Oligonucleotide-ligand conjugates and process for their preparation

Abstract

To provide ligand conjugates of oligonucleotides (e.g., iRNA agents) and methods for their preparation.SOLUTION: The ligands are derived primarily from monosaccharides These conjugates are useful for the in vivo delivery of oligonucleotides.SELECTED DRAWING: None

Description

Cross-reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 845,279, filed on July 11, 2013, which is hereby incorporated 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 mainly derived from monosaccharides. These conjugates are useful for in vivo delivery of oligonucleotides.

Background Art

[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 an iRNA agent. The targeting moiety serves to target the iRNA agent to the desired target site. One way in which the targeting moiety can improve delivery is through receptor-mediated endocytosis activity. This uptake mechanism involves the movement of the iRNA agent bound to the membrane receptor into the interior of the region enclosed by the membrane, by invagination of the membrane structure or fusion of the delivery system with the cell membrane. This process is initiated by activation of the cell surface or membrane receptor after binding of a specific ligand to the receptor. Many receptor-mediated endocytosis systems are known and studied, including those that recognize sugars such as galactose, mannose, 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 present in very large amounts in hepatocytes. ASGP-R exhibits a 50-fold higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal. Past studies have shown that multivalency is required to obtain nM affinity, while the spacing between sugars is also important.

[0004] Recently, certain carbohydrate conjugates have been shown to be useful for delivery instead of liposomes for siRNA delivery. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

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

[0006] In one embodiment, the present invention provides a compound of formula I: [Chemical formula] (wherein, the oligonucleotide is an oligonucleotide such as siRNA, microRNA, anti-miR, antagomir, microRNA mimetic, decoy, immunostimulatory, G-quadruplex, splicing change, 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 linkers in Table 1 or 1A; and the ligand is derived from a sugar, where (i) the ligand 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 ligand is (a) A ligand in Table 2 or 2A, (b) -R 2 -(R 3 ) k (where R 2 is absent (in which case k = 1), or R 3 is a spacer (also called a ligand backbone) having two or more binding sites for a group, R 3 is a targeting monomer selected from Table 3, and k is from 1 to 6 (preferably 1 to 5 or 1 to 3), and each R 3 may be attached to the same or different atoms in R 2 ); and (c) A ligand in Table 4 or 4A selected from ( ) of an oligonucleotide (e.g., an iRNA agent) or a ligand (e.g., a carbohydrate) conjugate of another biologically active substance.

[0007] The conjugate comprises at least one linker from Table 1 or 1A or an Example, one ligand from Table 2, 2A, 4, or 4A, or one targeting monomer from Table 3 or 3A. For example, the nucleoside linker described in the Examples can be used as the linker. In one embodiment, the conjugate comprises (i) at least one linker from Table 1 or 1A or an Example, (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-, polypeptide- (e.g., a dipeptide or tripeptide), heteroaryl- (e.g., a triazole), or sugar-containing group. In a preferred embodiment, each R 3 group is attached to R 2 via an amide, ether, or amino group. In one embodiment, R 3is linked 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 via a heteroatom such as a nitrogen atom (at the end of the spacer), or at the anomeric carbon, to the sugar group of the targeting monomer (as shown below). 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 rest of the ligand (right side). The spacers for the two ligands in Table 2A are shown in the following table (the arrow indicates the attachment point for targeting monomer R 3 (and the right side of the spacer is attached to the linker). Suitable spacers are also shown in Table 5 below. TIFF2025107295000002.tif85134

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

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

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

[0012] The oligonucleotide can be linked to a linker via a cleavable group (e.g., phosphate, phosphorothioate, or amide) or a non-cleavable group (e.g., ether, carbamate, or C-C (e.g., a bond between two carbon atoms or -CH2-CH2-)). As described herein, the cleavable or non-cleavable group is within the oligonucleotide of Formula I.

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

[0014] In the conjugates described herein (such as Formula (I)), the oligonucleotide or other biologically active substance can be replaced by a lipid nanoparticle (LNP) (such as PEG-lipid or cationic lipid) or a component of a polymer. The conjugated LNP component or conjugated polymer can be useful as a delivery agent to facilitate the delivery of the biologically active substance to the target site.

[0015] Table 1 - Linker groups a,b The following linkers are shown together with the protecting group DMTr. When conjugated, the DMTr group is removed and the adjacent oxygen atom is the site of attachment 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 a solid support (e.g.,

Chemical formula

Chemical formula

[0016]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0017] Table 1A - Linker Group 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 the linker may be a chemical moiety that does not contain an oligonucleotide. The wavy line indicates 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 present invention, X is compatible with solid-phase oligonucleotide synthesis and deprotection, or a solid support (e.g.,

Chemical Structure

Chemical Structure

[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

Table 3A-2

Table 3A-3

[0023] Ligand The ligand can also be selected from the following two general formulas

Chem.

Chem.

Chem.

[0024] The ligand can be represented by the formula shown in Table 4 or 4A below.

[0025]

Table 4

[0026]

Table 4A

[0027]

Table 5

[0028] In Table 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 at one or more of the indicated -OAc positions. Generally, the acetyl (Ac) group functions as a protecting group for the hydroxyl moiety. Thus, it will be understood by those skilled in the art that the corresponding hydroxy compounds are within the scope of the present invention and are intended to be used in the final conjugate with oligonucleotides or other biologically active substances.

[0029] Furthermore, the compounds of the present invention also include those in which any -NHAc substituents in the sugar moiety are replaced by hydroxy groups (for example, in that case, the -NHAc group at the 2-position of the sugar moiety is replaced by two -OH groups). In one embodiment, in addition to the replacement of the -NHAc group by a hydroxy group, any -OAc substituents in the sugar moiety are also replaced by hydroxy groups.

[0030] In one embodiment, the ligand is

Chemical formula

Chemical formula

[0031] Yet another embodiment is an intermediate compound of the formula

Chemical formula

Chemical formula

Chemical formula

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

[0033] Yet another embodiment is intermediate compound of formula IIIA

Chemical formula

Chemical formula

Chemical formula

[0034] In a preferred embodiment, the substitutions at the 3- and 4-positions 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 encompasses compounds of formula IIIA, wherein X is replaced by an oligonucleotide or other biologically active substance 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 invention provides a compound of formula IV

Chemical formula

[0038] In formula IV, the linker attaches two portions of the oligonucleotide (oligonucleotide 1 and 2) via two bonds. Each portion of the oligonucleotide represents at least one nucleoside moiety.

[0039] Yet another embodiment provides a compound of formula V:

Chemical formula

Chem.

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

[0041] In a preferred embodiment, the oligonucleotide in the conjugate described herein is linked to the linker via phosphate, phosphorothioate, or a combination thereof.

[0042] In one embodiment, the conjugate is of the formula:

Chem.

[0043] The present invention also relates to a ligand conjugate of an oligonucleotide, 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 carbohydrate conjugate of an oligonucleotide, wherein at least one nucleoside in the oligonucleotide is conjugated to a carbohydrate-containing ligand (e.g., a sugar-containing ligand) via a nitrogen atom in the nucleobase of the nucleoside. Any ligand described herein can be used. In one embodiment, the nucleoside in the conjugate has the formula VI:

Chemical formula

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

[0046] In another embodiment, R 6is cytosine substituted at the 5-position with an amide group -C(O)NH-, where R 7 is attached to R 6 through the nitrogen atom of the amide group.

[0047] Another embodiment is a carbohydrate conjugate of an oligonucleotide, wherein at least one nucleoside in the oligonucleotide is conjugated at its 2'-position to a ligand (e.g., a carbohydrate-containing ligand). Any ligand described herein can be used. In one embodiment, the nucleoside in the conjugate has the formula VII:

Chemical formula

[0048] The ligand moiety (e.g., carbohydrate moiety) promotes the delivery of the oligonucleotide to the target site. One way in which the ligand moiety can improve delivery is by receptor-mediated endocytosis activity. Without being bound by a particular theory, this uptake mechanism is thought to involve the movement of the oligonucleotide conjugated to the membrane receptor into the region enclosed by the membrane, through invagination of the membrane structure or fusion of the delivery system with the cell membrane. This process is initiated by the activation of the cell surface or membrane receptor after binding of the 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 both 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 the iRNA agent of the present invention, wherein the strand of the iRNA agent comprises the therapeutic RNA.

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

Brief Description of the Drawings

[0052]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

BEST MODE FOR CARRYING OUT THE INVENTION

[0053] Definitions 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 having a hairpin loop, or (iii) a DNA / RNA hybrid. Non-limiting examples of double-stranded RNA include siRNA (small interfering RNA). Examples of 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] As used herein, the term "solid support" refers to any particle, bead, or surface on which the synthesis of oligonucleotides occurs. Solid supports that can be used in different embodiments of the methods described herein can be selected, for example, from inorganic supports and organic supports. The inorganic support is preferably selected from silica gel and controlled pore glass (CPG). The organic support is preferably selected from highly cross-linked polystyrene, Tentagel (a graft copolymer consisting of a low cross-linked polystyrene matrix grafted with polyethylene glycol (PEG or POE)), polyvinyl acetate (PVA), Poros - a copolymer of polystyrene / divinylbenzene, 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] As used herein, the term "hydroxy protecting group" refers to a labile chemical moiety that protects a hydroxyl group from unwanted reactions during a synthetic procedure. After the synthetic procedure, 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. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). Examples of hydroxy 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-furfuryl oxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, 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)CH3), benzoyl (Bz or --C(O)C6H5), and trimethylsilyl (TMS or --Si(CH3)3).

[0057] As used herein, the term "amino protecting group" refers to a labile chemical moiety that protects an amino group from unwanted reactions during synthetic procedures. After the synthetic procedure, 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 that is used to block or protect a carboxylic acid functional group while reactions involving other functional sites of the compound are carried out. Such carboxy protecting groups are known by the ease of cleavage, either by hydrolysis methods to the corresponding carboxylic acid or by hydrogenolysis methods. 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 the carboxy group, 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 such as those described in E. Haslam in Protective Groups in Organic Chemistry (supra), Chapter 5 are suitable. Ester-forming protecting groups are preferred.

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

[0060] Suitable peptide coupling reagents include, but are not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazolediethyl phosphate-1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethylfluoromethamidinium hexafluorophosphate), DPPA (diphenyl azidophosphate), BOP (benzotriazol-1-yloxytri(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)-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 acid chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium tetrafluorophosphate), BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP (bromotris(pyrrolidino)phosphonium hexafluorophosphate). EDC, HOAT, BOP-Cl and PyBrOP are preferred peptide coupling reagents. The amount of the peptide coupling reagent ranges from about 1.0 to about 10.0 equivalents.Optional reagents that can be used in the amide bond formation reaction include an amount in the range of about 1.0 to about 10.0 equivalents of DMAP (4-dimethylaminopyridine) or HOBT (1-hydroxybenzotriazole), HOAT (hydroxyazabenzotriazole), HOSu (hydroxysuccinimide), HONB (endo-N-hydroxy-5-norbornene-2,3-dicarboxamide), and other active ester reagents.

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

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

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

[0064] The term "alkylenedioxo" refers to a divalent species of the structure -O-R-O- (wherein R represents alkylene).

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

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

[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 with substituents. 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] As used herein, the term "cycloalkyl" includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, for example, 3 to 6 carbons, and the cycloalkyl group may further be optionally substituted. Examples of 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 case, 1 to 6 heteroatoms in the bicyclic case, or 1 to 9 heteroatoms in the tricyclic case, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and, in the monocyclic, bicyclic, or tricyclic cases, 1 to 3, 1 to 6, or 1 to 9 heteroatoms of N, O, or S, respectively), and 0, 1, 2, 3, or 4 atoms of each ring may be substituted with 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 case, 1 to 6 heteroatoms in the bicyclic case, or 1 to 9 heteroatoms in the tricyclic case, where the heteroatoms are selected from O, N, or S (e.g., carbon atoms and, in the monocyclic, bicyclic, or tricyclic cases, 1 to 3, 1 to 6, or 1 to 9 heteroatoms of N, O, or S, respectively), and 0, 1, 2, or 3 atoms of each ring may be substituted with substituents. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl.

[0076] The term "oxo" refers to an oxygen atom that forms a carbonyl when bonded to carbon, an N-oxide when bonded to nitrogen, and a sulfoxide or sulfone when bonded 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" refers to 4,4'-dimethoxytrityl, unless otherwise specified.

[0079] The term "substitution" refers to the replacement of one or more hydrogen radicals in a given structure by radicals of defined substituents 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 substituents may be further substituted.

[0080] The term "monosaccharide" encompasses radicals of allose, altrose, arabinose, cladinosose, 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, glucosyl glyceraldehyde, L-glycero-D-manno-heptose, glycerol, glycerone, glucosulose, 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 (alcohol hydroxy groups substituted with hydrogen), amino sugars (alcohol hydroxy groups substituted with amino groups), thiosugars (alcohol hydroxy groups substituted with thiol, or C=O substituted with C=S, or cyclic form ring oxygen substituted with sulfur), selenosugars, telluronic sugars, azasugars (ring carbons substituted with nitrogen), iminosugars (ring oxygen substituted with nitrogen), phosphanosugars (ring oxygen substituted with phosphorus), phosphasugars (ring carbons substituted with phosphorus), C-substituted monosaccharides (hydrogen at non-terminal carbon atoms substituted with carbon), unsaturated monosaccharides, alditols (carbonyl groups substituted with CHOH groups), aldonic acids (aldehyde groups substituted with carboxy groups), ketoaldonic acids, uronic acids, aldaric acids, etc. Amino sugars include amino monosaccharides, preferably galactosamine, glucosamine, mannosamine, fucosamine, quinovosamine, neuraminic acid, muramic acid, lactosamine, acosamine, bacillosamine, daunosamine, desosamine, folosamine, galosamine, canosamine, kansosamine, mycarose, mycosamine, perosamine, purosamine, pullulanamine, rhodosamine. It is understood that monosaccharides etc. can be further substituted.

[0081] The terms "disaccharide", "trisaccharide" and "polysaccharide" include radicals such as abequose, acarbose, amicetose, amylopectin, amylose, apiose, alkanose, ascarylose, ascorbic acid, boivinose, cellobiose, cellotriose, cellulose, cacotriose, calose, chitin, colitose, cyclodextrin, cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, digitnose, digitallose, digitoxose, evanose, evemitrose, fructooligosaccharide, galactooligosaccharide, gentianose, gentiobiose, glucan, glycogen, glycogen, hamamelose, heparin, inulin, isolevoglucosenone, isomaltose, isomaltotriose, isopanose, kojibiose, lactose, lactosamine, lactosediamine, laminaribiose, levoglucosan, levoglucosenone, β-maltose, maltotriose, mannan-oligosaccharide, manninotriose, melezitose, melibiose, muramic acid, mycaminose, mycose, neuraminic acid, nigerose, nocardicin, novobiose, oleandrose, panose, paratose, planteose, primeverose, raffinose, rhodinose, rutinose, salmiento, sedoheptulose, sedoheptulosan, soratriose, sophorose, stachyose, streptose, sucrose, α,α-trehalose, trehalosamine, turanose, tibulose, xylobiose, umbelliferose and the like. Further, it is understood that "disaccharide", "trisaccharide" and "polysaccharide" and the like can be further substituted. Disaccharides also include amino sugars and their derivatives, in particular mycaminose derivatized at the C-4' position or 4-deoxy-3-amino-glucose derivatized at the C-6' position. Oligonucleotide

[0082] The oligonucleotide can be siRNA, microRNA, anti-microRNA, microRNA mimics, anti-miR, antagomir, dsRNA, ssRNA, aptamer, immunostimulatory, decoy oligonucleotide, splicing-changing oligonucleotide, triple-strand-forming oligonucleotide, G-quadruplex or antisense. In one embodiment, the oligonucleotide is an iRNA agent.

[0083] In certain embodiments, the oligonucleotides of the invention include one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA refers to an unlocked acyclic nucleic acid where at least one of the sugar linkages has been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers where the bond between C1’-C4’ (i.e., the carbon-oxygen-carbon covalent bond between the C1’-C4’ carbons) has been removed. In another example, the C2’-C3’ bond of the sugar (i.e., the carbon-carbon covalent bond between the C2’ and C3’ carbons) has been removed (see Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which is incorporated herein by reference).

[0084] The term "iRNA agent" refers to an RNA agent (or an agent cleavable into an RNA agent) that can downregulate the expression of a target gene (e.g., siRNA), preferably an endogenous or pathogen target RNA. Without wishing to be bound by theory, the iRNA agent can act by post-transcriptional cleavage of the target mRNA (referred to as RNAi in the art), or by one or more of several mechanisms including pre-transcriptional or pre-translational mechanisms. The iRNA agent can include a single strand or can include two or more strands, e.g., the iRNA agent can be a double-stranded iRNA agent. When the iRNA agent is single-stranded, the iRNA agent can include a 5’ modification that includes 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 contains a region that is sufficiently homologous to the target gene and has a sufficient length with respect to nucleotides, whereby the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. The iRNA agent is at least partially, and in some embodiments completely, a region that is complementary to or contains a region complementary to the target RNA. Complete complementarity between the iRNA agent and the target is not necessary, but it is preferred that the iRNA agent, or a cleavage product thereof, has sufficient identity such that it can direct sequence-specific silencing, for example, by RNAi cleavage of the target RNA, such as mRNA.

[0086] The nucleotides in the iRNA agent may be modified (for example, one or more nucleotides may contain a 2'-F or 2'-OCH3 group), or may be nucleotide surrogates. The single-stranded regions of the iRNA agent may be modified or may contain nucleoside surrogates. For example, unpaired regions or regions of hairpin structures, such as regions that join two complementary regions, may have modifications or nucleoside surrogates. Modifications include, for example, modifications that stabilize one or more 3' or 5' ends of the iRNA agent against exonucleases. Modifications result as 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 may include special biotin or fluorescein reagents that have another DMT-protected hydroxyl group and allow multiple linkages during RNA synthesis. Modifications may also include, for example, the use of modifications at the 2'OH group of the ribose sugar, such as the use of deoxythymidine instead of ribonucleotides, and modifications at the phosphate group, such as phosphorothioate modifications. In some embodiments, different strands contain different modifications.

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

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

[0089] The oligonucleotides can 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] As used herein, a "single-stranded siRNA compound" is an siRNA compound composed of a single molecule. It may contain a double-stranded region formed by intrastrand pairing, e.g., it can be or contain a hairpin or panhandle structure. A single-stranded siRNA compound can be antisense to a target molecule.

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

[0092] The hairpin siRNA compound has 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 or less in length. In certain embodiments, the double-stranded region ranges in length from 15 to 30, 17 to 23, 19 to 23, 19 to 21 nucleotide pairs. The hairpin can have a single-stranded overhang or a 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, on the antisense side of the hairpin.

[0093] As used herein, "double-stranded siRNA compound" is an siRNA compound comprising two or more, optionally two strands, in which intermolecular hybridization can form a region of double-stranded structure.

[0094] The antisense strand of the 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, 19 to 21 nucleotides in length. As used herein, the term "antisense strand" means the strand of the siRNA compound that is sufficiently complementary to a target molecule, e.g., a target RNA.

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

[0096] The double-stranded portion of the 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 longer. It can be 200, 100, or 50 nucleotide pairs or shorter. The 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 to be cleaved by an endogenous molecule, such as Dicer, to produce a smaller siRNA compound, e.g., an siRNA agent.

[0098] The sense and antisense strands can be selected such that the double-stranded siRNA compound includes single-stranded or unpaired regions at one or both ends of the molecule. Thus, the double-stranded siRNA compound can contain sense and antisense strands paired such that it contains overhangs of 1 - 3 nucleotides, e.g., one or two 5' or 3' overhangs, or a 3' overhang. The overhang can result from one strand being longer than the other or from two strands of the same length being staggered. Some embodiments have at least one 3' overhang. In one embodiment, both ends of the siRNA molecule have 3' overhangs. In some embodiments, the overhang is 2 nucleotides.

[0099] In certain embodiments, the length of the double-stranded region is, for example, within the range of the ssiRNA compounds described above, 15 to 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length. The ssiRNA compounds can be similar in length and structure to products processed from long dsiRNA by native Dicer. Embodiments are also included where the two strands of the ssiRNA compound are joined, for example, covalently. Hairpins, or other single-stranded structures that provide the required double-stranded region and 3' overhangs are also contemplated.

[0100] The siRNA compounds described herein, including double-stranded siRNA compounds and single-stranded siRNA compounds, can mediate the silencing of a target RNA, for example, an mRNA, for example, the transcription product of a gene encoding a protein. For convenience, such an mRNA is also referred to herein as the mRNA to be silenced. Such a gene is also referred to as the target gene. Generally, the RNA to be silenced is an endogenous gene or a pathogen gene. Furthermore, RNAs other than mRNA, such as tRNA, and viral RNA can also be targeted.

[0101] As used herein, the phrase "mediating RNAi" refers to the ability to sequence-specifically silence a target RNA. Without wishing to be bound by theory, silencing is thought to use the mechanism or process of RNAi and a guide RNA, for example, an ssiRNA compound of 21 to 23 nucleotides.

[0102] In one embodiment, the siRNA compound is "sufficiently complementary" to a target RNA, e.g., a target mRNA, whereby the siRNA compound silences the production of the 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 consisting only of Watson-Crick base pairs in the region of exact complementarity. A "sufficiently complementary" target RNA may include an internal region that is exactly complementary (e.g., at least 10 nucleotides) to the target RNA. Further, in certain embodiments, the siRNA compound specifically discriminates a single nucleotide difference. In this case, the siRNA compound mediates RNAi only when exact complementarity is found in a region with a single nucleotide difference (e.g., within 7 nucleotides).

