( 2h , m ), 2 . 15 - 2 . 35 ( 4h , m ), 2 . 66 - 2 . 77 ( 2h , m ), 4 . 00 - 4 . 10 ( 2h , m ), 7 . 35 ( 2h , d , j = 8 . 8 hz ), 7 . 41 ( 2h , d , j = 8 . 8 hz ), 8 . 54 ( 1h , s ). to a degassed solution of 4 - piperidin - 1 - ylmethyl - piperidine - 1 - carboxylic acid ( 4 - bromo - phenyl )- amide ( 100 mg , 0 . 26 mmol ) in dme / h 2 o ( 1 . 8 ml / 0 . 3 ml ) 2 - fluorophenyl boronic acid ( 55 mg , 0 . 39 mmol ), na 2 co 3 ( 55 mg , 0 . 52 mmol ), pd ( oac ) 2 ( 6 mg , 10 % mol ) and tri - o - tolylphosphine ( 34 mg , 20 % mol ), were added . the solution was irradiated under microwave conditions for 20 minutes with power 200 w . the organic phase was then diluted with 1 ml of acoet , separated and loaded on a scx column and eluted with 10 ml of meoh to remove triphenylphosphinoxide and then with nh3 ( 2n meoh solution ) to recover the pure product ( 55 mg , 56 % yield ). 1 h - nmr ( 400 mhz , d6 - dmso ): 0 . 92 - 1 . 09 ( m , 2h ), 1 . 28 - 1 . 40 ( m , 2h ), 1 . 41 - 1 . 56 ( m , 4h ), 1 . 61 - 1 . 79 ( m , 3h ), 2 . 01 - 2 . 11 ( m , 2h ), 2 . 12 - 2 . 37 ( m , 4h ), 2 . 69 - 2 . 84 ( m , 2h ), 4 . 00 - 4 . 19 ( m , 2h ), 7 . 22 - 7 . 29 ( m , 2h ), 7 . 31 - 7 . 36 ( m , 1h ), 7 . 38 - 7 . 44 ( m , 2h ), 7 . 43 - 7 . 56 ( m , 1h ), 7 . 51 - 7 . 59 ( m , 2h ), 8 . 57 ( s , 1h ). to 2 . 5 ml of neat piperidine ( 26 mmol ) a solution of 4 . 9 g of 4 -[ 2 -( toluene - 4 - sulfonyloxy )- ethyl ]- piperidine - 1 - carboxylic acid tert - butyl ester in 20 ml of ch 3 cn was added and the reaction was heated at 80 ° c . for about 6 hours . when tlc showed complete conversion the mixture was cooled down to room temperature and washed with 20 ml of saturated nacl water solution . the organic phase was dried over na 2 so 4 and then concentrated under reduced pressure . the oil obtained was purified by sio 2 column eluting with gradient starting from 100 % dcm to dcm - nh3 ( 2n meoh solution ) 9 - 1 , affording 1 . 8 g of pure product ( yield 46 %). 1 h - nmr ( 400 mhz , cdcl3 ): 1 . 08 - 1 . 14 ( m , 2h ), 1 . 38 - 1 . 46 ( m , 15h ), 1 . 52 - 1 . 67 ( m , 7h ), 2 . 26 - 2 . 42 ( m , 6h ), 2 . 61 - 2 . 74 ( m , 2h ), 3 . 91 - 4 . 15 ( m , 2h ). to 44 ml of a solution of 6n hcl 4 -( 2 - piperidin - 1 - yl - ethyl )- piperidine - 1 - carboxylic acid tert - butyl ester was added and the reaction was stirred at room temperature for 10 minutes . the mixture was basified to ph 12 and the product was extracted with 20 ml of dcm . the organic phase was dried over na 2 so 4 and then concentrated under reduced pressure affording 440 mg of the pure product ( yield 37 %). to a cooled solution of 4 -( 2 - piperidin - 1 - yl - ethyl )- piperidine ( 440 mg , 2 . 2 mmol ) in dichloromethane ( 10 ml ) p - bromophenylisocyanate ( 442 mg , 2 . 2 mmol ) was added and the mixture stirred at 0 ° c . for 2 hours . the mixture was concentrated under reduced pressure and the residue was washed with et 2 o . the solid obtained was filtered giving 750 mg of pure product ( yield 85 %). to a degassed solution of 4 -( 2 - piperidin - 1 - yl - ethyl )- piperidine - 1 - carboxylic acid ( 4 - bromo - phenyl )- amide ( 100 mg , 0 . 25 mmol ) in dme / h 2 o ( 1 . 8 ml / 0 . 3 ml ) 2 - fluorophenyl boronic acid ( 55 mg , 0 . 39 mmol ), na 2 co 3 ( 55 mg , 0 . 52 mmol ), pd ( oac ) 2 ( 6 mg , 10 % mol ) and tri - o - tolylphosphine ( 34 mg , 20 % mol ), were added . the solution was irradiated under microwave conditions for 20 minutes with power 200 w . the organic phase was diluted with 1 ml of acoet and separated . the organic phase was loaded on scx column and eluted with 10 ml of meoh to remove triphenylphosphine oxide and then with nh3 ( 2n meoh solution ) to recover the pure product ( 25 mg , 25 % yield ). 1 h - nmr ( 400 mhz , cdcl3 ): 0 . 97 - 1 . 10 ( m , 2h ), 1 . 27 - 1 . 32 ( m , 3h ), 1 . 43 - 1 . 49 ( m , 4h ), 1 . 58 - 1 . 70 ( m , 2h ), 2 . 18 - 2 . 37 ( m , 4h ), 2 . 67 - 2 . 78 ( m , 2h ), 4 . 03 - 4 . 11 ( m , 2h ), 7 . 19 - 7 . 28 ( m , 2h ), 7 . 29 - 7 . 36 ( m , 1h ), 7 . 36 - 7 . 41 ( m , 2h ), 7 . 43 - 7 . 49 9m , 1h ), 7 . 51 - 7 . 56 ( m , 2h ), 8 . 