Oligonucleotide-ligand conjugates and methods for their preparation

Abstract

The present invention relates to ligand conjugates of oligonucleotides (e.g., iRNA agents) and methods for their preparation. These ligands are primarily derived from monosaccharides, and these conjugates are useful for the in vivo delivery of oligonucleotides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 845,279, filed July 11, 2013, which is incorporated herein by reference in its entirety. [Technical Field]

[0002] The present invention relates to ligand conjugates of oligonucleotides (e.g., iRNA agents) and methods for their preparation. These ligands are primarily derived from monosaccharides. These conjugates are useful for in vivo delivery of oligonucleotides. [Background technology]

[0003] Efficient delivery to cells in vivo requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One way to achieve specific targeting is to conjugate a targeting moiety to the iRNA agent. The targeting moiety serves to target the iRNA agent to the desired target site. One way that a targeting moiety can improve delivery is through receptor-mediated endocytosis activity. This uptake mechanism involves the movement of an iRNA agent bound to a membrane receptor into a membrane-enclosed region by invagination of a membrane structure or fusion of the delivery system with the cell membrane. This process is initiated by activation of a cell surface or membrane receptor after binding of a specific ligand to the receptor. Many receptor-mediated endocytosis systems are known and have been studied, including those that recognize sugars such as galactose, mannose, and mannose-6-phosphate, peptides, and proteins such as transferrin, asialoglycoprotein, vitamin B12, insulin, and epidermal growth factor (EGF). The asialoglycoprotein receptor (ASGP-R) is a high-capacity receptor that is highly abundant in hepatocytes. ASGP-R exhibits 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal. Previous studies have shown that multivalency is required to achieve nM affinity, while intersaccharide spacing is also important.

[0004] Recently, certain carbohydrate conjugates have been shown to be useful alternatives to liposomes for siRNA delivery. Summary of the Invention [Means for solving the problem]

[0005] The present invention relates to ligand conjugates of oligonucleotides or other biologically active substances that have one or more advantageous properties, such as improved delivery of the oligonucleotides or other biologically active substances, lower manufacturing costs or fewer manufacturing problems, or better chemical stability. These conjugates provide for effective delivery of oligonucleotides and other biologically active substances.

[0006] In one embodiment, the present invention provides a compound of formula I: [ka] (In the formula, the oligonucleotide is an oligonucleotide such as a siRNA, microRNA, anti-miR, antagomir, microRNA mimic, decoy, immunostimulatory, G-quartet, splicing alteration, ssRNA, antisense, aptamer, stem-loop RNA or DNA or one of the two strands of any double-stranded RNA or DNA or the shorter of the double-stranded RNA or DNA (e.g., siRNA); The biologically active substance is any biologically active substance; The linker is a linking group between the ligand and the oligonucleotide or other biologically active substance, where the linker may be selected from the linking groups in Table 1 or 1A; and The ligands are derived from sugars, where (i) the ligands may be attached to the same or different atoms in the linker, and (ii) the conjugate contains 1 to 12 ligands (preferably 1 to 5 or 1 to 3 ligands), and (iii) the ligands are (a) a ligand in Table 2 or 2A; (b)-R 2 -(R 3 ) k (where, R 2 does not exist (in which case k=1) or R 3 a spacer (also called a ligand scaffold) having two or more binding sites for groups; R 3 is a targeting monomer selected from Table 3, and k is 1 to 6 (preferably 1 to 5 or 1 to 3), and each R 3 But R 2 may be attached to the same or different atoms in (c) a ligand in Table 4 or 4A may be selected from The present invention relates to ligand (e.g., carbohydrate) conjugates of oligonucleotides (e.g., iRNA agents) or other biologically active substances.

[0007] The conjugate comprises at least one linker from Table 1 or 1A or the Examples, one ligand from Table 2, 2A, 4, or 4A, or one targeting monomer from Table 3 or 3A. For example, the nucleoside linkers described in the Examples can be used as linkers. In one embodiment, the conjugate comprises (i) at least one linker from Table 1 or 1A or the Examples, (ii) one ligand from Table 2, 2A, 4, or 4A, and (iii) one targeting monomer from Table 3 or 3A.

[0008] R 2 can be an amino acid, a polypeptide (e.g., a dipeptide or tripeptide), a heteroaryl (e.g., a triazole), or a sugar-containing group. 3 The group is connected to the R 2 In one embodiment, R 3is attached via an amide group. Each entry in Tables 2, 2A, 4, and 4A shows a spacer attached to at least one targeting monomer. The spacer is attached to the targeting monomer through a heteroatom, such as a nitrogen atom (at the end of the spacer), or to the sugar group of the targeting monomer (as shown below) at the anomeric carbon. The heteroatom attachment point in the structures shown in Tables 2, 2A, 4, and 4A is the first nitrogen atom when moving along the chain from the sugar group (left side of the ligand) to the remainder of the ligand (right side). The spacers for the two ligands in Table 2A are shown in the table below (arrows indicate the targeting monomer R 3 (The right side of the spacer is attached to the linker.) Suitable spacers are also shown in Table 5 below. TIFF0007676460000002.tif85134

[0009] The oligonucleotide is preferably attached to the linker via (i) the 3' or 5' end of the oligonucleotide, (ii) one or more sugar moieties of a nucleoside present in the oligonucleotide regardless of position, or (iii) one or more base moieties of a nucleoside present in the oligonucleotide regardless of position.

[0010] In one embodiment, the ligand is conjugated to one of the two strands of the double-stranded siRNA via a linker.

[0011] In one embodiment, the ligand targets the asialoglycoprotein receptor (ASGPR). In another embodiment, the ligand targets the liver, such as liver parenchymal cells. The ligand can be an unmodified or modified monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or higher polysaccharide.

[0012] The oligonucleotide can be attached to the linker via a cleavable group (e.g., phosphate, phosphorothioate, or amide) or a non-cleavable group (e.g., ether, carbamate, or CC (e.g., a bond between two carbon atoms or -CH2-CH2-)). As described herein, cleavable or non-cleavable groups are present in the oligonucleotides of Formula I.

[0013] In one embodiment, the -linker-ligand is not L96 (shown in the examples).

[0014] In the conjugate formulas described herein (such as Formula (I)), oligonucleotides or other biologically active substances can be replaced by lipid nanoparticle (LNP) (such as PEG-lipid or cationic lipid) or polymer components. The conjugated LNP components or conjugated polymers can be useful as delivery agents to facilitate delivery of biologically active substances to target sites.

[0015] Table 1 - Linker groups a,b The following linker is shown with the protecting group DMTr. When conjugated, the DMTr group is removed and the adjacent oxygen atom is the attachment site of the linker to the oligonucleotide (e.g., to a cleavable group of the oligonucleotide). The wavy line is the point of attachment of the ligand. X can be hydrogen, a leaving group, -OH, or -NH2. When the linker group is incorporated into an intermediate compound useful for preparing the conjugates of the invention, X is compatible with solid phase oligonucleotide synthesis and deprotection, or can be attached to a solid support (e.g., [ka] ) coupled to a reactive phosphoramidite (e.g., [ka] ) can be.

[0016] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0017] Table 1A - Linker Groups The linkers shown below in Table 1A are shown with one or more oligonucleotides attached thereto. It will be understood by those skilled in the art that a linker is a chemical moiety that does not comprise an oligonucleotide. The wavy line is the point of attachment of the ligand. X can be hydrogen, a leaving group, -OH, or -NH2. When the linker group is incorporated into an intermediate compound useful for preparing the conjugates of the invention, X can be attached to a solid support (e.g., a solid support) that is compatible with solid phase oligonucleotide synthesis and deprotection, or that allows for solid phase oligonucleotide synthesis (e.g., [ka] ) coupled to a reactive phosphoramidite (e.g., [ka] The phrase "oligonucleotide / nucleotide" is intended to refer to a single nucleotide or an oligonucleotide.

[0018] [Table 1A-1] [Table 1A-2] [Table 1A-3] [Table 1A-4]

Table 1A-5

[0019]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0020]

Table 2A-1

Table 2A-2

Table 2A-3

Table 2A-4

[0021]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0022]

Table 3A-1

[0023] Ligand The ligands can also be represented by the following two general formulas: [ka] where the arrow indicates the point of attachment to the oligonucleotide conjugate (i.e., the ligand is attached via its carbonyl group). Useful intermediates for introducing the ligand include the compounds shown above. In the above formula, the variables have the definitions shown below: R 6 is H or Ac; R 7 -OH or -NHR 9 and; R 8 But Ac or R 9 where R 7 and R 8 at least one of is a nitrogen-containing moiety; R 9 but, [ka] and; Q 1 H, C1-C4 alkyl, [ka] and; Q 2 is H or C1-C4 alkyl; X is H or Me; Y is H, Ac, or COCF3; and n is 1 to 8 (for example, 1 to 4).

[0024] The ligands may be represented by the formulas shown in Table 4 or 4A below.

[0025] [Table 4]

[0026] [Table 4A]

[0027] [Table 5]

[0028] In Tables 2, 2A, 3, 3A, 4, and 4A and other formulas containing one or more -OAc substituents in the sugar moiety, the compounds of the present invention include the same compounds containing -OH substituents in the position of one or more of the -OAc shown. Generally, the acetyl (Ac) group functions as a protecting group for the hydroxyl moiety. Therefore, those skilled in the art will understand that the corresponding hydroxy compounds are within the scope of the present invention and are intended to be used in the final conjugate with an oligonucleotide or other biologically active substance.

[0029] Additionally, compounds of the invention include those in which any -NHAc substituents on the sugar moiety are replaced with hydroxy groups (e.g., where the -NHAc group at position 2 of the sugar moiety is replaced with two -OH groups). In one embodiment, in addition to replacing the -NHAc group with a hydroxy group, any -OAc substituents on the sugar moiety are also replaced with hydroxy groups.

[0030] In one embodiment, the ligand is [ka] is selected from: A is the point of attachment to the linker and is a bond or chemical linking group (e.g., amide, carbamate, urea, -CN- (e.g., -CH-NH- or -C(R a )(R b )-N(R c )-, where R a , R b , and R c are independently selected from hydrogen, alkyl, and aryl), C=NH, ether, thioether, triazole, oxime, or hydrazine; Y is any functional group (e.g., when used as a divalent group, it can be -CONH-, -NHCO-, or S, or, e.g., when used as a monovalent group, it can be -OH, -SH, or a halogen), -CH2-, a protecting group, or a chemically inert cap; Q is OH or any modification to the C6 position of the sugar described herein; and n is 1 to 6. In yet another embodiment, the sugar moiety of any of the above ligands or targeting monomers (such as in Tables 2, 2A, 3, 3A, 4, and 4A) is represented by the following formula III: [ka] (In the formula, R 6 each independently as defined above (e.g., H or Ac); R 7 and R" are independently -ZR 10 , unsubstituted and substituted heteroaryl (e.g., triazole or imidazole), -N3, -CN, and substituted and unsubstituted acetylene; each Z is independently O, NH, or S; R 10 each independently represents H, unsubstituted or substituted alkyl, unsubstituted acyl (e.g., -COCH), substituted acyl (e.g., -COCF), -OC(O)OR 11 , -NHC(O)OR 11, -NHC(O)NHR 11 or an amino acid; and R 11 are each independently H or unsubstituted or substituted alkyl; However, (i) R 6 is H, or (ii) when R″ is OH or NHAc, R 7 is not -OH or -OAc) The sugar moiety may be substituted with:

[0031] Yet another embodiment is a compound of the formula [ka] is an intermediate compound of the formula the ligand and linker are as defined above, X is, [ka] (wherein the sphere represents the solid support), [ka] , a leaving group, H, -OH, or -NH2.

[0032] These intermediates are useful for preparing the oligonucleotide-ligand conjugates of the present invention.

[0033] Yet another embodiment is a compound of formula IIIA [ka] is an intermediate compound of the formula R 52 is a divalent chemical group 1 to 12 atoms in length; the linker is as defined above; R 6 each independently as defined above (e.g., H or Ac); R 7 and R" are independently -ZR10 , unsubstituted and substituted heteroaryl (e.g., triazole or imidazole), -N3, -CN, and substituted and unsubstituted acetylene; each Z is independently O, NH, or S; R 10 each independently represents H, unsubstituted or substituted alkyl, unsubstituted acyl (e.g., -COCH), substituted acyl (e.g., -COCF), -OC(O)OR 11 , -NHC(O)OR 11 , -NHC(O)NHR 11 , or an amino acid; R 11 each of is independently H or unsubstituted or substituted alkyl; and X is, [ka] (wherein the sphere represents the solid support), [ka] , a leaving group, H, -OH, or -NH2; However, (i) R 6 is H, or (ii) when R″ is OH or NHAc, R 7 is not —OH or —OAc.

[0034] In a preferred embodiment, the substitutions at positions 3 and 4 of the sugar group in formula III or IIIA are equatorial and axial, respectively.

[0035] In one embodiment, the sugar in formula III or IIIA is in the α configuration. In another embodiment, the sugar is in the β configuration.

[0036] The present invention also includes compounds of Formula IIIA, where X is replaced with an oligonucleotide or other biologically active agent described herein.

[0037] The same bond or combination of bonds described herein can be used to attach two or more ligand / linker moieties to an oligonucleotide or other biologically active substance. For example, in one embodiment, the present invention provides a compound of formula IV [ka] (In the formula, the ligand and linker are as defined above; Oligonucleotide-1 and Oligonucleotide-2 have the same definition as oligonucleotide above; Biologically active substance-1 and biologically active substance-2 have the same definition as biologically active substance above; and Each ligand may be the same or different, and each oligonucleotide may be the same or different. of an oligonucleotide (e.g., an iRNA agent) or other biologically active substance.

[0038] In Formula IV, the linker connects the two portions of the oligonucleotide (oligonucleotides 1 and 2) via two bonds, each portion of the oligonucleotide representing at least one nucleoside moiety.

[0039] Yet another embodiment is a compound of formula V: [ka] (In the formula, each oligonucleotide (or biologically active substance) and ligand is independently as defined herein; Each linker can independently be any of those described herein (e.g., in Table 1 or 1A), or can be of the formula [ka] where R 4is the site of attachment of the oligonucleotide (e.g., via a cleavable group on the oligonucleotide) and the hydroxy group in the hydroxyproline is the site of attachment of a further linker-ligand group; and t is in the range of 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) of an oligonucleotide (e.g., an iRNA agent) or other biologically active substance.

[0040] In one preferred embodiment, t is 2.

[0041] In a preferred embodiment, the oligonucleotides in the conjugates described herein are attached to the linker via a phosphate, phosphorothioate, or a combination thereof.

[0042] In one embodiment, the conjugate has the formula: [ka] wherein each R is independently a ligand (such as those described herein). In a preferred embodiment, the ligands R are the same.

[0043] The present invention also relates to ligand conjugates of oligonucleotides, wherein at least one nucleoside is conjugated to a carbohydrate-containing ligand (i) via the nucleobase of the nucleoside or (ii) at the 2' position of the nucleoside.

[0044] One embodiment is a sugar conjugate of an oligonucleotide, wherein at least one nucleoside in the oligonucleotide is conjugated to a sugar-containing ligand (e.g., a sugar-containing ligand) via a nitrogen atom in the nucleoside's nucleobase. Any of the ligands described herein can be used. In one embodiment, the nucleoside in the conjugate is represented by Formula VI: [ka] (In the formula, The 5' and 3' ends of the nucleoside in Formula VI are each linked to another nucleoside or terminus of an oligonucleotide; R 6 is a nucleobase (e.g., uracil, cytosine, adenosine, or guanine) and optionally has a nitrogen-containing moiety attached to the nucleobase; R 7 is the linker, where R 7 But R 6 is bonded to a nitrogen atom (e.g., an amino group) in Each R 8 are, independently, ligands) Each R 8 But Linker R 7 The ligands R may be bonded to the same or different atoms in 8 For example, -R 2 -R 3 or may be a ligand in Tables 2, 2A, 4, and 4A.

[0045] In one embodiment, R 6 is uracil substituted at its 5-position with an amide group -C(O)NH-, where R 7 is connected to R via the nitrogen atom of the amide group 6 is combined with

[0046] In another embodiment, R 6 is a cytosine substituted at its 5-position with an amide group —C(O)NH—, where R 7 is connected to R via the nitrogen atom of the amide group 6 is combined with

[0047] Another embodiment is a sugar conjugate of an oligonucleotide, wherein at least one nucleoside in the oligonucleotide is conjugated to a ligand (e.g., a sugar-containing ligand) at its 2' position. Any of the ligands described herein can be used. In one embodiment, the nucleoside in the conjugate is represented by Formula VII: [ka] (In the formula, The 5' and 3' ends of the nucleoside in Formula VII are each linked to another nucleoside or terminus of the oligonucleotide; R 6 is a nucleobase; R 7 is the linker; Each R 8 are, independently, ligands) Each R 8 But Linker R 7 The oligonucleotide may be linked to the same or different atoms in the linker.In a preferred embodiment, the oligonucleotide is linked to the linker via phosphate, phosphorothioate, or a combination thereof.For example, the oligonucleotide may be linked to the linker at the 3' end via phosphate and / or at the 5' end via phosphorothioate, or vice versa.

[0048] Ligand moieties (e.g., carbohydrate moieties) facilitate delivery of oligonucleotides to target sites. One way in which ligand moieties can enhance delivery is through receptor-mediated endocytosis. Without being bound by any particular theory, this uptake mechanism is thought to involve the movement of membrane receptor-bound oligonucleotides into the interior of a membrane-enclosed region by invagination of a membrane structure or fusion of the delivery system with the cell membrane. This process is initiated by activation of a cell surface or membrane receptor following binding of a specific ligand to the receptor. Receptor-mediated endocytosis systems include those that recognize sugars such as galactose. Thus, the ligand moiety can include one or more monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides, such as those described above. In a preferred embodiment, the ligand moiety can be a moiety recognized by a human asialoglycoprotein receptor (ASGPR), such as human asialoglycoprotein receptor 2 (ASGPR2). Such carbohydrate moieties can include, for example, sugars (e.g., galactose or N-acetyl-D-galactosylamine).

[0049] Yet another embodiment is an oligonucleotide in which two or more nucleotides each have a -linker-ligand moiety. The -linker-ligand moieties in the oligonucleotide may be the same or different. In one embodiment, the first, third, and fifth nucleotides from the 5' end are each conjugated to a -linker-ligand moiety. In another embodiment, the first, third, and fifth nucleotides from the 3' end are each conjugated to a -linker-ligand moiety. In yet another embodiment, the first, third, and fifth nucleotides from the 3' and 5' ends of the oligonucleotide are each conjugated to a -linker-ligand moiety.

[0050] Yet another embodiment is a method of formulating a therapeutic RNA by preparing a conjugate of an iRNA agent of the invention, wherein the iRNA agent strand comprises a therapeutic RNA.

[0051] Yet another embodiment is a method of delivering a therapeutic RNA to a patient in need thereof by administering to the patient an iRNA agent conjugate of the invention, wherein the iRNA agent strand comprises a therapeutic RNA. Preferred routes of administration include subcutaneous and intravenous routes. [Brief explanation of the drawings]

[0052] [Figure 1] 1 is a bar graph showing TTR protein levels 48 and 144 hours after administration of TTR siRNA conjugates in mice compared to control mice according to the procedure in Example 33. [Figure 2] 10 is a bar graph showing AT3 protein levels in mice following administration of AT3 siRNA conjugates compared to control mice according to the procedure in Example 34. [Figure 3A] 6 is a bar graph showing mTTR protein levels 72 hours after a single subcutaneous administration of conjugates 57727, 63189, 63192, 63190, and 63191 to mice according to the procedure in Example 62. [Figure 3B] 6 is a bar graph showing mTTR protein levels 144 hours after a single subcutaneous administration of conjugates 57727, 63189, 63192, 63190, and 63191 to mice according to the procedure in Example 62. [Figure 4] 1 is a bar graph showing the binding affinity (Ki) of TTR siRNA conjugates 56718-56727, 56729, and 55727 in Example 42. [Figure 5] 1 is a graph showing the median fluorescence intensity (MFI) at various concentrations for TTR siRNA conjugates 56727, 56729, and 55727 in Example 42 (binding affinity curves). [Figure 6] 1 is a graph showing the median fluorescence intensity (MFI) at various concentrations for TTR siRNA conjugates 56721, 56722, 56723, and 55727 in Example 42. [Figure 7]1 is a graph showing the median fluorescence intensity (MFI) at various concentrations for TTR siRNA conjugates 56724, 56725, 56726, 56718, 56719, 56720, and 55727 in Example 42. [Figure 8] 1 is a graph showing the median fluorescence intensity (MFI) at various concentrations for TTR siRNA conjugates 56876, 66875, 56874, 54944, and 56877 in Example 44. [Figure 9] 1 is a graph showing the median fluorescence intensity (MFI) at various concentrations for TTR siRNA conjugates 66878, 56880, 56879, 54944, 56881, and 56882 in Example 44. [Figure 10] 1 is a bar graph showing TTR protein levels 48 and 144 hours after administration of TTR siRNA conjugates 43527, 60126, 60138, 60128, 60127, 60316, and 60123 (at doses of 15 mg / kg and 5 mg / kg) in mice compared to control mice, as described in Example 45. [Figure 11] 1 is a bar graph showing AT3 protein levels following administration of AT3 siRNA conjugates 54944, 56881, and 58137 in mice compared to control mice, as described in Example 46. [Figure 12] 10 is a bar graph showing mTTR protein levels following administration of mTTR siRNA conjugates 55727, 58138, and 58139 in mice compared to control mice, as described in Example 46. [Figure 13] 6 is a graph showing the median fluorescence intensity (MFI) at various concentrations for TTR siRNA conjugates 61696, 61695, 61692, 61694, 61697, 61693, 43527, and 61698 in Example 61. DETAILED DESCRIPTION OF THE INVENTION

[0053] definition The term "oligonucleotide" refers to a chemically modified or unmodified nucleic acid molecule (RNA or DNA) having a length of less than about 100 nucleotides (e.g., less than about 50 nucleotides). The nucleic acid can be, for example, (i) single-stranded DNA or RNA, (ii) double-stranded DNA or RNA, including double-stranded DNA or RNA with a hairpin loop, or (iii) a DNA / RNA hybrid. Non-limiting examples of double-stranded RNA include siRNA (small interfering RNA). Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, microRNAs, and triplex-forming oligonucleotides. In one embodiment, the oligonucleotide has a length in the range of about 5 to about 50 nucleotides, such as about 10 to about 50 nucleotides. In another embodiment, the oligonucleotide has a length in the range of about 6 to about 30 nucleotides, such as about 15 to about 30 nucleotides. In yet another embodiment, the oligonucleotide has a length in the range of about 18 to about 23 nucleotides.

[0054] The term "GalNAc" refers to N-acetyl-galactosamine.

[0055] The term "solid support" as used herein particularly refers to any particle, bead, or surface on which oligonucleotide synthesis occurs. Solid supports that can be used in different embodiments of the methods described herein can be selected from, for example, inorganic and organic supports. Inorganic supports are preferably selected from silica gel and controlled pore glass (CPG). Organic supports are preferably selected from highly cross-linked polystyrene, Tentagel (a grafted copolymer consisting of a low-cross-linked polystyrene matrix to which polyethylene glycol (PEG or POE) is grafted), polyvinyl acetate (PVA), Poros-polystyrene / divinylbenzene copolymer, aminopolyethylene glycol, and cellulose. Preferred solid supports suitable for the present invention include those that are hydrophobic. A preferred embodiment of the present invention uses a polystyrene-based solid support. Many other solid supports are commercially available and suitable for the present invention.

[0056] The term "hydroxy protecting group" as used herein refers to a labile chemical moiety that protects a hydroxyl group from undesired reactions during synthetic procedures. After synthetic procedures, the hydroxy protecting group can be selectively removed. Hydroxy protecting groups known in the art are generally described in T.H. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). Examples of hydroxyl protecting groups include, but are not limited to, benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxy. Examples of protecting groups include acetyl, benzoyl, methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 1,1-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl, triphenylmethyl (trityl), tetrahydrofuryl, methoxymethyl, methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl, trimethylsilyl, triethylsilyl, and triisopropylsilyl. Preferred hydroxyl protecting groups for the present invention are acetyl (Ac or —C(O)CH), benzoyl (Bz or —C(O)CH), and trimethylsilyl (TMS or —Si(CH)).

[0057] As used herein, the term "amino-protecting group" refers to a labile chemical moiety that protects an amino group from undesired reactions during synthetic procedures. After synthetic procedures, the amino-protecting groups described herein can be selectively removed. Amino-protecting groups known in the art are generally described in T.H. Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). Examples of amino-protecting groups include, but are not limited to, acetyl, t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, and benzyloxycarbonyl.

[0058] The term "carboxylic acid protecting group" refers to a carboxylic acid protecting group used to block or protect a carboxylic acid functionality while reactions involving other functional sites of a compound are carried out. Such carboxy protecting groups are known for their ease of cleavage by hydrolysis or hydrogenolysis to the corresponding carboxylic acid. Examples of carboxylic acid ester protecting groups include, but are not limited to, methyl, tert-butyl, benzyl, 4-methoxybenzyl, C2-C6 alkanoyloxymethyl, 2-iodoethyl, 4-nitrobenzyl, diphenylmethyl (benzhydryl), phenacyl, 4-halophenacyl, dimethylallyl, 2,2,2-trichloroethyl, tri(C1-C3 alkyl)silyl, succinimidomethyl, and similar ester-forming moieties. In addition to ester protection of carboxy groups, such groups can also be protected as mixed anhydrides, such as those formed with acetyl chloride, propionyl chloride, isobutyryl chloride, and other acid chlorides in the presence of a tertiary amine base. Other known carboxy protecting groups are suitable, such as those described by E. Haslam in Protective Groups in Organic Chemistry (ibid), Chapter 5. Ester-forming protecting groups are preferred.

[0059] In the above definitions, hydroxy and carboxy protecting groups are not exhaustively defined. The function of such groups is to protect reactive functional groups during the preparation process and then be removed at some later time without disrupting the rest of the molecule. Many protecting groups are known in the art, and the use of other protecting groups not specifically mentioned herein above is equally applicable.

[0060] Suitable peptide coupling reagents include, but are not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazole diethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethylfluoroformamide), nium hexafluorophosphate), DPPA (diphenyl azidophosphate), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate), TSTU (O-(N-succinimidyl) (1-H-1,2,3-benzotriazol-1-yloxy)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HATU (N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide), BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinic chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium tetrafluoroborate), Examples of suitable peptide coupling reagents include BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), and PyBrOP (bromotris(pyrrolidino)phosphonium hexafluorophosphate). EDC, HOAT, BOP-Cl, and PyBrOP are preferred peptide coupling reagents. The amount of peptide coupling reagent ranges from about 1.0 to about 10.0 equivalents.Optional reagents that may be used in the amide bond forming reaction include DMAP (4-dimethylaminopyridine) or activated ester reagents such as HOBT (1-hydroxybenzotriazole), HOAT (hydroxyazabenzotriazole), HOSu (hydroxysuccinimide), HONB (endo-N-hydroxy-5-norbornene-2,3-dicarboxamide), etc., in amounts ranging from about 1.0 to about 10.0 equivalents.

[0061] The term "halo" refers to any radical of fluorine, chlorine, bromine or iodine.

[0062] The term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains, which may be straight or branched, containing the indicated number of carbon atoms (including, but not limited to, propyl, allyl, or propargyl), which may optionally be interrupted by N, O, or S. For example, C1-C 10 indicates that the group may have from 1 to 10 carbon atoms in it. The term "alkylene" refers to a divalent alkyl (ie, -R-).

[0063] The term "alkoxy" refers to an --O-alkyl group.

[0064] The term "alkylenedioxo" refers to a divalent species of the structure -ORO-, where R represents alkylene.

[0065] The term "aminoalkyl" refers to an alkyl substituted with an amino group.

[0066] The term "mercapto" refers to the group --SH.

[0067] The term "thioalkoxy" refers to an --S-alkyl group.

[0068] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system in which 0, 1, 2, 3, or 4 atoms of each ring may be substituted. Examples of aryl groups include phenyl and naphthyl.

[0069] The terms "arylalkyl" and "aralkyl" refer to an alkyl substituted with an aryl.

[0070] The term "arylalkoxy" refers to an alkoxy substituted with an aryl.

[0071] The term "cycloalkyl" as used herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons, e.g., 3 to 6 carbons, and the cycloalkyl groups can be further optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0072] The term "heteroaryl" refers to an aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms in the monocyclic ring, 1 to 6 heteroatoms in the bicyclic ring, or 1 to 9 heteroatoms in the tricyclic ring, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms in the monocyclic, bicyclic, or tricyclic ring, respectively), and 0, 1, 2, 3, or 4 atoms in each ring can be substituted by substituents. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, and thiazolyl.

[0073] The terms "heteroarylalkyl" and "heteroaralkyl" refer to an alkyl substituted with a heteroaryl.

[0074] The term "heteroarylalkoxy" refers to an alkoxy substituted with a heteroaryl.

[0075] The term "heterocyclyl" refers to a non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms in the monocyclic ring, 1 to 6 heteroatoms in the bicyclic ring, or 1 to 9 heteroatoms in the tricyclic ring, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms in the monocyclic, bicyclic, or tricyclic ring, respectively), and 0, 1, 2, or 3 atoms in each ring can be substituted by substituents. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl.

[0076] The term "oxo" refers to an oxygen atom which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0077] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted with one or more substituents.

[0078] The term "DMTr," unless otherwise specified, refers to 4,4'-dimethoxytrityl.

[0079] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a defined substituent, including, but not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent may be further substituted.

[0080] The term "monosaccharide" encompasses radicals of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fuculose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate, guloseglyceraldehyde, L-glycero-D-mannos-heptose, glycerol, glycerone, gulose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose. Monosaccharides can be in the D- or L-configuration. Monosaccharides can further be deoxysugars (an alcohol hydroxy group replaced with a hydrogen), aminosugars (an alcohol hydroxy group replaced with an amino group), thiosugars (an alcohol hydroxy group replaced with a thiol, or a C=O replaced with a C=S, or a ring oxygen in a cyclic form replaced with a sulfur), selenosugars, tellurosugars, azasugars (a ring carbon replaced with a nitrogen), iminosugars (a ring oxygen replaced with a nitrogen), phosphanosugars (a ring oxygen replaced with a phosphorus), phosphasugars (a ring carbon replaced with a phosphorus), C-substituted monosaccharides (a hydrogen at a non-terminal carbon atom replaced with a carbon), unsaturated monosaccharides, alditols (a carbonyl group replaced with a CHOH group), aldonic acids (an aldehyde group replaced with a carboxy group), ketoaldonic acids, uronic acids, aldaric acids, and the like. Amino sugars include amino monosaccharides, preferably galactosamine, glucosamine, mannosamine, fucosamine, quinovosamine, neuraminic acid, muramic acid, lactosediamine, acosamine, bacillosamine, daunosamine, desosamine, forosamine, galosamine, kanosamine, kansosamine, mycaminose, mycosamine, perosamine, pneumosamine, purpurosamine, and rhodosamine. It is understood that the monosaccharides may be further substituted.

[0081] The terms "disaccharide," "trisaccharide," and "polysaccharide" refer to abequase, acrabose, amicetose, amylopectin, amylose, apiose, alkanose, ascarylose, ascorbic acid, boivinose, cellobiose, cellotriose, cellulose, chacotriose, chalcose, chitin, colitose, cyclodextrin, cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, diginose, digitalose, digitoxose, evalose, evemitrose, fructooligosaccharides, galactooligosaccharides, gentianose, gentiobiose, glucan, glucogen, glycogen, hamamelose, heparin, inulin, isolevoglucosenone, isomaltose, isomaltotriose, isomalt ... Radicals include sopanose, kojibiose, lactose, lactosamine, lactosediamine, laminarabiose, levoglucosan, levoglucosenone, β-maltose, maltotriose, mannan-oligosaccharide, manninotriose, melezitose, melibiose, muramic acid, mycarose, mycinose, neuraminic acid, nigerose, nojirimycin, nobiose, oleandrose, panose, paratose, planteose, primeverose, raffinose, rhodinose, rutinose, sarmentose, sedoheptulose, sedoheptulosan, solatriose, sophorose, stachyose, streptose, sucrose, α,α-trehalose, trehalosamine, turanose, tyvelose, xylobiose, and umbelliferose. Furthermore, it is understood that "disaccharides," "trisaccharides," "polysaccharides," and the like, can be further substituted. Disaccharides also include amino sugars and their derivatives, particularly mycaminose derivatized at the C-4' position or 4-deoxy-3-amino-glucose derivatized at the C-6' position. Oligonucleotides

[0082] The oligonucleotide can be an siRNA, microRNA, anti-microRNA, microRNA mimic, anti-miR, antagomir, dsRNA, ssRNA, aptamer, immunostimulatory, decoy oligonucleotide, splicing altering oligonucleotide, triplex-forming oligonucleotide, G-quartet, or antisense. In one embodiment, the oligonucleotide is an iRNA agent.

[0083] In some embodiments, the oligonucleotides of the present invention contain one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA refers to an unlocked, non-cyclic nucleic acid, in which at least one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1'-C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) of the sugar has been removed (see Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference).

[0084] The term "iRNA agent" refers to an RNA agent (or capable of being cleaved into an RNA agent) that can downregulate the expression of a target gene (e.g., an siRNA), preferably an endogenous or pathogen target RNA. Without wishing to be bound by theory, an iRNA agent may act by one or more of several mechanisms, including post-transcriptional cleavage of the target mRNA (referred to in the art as RNAi), or a pre-transcriptional or pre-translational mechanism. An iRNA agent can comprise a single strand, or can comprise two or more strands; for example, an iRNA agent can be a double-stranded iRNA agent. If the iRNA agent is single-stranded, it can include a 5' modification including one or more phosphate groups or one or more analogs of a phosphate group. In a preferred embodiment, the iRNA agent is double-stranded.

[0085] An iRNA agent typically comprises a region sufficiently homologous to a target gene and has sufficient length in terms of nucleotides so that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. An iRNA agent is or comprises a region that is at least partially, and in some embodiments completely, complementary to the target RNA. While perfect complementarity between the iRNA agent and the target is not required, there is preferably sufficient correspondence so that the iRNA agent, or its cleavage product, can direct sequence-specific silencing, for example, by RNAi cleavage of the target RNA, e.g., mRNA.

[0086] Nucleotides in an iRNA agent can be modified (e.g., one or more nucleotides can contain a 2'-F or 2'-OCH3 group) or can be nucleotide surrogates. Single-stranded regions of an iRNA agent can be modified or contain nucleoside surrogates; for example, unpaired regions or regions of hairpin structures, such as regions connecting two complementary regions, can have modifications or nucleoside surrogates. For example, modifications that stabilize one or more 3' or 5' ends of an iRNA agent against exonucleases. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), phosphoramidites, and special biotin or fluorescein reagents that have additional DMT-protected hydroxyl groups, allowing for multiple conjugations during RNA synthesis. Modifications can also include, for example, the use of modifications at the 2'OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine in place of ribonucleotides, and modifications at the phosphate group, e.g., phosphorothioate modifications. In certain embodiments, different strands contain different modifications.

[0087] In certain embodiments, the strands are preferably selected so that the iRNA agent contains single-stranded or unpaired regions at one or both ends of the molecule. Double-stranded iRNA agents preferably have paired strands with overhangs, e.g., one or two 5' or 3' overhangs (preferably at least a 3' overhang of 2-3 nucleotides). Preferred iRNA agents have single-stranded overhangs, preferably 3' overhangs, of one or preferably two or three nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.

[0088] The preferred length of the double-stranded region between the strands of an iRNA agent is 6 to 30 nucleotides in length. Preferred double-stranded regions are 15 to 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length. Other preferred double-stranded regions are 6 to 20 nucleotides in length, most preferably 6, 7, 8, 9, 10, 11, and 12 nucleotides in length.

[0089] The oligonucleotides may be those described in U.S. Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, each of which is incorporated herein by reference.

[0090] " single-stranded siRNA compound " as used herein is the siRNA compound that is composed of a single molecule.It can comprise the double-stranded region that is formed by intrastrand pairing, for example, it can be or comprise the structure of hairpin or frying pan handle.Single-stranded siRNA compound can be antisense to target molecule.

[0091] Single-stranded siRNA compound can be long enough to enter RISC and participate in the cleavage of target mRNA via RISC.Single-stranded siRNA compound is at least 14 nucleotides long, and in other embodiments, at least 15, 20, 25, 29, 35, 40 or 50 nucleotides long.In certain embodiments, single-stranded siRNA compound is less than 200, 100 or 60 nucleotides long.

[0092] Hairpin siRNA compounds have a double-stranded region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs or more. The double-stranded region can be 200, 100, or 50 nucleotide pairs or less in length. In certain embodiments, the double-stranded region ranges from 15 to 30, 17 to 23, 19 to 23, or 19 to 21 nucleotide pairs in length. The hairpin can have a single-stranded overhang or terminal unpaired region. In certain embodiments, the overhang is 2 to 3 nucleotides in length. In some embodiments, the overhang is on the sense side of the hairpin, and in some embodiments, it is on the antisense side of the hairpin.

[0093] A "double-stranded siRNA compound" as used herein is an siRNA compound that contains two or more, and in some cases two, strands, where interstrand hybridization can form a region of double-stranded structure.

[0094] The antisense strand of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides or more in length. It can be 200, 100, or 50 nucleotides or less in length. The range can be 17 to 25, 19 to 23, or 19 to 21 nucleotides in length. As used herein, the term "antisense strand" refers to the strand of an siRNA compound that is sufficiently complementary to a target molecule, such as a target RNA.

[0095] The sense strand of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides or more in length. It can be 200, 100, or 50 nucleotides or less in length. Ranges can be 17-25, 19-23, and 19-21 nucleotides in length.

[0096] The double-stranded portion of a double-stranded siRNA compound can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs or more in length. It can be 200, 100, or 50 nucleotide pairs or less in length. Ranges can be 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0097] In many embodiments, the siRNA compound is large enough that it can be cleaved by endogenous molecules, eg, by Dicer, to produce smaller siRNA compounds, eg, siRNA agents.

[0098] The sense strand and the antisense strand can be selected so that the double-stranded siRNA compound contains a single-stranded or unpaired region at one or both ends of the molecule.Therefore, the double-stranded siRNA compound can contain a paired sense strand and an antisense strand that contain an overhang of 1 to 3 nucleotides, for example, one or two 5' or 3' overhangs, or a 3' overhang.The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being crossed one another.Some embodiments have at least one 3' overhang.In one embodiment, both ends of the siRNA molecule have a 3' overhang.In some embodiments, the overhang is 2 nucleotides.

[0099] In certain embodiments, the length of the double-stranded region is 15 to 30, or 18, 19, 20, 21, 22, and 23 nucleotides, for example, within the range of ssiRNA compounds described above. ssiRNA compounds may be similar in length and structure to the products processed from long dsiRNAs by natural Dicer. Also included are embodiments in which the two strands of the ssiRNA compound are linked, e.g., covalently linked. Hairpins or other single-stranded structures that provide the required double-stranded region and 3' overhang are also contemplated.

[0100] siRNA compounds described herein, including double-stranded siRNA compounds and single-stranded siRNA compounds, can mediate the silencing of target RNA, for example, mRNA, for example, the transcription product of the gene that codes for protein.For convenience, this mRNA is also referred to herein as the mRNA that is silenced.This gene is also referred to as target gene.Generally, the RNA that is silenced is endogenous gene or pathogen gene.In addition, RNA other than mRNA, for example, tRNA and viral RNA can also be targeted.