[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 not translated into proteins. The processed miRNAs are incorporated into the RNA-induced silencing complex (RISC) and are single-stranded ~17-25 nucleotide (nt) RNA molecules that are recognized as major regulators of development, cell proliferation, apoptosis, and differentiation. They are thought to play a role in the regulation of gene expression by binding to the 3'-untranslated region of specific mRNAs. RISC mediates the downregulation of gene expression by translational inhibition, transcript cleavage, or both. RISC is also involved in transcriptional silencing in the nuclei of a wide range of eukaryotes.

[0104] The number of miRNA sequences identified so far is large and is increasing, and exemplary examples thereof are, for example, "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 found at http: / / microrna.sanger.ac.uk / sequences / .

[0105] Antisense oligonucleotide In one embodiment, the nucleic acid is an antisense oligonucleotide directed to a target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" is intended to include oligonucleotides complementary to a targeted polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA complementary to the selected sequence, e.g., the single strand of the target gene mRNA. An antisense oligonucleotide is thought to inhibit gene expression by binding to the complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression by preventing translation of the complementary mRNA strand by binding to it, or by causing degradation of the target mRNA. Antisense DNA can be used to target a specific, complementary (coding or non-coding) RNA. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, the antisense oligonucleotide contains from about 10 to about 50 nucleotides, more preferably from 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 seen with antisense, or that an antisense sequence containing one or more mismatches with the target sequence may be most preferred for a particular application.

[0106] Antisense oligonucleotides have been demonstrated to be effective targeted inhibitors of protein synthesis and, as a result, can be used to specifically inhibit protein synthesis by a targeted gene. The effectiveness of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalacturonase and the muscarinic 2-type acetylcholine receptor are inhibited by antisense oligonucleotides directed against their respective mRNA sequences (U.S. Patent Nos. 5,739,119 and 5,759,829, each incorporated by reference). Further, examples of antisense inhibition have been demonstrated for the nuclear protein cyclin, the multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA A 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. Patent No. 5,801,154; U.S. Patent No. 5,789,573; U.S. Patent No. 5,718,709 and U.S. Patent No. 5,610,288, each incorporated by reference). Further, it has also been described that antisense constructs can be used to inhibit and treat various abnormal cell proliferations, such as cancer (U.S. Patent Nos. 5,747,470; 5,591,317 and 5,783,683, each incorporated by reference).

[0107] Methods for producing antisense oligonucleotides are known in the art and can be readily adapted to produce antisense oligonucleotides that target any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific for a given target sequence involves the analysis of the selected target sequence as well as secondary structure, T mBased on the determination of binding energy and relative stability. Antisense oligonucleotides can be selected based on their relatively low ability to form dimers, hairpins, or other secondary structures that can reduce or prevent specific binding to the target mRNA in the host cell. Highly preferred target regions of mRNA include sequences substantially complementary to regions at or near the AUG translation start codon and the 5' region of the mRNA. Analysis of these secondary structures and selection of target sites can be performed, for example, using v.4 of the OLIGO primer analysis software (Molecular Biology Insights) and / or the 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 cell uptake. They differ from normal RNA, for example, by sugars, phosphorothioate backbones, and complete 2'-O-methylation of, for example, cholesterol moieties at the 3' end. Antagomirs can be used to efficiently silence endogenous miRNAs by forming duplexes containing the antagomir and the endogenous miRNA, thereby preventing gene silencing induced by the miRNA. An example of miRNA silencing via antagomirs is the silencing of miR-122 described in Krutzfeldt et al, Nature, 2005, 438:685 - 689, which is hereby expressly incorporated by reference in its entirety. Antagomir RNAs can be synthesized using standard solid-phase oligonucleotide synthesis protocols. See U.S. Patent Application Publication Nos. 2007 / 0123482 and 2007 / 0213292, each of which is hereby incorporated by reference.

[0109] Antagomirs can include monomer subunits and monomers conjugated to ligands 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 International Publication No. 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 International Publication No. 2004 / 080406, which is incorporated by reference in its entirety.

[0110] Aptamer An aptamer is a nucleic acid or peptide molecule that binds to a specific molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990), each incorporated by reference in its entirety). The generation of DNA or RNA aptamers that bind to a wide variety of entities, from large proteins to small organic molecules, has been successful. See, respectively, 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 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 the binding of the target affects the activity of the gene. Thus, an mRNA containing a riboswitch is directly involved in regulating its own activity in response to the presence or absence of its target molecule. In general, aptamers are engineered by iterative 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 may be used alone or in combination with other aptamers specific for the same target. Further, as described in more detail herein, the term "aptamer" specifically includes "secondary aptamers" that contain a consensus sequence derived from comparing two or more known aptamers to a given target.

[0111] Ribozyme According to another embodiment, the nucleic acid-lipid particle is bound to a ribozyme. A ribozyme is an RNA molecular complex having 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 often accelerate the phosphoester transfer reaction with a high degree of specificity that cleaves only one of several phosphoesters 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 is thought to result from the need for the substrate to bind to the internal guide sequence ("IGS") of the ribozyme by base-pairing interactions prior to the chemical reaction.

[0112] At least six basic types of natural enzymatic RNAs are currently known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Generally, an enzymatic nucleic acid acts by first binding to a target RNA. Such binding occurs by the target-binding portion of the enzymatic nucleic acid that is kept in proximity to the enzymatic portion of the molecule that functions to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA by complementary base-pairing, and when bound to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA will impair its ability to direct the synthesis of the encoded protein. After binding to its RNA target and cleaving that RNA target, the enzymatic nucleic acid can be released from that RNA to search for another target and repeat the binding and cleavage with a new target.

[0113] The enzymatic nucleic acids can be formed, for example, from hammerhead, hairpin, hepatitis delta virus, group I intron or RNaseP RNA (associated with an RNA guide sequence) or Neurospora VS RNA motif. Specific examples of the hammerhead motif are described by Rossi et al. Nucleic Acids Res. 1992 Sep 11;20(17):4559-65. Examples of the hairpin motif 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. Examples of the hepatitis delta virus motif are described by Perrotta and Been, Biochemistry. 1992 Dec 1;31(47):11843-52; examples of the RNaseP motif are described by Guerrier-Takada et al., Cell. 1983 Dec;35(3 Pt 2):849-57; the 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); examples of group I introns are described in U.S. Patent No. 4,987,071. An important property of the enzymatic nucleic acids used is that they have specific substrate binding sites that are complementary to one or more of the target gene DNA or RNA regions, and that they have nucleotide sequences within or surrounding the substrate binding sites that confer RNA cleavage activity on the molecule. Thus, ribozyme constructs need not be limited to the specific motifs described herein.

[0114] Methods for producing ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed as described in International Patent Application Publication Nos. WO 93 / 23569 and WO 94 / 02595, each of which is specifically incorporated herein by reference, and synthesized as described in those references and tested in vitro and in vivo.

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

[0116] Immunostimulatory oligonucleotide Nucleic acids that are bound to lipid particles and contain immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that are capable of inducing an immune response when administered to a subject, which can be a mammal or other patient, can be immunostimulatory. ISS include, for example, specific palindromes that result in a hairpin secondary structure (see Yamamoto S., et al. (1992) J. Immunol. 148:4072-4076, which is incorporated herein by reference in its entirety), or CpG motifs, as well as other known features of ISS (such as multiple G domains, see International Patent Application Publication No. WO 96 / 11266, which is incorporated herein by reference in its entirety).

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

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

[0119] An immunostimulatory nucleic acid is considered to be non-sequence specific if it specifically binds to a target polynucleotide to induce an immune response and does not need to reduce the expression of the target polynucleotide. Thus, certain immunostimulatory nucleic acids can contain sequences corresponding to regions of natural genes or mRNAs, but can still be considered non-sequence specific immunostimulatory nucleic acids.

[0120] In one embodiment, an immunostimulatory nucleic acid or oligonucleotide contains at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may or may not be methylated. In another embodiment, the immunostimulatory nucleic acid contains at least one CpG dinucleotide having methylated cytosine. In one embodiment, the nucleic acid contains a single CpG dinucleotide, where the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid contains at least two CpG dinucleotides, where at least one of the cytosines in the CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotides present in the sequence is methylated. In another embodiment, the nucleic acid contains a plurality of CpG dinucleotides, where at least one of the CpG dinucleotides contains methylated cytosine.

[0121] Linker The linker can be any suitable group for attaching the oligonucleotide to the ligand. Other examples of linkers are described in WO 2009 / 082607 and US Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, each of which is incorporated herein by reference.

[0122] Attachment point of the oligonucleotide to the linker The oligonucleotide can be attached to the linker via any suitable group for joining 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 WO 2009 / 082607 and US Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 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, 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, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, etc., including chains of atoms, each of which may be substituted or unsubstituted, and one or more methylenes may be intervened or terminated by O, S, S(O), SO2, N(R 8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic, where R 8 is hydrogen, acyl, aliphatic or substituted aliphatic.

[0123] The cleavable group is one that is sufficiently stable extracellularly but is cleaved once it enters the target cell, releasing the two moieties to which the group is attached. In a preferred embodiment, the cleavable group is cleaved at least 10-fold, preferably at least 100-fold faster in the target cell or under a first reference condition (e.g., selected to mimic or represent intracellular conditions) than in the blood of the subject or under a second reference condition (e.g., 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 degrading molecules. Generally, cleavage factors are present at higher levels or activities and are more widespread or numerous intracellularly than in serum or blood. Examples of such degrading factors include, for example, redox enzymes or reducing agents such as mercaptans present intracellularly that can decompose redox-cleavable groups by reduction, redox agents selected for or having substrate specificity for a particular substrate; esterases; agents capable of creating an endosomal or acidic environment, such as agents that create a pH of 5 or less; enzymes that can hydrolyze or decompose acid-cleavable groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0125] Cleavable groups such as disulfide bonds can be susceptible to the influence of pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, in the range of about 7.1 - 7.3. Endosomes have a more acidic pH in the range of 5.5 - 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 cationic lipids from intracellular ligands or into a desired compartment of the cell.

[0126] The conjugate may contain a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the conjugate is determined according to the target cell. For example, a liver-targeting ligand can be conjugated to a cationic lipid via a chemical moiety containing an ester group. Hepatocytes are rich in esterases, and thus the group is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0127] Linker groups containing peptide bonds can be used when targeting cell types rich in peptidases, such as hepatocytes and synoviocytes.

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

[0129] i. Cleavable groups by redox One type of cleavable group is a redox-cleavable group that is cleaved by reduction or oxidation. An example of a reduction-cleavable group is a disulfide linkage group (-S-S-). Attention can be paid to the methods described herein to determine whether a candidate cleavable group is a suitable "reduction-cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the rate of cleavage observed intracellularly, e.g., within a target cell. A candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved by 10% or less in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2, 4, 10, or 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to 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 kinetics assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0130] ii. Phosphate-based cleavable groups Phosphate-based cleavable groups are cleaved by agents that decompose or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups intracellularly are enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups are -O-P(O)(OR k )-O-, -O-P(S)(OR k )-O-, -O-P(S)(SR k )-O-, -S-P(O)(OR k )-O-, -O-P(O)(OR k )-S-, -S-P(O)(OR k )-S-, -O-P(S)(OR k )-S-, -S-P(S)(OR k )-O-, -O-P(O)(R k )-O-, -O-P(S)(Rk )-O-,-S-P(O)(R k )-O-,-S-P(S)(R k )-O-,-S-P(O)(R k )-S-,-O-P(S)(R k )-S-. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, and -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using a method similar to that described above.

[0131] iii. Acid-cleavable group An acid-cleavable group is a linking group that is cleaved under acidic conditions. In preferred embodiments, the acid-cleavable group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by an agent such as an enzyme that can act as a general acid. Inside cells, 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. The acid-cleavable group can be represented by the general formula -C=NN- 、 C(O)O, or -OC(O). Preferred embodiments are those in which the carbon (alkoxy group) bonded to the oxygen of the ester 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 a method similar to that described above.

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

[0133] v. Peptide-cleavable groups Peptide-cleavable groups are cleaved by enzymes such as intracellular peptidases and proteases. Peptide-cleavable groups are peptide bonds formed between amino acids that give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-cleavable groups do not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. The peptide bond is a special type of amide bond formed between amino acids that gives rise to peptides and proteins. Peptide-cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that give rise to peptides and proteins and do not include the entire amide functional group. The linking group cleavable by a peptide is represented by the general formula -NHCHR A C(O)NHCHR B C(O)-, where R A and R Bare the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to that described above. As used herein, "carbohydrate" refers to a compound that is a carbohydrate in itself, composed of one or more monosaccharide units (which can be linear, branched, or cyclic) having at least 6 carbon atoms, with oxygen, nitrogen, or sulfur atoms bonded to each carbon atom; or a compound having as part of it a carbohydrate moiety composed of one or more monosaccharides (which can be linear, branched, or cyclic), each having at least 6 carbon atoms, with oxygen, nitrogen, or sulfur atoms bonded 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 C5 or higher (preferably, C5 - C8) sugars; disaccharides and trisaccharides include sugars having 2 or 3 monosaccharide units (preferably, C5 - C8).

[0134] Ligand The ligand can be any ligand 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 The conjugates described herein can be formulated for administration to a subject. For ease of explanation, the formulations, compositions, and methods in this section are mainly described with respect to conjugates of unmodified iRNA agents. However, it will be understood that these formulations, compositions, and methods can be carried out with conjugates of other oligonucleotides, such as modified iRNA agents, and such implementations are within the scope of the present invention.

[0136] Formulated iRNA conjugates can take various states. In one example, the conjugate is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA conjugate is in an aqueous phase, e.g., in a solution containing water.

[0137] The aqueous or crystalline conjugate can be incorporated, for example, into a delivery vehicle, such as a liposome (particularly in the case of an aqueous phase) or a particle (e.g., microparticles, as may be appropriate for a crystalline composition). Generally, the iRNA conjugate is formulated in a manner compatible with the intended method of administration. The iRNA conjugate can be incorporated into nucleic acid lipid nanoparticles. In one embodiment, each nanoparticle comprises a conjugate, a cationic lipid (e.g., a cationic lipid having a pK in the range of about 4 to about 11, such as about 5 to about 7) a 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 the following methods, namely spray drying, freeze drying, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or by at least one of sonication with lipids, freeze drying, condensation, and other self-assembly.

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

[0140] In one embodiment, the iRNA composition comprises at least one second therapeutic agent (e.g., an agent other than RNA or DNA). For example, an iRNA composition for the treatment of a viral disease, such as HIV, may comprise a known antiviral agent (e.g., a protease inhibitor or a reverse transcriptase inhibitor). In another example, an iRNA composition for the treatment of cancer may further comprise 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 cells. The agent may be administered before, after, or simultaneously with the administration of the iRNA agent. The agent may also be covalently attached to the iRNA agent. The agent may be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The agent may transiently affect the cell.

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

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

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

Examples

[0145] Abbreviations: TBAHS is tetrabutylammonium hydrogensulfate; 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 Trinucleotide GalNAc Monomer with an Acyclic Linker-1 The trinucleotide GalNAc moieties 211a-d are synthesized as shown in Scheme 1 below.

Chemical formula

[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), solid sodium borohydride (0.4 g) is added at 0-5 °C. After addition, the reaction mixture is warmed to room temperature and stirred for 20 minutes. The reaction mixture is diluted with saturated ammonium chloride and extracted with dichloromethane. The organic layer is dried over Na2SO4 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), triethylamine (1.5 mL) was added and the mixture was cooled with stirring. To this stirred solution, methanesulfonyl chloride (1.3 g) in dichloromethane (30 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. Then, the reaction mixture was washed with saturated NaHCO3 solution (100 mL) and then with brine (100 mL). 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 the general procedure, the nucleobase (2 equivalents) was dissolved in anhydrous DMF (100 mL) at 110 °C. To this stirred solution, solid CsCO3 (2 g) and sodium iodide (2 g) were added and the mixture was stirred vigorously. To this stirred solution, a solution of mesylate 202 (1 equivalent) in anhydrous DMF (10 mL) was added dropwise. The reaction mixture was stirred at 110 °C for an additional 30 minutes and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate and the organic layer was washed with saturated NaHCO3 (100 mL), brine (100 mL), and dried (anhydrous Na2SO4). Concentration of the organic layer gave the crude product, which was purified by flash column chromatography to isolate the 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 the succinimidyl esters, which were then treated with monophthalimide - protected hexanediamine in the presence of pyridine. After column purification, the amine - substituted products 204a - d were obtained.

[0151] v. Synthesis of 205a - d Deprotection of the acetonide in 204a - d by treatment with acetic acid under the reported conditions gave 205a - d, which were used directly in the next step.

[0152] vi. Synthesis of 206a - d Treatment of diols 205a - d with DMTrCl in pyridine will afford 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 (20 - fold) at room temperature overnight. Deprotected amines 207a - d are obtained by concentration of the reaction mixture followed by column purification.

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

[0155] ix. Synthesis of 209a - d Using a similar coupling procedure, coupling of the amine 207a - d with the triple - stranded GalNAc - containing carboxylic acid can yield the triple - stranded 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 gives the corresponding amidites 210a - d and 211a - d after flash column purification.

[0157] Example 2: Synthesis of Triple - Stranded GalNAc Monomers Using Acyclic Linker - 2 The triple - stranded GalNAc moiety 219 is synthesized as shown in Scheme 2 below.

Chemical Structure

[0158] Example 3: Synthesis of a Trinucleotide GalNAc Monomer Using Non-Cyclic Linker-3 The trinucleotide GalNAc moiety 215 is synthesized as shown in Scheme 3 below.

Chemical formula

[0159] i. Synthesis of 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 eq) at 110 °C under microwave irradiation for 30 minutes gave the ring-opened product 222 in 90% yield.

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

[0161] iii. Synthesis of 225 Phosphorylation of the 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 Trinucleotide α-Anomer-Conjugate Building Unit A trinucleotide α-anomer represented by the following formula is prepared.

Chemical formula

[0163] For example, R, R', and R" can each independently be C6-C 10 aryl, C6-C 10 heterocyclic aromatic, C1-C 20 alkyl, or a sugar (e.g., galactose or GalNAc).

[0164] Prepare triple-stranded α-anomers represented by the following formula.

Chem.

[0165] For example, R, R', and R” can each independently be C6~C 10 aryl, C6~C 10 heterocyclic aromatic, C1~C 20 alkyl, or a sugar (e.g., galactose or GalNAc).

[0166] Example 5: General structure of triple-stranded β-anomer-conjugate structural units Prepare triple-stranded β-anomers represented by the following formula.

Chem.

[0167] For example, R, R', and R” can each independently be C6~C 10 aryl, C6~C 10 heterocyclic aromatic, C1~C 20 alkyl, or a sugar (e.g., galactose or GalNAc).

[0168] Example 6: General structure of double-stranded α-anomer-conjugate structural units Prepare double-stranded α-anomers represented by the following formula.

Chem.

[0169] For example, R, R', and R” can each independently be C6~C 10 aryl, C6~C 10 heterocyclic aromatic, C1~C 20 alkyl, or a sugar (e.g., galactose or GalNAc).

[0170] Example 7: Synthesis of Mono-, Bi- and Tri-conjugate Structural Units The mono-, bi- and tri-conjugate structural units can be prepared from azide intermediates as shown below.

Chem.

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

[0172]

Chem.

[0173] Synthesis of Compound 9: N-Acetylglucosamine (50 g, 226 mmol) is incorporated into allyl alcohol and heated at 90 °C for 24 h. The reaction mixture is cooled to room temperature and allyl alcohol is removed by distillation. The residue is dissolved in pyridine and reacted with pivaloyl chloride to give Compound 3. The pivaloyl ester is reacted with trifluoromethanesulfonic anhydride in pyridine for 2 h and the mixture is quenched by adding water. The mixture is heated at 90 °C for 24 h to give Product 4. The ester group is removed by treatment with sodium methoxide to isolate Compound 5. The hydroxyl group is protected with dimethoxypropane to give Product 6. Next, it is reacted with tosyl chloride and sodium azide in DMF to give Compound 7. Compound 7 is subjected to click cyclo-addition and epoxidation with MCPBA to produce Compound 9.

[0174]

Chem.

[0175] Synthesis of Compound 13: When epoxide 13 was reacted with ammonia in the presence of LiClO4, amino alcohol 10 was produced. When this was combined with N-Cbz aminohexanoic acid under peptide bond conditions, compound 11 was obtained. By removing the acetonide protection and benzoylating the hydroxyl group, compound 12 was produced. Hydrogenation of compound 12 using Pd / C in MeOH produced amine 13 as the TFA salt.

[0176]

Chem.

[0177] Synthesis of Compound 17: When amine 13 was reacted with tricarboxylic acid 14 under peptide bond conditions, compound 15 was produced. By hydrogenation, compound 16 was obtained, and then when this was reacted with monobenzyl dodecanedioic acid, protected triple-stranded intermediate 17 was obtained.

[0178]

Chem.

[0179] Synthesis of Hydroxyproline Intermediate 20: When compound 17 was hydrogenated under balloon pressure, carboxylic acid 18 was obtained, and then when this was reacted with amine 19, compound 20 was produced under peptide bond conditions.

[0180]

Chem.

[0181] Synthesis of Solid Support 21: When hydroxyproline 20 was reacted with succinic anhydride and DMAP, a succinate derivative was produced. When this succinate derivative was loaded onto a solid support using peptide bond conditions, support 21 was obtained.

