Ligand conjugates for the delivery of therapeutically active agents - Patents.com

Abstract

Compounds of formula (I) as novel ligand conjugates and their use for the delivery of therapeutically active agents are provided. These compounds have advantages for the ex vivo and / or in vivo delivery of therapeutically active agents, such as iRNA agents.

Description

[Technical field]

[0001] The present invention relates to the field of delivery of therapeutically active agents using ligand conjugates. In particular, the present invention discloses novel ligand conjugates having advantages for the ex vivo and / or in vivo delivery of therapeutically active agents, and their uses and compositions. [Background technology]

[0002] RNA interference (RNAi) is an RNA-dependent gene silencing process controlled by the RNA-induced silencing complex (RISC) and initiated by short double-stranded RNA molecules in the cytoplasm that interact with the catalytic RISC component Argonaute to regulate the expression of protein-coding genes. This sequence-specific gene silencing mechanism has been identified as a promising strategy for therapeutic intervention.

[0003] Efficient delivery of RNAi compounds to target organs in the body requires specific targeting and sufficient protection from the extracellular environment, especially from exonucleases. One way to achieve organ specificity is to conjugate the RNAi compound with a targeting ligand that selectively binds to membrane receptor surfaces abundant in the target tissue and initiates endocytic activity.

[0004] The asialoglycoprotein receptor (ASGPR) is a transmembrane receptor that is expressed primarily in hepatocytes and minimally in extrahepatic cells. ASGPR promotes internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrate compounds such as galactose, N-acetylgalactosamine, and glucose. These characteristics make it particularly attractive for receptor-mediated drug delivery, where toxicity concerns are minimal.

[0005] Liver diseases (e.g., nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatitis B virus (HBV) infection, liver fibrosis, and cirrhosis) are a significant burden to modern society, and there is a large unmet medical need in this area. Therefore, there is a clear need for safe and effective therapeutically active agents to improve the quality of life of patients with liver disease.

[0006] Many ligand conjugates for delivering therapeutically active iRNA agents have been reported in the literature (see, for example, International Patent Application Publication No. WO2009 / 073809A2, International Patent Application Publication No. WO2016 / 077321A1, International Patent Application Publication No. WO2021 / 113851A2, International Patent Application Publication No. WO2019 / 105419A1, International Patent Application Publication No. WO2009 / 134487A2, International Patent Application Publication No. WO2011 / 091396A1, International Patent Application Publication No. WO2017 / 157899A1). Given the complexity of drug discovery and the huge unmet medical demand, it is meaningful to design new ligand conjugates for delivering therapeutically active agents. Summary of the Invention

[0007] The present invention relates to novel ligand conjugate compounds that are effective in delivering therapeutically active agents, such as iRNA agents, and are therefore useful in regulating the expression of genes of interest in cells and treating various diseases and conditions.

[0008] In one embodiment, a compound having the structure shown in formula (I): [ka] or a pharma- ceutically acceptable salt or solvate thereof, A and B, at each occurrence, are independently O, N(RN), or S; R N is H or C 1-6 is alkyl; X and W, at each occurrence, are independently H, a protecting group, a phosphate group, a phosphate ester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid ligand, -P(Z')(Z")O-nucleoside, -P(Z')(Z")O-oligonucleotide, a lipid, PEG, a steroid, a polymer, a nucleotide, a nucleoside, an oligonucleotide, or a therapeutically active agent. Z′ and Z″ are each independently O or S; L is a covalent linker; Z is a carbohydrate mimetic, or a disaccharide, trisaccharide, or oligosaccharide; each Y is independently -L'-T; each T is a ligand selected from the group consisting of carbohydrate ligands, polypeptide ligands, and lipophilic ligands; each L' is independently a covalent linker; and p and q are each independently 1, 2, 3, 4, or 5.

[0009] In certain embodiments, Z is a carbohydrate mimetic of a monosaccharide.

[0010] In certain embodiments, Z is a monosaccharide carbohydrate mimetic selected from the group consisting of a deoxy sugar, an amino sugar, an N-glycoside, an imino sugar, an unsaturated sugar, a carboxylated sugar, an amidated sugar, a fused cyclic sugar, and a monosaccharide carbasugar.

[0011] In certain embodiments, Z is a monosaccharide imino sugar.

[0012] In certain embodiments, the monosaccharide is a tetrose, pentose, hexose, heptose, or octose.

[0013] In certain embodiments, Z has the structure: [ka] Where: R 1 is H, C 1-6Alkyl, halogen, or -NH(R 2 ) and R 2 is H or acetyl; Each R is independently H, halogen, -CN, -C≡CH, -NH2, -OC 1-6 Alkyl, or C 1-6 alkyl, 1-6 Alkyl and -OC 1-6 The alkyl of the alkyl is substituted with 0 to 5 halogen atoms. Or, two R's together with the carbon to which they are attached form C 3-6 A cycloalkyl or 3- to 6-membered heterocycloalkyl group is formed, 3-6 the cycloalkyl of the cycloalkyl and the heterocycloalkyl of the 3-6 membered heterocycloalkyl are substituted with 0 to 5 halogen atoms; and n is 0, 1, 2, or 3, as permitted by the valence.

[0014] In certain embodiments, n is 0.

[0015] In certain embodiments, Z has the structure: [ka] In certain embodiments, (Y)pZ- has the structure: [ka] In certain embodiments, Z is a disaccharide, a trisaccharide, or a carbohydrate mimetic of a disaccharide or trisaccharide.

[0016] In certain embodiments, Z is a disaccharide or a carbohydrate mimetic of a disaccharide.

[0017] In certain embodiments, Z is a disaccharide selected from the group consisting of gentiobiose, isomaltose, melibiose, trehalose, sucrose, lactose, maltose, cellobiose, or a carbohydrate mimetic thereof.

[0018] In certain embodiments, Z has the structure: [ka] In certain embodiments, Z has the structure: [ka] In certain embodiments, each [ka] is independently a group having the following structure: [ka] Where: s is an integer from 1 to 20, Each Q 3 are not independently present and are -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; Each Q 4 does not exist independently and is a substituted or unsubstituted C 1-12 Alkylene, substituted or unsubstituted C 2-12 Alkenylene, substituted or unsubstituted C 2-12 Alkynylene, substituted or unsubstituted C 2-12 heteroarylene, a substituted or unsubstituted 6- to 12-membered cyclic arylene, a substituted or unsubstituted 5- to 12-membered cyclic heteroarylene, or a substituted or unsubstituted 5- to 12-membered cyclic heterocyclylene; and Each Q 5 do not exist independently, and -CO-, -NH-, -O-, -S-, -SO2-, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -NH-CH(R a )-C(O)-, -C(O)-CH(R a )-NH-, -OP(O)(OH)O-, or -OP(S)(OH)O-, where each Ra are independently H or substituted or unsubstituted C 1-12 is alkyl, However, at least one Q 4 exists.

[0019] In certain embodiments, s is an integer from 1 to 5; Each Q 3 are not independently present and are -CO-, -NH-, -O-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; Each Q 4 does not exist independently and is a substituted or unsubstituted C 1-12 Alkylene, substituted or unsubstituted C 2-12 Alkenylene, or substituted or unsubstituted C 2-12 alkynylene, and Each Q 5 are not independently present and are -CO-, -NH-, -O-, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, or -NHC(O)-; However, at least one Q 4 exists.

[0020] In certain embodiments, s is 1 or 2; Each Q 3 are independently absent and are -CO-, -NH-, -CH2-, or -NHC(O)-; Each Q 4 does not exist independently, or C 1-12 is alkylene, and Each Q 5 are independently absent and are -CO-, -CH2-, or -NHC(O)-; However, at least one Q 4 exists.

[0021] In certain embodiments, each [ka] is independently a group having the structure [ka] Where: Each Q 5 are independently -CO-, -NH-, -O-, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, or -NHC(O)-, and Each j1 and j2 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0022] In certain embodiments, each [ka] is independently a group having the structure [ka] [ka] In certain embodiments, each -L'-T is independently a group having the structure: [-Q 3 -Q 4 -Q 5 ]sQ 6 -T, Where: s is an integer from 0 to 20. Each Q 3 and Q 6 are not independently present and are -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; Each Q 4 does not exist independently and is a substituted or unsubstituted C 1-12 Alkylene, substituted or unsubstituted C 2-12 Alkenylene, substituted or unsubstituted C2-12 Alkynylene, substituted or unsubstituted C 2-12 heteroarylene, a substituted or unsubstituted 6- to 12-membered cyclic arylene, a substituted or unsubstituted 5- to 12-membered cyclic heteroarylene, or a substituted or unsubstituted 5- to 12-membered cyclic heterocyclylene; and Each Q 5 do not exist independently, and -CO-, -NH-, -O-, -S-, -SO2-, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -NH-CH(R a )-C(O)-, -C(O)-CH(R a )-NH-, -OP(O)(OH)O-, or -OP(S)(OH)O-, where each R a are independently H or substituted or unsubstituted C 1-12 is alkyl, However, Q 3 , Q 4 , Q 5 , and Q 6 At least one of the following must be present:

[0023] In certain embodiments, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0024] In certain embodiments, each -L'-T is independently a group having the structure: [ka] Where: Each Q 7 are not independently present and are -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH2-, -CH2NH-, -NHCH2-, -CH2O-, or -OCH2-; each k1 and k2 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and Each n1, n2, and n3 is independently 1, 2, 3, 4, or 5.

[0025] In certain embodiments, each Q7 are independently -NHC(O)- or -C(O)NH-.

[0026] In certain embodiments, each -L'-T is independently a group having the structure: [ka] Where: each k1 and k2 is independently 0, 1, 2, or 3; each n1, n2, and n3 is independently 1, 2, 3, 4, or 5; One of t1 and t2 is 0 and the other is 1.

[0027] In certain embodiments, each -L'-T is independently a group having the structure: [ka] In certain embodiments, each -L'-T is independently a group having the structure: [ka] Where: each k1 and k2 is independently 0, 1, 2, or 3; each n1 and n2 is independently 1, 2, 3, 4, or 5; and One of t1 and t2 is 0 and the other is 1.

[0028] In certain embodiments, each -L'-T is independently a group having the structure: [ka] In certain embodiments, each T is independently a carbohydrate ligand.

[0029] In certain embodiments, each T is N-acetylgalactosamine (GalNAc), allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fuculose, galactosamine, D-galactosaminitol, galactose, glucosamine, N-acetylglucosamine, glucosaminitol, glucose, glucose-6-phosphate, gulose, glyceraldehyde, L-glycosyltransferase ... - a carbohydrate ligand in protected or unprotected form selected from the group consisting of glycero-D-mannoheptose, glycerol, glycerone, gulose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylose.

[0030] In certain embodiments, each T is independently N-acetylgalactosamine (GalNAc) or N-acetylgalactosamine triacetate.

[0031] In certain embodiments, the therapeutically active agent is selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), microRNA mimics, anti-microRNA oligonucleotides (AMOs), long non-coding RNAs, peptide nucleic acids (PNAs), helper lipids, and phosphorodiamidate morpholino oligomers (PMOs), which nucleic acids are either modified or unmodified.

[0032] In certain embodiments, the therapeutically active agent is a small interfering RNA (siRNA).

[0033] In certain embodiments, the compound comprises a structure selected from the following group: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] In certain embodiments, the compound comprises a structure selected from the following group: [ka] [ka] [ka] [ka] [ka] In certain embodiments, the compound is selected from the group consisting of the following compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt or solvate thereof.

[0034] In certain embodiments, the compound is selected from the group consisting of the following compounds: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Where: [ka] is an oligonucleotide (e.g., an iRNA agent), or a pharma- ceutically acceptable salt or solvate thereof.

[0035] In one embodiment, there is provided a method for regulating expression of a gene of interest in a cell, comprising delivering to said cell a compound according to the invention, or a pharma- ceutically acceptable salt or solvate thereof.

[0036] In certain embodiments, the gene of interest is associated with a liver disease.

[0037] In certain embodiments, the liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), HBV infection, liver fibrosis, and liver cirrhosis.

[0038] In one embodiment, there is provided a pharmaceutical composition comprising a compound according to the invention or a pharma- ceutically acceptable salt or solvate thereof, alone or in combination with a pharma- ceutically acceptable carrier or excipient. [Brief description of the drawings]

[0039] The summary will be better understood by reference to the accompanying drawings in conjunction with the following detailed description, although the invention is not limited to the specific disclosures in the drawings.

[0040] [Figure 1] In the drawings: FIG. 1 shows the activity of certain indicated GalNAc-siRNA conjugates provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] This specification provides a detailed description of the embodiments and examples set forth in the accompanying detailed description. Although enumerated embodiments are described, it should be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents included within the scope of the present invention as defined by the claims.

[0042] definition Terms used in this specification have their ordinary meanings, and each occurrence of a term has its meaning independently. Nevertheless, unless otherwise stated, the following definitions apply throughout the specification and claims.

[0043] As used herein, the singular forms "a," "an," and "the" include plural referents unless expressly stated to the contrary.

[0044] As used in this specification, "comprise" and "include" specify the presence of stated features, integers, components, or steps, but do not exclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

[0045] As used herein, the term "about" means approximately, in the vicinity, roughly, or around. When the term "about" is used with a numerical range, it modifies that range by expanding the boundaries above and below the set numerical value. In general, the term "about" is used herein to modify a given value above and below by a variance of 20%, typically 10%, more typically 5%, and even more typically 1%. Sometimes such ranges can be within the experimental error, the type of standard method used to measure and / or determine the given value or range.

[0046] Specific examples and definitions of chemical terms are described in more detail below. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific examples are generally defined as described therein. In addition, general principles of organic chemistry, specific examples and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic TRansformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0047] All ranges cited herein are inclusive and include everything within the range unless expressly stated to the contrary.

[0048] When a range of values ​​is listed, it is intended to encompass each value and subrange within the range. For example, "C 1-6 ” is C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C4-5 , and C 5-6 is intended to encompass.

[0049] When any variable occurs more than one time in any constituent of Formula I, or other formulas illustrating and describing compounds of the invention, its definition at each occurrence is independent of its definition at every other occurrence, and combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0050] The term "alkyl" as used herein, either as part of other terms or independently, refers to an acyclic, straight or branched chain saturated hydrocarbon group, which may be optionally substituted (i.e., substituted or unsubstituted) with one or more substituents described below. The term "Ci-j alkyl" refers to an alkyl having i to j carbon atoms. In certain embodiments, an alkyl group contains 1 to 12 carbon atoms. In certain embodiments, an alkyl group contains 1 to 11 carbon atoms. In certain embodiments, an alkyl group contains 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n- and isopropyl, n-, sec-, iso-, and tert-butyl, neopentyl, and the like. Alkyl groups may be optionally substituted, where valence allows, with one, two, three, or, in the case of alkyl groups having two or more carbons, four or more substituents independently selected from alkoxy, acyloxy, amino, aryl, aryloxy, azido, cycloalkyl, cycloalkoxy, halo, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, silyl, cyano, =O, =S, and =NR', where R' is H, alkyl, aryl, or heterocyclyl. In certain embodiments, alkyl groups may be optionally substituted with halo, amino, hydroxy, methoxy, nitro, cyano, etc. Each substituent may itself be unsubstituted or, where valence allows, substituted with an unsubstituted substituent defined herein for each group.

[0051] The term "alkylene" as used herein, whether as part of other terms or independently, refers to a divalent substituent that is a monovalent alkyl with one hydrogen atom replaced with a valence. An alkylene group can be substituted or unsubstituted. An optionally substituted alkylene is an alkylene that is optionally substituted as described herein for alkyl.

[0052] The term "alkenyl" as used herein, either as part of or independent of other terms, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond, which may be optionally substituted (i.e., substituted or unsubstituted) with one or more substituents described herein, including "cis" and "tRans" orientations, or alternatively, "E" and "Z" orientations. In certain embodiments, an alkenyl group contains 2-12 carbon atoms. In certain embodiments, an alkenyl group contains 2-11 carbon atoms. In certain embodiments, an alkenyl group contains 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3 carbon atoms. In certain embodiments, an alkenyl group contains 2 carbon atoms. Non-limiting examples of alkenyl groups include ethylenyl (or vinyl), propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, 5-hexenyl, and the like. Optionally substituted alkenyl is optionally substituted alkenyl as described herein for alkyl.

[0053] The term "alkenylene" as used herein, either as part of other terms or independently, refers to a divalent substituent that is a monovalent alkenyl with one hydrogen atom replaced with a valence. An alkenylene group can be substituted or unsubstituted. An optionally substituted alkenylene is an optionally substituted alkenylene as described herein for alkyl.

[0054] The term "alkynyl" as used herein, either as part of or independent of other terms, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond, which may be optionally substituted (i.e., substituted or unsubstituted) with one or more substituents described herein. In certain embodiments, an alkynyl group contains 2-12 carbon atoms. In certain embodiments, an alkynyl group contains 2-11 carbon atoms. In certain embodiments, an alkynyl group contains 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 carbon atoms. In certain embodiments, an alkynyl group contains 2 carbon atoms. Non-limiting examples of alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, and the like. An optionally substituted alkynyl is an optionally substituted alkynyl as described herein for alkyl.

[0055] The term "alkynylene" as used herein, either as part of other terms or independently, refers to a divalent substituent that is a monovalent alkynyl with one hydrogen atom replaced with a valence. An alkynylene group can be substituted or unsubstituted. An optionally substituted alkynylene is an optionally substituted alkynylene as described herein for alkyl.

[0056] The term "cycloalkyl" as used herein, whether as part of or independent of other terms, refers to a non-aromatic, saturated or partially unsaturated, monocyclic or polycyclic ring system, all ring atoms being carbon, and containing at least three ring-forming carbon atoms, all of which are monovalent. In certain embodiments, cycloalkyl may contain 3-12 ring-forming carbon atoms, 3-10 ring-forming carbon atoms, 3-9 ring-forming carbon atoms, 3-8 ring-forming carbon atoms, 3-7 ring-forming carbon atoms, 3-6 ring-forming carbon atoms, 3-5 ring-forming carbon atoms, 4-12 ring-forming carbon atoms, 4-10 ring-forming carbon atoms, 4-9 ring-forming carbon atoms, 4-8 ring-forming carbon atoms, 4-7 ring-forming carbon atoms, 4-6 ring-forming carbon atoms, 4-5 ring-forming carbon atoms. In particular, cycloalkyl groups may contain 3-10 ring-forming carbon atoms (i.e., C3-10 cycloalkyl). In particular, cycloalkyl groups may be monocyclic or bicyclic. Bicyclic cycloalkyl groups may be of the type bicyclic[pq0]alkyl, where each p and q is independently 1, 2, 3, 4, 5, 6, or 7, and the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, bicyclic cycloalkyl groups may contain bridged cycloalkyl structures, such as bicyclic[pqr]alkyl, where r is 1, 2, or 3, each p and q is independently 1, 2, 3, 4, 5, or 6, and the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. Cycloalkyl groups may be spirocyclic groups, such as spiro[pq]alkyl, where each p and q is independently 2, 3, 4, 5, 6, or 7, and the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and decalinyl.Cycloalkyl groups may be optionally substituted (i.e., substituted or unsubstituted), where valence allows, with one, two, three, four, or five substituents independently selected from the group: alkyl, alkoxy, acyloxy, amino, aryl, aryloxy, azido, cycloalkyl, cycloalkoxy, halo, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, silyl, cyano, =0, =S, =NR' (where R' is H, alkyl, aryl, or heterocyclyl), etc. Each substituent may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each group.

[0057] The term "cycloalkylene" as used herein, whether as part of other terms or independently, refers to a divalent substituent that is a cycloalkyl in which one hydrogen atom is replaced with a valence. Cycloalkylene groups can be substituted or unsubstituted. Optionally substituted cycloalkylene is cycloalkylene that is optionally substituted as described herein for cycloalkyl.

[0058] The term "cycloalkoxy" as used herein, either as part of other terms or independently, refers to the group -OR, where R is cycloalkyl. Cycloalkoxy groups can be substituted or unsubstituted. Optionally substituted cycloalkoxy is cycloalkoxy that is optionally substituted as described herein for cycloalkyl.

[0059] The term "aryl" as used herein, either as part of or independent of other terms, refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having at least one aromatic ring. Aryl groups can be 6-12 membered, e.g., 8-12 membered, 6-10 membered, 6 membered. All atoms in an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, and the like. Aryl groups, when valence allows, may be optionally substituted (i.e., substituted or unsubstituted) with one, two, three, four, or five substituents independently selected from the group, such as alkyl, alkoxy, acyloxy, amino, aryl, aryloxy, azido, cycloalkyl, cycloalkoxy, halo, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, silyl, and cyano. Each substituent may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each group.

[0060] The term "arylene" as used herein, whether as part of other terms or independently, refers to a divalent substituent that is an aryl with one hydrogen atom replaced by a valence. The arylene group can be substituted or unsubstituted. An optionally substituted arylene is an arylene that is optionally substituted as described herein for aryl.

[0061] The term "acyl," as used herein, either as part of other terms or independently, refers to a chemical substituent having the formula -C(O)-R, where R is alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or heteroarylalkyl. Optionally substituted acyl is an acyl that is optionally substituted as described herein for each group R.

[0062] The term "acyloxy" as used herein, whether as part of other terms or independently, refers to a chemical substituent having the formula -OR, where R is acyl. Optionally substituted acyloxy is optionally substituted acyloxy as described herein for acyl.

[0063] The term "alkoxy" as used herein, whether as part of other terms or independently, refers to a chemical substituent having the formula -OR, where R is specifically C 1-12 Alkyl, C 1-10 Alkyl, C 1-6 Alkyl groups include alkyl groups such as alkyl. Alkoxy groups can be substituted or unsubstituted. Optionally substituted alkoxy is an alkoxy group optionally substituted as defined herein for alkyl.

[0064] The term "heteroalkyl" as used herein, whether as part of other terms or independently, refers to an alkyl group (e.g., an alkyl group as defined herein) that is interrupted one or more times, each time by one or two heteroatoms. Each heteroatom is independently O, N, or S. A heteroalkyl group does not include two oxygen atoms adjacent to each other. A heteroalkyl group can be substituted or unsubstituted (e.g., an optionally substituted heteroalkyl). When a heteroalkyl is substituted and a substituent is attached to a heteroatom, the substituent is selected according to the nature and valence of the heteroatom. Thus, substituents attached to a heteroatom can include, where valence permits, =O, -N(R N2 )2, -SO2OR N3 , -SO2R N2 , -SOR N3 , -COOR N3 , an N-protecting group, alkyl, aryl, cycloalkyl, heterocyclyl, or cyano, wherein each R N2 is independently H, alkyl, cycloalkyl, aryl, or heterocyclyl, and each R N3is independently alkyl, cycloalkyl, aryl, or heterocyclyl. These substituents can themselves be unsubstituted or substituted with unsubstituted substituents for each corresponding group defined herein. When heteroalkyl is substituted and the substituent is bonded to a carbon atom, it is selected from those described for alkyl, except that the substituent on the carbon atom bonded to the heteroatom is not Cl, Br, or I. In certain embodiments, the carbon atom is at the terminus of the heteroalkyl group. In certain embodiments, the heteroalkyl is PEG.

[0065] The term "heteroalkylene" as used herein, whether as part of another term or used independently, refers to a divalent substituent that is a heteroalkyl in which a hydrogen atom is replaced by a valence. Heteroalkylene groups can be substituted or unsubstituted. Optionally substituted heteroalkylene is optionally substituted as described herein for heteroalkyl.

[0066] The term "heteroaryl" as used herein refers to a monocyclic ring system or a fused or bridged bicyclic, tricyclic, or tetracyclic ring system, the ring system containing 1, 2, 3, or 4 independently selected heteroatoms (selected from the group consisting of nitrogen, oxygen, and sulfur), and at least one ring is aromatic. Heteroaryl groups can be 5-12 membered rings, e.g., 8-12 membered rings, 5-10 membered rings, 5-6 membered rings. Heteroaryl groups have 1-16 carbon atoms, unless otherwise specified. Certain heteroaryl groups can have up to 9 carbon atoms. Non-limiting example heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, quinazolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl (e.g., 1H-1,2,3-triazolyl), tetrazolyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, etc. Bicyclic, tricyclic, and tetracyclic heteroaryl groups contain at least one aromatic ring containing at least one heteroatom as described above. For example, a ring containing at least one heteroatom is fused to one, two, or three carbocyclic rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring. Non-limiting examples of fused heteroaryl groups include 1,2,3,5,8,8a-hexahydroindolizine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, and 2,3-dihydrobenzothiophene. Heteroaryl groups include alkyl, alkoxy, acyloxy, aryloxy, amino, arylalkoxy, cycloalkyl, cycloalkoxy, halogen, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, cyano, =O, -NR 2wherein each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; -COOR A (R A is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl), -CON(R B ) 2 (each R may be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl). Each substituent may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each group.

[0067] The term "heteroarylene" as used herein, whether as part of another term or used independently, refers to a divalent substituent in which one hydrogen atom of a heteroaryl is replaced with a valence. Heteroarylene groups may be substituted or unsubstituted. Optionally substituted heteroarylene is optionally substituted as described herein for heteroaryl.

[0068] The term "heteroaryloxy" as used herein, whether used as part of another term or independently, refers to the structure -OR, where R is heteroaryl. Heteroarylene groups may be substituted or unsubstituted. Optionally substituted heteroaryloxy is optionally substituted as defined for heteroaryl.

