Compounds for targeted delivery of oligonucleotide drugs, conjugates and uses thereof

Abstract

The invention provides a novel compound for targeted delivery of an oligonucleotide drug and a conjugate combined with the oligonucleotide drug, the novel compound can deliver one or more different oligonucleotide drugs, and the compound has a structure shown as a formula (I), a formula (II) or a formula (III). # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to compounds, conjugates and applications thereof for targeted delivery of oligonucleotide drugs. Background Art

[0002] Oligonucleotide drugs (such as siRNA, antisense oligonucleotides, and plasmid DNA, etc.), as emerging and promising therapies, have performed outstandingly in regulating gene expression and treating many diseases. However, their high molecular weight and polyanionic properties lead to poor cell membrane permeability, and the influence of factors such as nucleases has greatly limited the practical application of oligonucleotide drugs.

[0003] Nowadays, using receptor-mediated endocytosis to conjugate target ligands to oligonucleotide drugs to enhance cellular uptake and improve the specificity of in vivo delivery tissues has become a hot topic in the field of oligonucleotide drug research and development. It is worth noting that sometimes multivalent ligand clusters may perform better than single ligands in promoting targeted tissue delivery, such as asialoglycoprotein receptor (ASGPR). In addition, studies have found that the ASGPR ligand N-acetylgalactosamine (GalNAc) can effectively enhance the promotion of oligonucleotide drugs into hepatocytes. Further experiments confirmed that compared with a single GalNAc ligand, the multivalent GalNAc ligand cluster has a stronger binding affinity to ASGPR, thereby improving the delivery efficiency of oligonucleotides targeting hepatocytes (Chem. Soc. Rev., 2023, 52, 1273; Bioconjugate Chem. 2017, 28, 2, 283-295).

[0004] At present, there are many patent documents reporting research in this area, such as US8106022B2, CN116832169A, WO2023045995A1, CN110959011B, CN117858948A, WO2024061157, etc., all involve the use of GalNAc ligands to deliver oligonucleotide drugs. However, most of the disclosures in these documents use multivalent ligands GalNAc to deliver single-target single oligonucleotide drugs. Some single genes in the body have multiple transcripts with sequence differences, and the occurrence of diseases usually involves the interaction of multiple genes. Therefore, it is necessary to develop compounds with new scaffolds for targeted delivery of oligonucleotide drugs, which can simultaneously deliver two nucleotide drugs for a single target or dual-target nucleotide drugs, which will have broad application prospects. Summary of the invention

[0005] Based on this, the object of the present invention is to provide a new compound for targeted delivery of oligonucleotide drugs and a conjugate thereof after combining with an oligonucleotide drug, which can deliver one or more different oligonucleotide drugs.

[0006] The first aspect of the present invention provides a compound or a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the compound has a structure represented by formula (I), formula (II) or formula (III):

[0007]

[0008] Wherein: the targeting binding part T is a group containing a ligand, and the ligand can specifically bind to a cell membrane protein receptor having endocytosis;

[0009] W 1 Selected from: a group capable of binding to an oligonucleotide drug via a covalent bond;

[0010] W 2 Selected from: groups capable of undergoing click chemistry reactions or hydrogen;

[0011] W 3 Selected from: W 1 or W 2 , and W 3 Not hydrogen;

[0012] Y1 and Y2 are independently selected from: or their chiral isomers; n is an integer from 1 to 4;

[0013] L1, L2, L3, L4, L5, L6, L7, L8 are independently selected from one or more of the following groups connected without violating the principle of chemical bonding: 1 -C 15 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O) 2 -, -S(=O)-, -CH=CH-, ethynyl, -NR C(=O)-, -NR C(=O)NR-, -C(=O)CRR 1 NR-, -OC(=O)-, -SC(=O)-, -NRS(=O) 2 -、C 6 -C 10 Aryl, 3-20 membered heterocyclic group, C 3 -C 10 Cycloalkyl, 5-10 membered heteroaryl, the linking unit may be substituted by one or more R;

[0014] R is selected from: H, C 1 -C 6 Alkyl, halogen, hydroxyl, hydroxy-substituted C 1 -C 6 Alkyl, C 1-C 6 Alkoxy, halogen substituted C 1 -C 6 Alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0015] R 1 Selected from: H, C 1 -C 6 Alkyl, amino acid side chain;

[0016] X is selected from: -O-, -S-, -NR-, -C(=O)-,

[0017] In some embodiments, Y1 is selected from: or their chiral isomers; n is: 1, 2, 3 or 4;

[0018] Y2 is selected from: or their chiral isomers;

[0019] R in the structures of Y1 and Y2 are independently selected from: H, C 1 -C 3 Alkyl, halogen substituted C 1 -C 3 alkyl.

[0020] In some embodiments, Y1 is selected from: or their chiral isomers;

[0021] Y2 is selected from: or their chiral isomers.

[0022] In some embodiments, W 2 Selected from: 5-20 membered heterocyclic group containing alkynyl, -SR 26 , alkynyl, azido, alkenyl, 5-20 membered heterocyclic group containing alkenyl, hydrogen;

[0023] R 26 Selected from: hydrogen, a thiol protecting group.

[0024] In some embodiments, W 2 Selected from:

[0025] -SR 26 , azido, hydrogen.

[0026] In some embodiments, L1, L2, L3, L4, L5, L6, L7, and L8 are independently selected from linking units formed by connecting one or more of the following groups without violating the principle of chemical bonding:

[0027] C1 -C 12 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O) 2 -、-

[0028] S(=O)-, -CH=CH-, ethynyl, -NR C(=O)-, -NR C(=O)NR-, -C(=O)CRR 1 NR-、-OC(=O)-

[0029] 、-SC(=O)-、-NRS(=O) 2 -、-P(=O) 2 O-, -P(=O)(=S)O-, C 6 -C 10 Aryl, 3-8 membered heteromonocyclic group, C 3 -C 8 Cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospiro ring group, 5-20 membered heterocyclic ring group, C 5 -C 10 Cycloalkyl, 5-6 membered heteroaryl, the linking unit may be substituted by one or more R;

[0030] R is selected from: H, C 1 -C 3 Alkyl, halogen, hydroxyl, hydroxy-substituted C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, halogen substituted C 1 -C 3 Alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0031] R 1 Selected from: H, C 1 -C 3 Alkyl, amino acid side chain.

[0032] In some embodiments, L1, L2, L3, L4, L5, L6, L7, and L8 are independently selected from linking units formed by connecting one or more of the following groups without violating the principle of chemical bonding:

[0033]

[0034] or their chiral isomers;

[0035] Among them, j 1 Selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0036] R is selected from: H, C 1 -C 3 Alkyl, halogen substituted C 1 -C 3 alkyl;

[0037] R 1 Selected from: H, C 1 -C 3 Alkyl, amino acid side chain.

[0038] In some embodiments, L1, L2, L3, L4, L5, L6, L7, and L8 are independently selected from linking units formed by connecting one or more of the following groups without violating the principle of chemical bonding: or their chiral isomers;

[0040] Among them, j 1 Selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0041] R is selected from: H, methyl, ethyl, propyl;

[0042] R 1 Selected from: H, methyl, ethyl, propyl, amino acid side chain.

[0043] In some embodiments, L1 is selected from:

[0044]

[0045] Wherein, each A in L1 is independently selected from: -C(R 2 ) 2 -、-NR 2 -、-C(=O)NR 2 -, -O-, -S-, where the connection of a A does not violate the principle of chemical bonding;

[0046] Each R 2 are independently selected from: H, C 1 -C 3 alkyl;

[0047] a is selected from: an integer between 0 and 20;

[0048] b is selected from: an integer between 0-10.

[0049] In some embodiments, L1 is selected from:

[0050]

[0051] wherein a is selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0052] b is selected from: 0, 1, 2, 3, 4, 5, 6.

[0053] In some embodiments, L1 is selected from: -NH(CH 2 ) 10 C(=O)-, -NH(CH 2 ) 4 C(=O)-, -NH(CH 2 ) 5 C(=O)-, -NH(CH 2 ) 6 C(=O)-、-(CH 2 ) 2 NHC(=O)(CH 2 ) 10 C(=O)-, -NH(CH 2 ) 4 C(=O)NH(CH 2 ) 4 C(=O)-、-(CH 2 ) 2 NHC(=O)(CH 2 ) 5 C(=O)-, -C(=O)(CH 2 ) 5 C(=O)-, -C(=O)(CH 2 ) 9 C(=O)-,

[0054] -C(=O)(CH 2 ) 10 C(=O)-, -NH(CH 2 ) 2 OCH 2 C(=O)-,

[0055] -C(=O)(CH 2 ) 2 NH(CH 2 ) 2 OCH 2 C(=O)-.

[0056] In some embodiments, L2 is selected from:

[0057]

[0058] Each A in L2 is independently selected from: -C(R 2 ) 2 -、-NR2 -, -O-, -S-, where the connection of c A's does not violate the principle of chemical bonding;

[0059] Each B in L2 is independently selected from: -NR 2 -, -O-, -S-;

[0060] Each R 2 are independently selected from: H, C 1 -C 6 Alkyl, preferably selected from: hydrogen, C 1 -C 3 alkyl;

[0061] c is selected from: an integer between 0 and 20;

[0062] d is selected from: an integer between 0 and 10;

[0063] e is selected from: an integer between 0-10.

[0064] In some embodiments, L2 is selected from:

[0065] Among them, each R 2 Each is independently selected from: H, methyl, ethyl;

[0066] c is selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0067] d is selected from: 0, 1, 2, 3, 4, 5, 6;

[0068] e is selected from: 0, 1, 2, 3, 4, 5, 6.

[0069] In some embodiments, L2 is selected from: -NH(CH 2 ) 2 NHC(=O)(CH 2 ) 5 -、-NH(CH 2 ) 2 NHC(=O)(CH 2 ) 3 -、-(CH 2 ) 2 NH(CH 2 ) 2 -、-(CH 2 ) 2 N(CH 3 )(CH 2 ) 2 -, -C(=O)(CH 2 ) 5-, -C(=O)(CH 2 ) 4 -、-NH(CH 2 ) 6 -、-NH(CH 2 ) 5 -、-NH(CH 2 )-、-NH(CH 2 ) 2 -、

[0070] -(CH 2 ) 2 O(CH 2 ) 5 -、-CH 2 (=O)NH(CH 2 ) 2 NHC(=O)(CH 2 ) 5 -、

[0071] In some embodiments, L3 and L4 are independently selected from: -NR 6 -R 7 -、-NR 6 -R 7 -OR 7 -、-C(=O)-R 7 -;

[0072] Each R 6 are independently selected from: hydrogen, C 1 -C 6 alkyl;

[0073] Each R 7 Each independently selected from: C 1 -C 8 Alkylene.

[0074] In some embodiments, each R 6 are independently selected from: hydrogen, C 1 -C 3 alkyl;

[0075] Each R 7 Each independently selected from: C 1 -C 4 Alkylene.

[0076] In some embodiments, L3 and L4 are independently selected from: -NHCH 2 CH 2 -、-NHCH 2 CH 2 OCH 2 -、-NHCH 2CH 2 OCH 2 CH 2 -, -C(=O)CH 2 CH 2 CH 2 -.

[0077] In some embodiments, L5 is selected from: -R 8 -(CR 9 R 10 ) n1 -、-R 8 -(CR 9 R 10 ) n2 -R 8 -(CR 9 R 10 ) n3 -、-(CR 9 R 10 ) n2 -R 8 -(CR 9 R 10 ) n3 -;

[0078] Among them, R 8 Selected from:-NR 9 -, -O-, -S-, -C(=O), -NR 9 C(=O)-,

[0079] R 9 , R 10 are independently selected from: hydrogen, C 1 -C 6 Alkyl, preferably selected from: hydrogen, C 1 -C 3 alkyl;

[0080] Each n 1 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0081] Each n 2 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8;

[0082] Each n 3 Respectively and independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8.

[0083] In some embodiments, L5 is selected from: -C(=O)(CH 2 ) n1 -, -C(=O)(CH 2 ) n2-NHC(=O)(CH 2 ) n3 -、

[0084] -C(=O)(CH 2 ) n2 -C(=O)NH(CH 2 ) n3 -、-(CH 2 ) n2 -NHC(=O)(CH 2 ) n3 -、

[0085] -(CH 2 ) n2 -O(CH 2 ) n3 -, -C(=O)(CH 2 ) n2 -C(=O)NH-,

[0086] Each n 1 Each independently selected from: 1, 2, 3, 4, 5;

[0087] Each n 2 Each independently selected from: 1, 2, 3, 4, 5;

[0088] Each n 3 Respectively and independently selected from: 1, 2, 3, 4, 5, 6.

[0089] In some embodiments, L5 is selected from: -C(=O)CH 2 -, -C(=O)CH 2 CH 2 -, -C(=O)CH 2 CH 2 CH 2 -, -C(=O)CH 2 CH 2 NHC(=O)CH 2 CH 2 -、-CH 2 CH 2 NHC(=O)CH 2 CH 2 -、-CH 2 CH 2 OCH 2 CH 2 -、-CH 2 CH 2 OCH 2 -, -C(=O)CH 2 CH 2 C(=O)NH(CH2 ) 4 -, -C(=O)CH 2 CH 2 C(=O)NH-,

[0090]

[0091] In some embodiments, L6 is selected from: -R 11 -(R 12 ) n4 -、-R 11 -(R 12 ) n4 -R 11 -、-R 11 -(R 12 ) n5 -R 11 -(R 12 ) n6 -R 11 -;

[0092] Among them, R 11 Selected from: -C(=O), -NR 13 C(=O)-;

[0093] Each R 12 Each independently selected from: -C(R 14 ) 2 -, O, S, -NR 13 , where n 4 or 5 R 12 The connection does not violate the chemical bonding principle;

[0094] Each R 13 are independently selected from: hydrogen, C 1 -C 6 Alkyl, preferably selected from: hydrogen, C 1 -C 3 alkyl;

[0095] Each R 14 are independently selected from: hydrogen, C 1 -C 6 Alkyl, amino acid side chain, preferably selected from: hydrogen, C 1 -C 3 Alkyl groups, side chains of amino acids;

[0096] Each n 4 Each of the following is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0097] Each n 5Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0098] Each n 6 Each of the following is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0099] In some embodiments, L6 is selected from:

[0100] -C(=O)NH(CH 2 ) n4 C(=O)-, -C(=O)NH(CH 2 ) n4 C(=O)-,

[0101] -C(=O)NH(CH 2 ) n5 O(CH 2 ) n7 C(=O)-, -C(=O)NH(CH 2 ) n5 O(CH 2 ) n7 C(=O)-,

[0102] -C(=O)NH(CH 2 ) n4 -、-C(=O)NH(CH 2 ) n4 NHC(=O)-,

[0103] -C(=O)(CH 2 ) n5 -C(=O)NH(CH 2 ) n6 C(=O)-,

[0104] Each n 4 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0105] Each n 5 Each independently selected from: 1, 2, 3, 4, 5, 6;

[0106] Each n 6 Each independently selected from: 1, 2, 3, 4, 5, 6;

[0107] Each n 7 Respectively and independently selected from: 1, 2, 3, 4, 5, 6.

[0108] In some embodiments, L6 is selected from:

[0109] -C(=O)NH(CH2 ) 5 C(=O)-, -C(=O)NH(CH 2 ) 4 C(=O)-,

[0110] -C(=O)NH(CH 2 ) 2 OCH 2 C(=O)-, -C(=O)NH(CH 2 ) 2 O(CH 2 ) 2 C(=O)-,

[0111] -C(=O)NH(CH 2 ) 4 -、-C(=O)NH(CH 2 ) 3 NHC(=O)-,

[0112] -C(=O)(CH 2 ) 2 -C(=O)NH(CH 2 ) 5 C(=O)-,

[0113] In some embodiments, L7 is selected from: -(CH 2 ) n’8 -R 15 -(R 16 ) n8 -;

[0114] Among them, R 15 Selected from: -C(=O), -NR 13 C(=O)-;

[0115] Each R 16 Each independently selected from: -C(R 17 ) 2 -, O, S, where n 8 R 16 The connection does not violate the principle of chemical bonding;

[0116] Each R 13 are independently selected from: hydrogen, C 1 -C 6 Alkyl, preferably selected from: hydrogen, C 1 -C 3 alkyl;

[0117] Each R 17 are independently selected from: hydrogen, C 1 -C 6Alkyl, preferably selected from: hydrogen, C 1 -C 3 alkyl;

[0118] Each n 8 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0119] Each n' 8 Respectively and independently selected from: 0, 1, 2, 3, 4, 5, 6.

[0120] In some embodiments, L7 is selected from: -C(=O)(CH 2 ) n8 -、-C(=O)NH(CH 2 ) n8 -, -NHC(=O)(CH 2 ) n8 -, -C(=O)CH 2 O(CH 2 ) n9 -、-(CH 2 ) 4 NHC(=O)(CH 2 ) n8 -;

[0121] Each n 8 Each independently selected from: 1, 2, 3, 4, 5, 6;

[0122] Each n 9 Independently selected from: 1, 2, 3, 4, 5.

[0123] In some embodiments, L7 is selected from: -C(=O)(CH 2 ) 5 -, -C(=O)(CH 2 ) 4 -、

[0124] -C(=O)CH 2 O(CH 2 ) 3 -、-C(=O)NH(CH 2 ) 5 -、-C(=O)NH(CH 2 ) 4 -, -NHC(=O)(CH 2 ) 5 -, -NHC(=O)(CH 2 ) 4 -、-(CH 2 ) 4 NHC(=O)(CH 2 ) 5 -.

[0125] In some embodiments, L8 is selected from: -R 18 -(R 19 ) n10 -R 18 -;

[0126] Among them, R 18 Selected from: -C(=O), -NR 13 C(=O)-;

[0127] Each R 19 Each independently selected from: -C(R 14 ) 2 -, O, S, -NR 13 , C 6 -C 10 Aryl, 5-6 membered heteromonocyclic group, C 5 -C 6 Cycloalkyl, 6-8 membered heterobridged ring group, 6-8 membered heterospiro ring group, 6-10 membered heterocyclic ring group, C 6 -C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl, wherein n 10 R 19 The connection does not violate the principle of chemical bonding;

[0128] Each R 13 are independently selected from: hydrogen, C 1 -C 6 Alkyl, preferably selected from: hydrogen, C 1 -C 3 alkyl;

[0129] Each R 14 are independently selected from: hydrogen, C 1 -C 6 Alkyl, amino acid side chain, preferably selected from: hydrogen, C 1 -C 3 Alkyl groups, side chains of amino acids;

[0130] Each n 10 are independently selected from: an integer between 1 and 20;

[0131] Or L8 is selected from the above L7.

[0132] In some embodiments, R 18 Selected from: -C(=O), -NHC(=O)-;

[0133] Each R 19 Each independently selected from: -CH 2 -, -O-, -S-, -NH-, phenyl, 5-6 membered heteromonocyclic group, C 5-C 6 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl;

[0134] Each n 10 Each of the following is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0135] In some embodiments, L8 is selected from: -C(=O)(CH 2 ) 10 C(=O)-,

[0136] -C(=O)(CH 2 ) 11 -N(H)-(CH 2 CH 2 O)n 12 (CH 2 ) 13 C(=O)-,

[0137] R 19 Selected from: phenyl, 5-6 membered heteromonocyclic group, C 5 -C 6 Cycloalkyl, 5-6 membered nitrogen heteroaryl, ethynyl, vinyl; R 19 Preferably:

[0138] n 10 Selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0139] n 11 Selected from: 1, 2, 3, 4, 5, 6;

[0140] n 12 Selected from: 1, 2, 3, 4;

[0141] n 13 Selected from: 1, 2, 3, 4;

[0142] n 14 Selected from: 1, 2, 3, 4, 5, 6, 7, 8.

[0143] In some embodiments, L8 is selected from: -C(=O)(CH 2 ) 10 C(=O)-,

[0144] -C(=O)(CH 2 ) 2 -N(H)-(CH 2 CH 2 O) 2CH 2 C(=O)-,

[0145] In some embodiments, W 1 Selected from: a group capable of being linked to an oligonucleotide via a phosphodiester bond or a phosphorothioate diester bond.

[0146] In some embodiments, W 1 Selected from hydroxyl, protected hydroxyl or a group having the following structure:

[0147]

[0148] or a chiral isomer thereof;

[0149] Wherein, R' is hydrogen or a hydroxyl protecting group.

[0150] In some embodiments, the cell membrane protein receptor with endocytosis function is selected from: cell protein receptors, cell channel receptors, cell transporter protein receptors and enzyme protein receptors.

[0151] In some embodiments, the cell membrane protein receptor is selected from: asialoglycoprotein receptor (ASGPR), folate receptor, integrin receptor, transferrin receptor, G protein coupled receptor (GPCR).

