Novel ligands and methods for preparing same

Abstract

A process is disclosed for modifying a parent ligand by attaching to the parent ligand a conjugation agent that is reactive with a moiety of a target receptor to which the parent ligand binds such that a covalent bond is formable between the conjugation agent and the receptor moiety. Also disclosed are compositions, probes and methods of detecting and/or quantifying receptors using the modified ligands of the invention.

1 . A process for modifying a parent ligand, comprising attaching to said parent ligand a conjugation agent that is reactive with a moiety of a target receptor to which said parent ligand binds, wherein when said parent ligand binds to the receptor a covalent bond is formed between said conjugation agent and said moiety, and wherein the parent ligand binds specifically with a nucleoside transporter.  
   
   
       2 . The process of  claim 1 , wherein the conjugation agent is attached to the parent ligand through a spacer.  
   
   
       3 . The process of  claim 2 , wherein the spacer comprises a group selected from the group consisting of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl, heterooxoalkyl, alkenyl, hetero alkenyl, aralkyl, hetero aralkyl, aryl and heteroaryl radicals.  
   
   
       4 . The process of  claim 1 , wherein the conjugation agent is selected from the group consisting of a sulfhydryl group specific conjugation agent, an amino group specific conjugation agent, a carboxyl group specific conjugation agent, a tyrosine specific conjugation agent, an arginine specific conjugation agent, a histidine specific conjugation agent, a methionine specific conjugation agent, a tryptophan specific conjugation agent, and a serine specific conjugation agent.  
   
   
       5 . The process of  claim 1 , wherein the conjugation agent is a sulfhydryl group specific conjugation agent selected from the group consisting of N-maleimide and N-maleimide derivatives.  
   
   
       6 . The process of  claim 5 , wherein the N-maleimide derivatives are selected from the group consisting of disulfide reagents including 5′-dithiobis-2-nitrobenzoic acid), 4,4′-dithiodipyridine, methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl disulfide.  
   
   
       7 . The process of  claim 1 , wherein the conjugation agent is selected from the group consisting of alkylating agents and acylating agents.  
   
   
       8 . A process for modifying a parent ligand, comprising attaching to said parent ligand a sulfhydryl group specific conjugation agent that is reactive with a sulfhydryl group of a target receptor to which said parent ligand binds, wherein when said parent ligand binds to the receptor a covalent bond is formed between said conjugation agent and said sulfhydryl group, and wherein the parent ligand binds specifically with a serotonin receptor.  
   
   
       9 . A modified ligand produced by the process of  claim 1 .  
   
   
       10 . A modified ligand produced by the process of  claim 8 .  
   
   
       11 . A modified ligand having the general formula:  
       L-R 1 -A   (I)  wherein L is a parent ligand that binds specifically with a target receptor comprising a nucleoside transporter;    wherein A is a conjugation agent that is reactive with a moiety of the target receptor to which the parent ligand binds, such that when said parent ligand binds to the receptor a covalent bond is formed between said conjugation agent and said moiety; and    R 1  is an optional spacer.    
   
   
       12 . The modified ligand of  claim 11 , wherein the spacer comprises a non-hydrolysable radical selected from the group consisting of alkyl, heteroalkyl, cycloalkyl heterocycloalkyl, oxoalkyl, heterooxoalkyl, alkenyl, hetero alkenyl, aralkyl, hetero aralkyl aryl and heteroaryl radicals.  
   
   
       13 . The modified ligand of  claim 11 , wherein the conjugation agent is a sulthydryl group specific conjugation agent.  
   
   
       14 . The modified ligand of  claim 12 , wherein the sulfhydryl group specific conjugation agent is selected from the group consisting of N-maleimide and N-maleimide derivatives.  
   
   
       15 . The modified ligand of  claim 11 , wherein the parent ligand binds specifically with an es nucleoside tporter.  
   
   
       16 . The modified ligand of  claim 11 , which is interactive with es nucleoside transporter and/or nucleoside/nucleotide/nucleobase-sensitive proteins, wherein said modified ligand has a general formula selected from the group consisting of:  
     
       
         
         
             
             
         
       
       wherein A is N-maleimide, 2-pyridyldithio, or halogen;  
       X is NH, S, or O;  
       Y is H, halogen, NH 2 , or O;  
       Z is H, halogen, or CH 3 ;  
       R 1  is spacer arm; and  
       R 2  is H, β- D -ribose, β- D -2-deoxyribose, or their 5′-mono-, 5′di-, and 5′tri-phosphate.  
     