[0101] As used herein, the phrase " mediate RNAi " refers to the ability to silence target RNA in a sequence-specific manner.Without wishing to be bound by theory, it is believed that silencing is achieved by using the mechanism or process of RNAi and guide RNA, for example, the ssiRNA compound of 21-23 nucleotides.

[0102] In one embodiment, an siRNA compound is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the siRNA compound silences the production of a protein encoded by the target mRNA. In another embodiment, the siRNA compound is "exactly complementary" to the target RNA, e.g., the target RNA and the siRNA compound anneal to form a hybrid composed solely of Watson-Crick base pairs in the exact region of complementarity. A "sufficiently complementary" target RNA may include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to the target RNA. Furthermore, in certain embodiments, the siRNA compound specifically recognizes single nucleotide differences. In this case, the siRNA compound mediates RNAi only when exact complementarity is found in the region (e.g., within 7 nucleotides) of the single nucleotide difference.

[0103] microRNA MicroRNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals but are not translated into proteins. Processed miRNAs are single-stranded, approximately 17–25 nucleotide (nt) RNA molecules that are incorporated into the RNA-induced silencing complex (RISC) and recognized as master regulators of development, cell proliferation, apoptosis, and differentiation. They are thought to play a role in regulating gene expression by binding to the 3′-unsaturated regions of specific mRNAs. RISC mediates downregulation of gene expression through translational inhibition, transcript cleavage, or both. RISC is also involved in transcriptional silencing in the nuclei of a wide range of eukaryotic organisms.

[0104] The number of miRNA sequences identified to date is large and ever-increasing, illustrative examples of which can be found, for example, in "miRBase: microRNA sequences, targets and gene nomenclature" Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. NAR, 2006, 34, Database Issue, D140-D144; "The microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111; and further at http: / / microrna.sanger.ac.uk / sequences / .

[0105] antisense oligonucleotides In one embodiment, the nucleic acid is an antisense oligonucleotide directed against a target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" is intended to include oligonucleotides complementary to a targeted polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA, such as a target gene mRNA, that are complementary to a selected sequence. Antisense oligonucleotides are thought to inhibit gene expression by binding to complementary mRNA. Binding to a target mRNA can result in inhibition of gene expression by preventing translation of the complementary mRNA strand or by causing degradation of the target mRNA. Antisense DNA can be used to target specific, complementary (coding or non-coding) RNA. If binding occurs, the DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, antisense oligonucleotides contain about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, it is envisioned that non-target specific activity may be observed with the antisense, or that an antisense sequence containing one or more mismatches with the target sequence may be most preferable for a particular application.

[0106] Antisense oligonucleotides have been demonstrated to be effective target inhibitors of protein synthesis, and therefore can be used to specifically inhibit protein synthesis by targeting genes.The effectiveness of antisense oligonucleotides for inhibiting protein synthesis has been well established.For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (US Pat. No. 5,739,119 and US Pat. No. 5,759,829, each of which is incorporated by reference).In addition, examples of antisense inhibition include nuclear protein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A This has been demonstrated for the receptor and human EGF (Jaskulski et al., Science. 1988 Jun 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288, each of which is incorporated by reference). It has further been described that antisense constructs can be used to inhibit and treat various abnormal cell proliferations, such as cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317; and U.S. Pat. No. 5,783,683, each of which is incorporated by reference).

[0107] Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific for a given target sequence depends on the analysis of the selected target sequence as well as the secondary structure, T mThe selection of antisense oligonucleotides is based on the determination of their binding energy, binding energy, and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that may reduce or prevent specific binding to target mRNA in host cells. Highly preferred target regions of mRNA include the region at or near the AUG translation initiation codon and sequences that are substantially complementary to the 5' region of mRNA. Analysis of these secondary structures and selection of target sites can be performed, for example, using OLIGO primer analysis software v.4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0108] Antagomir Antagomirs are RNA-like oligonucleotides with various modifications for pharmacological properties such as RNAse protection and improved tissue and cellular uptake.They differ from normal RNAs, for example, by sugars, phosphorothioate backbones, and, for example, complete 2'-O-methylation of cholesterol moieties at the 3' end.Antagomirs can be used to efficiently silence endogenous miRNAs by forming a duplex containing antagomirs and endogenous miRNAs, thereby preventing miRNA-induced gene silencing.An example of antagomirs-mediated miRNA silencing is the silencing of miR-122 described in Krutzfeldt et al., Nature, 2005, 438:685-689, the entire contents of which are expressly incorporated herein by reference.Antagomirs RNAs can be synthesized using standard solid-phase oligonucleotide synthesis protocols. See US Patent Application Publication Nos. 2007 / 0123482 and 2007 / 0213292, each of which is incorporated herein by reference.

[0109] Antagomirs can include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis.Exemplary monomers are described in U.S. Patent Application Publication No. 2005 / 0107325, which is incorporated by reference in its entirety.Antagomirs can have a ZXY structure as described in WO 2004 / 080406, which is incorporated by reference in its entirety.Antagomirs can form complexes with amphiphilic moieties.Exemplary amphiphilic moieties for use with oligonucleotide agents are described in WO 2004 / 080406, which is incorporated by reference in its entirety.

[0110] Aptamers Aptamers are nucleic acid or peptide molecules that bind to a particular molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990), each of which is incorporated by reference in its entirety). DNA or RNA aptamers have been successfully produced that bind to many different entities, from large proteins to small organic molecules. See Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997); Famulok, Curr. Opin. Struct. Biol. 9:324-9 (1999); and Hermann and Patel, Science 287:820-5 (2000), each of which is incorporated by reference in its entirety. Aptamers may be RNA- or DNA-based and may include riboswitches. A riboswitch is a part of an mRNA molecule that can directly bind to a small target molecule, and target binding affects the activity of a gene. Thus, the mRNA containing the riboswitch is directly involved in regulating its own activity depending on the presence or absence of the target molecule. Generally, aptamers are engineered by repeated in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) for binding to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers may be prepared by any known method, including synthetic, recombinant, and purification methods, and can be used alone or in combination with other aptamers specific to the same target. Furthermore, as described in more detail herein, the term "aptamer" specifically includes "secondary aptamers," which contain consensus sequences derived from comparing two or more known aptamers with a given target.

[0111] Ribozymes In another embodiment, the nucleic acid-lipid particle is conjugated to a ribozyme, which is an RNA molecular complex containing a specific catalytic domain with endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20). For example, many ribozymes accelerate phosphoester transfer reactions, often with a high degree of specificity, cleaving only one of several phosphates in an oligonucleotide substrate (Cech et al., Cell. 1981 Dec;27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 Dec 5;216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14;357(6374):173-6). This specificity has been attributed to the requirement that the substrate bind, via base-pairing interactions, to the ribozyme's internal guide sequence ("IGS") prior to chemical reaction.

[0112] At least six basic species of naturally occurring enzymatic RNAs are currently known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans under physiological conditions (and thus can cleave other RNA molecules). Generally, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs via the target-binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that functions to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA through complementary base-pairing, and, once bound to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA would impair its ability to direct synthesis of the encoded protein. After binding and cleaving its RNA target, the enzymatic nucleic acid is released from that RNA to search for another target, where it can repeatedly bind and cleave new targets.

[0113] Enzymatic nucleic acid molecules can be formed, for example, with hammerhead, hairpin, hepatitis delta virus, group I intron, or RNase P RNA (linked with an RNA guide sequence), or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described by Rossi et al., Nucleic Acids Res. 1992 Sep 11;20(17):4559-65. Examples of hairpin motifs are described by Hampel et al. (European Patent Application Publication No. EP0360257), Hampel and Tritz, Biochemistry 1989 Jun 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan 25;18(2):299-304, and U.S. Patent No. 5,631,359. An example of a hepatitis delta virus motif is described by Perrotta and Been, Biochemistry. 1992 Dec 1;31(47):11843-52; an example of an RNase P motif is described by Guerrier-Takada et al., Cell. 1983 Dec;35(3 Pt 2):849-57; a Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991 Oct 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar 23;32(11):2795-9); and an example of a group I intron is described in U.S. Pat. No. 4,987,071. The important characteristics of the enzymatic nucleic acid molecules used are that they have a specific substrate binding site complementary to one or more regions of the target gene DNA or RNA, and that they have nucleotide sequences within or surrounding the substrate binding site that confer RNA cleavage activity to the molecule. Thus, ribozyme constructs need not be limited to the particular motifs described herein.

[0114] Methods for producing ribozymes targeted to any polynucleotide sequence are known in the art. Ribozymes can be designed as described in International Patent Application Publications WO 93 / 23569 and WO 94 / 02595, each of which is specifically incorporated herein by reference, and synthesized for in vitro and in vivo testing as described therein.

[0115] Ribozyme activity can be optimized by altering the length of the ribozyme binding arms or by chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see, e.g., International Patent Applications WO 92 / 07065, WO 93 / 15187, and WO 91 / 03162; European Patent Application WO 92110298.4; U.S. Pat. No. 5,334,711; and International Patent Application WO 94 / 13688, which describe various chemical modifications that can be made to the sugar portion of enzymatic RNA molecules), modifications that increase their effectiveness in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

[0116] Immunostimulatory Oligonucleotides When administered to a subject, which may be a mammal or other patient, the nucleic acid that is combined with lipid particles can be immunostimulatory, and contains immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that can induce an immune response.ISS can include, for example, a specific palindrome that results in a hairpin secondary structure (see Yamamoto S., et al. (1992) J.Immunol.148:4072-4076, which is incorporated by reference in its entirety), or a CpG motif, as well as other known ISS features (such as multiple G domains, see International Publication No. 96 / 11266, which is incorporated by reference in its entirety).

[0117] The immune response can be an innate or adaptive immune response. The immune system is further divided into an innate immune system in vertebrates, and an adaptive immune system, the latter of which is further divided into humoral and cellular components. In certain embodiments, the immune response can be mucosal.

[0118] In certain embodiments, the immunostimulatory nucleic acid is immunostimulatory only when administered in combination with a lipid particle, and is not immunostimulatory when administered in its "free form." Such oligonucleotides are considered to be immunostimulatory.

[0119] Immunostimulatory nucleic acid is considered to be non-sequence specific if it does not need to specifically bind to target polynucleotide and reduce the expression of target polynucleotide to induce immune response.Thus, certain immunostimulatory nucleic acid may comprise the sequence corresponding to the region of natural gene or mRNA, but still be considered to be non-sequence specific immunostimulatory nucleic acid.

[0120] In one embodiment, the immune stimulatory nucleic acid or oligonucleotide contains at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immune stimulatory nucleic acid contains at least one CpG dinucleotide with a methylated cytosine. In one embodiment, the nucleic acid contains a single CpG dinucleotide, wherein the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid contains at least two CpG dinucleotides, wherein at least one cytosine in the CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid contains multiple CpG dinucleotides, wherein at least one of the CpG dinucleotides contains a methylated cytosine.

[0121] Linker The linker can be any suitable group for connecting the oligonucleotide to the ligand.Other examples of linkers are described in International Publication No. 2009 / 082607 and U.S. Patent Application Publication No. 2009 / 0239814, U.S. Patent Application Publication No. 2012 / 0136042, U.S. Patent Application Publication No. 2013 / 0158824, or U.S. Patent Application Publication No. 2009 / 0247608, each of which is incorporated herein by reference.

[0122] Attachment point of oligonucleotide to linker The oligonucleotide can be linked to the linker via any suitable group for linking the two.The group can be cleavable or non-cleavable.Examples of linkers and suitable linking groups are described herein.Other examples of linking groups are described in International Publication No. 2009 / 082607 and US Patent Application Publication No. 2009 / 0239814, US Patent Application Publication No. 2012 / 0136042, US Patent Application Publication No. 2013 / 0158824 or US Patent Application Publication No. 2009 / 0247608, each of which is incorporated herein by reference.Suitable linking groups include, for example, NR 8, C(O), C(O)NH, SO, SO2, SO2NH or including but not limited to alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhetero and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, and the like, each of which may be substituted or unsubstituted, and wherein one or more methylenes are selected from the group consisting of O, S, S(O), SO, N(R 8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic, wherein R 8 is hydrogen, acyl, aliphatic or substituted aliphatic.

[0123] A cleavable group is one that is sufficiently stable outside a cell but is cleaved upon entry into a target cell to release the two moieties to which it is attached. In preferred embodiments, the cleavable group is cleaved at least 10 times faster, and preferably at least 100 times faster, in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0124] Cleavable groups are susceptible to the influence of cleavage factors, such as pH, redox potential, or the presence of decomposition molecules.Generally, cleavage factors are found at higher levels or activities, more widely, or more abundantly in cells than in serum or blood.Examples of such decomposition factors include: oxidizing or reducing enzymes or reducing agents present in cells, such as mercaptans, which can decompose redox-cleavable groups by reduction, and the redox agents that are selected for specific substrates or do not have substrate specificity; esterase; agents that can generate endosomes or acidic environments, such as agents that generate a pH of 5 or less; enzymes that can hydrolyze or decompose acid-cleavable groups by acting as general acids, peptidases (can be substrate specific), and phosphatases.

[0125] Cleavable groups, such as disulfide bonds, can be sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand intracellularly or into a desired compartment of the cell.

[0126] Conjugate can comprise a cleavable group that can be cleaved by specific enzyme.The type of cleavable group incorporated into conjugate depends on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid through chemical moiety that comprises ester group.Hepatocytes are rich in esterase, therefore, the group is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0127] Linking groups containing peptide bonds may be used when targeting cell types rich in peptidases, such as hepatocytes and synovial cells.

[0128] In general, the suitability of a candidate cleavable group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate group. It may also be desirable to test the candidate cleavable group for its ability to resist cleavage in blood or when in contact with other non-target tissues. In this way, the relative susceptibility to cleavage between a first condition and a second condition can be determined, where the first condition is selected to indicate cleavage within target cells, and the second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluations in a cell-free or cultured condition and confirm with further evaluations in a whole animal. In a preferred embodiment, a useful candidate compound is cleaved at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0129] i. Redox-cleavable groups One type of cleavable group is a redox-cleavable group, which is cleaved by reduction or oxidation. An example of a reductively cleavable group is a disulfide linkage (-SS-). To determine whether a candidate cleavable group is a suitable "reductively cleavable linkage" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can turn to the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved 10% or less in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0130] ii. Phosphate-based cleavable groups Phosphate-based cleavable groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. An example of a phosphate-based linking group is -OP(O)(OR k )-O-, -OP(S)(OR k )-O-, -OP(S)(SR k )-O-, -SP(O)(OR k )-O-, -OP(O)(OR k )-S-, -SP(O)(OR k )-S-, -OP(S)(OR k )-S-, -SP(S)(OR k )-O-, -OP(O)(R k )-O-, -OP(S)(Rk )-O-, -SP(O)(R k )-O-, -SP(S)(R k )-O-, -SP(O)(R k )-S-, -OP(S)(R k )-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0131] iii. Acid-cleavable groups Acid-cleavable groups are linking groups that are cleaved under acidic conditions. In a preferred embodiment, acid-cleavable groups are cleaved in an acidic environment with a pH of about 6.5 or less (for example, about 6.0, 5.5, 5.0, or less), or by agents such as enzymes that can act as general acids. Intracellularly, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can be represented by the general formula -C=NN- 、 It may be represented as -C(O)O, or -OC(O). A preferred embodiment is where the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0132] iv. Ester group Ester-based cleavable groups are cleaved by enzymes such as intracellular esterases and amidases. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups are represented by the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0133] v. Peptide-based cleavage groups Peptide-based cleavable groups are cleaved by enzymes such as intracellular peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula -NHCHR A C(O)NHCHR B C(O)—, wherein R A and R Bare the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above. As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate, composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom attached to each carbon atom; or a compound that has a carbohydrate moiety composed of one or more monosaccharides (which may be linear, branched, or cyclic), each having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of C5 or higher (preferably C5 to C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5 to C8).

[0134] Ligand The ligand can be any of the ligands described herein. Other suitable ligands are described in U.S. Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, each of which is incorporated herein by reference.

[0135] formulation Conjugate described herein can be formulated for administration to subject.For ease of explanation, the formulation, composition and method in this section are mainly described with respect to the conjugate of unmodified iRNA agent.However, it will be understood that these formulations, compositions and methods can be carried out with other oligonucleotides, for example, the conjugate of modified iRNA agent, and such implementation is within the scope of the present invention.

[0136] The formulated iRNA conjugate can be in a variety of states. In some instances, the conjugate is at least partially crystalline, homogeneously crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In other instances, the iRNA conjugate is in an aqueous phase, e.g., in a solution containing water.

[0137] Aqueous phase or crystalline conjugates can be incorporated, for example, into delivery vehicles, such as liposomes (particularly in the case of aqueous phase) or particles (e.g., microparticles, as may be appropriate for crystalline compositions). Generally, iRNA conjugates are formulated in a manner compatible with the intended method of administration. iRNA conjugates can be incorporated into nucleic acid-lipid nanoparticles. In one embodiment, each nanoparticle contains a conjugate, a cationic lipid (e.g., a pK ranging from about 4 to about 11, such as about 5 to about 7), and / or a soluble lipid (e.g., a pK ranging from about 4 to about 11, such as about 5 to about 7). a a cationic lipid having the formula (I), a non-cationic lipid (such as a neutral lipid), an aggregation-reducing agent (such as polyethylene glycol (PEG) or a PEG-modified lipid), and optionally a sterol (e.g., cholesterol).

[0138] In certain embodiments, the composition is prepared by at least one of the following methods: spray drying, freeze drying, vacuum drying, evaporation, fluidized bed drying, or a combination of these techniques; or sonication with lipids, freeze drying, condensation, and other self-assembly.

[0139] The iRNA conjugates can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes the iRNA, e.g., a protein that complexes with the iRNA to form an iRNP. Still other agents include chelating agents, e.g., EDTA (e.g., Mg 2+ (for removing divalent cations such as HCl), salts, RNAse inhibitors (e.g., broad specificity RNAse inhibitors such as RNAsin), etc.

[0140] In one embodiment, the iRNA composition includes at least one second therapeutic agent (e.g., an agent other than RNA or DNA). For example, an iRNA composition for treating a viral disease, such as HIV, may include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, an iRNA composition for treating cancer may further include a chemotherapeutic agent.

[0141] The iRNA conjugate may be formulated with an agent that affects (e.g., increases) the uptake of the iRNA agent into the cell. The agent may be administered before, after, or simultaneously with the iRNA agent. The agent may also be covalently attached to the iRNA agent. The agent may be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The agent may have a transient effect on the cell.

[0142] In one embodiment, the agent used increases the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The agent can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0143] Agents can also increase the uptake of iRNA conjugates into cells, for example, by activating an inflammatory response. Exemplary agents that can have such an effect include tumor necrosis factor alpha (TNF-α), interleukin-1β, or gamma interferon.

[0144] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. [Example]

[0145] Abbreviations: TBAHS is tetrabutylammonium hydrogen sulfate; DCM is dichloromethane; NHS is N-hydroxysuccinamide; DIEA is N,N-diisopropylethylamine; EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0146] Example 1: Synthesis of a triple-stranded GalNAc monomer with acyclic linker-1 Tri-stranded GalNAc moieties 211a-d are synthesized as shown in Scheme 1 below. [ka]

[0147] i. Synthesis of (S)-1-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol 201 To a stirred solution of commercially available (R)-methyl 2-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-hydroxyacetate 200 (1.9 g) in methanol (60 mL) is added solid sodium borohydride (0.4 g) at 0-5 °C. After the addition, the reaction mixture is warmed to room temperature and stirred for 20 min. The reaction mixture is diluted with saturated ammonium chloride and extracted with dichloromethane. The organic layer is dried over NaSO and concentrated to isolate the crude product (1.6 g), which is used directly in the next step without further purification.

[0148] ii. Synthesis of (S)-2-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-hydroxyethyl methanesulfonate 202 To a solution of alcohol 201 (1.6 g) in dichloromethane (60 mL) was added triethylamine (1.5 mL), and the mixture was cooled with stirring. To this stirred solution was added methanesulfonyl chloride (1.3 g) in dichloromethane (30 mL) dropwise, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then washed with saturated NaHCO3 solution (100 mL), followed by brine (100 mL), and the organic layer was dried over anhydrous Na2SO4 and then concentrated. The crude product thus obtained was purified using silica gel to give the pure product.

[0149] iii. General procedure for the synthesis of 203a-d In a typical procedure, nucleobase (2 equiv.) is dissolved in anhydrous DMF (100 mL) at 110 °C. To this stirred solution, solid CsCO (2 g) and sodium iodide (2 g) are added, and the mixture is vigorously stirred. To this stirred solution, a solution of mesylate 202 (1 equiv.) in anhydrous DMF (10 mL) is added dropwise. The reaction mixture is stirred at 110 °C for an additional 30 min before being concentrated under reduced pressure. The residue is dissolved in ethyl acetate, and the organic layer is washed with saturated NaHCO (100 mL), brine (100 mL), and dried (anhydrous NaSO). Concentration of the organic layer affords the crude product, which is purified by flash column chromatography to isolate pure products 203a–d.

[0150] iv. Synthesis of 204a-d Alcohols 203a–d were first treated with disuccinimidyl carbonate (DSC) in the presence of triethylamine to give succinimidyl esters, which were then treated with monophthalimide-protected hexanediamine in the presence of pyridine to give the amine-substituted products 204a–d after column purification.

[0151] v. Synthesis of 205a~d The acetonide protection in 204a-d was removed by treatment with acetic acid under the conditions reported to give 205a-d, which was used directly in the next step.

[0152] Synthesis of vi.206a~d Treatment of diols 205a-d with DMTrCl in pyridine would provide the mono-DMT protected alcohols 206a-d.

[0153] vii. Synthesis of 207a-d The phthalimide-protected amines 206a-d are treated with a solution of methylamine in methanol (20x) at room temperature overnight. Concentration of the reaction mixture followed by column purification affords the deprotected amines 207a-d.

[0154] viii. Synthesis of 208a-d Coupling of single-stranded GalNAc-containing carboxylic acids with amines 207a-d using EDC and Hunig's base can yield ligand-conjugated monomers 208a-d.

[0155] Synthesis of ix.209a~d Using a similar coupling procedure, attachment of a triple-armed GalNAc-containing carboxylic acid with amines 207a-d can give triple-armed GalNAc-conjugated monomers 209a-d.

[0156] x. Synthesis of 210a-d and 211a-d Phosphorylation of alcohols 208a–d and 209a–d with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of Hunig's base in CH2Cl2 gave the corresponding amidites 210a–d and 211a–d after flash column purification.

[0157] Example 2: Synthesis of a triple-stranded GalNAc monomer using acyclic linker-2 The triple-stranded GalNAc moiety 219 is synthesized as shown in Scheme 2 below. [ka]

[0158] Example 3: Synthesis of a triple-stranded GalNAc monomer using acyclic linker-3 The triple-stranded GalNAc moiety 215 is synthesized as shown in Scheme 3 below. [ka]

[0159] Synthesis of i.222 Commercially available (R)-glycidol was converted to the ODMTr-protected epoxide 220 as reported in the literature. Treatment of epoxide 220 (1.12 g, 3 mmol) with 1,6-dimethylaminohexane 221 (6 mmol, 2 equiv.) at 110 °C under microwave irradiation for 30 min afforded the epoxide ring-opened product 222 in 90% yield.

[0160] Synthesis of ii.224 Coupling of amine 222 with carboxylic acid 223 gave the coupled product 224 in good yield.

[0161] iii. Synthesis of 225 Phosphorylation of alcohol 224 with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of Hunig's base in CH2Cl2 gave the corresponding amidite 225 after flash column purification.

[0162] Example 4: General structure of a three-chain α-anomer-conjugate building block The triple-chain α-anomer represented by the formula: [ka]

[0163] For example, R, R', and R" may be independently C6 to C6 10 Aryl, C6-C 10 Heteroaromatic, C1-C 20 It can be an alkyl, or a sugar (eg, galactose or GalNAc).

[0164] The triple-stranded α-anomer represented by the formula: [ka]

[0165] For example, R, R', and R" may be independently C6 to C6 10 Aryl, C6-C 10 Heteroaromatic, C1-C 20 It can be an alkyl, or a sugar (eg, galactose or GalNAc).

[0166] Example 5: General structure of a three-stranded β-anomer-conjugate building block The triple-stranded β-anomer represented by the formula: [ka]

[0167] For example, R, R', and R" may be independently C6 to C6 10 Aryl, C6-C 10 Heteroaromatic, C1-C 20 It can be an alkyl, or a sugar (eg, galactose or GalNAc).

[0168] Example 6: General structure of a two-stranded α-anomeric conjugate building block A double-chain α-anomer represented by the formula: [ka]

[0169] For example, R, R', and R" may be independently C6 to C6 10 Aryl, C6-C 10 Heteroaromatic, C1-C 20 It can be an alkyl, or a sugar (eg, galactose or GalNAc).

[0170] Example 7: Synthesis of mono-, bi-, and tri-conjugate building blocks Mono-, bi-, and tri-conjugate building blocks can be prepared from azide intermediates as shown below. [ka]

[0171] Example 8: Synthesis of ASGPR Ligand Intermediates (Schemes 4-8) Intermediates useful for preparing ASGPR ligands can be synthesized as shown in Schemes 4-8 below.

[0172] [ka]

[0173] Synthesis of Compound 9: N-acetylglucosamine (50 g, 226 mmol) was taken up in allyl alcohol and heated at 90°C for 24 hours. The reaction mixture was cooled to room temperature, and the allyl alcohol was removed by distillation. The residue was dissolved in pyridine and reacted with pivaloyl chloride to give compound 3. The pivaloyl ester was reacted with trifluoromethanesulfonic anhydride in pyridine for 2 hours, and the mixture was quenched by adding water. The mixture was heated at 90°C for 24 hours to give product 4. The ester group was removed by treatment with sodium methoxide, and compound 5 was isolated. The hydroxyl group was protected with dimethoxypropane to give product 6, which was then reacted with tosyl chloride and sodium azide in DMF to give compound 7. Compound 7 was subjected to click cycloaddition and epoxidation with MCPBA to give compound 9.

[0174] [ka]

[0175] Synthesis of Compound 13: Reaction of epoxide 13 with ammonia in the presence of LiClO4 yields amino alcohol 10, which is coupled with N-Cbz aminohexanoic acid under peptide coupling conditions to give compound 11. Removal of the acetonide protection and benzoylation of the hydroxyl group yields compound 12. Hydrogenation of compound 12 with Pd / C in MeOH yields amine 13 as the TFA salt.

[0176] [ka]

[0177] Synthesis of compound 17: Amine 13 was reacted with tricarboxylic acid 14 under peptide coupling conditions to give compound 15. Hydrogenation gave compound 16, which was then reacted with monobenzyldodecanedioic acid to give the protected triple-stranded intermediate 17.

[0178] [ka]

[0179] Synthesis of hydroxyproline intermediate 20: Compound 17 was hydrogenated under balloon pressure to give carboxylic acid 18, which was then reacted with amine 19 under peptide coupling conditions to produce compound 20.

[0180] [ka]

[0181] Synthesis of solid support 21: Hydroxyproline 20 was reacted with succinic anhydride and DMAP to produce the succinate derivative, which was loaded onto the solid support using peptide coupling conditions to give support 21.

[0182] Example 9: Synthesis of triple-stranded β-anomer-conjugate building blocks (Schemes 9-10)

[0183] [ka]

[0184] Synthesis of carboxylic acid 29: Compound 22 was reacted with alcohol 23 in the presence of TMSOTf in DCE to give carboxylate 24. Removal of the acetate group with TEA in MeOH gave compound 25. Introduction of DMTr at the O-6 position and benzoylation of the hydroxyl group gave compound 26. Removal of the DMTr group under acidic conditions and reaction with Ms-Cl gave the mesyl derivative. Reaction of this mesyl derivative with sodium azide gave the C-6 azido derivative 27. Click reaction and deprotection of the methyl ester gave carboxylic acid 29.

[0185] [ka]

[0186] Synthesis of solid support 32: Carboxylic acid 29 was reacted with a triple-stranded amine under peptide coupling conditions to produce triple-stranded derivative 31, which was hydrogenated and the carboxylic acid reacted with hydroxyproline. This intermediate was reacted with succinic anhydride, and the succinate was loaded onto the solid support to give compound 32.

[0187] Example 10: Synthesis of double-stranded α-anomer-conjugate building blocks (Schemes 11-14)

[0188] [ka]

[0189] Synthesis of carboxylic acid 36: Hydroxyproline derivative 33 was reacted with monobenzylhexanedioic acid using HBTU / DIEA to produce carboxylate 35, which was further hydrogenated to give carboxylic acid 36.

[0190] [ka]

[0191] Synthesis of double-stranded solid support 41: N-Boc glutamic acid was reacted with amine 13 using HBTU / DIEA to give compound 38. Deprotection of the Boc protecting group and reaction of this amine with carboxylic acid 36 gave the hydroxyproline derivative 39. Removal of TBDMS and reaction of the hydroxyl group with succinic anhydride produced the succinate derivative, which was loaded onto the solid support to give double-stranded solid support 41.

[0192] Similarly, compound 54 can be prepared as shown in Schemes 13 and 14 below.

[0193] [ka]

[0194] [ka]

[0195] Example 11: Synthesis of triple-chain α-anomeric building blocks The triple-chain α-anomeric building block 55 can be prepared according to Scheme 15 below. [ka]

[0196] Example 12: Synthesis of triple-stranded α-anomeric siRNA conjugates: Using the conjugate building block 21 described above (see Scheme 8), RNA is synthesized according to known procedures with a ligand attached to the 3' end of the sense strand. This is annealed with the antisense strand. The product is shown below. [ka]

[0197] Example 13: Synthesis of triple-stranded β-anomeric siRNA conjugates: Using the conjugate building block 32 described above (see Scheme 10), RNA is synthesized according to known procedures with a ligand attached to the 3' end of the sense strand. This is annealed with the antisense strand. The product is shown below. [ka]

[0198] Example 14: Synthesis of double-stranded α-anomeric siRNA conjugates: Using the conjugate building block 41 described above (see Scheme 12), RNA is synthesized according to known procedures with a ligand attached to the 3' end of the sense strand, which is then annealed with the antisense strand. [ka]

[0199] Example 15: Synthesis of mono-GalNAc building blocks for oligonucleotide conjugation Mono-GalNAc building blocks 104 and 105 are prepared as shown in Scheme 16. [ka]

[0200] Synthesis of 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyprolinamine (10.00 g, 18.77 mmol) were combined in dichloromethane. HBTU (10.68 g, 28.12 mmol) and DIEA (9.80 mL, 3 equiv.) were added, and the mixture was stirred at ambient temperature for 2 h. The product was checked by TLC, and the reaction mixture was transferred to a separatory funnel and washed with water and brine. The organic layer was dried over sodium sulfate, and the solvent was removed. The crude product was purified by silica gel chromatography using dichloromethane and MeOH as solvents to give compound 102 as a pale yellow fluffy solid (11.77 g, 63%). 1 H NMR (400MHz, DMSO) δ7.80(d,J=9.2Hz, 1H), 7.69(t,J=5.6Hz, 1H), 7.39-7.09(m,9H), 6.86(ddd,J=9.0, 5.4, 2.1Hz, 4H), 5.20(d,J=3.4Hz, 1H), 5.03-4.83(m,2H), 4.47(d,J=8.5Hz, 1H), 4.41-4.07(m,2H), 4.04-3.95(m ,3H), 3.86(dt,J=11.2, 8.9Hz, 1H), 3.79-3.68(m,6H), 3.68-3.36(m,3H), 3.21-2.88(m,5H), 2.26-2.14(m, 2H), 2.09(s,3H), 2.02(t,J=6.7Hz, 2H), 1.98(s,3H), 1.87(d,J=7.5Hz, 3H), 1.76(s,3H), 1.53-1.29(m,7H).

[0201] Synthesis of 104: Hydroxyproline derivative 102 (6.00 g, 6.24 mmol) was dissolved in dichloromethane (100 mL). DIEA (2.20 mL, 3 equiv.) and chloroamidite reagent were added. The reaction mixture was stirred for 30 min and checked by TLC. It was transferred to a separatory funnel and washed with water and sodium bicarbonate solution. The organic layer was dried over sodium sulfate, and the crude product was purified by silica gel chromatography using dichloromethane and MeOH as eluents to give the compound as a white fluffy solid. 1H NMR (400MHz, DMSO) δ7.80(d,J=9.2Hz, 1H), 7.68(s,1H), 7.42-7.06(m,8H), 7.01-6.73(m,4H), 5.20(d,J=3.3Hz, 1H), 4 .96(dd,J=11.2, 3.3Hz, 1H), 4.63(d,J=4.7Hz, 1H), 4.47(d,J=8.5Hz, 1H), 4.15(s,1H), 4.01(s,3H), 3.86(d,J=11.0Hz, 1H), 3.70(d,J=16.5Hz, 9H), 3.45(ddd,J=37.0, 23.3, 16.4Hz, 6H), 2.99(dd,J=12.3, 6.4Hz, 3H), 2.74(dd,J=9.2, 5.8H z, 2H), 2.21(s,2H), 2.09(s,3H), 2.05-1.95(m,5H), 1.88(s,3H), 1.76(s,3H), 1.52-1.16(m,11H), 1.16-1.02(m,11H). 31 P NMR δ=151.78, 151.61, 151.50, 151.30.

[0202] Synthesis of 105: Compound 102 (2.10 g, 2.18 mmol) was dissolved in DCM (20 mL). To this mixture, succinic anhydride (0.441 g, 4.36 mmol) and DMAP (0.532 g, equiv.) were added, followed by TEA (1 mL). The reaction mixture was stirred overnight at room temperature. The TLC checked the reaction mixture, and the reaction mixture was washed with water and brine. The organic layer was dried over sodium sulfate, and the crude product was filtered through a small pad of silica gel. The solvent was removed, and this material was used in the next reaction. The succinate from the above reaction was dissolved in anhydrous acetonitrile. HBTU (1.59 g, 4.20 mmol) and DIEA (1.10 mL) were added, and the mixture was rotated for 5 minutes. A polystyrene solid support was added to the reaction mixture, and the mixture was shaken at ambient temperature overnight. The solid support was filtered, washed, and capped with an acetic anhydride / Py mixture. The solid support was washed again with dichloromethane, MeOH / DCM and ether (27.10 g, 55 umol / g).

[0203] Example 16: Synthesis of single-stranded alpha anomeric siRNA: Using the conjugate building blocks 104 and 105 described above (see Scheme 16), RNA is synthesized according to known procedures with a ligand attached to the 3' end of the sense strand. This is annealed with the antisense strand. The product is shown below. [ka]

[0204] Example 17: Synthesis of trifluoroacetamide derivatives for post-synthetic conjugation Intermediate compound 8 is prepared as shown in Scheme 17 below. [ka]

[0205] Synthesis of Compound 2: Z-aminocaproic acid (22.2 g, 82.50 mmol) was dissolved in DMF (250 mL) and cooled to 0 °C. To the solution, diisopropylethylamine (44.4 mL, 275 mmol), HBTU (40.4 g, 106.7 mmol), and HOBT (30.0 g, 220 mmol) were added. After stirring at 0 °C for 20 minutes under argon, 4-hydroxy-l-proline methyl ester hydrochloride (20.0 g, 110 mmol) was added, and stirring was continued at room temperature overnight under argon. The reaction mixture was evaporated to dryness. To the residue, ethyl acetate (250 mL) was added. The organic layer was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude Compound 2 (Rf = 0.5 in 10% MeOH / DCM, 24.30 g) was obtained. To remove impurities, compound 2 was purified by column chromatography by eluting first with 2% methanol / dichloromethane followed by 5% methanol / dichloromethane to give 21.36 g (65%). 1H NMR (400 MHz, DMSO-d): Rotamers observed due to amide bond in the ring. δ 7.35 (m, 5H), 5.15 (d, OH, DO interchangeable), 4.99 (s, 2H), 4.27 (m, 1H), 3.97 (m, 1H), 3.58 (s, 1H), 3.20-3.47 (m, 5H), 2.94-3.02 (m, 2H), 2.10-2.32 (m, 2H), 1.74-2.01 (m, 2H), 1.35-1.4 (m, 4H), 1.22-1.28 (m, 4H).

[0206] Synthesis of Compound 3: Compound 2 (21.36 g, 54.43 mmol) was dissolved in THF (200 mL). The reaction mixture was stirred at 0° C. for 20 minutes under argon. Lithium borohydride (1.19 g, 54.43 mmol) was then added to the solution at 0° C. for 20 minutes, and stirring was continued at room temperature overnight under argon. The reaction mixture was cooled to 0° C. Excess lithium borohydride was quenched with 5 M NaOH (30 mL). After stirring for 30 minutes, the reaction mixture was evaporated to dryness. Dichloromethane (200 mL) was added to the residue. The organic layer was washed with water and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude compound 3 (Rf=0.4 in 10% MeOH / DCM, 35.88 g) was obtained. To remove impurities, compound 3 was purified by column chromatography by eluting with 3% methanol / dichloromethane followed by 5% methanol / dichloromethane to give 9.21 g (49%). 1 H NMR (400 MHz, DMSO-d): Rotamers observed due to amide bond in the ring. δ 7.35 (m, 5H), 4.99 (s, H), 4.91 (d, OH, DO exchangeable), 4.77 (t, OH, DO exchangeable), 4.27 (m, 1H), 3.97 (m, 1H), 3.20-3.47 (m, 5H), 2.94-3.02 (m, 2H), 2.10-2.32 (m, 2H), 1.74-2.01 (m, 2H), 1.35-1.4 (m, 4H), 1.22-1.28 (m, 4H). 13C NMR (100 mMHz, DMSO-d): δ 171.4, 171.1 (small due to rotamers), 156.1, 137.3, 128.9, 128.3, 128.2, 127.7, 125.3, 68.2, 67.4, 65.1, 63.4, 62.0, 57.6, 55.1, 54.9, 53.3, 40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 37.4, 36.1, 34.2, 32.6, 29.3, 26.1, 26.0, 24.6, 24.1, 21.0.

[0207] Synthesis of Compound 4: Compound 3 (9.21 g, 25.27 mmol) was coevaporated twice with anhydrous pyridine (80 mL). The compound was then placed under high vacuum overnight to dry. Compound 3 was removed from the high vacuum and dissolved in anhydrous pyridine (200 mL). A catalytic amount of dimethylaminopyridine (0.35 g, 2.53 mmol) was added to this solution. The reaction mixture was stirred at 0° C. for 30 minutes under argon. DMT-Cl (9.0 g, 26.53 mmol) was then added to the solution at 0° C. The mixture was stirred under reduced pressure, then under argon, and continued to stir at room temperature overnight under argon. Excess DMT-Cl was quenched by the addition of methanol (15 mL). The reaction mixture was evaporated to dryness, and dichloromethane (200 mL) was added to the residue. The organic layer was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 4 (Rf=0.6 in 100% EtOAc, 14.02 g). To remove impurities, compound 4 was purified by column chromatography by first eluting with 50% ethyl acetate (1% TEA) in hexane, followed by 100% ethyl acetate (1% TEA), to give 12.36 g (73.4%) of a white foamy solid. 1H NMR (400MHz, DMSO-d6): δ7.17-7.33(m,14H), 4.99(s,2H), 4.91(d,OH, D2O exchangeable), 4.37(m,1H), 4.01(m,1H), 3.72( s,6H)3.56(m,1H)3.29(m,1H), 3.14(m,1H), 2.93-3.02(m,4H), 2.18(m,2H)1.74-2.01(m,2H), 1.37-1.41(m,6H).

