Patent Application: US-16363302-A

Abstract:
compounds used for treating dependence on or withdrawal from a drug of abuse , for an eating disorder or for a cns disease or pathology having the following formulas :

Description:
the 2 , 6 - disubstituted piperidine analogs of the present invention include those contemplated by the following formula ( i ), without regard to chirality : r 1 represents a hydrogen , methyl , deuteromethyl ( cd 3 ), tritiomethyl ( ct 3 ), ethyl , or c 3 - c 7 straight chain or branched alkyl ( preferably methyl or ethyl ), c 3 - c 6 cycloalkyl , vinyl , allyl , c 4 - c 7 alkenyl ( including cis and trans geometrical forms ), benzyl , and phenylethyl . r 2 and r 3 are each independently ortho -, meta -, orpara - substituted moieties , where the substituent is described as hydrogen , methyl , ethyl , or c 3 - c 7 straight chain or branched alkyl , c 3 - c 6 cycloalkyl , vinyl , allyl , c 4 - c 7 alkenyl ( including cis and trans geometrical forms ), benzyl , and phenylethyl . further , the substitute moieties can be n - methylamino , n , n - dimethylamino , carboxylate , methylcarboxylate , ethylcarboxylate , propylcarboxylate , isopropylcarboxylate , carboxaldehyde , acetoxy , propionyloxy , isopropionyloxy , cyano , aminomethyl , n - methylaminomethyl , n , n - dimethylaminomethyl , carboxamide , n - methylcarboxamide , n , n - dimethylcarboxamide , acetyl , propionyl , formyl , benzoyl , sulfate , methylsulfate , hydroxyl , methoxy , ethoxy , propoxy , isopropoxy , thiol , methylthio , ethylthio , propiothiol , fluoro , chloro , bromo , iodo , trifluoromethyl , propargyl , nitro , carbamoyl , ureido , azido , isocyanate , thioisocyanate , hydroxylamino , and nitroso . the above 2 , 6 - substituted piperidino analogs are preferred in their 2 , 6 - cis geometrical isomeric forms , or in their 2 , 6 - trans geometric forms , including all possible geometric , racemic , diasteriomeric , and enantiomeric forms thereof . the above 2 , 6 - disubstituted piperidines as well as their analogs can be administered in their free base form or as a soluble salt . whenever it is desired to employ a salt of a 2 , 6 - substituted piperidine or its analog , it is preferred that a soluble salt be employed . some preferred salts include hydrochloride , hydrobromide , nitrate , sulfate , phosphate , tartrate , galactarate , fumarate , citrate , maleate , glycolate , malate , ascorbate , lactate , aspartate , glutamate , methanesulfonate , p - toluenesulfonate , benzenesulfonate , salicylate , proprionate , and succinate salts . the above salt forms may be in some cases hydrates or solvates with alcohols and other solvents . a pharmaceutical composition containing a compound of the invention is also contemplated , which may include a conventional additive , such as a stabilizer , buffer , salt , preservative , filler , flavor enhancer and the like , as known to those skilled in the art . representative buffers include phosphates , carbonates , citrates and the like . exemplary preservatives include edta , egta , bha , bht and the like . a composition of the invention may be administered by inhalation , i . e ., intranasally as an aerosol or nasal formulation ; topically , i . e ., in the form of an ointment , cream or lotion ; orally , i . e ., in solid or liquid form ( tablet , gel cap , time release capsule , powder , solution , or suspension in aqueous or non aqueous liquid ; intravenously as an infusion or injection , i . e ., as a solution , suspension or emulsion in a pharmaceutically acceptable carrier ; transdermally , e . g ., via a transdermal patch ; rectally as a