Pyrazolotriazolyl nucleoside analogues and oligonucleotides comprising them

Abstract

The present invention relates to pyrazolotriazolyl nucleoside analogues, oligonucleotide comprising them, and uses thereof. Further the invention relates to a method for reducing gene expression in a cell comprising transfecting the cell with such an oligonucleotide.

1 . An oligonucleotide comprising one or more of 9-adeninyl or 9-guaninyl PT nucleoside or nucleotide analogues each independently of the general formula II: 
       
         
           
           
               
               
           
         
         wherein 
       
       R 1  and R 4  each independently is selected from H, halogen, —CN, —SCN, —NO 2 , —O-hydrocarbyl, —S-hydrocarbyl, —CO—H, —CO-hydrocarbyl, —NR 8 R 9 , heteroaryl, and hydrocarbyl optionally substituted by one or more groups each independently is halogen, —CN, —SCN, or —NO 2 , wherein R 8  and R 9  are each independently H, hydrocarbyl, and benzoyl, provided that at least one of R 8  and R 9  is not H; or R 8  and R 9  together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from oxygen, nitrogen and sulfur; 
       R2 is H or absent; 
       R3 is O or —NR 10 R 10′ , wherein R 10  and R 10′  are each independently H, hydrocarbyl, or —CO-hydrocarbyl, provided that at least one of R 10  and R 10′  is not H; 
       provided that at least one of R 1  and R 3  is —NR 8 benzoyl or —NR 10 benzoyl, respectively;
 R 5  is halogen or —OR 11 ; 
 R 6  is —OR 11 ; 
 R 7  is —OR 11 , a monophosphate moiety, or a phosphate linking moiety; 
 R 11  each independently is (C 1 -C 8 )alkyl, (C 1 -C 8 )alkylene-OR 12 , (C 1 -C 8 )alkylene-SR 12 , or (C 1 -C 8 )alkylene-NR 12 R 13 , a hydroxyl protecting group, or a phosphoramidite moiety of the formula —P(OR 14 )NR 15 R 16 , wherein R 12  and R 13  each independently is H or (C 1 -C 8 )alkyl, provided that at least one of R 12  and R 13  is not H, wherein R 14  is H or cyano-(C 1 -C 8 )alkyl, and R 15  and R 16  each independently is (C 1 -C 8 )alkyl; and 
 
       the dotted line represents a potential double bond between the carbon atom at position 4 and either the nitrogen atom at position 3 or the radical R 3 , provided that, when R 2  is H, there is a double bond between the carbon atom at position 4 and R 3 , and when R 2  is absent, there is a double bond between the carbon atom at position 4 and the nitrogen atom at position 3. 
     
     
         2 . The oligonucleotide of  claim 1 , wherein in each one of said one or more 9-adeninyl PT nucleoside or nucleotide analogues:
 (i) R 1  and R 4  each independently is H, halogen, (C 1 -C 4 )alkyl, —O—(C 1 -C 4 )alkyl, —S—(C 1 -C 4 )alkyl, —CO—H, —CO—(C 1 -C 4 )alkyl, or —NR 8 R 9 , wherein R 8  and R 9  are each independently H or (C 1 -C 4 )alkyl, provided that at least one of R 8  and R 9  is not H;   (ii) R 3  is —NR 10 benzoyl;   (iii) R 5  independently is halogen or —OR 11 ;   (iv) R 6  is —OR 11 ; and   (v) R 11  each independently is (C 1 -C 2 )alkyl, (C 1 -C 2 )alkylene-OH, (C 1 -C 2 )alkylene-OCH 3 , (C 1 -C 2 )alkylene-SH, (C 1 -C 2 )alkylene-SCH 3 , or (C 1 -C 2 )alkylene-NH 2 .   
     