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

[0183] [Chemical formula]

[0184] Synthesis of carboxylic acid 29: When compound 22 was reacted with alcohol 23 in the presence of TMSOTf in DCE, carboxylate 24 was formed. When the acetate group was removed with TEA in MeOH, compound 25 was obtained. When DMTr was introduced at the O-6 position and the hydroxyl group was benzoylated, compound 26 was obtained. By removing the DMTr group under acidic conditions and reacting with Ms-Cl, a mesyl derivative was formed. By reacting this mesyl derivative with sodium azide, a C-6 azide derivative 27 was obtained. By click reaction and deprotection of the methyl ester, carboxylic acid 29 was obtained.

[0185] [Chemical formula]

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

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

[0188] [Chemical formula]

[0189] Synthesis of carboxylic acid 36: When hydroxyproline derivative 33 was reacted with monobenzyl hexanedioic acid using HBTU / DIEA, carboxylate 35 was produced. Further hydrogenation of it gave carboxylic acid 36.

[0190]

Chem.

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

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

[0193]

Chem.

[0194]

Chem.

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

Chem.

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

[0197] Example 13: Synthesis of Triplex β-Anomer siRNA Conjugate: Using the conjugate building unit 32 described above (see Scheme 10), RNA is synthesized with a ligand attached to the 3'-end of the sense strand according to a known procedure. This is annealed with the antisense strand. The product is shown below. [Chemical Formula]

[0198] Example 14: Synthesis of Duplex α-Anomer siRNA Conjugate: Using the conjugate building unit 41 described above (see Scheme 12), RNA is synthesized with a ligand attached to the 3'-end of the sense strand according to a known procedure. This is annealed with the antisense strand. [Chemical Formula]

[0199] Example 15: Synthesis of Mono-GalNAc Building Unit for Oligonucleotide Conjugation Mono-GalNAc building units 104 and 105 are prepared as shown in Scheme 16. [Chemical Formula]

[0200] Synthesis of 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyproline amine (10.00 g, 18.77 mmol) were combined 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 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, and compound 102 was obtained as a pale yellow fluffy solid (11.77 g, 63%). 1 H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.2 Hz, 1H), 7.69 (t, J = 5.6 Hz, 1H), 7.39 - 7.09 (m, 9H), 6.86 (ddd, J = 9.0, 5.4, 2.1 Hz, 4H), 5.20 (d, J = 3.4 Hz, 1H), 5.03 - 4.83 (m, 2H), 4.47 (d, J = 8.5 Hz, 1H), 4.41 - 4.07 (m, 2H), 4.04 - 3.95 (m, 3H), 3.86 (dt, J = 11.2, 8.9 Hz, 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 = 6.7 Hz, 2H), 1.98 (s, 3H), 1.87 (d, J = 7.5 Hz, 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 equivalents) and a chloroamidite reagent were added. The reaction mixture was stirred for 30 minutes and examined 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, and the compound was obtained as a white fluffy solid. 11H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.2 Hz, 1H), 7.68 (s, 1H), 7.42 - 7.06 (m, 8H), 7.01 - 6.73 (m, 4H), 5.20 (d, J = 3.3 Hz, 1H), 4.96 (dd, J = 11.2, 3.3 Hz, 1H), 4.63 (d, J = 4.7 Hz, 1H), 4.47 (d, J = 8.5 Hz, 1H), 4.15 (s, 1H), 4.01 (s, 3H), 3.86 (d, J = 11.0 Hz, 1H), 3.70 (d, J = 16.5 Hz, 9H), 3.45 (ddd, J = 37.0, 23.3, 16.4 Hz, 6H), 2.99 (dd, J = 12.3, 6.4 Hz, 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 31P 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, equivalent), followed by TEA (1 mL) were added. The reaction mixture was stirred overnight at room temperature. Its TLC was examined 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. The polystyrene solid support was added to the reaction mixture and the mixture was shaken overnight at ambient temperature. The solid support was filtered, washed and capped using acetic anhydride / Py mixture. The solid support was washed again with dichloromethane, MeOH / DCM and ether (27.10 g, 55 μmol / g).

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

[0204] Example 17: Synthesis of a Trifluoroacetamide Derivative for Post-Synthesis Conjugation Intermediate compound 8 is prepared as shown in Scheme 17 below. [Chemical Formula]

[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 under argon at 0 °C for 20 minutes, 4-hydroxy-l-proline methyl ester hydrochloride (20.0 g, 110 mmol) was added and stirring was continued under argon at room temperature overnight. 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. Compound 2 was purified by column chromatography by first eluting with 2% methanol / dichloromethane and then 5% methanol / dichloromethane to remove impurities, and 21.36 g (65%) was obtained. 11H NMR (400 MHz, DMSO-d6): Rotational isomers observed due to amide bonds in the ring. δ 7.35 (m, 5H), 5.15 (d, OH, exchangeable with D2O), 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 under argon at 0 °C for 20 minutes. Next, lithium borohydride (1.19 g, 54.43 mmol) was added to the solution at 0 °C over 20 minutes, and stirring was continued under argon at room temperature overnight. 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. Compound 3 was purified by column chromatography by eluting with 3% methanol / dichloromethane, followed by 5% methanol / dichloromethane to remove impurities, and 9.21 g (49%) was obtained. 1 1H NMR (400 MHz, DMSO-d6): Rotational isomers observed due to amide bonds in the ring. δ 7.35 (m, 5H), 4.99 (s, H), 4.91 (d, OH, exchangeable with D2O), 4.77 (t, OH, exchangeable with D2O), 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). 1313C NMR (100 MHz, DMSO-d6): δ 171.4, 171.1 (small due to rotamer), 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 co-evaporated twice with anhydrous pyridine (80 mL). Next, the compound was dried under high vacuum overnight. Compound 3 was removed from 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 under argon at 0 °C for 30 minutes. Next, DMT-Cl (9.0 g, 26.53 mmol) was added to the solution at 0 °C. The mixture was stirred under reduced pressure and then under argon, and stirring was continued under argon at room temperature overnight. The 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, again with water, and saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Crude Compound 4 (Rf = 0.6 in 100% EtOAc, 14.02 g) was obtained. To remove impurities, Compound 4 was purified by column chromatography by first eluting with 50% ethyl acetate in hexane (1% TEA), followed by 100% ethyl acetate (1% TEA), and 12.36 g (73.4%) was obtained as a white foamy solid. 11H NMR (400 MHz, DMSO-d6): δ 7.17 - 7.33 (m, 14H), 4.99 (s, 2H), 4.91 (d, OH, exchangeable with D2O), 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. 10 wt% palladium supported on wet activated carbon Degussa type (1.3 g) was added to the reaction mixture. The flask was repurged 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 continuously stirred under hydrogen at room temperature overnight. The reaction mixture was decanted onto a sintered funnel filled with celite and washed twice with methanol. Evaporation of the organic layer to dryness gave Compound 5 (Rf = 0.05 in 10% MeOH / DCM, 9.16 g, 93%) as a white solid, which did not require further purification. 1 1H NMR (400 MHz, DMSO-d6): δ 7.15 - 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), 3.29 (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 under argon at 10 °C for 10 minutes. Triethylamine (4.80 mL, 34.4 mmol) was added dropwise to the reaction mixture, and the mixture was continuously stirred under argon at 10 °C for 20 minutes. Ethyl trifluoroacetate (3.05 mL, 25.8 mmol) was added dropwise to the reaction mixture, and the mixture was continuously stirred under argon at 10 °C for 10 minutes. The reaction mixture was continuously stirred under argon at room temperature overnight. 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 in 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 1H NMR (400 MHz, DMSO-d6): δ 7.56 - 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, 6H), 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 19F NMR (400 MHz, 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. Next, succinic anhydride (2.80 g, 27.86 mmol) was added, and the mixture was continuously stirred under argon at room temperature overnight. The reaction mixture was washed twice with a slightly saturated sodium chloride solution. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 7 (Rf = 0.9 in 10% MeOH / DCM, 10.87 g, 94%) was obtained as a white solid and 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 continuously 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, and further washed with 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 de-blocking. To the remaining CPG compound, 25% acetic anhydride / pyridine (100 mL) was added, and the mixture was shaken overnight. The CPG compound and the reaction mixture were placed on a sintered funnel and washed in the same manner 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 de-blocking. Spectrophotometer: Before capping, 0.9892 Abs (502.0 nm), 65 micromoles / g; after capping, 1.4403 (502.0 nm), 67 micromoles / 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. Next, N,N-diisopropylaminocyanethyl phosphoramidate chloride (5.63 mL, 16.26 mmol) was added to the reaction mixture. The reaction mixture was continuously stirred under argon at room temperature 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, and crude compound 9 (Rf = 0.44 in 5% MeOH / DCM, 11.02 g) was obtained. Column purification by elution with 3% methanol / dichloromethane (1% TEA) gave compound 9 (6.31 g, 54%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.37 (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 (400 MHz, DMSO-d6): 151.68 (d, 1P).

[0213] Example 18: Synthesis of a Carbamate Linker for Post-Synthesis Conjugation

Chemical Structure

[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 under argon at 10 °C for 20 minutes. Next, at 10 °C, while continuously stirring the reaction mixture under argon, triethylamine (78 mL, 560 mmol) was added to the solution. The reaction mixture was continuously stirred under argon at room temperature overnight. 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, and a crude compound 2 (Rf = 0.5 10% MeOH / DCM, 71.97 g) was obtained as a white solid. This was used in the next step without further purification.

[0215] 4-Z-Aminobutanol 14 Activated by DSC: 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 under argon at 0 °C for 30 minutes. Triethylamine (7.88 mL, 53.74 mmol) was added dropwise, and the mixture was stirred under argon at 0 °C for 5 minutes. The reaction mixture was continuously stirred under argon at room temperature overnight. 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, and a crude compound 14 (Rf = 0.85 10% MeOH / DCM, 11.14 g) was obtained as a light brown solid. This 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 wt% palladium supported on wet activated carbon Degussa type (0.5 g). The flask was repurged 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 continuously stirred under hydrogen at room temperature overnight. The reaction mixture was decanted onto a sintered funnel filled with Celite and washed twice with methanol. Evaporation of the organic layer to dryness gave 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 (400 MHz, DMSO-d6) δ 7.41 - 7.14 (m, 9H), 6.99 - 6.72 (m, 4H), 4.96 (d, 1H), 4.30 (s, 1H), 4.04 - 3.58 (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 under argon at 10 °C for 10 minutes. Triethylamine (1.67 mL, 11.96 mmol) was added dropwise to the reaction mixture and the mixture was continuously stirred under argon at 10 °C for 20 minutes. Ethyl trifluoroacetate (1.06 mL, 8.97 mmol) was added dropwise to the reaction mixture and the mixture was continuously stirred under argon at 10 °C for 10 minutes. The reaction mixture was continuously stirred under argon at room temperature overnight. 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. 11H NMR (400 MHz, DMSO-d6) δ 7.46 - 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 19F NMR (400 MHz, 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. Next, succinic anhydride (0.63 g, 6.34 mmol) was added and the mixture was stirred under argon at room temperature overnight. The reaction mixture was washed twice with a slightly saturated sodium chloride solution. 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 elution with 5% methanol / dichloromethane (1% TEA) gave compound 12 (2.58 g, 98%) as a white solid. 1 1H NMR (400 MHz, DMSO-d6): δ 9.43 (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 continuously 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, and further washed with 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 (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 the reaction mixture were placed on a sintered funnel and washed in the same manner 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 micromol / g; after capping, 1.8798 Abs (502.0 nm), 89.6 micromol / 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. Next, N,N - diisopropylaminocyanethyl phosphoramidate chloride (1.73 mL, 7.76 mmol) was added to the reaction mixture. The reaction mixture was continuously stirred under argon at room temperature 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 in 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 (400 MHz, DMSO - d6) δ 7.41 - 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 the chemical action of palladium binding, GalNAc ligands and cationic molecules can be introduced at the C-5 position of pyrimidine nucleosides having various substituents at the 2'-position. As shown in the following schemes (Schemes 19 to 27), nucleoside building blocks can be synthesized accordingly. In Schemes 28 and 29, designed purine nucleoside analogs containing GalNAc ligands are shown. In Scheme 30, the GalNAc ligand is introduced at the C-1'-position. Using the click chemical action, GalNAc can be introduced at the abasic site as shown in Schemes 31 and 32. Scheme 33 shows an exchangeable nucleoside approach for introducing a GalNAc ligand at the C-2 position on the purine ring.

[0222]

Chem.

[0223]

Chem.

[0224]

Chem.

[0225]

Chem.

[0226]

Chem.

[0227]

Chem.

[0228]

Chem.

[0229]

Chem.

[0230]

Chem.

[0231]

Chem.

[0232]

Chem.

[0233]

Chem.

[0234]

Chem.

[0235]

Chem.

[0236]

Chem.

[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 CH2Cl2 and saturated aqueous NaHCO3 and dried over anhydrous Na2SO4. The crude material was purified by silica gel column chromatography (5% MeOH in CH2Cl2, R f = 0.23) to afford compound 103 (17.8 g, 26.4 mmol, 88%). 1 H NMR (DMSO - d6, 400 MHz): δ 11.81 (s, 1H), 8.07 (s, 1H), 7.46 - 7.14 (m, 10H), 6.88 (dd, J = 8.9, 1.8 Hz, 4H), 5.83 (d, J = 20.5 Hz, 1H), 5.60 (d, J = 7.0 Hz, 1H), 5.16 (dd, J = 53.4, 4.8 Hz, 1H), 4.43 - 4.20 (m, 1H), 4.08 - 3.90 (m, 1H), 3.74 (s, 7H), 3.23 (d, J = 2.7 Hz, 2H). 13 C NMR (100 MHz, DMSO - d6): δ 160.68, 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) were 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 CH2Cl2 and saturated aqueous NaHCO3. The organic layer was separated and dried over anhydrous Na2SO4. The filtrate was concentrated and the resulting crude material was purified by silica gel column chromatography (0 - 5% MeOH in CH2Cl2) to give 104 (4.30 g, 6.37 mmol, 67%, R f = CH2Cl2 developed with 5% MeOH in CH2Cl2, 0.32) was obtained. 1 1H NMR (400 MHz, DMSO-d6): δ 11.78 (s, 1H), 8.52 (s, 1H), 7.41 (d, J = 7.4 Hz, 2H), 7.34 - 7.16 (m, 8H), 6.93 - 6.80 (m, 4H), 5.90 (d, J = 20.1 Hz, 1H), 5.63 (d, J = 7.1 Hz, 1H), 5.26 (d, J = 4.4 Hz, 1H), 5.12 (d, J = 4.5 Hz, 1H), 4.50 - 4.17 (m, 3H), 4.07 (dd, J = 7.3, 5.0 Hz, 1H), 3.73 (d, J = 1.2 Hz, 7H), 3.31 - 3.17 (m, 2H). 13 13C NMR (100 MHz, DMSO-d6): δ 160.70, 158.96, 158.08, 158.06, 149.18, 148.89, 144.76, 135.47, 135.33, 129.70, 129.66, 127.80, 127.61, 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 19F NMR (376 MHz, 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 C4H4N2NaO9+ 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) overnight at room temperature. The reaction mixture was extracted with CH2Cl2 and saturated aqueous NaHCO3, and the organic layer was dried over anhydrous Na2SO4 to give the crude material 105. C 36 H 42 Molecular weight (M + H) for FN4O8 + Calculated value 677.2987, measured value 677.1.

[0240] Example 20. GalNAc Conjugation in the Ribose Ring (Schemes 34 - 44) Synthetic methods for conjugating GalNAc and its derivatives to the ribose ring in nucleosides are shown below. The tin-modified nucleoside can be coupled with an alkyl bromide to produce 2'- and 3'-linked products. Each of the resulting primary amines or activated esters and terminal alkenes can be coupled with a GalNAc ligand using appropriate reaction conditions. These building blocks are incorporated into oligonucleotides using the chemistry of standard phosphoramidites. The GalNAc ligand can also be conjugated to oligonucleotides by post-synthetic methods.

[0241]

Chem.

[0242]

Chem.

[0243]

Chem.

[0244] [Chemistry]

[0245] [Chemistry]

[0246] [Chemistry]

[0247] [Chemistry]

[0248] [Chemistry]

[0249] [Chemistry]

[0250] [Chemistry]

[0251] [Chemistry]