[0152] In some embodiments, the targeting binding moiety T has a structure as shown in the following formula (IV), formula (V), formula (VI) or formula (VII):

[0153]

[0154] Wherein: D, E, J, M are independently selected from:

[0155] or their chiral isomers;

[0156] G, V, W, and U are independently selected from: or their chiral isomers;

[0158] r, t are independently selected from: 0, 1, 2, 3, 4, 5, 6;

[0159] X 1 , X' 1 , X 2 , X 3 , X 4 , X 5 are independently selected from: a single bond, C1 -C 6 Alkylene, -C(=O)-C 1 -C 6 Alkylene, -C(=O)-NH-C 1 -C 6 Alkylene, -NH-C(=O)-C 1 -C 6 Alkylene, -NH-C 1 -C 6 Alkylene, -C(=O)-NH-C 1 -C 6 Alkylene-OC 1 -C 6 Alkylene, -NH-C(=O)-C 1 -C 6 Alkylene-OC 1 -C 6 Alkylene, or their chiral isomers;

[0160] The connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from one or more of the following groups, and are connected to form a connecting unit that does not violate the principle of chemical bonding: C 1 -C 15 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O) 2 -, -S(=O)-, -CH=CH-, ethynyl, -NR C(=O)-, -NR C(=O)NR-, -C(=O)CRR 1 NR-, -OC(=O)-, -SC(=O)-, -NRS(=O) 2 -、C 6 -C 10 Aryl, 3-20 membered heterocyclic group, C 3 -C 10 Cycloalkyl, 5-10 membered heteroaryl, the linking unit may be substituted by one or more R;

[0161] R is selected from: H, C 1 -C 6 Alkyl, halogen, hydroxyl, hydroxy-substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, halogen substituted C 1 -C 6 Alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0162] R1 Selected from: H, C 1 -C 6 Alkyl, amino acid side chain.

[0163] In some embodiments, the linkers K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from linking units formed by connecting one or more of the following groups without violating the principle of chemical bonding: 1 -C 12 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O) 2 -, -S(=O)-, -CH=CH-, ethynyl, -NR C(=O)-, -NR C(=O)NR-, -C(=O)CRR 1 NR-, -OC(=O)-, -SC(=O)-, -NRS(=O) 2 -、C 6 -C 10 Aryl, 3-8 membered heteromonocyclic group, C 3 -C 8 Cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospiro ring group, 5-20 membered heterocyclic ring group, C 5 -C 10 Cycloalkyl, 5-6 membered heteroaryl, the linking unit may be substituted by one or more R;

[0164] R is selected from: H, C 1 -C 3 Alkyl, halogen, hydroxyl, hydroxy-substituted C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy, halogen substituted C 1 -C 3 Alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0165] R 1 Selected from: H, C 1 -C 3 Alkyl, amino acid side chain.

[0166] In some embodiments, the linkers K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from linking units formed by connecting one or more of the following groups without violating the principle of chemical bonding:

[0167]

[0168] or their chiral isomers;

[0169] Among them, j 1 Selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0170] R is selected from: H, C 1 -C 3 Alkyl, halogen substituted C 1 -C 3 alkyl;

[0171] R 1 Selected from: H, C 1 -C 3 Alkyl, amino acid side chain.

[0172] In some embodiments, the linkers K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from linking units formed by connecting one or more of the following groups without violating the principle of chemical bonding:

[0173]

[0174] or their chiral isomers;

[0175] Among them, j 1 Selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0176] R is selected from: H, methyl, ethyl, propyl;

[0177] R 1 Selected from: H, methyl, ethyl, propyl, amino acid side chain.

[0178] In some embodiments, the linkers K1, K3, K6, and K7 are independently selected from: -(R 20 ) n15 -R 21 -(R 20 ) n16 -R 22 -;

[0179] Among them, each R 20 Each independently selected from: -CH 2 -, -O-, -S-, where n 15 or n16 R 20 The connection does not violate the principle of chemical bonding;

[0180] R 21Selected from: -C(=O)NH-, -C(=O)-, C 6 -C 10 Aryl, 3-8 membered heteromonocyclic group, C 3 -C 8 Cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospiro ring group, 5-20 membered heterocyclic ring group, C 5 -C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl, -C(=O)-3-8 membered heteromonocyclic group, -C(=O)-C 3 -C 8 Cycloalkyl, or none;

[0181] R 22 Selected from: -C(=O)NH-, -NH-, -C(=O)-, or none;

[0182] Each n 15 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0183] Each n 16 Each of the following is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0184] In some embodiments, R 21 Selected from: -C(=O)NH-, -C(=O)-, phenyl,

[0185]

[0186] Or not.

[0187] In some embodiments, linkers K1, K3, K6, and K7 are independently selected from: -(CH 2 ) n15 -R 22 -、-(CH 2 ) n15 -R 21 -(CH 2 ) n16 -R 22 -、-(CH 2 ) n15 -OR 21 -CH 2 O-(CH 2 ) n16 -R 22 -、-(CH 2 ) n15 -R 21 -CH 2 CH 2 O-(CH2 ) n16 -R 22 -、-(CH 2 ) n15 -R 21 -(CH 2 CH 2 O) 2 -、-(CH 2 ) n15 -R 21 -(CH 2 ) n16 -;

[0188] R 21 Selected from: -C(=O)NH-, -C(=O)-, phenyl,

[0189] R 22 Selected from: -C(=O)NH-, -NH-, -C(=O)-;

[0190] Each n 15 Each independently selected from: 1, 2, 3, 4, 5, 6;

[0191] Each n 16 Each of the following is independently selected from: 0, 1, 2, 3, and 4.

[0192] In some embodiments, connectors K1, K3, K6, and K7 are independently selected from:

[0193] In some embodiments, the linkers K2, K4, K5, K8, K9, K10, K11, K12, K13, and K14 are independently selected from: -(R 23 ) n17 -R 24 -(R 23 ) n18 -R 25 -;

[0194] Among them, each R 23 Each independently selected from: -CH 2 -, -O-, -S-, where n 18 R 23 The connection does not violate the principle of chemical bonding;

[0195] R 24 Selected from: -C(=O)NH-, -C(=O)-, C 6 -C 10 Aryl, 3-8 membered heteromonocyclic group, C 3 -C 8Cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospiro ring group, 5-20 membered heterocyclic ring group, C 5 -C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl, -C(=O)-3-8 membered heteromonocyclic group, -C(=O)-C 3 -C 8 Cycloalkyl, or none;

[0196] R 25 Selected from: -C(=O)NH-(CH 2 ) n19 -C(=O)-, -NHC(=O)-(CH 2 ) n19 -C(=O)-, -C(=O)NH-(CH 2 ) n19 -

[0197] 、-NHC(=O)-(CH 2 ) n19 -、-C(=O)-(CH 2 ) n19 -, or not;

[0198] Each n 17 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0199] Each n 18 Each is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0200] Each n 19 Independently selected from: 1, 2, 3.

[0201] In some embodiments, R 24 Selected from: -C(=O)NH-, -C(=O)-, phenyl,

[0202]

[0203] Or not.

[0204] In some embodiments, linkers K2, K4, K5, K8, K9, K10, K11, K12, K13, and K14 are independently selected from: -(CH 2 ) n17 -R 25 -、-(CH 2 ) n17 -R 24 -(CH 2 ) n18 -R25 -、-(CH 2 ) n17 -OCH 2 -R 24 -CH 2 O-(CH 2 ) n18 -R 25 -、-(CH 2 ) n17 -R 24 -(CH 2 ) n18 -、-(CH 2 ) n17 -R 24 -CH 2 CH 2 O-(CH 2 ) n18 -R 25 -、-(CH 2 ) n17 -R 24 -(CH 2 CH 2 O) 2 -(CH 2 ) n18 -、-(CH 2 ) n17 -;

[0205] R 24 Selected from: -C(=O)NH-, -C(=O)-, phenyl,

[0206] R 25 Selected from: -C(=O)NH-(CH 2 ) 2 -C(=O)-, -NHC(=O)-(CH 2 ) 2 -C(=O)-, -C(=O)NH-(CH 2 ) 2 -, -NHC(=O)-(CH 2 ) 2 -、-C(=O)-(CH 2 ) 2 -;

[0207] Each n 17 Each independently selected from: 1, 2, 3, 4, 5, 6;

[0208] Each n 18 Each of the following is independently selected from: 0, 1, 2, 3, and 4.

[0209] In some embodiments, connectors K2, K4, K5, K8, K9, K10, K11, K12, K13, and K14 are independently selected from:

[0210] In some embodiments, X 1 , X' 1 , X 2 , X 3 , X 4 , X 5 are independently selected from: a single bond, C 1 -C 3 Alkylene, -C(=O)-C 1 -C 3 Alkylene, -C(=O)-NH-C 1 -C 3 Alkylene, -NH-C(=O)-C 1 -C 3 Alkylene, -NH-C 1 -C 3 Alkylene, -C(=O)-NH-C 1 -C 3 Alkylene-OC 1 -C 3 Alkylene, -NH-C(=O)-C 1 -C 3 Alkylene-OC 1 -C 3 Alkylene, or chiral isomers thereof.

[0211] In some embodiments, X 1 , X' 1 , X 2 , X 3 , X 4 , X 5 Each of the following is independently selected from the group consisting of methylene, ethylene, propylene, -C(=O)-methylene, -C(=O)-ethylene, -C(=O)-propylene, -NH-C(=O)-methylene, -NH-C(=O)-ethylene, and -NH-C(=O)-propylene.

[0212] In some embodiments, the ligand is selected from: D-pyranose, L-pyranose, D-arabinose, D-furanose, L-furanose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-furanose, β-D-furanose, α-D-pyranose, β-D-pyranose, α-D-glucose, β-D-pyranose, α-D-glucose, β-D-glucose, α-D-furanose, β-D-glucose , α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, 4-fluorodeoxy-N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-difluoropropionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino -3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-tri Benzyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allosenitrile, D-ribose, D-4-thioribose, L-ribose and L-4-thioribose, RGD peptide, folic acid molecule, TfR1 peptide, integrin ligand small molecule, lipids, fatty acids, bile acid and cholesterol.

[0213] In some of the embodiments, the ligand is selected from: N-acetylgalactosamine, 4-fluorodeoxy-N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-difluoropropionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, folic acid molecules, integrin ligand small molecules.

[0214] In some embodiments, the ligand is selected from:

[0215]

[0216] or the hydrogen on its hydroxyl group is replaced by an acetyl group.

[0217] In some embodiments, the targeting binding moiety T is selected from:

[0218]

[0219]

[0220]

[0221] or the hydrogen on its hydroxyl group is replaced by an acetyl group.

[0222] In a second aspect, the present invention provides a conjugate obtained by connecting the compound of the present invention with an oligonucleotide drug, wherein W1 in the compound is connected to the oligonucleotide drug via a covalent bond; W2 in the compound is connected to the oligonucleotide drug by reacting with Q; Q is selected from: L9-L10-L11-Z 2 , Z 2 The residue after removing one hydroxyl group from the oligonucleotide drug;

[0223] L9 is selected from: Can be with W 2 The active group of the group undergoes click chemistry reaction;

[0224] L10 is selected from a connecting unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C 1 -C 15 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O) 2 -, -S(=O)-, -CH=CH-, ethynyl, -NR C(=O)-, -NR C(=O)NR-, -C(=O)CRR 1 NR-, -OC(=O)-, -SC(=O)-, -NRS(=O) 2 -、-P(=O) 2 O-, -P(=O)(=S)O-, C 6 -C 10 Aryl, 3-20 membered heterocyclic group, C 3 -C 10 Cycloalkyl, 5-10 membered heteroaryl, the linking unit may be substituted by one or more R;

[0225] R is selected from: H, C 1 -C 6 Alkyl, halogen, hydroxyl, hydroxy-substituted C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, halogen substituted C 1 -C 6 Alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0226] R 1 Selected from: H, C1 -C 6 Alkyl, amino acid side chain;

[0227] L11 is selected from: W1 without a hydroxyl hydrogen or a hydroxyl protecting group.

[0228] In some embodiments, L9 is selected from: 5-20 membered heterocyclic group containing alkynyl, -SR 26 , alkynyl, azido; R 26 Selected from: H or a thiol protecting group.

[0229] In some embodiments, L9 is selected from:

[0230] Azide, -SR 26 .

[0231] In some embodiments, L11 is selected from: -O-, or a group having the following structure:

[0232]

[0233] or their chiral isomers.

[0234] In some embodiments, L10 is selected from:

[0235]

[0236] Wherein, each A in L10 is independently selected from: -C(R 2 ) 2 -、-NR 2 -, -O-, -S-;

[0237] Each R 2 are independently selected from: H, C 1 -C 3 alkyl;

[0238] Each R 3 Independently selected from: R 5 , or not;

[0239] Each R 4 Each independently selected from: -C(R 2 ) 2 -、-NR 2 -, -O-, -S-, -C(=O), -C(=S)-, where f 4 R 4 The connection does not violate the principle of chemical bonding;

[0240] Each R 5Each independently selected from: C 6 -C 10 Aryl, 3-8 membered heteromonocyclic group, C 3 -C 8 Cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospiro ring group, 5-20 membered heterocyclic ring group, C 5 -C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl;

[0241] Each f 1 Each is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0242] Each f 2 Each is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0243] Each f 3 Each is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0244] Each f 4 are independently selected from: an integer between 0-20.

[0245] In some embodiments, each A in L10 is independently selected from: -CH 2 -, -NH-, -O-, -S-;

[0246] Each R 3 Independently selected from: R 5 , or not;

[0247] Each R 4 Each independently selected from: -CH-, -NH-, -O-, -S-, -C(=O);

[0248] Each R 5 Each independently selected from: C 6 -C 10 Aryl, 5-6 membered heteromonocyclic group, C 5 -C 6 Cycloalkyl, 6-8 membered heterobridged ring group, 6-8 membered heterospiro ring group, 6-10 membered heterocyclic ring group, C 6 -C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl;

[0249] Each f 1 Each is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0250] Each f 2 Each independently selected from: 0, 1, 2, 3, 4;

[0251] Each f 3 Each is independently selected from: 0, 1, 2, 3, 4;

[0252] Each f 4 Each of the following is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0253] In some embodiments, each R 5 Independently selected from: or their chiral isomers.

[0254] In some embodiments, L10 is selected from:

[0255]

[0256]

[0257] or their chiral isomers.

[0258] In some embodiments, Q is selected from:

[0259]

[0260]

[0261] In some embodiments, the compound forms a phosphate bond or a phosphorothioate bond with the phosphate group or the phosphorothioate group at the 3' and / or 5' end of the sense strand in the oligonucleotide drug through the hydroxyl group in W1.

[0262] In some embodiments, L11 is linked to the 3' and / or 5' end of the sense strand of the oligonucleotide drug via a phosphate bond or a phosphorothioate bond.

[0263] In some of these embodiments, the oligonucleotide drug includes small interfering RNA (siRNA), antisense oligonucleotide (ASO), small activating RNA (saRNA), micro RNA (miRNA), peptide nucleic acid (PNA), and aptamer.

[0264] In some embodiments, the conjugate has a structure as shown in the following formula (A), formula (B) or formula (C):

[0265]

[0266] Wherein, the targeting binding parts T, L1, L2, L3, L4, L6, L5, L7, and L8 are as described above, and the L10, L11, and Z 2 As mentioned before;

[0267] Z 1The residue after removing one hydroxyl group from the oligonucleotide drug;

[0268] W 4 is the W 1 A group after removing a hydroxyl hydrogen or a hydroxyl protecting group;

[0269] W 5 Selected from: W 2 and the group formed after the reaction of L9;

[0270] W 6 Selected from: W 4 or -W 5 -L10-L11-.

[0271] In some embodiments, W 4 Selected from: -O-, or a group having the following structure:

[0272]

[0273] or their chiral isomers.

[0274] In some embodiments, each W 5 Each of them is independently selected from: a 3-20 membered heterocyclic group, a 5-20 membered heteroaromatic ring group, a 3-20 membered heterocyclic thio group, and -SS-.

[0275] In some embodiments, each W 5 Each of the following is independently selected from: 5-20 membered heterocyclic group, 5-6 membered heteroaromatic ring group, 5-6 membered heterocyclic thio group, -SS-;

[0276] In some embodiments, each W 5 Independently selected from:

[0277] -SS-.

[0278] In a third aspect, the present invention further provides a carrier compound, the molecular structure of which is the same as the molecular structure of the conjugate of the present invention after the oligonucleotide drug is removed.

[0279] The fourth aspect of the present invention provides the use of the compounds and conjugates, including the following technical solutions.

[0280] Use of the compound of the present invention or its pharmaceutically acceptable salt or stereoisomer in preparing a carrier for delivering one or more oligonucleotide drugs.

[0281] In some of these embodiments, the oligonucleotide drug includes small interfering RNA (siRNA), antisense oligonucleotide (ASO), small activating RNA (saRNA), micro RNA (miRNA), peptide nucleic acid (PNA), and aptamer.

[0282] The conjugate of the present invention is used as an active ingredient in the preparation of a drug for regulating the expression of one or more target genes.

[0283] The compound of the present invention or its pharmaceutically acceptable salt or stereoisomer thereof is used as a carrier in the preparation of a drug for regulating the expression of one or more target genes.

[0284] The carrier compound of the present invention is used as a carrier in the preparation of a drug for regulating the expression of one or more target genes.

[0285] The fifth aspect of the present invention provides a pharmaceutical preparation for regulating the expression of one or more target genes, which is prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the conjugate described in the present invention.

[0286] The present invention provides a novel scaffold compound and a conjugate thereof combined with an oligonucleotide drug, which can be used for targeted delivery of oligonucleotide drugs, can simultaneously deliver two nucleotide drugs of a single target or dual-target nucleotide drugs, can enhance the therapeutic effect of oligonucleotide drugs on corresponding diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0287] Figure 1 This is the curve showing the inhibitory effect of conjugate 9A on the AGT target in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg over time.

[0288] Figure 2 This is the curve showing the inhibitory effect of conjugate 9A on the PCSK9 target in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg over time. DETAILED DESCRIPTION

[0289] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0290] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0291] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0292] In the compounds described in the present invention, when any variable (such as R 1 , etc.) occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permissible so long as such combinations result in stable compounds. It is to be understood that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the invention to provide chemically stable compounds that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

[0293] To facilitate understanding of the present technology, certain terms and phrases are defined below.

[0294] The term "alkyl" as used herein is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. 1 -C 6 "C" in "alkyl" 1 -C 6 The definition of " includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched chain arrangement. For example, "C 1 -C 6 The "alkyl group" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl and hexyl.

[0295] The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic hydrocarbon group whose ring atoms are composed of carbon atoms, and the bicyclic or polycyclic rings include spirocyclic, fused and bridged rings. For example, "cycloalkyl" includes but is not limited to the following groups: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, wait.

[0296] The term "alkoxy" as used herein refers to a group having an -O-alkyl structure, such as -OCH 3 、-OCH 2 CH 3 、-OCH 2 CH 2 CH 3 、-O-CH 2 CH(CH 3 ) 2 、-OCH 2 CH 2 CH 2 CH 3 、-O-CH(CH 3 ) 2 wait.

[0297] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic cyclic substituent, wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon, and the bicyclic or polycyclic rings include spirocyclic, fused and bridged rings. For example: oxetanyl, azetidinyl, morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuranyl, tetrahydrothienyl, etc., and N-oxides thereof. Attachment of the heterocyclic substituents may be via a carbon atom or via a heteroatom.

[0298] The term "heteroaryl" as used herein refers to an aromatic ring containing one or more heteroatoms selected from O, N or S, which may be monocyclic, bicyclic or polycyclic, including but not limited to: quinolyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, etc.; "heteroaryl" is also understood to include any N-oxide derivatives of heteroaryl containing nitrogen. The connection of the heteroaryl group can be achieved through a carbon atom or through a heteroatom.

[0299] As will be appreciated by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0300] The present invention includes free forms of compounds of formula (I), as well as pharmaceutically acceptable salts and stereoisomers thereof. The stereoisomers of the present invention are, depending on their structure, enantiomers, diastereomers, syn- / anti-isomers, cis- / trans-isomers, epimers and (E)- / (Z)-isomers. The compounds of formula (A) can be used in the context of the present invention in the form of pure stereoisomers or in the form of any mixture of stereoisomers, in the latter case racemates being preferred.

[0301] In the nucleic acid sequence table of the present invention, the abbreviations and structures of the nucleotide monomers used are as follows:

[0302]

[0303]

[0304]

[0305] According to existing techniques, a person skilled in the art can determine that the pharmaceutically acceptable salt of the siRNA may be a sodium salt or a potassium salt. For example, during purification, a sodium salt of the siRNA is produced.

[0306] The present invention is further described in detail below with reference to specific embodiments.

