CARBOHYDRATE CONJUGATES AS DELIVERY AGENTS FOR OLIGONUCLEOTIDES
The present invention provides iRNA agents comprising at least one subunit of the formula (I): wherein: A and B are each independently for each occurrence O, N(R N ) or S; X and Y are each independently for each occurrence H, OH, a hydroxyl protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, a polymer, —P(Z′)(Z″)O-Linker-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, an oligonucleotide, —P(Z′)(Z″)-formula (I), —P(Z′)(Z″)— or -Linker-R; R is L G , -Linker-L G , or has the structure shown below: L G is independently for each occurrence a carbohydrate, e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide; R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; and Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S.
1 - 20 . (canceled)
21 . A process of making a ligand-containing conjugate having the structure of
or a Salt thereof, the process comprising the step of:
reacting a ligand moiety containing L G with a branched linker compound that is a precursor to the Structure of
wherein L G is independently for each occurrence a ligand.
22 . The process of claim 21 , wherein the ligand-containing conjugate has the structure shown in Formula (II)-(V):
wherein:
L G is represented by L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C in Formula (II)-(V), each independently for each occurrence a carbohydrate;
q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B and q 5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , and T 5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O;
Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , and Q 5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO 2 , N(R N ), C(R′)═C(R″), C≡C or C(O);
R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , and R 5C are each independently for each occurrence absent, NH, O, S, CH 2 , C(Q)Q, C(Q)NH, NHCH(R a )C(Q), —C(Q)-CH(R a )—NH—, CO, CH═N—O,
or heterocyclyl;
R′ and R″ are each independently H, C 1 -C 6 , alkyl, OH, SH or N(R N ) 2 ;
R N is independently for each occurrence methyl, ethyl, propyl, isopropyl, butyl or benzyl; and
R a is H or amino acid side chain.
23 . The process of claim 21 , wherein L G is for each occurrence a monosaccharide selected from the group consisting of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucose, fucosamine, fuculose, galactose, D-galactosaminitol, galactosamine, N-acetyl-galactosamine (GalNAc), glucose, glucose-6-phosphate, glucosaminitol, glucosamine, N-acetyl-glucosamine (GluNAc), gulose glyceraldehyde, L-glycero-D-mannos-heptose, glycerol, glycerone, gulose, idose, lyxose, mannose, mannose-6-phosphate, mannosamine, lactose, psicose, quinovose, quinovosamine, rhamnose, rhamnitol, rhamnosamine, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose.
24 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein each n independently for each occurrence is an integer from 1 to 5,
wherein the reacting step comprises:
reacting a carbohydrate acid
or a salt thereof with a branched linker compound
under conditions effective to form a ligand-containing compound
and
deprotecting the Amine protecting group 1 of the formed ligand-containing compound under conditions effective to form the ligand-containing conjugate or a salt thereof.
25 . The process of claim 24 , wherein the branched linker compound
is prepared by:
reacting a branched linker compound
with a linker compound
to form a branched linker compound
and
deproctecting the Amine protecting group 2 of the branched linker compound
under conditions effective to form the branched linker compound
26 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein the reacting step comprises:
reacting a GalNAc acid
or a salt thereof with a branched linker compound
under conditions effective to form a ligand-containing compound
and
hydrogenating the formed ligand-containing compound under conditions effective to form the ligand-containing conjugate or a salt thereof.
27 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein each n independently for each occurrence is an integer from 1 to 5,
wherein the reacting step comprises:
reacting a carbohydrate amine
or a salt thereof with a branched linker compound
under conditions effective to form a ligand-containing compound
and
deprotecting the Amine protecting group 1 of the formed ligand-containing compound under conditions effective to form the ligand-containing conjugate or a salt thereof.
28 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein the reacting step comprises:
reacting a GalNAc amine
or a salt thereof with a branched linker compound
under conditions effective to form a ligand-containing compound
and
hydrogenating the formed ligand-containing compound under conditions effective to form the ligand-containing conjugate or a salt thereof.
29 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein each n independently for each occurrence is an integer from 1 to 5,
wherein the reacting step comprises:
reacting a carbohydrate acid
or a salt thereof with a branched linker compound
under conditions effective to form a ligand-containing compound
and
deprotecting the Carboxylic acid protecting group of the ligand-containing compound under conditions effective to from the ligand-containing conjugate or a salt thereof.
30 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein the reacting step comprises:
reacting a GalNAc acid
or a salt thereof with a branched linker
under conditions effective to form a ligand-containing compound
and
converting the ligand-containing compound to the ligand-containing conjugate or a salt thereof.
31 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein each n independently for each occurrence is an integer from 1 to 5, wherein the reacting step comprises:
reacting a carbohydrate compound
with a branched linker compound
under conditions effective to form a ligand-containing compound
and
deprotecting the Amine protecting group 1 of the formed ligand-containing compound under conditions effective to form the ligand-containing conjugate or a salt thereof.