[0252] 2'- and 3'-O-phthalimidohexyl-5-methyluridine (2a, 2b). A solution of the obtained 2',3'-O-dibutylstannylen-5-methyluridine (28 g, 57.24 mmol) (J. Org. Chem., 1974, 24 - 30), 6-bromohexyl phthalimide (35.5 g, 114.48 mmol) and NaI (1.72 g, 11.45 mmol) in DMF (105 mL) was heated in a microwave at 100 °C for 3.5 h as reported. After removing DMF, the residue was purified by silica gel column chromatography (R f = 0.26 in 5% MeOH in CH2Cl2), and the 2'- and 3'-isomers of O-phthalimidohexyl-5-methyluridine were obtained as an inseparable mixture (10.1 g, 20.7 mmol, 36%). MS m / z 488.0 (M+H) + , 510.2 (M+Na) + , 486.2 (M-H). 1 H NMR (400 MHz, DMSO-d6): δ 11.29 (s, 1H), 7.88 - 7.79 (m, 4H), 7.78 - 7.70 (m, 1H), 5.81 (d, J = 5.3 Hz, 1H), 5.72 (d, J = 5.6 Hz, 1H), 5.25 (d, J = 6.2 Hz, 1H), 5.12 (t, J = 5.0 Hz, 1H), 5.00 (d, J = 5.9 Hz, 1H), 4.14 (dd, J = 11.3, 5.6 Hz, 1H), 4.07 (dd, J = 10.1, 5.0 Hz, 1H), 3.89 - 3.79 (m, 2H), 3.76 - 3.72 (m, 1H), 3.63 (ddd, J = 11.1, 8.8, 5.4 Hz, 1H), 3.59 - 3.47 (m, 4H), 3.41 (dt, J = 11.6, 6.6 Hz, 1H), 3.28 (s, 1H), 1.75 (d, J = 3.7 Hz, 3H), 1.64 - 1.41 (m, 5H), 1.27 (d, J = 14.2 Hz, 5H). 1313C NMR (100 MHz, DMSO-d6): δ 167.99, 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 co-evaporated with pyridine (50 mL), then dissolved in pyridine (80 mL) and cooled to 0 °C in an ice bath. To this mixture was added dimethoxytrityl chloride (7.73 g, 22.80 mmol), and the reaction was stirred at 0 °C for 2 h and then at room temperature for 1 h. An additional 0.4 equivalent 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 hexane). MS m / z 812.3 (M+Na) + , 788.3 (M-H). 11H NMR (400 MHz, DMSO-d6): δ 11.35 (s, 1H), 7.87 - 7.77 (m, 4H), 7.48 (d, J = 0.8 Hz, 1H), 7.38 (d, J = 7.4 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.23 (dd, J = 12.1, 8.1 Hz, 5H), 6.89 (d, J = 8.0 Hz, 4H), 5.11 (d, J = 6.3 Hz, 1H), 4.18 (dd, J = 11.1, 5.4 Hz, 1H), 3.99 (ddd, J = 12.0, 11.2, 5.9 Hz, 3H), 3.72 (s, 6H), 3.62 - 3.46 (m, 4H), 3.21 (ddd, J = 12.9, 10.7, 3.3 Hz, 2H), 1.98 (d, J = 1.8 Hz, 1H), 1.60 - 1.44 (m, 5H), 1.38 (s, 3H), 1.35 - 1.19 (m, 5H), 1.16 (t, J = 7.1 Hz, 1H), 0.87 (dd, J = 10.1, 4.7 Hz, 1H). 13 13C NMR (100 MHz, DMSO-d6): δ 170.33, 167.92, 163.61, 158.19, 158.16, 150.41, 144.67, 135.45, 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 isolated by column chromatography (3.36 g, 20.5%, 4.26 mmol; R f = 0.18 in 60% EtOAc in hexane). MS m / z 812.0 (M+Na) + , 788.3 (M-H). 11H NMR (400 MHz, DMSO-d6): δ 11.34 (s, 1H), 7.88 - 7.76 (m, 4H), 7.50 (s, 1H), 7.36 (d, J = 7.5 Hz, 2H), 7.33 - 7.15 (m, 7H), 6.87 (d, J = 7.8 Hz, 4H), 5.75 - 5.69 (m, 1H), 5.37 (d, J = 6.0 Hz, 1H), 4.27 (dd, J = 10.5, 5.1 Hz, 1H), 4.05 - 3.94 (m, 2H), 3.91 (t, J = 5.2 Hz, 1H), 3.71 (s, 6H), 3.56 (ddd, J = 22.7, 11.8, 6.7 Hz, 3H), 3.21 (ddd, J = 24.1, 10.8, 3.3 Hz, 2H), 1.98 (t, J = 4.5 Hz, 1H), 1.60 - 1.37 (m, 7H), 1.34 - 1.13 (m, 5H), 0.94 - 0.83 (m, 1H). 13 13C NMR (100 MHz, DMSO-d6): δ 170.41, 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, 109.37, 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 for 3.5 h (66 °C). 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 (M-H) - . 1 H NMR (400 MHz, DMSO-d6): δ 7.48 (s, 1H), 7.38 (d, J = 7.5 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.24 (d, J = 8.9 Hz, 5H), 6.89 (d, J = 8.4 Hz, 4H), 5.84 (d, J = 5.0 Hz, 1H), 5.74 (s, 1H), 4.19 (t, J = 5.0 Hz, 1H), 3.97 (t, J = 4.9 Hz, 2H), 3.72 (s, 6H), 3.63 - 3.45 (m, 3H), 3.27 - 3.15 (m, 3H), 1.48 (d, J = 6.4 Hz, 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). The 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, washed with brine, and then the organic layer was dried over Na2SO4 and evaporated under reduced pressure. The crude material was purified by column chromatography (R f = 0.36 in 5% MeOH in DCM) to give 5a (9.56 g, 7.49 mmol, 83%). MS m / z 1298.3 (M+Na) + . 11H NMR (400 MHz, DMSO-d6): δ 11.36 (s, 1H), 7.99 - 7.86 (m, 5H), 7.73 - 7.52 (m, 8H), 7.48 (t, J = 7.7 Hz, 3H), 7.41 - 7.34 (m, 4H), 7.30 (t, J = 7.6 Hz, 2H), 7.23 (dd, J = 11.1, 8.1 Hz, 4H), 6.88 (d, J = 8.1 Hz, 4H), 5.83 (d, J = 4.8 Hz, 1H), 5.74 (d, J = 3.9 Hz, 1H), 5.35 (dd, J = 11.1, 3.3 Hz, 1H), 5.11 (d, J = 6.3 Hz, 1H), 4.72 (d, J = 8.5 Hz, 1H), 4.50 - 4.39 (m, 2H), 4.38 - 4.14 (m, 4H), 3.95 (t, J = 4.8 Hz, 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, 6.5 Hz, 2H), 2.03 (s, 2H), 1.69 (s, 3H), 1.49 (s, 6H), 1.40 - 1.15 (m, 9H). 13 13C NMR (100 MHz, DMSO-d6): δ 171.73, 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-succinate-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 co-evaporated with acetonitrile under reduced pressure, and 2.11 g (91%, 1.43 mmol) of 6a was obtained as the TEA salt (R f = 0.41 in 5% MeOH / 5% TEA in DCM). MS m / z 1397.4 (M+Na) + , 1373.4 (M-H) - . 1 H NMR (400 MHz, DMSO-d6): δ 11.44 (s, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.90 (dd, J = 10.2, 4.1 Hz, 4H), 7.75 (s, 1H), 7.72 - 7.52 (m, 7H), 7.51 - 7.44 (m, 3H), 7.34 (ddd, J = 24.0, 13.7, 7.9 Hz, 6H), 7.22 (d, J = 8.7 Hz, 5H), 6.89 (d, J = 7.9 Hz, 4H), 5.83 (d, J = 6.1 Hz, 1H), 5.73 (d, J = 3.4 Hz, 1H), 5.36 (dd, J = 11.1, 3.3 Hz, 1H), 5.26 - 5.22 (m, 1H), 4.75 (d, J = 8.5 Hz, 1H), 4.47 - 4.40 (m, 2H), 4.37 - 4.22 (m, 3H), 4.12 (d, J = 3.5 Hz, 1H), 3.83 - 3.69 (m, 6H), 3.53 - 3.19 (m, 15H), 2.97 (d, J = 7.8 Hz, 2H), 2.04 (s, 2H), 1.68 (s, 2H), 1.56 - 1.11 (m, 15H). 1313C NMR (100 MHz, DMSO-d6): δ 173.40, 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, 127.61, 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) was added HBTU (1.03 g, 2.72 mmol), DIEA (528 mg, 4.08 mmol) and CPG (16.0 g, 130 μmol / g, 540 Å), and the mixture was shaken for 24 h. The CPG was removed by filtration 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 removed by filtration and washed with the same solvents 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-cyanoethyl phosphoramidite-5-methyluridine (8a). 5a (2.90 g, 2.27 mmol) was co-evaporated twice with anhydrous acetonitrile and then placed under a complete 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 stirred mixture, followed by DCI (268 mg, 2.27 mmol). The mixture was stirred at 0 °C for 20 minutes and then at room temperature for 17 hours. The reaction mixture was diluted with DCM and then washed with brine and dried over Na2SO4, giving a pale yellow foam. The crude material was purified by silica gel column chromatography (φ = 4.2 cm × 19 cm; R f = 0.39 in EtOAc), giving 3.20 g of 8a (95%, 2.17 mmol). MS m / z 1498.3 (M+Na) + . 1 1H NMR (400 MHz, DMSO-d6): δ 11.39 (s, 1H), 8.06 - 7.84 (m, 5H), 7.78 - 7.16 (m, 21H), 6.93 - 6.82 (m, 4H), 5.82 (d, J = 4.3 Hz, 1H), 5.74 (s, 1H), 5.35 (dd, J = 11.1, 3.1 Hz, 1H), 4.73 (d, J = 8.5 Hz, 1H), 4.50 - 4.20 (m, 5H), 4.10 (dd, J = 12.8, 7.7 Hz, 2H), 3.74 (t, J = 11.5 Hz, 7H), 3.66 - 3.44 (m, 6H), 3.29 - 3.18 (m, 2H), 2.98 (d, J = 5.3 Hz, 2H), 2.76 (t, J = 5.8 Hz, 1H), 2.57 (dd, J = 10.0, 5.3 Hz, 1H), 2.49 (d, J = 1.5 Hz, 5H), 2.04 (s, 2H), 1.70 (s, 3H), 1.59 - 1.03 (m, 29H), 0.99 - 0.81 (m, 4H). 1313C NMR (125 MHz, DMSO-d6) δ 171.60, 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, 129.03, 128.90, 128.88, 128.87, 128.83, 128.56, 128.45, 127.75, 127.56, 127.50, 126.74, 118.72, 118.57, 113.08, 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, 61.89, 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 31P NMR (162 MHz, DMSO-d6) δ 154.01, 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 h. 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 (M-H) - . 11H NMR (400 MHz, DMSO-d6) δ 7.50 (s, 1H), 7.43 - 7.16 (m, 9H), 6.88 (d, J = 8.7 Hz, 4H), 5.73 (d, J = 4.6 Hz, 1H), 4.29 (t, J = 4.8 Hz, 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 13C NMR (100 MHz, DMSO-d6): δ 163.73, 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 h before an additional 0.2 equivalent of GalNAc-NHS ester was added and then stirred for a further 1 h. Following the same procedure 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) + . 11H NMR (400 MHz, DMSO-d6): δ 11.35 (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 = 8.8 Hz, 4H), 5.74 (dd, J = 5.9, 4.1 Hz, 2H), 5.41 - 5.33 (m, 2H), 4.73 (d, J = 8.5 Hz, 1H), 4.44 (t, J = 7.9 Hz, 2H), 4.31 (ddd, J = 16.9, 12.6, 7.9 Hz, 3H), 4.03 - 3.95 (m, 1H), 3.91 (t, J = 5.1 Hz, 1H), 3.83 - 3.75 (m, 1H), 3.72 (s, 6H), 3.63 - 3.45 (m, 2H), 3.22 (dd, J = 7.8, 2.9 Hz, 2H), 2.99 (d, J = 6.2 Hz, 2H), 2.87 (s, 1H), 2.72 (s, 1H), 2.04 (t, J = 6.4 Hz, 2H), 1.69 (s, 3H), 1.55 - 1.14 (m, 14H). 13 13C NMR (125 MHz, DMSO-d6): δ 171.74, 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-succinate-5-methyluridine (6b). To a solution of 5b (1.10 g, 0.86 mmol) in DCM (20 mL) were 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 the same manner as 6a and co-evaporated with acetonitrile under reduced pressure to afford 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 (M-H) - . 1 H NMR (400 MHz, DMSO-d6): δ 8.09 (d, J = 9.3 Hz, 1H), 7.91 (t, J = 6.9 Hz, 4H), 7.76 (t, J = 5.4 Hz, 1H), 7.69 (dd, J = 11.3, 7.5 Hz, 3H), 7.65 - 7.51 (m, 5H), 7.47 (t, J = 7.7 Hz, 2H), 7.41 - 7.33 (m, 4H), 7.32 - 7.16 (m, 7H), 6.87 (d, J = 8.7 Hz, 4H), 5.85 (d, J = 3.9 Hz, 1H), 5.74 (d, J = 3.2 Hz, 1H), 5.48 - 5.41 (m, 1H), 5.36 (dd, J = 11.1, 3.2 Hz, 1H), 5.29 (s, 1H), 4.75 (d, J = 8.5 Hz, 1H), 4.49 - 4.39 (m, 2H), 4.38 - 4.19 (m, 4H), 3.28 (ddd, J = 37.9, 12.7, 5.4 Hz, 11H), 3.08 - 2.90 (m, 3H), 2.69 (q, J = 7.2 Hz, 5H), 2.60 - 2.52 (m, 2H), 2.05 (d, J = 3.5 Hz, 2H), 1.76 (s, 1H), 1.69 (s, 3H), 1.56 - 0.94 (m, 28H). 1313C NMR (100 MHz, DMSO-d6): δ 173.63, 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, 128.65, 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 measured as in 7a, and CPG 7b was obtained 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 as described for 8a, and 1.89 g (83%, 1.28 mmol) of 8b was obtained by silica gel column chromatography. (R f = 0.43 in 100% EtOAc). MS m / z 1497.4 (M+Na) + , 1474.3 (M-H) - . 11H NMR (400 MHz, DMSO-d6): δ 11.38 (s, 1H), 8.00 - 7.87 (m, 5H), 7.75 - 7.18 (m, 22H), 6.88 (dd, J = 8.9, 2.7 Hz, 4H), 5.88 (dd, J = 9.2, 5.1 Hz, 1H), 5.75 (d, J = 3.2 Hz, 1H), 5.36 (dd, J = 11.1, 3.2 Hz, 1H), 4.73 (d, J = 8.5 Hz, 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, 4.8 Hz, 3H), 3.85 - 3.47 (m, 14H), 2.98 (s, 2H), 2.71 (ddd, J = 11.6, 8.9, 3.9 Hz, 2H), 2.10 - 1.95 (m, 4H), 1.69 (s, 3H), 1.57 - 0.99 (m, 31H), 0.94 - 0.83 (m, 2H). 13 13C NMR (125 MHz, DMSO-d6): δ 171.72, 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 31P NMR (162 MHz, DMSO-d6) δ 155.08, 154.60.

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

[0266]

Chem.

[0267]

Chem.

[0268]

Chem.

[0269]

Chem.

[0270]

Chem.

[0271]

Chem.

[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. C 14 H 20 Molecular weight (M + H) for NO8 + Calculated value 330.12, found 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. After aqueous work-up and purification by silica gel column, Compound 3 (380 mg, 0.731 mmol, 15%) was obtained. C 23 H 37 NNaO 10 Molecular weight (M+H) for S + Calculated value 542.20, found 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 (M+H) for S + Calculated value 464.1590, found value 464.1.

[0275] Synthesis of Compound 8: Compound 7 (ca. 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 CH2Cl2 (5 mL) over 14 h. After aqueous work-up and then column chromatography, Compound 8 (284 mg, 0.507 mmol, 69% over two steps) was obtained. C 23 H 33 N2O 12 Molecular weight (M+H) for S + Calculated value 561.1754, found value 561.1.

[0276] Compound 9 is obtained by S-alkylation of compound 6 with an alkyne bromide. 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 CH2Cl2 (30 mL) over 14 h. After aqueous workup and then column chromatography, compound 13 (1.83 g, 3.26 mmol, 63%) was obtained. C 23 H 33 N2O 12 Molecular weight (M + H) for S + Calculated 561.1754, found 561.2.

[0278] Compound 16 was prepared using the reported procedure (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 CH2Cl2 (20 mL) were heated at 40 °C for 40 h. 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%). C 27 H 36 NO 10 Molecular weight (M + H) for + Calculated 534.2339, found 534.2.

[0280] Synthesis of Compound 23: Palladium carbon was added to a solution of Compound 22 (1.85 g, 3.47 mmol) in EtOAc (30 mL) (Aldrich: 330108 - 50G, 10 wt%, Degussa type E101 NE / W: 185 mg). The reaction mixture was stirred under a H2 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%). C 20 H 32 NO 10 Molecular weight (M + H) for + Calculated 446.2026, found 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 CH2Cl2 (5 mL) for 14 hours. After aqueous workup and then column chromatography, Compound 24 (300 mg, 0.553 mmol, 76%) was obtained. C 24 H 35 N2O 12 Molecular weight (M + H) for + Calculated 543.2190, found 543.2.

[0282] Oxidative cleavage of 16 gives aldehyde 25. Reduction of it to alcohol, O - alkylation with benzyl - protected triflate, followed by deprotection and esterification gives 28. Reductive amination of 25 gives acid 29, and then esterification gives 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] When the activated esters 8, 13, 24, 38, 28, 42, 30, and 44 were combined with hydroxyprolinol containing triamine-(45) or monoamine-(48), 46 and 49 were obtained respectively. These compounds were converted to their corresponding phosphoramidites or packed onto a solid support.

[0285] When DMTr-protected di-azide 53 was combined with an alkyne-containing GalNAc derivative using click chemistry, compound 54 was obtained. When it was converted to its corresponding phosphoramidite or packed onto a solid support, 55 was obtained.

[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 the following Schemes 52 and 53.

[0287]

Chemical formula

[0288] Compound 58 was prepared in a manner similar to the reported procedure (see, for example, WO 96 / 39411). By O-alkylation followed by selective cleavage of the acetyl group, compounds 60 and 63 were obtained. NHS esters 61 and 64 were prepared by standard esterification. When the acetyl group of 65 was selectively removed and the resulting hydroxyl group was protected with a benzyl group, 66 was obtained. By oxidative cleavage of the terminal alkene, 67 was obtained. By esterification followed by hydrogenation, 61 / 64 was obtained.

[0289]

Chemical formula

[0290] The trifluoromethylacetamide-(TFA-)protected galactosamine (GalN-TFA) NHS ester is coupled with an amine-containing oligonucleotide (69 / 71) in a post-synthetic procedure to produce a Gal-TFA-containing oligonucleotide (70 / 72).

[0291] Example 23. Synthesis of ASGPR ligand mimics containing a pseudouridine backbone (Schemes 55 - 56) The pseudouridine ligand can be prepared as shown in Schemes 55 and 56.

[0292]

Chemical formula

[0293] Compound 452: Methyl acrylate (235 mL, 2.61 mol) was added dropwise 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). The reaction mixture was stirred for 16 hours. After removal of the solvent, the crude material was purified by silica gel column chromatography (10% MeOH in CH2Cl2, R f = 0.23), and compound 452 (26.6 g, 80.5 mmol, 98%) was obtained. 1 1H NMR (MeOH-d4, 400 MHz): δ 7.77 (d, J = 0.8 Hz, 1H), 4.58 (d, J = 4.8 Hz, 1H), 4.15 (t, J = 5.2 Hz, 1H), 4.05 (t, J = 5.0 Hz, 1H), 3.98 - 4.02 (m, 2H), 3.91 - 3.94 (m, 1H), 3.80 (dd, J = 12.0 Hz, 3.3 Hz, 1H), 3.67 (s, 3H), 3.66 (dd, J = 12.0 Hz, 3.3 Hz, 1H), 2.73 - 2.77 (m, 2H). 13 13C NMR (CDCl3, 100 MHz): δ 173.1, 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) was added dropwise di-tert-butylsilylbis(trifluoromethanesulfonate) (15.46 mL, 42.4 mmol) with stirring at 0 °C. The reaction mixture was stirred at 0 °C for an additional 30 minutes and then treated with imidazole (12.0 g, 176.5 mmol). The mixture was stirred at 0 °C for 10 minutes and then at room temperature for 30 minutes. TBDMSCl (7.98 g, 53.0 mmol) was added and the reaction mixture was heated at 75 °C for 6 hours. The reaction mixture was extracted with Et2O and saturated aqueous NaHCO3, dried over anhydrous Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (hexane:EtOAc = 1:1, R f = 0.50) to afford compound 453 (15.0 g, 25.6 mmol, 73%). 1 H NMR (DMSO-d6, 400 MHz): δ 11.39 (s, 1H), 7.54 (s, 1H), 4.55 (s, 1H), 4.34 - 4.38 (m, 1H), 4.18 (d, J = 4.4 Hz, 1H), 3.86 - 4.00 (m, 5H), 3.58 (s, 3H), 2.67 (t, J = 6.6 Hz, 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 CH2Cl2 and saturated aqueous NaHCO3, dried over anhydrous Na2SO4, and concentrated to afford 454 as a white solid (1.16 g, 1.89 mmol, 89%). 11H NMR (MeOD-d4, 400 MHz): δ 7.49 (s, 1H), 4.63 (s, 1H), 4.39 - 4.41 (m, 1H), 4.29 (d, J = 3.6 Hz, 1H), 4.00 - 4.04 (m, 5H), 3.18 - 3.26 (m, 2H), 2.69 (t, J = 6.2 Hz, 2H), 2.56 - 2.61 (m, 2H), 1.07 (s, 9H), 1.04 (s, 9H), 0.94 (s, 9H), 0.17 (s, 3H), 0.13 (s, 3H). C 28 H 53 MW (M + H) for N4O7Si2 + Calculated value: 613.35, Measured value: 613.2.

[0296] Compound 455: To a solution of GalNAc acid (930 mg, 2.08 mmol) in DMF (10 mL) were added HBTU (789 mg, 2.08 mmol) and iPr2NEt (1.65 mL, 9.45 mmol). After 10 minutes, a solution of compound 454 in DMF (15 mL) was added and stirred overnight. The reaction mixture was extracted with Et2O and saturated aqueous NaHCO3 and dried over anhydrous Na2SO4. After evaporation, the crude material was purified by silica gel column chromatography (10% MeOH in CH2Cl2, R f = 0.43), and compound 455 (1.83 g, 1.76 mmol, 93%) was obtained. 11H NMR (DMSO-d6, 400 MHz): δ 11.36 (s, 1H), 7.98 (s, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.77 (s, 1H), 7.51 (s, 1H), 5.21 (d, J = 3.6 Hz, 1H), 4.96 (dd, J = 11.4 Hz, 3.4 Hz, 1H), 4.53 (s, 1H), 4.48 (d, J = 8.4 Hz, 1H), 4.33 - 4.36 (m, 1H), 4.18 (d, J = 4.4 Hz, 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 13C NMR (DMSO-d6, 100 MHz): δ 172.0, 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 (M + Na) for Si2 + Calculated value: 1064.49, Measured value: 1064.2.

[0297] Compound 456: Hydrogen fluoride - pyridine (approx. 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 CH2Cl2 at 0 °C, and the mixture was stirred at 0 °C for 2 hours. The reaction solution was diluted with CH2Cl2, washed with saturated aqueous NaHCO3, and dried over anhydrous Na2SO4. 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), DMTrCl (596 mg, 1.76 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours and then evaporated. The residue was extracted with CH2Cl2 and saturated aqueous NaHCO3 and dried over anhydrous Na2SO4. The crude material was purified by silica gel column chromatography (10% MeOH in CH2Cl2, R f = 0.57) to give compound 456 (1.65 g, 1.37 mmol, 78%). 1 1H NMR (DMSO-d6, 400 MHz): δ 11.33 (s, 1H), 7.92 (s, 1H), 7.81 (d, J = 9.6 Hz, 1H), 7.75, (s, 1H), 7.42 - 7.44 (m, 3H), 7.19 - 7.32 (m, 7H), 6.87 - 6.90 (m, 4H), 5.21 (d, J = 3.2 Hz, 1H), 4.96 (dd, J = 11.4 Hz, 3.4 Hz, 1H), 4.63 (d, J = 6.4 Hz, 1H), 4.53 (d, J = 2.4 Hz, 1H), 4.48 (d, J = 8.4 Hz, 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 (M + Na) for C + Calculated value 1226.52, found value 1226.4.

[0298] Compound 457: To a solution of Compound 456 (1.86 g, 1.54 mmol) in CH2Cl2 (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 for 20 h under an argon atmosphere. The reaction mixture was diluted with CH2Cl2 (300 mL) and washed with saturated NaHCO3 (100 mL). The organic layer was separated and dried over anhydrous Na2SO4. The filtrate was concentrated and the resulting crude material was purified by silica gel column chromatography (EtOAc, then 0 - 3% MeOH in CH2Cl2), and 457 (1.80 g, 1.28 mmol, 83%, R f = 0.43) was obtained, which was developed with 10% MeOH in CH2Cl2. 1 1H NMR (400 MHz, DMSO-d6): δ 11.34 (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 - 3.60 (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 31P NMR (DMSO-d6, 162 MHz) δ 147.92, 147.70. C69 H 98 N7NaO 20 MW (M+Na) for PSi + Calculated value 1426.63, measured value 1426.5.