   
   
       17 . The modified ligand of  claim 11 , which has a general formula selected from the group consisting of:  
     
       
         
         
             
             
         
       
     
     wherein R 4  is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate], 4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], or N-[ε-caproylate];  
     
       
         
         
             
             
         
       
       wherein R 4  is 4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate], 4-[p-phenyl]butyrate, N-[γ-butyrate], N-[α-acetate], or N-[ε-caprylate];  
       
         
           
           
               
               
           
         
       
       wherein R 5  is 4-carbonyl-α-methyl-α-toluene, 6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or 6-[3′-propioarido]hexanoate; and  
       
         
           
           
               
               
           
         
       
       wherein R 5  is 4-carbonyl-α-methyl-α-toluene, 6-[α-methyl-α-tuloamido]-hexanoate, N-[3-propionate], or 6-[3′-propioamido]hexanoate.  
     
   
   
       18 . A modified litany having the general formula:  
       L-R 1 -A   (I)  wherein L is a parent ligand that binds specifically with a target serotonin receptor;    wherein A is a conjugation agent that is reactive with a sulfhydryl group of said target receptor to which the parent ligand binds, such that when said parent ligand binds to the receptor a covalent bond is formed between said conjugation agent and said sulfhydryl group; and    R 1  is an optional spacer.    
   
   
       19 . The modified ligand of  claim 18 , wherein the parent ligand comprises serotonin or a precursor or analog thereof.  
   
   
       20 . The modified ligand of  claim 18 , wherein the modified ligand has a structure selected from the group consisting of the structures shown below:  
     
       
         
         
             
             
         
       
     
     LBT3001 (1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione)  
     
       
         
         
             
             
         
       
     
     LBT3002 (4-2,5-dioxo-2,5-dihydro-pyrrol-1-yl)N-[2-5-hydroxy-1H-indol-3-yl)-ethyl]-butyramide)  
     
       
         
         
             
             
         
       
     
     LBT3004 (3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxy- 1H-indol-3-yl)-ethyl]-propionamide); and  
     
       
         
         
             
             
         
       
     
     LBT3005 (4-(2,5dioxo-2,5dihydro-pyrrol-1-ylmethyl)-cyclohexane carboxylic acid [2-5-hydroxy-1H-indol-3-yl)-ethyl]-amide)  
   
   
       21 . A composition comprising the modified ligand of  claim 11  and a pharmaceutically acceptable carrier.  
   
   
       22 . A composition comprising the-modified ligand of  claim 18  and a pharmaceutically acceptable carrier.  
   
   
       23 . A method of detecting the presence of a target receptor in a test sample, comprising: 
 contacting said sample with the modified ligand of  claim 11 , wherein said modified ligand binds said target receptor if present in said test sample; and    detecting the presence of a complex comprising said modified ligand and said receptor in said contacted sample.    
   
   
       24 . A method of quantifying the presence of a target receptor in a test sample, comprising: 
 contacting said sample with the modified ligand of  claim 11 , wherein said modified ligand binds said target receptor if present in said test sample;    measuring the concentration of a complex comprising said modified ligand and said receptor in said contacted sample; and    relating said measured complex concentration to the concentration of said receptor in said sample.    
   
   
       25 . A method of detecting the presence of a target receptor in a test sample, comprising: 
 contacting said sample with the modified ligand of  claim 18 , wherein said modified ligand binds said target receptor if present in said test sample; and    detecting the presence of a complex comprising said modified ligand and said receptor in said contacted sample.    
   
   
       26 . A method of quantifying the presence of a target receptor in a test sample, comprising: 
 contacting said sample with the modified ligand of  claim 18 , wherein said modified ligand binds said target receptor if present in said test sample;    measuring the concentration of a complex comprising said modified ligand and said receptor in said contacted sample; and    relating said measured complex concentration to the concentration of said receptor in said sample.    
   
   
       27 . A probe that covalently binds to a target receptor, said probe comprising the modified ligand of  claim 11  having a reporter molecule associated therewith.  
   
   
       28 . Use of the modified ligand of  claim 11  or of the probe of  claim 27  in the study of target receptor function.  
   
   
       29 . A probe that covalently binds to a target receptor, said probe comprising the modified ligand of  claim 18  having a reporter molecule associated therewith.  
   
   
       30 . Use of the modified ligand of  claim 18  or of the probe of  claim 29  in the study of target receptor function.