Synthetic methods and derivatives of triphosphate oligonucleotides

Abstract

The invention features a oligonucleotide of formula I, or pharmaceutically acceptable salts, or prodrugs thereof: which are capable of inducing an anti-viral or an antibacterial response, in particular, the induction of type I IFN, IL-18 and/or IL-1β by binding to RIG-I. The invention relates to methods of making and using modified oligonucleotide comprising at least one triphosphate or analogs thereof. The invention further relates to methods for treating various disorders and diseases such as viral infections, bacterial infections, parasitic infections, tumors, allergies, autoimmune diseases, immunodeficiencies and immunosuppression.

1 - 31 . (canceled) 
     
     
         32 . An oligonucleotide of formula I, or pharmaceutically acceptable salts or prodrugs thereof: 
       
         
           
           
               
               
           
         
         wherein: 
         Q 2  and Q 3  are each, independently NH, O or S; 
         X and Y are each, independently, OH, O − , OR 1 , O − , SH, S − , Se, BH 3 , BH 3   − , H, N(R 2 ) 2 , alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 1  is independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 2  is independently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         Q 4  and Q 5  are each independently O, CH 2 , CH(Me), C(Me) 2 , CHF, CF 2 , NH, NR 1 , or S; 
         Q 1  is OH, O − , OR 1 , S − , SH, or SR 1 ; 
         n is 0, 1, 2, 3, 4 or 5; wherein each repeating unit can be the same or different; 
         A is absent or selected from the group consisting of single-stranded oligonucleotide and double-stranded oligonucleotide, each of which may be chemically modified; 
         B is absent or a linker/spacer; and 
         E is a single-stranded oligonucleotide or a double-stranded oligonucleotide, each of which may be chemically modified and/or conjugated with a ligand; 
         with the proviso that when A and B are both absent and n is 0, 1 or 3, then Q 1 , Q 2 , Q 3 , Q 4 , Q 5 , X and Y cannot all be oxygen. 
       
     
     
         33 . The oligonucleotide of  claim 32 , represented by formula (II) or a pharmaceutically acceptable salt or prodrug thereof: 
       
         
           
           
               
               
           
         
         Q 2 , Q 3  and Q 30  are each, independently NH, O or S; 
         X and Y and Y 10  are each, independently, OH, O − , OR 1 , O − , SH, S − , Se, BH 3 , BH 3   − , H, N(R 2 ) 2 , alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 1  is independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 2  is independently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         Q 4  and Q 5  are each independently O, CH 2 , CH(Me), C(Me) 2 , CHF, CF 2 , NH, NR 1 , or S; 
         Q, is OH, O − , OR 1 , S − , SH, or SR 1 ; and 
         W is H, OH or -G-L; where G is selected from the group consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where L is a ligand; 
         provided that when W is OH, then Q 1 , Q 2 , Q 3 , Q 4 , Q 5 , Q 30 , X, Y and Y 10  cannot all be oxygen. 
       
     
     
         34 . The oligonucleotide of  claim 32 , represented by formula (III) or a pharmaceutically acceptable salt or prodrug thereof: 
       
         
           
           
               
               
           
         
         Q 2  and Q 3  are each, independently NH, O or S; 
         X and Y and Y 10  are each, independently, OH, O − , OR 1 , O − , SH, S − , Se, BH 3 , BH 3   − , H, N(R 2 ) 2 , alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 1  is independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 2  is independently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         Q 4  and Q 5  are each independently O, CH 2 , CH(Me), C(Me) 2 , CHF, CF 2 , NH, NR 1 , or S; 
         Q 1  is OH, O − , OR 1 , S − , SH, or SR 1 ; and 
         W is each independently H, OH or -G-L; where G is selected from the group consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where L is a ligand. 
       
     
     
         35 . The oligonucleotide of  claim 32 , represented by formula (IV) or a pharmaceutically acceptable salt or prodrug thereof: 
       
         
           
           
               
               
           
         
         Q 2 , Q 3  and Q 30  are each, independently NH, O or S; 
         X and Y and Y 10  are each, independently, OH, O − , OR 1 , O − , SH, S − , Se, BH 3 , BH 3   − , H, N(R 2 ) 2 , alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 1  is independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 2  is independently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         Q 4  and Q 5  are each independently O, CH 2 , CH(Me), C(Me) 2 , CHF, CF 2 , NR 1 , or S; 
         Q 1  is OH, O − , OR 1 , S − , SH, or SR 1 ; 
         R is H, phosphate or phosphorothioate; 
         W and W 1  are each independently H, OH, phosphate, phosphorothioate or -G-L; where G is selected from the group consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where L is a ligand; 
         provided that when W is OH, then Q 1 , Q 2 , Q 3 , Q 4 , Q 5 , Q 30 , X, Y and Y 10  cannot all be oxygen. 
       
