Targeting Ligands

Abstract

Described are novel targeting ligands that may be linked to compounds, such therapeutic compounds that are useful in directing the compounds to the in vivo target. The targeting ligands disclosed herein can serve to target expression-inhibiting oligomeric compounds, such as RNAi agents, to liver cells to modulate gene expression. The targeting ligands disclosed herein, when conjugated to a therapeutic compound, may be used in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Compositions including the targeting ligands disclosed herein when linked to expression-inhibiting oligomeric compounds are capable of mediating expression of target nucleic acid sequences in liver cells, such as hepatocytes, which may be useful in the treatment of diseases or conditions that respond to inhibition of gene expression or activity in a cell, tissue, or organism.

1 . A targeting ligand comprising a structure of Formula I: 
       
         
           
           
               
               
           
         
         comprising a linker, a branch point group, one or more tethers, and one or more targeting moieties, wherein n is an integer from 1 to 4, and wherein the linker is a structure selected from the group consisting of: 
         (Structure 2); 
       
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein n′ is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and when present, each Z′ is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted amino, carboxyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, substituted C1-C6 alkoxy, substituted C1-C6 aminoalkyl, halogen (e.g., F), hydroxyl, amido, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl, and sulfhydryl (Structure 7); 
       
         
           
           
               
               
           
         
       
       wherein n″ is 0, 1, 2, 3, 4, and when present, each Z″ is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, substituted or unsubstituted amino, carboxyl, substituted C1-C6 alkoxy, substituted C1-C6 aminoalkyl, halogen (e.g., F), hydroxyl, amido, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl, and sulfhydryl (Structure 8); and 
       
         
           
           
               
               
           
         
       
       wherein V comprises one or more substituted or unsubstituted cycloalkyl (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cycloocty, etc.), substituted or unsubstituted cycloalkenyl (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (e.g., phenyl, naphthyl, binapthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryl (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), or substituted or unsubstituted heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.), or any covalently linked combination thereof. (Structure 9). 
     
     
         2 . The targeting ligand of  claim 1 , wherein the linker is a structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         3 . The targeting ligand of  claim 1 , wherein the linker is: 
       
         
           
           
               
               
           
         
       
     
     
         4 . The targeting ligand of  claim 1 , wherein the branch point group is a structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein n is an integer from 1 to 20 (Structure 209); 
       
         
           
           
               
               
           
         
       
       wherein m is an integer from 0 to 20, and n is an integer from 0 to 20 (Structure 210); 
       
         
           
           
               
               
           
         
       
       wherein m is an integer from 0 to 20; n is an integer from 0 to 20; x is an integer from to 1 to 10; y is an integer from 1 to 10; z is an integer from 1 to 4; and K is selected from the group consisting of substituted or unsubstituted cycloalkyl (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cycloocty, etc.), substituted or unsubstituted cycloalkenyl (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (e.g., phenyl, naphthyl, binapthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryl (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), and substituted or unsubstituted heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc. or an combination thereof structure 211); 
       
         
           
           
               
               
           
         
       
       wherein m is an integer from 0 to 20; n is an integer from 0 to 20; x is an integer from to 1 to 10; and y is an integer from 1 to 10 (Structure 212); 
       
         
           
           
               
               
           
         
       
       wherein m is an integer from 0 to 20; n is an integer from 0 to 20; x is an integer from to 1 to 10; y is an integer from 1 to 10; G is selected from the group c; 
       
         
           
           
               
               
           
         
       
       or any substituted or unsubstituted cyclic or heterocyclic structure having a ring size of 5, 6, 7, 8, or 9 atoms, for example, substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl (Structure 213); 
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 0 to 20 (Structure 214); 
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 0 to 20, and Q is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       wherein n is an integer selected from 1 to 7 (Structure 219). 
     
     
         5 . The targeting ligand of  claim 1 , wherein the branch point group is: 
       
         
           
           
               
               
           
         
       
     
     
         6 . The targeting ligand of  claim 1 , wherein the tether is independently selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH (Structure 301); 
       
         
           
           
               
               
           
         
       
       wherein X is O, S, or NH (Structure 302); 
       
         
           
           
               
               
           
         
       
       (Structure 302a); 
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH (Structure 303); 
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH (Structure 304); 
       
         
           
           
               
               
           
         
       
       wherein X is O, S, or NH (Structure 305); and 
       
         
           
           
               
               
           
         
       
       wherein X is O, S, or NH (Structure 306). 
     
     
         7 . The targeting ligand of  claim 1 , wherein the targeting moiety is independently selected from the group consisting of: N-acetyl-galactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine. 
     
     
         8 . The targeting ligand of  claim 7 , wherein the targeting moiety is N-acetyl-galactosamine. 
     
     
         9 . The targeting ligand of  claim 7 , wherein the targeting moiety is independently selected from glycans, haptens, vitamins, folate, biotin, aptamers, and peptides, such as RGD-containing peptides, insulin, EGF, and transferrin. 
     
     
         10 . The targeting ligand of  claim 1 , wherein n is 3. 
     
     
         11 . The targeting ligand of  claim 1 , wherein n is 4. 
     
     
         12 . The targeting ligand of  claim 1 , wherein the targeting ligand is linked to an expression-inhibiting oligomeric compound. 
     
     
         13 . The targeting ligand of  claim 12 , wherein the expression-inhibiting oligomeric compound is an RNAi agent. 
     
     
         14 . The targeting ligand of  claim 13 , wherein the RNAi agent is double-stranded. 
     