suppository and the like . generally , the pharmacologically effective dose of a present compound is in the amount ranging from about 1 × 10 − 5 to about 1 mg / kg body weight / day . the amount to be administered depends to some extent on the lipophilicity of the specific compound selected , since it is expected that this property of the compound will cause it to partition into fat deposits of the subject . the precise amount to be administered can be determined by the skilled practitioner in view of desired dosages , side effects and medical history of the patient and the like . the 2 , 6 - disubstituted piperidino analogs of the present invention exhibit activity at either nachrs and / or the serotonin transporter protein ( sert ) and / or the vesicular monoamine transporter ( vmat2 ). table 1 below summarizes the interaction of cis - 2 , 6 - di - trans - styrylpiperidine ( compound 1 ) and trans - 2 , 6 - di - trans - styrylpiperidine ( compound 2 ) with nicotinic receptors , sert and vmat2 . the two 2 , 6 - disubstituted piperidino derivatives in table 1 have the chemical structure of formula i , and were assayed for interaction with α4β *, α7 *, α3β2 * and α3β4 * subtypes of nachrs , interaction with vmat2 located on vesicle membranes and inhibition of sert function . it shall be noted that the nachr subtypes for the activities described herein have not been elucidated conclusively , and thus , the asterisk is an indication of the putative nature of the receptor subtype mediating the action . compound 1 exhibits good selectivity ( 43 - fold ) for the α3β2 * subtype of nachr relative to its interaction with the mtbz site on vmat2 , as indicated by its ability to inhibit nicotine - evoked [ 3 h ] da release . moreover , compound 1 exhibits very good selectivity (& gt ; 370 - fold ) for the α3β2 * and α3β4 * subtypes of nachrs compared to its interaction with either α4β2 * or α7 * subtypes of nachr . furthermore , compound 1 was 770 - fold more selective as an inhibitor of the α3β2 * and α3β4 * subtypes of nachr compared to its inhibition of sert function . finally , compound 1 interacted with vmat2 18 - fold more selectively than it inhibited the function of sert . compound 2 was also assayed for interaction with nachrs subtypes , interaction with vmat2 located on vesicle membranes and inhibition of sert function . compound 2 exhibits good selectivity ( 9 . 5 - fold ) for the α3β2 * subtype of nachr relative to its interaction with the mtbz site on vmat2 , as indicated by its ability to inhibit nicotine - evoked [ 3 h ] da release . moreover , compound 2 exhibits very good selectivity (& gt ; 24 - fold ) for the α3β2 * subtype of nachr compared to its interaction with either α4β2 * or α7 * subtypes of nachr . furthermore , compound 2 was only 2 . 2 - fold more selective as an inhibitor of the α3β2 * subtypes of nachr compared to its inhibition of sert function . finally , compound 2 interacted with sert 4 . 3 - fold more selectively than it interacted with vmat2 . compound 1 was 18 - fold more potent at inhibiting nicotine - evoked [ 3 h ] da and [ 3 h ] ne release from rat striatal and hippocampal slices , indicated a higher affinity for the α3β2 * and α3β4 * subtypes of nachrs , compared to compound 2 . on the other hand , compound 2 was 19 - fold more potent inhibiting the function of sert than was compound 1 . finally , compound 1 was only 4 - fold more potent interacting with vmat2 compared to compound 2 . thus , alteration of stereochemistry at c2 and c6 from cis to trans resulted in a diminished affinity for the α3β2 * subtypes of nachr and for vmat2 , and enhanced the affinity for sert , whereas there was no change in affinity for either α4β2 * or α7 * subtypes of nar . therefore , cis - analogs have higher affinity for vmat2 and α3β2 * subtype of nachrs . the invention will now be discussed by certain examples which illustrate but do not limit the invention . to a suspension of l - lobeline hemisulfate salt ( 85 %, 10 . 