novel compounds are described which are used in the generation of antibodies and in the immunoassay process generally . the compounds are derivatives of methadone and the methadone metabolites . the elaboration of methadone can be performed at either end of the methadone molecule ; that is , the ethyl keto group may be substituted with another aliphatic keto group which includes an amine , carboxylic acid or thiol function to aid in the attachment of the derivative to the protein , polypeptide or label . in addition , the opposite end of the methadone molecule can be elaborated , for example , starting with normethadone and alkylating the secondary amine with a haloalkyl thioester or acylating with a carboxylic acid thioester . the elaboration of methadone metabolites , for example , 2 - ethylidene - 1 , 5 - dimethyl - 3 , 3 - diphenylpyrrolidine ( eddp ) and 2 - ethyl - 5 - methyl - 3 , 3 - diphenylpyrroline ( emdp ) can be performed on the pyrrolidine or the pyrroline rings , respectively . the synthesis of a particular derivative should allow for the character of the methadone or methadone metabolite derivative to be properly presented to the antibody or receptor in a manner which allows for the desired binding interaction . the synthesis of the linking group between the protein , polypeptide or label and the methadone or methadone metabolite derivative is designed to achieve the desired binding of the drug derivative and the receptor . for example , the derivative may be displaced from the surface of the protein , polypeptide or label to allow the derivative to present itself to the binding domain of receptors . in general , the compounds of this invention have the following formula : ## str4 ## where r is a linking group comprising one of the following : ## str5 ## where a is a linking group of from 1 to 20 carbons and from 0 to 10 heteroatoms ( h , o , s ), either branched or straight chain . in addition , the general form of the immunogenic protein or polypeptide molecule or the protein or polypeptide molecule or label which is derivatized via an amide , disulfide , thioether , or ester bond to the molecule or label also to a compound of the formula , is of the following : ## str6 ## where p is an antigenic protein or polypeptide or a protein , polypeptide or label ; where x is at least one and not greater than 100 ; where r is a linking group comprising one of the following : ## str7 ## where a is a linking group of from 1 to 20 carbons and 0 to 10 heteroatoms ( hn , o , s ) either branched or straight chain ; where b is a linking group ultimately attached to a protein , polypeptide or label selected from the group comprising : ## str8 ## where z is a linking group of from 1 to 20 carbons and 0 to 10 heteroatoms ( nh , o , s ) and may be branched or straight chain . in general , the compounds of this invention also have the following formula : ## str9 ## where r &# 39 ; is a saturated or unsaturated aliphatic group of between 0 and 5 carbon atoms ; ( or hydrogen when r &# 39 ; is o ). where r is a linking group comprising one of the following : ## str10 ## where a is a linking group of from 1 to 20 carbons and from 0 to 10 heteroatoms ( nh , o , s ), either branched or straight chain . in addition , the general form of the immunogenic protein or polypeptide molecule or the protein or polypeptide molecule or label which is derivatized via an amide , disulfide , thioether , or ester bond to the molecule or label also to a compound of the formula , is of the following : ## str11 ## where p is an antigenic protein or polypeptide or a protein , polypeptide or label ; where x is at least one and not greater than 100 ; where r &# 39 ; is a saturated or unsaturated aliphatic group of between 0 and 5 carbon atoms . where r is a linking group comprising one of the following : ## str12 ## where a is a linking group of from 1 to 20 carbons and 0 to 10 heteroatoms ( nh , o , s ), either branched or straight chain ; where b is a linking group ultimately attached to a protein , polypeptide or label selected from the group consisting of : ## str13 ## where z is a linking group of from 1 to 20 carbons and 0 to 10 heterocarbons ( nh , o , s ) and may be branched or straight chain . in general , the compounds of this invention also have the following formula : ## str14 ## where r is a saturated or unsaturated linking group comprising one of