55 ( s , 1h ). table 1 — examples 6 - 20 table 1 shows a selection of the compounds synthesised , which were prepared according to the method indicated in the last column of the table and discussed in detail in the experimental procedures with the synthesis of examples 1 - 5 . when the compound is indicated as the hcl salt , the salt was formed by dissolution of the free base in methanol and addition of 1 eq 1m hcl in ether followed by evaporation of the solvents . when the compound is indicated as hcooh ( formic acid ) salt , the compound was purified by preparative hplc . cloning of alpha7 nicotinic acetylcholine receptor and generation of stable recombinant alpha7 nachr expressing cell lines full length cdnas encoding the alpha7 nicotinic acetylcholine receptor were cloned from a rat brain cdna library using standard molecular biology techniques . rat gh4c1 cells were then transfected with the rat receptor , cloned and analyzed for functional alpha7 nicotinic receptor expression employing a flipr assay to measure changes in intracellular calcium concentrations . cell clones showing the highest calcium - mediated fluorescence signals upon agonist ( nicotine ) application were further subcloned and subsequently stained with texas red - labelled α - bungarotoxin ( bgtx ) to analyse the level and homogeneity of alpha7 nicotinic acetylcholine receptor expression using confocal microscopy . three cell lines were then expanded and one characterised pharmacologically ( see table 2 below ) prior to its subsequent use for compound screening . a robust functional flipr assay ( z ′= 0 . 68 ) employing the stable recombinant gh4c1 cell line was developed to screen the alpha7 nicotinic acetylcholine receptor . the flipr system allows the measurements of real time ca 2 + - concentration changes in living cells using a ca 2 + sensitive fluorescence dye ( such as fluo4 ). this instrument enables the screening for agonists and antagonists for alpha 7 nachr channels stably expressed in gh4c1 cells . gh4c1 cells stably transfected with rat - alphα7 - nachr ( see above ) were used . these cells are poorly adherent and therefore pretreatment of flasks and plates with poly - d - lysine was carried out . cells are grown in 150 cm 2 t - flasks , filled with 30 ml of medium at 37 ° c . and 5 % co 2 . ec 50 and ic 50 values were calculated using the idbs xlfit4 . 1 software package employing a sigmoidal concentration - response ( variable slope ) equation : the functional flipr assay was validated with the alpha7 nachr agonists nicotine , cytisine , dmpp , epibatidine , choline and acetylcholine . concentration - response curves were obtained in the concentration range from 0 . 001 to 30 microm . the resulting ec 50 values are listed in table 2 and the obtained rank order of agonists is in agreement with published data ( quik et al ., 1997 )( 22 ). the assay was further validated with the specific alpha7 nachr antagonist mla ( methyllycaconitine ), which was used in the concentration range between 1 microm to 0 . 01 nm , together with a competing nicotine concentration of 10 microm . the ic 50 value was calculated as 1 . 31 ± 0 . 43 nm in nine independent experiments . functional flipr assays were developed in order to test the selectivity of compounds against the alpha1 ( muscular ) and alpha3 ( ganglionic ) nach receptors and the structurally related 5 - ht3 receptor . for determination of activity at alpha1 receptors natively expressed in the rhabdomyosarcoma derived te 671 cell line an assay employing membrane potential sensitive dyes was used , whereas alpha3 selectivity was determined by a calcium - monitoring assays using the native sh - sy5y cell line . in order to test selectivity against the 5 - ht3 receptor , a recombinant cell line was constructed expressing the human 5 - ht3a receptor in hek 293 cells and a calcium - monitoring flipr assay employed . the compounds were tested using the functional flipr primary screening assay employing the stable recombinant gh4c1 cell line expressing the alpha7 nachr . hits identified were validated further by generation of concentration - response curves . the potency of compounds from examples 1 - 20 as measured in the functional flipr screening assay was found to range between 10 nm and 30 microm , with the majority showing a potency ranging between 10 nm and 10 microm . 1 . prendergast , m . a ., harris , b . r ., mayer , s ., holley , r . c ., pauly , j . r ., littleton , j . m . 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