5 g ) in absolute ethanol ( 300 ml ) was added nabh 4 ( 1 . 5 eq .) portionwise at 0 ° c . the mixture was stirred at 0 ° c . for 1 h , and then quenched with acetone . the mixture was evaporated under reduced pressure . water ( 100 ml ) was added to the residue and extracted with chloroform ( 80 ml × 3 ). the combined organic extract was dried ( mgso 4 ), filtered and evaporated to afford lobelanidine as a white solid which was used directly . an analytical sample was recrystallized from acetone / hexane as a needle crystal . mp 142 - 143 ° c ; 1 h nmr ( 300 mhz , cdcl 3 ) δ 1 . 15 ( m , 2h ), 1 . 44 - 1 . 82 ( m , 6h ), 2 . 04 ( ddd , j = 14 . 4 , 10 . 2 , 8 . 7 hz , 2h ), 2 . 35 ( s , 3h ), 2 . 96 ( m , 2h ), 4 . 89 ( dd , j = 8 . 7 , 5 . 5 hz , 2h ), 7 . 23 - 7 . 50 ( m , 10h ); 13 c nmr ( 75 mhz , cdcl 3 ) δ 23 . 48 , 25 . 20 , 25 . 96 , 41 . 79 , 62 . 37 , 74 . 30 , 125 . 98 , 127 . 50 , 128 . 55 , 144 . 88 ppm . crude lobelanidine was dissolved in 130 ml 85 % h 3 po 4 and allowed to stir at 60 ° c . for 24 h . the reaction mixture was cooled to room temperature , diluted with 250 ml water and made basic with solid naoh ( ph ˜ 10 ). the aqueous solution was extracted with etoac ( 150 ml × 3 ) the combined organic extract was dried ( mgso 4 ), filtered and concentrated under reduced pressure . the crude product was recrystallized from acetone affording 2 . 2 g of pure cis - 2 , 6 - di - trans - styrylpiperidine as a white solid . mp : 105 - 106 ° c . ( hcl salt ); 1 h nmr ( 300 mhz , cdcl 3 ) δ 1 . 42 - 1 . 88 ( m , 6h ), 2 . 25 ( s , 3h ), 2 . 63 ( m , 2h ), 6 . 21 ( dd , j = 15 . 9 , 9 . 0 , 2h ), 6 . 51 ( d , j = 15 . 9 , 2h ), 7 . 19 - 7 . 41 ( m , 10h ); 13 c nmr ( 75 mhz , cdcl 3 ) δ 24 . 14 , 33 . 94 , 42 . 61 , 68 . 56 , 126 . 32 , 127 . 46 , 128 . 69 , 130 . 51 , 134 . 03 , 137 . 17 ppm . the remaining mother liquor was evaporated and the residue was chromatographed ( sio 2 , etoac / hexanes , 1 / 10 ) to afford cis - 2 , 6 - di - trans - styrylpiperidine 1 . 4g and trans - 2s , 6s - di - trans - styrylpiperidine 230 mg . mp : 200 - 202 ° c . ( hcl salt ); 1 h nmr ( 300 mhz , cdcl 3 ) δ 1 . 60 - 1 . 75 ( m , 4h ), 1 . 82 - 1 . 97 ( m , 2h ), 2 . 27 ( s , 3h ), 3 . 38 ( m , 2h ), 6 . 38 ( dd , j = 15 . 9 , 8 . 7 , 2h ), 6 . 52 ( d , j = 15 . 9 , 2h ), 7 . 17 - 7 . 42 ( m , 10h ); 13 c nmr ( 75 mhz , cdcl 3 ) δ 19 . 57 , 32 . 92 , 41 . 96 , 62 . 29 , 126 . 39 , 127 . 50 , 128 . 69 , 130 . 49 , 131 . 78 , 137 . 20 ppm . the ability to displace s (−)[ 3 h ] nicotine binding from rat striatal membranes which assessed the α4β2 * subtype was determined . the [ 3 h ] nicotine binding assay was performed according to previously published methods ( romano et al ., 1980 ; marks et al ., 1986 ; crooks et al ., 1995 ). striata from two rats were dissected , pooled and homogenized with a tekmar polytron in 10 vol of ice - cold modified krebs - hepes buffer ( 20 mm hepes , 118 nim nacl , 4 . 8 mm kcl , 2 . 5 mm cacl 2 , 1 . 2 mm mgso 4 , adjusted to ph to 7 . 