the following : ## str15 ## where a is a linking group of from 1 to 20 carbons and from 0 to 10 heterocarbons ( nh , o , s ), either branched or straight chain . in addition , the general form of the immunogenic protein or polypeptide molecule or the protein or polypeptide molecule or label which is derivative via an amide , disulfide , thioether , or ester bond to the molecule or label also to a compound of the formula is of the following : ## str16 ## where p is an antigenic protein or polypeptide or a protein , polypeptide or label ; where x is at least one and not greater than 100 ; where r is a linking group comprising one of the following : ## str17 ## where a is a linking group of from 1 to 20 carbons and 0 to 10 heterocarbons ( nh , o , s ), either branched or straight chain ; where b is a linking group ultimately attached to a protein , polypeptide , or label selected from the group comprising : ## str18 ## where z is a linking group of from 1 to 20 carbons and 0 to 10 heterocarbons ( nh , o , s ) and may be branched or straight chain . in general , the compounds of this invention also have the following formula : ## str19 ## where r is a saturated or unsaturated linking group comprising one of the following : ## str20 ## in addition , an immunogenic protein or polypeptide molecule or the protein or polypeptide molecule or label which is derivatized via an amide or ester bond to the molecule or label to a compound of the formula , is of the following : ## str21 ## where p is an antigenic protein or polypeptide or a protein , polypeptide or label ; where x is at least one and not greater than 100 ; where r is a linking group comprising of one of the following : ## str22 ## where a is the linking group of 1 to 20 carbons and 0 to 10 heterocarbons ( nh , o , s ), either branched or straight chain ; where b is a linking group ultimately attached to a protein , polypeptide or label selected from the group comprising : ## str23 ## where z is a linking group of from 1 to 20 carbons and 0 to 10 heterocarbons ( nh , o , s ) and may be branched or straight chain . the preferred ( best mode ) compounds of this invention have the following formula : ## str24 ## where r is a linking group comprising one of the following : ## str25 ## in addition , the preferred ( best mode ) immunogenic protein or polypeptide molecule or the protein or polypeptide molecule or label derivatized via an amide or ester bond to the molecule or label to a compound of the formula is of the following : ## str26 ## where p is an antigenic protein or polypeptide or a protein , polypeptide or label ; where x is at least one and not greater than 100 ; where z is a linking group of from 1 to 20 carbons and 0 to 10 heterocarbons ( nh , o , s ) and may be branched or straight chain . of particular interest are methadone derivatives which have been synthesized by substitution of the ethyl keto group of methadone with a linking group containing a thioester function . methadone derivatives can also be synthesized by alkylation or acylation of the secondary amine of a precursor of normethadone . the alkylation reactions can be performed using various chain length alkyl halide carboxylic acids , for example , 3 - iodopropionic acid or ethyl - 3 - iodo propionate can be reacted to form an n - alkylated carboxylic acid methadone derivative , which can then be further reacted with an amino alkyl thiol ester , such as homocysteine thiolactone , to synthesize the thiol ester derivative of methadone . acylation reactions can also be performed with normethadone and various chain length alkyl thioester carboxylic acids , for example , 3 - acetylthio propionic acid , to synthesize an amide thio ester derivative of methadone . the attachment of linking arms to methadone metabolites , for example , the eddp and the emdp , can be performed through derivatization of the pyrrolidine and pyrroline rings , respectively . in the case of the synthesis of thiol ester derivatives of the eddp molecule , for example , elaboration of the ethyl keto function of methadone to an alkyl carboxylic acid ( example 2 ), or alkyl thio ester ( example 3 ) is first performed and then the methadone thio ester derivative is converted into the normethadone thio ester derivative so that a schiff base of the secondary amine and the keto function can form . further isomerization of the schiff base to the