[0299]

Chemical formula

[0300] Compound 458: To a solution of 452 (21.5 g, 65.1 mmol) in pyridine (400 mL) were 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 h and then evaporated. The residue was extracted with EtOAc and saturated aqueous NaHCO3, dried over anhydrous Na2SO4, and purified by silica gel column chromatography (5% MeOH in CH2Cl2, R f = 0.30) to give 458 (36.2 g, 57.2 mmol, 88%). 1 H NMR (DMSO-d6, 400 MHz): δ 11.37 (s, 1H), 7.48 (s, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.27 - 7.32 (m, 6H), 7.20 - 7.23 (m, 1H), 6.87 - 6.90 (m, 4H), 5.06 (d, J = 4.8 Hz, 1H), 4.80 (d, J = 6.4 Hz, 1H), 4.54 (d, J = 2.8 Hz, 1H), 3.84 - 3.93 (m, 1H), 3.73 (s, 6H), 3.56 - 3.69 (m, 2H), 3.53 (s, 3H), 3.15 - 3.17 (m, 2H), 2.58 (t, J = 6.6 Hz, 2H). 13 C NMR (MeOH-d4, 100 MHz): δ 172.7, 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) for +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 h. Ethylenediamine was removed by evaporation and co-evaporated with toluene. The residue was extracted with CH2Cl2 / MeOH (180 mL / 20 mL) and H2O (50 mL), the organic layer was dried over anhydrous Na2SO4 and then concentrated. The crude material was crystallized from hexane and CH2Cl2 to give 459 as a pale yellow solid (11.7 g, 17.7 mmol, 80%). 1 1H NMR (MeOD-d4, 400 MHz): δ 7.57 (s, 1H), 7.21 - 7.48 (m, 9H), 6.86 - 6.88 (m, 4H), 4.71 (d, J = 3.2 Hz, 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 = 6.2 Hz, 2H), 2.63 (t, J = 6.0 Hz, 2H), 2.41 (t, J = 6.2 Hz, 2H). C 35 H 40 MW (M + H) for C18H14N4NaO9 + Calculated value: 683.27, measured 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 iPr2NEt (10.3 mL, 59.3 mmol). After 10 min, a solution of compound 459 in DMF (50 mL) was added and the mixture was stirred overnight. The reaction mixture was extracted with EtOAc and H2O, dried over anhydrous Na2SO4. After evaporation, the crude material was purified by silica gel column chromatography (10% MeOH in CH2Cl2, R f = 0.50) to give compound 460 (6.85 g, 6.28 mmol, 59%). 11H NMR (DMSO-d6, 400 MHz): δ 11.33 (s, 1H), 7.93 (s, 1H), 7.81 (d, J = 9.2 Hz, 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 = 3.2 Hz, 1H), 5.03 (d, J = 4.8 Hz, 1H), 4.96 (dd, J = 11.2 Hz, 3.6 Hz, 1H), 4.78 (d, J = 6.4 Hz, 1H), 4.51 (d, J = 2.8 Hz, 1H), 4.48 (d, J = 8.4 Hz, 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 = 6.6 Hz, 2H), 2.09 (s, 3H), 2.02 (t, J = 7.0 Hz, 2H), 1.99 (s, 3H), 1.88 (s, 3H), 1.76 (s, 3H), 1.43 - 1.49 (m, 4H). 13 13C NMR (DMSO-d6, 100 MHz): δ 172.0, 169.9, 169.8, 169.5, 169.4, 169.2, 162.6, 157.9, 150.3, 144.9, 143.2, 135.7, 135.6, 129.7, 127.7, 126.5, 113.0, 111.3, 100.9, 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 MW (M + Na) for + Calculated value: 1112.43, Measured value: 1112.2.

[0303] Compound 461: To a solution of Compound 460 (1.55 g, 1.42 mmol) in pyridine (10 mL) were 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 CH2Cl2 and saturated aqueous NaHCO3, and dried over anhydrous Na2SO4. The crude material was purified by silica gel column chromatography (5% MeOH in CH2Cl2, 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 1H NMR (DMSO-d6, 400 MHz): δ 11.32 (s, 1H), 7.94 (s, 1H), 7.82 (d, J = 9.2 Hz, 1H), 7.75, (s, 1H), 7.54 (s, 1H), 7.40 - 7.41 (m, 2H), 7.21 - 7.32 (m, 7H), 6.87 - 6.89 (m, 4H), 5.21 (d, J = 3.2 Hz, 1H), 4.96 (dd, J = 11.2 Hz, 3.6 Hz, 1H), 4.73 (d, J = 4.8 Hz, 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 = 6.6 Hz, 2H), 2.10 (s, 3H), 2.02 (t, J = 7.0 Hz, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.45 - 1.50 (m, 4H), 0.74 (s, 9H), -0.034, (s, 3H), -0.11 (s, 3H). C 60 H 81 N5NaO 19 MW (M+Na) for Si + Calculated 1226.52, Found 1227.4.

[0304] Compound 462: To a solution of Compound 461 (2.28 g, 1.89 mmol) in CH2Cl2 (60 mL) were 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% Et3N in CH2Cl2, R f = 0.44) gave Compound 462 as the corresponding triethylammonium salt (2.50 g, 1.78 mmol, 94%). 1 H NMR (DMSO-d6, 400 MHz): δ 8.42 (s, 1H), 8.18 (s, 1H), 8.05 (d, J = 9.2 Hz, 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 = 11.2 Hz, 3.6 Hz, 1H), 4.70 (d, J = 4.0 Hz, 1H), 4.60 (d, J = 8.4 Hz, 1H), 4.37 (t, J = 5.8 Hz, 1H), 4.09 - 4.13 (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 = 7.2 Hz, 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). 1313C NMR (DMSO-d6, 100 MHz): δ 173.8, 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 (M - H) 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) were successively added HBTU (30 mg, 0.077 mmol), iPr2NEt (0.061 mL, 0.35 mmol), and aminomethyl polystyrene support (ARTVISION, 70 mol / g, 1.10 g, assumed to be 0.077 mmol). The mixture was shaken for 24 h, then filtered, washed with CH2Cl2, and dried under reduced pressure. 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, it was washed with CH2Cl2 (100 mL), then 50% MeOH / CH2Cl2 (100 mL), and dried under reduced pressure to give Compound 463 (1.12 g). Loading amount: 47 mol / g.

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

[0307] [Chemistry]

[0308] [Chemistry]

[0309] [Chemistry]

[0310] [Chemistry]

[0311] [Chemistry]

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

[0313] [Chemistry]

[0314] [Chemistry]

[0315] [Chemistry]

[0316] [Chemistry]

[0317] [Chemistry]

[0318] [Chemical]

[0319] Example 26. Amino Linker for Conjugating a Ligand to an Oligonucleotide (Schemes 74 - 76) An amino linker for conjugating a ligand to an oligonucleotide can be prepared according to the following Schemes 74 - 76.

[0320] [Chemical]

[0321] [Chemical]

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

[0323] Example 28. Conjugation of GalNAc Ligand to C2 of Purine Base The GalNAc ligand can be prepared at the C2 position of the purine base as shown in the following Scheme 78. [Chemical]

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

[0325] Example 29. siRNA-Ligand Conjugate RNA Synthesis and Double-Strand Annealing

[0326] 1. Oligonucleotide Synthesis All oligonucleotides were synthesized using an AKTA oligopilot synthesizer or an ABI 394 synthesizer. Unless otherwise specified, commercially available controlled pore glass solid supports (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5'-O-dimethoxytrityl N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O-N,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N2-isobutyl-2'-t-butyldimethylsilyl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O-N,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'-O-N,N'-diisopropyl-2-cyanoethyl-phosphoramidite and 5'-O-dimethoxytrityl-2'-fluoro-uridine-3'-O-N,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 coupling / recycle time of 16 minutes was used. The activator was 5-ethylthiotetrazole (0.75 M, American International Chemicals), iodine / water / pyridine was used for PO-oxidation, and PADS (2%) in 2,6-lutidine / ACN (1:1 v / v) was used for PS-oxidation.

[0327] The chain conjugated with the ligand was synthesized using a solid support containing the corresponding ligand. For example, the introduction of the carbohydrate moiety / ligand (e.g., in the case of GalNAc) at the 3'-end of the sequence was carried out by initiating the synthesis using the corresponding carbohydrate solid support. Similarly, the cholesterol moiety at the 3'-end was introduced by initiating the synthesis on a cholesterol support. Generally, the ligand moiety was linked to trans-4-hydroxyproline via the optimal tether described in the previous example to obtain a hydroxyproline-ligand moiety. Next, the hydroxyproline-ligand moiety was either coupled to the solid support via a succinate linker or converted to a phosphoramidite under standard phosphitylation conditions to obtain the desired carbohydrate conjugate building block. The fluorophore-labeled siRNA was synthesized from the corresponding phosphoramidite or solid support (purchased from Biosearch Technologies). The oleoyl lithocholic acid (GalNAc)3 polymer support was prepared in-house at a loading of 38.6 μmol / gram. The mannose (Man)3 polymer support was also prepared in-house at a loading of 42.0 μmol / gram.

[0328] Conjugation of the desired ligand, e.g., at the 5’ end of the array, was carried out by coupling the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions, unless otherwise specified. Extended 15-minute coupling of a 0.1 M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to the solid-supported oligonucleotide. Oxidation of internucleotide phosphite to phosphate was performed using standard iodine-water or by treatment with tert-butyl hydroperoxide / acetonitrile / water (10:87:3) with a 10-minute oxidation waiting time for the conjugated oligonucleotide as reported in (1) Beaucage, S.L. (2008) Solid-phase synthesis of siRNA oligonucleotide. Curr. Opin. Drug Discov. Devel., 11, 203-216; (2) Mueller, S., Wolf, J. and Ivanov, S.A. (2004) Current Strategies for the Synthesis of RNA. Curr. Org. Synth., 1, 293-307 and (3) Xia, J., Noronha, A., Toudjarska, I., Li, F., Akinc, A., Braich, R., Frank-Kamenetsky, M., Rajeev, K.G., Egli, M. and Manoharan, M. (2006) Gene Silencing Activity of siRNAs with a Ribo-difluorotoluyl Nucleotide. ACS Chem. Biol., 1, 176-183. Phosphorothioates were introduced by oxidation of phosphite to phosphorothioate using a sulfur transfer agent such as DDTT (purchased from AM Chemicals), PADS and / or Beaucage reagent. Cholesterol phosphoramidite was synthesized in-house and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterol phosphoramidite 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 while simultaneously deprotecting the bases and phosphate groups using 80 mL of a mixture of ethanol and ammonia [ammonia:ethanol (3:1)] at 55 °C for 6.5 hours. The bottle was cooled briefly on ice and then the ethanol-ammonia mixture was filtered and placed into a new 250 mL bottle. The CPG was washed with 2 x 40 mL portions of ethanol / water (1:1 v / v). Next, the volume of the mixture was reduced to approximately 30 mL by means of a rotary evaporator (roto-vap). Next, the mixture was frozen on dry ice and dried under reduced pressure using a speed vac.

[0330] 3. Deprotection-II (Removal of 2’ TBDMS Groups) 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 minutes 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 stored in the freezer until purification.

[0331] 4. Analysis Prior to purification, the oligonucleotide was analyzed by high performance liquid chromatography (HPLC), and the choice of buffer and column was determined by the sequence and / or the nature of the ligand to be conjugated.

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

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

[0334] Example 30: Synthesis of siRNA-ligand conjugate using post-synthesis method Single-stranded oligonucleotides containing the desired amino linker were synthesized using the corresponding amino linker monomer compatible with the solid-phase oligonucleotide synthesis and deprotection conditions described in Example 30 (Scheme 79). After deprotection, the oligonucleotide with the amino group attached was reacted with the NHS ester of the ligand shown in the table under Scheme 79, followed by treatment with ammonia and HPLC purification. When each purified ligand-conjugated single-stranded oligonucleotide was annealed with an equimolar mixture of the complementary strand, the siRNA shown in Table 5 was obtained. The (1 + 1 + 1) design shown in Scheme 79 and Table 5 was obtained by successively coupling the amino linker phosphoramidite to the amino linker solid support (two synthesis cycles), and then successively coupling the nucleoside phosphoramidite monomers as described in Example 29.

[0335] Post - synthesis conjugation of ligands to oligonucleotides [Chemical formula]

[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] TIFF2025107295000130.tif162166TIFF2025107295000131.tif192166TIFF2025107295000132.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 at 37°C in an atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC), 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 duplex per well in a 96 - well plate, and incubating at room temperature for 15 minutes. Next, 80 μl of antibiotic - free complete growth medium containing approximately 2×104 Hep3B cells was added to the siRNA mixture. The cells were incubated for 24 hours 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 - fold serial dilutions at the maximum dose of 10 nM final duplex concentration.

[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, and then mixed at 850 rpm for 5 minutes using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). 10 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 captured again, 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 at 70 °C for 5 minutes. The beads were captured on the magnet for 5 minutes. 40 μl of the supernatant was removed and added to another 96 - well plate.

[0340] cDNA synthesis using ABI High - Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat#4368813) For each reaction, a master mix of 1 μl of 10x buffer, 0.4 μl of 25x dNTP, 1 μl of random primers, 0.5 μl of reverse transcriptase, 0.5 μl of RNase inhibitor, and 1.6 μl of H2O was added to 5 μl of total RNA. cDNA was generated using a Bio - Rad C - 1000 or S - 1000 thermocycler (Hercules, CA) by the following steps: 10 minutes at 25 °C, 120 minutes at 37 °C, 5 seconds at 85 °C, hold at 4 °C.

[0341] Real - time PCR 2 μl 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 on a Roche LC 480 real-time PCR machine (Roche). Unless otherwise specified, each duplex was tested in at least two independent transfections and each transfection was assayed in duplicate.

[0342] To calculate the relative fold change, real-time data were analyzed using the ΔΔCt method and normalized to assays performed on cells transfected with 10 nM of AD-1955 or mock-transfected cells. IC 50 was calculated using a four-parameter fit model with XLFit and normalized to cells transfected with a non-target specific control or un-sensitized cells over the same dose range or to its own lowest dose. IC 50 was calculated for each individual transfection and in combination, where one IC 50 was fit 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 effect of chemical modifications including the introduction of a triple repeat motif together with a GalNAc3 ligand on the silencing activity of RNAi agents targeting TTR.

[0344] IC in Hep3B cells50 Protocol for the evaluation of The IC of each modified siRNA 50 was determined in Hep3B cells by standard reverse transfection using Lipofectamine RNAiMAX. Briefly, 5 μL of Opti-MEM was added to 5 μL of siRNA duplex per well in a 96-well plate together with 10 μL of Opti-MEM and 0.5 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA, cat# 13778-150) per well to perform reverse transfection and incubated at room temperature for 15 - 20 minutes. After incubation, next, 100 μL of complete growth medium without antibiotics containing 12,000 - 15,000 Hep3B cells was added to each well. The 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 in the range of 10 nM - 0.6 pM were evaluated for the determination of IC 50 and the TTR / GAPDH of siRNA-transfected cells was normalized to cells transfected with 10 nM of Luc siRNA.

[0345] Free-uptake IC 50 Protocol for the evaluation of Free-uptake silencing in primary cynomolgus monkey or mouse hepatocytes was evaluated after incubation with TTR siRNA for 4 hours or 24 hours. Silencing was measured at the 24-hour time point from the first exposure.

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

[0347] The effectiveness of the treatment was evaluated by (i) measuring TTR mRNA in the liver at the time points 48 and 144 hours after administration, and (ii) measuring TTR protein in plasma at the time points before bleeding and 48 / 144 hours after administration. TTR liver mRNA levels were assayed using the branched DNA assay - QuantiGene 2.0 (Panomics cat#: QS0011). Briefly, mouse liver samples were homogenized to prepare tissue lysates. The liver lysis mixture (a mixture of 1 volume of lysis mixture, 2 volumes of nuclease-free water and 10 ul of proteinase-K / ml to a final concentration of 20 mg / ml) was incubated at 65 °C for 35 minutes. Next, 20 μl of the Working Probe Set (TTR probe for the gene target and GAPDH for the endogenous control) and 80 ul of the tissue lysate were added to the Capture Plate. The Capture Plate was incubated at 55 °C ± 1 °C (for about 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 centrifuging at 240 g for 1 minute. 100 μl of the pre-Amplifier Working Reagent was added to the Capture Plate sealed with aluminum foil and incubated at 55 °C ± 1 °C for 1 hour. After 1 hour of incubation, the washing step was repeated, and then 100 μl of the Amplifier Working Reagent was added. After 1 hour, the washing and drying steps were repeated, and 100 μl of the Label Probe was added. The Capture Plate was incubated at 50 °C ± 1 °C for 1 hour. Next, the plate was washed with 1X wash buffer and dried, and 100 μl of the substrate was added to the Capture Plate. After incubating the Capture Plate for 5 - 15 minutes, it was read using a SpectraMax Luminometer. The average background was subtracted from each of the three samples, the three resulting GAPDH (control probe) and TTR (experimental probe) values were averaged, and then the bDNA data were analyzed by calculating the ratio of (experimental probe - background) / (control probe - 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 1-fold mixture diluent, added to plates pre-coated with the kit standard, incubated at room temperature for 2 hours, and then washed 5 times with the kit wash buffer. 50 microliters of biotinylated prealbumin antibody was added to each well, incubated at room temperature for 1 hour, and then washed 5 times with the wash buffer. 50 microliters of streptavidin-peroxidase conjugate was added to each well, the plate was incubated at room temperature for 30 minutes, and then washed as described above. The reaction was developed by the addition of 50 μl / well of chromogenic substrate, incubated at room temperature for 10 minutes, and the reaction was stopped by the addition of 50 μl / well of stop solution. Absorbance at 450 nm was read using a Versamax microplate reader (Molecular Devices, Sunnyvale, CA), and the data were analyzed using the Softmax 4.6 software package (Molecular Devices).

[0349] Results of the efficacy of representative siRNA-conjugates are shown in Figure 1. Most of the anomeric-linked 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 single subcutaneous administration of an AT3 siRNA-ligand conjugate according to a protocol similar to that described in Example 33 for in vivo TTR gene silencing. In vitro gene silencing was evaluated according to a protocol similar to that described in Examples 31 and 32.

[0351] The 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 the AT3 protein levels were measured 72 hours after administration and compared to a PBS control. The results are shown in Figure 2. The conjugates 54944 and 56881 are referred to as 70001 and 70002, respectively, in the above and in Figure 2.

[0352] Example 35: Synthesis of Mono-GalNAc Building Blocks for Oligonucleotide Conjugation The mono-GalNAc building blocks shown can be prepared as shown in Scheme 80.

Chemical formula

[0353] Synthesis of 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyproline amine (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. TLC was examined 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, and compound 102 was obtained as a pale yellow fluffy solid (11.77 g, 63%). 11H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.2 Hz, 1H), 7.69 (t, J = 5.6 Hz, 1H), 7.39 - 7.09 (m, 9H), 6.86 (ddd, J = 9.0, 5.4, 2.1 Hz, 4H), 5.20 (d, J = 3.4 Hz, 1H), 5.03 - 4.83 (m, 2H), 4.47 (d, J = 8.5 Hz, 1H), 4.41 - 4.07 (m, 2H), 4.04 - 3.95 (m, 3H), 3.86 (dt, J = 11.2, 8.9 Hz, 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 = 6.7 Hz, 2H), 1.98 (s, 3H), 1.87 (d, J = 7.5 Hz, 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 this, DIEA (2.20 mL, 3 equivalents) and a chloroamidite reagent were added. The reaction mixture was stirred for 30 minutes and examined 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, whereby the compound was obtained as a white fluffy solid. 11H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.2 Hz, 1H), 7.68 (s, 1H), 7.42 - 7.06 (m, 8H), 7.01 - 6.73 (m, 4H), 5.20 (d, J = 3.3 Hz, 1H), 4.96 (dd, J = 11.2, 3.3 Hz, 1H), 4.63 (d, J = 4.7 Hz, 1H), 4.47 (d, J = 8.5 Hz, 1H), 4.15 (s, 1H), 4.01 (s, 3H), 3.86 (d, J = 11.0 Hz, 1H), 3.70 (d, J = 16.5 Hz, 9H), 3.45 (ddd, J = 37.0, 23.3, 16.4 Hz, 6H), 2.99 (dd, J = 12.3, 6.4 Hz, 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 31P 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, equivalent), followed by TEA (1 mL) were added. The reaction mixture was stirred overnight at room temperature. The TLC of the reaction mixture was examined 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. The polystyrene solid support was added to the reaction mixture and the mixture was shaken overnight at ambient temperature. The solid support was filtered, washed and capped using acetic anhydride / Py mixture. The solid support was washed again with dichloromethane, MeOH / DCM and ether (27.10 g, 55 μmol / g).

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

[0357]

Chemical formula

[0358]

Chemical formula

[0359]

Chemical formula

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

Chemical formula

[0361] Synthesis of Compound 5001: To a stirred solution of Compound 5000 (23.22 g, 63.6 mmol) in DCM, NaN3 (12.4 g, 190.8 mmol) and TBAHS (21.6 g, 63.6 mmol) were added, followed by 150 mL of saturated NaHCO3 solution. The resulting mixture was stirred for 14 h. Next, the reaction mixture was extracted with ethyl acetate (3 × 250 ml), washed with water and brine, and dried over anhydrous Na2SO4. Concentration of the solvent gave a crude material. This material was dissolved in ethyl acetate (150 mL), and upon addition of 150 mL of hexane, precipitation of the product as a white solid occurred. Drying the solid under reduced pressure gave Compound 5001 (16.2 g, 68.4%). LCMS for Compound 5001: Calculated: 372.33 (M + ), Found: 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 PtO2 (0.6 g), and the reaction mixture was stirred under a hydrogen atmosphere at room temperature 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) were 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 + )、Measured: 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 gave Compound 5004 (6 g, 65%). LCMS for Compound 5004: Calculated: 557.5 (M + )、Measured: 558.0 (M + +1).