5 ). the homogenate was incubated at 37 ° c . for 5 min to promote hydrolysis of endogenous acetylcholine , and centrifuged at 27 , 000 g for 20 min and the pellet was resuspended in 10 vol of ice - cold distilled water and incubated at 37 ° c . for 5 min , followed by centrifugation at 27 , 000 g for 20 min . the pellet containing the striatal membranes was resuspended in 10 vol of fresh ice - cold 10 % krebs - hepes buffer and incubated at 37 ° c . for 10 min after which it was centrifuged at 27 , 000 g for 20 min . the latter sequence of resuspension , incubation and centrifugation was repeated . the pellet was frozen under fresh krebs - hepes buffer and stored at − 40 ° c . until assay . upon assay , the pellet was resuspended in krebs - hepes buffer , incubated at 37 ° c . for 5 min and centrifuged at 27 , 000 g for 20 min . the final pellet was resuspended in 3 . 6 ml ice - cold water , which provides for approximately 200 μg protein / 100 μl aliquot , as defined by the bradford dye binding method ( bradford , 1976 ) using bovine serum albumin ( bsa ) as the standard . competition assays were performed in duplicate in a final vol of 200 μl krebs - hepes buffer containing 250 mm tris buffer ( ph 7 . 5 at 4 ° c .). reactions were initiated by addition of 100 μl of membrane suspension to 3 nm [ 3 h ] nicotine ( l -(−)-[ n - methyl - 3 h ] nicotine ; 50 μl , specific activity 69 . 5 ci / mmol ) and 1 of at least 9 concentrations of analog ( 50 μl ). after a 90 min incubation at 4 ° c ., reactions were terminated by dilution of the samples with 3 ml of ice - cold buffer followed immediately by filtration through a whatman gf / b glass fiber filters ( presoaked in 0 . 5 % polyethyleneimine using a brandel cell harvester ). filters were rinsed 3 times with 3 ml of ice - cold buffer , transferred to scintillation vials and 5 ml scintillation cocktail added . nonspecific binding was defined as binding in the presence of 10 μm s (−)- nicotine . for competition curves , the ic 50 values were corrected for ligand concentration to obtain ki values for each analog ( cheng et al ., 1973 ). whole rat brain ( minus cortex , striatum and cerebellum ) was homogenized in 20 vol of ice - cold hypotonic buffer ( 2 mm hepes , 14 . 4 mm nacl , 0 . 15 mm kcl , 0 . 2 mm cacl 2 and 0 . 1 mm mgso 4 , ph 7 . 5 ). homogenates were incubated at 37 ° c . for 10 min and centrifuged at 25 , 000 g for 15 min at 4 ° c . pellets were washed 3 times by resuspension in 20 vol of the same buffer and centrifugation using the above parameters . final pellets were resuspended in incubation buffer to provide ˜ 150 μg protein / 100 μl . binding assays were performed in duplicate , in a final vol of 250 μl incubation buffer , containing 20 mm hepes , 144 mm naci , 1 . 5 mm kcl , 2 mm cacl 2 , 1 mm mgso 4 and 0 . 05 % bsa , ph 7 . 5 . assays were initiated by the addition of 100 μl membrane suspension to 150 μl of sample containing 2 . 5 nm [ 3 h ] mla ([ 1α , 4 ( s ), 6β , 14α , 16β ]- 20 - ethyl - 1 , 6 , 14 , 16 - tetramethoxy - 4 [[[ 2 -(-[ 3 - 3 h ]- methyl - 2 , 5 - dioxo - 1 - pyrrolidinyl ) benzoyl ] oxy ] methyl ] aconitane - 7 , 8 - diol ; specific activity 26 . 2 ci / mmol ) and at least 6 concentrations ( 30 nm - 100 μm ) synthetic analog ( final concentrations ), and incubated for 2 h at room temperature . nonspecific binding was determined in the presence of 10 μm mla . assays were terminated by dilution of samples with 3 ml ice - cold incubation buffer followed by immediate filtration through schleicher & amp ; schuell # 32 glass fiber filters ( keene , nh ; presoaked with 0 . 5 % pei ) using the cell harvester . filters were rinsed 3 times with 3 ml of ice - cold buffer , transferred to scintillation vials , 4 ml of scintillation cocktail added , and bound radiolabel determined by liquid scintillation spectroscopy . protein was measured using the bradford dye - binding method ( bradford , 1976 ) with bsa as the standard . synaptic vesicles were prepared from rat brain using a modification of a previously described procedure ( teng et al ., 1998 ). briefly , fresh whole brain ( excluding cerebellum ) was homogenized using a teflon pestle ( clearance 0 . 