unsaturated alkyl substitutent , mimicking the eddp structure , then forms the 2ethylenealkyl carboxylic acid derivative of eddp ( fig1 ) or the 2 - ethylenealkyl thio ester derivative of eddp ( fig2 ), respectively . the ethylene carboxylic acid derivative of eddp ( fig1 ) can be further reacted with an amino alkyl thioester , such as homocysteine thiolactone to form the thio ester derivative of eddp ( fig2 ). in another aspect of the instant invention , the synthesis of thio ester derivatives of emdp can be accomplished . the schiff base of the emdp is reduced with , for example , sodium cyanoborohydride , and the resulting secondary amine of the emdp derivative may be either alkylated with various chain length alkyl halide carboxylic acids , for example , 3 - iodopropionic acid , to form the n - alkylated carboxylic acid of emdp derivative , which can be further reacted with an amino alkyl thio ester , such as homocysteine thiolactone , to synthesize the thio ester derivative of emdp ( fig3 ), or acylated with various chain lengths of carboxylic acid alkyl thio esters , such as 3 - acetylthiopropionic acid to form the thio ester derivative of emdp ( fig4 ). the thio esters of the resulting methadone or methadone metabolite derivatives are hydrolyzed in dilute base , for example , 0 . 01m potassium hydroxide , to generate the thiol group which is reacted with the thiol reactive group , such as a maleimide , an alkyl halide or a thiol . those skilled in the art can recognize the versatility of synthetic strategies described herein . the compounds are synthesized as thiols or thiol esters so that their covalent attachment to proteins , polypeptides or labels can easily be performed under mild conditions , for example , ph 7 in a protein solution . the protein , polypeptide or label is reacted with a reagent which incorporates a maleimide or an alkylhalide or a thiol into the molecule . these reagents and methods for their use are available from pierce , rockford , ill ., for example , for incorporation of maleimide groups onto proteins , polypeptides or labels one can use succinimidyl 4 -( n - maleimidomethyl ) cyclohexane - 1 - carboxylate ( smcc ), succinimidyl 4 -( p - maleimidophenyl ) butyrate ( smpb ) or m - maleimidobenzoyl - n - hydroxysuccinimide ester ( mbs ). for introduction of an alkyl halide into a protein , polypeptide or label one can use n - succinimidyl ( 4 - iodoacetyl ) aminobenzoate ( siab ) also from pierce . for introduction of a thiol group into a protein , polypeptide or label , one can use n - succinimidyl 3 -( 2 - pyridyldithio ) propionate ( spdp ) also from pierce . the thiol reactive group , such as maleimide , an alkyl halide or a thiol can be incorporated into the protein , polypeptide or label prior to reaction with the drug thiol but the drug thiol can also be reacted with the thiol reactive compound prior to reaction with the protein , polypeptide or label . also , bis - maleimide compounds of varying length can be reacted with thiol containing proteins , polypeptides or labels for covalent coupling of the methadone derivatives . conversely , the bis - maleimide compound can be reacted with the thiol derivative and subsequently to the thiol containing protein , polypeptide or label . common bis - maleimides are bis - maleimidohexane from pierce , n , n &# 39 ;- bis ( 3maleimidopropionyl )- 2 - hydroxy - 1 , 3 - propanediamine from sigma chemical co ., st . louis , mo ., and 1 , 1 &# 39 ;-( methylenedi - 4 , 1 - phenylene )- bismaleimide from aldrich chem . co ., milwaukee , wis . the thiol methadone derivatives can also form disulfides with thiol containing polypeptide , protein or label molecules as a means to incorporate the derivative into the molecule . the use of drug derivatives , immunogens and protein and polypeptide conjugates for generating antibodies and for use in the immunoassay process is described , for example , in u . s . pat . nos . 4 , 067 , 774 , 4 , 952 , 336 , 5 , 028 , 535 and 5 , 089 , 391 . sodium hydride ( 19 . 5 g of an 80 % dispersion in oil , 0 . 65 mol ) was added to a 1 l 3 - neck round bottom flask and triturated twice with hexane ( 150 ml ) to remove the oil ). tetrahydrofuran ( thf )( 150 ml ) was added to the flask followed by a stir bar and the resulting suspension stirred under argon whilst adding a solution of diphenylacetonitrile ( 58 g , 0 . 