[0365]

Chemical Structure

[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 h). The solvent was concentrated, and then the product was extracted with ethyl acetate (3 × 50 mL), washed with water, 10% citric acid, and brine, and dried over anhydrous Na2SO4. Concentration of the solvent gave Compound 5005 (3.8 g, 95%). C 19 H 25 N3O 12 LCMS calculated value for: 487.41 (M + ), found: 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) were added amine (0.526 g, 1.5 mmol) and triethylamine (0.4 mL). The solvent was concentrated, and then the product was extracted with ethyl acetate (3 × 50 mL), washed with water, 10% citric acid, and brine, and dried over anhydrous Na2SO4. Concentration of the solvent gave Compound 5006 (0.7 g, 93%). C 25 H 33 N3O 11 LCMS calculated value for 551.54 (M + ), found: 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) was added Pd / C (0.1 g), and the resulting mixture was stirred under a hydrogen atmosphere overnight (14 hours). The catalyst was removed by filtration through 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) were added EDC (488 mg, 2.56 mmol), NHS (730 mg, 6.35 mmol) and DIEA (0.88 mL, 5.07 mmol). The reaction mixture was stirred overnight. Concentration of the reaction mixture followed by column chromatography gave Compound 5007 (250 mg, 35%). C 22 H 30 N4O 13 LCMS calculated value for: 558.49 (M + ), found: 559.2 (M + +1), 581.1 (M + +Na + ).

[0369] Example 38: Synthesis of S- and C-linked GalNAc Derivatives and Building Blocks

Chemical Structure

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

[0371] Synthesis of Compound 5013: TFA (1 mL) was added to a solution of Compound 5012 (380 mg, 0.731 mmol) in CH2Cl2 (4 mL) at 0 °C. The mixture was stirred at 0 °C for 2 hours and then at room temperature for 3 hours. 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 (M+H) for S + Calculated value 464.1590, measured value 464.1.

[0372] Synthesis of Compound 5014: Compound 5013 (approx. 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 CH2Cl2 (5 mL) over 14 hours. Aqueous workup followed by column chromatography gave Compound 5014 (284 mg, 0.507 mmol, 69% over two steps). C 23 H 33 N2O 12 Molecular weight (M+H) for S + Calculated value 561.1754, measured value 561.1.

[0373] Compound 5010 is obtained by S-alkylation of Compound 5009 with an alkyne bromide.

[0374]

Chemical Structure

[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 CH2Cl2 (30 mL) in the presence of EDCI (1.19 g, 6.22 mmol) and DIEA (2.70 mL, 15.5 mmol) over 14 hours. After aqueous workup followed by column chromatography, Compound 5018 (1.83 g, 3.26 mmol, 63%) was obtained. C 23 H 33 N2O 12 Molecular weight (M + H) for S + Calculated value 561.1754, found value 561.2.

[0377]

Chemical Structure

[0378] Compound 5024 was prepared using the 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 CH2Cl2 (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%). C 27 H 36 NO 10 Molecular weight (M + H) for + Calculated value 534.2339, found 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 a hydrogen atmosphere for 14 h. 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%). C 20 H 32 NO 10 Molecular weight (M + H) for + Calculated 446.2026, found 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 CH2Cl2 (5 mL) for 14 h. After aqueous workup followed by column chromatography, Compound 5031 (300 mg, 0.553 mmol, 76%) was obtained. C 24 H 35 N2O 12 Molecular weight (M + H) for + Calculated 543.2190, found 543.2.

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

[0383]

Chemical Structure

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

[0385] Example 39

Chemical Structure

[0386] Synthesis of Compound 5050: N - Acetylglucosamine (10 g) was refluxed with excess hexanol in the presence of BF3·Et2O to obtain Compound 5050.

[0387] Synthesis of Compound 5052: Compound 5050 was treated with pivaloyl chloride as reported in the literature. This pivaloyl ester was treated with trifluoromethanesulfonic anhydride and then with water under reflux to obtain Compound 5052.

[0388] Synthesis of Compound 5055: Compound 5052 was first treated with sodium hydroxide to remove the pivaloyl ester, and then the resulting trihydroxyl derivative was treated with benzoic anhydride to obtain Compound 5054. Oxidation of the double bond afterwards 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 CH2Cl2 (5 mL) over 14 hours. Aqueous workup followed by column chromatography gave Compound 5056 (400 mg, 0.553 mmol, 76%). C 38 H 38 N2O 13 Regarding the molecular weight (M + H) + Calculated value 730.24, measured value 730.25.

[0390] Example 40: Synthesis of a Novel GalNAc Conjugate Using a Post-Synthesis Method

[0391]

Chemical Structure

[0392]

Chemical Structure

[0393] The GalNAc groups in the following table were conjugated to siRNA by the procedures described in Scheme 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 were tested in mice as described in Example 33. The results are shown in Figure 1.

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

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

[0398] Example 43: siRNA-ligand conjugate The siRNA-ligand conjugates in the following table were prepared. The siRNA in each conjugate was the same and targeted AT3. The following ligands were conjugated 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 conjugated to the sense strand at the position shown 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 Ki values of these conjugates are reported below. [Table 10]

[0401] Example 45: siRNA (triple-stranded derivative) with different GalNAc 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 left side of the ligand designation indicates the binding site to the 3'-end of the sense strand.

Table 11

[0402] Figure 10 shows the in vivo efficacy of the 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: siRNAs 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] siRNA conjugates directed against AT3 (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 the triple-stranded GalNAc ligands 54944, 56881, and 58137 [(1+1+1) design].

Table 13

[0405] siRNA conjugates targeting 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 [Chemical formula]

[0407] Synthesis of Compound 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyproline amine (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, and Compound 102 was obtained as a pale yellow fluffy solid (11.77 g, 63%). 11H NMR (400 MHz, DMSO-d6): δ 7.80 (d, J = 9.2 Hz, 1H), 7.69 (t, J = 5.6 Hz, 1H), 7.39 - 7.09 (m, 9H), 6.86 (ddd, J = 9.0, 5.4, 2.1 Hz, 4H), 5.20 (d, J = 3.4 Hz, 1H), 5.03 - 4.83 (m, 2H), 4.47 (d, J = 8.5 Hz, 1H), 4.41 - 4.07 (m, 2H), 4.04 - 3.95 (m, 3H), 3.86 (dt, J = 11.2, 8.9 Hz, 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 = 6.7 Hz, 2H), 1.98 (s, 3H), 1.87 (d, J = 7.5 Hz, 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-cyanoethyl diisopropyl chlorophosphoramidite 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, and the compound was obtained as a white fluffy solid. 11H NMR (400 MHz, DMSO-d6): δ 7.80 (d, J = 9.2 Hz, 1H), 7.68 (s, 1H), 7.42 - 7.06 (m, 8H), 7.01 - 6.73 (m, 4H), 5.20 (d, J = 3.3 Hz, 1H), 4.96 (dd, J = 11.2, 3.3 Hz, 1H), 4.63 (d, J = 4.7 Hz, 1H), 4.47 (d, J = 8.5 Hz, 1H), 4.15 (s, 1H), 4.01 (s, 3H), 3.86 (d, J = 11.0 Hz, 1H), 3.70 (d, J = 16.5 Hz, 9H), 3.45 (ddd, J = 37.0, 23.3, 16.4 Hz, 6H), 2.99 (dd, J = 12.3, 6.4 Hz, 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 31P NMR: δ 151.78, 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), DMAP (0.532 g, equivalent), and then TEA (1 mL) were added. The reaction mixture was stirred at room temperature overnight. 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. The 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 an Amino Linker for Post-Synthesis Conjugation [Chemical formula]

[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 under argon at 0 °C for 20 minutes, 4-Hydroxy-L-proline methyl ester hydrochloride (20.0 g, 110 mmol) was added and stirring was continued under argon at room temperature overnight. 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 2% methanol / dichloromethane, 24.30 g) was obtained. Compound 101 was purified by column chromatography by eluting first with 2% methanol / dichloromethane and then with 5% methanol / dichloromethane to remove impurities, and 21.36 g (65%) of the product was obtained. 1H NMR (400 MHz, DMSO-d6): δ 7.35 (m, 5H), 5.15 (d, OH, exchangeable with D2O), 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 under argon at 0 °C for 20 minutes. Next, lithium borohydride (1.19 g, 54.43 mmol) was added to the solution at 0 °C over 20 minutes, and stirring was continued under argon at room temperature overnight. The reaction mixture was cooled to 0 °C. The 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 3% methanol / dichloromethane, 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, and 9.21 g (49%) of the product was obtained. 1 H NMR (400 MHz, DMSO-d6): δ 7.35 (m, 5H), 4.99 (s, 2H), 4.91 (d, OH, exchangeable with D2O), 4.77 (t, OH, exchangeable with D2O), 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 co-evaporated twice with anhydrous pyridine (80 mL). Next, the compound was dried under high vacuum overnight. Compound 102 was removed from 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 under argon at 0 °C for 30 minutes. Next, DMT-Cl (9.0 g, 26.53 mmol) was added to the solution at 0 °C. The mixture was stirred under reduced pressure and then under argon, and stirring was continued under argon at room temperature overnight. 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. Crude compound 103 (Rf = 0.6 in 100% EtOAc, 14.02 g) was obtained. To remove impurities, compound 103 was purified by column chromatography by eluting first with 50% ethyl acetate in hexane (1% TEA), followed by 100% ethyl acetate (1% TEA), and 12.36 g (73.4%) of the product was obtained as a white foamy solid. 1 1H NMR (400 MHz, DMSO-d6): δ 7.17 - 7.33 (m, 14H), 4.99 (s, 2H), 4.91 (d, OH, exchangeable with D2O), 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. 10 wt% palladium supported on wet activated carbon Degussa type (1.3 g) was added to the reaction mixture. The flask was repurged 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 continuously stirred under hydrogen at room temperature overnight. The reaction mixture was decanted onto a sintered funnel filled with celite and washed twice with methanol. Evaporation of the organic layer to dryness gave Compound 104 (10% MeOH in eluent DCM, 9.16 g, 93%) as a white solid, which did not require further purification. 1 H NMR (400 MHz, DMSO-d6): δ 7.15 - 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) 3.29 (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 under argon at 10 °C for 10 minutes. Triethylamine (4.80 mL, 34.4 mmol) was added dropwise to the reaction mixture, and the mixture was continuously stirred under argon at 10 °C for 20 minutes. Ethyl trifluoroacetate (3.05 mL, 25.8 mmol) was added dropwise to the reaction mixture, and the mixture was continuously stirred under argon at 10 °C for 10 minutes. The reaction mixture was continuously stirred under argon at room temperature overnight. 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 (400 MHz, DMSO-d6): δ 7.56 - 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, 6H), 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. Next, succinic anhydride (2.80 g, 27.86 mmol) was added, and the mixture was continuously stirred under argon at room temperature overnight. 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 and did not require further purification. MS: 727.2 (-H), 763.2 (+Cl). The succinate thus obtained (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. Long-chain aminoalkyl controlled pore glass support (CPG) (27 g) was added to the flask, and the mixture was continuously 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 in dichloromethane, and further washed with 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 reduced pressure 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 under argon for 5 minutes. Next, 2-cyanoethyl diisopropyl chlorophosphoramidite (5.63 mL, 16.26 mmol) was added to the reaction mixture. The reaction mixture was continuously stirred under argon at room temperature for 30 minutes. 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, giving the 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 1H NMR (400 MHz, DMSO-d6): δ 9.37 (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 31P (DMSO-d6): 151.68 (d, 1P).

[0418]

Chemical Structure

[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, and it was stirred at room temperature overnight. The reaction mixture was evaporated to dryness, and the residue was dissolved in ethyl acetate (120 mL). Next, it was 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 in 15% MeOH in DCM) was purified by silica gel column chromatography using 10% methanol in dichloromethane as the eluent, and compound 118 (4.98 g, 78%) was obtained as a white foam. m / z: 324.13 (+Na). 1H NMR (400 MHz, DMSO-d6): δ 9.39 (s, 2NH, exchangeable with D2O), 3.34 - 3.06 (m, 7H), 3.06 - 2.91 (m, 2OH, exchangeable with D2O), 2.30 - 2.00 (m, 2H), 1.97 - 0.86 (m, 6H). 13 13C NMR (101 MHz, CD3CN): δ 172.18 (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 co-evaporated twice with anhydrous pyridine (30 mL) and then dried under high vacuum overnight. Next, it was dissolved in anhydrous pyridine (90 mL). To this solution, a catalytic amount of DMAP (0.15 g, 1.23 mmol) was added and the mixture was stirred under argon at 0 °C for 30 minutes. 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. Next, the volatiles were 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 (400 MHz, DMSO-d6): δ 9.38 (s, 2NH, exchangeable with D2O), 7.31 - 7.13 (m, 7H), 6.86 (d, J = 8.9 Hz, 6H), 4.60 (t, J = 5.2 Hz, 1H), 3.72 (s, 6H), 3.54 - 3.23 (m, 6H), 2.95 (ddd, J = 36.9, 8.8, 5.8 Hz, 1OH, exchangeable with D2O), 2.08 (t, J = 7.4 Hz, 2H), 1.47 (tt, J = 14.6, 7.4 Hz, 2H), 1.34 - 1.12 (m, 4H). 1313C NMR (101 MHz, CD3CN): δ 172.83 (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 = 4.0 Hz), 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 = 21.0 Hz), 40.72 (s), 40.68 (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 under argon for 5 minutes. Next, succinic anhydride (340 mg, 3.4 mmol) was added, and the mixture was stirred continuously under argon at room temperature overnight. The reaction mixture was diluted with dichloromethane (100 mL) and washed with saturated sodium chloride (2 × 25 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. 1.01 g (98%) of the crude compound (Rf = 0.3 50% EtOAc in Hex) was obtained as a white solid and used in the next reaction without further purification. MS: 702.3 (-H), 737.5 (+Cl). 1 1H NMR (400 MHz, DMSO-d6): δ 9.42 (t, J = 5.2 Hz, 2NH, exchangeable with D2O), 7.87 (s, 1OH, exchangeable with D2O), 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). Diisopropylethylamine (1.15 mL, 5.6 mmol) and HBTU (1.26 g, 2.8 mmol) were added to this solution. The reaction mixture was rotated until all the 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 packed 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 dried overnight under high vacuum to obtain Compound 120 (14 g, 77 μmol / g).

[0423]

Chem.

[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 and it was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in ethyl acetate (120 mL). Next, it was 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 as the eluent, followed by 100% ethyl acetate to obtain Compound 122 (3.20 g, 87%) as a yellow foam. m / z: 315.1 (+H). 11H NMR (400 MHz, DMSO-d6): δ 8.76 (t, 2NH, exchangeable with D2O), 3.93 - 3.21 (m, 1H), 3.21 - 3.01 (m, 2OH, exchangeable with D2O), 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 13C NMR (101 MHz, DMSO-d6): δ 176.1 (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 co-evaporated twice with anhydrous pyridine (15 mL) and dried under high vacuum overnight. Next, it was dissolved in anhydrous pyridine (90 mL). To this solution was added a catalytic amount of DMAP (0.12 g, 0.96 mmol), and the mixture was stirred under argon at 0 °C for 30 minutes. 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. Next, the volatile substances were 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). 11H NMR (400 MHz, DMSO-d6): δ 8.76 (t, 2NH, exchangeable with D2O), 7.87 - 6.93 (m, 7H), 6.93 - 6.63 (m, 6H), 4.19 - 3.63 (m, 10H), 3.61 (m, 1OH, exchangeable with D2O), 2.74 - 1.98 (m, 4H), 1.71 - 1.10 (m, 6H), 1.08 - 0.76 (m, 3H). 13 13C NMR (101 MHz, DMSO-d6): δ 172.12 (d, J = 9.4 Hz), 157.88 (d, J = 15.4 Hz), 145.13 (s), 140.22 (s), 135.86 (d, J = 6.4 Hz), 131.07 - 130.03 (m), 130.03 - 129.28 (m), 128.90 (s), 127.66 (d, J = 7.1 Hz), 127.38 (s), 126.45 (d, J = 9.7 Hz), 112.89 (d, J = 30.1 Hz), 85.08 (s), 65.00 (s), 63.22 (d, J = 48.5 Hz), 54.97 (s), 53.72 (s), 45.56 (s), 40.02 (d, J = 21.0 Hz), 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 = 13.6 Hz), 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 under argon for 5 minutes. Next, succinic anhydride (230 mg, 2.0 mmol) was added, and stirring was continued under argon at room temperature overnight. The reaction mixture was diluted with dichloromethane (50 mL) and washed with saturated sodium chloride (2 × 25 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. Compound 124 (Rf = 0.6 in 25% EtOAc in hexane) 760 mg (99%) was obtained as a gray foam and 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 rotated until all the 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 dried overnight under high vacuum to give Compound 125 (10.2 g, 0.71 μmol / g).

[0428] [Chemical formula] 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 under argon at 0 °C for 30 minutes. Next, DMTr-Cl (18 g, 53.0 mmol) was added to the solution at 0 °C. The mixture was stirred under reduced pressure and then under argon, and stirring was continued under argon at room temperature for 4 hours. The reaction mixture was washed twice with water and then with 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 hexane) (18.9 g, 95%) as a yellow oil. 11H NMR (400 MHz, DMSO-d6): δ 7.42 - 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, 14.7 Hz, 2H), 2.49 (dd, J = 3.5, 1.7 Hz, 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, meta-chloroperbenzoic acid (16 g, 31.25 mmol) was added and the mixture was stirred at room temperature overnight. The reaction mixture was quenched by the addition of sodium bisulfite (500 mg) and stirred 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 in 5% EtOAc in hexane) (5.06 g, 97.3%) as a yellow solid, which did not require further purification. 1 1H NMR (400 MHz, DMSO-d6): δ 7.48 - 7.08 (m, 7H), 6.88 (t, J = 5.9 Hz, 6H), 3.74 (d, J = 19.4 Hz, 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 was added to the reaction mixture and it was stirred at 85 °C in an oil bath in a pressure vessel overnight. The reaction mixture was evaporated to dryness and then co-evaporated twice with toluene (10 mL). Next, the compound was co-evaporated with dichloromethane (50 mL) to give crude Compound 129 (Rf = 0.1 in 25% EtOAc in hexane, 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 under argon at 10 °C for 10 minutes. Triethylamine (4.0 mL, 19.1 mmol) was added dropwise to the reaction mixture, and the mixture was continuously stirred under argon at 10 °C for 20 minutes. Ethyl trifluoroacetate (5.0 mL, 28.7 mmol) was added dropwise to the reaction mixture at 10 °C. The reaction mixture was continuously stirred under argon at room temperature overnight. Next, the reaction mixture was washed twice with water and then with saturated sodium chloride. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness, and crude compound 130 (Rf = 0.43, 50% EtOAc / Hex, 6.12 g) was obtained. To remove impurities, first 5% ethyl acetate in hexane (1% TEA), and then 10% ethyl acetate in hexane (1% TEA) to elute the product from further impurities, purification of compound 130 by silica gel column chromatography gave 1.51 g (30% from compound 129) as a white foam. 1 1H NMR (400 MHz, DMSO-d6): δ 7.69 - 7.12 (m, 7H, 1NH, exchangeable with D2O), 7.10 - 6.70 (m, 6H), 4.12 - 3.48 (m, 9H, 1OH, exchangeable with D2O), 3.29 (dd, J = 19.7, 12.9 Hz, 2H), 2.1.57 - 1.17 (m, 6H). 13 13C NMR (101 MHz, DMSO-d6): δ 157.97 (s), 156.56 (s), 156.20 (s), 145.27 (s), 136.08 (s), 129.58 (s), 127.69 (d, J = 7.7 Hz), 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. Next, succinic anhydride (600 mg, 5.6 mmol) was added, and the mixture was continuously stirred under argon at room temperature overnight. The reaction mixture was diluted with dichloromethane (100 mL) and then washed twice with 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 in 25% EtOAc in hexane) (1.77 g, 99%) was obtained as a white foam and 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 rotated until all the 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, washed with 1% triethylamine / dichloromethane, followed by two washes with 10% methanol in dichloromethane, and further washed with 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. Next, it was 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 in the same manner 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] [Chemical formula]

[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). Triethylamine (14 mL, 151.5 mmol) was added to this solution. The reaction mixture was stirred under argon for 10 minutes. Next, methanesulfonyl chloride (4.25 mL, 50.5 mmol) was added dropwise to the solution over 20 minutes. Stirring was continued under argon at room temperature for 2 days. The reaction mixture was decanted into ice water and then washed with 5 × 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, first eluting with dichloromethane and then with 2% methanol / dichloromethane to remove impurities, gave 4.6 g (72%) of a clear liquid. 1 1H NMR (400 MHz, DMSO-d6): δ 5.78 (ddt, J = 16.9, 10.2, 6.6 Hz, 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. NaN3 (10 g) was added to this mixture and the mixture was heated at 80 °C overnight. The solid was then removed by filtration. The volatile substances 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 mixture 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 stirred 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 co-evaporated twice with anhydrous pyridine (10 mL). Next, the compound was dried under high vacuum overnight. Next, the compound was removed from high vacuum and dissolved in anhydrous pyridine (60 mL). A catalytic amount of DMAP (0.11 g, 0.94 mmol) was added to this solution. The reaction mixture was stirred under argon at 0 °C for 30 minutes. Next, DMT-Cl (3.35 g, 10.06 mmol) was added to the solution at 0 °C. The mixture was stirred under reduced pressure and then under argon, and stirring was continued at room temperature for 1.5 hours under argon. The reaction mixture was evaporated to dryness and dichloromethane (100 mL) was added to the residue. The organic layer was washed twice with water and then with saturated brine. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness to give crude Compound 137 (Rf = 0.8 in 1:1 EtOAc / hexane, 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. Next, water (1 mL) was added and the mixture was stirred at room temperature overnight. When the completion of the reaction was 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 under argon at 10 °C for 10 minutes. Triethylamine (1.5 mL, 10.75 mmol) was added dropwise to the reaction mixture and the mixture was stirred under argon at 10 °C for 20 minutes. Ethyl trifluoroacetate (6.5 mL, 54.0 mmol) was added dropwise to the reaction mixture and the mixture was stirred under argon at 10 °C for 10 minutes. The reaction mixture was stirred under argon at room temperature overnight. 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, first eluting 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 (400 MHz, DMSO-d6): δ 9.36 (s, 1NH, exchangeable with D2O), 7.47 - 7.14 (m, 7H), 6.87 (d, J = 8.8 Hz, 6H), 3.86 - 3.48 (m, 7H), 3.33 (s, 1OH, exchangeable with D2O), 3.13 (dd, J = 12.1, 6.4 Hz, 4H), 1.54 - 1.06 (m, 6H). 19 F NMR (376 MHz, DMSO-d6): δ -82.54 (s). 1313C NMR (101 MHz, DMSO-d6): δ 161.42 (s), 159.73 (s), 159.37 (s), 148.65 (s), 139.41 (d, J = 6.7 Hz), 136.65 (s), 135.74 - 135.30 (m), 134.92 (d, J = 9.8 Hz), 133.16 (s), 132.18 (d, J = 11.8 Hz), 131.18 (d, J = 6.0 Hz), 129.96 (s), 116.51 (s), 88.50 (s), 72.39 (s), 71.10 (s), 63.20 (s), 58.43 (s), 43.58 (s), 43.37 (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. Next, succinic anhydride (380 mg, 3.8 mmol) was added and the mixture was stirred continuously under argon at room temperature overnight. 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. 1.18 g (99%) of Compound 139 (eluent 1:1 EtOAc / hexane) was obtained as a pink oil and did not require further purification.