[0208] Synthesis of Compound 5: Compound 4 (12.36 g, 18.54 mmol) was dissolved in 10% methanol / ethyl acetate (300 mL) and purged with argon. To the reaction mixture was added 10% by weight palladium supported on wet activated carbon, Degussa type (1.3 g). The flask was re-purged with argon. The flask was purged twice with hydrogen, and then hydrogen was bubbled through the reaction mixture for 10 seconds. The reaction mixture was allowed to stir under hydrogen at room temperature overnight. The reaction mixture was decanted onto a sintered funnel filled with Celite and washed twice with methanol. The organic layer was evaporated to dryness to give compound 5 (Rf = 0.05 10% MeOH / DCM, 9.16 g, 93%) as a white solid, which did not require further purification. 1 H NMR (400MHz, DMSO-d6): δ7.15-7.31(m,9H), 6.86(m,4H)4.99(s,1H), 4.37(m,1H), 4.01(m,2H), 3.72(s,6H)3.56(m,1H)3.2 9(m,1H), 3.14(m,1H), 2.93-3.02(m,2H), 2.45(m,2H), 2.18(m,2H)1.74-2.01(m,2H), 1.37-1.41(m,3H)1.13-1.38(m,4H).

[0209] Synthesis of Compound 6: Compound 5 (9.16 g, 17.2 mmol) was dissolved in dichloromethane (200 mL). The reaction mixture was stirred at 10° C. for 10 minutes under argon. Triethylamine (4.80 mL, 34.4 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 20 minutes under argon. Ethyl trifluoroacetate (3.05 mL, 25.8 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 10 minutes under argon. The reaction mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed with water and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude Compound 6 (Rf=0.6 10% MeOH / DCM, 10.89 g) was obtained. Column purification by eluting with 5% methanol / dichloromethane (1% TEA) gave compound 6 (8.76 g, 81%) as a yellow foamy solid. 1 H NMR (400MHz, DMSO-d6): δ7.56-7.09(m,9H), 7.01-6.52(m,4H), 5.34-5.04(m,1H), 4.99-4.78(m,1H), 4.48-4.25(m,2H), 3.83-3.67(m,6) H), 3.60-3.50(m,1H), 3.49-3.18(m,2H), 3.16-2.91(m,2H), 2.89-2.56(m,2H), 2.54-2.32(m,2H), 2.32-1.69(m,3H), 1.59-1.03(m,4H). 19 F NMR (400MHz, DMSO-d6): -77.14(s,3F).

[0210] Synthesis of Compound 7: Compound 6 (8.76 g, 13.93 mmol), dimethylaminopyridine (5.10 g, 41.79 mmol), and triethylamine (3.90 mL, 27.86 mmol) were dissolved in dichloromethane (300 mL). The reaction mixture was stirred under argon for 10 minutes. Succinic anhydride (2.80 g, 27.86 mmol) was then added, and the mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed twice with a slightly saturated solution of sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 7 (Rf=0.9 10% MeOH / DCM, 10.87 g, 94%) was obtained as a white solid, which did not require further purification.

[0211] Synthesis of Compound 8: Compound 7 (2.00 g, 2.41 mmol) was dissolved in acetonitrile (100 mL). Diisopropylethylamine (1.68 mL, 9.64 mmol) and HBTU (1.83 g, 4.82 mmol) were added to the solution. The reaction mixture was shaken for 10 minutes. CPG (27 g) was added to the flask, and the mixture was shaken overnight. The CPG compound and the reaction mixture were decanted onto a sintered funnel. The reaction mixture was washed with 1% triethylamine / dichloromethane, followed by two washes with 10% methanol / dichloromethane, 1% triethylamine / dichloromethane, and anhydrous diethyl ether. The CPG compound was dried under vacuum for 1 hour, then recovered from the funnel and placed under high vacuum for 2 hours. The CPG compound (5.0 mg) was sent for deblocking. To the remaining CPG compound, 25% acetic anhydride / pyridine (100 mL) was added and the mixture was shaken overnight. The CPG compound and reaction mixture were placed on a sintered funnel and washed as described above. The CPG compound was dried under vacuum for 1 hour, removed from the funnel, and placed under high vacuum for 2 hours. The CPG compound (7.1 mg) was sent for deblocking. Spectrophotometer: Before capping 0.9892 Abs (502.0 nm) 65 μmol / g, after capping 1.4403 (502.0 nm) 67 μmol / g.

[0212] Synthesis of Compound 9: Compound 6 (8.89 g, 14.14 mmol) and diisopropylethylamine (4.93 mL, 28.28 mmol) were dissolved in anhydrous dichloromethane (60 mL). The reaction mixture was stirred under argon for 5 minutes. Then, N,N-diisopropylaminocyanoethyl phosphoramido acid chloride (5.63 mL, 16.26 mmol) was added to the reaction mixture. The reaction mixture was continued to stir at room temperature under argon for 30 minutes. Completion of the reaction was observed by TLC. The reaction mixture was diluted with dichloromethane (100 mL). The organic layer was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 9 (Rf=0.44 5% MeOH / DCM, 11.02 g). Column purification by eluting with 3% methanol / dichloromethane (1% TEA) gave compound 9 (6.31 g, 54%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ9.37(s,1H), 7.63(d 1H), 7.42-6.98(m,8H), 6.92-6.77(m,4H), 4.25-3.90(m,2H), 3.78-3.64(m,7H), 3.48(d,3H), 3.29(d,1H), 3.23 -2.92(m,4H), 2.86(d,1H), 2.73(t,1H), 2.58(t,1H), 2.53-2.47(m,4H), 2.33-1.87(m,4H), 1.55-0.97(m,12H). 31 P(400MHz, DMSO-d6): 151.68(d,1P).

[0213] Example 18: Synthesis of carbamate linkers for post-synthetic conjugation [ka]

[0214] 4-Z-aminobutanol 13: 4-Aminobutan-1-ol (26 mL, 280 mmol) and Cbz-O succinate (104.7 g, 420 mmol) were dissolved in dichloromethane (200 mL). The reaction mixture was stirred at 10° C. for 20 minutes under argon. Next, triethylamine (78 mL, 560 mmol) was added to the solution while continuing to stir the reaction mixture under argon at 10° C. The reaction mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 2 (Rf=0.5 10% MeOH / DCM, 71.97 g) as a white solid, which was used in the next step without further purification.

[0215] DSC-activated 4-Z-aminobutanol 14: Crude compound 13 (6.0 g) and DSC (10.33 g, 40.31 mmol) were dissolved in dichloromethane (100 mL). The reaction mixture was stirred at 0° C. for 30 minutes under argon. Triethylamine (7.88 mL, 53.74 mmol) was added dropwise, and the mixture was stirred at 0° C. for 5 minutes under argon. The reaction mixture was continued to stir at room temperature overnight under argon. The reaction mixture was diluted with dichloromethane (100 mL). The organic layer was washed with water, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 14 (Rf=0.85 10% MeOH / DCM, 11.14 g) as a light brown solid, which was used in the next step without further purification.

[0216] Compound 17: Compound 16 (5.00 g, 7.48 mmol) was dissolved in 10% methanol / ethyl acetate (100 mL) and purged with argon. To the reaction mixture was added 10% by weight palladium supported on wet activated carbon, Degussa type (0.5 g). The flask was re-purged with argon. The flask was purged twice with hydrogen, and then hydrogen was bubbled through the reaction mixture for 10 seconds. The reaction mixture was allowed to stir under hydrogen at room temperature overnight. The reaction mixture was decanted onto a sintered funnel filled with Celite and washed twice with methanol. The organic layer was evaporated to dryness to give compound 17 (Rf = 0.03 5% MeOH / DCM, 3.4 g, 87%) as a white solid, which did not require further purification. 1 H NMR (400MHz, DMSO-d6) δ7.41-7.14(m,9H), 6.99-6.72(m,4H), 4.96(d,1H), 4.30(s,1H), 4.04-3.5 8(m,6H), 3.46-3.25(m,1H), 3.21-2.81(m,3H), 2.52-2.46(m,2H), 1.97(d,3H), 1.63-1.42(m,4H).

[0217] Compound 18: Compound 17 (3.2 g, 5.98 mmol) was dissolved in dichloromethane (80 mL). The reaction mixture was stirred at 10° C. for 10 minutes under argon. Triethylamine (1.67 mL, 11.96 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 20 minutes under argon. Ethyl trifluoroacetate (1.06 mL, 8.97 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 10 minutes under argon. The reaction mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 18 (Rf=0.45 5% MeOH / DCM, 3.33 g, 88%) was obtained as a yellow foamy solid, which did not require further purification. 1H NMR (400MHz, DMSO-d6) δ7.46-7.03(m,9H), 6.86(d,4H), 4.93(s,1H), 4.29(s,1H), 4.11-3.63 (m,2H), 3.53-2.79(m,5H), 2.53(d,2H), 2.30(d,2H), 1.99(d,3H), 1.76(s,1H), 1.36(t,4H). 19 F NMR (400MHz, DMSO-d6): -77.11(s,3F).

[0218] Compound 19: Compound 18 (2.0 g, 3.17 mmol), dimethylaminopyridine (1.16 g, 3.48 mmol), and triethylamine (0.88 mL, 6.34 mmol) were dissolved in dichloromethane (50 mL). The reaction mixture was stirred under argon for 10 minutes. Succinic anhydride (0.63 g, 6.34 mmol) was then added, and the mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed twice with a slightly saturated solution of sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude compound 19 (Rf = 0.9 10% MeOH / DCM, 3.23 g) was obtained. Column purification by eluting with 5% methanol / dichloromethane (1% TEA) gave compound 12 (2.58 g, 98%) as a white solid. 1 H NMR (400MHz, DMSO-d6): δ9.43(d,1H), 7.45-7.04(m,9H), 7.02-6.71(m,4H), 5.51-4.91(m ,2H), 4.16-2.95(m,9H), 2.67(q,4H), 2.58-2.36(m,6H), 2.31-2.02(m,2H), 1.33(d,4H).

[0219] Compound 20: Compound 19 (2.03 g, 2.40 mmol) was dissolved in acetonitrile (100 mL). Diisopropylethylamine (1.67 mL, 9.64 mmol) and HBTU (1.82 g, 4.82 mmol) were added to the solution. The reaction mixture was shaken for 10 minutes. CPG (27 g) was added to the flask, and the mixture was shaken overnight. The CPG compound and reaction mixture were decanted onto a sintered funnel. The reaction mixture was washed with 1% triethylamine / dichloromethane, followed by two washes with 10% methanol / dichloromethane, 1% triethylamine / dichloromethane, and anhydrous diethyl ether. The CPG compound was dried under vacuum for 1 hour, then collected from the funnel and placed under high vacuum for 2 hours. The CPG compound (6.7 mg) was sent for deblocking. To the remaining CPG compound, 25% acetic anhydride / pyridine (150 mL) was added and the mixture was shaken overnight. The CPG compound and reaction mixture were placed on a sintered funnel and washed as described above. The CPG compound was dried under vacuum for 1 hour, removed from the funnel, and placed under high vacuum for 2 hours. The CPG compound (6.9 mg) was sent for deblocking. Spectrophotometer: Before capping, 1.8396 Abs (502.0 nm) 90.3 μmol / g; after capping, 1.8798 Abs (502.0 nm) 89.6 μmol / g.

[0220] Compound 21: Compound 18 (4.26 g, 6.75 mmol) and diisopropylethylamine (2.35 mL, 13.5 mmol) were dissolved in anhydrous dichloromethane (40 mL). The reaction mixture was stirred under argon for 5 minutes. N,N-diisopropylaminocyanoethyl phosphoramido acid chloride (1.73 mL, 7.76 mmol) was then added to the reaction mixture. The reaction mixture was continued to stir at room temperature under argon for 30 minutes. Completion of the reaction was observed by TLC. The reaction mixture was diluted with dichloromethane (100 mL). The organic layer was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 14 (Rf=0.44 5% MeOH / DCM, 11.02 g). Column purification by eluting with 3% methanol / dichloromethane (1% TEA) gave compound 14 (6.31 g, 54%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ7.41-6.99(m,8H), 6.85(d,4H), 4.10-3.62(m,2H), 3.59-3.24(m,7H), 3 .13(d,3H), 2.75-2.68(m,1H), 2.59-2.45(m,4H), 2.10(d,1H), 1.37(t,1H), 1.25-1.00(m,10H).

[0221] Example 19: GalNAc conjugation at pyrimidine, purine, and abasic sites (Schemes 19-33) Using palladium-based coupling chemistry, GalNAc ligands and cationic molecules can be introduced at the C-5 position of pyrimidine nucleosides bearing various substituents at the 2' position. Nucleoside building blocks can be synthesized accordingly, as shown in the following schemes (Schemes 19-27). In Schemes 28 and 29, designed purine nucleoside analogs containing GalNAc ligands are shown. In Scheme 30, a GalNAc ligand is introduced at the C-1' position. Using click chemistry, GalNAc can be introduced at the abasic site, as shown in Schemes 31 and 32. Scheme 33 illustrates an interchangeable nucleoside approach to introducing a GalNAc ligand at the C-2 position on the purine ring.

[0222] [ka]

[0223] [ka]

[0224] [ka]

[0225] [ka]

[0226] [ka]

[0227] [ka]

[0228] [ka]

[0229] [ka]

[0230] [ka]

[0231] [ka]

[0232] [ka]

[0233] [ka]

[0234] [ka]

[0235] [ka]

[0236] [ka]

[0237] i. Synthesis of Compound 103 (R=F) To a solution of 102 (R = F; 11.2 g, 30.1 mmol; Antiviral Chemistry & Chemotherapy, 2010, 21, 15-31) in pyridine (70 mL) was added DMTrCl (11.2 g, 32.1 mmol). The reaction mixture was stirred at room temperature for 14 h and then evaporated. The residue was extracted with CHCl and saturated aqueous NaHCO and dried over anhydrous NaSO. The crude material was purified by silica gel column chromatography (5% MeOH in CHCl, R f =0.23) to give compound 103 (17.8 g, 26.4 mmol, 88%). 1 H NMR (DMSO-d6, 400MHz): δ11.81(s,1H), 8.07(s,1H), 7.46-7.14(m,10H), 6.88(dd,J=8.9, 1.8Hz, 4H), 5.83(d,J=20.5Hz, 1H), 5.60(d,J=7.0Hz, 1H), 5.16(dd,J=53.4, 4.8Hz, 1H), 4.43-4.20(m,1H), 4.08-3.90(m,1H), 3.74(s,7H), 3.23(d,J=2.7Hz, 2H). 13 C NMR (100MHz, DMSO-d6): δ160.68, 158.09, 158.07, 149.89, 145.13, 144.72, 136.12, 135.46, 135.37, 129.71, 127.93, 127.67, 126.70, 123.90, 85.61, 81.30, 69.75, 62.47, 55.06, 55.03.

[0238] ii. Synthesis of Compound 104 (R=F) To a solution of compound 103 (R = F; 6.40 g, 9.49 mmol) in CH3CN (75 mL) was added PdCl2(PhCN)2 (73 mg, 0.190 mmol), Et3N (2.65 mL, 19.0 mmol), and CF3CH2OH (6.91 mL, 94.9 mmol). The mixture was stirred at 60 °C under a CO gas atmosphere. After evaporation, the residue was extracted with CHCl2 and saturated aqueous NaHCO3. The organic layer was separated and dried over anhydrous Na2SO4. The filtrate was concentrated, and the crude material obtained was purified by silica gel column chromatography (0-5% MeOH in CHCl2) to give 104 (4.30 g, 6.37 mmol, 67%, R = F). f =0.32) was obtained by development with 5% MeOH in CH2Cl2. 1 H NMR (400MHz, DMSO-d6): δ11.78(s,1H), 8.52(s,1H), 7.41(d,J=7.4Hz, 2H), 7.34-7.16(m,8H), 6.93-6.80(m,4H), 5.90(d,J=20.1Hz, 1H), 5.63(d,J =7.1Hz, 1H), 5.26(d,J=4.4Hz, 1H), 5.12(d,J=4.5Hz, 1H), 4.50-4.17(m,3 H), 4.07(dd,J=7.3, 5.0Hz, 1H), 3.73(d,J=1.2Hz, 7H), 3.31-3.17(m,2H). 13 C NMR (100MHz, DMSO-d6): δ160.70, 158.96, 158.08, 158.06, 149.18, 148.89, 144.76, 135.47, 135.33, 129.70, 129.66, 127.80, 1 27.61, 126.66, 124.62, 121.86, 102.57, 94.10, 92.27, 90.68, 90.32, 85.56, 81.36, 68.11, 67.95, 62.36, 59.26, 58.91, 54.98. 19 F NMR (376MHz, DMSO-d6): δ-74.94, -74.96, -74.99, -201.81, -201.86, -201.87, -201.93, -201.95, -202.01, -202.07.C 33 H 30 Molecular weight (M+Na) for F4N2NaO9+ Calculated value 697.1785, measured value 697.2.

[0239] iii. Synthesis of Compound 105 (R=F) Compound 104 (R = F; 4.15 g, 6.15 mmol) was treated with 3-(dimethylamino)-1-propylamine (25 mL) at room temperature overnight. The reaction mixture was extracted with CHCl and saturated aqueous NaHCO, and the organic layer was dried over anhydrous NaSO to give crude material 105. 36 H 42 Molecular weight (M+H) for FN4O8 + Calculated value 677.2987, measured value 677.1.

[0240] Example 20. GalNAc conjugation at the ribose ring (Schemes 34-44) Synthetic approaches for conjugating GalNAc and its derivatives to the ribose ring in nucleosides are shown below. Tin-modified nucleosides can be coupled with alkyl bromides to generate 2'- and 3'-linked products. The resulting primary amines or activated esters and terminal alkenes, respectively, can be coupled with GalNAc ligands using appropriate reaction conditions. These building blocks are incorporated into oligonucleotides using standard phosphoramidite chemistry. GalNAc ligands can also be conjugated to oligonucleotides by post-synthetic approaches.

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[0252] 2'- and 3'-O-phthalimidohexyl-5-methyluridine (2a, 2b). A solution of 2',3'-O-dibutylstannylene-5-methyluridine (28 g, 57.24 mmol) obtained as reported (J. Org. Chem., 1974, 24-30), 6-bromohexylphthalimide (35.5 g, 114.48 mmol), and NaI (1.72 g, 11.45 mmol) in DMF (105 mL) was heated at 100 °C in a microwave for 3.5 h. After removing the DMF, the residue was purified by silica gel column chromatography (R f Purification by HCl (0.26 in 5% MeOH in CH₂Cl₂) gave the 2'- and 3'-isomers of O-phthalimidohexyl-5-methyluridine as an inseparable mixture (10.1 g, 20.7 mmol, 36%). MS m / z 488.0 (M+H). + , 510.2(M+Na) + , 486.2(MH). 1 H NMR (400MHz, DMSO-d6): δ11.29(s,1H), 7.88-7.79(m,4H), 7.78-7.70(m,1H), 5.81(d,J=5.3Hz, 1H), 5.72(d,J=5 .6Hz, 1H), 5.25(d,J=6.2Hz, 1H), 5.12(t,J=5.0Hz, 1H), 5.00(d,J=5.9Hz, 1H), 4.14(dd,J=11.3, 5.6Hz, 1H), 4.07 (dd,J=10.1, 5.0Hz, 1H), 3.89-3.79(m,2H), 3.76-3.72(m,1H), 3.63(ddd,J=11.1, 8.8, 5.4Hz, 1H), 3.59-3.47(m, 4H), 3.41(dt,J=11.6, 6.6Hz, 1H), 3.28(s,1H), 1.75(d,J=3.7Hz, 3H), 1.64-1.41(m,5H), 1.27(d,J=14.2Hz, 5H). 13C NMR (100MHz, DMSO-d6): δ167.99, 167.97, 163.78, 163.73, 150.79, 150.57, 136.22, 136.07, 134.40, 131.60, 131.56, 123.02, 109.38, 109.26, 87.73, 85.89, 85.07, 82. 71, 80.82, 77.49, 72.43, 69.69, 69.51, 68.38, 60.86, 60.61, 54.92, 37.39, 37.35, 29.23, 28.96, 27.93, 27.76, 26.15, 26.07, 25.76, 25.39, 25.16, 24.96, 21.27, 12.26.

[0253] 5'-O-Dimethoxytrityl-2'-O-phthalimidohexyl-5-methyluridine (3a). A mixture of 2a and 2b (10.11 g, 20.73 mmol) was coevaporated with pyridine (50 mL), then dissolved in pyridine (80 mL) and cooled to 0 °C in an ice bath. To this mixture, dimethoxytrityl chloride (7.73 g, 22.80 mmol) was added, and the reaction was stirred at 0 °C for 2 h and then at room temperature for 1 h. An additional 0.4 equivalents of dimethoxytrityl chloride was added, and the reaction was stirred overnight. The reaction was quenched with MeOH (5 mL) and then evaporated under reduced pressure. The reaction mixture was diluted with DCM and washed twice with brine. The organic layer was dried over Na2SO4, evaporated under reduced pressure, and then purified by silica gel column chromatography to give 4.48 g of 3a (27.4%, 5.67 mmol; R f = 0.35 in 60% EtOAc in hexanes). MS m / z 812.3 (M+Na) + , 788.3(MH). 1H NMR (400MHz, DMSO-d6): δ11.35(s,1H), 7.87-7.77(m,4H), 7.48(d,J=0.8Hz, 1H), 7.38(d,J=7.4Hz, 2H), 7.30(t,J=7 .6Hz, 2H), 7.23(dd,J=12.1, 8.1Hz, 5H), 6.89(d,J=8.0Hz, 4H), 5.11(d,J=6.3Hz, 1H), 4.18(dd,J=11.1, 5.4Hz, 1H), 3.99(ddd,J=12.0, 11.2, 5.9Hz, 3H), 3.72(s,6H), 3.62-3.46(m,4H), 3.21(ddd,J=12.9, 10.7, 3.3Hz, 2H), 1.98(d,J =1.8Hz, 1H), 1.60-1.44(m,5H), 1.38(s,3H), 1.35-1.19(m,5H), 1.16(t,J=7.1Hz, 1H), 0.87(dd,J=10.1, 4.7Hz, 1H). 13 C NMR (100MHz, DMSO-d6): δ170.33, 167.92, 163.61, 158.19, 158.16, 150.41, 144.67, 1 35.45, 135.32, 135.11, 134.33, 131.58, 129.74, 127.93, 127.65, 126.84, 122.97, 113 .27, 109.60, 86.46, 85.91, 83.09, 80.53, 69.63, 68.76, 63.48, 63.19, 59.75, 55.06, 37.31, 30.16, 28.89, 27.92, 26.07, 24.95, 20.76, 20.72, 18.60, 14.08, 13.55, 11.66.

[0254] 5'-O-Dimethoxytrityl-3'-O-phthalimidohexyl-5-methyluridine (3b). The 3'-isomer (3b) was purified by column chromatography (3.36 g, 20.5%, 4.26 mmol; R f = 0.18 in 60% EtOAc in hexanes). MS m / z 812.0 (M+Na) + , 788.3(MH). 1H NMR (400MHz, DMSO-d6): δ11.34(s,1H), 7.88-7.76(m,4H), 7.50(s,1H), 7.36(d,J=7.5Hz, 2H), 7.33-7.1 5(m,7H), 6.87(d,J=7.8Hz, 4H), 5.75-5.69(m,1H), 5.37(d,J=6.0Hz, 1H), 4.27(dd,J=10.5, 5.1Hz, 1H), 4.05-3.94(m,2H), 3.91(t,J=5.2Hz, 1H), 3.71(s,6H), 3.56(ddd,J=22.7, 11.8, 6.7Hz, 3H), 3.21(ddd,J =24.1, 10.8, 3.3Hz, 2H), 1.98(t,J=4.5Hz, 1H), 1.60-1.37(m,7H), 1.34-1.13(m,5H), 0.94-0.83(m,1H). 13 C NMR (100MHz, DMSO-d6): δ170.41, 167.93, 163.69, 158.17, 158.16, 150.57, 144.63, 135. 73, 135.30, 135.17, 134.35, 131.58, 129.71, 127.91, 127.63, 126.82, 122.97, 113.24, 10 9.37, 88.69, 85.91, 80.60, 77.27, 72.18, 69.67, 63.49, 62.95, 59.76, 55.03, 54.90, 37.33, 30.17, 29.09, 27.88, 26.08, 25.08, 20.76, 20.72, 18.87, 18.61, 14.09, 13.55, 11.74.

[0255] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-5-methyluridine (4a). 3a (15.0 g, 18.99 mmol) was dissolved in 190 mL of MeOH. Hydrazine (3.04 g, 94.52 mmol) was added to the heterogeneous mixture and heated to reflux (66 °C) for 3.5 h. The mixture was cooled to room temperature and evaporated under reduced pressure to give a white powder. The product was dissolved in DCM and washed twice with ammonium hydroxide and saturated NaCl solution. The DCM layer was dried over MgSO4 and evaporated under reduced pressure to give 11.8 g. The crude material was used in the next step. (R f= 0.02 in 5% MeOH in DCM). MS m / z 660.2 (M+H) + , 682.1(M+Na) + , 658.1(MH) - . 1 H NMR (400MHz, DMSO-d6): δ7.48(s,1H), 7.38(d,J=7.5Hz, 2H), 7.30(t,J=7.5Hz, 2H), 7.24(d,J=8.9Hz, 5H), 6.89(d,J=8.4Hz, 4H), 5.84(d,J=5.0Hz, 1H), 5.74( s,1H), 4.19(t,J=5.0Hz, 1H), 3.97(t,J=4.9Hz, 2H), 3.72(s,6H), 3.63-3.45(m ,3H), 3.27-3.15(m,3H), 1.48(d,J=6.4Hz, 2H), 1.38(s,3H), 1.34-1.18(m,6H).

[0256] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-5-methyluridine (5a). Crude material 4a (6.00 g) was dissolved in DCM (250 mL) and triethylamine (3.8 mL, 27.30 mmol) and stirred for 10 minutes. GalNAc-C5-NHS ester (7.31 g, 10.0 mmol) was added and the reaction mixture was stirred for 2 hours. The reaction mixture was diluted with DCM and washed with brine, and the organic layer was then dried over Na2SO4 and evaporated under reduced pressure. The crude material was purified by column chromatography (R f Purification by HCl (0.36 in 5% MeOH in DCM) gave 5a (9.56 g, 7.49 mmol, 83%). MS m / z 1298.3 (M+Na). + . 1H NMR(400MHz、DMSO-d6):δ11.36(s,1H)、7.99-7.86(m,5H)、7.73-7.52(m,8H)、7.48(t,J=7.7Hz、3H)、7.41-7.34(m,4H)、7.30(t,J=7.6Hz、2H)、7.23(dd,J=11.1、8.1Hz、4H)、6.88(d,J=8.1Hz、4H)、5.83(d,J=4.8Hz、1H)、5.74(d,J=3.9Hz、1H)、5.35(dd,J=11.1、3.3Hz、1H)、5.11(d,J=6.3Hz、1H)、4.72(d,J=8.5Hz、1H)、4.50-4.39(m,2H)、4.38-4.14(m,4H)、3.95(t,J=4.8Hz、2H)、3.82-3.68(m,7H)、3.63-3.43(m,4H)、3.28-3.15(m,3H)、2.98(dd,J=12.9、6.5Hz、2H)、2.03(s,2H)、1.69(s,3H)、1.49(s,6H)、1.40-1.15(m,9H). 13 C NMR(100MHz、DMSO-d6):δ171.73、169.40、165.20、165.16、164.86、163.62、158.18、158.15、150.41、144.64、135.31、135.10、133.50、129.73、129.16、129.00、128.70、128.59、127.91、127.64、126.82、113.26、109.59、100.89、85.91、83.09、80.60、71.86、69.97、69.74、68.77、67.92、63.19、62.03、55.04、49.74、35.03、29.17、29.04、28.59、26.25、25.12、22.69、21.85、11.66。

[0257] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-3'-O-succinato-5-methyluridine (6a). 5a (2.00 g, 1.57 mmol) was dissolved in DCM (50 mL). DMAP (574 mg, 4.70 mmol) and succinic anhydride (313 mg, 3.14 mmol) were added, and the reaction mixture was stirred at room temperature for 17 h. The product was purified by silica gel column chromatography (φ=4.2 cm × 15 cm) and treated with 2% TEA in DCM. The product was eluted with 0-5% MeOH and 2-5% TEA in DCM and coevaporated with acetonitrile under reduced pressure to give 2.11 g (91%, 1.43 mmol) of 6a as the TEA salt (R f = 0.41 in 5% MeOH / 5% TEA in DCM). MS m / z 1397.4 (M+Na) + , 1373.4(MH) - . 1 H NMR (400MHz, DMSO-d6): δ11.44(s,1H), 8.11(d,J=9.0Hz, 1H), 7.90(dd,J=10.2, 4.1Hz, 4H), 7.75(s,1H), 7.72-7.52(m,7H), 7.5 1-7.44(m,3H), 7.34(ddd,J=24.0, 13.7, 7.9Hz, 6H), 7.22(d,J=8.7Hz, 5H), 6.89(d,J=7.9Hz, 4H), 5.83(d,J=6.1Hz, 1H), 5.73(d ,J=3.4Hz, 1H), 5.36(dd,J=11.1, 3.3Hz, 1H), 5.26-5.22(m,1H), 4.75(d,J=8.5Hz, 1H), 4.47-4.40(m,2H), 4.37-4.22(m,3H), 4. 12(d,J=3.5Hz, 1H), 3.83-3.69(m,6H), 3.53-3.19(m,15H), 2.97(d,J=7.8Hz, 2H), 2.04(s,2H), 1.68(s,2H), 1.56-1.11(m,15H). 13C NMR (100MHz, DMSO-d6): δ173.40, 171.76, 171.63, 169.39, 165.20, 165.15, 164.86, 163.50, 158.22 , 150.50, 144.48, 135.12, 134.95, 133.48, 129.70, 129.16, 129.03, 128.70, 128.58, 127.95, 127.6 1, 113.30, 110.16, 100.91, 86.13, 69.97, 67.91, 62.04, 55.04, 49.73, 45.48, 40.12, 39.92, 39.71, 39.50, 39.29, 39.08, 38.87, 38.33, 34.98, 29.13, 28.93, 26.19, 25.08, 22.68, 21.82, 11.69, 10.48.

[0258] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-3'-O-CPG-5-methyluridine (7a). To a solution of 6a (2.01 g, 1.36 mmol) in acetonitrile (100 mL), HBTU (1.03 g, 2.72 mmol), DIEA (528 mg, 4.08 mmol), and CPG (16.0 g, 130 μmol / g, 540 Å) were added, and the mixture was shaken for 24 h. The CPG was filtered off and washed with DCM, 20% MeOH in DCM, and then ether. The CPG was then treated with acetic anhydride (25 mL) in pyridine (75 mL) and TEA (1 mL) for 1 h. The CPG was filtered off and washed with the same solvent as above. The loading was measured twice using a spectrophotometer and the average loading was calculated (73.1 μmol / g).

[0259] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-3'-O-(N,N-diisopropyl)-β-cyanoethylphosphoramidite-5-methyluridine (8a). 5a (2.90 g, 2.27 mmol) was coevaporated twice with anhydrous acetonitrile and then placed under a full argon atmosphere. 5a was dissolved in anhydrous DCM (35 mL) and cooled to 0 °C. 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.37 g, 4.55 mmol) was added to the stirring mixture, followed by DCI (268 mg, 2.27 mmol). The mixture was stirred at 0 °C for 20 min and then at room temperature for 17 h. The reaction mixture was diluted with DCM, washed with brine, and dried over Na2SO4 to give a pale yellow foam. The crude material was purified by silica gel column chromatography (φ = 4.2 cm × 19 cm; R f Purification by HCl (0.3g in EtOAc) gave 3.20g of 8a (95%, 2.17mmol). MS m / z 1498.3 (M+Na). + . 1 H NMR (400MHz, DMSO-d6): δ11.39(s,1H), 8.06-7.84(m,5H), 7.78-7.16(m,21H), 6.93-6.82(m,4H), 5.82(d,J=4.3Hz) , 1H), 5.74(s,1H), 5.35(dd,J=11.1, 3.1Hz, 1H), 4.73(d,J=8.5Hz, 1H), 4.50-4.20(m,5H), 4.10(dd,J=12.8, 7.7Hz, 2H), 3.74(t,J=11.5Hz, 7H), 3.66-3.44(m,6H), 3.29-3.18(m,2H), 2.98(d,J=5.3Hz, 2H), 2.76(t,J=5.8Hz, 1H), 2. 57(dd,J=10.0, 5.3Hz, 1H), 2.49(d,J=1.5Hz, 5H), 2.04(s,2H), 1.70(s,3H), 1.59-1.03(m,29H), 0.99-0.81(m,4H). 13C NMR (125MHz, DMSO-d6) δ171.60, 169.26, 165.07, 165.03, 164.73, 163.48, 158.10, 158.07, 150.24, 150.22, 144.39, 144.34, 135.32, 135.08, 134.99 , 134.97, 134.87, 134.80, 133.64, 133.37, 133.34, 129.65, 129.07, 129.0 3, 128.90, 128.88, 128.87, 128.83, 128.56, 128.45, 127.75, 127.56, 127.5 0, 126.74, 118.72, 118.57, 113.08, 109.63, 109.54, 100.76, 87.02, 86.62, 85.92, 85.90, 82.13, 79.89, 71.72, 69.84, 68.61, 67.78, 62.57, 62.23, 6 1.89, 58.43, 57.81, 57.65, 54.91, 49.60, 46.05, 45.53, 42.42, 34.88, 29.02, 28.97, 28.45, 26.17, 25.04, 24.19, 24.13, 24.03, 22.55, 21.71, 11.50. 31 P NMR (162MHz, DMSO-d6) δ154.01, 153.65.

[0260] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-5-methyluridine (4b). 3b (3.64 g, 4.61 mmol) was dissolved in MeOH. Hydrazine (738 mg, 23.04 mmol) was added and the reaction mixture was refluxed for 5.5 hours. Following the same procedure as described for 4a, 2.93 g of crude 4b was obtained. (R f = 0.02 in 5% MeOH in DCM). MS m / z 660.2 (M+H) + , 682.1(M+Na) + , 658.1(MH) - . 1H NMR (400MHz, DMSO-d6) δ7.50(s,1H), 7.43-7.16(m,9H), 6.88(d,J=8.7Hz, 4H), 5.73(d,J=4.6Hz, 1H), 4.29( t,J=4.8Hz, 1H), 4.02-3.89(m,3H), 3.46-3.15(m,5H), 2.24-2.16(m,1H), 2.05(s,1H), 1.58-1.10(m,13H). 13 C NMR (100MHz, DMSO-d6): δ163.73, 158.04, 158.02, 150.58, 144.49, 135.54, 135.18, 135.06, 129.56, 127.78, 127.50, 126.69 , 113.11, 109.23, 88.49, 85.78, 80.47, 77.20, 72.02, 69.62, 62.84, 54.91, 41.44, 33.12, 29.17, 26.24, 26.11, 25.31, 11.62.

[0261] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-5-methyluridine (5b). To a solution of 4b (2.85 g, 4.32 mmol) in DCM (45 mL) and TEA (1.8 mL) was added GalNAc-NHS ester (3.47 g, 4.75 mmol). The reaction was stirred for 2 hours before adding an additional 0.2 equivalents of GalNAc-NHS ester, then stirred for an additional hour. Following the same procedure as described for 5a, 3.62 g of 5b (2.84 mmol, 66%) was obtained. (R f = 0.24 in 5% MeOH in DCM). MS m / z 1297.4 (M+Na) + . 1H NMR(400MHz、DMSO-d6):δ11.35(s,1H)、8.02-7.87(m,5H)、7.74-7.44(m,11H)、7.41-7.34(m,4H)、7.33-7.19(m,7H)、6.88(d,J=8.8Hz、4H)、5.74(dd,J=5.9、4.1Hz、2H)、5.41-5.33(m,2H)、4.73(d,J=8.5Hz、1H)、4.44(t,J=7.9Hz、2H)、4.31(ddd,J=16.9、12.6、7.9Hz、3H)、4.03-3.95(m,1H)、3.91(t,J=5.1Hz、1H)、3.83-3.75(m,1H)、3.72(s,6H)、3.63-3.45(m,2H)、3.22(dd,J=7.8、2.9Hz、2H)、2.99(d,J=6.2Hz、2H)、2.87(s,1H)、2.72(s,1H)、2.04(t,J=6.4Hz、2H)、1.69(s,3H)、1.55-1.14(m,14H). 13 C NMR(125MHz、DMSO-d6):δ171.74、169.40、165.20、165.16、164.86、163.65、162.27、158.16、150.57、144.58、135.70、135.32、135.17、133.74、133.45、129.68、129.15、129.01、128.94、128.67、128.57、127.89、127.63、126.81、113.23、109.37、100.89、88.58、85.91、80.64、77.36、72.17、71.84、69.98、69.73、68.74、67.92、62.98、62.03、55.02、54.85、49.75、40.33、40.05、39.77、39.49、39.21、38.94、38.66、38.34、35.74、35.02、30.74、29.23、29.12、28.58、26.26、25.21、22.66、21.84、11.68。

[0262] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-2'-O-succinato-5-methyluridine (6b). To a solution of 5b (1.10 g, 0.86 mmol) in DCM (20 mL) was added DMAP (315 mg, 2.58 mmol) and succinic anhydride (172 mg, 1.73 mmol). The reaction mixture was stirred for 23 h and then purified in a similar manner as for 6a and coevaporated with acetonitrile under reduced pressure to give 1.03 g of 6b (81%, 0.70 mmol). (R f = 0.18 in 5% MeOH in DCM). MS m / z 1397.4 (M+Na) + , 1373.4(MH) - . 1 H NMR (400MHz, DMSO-d6): δ8.09(d,J=9.3Hz, 1H), 7.91(t,J=6.9Hz, 4H), 7.76(t,J=5.4Hz, 1H), 7.69(dd,J=11.3, 7.5Hz, 3H), 7.65-7.51(m,5H) ), 7.47(t,J=7.7Hz, 2H), 7.41-7.33(m,4H), 7.32-7.16(m,7H), 6.87(d,J=8.7Hz, 4H), 5.85(d,J=3.9Hz, 1H), 5.74(d,J=3.2Hz, 1H), 5.48-5.4 1(m,1H), 5.36(dd,J=11.1, 3.2Hz, 1H), 5.29(s,1H), 4.75(d,J=8.5Hz, 1H), 4.49-4.39(m,2H), 4.38-4.19(m,4H), 3.28(ddd,J=37.9, 12.7, 5. 4Hz, 11H), 3.08-2.90(m,3H), 2.69(q,J=7.2Hz, 5H), 2.60-2.52(m,2H), 2.05(d,J=3.5Hz, 2H), 1.76(s,1H), 1.69(s,3H), 1.56-0.94(m,28H). 13C NMR (100MHz, DMSO-d6): δ173.63, 173.44, 172.79, 171.92, 171.52, 169.54, 165.29, 165.24, 164.94, 163. 78, 158.23, 150.33, 144.57, 136.18, 135.27, 135.18, 133.84, 133.58, 129.77, 129.24, 129.05, 128.76, 12 8.65, 127.96, 127.70, 126.90, 113.29, 109.73, 100.98, 85.97, 80.75, 73.31, 71.96, 70.48, 62.40, 55.10, 52.06, 51.36, 45.45, 35.04, 33.35, 29.20, 29.09, 28.95, 28.77, 22.70, 21.89, 11.81, 10.05, 7.26, 7.17.