[0069] The term "heterocyclyl" as used herein, whether as part of another term or used independently, refers to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused or bridged 4-, 5-, 6-, 7-, or 8-membered rings, unless otherwise specified. The ring system contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The heterocyclyl group may be 3-12 membered, e.g., 4-12 membered, 4-10 membered, 5-12 membered, 5-10 membered, 5-8 membered. The heterocyclyl may be aromatic or non-aromatic. An aromatic heterocyclyl is a heteroaryl as described herein. Non-aromatic 5-membered heterocyclyls have zero or one double bond, non-aromatic 6- and 7-membered heterocyclyl groups have zero to two double bonds, and non-aromatic 8-membered heterocyclyl groups have zero to two double bonds and / or zero or one carbon-carbon triple bond. Heterocyclyl groups have 1 to 16 carbon atoms, unless otherwise specified. Certain heterocyclyl groups may have up to 9 carbon atoms. Non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyranyl, dihydropyranyl, dithiazolyl, and the like. The term "heterocyclyl" also refers to heterocyclic compounds having bridged polycyclic structures in which one or more carbon and / or heteroatoms bridge two non-adjacent portions of a monocyclic ring. For example, quinuclidine, tropane, diazabicyclo[2.2.2]octane, and the like. "Heterocyclyl" includes a class of bicyclic, tricyclic, and tetracyclic rings in which any of the above heterocycles are fused to one, two, or three carbocyclic rings, such as a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or other heterocyclic rings.Non-limiting examples of fused heterocyclyls include 1,2,3,5,8,8a-hexahydroindolizine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, 2,3-dihydrobenzothiophene, etc. Heterocyclyl groups include alkyl, alkoxy, acyloxy, aryloxy, amino, arylalkoxy, cycloalkyl, cycloalkoxy, halogen, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, heterocyclyloxy, heteroaryloxy, hydroxy, nitro, thiol, cyano, =O, =S, -NR. 2 wherein each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; -COOR A (R A is hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl), -CON(R B ) 2 (each R B may be optionally substituted or unsubstituted with 1, 2, 3, 4, or 5 substituents independently selected from hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heteroaryl).

[0070] The term "heterocyclylalkyl" as used herein, whether as part of another term or used independently, refers to an alkyl group substituted with a heterocyclyl group. The heterocyclylalkyl group can be substituted or unsubstituted. The heterocyclyl and alkyl portions of an optionally substituted heterocyclylalkyl can be optionally substituted as described for heterocyclyl and alkyl, respectively.

[0071] The term "heterocyclylene" as used herein, whether as part of another term or used independently, refers to a divalent substituent that is a heterocyclyl in which one hydrogen atom is replaced by a valence. The heterocyclylene group can be substituted or unsubstituted. An optionally substituted heterocyclylene is an optionally substituted heterocyclylene as described herein for heterocyclyl.

[0072] As used herein, the term "thioheterocyclylene" refers to the divalent group -S-R'-, where R' is heterocyclylene as defined herein.

[0073] As used herein, the term "thiol" refers to a --SH group.

[0074] The term "triazolocycloalkenylene" as used herein refers to a cycloalkenylene containing a 1,2,3-triazole ring fused to an 8-membered ring. The inner ring atoms of a triazolocycloalkenylene group are all carbon atoms, and the bridgehead atoms are sp2 hybridized carbon atoms. A triazolocycloalkenylene group can be substituted or unsubstituted. An optionally substituted triazolocycloalkenylene is a triazolocycloalkenylene that is optionally substituted in the manner described for cycloalkenyl.

[0075] The term "triazoloheterocyclylene" as used herein refers to a heterocyclylene that includes a 1,2,3-triazole ring fused to an 8-membered ring that includes at least one heteroatom. The bridgehead atom of the triazoloheterocyclylene is a carbon atom. The triazoloheterocyclylene group can be substituted or unsubstituted. An optionally substituted triazoloheterocyclylene is a triazoloheterocyclylene that is optionally substituted in the manner described for heterocyclyl.

[0076] The term "oxo" as used herein refers to a divalent oxygen atom, for example, the structure of oxo is depicted as =O.

[0077] The terms "halogen" or "halo" as used herein refer to fluoride, chloride, bromide, and iodide, particularly fluoride and chloride, and more particularly fluoride.

[0078] The term "substituted" as used herein means that a chemical group has one or more hydrogen atoms removed and replaced with a substituent. The term "substituent" as used herein has its ordinary meaning and refers to a chemical functional group that is covalently attached to, or fused, where appropriate, to a parent group. It is understood that substitution at a particular atom is limited by valence. Examples of substituents include, but are 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 groups, and aliphatic groups. It is understood that the substituents may be further substituted.

[0079] When a functional group in chemical formula (I) or any of its embodiments is described as being "optionally substituted," it is meant that chemical formula (I) or its embodiments encompass both compounds having the substituent (or substituents) described on the functional group and compounds that do not have the substituent (or substituents) described on the functional group (i.e., the functional group is unsubstituted).

[0080] The term "protecting group" as used herein includes protecting groups that are well known in the chemical arts and are detailed in Greene's Protective Groups in Organic Synthesis, PGM Wuts and TW Greene, 4th Edition, Wiley-Inter science, 2006, which is incorporated herein by reference in its entirety.

[0081] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group, also referred to herein as a "hydroxy protecting group", which refers to a labile chemical functional group that protects the hydroxy group against undesired reactions during synthetic procedures. After synthetic procedures, the hydroxy protecting group can be selectively removed. Suitable hydroxy protecting groups are well known in the chemical arts and include those detailed in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference in its entirety. Non-limiting examples of hydroxy protecting groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (p-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, ... thiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, l-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-Trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzyl doryl, 5-dibenzosuberyl, triphenylmethyl, -naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis (4',4"-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-Butyldimethylsilyl (TBDMS), t-Butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-Butylmethoxyphenylsilyl (TBMPS), formic acid, benzoylformate, acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, methoxyacetic acid, triphenylmethoxyacetic acid, phenoxyacetic acid, p-chlorophenoxyacetic acid, 3-phenylpropionic acid, 4-oxopentanoic acid (levulinic acid), 4,4-(Ethylenedithio)pentanoate (levulinoyl dithioacetal), pivalic acid, adamantic acid, crotonate, 4-methoxycrotonic acid, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoic acid), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), isobutyric acid, Alkyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro -4-Methylpentanoate, o-(dibromomethyl)benzoate, 2-formobenzenesulfonic acid, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid, chlorodiphenylacetic acid, isobutyrate, monosaccharomyces cerevisiae sinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamide, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, tosylate (Ts), 4,4'-dimethoxytriphenylmethyl (DMTr), and the like. In certain embodiments, non-limiting examples of hydroxy protecting groups include acetyl, benzyl, benzoyl, trimethylsilyl, 4,4'-dimethoxytriphenylmethyl (DMTr) and others.

[0082] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group, also referred to as a "thiol protecting group," which refers to a labile chemical functional group that protects the thiol group against undesired reactions during synthetic procedures. After synthetic procedures, the thiol protecting group can be selectively removed. Suitable sulfur protecting groups are well known in the chemical arts and include those detailed in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference in its entirety. Non-limiting examples of thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt), acetamidomethyl (Acm), and the like.

[0083] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group, also referred to as an "amino protecting group," which refers to a labile chemical functional group that protects the amino group against undesired reactions during synthetic procedures. After synthetic procedures, the amino protecting group can be selectively removed. Suitable amino protecting groups are well known in the chemical arts and include those detailed in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference in its entirety. Non-limiting examples of amino protecting groups include acetyl, tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (FMOC), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), and the like.

[0084] In the above definitions, the hydroxy, thiol, and amino protecting groups are not meant to be exhaustive. The function of these protecting groups is to protect a reactive functional group in a preparatory step and then to be removed at some point without affecting the remainder of the molecule. Many protecting groups are known in the chemical arts, and the use of other protecting groups not specifically mentioned here applies as well.

[0085] The term "carbohydrate" as used herein refers to a compound composed of carbon (C), hydrogen (H), and oxygen (O) and has the general formula Cx(H2O)y, where x and y may be the same or different. Carbohydrates include monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides. The term "monosaccharide" as used herein refers to a carbohydrate compound having a single carbon chain in a linear, branched, or cyclic form. Additionally, the terms "disaccharide" and "trisaccharide" refer to molecules in which two or three monosaccharide units are linked by glycosidic bonds, respectively. The terms "oligosaccharide" and "polysaccharide" refer to larger aggregates of about 4-9 and more monosaccharide units, respectively. Monosaccharides or monosaccharide units may have the D- or L-configuration and may contain more than 4 carbon atoms, preferably 4-8 carbon atoms, for example, 4 carbon atoms (telose), 5 carbon atoms (pentose), 6 carbon atoms (hexose), 7 carbon atoms (heptose), or 8 carbon atoms (octose). In certain embodiments, monosaccharides or monosaccharide units may contain 5 or 6 carbon atoms. In disaccharides, trisaccharides, oligosaccharides, and polysaccharides, each monosaccharide unit may be the same or different. Non-limiting examples of monosaccharides include, for example, glucose, fructose, galactose, and the like. Non-limiting examples of disaccharides include, for example, gentiobiose, isomaltose, melibiose, trehalose, sucrose, lactose, maltose, cellobiose, and the like. Non-limiting examples of trisaccharides include, for example, lactosucrose, raffinose, and the like. Non-limiting examples of polysaccharides include, for example, starch, cellulose, glycogen, and the like. The term "carbohydrate" may be used interchangeably herein as a synonym for "sugar" and "saccharide."

[0086] The term "carbohydrate mimetics" as used herein refers to derivatives of carbohydrates or other compounds with multiple hydroxy groups that have some modification in their structure and look like sugars or saccharides. For example, in relation to monosaccharides, carbohydrate mimetics include deoxysugars (where the alcoholic hydroxy group is replaced by a hydrogen), aminosugars (where the alcoholic hydroxy group is replaced by an amino group), thiosugars (where the alcoholic hydroxy group is replaced by a thiol, or where the C=O is replaced by a C=S, or where the ring oxygen in a cyclic form is replaced by a sulfur), selenosugars, tellurosugars, azasugars (where the ring carbon is replaced by a nitrogen), iminosugars (where the ring oxygen is replaced by a nitrogen), phosphanosugars (where the ring acid is replaced by a cyclic sugar), and the like. Examples of monosaccharides include monosaccharides with a carbon atom substituted for a hydrogen atom, phosphasugars (ring carbon substituted for phosphorus), C-substituted monosaccharides (hydrogen at a non-terminal carbon atom substituted for carbon), unsaturated monosaccharides, alditols (carbonyl group substituted for CHOH group), aldonic acids (aldehyde group substituted for carboxyl group), carbasugars (ring oxygen substituted for carbon), ketoaldonic acids, uronic acids, aldaric acids, C-glycosides (anomeric oxygen substituted for carbon), carboxylated sugars, amidated sugars, fused cyclic sugars, and the like. Mimetics may include one or more of such structural modifications. In particular, non-limiting examples of monosaccharide mimetics may include one or more modifications of carbohydrate structures selected from the group consisting of deoxy sugars, amino sugars, N-glycosides, imino sugars, unsaturated sugars, carboxylated sugars, amidated sugars, fused cyclic sugars, and carbasugars of monosaccharides. It should be understood that carbohydrates may be further substituted. Also included are amino sugars with cyclized amino groups such as triazoles. In certain embodiments, the monosaccharide mimetic may be 2-((1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-3,4,5-triol, which may be optionally further substituted. In the context of disaccharides, trisaccharides, oligosaccharides, or polysaccharides, carbohydrate mimetics refer to carbohydrates with one or more monosaccharide units replaced by a mimetic of the monosaccharide as described above.

[0087] In certain embodiments, non-limiting examples of carbohydrate mimetics may have the following structures: [ka] Where: R 1 is H, C 1-6 Alkyl, halogen, or -NH(R 2 ) and R 2 is H or acetyl.

[0088] Each R is independently H, halogen, -CN, -C≡CH, -NH2, -OC 1-6 Alkyl, or C 1-6 Alkyl, -C 1-6 Alkyl and -OC 1-6 The corresponding alkyl of the alkyl is substituted with 0 to 5 halogen atoms. Or, two R's together with the carbon to which they are attached form a C 3-6 Forms a cycloalkyl or 3- to 6-membered heterocycloalkyl group; -C 3-6 The corresponding cycloalkyl of the cycloalkyl and the corresponding heterocycloalkyl of the 3- to 6-membered heterocycloalkyl are substituted with 0 to 5 halogen atoms.

[0089] n is 0, 1, 2, or 3, as permitted by the valence, and in particular n is 0.

[0090] In certain embodiments, non-limiting examples of carbohydrate mimetics may have the following structures: [ka] In certain embodiments, non-limiting examples of carbohydrate mimetics may have the following structures: [ka] In certain embodiments, non-limiting examples of carbohydrate mimetics conjugated to ligands may have the following structures: [ka] where Y is a ligand-linker functional group.

[0091] In certain embodiments, non-limiting examples of carbohydrate mimetics may have the following structures: [ka] In certain embodiments, non-limiting examples of carbohydrate mimetics may have the following structures: [ka] As used herein, the wavy line " [ka] " indicates the point at which one functional group is attached to another functional group.

[0092] For the ligand (i.e., T in formula (I)), both carbohydrates and carbohydrate mimetics may be used. Thus, the term "carbohydrate ligand" as used herein is meant to include carbohydrates, carbohydrate mimetics, or combinations thereof. In certain embodiments, the carbohydrate ligand is selected from the group consisting of N-acetylgalactosamine (GalNAc), allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fuculose, galactosamine, D-galactosaminitol, galactose, glucosamine, N-acetylglucosamine, glucosaminitol, glucose, glucose-6-phosphate, glucose glycerol, and the like. The carbohydrate ligand may be selected from the group consisting of aldehyde, L-glycero-D-mannose-heptose, glycerol, glycerone, gluose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptose, sorbose, tagatose, talose, tartaric acid, threose, xylose, quixulose, and the like, in unprotected or protected form. In a particular embodiment, the carbohydrate ligand is N-acetylgalactosamine (GalNAc) or N-acetylgalactosamine triacetate. Also, in a particular embodiment, the carbohydrate ligand is N-acetylgalactosamine (GalNAc).

[0093] The compounds of formula I may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomeric forms of the compounds of formula I. Each asymmetric center present in the compounds of formula I may independently have an (R) or (S) configuration. When the bonds to the chiral carbons in the structural formulas of the present invention are shown as straight lines, or when the names of the compounds are mentioned without the chiral designation (R) or (S) for the chiral carbons, it is understood that the (R) and (S) configurations of each chiral carbon, and each enantiomer or diastereoisomer and mixtures thereof, are encompassed by the formula or name. The production of specific stereoisomers or mixtures thereof may be specified in the examples in which such stereoisomers or mixtures were obtained, but this does not limit that all stereoisomers and mixtures thereof are within the scope of the present invention.

[0094] The present invention encompasses all possible enantiomers, diastereoisomers, and mixtures of two or more stereoisomers. For example, mixtures of enantiomers and / or diastereoisomers, in all ratios. Thus, enantiomers are subject to the present invention in optically pure form, both levorotary and dextrorotary antipodes, in racemic form, and mixtures of the two enantiomers in all ratios. In the case of cis / trans isomers, the invention includes mixtures of cis and trans forms, and of these forms in all ratios. The preparation of individual stereoisomers may be carried out, if necessary, by separation of the mixture, for example by chromatography or crystallization, by using a stereochemically uniform starting material for the synthesis, or by stereoselective synthesis. If necessary, derivatization may be carried out before the separation of stereoisomers. Separation of the mixture of stereoisomers may be carried out at an intermediate stage during the synthesis of the compound of formula I or on the final racemic product. The absolute stereochemistry may be determined by X-ray crystallography of a crystalline product or crystalline intermediate, derivatized, if necessary, with a reagent containing a stereoatomic center of known configuration. Alternatively, the absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopic analysis.

[0095] The term "therapeutically active agent" as used herein refers to compounds and classes of compounds known to be therapeutically active. For example, the therapeutically active agent may be a therapeutically active oligonucleotide. Non-limiting examples of therapeutically active agents include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), microRNA mimics, anti-miRNA oligonucleotides (AMOs), long non-coding RNAs, peptide nucleic acids (PNAs), helper lipids, and phosphorodiamidate morpholino oligomers (PMOs), where the nucleic acid is unmodified or modified. In certain embodiments, the therapeutically active agent may be an iRNA agent.

[0096] As used herein, the term "targeting functionality" refers to a functionality (e.g., an N-acetylgalactosamine cluster) that specifically binds to or reactively binds to or complexes with a receptor or other receptive functionality associated with a particular target cell population.

[0097] The term "linker" as used herein refers to an organic functional group that connects two parts of a compound. A linker may typically include a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, NHC(O), OC(O), C(O)O, SO, SO2, SO2NH, or a chain of atoms such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aryl alkyl, aryl alkenyl, aryl alkynyl, heteroaryl alkyl, heteroaryl alkenyl, heteroaryl alkynyl, heterocyclyl alkyl, heterocyclyl alkenyl, heterocyclyl alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, etc. These methylenes may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl. wherein R8 is hydrogen, acyl, an aliphatic or substituted aliphatic group. In certain embodiments, the linker is 1-24 atoms in length, preferably 4-24 atoms, more preferably 6-18 atoms, even more preferably 8-18 atoms, and most preferably 8-16 atoms. Other examples of linkers include those described in International Publication No. WO2009 / 082607, U.S. Patent Publication No. 2009 / 0239814, U.S. Patent Publication No. 2012 / 0136042, U.S. Patent Publication No. 2013 / 0158824, and U.S. Patent Publication No. 2009 / 0247608.

[0098] The term "nucleoside" as used herein refers to sugar and nucleobase compounds and known families (e.g., modified and unmodified ribofuranose-nucleobase and 2'-deoxyribofuranose-nucleobase compounds and families). The sugar may be ribofuranose. The sugar may be either modified or unmodified. An unmodified sugar nucleoside is a ribofuranose or 2'-deoxyribofuranose in which the anomeric carbon is bonded to a nucleobase. An unmodified nucleoside is a ribofuranose or 2'-deoxyribofuranose in which the anomeric carbon is bonded to an unmodified nucleobase. Non-limiting examples of unmodified nucleosides include adenosine, cytidine, guanosine, uridyl, 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the nucleobase modifications and sugar modifications described herein. A nucleobase modification is the replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification is, for example, 2'-substitution, locking, carbocyclization, or unlocking. A 2'-substitution is the replacement of the 2'-hydroxy of a ribofuranose with 2'-fluoro, 2'-methoxy, or 2'-(2-methoxy)ethoxy. A locking modification is the incorporation of a bridge between the 4'-carbon atom and the 2'-carbon atom of a ribofuranose. Nucleosides with locking modifications are known as bridged nucleosidic acids (BNAs), including, for example, locked nucleosidic acids (LNAs), ethylene-bridged nucleosidic acids (ENAs), and cEt nucleosidic acids. These bridged nucleosidic acids are commonly used as affinity-enhancing nucleosides.

[0099] As used herein, the term "nucleotide" refers to a nucleoside having the following structure: 1 -P(X 2 )(R 1 )2 or a monovalent functional group.1 is O, S, or NH, and X 2 is absent, =O, or =S, and each R 1 are independently -OH, -N(R 2 )2, or -O-CH2CH2CN, where each R 2 are independently optionally substituted alkyl, or both R 2 The groups, together with the nitrogen atom to which they are attached, form an optionally substituted heterocyclyl.

[0100] The term "oligonucleotide" refers to a structure in which 10 or more (e.g., 10-50) adjacent nucleosides are covalently linked together by internucleoside bonds. An oligonucleotide includes a 5' end and a 3' end. The 5' end of an oligonucleotide may be, for example, a hydroxyl group, a targeting functional group, a hydrophobic functional group, a 5' cap, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, a triphosphorothioate, a phosphorodithioate, a diphosphorodithioate, a triphosphorodithioate, a phosphonate, a phosphoramide, an intracellular internalization peptide, an endosomal escape functional group, or a neutral organic polymer. The 3' end of the oligonucleotide may be, for example, a hydroxyl group, a targeting functional group, a hydrophobic functional group, a phosphate, a diphosphate, a triphosphate, a phosphorothioate, a diphosphorothioate, a triphosphorothioate, a phosphorodithioate, a diphosphorodithioate, a triphosphorodithioate, a phosphonate, a phosphoramide, an internalization peptide, an endosomal escape functional group, or a neutral organic polymer (e.g., polyethylene glycol). An oligonucleotide having a 5'-hydroxyl group or a 5'-phosphate has an unmodified 5' end. An oligonucleotide having a 5' end other than a 5'-hydroxyl group or a 5'-phosphate has a modified 5' end. An oligonucleotide having a 3'-hydroxyl group or a 3'-phosphate has an unmodified 3' end. An oligonucleotide having a 3' end other than a 3'-hydroxyl group or a 3'-phosphate has a modified 3' end.

[0101] As used herein, " [ka] " refers to an oligonucleotide (e.g., an iRNA agent), unless otherwise indicated by context.

[0102] Unless otherwise stated, structures depicted herein are also intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the depicted structures in which a hydrogen is replaced by deuterium or tritium, or a carbon is replaced by 13 C or 14 It is within the scope of the invention that C is substituted. Such compounds may be useful, for example, as analytical tools, as probes in biological assays, or as therapeutically active agents in accordance with the present invention. Ligand

[0103] The ligand can be any ligand described herein, including, for example, ligands selected from groups such as carbohydrate ligands, polypeptide ligands, and aliphatic ligands. The ligand can be either protected or unprotected. Preferred exemplary ligands include N-acetylgalactosamine (GalNAc), such as N-acetyl-D-galactosylamine, and N-acetylgalactosamine triacetate.

[0104] Other suitable ligands are described in U.S. Patent Publication No. 2009 / 073809, U.S. Patent Publication No. 2012 / 0136042, U.S. Patent Publication No. 2013 / 0158824, U.S. Patent Publication No. 2009 / 0247608, U.S. Patent No. US8106022, International Patent Application No. WO2009 / 073809, and the like, which are incorporated herein by reference.

[0105] Ligand functional groups (e.g., carbohydrate functional groups) facilitate delivery of oligonucleotides to target sites. One way in which ligand functional groups improve delivery is through receptor-mediated endocytosis activity. Without being bound by theory, it is believed that this uptake mechanism involves the binding of oligonucleotides to membrane receptors and the movement into the interior of a membrane-enclosed envelope by invagination of the membrane structure or fusion of the delivery system with the cell membrane. This process is initiated by activation of a cell surface or membrane receptor after a specific ligand binds to the receptor. Receptor-mediated endocytosis systems include those that recognize sugars such as galactose. Thus, the ligand functional group may include one or more monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides, including those mentioned above. In a preferred embodiment, the ligand functional group may be a functional group recognized by a human asialoglycoprotein receptor (ASGPR), such as human asialoglycoprotein receptor 2 (ASGPR2). Such sugar functionalities may include, for example, sugars such as galactose or N-acetyl-D-galactosylamine. Oligonucleotides

[0106] An oligonucleotide is a chemically modified or unmodified nucleic acid molecule (RNA or DNA) that may have a length of less than 100 nucleotides. For example, less than 50 nucleotides. A nucleic acid may be (i) a single-stranded DNA or RNA, (ii) a double-stranded DNA or RNA (including a double-stranded DNA or RNA with a hairpin loop), or (iii) a DNA / RNA hybrid. Non-limiting examples of double-stranded RNA include siRNA (small interfering RNA). Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, microRNAs, and triplex-forming oligonucleotides. In certain embodiments, the length of the oligonucleotide ranges from about 5 to about 50 nucleotides. For example, from about 10 to about 50 nucleotides. In certain embodiments, the length of the oligonucleotide ranges from about 6 to about 30 nucleotides, for example, from about 15 to about 30 nucleotides, for example, from about 18 to about 23 nucleotides.

[0107] The oligonucleotides described herein may be siRNAs, microRNAs, anti-microRNAs, microRNA mimics, antimiRs, antagomirs, dsRNAs, ssRNAs, aptamers, immunostimulatory agents, decoy oligonucleotides, splice-altering oligonucleotides, triplex-forming oligonucleotides, G-quadruplexes, or antisense oligonucleotides. In certain embodiments, the oligonucleotide is an iRNA agent.

[0108] The term "iRNA agent" as used herein refers to an RNA agent (or an agent that degrades into an RNA agent) that can downregulate expression of a target gene, preferably an endogenous or pathogenic target RNA. Without being bound by theory, an iRNA agent may act by one or more of several mechanisms, including post-transcriptional cleavage of the target mRNA, known as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent may include a single strand or multiple strands. For example, it may be a double-stranded iRNA agent. If the iRNA agent is single-stranded, it may include a 5' modification that includes one or more phosphate groups or analogs of phosphate groups. In certain embodiments, the iRNA agent is double-stranded.

[0109] iRNA agents include those that have sufficient homology with the target gene and are of sufficient nucleic acid length so that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. RNA agents include or are those that are at least partially, and in certain embodiments, completely complementary to the target RNA. While perfect complementarity between an iRNA agent and a target is not required, sufficient correspondence is preferred so that the iRNA agent, or its cleavage product, directs sequence-specific silencing, e.g., by cleavage of the target RNA (e.g., mRNA) by RNAi.

[0110] The nucleotides of an iRNA agent may be modified (e.g., one or more nucleotides contain a 2'-F or 2'-OCH3 group) or may contain nucleotide substitutes. The single stranded segments of an iRNA agent may be modified or contain nucleoside substitutes. For example, the unpaired segments or segments of a hairpin structure, e.g., those that connect two complementary segments, may have modifications or nucleoside substitutes. Modifications to stabilize one or more 3' or 5' ends of an iRNA agent, e.g., against exonucleases. Modifications include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents provided as phosphoramidation reagents with additional DMT-protected hydroxy groups to allow multiple attachments during RNA synthesis, etc. Modifications include, for example, modifications at the 2'-OH group of the ribose sugar, eg, the use of deoxyribonucleotides, such as deoxythymidine, instead of ribonucleotides, modifications at the phosphate group, eg, the use of phosphothioate modifications, and the like.