     
         15 . The targeting ligand of  claim 14 , wherein the RNAi agent includes one or more modified nucleotides. 
     
     
         16 . The targeting ligand of  claim 1 , wherein the targeting ligand comprises the structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         17 . The targeting ligand of  claim 1 , wherein the structure is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         18 . The targeting ligand of  claim 1 , wherein the targeting ligand is linked to an RNAi agent. 
     
     
         19 . The targeting ligand of  claim 18 , wherein the RNAi agent is double-stranded. 
     
     
         20 . The targeting ligand of  claim 19 , wherein the double-stranded RNAi agent is linked to the targeting ligand at the 5′ terminal end of the sense strand of an RNAi agent. 
     
     
         21 . The targeting ligand of  claim 18 , wherein the RNAi agent is linked to the linker of the targeting ligand via a phosphate group, phosphorothioate group, or a phosphonate group. 
     
     
         22 . A composition comprising a targeting ligand of  claim 1 , wherein the targeting ligand is linked to an expression-inhibiting oligomeric compound, wherein the structure of the targeting ligand and expression-inhibiting oligomeric compound is represented by the structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein R in each structure comprises an expression-inhibiting oligomeric compound. 
       
     
     
         23 . The composition of  claim 22 , wherein the targeting ligand is linked to the expression-inhibiting oligomeric compound via a phosphate group, phosphorothioate group, or a phosphonate group. 
     
     
         24 . The composition of  claim 22 , wherein the expression-inhibiting oligomeric compound is an RNAi agent. 
     
     
         25 . The composition of  claim 24 , wherein the RNAi agent is a double-stranded RNAi agent. 
     
     
         26 . The composition of  claim 25 , wherein the targeting ligand is linked to the 5′ terminal end of the sense strand of an RNAi agent. 
     
     
         27 . A compound having the structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         28 . A targeting ligand comprising a structure of Formula II: 
       
         
           
           
               
               
           
         
       
       comprising a branch point group with linker replacement moiety, one or more tethers, and one or more targeting moieties, wherein n is an integer between 1 and 4, and wherein the linker replacement moiety includes one or more substituted or unsubstituted cycloalkyl (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cycloocty, etc.), substituted or unsubstituted cycloalkenyl (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (e.g., phenyl, naphthyl, binapthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryl (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), or substituted or unsubstituted heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.), or any combination thereof, is located within the branch point group. 
     
     
         29 . The targeting ligand of  claim 28 , wherein the branch point group with linker replacement moiety is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         30 . The targeting ligand of  claim 29 , comprising the structure selected from: 
       
         
           
           
               
               
           
         
       
     
     
         31 . The targeting ligand of  claim 28 , wherein the targeting ligand is linked to an expression-inhibiting oligomeric compound. 
     
     
         32 . The targeting ligand of  claim 31 , wherein the targeting ligand linked to an expression-inhibiting oligomeric compound has the following structure: 
       
         
           
           
               
               
           
         
       
       wherein R comprises the expression-inhibiting oligomeric compound (Structure 1027a). 
     
     
         33 . The targeting ligand of  claim 28 , wherein the expression-inhibiting oligomeric compound is an RNAi agent. 
     
     
         34 . The targeting ligand of  claim 33 , wherein the RNAi agent is double stranded. 
     
     
         35 . The targeting ligand of  claim 34 , wherein the RNAi agent includes one or more modified nucleotides. 
     
     
         36 . A method of inhibiting expression of a target nucleic acid in a subject, the method comprising administering to the subject a therapeutic amount of an expression-inhibiting oligomeric compound linked to any of the targeting ligands of  claim 1 . 
     
     
         37 . A method of introducing a therapeutic agent into a mammalian cell, the method comprising contacting a mammalian cell with the targeting ligand of  claim 1 , linked to the therapeutic agent. 
     
     
         38 . The method of  claim 37 , wherein the cell is present in a subject. 
     
     
         39 . The method of  claim 38 , wherein the subject is a human. 
     
     
         40 . The method of  claim 37 , wherein the therapeutic agent is an expression-inhibiting oligomeric compound. 
     
     
         41 . The method of  claim 40 , wherein the therapeutic agent is a double-stranded RNAi agent. 
     
     
         42 . A method of treating a disease or disorder that would benefit from administration of a therapeutic compound, the method comprising administering a therapeutic amount of the targeting ligand of on  claim 1  linked to a therapeutic compound to a subject in need of treatment thereof. 
     
     
         43 . A method of treating a disease or disorder that would benefit from administration of a therapeutic compound, the method comprising administering a therapeutic amount of the composition of  claim 22  to a subject in need of treatment thereof. 
     
     
         44 . A method of manufacturing a phosphoramidite compound including a targeting ligand of  claim 1 , the method comprising:
 (i) covalently linking the carboxylic acid moiety (or its activated ester) of the linker to a terminal amine located on the branch point group, and   (ii) linking the linker to a phosphorus atom of a phosphoramidite through a phosphitylation reaction with a phosphoramidite forming reagent; thereby forming a phosphoramidite compound that includes a targeting ligand.   
     
     
         45 . The method of  claim 44 , wherein the phosphoramidite reagent is selected from: 
       
         
           
           
               
               
           
         
       
     
     
         46 . A targeting ligand of  claim 1 , linked to a therapeutic agent, for use as a medicament. 
     
     
         47 . A targeting ligand of  claim 1 , linked to a therapeutic agent, for use in introducing a therapeutic agent into a mammalian cell, for inhibiting expression of a target nucleic acid in a subject, or for treating a disease or disorder that would benefit from administration of a therapeutic agent.