003 mm ) with 7 vertical strokes at 800 rpm in 20 vol of ice - cold 0 . 32 m sucrose and centrifuged at 1000 g for 12 min at 4 ° c . the resulting supernatant ( s 1 ) was then centrifuged at 22 , 000 g for 10 min at 4 ° c . the synaptosomal pellets ( p 2 ) were homogenized in 18 ml of ice - cold milli - q water and exposed for 5 min for lysing synaptosomes . osmolarity was restored by addition of 2 ml of 25 mm hepes with 100 mm dipotassium tartrate ( ph 7 . 5 ). samples were centrifuged at 20 , 000 g for 20 min at 4 ° c . to remove lysed synaptosomal membranes . mgso 4 ( 1 mm ) was added to the supernatant ( s 3 ), and was centrifuged at 100 , 000 g for 45 min at 4 ° c . the final vesicular pellets ( p 4 ) were resuspended in ice - cold assay buffer ( see below ) providing ˜ 15 μg protein / 100 μl , determined by the method of bradford ( 1976 ) using bovine serum albumin as a the standard . aliquot parts ( 100 μl ) of suspension of vesicle membrane protein were incubated in assay buffer ( 25 mm hepes , 100 mm dipotassium tartrate , 5 mm mgso 4 , 0 . 1 mm edta and 0 . 05 mm egta , ph 7 . 5 , at 25 ° c .) in the presence of 3 nm [ 3 h ] mtbz ([ o - methyl - 3 h ] methoxytetrabenazine ) and at least 7 concentrations ( 1 nm - 1 mm ) of analog for 1 hr at room temperature . nonspecific binding was determined in the presence of 20 μl tbz . assays were performed in duplicate using a 96 - well plate format . reactions were terminated by filtration of samples on a unifilter - 96 gf / b filter plates ( presoaked in 0 . 5 % polyethylenimine ), using a filtermate harvester ( packard bioscience co ., meriden , conn .). after washing 5 times with 350 μl of the ice - cold wash buffer ( 25 mm hyepes , 100 mm dipotassium tartrate , 5 mm mgso 4 and 10 mm nacl , ph 7 . 5 ), filter plates were dried , sealed and each well filled with 40 μl packard &# 39 ; s microscint 20 cocktail . bound [ 3 h ] mtbz was measured using a packard topcount nxt scintillation counter with a packard windows nt based operating system . [ 3 h ] 5 - ht uptake was assessed using modifications of a previously described method ( teng et al ., 1998 ). nonspecific uptake was determined in duplicate samples in the presence of excess ( 10 μm ) fluoxetine . rat hippocampus was homogenized in 20 ml cold 0 . 32 m sucrose containing 5 mm nahco 3 ( ph 7 . 4 ) with 12 vertical strokes of a teflon pestle homogenizer ( clearance ˜ 0 . 015 mm ). the homogenate was centrifuged ( 2 , 000 × g for 10 min at 4 ° c .). the supernatant was centrifuged ( 20 , 000 × g for 15 min at 4 ° c . ), and then the pellet was resuspended in 1 . 5 ml of kreb &# 39 ; s buffer ( 125 mm nacl , 5 mm kcl , 1 . 5 mm mgso 4 , 1 . 25 mm cacl 2 , 1 . 5 mm kh 2 po 4 , 10 mm α - d - glucose , 25 mm hepes , 0 . 1 mm disodium ethylenediamine tetraacetate , 0 . 1 mm pargyline and 0 . 1 mm ascorbic acid , saturated with 95 % o 2 / 5 % co 2 , ph 7 . 4 ). final protein concentration was 400 μg / ml , determined using bsa standard ( bradford , 1976 ). the assay was performed in duplicate in a total vol of 500 μl . aliquot parts of synaptosomal suspension ( 50 μl ) were added to tubes containing 350 μl kreb &# 39 ; s buffer and 50 μl of buffer containing 1 of 9 concentrations of analog . tubes were preincubated at 34 ° c . for 10 min before addition of 50 μl of [ 3 h ] 5 - ht ( 5 -[ 1 , 2 - 3 h ( n )]- hydroxytryptamine ; specific activity 25 . 