[0111] In certain embodiments, different strands contain different modifications. In certain embodiments, the strands are selected such that the iRNA agent contains single-stranded or unpaired regions at one or both ends of the molecule. A double-stranded iRNA agent is preferably paired with an overhanging strand, e.g., one or two 5' or 3' overhangs (preferably a 3' overhang of at least 2-3 nucleotides). For example, an iRNA agent may have a single-stranded overhang, such as a 3' overhang of 1, 2, or 3 nucleotides in length, at each end. The overhangs may be the result of one strand being longer than the other, or two strands of the same length being staggered.

[0112] Preferred lengths of duplexes between strands of iRNA agents range from 6 to 30 nucleotides. Preferred duplexes are 15 to 30 nucleotides in length, most preferably 18, 19, 20, 21, 22, or 23 nucleotides in length. Other preferred duplexes are 6 to 20 nucleotides in length, most preferably 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.

[0113] The oligonucleotides may be those described in U.S. Patent Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, 2009 / 0247608, all of which are incorporated by reference herein.

[0114] The term "single-stranded siRNA" as used herein refers to a siRNA compound that is composed of a single molecule.It may include the duplex domain formed by intrastrand pairing, and may include or be, for example, a hairpin structure or a panhandle structure.Single-stranded siRNA compounds may be antisense to target molecules.

[0115] Single-stranded siRNA may be long enough to enter RISC and participate in RISC-mediated cleavage of target mRNA.Single-stranded siRNA compound is at least 14 nucleotides, and in other embodiments is at least 15, 20, 25, 29, 35, 40 or 50 nucleotides long.In certain embodiments, it is less than 200, 100 or 60 nucleotides long.

[0116] Hairpin siRNA compounds have a duplex range of equal or at least 17, 18, 19, 29, 21, 22, 23, 24, 25 nucleotide pairs. The duplex range may be 200, 100, or 50 nucleotides or less in length. In certain embodiments, the duplex range is 15-30, 17-23, 19-23, 19-21 nucleotide pairs in length. The hairpin may have a single-stranded overhang or a terminal unpaired range. In certain embodiments, the overhang is 2-3 nucleotides in length. In certain embodiments, the overhang is on the sense side of the hairpin, or in certain embodiments, on the antisense side of the hairpin.

[0117] The term "duplex-stranded siRNA compound" as used herein refers to a siRNA compound that contains one or more, or in some cases two, strands, which can form a duplex structure upon hybridization between the strands.

[0118] The antisense strand of a double-stranded siRNA compound may be 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It may be 200, 100, or 50 nucleotides or less in length. The range may be 17-25, 19-23, or 19-21 nucleotides in length. The term "antisense strand" refers to the strand of a siRNA compound that has sufficient complementarity to a target molecule, such as a target RNA. The sense strand of a double-stranded siRNA compound may be 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It may be 200, 100, or 50 nucleotides or less in length. The range may be 17-25, 19-23, or 19-21 nucleotides in length.

[0119] The duplex portion of the duplex siRNA compound may be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It may be 200, 100, or 50 nucleotide pairs or less in length. The range may be 15-30, 17-23, 19-23, 19-21 nucleotide pairs in length. In many embodiments, the siRNA compound is large enough to be cleaved by endogenous molecules (e.g., Dicer) to generate smaller siRNA compounds (e.g., siRNA agents).

[0120] The sense and antisense strands may be selected such that the double-stranded siRNA compound contains a single strand or a non-paired region at one or both ends of the molecule. Thus, the double-stranded siRNA compound may be paired to contain a sense strand and an antisense strand and an overhang (e.g., one or two 5' or 3' overhangs, or a 3' overhang of 1-3 nucleotides). The overhang may be the result of one strand being longer than the other, or two strands of the same length being offset. In some embodiments, there is a 3' overhang at at least one end. In certain embodiments, there is a 3' overhang at both ends of the siRNA molecule. In certain embodiments, the overhang is 2 nucleotides.

[0121] In certain embodiments, the length of the duplex ranges from 15 to 30, or 18, 19, 20, 21, 22, 23 nucleotides (e.g., within the ranges of ssiRNA compounds listed above). ssiRNA compounds may resemble the length and structure of the natural products of Dicer processing of longer dsiRNAs. Also included are embodiments in which the two strands of the ssiRNA compound are linked, for example, by a covalent bond. Hairpins or other single-stranded structures that provide the required duplex range and 3' overhang are also contemplated.

[0122] The siRNA compounds, double-stranded siRNA compounds and single-stranded siRNA compounds described herein may mediate the silencing of target RNA, such as mRNA (e.g., the transcript of a gene coding for a protein).For convenience, such mRNA or gene is also referred to as target gene.It is also referred to herein as the mRNA to be silenced.Generally, the RNA to be silenced is an endogenous gene or a pathogen gene.In addition, RNA other than mRNA (e.g., tRNA, viral RNA) may also be targeted.

[0123] As used herein, the term "mediating RNAi" refers to the ability to silence a particular RNA in a sequence-specific manner. Without being bound by theory, it is believed that silencing occurs using the RNAi machinery or process and a guide RNA, such as a 21-23 base ssiRNA compound.

[0124] In certain embodiments, the siRNA compound is "sufficiently complementary" to, for example, silence the production of the protein encoded by the target mRNA. In certain embodiments, the siRNA compound is "fully complementary" by, for example, forming a perfect Watson-Crick base pair with the target RNA in its perfect complementary range, where the siRNA compound anneals with the target RNA. A "fully complementary" target RNA can include an internal range (e.g., at least 10 bases) that is completely complementary to the target RNA. Furthermore, in certain embodiments, the siRNA compound specifically discriminates between single nucleotide differences. In this case, the siRNA compound mediates RNAi only when perfect complementarity of a single nucleotide difference (e.g., within a range of 7 bases) is found.

[0125] MicroRNAs (miRNAs) are a class of highly conserved small RNA molecules that are transcribed from DNA in plant and animal genomes but are not translated into proteins. Processed miRNAs are single-stranded RNA molecules of ~17-25 nucleotides (nt) that are incorporated into RNA-induced silencing complexes (RISCs) and have been identified as important regulators of development, cell proliferation, apoptosis, and differentiation. They are believed to play a role in regulating gene expression by binding to the 3'-untranslated domain of specific mRNAs. RNA-induced silencing complexes (RISCs) mediate downregulation of gene expression by translational repression, transcript cleavage, or both. RISCs have also been implicated in transcriptional silencing in the nuclei of a wide range of eukaryotic organisms.

[0126] The number of miRNA sequences identified to date is large and growing, examples of which can be found in, 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 http: / / microrna.sanger.ac.uk / sequences / .

[0127] In certain embodiments, the nucleic acid is an antisense oligonucleotide opposite the target polynucleotide. As used herein, the term "antisense oligonucleotide" or simply "antisense" is meant to include oligonucleotides that are complementary to a target polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA that is complementary to a selected sequence, such as a target gene mRNA. Antisense oligonucleotides are believed to suppress gene expression by binding to complementary mRNA. Binding to the target mRNA may lead to suppression of gene expression by inhibiting translation of the complementary mRNA strand or by causing degradation of the target mRNA. Antisense DNA is used to target a specific, complementary (coding or non-coding) RNA. If this DNA / RNA hybrid is formed, it may be degraded by the RNase H enzyme. In certain embodiments, the antisense oligonucleotide contains about 10 to about 50 nucleotides, but more particularly about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be completely complementary to the desired target gene. Thus, it is contemplated that non-target specific activity may be found with antisense and that the most preferred antisense sequence for a particular use may contain one or more mismatches with the target sequence.

[0128] Antisense oligonucleotides have been demonstrated to be effective and target-specific inhibitors of protein synthesis and therefore may be used to specifically inhibit protein synthesis by a particular gene. The inhibitory effect of antisense oligonucleotides on protein synthesis is well established. Methods for producing antisense oligonucleotides are already known and antisense oligonucleotides can be easily adapted to target any polynucleotide sequence. Selection of antisense oligonucleotide sequences specific for a target sequence is based on analysis of the selected target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides may be selected based on their relative ability to form dimers, hairpins, or other secondary structures that reduce or inhibit specific binding to the target mRNA in the host cell. Highly preferred target regions of mRNA include those near the AUG translation initiation codon and those that are largely complementary to the 5' region of the mRNA.

[0129] Antagomirs are RNA-like oligonucleotides with various modifications that enhance RNAse protection and pharmacological properties, such as enhanced uptake into tissues and cells. Unlike normal RNA, they have, for example, complete 2'-O-methylation of sugars, a phosphorothioate backbone, and a cholesterol group at the 3'-terminus. Antagomirs can be used to efficiently silence endogenous miRNAs by forming a duplex containing the antagomir and the endogenous miRNA. An example of antagomir-mediated miRNA silencing is the silencing of miR-122 described in Krutzfeldt et al., Nature, 2005, 438: 685-689, which is expressly incorporated herein by reference in its entirety. Antagomir RNA can be synthesized using standard solid phase oligonucleotide synthesis protocols. See U.S. Patent Application Publication No. 2007 / 0123482 and U.S. Patent Application Publication No. 2007 / 0213292, each of which is incorporated herein by reference in its entirety.

[0130] Antagomires can include ligand-binding monomer subunits and monomers for oligonucleotide synthesis. Non-limiting example monomers are described in U.S. Patent Application Publication No. 2005 / 0107325, which is incorporated herein by reference in its entirety. Antagomires can have a ZXY structure, such as those described in International Patent Application No. WO2004 / 080406, which is incorporated herein by reference in its entirety. Antagomires can be conjugated to amphiphilic moieties. Non-limiting example amphiphilic moieties for use in oligonucleotide agents are described in International Patent Application No. WO2004 / 080406, which is incorporated herein by reference in its entirety.

[0131] Aptamers are nucleic acid or peptide molecules that bind with high affinity and specificity to a particular molecule of interest (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). DNA or RNA aptamers have been successfully generated that bind to a variety of entities, from large proteins to small organic molecules. See Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, 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). Aptamers can be RNA or DNA based and can include riboswitches. A riboswitch is a part of an mRNA molecule that directly binds a small target molecule, and the binding of the target affects the activity of the gene. Thus, the mRNA containing the riboswitch is directly involved in regulating its own activity depending on the presence or absence of its target molecule. In general, aptamers are engineered through repeated rounds of in vitro selection or its equivalent SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind 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. Additionally, the term "aptamer" specifically includes "secondary aptamers" that contain consensus sequences derived from comparing two or more known aptamers for a particular target.

[0132] In another embodiment, the nucleic acid-lipid particle is associated with a ribozyme, which is an RNA molecular complex that contains a specific catalytic domain and has endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987 Dec, 84(24): 8788-92; Forster and Symons, Cell. 1987 Apr 24, 49(2): 211-20). For example, many ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of multiple phosphates in an oligonucleotide substrate (Cech et al, Cell. 1981 Dec, 27 (3 Pt 2): 487-96; Michel and Westhof, J Mol Biol. 1990 Dec 5, 216(3): 585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14;357(6374): 173-6). This specificity is ascribed to the requirement that the substrate bind, via specific base pairing, to the ribozyme's internal guide sequence ("IGS") before the chemical reaction can occur.

[0133] Currently, at least six basic types of naturally occurring enzymatic RNAs are known. Each can catalyze the hydrolysis of phosphodiester bonds in RNA in trans (and can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding is achieved through the target binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule, which then acts to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes and binds to a target RNA through complementary base pairing, and after binding to the correct site, enzymatically cleaves the target RNA. Such strategic cleavage of the target RNA destroys its ability to direct the synthesis of its encoded protein. After an enzymatic nucleic acid has bound and cleaved an RNA target, it is released from that RNA and can search for another target, repeatedly binding and cleaving new targets.

[0134] Enzymatic nucleic acid molecules may be formed, for example, with hammerhead, hairpin, Hepatitis delta virus, group I intron, or RNaseP RNA (in combination with an RNA guide sequence) or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described by Rossi et al., Nucleic Acids Res. 1992 Sep 11, 20(17):4559-65. Examples of hairpin motifs are described by Hampel et al., (European Patent Application Publication No. EP0360257), Hampel and Tritz, Biochemistry 1989 Jun 13, 28(12): 4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan 25, 18(2): 299-304, and U.S. Patent No. 5,631,359. Examples of Hepatitis delta virus motifs are described by Perrotta and Been, Biochemistry, 1992 Dec 1, 31(47): 11843-52, examples of RNase P motifs are described by Guerrier-Takada et al., Cell. 1983 Dec, 35(3 Pt 2): 849-57, Neurospora VS RNA ribozyme motifs are described by Saville and Collins, Cell, 1990 May 18, 61(4): 685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991 Oct 1, 88(19): 8826-30; Collins and Olive, Biochemistry, 1993 Mar 23, 32(11): 2795-9, and examples of Group I introns are described by U.S. Patent No. 4,987,071. An important characteristic of the enzymatic nucleic acid molecule used is that it has a specific substrate binding site complementary to one or more of the DNA or RNA segments of the target gene, and that nucleotide sequences within or surrounding the substrate binding site confer RNA cleavage activity to the molecule. Thus, construction of ribozymes need not be limited to the specific motifs mentioned herein.

[0135] Methods for producing ribozymes to any polynucleotide sequence are known in the art. Ribozymes can be designed and synthesized for testing in vitro and in vivo, as described in International Patent Applications WO 93 / 23569 and WO 94 / 02595.

[0136] Ribozyme activity can be optimized by chemically synthesizing ribozymes with modifications to vary the length of the ribozyme binding arms or to prevent degradation by serum ribonucleases (e.g., various chemical modifications of the sugar moiety described in International Patent Application Publication Nos. WO 92 / 07065, WO 93 / 15187, WO 91 / 03162, European Patent Application Publication No. 92110298.4, U.S. Pat. No. 5,334,711, International Patent Application Publication No. WO 94 / 13688, etc.). These modifications include removing the stem II base to increase efficacy within the cell or to shorten RNA synthesis time and reduce chemical requirements.

[0137] The nucleic acids associated with the lipid particles are immunostimulatory and include single- or double-stranded immunostimulatory oligonucleotides (ISS) capable of inducing an immune response when administered to a particular subject, e.g., a mammal or other patient, including, for example, certain palindromes that form hairpin secondary structures (see Yamamoto S. et al., (1992) J. Immunol. 148: 4072-4076), CpG motifs, and other known ISS features (e.g., multiple G domains, see WO 96 / 11266).

[0138] The immune response may be an innate or adaptive immune response. The immune system is divided into a more innate immune system and the adaptive adaptive immune system of vertebrates, the latter being further divided into humoral cellular components. In certain embodiments, the immune response may be associated with mucosa. In certain embodiments, the immunostimulatory nucleic acid is only immunostimulatory when administered in combination with lipid particles, and not immunostimulatory when administered in "free form". Such oligonucleotides are considered to be immunostimulatory. An immunostimulatory nucleic acid is considered to be non-sequence specific when it is not required to specifically bind to and reduce the expression of a particular target polynucleotide. Thus, a particular immunostimulatory nucleic acid may contain a sequence that corresponds to a naturally occurring gene or mRNA category, yet still be considered a non-sequence specific immunostimulatory nucleic acid.

[0139] In certain embodiments, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may or may not be methylated. In certain embodiments, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide with a methylated cytosine. In certain embodiments, the nucleic acid comprises a single CpG dinucleotide, and the cytosine within the CpG dinucleotide is methylated. In another embodiment, the nucleic acid comprises at least two CpG dinucleotides, and at least one cytosine within the CpG dinucleotide is methylated. In a further embodiment, each cytosine within the CpG dinucleotide present in the sequence is methylated. In certain embodiments, the nucleic acid comprises a plurality of CpG dinucleotides, and at least one of the CpG dinucleotides comprises a methylated cytosine.

[0140] In nucleic acid sequences, "G", "C", "A", "T" and "U" refer to nucleotides containing guanine, cytosine, adenine, thymidine and uracil, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" may refer to modified nucleotides or alternative substitutes. Experts are well aware that guanine, cytosine, adenine and uracil may be substituted with other groups without significantly altering the base pairing properties of an oligonucleotide. For example, and without limitation, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine may be substituted in the nucleotide sequence of a dsRNA characterized in the present invention by, for example, a nucleotide containing inosine. Sequences containing such substitutes may be suitable for the compositions and methods described herein.

[0141] The term "solid support" as used herein specifically refers to a particle, bead, or surface on which synthesis is performed. Solid supports that can be used in different embodiments of the processes described herein may be selected from, for example, inorganic and organic supports. Non-limiting examples of inorganic supports include silica gel and controlled pore glass (CPG). Non-limiting examples of organic supports include highly cross-linked polystyrene, Tentagel (a graft copolymer of polyethylene glycol (PEG or POE) grafted onto a low cross-linked polystyrene matrix), polyvinyl acetate (PVA), Poros (polystyrene / divinylbenzene copolymer), aminopolyethylene glycol, cellulose, and the like. In certain embodiments, solid supports may include hydrophobic ones. Some embodiments of the present invention may utilize polystyrene-based solid supports. Many other solid supports are commercially available and suitable for the present invention.

[0142] Usage The present invention relates to ligand (e.g., carbohydrate ligand) conjugates of oligonucleotides (e.g., iRNA agents) or other therapeutically active agents that have one or more advantageous properties, such as improved delivery of the oligonucleotides or other therapeutically active agents in vivo and / or in vitro, reduced manufacturing costs, fewer manufacturing problems, or improved chemical stability. These conjugates provide for effective delivery of the oligonucleotides or other therapeutically active agents. In certain embodiments, the ligand conjugates of the present invention are prepared and used to deliver therapeutically active agents to cells, tissues, and organs. Non-limiting examples of therapeutically active agents include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), microRNA mimics, anti-miRNA oligonucleotides (AMOs), long non-coding RNAs, peptide nucleic acids (PNAs), helper lipids, and phosphorodiamidate morpholino oligomers (PMOs), which may be modified or unmodified.

[0143] In certain embodiments, the ligand conjugates of the invention are delivered to and contacted with cells. In certain embodiments of the invention, the contacted cells are in culture, and in other embodiments, in a subject. Non-limiting examples of cells that may be contacted with the ligand conjugates of the invention include hepatocytes, muscle cells, cardiomyocytes, circulating cells, neuronal cells, glial cells, adipocytes, skin cells, hematopoietic cells, epithelial cells, immune system cells, endocrine cells, exocrine cells, endothelial cells, sperm, oocytes, muscle cells, adipocytes, kidney cells, liver cells, or pancreatic cells. In certain embodiments, the cells contacted with the ligand conjugates of the invention are liver cells.

[0144] The ligand conjugates of the invention are useful for targeting genes whose expression is undesirable in a subject, such as TTR for hereditary transthyretin-mediated amyloidotic polyneuropathy, ALAS1 for acute hepatic porphyria (AHP), GO for primary hyperoxaluria type 1 (PH1), PCSK9 for hypercholesterolemia, AGT for hypertension, LPA for atherosclerotic cardiovascular disease (CVD), or ANGPTL3 for lipid disorders.

[0145] The ligand conjugates of the present invention may also be used to treat disorders in a subject, including disorders characterized by unwanted cell proliferation, hematological disorders, metabolic disorders, liver disorders, complement-mediated disorders, rare genetic diseases, and inflammation or chronic viral infections. For example, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH). In certain embodiments, the disorder in a subject is treated by administering one or more ligand conjugates of the present invention having a sequence substantially identical to the sequence of a gene involved in the disorder.

[0146] The ligand conjugates of the present invention may be useful for targeting genes whose expression in the liver is undesirable. For example, the ligand conjugates of the present invention may target nucleic acids expressed by hepatitis viruses (e.g., hepatitis C, hepatitis B, hepatitis A, hepatitis D, hepatitis E, hepatitis F, hepatitis G, or hepatitis H).

[0147] The ligand conjugates of the present invention may also be used to treat other liver disorders characterized by unwanted cell proliferation, hematological disorders, metabolic disorders, and inflammation. Liver proliferation disorders may be, for example, benign or malignant disorders, such as cancer, e.g., hepatocellular carcinoma (HCC), liver metastasis, or hepatoblastoma. Liver hematological or inflammatory disorders may be disorders involving clotting factors, complement-mediated inflammation, or fibrosis. Liver metabolic diseases include lipid disorders and irregularities in glucose regulation. In certain embodiments, liver disorders are treated by administering one or more ligand conjugates of the present invention having a sequence substantially identical to the sequence of a gene involved in liver disorders.

[0148] In certain embodiments, the ligand conjugates of the invention target a nucleic acid expressed in a subject, such as angiopoietin-like protein 3 RNA, apolipoprotein C3 RNA, proprotein convertase subtilisin / kexin type 9 (PCSK9) RNA, LAP RNA, angiotensinogen (AGT) RNA, c-jun RNA, beta-catenin RNA, or glucose-6-phosphatase RNA.

[0149] In certain embodiments, the ligand conjugates of the invention target nucleic acids expressed in the liver, such as ApoB RNA, c-jun RNA, beta-catenin RNA, or glucose-6-phosphatase mRNA.

[0150] Thus, in one aspect, the invention relates to a method of modulating expression of a target gene comprising delivering to a cell a ligand conjugate as described herein. In certain embodiments, the target gene is associated with a metabolic disease. In certain embodiments, the metabolic disease is lipid disorder. In certain embodiments, the target gene is associated with a liver disease. In certain embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), HBV, liver fibrosis, and liver cirrhosis.

[0151] In certain embodiments, a biological sample may be obtained and the delivery of a therapeutically active agent, such as a nucleic acid, may be evaluated using the ligand conjugates described herein. As used herein, the term "biological sample" refers to any sample, including a tissue sample (e.g., a tissue section or a needle biopsy of tissue), a cell sample (e.g., a cytology smear (e.g., a Pap smear or a blood smear) or a sample of cells obtained by microdissection), a whole biological sample (e.g., a yeast or bacterial sample), or a cell fraction, fragment, or organelle (a cell disrupted and its components separated, such as by centrifugation). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspirate, milk, vaginal fluid, saliva, swabs (e.g., buccal swabs), and the like, which are any material containing a biomolecule derived from a first biological sample.

[0152] In one aspect, the invention relates to the manufacture of a medicament using the ligand conjugate of the invention to regulate expression of a target gene in a cell. In a particular embodiment, the target gene is associated with a metabolic disease. In a particular embodiment, the metabolic disease is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis. In a particular embodiment, the target gene is associated with a liver disease. In a particular embodiment, the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), HBV, liver fibrosis, and liver cirrhosis.

[0153] In one aspect, the invention relates to the use of a ligand conjugate of the invention in a method for regulating expression of a target gene in a cell, wherein the ligand conjugate of the invention is delivered to the cell. In certain embodiments, the target gene is associated with a metabolic disease. In certain embodiments, the metabolic disease is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis. In certain embodiments, the target gene is associated with a liver disease. In certain embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), HBV, liver fibrosis, and liver cirrhosis.

[0154] Administration In certain embodiments, the ligand conjugates of the invention are administered to a subject. The ligand conjugates described herein may be used to deliver a therapeutically active agent to cells in a subject. In certain embodiments, the therapeutically active agent is an oligonucleotide. In certain embodiments, the oligonucleotide comprises an inhibitor RNA, or an siRNA molecule, selected to reduce expression of a target gene of the siRNA upon delivery. In certain embodiments, the invention relates to a method of treating a disease or condition associated with expression of a gene in a cell or group of cells of a subject, where administration of an iRNA agent reduces expression of the gene and treats the disease or condition in the subject. Administration of the ligand conjugates of the invention may be performed using routine methods.

[0155] The term "subject" as used herein refers to a human or vertebrate mammal, including, but not limited to, dogs, cats, horses, goats, cows, sheep, rodents, and primates (e.g., monkeys). Thus, the present invention may be used to treat diseases or conditions in human and non-human subjects. For example, the conjugates, compositions, and methods of the present invention may be used in veterinary applications as well as in human prophylactic and therapeutic regimens. In certain embodiments, the subject is a livestock animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human (e.g., male, female, or child). The human may be of either gender and at any stage of development. In certain embodiments, the subject has been diagnosed with a condition or disease to be treated. In other embodiments, the subject is at risk for developing a condition or disease. In certain embodiments, the subject is an experimental animal (e.g., a mouse, rat, rabbit, dog, pig, or primate).

[0156] The terms "administration" and "administering" as used herein refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound of the invention, or a pharmaceutical composition thereof. The terms "treatment" and "treating" refer to reversing, alleviating, delaying the onset, or inhibiting the progression of a "pathological state" (e.g., a disease, disorder, or condition, or one or more signs or symptoms thereof) as described herein. In certain embodiments, treatment may be administered after one or more signs or symptoms of a disease or condition have occurred or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease or condition. For example, treatment may be administered to susceptible individuals prior to the onset of symptoms, taking into account the history of the condition and genetic or other susceptibility factors. Treatment may be continued after symptoms have resolved, e.g., to delay or prevent recurrence. As used herein, the terms "disease," "disorder," "condition," and "pathological state" are used interchangeably.