[0307] Oligonucleotides were synthesized by conventional phosphoramidite method, which is briefly described as follows: 0.2-1 micromolar oligonucleotides were synthesized using solid phase oligonucleotide synthesis protocol on a 12-channel nucleic acid synthesizer of Beijing Qingke Biotechnology Co., Ltd., and siRNA sequences needed to be synthesized on a CPG pre-filled column. Aminolysis reagents were added to the synthesized oligonucleotides and incubated at 45-80°C to separate the oligonucleotides from the solid phase carrier to free the oligonucleotides. Then, crude oligonucleotides were precipitated with ethanol, and the supernatant was discarded by high-speed centrifugation. The crude oligonucleotides were obtained by repeating twice, and the precipitate was resuspended in DEPC water. The crude oligonucleotides were purified by ion pairing HPLC method, and the collected products were dried to powder in a vacuum centrifugal dryer. The purified products were dissolved in DEPC water and analyzed by TOF LC-MS. The concentration of oligonucleotides was determined, and the required volumes of equimolar sense and antisense chains were calculated. The equimolar sense and antisense chains were mixed evenly, and double strands were prepared by annealing method of heating at 95°C for 5 minutes and then naturally cooling to room temperature.

[0308] Preparation of intermediate M:

[0309]

[0310] To a mixture of 11-aminoundecanoic acid (5 g, 24.8 mmol, 1.0 eq.) and triethylamine (2.5 g, 24.8 mmol, 1.0 eq.) in methanol (50 ml) was added ethyl 2,2,2-trifluoroacetate (4.4 g, 31.0 mmol, 1.25 eq.). The mixture was stirred at room temperature overnight. Methanol was removed under vacuum, the residue was dissolved in water, and the pH was adjusted to 1 to 2 with 1 mol hydrochloric acid solution. The mixture was extracted with ethyl acetate, the organic phase was washed with saturated brine, and concentrated under vacuum to give a yellow oily product 11-(2,2,2-trifluoroacetamide)undecanoic acid (Intermediate M-1, 6.16 g, yield: 83.4%). LCMS (ESI): m / z=298[M+H] + .

[0311] To a mixture of 11-(2,2,2-trifluoroacetamide)undecanoic acid (6.06 g, 20.4 mmol, 1.0 eq), (3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol (8.55 g, 20.4 mmol, 1.0 eq) and N,N-diisopropylethylamine (7.8 g, 61.2 mmol, 3.0 eq) in N,N-dimethylformamide (60 ml) was added 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.1 g, 21.4 mmol, 1.05 eq). The mixture was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated under vacuum. The residue was purified by silica gel column (eluent: petroleum ether / ethyl acetate = 2 / 1 to 3 / 1, plus 0.5% triethylamine) to give the product N-(11-((2S, 4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)-11-oxoundecyl)-2,2,2-trifluoroacetamide (Intermediate M-2, 11.6 g, yield: 80.7%) as a white solid. LCMS (ESI): m / z = 699 [M+H] + .

[0312] A mixture of N-(11-((2S, 4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)-11oxonadecanyl)-2,2,2-trifluoroacetamide (11.6 g, 16.5 mmol, 1.0 eq.) and potassium hydroxide (9.3 g, 165 mmol, 10.0 eq.) in methanol (110 ml) was stirred at room temperature overnight. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated under vacuum. The residue was purified by silica gel column (eluent: dichloromethane / methanol=50 / 1, plus 0.5% triethylamine) to give yellow oily product intermediate M (9.24 g, yield: 92.4%). LCMS (ESI): m / z=625[M+Na] + .

[0313] Example 1: Preparation of conjugate 1A: (prepared according to Scheme 1a):

[0314]

[0315]

[0316] Step 1a.: Preparation of compound 1a-1: A mixture of (3R, 5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol (10 g, 23.84 mmol, 1.0 eq), 12-methoxy-12-oxodecanoic acid (5.8 g, 23.84 mmol, 1.0 eq), N,N-diisopropylethylamine (12.3 g, 95.36 mmol, 4.0 eq) in dichloromethane (100 ml) was stirred at room temperature for 2.0 hours. The mixture was washed with sodium carbonate solution, and the aqueous phase was extracted with dichloromethane (50 ml x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 20 / 1) to give a yellow oily product 1a-1 (16.7 g, yield: 79.3%).

[0317] Step 1b: Preparation of compound 1a-2: Add lithium hydroxide monohydrate (3.1 g, 64.73 mmol, 2.5 eq.) to a mixture of compound 1a-1 (16.7 g, 25.89 mmol, 1.0 eq.) in tetrahydrofuran / methanol / water (80 ml / 16 ml / 16 ml). The mixture is reacted at 40°C for 4 hours. The solvent is removed from the reaction solution under reduced pressure. The aqueous solution of the residue is freeze-dried. The residue is dissolved in dichloromethane and filtered. The filtrate is concentrated under reduced pressure to give a white solid product 1a-2 (14.3 g, yield: 85.3%). LCMS (ESI): m / z=630 [MH] - .

[0318] Step 1c: Preparation of compound 1a-3: To a mixture of compound (azadiylbis(ethane-2,1-diyl))carbamic acid tert-butyl ester (3.0 g, 9.9 mmol, 1.0 eq.) and sodium carbonate (1.57 g, 14.85 mmol, 1.5 eq.) in tetrahydrofuran (20 ml) and water (10 ml) was added benzyl chloroformate (2.03 g, 11.88 mmol, 1.2 eq.). The mixture was stirred at room temperature overnight. The mixture was diluted with water (50 ml) and then extracted with ethyl acetate (20 ml x 3). The combined organic layer was washed with saturated brine (20 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate = 3: 1) to give a white solid product 1a-3 (3.80 g, yield: 88%). LCMS (ESI): m / z = 438 [M+H] + .

[0319] Step 1d: Preparation of compound 1a-4: To a mixture of compound 1a-3 (3.74 g, 8.55 mmol, 1.0 eq.) in methanol (10 ml) was added a solution of hydrogen chloride-dioxane (4 mol / L, 15 ml). The mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was dried under vacuum to give a white solid product 1a-4 (2.92 g, crude). LCMS (ESI): m / z=238 [M+H] + .

[0320] Step 1e: Preparation of compound 1a-5: To a mixture of compound (S)-4-(tert-butoxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoic acid (45 g, 155.54 mmol, 1.0 eq.) and N-methylmorpholine (15.7 g, 155.54 mmol, 1.0 eq.) in tetrahydrofuran (220 ml) was added dropwise isobutyl chloroformate (20.17 ml, 155.54 mmol, 1.0 eq.) at -20°C. After the mixture was stirred for 10 minutes, the mixture was filtered. To the filtrate was slowly added a solution of sodium borohydride (11.8 g, 311.08 mmol, 2.0 eq.) in water (70 ml) at -30°C. The mixture was warmed to 0°C and stirred for 0.5 hours. The mixture was diluted with water (200 ml) and then extracted with ethyl acetate (100 ml x 3). The combined organic layers were washed with saturated brine (100 mL x 1), dried over anhydrous sodium sulfate and concentrated to give a colorless oil 1a-5 (43.67 g, crude product).

[0321] Step 1f: Preparation of compound 1a-6: To a mixture of compound 1a-5 (42.8 g, 155.54 mmol, 1.0 eq.) and sodium bicarbonate (39.2 g, 466.62 mmol, 3.0 eq.) in dichloromethane (300 ml) was added Dess-Martin reagent (79.2 g, 186.65 mmol, 1.2 eq.) at 0°C. The mixture was warmed to room temperature and stirred for 2 hours. The mixture was filtered. The filtrate was concentrated under reduced pressure. The residue was diluted with ethyl acetate (200 ml), washed successively with saturated sodium thiosulfate solution (100 ml x 1), saturated sodium bicarbonate solution (100 ml x 1) and saturated brine (100 ml x 1), dried over anhydrous sodium sulfate and concentrated to give a light yellow oil 1a-6 (40 g, crude product).

[0322] Step 1g: Preparation of compound 1a-7: A mixture of benzylamine (6.1 g, 57.01 mmol, 1.0 eq), compound 1a-6 (38 g, 139.2 mmol, 2.44 eq) and acetic acid (3.26 ml, 57.01 mmol, 1.0 eq) in methanol (200 ml) was stirred at room temperature for 10 minutes. The mixture was cooled to 0°C, and then sodium cyanoborohydride (12.53 g, 199.54 mmol, 3.5 eq) was slowly added. The mixture was warmed to room temperature and stirred overnight. The solvent was removed under reduced pressure, and the residue was diluted with water (150 ml). Solid sodium bicarbonate was added to adjust pH = 9, and the aqueous layer was extracted with ethyl acetate (60 ml x 3). The combined organic layers were washed with saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (petroleum ether: ethyl acetate = 50: 1 to 5: 1) to obtain a colorless oil 1a-7 (24.1 g, yield: 68%). LCMS (ESI): m / z 622 [M+H] + .

[0323] Step 1h: Preparation of compound 1a-8: To a mixture of compound 1a-7 (27.5 g, 44.23 mmol, 1.0 eq.) and N,N-diisopropylethylamine (1.54 ml, 8.85 mmol, 0.2 eq.) in acetonitrile (125 ml) was added dropwise 1-chloroethyl chloroformate (7.16 ml, 66.34 mmol, 1. eq.) at 0°C. The mixture was warmed to room temperature and stirred for 2 hours. The solvent was removed under reduced pressure. The residue was diluted with methanol (20 ml). The mixture was heated to 63°C and stirred for 1.5 hours. The solvent was removed under reduced pressure and the residue was diluted with dichloromethane (150 ml). The organic layer was washed with saturated sodium bicarbonate solution (30 ml x 1) and saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (dichloromethane: methanol = 200: 1 to 20: 1) to obtain a light yellow oil 1a-8 (11.5 g, yield: 49%). LCMS (ESI): m / z = 532 [M+H] + .

[0324] Step 1i: Preparation of compound 1a-9: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (9.84 g, 23.78 mmol, 1.1 eq.) was added to a mixture of compound 1a-8 (4.3 g, 22.7 mmol, 1.05 eq.) and 3-(tert-butyloxycarbonyl)amino)propionic acid (4.3 g, 22.7 mmol, 1.05 eq.) and N,N-diisopropylethylamine (5.58 g, 43.24 mmol, 2.0 eq.) in dichloromethane (70 ml). The mixture was stirred at room temperature for 2.5 hours. The solvent was removed under reduced pressure. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate = 4: 1 to 2: 1) to obtain a light yellow solid product 1a-9 (14.98 g, yield: 99%). LCMS (ESI): m / z=703 [M+H] + .

[0325] Step 1j: Preparation of compound 1a-10: A mixture of compound 1a-9 (14.9 g, 21.19 mmol, 1.0 eq) in hydrogen chloride-dioxane (4 mol per liter, 90 ml) was stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue was dissolved in tetrahydrofuran (100 ml) and water (100 ml). Sodium carbonate (13.48 g, 127.14 mmol, 6.0 eq) and di-tert-butyl carbonate (24.36 ml, 105.95 mmol, 5.0 eq) were added. The mixture was stirred at room temperature for 48 hours. Tetrahydrofuran was removed under reduced pressure. The residue was diluted with water (200 ml) and then washed with dichloromethane (60 ml x 4). A 2 mol per liter dilute hydrochloric acid solution was added to the aqueous layer to adjust the pH to 3, and then extracted with ethyl acetate (60 ml x 4). The combined organic layer was washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate and concentrated to obtain a light yellow solid product 1a-10 (10.5 g, yield: 84%). LCMS (ESI): m / z = 592 [M+H] + .

[0326] Step 1k: Preparation of compound 1a-11: To a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (6.58 g, 15.9 mmol, 2.5 eq) in dichloromethane (570 mL) was added dropwise a solution of compound 1a-10 (3.76 g, 6.36 mmol, 1.0 eq), compound 1a-4 (1.97 g, 6.36 mmol, 1.0 eq) and N,N-diisopropylethylamine (4.92 g, 38.16 mmol, 6.0 eq) in dichloromethane (60 mL) at 0°C over 30 min. The mixture was warmed to room temperature and stirred for 20 min. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium bicarbonate solution (50 mL) and then extracted with ethyl acetate (30 mL x 3). The combined organic layer was washed with saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (ethyl acetate: methanol = 50: 1 to 30: 1) to obtain a white solid product 1a-11 (1.97 g, yield: 39%). LCMS (ESI): m / z = 792.6 [M+H] + .

[0327] Step 11: Preparation of compound 1a-13: A mixture of 1a-11 (1.97 g, 2.49 mmol, 1.0 eq) in hydrogen chloride-dioxane (4 mol / L, 13 ml) was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (60 ml). Compound 1a-12 (3.34 g, 7.47 mmol, 3.0 eq), N,N-diisopropylethylamine (2.57 g, 19.92 mmol, 8.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.03 g, 7.97 mmol, 3.2 eq) were added. The mixture was stirred at room temperature for 1.5 hours. The mixture was washed with saturated sodium bicarbonate solution (30 ml x 2) and saturated brine (30 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane: methanol = 30: 1, plus 1% triethylamine) to obtain a light yellow solid product 1a-13 (3.43 g, yield: 77%). LCMS (ESI): m / z = 1782.2 [M+H] + .

[0328] Step 1m: Preparation of compound 1a-14: Palladium hydroxide / carbon (10%, 0.69 g) was added to a mixture of compound 1a-13 (3.47 g, 1.95 mmol, 1.0 eq.) and trifluoroacetic acid (0.15 ml, 1.95 mmol, 1.0 eq.) in isopropanol (50 ml) and ethanol (25 ml). The mixture was stirred at room temperature for 20 hours under hydrogen balloon pressure. The mixture was filtered. The filtrate was concentrated under reduced pressure to give a white solid product 1a-14 (3.45 g, crude product). LCMS (ESI): m / z=1646.9 [M+H] + .

[0329] Step 1n: Preparation of compound 1a-15 (Compound 31): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.26 g, 0.68 mmol, 1.2 eq.) was added to a mixture of compound 1a-14 (1.0 g, 0.57 mmol, 1.0 eq.), compound 1a-2 (0.396 g, 0.63 mmol, 1.1 eq.) and N,N-diisopropylethylamine (0.441 g, 3.42 mmol, 6.0 eq.) in dichloromethane (15 ml). The mixture was stirred at room temperature for 2.5 hours. The solvent was removed under reduced pressure. The residue was purified by preparative HPLC (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 50%-70% within 30 minutes) to obtain a white solid product 1a-15 (Compound 31) (0.45 g, yield: 35%). LCMS (ESI): m / z = 980.5 [(M-302) / 2+H] + .

[0330] Step 1o: Preparation of compound 1a-16: Succinic anhydride (34.5 mg, 0.348 mmol, 6.0 eq.) was added to a mixture of compound 1a-15 (130 mg, 0.058 mmol, 1.0 eq.), triethylamine (70 mg, 0.696 mmol, 12.0 eq.) and 4-dimethylaminopyridine (3.5 mg, 0.029 mmol, 0.5 eq.) in dichloromethane (1 ml). The mixture was stirred at room temperature for 2 days. The mixture was diluted with dichloromethane (10 ml), then washed with saturated sodium bicarbonate solution (5 ml x 2) and saturated brine (5 ml x 1), dried over anhydrous sodium sulfate and concentrated to give a white solid product 1a-16 (111 mg, yield: 81%). LCMS (ESI): m / z = 1030.5 [(M-302) / 2+H] + .1H NMR (500MHz, DMSO) δ8.42-8.17(m,2H),8.06(t,J=25.8Hz,2H),7.89-7.70(m,4H),7.30(dd,J=14.4,6.9Hz,4H),7.20(dd,J=10.1,6.9Hz,5H),6.9 3-6.85(m,4H),5.21(s,3H),4.97(d,J=13.1Hz,3H),4.50(d,J=8.2Hz,3H ),4.20(s,3H),4.02(s,9H),3.87(dd,J=18.8,9.5Hz,3H),3.80-3.63(m, 11H),3.49(dd,J=81.3,10.3Hz,11H),3.24(s,2H),3.20-3.12(m,3H),3. 03(dd,J=26.7,13.8Hz,3H),2.47(d,J=9.6Hz,3H),2.44-2.36(m,4H),2. 31-2.18(m,5H),2.11(d,J=10.6Hz,13H),2.07(s,2H),2.03(d,J=7.3Hz, 3H),1.99(s,9H),1.89(s,9H),1.77(s,12H),1.47(s,16H),1.25(s,11H).

[0331] Step 1p: Preparation of compound 1a-17: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21 mg, 0.056 mmol, 1.2 eq.) and 1-hydroxybenzotriazole (9 mg, 0.066 mmol, 1.4 eq.) were added to a mixture of compound 1a-16 (111 mg, 0.047 mmol, 1.0 eq.) and N,N-diisopropylethylamine (36 mg, 0.282 mmol, 6.0 eq.) in N,N-dimethylformamide (5.5 ml). The mixture was shaken at room temperature for 5 minutes. CPG-NH 2 (0.167 mmol / g, 550 mg, 0.092 mmol, 1.95 eq), the mixture was vibrated at room temperature for 22 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (3 ml / 1 ml) and vibrated at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to obtain a white solid product 1a-17 (578 mg, loading: 35 μmol / g).

[0332] Step 1q: Preparation of conjugate 1A-1: Compound 1a-17 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain conjugate 1A-1 (MS m / z: [MH] - :calcd.8279.0520Found 8278.826).

[0333] Step 1r: Preparation of conjugate 1A: Take equivalent amounts of conjugate 1A-1 and complementary antisense chain (1167AM25) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 1A.

[0334] Example 2: Preparation of conjugate 1B (preparation method is the same as conjugate 1A):

[0335] Step 2a: Conjugate 1B-1: Compound 1a-17 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain conjugate 1B-1 (MS m / z: [MH] - :calcd.9027.6065Found 9026.9963).

[0336] Step 2b: Preparation of conjugate 1B: Take equivalent amounts of conjugate 1B-1 and complementary antisense chain (1165AM8) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 1B.

[0337] Example 3: Preparation of Conjugate 2A and Conjugate 2B (prepared according to Scheme 2a and Scheme 2b):

[0338]

[0339]

[0340]

[0341] Step 3a: Preparation of compound 2a-2: A mixture of 6-heptynoic acid (3.00 g, 23.78 mmol, 1.0 eq), N,N-diisopropylethylamine (6.15 g, 47.56 mmol, 2.0 eq), compound 2a-1 (8.03 g, 24.97 mmol, 1.05 eq) and benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (9.92 g, 26.16 mmol, 1.1 eq) in acetonitrile (30 ml) was stirred at room temperature for 2 hours. The mixture was added to 60.0 ml of saturated aqueous ammonium chloride solution and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1) to obtain a colorless oil product 2a-2 (7.82 g, yield: 76.5%). LCMS (ESI): m / z=430 [M+H] + .

[0342] Step 3b: Preparation of compound 2a-3: Add trifluoroacetic acid (12 ml) to a solution of compound 2a-2 (7.82 g, 18.20 mmol, 1.0 eq.) in dichloromethane (60 ml), and stir the mixture at room temperature for 30 minutes. The mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 15:1) to give a light yellow oily product 2a-3 (4.90 g, yield: 72.1%). LCMS (ESI): m / z = 396 [M+Na] + .

[0343] Step 3c: Preparation of compound 2a-5: A mixture of compound 2a-4 (10.00 g, 26.00 mmol, 1.0 eq), N,N-diisopropylethylamine (6.70 g, 52.00 mmol, 2.0 eq), compound (3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol (11.40 g, 27.30 mmol, 1.05 eq) and benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (10.80 g, 28.6 mmol, 1.1 eq) in acetonitrile (120.0 ml) was stirred at room temperature for 2 hours. The mixture was added to 200 ml of saturated aqueous ammonium chloride solution and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane to dichloromethane / methanol = 100 / 1, with 2% triethylamine) to obtain a colorless oily product 2a-5 (17.41 g, yield: 85.2%). LCMS (ESI): m / z = 809 [M+Na] + .

[0344] Step 3d: Preparation of compound 2a-6: piperidine (10.0 ml) was added to a solution of 2a-5 (8.00 g, 10.20 mmol, 1.0 eq.) in acetonitrile (75.0 ml), and the mixture was stirred at room temperature for 15 minutes. The mixture was concentrated in vacuo, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1 to 10:1, with 1% triethylamine) to obtain a white foam solid product 2a-6 (3.8 g, yield: 66.2%). LCMS (ESI): m / z = 566 [M+H] + .

[0345] Step 3e: Preparation of compound 2a-7: A mixture of 2a-3 (2.50 g, 6.69 mmol, 1.0 eq), N,N-diisopropylethylamine (1.73 g, 13.39 mmol, 2.0 eq), 2a-6 (3.97 g, 7.03 mmol, 1.05 eq) and benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (3.05 g, 8.03 mmol, 1.2 eq) in acetonitrile (25.0 ml) was stirred at room temperature for 2 hours. The mixture was added to 60.0 ml of saturated aqueous ammonium chloride solution and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 50 / 1, 1% triethylamine) to obtain a colorless oily product 2a-7 (5.60 g, yield: 90.9%). LCMS (ESI): m / z = 921 [M+H] + .