3 mol ) in thf ( 150 ml ) dropwise via a dropping funnel over 40 minutes . it became necessary to cool the flask in a cold water bath during the addition due to the exotherm generated . after stirring a further 10 minutes at room temperature the flask was fitted with a reflux condenser and placed in an ice bath . the resulting green suspension was then treated with 2 - dimethylaminoisopropyl chloride hydrochloride ( 54 g , 0 . 34 mol ) portionwise , via an addition funnel , over 15 minutes with stirring . the yellow mixture was refluxed on a heating mantle for 5 hours , during which time it became thick and then thinned out . after cooling , the thf was evaporated , the residual yellow solid treated with water ( 150 ml ) and extracted with ether ( 2 × 150 ml ). the extract was washed with water ( 3 × 100 ml ) and dried over anhydrous magnesium sulfate . after evaporation of the ether , the thick oily orange residue was treated with ice - cold hexane to afford a white solid . the contents were stirred in an ice / water bath for 30 minutes and the solid collected by filtration washing with ice - cold hexane . after drying in a vacuum desiccator there was obtained 44 . 9 g ( 54 %) white solid m . p . 88 °- 89 ° c . this product was used without further purification in the next step . this compound was prepared according to a literature procedure ( u . s . pat . no . 3 , 843 , 696 ) starting with 2 , 2 - diphenyl 4 - dimethylaminopentanenitrile ( 10 g ) from the previous step to afford 8 . 5 g product . to a stirred suspension of homocysteine thiolactone hydrochloride ( 1 . 84 g , 11 . 96 mmol ) in dry dmf ( 100 ml ) there was added pyridine ( 3 ml , 37 . 08 mmol ). after 10 minutes , when complete solution was effected , a solution of 7 , 7 - diphenyl - 6 - keto - 9 - dimethylaminodecanoic acid hydrochloride ( 5 g , 11 . 96 m mol ) in dry dmf ( 20 ml ) was added followed by 1 -( 3 - dimethylaminopropyl )- 3 - ethylcarbodiimide hydrochloride ( 2 . 52 g , 13 . 16 mmol ). the yellow solution was stirred under argon for 5 hours . the dmf was evaporated , the residual oil treated with water ( 100 ml ) and extracted with methylene chloride ( 3 × 50 ml ). the extract was evaporated to an amber colored oil which was treated with 0 . 5m potassium phosphate / 0 . 1m potassium borate ph 7 buffer ( 100 ml ) and extracted with ethyl acetate ( 3 × 50 ml ). the extract was dried over anhydrous magnesium sulfate , filtered and evaporated . the gummy residue was treated with ether ( 100 ml ), decolorizing charcoal was added and the mixture filtered through celite . the light yellow filtrate was acidified to ph 1 - 2 by dropwise addition of a 1m solution of hydrogen chloride in ether ( 10 ml ) with stirring . the resulting cream colored solid which precipitated was collected by filtration washing with ether ( 50 ml ). after drying in a vacuum desiccator there was obtained 4 . 1 g cream colored , hygroscopic solid which was 85 % pure by hplc . a small portion of this product ( 60 mg ) was purified using a c18 reverse phase semi - preparative hplc column and eluting with 20 mm potassium phosphate ph 4 . 6 / methanol ( 0 - 100 % over 50 minutes ). the product fraction eluting at 34 - 37 minutes was evaporated . the residue was treated with methylene chloride ( 5 ml ) and filtered from insoluble buffer salts . the filtrate was evaporated to afford , after vacuum drying , 35 mg of 1 - n -( 2 - butyrothiolactone ) amido - 6 , 6 - diphenyl - 5 - keto - 8 - dimethylaminononane phosphate salt as a white hygroscopic solid . this product is now 100 % pure by hplc . nmr ( dmso d - 6 ) δ8 . 08 ( m , 1h ), δ7 . 32 ( m , 10h ), δ2 . 29 ( s , 6h ), δ1 . 20 ( m , 4h ), δ0 . 43 ( d , 3h ). 1 - n -( 2 - butrlothiolactone ) amido - 6 , 6 - diphenyl - 5 - keto - 8 - dimethylaminononane ( 1 . 0 mg , 1 . 6 μmol ) was dissolved in 70 / 30 , v / v dimethylformamide / water ( 65 μl ). 1n potassium hydroxide solution ( 16 μl ) was added and the solution allowed to stand at room temperature for 3 minutes . 0 . 5m potassium phosphate / 0 . 1m potassium borate ph 7 buffer ( which was then made 1n in hydrochloric acid ) was immediately added in 5 μl increments until ph 7 was attained . the title compound in solution was used as is to react with thiol reactive groups , such as maleimides , alkyl halides or thiols which are either free in solution or are coupled to proteins , polypeptides or labels .