[0157] Dosage levels may be determined by one skilled in the art by routine experimentation. In certain embodiments, the unit dose may include a range of about 0.01 mg / kg to about 100 mg / kg body weight for siRNA. Alternatively, the dose may range from 10 mg / kg to 25 mg / kg body weight, or 1 mg / kg to 10 mg / kg body weight, or 0.05 mg / kg to 5 mg / kg body weight, or 0.1 mg / kg to 5 mg / kg body weight, or 0.1 mg / kg to 1 mg / kg body weight, or 0.1 mg / kg to 0.5 mg / kg body weight, or 0.5 mg / kg to 1 mg / kg body weight. Clinical trials are routinely used to assess dosage levels of therapeutically active agents.

[0158] The ligand conjugates of the present invention may be formulated as pharma- ceutically acceptable salts. When used as pharmaceuticals, the salts should be pharma- ceutically acceptable, although non-pharmaceutical acceptable salts may be conveniently used to prepare pharma- ceutically acceptable salts and are not excluded from the scope of the present invention.

[0159] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that, within the scope of sound medical judgment, does not produce excessive toxicity, irritation, allergic reaction, or the like upon contact with the tissues of humans or other animals and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.

[0160] Pharmaceutically acceptable salts of the compounds of the present invention include those obtained from suitable inorganic and organic acids and bases. These salts may be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate acid with the free base form of the appropriate compound. Pharmacologically and pharmaceutically acceptable salts include, for example, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formate, malonate, succinic acid, etc. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, calcium salts.

[0161] Representative acid addition salts include acetate, adipate, alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentamiate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, malonate, and the like. The basic moieties of the compounds disclosed herein include methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids used to form pharma-ceutically acceptable salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid.

[0162] Representative base salts are formed from bases which form non-toxic salts. Examples include, but are not limited to, aluminum, arginine, benzathine, calcium, choline, diethylamine, bis(2-hydroxyethyl)amine (diolamine), glycine, lysine, magnesium, meglumine, 2-aminoethanol (olamine), potassium, sodium, 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris or tromethamine), and zinc salts. Hemi-salts of acids and bases may also be formed, such as hemisulfate and hemicalcium salts. For a review of suitable salts, see, for example, Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0163] The ligand conjugates of the present invention may be formulated in association with a solvent, typically by a solvation reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds of the present invention may be prepared, for example, in crystalline form and solvated. Suitable solvates include pharma- ceutically acceptable solvates, including both quantitative and non-quantitative solvates. In certain cases, the solvates may be separable, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. The term "solvate" encompasses both solution-phase and separable solvates. Representative solvates include hydrates, ethanolates, and methanolates. The term "hydrate" refers to a compound associated with water. Typically, the number of water molecules contained in a hydrate of a compound is a fixed ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound may be represented, for example, by the general formula RxH2O, where R is the compound and x is a number greater than 0. A given compound may form multiple types of hydrates, including monohydrates (x is 1), hypohydrates (x is greater than 0 and less than 1, e.g., hemihydrate (R0.5H2O)), and polyhydrates (x is greater than 1, e.g., dihydrate (R2H2O) or hexahydrate (R6H2O)).

[0164] A variety of administration routes are available for the ligand conjugates of the present invention. The choice of a particular delivery mode depends on the particular condition being treated and the dosage required to obtain a therapeutic effect. In general, the methods of the present invention may be practiced using any medically acceptable mode of administration. This means any mode that produces an effective level of treatment without causing clinically unacceptable side effects. In certain embodiments, the ligand conjugates of the present invention may be administered via oral, enteral, mucosal, transdermal, and / or parenteral routes. The term "parenteral" includes subcutaneous, intraspinal, intravenous, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal, dermal, vaginal, rectal, and sublingual. Delivery routes of the present invention may include intraspinal, intraventricular, or intracerebral. In certain embodiments, the ligand conjugates of the present invention may be administered via a parenteral route. In certain embodiments, the ligand conjugates of the present invention may be administered via a subcutaneous injection route.

[0165] In certain embodiments, the ligand conjugates of the invention may be administered directly to a tissue. Administration directly to a tissue may be accomplished by direct injection or other known means. The ligand conjugates of the invention may be administered once or, alternatively, multiple times. When administered multiple times, the ligand conjugates of the invention may be administered via different routes. For example, the first (or first few) administrations may be administered directly to the affected tissue or organ, and subsequent administrations may be administered systemically.

[0166] When it is desired to administer the ligand conjugates of the invention systemically, they can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Preparations for injection can be presented in unit dosage form, such as in ampoules or multidose containers, with or without added preservatives.

[0167] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic / aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's solution, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases. Lower dosages may result from other forms of administration, such as intravenous administration. If the subject does not respond adequately to the initial dosage, higher dosages (or substantially higher dosages via a different, more localized delivery route) may be used, as tolerated by the patient. To achieve appropriate systemic or local levels, multiple daily doses may be used as necessary to result in the desired level of a therapeutically active agent, e.g., a desired siRNA level.

[0168] In certain embodiments of the invention, the ligand conjugates of the invention can be delivered using biodegradable implants, either through diffusion or degradation of the polymer matrix. Non-limiting examples of synthetic polymers for such use are well known in the art. Biodegradable and non-biodegradable polymers can be used for delivery of one or more ligand conjugates of the invention using known methods. Such methods can also be used to deliver one or more ligand conjugates of the invention for therapy. Additional suitable delivery systems can include time-release, delayed release, or sustained release delivery systems. Such systems can avoid repeated administration of the ligand conjugates of the invention, increasing convenience for subjects and healthcare providers. Many types of release delivery systems are available and are well known to those skilled in the art. (See, e.g., U.S. Pat. Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686.) Additionally, pump-based hardware delivery systems, some of which are adapted for implantation, may also be used.

[0169] When delivering a therapeutically active agent (e.g., siRNA) using the ligand conjugates described herein to a subject at risk for prophylactic treatment or development of a recurrent disease or condition, the use of a long-term sustained release implant may also be appropriate. As used herein, long-term release means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for a period of at least 30, 60, 90 or more days. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems discussed above.

[0170] In certain embodiments, the compounds of the invention may be administered in combination with an additional therapeutically active agent. Non-limiting examples of additional therapeutically active agents include agents for treating liver disease. The additional therapeutically active agent may be administered before, after, or simultaneously with the administration of the ligand conjugate of the invention.

[0171] Pharmaceutical Compositions In certain aspects, the invention relates to pharmaceutical compositions comprising a ligand conjugate described herein and a pharma- ceutically acceptable ligand or excipient.

[0172] The term "pharmaceutically acceptable ligand or excipient" as used herein refers to ligands or excipients that are generally safe, non-toxic, and not biologically or otherwise undesirable and useful in preparing pharmaceutical compositions, including ligands or excipients that are acceptable for both veterinary and human pharmaceutical use. As used herein, "pharmaceutically acceptable ligand or excipient" includes one or more such ligands or excipients. The particular excipient, ligand, or diluent will depend on the means and purpose for which the compound of the invention is being applied. Suitable ligands and excipients are well known to those skilled in the art and are described in detail, for example, in Ansel, Howard C, et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al., Remington: The Science and PRactice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulation may also include other known additives, such as one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, glidants, processing aids, colorants, sweeteners, flavorings, flavorings, diluents, and the like. These may be to provide an elegant presentation of a pharmaceutical agent (ie, a compound or pharmaceutical composition described herein) or to aid in the manufacture of a pharmaceutical product (ie, a drug product).

[0173] The compositions of the present invention can be in a variety of forms, including, for example, liquid, semi-solid, and solid dosage forms, including liquid solutions (e.g., injectable and perfusable solutions), dispersions and suspensions, tablets, pills, powders, liposomes, and suppositories.

[0174] The form will depend on the intended mode of administration and therapeutic application.

[0175] The pharmaceutical composition of the present invention can be prepared by well-known techniques in pharmacy, such as effective formulation and administration procedures.The above considerations regarding effective formulation and administration procedures are well known to those of ordinary skill in the art and are described in standard textbooks.The formulation of pharmaceuticals is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

[0176] synthesis The compounds of the present invention can be prepared by those having ordinary skill in the art of synthetic organic chemistry using the general and specific methods described below. Such ordinary general knowledge can be found in standard reference books such as Comprehensive Organic Chemistry, Ed. Barton and Ollis, Elsevier; Comprehensive Organic TRansformations: A Guide to Functional Group Preparations, Larock, John Wiley and Sons; and Compendium of Organic Synthetic Methods, Vol. I-XII (published by Wiley-Interscience). The starting materials used herein are commercially available or can be prepared by methods generally known in the art.

[0177] In preparing the compounds of the present invention, some of the preparative methods described herein may require protection of remote functionalities. The need for such protection will vary depending on the nature of the remote functionality and the conditions of the preparative method. The need for such protection will be readily determined by one of ordinary skill in the art. The use of such protection / deprotection methods is also within the skill of the artisan. For a general description of protecting groups and their use, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0178] The schemes described below are intended to provide a general description of the methodology used to prepare the compounds of the present invention. Some of the compounds of the present invention may contain one or more chiral centers with stereochemical designation (R) or (S). As will be apparent to one of ordinary skill in the art, synthetic transformations may be carried out in a similar manner whether the materials are enantiomerically enriched or racemic. Furthermore, resolution to the desired optically active substances may be carried out at any desired point in the series using well-known methods such as those described herein and in the chemical literature. EXAMPLES

[0179] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the methods and compositions provided herein and should not be construed as limiting the scope thereof in any way.

[0180] All reagents and materials were purchased from commercial vendors or could be readily prepared by one of ordinary skill in the art. A list of the abbreviations of the reagents used can be found in Table 1 below. [Table 1]

[0181] Example 1 (3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol (Intermediate IA): [ka]

[0182] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0183] 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)-12-oxododecanoic acid (Intermediate IB): [ka]

[0184] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0185] Example 2 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (Intermediate II-A): [ka]

[0186] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0187] 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-bis(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (Intermediate II-B): [ka]

[0188] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0189] Example 3 (2R,3S,4R,5S)-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (intermediate III): [ka]

[0190] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0191] Example 4 (2R,3S,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride (intermediate IV): [ka]

[0192] The title compound was synthesized according to the following synthetic route. [ka]

[0193] Step 1. (2R,3S,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-chlorotetrahydro-2H-pyran-3,4-diyl diacetate (IV-1) (40.0 g, 109 mmol, 1.00 eq) was added to anhydrous toluene (800 mL), (n-Bu)3SnH (39.0 g, 134 mmol, 35.4 mL, 1.22 eq) was added, followed by AIBN (3.59 g, 21.9 mmol, 0.20 eq) at 15 °C. The corresponding mixture was then stirred at 120 °C under N2 for 3 h. TLC (petroleum ether / EtOAc = 1 / 2 (PMA)) showed complete consumption of IV-1, and LCMS showed the desired product MS. After the reaction was complete, the mixture was cooled to 20 °C and concentrated under vacuum at 45 °C. The crude product was triturated with (i-Pr)2O (200 mL) and toluene (50 mL) at 15 °C for 2 h, and the solid was collected to give (2R,3S,4R,5S)-5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (IV-2) (35.2 g). LCMS: calculated [M+H]: 332.1, found 332.2. 1 H NMR:(400 MHz CDCl3) δ 5.93 (d, J = 7.6 Hz, 1H), 5.06-4.99 (m, 1H), 4.98-4.90 (m, 1H), 4.23-4.05 (m, 4H), 3.59-3.48 (m, 1H), 3.22-3.08 (m, 1H), 2.05 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H).

[0194] Step 2. A mixture of compound IV-2 (39.0 g, 118 mmol, 1.00 eq) in hydroxide HCl (3.00 M, 825 mL, 21.0 eq) was stirred at 110 °C for 16 h. Compound IV-2 was completely consumed by TLC (petroleum ether / EtOAc = 0 / 1 (PMA)) and LCMS showed the desired product MS. The brown solution was extracted with ethyl acetate (300 mL x 3), and the aqueous layer was concentrated under reduced pressure at 45 °C to a residue, coevaporated with ACN (300 mL x 3) and toluene (300 mL x 3), and evaporated at 50 °C to remove HO / HCl. (2R,3S,4R,5S)-5-amino-2-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol hydrochloride (Intermediate IV) (23.6 g, crude) was obtained as a white solid and used directly in the next step without further purification. LCMS: calculated [M+H]: 164.08, found 164.2. 1 H NMR:(400 MHz D2O) δ 4.19 (dd, J = 4.8, 11.2 Hz, 1H), 3.90 (dd, J = 2.0, 12.4 Hz, 1H), 3.72 (dd, J = 5.2, 12.4 Hz, 1H), 3.65 (dd, J = 8.4, 10.0 Hz, 1H), 3.56 (t, J = 11.2 Hz, 1H), 3.49-3.38 (m, 2H), 3.35-3.25 (m, 1H).

[0195] Example 5 (2R,3R,4R)-2-(hydroxymethyl)piperidine-3,4-diol (intermediate V): [ka]

[0196] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0197] Example 6 (3S,4S,5R,Z)-1,2,3,4,5,8-Hexahydroazocine-3,4,5-triol (Intermediate VI): [ka]

[0198] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0199] Example 7 (3S,4S,5R)-Azocane-3,4,5-triol (Intermediate VII): [ka]

[0200] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0201] Example 8 (3R,4R,5S)-Piperidine-3,4,5-triol (Intermediate VIII): [ka]

[0202] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0203] Example 9 Tri-tert-butyl 3,3',3''-(((2R,3R,4R,5S)-2-(((12-methoxy-12-oxododecyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl)tris(oxy))tripropionate (Intermediate 1-7): [ka]

[0204] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0205] Example 10 Tri-tert-butyl 3,3',3''-(((2R,3R,4R,5S)-2-((((benzyloxy)carbonyl)amino)methyl)tetrahydro-2H-pyran-3,4,5-triyl)tris(oxy))tripropionate (Intermediate 2-3): [ka]

[0206] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0207] Example 11 Tri-tert-butyl 3,3',3''-(((2R,3R,4R,5S)-2-((2-(((benzyloxy)carbonyl)amino)ethoxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl)tris(oxy))tripropionate (Intermediate 3-4): [ka]

[0208] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0209] Example 12 Tri-tert-butyl 3,3',3''-(((2R,3R,4R,5S)-2-(((2-(((benzyloxy)carbonyl)amino)ethyl)amino)methyl)tetrahydro-2H-pyran-3,4,5-triyl)tris(oxy))tripropionate (Intermediate 4-4): [ka]

[0210] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0211] Example 13 Di-tert-butyl 3,3'-(((2R,3S,4R,5S)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-5-(12-methoxy-12-oxododecanoamido)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionate (Intermediate 5-4): [ka]

[0212] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0213] Example 14 Di-tert-butyl 3,3'-(((2R,3S,4R,5R,6R)-5-acetamido-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-6-(12-methoxy-12-oxododecanamido)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionate (Intermediate 6-4): [ka]

[0214] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0215] Example 15 Di-tert-butyl 3,3'-(((2R,3R,4S,5R,6R)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-5-fluoro-6-(12-methoxy-12-oxododecanamido)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionate (Intermediate 7-8): [ka]

[0216] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0217] Example 16 Di-tert-butyl 3,3'-(((2R,3S,4R,5R,6S)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-6-((tert-butyldiphenylsilyl)oxy)-5-(12-methoxy-12-oxododecanoamido)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionate (Intermediate 8-10): [ka]

[0218] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0219] Example 17 Di-tert-butyl 3,3'-(((2R,3R,4R)-1-((benzyloxy)carbonyl)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)piperidine-3,4-diyl)bis(oxy))dipropionate (Intermediate 9-2): The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0220] Example 18 Tri-tert-butyl 3,3',3''-(((3S,4S,5R,Z)-1-((benzyloxy)carbonyl)-1,2,3,4,5,8-hexahydroazocine-3,4,5-triyl)tris(oxy))tripropionate (Intermediate 10-2): [ka]

[0221] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0222] Example 19 Tri-tert-butyl 3,3',3''-(((3S,4S,5R)-1-((benzyloxy)carbonyl)azocane-3,4,5-triyl)tris(oxy))tripropionate (Intermediate 11-2): [ka]

[0223] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0224] Example 20 Tri-tert-butyl 3,3',3''-(((3S,4R,5R)-1-((benzyloxy)carbonyl)piperidine-3,4,5-triyl)tris(oxy))tripropionate (Intermediate 12-2): [ka]

[0225] The corresponding compounds may be synthesized according to the following synthetic route. [ka]

[0226] Example 21 Synthesis of Compounds 1-11 [ka]

[0227] Example 22 Synthesis of Compounds 1-14 [ka]

[0228] Example 23 Synthesis of compound 1 [ka]

[0229] Example 24 Synthesis of Compounds 2-8 [ka]

[0230] Example 25 Synthesis of compound 2 [ka]

[0231] The title compound was synthesized according to the following synthetic route. [ka] [ka]

[0232] Step 1: To a solution of Cpd. 2-a (7.50 g, 45.7 mmol, 1.00 eq) in pyridine (52.5 mL) was added TsCl (13.1 g, 68.5 mmol, 1.5 eq) at 0 °C. The mixture was stirred at 25 °C for 11 h. LCMS (RT for compound 5-1a = 1.368 min) showed that Cpd. 2-a was consumed and the main peak had the desired mass. The reaction mixture was concentrated under reduced pressure to give a residue. The resulting residue was purified by reverse phase HPLC (neutral conditions). Cpd. 2-b (13.6 g, 42.7 mmol, 46.7% yield) was obtained as a yellow solid. 1H NMR(400 MHz CD3OD)(product)δ 7.78(d, J = 8.4 Hz, 2H), 7.44(d, J = 8.0 Hz, 2H), 4.29(dd, J = 2.0, 10.8 Hz, 2H), 4.17-4.05(m, 1H), 3.85-3.75(m, 1H), 3.42-3.33(m, 1H), 3.30-3.00(m, 4H), 2.45(s, 3H).

[0233] Step 2: To a solution of Cpd.2-b (20.0 g, 62.8 mmol, 1.00 eq) in DMF (140 mL) was added NaN3 (12.3 g, 188 mmol, 3.00 eq) and TBAI (2.32 g, 6.28 mmol, 0.1 eq). The mixture was stirred at 80 °C for 24 h. TLC (petroleum ether / EtOAc = 0 / 1 (KMnO4), Rf of Cpd.2-c = 0.20) showed that Cpd.2-b was consumed and a major spot was detected. The reaction mixture was purified by column chromatography (Al2O3, petroleum ether / EtOAc = 1 / 1 ~ 0 / 1). Cpd.2-c (4.56 g, 24.1 mmol, 38.4% yield) was obtained as a yellow oil. 1 H NMR: ET52873-23-P1A1 (400 MHz CD3OD) (product) δ 3.95-3.85(m, 1H), 3.65-3.41(m, 2H), 3.40-3.32(m, 1H), 3.28-3.15(m, 4H).

[0234] Step 3: To a solution of Cpd. 2-c (400 mg, 2.11 mmol, 1.00 eq) in dichloromethane (6.00 mL) was added Cpd. 7-f (1.07 g, 8.46 mmol, 1.16 mL, 4.00 eq) and DMAP (155 mg, 1.27 mmol, 0.06 eq). The mixture was stirred at 25 °C for 2 h. Cpd. 2-c was consumed and a major spot was detected by TLC (petroleum ether / EtOAc = 2 / 1, Rf of Cpd. 2-d = 0.50). The reaction mixture was diluted with water (15 mL) and extracted with ethyl acetate (6 mL x 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (silica gel, petroleum ether / EtOAc = 1 / 0 to 2 / 1) to give Cpd. 2-d (600 mg, 1.06 mmol, 49.9% yield) as a brown oil. 1 H NMR (400 MHz CDCl3) (product) δ 7.40-7.35(m, 2H), 7.25-7.18(m, 1H), 5.35-5.25(m, 3H), 4.25-4.15(m, 1H), 4.10-3.85(m, 3H), 3.66-3.60(m, 1H), 3.51-3.44(m, 1H), 3.40-3.24(m, 1H), 1.50-1.44(m, 27H).

[0235] Step 4: To a suspension of Pd / C (1.00 g, 8.81 mmol, 10% purity) in methanol (35 mL) under Ar was added Cpd. 2-d (5.00 g, 8.81 mmol, 1.00 eq). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (50 psi) at 25 °C for 72 h. 1 Cpd. 2-d was consumed and product was detected by H NMR. The reaction mixture was filtered and concentrated under reduced pressure to give the residue. Cpd. 2-e (4.10 g, 7.49 mmol, 84.9% yield) was obtained as a brown oil. 1H NMR (400 MHz CDCl3) (product) δ 4.85-4.29(m, 1H), 4.11-3.58(m, 5H), 3.55-2.66(m, 8H), 2.60-2.37(m, 5H), 1.56-1.35(m, 27H).

[0236] Step 5: To a solution of Cpd. 7-i (2.11 g, 6.57 mmol, 1.20 eq) in DMF (30 mL) was added HBTU (4.15 g, 10.9 mmol, 2.00 eq) and DIPEA (2.83 g, 21.9 mmol, 3.82 mL, 4.00 eq) at 25 °C. Then, Cpd. 2-e (3.00 g, 5.48 mmol, 1.00 eq) was added and stirred at 25 °C for 2 h. LCMS (RT for Cpd. 2-f = 3.162 min) showed that Cpd. 2-e was consumed, many peaks had formed, and a new peak of the desired mass was detected. The reaction mixture was diluted with water (60.0 mL) and extracted with dichloromethane (20 mL × 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative HPLC (column: Welch Xtimate C18 250 × 70 mm#10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 65%-98%, 20 min). Cpd. 2-f (1.44 g, 1.69 mmol, 30.9% yield) was obtained as a brown oil. 1 H NMR (400 MHz CDCl3) (product) δ 7.52-7.28(m, 5H), 5.12(s, 2H), 4.16-3.50(m, 8H), 3.41-2.95(m, 6H), 2.61-2.19(m, 10H), 1.64-1.55(m, 4H), 1.50-1.38(m, 27H), 1.36-1.21(m, 12H).

[0237] Step 6: Cpd. 2-f (650 mg, 765 umol, 1.00 eq) was dissolved in anhydrous dichloromethane (13.0 mL) at 17 °C, then TFA (6.01 g, 52.7 mmol, 3.90 mL, 68.9 eq) was added and stirred at 17 °C for 4 h under N2. TLC (petroleum ether / EtOAc = 1 / 1 (PMA), R of Cpd. 2-f f = 0.60) and LCMS (RT for Cpd. 2-g = 0.610 min) showed the desired MS. The reaction was concentrated under reduced pressure at 35 °C to give a residue, which was then evaporated with 5 x anhydrous toluene / THF (30 mL / 30 mL) at 40 °C and used in the next step without further purification. Cpd. 2-g (600 mg, crude) was obtained as a brown gum and showed about 80.6% purity by LCMS (RT for Cpd. 2-g = 0.609 min). LCMS: [M+H] = 682.3 (product). 1 H NMR (400 MHz CDCl3) (product) δ 7.40-7.29(m, 5H), 5.11(s, 2H), 4.16-3.51(m, 8H), 3.49-2.99(m, 6H), 2.75-2.49(m, 6H), 2.48-2.26(m, 5H), 1.69-1.50(m, 4H), 1.39-1.19(m, 14H).

[0238] Step 7: Cpd. 2-g (540 mg, 792 umol, 1.00 eq) was dissolved in DMF (10.8 mL), then DIPEA (1.54 g, 11.9 mmol, 2.07 mL, 15.0 eq) was added and stirred for 10 min, HBTU (931 mg, 2.46 mmol, 3.10 eq), Cpd. 7-l (1.66 g, 2.46 mmol, 3.10 eq, TsOH salt) were added at 17 °C, and the mixture was stirred at 30 °C (oil bath) under N2 for 16 h. TLC (DCM / MeOH = 10 / 1, 1d AcOH, (PMA), R of R1) was confirmed. f= 0.30) and LCMS (RT = 0.768 min for Cpd. 2-h) showed the desired MS and de-1Ac (1049). The reaction was poured into stirring water (100 mL) and dichloromethane (80 mL), stirred, separated, the aqueous layer was extracted with dichloromethane (80 mL × 3), the combined organic layers were washed with brine (100 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure at 35 °C to give the residue. Three batches (54 mg & 216 mg & 60 mg scale) were carried out as above and combined for purification. The residue was purified by column chromatography (silica gel, DCM / MeOH = 20 / 1, 15 / 1, 10 / 1, 5 / 1, 0 / 1). The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×25 mm×5 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 15%-45%, 5 min). The solution was extracted with dichloromethane (300 mL, 200 mL, 200 mL). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure at 35° C. to give the residue. Cpd. 2-h (940 mg, crude) was obtained as a light brown solid. LCMS: ESI+, calculated [M+2H] / 2 = 1069.5, found 1069.9 (product).

[0239] Step 8: Cpd. 2-h (940 mg, 439 umol, 1.00 eq) was dissolved in THF (20 mL), followed by addition of wet Pd / C (500 mg, 10% purity) and stirring at 30°C (oil bath) under H2 (15 psi, balloon) for 4 h. TLC (DCM / MeOH = 5 / 1, (PMA), R of Cpd. 2-h f = 0.30, R for Cpd. 2-i f = 0.10) and LCMS (RT for Cpd. 2-i = 0.637 min) showed the desired MS. The mixture was filtered through diatomaceous earth and the filtrate was concentrated at 35 °C. Cpd. 2-i (840 mg, crude) was obtained as a white solid. LCMS: ESI+, calculated [M+2H] / 2 = 1024.5, found 1025.0 (product).