32 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein the reacting step comprises:
reacting a GalNAc compound
with a branched linker compound
under conditions effective to form a ligand-containing compound
and
hydrogenating the formed ligand-containing compound under conditions effective to form the ligand-containing conjugate or a salt thereof.
33 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein each n independently for each occurrence is an integer from 1 to 5,
wherein the reacting step comprises:
reacting a carbohydrate compound
with a branched linker compound
under conditions effective to form the ligand-containing conjugate or a salt thereof.
34 . The process of claim 21 , wherein the ligand-containing conjugate has the structure of
wherein the reacting step comprises:
reacting a GalNAc compound
with a branched linker compound
under conditions effective to form the ligand-containing conjugate or a salt thereof.
35 . A process of synthesizing a ligand-conjugated oligonucleotide, comprising the step of:
conjugating, to an oligonucleotide, a ligand-containing conjugate having the structure of
wherein L G is independently for each occurrence a ligand.
36 . The process of claim 35 , wherein the ligand-containing conjugate has the structure shown in formula (II)-(V):
wherein:
L G is represented by L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C in Formula (II)-(V), each independently for each occurrence a carbohydrate;
q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B and q 5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , and T 5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O;
Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , and Q 5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO 2 , N(R N ), C(R′)═C(R″), C≡C or C(O);
R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , and R 5C are each independently for each occurrence absent, NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), —C(O)—CH(R a )—NH—, CO, CH═N—O
or heterocyclyl;
R′ and R″ are each independently H, C 1 -C 6 , alkyl, OH, SH or N(R N ) 2 ;
R N is independently for each occurrence methyl, ethyl, propyl, isopropyl, butyl or benzyl; and
R a is H or amino acid side chain.
37 . The process of claim 35 , wherein the ligand-containing conjugate contains the structure of
wherein each n independently for each occurrence is an integer from 1 to 5.
38 . The process of claim 37 , wherein the ligand-containing conjugate contains the structure of
39 . The process of claim 35 , wherein the ligand-containing conjugate contains the structure of
wherein each n independently for each occurrence is an integer from 1 to 5.
40 . The process of claim 39 , wherein the ligand-containing conjugate contains the structure of
41 . The process of claim 35 , wherein the ligand-containing conjugate contains the structure of
wherein each n independently for each occurrence is an integer from 1 to 5.
42 . The process of claim 41 , wherein the ligand-containing conjugate contains the structure of
43 . The process of claim 35 , wherein the ligand-containing conjugate contains the structure of
wherein each n independently for each occurrence is an integer from 1 to 5.
44 . The process of claim 43 , wherein the ligand-containing conjugate contains the structure of
45 . The process of claim 35 , wherein the ligand-containing conjugate contains the structure of
wherein each n independently for each occurrence is an integer from 1 to 5.
46 . The process of claim 45 , wherein the ligand-containing conjugate contains the structure of
47 . The process of claim 35 , wherein at least one ligand-containing conjugate is conjugated to the 3′-end of the oligonucleotide.
48 . The process of claim 47 , wherein the ligand-containing conjugate further contains a solid support, wherein the conjugating step is carried out by starting the synthesis of the oligonucleotide on the solid support of the ligand-containing conjugate.
49 . The process of claim 35 , wherein at least one ligand-containing conjugate is conjugated to the 5′-end of the oligonucleotide.
50 . The process of claim 49 , wherein the ligand-containing conjugate further contains a phosphoramidite moiety, wherein the conjugating step is carried out by coupling the phosphoramidite of the ligand-containing conjugate to the growing chain of the oligonucleotide.
51 . A process of making a ligand-containing conjugate, comprising:
reacting a carbohydrate amine ligand having the formula
or a salt thereof, with a biantennary or triantennary branched linker compound containing a terminal carboxylic acid group in each branch of the branched linker compound, via an amidation reaction, under conditions effective to form the ligand-containing conjugate.
52 . The process of claim 51 , wherein the carbohydrate in the carbohydrate amine ligand is a monosaccharide selected from the group consisting of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucose, fucosamine, fuculose, galactose, D-galactosaminitol, galactosamine, N-acetyl-galactosamine (GalNAc), glucose, glucose-6-phosphate, glucosaminitol, glucosamine, N-acetyl-glucosamine (GluNAc), gulose glyceraldehyde, L-glycero-D-mannos-heptose, glycerol, glycerone, gulose, idose, lyxose, mannose, mannose-6-phosphate, mannosamine, lactose, psicose, quinovose, quinovosamine, rhamnose, rhamnitol, rhamnosamine, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose.
53 . The process of claim 51 , wherein the carbohydrate amine ligand is
and wherein the biantennary or triantennary branched linker compound is