Galnac phosphoramidites, nucleic acid conjugates thereof and their use

Abstract

This invention generally relates to the field of phosphoramidite derivatives. In particular, the invention relates to N-Acetylgalactosamine phosphoramidite molecules and to conjugates of nucleic acid molecules with N-Acetylgalactosamine containing molecules. Also provided are methods for preparation of these molecules and possible uses thereof, in particular in medicine.

1 . A compound having the general formula (I) 
       
         
           
           
               
               
           
         
       
       wherein
 NR′R″ is a secondary amino group, wherein R′ and R″ are independently selected from C 1 -C 6 -alkyl or R′ and R″ together form a five- or six-membered ring, which may be substituted and which may contain one further heteroatom selected from N and O; 
 A is a C 1 -C 6 -alkyl group or a protected hydroxy- or thio-group; and 
 G is represented by general formula (II) 
 
       
         
           
           
               
               
           
         
         
           wherein A 1  is a suitable hydroxyl protecting group, which may be the same or different at each occurrence; and 
           L is a linker group selected from the group consisting of C 2 -C 30 -alkenylene, —(CH 2 ) 2 —, —(CH 2 ) 3 —, —(CH 2 ) 4 —, —(CH 2 ) 5 —, —(CH 2 ) 7 —, —(CH 2 ) 8 —, —(CH 2 ) 9 —, —(CH 2 ) 10 —, —(CH 2 ) 11 —, —(CH 2 ) 12 —, —CH 2 CH 2 OCH 2 CH 2 —, —CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 —, and —CH 2 CH 2 (OCH 2 CH 2 ) 2 OCH 2 CH 2 —, more preferably from —(CH 2 ) 2 —, —(CH 2 ) 3 —, and —(CH 2 ) 8 —. 
         
       
     
     
         2 . The compound according to  claim 1 , wherein the linker group is selected from CH 2 CH 2 OCH 2 CH 2 —, —CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 —, and —CH 2 CH 2 (OCH 2 CH 2 ) 2 OCH 2 CH 2 —. 
     
     
         3 . The compound according to  claim 1 ,
 wherein the secondary amino group —NR′R″ is selected from the group consisting of dimethylamino, diethylamino, diisopropylamino, dibutylamino, pyrrolidino, piperidino, 2,6-dimethylpiperidino, morpholino, imidazolyl and 4-methylimidazolyl, and/or   wherein A is selected from the group consisting of 2-cyanoethoxy, 2-cyanoethylthio, methoxy, ethoxy, S-isobutanoyl-2-(2-mercaptoethoxy)ethoxy, S-pivaloyl-2-(2-mercaptoethoxy)ethoxy, S-pivaloyl-2-mercaptoethoxy, methyl and ethyl, and/or   wherein the hydroxyl protecting group A 1  is selected from an acyl group and a silyl group, preferably from the group consisting of acetyl, benzoyl, phenoxy-acetyl, pivaloyl, isobutyryl, t-butyl dimethyl silyl, t-butyldiphenylsilyl, triisopropylsilyl and isopropyldimethylsilyl.   
     
     
         4 . The compound according to  claim 1 , wherein
 A is —O—CH 2 CH 2 CN, A 1  is acetyl, R′ and R″ each are isopropyl, and L is selected from the group consisting of —(CH 2 ) 2 —, —(CH 2 ) 3 —, —(CH 2 ) 4 —, —(CH 2 ) 5 —, —(CH 2 ) 7 —, —(CH 2 ) 8 —, —(CH 2 ) 9 —, —(CH 2 ) 10 —, —(CH 2 ) 11 —, —(CH 2 ) 12 —, —CH 2 CH 2 OCH 2 CH 2 —, —CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 —, and —CH 2 CH 2 (OCH 2 CH 2 ) 2 OCH 2 CH 2 —, preferably from —(CH 2 ) 2 — and —(CH 2 ) 3 —.   
     
     
         5 . A compound having the general formula (III) 
       
         
           
           
               
               
           
         
       
       wherein
 Z is a nucleic acid molecule; 
 T is represented by general formula (IV) or alternatively by general formula (VI) 
 
       
         
           
           
               
               
           
         
         
           wherein in formula (IV) 
           R 2  represents H or —(CH 2 ) q —R 1 , 
           R 1  and R 3  at each occurrence are represented by general formula (V) 
         
       
       
         
           
           
               
               
           
         
         
           wherein independently at each occurrence 
           V is selected from —O—, —NH—CO— and —CO—NH—; 
           W is selected from the group consisting of —(CH 2 ) 2-15 — and  CH 2 CH 2 (OCH 2 CH 2 ) 0-4 OCH 2 CH 2 —; 
           Y is O or S; 
           m is an integer from 1 to 3; 
           n is an integer from 0 to 5; 
           p is an integer from 0 to 3; and 
           q is an integer from 1 to 2; 
           r is an integer from 1 to 5; 
         