[0263] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-2'-O-CPG-5-methyluridine (7b). To a solution of 6b (970 mg, 0.66 mmol) in acetonitrile (50 mL) was added HBTU (497 mg, 1.31 mmol), DIEA (339 mg, 1.97 mmol), and CPG (8.20 g, 130 μmol / g, 540 Å), and the mixture was shaken for 21 h. The CPG was filtered, washed, capped, and assayed similarly to 7a to give CPG 7b with an average loading of 56.1 μmol / g.

[0264] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-2'-O-(cyanoethyl N,N-diisopropyl)-phosphoramidite-5-methyluridine (8b). 5b (1.98 g, 1.55 mmol) was treated in the same manner as described for 8a, and silica gel column chromatography gave 1.89 g (83%, 1.28 mmol) of 8b. (R f = 0.43 in 100% EtOAc). MS m / z 1497.4 (M+Na) + , 1474.3(MH) - . 1H NMR(400MHz、DMSO-d6):δ11.38(s,1H)、8.00-7.87(m,5H)、7.75-7.18(m,22H)、6.88(dd,J=8.9、2.7Hz、4H)、5.88(dd,J=9.2、5.1Hz、1H)、5.75(d,J=3.2Hz、1H)、5.36(dd,J=11.1、3.2Hz、1H)、4.73(d,J=8.5Hz、1H)、4.61-4.50(m,1H)、4.49-4.40(m,2H)、4.38-4.23(m,2H)、4.01(ddd,J=19.3、11.6、4.8Hz、3H)、3.85-3.47(m,14H)、2.98(s,2H)、2.71(ddd,J=11.6、8.9、3.9Hz、2H)、2.10-1.95(m,4H)、1.69(s,3H)、1.57-0.99(m,31H)、0.94-0.83(m,2H). 13 C NMR(125MHz、DMSO-d6):δ171.72、169.37、165.18、165.14、164.85、163.58、163.50、158.18、158.16、150.48、150.43、144.52、135.15、134.98、133.74、133.48、133.44、129.66、129.14、129.01、128.99、128.98、128.93、128.67、128.56、127.91、127.88、127.57、126.84、118.81、118.71、113.25、113.21、109.72、109.68、100.86、86.17、86.02、71.82、69.95、69.86、68.72、67.88、63.45、62.00、55.00、49.72、39.80、39.63、39.46、39.30、39.13、38.96、38.33、35.00、30.12、30.03、29.26、29.14、29.11、28.56、26.29、25.27、25.25、24.28、24.22、24.08、24.02、22.65、21.84、20.67、19.80、19.75、19.67、19.62、18.56、14.04、13.50、11.68、11.55. 31 P NMR(162MHz、DMSO-d6)δ155.08、154.60。

[0265] Example 21. Synthesis of S- and C-linked GalNAc derivatives and building blocks (Schemes 45-51)

[0266] [ka]

[0267] [ka]

[0268] [ka]

[0269] [ka]

[0270] [ka]

[0271] [ka]

[0272] Synthesis of Compounds (Schemes 45-51) Compound 1 (15.6 g, 40.1 mmol) was treated with TMSOTf (7.98 mL, 44.1 mmol) in DCE to give compound 2. 14 H 20 Molecular weight (M+H) for NO8 + Calculated value: 330.12, measured value: 330.0.

[0273] Synthesis of Compound 3: Compound 2 (1.65 g, 5 mmol) and tert-butyl 5-mercaptopentanoate (1.0 g, 5.25 mmol) in DCE were treated with TMSOTf (0.181 mL, 1.0 mmol) overnight. Aqueous work-up and purification by silica gel column chromatography gave Compound 3 (380 mg, 0.731 mmol, 15%). 23 H 37 NNaO 10 Molecular weight for S (M+H) + Calculated value: 542.20, measured value: 542.1.

[0274] Synthesis of Compound 7: To a solution of Compound 3 (380 mg, 0.731 mmol) in CH2Cl2 (4 mL) was added TFA (1 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and then at room temperature for 3 h. The solvent was evaporated and the residue was co-evaporated with toluene to give crude Compound 7. This material was used in the next step without purification. C 19 H 30 NO 10 Molecular weight for S (M+H) + Calculated value: 464.1590, measured value: 464.1.

[0275] Synthesis of Compound 8: Compound 7 (approximately 0.731 mmol) from the previous step was treated with N-hydroxysuccinimide (168 mg, 1.46 mmol) in the presence of EDCI (280 mg, 1.46 mmol) and DIEA (0.764 mL, 4.38 mmol) in CHCl (5 mL) for 14 hours. Aqueous workup followed by column chromatography afforded compound 8 (284 mg, 0.507 mmol, 69% over two steps). 23 H 33 N2O 12 Molecular weight for S (M+H) + Calculated value: 561.1754, measured value: 561.1.

[0276] S-Alkylation of compound 6 with alkyne bromide gives compound 9. Similarly, compound 14 is prepared from compound 11 (J. Org. Chem., 2002, 67, 2995-2999). Acid 12 is prepared according to the reported procedure.

[0277] Synthesis of Compound 13: Compound 12 (2.40 g, 5.18 mmol) was treated with N-hydroxysuccinimide (716 mg, 6.22 mmol) in the presence of EDCI (1.19 g, 6.22 mmol) and DIEA (2.70 mL, 15.5 mmol) in CHCl (30 mL) for 14 hours. Aqueous workup followed by column chromatography afforded Compound 13 (1.83 g, 3.26 mmol, 63%). 23 H 33 N2O 12 Molecular weight for S (M+H) + Calculated value: 561.1754, measured value: 561.2.

[0278] Compound 16 was prepared using reported procedures (J. Org. Chem., 2006, 71, 3619-3622; Carbohydrate Research, 1998, 309, 319-330).

[0279] Synthesis of Compound 22: Compound 16 (1.94 g, 5.22 mmol), benzyl 4-pentenoate (2.99 g, 15.7 mmol), and second-generation Grubbs catalyst (433 mg, 0.522 mmol) in CHCl (20 mL) were heated at 40° C. for 40 hours. The solvent was removed, and the residue was purified by silica gel column chromatography to give Compound 22 (1.87 g, 3.50 mmol, 67%). 27 H 36 NO 10 Molecular weight (M+H) + Calculated value 534.2339, measured value 534.2.

[0280] Synthesis of Compound 23: To a solution of Compound 22 (1.85 g, 3.47 mmol) in EtOAc (30 mL) was added palladium on carbon (Aldrich: 330108-50G, 10 wt%, Degussa type E101 NE / W: 185 mg). The reaction mixture was stirred under an H atmosphere for 14 hours. After filtration through Celite, the filtrate was removed under reduced pressure. The residue was purified by silica gel column chromatography to give Compound 23 (903 mg, 2.03 mmol, 59%). 20 H 32 NO 10 Molecular weight (M+H) + Calculated value: 446.2026, measured value: 446.1.

[0281] Synthesis of Compound 24: Compound 23 (326 mg, 0.732 mmol) was treated with N-hydroxysuccinimide (127 mg, 1.10 mmol) in the presence of EDCI (211 mg, 1.10 mmol) and DIEA (0.383 mL, 2.20 mmol) in CHCl (5 mL) for 14 hours. Aqueous workup followed by column chromatography afforded Compound 24 (300 mg, 0.553 mmol, 76%). 24 H 35 N2O 12 Molecular weight (M+H) + Calculated value 543.2190, measured value 543.2.

[0282] Oxidative cleavage of 16 gives aldehyde 25, which is reduced to the alcohol and subjected to O-alkylation with a benzyl-protected triflate, followed by deprotection and esterification to give 28. Reductive amination of 25 gives acid 29, which is then esterified to give 30.

[0283] Compound 35 can be synthesized in a similar manner as reported in the literature (J. Org. Chem., 1996, 61, 6442-6445). Compounds 38, 42, and 44 are prepared as described above for Scheme 48.

[0284] Activated esters 8, 13, 24, 38, 28, 42, 30, and 44 were coupled with hydroxyprolinol containing triamine (45) or monoamine (48) to give 46 and 49, respectively. These compounds were converted to their corresponding phosphoramidites or loaded onto solid supports.

[0285] DMTr-protected di-azide 53 was coupled with an alkyne-containing GalNAc derivative using click chemistry to give compound 54, which was converted to its corresponding phosphoramidite or loaded onto a solid support to give 55.

[0286] Example 22. Synthesis of C2-derivatized galactosamine analogs for ASGPR binding (Schemes 52-53) C2-derivatized galactosamine analogs can be prepared as shown in Schemes 52 and 53 below.

[0287] [ka]

[0288] Compound 58 is prepared in a manner similar to reported procedures (see, for example, WO 96 / 39411). O-Alkylation followed by selective cleavage of the acetyl group gives compounds 60 and 63. NHS esters 61 and 64 are prepared by standard esterification. Selective removal of the acetyl group of 65 and protection of the resulting hydroxyl group with a benzyl group gives 66. Oxidative cleavage of the terminal alkene gives 67. Esterification followed by hydrogenation gives 61 / 64.

[0289] [ka]

[0290] Trifluoromethylacetamide-(TFA-) protected galactosamine (GalN-TFA) NHS ester is coupled to amine-containing oligonucleotides (69 / 71) in a post-synthetic manner to generate Gal-TFA-containing oligonucleotides (70 / 72).

[0291] Example 23. Synthesis of ASGPR Ligand Mimetics Containing a Pseudouridine Backbone (Schemes 55-56) Pseudouridine ligands can be prepared as shown in Schemes 55 and 56.

[0292] [ka]

[0293] Compound 452: To a solution of pseudouridine 451 (20 g, 81.9 mmol) in 1 M triethylammonium bicarbonate buffer (pH 8.5, 780 mL) and EtOH (940 mL) was added methyl acrylate (235 mL, 2.61 mol) dropwise. The reaction mixture was stirred for 16 h. After removal of the solvent, the crude material was purified by silica gel column chromatography (10% MeOH in CHCl, R f =0.23) to give compound 452 (26.6 g, 80.5 mmol, 98%). 1 H NMR (MeOH-d4, 400MHz): δ7.77(d,J=0.8Hz, 1H), 4.58(d,J=4.8Hz, 1H), 4.15(t,J=5.2Hz, 1H), 4.05(t,J=5.0Hz, 1H), 3.98-4.0 2(m,2H), 3.91-3.94(m,1H), 3.80(dd,J=12.0Hz, 3.3Hz, 1H), 3.67(s,3H), 3.66(dd,J=12.0Hz, 3.3Hz, 1H), 2.73-2.77(m,2H). 13 C NMR (CDCl3, 100MHz): δ173.1, 165.4, 152.5, 145.8, 112.9, 85.6, 81.5, 75.6, 72.6, 63.3, 52.5, 46.2, 33.7.C 13 H 19MW (M+H) for N2O8 + Calculated value: 330.11, measured value: 331.0.

[0294] Compound 453: To a solution of compound 452 (11.67 g, 35.3 mmol) in DMF (65 mL), di-tert-butylsilyl bis(trifluoromethanesulfonate) (15.46 mL, 42.4 mmol) was added dropwise with stirring at 0 °C. The reaction mixture was continued to stir at 0 °C for 30 min and treated with imidazole (12.0 g, 176.5 mmol). The mixture was stirred at 0 °C for 10 min and then at room temperature for 30 min. TBDMSCl (7.98 g, 53.0 mmol) was added, and the reaction mixture was heated at 75 °C for 6 h. The reaction mixture was extracted with EtO and saturated aqueous NaHCO, dried over anhydrous NaSO, and concentrated. The residue was purified by silica gel column chromatography (hexane: EtOAc = 1:1, R f =0.50) to give compound 453 (15.0 g, 25.6 mmol, 73%). 1 H NMR (DMSO-d6, 400MHz): δ11.39(s,1H), 7.54(s,1H), 4.55(s,1H), 4.34-4.38(m,1H), 4.18(d,J=4.4Hz, 1H), 3.86-4 .00(m,5H), 3.58(s,3H), 2.67(t,J=6.6Hz, 2H), 1.02(s,9H), 0.99(s,9H), 0.89(s,9H), 0.13(s,3H), 0.087(s,3H).C 27 H 49 MW (M+H) for N2O8Si2 + Calculated value: 585.30, measured value: 585.2.

[0295] Compound 454: Compound 453 (1.24 g, 2.12 mmol) was treated with ethylenediamine (10 mL) at room temperature for 2 hours. The ethylenediamine was removed by evaporation, and the residue was dried under reduced pressure. The crude material was extracted with CHCl and saturated aqueous NaHCO, dried over anhydrous NaSO, and concentrated to give 454 as a white solid (1.16 g, 1.89 mmol, 89%). 1H NMR (MeOD-d4, 400MHz): δ7.49(s,1H), 4.63(s,1H), 4.39-4.41(m,1H), 4.29(d,J=3.6Hz, 1H), 4.00-4.04(m,5H), 3.18-3 C 28 H 53 MW (M+H) for N4O7Si2 + Calculated value 613.35, actual value 613.2.

[0296] Compound 455: To a solution of GalNAc acid (930 mg, 2.08 mmol) in DMF (10 mL) was added HBTU (789 mg, 2.08 mmol) and iPrNEt (1.65 mL, 9.45 mmol). After 10 min, compound 454 in DMF (15 mL) was added to the solution and stirred overnight. The reaction mixture was extracted with EtO and saturated aqueous NaHCO and dried over anhydrous NaSO. After evaporation, the crude material was purified by silica gel column chromatography (10% MeOH in CHCl, R f =0.43) to give compound 455 (1.83 g, 1.76 mmol, 93%). 1H NMR (DMSO-d6, 400MHz): δ11.36(s,1H), 7.98(s,1H), 7.82(d,J=9.2Hz, 1H), 7.77, (s,1H), 7.51(s,1H), 5.21(d,J=3.6Hz, 1H), 4.96(dd,J=11.4Hz, 3.4Hz, 1H), 4.53(s,1H), 4.48(d,J=8.4Hz, 1H), 4.33-4.36(m,1H), 4.18(d,J=4.4Hz, 1H), 3.85- 4.02(m,9H), 3.67-3.73(m,1H), 3.37-3.43(m,1H), 3.04(s,4H), 2.39-2.44(m,2H), 2.10(s,3H), 2.02-2.05(m,2H), 1.99 (s,3H), 1.89(s,3H), 1.77(s,3H), 1.46-1.49(m,4H), 1.01(s,9H), 0.99(s,9H), 0.89(s,9H), 0.12(s,3H), 0.080(s,3H). 13 C NMR (DMSO-d6, 100MHz): δ172.0, 169.8, 169.7, 169.5, 169.2, 162.3, 150.3, 143.4, 110.6, 100.8, 83.4, 76.2, 74.7, 73.1, 70.3, 69.7, 68.5, 67.5, 66.6, 61.3, 54.9, 54.8, 49.3, 44.6, 38.3, 38.0, 34.9, 33.9, 28.5, 27.3, 26.7, 25.7, 25.6, 22.6, 22.0, 21.6, 20.4, 20.3, 19.8, 17.8, -4.5, -5.1.C 47 H 79 N5NaO 17 MW for Si2 (M+Na) + Calculated value 1064.49, actual value 1064.2.

[0297] Compound 456: Hydrogen fluoride-pyridine (approximately 70% HF, 0.165 mL, 6.34 mmol) was diluted with pyridine (2 mL) while cooling. The resulting solution was added to a solution of compound 455 in CHCl at 0° C., and the mixture was stirred at 0° C. for 2 hours. The reaction solution was diluted with CHCl, washed with saturated aqueous NaHCO, and dried over anhydrous NaSO. After evaporation, the crude material was dried under reduced pressure to give a white foam. To a solution of this material in pyridine (15 mL) was added DMTrCl (596 mg, 1.76 mmol). The reaction mixture was stirred at room temperature for 4 hours and then evaporated. The residue was extracted with CHCl and saturated aqueous NaHCO and dried over anhydrous NaSO. The crude material was purified by silica gel column chromatography (10% MeOH in CHCl, R f =0.57) to give compound 456 (1.65 g, 1.37 mmol, 78%). 1 H NMR (DMSO-d6, 400MHz): δ11.33(s,1H), 7.92(s,1H), 7.81(d,J=9.6Hz, 1H), 7.75, (s,1H), 7.42-7.44(m,3H), 7.19-7.32(m,7H), 6.87-6.9 0(m,4H), 5.21(d,J=3.2Hz, 1H), 4.96(dd,J=11.4Hz, 3.4Hz, 1H), 4.63(d,J=6.4Hz, 1H), 4.53(d,J=2.4Hz, 1H), 4.48(d,J=8.4Hz, 1H), 4.02 -4.07(m,4H), 3.81-3.91(m,3H), 3.73(s,6H), 3.68-3.70(m,2H), 3.53-3.63(m,1H), 3.23-3.40(m,2H), 3.02-3.14(m,5H), 2.32-2.35(m, 2H), 2.10(s,3H), 2.00-2.04(m,2H), 1.99(s,3H), 1.89(s,3H), 1.76( s,3H), 1.44-1.47(m,4H), 0.87(s,9H), 0.064(s,3H), 0.041(s,3H).C 60 H 81 N5NaO 19 MW for Si (M+Na) + Calculated value 1226.52, actual value 1226.4.

[0298] Compound 457: To a solution of compound 456 (1.86 g, 1.54 mmol) in CHCl (20 mL) was added 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.47 mL, 4.63 mmol) and 4,5-dicyanoimidazole (182 mg, 1.54 mmol) at 0 °C. The reaction mixture was stirred at room temperature under an argon atmosphere for 20 h. The reaction mixture was diluted with CHCl (300 mL) and washed with saturated NaHCO (100 mL). The organic layer was separated and dried over anhydrous NaSO. The filtrate was concentrated, and the resulting crude material was purified by silica gel column chromatography (EtOAc, followed by 0-3% MeOH in CHCl) to give 457 (1.80 g, 1.28 mmol, 83%, R f = 0.43) was obtained by development with 10% MeOH in CH2Cl2. 1 H NMR (400MHz, DMSO-d6): δ11.34(s,0.5H), 11.33(s,0.5H), 7.91(s,1H), 7.81 (d,J=9.2, 1H), 7.75(s,1H), 7.56(s,0.5H), 7.52(s,0.5H), 7.43(t,J=8.2, 2H ), 7.19-7.32(m,7H), 6.85-6.90(m,4H), 5.21(s,0.5H), 5.21(s,0.5H), 4.96 (dd,J=11.2, 3.4, 1H), 4.47-4.51(m,2H), 4.36-4.41(m,1H), 4.02-4.07(m,5H ), 3.83-3.90(m,1H), 3.73(s,3H), 3.72(s,3H), 3.69-3.71(m,3H), 3.31-3.6 0(m,xxH), 3.04-3.26(m,6H), 2.69-2.73(m,1H), 2.35(t,J=6.3, 2H), 2.10(s, 3H), 2.03(m,2H), 1.99(s,3H), 1.89(s,3H), 1.77(s,3H), 1.45-1.48(m,4H), 0 .91-1.08(m,12H), 0.85(s,9H), 0.063(s,1.5H), 0.046(s,1.5H), 0.035(3H). 31 P NMR (DMSO-d6, 162MHz) δ147.92, 147.70.C69 H 98 N7NaO 20 MW (M+Na) for PSi + Calculated value: 1426.63, measured value: 1426.5.

[0299] [ka]

[0300] Compound 458: To a solution of 452 (21.5 g, 65.1 mmol) in pyridine (400 mL) was added DMAP (1.59 g, 13.0 mmol) and DMTrCl (22.1 g, 65.1 mmol). The reaction mixture was stirred at room temperature for 6 hours and then evaporated. The residue was extracted with EtOAc and saturated aqueous NaHCO, dried over anhydrous NaSO, and purified by silica gel column chromatography (5% MeOH in CHCl, R f =0.30) to give 458 (36.2 g, 57.2 mmol, 88%). 1 H NMR (DMSO-d6, 400MHz): δ11.37(s,1H), 7.48(s,1H), 7.36(d,J=8.0Hz, 2H), 7 .27-7.32(m,6H), 7.20-7.23(m,1H), 6.87-6.90(m,4H), 5.06(d,J=4.8Hz, 1H ), 4.80(d,J=6.4Hz, 1H), 4.54(d,J=2.8Hz, 1H), 3.84-3.93(m,1H), 3.73(s,6 H), 3.56-3.69(m,2H), 3.53(s,3H), 3.15-3.17(m,2H), 2.58(t,J=6.6Hz, 2H). 13 C NMR (MeOH-d4, 100MHz): δ172.7, 165.5, 160.2, 152.7, 146.4, 144.5, 137.4, 137.3, 131.5, 131.4, 129.6, 128.9, 128.0, 114.2, 114.0, 87.5, 83.0, 81.1, 76.2, 72.4, 64.7, 55.8, 52.4, 46.2, 33.5.C 34 H 36 N2NaO 10 MW (M+Na) +Calculated value 655.23, measured value 655.2.

[0301] Compound 459: Compound 458 (13.9 g, 22.0 mmol) was treated with ethylenediamine (75 mL) at room temperature for 18 hours. The ethylenediamine was removed by evaporation and coevaporated with toluene. The residue was extracted with CHCl / MeOH (180 mL / 20 mL) and HO (50 mL), and the organic layer was dried over anhydrous NaSO and then concentrated. The crude material was crystallized from hexane and CHCl to give 459 as a pale yellow solid (11.7 g, 17.7 mmol, 80%). 1 H NMR (MeOD-d4, 400MHz): δ7.57(s,1H), 7.21-7.48(m,9H), 6.86-6.88(m,4H), 4.71(d,J=3.2Hz, 1H), 4.02-4.17(m,3H), 3 .79-3.82(m,1H), 3.78(s,6H), 3.31-3.36(m,3H), 3.15(t,J=6.2Hz, 2H), 2.63(t,J=6.0Hz, 2H), 2.41(t,J=6.2Hz, 2H).C 35 H 40 MW (M+H) for N4NaO9 + Calculated value 683.27, actual value 683.2.

[0302] Compound 460: To a solution of GalNAc acid (5.60 g, 12.5 mmol) in DMF (50 mL) was added HBTU (4.70 g, 12.4 mmol) and iPrNEt (10.3 mL, 59.3 mmol). After 10 min, compound 459 in DMF (50 mL) was added to the solution and stirred overnight. The reaction mixture was extracted with EtOAc and HO and dried over anhydrous NaSO. After evaporation, the crude material was purified by silica gel column chromatography (10% MeOH in CHCl, R f =0.50) to give compound 460 (6.85 g, 6.28 mmol, 59%). 1H NMR (DMSO-d6, 400MHz): δ11.33(s,1H), 7.93(s,1H), 7.81(d,J=9.2Hz, 1H), 7.75, (s,1H), 7.41-7.44(m,3H), 7.27-7.31(m,6H), 7.18 -7.22(m,1H), 6.87-6.89(m,4H), 5.21(d,J=3.2Hz, 1H), 5.03(d,J=4.8Hz, 1H), 4.96(dd,J=11.2Hz, 3.6Hz, 1H), 4.78(d,J=6.4Hz, 1H) , 4.51(d,J=2.8Hz,1H), 4.48(d,J=8.4Hz,1H), 4.02(m,3H), 3.82-3.92(m,4H), 3.73(s,6H), 3.54-3.70(m,3H), 3.36-3.42(m,1H), 3. 02-3.21(m,6H), 2.35(t,J=6.6Hz,2H), 2.09(s,3H), 2.02(t,J=7.0Hz,2H), 1.99(s,3H), 1.88(s,3H), 1.76(s,3H), 1.43-1.49(m,4H). 13 C NMR (DMSO-d6, 100MHz): δ172.0, 169.9, 169.8, 169.5, 169.4, 169.2, 162.6, 15 7.9, 150.3, 144.9, 143.2, 135.7, 135.6, 129.7, 127.7, 126.5, 113.0, 111.3, 1 00.9, 85.2, 80.7, 79.8, 73.5, 70.8, 70.4, 69.7, 68.5, 66.6, 64.1, 61.3, 54.9, 54.8, 49.3, 48.5, 44.8, 38.3, 38.1, 34.9, 33.9, 28.5, 22.7, 21.6, 20.4, 20.3.C 54 H 67 N5NaO 19 についてのMW(M+Na) + The calculated value is 1112.43 and the measured value is 1112.2.

[0303] Compound 461: To a solution of compound 460 (1.55 g, 1.42 mmol) in pyridine (10 mL) was added TBDMSCl (214 mg, 1.42 mmol) and imidazole (290 mg, 4.26 mmol). The reaction mixture was stirred overnight. After evaporation, the residue was extracted with CHCl and saturated aqueous NaHCO and dried over anhydrous NaSO. The crude material was purified by silica gel column chromatography (5% MeOH in CHCl, R f =0.15) to give compound 461 (550 mg, 0.457 mmol, 32%) and its 2'-O-TBDMS isomer 456 (390 mg, 0.324 mmol, 23%). 1 H NMR (DMSO-d6, 400MHz): δ11.32(s,1H), 7.94(s,1H), 7.82(d,J=9.2Hz, 1H), 7.75, (s,1H), 7.54(s,1H), 7.40-7.41(m,2H), 7.21-7.3 2(m,7H), 6.87-6.89(m,4H), 5.21(d,J=3.2Hz, 1H), 4.96(dd,J=11.2Hz, 3.6Hz, 1H), 4.73(d,J=4.8Hz, 1H), 4.47-4.49(m,2H), 3.95-4 .02(m,5H), 3.83-3.88(m,2H), 3.72(s,6H), 3.68-3.71(m,3H), 3.38-3.41(m,1H), 3.03-3.19(m,6H), 2.39(t,J=6.6Hz, 2H), 2.10(s C 60 H 81 N5NaO 19 MW for Si (M+Na) + Calculated value: 1226.52, measured value: 1227.4.

[0304] Compound 462: To a solution of compound 461 (2.28 g, 1.89 mmol) in CHCl (60 mL) was added DMAP (693 mg, 5.67 mmol) and succinic anhydride (378 mg, 3.78 mmol). The reaction mixture was stirred overnight at room temperature. Silica gel column chromatography of the crude mixture without aqueous workup (10% MeOH / 10% EtN in CHCl, R f =0.44) to give compound 462 as the corresponding triethylammonium salt (2.50 g, 1.78 mmol, 94%). 1 H NMR (DMSO-d6, 400MHz): δ8.42(s,1H), 8.18(s,1H), 8.05(d,J=9.2Hz, 1H), 7.71(s,1H), 7.48-7.50(m,2H), 7.29-7.40(m,7H), 6.95-6.97 (m,4H), 5.28-5.30(m,2H), 5.07(dd,J=11.2Hz, 3.6Hz, 1H), 4.70(d,J=4.0Hz, 1H), 4.60(d,J=8.4Hz, 1H), 4.37(t,J=5.8Hz, 1H), 4.09-4.1 3(m,3H), 3.91-3.97(m,2H), 3.81(s,6H), 3.78-3.85(m,3H), 3.42-3.49(m,2H), 3.27-3.30(m,1H), 3.10-3.16(m,5H), 2.43-2.53(m,5H) , 2.18(s,3H), 2.12(t,J=7.2Hz, 2H), 2.07(s,3H), 1.97(s,3H), 1.85(s,3H), 1.52-1.57(m,4H), 0.79(s,9H), 0.00(s,3H), -0.075(s,3H). 13C NMR (DMSO-d6, 100MHz): δ173.8, 172.2, 172.1, 171.5, 169.9, 169.6, 169.5, 169.3, 162.5, 158 .1, 150.4, 144.7, 144.6, 135.5, 135.4, 129.7, 127.7, 126.6, 113.1, 109.5, 100.9, 85.6, 81.6 , 77.5, 74.2, 71.0, 70.5, 69.8, 68.5, 66.7, 63.6, 61.4, 52.0, 49.3, 38.4, 38.2, 34.9, 34.0, 30 .0, 29.5, 28.5, 25.8, 25.5, 25.4, 22.7, 21.6, 21.4, 20.5, 20.4, 17.5, 14.7, 7.1, -5.1, -5.4.C 64 H 84 N5O 22 MW(MH) for Si - Calculated value: 1302.54, measured value: 1302.4.

[0305] Compound 463: To a solution of compound 462 (98 mg, 0.07 mmol) in DMF (10 mL), HBTU (30 mg, 0.077 mmol), iPrNEt (0.061 mL, 0.35 mmol), and aminomethyl polystyrene support (ARTVISION, 70 mol / g, 1.10 g, 0.077 mmol) were added sequentially. The mixture was shaken for 24 h, then filtered, washed with CHCl, and dried under vacuum. The remaining amino groups were capped by shaking with pyridine (15 mL), acetic anhydride (5 mL), and triethylamine (1 mL) for 1 h. After filtration, the mixture was washed with CHCl (100 mL), then 50% MeOH / CHCl (100 mL), and dried under vacuum to give compound 463 (1.12 g). Loading: 47 mol / g.

[0306] Example 24. Synthesis of ASGPR Ligand Mimetics Containing N-Glycosidic Linkages (Schemes 57-63) ASGPR ligands containing N-glycosidic bonds can be prepared as shown in Schemes 57-63.

[0307] [ka]

[0308] [ka]

[0309] [ka]

[0310] [ka]

[0311] [ka]

[0312] Example 25. ASGPR Ligand Mimetics (Schemes 64-73) The following ASGPR ligands can be prepared as shown in Schemes 64-73.

[0313] [ka]

[0314] [ka]

[0315] [ka]

[0316] [ka]

[0317] [ka]

[0318] [ka]

[0319] Example 26. Amino Linkers for Conjugating Ligands to Oligonucleotides (Schemes 74-76) Amino linkers for conjugating ligands to oligonucleotides can be prepared according to Schemes 74-76 below.

[0320] [ka]

[0321] [ka]

[0322] Example 27. ASGPR Ligand Mimics - Carbohydrate Backbone The following ASGPR ligands can be prepared as shown in Scheme 77: [ka]

[0323] Example 28. Conjugation of GalNAc Ligands to C2 of Purine Bases A GalNAc ligand can be prepared at the C2 position of the purine base as shown in Scheme 78 below. [ka]

[0324] Q A and Q B is any of the ASGPR ligands described herein.

[0325] Example 29. siRNA-ligand conjugates RNA synthesis and double-strand annealing

[0326] 1. Oligonucleotide Synthesis All oligonucleotides were synthesized on an AKTA oligopilot synthesizer or an ABI 394 synthesizer. Unless otherwise specified, oligonucleotides were synthesized on a commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime RNA phosphoramidites with standard protecting groups (Pierce Nucleic Acids Technologies) and 5'-O-dimethoxytrityl-N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N2-isobutyl-2'-t-butyldimethylsilyl-guanosine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite (Pierce Nucleic Acids Technologies) were used for oligonucleotide synthesis. 2'-F phosphoramidites, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluoro-cytidine-3'-ON,N'-diisopropyl-2-cyanoethyl-phosphoramidite, and 5'-O-dimethoxytrityl-2'-fluoro-uridine-3'-ON,N'-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from Promega. All phosphoramidites were used at a concentration of 0.2 M in acetonitrile (CH3CN), except for guanosine, which was used at a concentration of 0.2 M in 10% THF / ANC (v / v). A 16-minute coupling / recycling time was used. The activator was 5-ethylthiotetrazole (0.75 M, American International Chemicals); for PO-oxidation, iodine / water / pyridine was used, and for PS-oxidation, PADS (2%) in 2,6-lutidine / ACN (1:1 v / v) was used.

[0327] Ligand-conjugated chains were synthesized using solid supports containing the corresponding ligands. For example, the introduction of a carbohydrate moiety / ligand (e.g., in the case of GalNAc) at the 3' end of the sequence was carried out by initiating synthesis on the corresponding carbohydrate solid support. Similarly, the introduction of a cholesterol moiety at the 3' end was carried out by initiating synthesis on a cholesterol support. Generally, the ligand moiety was linked to trans-4-hydroxyprolinol via the optimal tether described in the previous example to obtain a hydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety was then attached to the solid support via a succinate linker or converted to a phosphoramidite using standard phosphitylation conditions to obtain the desired carbohydrate conjugate building block. Fluorophore-labeled siRNA was synthesized from the corresponding phosphoramidite or solid support (purchased from Biosearch Technologies). The oleyllithocholic acid (GalNAc)3 polymer support was produced in-house with a loading of 38.6 μmol / gram. A mannose (Man)3 polymeric carrier was also produced in-house with a loading of 42.0 μmol / gram.

[0328] Conjugation of the ligand of choice at the desired position, e.g., the 5' end of the sequence, was carried out by coupling the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions, unless otherwise specified: coupling of a 0.1 M solution of the phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to the solid-bound oligonucleotide for a prolonged 15 min period. (1)Beaucage,SL(2008)Solid-phase synthesis of siRNA oligonucleotide.Curr.Opin.Drug Discov.Devel.,11,203-216;(2)Mueller,S.,Wolf,J.and Ivanov,SA(2004)Current Strategies for the Synthesis of RNA.Curr.Org.Synth., 1,293-307 and (3) Ribo-difluorotoluyl Nucleotide.ACS Oxidation of internucleotide phosphites to phosphates was carried out using standard iodine-water as reported in Chem. Biol., 1, 176-183, or by treatment of the conjugated oligonucleotide with tert-butyl hydroperoxide / acetonitrile / water (10:87:3) with a 10-minute oxidation wait time. Phosphorothioates were introduced by oxidation of phosphites to phosphorothioates using sulfur transfer agents such as DDTT (purchased from AM Chemicals), PADS, and / or Beaucage reagent. Cholesterol phosphoramidites were synthesized in-house and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterol phosphoramidites was 16 minutes.

[0329] 2. Deprotection-I (nucleobase deprotection) After completion of the synthesis, the support was transferred to a 100 ml glass bottle (VWR). The oligonucleotide was cleaved from the support using 80 ml of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] at 55 °C for 6.5 hours, simultaneously deprotecting the base and phosphate groups. The bottle was briefly cooled on ice, and then the ethanolic ammonia mixture was filtered into a new 250 ml bottle. The CPG was washed with 2 x 40 ml portions of ethanol / water (1:1 v / v). The volume of the mixture was then reduced to approximately 30 ml by rotary evaporation (roto-vap). The mixture was then frozen on dry ice and dried under reduced pressure in a speed vac.

[0330] 3. Deprotection-II (Removal of 2'TBDMS group) The dried residue was resuspended in 26 ml of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60°C for 90 min to remove the tert-butyldimethylsilyl (TBDMS) group at the 2'-position. The reaction was then quenched with 50 ml of 20 mM sodium acetate, the pH was adjusted to 6.5, and the mixture was stored in a freezer until purification.

[0331] 4.Analysis Prior to purification, oligonucleotides are analyzed by high performance liquid chromatography (HPLC), with the choice of buffer and column depending on the sequence and or nature of the conjugated ligand.

[0332] 5.HPLC purification Ligand-conjugated oligonucleotides were purified by reverse-phase preparative HPLC. Unconjugated oligonucleotides were purified by anion-exchange HPLC on a TSK gel column packed in-house. The buffers were 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (Buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1 M NaBr (Buffer B). Fractions containing full-length oligonucleotides were pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotide was diluted to 150 μl with water and then pipetted into specialized vials for CGE and LC / MS analysis. The compounds were finally analyzed by LC-ESMS and CGE.

[0333] 6. Preparation of RNAi Agents To prepare the RNAi agent, equimolar amounts of the sense strand and antisense strand were heated in 1x PBS at 95°C for 5 minutes and then slowly cooled to room temperature. The integrity of the duplex was confirmed by HPLC analysis. Table 1 below shows the synthesized RNAi agents.

[0334] Example 30: Synthesis of siRNA-ligand conjugates using post-synthetic methods Single-stranded oligonucleotides containing the desired amino linker were synthesized using the corresponding amino linker monomers compatible with the solid-phase oligonucleotide synthesis and deprotection conditions described in Example 30 (Scheme 79). After deprotection, the amino-linked oligonucleotides were reacted with the NHS esters of the ligands shown in the table below Scheme 79, followed by treatment with ammonia and HPLC purification. Each purified ligand-conjugated single-stranded oligonucleotide was annealed with an equimolar mixture of complementary strands to yield the siRNAs shown in Table 5. The (1+1+1) design shown in Scheme 79 and Table 5 was obtained by sequentially attaching the amino linker phosphoramidite to the amino linker solid support (two synthesis cycles), followed by sequentially attaching the nucleoside phosphoramidite monomers, as described in Example 29.

[0335] Post-synthetic conjugation of ligands to oligonucleotides [ka]

[0336] The TTR siRNA in each conjugate was the same. Similarly, the AT3 siRNA in each conjugate was the same. The following ligands were attached to the 3' end of the sense strand of each siRNA. The left side of the ligand designation indicates the binding site to the 3' end of the sense strand.

[0337] TIFF0007676460000130.tif162166TIFF0007676460000131.tif192166TIFF0007676460000132.tif88166

[0338] Example 31: In vitro screening of RNAi agents Cell culture and transfection Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, VA) were grown to near confluence in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C in a 5% CO atmosphere and then released from the plate by trypsinization. Transfection was performed by adding 14.8 μl of Opti-MEM and 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cat# 13778-150) per well to 5 μl of siRNA duplexes per well in a 96-well plate and incubating at room temperature for 15 minutes. Next, 80 μl of complete growth medium without antibiotics containing approximately 2 × 10 Hep3B cells was added to the siRNA mixture. Cells were incubated for 24 or 120 hours before RNA purification. Single dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose response experiments were performed using 8, 4-fold serial dilutions with the highest dose at a final duplex concentration of 10 nM.

[0339] Total RNA isolation using DYNABEADS mRNA isolation kit (Invitrogen, part#:610-12) Cells were harvested and lysed in 150 μl of lysis / binding buffer, then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl of lysis / binding buffer mixture were added to a round-bottom plate and mixed for 1 minute. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were recaptured and the supernatant was removed. Next, the beads were washed with 150 μl of wash buffer B, captured, and the supernatant was removed. Next, the beads were washed with 150 μl of elution buffer, captured, and the supernatant was removed. The beads were dried for 2 minutes. After drying, 50 μl of elution buffer was added and mixed for 5 minutes at 70°C. The beads were captured on a magnet for 5 minutes. 40 μl of the supernatant was removed and added to another 96-well plate.

[0340] cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat# 4368813) A master mix of 1 μl of 10x buffer, 0.4 μl of 25x dNTPs, 1 μl of random primers, 0.5 μl of reverse transcriptase, 0.5 μl of RNase inhibitor, and 1.6 μl of HO was added to 5 μl of total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermocycler (Hercules, CA) by the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and a 4°C hold.

[0341] Real-time PCR Two microliters of cDNA was added to a master mix containing 0.5 μl of GAPDH TaqMan Probe (Applied Biosystems Cat#4326317E (human) Cat#4308313 (rodent)), 0.5 μl of TTR TaqMan probe (Applied Biosystems Cat#HS00174914_m1 (human) Cat#Rn00562124_m1 (rat)), and 5 μl of Lightcycler 480 Probe Master Mix (Roche Cat#04887301001) per well in a 384-well plate (Roche Cat#04887301001). Real-time PCR was performed in a Roche LC 480 real-time PCR machine (Roche). Unless otherwise noted, each duplex was tested in at least two independent transfections, and each transfection was assayed in duplicate.

[0342] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC was calculated using a four-parameter fit model using XLFit. 50 The IC was calculated and normalized to the lowest dose of non-target-specific control transfected or naive cells over the same dose range, or to the lowest dose of the compound itself. 50 was calculated for each individual transfection as well as in combination, where one IC 50 was fitted to the data from both transfections.

