6-acetylmorphine analogs, and methods for their synthesis and use

Abstract

The present invention relates to novel 6-acetylmorphine analogs, and methods for their synthesis and use. Such analogs are designed to provide a convenient linkage chemistry for coupling under mild conditions to a suitable group on a target protein, polypeptide, solid phase or detectable label.

We claim: 
     
         1 . A method of stimulating an immune response to 6-acetylmorphine, comprising:
 immunizing an animal with a conjugate comprising at least one compound or salt thereof, having a general formula selected from (I), (II), (III) or (IV):   
       
         
           
           
               
               
           
         
         covalently bound through the functional moiety of the compound to a corresponding coupling site or sites on a protein, polypeptide, detectable label, nucleic acid, or solid phase. 
         wherein: 
         R 1 , R 3 , R 4  or R 5  is a linkage chemistry which provides a terminal functional moiety selected from the group consisting of protected or unprotected sulfhydryl moieties, protected or unprotected amine moieties, primary amine-reactive moieties, sulfhydryl-reactive moieties, photoreactive moieties, carboxyl-reactive moieties, arginine-reactive moieties, and carbonyl-reactive moieties; 
         each Z is independently optionally substituted C 1-4  alkyl, C 1-4  alkoxy, N, O, S, and aryl, wherein substitution(s), when present, are independently selected from the group consisting of C 1-6  alkyl straight or branched chain, benzyl, halogen, trihalomethyl, C 1-6  alkoxy, —NO2, —NH2, —OH, =0, —COOR′ where R′ is H or lower alkyl, —CH 2 OH, and —CONH 2 ; 
         each Y is independently selected from the group consisting of 
       
       
         
           
           
               
               
           
         
         wherein each R 2  is independently optionally substituted C 1-4  alkyl, C 1-4  alkoxy, OH, and aryl, wherein substitution(s), when present, are independently selected from the group consisting of C 1-6  alkyl straight or branched chain, benzyl, halogen, trihalomethyl, C 1-6  alkoxy, —NO2, —NH2, —OH, =0, —COOR′ where R′ is H or lower alkyl, —CH20H, and —CONH2; and 
         each X is H or together form a covalent bond. 
       
     
     
         2 . A method according to  claim 1 , further comprising isolating one or more antibodies that specifically bind 6-AM, wherein said binding affinity for 6-AM is at least a factor of 30 greater than the affinity of the antibody for 6-acetylcodeine and heroin, at least a factor of 30,000 greater than the affinity of the antibody for hydromorphone and oxymorphone and at least a factor of 100,000 greater than the affinity of the antibody for, codeine, hydrocodone, morphine, morphine 3-D-glucuronide, morphine 6-D-glucuronide, oxycodone. 
     
     
         3 . A method according to  claim 2 , wherein said one or more antibodies are isolated directly from said animal. 
     
     
         4 . The method of  claim 1 , wherein the functional moiety is a sulfhydryl-reactive moiety. 
     
     
         5 . The method of  claim 4 , wherein the functional moiety is selected from the group consisting of a maleimide, an alkyl halide, an aryl halide, an alphahaloacyl, and a pyridyl disulfide. 
     
     
         6 . The method of  claim 1 , wherein said sulfhydryl-reactive moiety is a maleimide, whereby said compound(s) are covalently bound to said protein, polypeptide, detectable label, nucleic acid, or solid phase 
     
     
         7 . The method of  claim 1 , wherein said detectable label is selected from the group consisting of an enzyme, a fluorophore, biotin, avidin, streptavidin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, and a fluorescent latex particle. 
     
     
         8 . The method of  claim 1 , wherein said protein is keyhole limpet hemocyanin or bovine serum albumin. 
     
     
         9 . The method of  claim 1 , wherein said compound(s) are bound to a solid phase selected from the group consisting of a membrane, a cellulose-based paper, a polymeric particle, a latex particle, a paramagnetic particle, a glass substrate, a silicon substrate, a plastic substrate, and a multiple-well plate. 
     
     
         10 . An antibody raised by the method of  claim 1 , and specific for the compound 6-acetylmorphine (6AM). 
     
     
         11 . An antibody specific for 6-AM and having a cross-reactivity for 6-acetylcodeine or heroin of less than 3.5%. 
     
     
         12 . An antibody specific for 6-AM and having a cross-reactivity for hydromorphone or oxymorphone of less than 0.00035%. 
     
     
         13 . An antibody specific for 6-AM and having a cross-reactivity for codeine, hydrocodone, morphine, morphine 3-Dglucuronide, morphine 6-D-glucuronide, oxycodone of less than 0.000001%. 
     
     
         14 . An antibody raised by the method of  claim 1  having a cross-reactivity for 6-acetylcodeine or heroin of less than 3.5%. 
     
     
         15 . An antibody raised by the method of  claim 1  having a cross-reactivity for hydromorphone or oxymorphone of less than 0.00035%. 
     
     
         16 . An antibody raised by the method of  claim 1  having a cross-reactivity for codeine, hydrocodone, morphine, morphine 3-Dglucuronide, morphine 6-D-glucuronide, oxycodone of less than 0.000001%. 
     
     
         17 . An antibody specific for 6-AM and raised against a compound or salt thereof of formula (I), (II), (III), (IV): 
       
         
           
           
               
               
           
         
         wherein: 
         R 1 , R 3 , R 4  or R 5  is a linkage chemistry which provides a terminal functional moiety selected from the group consisting of protected or unprotected sulfhydryl moieties, protected or unprotected amine moieties, primary amine-reactive moieties, sulfhydryl-reactive moieties, photoreactive moieties, carboxyl-reactive moieties, arginine-reactive moieties, and carbonyl-reactive moieties; 
         each Z is independently optionally substituted C 1-4  alkyl, C 1-4  alkoxy, N, O, S, and aryl, wherein substitution(s), when present, are independently selected from the group consisting of C 1-6  alkyl straight or branched chain, benzyl, halogen, trihalomethyl, C 1-6  alkoxy, —NO2, —NH2, —OH, =0, —COOR′ where R′ is H or lower alkyl, —CH 2 OH, and —CONH 2 ; 
         each Y is independently selected from the group consisting of 
       
       
         
           
           
               
               
           
         
         wherein each R 2  is independently optionally substituted C 1-4  alkyl, C 1-4  alkoxy, OH, and aryl, wherein substitution(s), when present, are independently selected from the group consisting of C 1-6  alkyl straight or branched chain, benzyl, halogen, trihalomethyl, C 1-6  alkoxy, —NO2, —NH2, —OH, =0, —COOR′ where R′ is H or lower alkyl, —CH20H, and —CONH2; and 
         each X is H or together form a covalent bond. 
       
     
     
         18 . A method for detecting 6-AM in a sample, comprising the steps of:
 a) combining the sample with the antibody of  claim 17  under conditions that permit formation of a stable 6-AM-antibody complex; and   b) detecting any 6-AM-antibody complex formed in step a).   
     
     
         19 . The method of  claim 18 , comprising quantitating any 6-AM-antibody complex formed in step a). 
     
     
         20 . The method of  claim 18 , which is a competition assay method. 
     
     
         21 . The method of  claim 18 , further comprising the steps of:
 i) contacting the antibody with a labeled analog of 6-AM under conditions that permit formation of a stable analog-antibody complex; and   ii) separating any analog not forming a complex in step i).