[0240] Step 9: Cpd. 2-i (200 mg, 97.6 umol, 1.00 eq) was dissolved in DMF (4 mL) and DIPEA (37.9 mg, 293 umol, 51.0 uL, 3.00 eq) was added followed by HBTU (55.6 mg, 146 umol, 1.50 eq) and then Cpd. 7-o (49.2 mg, 117 umol, 1.20 eq) and stirred at 30° C. (oil bath) under N2 for 16 h. LCMS showed Cpd. 2-i was consumed and LCMS (RT for Cpd. 2-j = 0.804 min) showed desired MS. The reaction was poured into stirring water (40 mL) and DCM (40 mL), stirred, separated, the aqueous layer was extracted with DCM (40 mL × 3), the combined organic layers were washed with brine (50 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure at 35 °C to give a residue. It was combined with another batch (200 mg scale) and purified. The residue was purified by preparative HPLC (column: Phenomenex C18 75 × 30 mm × 3 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-55%, 8 min). Cpd. 2-j (210 mg) was obtained as a pale yellow solid. LCMS: ESI-, calculated [M-2H] / 2 = 1223.1, found 1223.6 (product).

[0241] Step 10: Cpd. 2-j (100 mg, 40.8 umol, 1.00 eq) was dissolved in anhydrous dichloromethane (4 mL), then DMAP (1.25 mg, 10.2 umol, 0.25 eq), DIPEA (21.1 mg, 163 umol, 28.4 uL, 4.00 eq) and succinic anhydride (16.3 mg, 163 umol, 4.00 eq) were added and stirred at 20 °C (oil bath) under N2 for 16 h. LCMS (RT for Cpd. 2-k = 0.788 min) showed ~33.3% desired MS and ~27.4% Cpd. 2-j. The reaction was poured into saturated NaHCO3 (15 mL) and DCM (15 mL), stirred, separated, the aqueous layer was extracted with DCM (10 mL × 4), and the combined organic layers were washed with brine (10 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure at 35 °C to give a residue, which was purified by preparative HPLC (column: Waters Xbridge BEH C18 100 × 30 mm × 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-55%, 8 min). Cpd. 2-k(4-(((3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(12-oxo-12-((((2R,3R,4R,5S)-3,4,5-tris(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-2-yl)methyl)amino)dodecanoyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid) (19.0 mg, 7.40 μmol, 18.1% yield, NH salt) was obtained as a white solid. LCMS: ESI-, calculated [M-2H] / 2 = 1273.1, found 1273.2 (product).

[0242] Step 11: Cpd. 2-k (19.4mg, 7.56umol, 1.00eq, NH4 salt) was dissolved in anhydrous MF (0.97mL) and HBTU (14.3mg, 37.8umol, 5.00eq), DIPEA (7.81mg, 60.4umol, 10.5uL, 8.00eq), DMAP (923ug, 7.56umol, 1.00eq) were added in one portion at 17°C. Then resin CPG-NH2 (149mg, 7.56umol; chemical name: Aminoalkyl-CPG, 500A, from Hebei DNAchem Biotechnology Co., Ltd.) was added to the mixture. The mixture was stirred at 40°C for 24 hours and LCMS showed the reaction was complete. After that, it was filtered and the filter cake was washed with methanol (5.00mL x 4) and DCM (5.00mL x 4). The filter cake was dried under N2 flow and obtained as a light yellow solid (222mg). Ac2O (327mg, 3.20mmol, 300uL, 36.8eq) was added to pyridine (Py, 1.5mL) and mixed, then added to the above light yellow solid (222mg) and stirred at 35℃ for 0.5h. After that, it was filtered and the filter cake was washed with DCM (5.00mL x 4) and methanol (5.00mL x 4). The obtained light yellow solid was dried under vacuum for 12h to obtain 132mg of solid capsule product (loading: 32.3umol / g).

[0243] Example 26 Synthesis of compound 3 [ka]

[0244] Compound 3 is synthesized according to the above synthesis route of compound 2 using intermediate 3-4 as the starting material.

[0245] Example 27 Synthesis of compound 4 [ka]

[0246] Compound 4 can be synthesized according to the above synthesis route for compound 2 using intermediate 4-4 as the starting material.

[0247] Example 28 Synthesis of compound 5 [ka]

[0248] Compound 5 was synthesized according to the procedure of compound 6 in Example 29, using 5-j as the starting material. The synthesis is shown below. Loading: 27.0 μmol / g.

[0249] 4-(((3R,5S)-1-(12-(((3S,4R,5S,6R)-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R,6R) -3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)tetrahydro-2H-pyran-3-yl)amino)-12-oxododecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (5-j): The target compound (5-j) was synthesized according to the procedure of compound 6-j in example 29, using 5-f as starting material. LCMS: Calculated value [M-2H] / 2: 1273.1, Found: 1273.7.

[0250] Synthesis of 3,3'-(((2R,3S,4R,5S)-5-(12-(benzyloxy)-12-oxododecanoamido)-2-((2-carboxyethoxy)methyl)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionic acid (5-f): [ka]

[0251] Step 1. Compound IV (27.2 g, 136 mmol, 1.00 equiv, HCl salt) was dissolved in dioxane (270 mL) and water (270 mL), NaHCO3 (41.9 g, 499 mmol, 19.4 mL, 3.66 equiv) was added, followed by Boc2O (37.7 g, 173 mmol, 39.6 mL, 1.27 equiv) at 15°C, and stirred at 30°C for 16 hours under N2. TLC (i-PrOH / H2O / NH3-H2O = 6 / 3 / 1) showed that compound IV (Example 4) was completely consumed, and LCMS showed the desired product MS. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the residue at 45°C. The residue was purified by column chromatography (silica gel, DCM / MeOH = 15 / 1, 10 / 1, 8 / 1, 4 / 1, 0 / 1) to give tert-butyl ((3S,4R,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)carbamate (5-f-1) (33.7 g; yield: 93.9%). LCMS: calculated [M-Boc+H]: 164.1, found: 164.1. 1 H NMR:(400 MHz CD3OD)δ 3.91(dd, J = 5.2, 11.2 Hz, 1H), 3.83(dd, J = 2.0, 11.6 Hz, 1H), 3.61(dd, J = 6.0, 12.0 Hz, 1H), 3.54-3.40(m, 1H), 3.36-3.23(m, 2H), 3.19-3.03(m, 2H), 1.44(s, 9H).

[0252] Step 2. Compound 5-f-1 (4.55 g, 17.3 mmol, 1.00 equiv) was dissolved in anhydrous DCM (68 mL) and DMAP (1.27 g, 10.4 mmol, 0.60 equiv) was added, followed by tert-butyl propiolate (8.72 g, 69.1 mmol, 9.49 mL, 4.00 equiv) to the white mixture and stirred at 25 °C (oil bath) under N2 for 16 h. TLC (DCM / MeOH = 5 / 1 (PMA)) showed compound 5-f-1 was completely consumed and LCMS showed the desired product MS was about 31.6%. The black solution was concentrated at 40 °C. The remainder was purified by column chromatography (silica gel, petroleum ether / EtOAc = 20 / 1, 10 / 1, 8 / 1, 5 / 1, 4 / 1, 1 / 1, 0 / 1) to provide di-tert-butyl 3,3'-(((2R,3S,4R,5S)-2-((((E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl)oxy)methyl)-5-((tert-butoxycarbonyl)amino)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))(2E,2'E)-diacrylate (5-f-2) (3.42 g; yield: 30.8%). LCMS: calculated [M+NH4]: 659.3, found: 659.4. 1 H NMR: (400 MHz CDCl3) δ 7.45 (d, J = 12.4 Hz, 1H), 7.35-7.17 (m, 2H), 5.36-5.21 (m, 2H), 5.15 (d, J = 12.4 Hz, 1H), 4.69 (d, J = 8.0 Hz, 1H), 4.25-3.83 (m, 6H), 3.77-3.37 (m, 3H), 1.54-1.33 (m, 27H).

[0253] Step 3. Compound 5-f-2 (2.42 g, 3.77 mmol, 1.00 equiv) was dissolved in THF (24 mL) and MeOH (24 mL), wet Pd / C (4.84 g, 156 umol, 10% purity) was added, and the mixture was stirred under H2 (15 psi, balloon) at 30°C for 48 hours. LCMS showed complete consumption of compound 5-f-2 and the desired product MS. The mixture was filtered through diatomaceous earth, the cake was washed with methanol, and the filtrate was concentrated at 40 °C to provide di-tert-butyl 3,3'-(((2R,3S,4R,5S)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)-5-((tert-butoxycarbonyl)amino)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionate (5-f-3) (2.40 g; yield: 98.3%; yellow syrup). 1 H NMR: (400 MHz CDCl3) δ 5.18-4.91 (m, 1H), 4.24-4.08 (m, 1H), 4.04-3.94 (m, 1H), 3.93-3.59 (m, 7H), 3.55-3.41 (m, 1H), 3.35-3.18 (m, 3H), 3.10 (t, J = 10.8 Hz, 1H), 2.62-2.38 (m, 6H), 1.54-1.33 (m, 36H).

[0254] Step 4. Compound 5-f-3 (2.40 g, 3.70 mmol, 1.00 equiv) was dissolved in anhydrous DCM (48.0 mL) at 15 °C, then TFA (37.0 g, 324 mmol, 24.0 mL, 87.5 equiv) was added and stirred under N2 at 15 °C for 7 h. LCMS showed complete consumption of compound 5-f-3 and desired product MS. The solution was concentrated under reduced pressure to give a residue at 40 °C, then co-evaporated with ACN (50 mL x 4) and toluene (50 mL x 4) to remove TFA at 45 °C to give 3,3'-(((2R,3S,4R,5S)-5-amino-2-((2-carboxyethoxy)methyl)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionic acid (5-f-4) (2.00 g, TFA salt, yellow gum). LCMS: calculated [M+H]: 380.1, found: 380.1. 1 H NMR: (400 MHz D2O) δ 4.25-4.08 (m, 2H), 4.05-3.71 (m, 7H), 3.68-3.41 (m, 4H), 3.39-3.24 (m, 1H), 2.83-2.61 (m, 6H), 2.06 (s, 2H).

[0255] Step 5. Compound 5-f-4 (2.00 g, 4.05 mmol, 1.00 equiv, TFA salt) was dissolved in anhydrous DMF (20 mL) and after addition of TEA (2.87 g, 28.4 mmol, 3.95 mL, 7.00 equiv), a solid was observed, the synthesis of which is shown below. 5-fa (1.86 g, 4.46 mmol, 1.10 equiv) was added and stirred at 25 °C (oil bath) under N2 for 16 h to give a pale yellow solution. LCMS showed the desired product MS was about 59.3%. The reaction was concentrated under reduced pressure at 45 °C to give the residue. This residue was roughly purified by column chromatography to provide compound 5-f (1.83 g, white solid), which was used in the next step without further purification. LCMS: Calculated [M+H]: 682.3, Found: 682.4.

[0256] Synthesis of 1-benzyl 12-(2,5-dioxopyrrolidin-1-yl)dodecanedioate (5-fa):

[0257] 12-(Benzyloxy)-12-oxododecanoic acid (5.00 g, 15.6 mmol, 1.00 equiv) was dissolved in DCM (35.0 mL) and N-hydroxysuccinimide (2.16 g, 18.7 mmol, 1.20 equiv) and DCC (4.19 g, 20.3 mmol, 4.10 mL, 1.30 equiv) were added in one portion at -5 °C under N2. The mixture was stirred at -5 °C for 2 h, then heated to 30 °C and stirred for an additional 10 h. TLC (petroleum ether:ethyl acetate = 1:1, R f = 0.7) indicated that 12-(benzyloxy)-12-oxododecanoic acid was completely consumed and one major new spot with low polarity was detected. It was filtered and concentrated under vacuum. The crude product was stirred in i-propanol:heptane = 1:1 at 25 °C for 60 min. Then the mixture was filtered and the liquid was concentrated under vacuum to provide compound 5-fa (6.00 g; yield: 92.1%). LCMS: Calculated [M+NH4]: 435.2, Found: 435.3.

[0258] Example 29 Synthesis of compound 6 [ka]

[0259] The title compound was synthesized according to the following synthetic route. [ka] [ka]

[0260] Step 1: Cpd.6-a (5.00 g, 13.4 mmol) was dissolved in MeOH (30.0 mL) and NaOMe (5.40 M, 149 μL) was added in one portion at 20 °C under N2. The mixture was stirred at 20 °C for 1 h. LCMS (product: RT = 0.220 min) showed that one major peak with the desired mass was detected. The pH of the mixture was adjusted to 6 with Amberlite IR120, Na resin (CAS: 78922-04-0), filtered and concentrated under vacuum to give a white solid as Cpd.6-b (3.20 g, 96.7% yield). 1 H NMR (400 MHz, CD3OD)δ 4.50(d, J = 9.2 Hz, 1H), 3.86-3.94(m, 1H), 3.63-3.75(m, 2H), 3.42-3.50(m, 1H), 3.35(s, 10H), 1.99(s, 3H).

[0261] Step 2: Cpd. 6-b (3.34 g, 13.5 mmol) was dissolved in DCM (50.0 mL) and DMAP (662 mg, 5.43 mmol) was added. Cpd. 7-f (6.85 g, 54.2 mmol, 7.5 mL) was added to the mixture and stirred at 20 °C for 5 h. TLC (petroleum ether / ethyl acetate, product: R f = 0.43) indicated that the starting material was completely consumed, and LCMS (product: RT = 0.220 min) showed one major peak with the desired mass was detected. The mixture was concentrated under vacuum. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 8 / 1 to 0 / 1) to give a yellow solid as Cpd. 6-c (2.10 g, 33.0% yield). 1 H NMR(400 MHz, CD3OD)δ 4.50(d, J = 6.2 Hz, 3H), 3.80-3.90(m, 2H), 3.74(br t, J = 6.3 Hz, 2H), 3.61-3.68(m, 2H), 3.53-3.61(m, 2H), 3.10-3.27(m, 4H), 1.82-1.93(m, 3H), 1.27-1.42 ppm(m, 28H).

[0262] Step 3: Cpd. 6-c (100 mg, 160 umol) was dissolved in MeOH (2.00 mL) and Pd / C (0.02 g, 10% purity) was added under Ar. The mixture was stirred at 25° C. under H2 (50 psi) for 48 h. LCMS (product: RT = 0.884 min) showed that one major peak with the desired mass was detected. The mixture was filtered and concentrated under vacuum to give a brown oil as Cpd.6-d (125 mg, 53.41% yield, 41.36% purity). 1 H NMR(400 MHz, CD3OD)δ 3.80-3.90(m, 2H), 3.74(br t, J = 6.3 Hz, 2H), 3.61-3.68(m, 2H), 3.53-3.61(m, 2H), 3.10-3.27(m, 4H), 2.27-2.49(m, 6H), 1.82-1.93(m, 3H), 1.27-1.42 ppm(m, 27H).

[0263] Step 4: Cpd. 7-i (244 mg, 763 umol) was dissolved in DMF (3.00 mL) and HBTU (289 mg, 763 umol) and DIPEA (179 mg, 1.39 mmol) were added. To this mixture was added Cpd. 6-d (420 mg, 694 umol) and stirred at 25 °C for 17 h. LCMS (product: RT = 1.07 min) showed that one major peak with the desired mass was detected. The mixture was poured into a mixture consisting of saturated NaHCO3(aq) (10 mL) and ice and extracted with ethyl acetate (10 mL x 4). The combined organic phase was washed with water (10 mL x 2) and saturated NaHCO3(aq) (10 mL x 2). The combined organic phase was then dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The mixture was concentrated under vacuum. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate=20 / 1, 0 / 1) to give a white solid as Cpd. 6-e (450 mg, 496 umol, 71.4% yield). LCMS: [MH] = 905.6.

[0264] Step 5: To a mixture of Cpd. 6-e (1.50 g, 1.65 mmol) was added formic acid (30.0 mL) at 25 °C under N2. The mixture was stirred at 20 °C for 2 h. LCMS (product: RT = 0.759 min) showed that the starting material was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Toluene (4 mL) was added to the residue and concentrated, and this procedure was repeated three times. Finally, Cpd. 6-f (1.15 g, 1.56 mmol, 94.1% yield) was obtained as a yellow solid. 1 H NMR: (400 MHz, D6-DMSO) (product) δ = 8.13(d, J = 9.3 Hz, 1H), 7.86(br d, J = 9.3 Hz, 1H), 7.28-7.43(m, 5H), 5.08(s, 2H), 4.83(s, 1H), 3.68-3.88(m, 4H), 3.48-3.67(m, 5H), 3.27-3.37(m, 1H), 3.18-3.25(m, 1H), 3.09(s, 1H), 2.25-2.56(m, 10H), 1.92-2.13(m, 2H), 1.77(s, 3H), 1.52(br d, J = 6.8 Hz, 5H), 1.21 ppm(br d, J = 7.8 Hz, 12H).

[0265] Step 6: Cpd. 7-l (3.41 g, 5.05 mmol, TsOH salt) was dissolved in DCM (8.00 mL) and HBTU (1.80 g, 4.74 mmol) and DIPEA (1.98 g, 15.29 mmol, 2.66 mL) were added in one portion at 25 °C under N2, then the mixture was stirred at 25 °C under N2 for 10 min. Cpd. 6-f (1.13 g, 1.53 mmol) was added to this mixture and stirred at 25 °C for 20 h. LCMS (product: RT = 2.755 min) showed that starting material was completely consumed. The reaction mixture was suspended in ethyl acetate (35 mL) and washed with saturated NaHCO3 (35 mL). The organic phase was collected and washed with saturated NaCl (35 mL x 2). The organic phase (suspension) was collected and centrifuged to give Cpd. 6-g (1.35 g, 0.614 mmol, 40.2% yield) as a white solid. LCMS: ESI-, calculated [M-2H] / 2 = 1096.0, found 1096.6 (product).

[0266] Step 7: Cpd. 6-g (500 mg, 227 umol) was dissolved in THF (2.5 mL) and Pd / C (600 mg, 10% purity) was added under Ar. The mixture was stirred at 25° C. under H2 (15 psi) for 48 h. LCMS (product: RT = 2.1 min) showed that one major peak with the desired mass was detected. The mixture was filtered and concentrated under vacuum to give Cpd. 6-h (430 mg, crude) as a white solid, which was used in the next step without further purification. LCMS: ESI-, calculated [M-2H] / 2 = 1051.01, found 1051.1 (product).

[0267] Step 8: To a mixture of Cpd. 6-h (170 mg, 80.7 umol) was added HBTU (45.9 mg, 121 umol) and DIPEA (20.8 mg, 161 umol) in one portion at 25°C under N2 and stirred at 25°C for 10 min under N2 atmosphere. To this mixture was added Cpd. 7-o (50.8 mg, 121 umol) and stirred at 17°C for 17 h. LCMS (product: RT = 2.86 min) showed complete consumption of starting material. The remainder was purified by precipitation with ACN (3.0 mL). Finally, Cpd. 6-i (150 mg, 59.8 umol, 74.1% yield) was obtained as a white solid. LCMS: ESI-, calculated [M-2H] / 2 = 1251.61, found 1252.1 (product).

[0268] Step 9: Succinic anhydride (35.9 mg, 359 umol) was added to DCM (3.00 mL) and DIPEA (46.4 mg, 359 umol) and DMAP (1.83 mg, 14.9 umol) were added in one portion at 25°C under N2. With stirring, Cpd. 6-i (150 mg, 59.8 umol) was added and stirred at 20°C for 4 h. LCMS (product: RT = 2.19 min) showed that the starting material was completely consumed. The mixture was poured into DCM (20 mL). The combined organic phase was washed with TEBA (aq) (20 mL x 2). The combined organic phase was then dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by reverse-phase preparative HPLC (0.1M TEAB-ACN; B%: 20%-45%, 20 min) to give Cpd. 6-j(4-(((3R,5S)-1-(12-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5 R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)tetrahydro-2H-pyran-2-yl)amino)-12-oxododecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid) (35.0 mg, 13.4 umol, 22.4% yield) was obtained as a white solid. LCMS: ESI-, calculated [M-2H] / 2 = 1301.62, found 1301.1 (product).

[0269] Step 10: Cpd. 6-j (20 mg, 7.67 umol) was dissolved in DMF (1.50 mL) and HBTU (14.5 mg, 38.3 umol), DIEA (7.93 mg, 61.3 umol, 10.6 uL), and DMAP (937 ug, 7.67 umol) were added at once at 25°C. Then, CPG (130 mg; Chemical name: Aminoalkyl-CPG, 500A, from Hebei DNAchem Biotechnology Co., Ltd.) was added to the mixture. The mixture was stirred at 40°C for 19 hours. Then, it was filtered and the filter cake was washed with methanol (5.00 mL × 4) and DCM (5.00 mL × 4). The filter cake was dried under N2 stream to give a light yellow solid. The above light yellow solid was added to a mixture of dry pyridine (1.50 mL) and acetic anhydride (Ac2O) (0.30 mL). The mixture was then stirred at 40°C for 0.5 h. It was then filtered and the filter cake was washed with DCM (5.00 mL x 4) and methanol (5.00 mL x 4). The obtained light yellow solid was dried under vacuum for 12 h to give 130 mg of solid liquid product (loading: 32 umol / g).

[0270] Example 30 Synthesis of compound 7 [ka]

[0271] The title compound was synthesized according to the following synthetic route. [ka] [ka]

[0272] Step 1: A solution of Cpd. 7-a (5.00 g, 14.4 mmol, 1.00 eq) was dissolved in DCM (28.0 mL) and DAST (9.26 g, 57.4 mmol, 7.59 mL, 4.00 eq) was added at -40 °C and stirred at 25 °C for 12 h. TLC (petroleum ether: ethyl acetate = 2:1, R f =0.4), which indicated the reaction was complete. The mixture was quenched with aqueous NH4Cl (50.0 mL), then extracted with EtOAc (50.0 mL × 3) and concentrated under reduced pressure to give the residue. Four batches were run in parallel as above and combined for purification. The residue was purified by column chromatography (silica gel, petroleum ether: ethyl acetate = 20:1 to 3:1). Cpd. 7-b (4.65 g, 13.3 mmol, 23.3% yield) was obtained as a yellow oil. 1 H NMR (400 MHz, CDCl3) (product) δ 5.77-5.80(m, 1H), 5.33-5.42(d, 1H), 5.07(t, J = 8 Hz, 1H), 4.36-4.53(m, 1H), 4.28-4.32(m, 1H), 4.08-4.12(m, 1H), 3.84-3.88(m, 1H), 2.18(s, 3H), 2.04-2.09(m, 9H).

[0273] Step 2: To a solution of Cpd. 7-b (9.50 g, 27.1 mmol, 1.00 eq) dissolved in CHCl3 (20.0 mL) was added hydrogen bromide (99.7 g, 407 mmol, 66.9 mL, 33% purity, 15.0 eq) at 0 °C and stirred at 25 °C for 3 h. TLC (petroleum ether:ethyl acetate = 2:1, R f=0.6), indicating the reaction was complete. The mixture was poured into ice water (100 mL) and stirred briefly before being extracted with CHCl3 (3 portions of 100 mL). The yellow extract was washed several times with ice-cold saturated NaHCO3 solution, first until the evolution of CO2 ceased and then until the organic layer became colorless. The layers were then washed once with water, dried over Na2SO4 and concentrated to dryness. Cpd. 7-c (9.2 g, crude) was obtained as a brown oil. 1 H NMR(400 MHz, CDCl3) (product) δ 6.54(d, J = 8 Hz, 1H), 5.59-5.67(m, 1H), 5.09-5.14(m, 1H), 4.46-4.62(m, 1H), 4.32-4.53(m, 2H), 4.11-4.15(m, 1H), 2.09(d, J = 4 Hz, 6H), 2.06(s, 3H).

[0274] Step 3: To a solution of Cpd. 7-c (9.20 g, 24.8 mmol, 1.00 eq) in acetone (108 mL) was added NaN3 (6.50 g, 100 mmol, 4.03 eq) in H2O (90.0 mL) dropwise at 0 °C, and the mixture was stirred at 20 °C for 3 h. TLC (petroleum ether:ethyl acetate = 2:1, R f =0.5), indicating that starting material had been consumed and a new spot had formed. The white precipitate was filtered, washed thoroughly with HO (150 mL), and dried under vacuum to give the product. Cpd. 7-d (6.60 g, 19.8 mmol, 79.9% yield) was obtained as a white solid. 1 H NMR(400 MHz, CDCl3) (product) δ 5.31-5.37(m, 1H), 5.05(t, J = 8 Hz, 1H), 4.81-4.84(m, 1H), 4.14-4.34(m, 3H), 3.80-3.87(m, 1H), 2.09(d, J = 4Hz, 6H), 2.04(s, 3H).

[0275] Step 4: Cpd. 7-d (6.50 g, 19.5 mmol, 1.00 eq) dissolved in MeOH (39.0 mL) was added with NaOMe (211 mg, 1.17 mmol, 30% purity, 0.06 eq) and stirred at 20 °C for 1 h. TLC (petroleum ether:ethyl acetate = 1:1, R f =0.1), indicating that the starting material had been consumed and a new spot had formed. The reaction was adjusted to pH 7 with Amberlite IR120, Na resin (CAS: 78922-04-0), filtered, and concentrated under reduced pressure to give the residue. Cpd. 7-e (4.00 g, crude) was obtained as a yellow oil. 1 H NMR (400 MHz, D6-DMSO) (product) δ 5.58(s, 1H), 5.32(s, 1H), 4.97-4.99(m, 1H), 4.70(s, 1H), 3.81-3.99(m, 1H), 3.68-3.71(m, 1H), 3.34-3.53(m, 2H), 3.32-3.36(m, 1H), 3.15(t, J = 8 Hz, 1H).