[0343] Example 32: In vitro silencing activity of chemically modified RNAi agents targeting TTR The following experiments demonstrated the beneficial effects of chemical modifications, including the introduction of triplet repeat motifs together with GalNAc3 ligands, on the silencing activity of RNAi agents targeting TTR.

[0344] IC in Hep3B cells50 Protocol for the assessment of IC of each modified siRNA 50 The IC was determined in Hep3B cells by standard reverse transfection using Lipofectamine RNAiMAX. Briefly, reverse transfection was performed by adding 5 μL of Opti-MEM to 5 μL of siRNA duplex per well in a 96-well plate, along with 10 μL of Opti-MEM and 0.5 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cat# 13778-150) per well, and incubated at room temperature for 15–20 minutes. After incubation, 100 μL of complete growth medium without antibiotics containing 12,000–15,000 Hep3B cells was then added to each well. Cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours before lysis and analysis of TTR and GAPDH mRNA by bDNA (Quantigene). Seven different siRNA concentrations ranging from 10 nM to 0.6 pM were used to determine the IC. 50 The TTR / GAPDH levels in siRNA-transfected cells were normalized to those in cells transfected with 10 nM Luc siRNA.

[0345] Free-uptake IC 50 Protocol for the assessment of Free uptake silencing in primary cynomolgus monkey or mouse hepatocytes was assessed after 4 or 24 hours of incubation with TTR siRNA. Silencing was measured 24 hours after initial exposure.

[0346] Example 33: TTR mRNA silencing and TTR protein suppression in mice To evaluate the efficacy of RNAi agents, these agents were administered to mice. Mice were administered a single subcutaneous dose of RNAi agent or PBS control at 5 mg / kg or 1 mg / kg. Approximately 48 hours later, mice were anesthetized with 200 μl of ketamine and then exsanguinated by cutting the right tail artery. Whole blood was isolated, and plasma was isolated and stored at -80°C until assay. Liver tissue was harvested, flash-frozen, and stored at -80°C until processing.

[0347] Treatment efficacy was assessed by (i) measuring TTR mRNA in the liver at 48 and 144 hours post-dose, and (ii) measuring TTR protein in plasma before exsanguination and at 48 and 144 hours post-dose. TTR liver mRNA levels were assayed using the branched DNA assay - QuantiGene 2.0 (Panomics cat#: QS0011). Briefly, mouse liver samples were mashed and tissue lysates were prepared. The liver lysis mixture (a mixture of 1 volume of lysis mixture, 2 volumes of nuclease-free water, and 10 μl of proteinase-K / ml, resulting in a final concentration of 20 mg / ml) was incubated at 65°C for 35 minutes. Next, 20 μl of the Working Probe Set (TTR probes for gene targets and GAPDH as an endogenous control) and 80 μl of tissue lysate were added to a capture plate. The capture plate was incubated at 55°C ± 1°C (approximately 16–20 hours). The next day, the capture plate was washed three times with 1x wash buffer (nuclease-free water, buffer component 1, and wash buffer component 2) and then dried by centrifugation at 240g for 1 minute. 100 μl of pre-amplifier working reagent was added to the aluminum foil-sealed capture plate and incubated for 1 hour at 55°C ± 1°C. After the 1-hour incubation, the wash step was repeated, and then 100 μl of amplifier working reagent was added. After 1 hour, the wash and drying steps were repeated, and 100 μl of Label Probe was added. The capture plate was incubated for 1 hour at 50°C ± 1°C. The plate was then washed with 1x wash buffer, dried, and 100 μl of substrate was added to the capture plate. The capture plate was read using a SpectraMax Luminometer after a 5-15 minute incubation. The bDNA data were analyzed by subtracting the average background from each of the three samples, averaging the three resulting GAPDH (control probe) and TTR (experimental probe) values, and then calculating the ratio (experimental probe minus background) / (control probe minus background).

[0348] Plasma TTR levels were assayed using a commercially available kit according to the manufacturer's guidelines. Briefly, mouse plasma was diluted 1:10,000 in 1x mixture diluent and added to a pre-coated plate along with the kit standard. After incubation at room temperature for 2 hours, the plate was washed five times with the kit's wash buffer. Fifty microliters of biotinylated prealbumin antibody was added to each well, incubated at room temperature for 1 hour, and then washed five times with the wash buffer. Fifty microliters of streptavidin-peroxidase conjugate was added to each well, and the plate was incubated at room temperature for 30 minutes before being washed as described above. The reaction was developed by adding 50 μl / well of chromogenic substrate, incubated at room temperature for 10 minutes, and stopped by adding 50 μl / well of stop solution. Absorbance at 450 nm was read on a Versamax microplate reader (Molecular Devices, Sunnyvale, Calif.) and data were analyzed using the Softmax 4.6 software package (Molecular Devices).

[0349] Representative siRNA-conjugate efficacy results are shown in Figure 1. Most of the anomeric binding modified ligands showed similar TTR protein inhibition at a dose of 5 mg / kg.

[0350] Example 34: AT3 mRNA silencing in vitro and in vivo In vivo gene silencing of AT3 in mice was determined by a single subcutaneous administration of the AT3 siRNA-ligand conjugate, following a protocol similar to that described for in vivo TTR gene silencing in Example 33. In vitro gene silencing was assessed following a protocol similar to that described in Examples 31 and 32.

[0351] siRNA conjugates 54944 and 56881 were administered subcutaneously to C57 / BL6 mice at three different single dose levels: 5, 10, and 25 mg / kg, and AT3 protein levels compared to PBS controls were measured 72 hours after administration. The results are shown in Figure 2. Conjugates 54944 and 56881 are referred to above and in Figure 2 as 70001 and 70002, respectively.

[0352] Example 35: Synthesis of mono-GalNAc building blocks for oligonucleotide conjugation The mono-GalNAc building block shown can be prepared as shown in Scheme 80. [ka]

[0353] Synthesis of 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyprolinamine (10.00 g, 18.77 mmol) were placed together in dichloromethane. HBTU (10.68 g, 28.12 mmol) and DIEA (9.80 mL, 3 equiv.) were added, and the mixture was stirred at ambient temperature for 2 h. TLC checked, and the reaction mixture was transferred to a separatory funnel and washed with water and brine. The organic layer was dried over sodium sulfate, and the solvent was removed. The crude product was purified by silica gel chromatography using dichloromethane and MeOH as solvents to give compound 102 as a pale yellow fluffy solid (11.77 g, 63%). 1H NMR (400MHz, DMSO) δ7.80(d,J=9.2Hz, 1H), 7.69(t,J=5.6Hz, 1H), 7.39-7.09(m,9H), 6.86(ddd,J=9.0, 5.4, 2.1Hz, 4H), 5.20(d,J=3.4Hz, 1H), 5.03-4.83(m,2H), 4.47(d,J=8.5Hz, 1H), 4.41-4.07(m,2H), 4.04-3.95(m ,3H), 3.86(dt,J=11.2, 8.9Hz, 1H), 3.79-3.68(m,6H), 3.68-3.36(m,3H), 3.21-2.88(m,5H), 2.26-2.14(m, 2H), 2.09(s,3H), 2.02(t,J=6.7Hz, 2H), 1.98(s,3H), 1.87(d,J=7.5Hz, 3H), 1.76(s,3H), 1.53-1.29(m,7H).

[0354] Synthesis of 104: Hydroxyproline derivative 102 (6.00 g, 6.24 mmol) was dissolved in dichloromethane (100 mL). To it was added DIEA (2.20 mL, 3 equiv.) and chloroamidite reagent. The reaction mixture was stirred for 30 min and checked by TLC. It was transferred to a separatory funnel and washed with water and sodium bicarbonate solution. The organic layer was dried over sodium sulfate, and the crude product was purified by silica gel chromatography using dichloromethane and MeOH as eluents to give the compound as a white fluffy solid. 1H NMR (400MHz, DMSO) δ7.80(d,J=9.2Hz, 1H), 7.68(s,1H), 7.42-7.06(m,8H), 7.01-6.73(m,4H), 5.20(d,J=3.3Hz, 1H), 4 .96(dd,J=11.2, 3.3Hz, 1H), 4.63(d,J=4.7Hz, 1H), 4.47(d,J=8.5Hz, 1H), 4.15(s,1H), 4.01(s,3H), 3.86(d,J=11.0Hz, 1H), 3.70(d,J=16.5Hz, 9H), 3.45(ddd,J=37.0, 23.3, 16.4Hz, 6H), 2.99(dd,J=12.3, 6.4Hz, 3H), 2.74(dd,J=9.2, 5.8H z, 2H), 2.21(s,2H), 2.09(s,3H), 2.05-1.95(m,5H), 1.88(s,3H), 1.76(s,3H), 1.52-1.16(m,11H), 1.16-1.02(m,11H). 31 P NMR δ=151.78, 151.61, 151.50, 151.30.

[0355] Synthesis of 105: Compound 102 (2.10 g, 2.18 mmol) was dissolved in DCM (20 mL). To this mixture, succinic anhydride (0.441 g, 4.36 mmol) and DMAP (0.532 g, equiv.) were added, followed by TEA (1 mL). The reaction mixture was stirred overnight at room temperature. TLC of the reaction mixture was checked, and the reaction mixture was washed with water and brine. The organic layer was dried over sodium sulfate, and the crude product was filtered through a small pad of silica gel. The solvent was removed, and this material was used in the next reaction. The succinate from the above reaction was dissolved in anhydrous acetonitrile. HBTU (1.59 g, 4.20 mmol) and DIEA (1.10 mL) were added, and the mixture was rotated for 5 minutes. A polystyrene solid support was added to the reaction mixture, and the mixture was shaken at ambient temperature overnight. The solid support was filtered, washed, and capped with an acetic anhydride / Py mixture. The solid support was washed again with dichloromethane, MeOH / DCM and ether (27.10 g, 55 umol / g).

[0356] Example 36: Synthesis of mono-GalNAc NHS ester for oligonucleotide conjugation Mono-GalNAc NHS esters useful for oligonucleotide conjugation can be prepared as shown in Schemes 81-86 below.

[0357] [ka]

[0358] [ka]

[0359] [ka]

[0360] Example 37: Synthesis of Amide and Carbamate Linked Building Blocks for Oligonucleotide Conjugation [ka]

[0361] Synthesis of compound 5001: To a stirred solution of compound 5000 (23.22 g, 63.6 mmol) in DCM, NaN (12.4 g, 190.8 mmol) and TBAHS (21.6 g, 63.6 mmol) were added, followed by 150 mL of saturated NaHCO solution. The resulting mixture was stirred for 14 hours. The reaction mixture was then extracted with ethyl acetate (3×250 ml), washed with water, brine, and dried over anhydrous NaSO. Concentration of the solvent afforded the crude material. This material was dissolved in ethyl acetate (150 mL), and 150 mL of hexane was added, resulting in precipitation of the product as a white solid. The solid was dried under reduced pressure to afford compound 5001 (16.2 g, 68.4%). LCMS calculated for compound 5001: 372.33 (M + ), Actual measurement: 407.1 (M - +Cl- ).

[0362] Synthesis of compound 5002: To a stirred solution of compound 5001 (13.2 g, 35.5 mmol) in THF (600 mL) was added PtO (0.6 g), and the reaction mixture was stirred at room temperature under a hydrogen atmosphere for 14 hours. The catalyst was removed by filtration. Concentration of the solvent gave compound 5002 (13.0 g).

[0363] Synthesis of compound 5003: To a stirred solution of compound 5002 (10.0 g, 28.89 mmol) and glutaric anhydride (3.29 g, 28.89 mmol) in DCM (100 mL) was added pyridine (4.6 g) and DMAP (0.176 g). The reaction mixture was stirred for 14 hours. The resulting mixture was concentrated and then passed through a filter column to give compound 5003 (11.45 g, 86%). LCMS for compound 5003: calculated: 460.43 (M + ), Actual measurement: 459.3 (M - -1), 495.0(M - +Cl - ).

[0364] Synthesis of compound 5004: To a stirred solution of compound 5003 (7.6 g, 16.51 mmol), NHS (2.09 g, 18.16 mmol), and EDC (3.8 g, 19.8 mmol) in DCM (100 mL) was added dropwise DIEA (7.16 mL, 41.27 mmol). The resulting mixture was stirred for 14 hours. Then, 100 mL of water was added, and the product was extracted with DCM (2×50 mL), washed with citric acid (20%), saturated NaHCO3, brine, and then dried over anhydrous Na2SO4. Concentration of the solvent afforded compound 5004 (6 g, 65%). LCMS calculated for compound 5004: 557.5 (M + ), Actual measurement: 558.0 (M + +1).

[0365] [ka]

[0366] Synthesis of compound 5005: To a stirred solution of compound 5002 (3.0 g, 8.66 mmol) in acetonitrile (50 mL) was added DSC (2.22 g, 8.66 mmol), and the resulting mixture was stirred at room temperature overnight (14 hours). The solvent was concentrated, and the product was then extracted with ethyl acetate (3 x 50 mL), washed with water, 10% citric acid, brine, and dried over anhydrous NaSO. Concentration of the solvent afforded compound 5005 (3.8 g, 95%). 19 H 25 N3O 12 LCMS calculated for: 487.41 (M + ), Actual measurement: 488.1 (M + +1), 510.1(M + +Na + ).

[0367] Synthesis of compound 5006: To a stirred solution of compound 5005 (0.663 g, 1.36 mmol) in DCM (15 mL) was added amine (0.526 g, 1.5 mmol) and triethylamine (0.4 mL). The solvent was concentrated, and the product was then extracted with ethyl acetate (3 x 50 mL), washed with water, 10% citric acid, brine, and dried over anhydrous NaSO. Concentration of the solvent afforded compound 5006 (0.7 g, 93%). 25 H 33 N3O 11 LCMS calculated value of 551.54 (M + ), Actual measurement: 552.2 (M + +1), 574.2(M + +Na + ).

[0368] Synthesis of compound 5007: To a stirred solution of compound 5005 (0.7 g, 1.26 mmol) in EtOH (15 mL), Pd / C (0.1 g) was added, and the resulting mixture was stirred overnight (14 hours) under a hydrogen atmosphere. The catalyst was removed by filtration over Celite, and the mixture was washed with EtOH (950 mL) and concentrated to give the product, which was used in the next step without purification. To a stirred solution of the above acid in DCM (20 mL), EDC (488 mg, 2.56 mmol), NHS (730 mg, 6.35 mmol), and DIEA (0.88 mL, 5.07 mmol) were added. The reaction mixture was stirred overnight. Concentration of the reaction mixture, followed by column chromatography, gave compound 5007 (250 mg, 35%). 22 H 30 N4O 13 LCMS calculated for: 558.49 (M + ), Actual measurement: 559.2 (M + +1), 581.1(M + +Na + ).

[0369] Example 38: Synthesis of S- and C-linked GalNAc derivatives and building blocks [ka]

[0370] Synthesis of compound 5012: Compound 4999 (15.6 g, 40.1 mmol) was treated with TMSOTf (7.98 mL, 44.1 mmol) in DCE to give compound 5011. 14 H 20 Molecular weight (M+H) for NO8 + Calculated 330.12, found 330.0. Compound 5011 (1.65 g, 5 mmol) and tert-butyl 5-mercaptopentanoate (1.0 g, 5.25 mmol) in DCE were treated with TMSOTf (0.181 mL, 1.0 mmol) overnight. Aqueous workup and purification by silica gel column afforded compound 5012 (380 mg, 0.731 mmol, 15%). 23 H 37 NNaO10 Molecular weight for S (M+H) + Calculated value: 542.20, measured value: 542.1.

[0371] Synthesis of compound 5013: To a solution of compound 5012 (380 mg, 0.731 mmol) in CH2Cl2 (4 mL) was added TFA (1 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and then at room temperature for 3 h. The solvent was evaporated and the residue was co-evaporated with toluene to give crude compound 5013. This material was used in the next step without purification. C 19 H 30 NO 10 Molecular weight for S (M+H) + Calculated value: 464.1590, measured value: 464.1.

[0372] Synthesis of compound 5014: Compound 5013 (approximately 0.731 mmol) from the previous step was treated with N-hydroxysuccinimide (168 mg, 1.46 mmol) in the presence of EDCI (280 mg, 1.46 mmol) and DIEA (0.764 mL, 4.38 mmol) in CHCl (5 mL) for 14 hours. Aqueous workup followed by column chromatography afforded compound 5014 (284 mg, 0.507 mmol, 69% over two steps). 23 H 33 N2O 12 Molecular weight for S (M+H) + Calculated value: 561.1754, measured value: 561.1.

[0373] S-alkylation of compound 5009 with an alkyne bromide provides compound 5010.

[0374] [ka]

[0375] Compound 5016 is prepared from compound 5015 (see J. Org. Chem., 67, 2995-2999, 2002). Acid 5017 is prepared according to the reported procedure.

[0376] Synthesis of compound 5018: Compound 5017 (2.40 g, 5.18 mmol) was treated with N-hydroxysuccinimide (716 mg, 6.22 mmol) in the presence of EDCI (1.19 g, 6.22 mmol) and DIEA (2.70 mL, 15.5 mmol) in CHCl (30 mL) for 14 hours. Aqueous workup followed by column chromatography afforded compound 5018 (1.83 g, 3.26 mmol, 63%). 23 H 33 N2O 12 Molecular weight for S (M+H) + Calculated value: 561.1754, measured value: 561.2.

[0377] [ka]

[0378] Compound 5024 was prepared using reported procedures (see J. Org. Chem., 71, 3619-362, 2006 and Carbohydrate Research, 309, 319-330, 1998).

[0379] Synthesis of compound 5025: Compound 5024 (1.94 g, 5.22 mmol), benzyl 4-pentenoate (2.99 g, 15.7 mmol), and second-generation Grubbs catalyst (433 mg, 0.522 mmol) in CHCl (20 mL) were heated at 40° C. for 40 hours. The solvent was removed, and the residue was purified by silica gel column chromatography to give compound 5025 (1.87 g, 3.50 mmol, 67%). 27 H 36 NO 10 Molecular weight (M+H) + Calculated value 534.2339, measured value 534.2.

[0380] Synthesis of compound 5028: To a solution of compound 5025 (1.85 g, 3.47 mmol) in EtOAc (30 mL) was added palladium on carbon (Aldrich: 330108-50G, 10 wt%, Degussa type E101 NE / W: 185 mg). The reaction mixture was stirred under an atmosphere of hydrogen for 14 hours. After filtration through Celite, the filtrate was removed under reduced pressure. The residue was purified by silica gel column chromatography to give compound 5028 (903 mg, 2.03 mmol, 59%). 20 H 32 NO 10 Molecular weight (M+H) + Calculated value: 446.2026, measured value: 446.1.

[0381] Synthesis of compound 5031: Compound 5028 (326 mg, 0.732 mmol) was treated with N-hydroxysuccinimide (127 mg, 1.10 mmol) in the presence of EDCI (211 mg, 1.10 mmol) and DIEA (0.383 mL, 2.20 mmol) in CHCl (5 mL) for 14 hours. Aqueous workup followed by column chromatography afforded compound 5031 (300 mg, 0.553 mmol, 76%). 24 H 35 N2O 12 Molecular weight (M+H) + Calculated value 543.2190, measured value 543.2.

[0382] Oxidative cleavage of compound 5024 gives aldehyde 5026, which is reduced to the alcohol, followed by O-alkylation with a benzyl-protected triflate, followed by deprotection and esterification to give compound 5032. Reductive amination of compound 5026 gives acid compound 5030, which is esterified to give compound 5033.

[0383] [ka]

[0384] Compound 5038 was prepared in a manner similar to that reported in the literature (see J. Org. Chem., 61, 6442-6445, 1996). Compounds 5046, 5047, and 5048 were prepared in a manner similar to that shown in Scheme 91.

[0385] Example 39 [ka]

[0386] Synthesis of compound 5050: N-acetylglucosamine (10 g) was refluxed with hexanol (excess) in the presence of BF3.Et2O to give compound 5050.

[0387] Synthesis of compound 5052: Compound 5050 was treated with pivaloyl chloride as reported in the literature. The pivaloyl ester was treated with trifluoromethanesulfonic anhydride followed by water at reflux to give compound 5052.

[0388] Synthesis of compound 5055: Compound 5052 was first treated with sodium hydroxide to remove the pivaloyl ester, and the resulting trihydroxyl derivative was then treated with benzoic anhydride to give compound 5054. Subsequent oxidation of the double bond gave carboxylic acid 5055.

[0389] Synthesis of compound 5056: Compound 5055 (502 mg, 0.732 mmol) was treated with N-hydroxysuccinimide (127 mg, 1.10 mmol) in the presence of EDCI (211 mg, 1.10 mmol) and DIEA (0.383 mL, 2.20 mmol) in CHCl (5 mL) for 14 hours. Aqueous workup followed by column chromatography afforded compound 5056 (400 mg, 0.553 mmol, 76%). 38 H 38 N2O 13 Molecular weight (M+H) + Calculated value: 730.24, actual value: 730.25.

[0390] Example 40: Synthesis of novel GalNAc conjugates using post-synthetic methods

[0391] [ka]

[0392] [ka]

[0393] The GalNAc groups in the table below were conjugated to siRNA by the procedures described in Schemes 94, 95 or 96 above. [Table 6]

[0394] Example 41: siRNA-ligand conjugates siRNA-ligand conjugates were prepared. The siRNA in each conjugate was the same and targeted TTR. The following ligands were attached to the 3' end of the sense strand of each siRNA. The left side of the ligand designation indicates the binding site to the 3' end of the sense strand. [Table 7]

[0395] siRNA-ligand conjugates 43527, 60148, 60146, 60142, 60133, 60139, 60134, 60132 and / or 60125 are tested in mice as described in Example 33. The results are shown in FIG.

[0396] Example 42: siRNA-ligand conjugates siRNA-ligand conjugates were prepared. The siRNA in each conjugate targeted TTR. The following ligands were attached to the sense strand of the siRNA at the positions indicated: [Table 8]

[0397] 4-7 show the binding affinities of siRNA-ligand conjugates 56718-56727, 56729 and 55727.

[0398] Example 43: siRNA-ligand conjugates The siRNA-ligand conjugates in the table below were prepared. The siRNA in each conjugate was the same and targeted AT3. The following ligands were attached to the sense strand of each siRNA as indicated: [Table 9]

[0399] Example 44: The siRNA-ligand conjugates in the table were prepared. The siRNA in each conjugate was the same and targeted AT3. The ligand was attached to the sense strand at the position indicated in the table.

[0400] Figures 8 and 9 show the binding affinities of SiRNA-ligand conjugates 56876, 66875, 56874, 66878, 56880, 56879, 54944, 56877, 56881 and / or 56882. The K values ​​for these conjugates are reported below. [Table 10]

[0401] Example 45: siRNA with different GalNAc ligands (triple-stranded derivatives) siRNA-ligand conjugates were prepared. The siRNA in each conjugate was the same and targeted TTR. The following ligands were attached to the 3' end of the sense strand of each siRNA. The left side of the ligand designation indicates the binding site to the 3' end of the sense strand. [Table 11]

[0402] FIG. 10 shows the in vivo efficacy of triple-stranded GalNAc ligands 43527, 60126, 60138, 60128, 60127, 60316, and 60123 (at doses of 15 mg / kg and 5 mg / kg) after 72 and 144 hours.

[0403] Example 46: siRNA with different GalNAc ligands (triple-stranded derivatives (1+1+1)) siRNA-ligand conjugates were prepared. The siRNA in each conjugate was the same and targeted TTR. The following ligands were attached to the 3' end of the sense strand of each siRNA. The left side of the ligand designation indicates the binding site to the 3' end of the sense strand. [Table 12]

[0404] AT3-directed siRNA conjugates (two of which are described in Examples 30 and 34) were prepared and tested. The structure of 58137 is shown below. Each conjugate had the same AT3 siRNA sequence. Figure 11 shows the in vivo efficacy of triple-stranded GalNAc ligands 54944, 56881, and 58137 [(1+1+1) design]. [Table 13]

[0405] siRNA conjugates directed against TTR were prepared and tested. Each conjugate had the same TTR siRNA sequence. The following ligands were attached to the 3' end of the sense strand of each siRNA. The left side of the ligand designation indicates the binding site to the 3' end of the sense strand. Figure 12 shows the in vivo efficacy of the triple-stranded GalNAc ligands 55727, 58138, and 58139 [(1+1+1) design]. The ligand designs for the double-stranded 55727, 58138, and 58139 are shown below. [Table 14]

[0406] Example 47 [ka]

[0407] Synthesis of compound 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyprolinamine (10.00 g, 18.77 mmol) were placed together in dichloromethane. HBTU (10.68 g, 28.12 mmol) and DIEA (9.80 mL, 3 equivalents) were added, and the mixture was stirred at ambient temperature for 2 hours. The product was examined by thin-layer chromatography, and the reaction mixture was transferred to a separatory funnel and washed with water and brine. The organic layer was dried over sodium sulfate, and the solvent was removed. The crude product was purified by silica gel chromatography using dichloromethane and MeOH as solvents to give compound 102 as a pale yellow fluffy solid (11.77 g, 63%). 1H NMR (400MHz, DMSO-d6): δ7.80(d,J=9.2Hz, 1H), 7.69(t,J=5.6Hz, 1H), 7.39-7.09(m,9H), 6.86(ddd,J=9.0, 5 .4, 2.1Hz, 4H), 5.20(d,J=3.4Hz, 1H), 5.03-4.83(m,2H), 4.47(d,J=8.5Hz, 1H), 4.41-4.07(m,2H), 4.04-3.95 (m,3H), 3.86(dt,J=11.2, 8.9Hz, 1H), 3.79-3.68(m,6H), 3.68-3.36(m,3H), 3.21-2.88(m,5H), 2.26-2.14(m ,2H), 2.09(s,3H), 2.02(t,J=6.7Hz, 2H), 1.98(s,3H), 1.87(d,J=7.5Hz, 3H), 1.76(s,3H), 1.53-1.29(m,7H).

[0408] Synthesis of compound 104: Hydroxyproline derivative 102 (6.00 g, 6.24 mmol) was dissolved in dichloromethane (100 mL). DIEA (2.20 mL, 3 equivalents) and 2-cyanoethyldiisopropylchlorophosphoramidite were added. The reaction mixture was stirred for 30 minutes and examined by thin-layer chromatography. The mixture was transferred to a separatory funnel and washed with water and sodium bicarbonate solution. The organic layer was dried over sodium sulfate, and the crude product was purified by silica gel chromatography using dichloromethane and MeOH as eluents to give the compound as a white fluffy solid. 1H NMR (400MHz, DMSO-d6): δ7.80(d,J=9.2Hz, 1H), 7.68(s,1H), 7.42-7.06(m,8H), 7.01-6.73(m,4H), 5.20(d,J=3.3Hz, 1H) ), 4.96(dd,J=11.2, 3.3Hz, 1H), 4.63(d,J=4.7Hz, 1H), 4.47(d,J=8.5Hz, 1H), 4.15(s,1H), 4.01(s,3H), 3.86(d,J=11.0H) z, 1H), 3.70(d,J=16.5Hz, 9H), 3.45(ddd,J=37.0, 23.3, 16.4Hz, 6H), 2.99(dd,J=12.3, 6.4Hz, 3H), 2.74(dd,J=9.2, 5.8 Hz, 2H), 2.21(s,2H), 2.09(s,3H), 2.05-1.95(m,5H), 1.88(s,3H), 1.76(s,3H), 1.52-1.16(m,11H), 1.16-1.02(m,11H). 31 P NMR: δ151.78, 151.61, 151.50, 151.30.

[0409] Synthesis of Compound 105: Compound 102 (2.10 g, 2.18 mmol) was dissolved in DCM (20 mL). To this mixture, succinic anhydride (0.441 g, 4.36 mmol) and DMAP (0.532 g, equivalents) were added, followed by TEA (1 mL). The reaction mixture was stirred overnight at room temperature. The reaction was examined by thin layer chromatography, and then the reaction mixture was washed with water and brine. The organic layer was dried over sodium sulfate, and the crude product was filtered through a small pad of silica gel. The solvent was removed, and this material was used in the next reaction. The succinate from the above reaction was dissolved in anhydrous acetonitrile. HBTU (1.59 g, 4.20 mmol) and DIEA (1.10 mL) were added, and the mixture was rotated for 5 minutes. A polystyrene solid support was added to the reaction mixture, and the mixture was shaken at ambient temperature overnight. The solid support was filtered, washed, and capped with an acetic anhydride / pyridine mixture. The solid support was washed again with dichloromethane, MeOH / DCM and ether (27.10 g, 55 μmol / g).

[0410] Example 48: Synthesis of amino linkers for post-synthetic conjugation [ka]

[0411] Compound 101: Z-aminocaproic acid (22.2 g, 82.50 mmol) was dissolved in DMF (250 mL) and cooled to 0 °C. To the solution, diisopropylethylamine (44.4 mL, 275 mmol), HBTU (40.4 g, 106.7 mmol), and HOBT (30.0 g, 220 mmol) were added. After stirring at 0 °C for 20 minutes under argon, 4-hydroxy-L-proline methyl ester hydrochloride (20.0 g, 110 mmol) was added, and stirring was continued at room temperature overnight under argon. The reaction mixture was evaporated to dryness. To the residue, ethyl acetate (250 mL) was added. The organic layer was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude compound 101 (Rf=0.5 in 10% MeOH / DCM, 24.30 g) was obtained. Compound 101 was purified by column chromatography by eluting first with 2% methanol / dichloromethane followed by 5% methanol / dichloromethane to remove impurities, affording 21.36 g (65%) of the product. 1H NMR (400MHz, DMSO-d6): δ7.35(m,5H), 5.15(d,OH, D2O exchangeable), 4.99(s,2H), 4.27(m,1H), 3.97(m,1H), 3.58(s,1H) 3.20-3.47(m,5H), 2.94-3.02(m,2H), 2.10-2.32(m,2H), 1.74-2.01(m,2H), 1.35-1.4(m,4H), 1.22-1.28(m,4H).

[0412] Compound 102: Compound 101 (21.36 g, 54.43 mmol) was dissolved in THF (200 mL). The reaction mixture was stirred at 0° C. for 20 minutes under argon. Lithium borohydride (1.19 g, 54.43 mmol) was then added to the solution over 20 minutes at 0° C., and stirring was continued at room temperature overnight under argon. The reaction mixture was cooled to 0° C. Excess lithium borohydride was quenched with 5 M NaOH (30 mL). After stirring for 30 minutes, the reaction mixture was evaporated to dryness. Dichloromethane (200 mL) was added to the residue. The organic layer was washed with water and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude compound 102 (Rf=0.4 in 10% MeOH / DCM, 35.88 g) was obtained. To remove impurities, compound 102 was purified by column chromatography by eluting with 3% methanol / dichloromethane followed by 5% methanol / dichloromethane to give 9.21 g (49%) of product. 1 H NMR (400MHz, DMSO-d6): δ7.35(m,5H), 4.99(s,2H), 4.91(d,OH, D2O exchangeable), 4.77(t,OH, D2O exchangeable), 4.27(m,1H), 3.97(m, 1H), 3.20-3.47(m,5H), 2.94-3.02(m,2H), 2.10-2.32(m,2H), 1.74-2.01(m,2H), 1.35-1.4(m,4H), 1.22-1.28(m,4H).

[0413] Compound 103: Compound 102 (9.21 g, 25.27 mmol) was coevaporated twice with anhydrous pyridine (80 mL). The compound was then placed under high vacuum overnight to dry. Compound 102 was removed from the high vacuum and dissolved in anhydrous pyridine (200 mL). To this solution, a catalytic amount of dimethylaminopyridine (0.35 g, 2.53 mmol) was added. The reaction mixture was stirred at 0° C. for 30 minutes under argon. DMT-Cl (9.0 g, 26.53 mmol) was then added to the solution at 0° C. The mixture was stirred under reduced pressure, then under argon, and continued to stir at room temperature overnight under argon. Excess DMT-Cl was quenched by the addition of methanol (15 mL). The reaction mixture was evaporated to dryness, and dichloromethane (200 mL) was added to the residue. The organic layer was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 103 (Rf=0.6 in 100% EtOAc, 14.02 g). To remove impurities, compound 103 was purified by column chromatography by first eluting with 50% ethyl acetate (1% TEA) in hexane, followed by 100% ethyl acetate (1% TEA), to give 12.36 g (73.4%) of the product as a white foamy solid. 1 H NMR (400MHz, DMSO-d6): δ7.17-7.33(m,14H), 4.99(s,2H), 4.91(d,OH, D2O exchangeable), 4.37(m,1H), 4.01(m,1H), 3.72( s,6H)3.56(m,1H)3.29(m,1H), 3.14(m,1H), 2.93-3.02(m,4H), 2.18(m,2H)1.74-2.01(m,2H), 1.37-1.41(m,6H).

[0414] Compound 104: Compound 103 (12.36 g, 18.54 mmol) was dissolved in 10% methanol / ethyl acetate (300 mL) and purged with argon. To the reaction mixture was added 10% by weight palladium supported on wet activated carbon, Degussa type (1.3 g). The flask was purged again with argon. The flask was purged twice with hydrogen, and then hydrogen was bubbled through the reaction mixture for 10 seconds. The reaction mixture was left stirring under hydrogen at room temperature overnight. The reaction mixture was decanted onto a sintered funnel filled with Celite and washed twice with methanol. The organic layer was evaporated to dryness to give compound 104 (eluent 10% MeOH in DCM, 9.16 g, 93%) as a white solid, which did not require further purification. 1 H NMR (400MHz, DMSO-d6): δ7.15-7.31(m,9H), 6.86(m,4H)4.99(s,1H), 4.37(m,1H), 4.01(m,2H), 3.72(s,6H)3.56(m,1H)3.2 9(m,1H), 3.14(m,1H), 2.93-3.02(m,2H), 2.45(m,2H), 2.18(m,2H)1.74-2.01(m,2H), 1.37-1.41(m,3H)1.13-1.38(m,4H). MS: 533.4(+H), 555.3(+Na).

[0415] Compound 105: Compound 104 (9.16 g, 17.2 mmol) was dissolved in dichloromethane (200 mL). The reaction mixture was stirred at 10° C. for 10 minutes under argon. Triethylamine (4.80 mL, 34.4 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 20 minutes under argon. Ethyl trifluoroacetate (3.05 mL, 25.8 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 10 minutes under argon. The reaction mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed with water and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude compound 105 (Rf=0.6 10% MeOH in DCM, 10.89 g) was obtained. Column purification by eluting with 5% methanol / dichloromethane (1% TEA) gave compound 105 (8.76 g, 81%) as a yellow foamy solid. 1 H NMR (400MHz, DMSO-d6): δ7.56-7.09(m,9H), 7.01-6.52(m,4H), 5.34-5.04(m,1H), 4.99-4.78(m,1H), 4.48-4.25(m,2H), 3.83-3.67(m,6) H), 3.60-3.50(m,1H), 3.49-3.18(m,2H), 3.16-2.91(m,2H), 2.89-2.56(m,2H), 2.54-2.32(m,2H), 2.32-1.69(m,3H), 1.59-1.03(m,4H). 19 F NMR(DMSO-d6):-77.14(s,3F).MS:627.3(-H), 663.3(+Cl).

[0416] Compound 106: Compound 105 (8.76 g, 13.93 mmol), DMAP (5.10 g, 41.79 mmol), and triethylamine (3.90 mL, 27.86 mmol) were dissolved in dichloromethane (300 mL). The reaction mixture was stirred under argon for 10 minutes. Succinic anhydride (2.80 g, 27.86 mmol) was then added, and the mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed twice with saturated sodium chloride solution. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 106 (Rf = 0.9 10% MeOH in DCM, 10.87 g, 94%) was obtained as a white solid, which did not require further purification. MS: 727.2 (-H), 763.2 (+Cl). The succinate (2.00 g, 2.41 mmol) thus obtained was dissolved in acetonitrile (100 mL). Diisopropylethylamine (1.68 mL, 9.64 mmol) and HBTU (1.83 g, 4.82 mmol) were added to the solution. The reaction mixture was shaken for 10 minutes. Long-chain aminoalkyl-controlled pore glass (CPG) (27 g) was added to the flask, and the mixture was shaken overnight. The CPG compound and reaction mixture were decanted onto a sintered funnel. The reaction mixture was washed with 1% triethylamine / dichloromethane, followed by two washes with 10% methanol in dichloromethane, 1% triethylamine in dichloromethane, and anhydrous diethyl ether. The CPG compound was dried under vacuum for 1 hour, then recovered from the funnel and placed under high vacuum for 2 hours. The CPG was capped with 25% acetic anhydride in pyridine (100 mL), and the mixture was shaken for 4 hours. The washing procedure was repeated as above and the compound was dried under vacuum to give CPG 106 (28 g, 67 μmol / g).

[0417] Compound 107: Compound 105 (8.89 g, 14.14 mmol) and diisopropylethylamine (4.93 mL, 28.28 mmol) were dissolved in anhydrous dichloromethane (60 mL). The reaction mixture was stirred for 5 minutes under argon. Then, 2-cyanoethyldiisopropylchlorophosphoramidite (5.63 mL, 16.26 mmol) was added to the reaction mixture. The reaction mixture was continued to stir at room temperature for 30 minutes under argon. The reaction mixture was diluted with dichloromethane (100 mL). The organic layer was washed with water, saturated sodium bicarbonate, water again, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 107 (Rf=0.44 5% MeOH in DCM, 11.02 g). Column purification by eluting with 3% methanol (1% TEA) in DCM gave compound 107 (6.31 g, 54%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6): δ9.37(s,1H), 7.63(d 1H), 7.42-6.98(m,8H), 6.92-6.77(m,4H), 4.25-3.90(m,2H), 3.78-3.64(m,7H), 3.48(d,3H), 3.29(d,1H), 3.23 -2.92(m,4H), 2.86(d,1H), 2.73(t,1H), 2.58(t,1H), 2.53-2.47(m,4H), 2.33-1.87(m,4H), 1.55-0.97(m,12H). 31 P(DMSO-d6):151.68(d,1P).

[0418] [ka]

[0419] Compound 118: Serinol 116 (2.05 g, 22.5 mmol) and compound 117 (8.03 g, 24.75 mmol) were dissolved in DMF (120 mL) under argon. Triethylamine (5.0 mL, 67.5 mmol) was added to the reaction mixture, which was stirred at room temperature overnight. The reaction mixture was evaporated to dryness, and the residue was dissolved in ethyl acetate (120 mL). It was then washed with water (30 mL) and saturated sodium chloride (2 × 50 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. The crude compound (5.33 g, Rf = 0.21 15% MeOH in DCM) was purified by silica gel column chromatography using 10% methanol in dichloromethane as the eluent to give compound 118 (4.98 g, 78%) as a white foam. m / z: 324.13 (+Na). 1H NMR (400MHz, DMSO-d6): δ9.39 (s,2NH, D2O exchangeable), 3.34-3.06 (m,7H), 3.06-2.91 (m,2OH, D2O exchangeable), 2.30-2.00 (m,2H), 1.97-0.86 (m,6H). 13 C NMR (101MHz, CD3CN): δ172.18(s), 156.41(s), 117.50(s), 60.30(s), 52.85(s), 38.88(s), 35.35(s), 28.11(s), 25.90(s), 25.29(s).