[0441] Compound 140: Compound 139 (1.18 g, 1.9 mmol) was dissolved in acetonitrile (60 mL). Diisopropylethylamine (1.3 mL, 7.6 mmol) and HBTU (1.43 g, 3.8 mmol) were added to the solution. The reaction mixture was rotated until all the contents were dissolved. CPG (12 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 in dichloromethane, followed by two washes with 10% methanol / dichloromethane, and further washed with 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 thus obtained 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]

Chemical Structure

[0443] Compound 142: Triethylamine (5 mL, 36.5 mmol) was added to a stirred solution of (R)-glycidol (2.3 g, 31 mmol) in DCM, and then a 1 M solution of DMTrCl (10.66 g, 31.5 mmol) in DCM was added at room temperature. The reaction was stirred until the starting material indicated by TLC disappeared. A few drops of MeOH were added to hydrolyze the unreacted DMTrCl, and the mixture was stirred for 10 minutes. The product was washed with H2O, brine, and dried over Na2SO4. The product was purified by column chromatography using a gradient of hexane / EtOAc (9:1), and 6 g (52%) of pure product 142 was obtained. 11H NMR (400 MHz, DMSO-d6): δ 7.38 (d, J = 7.6 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.27 - 7.12 (m, 5H), 6.87 (d, J = 6.1 Hz, 4H), 3.72 (s, 6H), 3.24 (dd, J = 10.9, 2.4 Hz, 1H), 3.15 - 3.08 (m, 1H), 2.86 (dd, J = 10.9, 6.0 Hz, 1H), 2.70 (t, J = 4.6 Hz, 1H), 2.54 (m, 1H). 13 13C NMR (101 MHz, DMSO-d6): δ 158.07, 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 compound 142 was consumed. 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 1H NMR (500 MHz, DMSO-d6): δ 7.40 (d, J = 7.6 Hz, 2H), 7.35 - 7.14 (m, 7H), 6.88 (d, J = 8.5 Hz, 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, 7.6 Hz, 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). 1313C NMR (DMSO-d6): δ 158.03, 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. Compound 144 was added to this solution. 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 H2O, saturated NaHCO3, and brine, dried over Na2SO4, and then purified by column chromatography using a gradient of EtOAc (1% NEt3):MeOH (15%) to give 5.35 g (36%) of pure compound 146. 1H NMR (400 MHz, DMSO-d6): δ 8.08 - 7.85 (m, 4H), 7.82 - 7.11 (m, 20H), 6.86 (d, J = 8.8 Hz, 4H), 5.76 (d, J = 3.3 Hz, 1H), 5.36 (dt, J = 26.7, 13.3 Hz, 1H), 4.75 (d, J = 8.5 Hz, 1H), 4.61 - 4.40 (m, 3H), 4.32 (ddd, J = 25.2, 16.9, 9.1 Hz, 2H), 3.88 - 3.60 (m, 1H), 3.71 (s, 6H), 3.63 - 3.45 (m, 1H), 3.18 (dd, J = 14.6, 7.4 Hz, 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). 1313C NMR (DMSO-d6): δ 171.45, 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, 129.57, 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, 67.72, 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 H2O and then brine and dried over Na2SO4 to give 850 mg (69%) of compound 147. 11H NMR (500 MHz, DMSO-d6): δ 8.11 (dd, J = 15.3, 9.3 Hz, 1H), 7.98 - 7.84 (m, 4H), 7.73 - 7.16 (m, 19H), 6.87 (d, J = 8.7 Hz, 4H), 5.74 (d, J = 3.1 Hz, 1H), 5.36 (dd, J = 11.1, 2.6 Hz, 1H), 5.01 (s, 1H), 4.76 (dd, J = 8.5, 2.6 Hz, 1H), 4.45 (m, 2H), 4.37 - 4.23 (m, 2H), 3.71 (s, 6H), 3.54 (dd, J = 13.7, 10.4 Hz, 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 13C NMR (126 MHz, DMSO-d6): δ 173.83, 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. Next, the solid was 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 obtain 5.2 g (filling amount of 70.4 μmol / g) of Compound 148.

[0448] Compound 149: Compound 146 (2.3 g, 2.025 mmol) was azeotroped (three times) in pyridine to ensure complete removal of all moisture and was kept under argon from this point forward. 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-cyanoethyl diisopropyl chlorophosphoramidite (1 g, 4.2 mmol) was added. The mixture was stirred until TLC indicated the disappearance of all starting materials. The reaction mixture was concentrated, dissolved in a solution of EtOAc:NEt3 (1%):DCM (20%), and passed through a flash filtration column of silica gel using EtOAc:NEt3 (3%):DCM (25%) to remove all salts by filtration. 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 obtain 2.2 g (79%) of Compound 149. 311P NMR (162 MHz, CD3CN) δ 148.03, 147.94. 1H NMR (400 MHz, CD3CN): δ 8.00 - 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 = 3.0 Hz, 1H), 5.45 - 5.37 (m, 1H), 4.80 (d, J = 8.6 Hz, 1H), 4.51 (ddd, J = 10.7, 6.4, 2.3 Hz, 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, 2.5 Hz, 3H), 1.77 - 1.73 (m, 3H), 1.64 - 1.56 (m, 4H), 1.36 - 1.10 (m, 16H), 1.09 (d, J = 6.8 Hz, 2H), 0.99 (d, J = 6.2 Hz, 1H). 1313C NMR (101 MHz, CD3CN): δ 173.16, 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

Chemical Structure

[0450] 3,4 - Di - acetyl - 6 - mesyl GalNAc ester 2: To a solution of 6 - hydroxy derivative 1 (2.00 g, 4.3 mmol) in anhydrous DCM (20 mL) was successively added DIEA (1.2 mL, 6.5 mmol) and mesyl chloride (0.5 mL, 6.5 mmol) under an Ar atmosphere. The mixture was stirred at room temperature overnight and then quenched by the addition of 5% aqueous NaCl solution (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 foamy 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 (M - t - Bu); (-) mode: 598 (M + AcOH). 1 H 1 NMR (400 MHz), DMSO - d6, J (Hz): 1.38 (s, 9H); 1.47 (m, 4H); 1.76 (s, 3H); 1.88 (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 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 (M - t - Bu); (-) mode: 490 (M + Cl). H 1NMR (400 MHz), 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), and then methanesulfonic acid (2 drops) was added. 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, evaporated under reduced pressure, co-evaporated 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 (M - t-Bu); (-) mode: 530 (M + Cl), 554 (M + AcOH); H 1 NMR (400 MHz), 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 for 23 hours under an argon atmosphere. 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, J1 = 6.2, J2 = 10.0); 3.64 (m, 2H); 3.99 (m, 1H); 4.03 (dd, 1H, J1 = 2.0, J2 = 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 and saturated NaCl, dried over anhydrous sodium sulfate, and evaporated. The residue was chromatographed on a silica gel column using a gradient of MeOH in AcOEt (0 - 30%), yielding 63 mg (18%) of compound 5b. MS (in AcOEt): (+) mode: 574 (M); (-) mode: 608 (M + Cl), 532 (M + AcOH). 1 H NMR (400 MHz), ACN-d3, J (Hz): 1.31 (s, 3H); 1.40 (s, 9H); 1.47 (m, 4H); 1.50 (s, 3H); 1.85 (s, 3H); 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. The methanol was removed under reduced pressure, and the residue was co-evaporated three times with 3 mL of pyridine, giving 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 (M-H + ). 1 H NMR (400 MHz), 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 (M-H + ). H 1 NMR (400 MHz), 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, J1 = 1.4, J2 = 2.5, J3 = 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

Chem.

[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 (400 MHz), 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 (s broad, 1H); 6.40 (d, 1H, J = 9.4); 6.81 (s broad, 1H); 7.49 (s broad, 1H).

[0458] Deprotected Histamine Derivative 9: A solution of 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 at 65 °C overnight, filtered, evaporated, and the residue was co-evaporated three times with pyridine (3 mL) and once with ACN (5 mL) and dried under high vacuum, whereupon 50 mg (quantitative) of compound 9 was obtained as a very hygroscopic white solid. MS (in MeOH): (-) mode: 457 (M-H). 1 H NMR (400 MHz), 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

Chemical Structure

[0460] Synthesis of Compound 102: Compound 101 (5 g, 22.6 mmol) was heated at 100 °C for 16 h with 5-hexen-1-ol (80 mL) and boron trifluoride diethyl etherate (0.5 mL). Trituration with Et2O gave compound 102 (4.0 g, 13.2 mmol, 58%). C 14 H 26 Molecular weight (M + H) for C +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 Ac2O (10 mL). After aqueous workup and purification by silica gel column, compound 103 (1.48 g, 3.45 mmol, 70%) was obtained. C 20 H 32 Molecular weight (M+H) for C + 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 derived from Candida rugosa (3.43 g) in dioxane (13.7 mL) and potassium hydrogen phthalate buffer (54.8 mL, pH = 4) over 64 hours. After aqueous workup and purification by silica gel column, compound 104 (680 mg, 1.76 mmol, 55%) was obtained. 1 H NMR (400 MHz, CDCl3) δ 5.85 - 5.75 (m, 1H), 5.59 (d, J = 9.7 Hz, 1H), 5.33 - 5.32 (m, 1H), 5.21 (dd, J = 11.3 Hz, 3.2 Hz, 1H), 5.06 - 4.96 (m, 2H), 4.86 (d, J = 3.7 Hz, 1H), 4.63 - 4.57 (m, 1H), 4.01 (td, J = 6.5 Hz, 1.2 Hz, 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) over 18 hours. After aqueous workup and purification by silica gel column, Compound 105 (752 mg, quantitative) was obtained. 1 H NMR (400 MHz, CDCl3) δ 5.85 - 5.75 (m, 1H), 5.56 (d, J = 9.7 Hz, 1H), 5.30 (d, J = 2.7 Hz, 1H), 5.15 (dd, J = 11.3, 3.3 Hz, 1H), 5.05 - 4.97 (m, 2H), 4.88 (d, J = 3.7 Hz, 1H), 4.60 - 4.54 (m, 1H), 4.06 (dd, J = 8.8, 3.6 Hz, 1H), 3.77 - 3.71 (m, 1H), 3.49 - 3.40 (m, 2H), 3.13 (dd, J = 12.8, 4.1 Hz, 1H), 2.17 (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

Chemical Structure

[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 CH2Cl2 (5 mL), CH3CN (5 mL), and H2O (7 mL) over 18 hours. After aqueous workup and purification by silica gel column, Compound 106 (677 mg, 1.57 mmol, 89%) was obtained. C 17 H 27 Molecular weight (M + H) for C + 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 CH2Cl2 (3 mL) for 18 h. After aqueous workup and purification by silica gel column, Compound 107 (183 mg, 0.347 mmol, 75%) was obtained. C 21 H 30 N5O 11 Molecular weight (M+H) for + Calculated value 528.1942, found 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 CuSO4·5H2O (1.3 mg, 0.00527 mmol) in H2O (0.1 mL) and a solution of sodium ascorbate (10 mg, 0.0527 mmol) in H2O (0.1 mL). The reaction mixture was stirred at room temperature for 18 h. After aqueous workup and purification by silica gel column, Compound 108 (264 mg, 0.469 mmol, 89%) was obtained. C 26 H 35 N4O 10 Molecular weight (M+H) for + Calculated value 563.2353, found 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 CH2Cl2 (4 mL) for 18 h. After aqueous workup and purification by silica gel column, Compound 109 (100 mg, 0.152 mmol, 37%) was obtained. C30 H 38 N5O 12 Molecular weight (M+H) of + 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) was added a solution of THPTA (11.2 mg, 0.0259 mmol) and CuSO4·5H2O (1.3 mg, 0.00518 mmol) in H2O (0.1 mL) and a solution of sodium ascorbate (10.3 mg, 0.0518 mmol) in H2O (0.1 mL). The reaction mixture was stirred at room temperature for 18 h. After aqueous workup and purification by silica gel column, compound 110 (320 mg, 0.510 mmol, 98%) was obtained. C 30 H 38 N5O 10 Molecular weight (M+H) of + 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 CH2Cl2 (4 mL) over 18 h. After aqueous workup and purification by silica gel column, compound 111 (155 mg, 0.207 mmol, 42%) was obtained. C 34 H 41 N6O 12 Molecular weight (M+H) of + Calculated value 725.2782, measured value 725.1.

[0471] Synthesis of Compound 112: When Compound 106 (240 mg, 0.558 mmol) was treated in MeOH (9 mL) and Et3N (1 mL) over 5 days, Compound 112 (250 mg, 0.558 mmol, quantitative) was obtained. C 13 H 23 Molecular weight (M+H) for N4O7 + Calculated value: 347.1567, Measured value: 347.1.

[0472] Example 53

Chemical Structure

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

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

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

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

[0477] Synthesis of Compound 125: Using Compound 120 instead of Compound 106 and following a procedure similar to that described for Compound 108, Compound 125 (323 mg, 0.54 mmol, 83%) was obtained. C 29 H 41 N4O 10 Molecular weight (M+H) for + 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 yielded Compound 126 (241 mg, 0.438 mmol, 92%). C 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 yielded Compound 127 (318 mg, 0.530 mmol, 85%). C 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) over 18 hours. After removing the solvent, purification by silica gel column gave Compound 128 (281 mg, 0.504 mmol, 93%). C 26 H 32 Molecular weight (M+H) for C + Calculated value 558.2200, found value 558.2.

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

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

[0483] Example 54

Chemical Structure

[0484] The NHS ester compounds 131, 132, 133, 134 and 135 are prepared from the compounds 126, 127, 128, 129 and 130 respectively, by a standard esterification process using N-hydroxysuccinimide. The fully deprotected GalNAc derivatives 136, 137, 138, 139 and 140 are prepared from the compounds 126, 127, 128, 129 and 130 by treatment with Et3N / MeOH.

[0485] Example 55 [Chemical formula]

[0486] Synthesis of 201: Compound 200 (20 g, 44.74 mmol) was stirred in dichloromethane (150 mL). Next, EDAC (12.8 g, 65 mmol), DMAP (2 g, catalyst), and t-butanol (20 mL) were added. The mixture was stirred at room temperature for 2 days. Next, the solvent was removed under reduced pressure. The residue was extracted with dichloromethane (3 × 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%). C 23 H 37 NO 11 Calculated molecular weight for 503.24, found 526.23 (M+Na).

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

[0488] Synthesis of compound 203: Using the same procedure as described for the synthesis of compound 105, compound 203 was synthesized (5.2 g, 64%). C 21 H 34Calculated molecular weight of N4O9: 486.23, measured value: 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, and Compound 204 (4.6 g, 82%) was obtained. C 17 H 26 Calculated molecular weight of N4O9: 430.17, measured value: 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 equiv), 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 obtain Compound 205.

[0491] Synthesis of Compound 206: Compound 204 (1.00 g, 2.32 mmol) was stirred in a methanol / water mixture (2:1). Next, 1-ethynyl-3-methoxybenzene (204A) (0.367 g, 2.7 mmol), CuSO4·xH2O (0.050 g, catalytic amount), and sodium ascorbate (0.25 g, 1 mmol) were added, and the mixture was stirred overnight. The solvent was removed, and the residue was dissolved in dichloromethane and then washed with water, brine, and dried over sodium sulfate. The crude product was purified by silica gel chromatography to obtain Compound 206. C 26 H 34 N4O 10 Calculated molecular weight: 562.23, measured value: 585.22 (M+Na).

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

[0493] Example 56

Chemical Structure

[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. Next, the reaction mixture was 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: The 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. Next, the solvent was 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 obtain Compound 208 (0.300 g, 65%). C 29 H 42 N2O 11 Calculated molecular weight for C H N2O is 594.28, measured value is 595.29 (M+).

[0496] Synthesis of Compound 209: DSC derivative compound 207 (0.5 g, 0.83 mmol) was stirred in dichloromethane (20 mL). Next, ethanolamine (0.07 g, 1 mmol) and pyridine (5 mL) were added, and the mixture was stirred overnight. Next, the solvent was 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 obtain compound 209 (0.25 g, 42%). C 24 H 40 N2O 12 Calculated molecular weight for 548.26, measured value 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. Next, the solvent was 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 obtain compound 210 (200 mg, 87%). Next, this compound was 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 with pyridine twice. The product was further dissolved in water and then lyophilized to obtain compound 210 as a white powder. C 21 H 30 Calculated molecular weight for 454.20, measured value 477.21 (M+Na) for N2O9.

[0498] Synthesis of Compound 211: Compound 211 was prepared from compound 209 using the same method as used for the preparation of compound 210. C 16 H 28 N2O 10 Calculated molecular weight for 408.17, measured value 431.20 (M+Na).

[0499] Example 57

Chemical Structure

[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. Next, the solvent was removed under reduced pressure. The crude compound was purified by silica gel chromatography using dichloromethane / methanol, and Compound 213 (0.350 g, 57%) was obtained. C 23 H 40 N4O 10 Calculated molecular weight for it is 532.27, measured value is 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 at room temperature overnight. The solvent was removed under reduced pressure, and the residue was co-evaporated with toluene twice. This residue was dissolved in water and lyophilized, and Compound 214 (200 mg, 74%) was obtained as a white powder. C 19 H 32 N4O 10 Calculated molecular weight for it is 476.21, measured value is 477.20 (M+H).

[0502] Synthesis of Compound 215: Compound 215 was prepared from Compound B (140 mg) by a similar procedure. C 27 H 48 N4O 10 Calculated molecular weight for it is 588.34, measured value is 611.33 (M+Na).

[0503] Synthesis of Compound 216: Compound 216 was prepared from Compound 215 using the same procedure as that used to prepare Compound 214 (0.125 g, 35%). C 23 H 40 N4O 10 Calculated molecular weight for it is 532.27, measured value is 555.25 (M+Na).

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

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

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

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

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

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

[0510] Example 58

Chem.

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

[0512]

Chem.

[0513] The trifluoromethylacetamide (TFA)-protected galactosamine (GalN-TFA) NHS ester is coupled with amine-containing oligonucleotides (compounds 69 and 71) in a post-synthetic procedure to produce Gal-TFA-containing oligonucleotides (compounds 70 and 72).

[0514] Example 59

Chem.

[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 h). The solvent was concentrated, and the product was extracted with ethyl acetate (3 × 50 mL), washed with water and brine, and dried over anhydrous Na2SO4. Concentration of the solvent gave crude Compound 5009 (25 g). C 21 H 29 LCMS calculated value for CNO8S: 455.16 (M + ), found: 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 NaN3 (16 g, 246 mmol). The resulting mixture was stirred at 80 °C for 3 days. Then, an additional 8 g of NaN3 was added, and the solution was heated to 100 °C for 14 h. The solvent was concentrated, and the product was extracted with ethyl acetate (3 × 50 mL), washed with water and brine, and dried over anhydrous Na2SO4. Concentration of the solvent gave crude Compound 5010, which was purified by column chromatography (5 g, 37%). C 14 H 22 LCMS calculated value for CN4O5: 326.35 (M + ), found: 327.1 (M + + 1).

[0517] Synthesis of Compound 5011: A solution of Compound 5010 (574 mg, 1.76 mmol) in THF (10 mL) was added dropwise to a stirred solution of LAH (139 mg, 3.52 mmol) in THF (10 mL) at 0 °C. The mixture was stirred at room temperature overnight (14 h). The reaction mixture was quenched with 1 mL of water, filtered through Celite, and washed with ethyl acetate (25 mL). Concentration of the solvent gave a 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 a 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). C 16 H 26 LCMS calculated value for N2O8: 374.39 (M + ), found value: 373.1 (M - -1).

[0518] Synthesis of Compound 5012: DCC (218 mg, 1.06 mmol) was added to a stirred solution of Compound 5011 (200 mg, 0.53 mmol) and NHS (112 mg, 1.06 mmol) in DMF (10 mL). The mixture was stirred at room temperature for 14 h. Next, 20 mL of ethyl acetate was added. Filtration of the solid gave Compound 5012 (150 mg, 60%). C 20 H 29 N3O 10 LCMS calculated value for : 471.46 (M + ), found value: 506 (M - +Cl - ).