[0276] Step 5: Cpd. 7-e (4.00 g, 19.3 mmol, 1.00 eq) dissolved in DCM (40.0 mL) was added with Cpd. 7-f (9.74 g, 77.2 mmol, 10.6 mL, 4.00 eq) and DMAP (1.42 g, 11.6 mmol, 0.60 eq) and stirred at 20 °C for 12 h. TLC (petroleum ether: ethyl acetate = 1:1, R f =0.9), indicating that the starting material had been consumed and a new spot had formed. The solvent was removed under vacuum and water (50.0 ml) was added to the residue. The aqueous layer was extracted with dichloromethane (3×50.0 ml) and the organic layer was dried over Na2SO4. The residue was purified by column chromatography (silica gel, petroleum ether:ethyl acetate=20:1 to 8:1). Cpd. 7-g (5.20 g, 8.88 mmol, 46.0% yield) was obtained as an orange solid. 1H NMR(400 MHz, CDCl3) (product) δ 7.44(d, J = 12 Hz, 1H), 7.28(d, J = 12 Hz, 1H), 5.27-5.36(m, 2H), 5.15(d, J = 12 Hz, 1H), 4.81(m, 1H), 4.04-4.32(m, 4H), 1.44-1.45(m, 27H).

[0277] Step 6: Cpd. 7-e (1.00 g, 1.71 mmol, 1.00 eq) dissolved in MeOH (12 mL) was added Pd / C (0.6 g, 10% purity, 1.00 eq) and stirred at 25 °C under H2 (800 mg, 1.00 eq) for 14 h. 1 H NMR indicated complete consumption of starting material. Two batches were run in parallel as described above and combined after workup. The mixture was filtered and concentrated under reduced pressure to give the residue. Cpd. 7-h (1.80 g, 3.18 mmol, 93.2% yield) was obtained as a white solid. 1 H NMR (400 MHz, CDCl3) (product) δ 3.47-4.15(m, 9H), 3.14-3.47(m, 3H), 2.36-2.47(m, 6H), 1.89(s, 1H), 1.33-1.38(m, 27H).

[0278] Step 7: Cpd.7-h (2.10 g, 3.71 mmol, 1.00 eq) dissolved in DMF (20.0 mL) was added with cpd.7-i (1.31 g, 4.08 mmol, 1.10 eq), HBTU (2.82 g, 7.42 mmol, 2.00 eq), DIEA (1.44 g, 11.1 mmol, 1.94 mL, 3.00 eq) and stirred at 25 °C for 12 h. LCMS showed complete consumption of starting material. DCM (150 ml) was added, washed with water (120 ml × 3), dried over anhydrous Na2SO4, filtered and concentrated in vacuum. Two batches were run in parallel as described above and combined for purification. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate=20 / 1-5 / 1) to give Cpd. 7-j (4.1 g, 4.72 mmol, 63.6% yield) as a yellow solid. 1 H NMR (400 MHz, CDCl3) (product) δ 7.35(s, 5H), 5.96(d, J = 9.2 Hz, 1H), 5.20(t, J = 8 Hz, 1H), 5.11(s, 2H), 3.65-4.13(m, 13H), 3.39-3.52(m, 5H), 2.41-2.52(m, 8H), 2.33-2.45(m, 3H), 2.20(t, J = 8 Hz, 2H), 1.62-1.64(m, 6H), 1.45(s, 27H), 1.26(s, 12H).

[0279] Step 8: Cpd. 7-j (4.10 g, 4.72 mmol, 1.00 eq) was added to FA (80.0 mL) and stirred at 25° C. for 2 h. LCMS showed that the starting material was completely consumed. The reaction solution was then concentrated under vacuum. The residue was dissolved in DCM (20.0 mL), followed by addition of toluene (60.0 mL) and THF (60.0 mL) and drying under vacuum (this operation was repeated three times). Cpd. 7-k (2.90 g, crude product) was obtained as a yellow solid. 1H NMR (400 MHz, D6-DMSO) (product) δ 12.1(s, 3H), 8.62(d, J = 8 Hz, 1H), 7.32-7.39(m, 5H), 5.04-5.08(m, 3H), 3.98-4.16(m, 1H), 3.870-3.91(m, 5H), 3.48-3.66(m, 6H), 3.40(d, J = 12 Hz, 1H), 3.13(t, J = 8 Hz, 1H), 2.69(s,1H), 2.41-2.46(m, 6H), 2.34(t, J = 8 Hz, 2H), 2.10(t, J = 8 Hz, 2H), 1.48-1.54(m, 4H), 1.22(s, 12H).

[0280] Step 9: To a mixture of Cpd. 7-k (1.50 g, 2.14 mmol, 1.00 eq) dissolved in DMF (30.0 mL), HBTU (2.60 g, 6.86 mmol, 3.2 eq) and DIPEA (3.60 g, 27.9 mmol, 4.85 mL, 13.0 eq) was added. Cpd.7-l (4.49 g, 6.65 mmol, 3.10 eq, TsOH salt) (synthesis of Cpd. 7-l is shown below) was then added to the solution and stirred at 25 °C for 12 h. LCMS showed complete consumption of starting material. The aqueous phase was diluted with DCM (100 mL). The combined organic layers were washed with half-saturated brine (100 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The remainder was purified by column chromatography (silica gel, DCM / MeOH=20 / 1~5 / 1). The crude product was purified by reversed-phase HPLC. Column: Welch Xtimate C18 250*70mm#10um; Mobile phase: [water(NH4HCO3)-ACN]; B%: 30%-60%, 20min. Cpd.7-m (1.50 g, 696 umol, 32.5% yield) was obtained as a white solid. Synthesis of Cpd. 7-l [ka]

[0281] Step A: To Cpd. 7-l-1 (20.0 g, 44.7 mmol, 1.00 eq) dissolved in DMF (140 mL) was added DIEA (23.11 g, 179 mmol, 31.1 mL, 4.00 eq), HBTU (18.7 g, 49.2 mmol, 1.10 eq) and Cpd. G-2 (10.2 g, 49.2 mmol, 1.10 eq) at 0-5 °C. The mixture was then stirred at 15 °C for 12 h. TLC (DCM: MeOH = 10:1, R f = 0.3) consumed Cpd. 7-l-1 and two major spots were detected. The aqueous phase was diluted with DCM (400 mL). The combined organic layers were washed with half-saturated brine (200 mL × 3) and dried over anhydrous Na2SO4, then filtered and concentrated under vacuum. The crude product (30.0 g) was purified by prep HPLC. Column: Agela DuRaShell C18 250*70mm*10um; Mobile phase: [water(NH4HCO3)-ACN]; B%: 20%-50%, min. Cpd. 7-l-2 (26.0 g, 40.8 mmol, 91.2% yield) was obtained as a yellow oil. 1 H NMR: (400 MHz, DMSO) (product) δ7.81(d, J = 8 Hz, 1H), 7.73(t, J = 8 Hz, 1H), 7.30-7.38(m, 5H), 7.20-7.22(m, 1H), 5.22(s, 1H), 4.96-5.01(m, 3H), 4.48-4.50(d, J = 8 Hz, 1H), 4.03(s, 3H), 3.84-3.92(m, 1H), 3.70-3.72(m, 1H), 3.40-3.43(m, 1H), 2.97-3.06(m, 4H), 2.10(s, 2H), 1.99-2.07(m, 5H), 1.89(s, 3H), 1.77(s, 3H), 1.46-1.54(m, 6H). [M+H] = 638.3(product).

[0282] Step B: Cpd. 7-l-2 (26.0 g, 40.8 mmol, 1.00 eq) dissolved in THF (260 mL) was added to Pd / C (10.0 g, 10% purity) and stirred at 25 °C under H2 for 12 h. TLC (DCM:MeOH = 10:1, R 1 , R f =0.3, P1, R 1 =0.2), indicating that the starting material had been consumed and a new spot had formed. The mixture was filtered and concentrated under reduced pressure to give a residue. Cpd. 7-l (27.0 g, 40.0 mol, 98% yield, PTSA salt) was obtained as a white solid. 1 H NMR: (400 MHz, DMSO) (product) δ7.93(t, J = 4 Hz, 1H), 7.85(d, J = 8 Hz, 1H), 7.69(s, J = 8 Hz, 3H), 7.49(d, J = 8 Hz, 2H), 7.13(d, J = 8 Hz, 2H), 5.22(s, 1H), 4.96-4.99(m, 1H), 4.48-4.51(d, J = 12 Hz, 1H), 4.03(s, 3H), 3.84-3.92(m, 1H), 3.70-3.72(m, 1H), 3.38-3.43(m, 1H), 3.07-3.11(m, 2H), 2.29(s, 3H), 2.05-2.10(m, 5H), 2.00(s, 3H), 1.89(s, 3H), 1.76(s, 1H), 1.64-1.67(m, 2H), 1.44-1.50(m, 4H).

[0283] Step 10: Cpd. 7-m (850 mg, 394 umol, 1.00 eq) dissolved in THF (20 ml) was added with Pd / C (850 mg, 10% purity, 1.00 eq) and stirred at 25° C. under H2 for 12 h. LCMS showed complete consumption of starting material. The mixture was filtered and concentrated under reduced pressure to give a residue (THF: MeOH = 1:1, 100 mL). Cpd. 7-n (730 mg, crude) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1031.5, found 1031.9 (product).

[0284] Step 11: To a mixture of Cpd. 7-n (620 mg, 300.07 umol, 1.00 eq) dissolved in DMF (6.00 mL), HBTU (171 mg, 450 umol, 1.50 eq) and DIPEA (78 mg, 600 umol, 105 uL, 2.00 eq) was added. Cpd.7-o (189 mg, 450 umol, 1.5 eq) was then added to the solution and stirred at 30 °C for 12 h. LCMS showed complete consumption of starting material. The aqueous phase was diluted with DCM (50.0 mL). The combined organic layers were washed with half-saturated brine (40.0 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under vacuum. Two batches of the crude reaction mixture (100 mg and 620 mg scales) were combined and purified. The crude product was purified by reversed-phase HPLC. Column: Waters Xbridge Prep OBD C18 150*40mm*10um; Mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-60%, 8 min. Cpd. 7-p (500 mg, 203 umol, 55.6% yield) was obtained as a white solid.

[0285] Step 12: To a mixture of Cpd. 7-p (100 mg, 40.5 umol, 1.00 eq) dissolved in DCM (2.00 mL), DMAP (1.24 mg, 10.1 umol, 0.25 eq) and DIPEA (31.4 mg, 243 umol, 42.4 uL, 6.00 eq) was added, then succinic anhydride (24.3 mg, 243 umol, 6.00 eq) was added to the solution and stirred at 15 °C for 12 hours. LCMS showed that the starting material was completely consumed. The residue was diluted with DCM (5.00 mL) and extracted with TEAB (15 mL × 3). The combined organic layers were washed with water (15 mL × 2), dried over sodium sulfate, filtered and concentrated under reduced pressure to give the residue. The crude product was purified by reversed phase HPLC. Column: C18-2 100 x 30mm x 5um; Mobile phase: [0.1M TEAB-ACN]; B%: 20%-45%, 20min. Cpd. 7-q(4-(((3R,5S)-1-(12-(((2R,3R,4S,5R,6R)-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R,6R)-3 -acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-3-fluorotetrahydro-2H-pyran-2-yl)amino)-12-oxododecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid) (55 mg, 21.4 umol, 52.9% yield) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1282.1, found 1282.6 (product).

[0286] Step 13: To Cpd. 7-q (30.0 mg, 11.7 umol, 1.00 eq) dissolved in DMF (1.50 mL), HBTU (22.1 mg, 58.4 umol, 5.00 eq), DIEA (12.1 mg, 93.5 umol, 16.3 uL, 8.00 eq), and DMAP (1.43 mg, 11.7 umol, 1.00 eq) were added in one portion at 25 °C. Then, CPG (210 mg; Chemical name: Aminoalkyl-CPG, 500A, Hebei DNAchem Biotechnology Co., Ltd.) was added to the mixture. The mixture was stirred at 40 °C for 48 h. Then, it was filtered and the filter cake was washed with methanol (5.00 mL × 4) and DCM (5.00 mL × 4). The filter cake was dried under N2 stream to give a pale yellow solid. The above pale yellow solid was added to a mixture of dry pyridine (1.50 mL) and acetic anhydride (Ac2O) (0.30 mL). The mixture was then stirred at 40°C for 0.5 h. It was then filtered and the filter cake was washed with DCM (5.00 mL x 4) and methanol (5.00 mL x 4). The obtained pale yellow solid was dried under vacuum for 12 h to give 180 mg of solid liquid product (loading: 36 umol / g).

[0287] Example 31 Synthesis of compound 8 [ka]

[0288] Compound 8 is synthesized according to the above-mentioned synthetic route of compound 1 using intermediate 8-10 as the starting material.

[0289] Example 32 Synthesis of compound 9 [ka]

[0290] Compound 9 is synthesized according to the above-mentioned synthetic route of compound 2 using intermediate 9-2 as the starting material.

[0291] Example 33 Synthesis of compound 10 [ka]

[0292] Compound 10 is synthesized according to the above-mentioned synthetic route of compound 2 using intermediate 10-2 as the starting material.

[0293] Example 34 Synthesis of compound 11 [ka]

[0294] Compound 11 is synthesized according to the above-mentioned synthetic route of compound 2 using intermediate 11-2 as a starting material.

[0295] Example 35 Synthesis of compound 12 [ka]

[0296] Compound 12 is synthesized according to the above-mentioned synthetic route of compound 2 using intermediate 12-2 as a starting material.

[0297] Example 36 Synthesis of compound 13-7 [ka]

[0298] Example 37 Synthesis of compounds 13-11 [ka]

[0299] Example 38 Synthesis of compound 13 [ka]

[0300] Example 39 Synthesis of compound 14 [ka]

[0301] Compound 14 is synthesized using intermediate 14-4 as the starting material according to the above-mentioned synthesis route of compound 1. The synthesis is shown below. [ka]

[0302] Example 40 Synthesis of compound 15 [ka]

[0303] Compound 15 is synthesized using intermediate 15-3 as the starting material according to the above-mentioned synthesis route of compound 1. The synthesis is shown below. [ka]

[0304] Example 41 Synthesis of compound 16 [ka]

[0305] Compound 16 is synthesized according to the above-mentioned synthetic route of compound 2 using (2R,3S,4s,5R,6S)-2,6-bis(aminomethyl)tetrahydro-2H-pyran-3,4,5-triol as a starting material.

[0306] Examples 42-49 (omission)

[0307] Example 50 Synthesis of compound 25 [ka]

[0308] Compound 25 is synthesized by using intermediate 25-7 as the starting material and following the above-mentioned synthetic route of compound 2. The synthesis is shown below. [ka]

[0309] Example 51 Synthesis of GalNAc-siRNA conjugate compound 1-F: [ka]

[0310] Using the conjugate building block compound 1 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0311] Example 52 Synthesis of GalNAc-siRNA conjugate compound 2-F: [ka]

[0312] Using the conjugate building block compound 2 previously described (described in Example 25), RNA is synthesized according to known procedures with a ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0313] Example 53 Synthesis of GalNAc-siRNA conjugate compound 3-F: [ka]

[0314] Using the conjugate building block compound 3 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0315] Example 54 Synthesis of GalNAc-siRNA conjugate compound 4-F: [ka]

[0316] Using the conjugate building block compound 4 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0317] Example 55 Synthesis of GalNAc-siRNA conjugate compound 5-F: [ka]

[0318] Using the conjugate building block compound 5 previously described (described in Example 28), RNA is synthesized according to known procedures with a ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0319] Example 56 Synthesis of GalNAc-siRNA conjugate compound 6-F: [ka]

[0320] Using the conjugate building block compound 6 previously described (described in Example 29), RNA is synthesized according to known procedures with a ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0321] Example 57 Synthesis of GalNAc-siRNA conjugate compound 7-F: [ka]

[0322] Using the conjugate building block compound 7 previously described (described in Example 30), RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0323] Example 58 Synthesis of GalNAc-siRNA conjugate compound 8-F: [ka]

[0324] Using the conjugate building block compound 8 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0325] Example 59 Synthesis of GalNAc-siRNA conjugate compound 9-F: [ka]

[0326] Using the conjugate building block compound 9 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0327] Example 60 Synthesis of GalNAc-siRNA conjugate compound 10-F: [ka]

[0328] Using the conjugate building block compound 10 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0329] Example 61 Synthesis of GalNAc-siRNA conjugate compound 11-F: [ka]

[0330] Using the conjugate building block compound 11 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0331] Example 62 Synthesis of GalNAc-siRNA conjugate compound 12-F: [ka]

[0332] Using the conjugate building block compound 12 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0333] Example 63 Synthesis of GalNAc-siRNA conjugate compound 13-F: [ka]

[0334] Using the conjugate building block compound 13 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0335] Example 64 Synthesis of GalNAc-siRNA conjugate compound 14-F [ka]

[0336] Using the conjugate building block compound 14 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0337] Example 65 Synthesis of GalNAc-siRNA conjugate compound 15-F [ka]

[0338] Using the conjugate building block compound 15 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0339] Example 66 Synthesis of GalNAc-siRNA conjugate compound 16-F [ka]

[0340] Using the conjugate building block compound 16 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0341] Examples 67-74 (omission)

[0342] Example 75 Synthesis of GalNAc-siRNA conjugate compound 25-F [ka]

[0343] Using the conjugate building block compound 25 described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0344] Example 76 Synthesis of compound 26 [ka]

[0345] The corresponding compounds may be synthesized according to the following synthetic route. [ka] [ka]

[0346] Step 1: To Cpd. 26-a (5.00 g, 9.11 mmol) (synthesis of Cpd. 26-a is shown below) dissolved in pyridine (30.0 mL) was added DMAP (4.45 g, 36.4 mmol) and Cpd. 26-b (7.35 g, 36.4 mmol) at 20 °C. The reaction was stirred at 20 °C for 2 h. LCMS (RT = 1.11 min) showed that Cpd. 26-c was formed. The two reactions were now combined. The liquid was diluted with water (40.0 mL) and extracted with ethyl acetate (50.0 mL × 3). The combined organic layers were washed with 1N hydrochloric acid (15.0 ml), sodium bicarbonate (15.0 ml), and saturated brine (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give Cpd. 26-c (11.5 g, crude) as a yellow oil. 1 H NMR (400 MHz CDCl3) (product) δ 8.16(d, J = 2.4 Hz, 2H), 7.43-7.40(m, 2H), 4.52(d, J = 4.0 Hz, 1H), 4.48-4.31(m, 1H), 4.07-4.04(m, 3H), 4.03-4.01(m, 1H), 3.83-3.81(m, 3H), 3.27-3.10(m, 6H), 2.53-2.46(m, 6H), 1.45(s,9H). Synthesis of Cpd. 26-a [ka]

[0347] Step A: Sodium methoxide (9.23 g, 170.93 mmol) was added to Cpd. 26-a-1 (142 g, 427.32 mmol) dissolved in methanol (994 mL). The mixture was stirred at 25° C. for 1 h. TLC (dichloromethane:methanol = 3:1, product: R f = 0.40) (starting material: R f= 0.80) was completely consumed and a new spot was formed. Amberlite (H+) was added to the above mixture and stirred until the pH of the solution reached 7. The Amberlite was filtered and washed with methanol. Cpd. 26-a-2 (70 g, 426.42 mmol, 99.79% yield) was obtained as a colorless oil. 1 H NMR (400 MHz CD3OD) (product) δ 3.87-3.79(m, 2H), 3.29-3.28(m, 1H), 3.28-3.27(m, 2H), 3.16-3.13(m, 2H).

[0348] Step B: To a solution of Cpd. 26-a-2 (7 g, 42.64 mmol) in pyridine (49 mL) was added chloro(triisopropyl)silane (9.04 g, 46.91 mmol) at 0° C. The mixture was stirred at 25° C. for 12 h. TLC (ethyl acetate, product: R f = 0.35) (starting material: R f = 0.80), indicating complete consumption and the formation of a new major spot. The reaction mixture was diluted with 50 mL of water and extracted with ethyl acetate (50 mL x 2). The combined organic layers were washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 1000 / 1~1 / 1, TLC: ethyl acetate, R f = 0.35) to give Cpd. 26-a-3 (6.1 g, 19.03 mmol, 44.63% yield) as a white solid. 1 H NMR (400 MHz CD3OD) (product) δ 4.01-4.00(d, 1H), 3.87-3.79(m, 2H), 3.29-3.28(m, 1H), 3.28-3.27(m, 2H), 3.16-3.13(m, 2H), 1.01(s 21H).

[0349] Step C: To Cpd.26-a-3 (14.2 g, 44.31 mmol) dissolved in DCM (99.4 mL) was added tert-butylpropionate (22.36 g, 177.24 mmol) and DMAP (3.25 g, 26.59 mmol). The mixture was stirred at 25° C. for 6 h. TLC (petroleum ether: ethyl acetate=4:1, product: R f = 0.40 & 0.50) (Starting material: R f = 0.02) was completely consumed and two new spots were formed. The reaction mixture was diluted with 100 mL of water and extracted with DCM (100 mL x 2). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 1000 / 1 to 1 / 1, TLC: petroleum ether: ethyl acetate = 4 / 1, product: R f = 0.40 & 0.50) to give Cpd. 26-a-4 (30.5 g, 43.64 mmol, 98.48% yield) as a yellow oil. 1 H NMR (400 MHz CD3OD) (product) δ 7.47-7.34(m, 2H), 5.27-5.22(m, 2H), 4.34-4.23(m,2H), 3.95-3.98(d, J = 1.2 Hz,1H), 3.50-3.42(m, 2H), 1.45-1.44(s, 28H), 1.11-1.08(s, 21H).

[0350] Step D: To Pd / C (3.00 g, 10% purity) dissolved in methanol (20 mL) was added Cpd. 26-a-4 (30.5 g, 43.64 mmol) dissolved in methanol (190 mL) under argon atmosphere. The suspension was degassed and purged with H2 three times. The mixture was stirred under H2 (50 Psi) at 50 °C for 12 h. HNMR (ET52646-51-P1A2) showed complete consumption of starting material. The suspension was filtered through a pad of Celite or silica gel, and the pad or filter cake was washed with 1.00 L of methanol. Cpd. 26-a-5 (30.0 g, 42.55 mmol, 97.52% yield) was obtained as a colorless oil. 1 H NMR (400 MHz CDCl3) (product) δ 3.98-3.96(m, 4H), 3.95-3.80(m, 4H), 3.27-3.20(m, 3H), 3.18-3.05(m, 2H), 1.46-1.44(s, 27H), 1.11-1.06(s, 21H).

[0351] Step E: To a solution of Cpd. 26-a-5 (30 g, 42.55 mmol) in THF (210 mL) was added TBAF (1M, 42.55 mL). The mixture was stirred at 25° C. for 2 h. TLC (petroleum ether: ethyl acetate = 2:1) showed (starting material: R f =0.85) is completely consumed and a new spot (R f The reaction mixture was diluted with 200 mL of water and extracted with ethyl acetate (200 mL x 2). The combined organic layers were washed with 200 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate=1000 / 1~1 / 1, TLC: petroleum ether: ethyl acetate=2 / 1, product: R f =0.35) to give Cpd.26-a (17.9 g, 32.62 mmol, 76.67% yield) as a colorless oil. 1H NMR (400 MHz CDCl3) (product) δ 3.98-3.96(m,4H), 3.95-3.80(m, 4H), 3.27-3.20(m, 3H), 3.18-3.05(m, 2H), 1.46-1.44(s, 27H).

[0352] Step 2: To Cpd. 26-c (4.00 g, 5.60 mmol) dissolved in MeCN (80.0 mL) at 25°C, Cpd. 26-d (2.34 g, 8.41 mmol) and DMAP (753 mg, 6.16 mmol) were added. The reaction was stirred at 25°C for 16 hours. LCMS (RT=0.861 min) showed the formation of Cpd.26-e. The mixture was concentrated under reduced pressure to give the residue. The residue was first purified by column chromatography (silica gel, petroleum ether / ethyl acetate=25 / 1-10 / 1) and then by prep HPLC column: Phenomenex luna C18 250*150mm*15um; mobile phase: [water (TFA)-ACN]; B%: 60%-98%, 25 min. Cpd. 26-e (4 g, 4.69 mmol, 83.7% yield) was obtained as a yellow oil. 1 H NMR (400 MHz CDCl3) (product) δ 7.37-7.32(m, 5H), 5.12(s, 2H), 4.38(d, J=1.6 Hz, 1H), 4.13-4.10(m, 1H), 4.03-4.01(m,1H), 4.00-3.99(m,2H), 3.97-3.88(m, 5H), 3.80-3.79(m,4H), 3.30-3.14(m,6H), 2.52-2.45(m,8H), 2.37-2.33(m, 2H), 1.65-1.63(m, 6H), 1.62(m,34H), 1.45-1.28(m,11H).

[0353] Step 3: Cpd. 26-e (4.00 g, 5.60 mmol) dissolved in HCOOH (15.0 g, 312 mmol) at 25 °C was added. The reaction was stirred at 25 °C for 4 h. LCMS (RT = 0.824 min) showed the formation of Cpd. 26-f. The mixture was treated under reduced pressure to give Cpd. 26-f (4.00 g, 4.69 mmol, 83.6% yield) as a colorless oil. 1 H NMR (400 MHz CDCl3) (product) δ 7.38-7.27(m, 4H), 5.11(s, 2H), 4.38(d, J=1.6 Hz, 1H), 4.37-3.78(m, 10H), 3.33-3.08(m, 5H), 2.50-2.36(m, 5H), 2.35-2.01(t, J=1.36, 3H), 1.63(s, 4H), 1.27(s,10H).