[0420] Compound 119: Compound 118 (3.70 g, 12.3 mmol) was coevaporated twice with anhydrous pyridine (30 mL) and then dried overnight under high vacuum. It was then dissolved in anhydrous pyridine (90 mL). A catalytic amount of DMAP (0.15 g, 1.23 mmol) was added to this solution, and the mixture was stirred at 0 °C for 30 minutes under argon. DMTr-Cl (4.38 g, 12.9 mmol) was added to the solution at 0 °C, and stirring was continued at room temperature for 2 hours. Volatiles were then removed under reduced pressure. The residue was dissolved in dichloromethane (150 mL) and washed with water (2 × 100 mL), followed by saturated sodium chloride (100 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. The crude compound (6.83 g, Rf = 0.44 in 5% MeOH in DCM) was purified by silica gel column chromatography by eluting first with dichloromethane (containing 1% TEA) followed by 3% methanol in dichloromethane (containing 1% TEA) to give compound 119 (2.32 g, 23%) as a white foam. m / z: 601.2 (-1), 637.2 (+Cl). 1 H NMR (400MHz, DMSO-d6): δ9.38(s,2NH, D2O exchangeable), 7.31-7.13(m,7H), 6.86(d,J=8.9Hz, 6H), 4.60(t,J=5.2Hz, 1H), 3.72(s,6H), 3.54 -3.23(m,6H), 2.95(ddd,J=36.9, 8.8, 5.8Hz, 1OH, D2O exchangeable), 2.08(t,J=7.4Hz, 2H), 1.47(tt, J=14.6, 7.4Hz, 2H), 1.34-1.12(m,4H). 13C NMR (101MHz, CD3CN): δ172.83(s), 158.97(s), 157.28(s), 156.93(s), 146.13(s), 136.82(s), 136.52(s), 130.70(s), 128.68(d,J=4.0Hz), 127.49(s), 118.41(s), 115.54(s), 114.02(s), 86 .08(s), 63.66(s), 61.84(s), 55.93(s), 51.77(s), 46.68(s), 40.99(d,J=21.0Hz), 40.72(s), 4 0.68(s), 40.47(s), 40.26(s), 40.05(s), 39.84(s), 36.31(s), 29.04(s), 26.85(s), 25.95(s).

[0421] Compound 120: Compound 119 (1.0 g, 1.7 mmol), DMAP (620 mg, 5.1 mmol), and triethylamine (0.5 mL, 3.4 mmol) were dissolved in dichloromethane (15 mL). The reaction mixture was stirred for 5 minutes under argon. Succinic anhydride (340 mg, 3.4 mmol) was then added, and stirring was continued at room temperature overnight under argon. The reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated sodium chloride (2 x 25 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. 1.01 g (98%) of crude compound (Rf = 0.3 50% EtOAc in Hex) was obtained as a white solid, which was used in the next reaction without further purification. MS: 702.3 (-H), 737.5 (+Cl). 1 H NMR (400MHz, DMSO-d6): δ9.42 (t,J=5.2Hz, 2NH, D2O exchangeable), 7.87 (s,1OH, D2O exchangeable), 7.47-7.15 (m,7H), 6.99- 6.74(m,6H), 4.32-3.94(m,3H), 3.90-3.42(m,8H), 3.31-2.72(m,6H), 2.57-2.29(m,2H), 1.30-0.87(m,6H).

[0422] Compound 121: Succinate 120 (1.0 g, 1.4 mmol) was dissolved in acetonitrile (60 mL). To this solution, diisopropylethylamine (1.15 mL, 5.6 mmol) and HBTU (1.26 g, 2.8 mmol) were added. The reaction mixture was swirled until all contents were dissolved. CPG (14 g) was added to the flask, and the mixture was shaken overnight. The CPG was filtered and washed successively with dichloromethane, 10% methanol in dichloromethane, dichloromethane, and anhydrous diethyl ether. The CPG was dried under vacuum for 1 hour, then recovered from the funnel and placed under high vacuum for 2 hours. The loaded CPG was capped with 25% acetic anhydride / pyridine (100 mL) for 3 hours. The CPG was filtered, and the same washing procedure as described above was repeated. The CPG was dried under vacuum for 1 hour and then under high vacuum overnight to give compound 120 (14 g, 77 μmol / g).

[0423] [ka]

[0424] Compound 122: L-Threoninol (3.54 g, 33.7 mmol) and Compound 117 (12.0 g, 37.1 mmol) were dissolved in DMF (90 mL) under argon. Triethylamine (14.0 mL, 101.1 mmol) was added to the reaction mixture, which was stirred at room temperature overnight. The reaction mixture was evaporated to dryness, and the residue was dissolved in ethyl acetate (120 mL). It was then washed with water (50 mL) and saturated sodium chloride (2 × 50 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. The crude compound (3.60 g, Rf = 0.43 50% EtOAc in hexane) was purified by silica gel column chromatography using 50% ethyl acetate in hexane followed by 100% ethyl acetate as the eluent to give Compound 122 (3.20 g, 87%) as a yellow foam. m / z: 315.1 (+H). 1H NMR (400MHz, DMSO-d6): δ8.76 (t,2NH, D2O exchangeable) 3.93-3.21 (m,1H), 3.21-3.01 (m,2OH, D2O exchangeable), 3.05-2. 83(m,3H), 2.75-2.51(m,1H), 2.55-2.37(m,1H), 2.20-1.98(m,2H), 1.70-1.39(m,7H), 1.28-1.06(m,3H). 13 C NMR (101MHz, DMSO-d6): δ176.1(s), 156.3(s), 158.33(s), 65.36(s), 64.20(s) , 60.51, 38.88(s), 36.34(s), 27.98(s), 25.79(s), 25.70-24.33(m), 20.05(s).

[0425] Compound 123: Compound 122 (3.00 g, 9.60 mmol) was coevaporated twice with anhydrous pyridine (15 mL) and dried overnight under high vacuum. It was then dissolved in anhydrous pyridine (90 mL). A catalytic amount of DMAP (0.12 g, 0.96 mmol) was added to this solution, and the mixture was stirred at 0 °C for 30 minutes under argon. DMTr-Cl (3.39 g, 10.08 mmol) was added to the solution at 0 °C, and stirring was continued at room temperature for 2 hours. The volatiles were then removed under reduced pressure. The residue was dissolved in dichloromethane (150 mL) and washed with water (2 × 100 mL), followed by saturated sodium chloride (100 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. The crude compound (7.02 g, Rf = 0.6 in 25% EtOAc in hexane) was purified by silica gel column chromatography by eluting first with 5% ethyl acetate in hexane (containing 1% TEA) followed by 15% ethyl acetate in hexane (containing 1% TEA) to give compound 123 (700 mg, 11%) as a white foam. m / z: 615.2 (-1), 651.2 (+Cl). 1H NMR (400MHz, DMSO-d6): δ8.76 (t,2NH, D2O exchangeable) 7.87-6.93(m,7H), 6.93-6.63(m,6H), 4.19-3 .63(m,10H), 3.61(m,1OH, D2O exchangeable), 2.74-1.98(m,4H), 1.71-1.10(m,6H), 1.08-0.76(m,3H). 13 C NMR (101MHz, DMSO-d6): δ172.12(d,J=9.4Hz), 157.88(d,J=15.4Hz), 145.13(s), 140.22(s), 135.86(d,J=6.4Hz), 131.07-1 30.03(m), 130.03-129.28(m), 128.90(s), 127.66(d,J=7.1Hz), 127.38(s), 126.45(d,J=9.7Hz), 112.89(d,J=30.1Hz), 85.0 8(s), 65.00(s), 63.22(d,J=48.5Hz), 54.97(s), 53.72(s), 45.56(s), 40.02(d,J=21.0Hz), 39.85-39.80(m), 39.71(s), 39. 50(s), 39.29(s), 39.08(s), 38.88(s), 35.24(s), 28.02(s), 25.86(s), 25.13(d,J=13.6Hz), 21.15(s), 20.20(s), 10.35(s).

[0426] Compound 124: Compound 123 (600 mg, 1.0 mmol), DMAP (420 mg, 3.0 mmol), and triethylamine (0.4 mL, 2.0 mmol) were dissolved in dichloromethane (15 mL). The reaction mixture was stirred for 5 minutes under argon. Succinic anhydride (230 mg, 2.0 mmol) was then added, and stirring was continued at room temperature overnight under argon. The reaction mixture was diluted with dichloromethane (50 mL) and washed with saturated sodium chloride (2 x 25 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 124 (Rf = 0.6 25% EtOAc in hexanes) 760 mg (99%) was obtained as a gray foam, which was used in the next reaction without further purification. MS: 715.3 (-H).

[0427] Compound 125: Compound 124 (760 mg, 1.0 mmol) was dissolved in acetonitrile (60 mL). To this solution, diisopropylethylamine (0.74 mL, 4.0 mmol) and HBTU (800 mg, 2.0 mmol) were added. The reaction mixture was swirled until all contents were dissolved. CPG (10 g) was added to the flask, and the mixture was shaken overnight. The CPG was filtered and washed successively with dichloromethane, 10% methanol in dichloromethane, dichloromethane, and anhydrous diethyl ether. The CPG was dried under vacuum for 1 hour, then recovered from the funnel and placed under high vacuum for 2 hours. The loaded CPG was capped with 25% acetic anhydride in pyridine (100 mL) for 3 hours. The CPG was filtered, and the same washing procedure as described above was repeated. The CPG was dried under vacuum for 1 hour and then under high vacuum overnight to give compound 125 (10.2 g, 0.71 μmol / g).

[0428] [ka] Compound 127: 5-Hexenol (6.0 mL, 50.5 mmol) was dissolved in dichloromethane (120 mL). To this solution, triethylamine (14 mL, 151.5 mmol) was added. The reaction mixture was stirred at 0° C. for 30 minutes under argon. DMTr-Cl (18 g, 53.0 mmol) was then added to the solution at 0° C. The mixture was stirred under reduced pressure, then under argon, and continued to stir at room temperature for 4 hours under argon. The reaction mixture was washed twice with water, followed by saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give compound 127 (Rf = 0.85 in 25% EtOAc in hexanes) (18.9 g, 95%) as a yellow oil. 1H NMR (400MHz, DMSO-d6): δ7.42-7.03(m,7H), 7.01-6.74(m,6H), 5.85-5.63(m,1H), 5.05-4.77(m,2H) ), 3.93-3.47(m,6H), 2.93(dd,J=21.1, 14.7Hz, 2H), 2.49(dd,J=3.5, 1.7Hz, 2H), 1.66-1.47(m,4H).

[0429] Compound 128: Compound 127 (5.0 g, 12.5 mmol) and sodium bicarbonate (4.2 g, 25 mmol) were mixed in dichloromethane (250 mL). The reaction mixture was stirred at room temperature for 5 minutes. Next, metachloroperbenzoic acid (16 g, 31.25 mmol) was added, and the mixture was continued to stir at room temperature overnight. The reaction mixture was quenched by the addition of sodium bisulfite (500 mg) and allowed to stir at room temperature for 30 minutes. The reaction mixture was washed with water, saturated bicarbonate solution, water, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give compound 128 (Rf = 0.25 5% EtOAc in hexanes) (5.06 g, 97.3%) as a yellow solid, which did not require further purification. 1 H NMR (400MHz, DMSO-d6): δ7.48-7.08(m,7H), 6.88(t,J=5.9Hz, 6H), 3.74(d,J=19 .4Hz, 3H), 3.32(s,6H), 3.04-2.77(m,1H), 2.65-2.35(m,2H), 1.69-1.09(m,6H).

[0430] Compound 129: Compound 128 (5.0 g, 9.56 mmol) was dissolved in ethanol (15 mL). 30% ammonium hydroxide in water (3 mL) was added to the reaction mixture, which was stirred overnight at 85° C. in an oil bath in a pressure vessel. The reaction mixture was evaporated to dryness and then co-evaporated twice with toluene (10 mL). The compound was then co-evaporated with dichloromethane (50 mL) to give crude compound 129 (Rf=0.1 25% EtOAc in hexanes, 5.48 g) as a brown oil, which was used without purification.

[0431] Compound 130: Compound 129 (5.48 g, crude material) was dissolved in dichloromethane (100 mL). The reaction mixture was stirred at 10° C. for 10 minutes under argon. Triethylamine (4.0 mL, 19.1 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 20 minutes under argon. Ethyl trifluoroacetate (5.0 mL, 28.7 mmol) was added dropwise to the reaction mixture at 10° C. The reaction mixture was continued to stir at room temperature overnight under argon. The reaction mixture was then washed twice with water, followed by saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 130 (Rf=0.43 50% EtOAc / Hex, 6.12 g). Purification of compound 130 by silica gel column chromatography by eluting first with 5% ethyl acetate in hexane (1% TEA) to remove impurities, followed by 10% ethyl acetate in hexane (1% TEA) to elute the product from additional impurities, afforded 1.51 g (30% from compound 129) as a white foam. 1 H NMR (400MHz, DMSO-d6): δ7.69-7.12 (m,7H, 1NH, D2O exchangeable), 7.10-6.70 (m,6H), 4.12-3.48 (m,9H, 1OH, D2O exchangeable), 3.29 (dd,J=19.7, 12.9Hz, 2H), 2 1.57-1.17(m,6H). 13 C NMR (101MHz, DMSO-d6): δ157.97(s), 156.56(s), 156.20(s), 145.27(s), 136.08( s), 129.58(s), 127.69(d,J=7.7Hz), 126.51(s), 117.45(s), 114.58(s), 113.09(s ), 85.15(s), 67.93(s), 62.78(s), 54.96(s), 45.67(s), 40.13(s), 39.92(s), 39. 71(s), 39.50(s), 39.30(s), 39.09(s), 38.88(s), 34.28(s), 29.51(s), 21.92(s).

[0432] Compound 131: Compound 130 (1.49 g, 2.8 mmol), DMAP (1.02 g, 8.4 mmol), and triethylamine (0.8 mL, 5.6 mmol) were dissolved in dichloromethane (30 mL). The reaction mixture was stirred under argon for 5 minutes. Succinic anhydride (600 mg, 5.6 mmol) was then added, and the mixture was continued to stir at room temperature overnight under argon. The reaction mixture was diluted with dichloromethane (100 mL) and then washed with two 50 mL portions of slightly saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 131 (Rf = 0.24 25% EtOAc in hexanes) (1.77 g, 99%) was obtained as a white foam, which did not require further purification.

[0433] Compound 132: Compound 131 (1.77 g, 2.8 mmol) was dissolved in acetonitrile (120 mL). Diisopropylethylamine (1.95 mL, 11.2 mmol) and HBTU (2.13, 5.6 mmol) were added to the solution. The reaction mixture was swirled until all contents were dissolved. CPG (26 g) was added to the flask, and the mixture was shaken overnight. The reaction mixture was decanted onto a sintered funnel and washed with 1% triethylamine / dichloromethane, followed by two washes with 10% methanol in dichloromethane, 1% triethylamine in dichloromethane, and anhydrous diethyl ether. The CPG was dried under vacuum for 1 hour, then removed from the funnel and placed under high vacuum for 2 hours. It was then capped with 25% acetic anhydride in pyridine (200 mL), and the mixture was shaken for 3 hours. The reaction mixture was then placed on a sintered funnel and washed as described above. The CPG was dried under vacuum for 1 hour, removed from the funnel, and placed under high vacuum overnight (27 g, 83 μmol / g).

[0434] [ka]

[0435] Compound 134: 5-Hexenol (6.0 mL, 50.5 mmol) and sodium azide (17 g, 252.5 mmol) were dissolved in DMF (120 mL). To this solution, triethylamine (14 mL, 151.5 mmol) was added. The reaction mixture was stirred under argon for 10 minutes. Methanesulfonyl chloride (4.25 mL, 50.5 mmol) was then added dropwise to the solution over 20 minutes. Stirring was continued at room temperature under argon for 2 days. The reaction mixture was decanted into ice water, which was then washed with 5 x 50 mL portions of diethyl ether. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude Compound 134 (Rf = 0.67 in 5% MeOH / DCM, 6.0 g). Purification of compound 134 by column chromatography by eluting first with dichloromethane followed by 2% methanol / dichloromethane to remove impurities gave 4.6 g (72%) of a clear liquid. 1 H NMR (400MHz, DMSO-d6): δ5.78(ddt,J=16.9, 10.2, 6.6Hz, 1H), 5.16-4.74(m,2H), 2.68-2.14(m,2H), 1.64-1.27(m,4H), 1.24-0.92(m,2H).

[0436] Compound 135: Compound 134 (4.5 g, 25.5 mmol) was taken up in DMF (100 mL) in a pressure bottle. To this mixture, NaN3 (10 g) was added, and the mixture was heated at 80 °C overnight. The solid was then removed by filtration. The volatiles were removed under reduced pressure, and the residue was extracted with ethyl acetate and washed with water and brine. The organic layer was dried over sodium sulfate, and the solvent was removed to give compound 134134 as a pale yellow liquid (3.6 g, 82%). m / z: 132.1 (-N2).

[0437] Compound 136: Compound 135 (2.0 g, 16 mmol) and N-methylmorpholine-N-oxide (2.25 g, 19.2 mmol) were dissolved in 10% water in acetone (60 mL). The reaction was stirred at room temperature for 5 minutes. Next, osmium tetroxide in 10% water in acetone (1.3 mL, 0.16 mmol) was added, and the mixture was continued to stir at room temperature overnight. The reaction mixture was decanted onto Celite and washed with two 50 mL portions of acetone. The reaction mixture was evaporated to dryness and then dissolved in 100 mL of ethyl acetate. The organic layer was washed with 50 mL portions of 1 N HCl. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give 1.70 g (67%) of compound 136 as a brown liquid, which did not require further purification.

[0438] Compound 137: Compound 136 (1.5 g, 9.40 mmol) was coevaporated twice with anhydrous pyridine (10 mL). The compound was then placed under high vacuum overnight to dry. The compound was then removed from the high vacuum and dissolved in anhydrous pyridine (60 mL). To this solution was added a catalytic amount of DMAP (0.11 g, 0.94 mmol). The reaction mixture was stirred at 0° C. for 30 minutes under argon. DMT-Cl (3.35 g, 10.06 mmol) was then added to the solution at 0° C. The mixture was stirred under reduced pressure, then under argon, and continued to stir at room temperature under argon for 1.5 hours. The reaction mixture was evaporated to dryness, and dichloromethane (100 mL) was added to the residue. The organic layer was washed twice with water, followed by saturated brine. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 137 (Rf=0.8 1:1 EtOAc / hexanes, 5.3.3 g) as an orange oil, which was used without purification.

[0439] Compound 138: Compound 137 (3.3 g, crude material) and triphenylphosphine (1.7 g, 6.48 mmol) were dissolved in tetrahydrofuran. The reaction mixture was stirred at room temperature for 5 minutes. Water (1 mL) was then added, and the mixture was continued to stir at room temperature overnight. When the reaction was complete as observed by TLC, the reaction mixture was evaporated to dryness and then co-evaporated with toluene. The reaction mixture was dissolved in dichloromethane (60 mL). The reaction mixture was stirred at 10° C. for 10 minutes under argon. Triethylamine (1.5 mL, 10.75 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 20 minutes under argon. Ethyl trifluoroacetate (6.5 mL, 54.0 mmol) was added dropwise to the reaction mixture, and the mixture was continued to stir at 10° C. for 10 minutes under argon. The reaction mixture was continued to stir at room temperature overnight under argon. The reaction mixture was washed with water, saturated bicarbonate solution, water, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude compound 138 (Rf=0.5 50% EtOAc / Hex, 3.40 g). Purification of the compound by column chromatography, eluting first with 5% ethyl acetate in hexane, followed by 15% ethyl acetate in hexane to remove impurities, gave 2.1 g (42% from compound 136) as a yellow oil. 1 H NMR (400MHz, DMSO-d6): δ9.36 (s, 1NH, D2O exchangeable), 7.47-7.14 (m, 7H), 6.87 (d, J = 8.8Hz, 6H), 3.86-3.48(m,7H), 3.33(s,1OH, D2O exchangeable), 3.13(dd,J=12.1, 6.4Hz, 4H), 1.54-1.06(m,6H). 19 F NMR (376MHz, DMSO-d6): δ-82.54(s). 13C NMR (101MHz, DMSO-d6): δ161.42(s), 159.73(s), 159.37(s), 148.65(s), 139.41(d,J=6.7Hz), 136. 65(s), 135.74-135.30(m), 134.92(d,J=9.8Hz), 133.16(s), 132.18(d,J=11.8Hz), 131.18(d,J=6. 0Hz), 129.96(s), 116.51(s), 88.50(s), 72.39(s), 71.10(s), 63.20(s), 58.43(s), 43.58(s), 43.3 7(s), 43.16(s), 42.95(s), 42.80-42.53(m), 42.33(s), 36.88(s), 31.79(s), 25.67(s), 17.51(s).

[0440] Compound 139: Compound 138 (1.0 g, 1.9 mmol), DMAP (700 mg, 5.7 mmol), and triethylamine (0.55 mL, 3.8 mmol) were dissolved in dichloromethane (20 mL). The reaction mixture was stirred under argon for 5 minutes. Succinic anhydride (380 mg, 3.8 mmol) was then added, and the mixture was continued to stir at room temperature overnight under argon. The reaction mixture was diluted with dichloromethane (50 mL) and then washed with two 25 mL portions of slightly saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 139 (eluent 1:1 EtOAc / hexane) 1.18 g (99%) was obtained as a pink oil, which did not require further purification.

[0441] Compound 140: Compound 139 (1.18 g, 1.9 mmol) was dissolved in acetonitrile (60 mL). To the solution, diisopropylethylamine (1.3 mL, 7.6 mmol) and HBTU (1.43 g, 3.8 mmol) were added. The reaction mixture was swirled until all contents were dissolved. CPG (12 g) was added to the flask, and the mixture was shaken overnight. The CPG compound and reaction mixture were decanted onto a sintered funnel. The reaction mixture was washed with 1% triethylamine in dichloromethane, followed by two washes with 10% methanol / dichloromethane, 1% triethylamine in dichloromethane, and anhydrous diethyl ether. The CPG was dried under vacuum for 1 hour, then recovered from the funnel and placed under high vacuum for 2 hours. The CPG was capped with 25% acetic anhydride / pyridine (100 mL), and the mixture was shaken for 3 hours. The reaction mixture was placed on a sintered funnel and washed as described above. The resulting CPG 140 was dried under vacuum for 1 hour, removed from the funnel, and placed under high vacuum overnight (12 g, 77 μmol / g).

[0442] [ka]

[0443] Compound 142: To a stirred solution of (R)-glycidol (2.3 g, 31 mmol) in DCM, triethylamine (5 mL, 36.5 mmol) was added, followed by a 1 M solution of DMTrCl (10.66 g, 31.5 mmol) in DCM at room temperature. The reaction was stirred until no starting material was present, as indicated by TLC. A few drops of MeOH were added to hydrolyze unreacted DMTrCl, and the mixture was stirred for 10 min. The product was washed with HO, brine, and dried over NaSO. The product was purified by column chromatography using a gradient of hexane / EtOAc (9:1) to give 6 g (52%) of pure product 142. 1H NMR (400MHz, DMSO-d6): δ7.38(d,J=7.6Hz, 2H), 7.31(t,J=7.6Hz, 2H), 7.27-7.12(m,5H), 6.87(d,J=6.1Hz, 4H), 3.72(s ,6H), 3.24(dd,J=10.9, 2.4Hz, 1H), 3.15-3.08(m,1H), 2.86(dd,J=10.9, 6.0Hz, 1H), 2.70(t,J=4.6Hz, 1H), 2.54(m,1H). 13 C NMR (101MHz, DMSO-d6): δ158.07, 144.75, 135.48, 135.45, 129.60, 127.85, 127.59, 126.69, 113.46, 113.21, 85.46, 64.52, 55.01, 50.37, 43.59.

[0444] Compound 144: To a stirred solution of N1,N6-dimethylhexane-1,6-diamine 143 (4.225 g, 29 mmol) and K2CO3 (0.15 g, 1 mmol) dissolved in DMF and heated to 90 °C, compound 142 (5.06 g, 13.5 mmol) was added dropwise. The reaction mixture was stirred overnight until no compound 142 remained. The reaction was quenched with ice, extracted with DCM, dried over Na2SO4, and purified by column chromatography using a gradient of DCM (2.5% NEt3) MeOH (30%) to give 7.8 g (63%) of pure compound 144. 1 H NMR (500MHz, DMSO-d6): δ7.40(d,J=7.6Hz, 2H), 7.35-7.14(m,7H), 6.88(d,J=8.5Hz, 4H), 4.12(s,1H), 3.80-3.64(s,6H), 3.37(m, 2H), 3.11-2.89(m,5H), 2.85(dt,J=15.1, 7.6Hz, 1H), 2.82-2.76(m,2H), 2.69(s,3H), 2.48-2.38(s,3H), 1.61(m,4H), 1.28(m,4H). 13C NMR (DMSO-d6): δ158.03, 144.82, 135.54, 135.48, 129.72, 127.79, 127.69, 126.63, 113.15, 85.41, 65.77, 64.74, 58.49, 55.03, 54.92 , 52.02, 47.83, 45.17, 40.00, 39.92, 39.83, 39.76, 39.66, 39.50, 39.33, 39.16, 39.00, 32.11, 25.50, 25.40, 24.99, 23.29, 8.37, 7.23.

[0445] Compound 146: (OBz)GalNAc acid 145 (6.95 g, 13.3 mmol), HOBt (3.59 g, 26.6 mmol), HBTU (5.04 g, 13.3 mmol), and DIPEA (5 mL, 28.5 mmol) were stirred in anhydrous DMF (80 mL) for 15 minutes. To this solution, compound 144 was added. The mixture was stirred for 45 minutes until no starting material remained. The product was dissolved in ethyl acetate (100 mL), and the organic layer was washed with HO, saturated NaHCO, brine, dried over NaSO, and then purified by column chromatography using a gradient of EtOAc (1% NEt):MeOH (15%) to give 5.35 g (36%) of pure compound 146. NMR (400MHz, DMSO-d6): δ8.08-7.85(m,4H), 7.82-7.11(m,20H), 6.86(d,J=8.8Hz, 4H), 5.76(d,J=3.3Hz) , 1H), 5.36(dt,J=26.7, 13.3Hz, 1H), 4.75(d,J=8.5Hz, 1H), 4.61-4.40(m,3H), 4.32(ddd,J=25.2, 16.9, 9 .1Hz, 2H), 3.88-3.60(m,1H), 3.71(s,6H), 3.63-3.45(m,1H), 3.18(dd,J=14.6, 7.4Hz, 2H), 3.03-2.81( m,4H), 2.74(s,3H), 2.42-2.29(m,1H), 2.31-2.12(m,5H), 2.07(s,3H), 1.69(s,3H), 1.60-0.99(m,12H). 13C NMR (DMSO-d6): δ171.45, 171.37, 170.36, 170.29, 169.32, 165.19, 165.14, 164. 86, 157.91, 145.23, 145.22, 136.01, 135.98, 133.75, 133.49, 133.45, 129.70, 12 9.57, 129.20, 129.15, 129.02, 128.99, 128.95, 128.68, 128.57, 127.78, 127.62, 126.45, 113.10, 112.98, 100.85, 85.01, 71.88, 69.98, 68.80, 68.73, 67.94, 67.7 2, 66.39, 63.45, 62.08, 60.72, 59.72, 57.68, 57.64, 54.96, 54.88, 49.72, 48.89, 46.60, 42.83, 40.09, 40.00, 39.92, 39.83, 39.76, 39.67, 39.59, 39.50, 39.33, 39. .17, 39.00, 34.67, 32.69, 32.17, 31.51, 30.14, 28.65, 28.54, 27.90, 26.76, 26.74, 26.48, 26.22, 26.06, 22.67, 21.41, 21.11, 20.73, 20.69, 18.58, 14.06, 13.52.

[0446] Compound 147: To a stirred solution of compound 146 (1.136 g, 1 mmol) and DIPEA (1 mL, 6 mmol) in DCM was added succinic anhydride (220 mg, 2.2 mmol). The reaction was stirred overnight until no starting material remained. The product was washed with HO, then brine, and dried over NaSO to give 850 mg (69%) of compound 147. 1H NMR(500MHz、DMSO-d6):δ8.11(dd,J=15.3、9.3Hz、1H)、7.98-7.84(m,4H)、7.73-7.16(m,19H)、6.87(d,J=8.7Hz、4H)、5.74(d,J=3.1Hz、1H)、5.36(dd,J=11.1、2.6Hz、1H)、5.01(s,1H)、4.76(dd,J=8.5、2.6Hz、1H)、4.45(m,2H)、4.37-4.23(m,2H)、3.71(s,6H)、3.54(dd,J=13.7、10.4Hz、1H)、3.21-3.02(m,4H)、3.02-2.93(m,4H)、2.87(s,2H)、2.73(s,1H)、2.55-2.30(m,8H)、2.30-2.12(m,5H)、2.07(s,3H)、1.68(s,3H)、1.58-1.07(m,8H). 13 C NMR(126MHz、DMSO-d6):δ173.83、172.03、171.45、171.38、169.31、165.19、165.13、164.85、158.01、144.84、135.54、133.74、133.45、129.57、129.19、129.14、129.02、128.99、128.94、128.67、128.56、127.75、127.59、126.59、113.10、100.84、85.14、71.92、70.73、69.97、68.72、67.93、63.16、62.09、57.39、57.33、56.90、54.97、49.69、48.89、47.86、46.59、42.65、40.00、39.92、39.83、39.76、39.67、39.59、39.50、39.33、39.16、39.00、38.23、34.68、32.72、32.68、32.15、31.49、30.09、29.89、29.84、28.62、28.53、27.89、26.72、26.34、26.16、26.01、22.66、21.40、21.09、20.46、16.64。

[0447] Compound 148: Compound 147 (0.85 g, 0.64 mmol), CPG (5 g, 0.67 mmol), HBTU (0.485 g, 1.3 mmol), and DIPEA (0.4 mL, 2 mmol) were dissolved in acetonitrile and shaken for 2 hours. The product was filtered, washed with DCM, a solution of DCM:MeOH (9:1), and then dried. The solid was then shaken with a solution of pyridine:acetic anhydride (35%) for 3 hours for capping. The product was filtered, washed with DCM, DCM:MeOH (9:1), hexane, and DCM, and then dried under reduced pressure to give 5.2 g (70.4 μmol / g loading) of Compound 148.

[0448] Compound 149: Compound 146 (2.3 g, 2.025 mmol) was azeotroped in pyridine (three times) to ensure all water was removed and kept under argon from this point onward. All solvents used were degassed with argon. To a stirred solution of compound 4 and DIPEA (1.3 mL, 7.2 mmol) in pyridine at 0 °C, 2-cyanoethyldiisopropylchlorophosphoramidite (1 g, 4.2 mmol) was added. The mixture was stirred until TLC showed the disappearance of all starting material. The reaction mixture was concentrated, dissolved in a solution of EtOAc:NEt3 (1%):DCM (20%), and passed through a silica gel rapid filtration column with EtOAc:NEt3 (3%):DCM (25%) to filter out all salts. The filtrate was concentrated, redissolved in EtOAc, and heptane was added until the product oiled out. The supernatant layer was decanted, and the product was concentrated. The product was redissolved in EtOAc and added dropwise to a solution of heptane at 0° C. until the product precipitated. The heptane was decanted and the solid was dried to give 2.2 g (79%) of compound 149. 31P NMR(162MHz、CD3CN)δ148.03、147.94。1H NMR(400MHz、CD3CN):δ8.00-7.95(m,4H)、7.80-7.76(m,2H)、7.69-7.42(m,10H)、7.38-7.18(m,10H)、6.89-6.66(m,5H)、5.83(d,J=3.0Hz、1H)、5.45-5.37(m,1H)、4.80(d,J=8.6Hz、1H)、4.51(ddd,J=10.7、6.4、2.3Hz、1H)、4.41-4.31(m,3H)、3.77-3.75(m,5H)、3.31-3.07(m,4H)、2.93-2.34(m,8H)、2.32-2.18(m,5H)、2.14(m,4H)、1.94(dt,J=4.9、2.5Hz、3H)、1.77-1.73(m,3H)、1.64-1.56(m,4H)、1.36-1.10(m,16H)、1.09(d,J=6.8Hz、2H)、0.99(d,J=6.2Hz、1H). 13C NMR (101MHz, CD3CN): δ173.16, 171.09, 166.76, 166.71, 166.43, 159.80, 159.75, 146. 11, 137.19, 136.98, 136.93, 134.74, 134.45, 134.36, 131.12, 131.06, 130.87, 130.59 , 130.56, 130.50, 130.46, 130.38, 129.94, 129.66, 129.59, 129.16, 129.07, 128.97, 128.90, 128.84, 128.78, 127.96, 127.90, 118.73, 118.37, 114.19, 114.14, 114.06, 114. 01, 101.98, 87.45, 87.15, 73.13, 71.75, 70.13, 69.21, 63.22, 59.00, 56.00, 55.98, 51.70, 50.42, 48.02, 46.06, 43.01, 42.79, 35.70, 33.56, 32.93, 32.69, 29.84, 29.75, 29 .19, 28.25, 28.09, 27.96, 27.87, 27.50, 27.36, 25.12, 23.48, 23.31, 23.18, 22.55, 22.28, 20.94, 20.89, 20.66, 14.47, 2.21, 2.02, 1.81, 1.61, 1.40, 1.19, 0.99, 0.89, 0.78.

[0449] Example 49 [ka]

[0450] 3,4-Diacetyl-6-mesyl GalNAc ester 2: To a solution of the 6-hydroxy derivative 1 (2.00 g, 4.3 mmol) in anhydrous DCM (20 mL) under an Ar atmosphere, DIEA (1.2 mL, 6.5 mmol) and mesyl chloride (0.5 mL, 6.5 mmol) were added sequentially. The mixture was stirred overnight at room temperature and then quenched by the addition of 5% aqueous NaCl (60 mL). The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was co-evaporated once with anhydrous ACN, and the remaining foam-like amorphous solid was dried under high vacuum overnight to give 2.38 g (quantitative) of crude compound 2, which was used in the next step without further purification. MS (in AcOEt): (+) mode: 484 (Mt-Bu); (-) mode: 598 (M+AcOH). 1 H 1 NMR(400MHz), DMSO-d6, J(Hz):1.38(s,9H);1.47(m,4H);1.76(s,3H);1.8 8(s,3H);2.10(s,3H);2.16(t,2H, J=7.1);3.18(s,3H);3.41(m,1H), 3.71 (m,1H);3.87(q,1H,J=8.9);4.10(m,1H);4.19(m,2H);4.50(d,1H,J=8.5) ;4.96(dd,1H,J1=3.4,J2=11.2);5.25(d,1H,J=2.8);1.80(d,1H,J=9.2).

[0451] Unprotected 6-mesyl GalNAc ester 3: To a cold (0 °C) solution of the 3,4-diacetyl derivative 2 (1.29 g, 2.2 mmol) in anhydrous MeOH (10 mL) was added a 25 wt% solution of MeONa in MeOH (0.05 mL, 0.22 mmol) under an argon atmosphere. The mixture was stirred at 0 °C for 2.5 h, quenched by the addition of triethylamine hydrochloride (34 mg, 0.25 mmol), and evaporated under reduced pressure to give 1.20 g (quantitative) of crude compound 3, which was used in the next step without further purification. MS (in MeOH): (+) mode: 400 (Mt-Bu); (-) mode: 490 (M+Cl). 1NMR(400MHz), DMSO-d6, J(Hz):1.37(s,9H);1.46(m,4H);1.78(s,3H);2.16(t,2H, J=7.5);3.17(s,3H);3.36 (m,1H), 3.46(m,1H); 3.67(m,4H); 4.28(m,3H); 4.72(d,1H, J=6.2); 4.85(d,1H, J=4.0); 7.63(d,1H, J=9.0).

[0452] 3,4-Isopropylidene-6-mesyl GalNAc ester 4: The residue from the previous step containing crude compound 3 (1.20 g, 2.2 mmol) was dissolved in a mixture of 2,2-dimethoxypropane (10 mL) and acetone (2 mL), followed by the addition of methanesulfonic acid (2 drops). The mixture was stirred at room temperature for 2.5 h, neutralized by the addition of triethylamine (4 drops), and partitioned between ethyl acetate and saturated sodium bicarbonate. The organic phase was separated, washed with saturated NaCl, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was coevaporated with anhydrous ACN and dried under high vacuum to give 1.10 g (quantitative) of crude compound 4, which was used in the next step without further purification. MS (in AcOEt): (+) mode: 496 (M), 440 (Mt-Bu); (-) mode: 530 (M+Cl), 554 (M+AcOH); H 1 NMR(400MHz), DMSO-d6, J(Hz):1.23(s,3H);1.38(s,9H);1.40(s,3H);1.46(m,4H);1.79(s,3H);2.16(t,2H, J=7.0);3.21(s,3H);3.67(m,1H), 3.55(m,1H);3.68(m,1H);4.14(m,3H);4.27(dd,1H, J1=8.4, J2=10.7); 4.36(d,1H, J=8.7); 4.42(dd,1H, J1=3.3, J2=10.9); 7.86(d,1H, J=9.0).

[0453] 6-(1-Imidazolyl)ester 5a: A solution of mesylate compound 4 (200 mg, 0.4 mmol), imidazole (136 mg, 2 mmol), and DBU (0.075 mL, 0.5 mmol) in anhydrous DMA (3 mL) was heated at 140 °C under argon for 23 h. The mixture was cooled to room temperature, diluted with a 1:1 mixture of saturated ammonium chloride and water (40 mL), and extracted with AcOEt. The organic phase was separated, washed twice with saturated brine, dried over anhydrous sodium sulfate, evaporated, and the residue was chromatographed on a silica gel column using a gradient of MeOH (0–50%) in AcOEt to give 74 mg (40%) of compound 5a. MS (in AcOEt): (+) mode: 468 (M); (-) mode: 502 (M + Cl), 526 (M + AcOH). 1 H NMR (400 MHz), ACN-d3, J (Hz): 1.29 (s, 3H); 1.41 (s, 9H); 1.48 (s, 3H); 1.50 (m, 4H); 1.85 (s, 3H); 2.16 (t, 2H, J = 7.0); 3.31 (dt, 1H, J = 6.2, J = 10.0); 3.64 (m, 2H); 3.99 (m, 1H); 4.03 (dd, 1H, J = 2.0, J = 5.1); 4.20 (m, 3H); 4.30 (d, 1H, J = 8.8); 6.67 (d, 1H, J = 9.2); 6.92 (s split, 1H); 7.08 (s split, 1H); 7.52 (s split, 1H).

[0454] 6-(3-[3-Methoxyphenyl]-imidazolyl-1-ester 5b: A solution of mesylate compound 4 (300 mg, 0.6 mmol), 2-(3-methoxyphenyl)imidazole (520 mg, 3 mmol), and DBU (0.14 mL, 0.9 mmol) in anhydrous DMA (4 mL) was heated at 140 °C under an argon atmosphere for 76 h. The mixture was cooled to room temperature, diluted with a 1:1 mixture of saturated ammonium chloride and water (40 mL), and extracted with AcOEt. The organic phase was separated, washed successively with 5% aqueous NaCl, saturated NaCl, dried over anhydrous sodium sulfate, and evaporated. The residue was chromatographed on a silica gel column using a gradient of MeOH (0–30%) in AcOEt to give 63 mg (18%) of compound 5b. MS (in AcOEt): (+) mode: 574 (M); (-) mode: 608 (M+Cl), 532 (M+AcOH). 1 H NMR(400MHz), ACN-d3, J(Hz):1.31(s,3H);1.40(s,9H);1.47(m,4H);1.50(s,3H);1.85(s,3 H);2.13(t,2H, J=7.1);3.33(m,1H);3.65(m,2H);3.80(s,3H);4.03(m,1H);4.08(dd,1H, J1 =2.1, J2=5.2);4.20(m,3H);4.32(d,1H,J=8.8);6.48(d,1H,J=9.2);6.77(ddd,1H,J1=1.2, J2=2.5, J3=8.1);7.52(t,1H,J=8.2);7.33(m,2H);7.48(d,1H,J=1.3);7.56(d,1H,J=1.3).