       
       
         
           
           
               
               
           
         
         
           wherein independently at each occurrence in formula (V) and (VI) 
           s is 0 or 1 
           G is represented by general formula (II)′ 
         
       
       
         
           
           
               
               
           
         
         
           
             wherein A 1  is H or a suitable hydroxyl protecting group, which may be the same or different at each occurrence; and L is selected from the group consisting of C 2 -C 20 -alkenylene, —CH 2 CH 2 —(OCH 2 CH 2 ) 0-6 —OCH 2 CH 2 , —(CH 2 ) 2 —, —(CH 2 ) 3 —, —(CH 2 ) 4 —, —(CH 2 ) 5 —, —(CH 2 ) 7 —, —(CH 2 ) 8 —, —(CH 2 ) 9 —, —(CH 2 ) 10 —, —(CH 2 ) 11 —, —(CH 2 ) 12 -, —CH 2 CH 2 OCH 2 CH 2 —, —CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 —, and —CH 2 CH 2 (OCH 2 CH 2 ) 2 OCH 2 CH 2 —; and 
           
           independently at each occurrence in formula (III), (V) and (VI) 
           X 1  is —OH and X 2  is selected from ═O and ═S, or 
           X 1  is —O −  and X 2  is selected from ═O and ═S, or 
           X 1  is ═O and X 2  is selected from —CH 3 , —OR, —NHR, and —BH 3 , wherein R is independently at each occurrence a C 1 -C 6  alkyl group, or 
           X 1  is ═S and X 2  is selected from —CH 3  and —SH, 
         
         wherein the contiguous chain starting with the first atom of linker L in R1, R3 or, if R2 is not H, R2 or in formula (VI), and ending with the attachment point in formula (IV) or in the nucleic acid molecule if T is represented by formula (VI) has a minimum length of 8 atoms and a maximum length of 30 atoms. 
       
     
     
         6 . The compound according to  claim 5 , wherein s is 1, and wherein W is preferably selected from the group consisting of —CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 —, —CH 2 CH 2 (OCH 2 CH 2 ) 4 OCH 2 CH 2 —, —CH 2 CH 2 CH 2 —, and —(CH 2 ) 12 —. 
     
     
         7 . The compound according to  claim 5 , wherein s is 0. 
     
     
         8 . The compound according to  claim 5 , wherein independently at each occurrence
 X 1  is —OH and X 2  is ═O, or   X 1  is O −  and X 2  is ═O, or   X 1  is —OH and X 2  is ═S, or   X 1  is O −  and X 2  is ═S, or   X 1  is ═S and X 2  is —SH.   
     
     
         9 . The compound according to  claim 5 , wherein the compound is capable of binding to the asialoglycoprotein receptor (ASGPR). 
     
     
         10 . A process for the preparation of a compound according to  claim 1 , comprising the steps:
 (i) stereoselectively forming an internal oxazoline ring between carbon atoms 1 and 2 of N-Acetylgalactosamine (GalNAc);   (ii) reacting the product of step (i) with a compound having the general formula HO-L-O-A 3 , wherein   A 3  is a suitable protecting group, and   L is a linker group selected from the group consisting of C 2 -C 20 -alkenylene, —CH 2 CH 2 —(OCH 2 CH 2 ) 0-6 —OCH 2 CH 2 , —(CH 2 ) 2 —, —(CH 2 ) 3 —, —(CH 2 ) 4 —, —(CH 2 ) 5 —, —(CH 2 ) 7 —, —(CH 2 ) 8 —, —(CH 2 ) 9 —, —(CH 2 ) 10 , —(CH 2 ) 11 —, —(CH 2 ) 12 —, —CH 2 CH 2 OCH 2 CH 2 —, —CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 —, and —CH 2 CH 2 (OCH 2 CH 2 ) 2 OCH 2 CH 2 —,   thereby forming an ether bond at carbon atom 1 of the GalNAc ring;   (iii) deprotecting the —O-A 3  group in the product of step (ii), thereby providing a deprotected —OH group,   (iv) reacting the product of step (iii) with a phosphordiamidite, thereby providing a compound according to  claim 1 .   
     