5 ci / mmol , final concentration 10 nm ). accumulation proceeded for 10 min at 34 ° c . reactions were terminated by addition of 3 ml ice - cold kreb &# 39 ; s buffer . samples were rapidly filtered through a whatman gf / b filter using a cell harvester ( mp - 43rs , brandel inc ., gaithersburg , md . ), and the filter was subsequently washed 3 times with 4 ml ice - cold kreb &# 39 ; s buffer containing catechol ( 1 mm ). filters were previously soaked for 2 hours in the ice - cold kreb &# 39 ; s buffer containing catechol ( 1 mm ). radioactivity on filters was determined by liquid scintillation spectroscopy . alkaloid effects on [ 3 h ] overflow from rat striatal slices preloaded with [ 3h ] da and hippocampal slices preloaded with [ 3 h ] ne were determined using modifications of a previously published method ( dwoskin and zahniser , 1986 ). briefly , coronal striatal or hippocampal slices ( 500 μm , 6 - 8 mg ) were incubated in krebs &# 39 ; buffer ( 118 mm nacl , 4 . 7 mm kcl , 1 . 2 mm mgcl 2 , 1 . 0 mm nah 2 po 4 , 1 . 3 mm cacl 2 , 11 . 1 mm α - d - glucose , 25 mm nahco3 , 0 . 11 mm l - ascorbic acid and 4 . 0 mm disodium ethylenediaminetetraacetate ; ph 7 . 4 , and saturated with 95 % o 2 / 5 % co 2 ) in a metabolic shaker at 34 ° c . for 30 min . striatal or hippocampal slices were incubated in fresh buffer ( 6 - 8 slices / 3 ml ) containing 0 . 1 μm [ 3 h ] da ( 3 , 4 -[ 7 - 3 h ]- dihydroxyphenylethylamine ; specific activity 28 ci / mmol ) or 0 . 1 μm [ 3 h ] ne ( levo -[ 7 - 3 h ]- norepinephrine ; specific activity 14 . 4 ci / mmol ), respectively , for an additional 30 min . after rinsing , each slice was transferred to a glass superfusion chamber maintained at 34 ° c . and was superfused at 1 ml / min with oxygenated krebs &# 39 ; buffer containing pargyline ( 10 μm ) and nomifensine ( 10 μl ) or pargyline and desipramine ( 10 μl ) in experiments assessing [ 3 h ] da and [ 3 h ] ne overflow , respectively . after 60 min of superfusion , three 5 - min samples ( 5 ml ) were collected to determine basal [ 3 h ] outflow . after collection of the third basal sample , slices from an individual rat were superfused in the absence or presence of a single concentration of analog , which remained in the buffer until the end of the experiment . each slice was exposed to only one concentration of analog . after 30 min , s (−)- nicotine ( 10 μm ) was added to the buffer containing analog , and superfusion continued for an additional 60 min . these experiments utilized a repeated measures design , such that the analog concentration - effect was determined in both the absence and presence of s (−)- nicotine using striatal or hippocampal slices from a single rat . additionally , one striatal or hippocampal slice was superfused in the absence of analog and constituted the control condition . at the end of the experiment , each slice was solubilized with ts - 2 . the ph and volume of the solubilized tissue samples were adjusted to those of the superfusate samples . radioactivity in the superfusate and tissue samples was determined by liquid scintillation spectroscopy ( packard model b1600 tr scintillation counter , downer &# 39 ; s grove , ill .). analog effects on 86 rb + efflux were determined using a previously published method ( miller et al ., 2000 ). thalamus was homogenized and centrifuged at i1000 × g for 10 min at 4 ° c . the supernatant fraction was centrifuged at 12 , 000 × g for 20 min at 4 ° c . to obtain the synaptosomal fraction . synaptosomes were incubated for 30 min in 35 μl of uptake buffer ( 1 . 40 mm nacl , 1 . 5 mm kcl , 2 . 