     
     
         36 . The oligonucleotide of  claim 32 , represented by formula (V) or a pharmaceutically acceptable salt or prodrug thereof: 
       
         
           
           
               
               
           
         
         Q 2  and Q 3  are each, independently NH, O or S; 
         X and Y and Y 10  are each, independently, OH, O − , OR 1 , O − , SH, S − , Se, BH 3 , BH 3   − , H, N(R 2 ) 2 , alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 1  is independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 2  is independently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         Q 4  and Q 5  are each independently O, CH 2 , CH(Me), C(Me) 2 , CHF, CF 2 , NR 1 , or S; 
         Q 1  is OH, O − , OR 1 , S − , SH, or SR 1 ; 
         R is H, phosphate or phosphorothioate; 
         W and W 1  are each independently H, OH, phosphate, phosphorothioate or -G-L; where G is selected from the group consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where L is a ligand. 
       
     
     
         37 . The oligonucleotide of  claim 32 , represented by formula (VI) or a pharmaceutically acceptable salt or prodrug thereof: 
       
         
           
           
               
               
           
         
         Q 2 , Q 3  and Q 30  are each, independently NH, O or S; 
         X and Y and Y 10  are each, independently, OH, O − , OR 1 , O − , SH, S − , Se, BH 3 , BH 3   − , H, N(R 2 ) 2 , alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 1  is independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 2  is independently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         Q 4  and Q 5  are each independently O, CH 2 , CH(Me), C(Me) 2 , CHF, CF 2 , NR 1 , or S; 
         Q 1  is OH, O − , OR 1 , S − , SH, or SR 1 ; 
         W, W 1  and W 2  are each independently H, OH, phosphate, phosphorothioate or -G-L; where G is selected from the group consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where L is a ligand; and 
         Linker/spacer is selected from the group consisting of phosphate, phosphorothioate, phosphorodithioate, alkylphosphonate, amide, ester, disulfide, thioether, oxime, hydrazone, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, and heteroaryl. 
       
     
     
         38 . The oligonucleotide of  claim 32 , represented by formula (VII) or a pharmaceutically acceptable salt or prodrug thereof: 
       
         
           
           
               
               
           
         
         Q 2  and Q 3  are each, independently NH, O or S; 
         X and Y and Y 10  are each, independently, OH, O − , OR 1 , O − , SH, S − , Se, BH 3 , BH 3   − , H, N(R 2 ) 2 , alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 1  is independently alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         R 2  is independently hydrogen, alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted; 
         Q 4  and Q 5  are each independently O, CH 2 , CH(Me), C(Me) 2 , CHF, CF 2 , NR 1 , or S; 
         Q 1  is OH, O − , OR 1 , S − , SH, or SR 1 ; 
         W, W, and W 2  are each independently H, OH, phosphate, phosphorothioate or -G-L; where G is selected from the group consisting of —CONH—, —NHCO—, —S—S—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, acetal, ketal, —O—N═C—, —NH—N═C—, —S—, —O—, pyrrolidine, morpholine, piperazine and thiazolidine; and where L is a ligand; and 
         Linker/spacer is selected from the group consisting of phosphate, phosphorothioate, phosphorodithioate, alkylphosphonate, amide, ester, disulfide, thioether, oxime, hydrazone, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, and heteroaryl. 
       
     
     
         39 . The oligonucleotide of  claim 32 , wherein the single- or double-stranded oligonucleotide comprises at least one modified nucleotide. 
     
     
         40 . The oligonucleotide of  claim 39 , wherein at least one of said modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. 
     
     
         41 . The oligonucleotide of  claim 39 , wherein at least one of said modified nucleotides is selected from the group of a 2′-fluoro nucleotide, a 2′-O-alkyl nucleotide, a 2′-O-alkoxyalkyl nucleotide, a 2′-O-allyl nucleotide, a 2′ 0-propyl nucleotide, a 2′-O-methylated nucleotide (2′-O-Me), a 2′-deoxy nucleotide, a 2′-deoxyfluoro nucleotide, a 2′-O-methoxyethyl nucleotide (2′-O-MOE), a 2′-O—N-MeAcetamide nucleotide (2′-O-NMA), a 2′-β-dimethylaminoethyloxyethyl nucleotide (2′-O— DMAEOE), a 2′-aminopropyl, a 2′-hydroxy, a 2′-ara-fluoro, a 3′-amidate (3′—NH in place of 3′-0), a locked oligonucleotide (LNA), an extended ethylene oligonucleotide (ENA), a hexose oligonucleotide (HNA), or a cyclohexene oligonucleotide (CeNA). 
     
     
         42 . The oligonucleotide of  claim 32 , wherein the oligonucleotide is comprised in a viral vector. 
     
     
         43 . The oligonucleotide of  claim 32 , wherein the oligonucleotide binds to RIG-I. 
     
     
         44 . A composition comprising the oligonucleotide of  claim 32  and at least one agent selected from the group consisting of an immunostimulatory agent, an anti-viral agent, an anti-bacterial agent, an anti-tumor agent, a gene-silencing agent, an anti-tumor therapy, and combinations thereof. 
     