[0346] Step 3f: Preparation of compound 2a-8: Under nitrogen protection, add succinic anhydride (1.83 g, 18.26 mmol, 3.0 eq.) to a mixture of compound 2a-7 (2.50 g, 6.69 mmol, 1.0 eq.), 4-dimethylaminopyridine (74.36 mg, 608.62 μmol, 0.1 eq.) and triethylamine (3.70 g, 36.52 mmol, 6.0 eq.) in anhydrous dichloromethane (15.0 ml). The mixture was stirred at room temperature for 18 hours. 15.0 ml of dichloromethane and 30.0 ml of water were added, and the mixture was stirred for 5 minutes. The liquid was separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain lavender foamy product 2a-8 (4.21 g, yield: 61.7%). LCMS (ESI): m / z = 1021 [M+H] + .

[0347] Step 3g: Preparation of compound 2a-9: A mixture of compound 2a-8 (4.21 g, 3.75 mmol, 1.0 eq), benzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (2.14 g, 5.63 mmol, 1.5 eq) and N,N-diisopropylethylamine (0.97 g, 7.50 mmol, 2.0 eq) in anhydrous acetonitrile (42.0 ml) was shaken at room temperature for 10 minutes. CPG-NH 2 (13.50 g, 500 μmol / g, 1.8 eq.), the mixture was shaken at room temperature for 5 hours. The mixture was filtered, the filter cake was washed with acetonitrile and dichloromethane, respectively, and dried under vacuum for 1 hour. 30 ml of anhydrous pyridine and 10 ml of acetic anhydride were added to the solid, shaken at room temperature for 2 hours, the mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to obtain 2a-9 (17.8 g, loading of 182.5 μmol / g).

[0348] Step 3h: Preparation of compound 2a-10-A-ssRNA-2: Compound 2a-9 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 2a-10-A-ssRNA-2 (MS m / z: [MH] - :calcd.7317.9750Found7317.2356).

[0349] Step 3i: Preparation of compound 2b-2: A mixture of compound 2b-1 (22.02 g, 50.00 mmol, 1.0 eq), benzyl (2-aminoethyl)carbamate (9.71 g, 50.00 mmol, 1.0 eq), N,N-diisopropylethylamine (12.90 g, 10.00 mmol, 2.0 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (21.72 g, 52.50 mmol, 1.05 eq) in N,N-dimethylformamide (250 ml) was stirred at room temperature for 30 minutes. The mixture was diluted with water and then filtered. The filter cake was washed with water. The filter cake was dried to give a white solid product 2b-2 (crude product). LCMS (ESI): m / z=617 [M+H] + . 1 H NMR (500MHz, DMSO) δ7.87(t,J=8.1Hz,3H),7.67(d,J=6.9Hz,2H),7.42(d,J=7.4Hz,2H),7.37-7.26(m,7H),7.19(d,J=4.7Hz,2H),6.84(d,J=7.2H z,1H),5.00(s,2H),4.34-4.15(m,3H),3.90(d,J=5.0Hz,1H),3.19-2.94 (m,6H),1.78(d,J=6.6Hz,1H),1.62(dd,J=13.1,6.6Hz,1H),1.37(s,9H).

[0350] Step 3j: Preparation of compound 2b-3: A mixture of compound 2b-2 (crude product) in piperidine / acetonitrile (50 ml / 200 ml) was stirred at room temperature for 1 hour. The mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane to dichloromethane / methanol = 20 / 1) to obtain a yellow solid product 2b-3 (15.0 g, yield: 76.14%). LCMS (ESI): m / z = 395 [M+H] + .

[0351] Step 3k: Preparation of compound 2b-4: A mixture of compound 2b-3 (7.80 g, 19.80 mmol, 1.5 eq.), tert-butyl (2-bromoethyl)carbamate (2.96 g, 13.20 mmol, 1.0 eq.) and potassium carbonate (3.64 g, 26.40 mmol, 2.0 eq.) in N,N-dimethylformamide (80 ml) was stirred at room temperature overnight. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 50 / 1) to give a colorless oily product 2b-4 (4.0 g, yield: 56.43%). LCMS (ESI): m / z = 538 [M+H] + . 1 H NMR (500MHz, DMSO) δ7.92(s,1H),7.42-7.28(m,5H),7.19(s,1H),6.93(d,J=7.3Hz,1H),6.75(s,1H),5.01(s,2H),3.95(d,J=5.4Hz ,1H),3.14(ddd,J=18.3,12.5,6.1Hz,2H),3.05(d,J=5.5Hz,4H),2.68-2.56(m,4H),1.79(s,1H),1.72-1.60(m,1H),1.37(s,18H).

[0352] Step 31: Preparation of compound 2b-6: A mixture of compound 2b-4 (4.0 g, 7.45 mmol, 1.0 eq), compound 2b-5 (2.39 g, 7.08 mmol, 0.95 eq), N,N-diisopropylethylamine (1.92 g, 14.90 mmol, 2.0 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (3.08 g, 7.45 mmol, 1.0 eq) in N,N-dimethylformamide (50 ml) was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 60 / 1) to give a white solid product 2b-6 (4.4 g, yield: 72.52%). LCMS (ESI): m / z=858 [M+H] + . 1H NMR(500MHz,DMSO)δ7.88(d,J=12.6Hz,1H),7.32(d,J=22.7Hz,10H),7.26- 7.13(m,2H),7.01-6.65(m,2H),5.25-5.03(m,2H),5.00(s,2H),4.10-3.96( m,1H),3.82(s,1H),3.17(d,J=43.7Hz,6H),3.03(d,J=22.8Hz,4H),2.43-2. 21(m,2H),2.03-1.86(m,1H),1.79(s,2H),1.63(s,1H),1.44-1.29(m,27H).

[0353] Step 3m: Preparation of compound 2b-7: A solution of compound 2b-6 (4.4 g, 5.13 mmol, 1.0 eq.) in hydrogen chloride-dioxane (40 ml) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step without purification (crude product). LCMS (ESI): m / z=557 [M+H] + .

[0354] Step 3n: Preparation of compound 2b-8: A mixture of 2b-7 (crude), N,N-diisopropylethylamine (6.60 g, 51.16 mmol, 10.0 eq.), compound 1a-12 (7.10 g, 15.87 mmol, 3.1 eq.) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.20 g, 16.31 mmol, 3.2 eq.) in acetonitrile (70 ml) was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 25 / 1) to give a yellow solid product 2b-8 (6.0 g, yield: 63.39%). LCMS (ESI): m / z = 923 [M / 2+H] + .

[0355] Step 3o: Preparation of compound 2b-9: Under a hydrogen atmosphere, a mixture of compound 2b-8 (6.0 g, 3.25 mmol, 1.0 eq), palladium hydroxide / carbon (1.5 g, 25% mass fraction) and trifluoroacetic acid (371 mg, 3.25 mmol, 1.0 eq) in ethanol (100 ml) was stirred at room temperature overnight. The mixture was filtered through celite. The filter cake was washed with ethanol and the filtrate was concentrated. The residue was used directly in the next step without purification (5.0 g, yield: 94.93%). LCMS (ESI): m / z=811[M / 2+H] + .

[0356] Step 3p: Preparation of compound 2b-10: Under nitrogen protection, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (40.2 g, 210 mmol, 1.1 eq.) was added to a mixture of 6-azidohexanoic acid (30 g, 190.8 mmol, 1.0 eq.) and 1-hydroxypyrrolidine-2,5-dione (24 g, 210 mmol, 1.1 eq.) in dichloromethane / N,N-dimethylformamide (270 / 30 ml). The mixture was stirred at room temperature overnight. Dichloromethane was removed under vacuum, 1 mol hydrochloric acid solution and methyl tert-butyl ether were added, the liquids were separated, and the organic layer was washed with sodium bicarbonate solution and saturated brine. The organic phase was dried and concentrated under vacuum to obtain a yellow oily product 2b-10 (46.8 g, yield: 96.8%). LCMS (ESI): m / z=255[M+H] + .

[0357] Step 3q: Preparation of compound 2b-11: A mixture of compound 2b-9 (5.0 g, 3.09 mmol, 1.0 eq), compound 2b-10 (824 mg, 3.24 mmol, 1.05 eq) and N,N-diisopropylethylamine (1.19 g, 9.22 mmol, 3.0 eq) in acetonitrile (60 ml) was stirred at room temperature for 1 hour. The reaction was directly carried out to the next step without post-treatment. LCMS (ESI): m / z=881 [M / 2+H] + .

[0358] Step 3r: Preparation of compound 2b-12 (compound 54): A mixture of compound 2b-11 (crude), intermediate M (2.04 g, 3.39 mmol, 1.1 eq), N,N-diisopropylethylamine (796 mg, 6.17 mmol, 2.0 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (1.4 g, 3.39 mmol, 1.1 eq) in acetonitrile (60 ml) was stirred for 1 hour. The mixture was directly purified by high performance liquid chromatography (chromatographic column: UniSil 10-120C18, 30×250 mm) to obtain a white solid product 2b-12 (compound 54) (3.1 g, yield: 42.79%). LCMS (ESI): m / z=1022[(M-302) / 2+H] +.1H NMR (500MHz, DMSO) δ8.04-7.72(m,9H),7.30(dd,J=17.9,8.1Hz,4H),7.23-7.10(m,5H),6.93-6.81(m,4H),5.21(d,J=2.3Hz,3H),5.02-4.83( m,4H),4.49(d,J=8.2Hz,3H),4.38(s,1H),4.15(s,3H),4.02(s,9H),3. 87(q,J=9.5Hz,3H),3.72(d,J=12.9Hz,9H),3.41(s,3H),3.29(d,J=6.8 Hz,2H),3.07(ddd,J=23.5,22.3,17.2Hz,14H),2.22(dd,J=22.2,15.1Hz,4H),2.11(d,J=13.0Hz,13H),2.04(dd,J=14.3,6.9Hz,5H),1.99(s,1 0H),1.89(s,9H),1.84(d,J=13.3Hz,3H),1.77(s,9H),1.68(d,J=35.4H z,2H),1.50(dd,J=14.8,7.4Hz,19H),1.35(s,3H),1.30-1.18(m,13H).

[0359] Step 3s: Preparation of compound 2b-13: A mixture of compound 2b-12 (3.1 g, 1.32 mmol, 1.0 eq), succinic anhydride (793 mg, 7.93 mmol, 6.0 eq), triethylamine (1.60 g, 15.84 mmol, 12.0 eq) and 4-dimethylaminopyridine (16 mg, 0.13 mmol, 0.1 eq) in dichloromethane (25 ml) was stirred at room temperature for 6 hours. The mixture was washed with 10% sodium bicarbonate solution and acetonitrile. The organic phase was concentrated to give a light purple solid product 2b-13 (3.4 g, crude product). LCMS (ESI): m / z=1072[(M-302) / 2+H] + .

[0360] Step 3t: Preparation of compound 2b-14: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (56 mg, 0.15 mmol, 1.2 eq.), 1-hydroxybenzotriazole (23 mg, 0.17 mmol, 1.4 eq.) and N,N-diisopropylethylamine (63 mg, 0.49 mmol, 4.0 eq.) were added to a solution of compound 2b-13 (300 mg, 0.12 mmol, 1.0 eq.) in N,N-dimethylformamide, and the mixture was shaken at room temperature for 5 minutes. 1.5 g of CPG-NH 2The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (12 ml / 4 ml) and shaken at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour to obtain a light yellow solid product 2b-14 (1.61 g, loading: 41.1 μmol / g).

[0361] Step 3u: Preparation of compound 2b-15-A-ssRNA-1: Compound 2b-14 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 2b-15-A-ssRNA-1 (MS m / z: [MH] - :calcd.8981.5075Found8980.9595).

[0362] Step 3v: Preparation of conjugate 2A-1: Compound 2a-10-A-ssRNA-2 (182 nmol, 1 equivalent) and 2b-15-A-ssRNA-1 (200 nmol, 1.1 equivalent) were sequentially added with 53 μl PBS buffer (pH 8.0, 0.5 M), 259 μl DEPC water, 267 μl DMF, and 286 μl premixed CuSO 4 -THPTA (1:1) solution (conc. 14 nmol / μl), mixed well, added 1.6 mg sodium ascorbate, replaced with nitrogen, and reacted at 35°C for 2 h. After the reaction was complete, 15 μl of ammonia water and 16 mg EDTA were added, shaken for 5-10 minutes, and ethanol precipitation was performed to obtain about 2.3 mg of the crude product of conjugate 2A-1, which was then purified by HPLC to obtain conjugate 2A-1 (MS m / z: [MH] - :calcd.16299.4826Found16299.3828).

[0363] Step 3w: Preparation of conjugate 2A: Take an equivalent amount of conjugate 2A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 2A.

[0364] Step 3x: Preparation of conjugate 2B (preparation method is the same as conjugate 2A):

[0365] Compound 2a-10-B-ssRNA-2 (182 nanomoles, 1 equivalent) and 2b-15-B-ssRNA-1 (200 nanomoles, 1.1 equivalents) were added in sequence to 53 μl of PBS buffer (pH 8.0, 0.5 M), 259 μl of DEPC water, 267 μl of DMF, and 286 μl of premixed CuSO 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.0 mg of the crude product of conjugate 2B-1, which is then purified by HPLC to obtain conjugate 2B-1 (MS m / z: [MH] - :calcd.15682.1370Found 15682.1011).

[0366] Take an equivalent amount of conjugate 2B-1 and two complementary antisense strands (1167AM25 and 1167AM25) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 2B.

[0367] Example 4: Preparation of conjugate 3A (prepared according to Scheme 3a):

[0368]

[0369]

[0370]

[0371] Step 4a: Preparation of compound 3a-1: Glycine benzyl ester hydrochloride (6.04 g, 29.96 mmol, 2.0 eq), (9H-fluoren-9-yl)methyl (2-oxoethyl) carbamate (4.21 g, 14.98 mmol, 1.0 eq) and acetic acid (90 mg, 1.5 mmol, 0.1 eq) were added to 80 ml of methanol and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (1.4 g, 22.47 mmol, 1.5 eq) was added and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, saturated aqueous sodium carbonate solution was added and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1 to 1 / 2) to obtain colorless oil 3a-1 (3.22 g, yield: 49.9%). LCMS (ESI): m / z=431 [M+H] + .

[0372] Step 4b: Preparation of compound 3a-2: To a solution of compound 3a-1 (3.22 g, 7.44 mmol, 1.0 eq) in 70 ml of tetrahydrofuran was added a solution of sodium carbonate (3.16 g, 29.77 mmol, 4.0 eq) in 18 ml of water. Di-tert-butyl dicarbonate (3.24 g, 14.88 mmol, 2.0 eq) was added and the mixture was stirred at room temperature for 2 hours. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1 to 1 / 3) to give a yellow oil 3a-2 (3.41 g, yield: 86.4%). LCMS (ESI): m / z = 531 [M+H] + .

[0373] Step 4c: Preparation of compound 3a-3: Compound 3a-2 (3.41 g, 6.43 mmol, 1.0 eq) was dissolved in 90 ml methanol and 341 mg of 10% palladium on carbon was added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to give a yellow oil 3a-3 (2.65 g, yield: 93.6%). LCMS (ESI): m / z = 441 [M+H] + .

[0374] Step 4d: Preparation of compound 3a-4: Compound 3a-3 (2.65 g, 6.04 mmol, 1.0 eq), (2-aminoethyl) benzyl carbamate (1.17 g, 6.04 mmol, 1.0 eq), N,N-diisopropylethylamine (1.56 g, 12.08 mmol, 2.0 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (2.5 g, 6.04 mmol, 1.0 eq) were added to 20 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1 to 1 / 3) to obtain colorless syrup 3a-4 (2.80 g, yield: 75.2%). LCMS (ESI): m / z = 617 [M+H] + .

[0375] Step 4e: Preparation of compound 3a-5: Compound 3a-4 (2.8 g, 7.10 mmol, 1.0 eq.) and 1.5 ml of piperidine were added to 24 ml of acetonitrile. The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 to 10 / 1, with triethylamine) to obtain a colorless oil 3a-5 (1.25 g, yield: 69.8%). LCMS (ESI): m / z = 395 [M+H] + .

[0376] Step 4f: Preparation of compound 3a-6: Compound 3a-5 (1.9 g, 4.82 mmol, 1.0 eq), tert-butyl (2-oxoethyl)carbamate (768 mg, 4.82 mmol, 1.0 eq), acetic acid (29 mg, 0.48 mmol, 0.1 eq) and sodium cyanoborohydride (337 mg, 5.2 mmol, 1.1 eq) were added to 20 ml of methanol, and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, sodium carbonate solution was added, and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, triethylamine was added) to give a yellow oil 3a-6 (914 mg, yield: 35.2%). LCMS (ESI): m / z = 538 [M+H] + .

[0377] Step 4g: Preparation of compound 3a-7: Compound 3a-6 (914 mg, 1.7 mmol, 1.0 eq.), compound 2b-5 (631 mg, 1.87 mmol, 1.1 eq.), N,N-diisopropylethylamine (439 mg, 3.4 mmol, 2.0 eq.) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (774 mg, 1.87 mmol, 1.7 eq.) were added to 15 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, with triethylamine) to obtain yellow oil 3a-7 (1.23 g, yield: 84.2%). LCMS (ESI): m / z = 857.5 [M+H] + .

[0378] Step 4h: Preparation of compound 3a-8: Compound 3a-7 (1.23 g, 1.43 mmol, 1.0 eq) was added to 40 ml of 4 mol hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give a yellow solid 3a-8 (1.01 g, crude product). LCMS (ESI): m / z=557 [M+H] + .

[0379] Step 4i: Preparation of compound 3a-9: Compound 3a-8 (1.01 g, 1.43 mmol, 1.0 eq), compound 1a-12 (2.05 g, 4.58 mmol, 3.2 eq), N,N-diisopropylethylamine (1.85 g, 14.3 mmol, 10.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.74 g, 4.58 mmol, 3.2 eq) were added to 30 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, plus triethylamine) to give white solid compound 3a-9 (1.54 g, yield: 58.3%).

[0380] LCMS(ESI): m / z=923.7[M / 2+H] + .

[0381] Step 4j: Preparation of compound 3a-10: Compound 3a-9 (1.54 g, 0.84 mmol, 1.0 eq) was dissolved in 60 ml of ethanol, and trifluoroacetic acid (95 mg, 0.84 mmol, 1.0 eq) and 200 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a white solid compound 3a-10 (1.42 g, crude product). LCMS (ESI): m / z=1622 [M+H] + .

[0382] Step 4k: Preparation of compound 3a-11 (compound 55): Compound 3a-10 (1.42 g, 0.84 mmol, 1.0 eq), N,N-diisopropylethylamine (162 mg, 1.25 mmol, 1.5 eq) and compound 2b-10 (234 mg, 0.92 mmol, 1.1 eq) were added to 15 ml of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. N,N-diisopropylethylamine (162 mg, 1.25 mmol, 1.5 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (414 mg, 1.0 mmol, 1.2 eq) and intermediate M were added to the mixture, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by preparative HPLC (eluent: pH=7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to obtain white solid compound 3a-11 (compound 55) (550 mg, yield: 27.9%). LCMS (ESI): m / z=1022[(M-302) / 2+H] + .

[0383] Step 41: Preparation of compound 3a-12: Compound 3a-11 (550 mg, 0.235 mmol, 1.0 eq), N,N-diisopropylethylamine (285 mg, 2.82 mmol, 12.0 eq), succinic anhydride (141 mg, 1.41 mmol, 6.0 eq) and 4-dimethylaminopyridine (3 mg, 0.024 mmol, 0.1 eq) were added to 3 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give white solid compound 3a-12 (550 mg, crude product). LCMS (ESI): m / z=1072[(M-302) / 2+H] + .

[0384] Step 4m: Preparation of compound 3a-13: Compound 3a-12 (550 mg, 0.225 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (117 mg, 0.308 mmol, 1.4 eq), 1-hydroxybenzotriazole (49 mg, 0.36 mmol, 1.6 eq) and N,N-diisopropylethylamine (133 mg, 1.03 mmol, 4.6 eq) were added to 25 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 2.75 g of CPG-NH2 was added, and the mixture was shaken in a shaker at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 18 ml of pyridine and 6 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. Filter, wash the solid phase with dichloromethane and acetonitrile, and dry the solid phase under reduced pressure to obtain a yellow solid 3a-13 (2.5 g, loading: 45.7 μmol / g).

[0385] Step 4n: Preparation of compound 3a-14-A-ssRNA-1: Compound 3a-13 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 3a-14-A-ssRNA-1 (MS m / z: [MH] - :calcd.8981.5075Found8980.9588).