0 mm cacl 2 , 1 . 0 mm mgso 4 , 20 mm α - d - glucose ; ph 7 . 5 ) containing 4 μci of 86 rb + . 86 rb + uptake was terminated by filtration of the synaptosomes onto glass fiber filters ( 6 mm ; type a / e , gelman sciences , ann arbor , mich .) under gentle vacuum ( 0 . 2 atm ), followed by three washes with superfusion buffer ( 0 . 5 ml each ). subsequently , each filter with 86 rb + - loaded synaptosomes ( 40 μg protein / μl ) was placed on a 13 mm glass fiber filter ( type a / f ) mounted on a polypropylene platform . 86 rb + efflux assay buffer ( 125 mm nacl , 5 mm cscl , 1 . 5 mm kcl ; 2 mm cacl 2 , 1 mm mgso 4 , 25 mm hepes , 20 mm α - d - glucose , 0 . 1 μm tetrodotoxin , 1 . 0 g / l bovine serum albumin ; ph 7 . 5 ) was superfused onto the synaptosomes at a rate of 2 . 5 ml / min . tetrodotoxin and cscl were included in the buffer to block voltage - gated na + and k + channels , respectively , and to reduce the rate of basal 86 rb + efflux . the ability of analogs to inhibit 86 rb + efflux evoked by 1 μm nicotine was determined . after 8 min of superlusion , samples were collected ( sample / 18 s ) for 5 min to determine basal 86 rb + efflux . subsequently , synaptosomes were superfused for 3 min with analog followed by superfusion with buffer containing analog and nicotine for an additional 3 min . each aliquot part of thalamic synaptosomes was exposed to only one concentration of analog . in each experiment , one synaptosomal aliquot part was superfused in the absence of analog to determine basal 86 rb + efflux over the course of the superfusion period , and another aliquot part was superfused with nicotine ( 1 μm ) to determine the effect of nicotine on 86 rb + efflux in the absence of analog . samples were analyzed by liquid scintillation spectroscopy ( packard model b1600 tr scintillation counter ). the foregoing is considered as illustrative only of the principles of the invention . further , since numerous modifications and changes will readily occur to those skilled in the art , it is not desired to limit the invention to the exact construction and operation shown and described , and accordingly , all suitable modifications and equivalence thereof may be resorted to , falling within the scope of the invention claimed . the pertinent disclosures of the references listed below and as discussed above herein are incorporated herein by reference . barlow r . b . et al ., “ relations between structure and nicotine - like activity : x - ray crystal structure analysis of (−) cystine and (−) lobeline hydrochloride and a comparison with (−) nicotine and other nicotine - like compounds ,” br . j . pharmacol ., 1989 ; 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30 : 427 - 436 . miller d . k et al ., “ lobeline inhibits nicotine - evoked [ 3 h ] dopamine overflow from rat striatal slices and nicotine - evoked 86 rb + efflux from thalamic synaptosomes . neuropharmacology , 2000 ; 39 : 2654 - 2662 . olin b . r . et al ., drug facts and comparisons , jb lippincott co ., st . louis , mo ., pp 3087 - 3095 ( 1995 ). romano c . et al ., “ stereospecific nicotinic receptors on rat brain membranes ,” science , 1980 ; 210 : 647 - 650 . teng l . h . et al ., “ lobeline and nicotine evoke [ 3 h ]- overflow from rat striatal slices reloaded with [ 3 h ] dopamine : differential inhibition of synaptosomal and vesicular [ 3 h ] dopamine uptake ,” j . pharmacol . exp . therap ., 1997 ; 80 : 1432 - 1444 . teng l . h . et al , “ lobeline displaces [ 3 h ] dihydrotetrabenazine binding and releases [ 3 h ] dopamine from rat sriatal synaptic vesicles ,” j . neurochem ., 1998 ; 71 : 258 - 265 .