     
         3 . The oligonucleotide of  claim 1 , comprising one or more 9-adeninyl PT nucleotide analogues, each independently of the general formula II, wherein R 1  is and R 4  each independently is H, halogen, (C 1 -C 2 )alkyl, —O—(C 1 -C 2 )alkyl, —S—(C 1 -C 2 )alkyl, —CO—H, —CO—(C 1 -C 2 )alkyl, or —NR 8 R 9 , wherein R 8  and R 9  are each independently H or (C 1 -C 2 )alkyl, provided that at least one of R 8  and R 9  is not H; R 2  is absent; R 3  is —NH-benzoyl; R 5  is halogen or —OR 11 ; R 6  is —OR 11 ; R 7  is —OR 11 , a monophosphate moiety, or a phosphate linking moiety; and R 11  each independently is (C 1 -C 4 )alkyl, (C 1 -C 4 )alkylene-OH, (C 1 -C 4 )alkylene-OCH 3 , (C 1 -C 4 )alkylene-SH, (C 1 -C 4 )alkylene-SCH 3 , or (C 1 -C 4 )alkylene-NH 2 . 
     
     
         4 . The oligonucleotide of  claim 1 , comprising one or more 9-guaninyl PT nucleotide analogues each independently of the general formula II, wherein R 1  is —NH-benzoyl; R 2  is H; R 3  is O; R 4  is H, halogen, (C 1 -C 2 )alkyl, —O—(C 1 -C 2 )alkyl, —S—(C 1 -C 2 )alkyl, —CO—H, —CO—(C 1 -C 2 )alkyl, or —NR 8 R 9 , wherein R 8  and R 9  are each independently H or (C 1 -C 2 )alkyl, provided that at least one of R 8  and R 9  is not H; R 5  is halogen or —OR 11 ; R 6  is —OR 11 ; R 7  is —OR 11 , a monophosphate moiety, or a phosphate linking moiety; and R 11  each independently is (C 1 -C 4 )alkyl, (C 1 -C 4 )alkylene-OH, (C 1 -C 4 )alkylene-OCH 3 , (C 1 -C 4 )alkylene-SH, (C 1 -C 4 )alkylene-SCH 3 , or (C 1 -C 4 )alkylene-NH 2 . 
     
     
         5 . The oligonucleotide according to  claim 1 , wherein the oligonucleotide is a DNA oligonucleotide comprising one or more nucleoside or nucleotide analogues, each independently of the general formula II, wherein R 5  is halogen; R 6  is —OR 11 ; and R 11  is (C 1 -C 8 )alkyl, (C 1 -C 8 )alkylene-OR 12 , (C 1 -C 8 )alkylene-SR 12 , or (C 1 -C 8 )alkylene-NR 12 R 13 , wherein R 12  and R 13  each independently is H or (C 1 -C 8 )alkyl, provided that at least one of R 12  and R 13  is not H. 
     
     
         6 . The oligonucleotide according to  claim 1 , wherein the oligonucleotide is an RNA oligonucleotide comprising one or more nucleoside or nucleotide analogues, each independently of the general formula II, wherein R 5  is —OR 11 ; R 6  is —OR 11 ; and R 11  each independently is (C 1 -C 8 )alkyl, (C 1 -C 8 )alkylene-OR 12 , (C 1 -C 8 )alkylene-SR 12 , or (C 1 -C 8 )alkylene-NR 12 R 13 , wherein R 12  and R 13  each independently is H or (C 1 -C 8 )alkyl, provided that at least one of R 12  and R 13  is not H. 
     
     
         7 . A double-stranded DNA molecule, wherein at least one strand is a DNA oligonucleotide according to  claim 5 . 
     
     
         8 . A pharmaceutical composition comprising a DNA oligonucleotide according to  claim 5  and a pharmaceutically acceptable carrier. 
     
     
         9 . A pharmaceutical composition comprising an RNA oligonucleotide according to  claim 6  and a pharmaceutically acceptable carrier. 
     
     
         10 . A pharmaceutical composition comprising a double-stranded DNA molecule according to  claim 7  and a pharmaceutically acceptable carrier. 
     
     
         11 . A pharmaceutical composition comprising an oligonucleotide according to  claim 1 , and a pharmaceutically acceptable carrier.