[0519] Example 60: Pseudouridine Building Unit Conjugated with Trivalent GalNAc

Chemical Structure

[0520] Synthesis of Compound 143: To a solution of trivalent GalNAc acid compound 142 (1.80 g, 0.898 mmol) in DMF (12 mL) were added HBTU (341 mg, 0.898 mmol) and i-Pr2NEt (0.568 mL, 3.26 mmol). After 10 minutes, 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 CH2Cl2 and saturated aqueous NaHCO3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (0 - 15% MeOH in CH2Cl2) to give compound 143 (1.75 g, 0.673 mmol, 82%). 1 H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 7.96 (s, 1H), 7.84 - 7.81 (m, 6H), 7.76 - 7.72 (m, 4H), 7.50 (d, J = 0.9 Hz, 1H), 6.98 (s, 1H), 5.21 (d, J = 3.4 Hz, 3H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.53 (s, 1H), 4.48 (d, J = 8.5 Hz, 3H), 4.35 - 4.33 (m, 1H), 4.17 (d, J = 4.5 Hz, 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), 2.44 - 2.39 (m, 2H), 2.27 (t, J = 6.4 Hz, 6H), 2.10 (s, 9H), 2.04 (t, J = 7.3 Hz, 9H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.52 - 1.42 (m, 22H), 1.21 (s, 13H), 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 (approx. 70% HF, 0.173 mL, 6.66 mmol) was diluted with pyridine (2 mL) while cooling to 0 °C. The resulting solution was added to a solution of Compound 143 in CH2Cl2 (20 mL) at 0 °C, and the mixture was stirred at 0 °C for 2 hours. The reaction solution was diluted with CH2Cl2, washed with saturated aqueous NaHCO3, and then dried over anhydrous Na2SO4. After evaporating the volatile substances, the crude product was dried under reduced pressure, and the diol was obtained as a white foam. To a solution of this material in pyridine (15 mL), DMTrCl (691 mg, 2.04 mmol) was added. The reaction mixture was stirred at room temperature for 14 hours and then evaporated. The residue was extracted with CH2Cl2 and saturated aqueous NaHCO3, and then dried over anhydrous Na2SO4. The crude product was purified by silica gel column chromatography (0 - 10% MeOH in CH2Cl2), and Compound 144 (3.31 g, 1.20 mmol, 65%) was obtained.

[0522] Synthesis of Compound 145: To a solution of Compound 144 (3.25 g, 1.18 mmol) in CH2Cl2 (20 mL), DMAP (432 mg, 3.54 mmol) and succinic anhydride (236 mg, 2.36 mmol) were added. The reaction mixture was stirred at room temperature overnight. After concentration, the crude material was purified by silica gel column chromatography (8% MeOH / 8% Et3N in CH2Cl2), and Compound 145 (3.03 g, 1.78 mmol, 87%) was obtained. 11H NMR (400 MHz, DMSO-d6) δ 12.22 (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 = 3.4 Hz, 3H), 5.07 (t, J = 5.1 Hz, 1H), 4.96 (dd, J = 11.2, 3.4 Hz, 3H), 4.55 (t, J = 5.2 Hz, 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 = 6.4 Hz, 6H), 2.10 (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, 22H), 1.21 (s, 13H), 0.95 (t, J = 7.2 Hz, 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 CH3CN (5 mL) were added HBTU (26 mg, 0.0694 mmol), iPr2NEt (0.026 mL, 0.149 mmol), and CPG-NH2 (Prime Synthesis CPG-500, assumed NH2 loading of 80 μmol / g) (450 mg, 0.036 mmol). The mixture was shaken for 24 h, then filtered, washed with CH2Cl2, and dried under reduced pressure. The remaining amino groups were capped by shaking for 1 h with pyridine (7.5 mL), acetic anhydride (2.5 mL), and triethylamine (0.5 mL). Filtration, washing with CH2Cl2 (100 mL), then 50% MeOH / CH2Cl2 (100 mL), and drying under reduced pressure gave Compound 146. Loading: 49 μmol / g.

[0524] Example 61: Primary hepatocyte binding for triple-stranded and 1+1+1 ligand design 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 substitutions shown below.

Chemical formula

[0525] Figure 13 shows the primary hepatocyte binding affinities for siRNA-ligand conjugates 61696, 61695, 61692, 61694, 61697, 61693, 43527 and 61698, and their structures are shown below. The binding affinity Ki 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 structure of the ligand in the conjugate was the same as that in conjugate 57727, except for the sugar substitutions shown below. The structures of L224, L223, L221, L96 and L227 are shown below.

Table 16

Chemical formula

[0527] Figures 3A and 3B show the blood serum mTTR SiRNA levels 72 hours (Figure 3A) and 144 hours (Figure 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 blocks [Table 17]

[0529] Example 64: Compounds for T-3’-GalNAc building blocks [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: [Chemical Structure]

[0531] 2'- and 3'-O-phthalimidohexyl-5-methyluridine (2A, 2B). 2',3'-O-Dibutylstannylen-5-methyluridine (2.0 g, 4.1 mmol) was suspended in DMF (10 mL). 6-Bromohexyl phthalimide (2.5 g, 8.2 mmol) and NaI (120 mg, 0.82 mmol) were added to the suspension. The reagents were irradiated with microwave at 100 °C for 3.5 h, and a dark brown homogeneous mixture was obtained. DMF was evaporated under reduced pressure, and the residue was adsorbed on silica gel. The silica gel was packed into a cartridge for silica gel chromatography. The 2'- and 3'-isomers of O-phthalimidohexyl-5-methyluridine were eluted as a non-separable mixture, and 890 mg of 2A and 2B (1.8 mmol, 45%) were obtained. 1 H NMR (400 MHz, DMSO-d6) δ 11.29 (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 = 5.3 Hz, 1H), 5.72 (d, J = 5.6 Hz, 1H), 5.25 (d, J = 6.2 Hz, 1H, D2O-exchangeable), 5.12 (t, J = 5.0 Hz, 1H, D2O-exchangeable), 5.00 (d, J = 5.9 Hz, 1H, D2O-exchangeable), 4.14 (q, J = 5.6 Hz, 1H), 4.07 (q, J = 5.0 Hz, 1H), 3.90 - 3.79 (m, 2H), 3.74 (t, J = 4.6 Hz, 0H), 3.67 - 3.58 (m, 1H), 3.58 - 3.49 (m, 4H), 3.46 - 3.37 (m, 1H), 1.75 (d, J = 3.7 Hz, 3H), 1.62 - 1.42 (m, 4H), 1.37 - 1.20 (m, 4H). 1313C NMR (100 MHz, DMSO-d6) δ 168.00, 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, 109.26, 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 co-evaporated 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 equivalent of DMTrCl was added and the reaction was stirred for a further 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 Na2SO4 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 isomer was characterized by D2O exchange followed by identification of the 3'-OH by COSY. 11H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H, exchangeable with D2O), 7.88 - 7.77 (m, 4H), 7.48 (s, 1H), 7.38 (d, J = 7.4 Hz, 2H), 7.33 - 7.19 (m, 7H), 6.89 (d, J = 8.0 Hz, 4H), 5.82 (d, J = 4.8 Hz, 1H), 5.10 (d, J = 6.3 Hz, 1H, exchangeable with D2O), 4.18 (q, J = 5.5 Hz, 1H), 3.96 (q, J = 4.8, 4.4 Hz, 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 13C NMR (100 MHz, DMSO-d6) δ 167.91, 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 The calculated MS value for 789.3261, the measured value 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) in 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 in the same manner except for the identification of the 2'-OH by D2O exchange. 11H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H, exchangeable with D2O), 7.87 - 7.79 (m, 4H), 7.49 (s, 1H), 7.36 (d, J = 7.4 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 7.26 - 7.19 (m, 5H), 6.94 - 6.81 (m, 4H), 5.71 (d, J = 4.6 Hz, 1H), 5.36 (d, J = 6.0 Hz, 1H, exchangeable with D2O), 4.27 (q, J = 5.2 Hz, 1H), 3.99 - 3.95 (m, 1H), 3.90 (t, J = 5.3 Hz, 1H), 3.71 (s, 6H), 3.63 - 3.47 (m, 3H), 3.36 (t, J = 6.8 Hz, 1H), 3.26 - 3.15 (m, 2H), 1.59 - 1.39 (m, 7H), 1.28 - 1.20 (m, 4H). 13 13C NMR (100 MHz, DMSO-d6) δ 167.93, 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 value for 789.3261, measured value 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 *)。A solution of 3A (Lot #H1010 - 04, 15.0 g, 18.99 mmol, obtained from RI Chemicals) 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 aid in the removal of the emulsion. The organic layer was dried over MgSO4 and evaporated under reduced pressure to give 11.80 g of the crude product, which was used in the next step without purification. C 37 H 45 MS calculated for C20H21N3O8 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 = 5% MeOH / DCM v / v of 0.02. 1 1H NMR (400 MHz, DMSO - d6) δ 7.48 (s, 1H), 7.38 (d, J = 7.5 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.24 (d, J = 8.9 Hz, 5H), 6.89 (d, J = 8.4 Hz, 4H), 5.84 (d, J = 5.0 Hz, 1H), 5.74 (s, 1H), 4.19 (t, J = 5.0 Hz, 1H), 3.97 (t, J = 4.9 Hz, 2H), 3.72 (s, 6H), 3.63 - 3.45 (m, 3H), 3.27 - 3.15 (m, 3H), 1.48 (d, J = 6.4 Hz, 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 (6.00 g, 9.09 mmol), triethylamine (3.8 mL, 27.30 mmol) was added, and the mixture was stirred 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, 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 (400 MHz, DMSO-d6) δ 11.37 (s, 1H, exchangeable with D2O), 7.97 (d, J = 9.3 Hz, 1H, exchangeable with D2O), 7.91 (t, J = 6.8 Hz, 4H), 7.73 - 7.45 (m, 11H), 7.41 - 7.19 (m, 11H), 6.88 (d, J = 8.4 Hz, 4H), 5.84 (d, J = 4.7 Hz, 1H), 5.74 (d, J = 3.4 Hz, 1H), 5.36 (dd, J = 11.1, 3.3 Hz, 1H), 5.12 (d, J = 6.3 Hz, 1H, exchangeable with D2O), 4.73 (d, J = 8.5 Hz, 1H), 4.45 (q, J = 8.8, 7.6 Hz, 2H), 4.39 - 4.15 (m, 3H), 3.96 (t, J = 4.7 Hz, 2H), 3.79 (dd, J = 9.4, 3.8 Hz, 1H), 3.72 (s, 6H), 3.63 - 3.45 (m, 3H), 3.28 - 3.16 (m, 2H), 2.99 (q, J = 6.5 Hz, 2H), 2.04 (s, 2H), 1.69 (s, 3H), 1.55 - 1.43 (m, 6H), 1.36 (d, J = 17.6 Hz, 5H), 1.24 (s, 4H). 1313C NMR (100 MHz, DMSO-d6) δ 171.73, 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 value for 1274.5311, found 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-succinate-5-methyluridine (5A). To a solution of 4A (2.00 g, 1.57 mmol) in DCM (50 mL) were 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% Et3N (v / v) in DCM and co-evaporated with acetonitrile under reduced pressure to give 2.11 g (1.43 mmol, 91%) of 6a as the Et3N salt. 11H NMR (400 MHz, DMSO-d6) δ 11.44 (s, 1H), 8.05 (d, J = 9.3 Hz, 1H), 7.91 (t, J = 6.8 Hz, 4H), 7.74 - 7.45 (m, 11H), 7.40 - 7.27 (m, 6H), 7.23 (d, J = 8.7 Hz, 5H), 6.89 (d, J = 8.1 Hz, 4H), 5.84 (d, J = 6.1 Hz, 1H), 5.74 (d, J = 3.5 Hz, 1H), 5.36 (dd, J = 11.1, 3.3 Hz, 1H), 5.27 - 5.22 (m, 1H), 4.74 (d, J = 8.5 Hz, 1H), 4.48 - 4.40 (m, 2H), 4.38 - 4.22 (m, 3H), 4.13 (q, J = 3.5 Hz, 1H), 3.78 (d, J = 9.7 Hz, 1H), 3.72 (s, 6H), 3.54 - 3.28 (m, 5H), 3.25 - 3.19 (m, 1H), 2.97 (q, J = 6.5 Hz, 2H), 2.57 - 2.51 (m, 2H), 2.44 (t, J = 6.5 Hz, 2H), 2.04 (s, 2H), 1.69 (s, 3H), 1.57 - 1.27 (m, 11H), 1.18 (s, 4H). 13 13C NMR (100 MHz, DMSO-d6) δ 173.40, 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 - The calculated MS value for it is 1374.5472, and the measured value is 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 (v / v) in DCM, and then ether. The CPG was evaporated under reduced pressure and then treated with acetic anhydride (25 mL) in pyridine (75 mL) and Et3N (1 mL) and shaken for 1 hour. The CPG was filtered and washed with the same solvents as described above. The average loading was measured by trityl absorbance spectrometry 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-cyanoethyl phosphoramidite-5-methyluridine (7A). 4A (2.90 g, 2.27 mmol) was co-evaporated twice with anhydrous acetonitrile and then kept under a complete argon atmosphere. To a solution of 4A in anhydrous DCM (35 mL) at 0 °C was added 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite (1.37 g, 4.55 mmol), followed by DCI (268 mg, 2.27 mmol). The mixture was stirred at 0 °C for 20 minutes and then at room temperature for 17 hours. 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). The product 7A eluted with 100% EtOAc and again 3% MeOH. The fractions containing 7A were combined and evaporated under reduced pressure to give 3.20 g of 7A (2.17 mmol, 95%). 11H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.98 (d, J = 9.3 Hz, 1H), 7.91 (t, J = 7.2 Hz, 4H), 7.72 - 7.45 (m, 11H), 7.38 (t, J = 6.8 Hz, 4H), 7.33 - 7.20 (m, 7H), 6.88 (t, J = 5.4 Hz, 4H), 5.82 (d, J = 4.3 Hz, 1H), 5.75 (s, 1H), 5.35 (dd, J = 11.1, 3.1 Hz, 1H), 4.73 (d, J = 8.5 Hz, 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 = 5.8 Hz, 1H), 2.57 (q, J = 5.3 Hz, 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 = 6.7 Hz, 3H). 13 13C NMR (125 MHz, DMSO-d6) δ 171.60, 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. 311P NMR (160 MHz, DMSO-d6) δ 154.01, 153.65. C 80 H 95 N6O 19 MS calculated for P 1474.6390, found m / z 1497.4 (M + 23) Na+ , 1509.4 (M + 35) Cl- . R f = 0.39 in 100% EtOAc.

[0539] Example 66:

Chem.

[0540] 5'-O-dimethoxytrityl-3'-O-aminohexyl-5-methyluridine (4B * ). To a solution of 3B (3.64 g, 4.61 mmol ~1 g) in MeOH (46 ml) was added hydrazine (738 mg, 23.04 mmol) and the reaction mixture was refluxed for 5.5 h. 4B * was isolated using the work-up procedure described for 4A * . Co-evaporation with acetonitrile gave 2.93 g of crude 4B * . C 37 H 45 MS calculated for C H 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. 1H NMR of crude 4B * : 11H NMR (400 MHz, DMSO-d6) δ 7.50 (s, 1H), 7.43 - 7.16 (m, 9H), 6.88 (d, J = 8.7 Hz, 4H), 5.73 (d, J = 4.6 Hz, 1H), 4.29 (t, J = 4.8 Hz, 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 13C NMR (125 MHz, DMSO-d6) δ 163.73, 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 (2.85 g, 4.32 mmol) in DCM (45 mL) treated with TEA (1.8 mL) * (2.85 g, 4.32 mmol) was added to a solution of GalNAc-NHS ester (3.47 g, 4.75 mmol). The reaction was stirred for 2 hours and then an additional 0.2 equivalent 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). 11H NMR (400 MHz, DMSO-d6) δ 11.35 (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 = 8.8 Hz, 4H), 5.74 (d, J = 10.1 Hz, 2H), 5.42 - 5.33 (m, 2H), 4.73 (d, J = 8.5 Hz, 1H), 4.48 - 4.41 (m, 2H), 4.38 - 4.23 (m, 3H), 3.99 (d, J = 4.7 Hz, 1H), 3.91 (t, J = 5.1 Hz, 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 = 6.6 Hz, 2H), 2.04 (t, J = 6.4 Hz, 2H), 1.69 (s, 3H), 1.55 - 1.28 (m, 11H), 1.28 - 1.15 (m, 4H). 13 13C NMR (75 MHz, DMSO-d6) δ 171.72, 169.38, 165.19, 165.14, 164.85, 163.64, 162.25, 158.14, 150.55, 144.57, 135.68, 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, 126.80, 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 For MS calculated value of 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-succinate-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 co-evaporated with acetonitrile under reduced pressure to give 1.03 g of 5B (0.70 mmol, 81%). 1 H NMR (400 MHz, DMSO-d6) δ 8.09 (d, J = 9.3 Hz, 1H), 7.91 (t, J = 6.9 Hz, 4H), 7.76 (t, J = 5.4 Hz, 1H), 7.73 - 7.51 (m, 9H), 7.47 (t, J = 7.7 Hz, 2H), 7.40 - 7.19 (m, 12H), 6.87 (d, J = 8.7 Hz, 4H), 5.85 (d, J = 3.9 Hz, 1H), 5.74 (d, J = 3.2 Hz, 1H), 5.47 - 5.42 (m, 1H), 5.36 (dd, J = 11.1, 3.2 Hz, 1H), 4.75 (d, J = 8.5 Hz, 1H), 4.44 (q, J = 9.1, 7.7 Hz, 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, 5.7 Hz, 2H), 2.43 (d, J = 4.1 Hz, 3H), 2.07 - 2.01 (m, 2H), 1.69 (s, 3H), 1.48 (d, J = 15.0 Hz, 7H), 1.40 - 1.27 (m, 4H), 1.16 (s, 4H). 1313C NMR (100 MHz, DMSO-d6) δ 173.63, 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 value for 1374.5472, measured value 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 was continued for 21 hours. The CPG was removed by filtration, washed, capped, and the loading was determined as described for 6A, and CPG with an average loading of 56 μmol / g was obtained.

[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 and treated with the same reagents as described for 7A. The product 7B was loaded onto a column prepared as described for 7A and eluted with 70% EtOAc in hexane (4 CV), 80% EtOAc in hexane (10 CV), 100% EtOAc (2 CV), and then 3% MeOH in DCM (4 CV). The desired product eluted with 100% EtOAc and 3% MeOH. The 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 (400 MHz, DMSO-d6) δ 11.38 (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, 5.1 Hz, 1H), 5.75 (d, J = 3.3 Hz, 1H), 5.36 (dd, J = 11.1, 3.3 Hz, 1H), 4.73 (d, J = 8.5 Hz, 1H), 4.62 - 4.50 (m, 1H), 4.45 (q, J = 8.2, 7.2 Hz, 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, 2H), 2.98 (s, 2H), 2.71 - 2.66 (m, 1H), 2.04 (s, 2H), 1.69 (s, 3H), 1.47 (d, J = 28.1 Hz, 8H), 1.40 - 1.18 (m, 8H), 1.13 - 1.00 (m, 11H). 1313C NMR (125 MHz, DMSO-d6) δ 171.72, 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 31P NMR (162 MHz, DMSO-d6) δ 155.08, 154.60. C 80 H 95 N6O 19 Calculated MS value for P: 1474.6390, measured value m / z 1497.4 (M + 23) Na+ , 1474.3 (M - 1) - , 1509.4 (M + 35) Cl- R f = 100% in EtOAc (0.43).

[0545] Example 67:

Chem.

[0546] (2A / 2B)-Adenosine (20 g, 74.8 mmol) was treated with NaH (4.5 g, 112 mmol) in DMF (200 ml) at 0 °C for 20 minutes. N-(Iodohexyl)phthalimide (30.7 g, 86 mmol) was added to the solution and then heated to 80 °C for 2 days. Evaporation of the DMF under reduced pressure gave a pale orange gum containing 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 a mixture of 2A and positional isomers. 1 H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H), 8.12 (s, 1H), 7.95 - 7.68 (m, 4H), 7.32 (s, 2H, exchangeable with D2O), 5.96 (...

Claims

1. The following formula: 【Chemical 1】 A compound of, wherein The linker is as shown in Table 1: 【Table 1-1】 【Table 1-2】 (wherein the wavy line represents the binding site of the ligand, X represents the binding site of X, and DMTr is 4,4'-dimethoxytrityl) Selected from; X is 【Chemical Formula 2】 (wherein the sphere represents a solid support), 【Chemical Formula 3】 , a leaving group, H, -OH, or -NH ２ ; The ligand is selected from Table 2 or Table 2A: 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2A-1】 【Table 2A-2】 【Table 2A-3】 【Table 2A-4】 The compound as described above.

2. X is 【Chemical 4】 (wherein the sphere represents a solid support) The compound according to Claim 1.

3. X is 【Chemical Formula 5】 The compound according to Claim 1.

4. The linker is 【Chemical Formula 6】 The compound according to any one of Claims 1 to 3.

5. The linker is 【Chemical Formula 7】 The compound according to any one of Claims 1 to 3.

6. The ligand is selected from the following table: 【Table 3】 The compound according to any one of Claims 1 to 5.

Images