[0386] Step 4o: Preparation of conjugate 3A-1: 2a-10-A-ssRNA-2 (109 nmol, 1 equivalent) and 3a-14-A-ssRNA-1 (120 nmol, 1.1 equivalent) were added in sequence to 32 μl PBS buffer (pH 8.0, 0.5 M), 186 μl DEPC water, 160 μl DMF, and 171 μl premixed CuSO 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1 mg of sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is sufficient, add 9 μl of ammonia water and 10 mg of EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain conjugate 3A-1 (crude product, about 1.7 mg), and then HPLC purification to obtain conjugate 3A-1 (MS m / z: [MH] - :calcd.16299.4826Found

[0387] 16299.417).

[0388] Step 4p: Preparation of conjugate 3A: Take an equivalent amount of conjugate 3A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 3A.

[0389] Example 5: Preparation of conjugate 4A (prepared according to Scheme 4a):

[0390]

[0391]

[0392] Step 5a: Preparation of compound 4a-2: Under nitrogen protection, a mixture of compound 4a-1 (10 g, 62 mmol, 1.0 eq.), compound 2b-10 (17 g, 68 mmol, 1.1 eq.) and N,N-diisopropylethylamine (18 g, 136 mmol, 2.2 eq.) in acetonitrile (100 ml) was stirred at room temperature overnight. Acetonitrile was removed under vacuum, 1 mol hydrochloric acid solution and methyl tert-butyl ether were added, the liquids were separated, and the organic layer was washed with water and saturated brine. The organic phase was dried and concentrated under vacuum to obtain a yellow oily product 4a-2 (18.5 g, yield: 99.4%). LCMS (ESI): m / z=301[M+H] + .

[0393] Step 5b: Preparation of compound 4a-3: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (28 g, 67.6 mmol, 1.1 eq.) was added to a mixture of compound 4a-2 (18.5 g, 61.5 mmol, 1.0 eq.), tert-butyl 3-(2-aminoethoxy)propionate (11.6 g, 61.5 mmol, 1.0 eq.) and N,N-diisopropylethylamine (17.4 g, 135.2 mmol, 2.0 eq.) in N,N-dimethylformamide (180 ml). The mixture was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1 to dichloromethane / methanol = 20 / 1) to obtain the product 4a-3 (14.8 g, yield: 51.4%) as a white solid. LCMS (ESI): m / z = 472 [M+H] + .

[0394] Step 5c: Preparation of compound 4a-4: A mixture of compound 4a-3 (8.86 g, 20.7 mmol, 1.0 eq.) in trifluoroacetic acid / dichloromethane (10 / 50 ml) was stirred at room temperature for 5.0 hours. The mixture was concentrated under vacuum to give a yellow oily product 4a-4 (8 g, crude product). LCMS (ESI): m / z=416 [M+H] + .

[0395] Step 5d: Preparation of compound 4a-5: To a mixture of compound 4a-4 (8.86 g, 18.7 mmol, 1.0 eq), (3R, 5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol (7.8 g, 18.7 mmol, 1.0 eq) and N,N-diisopropylethylamine (9.6 g, 75 mmol, 4.0 eq) in N,N-dimethylformamide (90 ml) was added 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (8.1 g, 19.7 mmol, 1.1 eq). The mixture was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / methanol = 50 / 50 / 1 to 30 / 30 / 1) to give the product 4a-5 (7.5 g, yield: 49%) as a white solid. LCMS (ESI): m / z = 817 [M+H] + .

[0396] Step 5e: Preparation of compound 4a-6: A mixture of compound 4a-5 (7.5 g, 9.19 mmol, 1.0 eq) and lithium hydroxide monohydrate (1.1 g, 27.5 mmol, 3.0 eq) in tetrahydrofuran / methanol / water (60 / 30 / 30 ml) was stirred at room temperature overnight. The mixture was diluted with water and washed with methyl tert-butyl ether. The aqueous phase was extracted with dichloromethane. The organic phase was concentrated under vacuum to give a pale white solid product 4a-6 (4.68 g, yield: 63.4%) LCMS (ESI): m / z=801 [MH] - .

[0397] Step 5f: Preparation of compound 4a-7 (compound 53): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (148 mg, 0.39 mmol, 1.2 eq.) was added to a mixture of compound 1a-14 (570 mg, 0.32 mmol, 1.0 eq.), compound 4a-6 (260 mg, 0.32 mmol, 1.0 eq.) and N,N-diisopropylethylamine (146 mg, 1.13 mmol, 3.5 eq.) in N,N-dimethylformamide (2.5 ml). The mixture was stirred at room temperature overnight. The mixture was purified by preparative HPLC (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 30%-90% within 30 minutes) to obtain a white solid product 4a-7 (compound 53) (245 mg, yield: 31%). LCMS(ESI): m / z=1065.4[(M-302) / 2+H] + .

[0398] Step 5g: Preparation of compound 4a-8: Succinic anhydride (60.5 mg, 0.60 mmol, 6.0 eq.) was added to a mixture of compound 4a-7 (245 mg, 0.10 mmol, 1.0 eq.), triethylamine (121 mg, 1.2 mmol, 12.0 eq.) and 4-dimethylaminopyridine (12 mg, 0.10 mmol, 1.0 eq.) in dichloromethane (2 ml). The mixture was stirred at room temperature for 20 hours. The mixture was diluted with dichloromethane (10 ml), then washed with saturated sodium bicarbonate solution (10 ml x 2) and saturated brine (10 ml x 1), dried over anhydrous sodium sulfate and concentrated to give a white solid product 4a-8 (275 mg, crude). LCMS (ESI): m / z = 1115.4 [(M-302) / 2+H] + .

[0399] Step 5h: Preparation of compound 4a-9: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (57 mg, 0.15 mmol, 1.5 eq.) and 1-hydroxybenzotriazole (20 mg, 0.15 mmol, 1.5 eq.) were added to a mixture of compound 4a-8 (253 mg, 0.10 mmol, 1.0 eq.) and N,N-diisopropylethylamine (77 mg, 0.60 mmol, 6.0 eq.) in N,N-dimethylformamide (10 ml). The mixture was shaken at room temperature for 5 minutes. CPG-NH 2(0.167 mmol / g, 1.27 g, 0.21 mmol, 2.1 eq) and the mixture was shaken at room temperature for 22 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (12 ml / 4 ml) and shaken at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour to give 4a-9 (1.36 g, loading: 30 μmol / g) as a white solid.

[0400] Step 5i: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 4a-10-A-ssRNA-1 (MS m / z: [MH] - :calcd.9067.5135Found9066.8616).

[0401] Step 5j: Preparation of conjugate 4A-1: 2a-10-A-ssRNA-2 (293 nmol, 1 equivalent) and 4a-10-A-ssRNA-1 (322 nmol, 1.1 equivalent) were added in sequence to 86 μl PBS buffer (pH 8.0, 0.5 M), 189 μl DEPC water, 429 μl DMF, and 161 μl premixed CuSO 4 -THPTA (1:1) solution (conc. 40 nmol / μl), mixed well, added 2.6 mg sodium ascorbate, replaced with nitrogen, and reacted at 35°C for 2 h. After the reaction was complete, 25 μl of ammonia water and 26 mg EDTA were added, shaken for 5-10 minutes, and ethanol precipitation was performed to obtain about 2.6 mg of the crude product of conjugate 4A-1, which was then purified by HPLC to obtain conjugate 4A-1 (MS m / z: [MH] - :calcd.16385.4886Found16385.4753).

[0402] Step 5k: Preparation of conjugate 4A: Take an equivalent amount of conjugate 4A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 4A.

[0403] Example 6: Preparation of conjugate 6A (prepared according to Scheme 5a):

[0404]

[0405]

[0406] Step 6a: Preparation of compound 5a-1: Under nitrogen protection, a mixture of tert-butyl (2-(2-bromoethoxy)ethyl)carbamate (9.0 g, 33.58 mmol, 1.00 eq), benzylamine (1.80 g, 15.99 mmol, 0.5 eq) and potassium carbonate (13.9 g, 100.75 mmol, 3.0 eq) in acetonitrile (60 ml) was stirred at 75°C overnight. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / acetone = 10 / 10 / 1) to give a yellow oil 5a-1 (6.28 g, yield: 77.43%). LCMS (ESI): m / z = 482 [M+H] + .

[0407] Step 6b: Preparation of compound 5a-2: Under hydrogen balloon pressure, a mixture of compound 5a-1 (6.28 g, 13.03 mmol, 1.0 eq.) and palladium hydroxide / carbon (1.26 g, 20% mass fraction) in ethanol (50 ml) was stirred at room temperature overnight. The mixture was filtered and the filtrate was concentrated to obtain product 5a-2 (crude product). LCMS (ESI): m / z=392 [M+H] + .

[0408] Step 6c: Preparation of compound 5a-3: Under nitrogen protection, a mixture of compound 5a-2 (crude product), benzyl chloroformate (2.67 g, 15.63 mmol, 1.2 eq.) and sodium carbonate (2.07 g, 19.54 mmol, 1.5 eq.) in tetrahydrofuran / water (30 ml / 15 ml) was stirred at room temperature for three hours. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to give a colorless oily product 5a-3 (5.4 g, yield: 78.83%). LCMS (ESI): m / z = 526 [M+H] + .

[0409] Step 6d: Preparation of compound 5a-4: A mixture of compound 5a-3 (5.4 g, 10.27 mmol, 1.0 eq.) in hydrogen chloride-dioxane (40 ml) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step without purification (crude product). LCMS (ESI): m / z=326 [M+H] + .

[0410] Step 6e: Preparation of compound 5a-5: Compound 5a-4 (2.8 g, 7.05 mmol, 1.0 eq.), compound 1a-10 (4.17 g, 7.05 mmol, 1.0 eq.), N,N-diisopropylethylamine (4.55 g, 35.26 mmol, 5.0 eq.) in dichloromethane (50 ml) were added dropwise to a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (5.90 g, 15.52 mmol, 2.2 eq.) at 0°C over 30 minutes. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure. The residue was diluted with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 20 / 1) to obtain a white solid product 5a-5 (1.54 g, yield: 24.84%). LCMS (ESI): m / z = 880 [M+H] + .

[0411] Step 6f: Preparation of compound 5a-6: A mixture of compound 5a-5 (1.54 g, 1.75 mmol, 1.0 eq.) in hydrogen chloride-dioxane (10 ml) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step without purification (crude product). LCMS (ESI): m / z=580 [M+H] + .

[0412] Step 6g: Preparation of compound 5a-7: A mixture of compound 5a-6 (crude), N,N-diisopropylethylamine (1.8 g, 14.0 mmol, 8.0 eq), compound 1a-12 (2.35 g, 5.25 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.13 g, 5.60 mmol, 3.2 eq) in dichloromethane (20 ml) was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / methanol = 10 / 10 / 2 to dichloromethane / methanol / triethylamine = 30 / 1 / 0.2) to give a yellow solid product 5a-7 (1.9 g, yield: 58.28%). LCMS (ESI): m / z = 935 [M / 2+H] + .

[0413] Step 6h: Preparation of compound 5a-8: Under hydrogen balloon pressure, a mixture of compound 5a-7 (1.9 g, 1.02 mmol, 1.0 eq), palladium hydroxide / carbon (380 g, 20% mass fraction) and trifluoroacetic acid (116 mg, 1.02 mmol, 1.0 eq) in isopropanol (20 ml) was stirred at room temperature overnight. The mixture was filtered through celite. The filter cake was washed with ethanol and the filtrate was concentrated. The residue was used directly in the next step without further purification (2.08 g, crude product). LCMS (ESI): m / z=868[M / 2+H] + .

[0414] Step 6i: Preparation of compound 5a-9 (compound 51): A mixture of compound 5a-8 (792 mg, 0.46 mmol, 1.0 eq), compound 4a-6 (367 mg, 0.46 mmol, 1.0 eq), N,N-diisopropylethylamine (177 mg, 1.37 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (209 mg, 0.55 mmol, 1.2 eq) in N,N-dimethylformamide (10 ml) was stirred at room temperature for 1 hour. The mixture was purified by preparative high performance liquid chromatography (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 30%-90% within 30 minutes) to obtain a white solid product 5a-9 (compound 51) (210 mg, yield: 18.26%). LCMS(ESI): m / z=1109[(M-302) / 2+H] + .

[0415] Step 6j: Preparation of compound 5a-10: A mixture of compound 5a-9 (210 mg, 0.083 mmol, 1.0 eq), succinic anhydride (51 mg, 0.50 mmol, 6.0 eq), triethylamine (101 mg, 1.00 mmol, 12.0 eq) and 4-dimethylaminopyridine (2 mg, 0.008 mmol, 0.1 eq) in dichloromethane (5 ml) was stirred at room temperature overnight. The mixture was diluted with dichloromethane (10 ml) and washed with 10% sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a white solid product 5a-10 (210 mg, crude). LCMS (ESI): m / z=1159[(M-302) / 2+H] + .

[0416] Step 6k: Preparation of compound 5a-11: To a solution of compound 5a-10 (210 mg, 0.08 mmol, 1.0 eq) in N,N-dimethylformamide (8 ml) were added 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (36 mg, 0.096 mmol, 1.2 eq), 1-hydroxybenzotriazole (16 mg, 0.11 mmol, 1.4 eq) and N,N-diisopropylethylamine (42 mg, 0.32 mmol, 4.0 eq), and the mixture was shaken at room temperature for 5 min. CPG-NH 2 (850 mg), shake at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (6 ml / 2 ml) and shaken at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane, and dried under vacuum for 1 hour to obtain a light yellow solid product 5a-11 (990 mg, loading: 40 μmol / g).

[0417] Step 61: Preparation of compound 5a-12-A-ssRNA-1: Compound 5a-11 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 5a-12-A-ssRNA-1 (MS m / z: [MH] - :calcd.9155.6195Found9155.8439).

[0418] Step 6m: Preparation of conjugate 6A-1: 2a-10-A-ssRNA-2 (200 nmol, 1 equivalent) and 5a-12-A-ssRNA-1 (220 nmol, 1.1 equivalent) were added in sequence with 59 μl PBS buffer (pH 8.0, 0.5 M), 257 μl DEPC water, 293 μl DMF, and 73 μl premixed CuSO 4 -THPTA (1:1) solution (conc. 60 nmol / μl), mixed well, added 1.7 mg sodium ascorbate, replaced with nitrogen, and reacted at 35°C for 2 h. After the reaction was complete, 17 μl of ammonia water and 18 mg EDTA were added, shaken for 5-10 minutes, and ethanol precipitation was performed to obtain about 2.2 mg of the crude product of conjugate 6A-1, which was then purified by HPLC to obtain conjugate 6A-1 (MS m / z: [MH] - :calcd.16473.5946Found16471.1600).

[0419] Step 6n: Preparation of conjugate 6A: Take an equivalent amount of conjugate 6A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 6A.

[0420] Example 7: Preparation of conjugate 7A (prepared according to Scheme 6a):

[0421]

[0422]

[0423] Step 7a: Preparation of compound 6a-1: To a mixture of methyl piperidine-4-carboxylate (2.0 g, 13.97 mmol, 1.0 eq), 3-(tert-butyloxycarbonyl)amino)propionic acid (2.77 g, 14.67 mmol, 1.05 eq) and N,N-diisopropylethylamine (3.6 g, 27.94 mmol, 2.0 eq) in dichloromethane (15 ml) was added 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.11 g, 16.07 mmol, 1.15 eq). The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium carbonate solution (50 ml) and then extracted with ethyl acetate (30 ml x 2). The combined organic layer was washed with saturated brine (30 mL x 1), dried over anhydrous sodium sulfate and concentrated to obtain brown oil 6a-1 (8.64 g, crude product). LCMS (ESI): m / z = 315 [m+H] + .

[0424] Step 7b: Preparation of compound 6a-2: A mixture of compound 6a-1 (4.39 g, 13.97 mmol, 1.0 eq) in hydrogen chloride-dioxane (4 m, 17 ml) was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (40 ml). Hept-6-ynoic acid (1.76 g, 13.97 mmol, 1.0 eq), N,N-diisopropylethylamine (5.4 g, 41.91 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.84 g, 15.37 mmol, 1.1 eq) were added. The mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The mixture was diluted with saturated sodium carbonate solution (50 ml) and then extracted with ethyl acetate (30 ml x 2). The combined organic layer was washed with saturated brine (30 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (dichloromethane: methanol = 60: 1) to obtain a light yellow oil 6a-2 (4.5 g, yield: 100%). LCMS (ESI): m / z = 323 [M+H] + .

[0425] Step 7c: Preparation of compound 6a-3: To a mixture of compound 6a-2 (4.5 g, 13.97 mmol, 1.0 eq.) in methanol (7.5 ml) and water (7.5 ml) was added sodium hydroxide (0.84 g, 20.96 mmol, 1.5 eq.). The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. A 2 mol / L dilute hydrochloric acid solution was added to adjust the pH to 3, and then extracted with ethyl acetate (20 ml x 5). The combined organic layer was washed with saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated to give a light yellow oil 6a-3 (4.32 g, crude product). LCMS (ESI): m / z = 309 [M+H] + .

[0426] Step 7d: Preparation of compound 6a-4: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (400 mg, 1.05 mmol, 1.2 eq.) was added to a mixture of compound 2a-6 (415 mg, 1.35 mmol, 1.52 eq.), compound 6a-3 (500 mg, 0.886 mmol, 1.0 eq.) and N,N-diisopropylethylamine (286 mg, 2.22 mmol, 2.5 eq.) in N,N-dimethylformamide (3 ml). The mixture was stirred at room temperature for 1 hour. The mixture was purified by preparative HPLC (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 30%-90% within 30 minutes) to obtain a white solid product compound 6a-4 (363 mg, yield: 48%). LCMS (ESI): m / z = 877.7 [M+Na] + .

[0427] Step 7e: Preparation of compound 6a-5: Succinic anhydride (255 mg, 2.55 mmol, 6.0 eq.) was added to a mixture of compound 6a-4 (363 mg, 0.43 mmol, 1.0 eq.), triethylamine (515 mg, 5.1 mmol, 12.0 eq.) and 4-dimethylaminopyridine (26 mg, 0.21 mmol, 0.5 eq.) in dichloromethane (3 ml). The mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane (15 ml), then washed with saturated sodium bicarbonate solution (15 ml x 2) and saturated brine (15 ml x 1), dried over anhydrous sodium sulfate and concentrated to give a brown solid product 6a-5 (448 mg, crude product). LCMS (ESI): m / z = 978 [M+Na] + .

[0428] Step 7f: Preparation of compound 6a-6: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (125 mg, 0.33 mmol, 1.5 eq.) and 1-hydroxybenzotriazole (45 mg, 0.33 mmol, 1.5 eq.) were added to a mixture of compound 6a-5 (210 mg, 0.22 mmol, 1.0 eq.) and N,N-diisopropylethylamine (170 mg, 1.32 mmol, 6.0 eq.) in N,N-dimethylformamide (20 ml). The mixture was shaken at room temperature for 5 minutes. CPG-NH 2(2.37 g, 0.40 mmol, 1.8 eq.). The mixture was shaken at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (20 ml / 6 ml) and shaken at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour to give a light yellow solid product 6a-6 (2.48 g, loading: 67 μmol / g).

[0429] Step 7g: Preparation of compound 6a-7-A-ssRNA-2: Compound 6a-6 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 6a-7-A-ssRNA-2 (MS m / z: [MH] - :calcd.7252.9070Found7252.1811).

[0430] Step 7h: Preparation of conjugate 7A-1: 6a-7-A-ssRNA-2 (200 nmol, 1 equivalent) and 2b-15-A-ssRNA-1 (220 nmol, 1.1 equivalent) were added in sequence with 59 μl PBS buffer (pH 8.0, 0.5 M), 397 μl DEPC water, 293 μl DMF, and 73 μl premixed CuSO 4 -THPTA (1:1) solution (conc. 60 nmol / μl), mixed well, added 1.7 mg sodium ascorbate, replaced with nitrogen, and reacted at 35°C for 2 h. After the reaction was complete, 17 μl of ammonia water and 18 mg EDTA were added, shaken for 5-10 minutes, and ethanol precipitation was performed to obtain about 2.0 mg of the crude product of conjugate 7A-1, which was then purified by HPLC to obtain conjugate 7A-1 (MS m / z: [MH] - :calcd.16234.4146Found16234.1400).

[0431] Step 7i: Preparation of conjugate 7A: Take an equivalent amount of conjugate 7A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 7A.

[0432] Example 8: Preparation of conjugate 8A (prepared according to Scheme 7a):

[0433]

[0434]

[0435] Step 8a: Preparation of compound 7a-1: To a mixture of tert-butyl piperazine-1-carboxylate (2.0 g, 10.74 mmol, 1.0 eq.) and N,N-diisopropylethylamine (2.08 g, 16.11 mmol, 1.5 eq.) in dichloromethane (15 ml) was added dropwise 4-chloro-4-oxobutyric acid methyl ester (1.38 ml, 11.28 mmol, 1.05 eq.) at 0°C. The mixture was stirred at 0°C for 20 minutes. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium carbonate solution (30 ml), and then extracted with ethyl acetate (30 ml x 2). The combined organic layer was washed with saturated brine (30 ml x 1), dried over anhydrous sodium sulfate and concentrated to give a brown oily product 7a-1 (3.19 g, yield: 99%). LCMS (ESI): m / z=301[M+H] + .