     
         11 . A Process for the preparation of a GalNAc-nucleic acid conjugate, comprising the steps:
 (i) providing a nucleic acid molecule on a solid support;   (ii) optionally adding a brancher molecule to the nucleic acid molecule using phosphoramidite chemistry;   (iii) optionally adding a spacer phosphoramidite molecule to each of the branches of the brancher molecule using phosphoramidite chemistry;   (iv) reacting a compound according to  claim 1  with the reactive end of the nucleic acid molecule, if no brancher molecule is present, or reacting a compound according to  claim 1  with the reactive end of each of the branches, if a brancher molecule is present, or reacting a compound according to  claim 1  with the reactive end of each of the spacers, if spacers are present; and   (v) cleaving the product of step (iv) from the solid support;   wherein the contiguous chain starting with the first atom of linker L in the compound according to  claim 1  and ending with the phosphorus atom linking the conjugate moiety to the nucleic acid has a minimum length of 10 atoms and a maximum length of 34 atoms.   
     
     
         12 . The process according to  claim 11 , wherein said branched molecule after addition results in structures preferably represented by general formula (IV a) 
       
         
           
           
               
               
           
         
         
           wherein 
           A 4  is a suitable protecting group; 
           V 1  is selected from —O—, —NH—CO— and —CO—NH—; 
           V 2  is absent or is selected from —O—, —NH—CO— and —CO—NH—; 
           V 3  is selected from —O—, —NH—CO— and —CO—NH—; 
           X 1  is —OH and X 2  is selected from ═O and ═S, or 
           X 1  is O −  and X 2  is selected from ═O and ═S, or 
           X 1  is ═O and X 2  is selected from —CH 3 , —SH, —OR, —NHR, and —BH 3 , wherein R is independently at each occurrence a C 1 -C 6  alkyl group, or 
           X 1  is ═S and X 2  is selected from —CH 3  and —SH; 
           m is an integer from 1 to 3; 
           n is an integer from 0 to 5; 
           p is an integer from 0 to 3; and independently at each occurrence 
           q is an integer from 1 to 2; 
           q′ is an integer from 0 to 2; and 
           r is an integer from 1 to 5; 
           with the proviso that when V 2  is absent, q′ is 0 and —(CH 2 ) r —OA 4  attached to V 2  in formula (IV a) is also absent; and 
         
         said spacer phosphoramidite molecule after addition results in structures preferably represented by general formula (V a) 
       
       
         
           
           
               
               
           
         
         
           wherein independently at each occurrence 
           A 5  is a suitable protecting group; 
           W is selected from the group consisting of —(CH 2 ) 2-15 — and 
           —CH 2 CH 2 (OCH 2 CH 2 ) 0-4 OCH 2 CH 2 —; 
           X 1  is —OH and X 2  is selected from ═O and ═S, or 
           X 1  is O −  and X 2  is selected from ═O and ═S, or 
           X 1  is ═O and X 2  is selected from —CH 3 , —SH, —OR, —NHR, and —BH 3 , wherein R is independently at each occurrence a C 1 -C 6  alkyl group, or 
           X 1  is ═S and X 2  is selected from —CH 3  and —SH; and 
         
         wherein the contiguous chain starting with the first atom of linker L in the compound according to  claim 1   4 , and ending with the attachment point in formula (IVa) or the attachment point in the nucleic acid molecule has a minimum length of 8 atoms and a maximum length of 30 atoms. 
       
     
     
         13 . The process according to  claim 11 , wherein the brancher molecule is selected from the group consisting of: 1,3-bis-[5-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite; tris-2,2,2-[3- (4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; and tris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. 
     
     
         14 . The process according to  claim 11 , wherein the spacer phosphoramidite molecule is represented by general formula (VII) 
       
         
           
           
               
               
           
         
         wherein A 2  is a suitable protecting group, preferably —CH 2 CH 2 CN, A 6  is a suitable protecting group such as 4′,4′-Dimethoxytrityl and R 4  is selected from the group consisting of: —CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 —, —CH 2 CH 2 (OCH 2 CH 2 ) 4 OCH 2 CH 2 —, —CH 2 CH 2 CH 2 —, and —(CH 2 ) 12 —; and 
         NR′R″ is a secondary amino group, wherein R′ and R″ are independently selected from C 1 -C 6 -alkyl or R′ and R″ together form a five- or six-membered ring, which may be substituted and which may contain one further heteroatom selected from N and O. 
       
     
     
         15 . The process according to  claim 11 , wherein the nucleic acid molecule is selected from DNA, RNA and molecules comprising one or more nucleoside analogues, particularly the nucleic acid molecule is selected from antisense oligonucleotides, gapmers, small interfering RNAs, and microRNAs, and wherein the nucleic acid molecule preferably contains one or more locked nucleic acid nucleosides. 
     
     
         16 . Use of a compound according to  claim 1  for the preparation of compounds according to  claim 5 . 
     
     
         17 . Use of a compound according to  claim 5  as a medicament.