[0455] Deprotected imidazolyl derivatives 6a and 6b: Protected derivative 5a or 5b (0.11 mmol) was dissolved in 98% formic acid (2 mL) and water (0.05 mL) was added. The solution was left overnight at room temperature, the formic acid was evaporated under reduced pressure, and the residue was co-evaporated twice with a 1:1 mixture of ethanol and toluene (6 mL). The residue was dissolved in methanol (3 mL), triethylamine (0.15 mL) was added, and the solution was stirred at 65 °C for 4 h. Methanol was removed under reduced pressure, and the residue was co-evaporated three times with 3 mL of pyridine to give 49 and 73 mg of crude compounds 6a and 6b, respectively. The products were further purified by crystallization from an acetonitrile-methanol mixture. 6a: MS (in MeOH): (+) mode: 472 (MH + );(-)Mode:370(MH + ). 1 H NMR(400MHz), DMSO-d6, J(Hz):1.44(m,4H);1.78(s,3H);2.17(t,2H, J=7.0 );3.26(m,1H);3.43(dd,1H, J1=3.0, J2=10.6);3.51(d,1H, J=2.8);3.57(m ,2H);3.70(q,1H,J=10.4);4.11(m,2H);4.18(d,1H,J=8.4);6.86(s,1H);7 .14(s,1H);7.59(m,2H);12.0(s broad,COOH).6b:MS(in MeOH):(+)mode:478(MH + );(-)Mode:476(MH + ).H 1 NMR(400MHz), DMSO-d6, J(Hz):1.44(m,4H);1.78(s,3H);2.16(t,2H, J=7.0);3.28(m, 1H);3.45(m,1H);3.55(s broad,1H);3.62(m,2H);3.71(m,1H);3.76(s,3H);4.15(m,2H) ); 4.22 (d, 1H, J = 8.4); 4.71 (s broad, 1H, OH); 4.92 (s broad, 1H, OH); 6.73 (ddd, 1H, J = 1.4, J = 2.5, J = 8.0); 7.23 (t, 1H, J = 8.0); 7.28 (m, 2H); 7.63 (m, 3H), 12.0 (s broad, COOH).

[0456] Example 50 [ka]

[0457] Histamine derivative 8: A suspension of NHS ester compound 7 (200 mg, 0.33 mmol) and histamine base (52 mg, 0.47 mmol) in anhydrous DCM (3 mL) and pyridine (0.05 mL) was stirred at room temperature for 5 days. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution, and the product was extracted with ethyl acetate. The organic phase was separated, washed with saturated brine, and dried over anhydrous sodium sulfate. The product compound 8 was isolated by column chromatography on silica gel using a gradient of MeOH (0-30%) in DCM. Yield: 64 mg, 32%. MS (in MeOH): (+) mode: 599 (M); (-) mode: 633 (M+Cl). 1 H NMR(400MHz), ACN-d3, J(Hz):1.41(s,9H);1.54(m,4H);1.83(s,3H);1.91(s,3H);2.09(s,3H) ;2.18(t,2H, J=7.0);2.69(t,2H, J=6.5);3.29(m,2H);3.47(m,1H);3.78(m,1H);3.87(t,1H, J =6.3); 3.94 (q, 1H, J=11.0); 4.04 (m, 2H); 4.51 (d, 1H, J=8.5); 5.00 (dd, 1H, J1=3.2, J2=11.2); 5.26 (d, 1H, J=2.9); 5.91 (sbroad, 1H); 6.40 (d, 1H, J=9.4); 6.81 (sbroad, 1H); 7.49 (sbroad, 1H).

[0458] Deprotected histamine derivative 9: A solution of the protected histamine derivative 8 (63 mg, 0.11 mmol) in 98% formic acid (2 mL) was kept at room temperature overnight, then diluted with toluene (10 mL) and evaporated under reduced pressure. The residue was co-evaporated once with a MeOH-ACN mixture and once with a MeOH-pyridine mixture. The product was dried under high vacuum, dissolved in methanol (3 mL), and triethylamine (0.3 mL) was added. The mixture was stirred overnight at 65 °C, filtered, evaporated, and the residue was co-evaporated three times with pyridine (3 mL) and once with ACN (5 mL). It was then dried under high vacuum to give 50 mg (quantitative) of compound 9 as a highly hygroscopic white solid. MS (in MeOH): (-) mode: 457 (MH). 1 H NMR(400MHz), DMSO-d6, J(Hz):1.46(m,4H);1.78(s,3H);2.18(t,2H, J=7.3);2.61(t,2H, J=7.6);3.18(q,2H, J=6.9);3.34(m,2H);3.44(dd,1H, J1=3.0, J2=10.6);3.51(t,1H, J=5. 8); 3.60 (d, 1H, J = 2.4); 3.68 (m, 2H); 4.04 (d, 2H, J = 5.8); 4.23 (d, 1H, J = 8.4); 4.67 (s broad, 2H); 6.77 (s, 1H); 7.23 (t, 1H, J = 5.5); 7.52 (s, 1H); 7.60 (d, 1H, J = 9.0); 12.01 (s broad, 2H).

[0459] Example 51 [ka]

[0460] Synthesis of Compound 102: Compound 101 (5 g, 22.6 mmol) was heated at 100 °C with 5-hexen-1-ol (80 mL) and boron trifluoride diethyl etherate (0.5 mL) for 16 hours. Trituration with EtO gave Compound 102 (4.0 g, 13.2 mmol, 58%). 14 H 26 Molecular weight (M+H) for NO6 +Calculated value: 304.1760, measured value: 304.2.

[0461] Synthesis of Compound 103: Compound 102 (1.48 g, 4.88 mmol) was treated in pyridine (30 mL) and AcO (10 mL). Aqueous workup and purification by silica gel column afforded Compound 103 (1.48 g, 3.45 mmol, 70%). 20 H 32 Molecular weight (M+H) for NO9 + Calculated value: 430.2077, measured value: 430.2.

[0462] Synthesis of Compound 104: Compound 103 (1.37 g, 3.19 mmol) was treated with lipase from Candida rugosa (3.43 g) in dioxane (13.7 mL) and potassium hydrogen phthalate buffer (54.8 mL, pH = 4) for 64 h. Aqueous workup and purification by silica gel column chromatography gave Compound 104 (680 mg, 1.76 mmol, 55%). 1 H NMR (400MHz, CDCl3) δ5.85-5.75(m,1H), 5.59(d,J=9.7Hz, 1H), 5.33-5.32(m,1H), 5. 21(dd,J=11.3Hz, 3.2Hz, 1H), 5.06-4.96(m,2H), 4.86(d,J=3.7Hz, 1H), 4.63-4.57(m, 1H), 4.01(td, J=6.5Hz, 1.2Hz, 1H), 3.73-3.62(m,2H), 3.51-3.39(m,2H), 2.19(s,3H) ), 2.12-2.06(m,2H), 2.02(s,3H), 1.96(s,3H), 1.66-1.59(m,2H), 1.49-1.41(m,2H).

[0463] Synthesis of Compound 105: Compound 104 (747 mg, 1.74 mmol) was treated with triphenylphosphine (913 mg, 3.48 mmol), diisopropyl azodicarboxylate (0.674 mL, 3.48 mL), and diphenylphosphoryl azide (0.752 mL, 3.48 mL) in THF (17 mL) for 18 hours. Aqueous workup and purification by silica gel column afforded Compound 105 (752 mg, quantitative). 1 H NMR (400MHz, CDCl3) δ5.85-5.75(m,1H), 5.56(d,J=9.7Hz, 1H), 5.30(d,J=2.7Hz, 1H), 5.15( dd,J=11.3, 3.3Hz, 1H), 5.05-4.97(m,2H), 4.88(d,J=3.7Hz, 1H), 4.60-4.54(m,1H), 4.06(dd ,J=8.8, 3.6Hz, 1H), 3.77-3.71(m,1H), 3.49-3.40(m,2H), 3.13(dd,J=12.8, 4.1Hz, 1H), 2.1 7(s,3H), 2.12-2.07(m,2H), 1.99(s,3H), 1.96(s,3H), 1.65-1.60(m,2H), 1.50-1.44(m,2H).

[0464] Example 52 [ka]

[0465] Synthesis of Compound 106: Compound 105 (730 mg, 1.77 mmol) was treated with ruthenium(III) chloride hydrate (18 mg, 0.089 mmol) and sodium periodate (1.89 g, 8.85 mL) in CHCl (5 mL), CHCN (5 mL), and HO (7 mL) for 18 hours. Aqueous workup and purification by silica gel column afforded Compound 106 (677 mg, 1.57 mmol, 89%). 17 H 27 Molecular weight (M+H) for N4O9 + Calculated value: 431.1778, measured value: 431.1.

[0466] Synthesis of Compound 107: Compound 106 (200 mg, 0.465 mmol) was treated with N-hydroxysuccinimide (80 mg, 0.698 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (134 mg, 0.698 mmol), and DIEA (0.244 mL, 1.40 mmol) in CHCl (3 mL) for 18 hours. Aqueous workup and purification by silica gel column afforded Compound 107 (183 mg, 0.347 mmol, 75%). 21 H 30 N5O 11 Molecular weight (M+H) + Calculated value 528.1942, measured value 528.1.

[0467] Synthesis of Compound 108: To a solution of Compound 106 (227 mg, 0.527 mmol) and 3-ethynylanisole (0.080 mL, 0.632 mmol) in MeOH (2 mL) was added a solution of THPTA (11 mg, 0.0264 mmol) and CuSO4.5HO (1.3 mg, 0.00527 mmol) in HO (0.1 mL) and a solution of sodium ascorbate (10 mg, 0.0527 mmol) in HO (0.1 mL). The reaction mixture was stirred at room temperature for 18 hours. Aqueous workup and purification by silica gel column chromatography afforded Compound 108 (264 mg, 0.469 mmol, 89%). 26 H 35 N4O 10 Molecular weight (M+H) + Calculated value: 563.2353, measured value: 563.2.

[0468] Synthesis of Compound 109: Compound 108 (233 mg, 0.414 mmol) was treated with N-hydroxysuccinimide (72 mg, 0.621 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (119 mg, 0.621 mmol), and DIEA (0.216 mL, 1.24 mmol) in CHCl (4 mL) for 18 hours. Aqueous workup and purification by silica gel column afforded Compound 109 (100 mg, 0.152 mmol, 37%).30 H 38 N5O 12 Molecular weight (M+H) + Calculated value 660.2517, measured value 660.2.

[0469] Synthesis of Compound 110: To a solution of Compound 106 (223 mg, 0.518 mmol) and 2-methyl-8-(prop-2-yn-1-yloxy)quinoline (123 mg, 0.622 mmol) in MeOH (2 mL), a solution of THPTA (11.2 mg, 0.0259 mmol) and CuSO4.5HO (1.3 mg, 0.00518 mmol) in HO (0.1 mL) and a solution of sodium ascorbate (10.3 mg, 0.0518 mmol) in HO (0.1 mL) were added. The reaction mixture was stirred at room temperature for 18 hours. Aqueous workup and purification by silica gel column chromatography afforded Compound 110 (320 mg, 0.510 mmol, 98%). 30 H 38 N5O 10 Molecular weight (M+H) + Calculated value 628.2619, measured value 628.2.

[0470] Synthesis of Compound 111: Compound 110 (307 mg, 0.489 mmol) was treated with N-hydroxysuccinimide (85 mg, 0.734 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (141 mg, 0.734 mmol), and DIEA (0.256 mL, 1.47 mmol) in CHCl (4 mL) for 18 hours. Aqueous workup and purification by silica gel column afforded Compound 111 (155 mg, 0.207 mmol, 42%). 34 H 41 NO 12 Molecular weight (M+H) + Calculated value 725.2782, measured value 725.1.

[0471] Synthesis of Compound 112: Compound 106 (240 mg, 0.558 mmol) was treated in MeOH (9 mL) and EtN (1 mL) for 5 days to give Compound 112 (250 mg, 0.558 mmol, quantitative). 13 H 23 Molecular weight (M+H) for N4O7 + Calculated value: 347.1567, measured value: 347.1.

[0472] Example 53 [ka]

[0473] Synthesis of Compound 121: Following a procedure similar to that described for Compound 108, but using Compound 120 instead of Compound 106, Compound 121 (300 mg, 0.495 mmol, 80%) was obtained. 29 H 40 Molecular weight (M+H) for FN4O9 + Calculated value 607.2779, measured value 607.1.

[0474] Synthesis of Compound 122: Following a procedure similar to that described for Compound 108, but using Compound 120 instead of Compound 106, Compound 122 (417 mg, 0.635 mmol, 90%) was obtained. 30 H 40 Molecular weight (M+H) for F3N4O9 + Calculated value 657.2747, measured value 657.2.

[0475] Synthesis of Compound 123: Following a procedure similar to that described for Compound 108, but using Compound 120 instead of Compound 106, Compound 123 (343 mg, 0.559 mmol, 80%) was obtained. 30 H 40 Molecular weight (M+H) for N5O9 + Calculated value 614.2826, measured value 614.2.

[0476] Synthesis of Compound 124: Following a procedure similar to that described for Compound 108, but using Compound 120 instead of Compound 106, Compound 124 (340 mg, 0.537 mmol, 79%) was obtained. 29 H 40 N5O 11 Molecular weight (M+H) + Calculated value 634.2724, actual value 634.0.

[0477] Synthesis of Compound 125: Following a procedure similar to that described for Compound 108, but using Compound 120 instead of Compound 106, Compound 125 (323 mg, 0.54 mmol, 83%) was obtained. 29 H 41 N4O 10 Molecular weight (M+H) + Calculated value 605.2823, measured value 605.2.

[0478] Synthesis of Compound 126: Compound 121 (290 mg, 0.478 mmol) was treated with formic acid (10 mL) for 18 hours. After removing the solvent, purification by silica gel column gave Compound 126 (241 mg, 0.438 mmol, 92%). 25 H 32 Molecular weight (M+H) for FN4O9 + Calculated value: 551.2153, measured value: 551.0.

[0479] Synthesis of Compound 127: Compound 122 (407 mg, 0.620 mmol) was treated with formic acid (10 mL) for 18 hours. After removing the solvent, purification by silica gel column gave Compound 127 (318 mg, 0.530 mmol, 85%). 26 H 32 Molecular weight (M+H) for F3N4O9 + Calculated value 601.2121, measured value 601.0.

[0480] Synthesis of Compound 128: Compound 123 (333 mg, 0.543 mmol) was treated with formic acid (10 mL) for 18 hours. After removing the solvent, purification by silica gel column gave Compound 128 (281 mg, 0.504 mmol, 93%). 26 H 32 Molecular weight (M+H) for N5O9 + Calculated value 558.2200, measured value 558.2.

[0481] Synthesis of Compound 129: Compound 124 (330 mg, 0.521 mmol) was treated with formic acid (10 mL) for 18 hours. After removing the solvent, purification by silica gel column gave Compound 129 (277 mg, 0.480 mmol, 92%). 25 H 32 N5O 11 Molecular weight (M+H) + Calculated value: 578.2098, measured value: 578.0.

[0482] Synthesis of Compound 130: Compound 125 (313 mg, 0.518 mmol) was treated with formic acid (10 mL) for 18 hours. After removing the solvent, purification by silica gel column gave Compound 130 (253 mg, 0.461 mmol, 89%). 25 H 33 N4O 10 Molecular weight (M+H) + Calculated value: 549.2197, measured value: 549.0.

[0483] Example 54 [ka]

[0484] NHS ester compounds 131, 132, 133, 134, and 135 are prepared by a standard esterification process with N-hydroxysuccinimide using compounds 126, 127, 128, 129, and 130, respectively. Fully deprotected GalNAc derivatives 136, 137, 138, 139, and 140 are prepared from compounds 126, 127, 128, 129, and 130 by treatment with EtN / MeOH.

[0485] Example 55 [ka]

[0486] Synthesis of 201: Compound 200 (20 g, 44.74 mmol) was stirred in dichloromethane (150 mL). EDAC (12.8 g, 65 mmol), DMAP (2 g, catalytic), and t-butanol (20 mL) were then added. The mixture was stirred at room temperature for 2 days. The solvent was then removed under reduced pressure. The residue was extracted with dichloromethane (3 x 100 mL) and dried over sodium sulfate. The crude product was purified by silica gel chromatography using ethyl acetate and hexane to give compound 201 (15 g, 68%). 23 H 37 NO 11 Calculated molecular weight: 503.24, found: 526.23 (M+Na).

[0487] Synthesis of Compound 202: Using a procedure similar to that described for the synthesis of Compound 104, 10 g of Compound 201 was converted to Compound 202 (7.6 g, 78%). 21 H 35 NO 10 Calculated molecular weight: 461.23, found: 484.25 (M+Na).

[0488] Synthesis of Compound 203: Compound 203 was synthesized (5.2 g, 64%) using a procedure similar to that described for the synthesis of Compound 105. 21 H 34Calculated molecular weight for N4O9: 486.23, found: 509.24 (M+Na).

[0489] Synthesis of Compound 204: Compound 203 (6.34 g, 13.03 mmol) was stirred in formic acid (20 ml) overnight. The solvent was removed, and the residue was dissolved in dichloromethane and washed with water and brine. The crude product was purified by silica gel chromatography using ethyl acetate / hexane to give Compound 204 (4.6 g, 82%). 17 H 26 Calculated molecular weight for N4O9: 430.17, found: 453.18 (M+Na).

[0490] Synthesis of Compound 205: Compound 204 (0.50 g, 1.16 mmol) was dissolved in dichloromethane (50 mL). Next, N-hydroxysuccinimide (0.200 g, 1.5 equivalents), EDAC, and DIEA were added, and the resulting mixture was stirred overnight. The mixture was then washed with water and brine. The solvent was removed, and the residue was purified by filtration chromatography using ethyl acetate / hexane to give Compound 205.

[0491] Synthesis of Compound 206: Compound 204 (1.00 g, 2.32 mmol) was stirred in a mixture of methanol and water (2:1). 1-Ethynyl-3-methoxybenzene (204A) (0.367 g, 2.7 mmol), CuSO4.×H2O (0.050 g, catalytic amount), and sodium ascorbate (0.25 g, 1 mmol) were then added, and the mixture was stirred overnight. The solvent was removed, and the residue was dissolved in dichloromethane, washed with water, brine, and dried over sodium sulfate. The crude product was purified by silica gel chromatography to give Compound 206. 26 H 34 N4O 10 Calculated molecular weight: 562.23, found: 585.22 (M+Na).

[0492] Synthesis of Compound 207: Compound 207 was prepared using a similar procedure as described for the synthesis of compound 205 (320 mg, 95%).

[0493] Example 56 [ka]

[0494] Synthesis of Compound 207: Compound 202 (2.8 g, 6.07 mmol) was dissolved in dichloromethane (100 mL), and the mixture was cooled in an ice-water bath. To this mixture, DSC (1.3 g, 1.5 equivalents) and TEA (0.7 mL) were added, and the solution was stirred overnight. The reaction mixture was then diluted with dichloromethane and transferred to a separatory funnel. The mixture was washed with water and brine, and then dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue was dried under reduced pressure overnight. The resulting product was used in the next reaction step without further purification.

[0495] Synthesis of Compound 208: DSC derivative Compound 207 (0.5 g, 0.83 mmol) was stirred in dichloromethane (20 mL). Benzylamine (0.100 g, 1 mmol) and pyridine (5 mL) were added, and the mixture was stirred overnight. The solvent was then removed under reduced pressure. The residue was dissolved in dichloromethane and washed with water and brine. The crude product was purified by silica gel chromatography using dichloromethane / methanol to give Compound 208 (0.300 g, 65%). 29 H 42 N2O 11 Calculated molecular weight: 594.28, found: 595.29 (M+).

[0496] Synthesis of Compound 209: DSC derivative Compound 207 (0.5 g, 0.83 mmol) was stirred in dichloromethane (20 mL). Ethanolamine (0.07 g, 1 mmol) and pyridine (5 mL) were then added, and the mixture was stirred overnight. The solvent was then removed under reduced pressure. The residue was dissolved in dichloromethane and washed with water and brine. The crude product was purified by silica gel chromatography using dichloromethane / methanol to give Compound 209 (0.25 g, 42%). 24 H 40 N2O 12 Calculated molecular weight for 548.26, found 549.27 (M+H).

[0497] Synthesis of Compound 210: Compound 208 (250 mg, 0.42 mmol) was dissolved in formic acid (20 mL), and the solution was stirred overnight. The solvent was then removed under reduced pressure. The crude compound was dissolved in dichloromethane and washed with water and brine. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography using dichloromethane / methanol to give Compound 210 (200 mg, 87%). This compound was then dissolved in methanol, and TEA (2 mL) was added. After stirring overnight at room temperature, the solvent was removed under reduced pressure, and the residue was co-evaporated twice with pyridine. The product was further dissolved in water and then lyophilized to give Compound 210 as a white powder. 21 H 30 Calculated molecular weight for N2O9: 454.20, found: 477.21 (M+Na).

[0498] Synthesis of Compound 211: Compound 211 was prepared from compound 209 using a method similar to that used to prepare compound 210. 16 H 28 N2O 10 Calculated molecular weight: 408.17, found: 431.20 (M+Na).

[0499] Example 57 [ka]

[0500] Synthesis of Compound 213: Glutamate derivative A (0.174 g, 1 mmol) was dissolved in DMF (10 mL). HBTU (0.390 g, 1.05 mmol) and DIEA were added, and the mixture was stirred at room temperature for several minutes. Amino derivative compound 212 (0.400 g, 1 mmol) in DMF was added to this solution, and stirring was continued overnight. The solvent was then removed under reduced pressure. The crude compound was purified by silica gel chromatography using dichloromethane / methanol to give compound 213 (0.350 g, 57%). 23 H 40 N4O 10 Calculated molecular weight: 532.27, found: 555.28 (M+Na).

[0501] Synthesis of Compound 214: Compound 213 (300 mg, 0.56 mmol) was dissolved in formic acid, and the mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was co-evaporated twice with toluene. The residue was dissolved in water and lyophilized to give Compound 214 (200 mg, 74%) as a white powder. 19 H 32 N4O 10 Calculated molecular weight for 476.21, found 477.20 (M+H).

[0502] Synthesis of Compound 215: Compound 215 was prepared from Compound B (140 mg) in a similar manner. 27 H 48 N4O 10 Calculated molecular weight: 588.34, found: 611.33 (M+Na).

[0503] Synthesis of Compound 216: Compound 216 was prepared from compound 215 using a procedure similar to that used to prepare compound 214 (0.125 g, 35%). 23 H 40 N4O 10 Calculated molecular weight: 532.27, found: 555.25 (M+Na).

[0504] Synthesis of Compound 217: Compound 216 was prepared from compound C and the amino derivative 212 (0.250 mg, 65%). 25 H 41 Calculated molecular weight for N5O9: 555.29, found: 556.31 (M+H).

[0505] Synthesis of Compound 218: Compound 218 was prepared from compound 217 using a procedure similar to that used to prepare compound 214 (0.140 mg, 45%). 21 H 33 Calculated molecular weight for N5O9: 499.23, found: 500.25 (M+H).

[0506] Synthesis of Compound 219: Compound 219 was prepared from compound D and amino derivative 212 using a procedure similar to that used to prepare compound 213 (0.525 g, 43%). 24 H 43 Calculated molecular weight for N3O9: 517.30, found: 518.28 (M+H).

[0507] Synthesis of Compound 220: Compound 220 was prepared from compound 219 using a procedure similar to that used to prepare compound 214 (95 mg, 26%). 20 H 35 Calculated molecular weight for N3O9: 461.24, found: 462.26 (M+H).

[0508] Synthesis of Compound 221: Compound 221 was prepared from Compound E and the amino derivative Compound 212 (0.320 g, 65%). 21 H 37 Calculated molecular weight for N3O9: 475.25, found: 476.23 (M+H).

[0509] Synthesis of Compound 222: Compound 222 was prepared from compound 221 using a procedure similar to that used to prepare compound 214 (85 mg, 56%). 17 H 29 Calculated molecular weight for N3O9: 419.19, found: 420.20 (M+Na).

[0510] Example 58 [ka]

[0511] Compound 58 is prepared in a manner similar to that reported in WO 96 / 39411. O-glycosylation followed by hydrolysis gives compounds 60 and 63. NHS ester compounds 61 and 64 are prepared by standard esterification with NHS. The acetyl group of compound 65 is selectively removed and the resulting hydroxyl group is protected with a benzyl group to give compound 66. Oxidative cleavage of the terminal alkene gives compound 67. Esterification followed by hydrogenation gives compounds 61 and 64. O-glycosylation of compound 58 followed by oxidation and hydrolysis gives compound 60. Esterification of compound 60 gives compound 61.

[0512] [ka]

[0513] Trifluoromethylacetamide (TFA)-protected galactosamine (GalN-TFA) NHS ester is coupled to amine-containing oligonucleotides (compounds 69 and 71) in a post-synthetic manner to generate Gal-TFA-containing oligonucleotides (compounds 70 and 72).

[0514] Example 59 [ka]

[0515] Synthesis of compound 5009: To a stirred solution of compound 5008 (25.0 g, 83 mmol) in pyridine (100 mL) was added TsCl (19.8 g, 103.7 mmol). The resulting mixture was stirred at room temperature overnight (14 hours). The solvent was concentrated, and the product was extracted with ethyl acetate (3 x 50 mL), washed with water, brine, and dried over anhydrous Na2SO4. Concentration of the solvent afforded crude compound 5009 (25 g). C 21 H 29 LCMS calculated for NO8S: 455.16 (M + ), Actual measurement: 456.1 (M + +1), 478.0(M + +Na + ).

[0516] Synthesis of compound 5010: To a stirred solution of compound 5009 (19.0 g, 41.7 mmol) in DMF (200 mL) was added NaN (16 g, 246 mmol). The resulting mixture was stirred at 80 °C for 3 days. Next, an additional 8 g of NaN was added, and the solution was heated to 100 °C for 14 hours. The solvent was concentrated, and the product was extracted with ethyl acetate (3 × 50 mL), washed with water, brine, and dried over anhydrous NaSO. Concentration of the solvent gave crude compound 5010, which was purified by column chromatography (5 g, 37%). 14 H 22 LCMS calculated for N4O5: 326.35 (M + ), Actual measurement: 327.1 (M + +1).

[0517] Synthesis of compound 5011: To a stirred solution of LAH (139 mg, 3.52 mmol) in THF (10 mL) was added dropwise a solution of compound 5010 (574 mg, 1.76 mmol) in THF (10 mL) at 0° C. The mixture was stirred overnight (14 h) at room temperature. The reaction mixture was quenched with 1 mL of water, filtered over Celite, and washed with ethyl acetate (25 mL). Concentration of the solvent gave the crude product (0.5 g), which was dissolved in DCM (0 mL) and added to a stirred solution of glutaric anhydride (251 mg, 2.2 mmol). The reaction mixture was stirred at room temperature for 14 h. Concentration of the solvent gave the crude acid (285 mg), which was dissolved in 20 mL of 2N HCl in diethyl ether and stirred for 3 h. Concentration of the solvent gave compound 5011 (200 mg). 16 H 26 LCMS calculated for N2O8: 374.39 (M + ), Actual measurement: 373.1 (M - -1).

[0518] Synthesis of compound 5012: To a stirred solution of compound 5011 (200 mg, 0.53 mmol) and NHS (112 mg, 1.06 mmol) in DMF (10 mL) was added DCC (218 mg, 1.06 mmol). The mixture was stirred at room temperature for 14 hours. Then, 20 mL of ethyl acetate was added. Filtration of the solid afforded compound 5012 (150 mg, 60%). 20 H 29 N3O 10 LCMS calculated for: 471.46 (M + ), Actual measurement: 506 (M - +Cl - ).

[0519] Example 60: Trivalent GalNAc-conjugated pseudouridine building blocks [ka]

[0520] Synthesis of compound 143: To a solution of trivalent GalNAc acid compound 142 (1.80 g, 0.898 mmol) in DMF (12 mL) was added HBTU (341 mg, 0.898 mmol) and i-PrNEt (0.568 mL, 3.26 mmol). After 10 min, compound 141 (500 mg, 0.816 mmol) was added to the solution, and the mixture was stirred overnight. After removing DMF under reduced pressure, the residue was extracted with CHCl and saturated aqueous NaHCO. The organic layer was dried over anhydrous NaSO, filtered, and concentrated. The crude product was purified by silica gel column chromatography (0–15% MeOH in CHCl) to give compound 143 (1.75 g, 0.673 mmol, 82%). 1 H NMR (400MHz, DMSO-d6) δ11.35(s,1H), 7.96(s,1H), 7.84-7.81(m,6H), 7.76-7.72(m,4H), 7.50(d,J=0.9Hz, 1H), 6.98(s,1H), 5.21(d,J=3.4Hz, 3H), 4.96(dd,J=11.2, 3.4Hz, 3H), 4.53(s,1H), 4.48(d,J=8.5Hz, 3H), 4 .35-4.33(m,1H), 4.17(d,J=4.5Hz, 1H), 4.05-3.83(m,17H), 3.73-3.64 (m,3H), 3.57-3.52(m,12H), 3.43-3.38(m,3H), 3.06-3.00(m,16H), 2.4 4-2.39(m,2H), 2.27(t,J=6.4Hz, 6H), 2.10(s,9H), 2.04(t,J=7.3Hz, 9H ), 1.99(s,9H), 1.89(s,9H), 1.77(s,9H), 1.52-1.42(m,22H), 1.21(s,1 3H), 1.01(s,9H), 0.98(s,9H), 0.89(s,9H), 0.12(s,3H), 0.079(s,3H).

[0521] Synthesis of Compound 144: Hydrogen fluoride-pyridine (approximately 70% HF, 0.173 mL, 6.66 mmol) was diluted with pyridine (2 mL) while cooling at 0 °C. The resulting solution was added to a solution of Compound 143 in CHCl (20 mL) at 0 °C, and the mixture was stirred at 0 °C for 2 hours. The reaction solution was diluted with CHCl, washed with saturated aqueous NaHCO, and then dried over anhydrous NaSO. After evaporation of the volatiles, the crude product was dried under reduced pressure to give the diol as a white foam. To a solution of this material in pyridine (15 mL) was added DMTrCl (691 mg, 2.04 mmol). The reaction mixture was stirred at room temperature for 14 hours and then evaporated. The residue was extracted with CHCl and saturated aqueous NaHCO and then dried over anhydrous NaSO. The crude product was purified by silica gel column chromatography (0-10% MeOH in CH2Cl2) to give compound 144 (3.31 g, 1.20 mmol, 65%).

[0522] Synthesis of compound 145: To a solution of compound 144 (3.25 g, 1.18 mmol) in CHCl (20 mL) was added DMAP (432 mg, 3.54 mmol) and succinic anhydride (236 mg, 2.36 mmol). The reaction mixture was stirred at room temperature overnight. After concentration, the crude material was purified by silica gel column chromatography (8% MeOH / 8% EtN in CHCl) to give compound 145 (3.03 g, 1.78 mmol, 87%). 1H NMR (400MHz, DMSO-d6) δ12.22(brs, 1H), 11.41(s,1H), 7.94(brs, 1H), 7.85-7.81(m ,6H), 7.75-7.72(m,4H), 7.66(s,1H), 7.41-7.38(m,2H), 7.32-7.19(m,9H), 6.98(s, 1H), 6.89-6.87(m,4H), 5.21(d,J=3.4Hz, 3H), 5.07(t,J=5.1Hz, 1H), 4.96(dd,J=11 .2, 3.4Hz, 3H), 4.55(t,J=5.2Hz, 1H), 4.56-4.44(m,4H), 4.05-3.97(m,10H), 3.91-3 .83(m,3H), 3.73(s,6H), 3.71-3.64(m,4H), 3.56-3.52(m,14H), 3.43-3.38(m,3H), 3.23-3.14(m,2H), 3.06-3.00(m,16H), 2.46-2.38(m,4H), 2.27(t,J=6.4Hz, 6H), 2.1 0(s,9H), 2.06-2.01(m,9H), 1.99(s,9H), 1.89(s,9H), 1.77(s,9H), 1.52-1.43(m,22 H), 1.21(s,13H), 0.95(t,J=7.2Hz, 1H), 0.80(s,9H), -0.013(s,3H), -0.046(s,3H).

[0523] Synthesis of Compound 146: To a solution of Compound 145 (103 mg, 0.0347 mmol) in CHCN (5 mL) was added HBTU (26 mg, 0.0694 mmol), iPrNEt (0.026 mL, 0.149 mmol), and CPG-NH (Prime Synthesis CPG-500, NH loading estimated at 80 μmol / g) (450 mg, 0.036 mmol). The mixture was shaken for 24 h, then filtered, washed with CHCl, and dried under reduced pressure. The remaining amino groups were capped with pyridine (7.5 mL), acetic anhydride (2.5 mL), and triethylamine (0.5 mL) by shaking for 1 h. After filtration, washing with CHCl (100 mL), then 50% MeOH / CHCl (100 mL), and drying under reduced pressure, Compound 146 was obtained. Loading amount: 49μmol / g.

[0524] Example 61: Primary hepatocyte binding for triple-chain and 1+1+1 ligand designs siRNA-ligand conjugates were prepared. The siRNA in each conjugate was the same and targeted TTR. The following ligands were attached to the 3' end of the sense strand of each siRNA. The structure of the ligand in the conjugate was the same as that in conjugate 43527, except for the sugar group substitution shown below. [ka]

[0525] Figure 13 shows the primary hepatocyte binding affinity for siRNA-ligand conjugates 61696, 61695, 61692, 61694, 61697, 61693, 43527 and 61698, the structures of which are shown below. The binding affinity K values ​​are shown in the table below. [Table 15]

[0526] Example 62: SiRNA GalNAC conjugates with high affinity ligands siRNA-ligand conjugates were prepared. The siRNA in each conjugate was the same and targeted TTR. The following ligands were attached to the 3' end of the sense strand of each siRNA. The structures of the ligands in the conjugates were the same as those in conjugate 57727, except for the sugar group substitutions shown below. The structures of L224, L223, L221, L96, and L227 are shown below. [Table 16] [ka]

[0527] 3A and 3B show serum mTTR SiRNA levels in the blood 72 hours (FIG. 3A) and 144 hours (FIG. 3B) after a single subcutaneous administration of conjugates 57727, 63189, 63192, 63190, and 63191 to mice according to the protocol in Example 33.

[0528] Example 63: Compounds for T-2'-GalNAc Building Block [Table 17]

[0529] Example 64: Compounds for T-3'-GalNAc Building Block [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6] [Table 18-7] [Table 18-8]

[0530] Example 65: [ka]

[0531] 2'- and 3'-O-Phthalimidohexyl-5-methyluridine (2A, 2B). 2',3'-O-Dibutylstannylene-5-methyluridine (2.0 g, 4.1 mmol) was suspended in DMF (10 mL). 6-Bromohexylphthalimide (2.5 g, 8.2 mmol) and NaI (120 mg, 0.82 mmol) were added to the suspension. The reagents were microwaved at 100 °C for 3.5 h, resulting in a dark brown homogeneous mixture. DMF was evaporated under reduced pressure, and the residue was adsorbed onto silica gel. The silica gel was loaded into a silica gel chromatography cartridge. The 2'- and 3'-isomers of O-phthalimidohexyl-5-methyluridine eluted as an inseparable mixture, yielding 890 mg of 2A and 2B (1.8 mmol, 45%). 1 H NMR (400MHz, DMSO-d6) δ11.29(s,1H, D2O exchangeable), 7.88-7.80(m,4H), 7.78-7.76(m,1H), 7.73-7.70(m,0H), 5.81(d ,J=5.3Hz, 1H), 5.72(d,J=5.6Hz, 1H), 5.25(d,J=6.2Hz, 1H, D2O replaceable), 5.12(t,J=5.0Hz, 1H, D2O replaceable), 5.00(d,J= 5.9Hz, 1H, D2O replaceable), 4.14(q,J=5.6Hz, 1H), 4.07(q,J=5.0Hz, 1H), 3.90-3.79(m,2H), 3.74(t,J=4.6Hz, 0H), 3.67 -3.58(m,1H), 3.58-3.49(m,4H), 3.46-3.37(m,1H), 1.75(d,J=3.7Hz, 3H), 1.62-1.42(m,4H), 1.37-1.20(m,4H). 13C NMR (100MHz, DMSO-d6) δ168.00, 167.97, 163.78, 163.73, 150.79, 150.57 , 136.23, 136.07, 134.44, 134.40, 131.60, 131.56, 123.02, 109.38, 109.2 6, 87.73, 85.89, 85.07, 82.71, 80.82, 77.49, 72.43, 69.69, 69.51, 68.38 , 60.86, 60.62, 29.23, 28.96, 27.93, 26.15, 26.07, 25.16, 24.96, 12.26.C 24 H 29 MS calculated for N3O8: 487.1955, found m / z: 488.0 (M+1) + , 510.2 (M+23) Na+ , 486.2(M-1) - , 522.2(M+35) Cl- .R f = 0.26 in 5% MeOH / DCM v / v.

[0532] 5'-O-Dimethoxytrityl-2'-O-phthalimidohexyl-5-methyluridine (3A). A mixture of 2A and 2B (890 mg, 1.83 mmol) was coevaporated with pyridine and then dissolved in pyridine (10 mL) under an argon atmosphere and cooled to 0 °C in an ice bath. To this mixture was added DMTrCl (690 mg, 2.04 mmol), and the reaction was stirred overnight while warming to room temperature. An additional 0.55 equivalents of DMTrCl was added, and the reaction was stirred for an additional 2 hours. The reaction was quenched with MeOH and evaporated under reduced pressure. The crude 2' and 3' isomers (3A and 3B) were dissolved in DCM, and the organic layer was washed twice with brine. The organic layer was dried over NaSO and evaporated under reduced pressure. The compound was purified by silica gel chromatography and concentrated under reduced pressure to give 510 mg of 3A (0.65 mmol, 35%). The 2'-O-alkylated isomers were characterized by DO exchange followed by identification of the 3'-OH by COSY. 1H NMR (400MHz, DMSO-d6) δ11.35 (s, 1H, D2O exchangeable), 7.88-7.77 (m, 4H), 7.48 (s, 1H), 7.38 (d ,J=7.4Hz, 2H), 7.33-7.19(m,7H), 6.89(d,J=8.0Hz, 4H), 5.82(d,J=4.8Hz, 1H), 5.10(d, J=6.3Hz, 1H, D2O exchangeable), 4.18 (q, J=5.5Hz, 1H), 3.96 (q, J=4.8, 4.4Hz, 2H), 3.72 (s, 6H), 3 .62-3.46(m,4H), 3.27-3.14(m,2H), 1.59-1.45(m,4H), 1.38(s,3H), 1.34-1.20(m,4H). 13 C NMR (100MHz, DMSO-d6) δ167.91, 163.61, 158.19, 158.16, 150.41, 144.66, 135.45, 135.31, 135.11, 134.33, 131.58, 129.74, 127.93, 127. 65, 126.84, 122.97, 113.27, 109.60, 86.46, 85.91, 83.09, 80.53, 69. 63, 68.76, 63.19, 55.06, 37.31, 28.89, 27.91, 26.07, 24.95, 11.66.C 45 H 47 N3O 10 MS calculated for 789.3261, observed m / z 812.3 (M+23) Na+ , 788.3(M-1) - , 824.3(M+35) Cl- R f = 0.35 in 60% EtOAc / hexane v / v.