[0436] Step 8b: Preparation of compound 7a-2: A mixture of compound 7a-1 (3.19 g, 10.60 mmol, 1.0 eq) in hydrogen chloride-dioxane (4 mol / L, 12 ml) was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (30 ml). 2,2-dimethyl-4-oxo-3,8,11-trioxo-5-azatridecane-13-acid (2.93 g, 11.13 mmol, 1.05 eq), N,N-diisopropylethylamine (4.31 g, 33.39 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.64 g, 12.19 mmol, 1.15 eq) were added. The mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The mixture was diluted with saturated sodium carbonate solution (50 mL), and then extracted with ethyl acetate (30 mL × 2). The combined organic layer was washed with saturated brine (30 mL × 1), dried over anhydrous sodium sulfate and concentrated to give a brown oily product 7a-2 (7.57 g, crude). LCMS (ESI): m / z = 447 [M+H] + .

[0437] Step 8c: Preparation of compound 7a-3: A mixture of compound 7a-2 (4.73 g, 10.60 mmol, 1.0 eq) in hydrogen chloride-dioxane (4 mol / L, 12 ml) was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (30 ml). Hept-6-ynoic acid (1.34 g, 10.60 mmol, 1.0 eq), N,N-diisopropylethylamine (4.1 g, 31.8 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.43 g, 11.66 mmol, 1.1 eq) were added. The mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The mixture was diluted with saturated sodium carbonate solution (50 ml) and then extracted with ethyl acetate (30 ml x 2). The combined organic layer was washed with saturated brine (30 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane: methanol = 40: 1) to obtain a brown oily product 7a-3 (3.72 g, yield: 77%). LCMS (ESI): m / z = 455 [M+H] + .

[0438] Step 8d: Preparation of compound 7a-4: To a mixture of compound 7a-3 (3.72 g, 8.19 mmol, 1.0 eq.) in methanol (7.5 ml) and water (7.5 ml) was added sodium hydroxide (0.49 g, 12.29 mmol, 1.5 eq.). The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. A 2 mol / L dilute hydrochloric acid solution was added to adjust the pH to 3, and then extracted with ethyl acetate (20 ml x 5). The combined organic layers were washed with saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated to give a light yellow oil 7a-4 (3.65 g, crude product). LCMS (ESI): m / z = 441 [M+H] + .

[0439] Step 8e: Preparation of compound 7a-5: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (411 mg, 1.08 mmol, 1.2 eq.) was added to a mixture of compound 7a-4 (600 mg, 1.36 mmol, 1.52 eq.), compound 2a-6 (510 mg, 0.90 mmol, 1.0 eq.) and N,N-diisopropylethylamine (290 mg, 2.25 mmol, 2.5 eq.) in N,N-dimethylformamide (3 ml). The mixture was stirred at room temperature for 1 hour. The mixture was purified by preparative HPLC (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 30%-90% within 30 minutes) to obtain a white solid product 7a-5 (201 mg, yield: 23%). LCMS(ESI): m / z=684.5[M-302+H] + .

[0440] Step 8f: Preparation of compound 7a-6: Succinic anhydride (123 mg, 1.22 mmol, 6.0 eq.) was added to a mixture of compound 7a-5 (201 mg, 0.204 mmol, 1.0 eq.), triethylamine (247 mg, 2.45 mmol, 12.0 eq.) and 4-dimethylaminopyridine (12 mg, 0.102 mmol, 0.5 eq.) in dichloromethane (2 ml). The mixture was stirred at room temperature for 4 hours. The mixture was diluted with dichloromethane (15 ml), then washed with saturated sodium bicarbonate solution (15 ml x 2) and saturated brine (15 ml x 1), dried over anhydrous sodium sulfate and concentrated to give 7a-6 (243 mg, crude) as a white solid. LCMS (ESI): m / z = 784.6 [M-302+H] + .

[0441] Step 8g: Preparation of compound 7a-7: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (116 mg, 0.31 mmol, 1.5 eq.) and 1-hydroxybenzotriazole (41 mg, 0.31 mmol, 1.5 eq.) were added to a mixture of compound 7a-6 (222 mg, 0.20 mmol, 1.0 eq.) and N,N-diisopropylethylamine (158 mg, 1.22 mmol, 6.0 eq.) in N,N-dimethylformamide (20 ml). The mixture was shaken at room temperature for 5 minutes. CPG-NH 2(2.20 g, 0.37 mmol, 1.8 eq.). The mixture was shaken at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (20 ml / 6 ml) and shaken at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour to give a light yellow solid product 7a-7 (2.34 g, loading: 62 μmol / g).

[0442] Step 8h: Preparation of compound 7a-8-A-ssRNA-2: Compound 7a-7 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 7a-8-A-ssRNA-2 (MS m / z: [MH] - :calcd.7384.0380Found7383.3414).

[0443] Step 8i: Preparation of conjugate 8A-1: 7a-8-A-ssRNA-2 (200 nmol, 1 equivalent) and 2b-15-a-ssRNA-1 (220 nmol, 1.1 equivalent) were added in sequence with 59 μl PBS buffer (pH 8.0, 0.5 M), 290 μl DEPC water, 293 μl DMF, and 73 μl premixed CuSO 4 -THPTA (1:1) solution (conc. 60 nmol / μl), mixed well, added 1.7 mg sodium ascorbate, replaced with nitrogen, and reacted at 35°C for 2 h. After the reaction was complete, 17 μl of ammonia water and 18 mg EDTA were added, shaken for 5-10 minutes, and ethanol precipitation was performed to obtain about 2.1 mg of the crude product of conjugate 8A-1, which was then purified by HPLC to obtain conjugate 8A-1 (MS m / z: [MH] - :calcd.16365.5456Found16365.2100).

[0444] Step 8j: Preparation of conjugate 8A: Take an equivalent amount of conjugate 8A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 8A.

[0445] Example 9: Preparation of conjugate 9A (prepared according to Scheme 8a):

[0446]

[0447]

[0448] Step 9a: Preparation of compound 8a-2: A mixture of compound 8a-1 (2.72 g, 18.99 mmol, 1.0 eq), 4-piperidinyl carboxylate (2.72 g, 18.99 mmol, 1.0 eq), N,N-diisopropylethylamine (4.90 g, 37.98 mmol, 2.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.47 g, 22.28 mmol, 1.2 eq) in dichloromethane (50 ml) was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a yellow oily product 8a-2 (12.7 g, crude product). LCMS (ESI): m / z=389 [M+H] + .

[0449] Step 9b: Preparation of compound 8a-3: A solution of compound 8a-2 (crude) in hydrogen chloride-dioxane (40 ml) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step without purification (crude). LCMS (ESI): m / z=289 [M+H] + .

[0450] Step 9c: Preparation of compound 8a-4: A mixture of compound 8a-3 (crude), hept-6-ynoic acid (2.35 g, 18.69 mmol, 1.0 eq), N,N-diisopropylethylamine (7.23 g, 56.06 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.52 g, 22.42 mmol, 1.2 eq) in dichloromethane (50 ml) was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate to dichloromethane / methanol = 20 / 1) to give a yellow oily product 8a-4 (8.19 g, crude). LCMS (ESI): m / z=397 [M+H] + .

[0451] Step 9d: Preparation of compound 8a-5: A mixture of compound 8a-4 (8.19 g, 18.69 mmol, 1.0 eq) and sodium hydroxide (1.24 g, 30.94 mmol, 1.5 eq) in tetrahydrofuran / methanol / water (20 ml / 10 ml / 10 ml) was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, diluted with water, adjusted to pH = 3 with dilute hydrochloric acid and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the yellow oily product 8a-5 (6.0 g, crude product). LCMS (ESI): m / z = 383 [M+H] + .

[0452] Step 9e: Preparation of compound 8a-6: A mixture of compound 8a-5 (1.1 g, 2.87 mmol, 1.0 eq), compound 2a-6 (1.22 g, 2.15 mmol, 0.75 eq), N,N-diisopropylethylamine (1.11 g, 8.62 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.31 g, 3.45 mmol, 1.2 eq) in N,N-dimethylformamide (10 ml) was stirred at room temperature for 1 hour. The mixture was purified by preparative HPLC (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 30%-90% within 30 minutes) to obtain a white solid product 8a-6 (320 mg, yield: 16%). LCMS(ESI): m / z=627[(M-302)+H] + .

[0453] Step 9f: Preparation of compound 8a-7: A mixture of compound 8a-6 (320 mg, 0.34 mmol, 1.0 eq), succinic anhydride (207 mg, 2.07 mmol, 6.0 eq), triethylamine (418 mg, 4.14 mmol, 12.0 eq) and 4-dimethylaminopyridine (5 mg, 0.034 mmol, 0.1 eq) in dichloromethane (5 ml) was stirred overnight at room temperature. The mixture was diluted with dichloromethane and washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo to give a yellow solid product 8a-7 (380 mg, crude product). LCMS (ESI): m / z=727[(M-302)+H] + . 1H NMR(500MHz,MeOD)δ7.42-7.12(m,9H),6.93-6.71(m,4H),5.55-5.28(m,1H),4.40(dd,J=44.2,7.2 Hz,2H),4.32-4.16(m,3H),3.91-3.82(m,2H),3.77(s,6H),3.72-3.60(m,8H),3.54(td,J=13.4,6.2 Hz,6H),3.35(dd,J=14.8,9.4Hz,4H),3.18-3.05(m,6H),3.02-2.82(m,1H),2.64-2.41(m,6H),2.39 -2.24(m,1H),2.17(dtd,J=17.2,12.8,5.6Hz,5H),1.81-1.60(m,5H),1.51(dt,J=14.4,7.0Hz,3H).

[0454] Step 9g: Preparation of compound 8a-8: To a solution of compound 8a-7 (380 mg, 0.37 mmol, 1.0 eq) in N,N-dimethylformamide (10 ml) were added 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (169 mg, 0.44 mmol, 1.2 eq), 1-hydroxybenzotriazole (70 mg, 0.52 mmol, 1.4 eq) and N,N-diisopropylethylamine (191 mg, 1.48 mmol, 4.0 eq), and the mixture was shaken at room temperature for 5 minutes. CPG-NH 2 (1.9 g) and shaken at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (7.5 ml / 2.5 ml) and shaken at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to obtain a light yellow solid product 8a-8 (2.08 g, loading: 101 μmol / g).

[0455] Step 9h: Preparation of compound 8a-9-A-ssRNA-2: Compound 8a-8 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 8a-9-A-ssRNA-2 (MS m / z: [MH] - :calcd.7326.9860Found7326.2315).

[0456] Step 9i: Preparation of conjugate 9A-1: 8a-9-A-ssRNA-2 (200 nmol, 1 equivalent) and 2b-15-A-ssRNA-1 (220 nmol, 1.1 equivalent) were added in sequence with 59 μl PBS buffer (pH 8.0, 0.5 M), 590 μl DEPC water, 293 μl DMF, and 73 μl premixed CuSO 4 -THPTA (1:1) solution (conc. 60 nmol / μl), mixed well, added 1.7 mg sodium ascorbate, replaced with nitrogen, and reacted at 35°C for 2 h. After the reaction was complete, 17 μl of ammonia water and 18 mg EDTA were added, shaken for 5-10 minutes, and ethanol precipitation was performed to obtain about 2.1 mg of the crude product of conjugate 9A-1, which was then purified by HPLC to obtain conjugate 9A-1 (MS m / z: [MH] - :calcd.16308.4936Found 16308.1200).

[0458] Step 9j: Preparation of conjugate 9A: Take an equivalent amount of conjugate 9A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 9A.

[0459] Example 10: Preparation of conjugate 5A (prepared according to Scheme 9a):

[0460]

[0461]

[0462] Step 10a: Preparation of compound 9a-1: A mixture of compound 1a-8 (8.15 g, 15.32 mmol, 1.0 eq.) and hydrogen chloride-dioxane (4M, 60 ml,) was stirred at room temperature for 2 days. The solvent was removed under reduced pressure. The residue was dissolved in a mixed solvent of tetrahydrofuran (100 ml) and water (100 ml). Sodium carbonate (9.7 g, 91.92 mmol, 6.0 eq.) and di-tert-butyl carbonate (20.06 g, 91.92 mmol, 6.0 eq.) were added. The mixture was stirred at room temperature for 2 days. Tetrahydrofuran was removed under reduced pressure. The residue was diluted with water (200 ml) and then washed with dichloromethane (60 ml x 4). 2N dilute hydrochloric acid solution was added to the aqueous layer to adjust pH = 3, and then extracted with ethyl acetate (60 ml x 4). The combined organic layer was washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate and concentrated to obtain white solid compound 9a-1 (7.08 g, yield: 89%). LCMS (ESI): m / z 520 [M+H] +; TLC: Rf0.4 (dichloromethane:methanol=10:1).

[0463] Step 10b: Preparation of compound 9a-2: Compound 9a-1 (4.0 g, 6.76 mmol, 1.0 eq.), compound 1a-4 (2.1 g, 6.76 mmol, 1.0 eq.) and N,N-diisopropylethylamine (5.23 g, 40.56 mmol, 6.0 eq.) in dichloromethane (70 ml) were added dropwise to a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (6.99 g, 16.9 mmol, 2.5 eq.) and dichloromethane (600 ml) at 0°C over 30 min. The mixture was warmed to room temperature and stirred for 20 min. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium carbonate solution (50 ml) and then extracted with ethyl acetate (30 ml x 3). The combined organic layer was washed with saturated brine (30 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (ethyl acetate: methanol 50: 1 to 30: 1) to obtain a light yellow solid compound 9a-2 (4.88 g, yield: 100%). LCMS (ESI): m / z 721.7 [M+H] + ; TLC: Rf0.5 (ethyl acetate:dichloromethane=1:1).

[0464] Step 10c: Preparation of compound 9a-3: A mixture of compound 9a-2 (4.88 g, 6.76 mmol, 1.0 eq.) and hydrogen chloride-dioxane (4M, 25 ml,) was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (50 ml). 3-(tert-butyloxycarbonyl)amino)propionic acid (3.84 g, 20.28 mmol, 3.0 eq.), N,N-diisopropylethylamine (6.98 g, 54.08 mmol, 8.0 eq.) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.7 g, 20.28 mmol, 3.0 eq.) were added. The mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The mixture was diluted with saturated sodium carbonate solution (60 ml) and then extracted with ethyl acetate (30 ml x 3). The combined organic layer was washed with saturated brine (30 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (dichloromethane: methanol 40: 1) to obtain a light yellow solid compound 9a-3 (2.19 g, yield: 35%). LCMS (ESI): m / z 934.9 [M+H] + ; TLC: Rf0.5 (dichloromethane:methanol=20:1).

[0465] Step 10d: Preparation of compound 9a-4: A mixture of compound 9a-3 (2.19 g, 2.34 mmol, 1.0 eq.) and hydrogen chloride-dioxane (4M, 20 ml,) was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (100 ml). Compound 1a-12 (3.15 g, 7.03 mmol, 3.0 eq.), N,N-diisopropylethylamine (2.41 g, 18.72 mmol, 8.0 eq.) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.85 g, 7.49 mmol, 3.2 eq.) were added. The mixture was stirred at room temperature for 1 hour. The mixture was washed with saturated sodium bicarbonate solution (40 ml x 2) and saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane: methanol: triethylamine 30: 1: 0.2) to obtain a pale yellow solid compound 9a-4 (3.5 g, yield: 78%). LCMS (ESI): m / z 1922.6 [M+H] + ; TLC: Rf 0.5 (dichloromethane:methanol=10:1).

[0466] Step 10e: Preparation of compound 9a-5: Palladium hydroxide / carbon (10%, 0.7 g) was added to a mixture of compound 9a-4 (3.5 g, 1.82 mmol, 1.0 eq), trifluoroacetic acid (0.14 ml, 1.82 mmol, 1.0 eq), isopropanol (50 ml) and ethanol (50 ml). The mixture was stirred at room temperature for 2 days under hydrogen balloon pressure. The mixture was filtered. The filtrate was concentrated under reduced pressure to give white solid compound 9a-5 (3.48 g, crude product). LCMS (ESI): m / z 1790.4 [M+H] + ; TLC: Rf0.4 (dichloromethane:methanol=10:1).

[0467] Step 10f: Preparation of compound 9a-6 (compound 52): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol, 1.5 eq.) was added to a mixture of compound 9a-5 (735 mg, 0.39 mmol, 1.25 eq.), compound 4a-6 (250 mg, 0.312 mmol, 1.0 eq.), N,N-diisopropylethylamine (240 mg, 1.86 mmol, 6.0 eq.) and N,N-dimethylformamide (2 ml). The mixture was stirred at room temperature overnight. The mixture was purified by preparative liquid chromatography (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 30%-90% within 30 minutes) to obtain white solid compound 9a-6 (199 mg, yield: 25%). LCMS(ESI): m / z 1136.5[(M-302) / 2+H] + ; TLC: Rf0.5 (dichloromethane:methanol=10:1).

[0468] Step 10g: Preparation of compound 9a-7: Succinic anhydride (46 mg, 0.462 mmol, 6.0 eq.) was added to a mixture of compound 9a-6 (199 mg, 0.077 mmol, 1.0 eq.), triethylamine (93 mg, 0.92 mmol, 12.0 eq.), 4-dimethylaminopyridine (9 mg, 0.077 mmol, 1.0 eq.) and dichloromethane (2 ml). The mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane (10 ml), then washed with saturated sodium bicarbonate solution (10 ml × 2) and saturated brine (10 ml, × 1), dried over anhydrous sodium sulfate and concentrated to give white solid compound 9a-7 (212 mg, crude product). LCMS (ESI): m / z 1186.5 [(M-302) / 2+H] + ; TLC: Rf0.4 (dichloromethane:methanol=10:1).

[0469] Step 10h: Preparation of compound 9a-8: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (44 mg, 0.116 mmol, 1.5 eq.) and 1-hydroxybenzotriazole (16 mg, 0.116 mmol, 1.5 eq.) were added to a mixture of compound 9a-7 (206 mg, 0.077 mmol, 1.0 eq.), N,N-diisopropylethylamine (60 mg, 0.462 mmol, 6.0 eq.) and N,N-dimethylformamide (8 ml). The mixture was stirred at room temperature for 5 minutes. CPG-NH 2(0.167 mmol / g, 1.0 g, 0.167 mmol, 2.2 eq.). The mixture was stirred at room temperature for 22 hours. The mixture was filtered. The solid was washed with dichloromethane (8 ml x 5). Pyridine (6 ml) and acetic anhydride (2 ml) were added. The mixture was stirred at room temperature for 3 hours. The mixture was filtered. The solid was washed with dichloromethane (8 ml x 5) and dried under vacuum to give white solid compound 9a-8 (1.0 g, loading: 35 μmol / g, yield: 43%).

[0470] Step 10i: Preparation of compound 9a-9-A-ssRNA-1: Compound 9a-8 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 9a-9-A-ssRNA-1 (MS m / z: [MH]-: calcd 9209.6715.Found9208.8616).

[0471] Step 10j: Preparation of conjugate 5A-1: 53 μl of PBS buffer (pH 8.0, 0.5 M), 259 μl of DEPC water, 267 μl of DMF, and 286 μl of premixed CuSO were added to a mixture of compounds 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 9a-9-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain the crude product of conjugate 5A-1, about 2.3 mg, which is then purified by HPLC to obtain conjugate 5A-1 (MS m / z: [MH]-: calcd.16536.6576Found 16536.1842).

[0472] Step 10k: Preparation of conjugate 5A: Take an equivalent amount of conjugate 5A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 5A.

[0473] Example 11: Preparation of conjugate 12A (prepared according to Scheme 10a):

[0474]

[0475]

[0476] Step 11a: Preparation of compound 10a-1: Under nitrogen protection, ethyl chloroformate (11.26 g, 103.74 mmol, 3.5 eq) was added dropwise to a mixture of compound 2b-5 (10.0 g, 29.64 mmol, 1.0 eq), triethylamine (10.8 g, 106.7 mmol, 3.6 eq) and 160 ml of dry tetrahydrofuran at -5°C. The mixture was stirred at 0°C for 1 hour. 160 ml of water was added, and sodium borohydride (2.25 g, 59.2 mmol, 2.0 eq) was added in batches at 0°C. 80 ml of methanol was added and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (1.4 g, 22.47 mmol, 1.5 eq) was added. The mixture was stirred at 0°C for 1.5 hours. The mixture was extracted with ethyl acetate and washed with water and saturated brine. The organic phase was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 2) to obtain yellow oily compound 10a-1 (5.53 g, yield: 57.6%). LCMS (ESI): m / z = 324 (M+H) + .