[0354] Step 4: To Cpd. 7-l (1.47 g, 2.92 mmol) dissolved in DCM (2.50 mL) at 25 °C under N2, HBTU (858 mg, 2.26 mmol) and DIPEA (943 mg, 7.30 mmol) were added in one portion and then stirred at 25 °C under N2 atmosphere for 10 min. Cpd. 26-f (500 mg, 730 umol) was added to this mixture and stirred at 17 °C for 20 h. LCMS (product: RT = 2.884 min) showed complete consumption of starting material. The mixture was poured into DCM (20 mL). The combined organic phase was washed with saturated aqueous sodium bicarbonate (20.0 mL × 2) and saturated brine (20.0 mL × 3). The combined organic phase was then dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by reverse phase HPLC. Mobile phase: water (NH4HCO3)-ACN; B%: 40%-70%, 10 min. Cpd. 26-g (350 mg, 163 umol, 22.4% yield) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1069.0, found 1069.1 (product).

[0355] Step 5: To Cpd. 26-g (350 mg, 163 umol) dissolved in THF (6 mL) was added Pd / C (600 mg, 10% purity) under Ar. The mixture was stirred under H2 (15 psi) at 25°C for 19 h. LCMS (product: RT = 2.149 min) showed that a major peak with the desired mass was detected. The mixture was filtered and concentrated under vacuum to give Cpd.26-h (400 mg, crude), which was used as a white solid without further purification in the next step. LCMS: ESI-, calculated [[M-2H] / 2 = 1024.0, found 1024.2 (product).

[0356] Step 6: To Cpd. 26-h (400 mg, 195 umol) dissolved in DMF (3.00 mL) was added HBTU (110 mg, 292 umol) and DIPEA (50.41 mg, 390 umol) in one portion at 17 °C under N2, then stirred at 17 °C for 10 min under N2. To this mixture was added Cpd. 7-o (122 mg, 292 umol) and stirred at 17 °C for 17 h. LCMS (product: RT = 2.907 min) showed complete consumption of starting material. The crude product was purified by reverse phase HPLC (water (NH4HCO3)-ACN; B%: 25%-55%, 20 min). Cpd. 26-i (140 mg, 57.0 umol, 29.3% yield) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1224.6, found 1225.1 (product).

[0357] Step 7: To Cpd. 26-i (140 mg, 57.1 umol) dissolved in DCM (1.00 mL) was added DIPEA (44.3 mg, 342 umol) and DMAP (1.74 mg, 14.3 umol) in one portion at 25 °C under N2, then stirred at 20 °C for 10 min under N2. To this mixture was added succinic anhydride (34.3 mg, 342 umol) and stirred at 20 °C for 4 h. LCMS (product: RT =2.611 min) showed complete consumption of starting material. The mixture was poured into DCM (10 mL). The combined organic phase was washed with aqueous TEBA solution (5 mLx2). The combined organic phase was then dried over anhydrous Na2SO4, filtered and concentrated under vacuum to give Cpd. 26-j(4-(((3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(10-(((((2R,3R,4R,5S)-3,4,5-tris(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-2-yl)methoxy)carbonyl)oxy)decanoyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid) (90 mg, 35.3 umol, Yield 61.8%) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1274.6, found 1275.1 (product).

[0358] Step 8: To Cpd. 26-j (33 mg, 12.9 umol) dissolved in DMF (1.50 mL), HBTU (24.5 mg, 64.6 umol), DIEA (13.4 mg, 103.4 umol), and DMAP (1.58 mg, 12.9 umol) were added in one portion at 25 °C. Then, CPG (250 mg; chemical name: Aminoalkyl-CPG, 500A, from Hebei DNAchem Biotechnology Co., Ltd.) was added to the mixture. The mixture was stirred at 40 °C for 17 h, then filtered, and the filter cake was washed with methanol (5.00 mL × 4) and DCM (5.00 mL × 4). The filter cake was dried under N2 stream to give a light yellow solid. The above light yellow solid was added to a mixture of dry pyridine (1.50 mL) and acetic anhydride (AcO) (0.30 mL). The mixture was then stirred at 40° C. for 0.5 h. It was then filtered, and the filter cake was washed with DCM (5.00 mL × 4) and methanol (5.00 mL × 4). The resulting light yellow solid was dried under vacuum for 12 h to give 225 mg of solid liquid product (loading 32 μmol / g).

[0359] Example 77 Synthesis of compound 27 [ka]

[0360] The corresponding compounds may be synthesized according to the following synthetic route. [ka] [ka]

[0361] Step 1: Cpd. 26-c (4.00 g, 5.60 mmol, 1.00 equiv.), Cpd. 27-a (1.71 g, 6.16 mmol, 1.10 equiv.) and DMAP (3.42 g, 28.0 mmol, 5.00 equiv.) were added to CH3CN (80.0 mL), degassed and purged with N2 three times, and then stirred under N2 atmosphere at 25 °C for 16 h. LCMS (RT = 1.084 min) showed that Cpd. 26-c was completely consumed. The reaction was concentrated. The residue was purified by column chromatography (silica gel, dichloromethane:methanol = 1:0 to 0:1). Cpd. 27-b (4.00 g, 4.47 mmol, 79.8% yield, 95.3% purity) was obtained as a yellow oil. 1 H NMR (400 MHz CD3OD) (product) δ 7.34(m, 5H), 5.10(s, 2H), 4.09-4.26(m, 1H), 3.98-4.01(m, 6H), 3.85-3.75(m, 1H), 3.94-3.96(m, 3H), 3.07-3.31(m, 8H), 2.37-2.51(m, 9H), 1.45(t, J = 1.6 Hz, 27H), 1.29(t, J = 1.6 Hz, 12H).

[0362] Step 2: Cpd. 27-b (4.00 g, 4.69 mmol, 1.00 equiv) dissolved in HCOOH (80.0 g, 1.67 mol, 20V) was added. The mixture was stirred at 25 °C for 3 h. LCMS (RT = 0.789&0.810 min) showed that Cpd. 27-b was completely consumed. The reaction mixture was concentrated. Cpd. 27-c (3.00 g, 4.39 mmol, 93.5% yield) was obtained as a yellow oil. 1 H NMR (product) δ 7.19-7.31(m, 5H), 7.10-14(m, 4H), 5.04(s, 2H), 4.12(d, J = 10.8 Hz, 1H), 3.80-3.87(m, 5H), 3.67-3.69(m, 3H), 2.90-3.12(m, 4H), 2.26-2.43(m, 8H), 1.17-1.50(m, 12H).

[0363] Step 3: To Cpd. 27-c (0.60 g, 877 umol, 1.00 equiv.) dissolved in DMF (12.0 mL), HBTU (1.10 g, 2.90 mmol, 3.30 equiv.) and DIPEA (1.47 g, 11.4 mmol, 1.99 mL, 13.0 equiv.) were added dropwise over 30 min at 25°C. After addition, Cpd. 7-l (1.46 g, 2.90 mmol, 3.30 equiv.) was added and stirred at this temperature for 12 h. LCMS showed that Cpd. 27-c was completely consumed. The crude product was purified by reversed phase HPLC. Column: Phenomenex luna C18 (250*70mm, 15 um); Mobile phase: [water-ACN]; B%: 30%-60%, 20 min. Cpd. 27-d (1.5 g, 700.83 umol, 79.86% yield) was obtained as a yellow oil. LCMS: ESI+, calculated [[M+2H] / 2 = 1070.5, found 1070.5 (product).

[0364] Step 4: To Cpd. 27-d (900 mg, 420 umol, 1.00 equiv.) dissolved in methanol (10.0 ml) was added Pd / C (900 mg, 10% purity, 1.00 equiv.) and the reaction was stirred at 25° C. under H2 for 12 h. LCMS (ET54421-53-P1A1, RT = 1.820 min) showed complete consumption of starting material. The mixture was filtered and concentrated under reduced pressure to give a residue (THF:methanol=1:1, 50.0 mL). Cpd. 27-e (700 mg, crude) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1023.5, found 1023.6 (product).

[0365] Step 5: To Cpd. 27-e (200 mg, 97.5 umol, 1.00 equiv.) dissolved in DMF (3.00 mL), HBTU (171 mg, 450 umol, 1.50 equiv.) and DIPEA (25.2 mg, 195 umol, 34 uL, 2.00 equiv.) were added to the mixture. Cpd. 7-o (61.4 mg, 146 umol, 1.5 equiv.) was then added to the solution and stirred at 30° C. for 12 h. LCMS (RT = 0.738 min) showed complete consumption of starting material. The aqueous phase was diluted with DCM (30.0 mL). The combined organic phase was washed with half-saturated brine (20.0 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by reverse phase HPLC. Column: Waters Xbridge BEH C18 100*30mm*10um; Mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-55%, 8min. Cpd. 27-f (140 mg, 57.1 umol, 31.0% yield) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1224.1, found 1224.1 (product).

[0366] Step 6: To Cpd. 27-f (135 mg, 55.0 umol, 1.00 equiv.) dissolved in DCM (1.50 mL), DMAP (1.68 mg, 13.7 umol, 0.25 equiv.) and DIPEA (35.5 mg, 275 umol, 5.00 equiv.) were added to the mixture. Tetrahydrofuran-2,5-dione (27.5 mg, 275 umol, 5.00 equiv.) was then added to the solution and stirred at 15° C. for 12 h. LCMS showed that the starting material was completely consumed. The residue was diluted with DCM (5.00 mL) and extracted with TEAB (15 mL × 3). The combined organic layers were washed with water (15 mL × 2), dried over sodium sulfate, filtered and concentrated under reduced pressure to give the residue. The crude product was purified by reverse phase HPLC. Column: C18-2 100*30mm*5um; Mobile phase: [0.1M TEAB-ACN]; B%: 20%-45%, 20 minutes. Cpd. 27-g (4-(((3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(10-(((((2R,3R,4R,5S)-3,4,5-tris(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-2-yl)methoxy)carbonyl)amino)decanoyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid) (50.0 mg, 19.6 umol, 35.7% yield) was obtained as a white solid. LCMS: ESI-, calculated [[M-2H] / 2 = 1274.1, found 1274.7 (product).

[0367] Step 7: To Cpd. 27-g (30.0 mg, 11.7 umol, 1.00 equiv.) dissolved in DMF (1.50 mL), HBTU (22.2 mg, 58.7 umol, 5.00 equiv.), DIEA (12.1 mg, 94.0 umol, 16.3 uL, 8.00 equiv.), and DMAP (1.44 mg, 11.7 umol, 1.00 equiv.) were added in one portion at 25°C. Then, CPG (220 mg; Chemical name: Aminoalkyl-CPG, 500A, Hebei DNAchem Biotechnology Co., Ltd.) was added to the mixture. The mixture was stirred at 40°C for 48 hours. Then, it was filtered and the filter cake was washed with methanol (5.00 mL × 4) and DCM (5.00 mL × 4). The filter cake was dried under a stream of N2 to give a pale yellow solid. The above pale yellow solid was added to a mixture of dry pyridine (1.50 mL) and acetic anhydride (Ac2O) (0.30 mL). The mixture was then stirred at 40 °C for 0.5 h. It was then filtered and the filter cake was washed with DCM (5.00 mL × 4) and methanol (5.00 mL × 4). The resulting pale yellow solid was dried under vacuum for 12 h to give 195 mg of solid liquid product (loading 32 μmol / g).

[0368] Example 78 Synthesis of GalNAc-siRNA conjugate compound 27-F: [ka]

[0369] Using the conjugated building block compound 27 (Example 77) described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0370] Example 79 Synthesis of compound 28 [ka]

[0371] Compound 28 was synthesized starting from 28-j according to the procedure of compound 6 in Example 29. The synthesis is as follows: Loading: 34.0 μmol / g.

[0372] 4-(((3R,5S)-1-(10-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R, 6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)tetrahydro-2H-pyran-2-yl)amino)-10-oxodecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (28-j): The title compound was synthesized according to the procedure of compound 6-j in example 29. LCMS: ESI-, calculated [[M-2H] / 2 = 1287.6, found 1288.2.

[0373] Example 80 Synthesis of GalNAc-siRNA conjugate compound 28-F: [ka]

[0374] Using the conjugated building block compound 28 (Example 79) described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0375] Example 81 Synthesis of compound 29 [ka]

[0376] Compound 29 was synthesized starting from 29-j according to the procedure of compound 6 in Example 29. The synthesis is as follows: Loading: 22.0 μmol / g.

[0377] 4-(((3R,5S)-1-(14-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R,6R )-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)tetrahydro-2H-pyran-2-yl)amino)-14-oxotetradecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (29-j): The title compound was synthesized according to the procedure of compound 6-j in example 29. LCMS: ESI-, calculated [[M-2H] / 2 = 1315.6, found 1316.2.

[0378] Example 82 Synthesis of GalNAc-siRNA conjugate compound 29-F: [ka]

[0379] Using the conjugated building block compound 29 (Example 81) described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0380] Example 83 Synthesis of compound 30 [ka]

[0381] Compound 30 was synthesized starting from 30-j according to the procedure of compound 6 in Example 29. The synthesis is as follows: Loading: 23.0 μmol / g.

[0382] 4-(((3R,5S)-1-(12-(((2R,3R,4R,5S,6R)-3-acetamido-4,5-bis(3-((4-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)butyl)amino)-3-oxopropoxy)-6-((3-((4-(5-(((2R,3R,4R,5R,6 R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)butyl)amino)-3-oxopropoxy)methyl)tetrahydro-2H-pyran-2-yl)amino)-12-oxododecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (30-j): The title compound was synthesized according to the procedure of compound 6-j in Example 29. LCMS: ESI-, calculated [[M-2H] / 2 = 1322.65, found 1323.2.

[0383] Example 84 Synthesis of GalNAc-siRNA conjugate compound 30-F: [ka]

[0384] Using the conjugated building block compound 30 (Example 83) described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0385] Example 85 Synthesis of compound 31 [ka]

[0386] Compound 31 was synthesized starting from 31-j according to the procedure of compound 6 in Example 29. The synthesis is as follows: Loading: 25.0 μmol / g.

[0387] 4-(((3R,5S)-1-(2-(2-(2-((2R,3R,4R,5S,6R)-3-acetamido-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R,6R) -3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)tetrahydro-2H-pyran-2-yl)amino)-2-oxoethoxy)ethoxy)ethoxy)acetyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (31-j): The title compound was synthesized according to the procedure of compound 6-j in Example 29. LCMS: ESI-, calculated [[M-2H] / 2 = 1297.58, found 1298.1.

[0388] Example 86 Synthesis of GalNAc-siRNA conjugate compound 31-F: [ka]

[0389] Using the conjugated building block compound 31 (Example 85) described above, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0390] Example 87 Synthesis of compound 32 [ka]

[0391] Compound 32 was synthesized starting from 32-j according to the procedure of compound 6 in Example 29. The synthesis is as follows: Loading: 30.0 μmol / g.

[0392] 4-(((3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(9-(1-(((2R,3R,4R,5S)-3,4,5-tris(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)nonanoyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (32-j): The title compound was synthesized starting from 32-e according to the procedure of compound 6-j in Example 29. LCMS: ESI-, calculated [[M-2H] / 2 = 1271.1, found 1271.7.

[0393] Synthesis of compound 32-e [ka]

[0394] Step 1: To a solution of compound 2-d (4.50 g, 7.93 mmol, 1.00 eq) in t-BuOH (22.5 mL) and H2O (22.5 mL) was added undec-10-inoic acid (1.59 g, 8.72 mmol, 1.10 eq) at 25 °C (solution 1). Sodium ascorbate (47.1 mg, 238 μmol, 0.03 eq) was then added to a solution of CuSO4 (19.0 mg, 119 μmol, 0.015 eq) in H2O (1.80 mL) (solution 2). Solution 2 was added to solution 1 at 25 °C and stirred at 85 °C for 2 h. TLC (petroleum ether / ethyl acetate = 2 / 1, R f =0.75), indicating that the starting material had been consumed. The reaction mixture was poured into water (30.0 mL) and extracted with ethyl acetate (40.0 mL×2). The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate=20 / 1-3 / 1). The crude product was purified by reverse phase HPLC. Column: Welch xtimate C18 × 250 70mm#10um; Mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 20 min gave 9-(1-(((2R,3R,4R,5S)-3,4,5-tris(((E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)nonanoic acid (32-e-1) (2.20 g; yield: 61.1%). LCMS: Calculated [M+H]= 750.4, found 750.5.

[0395] Step 2: To a solution of compound 32-e-1 (2.00 g, 2.67 mmol, 1.00 eq) in EtOH (20.0 mL), Pd(OH)2 (3.00 g, 10% purity) was added and stirred at 25 °C under H2 (15 psi) for 2 h. LCMS showed complete consumption of starting material. The reaction mixture was filtered and concentrated under reduced pressure to give 9-(1-(((2R,3R,4R,5S)-3,4,5-tris(3-(tert-butoxy)-3-oxopropoxy)tetrahydro-2H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-yl)nonanoic acid (32-e-2) (1.98 g; yield: 98%). 1 H NMR: (400 MHz, CDCl3) δ 7.37 (s, 1H), 4.67 (d, J = 12 Hz, 3H), 4.41-4.46 (m, 1H), 3.88-4.05 (m, 5H), 3.70-3.81 (m, 3H), 3.38-3.41(m, 1H), 3.20-3.27(m, 2H), 2.86-3.03(m, 2H), 2.69(t, J = 8 Hz, 2H), 2.44-2.58(m, 6H), 2.29(t, J = 8 Hz, 2H), 1.60-1.67(m, 4H), 1.44(t, J = 8 Hz, 27H), 1.31(s, 8H).

[0396] Step 3: To a solution of compound 32-e-2 (1.60 g, 2.12 mmol, 1.00 eq) in DMF (16.0 mL) and HO (22.5 mL), KCO (878 mg, 6.35 mmol, 3.00 eq) and BnBr (398 mg, 2.33 mmol, 277 μL, 1.10 eq) were added dropwise with stirring for 2 h at 25 °C. TLC (petroleum ether / ethyl acetate = 2 / 1, R f=0.3), indicating that the starting material had been consumed. The reaction mixture was extracted with EtOAc (200 mL), washed with saturated brine (200 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 20 / 1 to 2 / 1) to give tri-tert-butyl 3,3',3''-(((2R,3R,4R,5S)-2-((4-(9-(benzyloxy)-9-oxononyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-3,4,5-triyl)tris(oxy))tripropionate (32-e) (1.30 g; yield: 72.6%). 1 H NMR: (400 MHz, CDCl3) δ 7.35 (s, 5H), 5.11 (s, 2H), 4.67 (d, J = 12 Hz, 1H), 4.41-4.46 (m, 1H), 3.93-4.04 (m, 5H), 3.78-3.79(m, 2H), 3.40(s, 1H), 3.24-3.25 (m, 2H), 2.89-3.03(t, 2H), 2.67-2.69(m, 2H), 2.46-2.51(m, 6H), 2.34-2.37(m, 2H), 1.66(s, 4H), 1.45-1.46(m, 27H), 1.31(s, 8H).

[0397] Example 88 Synthesis of GalNAc-siRNA conjugate compound 32-F: [ka]

[0398] Using the conjugate building block compound 32 described above in Example 87, RNA is synthesized according to known procedures with a ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is shown above.

[0399] Example 89 Synthesis of compound 33 [ka]

[0400] Compound 33 was synthesized according to the procedure of compound 6 in Example 29, using 33-j as the starting material, as shown in the synthesis method below. Loading: 42.1 μmol / g.

[0401] 4-(((3R,5S)-1-(12-((3R,4R,5R)-3,4-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-5-((3-((3-(5-(((2R,3R,4R,5 R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)piperidin-1-yl)-12-oxododecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (33-j): The title compound was synthesized according to the procedure of compound 6-j in Example 29, using 33-e as starting material, as shown below in the synthesis method. LCMS: ESI-, calculated value [M-2H] / 2 = 1265.1, found: 1265.1. 1H NMR: (400 MHz, CDCl3) δ 7.60-7.28 (m, 8H), 7.26-7.15 (m, 4H), 7.12-6.93 (m, 4H), 6.83-6.75 (m, 6H), 5.42-5.31 (m, 3H), 5.25-5.15 (m, 3H), 4.70-4.32 (m, 6H), 4.23-4.05 (m, 12H), 3.96-3.85 (m, 9H), 3.80-3.56 (m, 14H), 3.55-3.40 (m, 7H), 3.35-3.15 (m, 17H), 3.12-3.03 (m, 2H), 2.58-2.50 (m, 8H), 2.45-2.29 (m, 15H), 2.18 (s, 9H), 2.15 (s, 9H), 2.05 (s, 9H), 2.00 (s, 9H), 1.76-1.50 (m, 28H), 1.35-1.17 (m, 18H).

[0402] Synthesis of di-tert-butyl 3,3'-(((3R,4R,5R)-1-(12-(benzyloxy)-12-oxododecanoyl)-5-((3-(tert-butoxy)-3-oxopropoxy)methyl)piperidine-3,4-diyl)bis(oxy))dipropionate (33-e): [ka]

[0403] Step 1. Compound 33-e-1 (3.37 g, 11.4 mmol, 1.00 eq) and TEA (2.87 g, 28.4 mmol, 3.95 mL, 2.50 eq) were dissolved in DMF (33.0 mL) and 1-benzyl 12-(2,5-dioxopyrrolidin-1-yl)dodecanedioate (5-fa) (5.21 g, 12.5 mmol, 1.10 eq) was added in one portion at 15 °C under N2 and stirred at 25 °C for 12 h. LCMS showed the reaction was complete. The reaction solution was concentrated under reduced pressure to give a residue. The reaction solution was concentrated under reduced pressure to give a yellow oily residue. The residue was purified by column chromatography (silica gel, DCM / MeOH = 30 / 1 to 20 / 1) to give benzyl 12-((3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)piperidin-1-yl)-12-oxododecanoate (33-e-2) (2.30 g, yield: 90.2%). LCMS: calculated [M+H] = 450.3, found 450.3. 1 H NMR: (400 MHz, MeOD) δ 7.18-7.28 (m, 5H), 5.03 (s, 2H), 4.40-4.57 (m, 2H), 3.69-3.88 (m, 4H), 3.40-3.43 (m, 4H), 2.83-2.89 (m, 1H), 2.32-2.40 (m, 1H), 2.27 (t, J = 8 Hz, 4H), 1.51-1.57 (m, 4H), 1.18-1.20 (m, 12H).

[0404] Step 2. Compound 33-e-2 (4.60 g, 10.2 mmol, 1.00 eq) in DCM (46.0 mL) was added with tert-butyl propiolate (5.16 g, 40.9 mmol, 5.62 mL, 4.00 eq) and DMAP (750 mg, 6.14 mmol, 0.60 eq) and stirred at 20 °C for 12 h. TLC (petroleum ether / ethyl acetate = 1 / 1, R f= 0.6), indicating that the starting material had been consumed and a new spot had formed. The solvent was removed in vacuo and water (30.0 mL) was added to the residue. The aqueous layer was extracted with dichloromethane (3 x 50.0 mL) and the organic layer was dried over Na2SO4. The remainder was purified by column chromatography (silica gel, petroleum ether:ethyl acetate=30:1 to 8:1) to give di-tert-butyl 3,3'-(((3R,4R,5R)-1-(12-(benzyloxy)-12-oxododecanoyl)-5-((((E)-3-(tert-butoxy)-3-oxoprop-1-en-1-yl)oxy)methyl)piperidine-3,4-diyl)bis(oxy))(2E,2'E)-diacrylate (33-e-3) (4.70 g; yield: 55.5%). LCMS: calculated [M+H] = 845.5, found 845.6. 1 H NMR: (400 MHz, CDCl3) δ 7.45-7.50 (m, 1H), 7.27-7.37 (m, 7H), 5.25-5.31 (m, 2H), 5.14-5.17 (m, 1H), 5.12(s, 2H), 4.43-4.83 (m, 1H), 3.81-4.08 (m, 5H), 3.08-3.19(m, 1H), 2.65-2.93(m, 1H), 2.30-2.37 (m, 4H), 1.61-1.68 (m, 6H), 1.47(d, J = 4 Hz, 27H), 1.27-1.28 (m, 12H).

[0405] Step 3. Compound 33-e-3 (2.70 g, 3.26 mmol, 1.00 eq) in ethanol (100 mL) was added with Pd / (OH)2 (2.70 g, 3.85 mmol, 20% purity, 1.18 eq) and stirred at 25 °C under H2 (15 psi) for 5 h. No raw material was observed by HNMR. The mixture was filtered and the filtrate was washed with methanol (100.0 mL x 3) and concentrated under reduced pressure to give 12-((3R,4R,5R)-3,4-bis(3-(tert-butoxy)-3-oxopropoxy)-5-((3-(tert-butoxy)-3-oxopropoxy)methyl)piperidin-1-yl)-12-oxododecanoic acid (33-e-4) (2.30 g, crude).

[0406] Step 4. Compound 33-e-4 (2.30 g, 3.09 mmol, 1.00 eq) in DMF (23.0 mL) was added with K2CO3 (1.28 g, 9.27 mmol, 3.00 eq) and BnBr (634.5 mg, 3.71 mmol, 440.63 uL, 1.20 eq) and stirred at 25 °C for 2 h. TLC (petroleum ether: ethyl acetate = 2:1, R f No starting material was observed by HPLC (product = 0.3). The mixture was extracted with ethyl acetate (120 mL), washed with brine (120 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 10 / 1~2 / 1) to give compound 33-e (2.20 g; yield: 85.3%). 1H NMR: (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.29-7.40 (m, 5H), 4.53-4.20 (m, 1H), 3.95-3.40 (m, 9H), 3.35-3.10 (m, 2H), 2.96 (s, 3H), 2.88 (s, 3H), 2.85-2.60 (m, 1H), 2.50-2.38 (m, 6H), 2.34-2.25 (m, 4H), 1.70-1.55 (m, 5H), 1.45 (s, 27H), 1.35-1.20 (m, 12H).