     
         45 . The composition of  claim 44 , wherein the agent is retinoic acid, type I IFN, or a combination thereof. 
     
     
         46 . A method of using the oligonucleotide of  claim 32  for inducing apoptosis of tumor cells, inducing an anti-viral response, inducing an anti-bacterial response, and/or inducing an anti-tumor response in a vertebrate animal. 
     
     
         47 . The method of  claim 46 , wherein the anti-viral response, the anti-bacterial response and/or the anti-tumor response comprise type I IFN production, IL-18 production, and/or IL-1β production. 
     
     
         48 . A composition comprising the oligonucleotide of  claim 32  and at least one antigen for inducing an immune response against an antigen in a vertebrate animal. 
     
     
         49 . The composition of  claim 48 , wherein the oligonucleotide is covalently linked to the at least one antigen. 
     
     
         50 . A method of using the oligonucleotide of  claim 32  for the preparation of a medicament for preventing and/or treating a disease and/or disorder selected from the group consisting of viral infection, bacterial infection, parasitic infection, tumor, multiple sclerosis, allergy, autoimmune diseases, immunosuppression, and immunodeficiency in a vertebrate animal. 
     
     
         51 . A method of using the oligonucleotide of  claim 32  for the preparation of a medicament for inducing apoptosis of tumor cells, inducing an anti-viral response, inducing an anti-bacterial response, and/or inducing an anti-tumor response in a vertebrate animal. 
     
     
         52 . The method of  claim 51 , wherein the anti-viral response, the anti-bacterial response, and/or the anti-tumor response comprise type I IFN production, IL-18 production, and/or IL-1β production. 
     
     
         53 . A pharmaceutical composition comprising the oligonucleotide of  claim 32  and a pharmaceutically acceptable carrier. 
     
     
         54 . A pharmaceutical package, comprising the pharmaceutical composition of  claim 53  and an instruction for use. 
     
     
         55 . A combined preparation, comprising the oligonucleotide of  claim 32  and at least one agent selected from the group consisting of an immunostimulatory agent, an anti-viral agent, an anti-bacterial agent, an anti-tumor agent, and a gene silencing agent, wherein the oligonucleotide and the at least one agent are administered simultaneously, separately, or sequentially. 
     
     
         56 . The combined preparation of  claim 55 , wherein the agent is retinoic acid, type I IFN, or a combination thereof. 
     
     
         57 . A pharmaceutical package, comprising the combined preparation of  claim 55  and an instruction for use. 
     
     
         58 . A method of using the oligonucleotide of  claim 32  for the preparation of a bacterial RNA for preventing and/or treating a disease and/or disorder selected from the group consisting of viral infection, bacterial infection, parasitic infection, tumor, multiple sclerosis, allergy, autoimmune diseases, immunosuppression, and immunodeficiency in a vertebrate animal. 
     
     
         59 . A process of preparing an oligonucleotide molecule having one or more ribonucleotides that contain a triphosphosphate or a triphosphate analog, comprising the steps of:
 (a) protecting the 2′ hydroxyl moiety of one or more ribonucleotides with a fluoride labile group or a fluoride non-labile group;   (b) converting the desired terminal hydroxyl moiety to a triphosphate or triphosphate analog with a reagent selected from the group consisting of:   
       
         
           
           
               
               
           
         
       
       wherein: 
       R 100  is independently electron withdrawing group (EWG); 
       R 200  and R 300  are each independently haloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycle, substituted heterocyclo; 
       Z 10  is O, S, Se, BH 3  or NR; 
       X 40  is Cl, dialkylamine or cyclic amine; 
       X 10  is Cl, O-aryl or O-substituted aryl; 
       Y 10  and Y 20  are independently O-substituted alkyl; dialkylamine or cyclic amine, wherein the nitrogen is connected to the phosphorus; 
       X 20  and X 30  is independently O, CH 2 , S, NR′, wherein R′ is H or aliphatic; 
       n is 1, 2, 3, 4, or 5; and 
       s is 0, 1, 2 or 3;
 (c) synthesizing said oligonucleotide molecule using a method selected from the group consisting of solid phase phosphoramidite, solution phase phosphoramidite, solid phase H-phosphonate, solution phase H-phosphonate, hybrid phase phosphoramidite, and hybrid phase H-phosphonate-based synthetic methods; and (d) removing the protecting group(s) and/or solid support. 
 
     
     
         60 . The pharmaceutical composition of  claim 53 , adapted for delivery by a mode selected from the group consisting of intraocular injection, oral ingestion, enteral application, inhalation, topical application, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrathecal injection, intratrachael injection, and intravenous injection. 
     
     
         61 . A kit comprising a oligonucleotide of  claim 32  in a labeled package, wherein the label on said package indicates that said oligonucleotide can be used against at least one virus. 
     
     
         62 . The kit of  claim 61 , wherein said kit is approved by a regulatory agency for use in humans. 
     
     
         63 . An assay for identifying an anti-viral or an antibacterial response, comprising a test sample and a test agent, wherein the test agent comprises an oligonucleotide of  claim 32 .