[0477] Step 11b: Preparation of compound 10a-2: To a solution of compound 10a-1 (5.1 g, 17.1 mmol, 1.0 eq.) in dimethyl sulfoxide (50 ml) was added 2-iodobenzoic acid (5.73 g, 20.47 mmol, 1.2 eq.) at room temperature. The mixture was stirred overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was concentrated to dryness under reduced pressure to obtain yellow oily compound 10a-2 (5.11 g, crude product). LCMS (ESI): m / z=322 (M+H) + .

[0478] Step 11c: Preparation of compound 10a-4: A solution of benzyl (2-aminoethyl)carbamate (10.16 g, 52.3 mmol, 0.95 eq) in N,N-dimethylformamide was added to a solution of compound 10a-3 (25 g, 55.0 mmol, 1.0 eq), N,N-diisopropylethylamine (10.64 g, 82.5 mmol, 1.5 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (27.3 g, 66.0 mmol, 1.2 eq) in N,N-dimethylformamide (160 ml), and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and the solid was collected by filtration. The solid was dried under reduced pressure to give a yellow solid compound 10a-4 (37.97 g, crude product). LCMS (ESI): m / z=631 (M+H) + .

[0479] Step 11d: Preparation of compound 10a-5: Compound 10a-4 (38 g, 60.23 mmol, 1.0 eq.) and 30 ml of diethylamine were added to 600 ml of acetonitrile. The mixture was stirred at room temperature for 6 hours. The mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1, triethylamine was added) to obtain yellow oily compound 10a-5 (16.7 g, yield: 67.8%). LCMS (ESI): m / z = 409 (M+H) + .

[0480] Step 11e: Preparation of compound 10a-6: Compound 10a-5 (6.4 g, 15.58 mmol, 1.0 eq), compound 10a-2 (5.0 mg, 15.58 mmol, 1.0 eq), acetic acid (935 mg, 15.58 mmol, 1.0 eq) and sodium cyanoborohydride (2.94 g, 46.74 mmol, 3.0 eq) were added to 75 ml of methanol. The mixture was stirred at room temperature overnight, concentrated under reduced pressure, sodium carbonate solution was added, and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1, triethylamine was added) to give white solid compound 10a-6 (2.35 g, yield: 20.8%). LCMS (ESI): m / z=714.5 (M+H) + .

[0481] Step 11f: Preparation of compound 10a-7: Compound 10a-6 (2.3 g, 3.22 mmol, 1.0 eq), 3-(tert-butyloxycarbonyl)amino)propionic acid (700 mg, 3.70 mmol, 1.15 eq), N,N-diisopropylethylamine (831 mg, 6.44 mmol, 2.0 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (1.53 g, 3.70 mmol, 1.15 eq) were added to 20 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1, triethylamine was added) to obtain yellow oil solid compound 10a-7 (2.2 g, yield: 77.2%). LCMS (ESI): m / z = 885.5 (M+H) + .

[0482] Step 11g: Preparation of compound 10a-8: Compound 10a-7 (2.2 g, 2.26 mmol, 1.0 eq) was added to 25 ml of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain yellow solid compound 10a-8 (2.7 g, crude product). LCMS (ESI): m / z=585.5 (M+H) + .

[0483] Step 11h: Preparation of compound 10a-9: Compound 10a-8 (2.7 g, 2.26 mmol, 1.0 eq), compound 1a-12 (3.24 g, 7.23 mol, 3.2 eq), N,N-diisopropylethylamine (2.92 g, 22.6 mmol, 10.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.75 g, 7.23 mmol, 3.2 eq) were added to 40 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 15 / 1, triethylamine was added) to obtain white solid compound 10a-9 (2.46 g, yield: 58.1%). LCMS (ESI): m / z = 937.7 (M / 2+H) + .

[0484] Step 11i: Preparation of compound 10a-10: Compound 10a-9 (2.46 g, 1.31 mmol, 1.0 eq) was dissolved in 50 ml of ethanol, and trifluoroacetic acid (150 mg, 1.31 mmol, 1.0 eq) and 300 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a white solid compound 10a-10 (2.31 g, crude product). LCMS (ESI): m / z=825 (M / 2+H) + .

[0485] Step 11j: Preparation of compound 10a-11 (compound 60): Compound 10a-10 (2.31 g, 1.31 mmol, 1.0 eq), N,N-diisopropylethylamine (507 mg, 3.93 mmol, 3.0 eq) and compound 2b-10 (383 mg, 1.51 mmol, 1.15 eq) were added to 20 ml of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then, N,N-diisopropylethylamine (254 mg, 1.96 mmol, 1.5 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (662 mg, 1.60 mmol, 1.22 eq) and intermediate M (963 mg, 1.60 mmol, 1.22 eq) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine, and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to obtain white solid compound 10a-11 (1.81 g, yield: 58.1%). LCMS (ESI): m / z = 1036 ((M-302) / 2+H) + .

[0486] Step 11k: Preparation of compound 10a-12: Under nitrogen protection, compound 10a-11 (300 mg, 0.126 mmol, 1.0 eq), triethylamine (154 mg, 1.52 mmol, 12.0 eq), succinic anhydride (76 mg, 0.76 mmol, 6.0 eq) and p-dimethylaminopyridine (1.5 mg, 0.0126 mmol, 0.1 eq) were added to 1.5 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane / acetonitrile, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound 10a-12 (350 mg, crude) as a white solid. LCMS (ESI): m / z=1086 ((M-302) / 2+H) + .

[0487] Step 111: Preparation of compound 10a-13: Compound 10a-9 (350 mg, 0.126 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (64.8 mg, 0.170 mmol, 1.35 eq), 1-hydroxybenzotriazole (27 mg, 0.20 mmol, 1.6 eq) and N,N-diisopropylethylamine (74 mg, 0.57 mmol, 4.5 eq) were added to 16 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 1.7 g of CPG-NH2 was added, and the mixture was shaken at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 12 ml of pyridine and 4 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. The solid phase was filtered, washed with dichloromethane and acetonitrile, and dried under reduced pressure to obtain yellow solid compound 10a-13 (32.1 μmol / g).

[0488] Step 11m: Preparation of compound 10a-14-A-ssRNA-1: Compound 10a-13 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 10a-14-A-ssRNA-1 (MS m / z: [MH]-: calcd.9009.5615Found9008.8216).

[0489] Step 11n: Preparation of conjugate 12A-1: 53 μl PBS buffer (pH 8.0, 0.5 M), 259 μl DEPC water, 267 μl DMF, and 286 μl premixed CuSO were added to a mixture of compounds 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 10a-14-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 12A-1, which is then purified by HPLC to obtain conjugate 12A-1 (MS m / z: [MH]-: calcd.16336.5476Found16336.1821).

[0490] Step 11o: Preparation of conjugate 12A: Take an equivalent amount of conjugate 12A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 12A.

[0491] Example 12: Preparation of conjugate 11A (prepared according to the route of Scheme 11a):

[0492]

[0493]

[0494] Step 12a: Preparation of compound 11a-1: Compound 10a-6 (3.03 g, 5.49 mmol, 1.0 eq), compound 2b-5 (2.01 g, 6.04 mmol, 1.1 eq), N,N-diisopropylethylamine (1.42 g, 10.98 mmol, 2.0 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (2.5 g, 6.04 mmol, 1.1 eq) were added to 15 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, triethylamine was added) to obtain white solid compound 11a-1 (4.75 g, yield: 99.37%). LCMS (ESI): m / z = 871 (M+H) + .

[0495] Step 12b: Preparation of compound 11a-2: Compound 11a-1 (4.75 g, 5.49 mmol, 1.0 eq.) was added to 20 ml of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain yellow solid compound 11a-2 (4.85 g, crude product). LCMS (ESI): m / z=571 (M+H) + .

[0496] Step 12c: Preparation of compound 11a-3: Compound 11a-2 (4.85 g, 5.49 mmol, 1.0 eq), compound 1a-12 (7.6 g, 17.02 mmol, 3.1 eq), N,N-diisopropylethylamine (7.08 g, 54.9 mmol, 10.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.67 g, 17.57 mmol, 3.2 eq) were added to 30 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, triethylamine was added) to obtain white solid compound 11a-3 (6.92 g, yield: 67.84%). LCMS (ESI): m / z = 930.6 (M / 2+H) + .

[0497] Step 12d: Preparation of compound 11a-4: Compound 11a-3 (2 g, 1.07 mmol, 1.0 eq) was dissolved in 10 ml of ethanol, and trifluoroacetic acid (123 mg, 1.07 mmol, 1.0 eq) and 200 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a yellow solid compound 11a-4 (1.77 g, crude product). LCMS (ESI): m / z=1635 (M+H) + .

[0498] Step 12e: Preparation of compound 11a-5 (compound 59): Compound 11a-4 (1.77 g, 1.07 mmol, 1.0 eq), N,N-diisopropylethylamine (417 mg, 3.23 mmol, 3 eq) and compound 2b-10 (302 mg, 1.18 mmol, 1.1 eq) were added to 10 ml of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then N,N-diisopropylethylamine (417 mg, 3.23 mmol, 3 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (535 mg, 1.29 mmol, 1.2 eq) and intermediate M (778 mg, 1.29 mmol, 1.2 eq) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to obtain white solid compound 11a-5 (1.5 g, yield: 58.82%). LCMS (ESI): m / z=1029 ((M-302) / 2+H) +.1H NMR (500MHz, DMSO) δ7.85(dt,J=23.3,19.9Hz,9H),7.31(p,J=8.0Hz,4H),7.20(dt,J=11.3,6.5Hz,5H),6.91-6.79(m,4H),5.21(d,J=2.7Hz, 3H),5.03-4.88(m,4H),4.49(d,J=8.4Hz,3H),4.39(d,J=4.2Hz,1H),4.15(s,3H),4.02(s,9H),3.87(q,J=9.6Hz,3H),3.78-3.64(m,9H),3.4 1(s,3H),3.29(d,J=6.9Hz,2H),3.26-2.96(m,14H),2.50-2.47(m,3H),2.31-2.18(m,3H),2.11(d,J=12.1Hz,13H),2.05(dd,J=13.6,6.5Hz, 5H),1.99(s,10H),1.89(s,9H),1.87-1.81(m,2H),1.77(s,9H),1.72( s,1H),1.55-1.41(m,19H),1.36(d,J=6.4Hz,4H),1.31-1.16(m,14H).

[0499] Step 12f: Preparation of compound 11a-6: Compound 11a-5 (200 mg, 0.09 mmol, 1.0 eq), triethylamine (108 mg, 1.08 mmol, 12.0 eq), succinic anhydride (54 mg, 0.54 mmol, 6.0 eq) and p-dimethylaminopyridine (1 mg, 0.009 mmol, 0.1 eq) were added to 2 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound 11a-6 (229 mg, crude) as a white solid. LCMS (ESI): m / z=1079 ((M-302) / 2+H) + .

[0500] Step 12g: Preparation of compound 11a-7: Compound 11a-6 (229 mg, 0.09 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41 mg, 0.11 mmol, 1.2 eq), 1-hydroxybenzotriazole (17 mg, 0.13 mmol, 1.4 eq) and N,N-diisopropylethylamine (46 mg, 0.36 mmol, 4.0 eq) were added to 10 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 1.14 g of CPG-NH2 was added, and the mixture was shaken at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 9 ml of pyridine and 3 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. Filter, wash the solid phase with dichloromethane and acetonitrile, and dry the solid phase under reduced pressure to obtain yellow solid compound 11a-7 (1.2 g, loading: 41.7 μmol / g).

[0501] Step 12m: Preparation of compound 11A-8-A-ssRNA-1: Compound 11a-7 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 11A-8-A-ssRNA-1 (MS m / z: [MH]-: calcd.8995.5345Found8997.8616).

[0502] Step 12n: Preparation of conjugate 11A-1: 53 μl of PBS buffer (pH 8.0, 0.5 M), 259 μl of DEPC water, 267 μl of DMF, and 286 μl of premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 11A-8-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 11A-1, which is then purified by HPLC to obtain conjugate 11A-1 (MS m / z: [MH]-: calcd.16322.5206Found16322.1033).

[0503] Step 12o: Preparation of conjugate 11A: Take an equivalent amount of conjugate 11A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 11A.

[0504] Example 13: Preparation of conjugate 10A (prepared according to Scheme 12a):

[0505]

[0506]

[0507] Step 13a: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 4a-10-A-ssRNA-1 (MS m / z: [MH] - :calcd.Found).

[0508] Step 13b: Preparation of conjugate 10A-1: 53 μl PBS buffer (pH 8.0, 0.5 M), 259 μl DEPC water, 267 μl DMF, and 286 μl premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 4a-10-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 10A-1, which is then purified by HPLC to obtain conjugate 10A-1 (MS m / z: [MH]-: calcd.16394.4996Found16394.0447).

[0509] Step 13c: Preparation of conjugate 10A: Take an equivalent amount of conjugate 10A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 10A.

[0510] Example 14: Preparation of conjugate 13A (prepared according to the route of Scheme 13a):

[0511]

[0512]

[0513] Step 14a: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 4a-10-A-ssRNA-1 (MS m / z: [MH]-: calcd. Found).

[0514] Step 14b: Preparation of conjugate 13A-1: 53 μl PBS buffer (pH 8.0, 0.5 M), 259 μl DEPC water, 267 μl DMF, and 286 μl premixed CuSO were added to a mixture of compound 6a-7-A-ssRNA-2 (182 nmol, 1 eq.) and 4a-10-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 13A-1, which is then purified by HPLC to obtain conjugate 13A-1 (MS m / z: [MH]-: calcd.16320.4206Found16319.8622).

[0515] Step 14c: Preparation of conjugate 13A: Take an equivalent amount of conjugate 13A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 13A.

[0516] Example 15: Preparation of conjugate 79A (prepared according to Scheme 14a):

[0517]

[0518]

[0519] Step 15a: Preparation of compound 14a-1: A mixture of compound 2b-7 (2.6 g, 3.0 mmol, 1.0 eq), 2-(2-((tert-butyloxycarbonyl)amino)ethoxy)acetic acid (2 g, 9.4 mmol, 3.1 eq), N,N-diisopropylethylamine (4 g, 30.4 mmol, 10.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.7 g, 9.7 mmol, 3.2 eq) in acetonitrile (30 ml) was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel (eluent: petroleum ether / ethyl acetate = 1 / 2, 0.5% triethylamine) to obtain white solid compound 14a-1 (2.08 g, yield: 59.4%). LCMS (ESI): m / z = 1161.5 [M+H] + .

[0520] Step 15b: Preparation of compound 14a-2: A solution of compound 14a-1 (2.08 g, 1.79 mmol, 1.0 eq.) in hydrogen chloride-dioxane (5 ml) was stirred at room temperature for 1 hour. The mixture was concentrated to obtain crude compound 14a-2. LCMS (ESI): m / z = 860.7 [M+H] + .

[0521] Step 15c: Preparation of compound 14a-3: A mixture of compound 14a-2 (2.08 g, 1.79 mmol, 1.0 eq), compound 1a-12 (2.5 g, 5.5 mmol, 3.1 eq), N,N-diisopropylethylamine (2.3 g, 17.9 mmol, 10.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.2 g, 5.7 mmol, 3.2 eq) in acetonitrile (20 ml) was stirred at room temperature for 1 hour. The mixture was diluted with water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified with silica gel (eluent: dichloromethane / methanol = 50 / 1 to 20 / 1) to give a white solid compound 14a-3 (1.46 g, yield: 38.4%). LCMS(ESI): m / z=1075.4[M / 2+H] + .

[0522] Step 15d: Preparation of compound 14a-4: Under a hydrogen atmosphere, a mixture of compound 14a-3 (1.46 g, 3.25 mmol, 1.0 eq), palladium hydroxide / carbon (146 mg, 25% mass fraction) and trifluoroacetic acid (77.5 mg, 0.64 mmol, 1.0 eq) in ethanol (60 ml) was stirred at room temperature overnight. The mixture was filtered through celite. The filter cake was washed with ethanol and the filtrate was concentrated. The residue was used directly in the next step without purification (1.23 g, yield: 94.61%). LCMS (ESI): m / z=963[M / 2+H] + .

[0523] Step 15e: Preparation of compound 14a-5: A mixture of compound 14a-4 (1.23 g, 0.6 mmol, 1.0 eq), compound 2b-10 (186.8 mg, 0.7 mmol, 1.15 eq) and N,N-diisopropylethylamine (247.5 mg, 1.9 mmol, 3.0 eq) in acetonitrile (10 ml) was stirred at room temperature for 1 hour. The reaction was directly carried out to the next step without post-treatment. LCMS (ESI): m / z=1032.9 [M / 2+H]+.

[0524] Step 15f: Preparation of compound 14a-6 (compound 62): A mixture of crude compound 14a-5, intermediate M (469.7 mg, 0.78 mmol, 1.22 eq), N,N-diisopropylethylamine (123.7 mg, 0.96 mmol, 1.5 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (322.9 mg, 0.78 mmol, 1.22 eq) in acetonitrile (60 ml) was stirred at room temperature for 1 hour. The mixture was directly purified by HPLC (chromatographic column: UniSil 10-120C18, 30×250 mm) to obtain a white solid product 14a-6 (360 mg, yield: 21.3%). LCMS (ESI): m / z=1174[(M-302) / 2+H] + .

[0525] Step 15g: Preparation of compound 14a-7: A mixture of compound 14a-6 (330 mg, 0.12 mmol, 1.0 eq), succinic anhydride (74.8 mg, 0.75 mmol, 6.0 eq), triethylamine (151.1 mg, 1.5 mmol, 12.0 eq) and 4-dimethylaminopyridine (1.5 mg, 0.012 mmol, 0.1 eq) in dichloromethane (1.6 ml) was stirred at room temperature for 6 hours. The mixture was washed with 10% sodium bicarbonate solution and acetonitrile. The organic phase was concentrated to give a light purple solid compound 14a-7 (330 mg, crude product). LCMS (ESI): m / z=1224[(M-302) / 2+H] + .

[0526] Step 15h: Preparation of compound 14a-8: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (57.8 mg, 0.14 mmol, 1.2 eq.), 1-hydroxybenzotriazole (22.7 mg, 0.17 mmol, 1.4 eq.) and N,N-diisopropylethylamine (62 mg, 0.49 mmol, 4.0 eq.) were added to a solution of compound 14a-7 (330 mg, 0.12 mmol, 1.0 eq.) in N,N-dimethylformamide, and the mixture was shaken at room temperature for 5 minutes. 1.5 g of CPG-NH 2 The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour. The residue was added with pyridine / acetic anhydride (12 ml / 4 ml) and shaken at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour to give a light yellow solid product 14a-8 (1.7 g, loading: 23.1 μmol / g).

[0527] Step 15i: Preparation of compound 14a-9-A-ssRNA-1: Compound 14a-8 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 14a-9-A-ssRNA-1 (MS m / z: [MH]-: calcd.9284.8225Found9284.8267).

[0528] Step 15j: Preparation of conjugate 79A-1: 53 μl PBS buffer (pH 8.0, 0.5 M), 259 μl DEPC water, 267 μl DMF, and 286 μl premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 14a-9-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4-THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 79A-1, which is then purified by HPLC to obtain conjugate 79A-1 (MS m / z: [MH]-: calcd.16611.8086Found16611.1957).

[0529] Step 15k: Preparation of conjugate 79A: Take an equivalent amount of conjugate 79A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 79A.

[0530] Example 16: Preparation of conjugate 82A (prepared according to Scheme 15a):

[0531]

[0532]

[0533] Step 16a: Preparation of compound 15a-1: To a mixture of compound 10a-5 (6.0 g, 14.7 mol, 1.5 eq.), (2-bromoethyl) tert-butyl carbamate (2.2 g, 9.8 mmol, 1.0 eq.) and 30 ml of N,N-dimethylformamide, potassium carbonate (5.07 g, 36.75 mmol, 2.5 eq.) was added at room temperature and stirred overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 30 / 1 to 10 / 1, with ammonia water) to give a white solid compound 15a-1 (3.29 g, yield: 60.8%). LCMS (ESI): m / z = 552 (M+H) + .

[0534] Step 16b: Preparation of compound 15a-2: Compound 15a-1 (1.7 g, 3.08 mmol, 1.0 eq), compound 10a-2 (3.2 g, 9.97 mmol, 3.2 eq), acetic acid (185 mg, 3.08 mmol, 1.0 eq) and sodium cyanoborohydride (626 mg, 9.97 mmol, 3.2 eq) were added to 24 ml of methanol. The mixture was stirred at room temperature for 96 hours, concentrated under reduced pressure, sodium carbonate solution was added, and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1, ammonia water was added) to obtain white solid compound 15a-2 (1.72 g, yield: 54.0%). LCMS (ESI): m / z = 857.5 (M+H) + .