[0407] Example 90 Synthesis of GalNAc-siRNA conjugate compound 33-F: [ka]

[0408] Using the conjugate building block compound 33 described in Example 89, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0409] Examples 91-92 (omission)

[0410] Example 93 Synthesis of compound 35 [ka]

[0411] Compound 35 was synthesized according to the procedure of compound 7 in Example 30, using 35-q as the starting material, as shown in the synthesis method below. Loading: 24.0 μmol / g.

[0412] 4-(((3R,5S)-1-(14-(((2R,3R,4S,5R,6R)-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R,6R)-3- Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-3-fluorotetrahydro-2H-pyran-2-yl)amino)-14-oxotetradecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (35-q): The title compound was synthesized according to the procedure of compound 7-q in Example 30. LCMS: Calculated [M-2H] / 2: 1296.1, Found: 1296.7.

[0413] Example 94 Synthesis of GalNAc-siRNA conjugate compound 35-F: [ka]

[0414] Using the conjugate building block compound 35 described in Example 93, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0415] Example 95 Synthesis of compound 36 [ka]

[0416] Compound 36 was synthesized according to the procedure of compound 6 in Example 29, using 36-j as the starting material, as shown in the synthesis method below. Loading: 28.0 μmol / g.

[0417] 4-(((3R,5S)-1-(10-((((3S,4R,5S,6R)-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R,6R )-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)tetrahydro-2H-pyran-3-yl)carbamoyl)oxy)decanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (36-j): The title compound was synthesized according to the procedure of compound 5-j in Example 28, using 36-f as starting material, as shown below in the synthesis method. LCMS: Calculated value [M-2H] / 2: 1274.1, Found: 1274.7.

[0418] Synthesis of 3,3'-(((2R,3S,4R,5S)-5-((((10-(benzyloxy)-10-oxodecyl)oxy)carbonyl)amino)-2-((2-carboxyethoxy)methyl)tetrahydro-2H-pyran-3,4-diyl)bis(oxy))dipropionic acid (36-f): [ka]

[0419] Compound 5-f-4 (286.74 mg, 646.56 μmol, 1.1 eq) was dissolved in anhydrous DMF (6 mL) and TEA (416.34 mg, 4.11 mmol, 572.68 μL, 7 eq) was added, and a solid precipitated from the mixture. Then, 36-fa (0.29 g, 587.78 μmol) was added and stirred at 25 °C (oil bath) under N2 for 16 h, resulting in a pale yellow solution. LCMS showed that compound 5-f-4 was completely consumed. The mixture was filtered and concentrated under vacuum. The crude product was precipitated with DCM / Hexane=1 / 5 for 60 min at 25 °C, and then with ACN for 60 min at 25 °C to give compound 36-f (0.49 g, brown powder), which was used in the next step without further purification. LCMS: calculated [M+NH4]: 701.3, found: 701.3.

[0420] Synthesis of benzyl 10-(((4-nitrophenoxy)carbonyl)oxy)decanoate (36-fa): Benzyl 10-hydroxydecanoate (0.5 g, 1.80 mmol, 1 eq) was dissolved in THF (6 mL) and TEA (272.61 mg, 2.69 mmol, 374.98 μL, 1.5 eq) was added dropwise at 0 °C, followed by the addition of 4-NO2-C6H4O-COCl (615.44 mg, 3.05 mmol, 1.7 eq) in portions. The resulting solution was stirred at 20 °C for 17 h. LCMS showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure. The remainder was purified by column chromatography (silica gel, petroleum ether / ethyl acetate = 50 / 1 to 0 / 1) to obtain 36-fa (0.6 g, yield: 75.33%). 1 H NMR: (400 MHz CDCl3) δ = 8.31 - 8.22 (m, 2H), 7.44 - 7.27 (m, 5H), 5.11 (s, 2H), 4.28 (t, J = 6.8 Hz, 2H), 2.36 (t, J = 7.2 Hz, 2H), 1.70 - 1.19 (m, 14H).

[0421] Example 96 Synthesis of GalNAc-siRNA conjugate compound 36-F: [ka]

[0422] Using the conjugate building block compound 36 described in Example 95, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0423] Example 97 Synthesis of compound 37 [ka]

[0424] Compound 37 was synthesized according to the procedure of compound 7 in Example 30, using 37-q as the starting material, as shown in the synthesis method below. Loading: 37.0 μmol / g.

[0425] 4-(((3R,5S)-1-(10-(((2R,3R,4S,5R,6R)-4,5-bis(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)-6-((3-((3-(5-(((2R,3R,4R,5R,6R)- 3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)methyl)-3-fluorotetrahydro-2H-pyran-2-yl)amino)-10-oxodecanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (37-q): The title compound was synthesized according to the synthesis route of compound 7-q in Example 30. LCMS: Calculated value [M-2H] / 2: 1268.1, Found value: 1268.6.

[0426] Example 98 Synthesis of GalNAc-siRNA conjugate compound 37-F: [ka]

[0427] Using the conjugate building block compound 37 described in Example 96, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0428] Example 99 Synthesis of compound 38 [ka]

[0429] Compound 38 was synthesized according to the procedure of compound 27 in Example 77, using 38-g as the starting material, as shown in the synthesis method below. Loading: 27.0 μmol / g.

[0430] 4-(((3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(6-(((((2R,3R,4R,5S)-3,4,5-tris(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-2-yl)methoxy)carbonyl)amino)hexanoyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (38-g): The title compound was synthesized according to the procedure of compound 27-g in Example 77. LCMS: calculated [M-2H] / 2: 1246.1, found: 1246.6.

[0431] Example 100 Synthesis of GalNAc-siRNA conjugate compound 38-F: [ka]

[0432] Using the conjugate building block compound 38 described in Example 99, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0433] Example 101 Synthesis of compound 39 [ka]

[0434] Compound 39 was synthesized according to the procedure of compound 27 in Example 77, using 39-g as the starting material, as shown in the synthesis method below. Loading: 31.0 μmol / g.

[0435] 4-(((3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(8-(((((2R,3R,4R,5S)-3,4,5-tris(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)-3-oxopropoxy)tetrahydro-2H-pyran-2-yl)methoxy)carbonyl)amino)octanoyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (39-g): The title compound was synthesized according to the procedure of compound 27-g in Example 77. LCMS: calculated [M-2H] / 2: 1260.1, found: 1260.6.

[0436] Example 102 Synthesis of GalNAc-siRNA conjugate compound 39-F: [ka]

[0437] Using the conjugate building block compound 39 described in Example 101, RNA is synthesized according to known procedures with the ligand added to the 3' end of the sense strand, which is annealed with the antisense strand. The product is as shown above.

[0438] Biological Example 1 RNA synthesis and duplex annealing

[0439] Oligonucleotides were synthesized on an oligonucleotide synthesizer using commercially available nucleotides or chemically modified nucleotides with appropriate protecting groups.

[0440] The ligand-binding chains are synthesized using solid ligands containing the corresponding ligands. For example, the introduction of a sugar / ligand (e.g., GalNAc) at the 3' end of the sequence is achieved by starting the synthesis from the corresponding sugar solid ligand and following standard oligonucleotide synthesis procedures using an oligonucleotide synthesizer.

[0441] After synthesis is complete, the protecting groups and the protecting groups are removed from the oligonucleotide either individually or simultaneously using an appropriate deprotection system.

[0442] To prepare siRNA, equimolar amounts of sense and antisense strands are heated to 95°C for 5 min in 1x PBS and slowly cooled to room temperature. The integrity of the duplex is confirmed by HPLC analysis.

[0443] Table 2 lists the published siRNA sequences that were used to provide GalNAc-siRNA conjugates for biochemical characterization (see: "miR-145 Antagonizes SNAI1-Mediated Stemness and Radiation Resistance in Colorectal Cancer," Molecular Therapy, Vol. 26, No. 3, March 2018). [Table 2] Note: Lower case s indicates a PS linkage, lower case m indicates a 2'-O-methyl nucleotide, and lower case f indicates a 2'-fluoro nucleotide; TTR stands for transthyretin.

[0444] Table 3 lists the GalNAc-siRNA conjugates and their corresponding quality attributes. [Table 3] Notes: 1 siRNA bound to TTR; 2 The structure of L96 is shown below. [ka]

[0445] In vitro silencing activity of GalNAc-siRNA conjugates targeting TTR

[0446] Isolation of mouse primary hepatocytes C57BL / 6 mice were anesthetized and the liver was perfused with 100 ml of HBSS (GIBCO, 14025092) containing 1 mM EGTA (Aladdin, e104432) through an intravenous needle inserted into the inferior vena cava, followed by washing with collagenase-containing HBSS (collagenase type I, Solelybio, sy0535). The liver was removed, washed with PBS, and isolated in culture medium. The liver envelope was broken and the contents were removed. Cells were filtered through a 100 micron nylon mesh and centrifuged at 200 g for 3 min at 4 °C. Yield and viability were determined using a trypan blue exclusion test (Sigma, 0.08%). Hepatocytes with viability of 85% or more were available for subsequent manipulations.

[0447] Free Uptake 8 x 10 in 900 μL complete growth medium 4Mouse primary hepatocytes (PMH) were added and placed in a 24-well plate pretreated with type I collagenase. Free uptake was performed by adding 10 μL of test sample / siRNA duplex and 90 μL of Opti-MEM to PMH and mixing well. Cells were incubated at 37 °C in an atmosphere of 5% CO2 for 24 h before RNA purification. Experiments were performed at four doses: 2 nM, 1 nM, 0.5 nM, and 0.25 nM. Eight-point IC50 curve fitting was based on 10 nM, 2.5 nM, 0.63 nM, 0.16 nM, 39 pM, 9.8 pM, 2.4 pM, and 0.61 pM.

[0448] Total RNA isolation (Invitrogen, 610-12) and cDNA synthesis (TaKaRa, RR047A)

[0449] Cells are lysed with 300 μL lysis buffer and transferred to a 96 deep-well plate (plate 1). 20 μL of magnetic beads and 280 μL of absolute ethanol are added to each well in plate 1. MW2 is added to plate 2 and plate 3 in the same arrangement as 500 μL / well. Elution buffer is added to plate 4 in the same type set as 50 μL / well. Place each plate into the nucleic acid extraction instrument and run the isolation program. The concentration of each RNA sample is determined using a NanoDrop. A master mix of 1 μL gDNA eraser and 2 μL 5x gDNA eraser buffer is added to 7 μL of total RNA. The RT1 program is as follows: 42°C 2 min, hold at 4°C. For the RT2 program, add 4 μL 5 x PrimeScript buffer 2, 1 μL PrimeScript RT enzyme mix I, 1 μL RT primer mix, and 4 μL RNase-free dH2O to the above reaction. 37° C. 15 min, 85° C. 5 sec, hold at 4° C. For real-time PCR, add 180 μL of dilution buffer to each well.

[0450] Real-time PCR 4 μL of cDNA is added to a master mix containing 1 μL TBP / GAPDH (mouse) primer or 1 μL TTR (mouse) primer and 5 μL PCR buffer and added to each well of a 384-well plate. Real-time PCR is performed on a Roche LC480 real-time PCR machine. Each duplex is tested with two independent free uptakes, and each free uptake is analyzed in triplicate. To calculate relative changes, real-time PCR data are analyzed using the ΔΔCt method and normalized to assays performed with mock cells.

[0451] Activity results The activity of GalNAc-siRNA conjugates (listed in Table 3) is shown in Table 4 and Figure 1. In particular, Figure 1 shows GalNAc-siRNA conjugates by in vitro TTR gene silencing. [Table 4]

[0452] The foregoing description is considered as illustrative of the principles of the invention. Moreover, since numerous variations and modifications will be readily apparent to those skilled in the art, it is not desired to limit the invention to the exact structures and processes illustrated above. Accordingly, all suitable modifications and equivalents may be resorted to within the scope of the invention as defined by the following claims.

[0453] All references, patents, patent applications, and publications cited or referred to in this application are hereby incorporated by reference in their entirety.

Claims

1. A compound having the structure shown in formula (I), 【Chemical 1】 or a pharmaceutically acceptable salt or solvate thereof, wherein A and B are each, independently for each occurrence, O, N(RN), or S, RN is H or C １－６ is alkyl, X and W are each, independently for each occurrence, H, a protecting group, a phosphate group, a phosphate ester group, an activated phosphate group, an activated phosphorous acid group, a phosphoramidite, a solid support, -P(Z′)(Z″)O-nucleoside, -P(Z′)(Z″)O-oligonucleotide, a lipid, PEG, a steroid, a polymer, a nucleotide, a nucleoside, an oligonucleotide, or a therapeutic active agent, Z′ and Z″ are each independently O or S, L is a covalent linker, Z is a carbohydrate mimetic, or a disaccharide, trisaccharide or oligosaccharide, each Y is independently -L′-T, each T is a ligand selected from the group consisting of a carbohydrate ligand, a polypeptide ligand, and a lipophilic ligand, each L′ is independently a covalent linker, and p and q are each independently 1, 2, 3, 4, or 5, said compound, or a pharmaceutically acceptable salt or solvate thereof.

2. The compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein Z is a monosaccharide carbohydrate mimetic.

3. The compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein Z is a monosaccharide carbohydrate mimetic selected from the group consisting of a deoxysaccharide, an amino sugar, an N-glycoside, an imino sugar, an unsaturated sugar, a carboxylated sugar, an amidated sugar, a condensed cyclic sugar, and a carba sugar of a monosaccharide.

4. The compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein Z is an imino sugar of a monosaccharide.

5. The compound according to claim 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the monosaccharide is threose, pentose, hexose, heptose, or octose.

6. Z has the following structure, 【Chemical 2】 wherein, R １ is H, C １－６ alkyl, halogen or -NH(R ２ ), where R ２ is H or acetyl, Each R is independently H, halogen, -CN, -C≡CH, -NH ２ , -OC １－６ alkyl, or C １－６ alkyl, where the -C １－６ alkyl and -OC １－６ alkyl are substituted with 0 to 5 halogen atoms. Or, two Rs together with the carbon to which they are attached form a C ３－６ cycloalkyl or 3- to 6-membered cyclic heteroalkyl group, where the C ３－６ cycloalkyl of the cycloalkyl and the heteroalkyl of the 3- to 6-membered cyclic heteroalkyl are substituted with 0 to 5 halogen atoms, and n is 0, 1, 2, or 3 within the range permitted by the valence, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

7. The compound according to claim 6, or a pharmaceutically acceptable salt or solvate thereof, wherein n is 0.

8. Z is the compound according to claim 1 having the following structure, or a pharmaceutically acceptable salt or solvate thereof. 【Chemical 3】

9. (Y)p-Z- is the compound according to claim 1 having the following structure, or a pharmaceutically acceptable salt or solvate thereof. [Chemical 4]

10. Z is a disaccharide, trisaccharide, or a carbohydrate mimetic of a disaccharide or trisaccharide, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

11. Z is a disaccharide or a carbohydrate mimetic of a disaccharide, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

12. Z is a disaccharide selected from the group consisting of gentiobiose, isomaltose, melibiose, trehalose, sucrose, lactose, maltose, cellobiose, or a carbohydrate mimetic thereof, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

13. Z is the compound according to claim 1 having the following structure, or a pharmaceutically acceptable salt or solvate thereof. 【Chemical Formula 5】

14. Z is the compound according to claim 1 having the following structure, or a pharmaceutically acceptable salt or solvate thereof. [[Chemical Formula 6]]

15. Each 【Chemical Formula 7】 is independently a group having the following structure, 【Chemical Formula 8】 where s is an integer from 1 to 20, Each Q ３ does not exist independently and is —CO—, —NH—, —O—, —S—, —SO ２ —, —OC(O)—, —C(O)O—, —NHC(O)—, —C(O)NH—, —CH ２ —, —CH ２ NH—, —NHCH ２ —, —CH ２ O—, or —OCH ２ — and Each Q ４ does not exist independently and is a substituted or unsubstituted C １－１２ alkylene, substituted or unsubstituted C ２－１２ alkenylene, substituted or unsubstituted C ２－１２ alkynylene, substituted or unsubstituted C ２－１２ heteroarylene, substituted or unsubstituted 6- to 12-membered cyclic arylene, substituted or unsubstituted 5- to 12-membered cyclic heteroarylene, or substituted or unsubstituted 5- to 12-membered cyclic heterocyclylene, and Each Q ５ does not exist independently and is —CO—, —NH—, —O—, —S—, —SO ２ —, —CH ２ —, —C(O)O—, —OC(O)—, —C(O)NH—, —NHC(O)—, —NH—CH(R ａ )—C(O)—, —C(O)—CH(R ａ )—NH—, —OP(O)(OH)O—, or —OP(S)(OH)O—, where each R ａ is independently H or substituted or unsubstituted C １－１２ alkyl, However, at least one Q ４ is present, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

16. s is an integer from 1 to 5, Each Q ３ does not exist independently and is —CO—, —NH—, —O—, —OC(O)—, —C(O)O—, —NHC(O)—, —C(O)NH—, —CH ２ —, —CH ２ NH—, —NHCH ２ —, —CH ２ O—, or —OCH ２ —, and Each Q ４ does not exist independently and is a substituted or unsubstituted C １－１２ alkylene, a substituted or unsubstituted C ２－１２ alkenylene, or a substituted or unsubstituted C ２－１２ alkynylene, and Each Q ５ does not exist independently and is —CO—, —NH—, —O—, —CH ２ —, —C(O)O—, —OC(O)—, —C(O)NH—, or —NHC(O)—, However, at least one Q ４ is present, the compound according to claim 15, or a pharmaceutically acceptable salt or solvate thereof.

17. s is 1 or 2, Each Q ３ does not exist independently and is —CO—, —NH—, —CH ２ —, or —NHC(O)—, and Each Q ４ does not exist independently or is C １－１２ alkylene, and Each Q ５ does not exist independently and is -CO-, -CH ２ -, or -NHC(O)-, and However, at least one Q ４ is present, the compound according to claim 15, or a pharmaceutically acceptable salt or solvate thereof.

18. Each 【Chemical Formula 9】 is independently a group having the following structure, 【Chemical 10】 where Each Q ５ is independently —CO—, —NH—, —O—, —CH ２ —, —C(O)O—, —OC(O)—, —C(O)NH—, or —NHC(O)—, and each j1 and j2 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, the compound according to claim 15, or a pharmaceutically acceptable salt or solvate thereof.

19. Each 【Chemical 11】 is independently a group having the following structure, the compound according to claim 15, or a pharmaceutically acceptable salt or solvate thereof. 【Chemical Formula 12-1】 【Chemical Formula 12-2】

20. Each -L'-T is independently a group having the following structure, [-Q ３ -Q ４ -Q ５ s - Q ６ -T where s is an integer from 0 to 20, Each Q ３ and Q ６ do not exist independently, and are -CO-, -NH-, -O-, -S-, -SO ２ -, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH ２ -, -CH ２ NH-, -NHCH ２ -, -CH ２ O-, or -OCH ２ -, and each Q ４ does not exist independently and is a substituted or unsubstituted C １－１２ alkylene, a substituted or unsubstituted C ２－１２ alkenylene, a substituted or unsubstituted C ２－１２ alkynylene, a substituted or unsubstituted C ２－１２ heteroarylene, a substituted or unsubstituted 6- to 12-membered cyclic arylene, a substituted or unsubstituted 5- to 12-membered cyclic heteroarylene, or a substituted or unsubstituted 5- to 12-membered cyclic heterocyclylene, and Each Q ５ does not exist independently and is -CO-, -NH-, -O-, -S-, -SO ２ -, -CH ２ -, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -NH-CH(R ａ )-C(O)-, -C(O)-CH(R ａ )-NH-, -OP(O)(OH)O-, or -OP(S)(OH)O-, where each R ａ is independently H or substituted or unsubstituted C １－１２ alkyl, However, Q ３ , Q ４ , Q ５ , and Q ６ The compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein at least one of them is present.

21. s is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, the compound according to claim 20, or a pharmaceutically acceptable salt or solvate thereof.

22. Each -L'-T is independently a group having the following structure, 【Chemical Formula 13】 where Each Q ７ does not exist independently and is —CO—, —NH—, —O—, —S—, —SO ２ —, —OC(O)—, —C(O)O—, —NHC(O)—, —C(O)NH—, —CH ２ —, —CH ２ NH—, —NHCH ２ —, —CH ２ O—, or —OCH ２ — and each k1 and k2 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and The compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein each of n1, n2, and n3 is independently 1, 2, 3, 4, or 5.

23. Each Q ７ is independently —NHC(O)— or —C(O)NH—, the compound according to claim 22, or a pharmaceutically acceptable salt or solvate thereof.

24. Each -L'-T is independently a group having the following structure 【Chemical Formula 14】 wherein each of k1 and k2 is independently 0, 1, 2, or 3, each of n1, n2, and n3 is independently 1, 2, 3, 4, or 5, and the compound according to claim 23, or a pharmaceutically acceptable salt or solvate thereof, wherein one of t1 and t2 is 0 and the other is 1.

25. Each -L'-T is independently a group having the following structure 【Chemical Formula 15】 the compound according to claim 24, or a pharmaceutically acceptable salt or solvate thereof.

26. Each -L'-T is independently a group having the following structure 【Chemical 16】 wherein each of k1 and k2 is independently 0, 1, 2, or 3, each of n1 and n2 is independently 1, 2, 3, 4, or 5, and the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein one of t1 and t2 is 0 and the other is 1.

27. Each -L'-T is independently a group having the following structure 【Chemical 17】 the compound according to claim 26, or a pharmaceutically acceptable salt or solvate thereof.

28. The compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, wherein each T is independently a carbohydrate ligand.

29. Each T is an unprotected or protected carbohydrate ligand independently selected from the group consisting of N-acetylgalactosamine (GalNAc), allose, altrose, arabinose, cladinosose, erythrose, erythritol, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fuculose, galactosamine, D-galactosaminol, galactose, glucosamine, N-acetyl-glucosamine, glucosaminol, glucose, glucose-6-phosphate, gulose, glyceraldehyde, L-glycero-D-manno-heptose, glycerol, glycerone, grucose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovosamine, laminitol, laminosamine, lamine, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, xylulose, the compound according to claim 28, or a pharmaceutically acceptable salt or solvate thereof.

30. Each T is independently N-acetylgalactosamine (GalNAc) or N-acetylgalactosamine triacetate, the compound according to claim 29, or a pharmaceutically acceptable salt or solvate thereof.

31. The therapeutic active agent is selected from the group consisting of antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), microRNA mimic, anti-miRNA oligonucleotide (AMO), long non-coding RNA, peptide nucleic acid (PNA), helper lipid, and phosphorodiamidate morpholino oligomer (PMO), wherein the nucleic acid is unmodified or modified, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

32. The therapeutic active agent is small interfering RNA (siRNA), the compound according to claim 31, or a pharmaceutically acceptable salt or solvate thereof.

33. The compound has a structure selected from the following group, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof. 【Chemical 18-1】 【Chemical Formula 18-2】 【Chemical 18-3】 【Chemical Formula 18-4】 【Chemical Formula 18-5】 【Chemical 18-6】 【Chemical Formula 18-7】 【Chemical 18-8】

34. The compound has a structure selected from the following group, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof. 【Chemical Formula 19-1】 【Chemical Formula 19-2】 【Chemical Formula 19-3】 【Chemical Formula 19-4】 【Chemical Formula 19-5】

35. W is a -P(Z')(Z")O-oligonucleotide, the compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof.

36. The compound according to claim 1, or a pharmaceutically acceptable salt or solvate thereof, having a structure selected from the following group. 【Chemical Formula 20-1】 【Chemical 20-2】 [Chemical 20-3] 【Chemical 20-4】 【Chemical 20-5】 ​ [Chemical 20-7] 【Chemical 20-8】 [Chemical 20-9]

37. The compound according to claim 36, or a pharmaceutically acceptable salt or solvate thereof, wherein the oligonucleotide is an iRNA agent, such as siRNA.

38. A method for regulating the expression of a target gene in a cell, comprising delivering the compound according to any one of claims 1 to 37, or a pharmaceutically acceptable salt or solvate thereof, to the cell.

39. The method according to claim 38, wherein the target gene is associated with a metabolic disease.

40. The method according to claim 38, wherein the target gene is associated with a liver disease.

41. The method according to claim 40, wherein the liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), HBV infection, liver fibrosis, and cirrhosis.

42. Use for preparing a medicament for treating and / or preventing a pathological condition or disease caused by the expression of a target gene in hepatocytes, the compound according to any one of claims 1 to 37, or a pharmaceutically acceptable salt or solvate thereof.

43. The use according to claim 42, wherein the target gene is associated with a metabolic disease.

44. The use according to claim 42, wherein the target gene is associated with a liver disease.

45. The target gene is selected from the hepatitis B virus gene, the angiopoietin-like protein 3 gene, or the apolipoprotein C3 gene, Optionally, the disease is selected from chronic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis, liver fibrosis, liver proliferative disease, and lipid disorder, Optionally, the lipid disorder is selected from hypercholesterolemia, hypertriglyceridemia, or arteriosclerosis. The use according to claim 42.

46. A pharmaceutical composition comprising the compound according to any one of claims 1 to 37, or a pharmaceutically acceptable salt or solvate thereof, alone or in combination with a pharmaceutically acceptable carrier or excipient.

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