[0533] 5'-O-Dimethoxytrityl-3'-O-phthalimidohexyl-5-methyluridine (3B). The 3'-isomer (3B) was separated from the 2'-isomer (3A) during silica gel chromatography and concentrated under reduced pressure to give 420 mg of 3B (29%, 0.53 mmol). The 3'-O-alkylated isomer was characterized identically, except for identification of the 2'-OH by DO exchange. 1H NMR (400MHz, DMSO-d6) δ11.34(s,1H, D2O exchangeable), 7.87-7.79(m,4H), 7.49(s,1H), 7.36(d,J=7.4Hz, 2H), 7.29(t,J=7.6Hz, 2H), 7.26-7.19(m,5H), 6.94-6.81(m,4H), 5.71(d,J=4.6Hz, 1H), 5.36(d,J= 6.0Hz, 1H, D2O replaceable), 4.27(q,J=5.2Hz, 1H), 3.99-3.95(m,1H), 3.90(t,J=5.3Hz, 1H), 3.71(s,6H), 3.63-3.47(m,3H), 3.36(t,J=6.8Hz, 1H), 3.26-3.15(m,2H), 1.59-1.39(m,7H), 1.28-1.20(m,4H). 13 C NMR (100MHz, DMSO-d6) δ167.93, 163.69, 158.17, 158.15, 150.57, 144.63, 135.72, 135.30, 135.17, 134.35, 131.58, 129.71, 127.91, 127. 63, 126.82, 122.97, 113.24, 109.37, 88.69, 85.91, 80.60, 77.27, 72. 18, 69.67, 55.03, 39.50, 37.33, 29.09, 27.88, 26.08, 25.08, 11.74.C 45 H 47 N3O 10 MS calculated for 789.3261, observed m / z 812.0 (M+23) Na+ , 788.3(M-1) - , 824.3(M+35) Cl- R f = 0.18 in 60% EtOAc / hexane v / v.

[0534] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-5-methyluridine (4A *). To a solution of 3A (from RI Chemicals, lot #H1010-04, 15.0 g, 18.99 mmol) in MeOH (190 mL) was added hydrazine (3.04 g, 94.52 mmol) and the heterogeneous mixture was heated to reflux for 3.5 h. The mixture was cooled to room temperature and evaporated under reduced pressure to give a white powder. The product was dissolved in DCM and washed with ammonium hydroxide. Brine was added to help remove the emulsion. The organic layer was dried over MgSO4 and evaporated under reduced pressure to give 11.80 g of crude product, which was used in the next step without purification. C 37 H 45 MS calculated for N3O8 659.3207, found m / z 660.2 (M+1) + , 682.1 (M+23) Na+ , 658.1(M-1) - , 694.1 (M+35) Cl- .R f = 0.02 in 5% MeOH / DCM v / v. 1 H NMR (400MHz, DMSO-d6) δ7.48(s,1H), 7.38(d,J=7.5Hz, 2H), 7.30(t,J=7.5Hz, 2 H), 7.24(d,J=8.9Hz, 5H), 6.89(d,J=8.4Hz, 4H), 5.84(d,J=5.0Hz, 1H), 5.74(s ,1H), 4.19(t,J=5.0Hz, 1H), 3.97(t,J=4.9Hz, 2H), 3.72(s,6H), 3.63-3.45(m, 3H), 3.27-3.15(m,3H), 1.48(d,J=6.4Hz, 2H), 1.38(s,3H), 1.34-1.18(m,6H).

[0535] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-5-methyluridine (4A). Crude 4A in DCM (250 mL) *To a solution of 4A (6.00 g, 9.09 mmol), triethylamine (3.8 mL, 27.30 mmol) was added and the mixture was allowed to stir for 10 minutes. GalNAc-C5-NHS ester (7.31 g, 10.00 mmol) was added and the reaction mixture was stirred for 2 hours. The reaction mixture was washed with saturated bicarbonate, and the organic layer was dried over Na2SO4 and then evaporated under reduced pressure. The crude product was purified by silica gel chromatography to give 9.56 g of 4A (7.49 mmol, 82%). 1 H NMR (400MHz, DMSO-d6) δ11.37(s,1H, D2O exchangeable), 7.97(d,J=9.3Hz, 1H, D2O exchangeable), 7.91(t,J=6.8Hz, 4H), 7.73-7.45(m,11H), 7.41-7.19(m, 11H), 6.88(d,J=8.4Hz, 4H), 5.84(d,J=4.7Hz, 1H), 5.74(d,J=3.4Hz, 1H), 5.36(dd,J=11.1, 3.3Hz, 1H), 5.12(d,J=6.3Hz, 1H, D2O replaceable), 4 .73(d,J=8.5Hz, 1H), 4.45(q,J=8.8, 7.6Hz, 2H), 4.39-4.15(m,3H), 3.96(t,J=4.7Hz, 2H), 3.79(dd,J=9.4, 3.8Hz, 1H), 3.72(s,6H), 3.63 -3.45(m,3H), 3.28-3.16(m,2H), 2.99(q,J=6.5Hz, 2H), 2.04(s,2H), 1.69(s,3H), 1.55-1.43(m,6H), 1.36(d,J=17.6Hz, 5H), 1.24(s,4H). 13C NMR (100MHz, DMSO-d6) δ171.73, 169.40, 165.20, 165.16, 164.86, 163.62, 158.18, 158.15, 150.41, 144.64, 135.47, 135.31, 135.10, 133.77, 133.49, 129.73, 129.20, 129.16, 129.03, 129.00, 128.97, 128.70, 128.59, 127.91, 127.64, 126.82, 113.26, 109.59, 100.89, 86.47, 85.91, 83.09, 80.60, 71.85, 69.97, 69.74, 68.77, 67.92, 63.19, 62.03, 55.04, 49.74, 38.34, 35.03, 29.17, 29.04, 28.59, 26.25, 25.12, 22.69, 21.85, 11.66.C 71 H 78 N4O 18 MS calculated for 1274.5311, observed m / z 1298.3 (M+23) Na+ , 1309.4 (M+35) Cl- .R f = 0.36 in 5% MeOH / DCM v / v.

[0536] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-3'-O-succinato-5-methyluridine (5A). To a solution of 4A (2.00 g, 1.57 mmol) in DCM (50 mL) was added DMAP (574 mg, 4.70 mmol) and succinic anhydride (313 mg, 3.14 mmol). The reaction mixture was stirred overnight at room temperature. The product was purified by silica gel chromatography (φ = 4.2 cm × 15 cm, pretreated with 2% TEA in DCM). The product was eluted with 0-5% MeOH and 2-5% EtN (v / v) in DCM and coevaporated with acetonitrile under reduced pressure to give 2.11 g (1.43 mmol, 91%) of 6a as the EtN salt. 1H NMR (400MHz, DMSO-d6) δ11.44(s,1H),8.05(d,J=9.3Hz,1H),7.91(t,J=6.8Hz,4H),7.74-7.45(m,11H),7.40-7.27(m,6H),7.23(d,J=8. 7Hz, 5H), 6.89(d,J=8.1Hz, 4H), 5.84(d,J=6.1Hz, 1H), 5.74(d,J=3.5Hz, 1H), 5.36(dd,J=11.1, 3.3Hz, 1H), 5.27-5.22(m,1H), 4.74(d,J =8.5Hz, 1H), 4.48-4.40(m,2H), 4.38-4.22(m,3H), 4.13(q,J=3.5Hz, 1H), 3.78(d,J=9.7Hz, 1H), 3.72(s,6H), 3.54-3.28(m,5H), 3.25-3 .19(m,1H), 2.97(q,J=6.5Hz,2H), 2.57-2.51(m,2H), 2.44(t,J=6.5Hz,2H), 2.04(s,2H), 1.69(s,3H), 1.57-1.27(m,11H), 1.18(s,4H). 13 C NMR (100MHz, DMSO-d6) δ173.40, 171.76, 171.63, 169.39, 165.20, 165.15, 164.86, 163.50, 158.22, 150.50, 144.48, 135.45, 135.12, 134.95, 133.76, 133.48, 129.70, 129.17, 129.03, 129.00, 128.97, 128.70, 128.58 ,127.95,127.61,113.30,110.16,100.91,86.13,80.75,78.22,71.89,70.67,70.23,69.97,68.73,67.91, 62.04, 55.04, 52.01, 49.73, 38.33, 34.98, 29.13, 28.92, 28.55, 26.19, 25.08, 22.68, 21.82, 11.69, 10.48.C 75 H 81 N4O 21 - についてのMS calculated value 1374.5472, measured value m / z 1397.4(M+23) Na+ 、1373.4(M-1) - 、1409.4(M+35) Cl- .Rf = 0.41 in 5% MeOH / 5% Et3N / DCM v / v.

[0537] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-3'-O-CPG-5-methyluridine (6A). To a solution of 5A (2.01 g, 1.36 mmol) in acetonitrile (100 mL) was added HBTU (1.03 g, 2.72 mmol) and DIEA (528 mg, 4.08 mmol). The mixture was shaken for 5 minutes, and then CPG (16.00 g, 130 μmol / g, 540 Å) was added. The mixture was shaken for 24 hours. The CPG was filtered and washed with DCM, 20% MeOH in DCM (v / v), and then ether. The CPG was evaporated under reduced pressure and then treated with acetic anhydride (25 mL) in pyridine (75 mL) and EtN (1 mL) and shaken for 1 hour. The CPG was filtered and washed with the same solvent as described above. The average loading was determined by trityl absorbance spectroscopy of two samples and calculated to be 73 μmol / g.

[0538] 5'-O-Dimethoxytrityl-2'-O-aminohexyl-C5-GalNAc(O-Bz)-3'-O-(N,N-diisopropyl)-β-cyanoethylphosphoramidite-5-methyluridine (7A). 4A (2.90 g, 2.27 mmol) was coevaporated twice with anhydrous acetonitrile and then kept under a full argon atmosphere. To a solution of 4A in anhydrous DCM (35 mL) at 0 °C, 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.37 g, 4.55 mmol) was added, followed by DCI (268 mg, 2.27 mmol). The mixture was stirred at 0 °C for 20 min and then at room temperature for 17 h. The product was washed with saturated bicarbonate and extracted with DCM. The organic layer was dried over Na2SO4 to give a pale yellow foam. Silica gel chromatography (φ=4.2 cm × 19 cm, pretreated with 50% EtOAc and 1% TEA in hexane) was performed. The column was washed with 80% EtOAc in hexane (8 CV), followed by 100% EtOAc (8 CV), and then 3% MeOH in DCM (5 CV). Product 7A was eluted with 100% EtOAc and again with 3% MeOH. Fractions containing 7A were combined and evaporated under reduced pressure to give 3.20 g of 7A (2.17 mmol, 95%). 1H NMR(400MHz、DMSO-d6)δ11.39(s,1H)、7.98(d,J=9.3Hz、1H)、7.91(t,J=7.2Hz、4H)、7.72-7.45(m,11H)、7.38(t,J=6.8Hz、4H)、7.33-7.20(m,7H)、6.88(t,J=5.4Hz、4H)、5.82(d,J=4.3Hz、1H)、5.75(s,1H)、5.35(dd,J=11.1、3.1Hz、1H)、4.73(d,J=8.5Hz、1H)、4.48-4.31(m,4H)、4.15-4.04(m,2H)、3.71(s,8H)、3.58-3.45(m,5H)、3.28-3.20(m,2H)、3.03-2.93(m,2H)、2.76(t,J=5.8Hz、1H)、2.57(q,J=5.3Hz、1H)、2.04(s,2H)、1.69(s,3H)、1.49(s,6H)、1.42-1.29(m,5H)、1.27-1.15(m,5H)、1.14-1.03(m,10H)、0.94(d,J=6.7Hz、3H). 13 C NMR(125MHz、DMSO-d6)δ171.60、169.26、165.07、165.03、164.73、163.48、158.10、158.08、158.07、150.24、150.22、144.39、144.34、134.99、134.97、134.87、134.80、133.63、133.37、133.34、129.65、129.62、129.60、129.07、129.05、129.03、128.90、128.88、128.87、128.83、128.56、128.45、127.75、127.56、127.50、126.74、118.72、118.57、113.10、113.08、109.63、109.54、100.76、85.92、85.90、71.72、69.84、68.61、67.78、61.89、54.91、49.60、45.53、42.53、42.42、42.31、38.23、38.21、34.88、29.05、29.02、28.96、28.45、26.17、25.08、25.04、24.18、24.13、24.09、24.03、22.55、21.71、19.71、19.66、19.62、11.51、11.49. 31P NMR (160MHz, DMSO-d6) δ154.01, 153.65. C 80 H 95 NO 19 MS calculated for P 1474.6390, observed m / z 1497.4 (M+23) Na+ , 1509.4 (M+35) Cl- .R f = 0.39 in 100% EtOAc.

[0539] Example 66: [ka]

[0540] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-5-methyluridine (4B * To a solution of 3B (3.64 g, 4.61 mmol, ca. 1 g) in MeOH (46 ml) was added hydrazine (738 mg, 23.04 mmol) and the reaction mixture was refluxed for 5.5 hours. * Using the work-up procedure described for crude 4B * Coevaporation with acetonitrile gave 2.93 g of crude 4B. * was obtained. C 37 H 45 MS calculated for N3O8 659.3207, found m / z 660.2 (M+1) + , 682.1 (M+23) Na+ , 658.1(M-1) - , 694.1 (M+35) Cl- .R f = 0.02 in 5% MeOH / DCM v / v. Crude 4B * NMR: 1H NMR (400MHz, DMSO-d6) δ7.50(s,1H), 7.43-7.16(m,9H), 6.88(d,J=8.7Hz, 4H), 5.73(d,J=4.6Hz, 1H), 4.29( t,J=4.8Hz, 1H), 4.02-3.89(m,3H), 3.46-3.15(m,5H), 2.24-2.16(m,1H), 2.05(s,1H), 1.58-1.10(m,13H). 13 C NMR (125MHz, DMSO-d6) δ163.73, 158.04, 158.02, 150.58, 144.49, 135.54, 135.18, 135.06, 129.56, 127.78, 127.50, 126.69, 113.11, 109.23, 88.49, 85.78, 80.47, 77.20, 72.02, 69.62, 62.85, 54.91, 54.90, 41.44, 33.12, 29.17, 26.11, 25.31, 11.62.

[0541] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-5-methyluridine (4B). 4B in DCM (45 mL) treated with TEA (1.8 mL). * To a solution of 4B (2.85 g, 4.32 mmol) was added GalNAc-NHS ester (3.47 g, 4.75 mmol). The reaction was stirred for 2 hours, and then an additional 0.2 equivalents of GalNAc-NHS ester was added. After 1 hour, the product was isolated as described for 5A. Silica column purification gave 3.62 g of 4B (2.84 mmol). 1H NMR (400MHz, DMSO-d6) δ11.35(s,1H), 8.00-7.89(m,5H), 7.73-7.45(m,11H), 7.40-7.35(m,4H), 7.33-7.20(m,7H), 6. 88(d,J=8.8Hz, 4H), 5.74(d,J=10.1Hz, 2H), 5.42-5.33(m,2H), 4.73(d,J=8.5Hz, 1H), 4.48-4.41(m,2H), 4.38-4.23(m, 3H), 3.99(d,J=4.7Hz,1H), 3.91(t,J=5.1Hz,1H), 3.82-3.76(m,1H), 3.72(s,6H), 3.62-3.47(m,2H), 3.41-3.36(m,1H) ), 3.27-3.17(m,2H), 2.99(q,J=6.6Hz,2H), 2.04(t,J=6.4Hz,2H), 1.69(s,3H), 1.55-1.28(m,11H), 1.28-1.15(m,4H). 13 C NMR (75MHz, DMSO-d6) δ171.72, 169.38, 165.19, 165.14, 164.85, 163.64, 162.25, 158.14, 150.55, 144.57, 135.6 8. 135.30, 135.16, 133.73, 133.45, 129.66, 129.18, 129.14, 129.00, 128.93, 128.66, 128.55, 127.88, 127.61, 12 6.80, 113.22, 109.36, 100.88, 88.57, 85.90, 80.63, 77.35, 72.16, 71.83, 69.97, 69.73, 68.73, 67.91, 62.97, 62 .02, 55.01, 54.84, 49.75, 38.34, 35.73, 35.02, 30.73, 29.23, 29.11, 28.57, 26.25, 25.21, 22.66, 21.84, 11.68.C 71 H 78 N4O 18 についてのMS calculated value 1274.5311, measured value m / z 1297.4(M+23) Na+ 、1309.4(M+35) Cl- .R f =0.24 in 5% MeOH in DCM).

[0542] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-2'-O-succinato-5-methyluridine (5B). To a solution of 4B (1.10 g, 0.86 mmol) in DCM (20 mL) and DMAP (315 mg, 2.58 mmol) was added succinic anhydride (172 mg, 1.73 mmol). The reaction mixture was stirred for 23 h and then purified using the procedure described for 5A and coevaporated with acetonitrile under reduced pressure to give 1.03 g of 5B (0.70 mmol, 81%). 1 H NMR (400MHz, DMSO-d6) δ8.09(d,J=9.3Hz, 1H), 7.91(t,J=6.9Hz, 4H), 7.76(t,J=5.4Hz, 1H), 7.73-7.51(m,9H), 7.47(t,J=7.7Hz, 2H), 7.40-7. 19(m,12H), 6.87(d,J=8.7Hz, 4H), 5.85(d,J=3.9Hz, 1H), 5.74(d,J=3.2Hz, 1H), 5.47-5.42(m,1H), 5.36(dd,J=11.1, 3.2Hz, 1H), 4.75(d,J=8. 5Hz, 1H), 4.44(q,J=9.1, 7.7Hz, 2H), 4.38-4.20(m,4H), 4.01-3.94(m, 2H), 3.80-3.76(m,2H), 3.71(s,6H), 3.40-3.16(m,10H), 3.07-2.94(m, 3H), 2.56(q,J=6.2, 5.7Hz, 2H), 2.43(d,J=4.1Hz, 3H), 2.07-2.01(m,2H ), 1.69(s,3H), 1.48(d,J=15.0Hz, 7H), 1.40-1.27(m,4H), 1.16(s,4H). 13C NMR (100MHz, DMSO-d6) δ173.63, 173.45, 172.79, 171.92, 171.52, 169.54, 168.92, 165.29, 165.24, 164.94, 163.78, 158. 23, 150.33, 144.57, 136.18, 135.27, 135.18, 133.84, 133.57, 129.77, 129.24, 129.09, 129.06, 129.04, 128.77, 128.65, 127.96, 127.70, 126.90, 113.29, 109.73, 100.97, 85.97, 80.75, 75.82, 73.31, 71.96, 70.48, 62.59, 62.40, 55.10, 55.07 ,52.06,51.40,51.36,45.45,38.42,35.04,33.35,29.20,29.09,28.95,28.77,22.70,21.90,11.81,10.08,7.26,7.17.C 75 H 81 N4O 21 - MS calculated for 1374.5472, observed m / z 1397.4 (M+23) Na+ , 1373.4(M-1) - .R f = 0.18 in 5% MeOH in DCM).

[0543] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-2'-O-CPG-5-methyluridine (6B). To a solution of 5B (970 mg, 0.66 mmol) in acetonitrile (50 mL) was added HBTU (497 mg, 1.31 mmol) and DIEA (339 mg, 1.97 mmol). After shaking for 5 minutes, CPG (8.20 g, 130 μmol / g, 540 Å) was added and shaking continued for 21 hours. The CPG was removed by filtration, washed, capped, and the loading determined as described for 6A, yielding CPG with an average loading of 56 μmol / g.

[0544] 5'-O-Dimethoxytrityl-3'-O-aminohexyl-C5-GalNAc(O-Bz)-2'-O-(cyanoethyl-N,N-diisopropyl)-phosphoramidite-5-methyluridine (7B). 4B (1.98 g, 1.55 mmol) was prepared in the same manner as described for 7A and treated with the same reagents. The product 7B was loaded onto a column prepared in the same manner as described for 7A and eluted with 70% EtOAc in hexanes (4 CV), 80% EtOAc in hexanes (10 CV), 100% EtOAc (2 CV), and then 3% MeOH in DCM (4 CV). The desired product eluted with 100% EtOAc and 3% MeOH. Fractions containing the desired product were combined and evaporated under reduced pressure to give 1.89 g (1.28 mmol, 83%) of 7B. 1 H NMR (400MHz, DMSO-d6) δ11.38(s,1H), 8.00-7.88(m,5H), 7.72-7.45(m,11H), 7.41-7.34(m,4H), 7.33-7.20(m,7H), 6.91-6. 85(m,4H), 5.88(dd,J=9.3, 5.1Hz, 1H), 5.75(d,J=3.3Hz, 1H), 5.36(dd,J=11.1, 3.3Hz, 1H), 4.73(d,J=8.5Hz, 1H), 4.62-4.5 0(m,1H), 4.45(q,J=8.2, 7.2Hz, 2H), 4.38-4.23(m,2H), 4.07-3.95(m,3H), 3.72(s,13H), 3.44-3.39(m,1H), 3.31-3.22(m,2 H), 2.98(s,2H), 2.71-2.66(m,1H), 2.04(s,2H), 1.69(s,3H), 1.47(d,J=28.1Hz, 8H), 1.40-1.18(m,8H), 1.13-1.00(m,11H). 13C NMR (125MHz, DMSO-d6) δ171.72, 171.70, 170.37, 170.29, 169.37, 165.18, 165.14, 164.85, 163.58, 163.50, 158.20 , 158.18, 158.16, 158.14, 154.86, 150.48, 150.43, 144.53, 144.52, 144.50, 135.18, 135.14, 135.05, 134.98, 133. 74, 133.48, 133.44, 129.69, 129.66, 129.63, 129.18, 129.16, 129.13, 129.01, 128.99, 128.98, 128.93, 128.67, 128.64, 128.56, 128.54, 128.52, 127.91, 127.88, 127.57, 118.81, 118.70, 113.25, 113.21, 109.72, 109.68, 100.87, 86.16, 86.15, 86.02, 71.83, 71.81, 69.95, 69.92, 68.72, 68.71, 67.89, 63.45, 59.71, 55.00, 54.98, 49.72, 42.79, 42.69, 42.64, 38.35, 38.33, 35.00, 30.12, 30.03, 29.26, 29.25, 29.14, 29.11, 29.09, 28.56, 28.54, 26.29, 25.27, 25.25, 24.34, 24.29, 24.24, 24.22, 24.08, 24.02, 22.66, 22.65, 21.84, 21.83, 21.36, 20.72, 20.67, 20.66, 19.80, 19.78, 19.74, 19.73, 19.67, 19.62, 18.82, 18.56, 16.57, 14.04, 13.65, 13.50, 11.69, 11.56, 11.56, 11.55, 11.53. 31 P NMR (162MHz, DMSO-d6) δ155.08, 154.60. C 80 H 95 NO 19 MS calculated for P 1474.6390, observed m / z 1497.4 (M+23) Na+ , 1474.3(M-1) - , 1509.4 (M+35) Cl- R f = 0.43 in 100% EtOAc).

[0545] Example 67: [ka] C 24 H 28 LRMS calculated for N6O6: 496.52, found m / z: 497.2 (M+1) + , 519.2 (M+23) Na+ , 486.2(M-1) - , 522.2(M+35) Cl- .

[0546] (2A / 2B)-Adenosine (20 g, 74.8 mmol) was treated with NaH (4.5 g, 112 mmol) in DMF (200 ml) for 20 minutes at 0° C. N-(iodohexyl)phthalimide (30.7 g, 86 mmol) was added to the solution, which was then heated to 80° C. for 2 days. Evaporation of the DMF under reduced pressure gave a pale orange gum containing the 2′- and 3′-O-alkylated isomers. The crude mixture was adsorbed onto silica gel and purified (5% MeOH / DCM v / v) to give 5.1 g (10.3 mmol, 14%) of 2A and a mixture of regioisomers. 1 H NMR (400MHz, DMSO-d6) δ8.36(s,1H), 8.12(s,1H), 7.95-7.68(m,4H), 7.32(s,2H, D2O exchangeable), 5.96 (d,J=6.2Hz, 1H), 5.42(t,J=7.1, 4.6Hz, 1H, D2O replaceable), 5.15(d,J=5.1Hz, 1H, D2O replaceable), 4.44(t,J =6.3, 4.7Hz, 1H), 4.27(q,J=4.9, 2.8Hz, 1H), 3.96(q,J=3.4Hz, 1H), 3.66(dt,J=12.2, 4.2Hz, 1H) , 3.60-3.42(m,4H), 3.30(dd,J=9.5, 6.4Hz, 1H), 1.41(dt,J=33.8, 6.9Hz, 4H), 1.25-1.04(m,4H). 13C NMR (100MHz, DMSO-d6) δ167.89, 150.68, 148.24, 148.19, 134.35, 131.54, 128.38, 127.61, 125.44, 122.96, 118.74, 86.39, 86.03, 81.49, 69.66, 68.64, 28.85, 27.81, 25.93, 24.84.C 24 H 28 LRMS calculated for N6O6: 496.2070, found m / z: 497.2 (M+1) + , 519.2 (M+23) Na+ , 531.2 (M+35) Cl- .R f = 0.28 in 5% MeOH / DCM v / v.

[0547] (3A)—Compound 2A (7.4 g, 14.9 mmol) was coevaporated with pyridine and then dissolved in pyridine (75 ml) under argon gas. Triethylamine (3.2 ml, 22.4 mmol) and 4-dimethylaminopyridine (45 mg, 0.37 mmol) were added, and the reaction mixture was stirred for 15 minutes. DMTrCl (5.6 g, 16.4 mmol) was then added and stirred overnight. An additional 1.5 g of DMTrCl was then added to complete the reaction. The reaction mixture was quenched with MeOH (5 ml) and evaporated under reduced pressure. The crude product was washed with saturated NaHCO3, extracted with EtOAc, and dried over Na2SO4. The crude product was purified by silica column (2.5% MeOH / DCM v / v) to give 7.8 g (13 mmol, 66%) of pure 3A. 1H NMR (400MHz, DMSO-d6) δ8.26(s,1H), 8.09(s,1H), 7.88-7.77(m,4H), 7.42-7.33(m,2H) ), 7.33-7.16(m,9H), 6.88-6.78(m,4H), 6.01(d,J=4.8Hz, 1H), 5.17(d,J=5.9Hz, 1H), 4 .56(t,J=5.0Hz, 1H), 4.38(q,J=5.2Hz, 1H), 4.06(q,J=4.6Hz, 1H), 3.72(s,6H), 3.62- 3.37(m,4H), 3.34(s,2H), 3.23(d,J=4.6Hz, 2H), 1.60-1.37(m,4H), 1.31-1.11(m,4H). 13 C NMR (100MHz, DMSO-d6) δ167.90, 158.02, 156.08, 152.63, 149.21, 144.85, 139.58, 135.55, 135.45, 134.31, 131.57, 129.70, 12 7.75, 127.68, 126.62, 122.95, 119.20, 113.10, 85.97, 85.49, 83.48, 80.10, 69.76, 69.12, 63.53, 28.92, 27.85, 25.99, 24.91.C 45 H 46 LRMS calculated for N6O8: 798.3377, found m / z: 799.2 (M+1) + , 821.1 (M+23) Na+ , 833.1 (M+35) Cl- R f = 0.33 in 5% MeOH / DCM v / v.

[0548] (4A)—Compound 3 (13 g, 16.2 mmol) was dissolved in MeOH (160 ml) and hydrazine (2.6 g, 81 mmol) was added to the solution. The reaction mixture was stirred at reflux for 3 h. TLC analysis showed the complete disappearance of 3A and the presence of a more polar spot, presumably 4A. * The MeOH was evaporated under reduced pressure and the crude foam was washed with NH4OH. The aqueous layer was extracted with DCM; saturated NaCl was required to remove the emulsion. The organic layer was dried over MgSO4 and evaporated under reduced pressure. 4A *The residue was co-evaporated with toluene and then dissolved in DCM (150 ml) and triethylamine (6.6 ml, 47.6 mmol). GalNAc(OBz)-C5-NHS ester (12.8 g, 17.4 mmol) was added to the mixture, and the reaction was continued at room temperature for 2 h. An additional 1.7 g, 2.4 mmol of GalNAc(OBz)-C5-NHS ester was added to the reaction and allowed to stir for an additional 2 h. The reaction mixture was evaporated under reduced pressure, and the crude product was washed with NaHCO. The aqueous layer was extracted with DCM and dried over NaSO. The crude 4A was adsorbed onto silica and purified (2.5-5% MeOH / DCM v / v) to give 16.7 g (13.1 mmol, 81%) of 4A. 1 H NMR (400MHz, DMSO-d6) δ8.26(s,1H), 8.09(s,1H), 8.02-7.85(m,5H), 7.76-7.45(m,10H), 7.44-7.17(m,13H), 6.83(dd,J=8.8, 5.1Hz, 4H ), 6.01(d,J=4.9Hz, 1H), 5.78-5.74(m,1H), 5.37(dd,J=11.1, 3.2Hz, 1H), 5.18(d,J=5.8Hz, 1H), 4.74(d,J=8.5Hz, 1H), 4.57(t,J=4.9Hz) , 1H), 4.46(q,J=8.6, 7.4Hz, 2H), 4.41-4.23(m,3H), 4.07(q,J=4.5Hz, 1H), 3.85-3.76(m,1H), 3.72(s,6H), 3.55(ddd,J=24.2, 9.5, 5.1H z, 2H), 3.46-3.38(m,1H), 3.23(d,J=4.4Hz, 2H), 2.97(q,J=6.5Hz, 2H), 2.04(s,2H), 1.70(s,3H), 1.58-1.36(m,6H), 1.33-1.12(m,6H). 13C NMR (100MHz, DMSO-d6) δ171.69, 169.38, 165.20, 165.15, 164.85, 158.00, 156.07, 152.62, 149.19, 144.82, 13 9.58, 135.53, 135.42, 133.77, 133.51, 133.47, 129.68, 129.19, 129.16, 129.02, 129.00, 128.97, 128.69, 128 .59, 127.73, 127.65, 126.61, 119.18, 113.08, 100.88, 85.94, 85.47, 83.49, 80.08, 71.84, 69.96, 69.80, 69.1 0, 68.75, 67.91, 63.53, 62.02, 54.98, 54.89, 49.73, 35.01, 29.11, 29.03, 28.58, 26.17, 25.05, 22.69, 21.83.C 37 H 44 LRMS calculated for N6O6: 668.3322, found m / z: 691.3 (M+23) Na+ , 703.2 (M+35) Cl- (4A * ).C 71 H 77 N7O 16 LRMS calculated value for 1283.5427, observed value m / z 1306.3 (M+23) Na+ , 1282.3(M-1) - , 1318.3 (M+35) Cl- (4A).R f = 0.00 (4A) in 5% MeOH / DCM v / v * ).R f = 0.24 (4A) in 5% MeOH / DCM v / v.

[0549] (5A)—Compound 4A (16 g, 12.5 mmol) was dissolved in DMF (50 ml), and N,N-dimethylformamide dimethyl acetal (7.4 g, 62.3 mmol) was added to the stirred solution. The reaction mixture was heated to 60° C. for 3 hours, and then the DMF was removed under reduced pressure. Traces of starting material were removed by silica gel column (2.5% MeOH / DCM v / v) to give 12.5 g (9.3 mmol, 75%) of 5A. 1 H NMR(400MHz、DMSO-d6)δ8.88(s,1H)、8.35(d,J=5.2Hz、2H)、7.98-7.88(m,5H)、7.59(ddt、J=60.0、30.9、7.5Hz、11H)、7.37(dd,J=16.2、8.2Hz、4H)、7.21(q,J=7.3Hz、8H)、6.81(dd,J=8.8、7.2Hz、4H)、6.05(d,J=5.0Hz、1H)、5.74(d,J=3.3Hz、1H)、5.35(dd,J=11.1、3.3Hz、1H)、5.18(d,J=5.9Hz、1H)、4.72(d,J=8.5Hz、1H)、4.58(t,J=5.0Hz、1H)、4.44(q,J=8.3、7.3Hz、2H)、4.39-4.22(m,3H)、4.06(q,J=4.6Hz、1H)、3.78(dd,J=9.9、4.5Hz、1H)、3.71(s,6H)、3.53(dtd、J=19.4、9.5、5.1Hz、2H)、3.40(dt,J=9.4、6.4Hz、1H)、3.22(d,J=4.5Hz、2H)、3.18(s,3H)、3.11(s,3H)、2.93(q,J=6.5Hz、2H)、2.02(s,2H)、1.68(s,3H)、1.53-1.36(m,7H)、1.30-1.08(m,7H). 13C NMR (125MHz, DMSO-d6) δ171.68, 169.38, 165.19, 165.15, 164.85, 159.23, 158.01, 157.98, 157.91, 151.93, 151.19, 144.80, 141.3 9, 135.49, 135.44, 133.76, 133.49, 133.46, 129.68, 129.63, 129.18, 129.17, 129.15, 129.02, 129.00, 128.99, 128.96, 128.68, 128 .58, 127.73, 127.65, 126.61, 125.76, 118.03, 100.88, 85.97, 85.47, 83.59, 80.01, 71.84, 69.96, 69.79, 69.11, 68.74, 67.90, 63.5 4,62.51,62.02,54.97,54.95,54.88,51.97,49.72,45.62,35.00,34.52,29.07,29.00,28.57,26.14,25.03,22.68,21.83,7.15.C 74 H 82 N8O 16 LRMS calculated value 1338.5849, observed values ​​m / z 1339.4 (M), 1361.4 (M+23) Na+ , 1338.4(M-1) - , 1373.4 (M+35) Cl- .R f = 0.29 in 5% MeOH / DCM v / v.

[0550] (6A) - Compound 5A (2 g, 1.5 mmol) was dissolved in DCM (15 ml) and 4-dimethylaminopyridine (550 mg, 4.5 mmol) was added to the stirring mixture. Succinic anhydride (300 mg, 3 mmol) was added, and the solution was stirred at room temperature for 3 h. The DCM was evaporated under reduced pressure, and the crude foam was loaded onto a manual column (φ = 4.6 × 17 mm) pretreated with 2% triethylamine in DCM (v / v). 6A was purified using a gradient of 1-5% MeOH / 2-5% triethylamine / DCM (v / v). 6A was obtained in quantitative yield from 3% MeOH / 3% triethylamine / DCM (v / v). 1H NMR(500MHz、DMSO-d6)δ8.89(s,1H)、8.42(s,1H)、8.30(s,1H)、8.09(d,J=9.3Hz、1H)、7.91(t,J=8.2Hz、4H)、7.75-7.65(m,4H)、7.65-7.53(m,4H)、7.47(t,J=7.6Hz、2H)、7.37(dd,J=13.9、7.3Hz、4H)、7.21(td、J=12.2、10.6、5.5Hz、7H)、6.82(t,J=8.3Hz、4H)、6.04(d,J=6.6Hz、1H)、5.74(d,J=3.1Hz、1H)、5.45-5.41(m,1H)、5.36(dd,J=11.1、3.2Hz、1H)、5.08-5.02(m,1H)、4.75(d,J=8.5Hz、1H)、4.48-4.39(m,3H)、4.38-4.19(m,5H)、3.78(d,J=9.4Hz、2H)、3.70(s,6H)、3.50(d,J=9.3Hz、2H)、3.40-3.26(m,6H)、3.18(s,3H)、3.11(s,3H)、2.59(q,J=6.8、6.3Hz、2H)、2.03(s,2H)、1.69(s,3H)、1.49(s,4H)、1.34-1.16(m,6H)、1.08-0.97(m,4H). 13 C NMR(125MHz、DMSO-d6)δ173.35、171.73、171.48、169.39、165.20、165.14、164.85、159.33、158.06、158.03、157.96、151.94、151.22、144.68、141.81、135.31、133.75、133.48、133.45、129.69、129.61、129.17、129.16、129.14、129.01、128.98、128.95、128.68、128.57、127.75、127.61、126.66、125.86、113.11、100.88、85.82、85.69、81.39、77.45、71.89、71.01、70.19、69.96、68.70、67.89、63.28、62.04、54.98、54.96、52.01、49.71、34.95、34.54、29.00、28.92、28.85、28.81、28.53、26.05、24.95、22.67、21.80、7.18.C 78 H86 N8O 19 LRMS calculated value 1438.6009, observed values ​​m / z 1439.4 (M), 1463.4 (M+23) Na+ , 1437.4(M-1) - .R f = 0.23 in 5% MeOH / 5% Et3N / DCM v / v.

[0551] (7A)—Compound 6A (2.2 g, 1.4 mmol) was dissolved in acetonitrile (110 ml), and HBTU (1.1 g, 2.9 mmol) and DIEA (550 mg, 4.3 mmol) were added. The mixture was shaken for 5 minutes, and then LCAA-CPG (18 g, 540 Å, 130 μmol / g) was added and shaken at room temperature overnight. The CPG was filtered, washed with 300 ml each of DCM, 20% MeOH / DCM (v / v), and diethyl ether, and then dried under reduced pressure. The CPG was shaken in acetic anhydride (25 ml), pyridine (75 ml), and triethylamine (1 ml) for 1 hour and then washed again under the same conditions as above. Compound 7A was dried under reduced pressure overnight, and the loading was measured by spectrophotometry (72 μmol / g).

[0552] (8A)—Compound 5A (1.0 g, 0.75 mmol) was coevaporated twice with ACN and placed under a full argon atmosphere. DCM (7.5 mL) was added to the flask and cooled to 0 °C, followed by the addition of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (450 mg, 1.5 mmol). The mixture was stirred for 20 minutes, and then 4,5-dicyanoimidazole (90 mg, 0.75 mmol) was added to the reaction. The reaction was allowed to warm slowly to room temperature overnight. The reaction was washed with saturated bicarbonate, and the aqueous layer was extracted with DCM. The organic layer was dried over Na2SO4 and evaporated under reduced pressure to give a pale yellow foam. The foam was loaded onto a pre-conditioned manual column (φ = 4....

Claims

1. A conjugate of the formula: 【Chemistry 1】 [In the formula, R Ａ is a single- or double-stranded oligonucleotide having a length of 6 to 30 nucleotide units and bound at its 3' end to the oxygen atom shown; R ３ is selected from the following: 【Table 1】 Where: The wavy lines represent the conjugation sites. R is -OH or -NHAc.

2. The following formula: 【Chemistry 2】 2. The conjugate of claim 1, having the formula:

3. R Ａ The conjugate of claim 1, wherein the oligonucleotide portion is double-stranded and has a length of 15 to 30 nucleotide units.

4. The conjugate of claim 3, wherein the oligonucleotide portion has a length of 18 to 23 nucleotide units.

5. A conjugate of the formula: 【Chemistry 3】 [In the formula, R Ａ is a single- or double-stranded oligonucleotide having a length of 6 to 30 nucleotide units and bound at its 3' end to the oxygen atom shown; R Ｂ is selected from Table 2 or Table 2A: 【Table 2】 (In Table 2, R represents the point of attachment to the remainder of the conjugate); 【Table 2A】 (In Table 2A, The wavy line represents the point of attachment to the remainder of the conjugate; each occurrence of R is independently OH or NHAc).

6. R Ａ The conjugate according to claim 5, wherein the oligonucleotide portion is double-stranded and has a length of 15 to 30 nucleotide units.

7. The conjugate of claim 6, wherein the oligonucleotide portion has a length of 18 to 23 nucleotide units.

8. A conjugate having the formula: 【Chemistry 4】 [In the formula, R Ａ are single- or double-stranded oligonucleotides having a length of 6 to 30 nucleotide units and bound at their 3' termini to the oxygen atom shown.

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