[0535] Step 16c: Preparation of compound 15a-3: Compound 15a-2 (1.72 g, 2.01 mmol, 1.0 eq.) was added to 20 ml of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain compound 15a-3 (1.57 g, crude product). LCMS (ESI): m / z=557.7 (M+H) + .

[0536] Step 16d: Preparation of compound 15a-4: Crude compound 15a-3 (1.57 g, 2.01 mmol, 1.0 eq), compound 1a-12 (2.87 g, 6.41 mol, 3.2 eq), N,N-diisopropylethylamine (2.58 g, 20.1 mmol, 10.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.87 g, 6.41 mmol, 3.2 eq) were added to 35 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 15 / 1, triethylamine was added) to obtain white solid compound 15a-4 (2.01 g, yield: 54.1%). LCMS (ESI): m / z = 923.7 (M / 2+H) + .

[0537] Step 16e: Preparation of compound 15a-5: Compound 15a-4 (2.0 g, 1.08 mmol, 1.0 eq) was dissolved in 45 ml of ethanol, and trifluoroacetic acid (160 mg, 1.41 mmol, 1.3 eq) and 300 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a white solid compound 15a-5 (2.0 g, crude product). LCMS (ESI): m / z=1621.5 (M+H) + .

[0538] Step 16f: Preparation of compound 15a-6 (compound 61): Compound 15a-5 (2.0 g, 1.08 mmol, 1.0 eq), N,N-diisopropylethylamine (418 mg, 3.24 mmol, 3.0 eq) and compound 2b-10 (307 mg, 1.21 mmol, 1.12 eq) were added to 15 ml of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then, N,N-diisopropylethylamine (209 mg, 1.62 mmol, 1.5 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (538 mg, 1.30 mmol, 1.2 eq) and intermediate M (783 mg, 1.30 mmol, 1.2 eq) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine, and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to obtain white solid compound 15a-6 (1.81 g, yield: 37.1%). LCMS (ESI): m / z = 1022.5 ((M-302) / 2+H) + .

[0539] Step 16g: Preparation of compound 15a-7: Under nitrogen protection, compound 15a-6 (300 mg, 0.128 mmol, 1.0 eq), triethylamine (155 mg, 1.54 mmol, 12.0 eq), succinic anhydride (77 mg, 0.768 mmol, 6.0 eq) and p-dimethylaminopyridine (1.6 mg, 0.013 mmol, 0.1 eq) were added to 1.5 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane / acetonitrile, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound 15a-7 (375 mg, crude) as a white solid. LCMS (ESI): m / z=1072 ((M-302) / 2+H) + .

[0540] Step 16h: Preparation of compound 15a-8: Compound 15a-7 (375 mg, 0.128 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (68 mg, 0.179 mmol, 1.4 eq), 1-hydroxybenzotriazole (28 mg, 0.205 mmol, 1.6 eq) and N,N-diisopropylethylamine (76 mg, 0.59 mmol, 4.6 eq) were added to 13 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 1.735 g of CPG-NH 2 The mixture was shaken at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 10 ml of pyridine and 3.4 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to obtain yellow solid compound 15a-8 (1.39 g, 37.5 μmol / g).

[0541] Step 16i: Preparation of compound 15a-9-A-ssRNA-1: Compound 15a-8 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 15a-9-A-ssRNA-1 (MS m / z: [MH]-: calcd.8981.5515Found8980.7234).

[0542] Step 16j: Preparation of conjugate 82A-1: 53 μl PBS buffer (pH 8.0, 0.5 M), 259 μl DEPC water, 267 μl DMF, and 286 μl premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 15a-9-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 82A-1, which is then purified by HPLC to obtain conjugate 82A-1 (MS m / z: [MH]-: calcd.16308.5376Found16307.9277).

[0543] Step 16k: Preparation of conjugate 82A: Take an equivalent amount of conjugate 82A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 82A.

[0544] Example 17: Preparation of conjugate 87A (prepared according to Scheme 16a):

[0545]

[0546]

[0547]

[0548] Step 17a: Preparation of compound 16a-1: Under nitrogen protection, a solution of 2,4-dinitrobenzenesulfonyl chloride (2.6 g, 9.7 mmol, 1.2 eq.) in tetrahydrofuran (10 ml) was added dropwise to a mixture of compound 2b-3 (3.2 g, 8.1 mmol, 1.0 eq.) and N,N-diisopropylethylamine (2.0 g, 16.2 mmol, 2.0 eq.) in tetrahydrofuran (40 ml) under ice bath conditions. The mixture was stirred for 1 hour under ice bath. Ethyl acetate was added to the mixture, washed with water, diluted hydrochloric acid, sodium bicarbonate aqueous solution, saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a brown solid 16a-1 (6.0 g, crude product). LCMS (ESI): m / z=582[M+H] + .

[0549] Step 17b: Preparation of compound 16a-2: Compound 16a-1 (1 g, 1.6 mmol, 1.0 eq), (tert-butyloxycarbonyl)-L-homoserine benzyl ester (1.24 g, 4.0 mmol, 2.5 eq) and triphenylphosphine (1.26 g, 4.8 mmol, 3.0 eq) were added to 30 ml of toluene and stirred at room temperature for 5 minutes. Diethyl azodicarboxylate (0.835 g, 4.8 mmol, 3.0 eq) was added dropwise under ice bath, and the mixture was stirred at room temperature for 2 hours. The mixture was filtered. The residue was dissolved in 30 ml of dichloromethane and 30 ml of petroleum ether was added. The mixture was stirred at room temperature for 1 hour. The mixture was filtered. The residue was washed with dichloromethane / petroleum ether (1 / 1). Drying gave yellow solid compound 16a-2 (1.42 g, yield: 96.59%). LCMS (ESI): m / z = 916 (M+H) + .

[0550] Step 17c: Preparation of compound 16a-3: Compound 16a-2 (1.6 g, 1.75 mmol, 1.0 eq) and triethylamine (530 mg, 5.25 mmol, 3.0 eq) were added to 10 ml of dichloromethane. 2-Mercaptoacetic acid (322 mg, 3.5 mmol, 2.0 eq) was added and the mixture was stirred at room temperature for 10 minutes. Ethyl acetate was added for extraction. The organic phase was washed with aqueous sodium carbonate solution and saturated brine. The organic phase was concentrated under reduced pressure to give compound 16a-3 (1.2 g, crude) as a yellow oil. LCMS (ESI): m / z=686 (M+H) + .

[0551] Step 17d: Preparation of compound 16a-4: Compound 16a-3 (1.2 g, 1.75 mmol, 1.0 eq), 3-(tert-butyloxycarbonyl)amino)propionic acid (331 mg, 1.75 mmol, 1.0 eq), N,N-diisopropylethylamine (451 mg, 3.5 mmol, 2.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (798 mg, 2.1 mmol, 1.2 eq) were added to 30 ml of dichloromethane, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, triethylamine was added) to obtain white solid compound 16a-4 (740 mg, yield: 49.33%). LCMS (ESI): m / z = 858 (M+H) + .

[0552] Step 17e: Preparation of compound 16a-5: Compound 16a-4 (740 mg, 0.86 mmol, 1.0 eq.) was added to 5 ml of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain yellow solid compound 16a-5 (482 mg, crude product). LCMS (ESI): m / z=557 (M+H) + .

[0553] Step 17f: Preparation of compound 16a-6: Compound 16a-5 (482 mg, 0.86 mmol, 1.0 eq), compound 1a-12 (1.2 g, 2.68 mmol, 3.1 eq), N,N-diisopropylethylamine (1.12 g, 8.64 mmol, 10.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.05 g, 2.76 mmol, 3.2 eq) were added to 10 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, triethylamine was added) to obtain white solid compound 16a-6 (879 mg, yield: 55.28%). LCMS (ESI): m / z = 1844 (M+H) + .

[0554] Step 17g: Preparation of compound 16a-7: Compound 16a-6 (879 mg, 0.476 mmol, 1.0 eq) was dissolved in 5 ml of ethanol, and trifluoroacetic acid (54 mg, 0.476 mmol, 1.0 eq) and 88 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain a white solid compound 16a-7 (800 mg, crude product). LCMS (ESI): m / z=1621 (M+H) + .

[0555] Step 17h: Preparation of compound 16a-8 (compound 65): Compound 16a-7 (800 mg, 0.476 mmol, 1.0 eq), N,N-diisopropylethylamine (184 mg, 1.428 mmol, 3 eq) and compound 2b-10 (133 mg, 0.523 mmol, 1.1 eq) were added to 10 ml of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then, N,N-diisopropylethylamine (184 mg, 1.428 mmol, 3 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (236 mg, 0.57 mmol, 1.2 eq) and intermediate M (343 mg, 0.57 mmol, 1.2 eq) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine, and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to obtain yellow solid compound 16a-8 (343 mg, yield: 30.90%).

[0556] Step 17i: Preparation of compound 16a-9: Compound 16a-8 (343 mg, 0.146 mmol, 1.0 eq), triethylamine (177 mg, 1.752 mmol, 12.0 eq), succinic anhydride (88 mg, 0.878 mmol, 6.0 eq) and p-dimethylaminopyridine (2 mg, 0.014 mmol, 0.1 eq) were added to 5 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give yellow solid compound 16a-9 (396 mg, crude product). LCMS (ESI): m / z=1072 ((M-302) / 2+H) + .

[0557] Step 17j: Preparation of compound 16a-10: Compound 16a-9 (396 mg, 0.162 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (74 mg, 0.194 mmol, 1.2 eq), 1-hydroxybenzotriazole (30 mg, 0.226 mmol, 1.4 eq) and N,N-diisopropylethylamine (84 mg, 0.648 mmol, 4.0 eq) were added to 10 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 1.58 g of CPG-NH 2 The mixture was shaken at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 9 ml of pyridine and 3 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to obtain yellow solid compound 16a-10 (1.68 g, loading: 45.7 μmol / g).

[0558] Step 17k: Preparation of compound 16a-11-A-ssRNA-1: Compound 16a-10 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 16a-11-A-ssRNA-1 (MS m / z: [MH]-: calcd.8981.5075Found8983.7126).

[0559] Step 171: Preparation of conjugate 87A-1: 53 μL PBS buffer (pH 8.0, 0.5 M), 259 μL DEPC water, 267 μL DMF, and 286 μL premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 16a-11-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4-THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 87A-1, which is then purified by HPLC to obtain conjugate 87A-1 (MS m / z: [MH]-: calcd.16308.4936Found16307.8157).

[0560] Step 17m: Preparation of conjugate 87A: Take an equivalent amount of conjugate 87A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 87A.

[0561] Example 18: Preparation of conjugate 81A (prepared according to Scheme 17a):

[0562]

[0563]

[0564]

[0565] Step 18a: Preparation of compound 17a-2: Compound 17a-1 (3.03 g, 10.0 mmol, 1.0 eq) was added to a mixture of 3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionic acid (3.43 g, 11.0 mmol, 1.1 eq), N,N-diisopropylethylamine (1.94 g, 15.0 mmol, 1.5 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.56 g, 12.0 mmol, 1.2 eq) in 80 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 2) to obtain yellow solid compound 17a-2 (6.88 g, crude product). LCMS (ESI): m / z = 597 (M+H) + .

[0566] Step 18b: Preparation of compound 17a-3: Compound 17a-2 (3.44 g, 5.76 mmol, 1.0 eq.) and 5 ml of piperidine were added to 35 ml of acetonitrile. The mixture was stirred at room temperature for 1 hour. The mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1, ammonia water was added) to obtain white solid compound 17a-3 (1.9 g, yield: 87.9%). LCMS (ESI): m / z = 375 (M+H) + .

[0567] Step 18c: Preparation of compound 17a-5: A mixture of compound 17a-4 (5.0 g, 19.1 mmol, 1.0 eq), (2-aminoethyl) benzyl carbamate (3.9 g, 20 mmol, 1.05 eq), N,N-diisopropylethylamine (4.9 g, 38.2 mmol, 2.0 eq) and 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (8.3 g, 20 mmol, 1.05 eq) in N,N-dimethylformamide (70 ml) was stirred at room temperature for 1 hour. The mixture was poured into water, extracted with ethyl acetate, and the organic phase was washed with saturated brine and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 50 / 1) to obtain white solid compound 17a-5 (7.1 g, yield: 85.5%). LCMS (ESI): m / z=438 [M+H] + .

[0568] Step 18d: Preparation of compound 17a-6: A mixture of compound 17a-5 (7.4 g, 17 mmol, 1.0 eq), lithium bromide (7.4 g, 85 mmol, 5.0 eq) and N,N-diisopropylethylamine (6.6 g, 51 mmol, 3.0 eq) in acetonitrile / water = 10 / 1 (100 ml) was stirred at room temperature for 6 hours. The mixture was filtered, the filter cake was washed with acetonitrile, dissolved in water, and the pH was adjusted to 5-6 with dilute hydrochloric acid. It was extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a white solid compound 17a-6 (6.0 g, yield: 83.8%). LCMS (ESI): m / z = 424 [M+H] + .

[0569] Step 18e: Preparation of compound 17a-7: A mixture of 17a-6 (1.6 g, 3.8 mmol, 1.0 eq), 1-hydroxypyrrolidine-2,5-dione (0.7 g, 5.7 mmol, 1.5 eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.1 g, 5.7 mmol, 1.5 eq) in dichloromethane (30 ml) was stirred overnight at room temperature. The mixture was washed with water, sodium bicarbonate aqueous solution, and saturated brine in turn, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a white solid compound 17a-7 (1.9 g, crude product). LCMS (ESI): m / z=521 [M+H] + .

[0570] Step 18f: Preparation of compound 17a-8: A mixture of compound 17a-7 (1.8 g, 3.46 mmol, 1.0 eq), (S)-2-amino-5-(benzyloxy)-5-oxopentanoic acid (0.8 g, 3.46 mmol, 1.0 eq) and N,N-diisopropylethylamine (1.3 g, 10.38 mmol, 3.0 eq) in acetonitrile (30 ml) was stirred at room temperature for 2 hours. Ethyl acetate and water were added, and dilute hydrochloric acid was added to adjust the pH to 5-6, and the layers were separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give a white solid compound 17a-8 (2.0 g, yield: 90.9%). LCMS (ESI): m / z=643[M+H] + .

[0571] Step 18g: Preparation of compound 17a-9: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (823 mg, 1.99 mmol, 1.1 eq) was added to a mixture of compound 17a-3 (678 mg, 1.81 mmol, 1.0 eq), compound 17a-8 (1.28 g, 1.99 mmol, 1.1 eq), N-methylimidazole (298 mg, 3.63 mmol, 2.0 eq) in 15 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1, ammonia water was added) to obtain yellow solid compound 17a-9 (1.12 g, yield: 61.8%). LCMS (ESI): m / z = 999.5 (M+H) + .

[0572] Step 18h: Preparation of compound 17a-10: Compound 17a-9 (1.12 g, 1.12 mmol, 1.0 eq) was added to 10 ml of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain yellow solid compound 17a-10 (854 mg, crude product). LCMS (ESI): m / z=699 (M+H) + .

[0573] Step 18i: Preparation of compound 17a-11: Compound 17a-10 (854 mg, 1.12 mmol, 1.0 eq), compound 1a-12 (1.65 g, 3.70 mol, 3.3 eq), N,N-diisopropylethylamine (1.45 g, 11.2 mmol, 10.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.41 g, 3.70 mmol, 3.3 eq) were added to 13 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 15 / 1, triethylamine was added) to obtain white solid compound 17a-11 (1.59 g, yield: 70.0%). LCMS (ESI): m / z = 994.7 (M / 2+H) + .

[0574] Step 18j: Preparation of compound 17a-12: Compound 17a-11 (1.59 g, 0.8 mmol, 1.0 eq) was dissolved in 60 ml of ethanol, and trifluoroacetic acid (100 mg, 0.88 mmol, 1.1 eq) and 230 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a white solid compound 17a-12 (2.31 g, crude product). LCMS (ESI): m / z=882.7 (M / 2+H) + .

[0575] Step 18k: Preparation of compound 17a-13 (compound 64): Compound 17a-12 (1.52 g, 0.8 mmol, 1.0 eq), N,N-diisopropylethylamine (413 mg, 3.2 mmol, 4.0 eq) and compound 2b-10 (227 mg, 0.90 mmol, 1.12 eq) were added to 10 ml of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then, N,N-diisopropylethylamine (155 mg, 1.2 mmol, 1.5 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (397 mg, 0.96 mmol, 1.2 eq) and intermediate M (578 mg, 0.96 mmol, 1.2 eq) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine, and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to obtain white solid compound 17a-13 (886 mg, yield: 44.6%). LCMS (ESI): m / z=1093 ((M-302) / 2+H) + .

[0576] Step 181: Preparation of compound 17a-14: Under nitrogen protection, compound 17a-13 (300 mg, 0.121 mmol, 1.0 eq), triethylamine (171 mg, 1.69 mmol, 14.0 eq), succinic anhydride (85 mg, 0.844 mmol, 7.0 eq) and p-dimethylaminopyridine (1.5 mg, 0.0121 mmol, 0.1 eq) were added to 2.5 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane / acetonitrile, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give white solid compound 17a-14 (269 mg, yield: 85.9%). LCMS (ESI): m / z=1143 ((M-302) / 2+H) + .

[0577] Step 18m: Preparation of compound 17a-15: Compound 17a-14 (269 mg, 0.104 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (56 mg, 0.146 mmol, 1.4 eq), 1-hydroxybenzotriazole (23 mg, 0.166 mmol, 1.6 eq) and N,N-diisopropylethylamine (62 mg, 0.478 mmol, 4.6 eq) were added to 10 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 807 mg of CPG-NH 2The mixture was shaken at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 7.5 ml of pyridine and 2.5 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to obtain yellow solid compound 17a-15 (770 mg, 43.71 μmol / g).

[0578] Step 18n: Preparation of compound 17a-16-A-ssRNA-1: Compound 17a-15 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 17a-16-A-ssRNA-1 (MS m / z: [MH]-: calcd.9123.6655Found9122.7623).

[0579] Step 18o: Preparation of conjugate 81A-1: 53 μl of PBS buffer (pH 8.0, 0.5 M), 259 μl of DEPC water, 267 μl of DMF, and 286 μl of premixed CuSO were added to a mixture of compounds 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 17a-16-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 81A-1, which is then purified by HPLC to obtain conjugate 81A-1 (MS m / z: [MH]-: calcd.16450.6516Found16450.1231).

[0580] Step 18p: Preparation of conjugate 81A: Take an equivalent amount of conjugate 81A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 81A.

[0581] Example 19: Preparation of conjugate 74A (prepared according to Scheme 18a):

[0582]

[0583]

[0584] Step 19a: Preparation of compound 18a-2: Benzyl bromide (13.1 ml, 109.89 mmol, 2.5 eq.) was added to a mixture of compound 18a-1 (4.0 g, 43.96 mmol, 1.0 eq.), potassium carbonate (18.2 g, 131.88 mmol, 3.0 eq.) and ethanol (40 ml). The mixture was heated to reflux for 3 hours. The solvent was removed under reduced pressure. The residue was diluted with water (60 ml) and then extracted with dichloromethane (50 ml x 2). The combined organic layer was washed with saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (dichloromethane: methanol 80: 1) to obtain white solid compound 18a-2 (9.89 g, yield: 83%). LCMS (ESI): m / z 272 [M+H] + ; TLC: Rf0.5 (dichloromethane:methanol ester=50:1).

[0585] Step 19b: Preparation of compound 18a-3: Sodium hydroxide (15 g, 375 mmol, 15 eq.) was added to a mixture of compound 18a-2 (6.7 g, 24.72 mmol, 1.0 eq.), tert-butyl bromoacetate (28.78 g, 147.55 mmol, 6.0 eq.), tetrabutylamine hydrogensulfate (1.26 g, 3.71 mmol, 0.15 eq.), water (50 ml) and toluene (65 ml). The mixture was heated to 50°C and reacted for 40 hours. The mixture was diluted with water (50 ml) and then extracted with dichloromethane (50 ml x 2). The combined organic layer was washed with saturated brine (50 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate 50: 1) to obtain colorless oily compound 18a-3 (3.61 g, yield: 29%). LCMS (ESI): m / z 500 [M+H] + ; TLC: Rf0.5 (petroleum ether:ethyl acetate=30:1).

[0586] Step 19c: Preparation of compound 18a-4: A mixture of compound 18a-3 (3.73 g, 7.46 mmol, 1.0 eq) in hydrogen chloride-dioxane (4M, 20 ml,) was stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (40 ml). Ammonium chloride (2.39 g, 44.76 mmol, 6.0 eq), N,N-diisopropylethylamine (7.7 g, 59.68 mmol, 8.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.51 g, 22.38 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for 7 hours. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium carbonate solution (50 ml) and then extracted with ethyl acetate (20 ml x 3). The combined organic layers were washed with saturated brine (30 mL x 1), dried over anhydrous sodium sulfate and concentrated to obtain a pale yellow oily compound 18a-4 (3.5 g, crude product). LCMS (ESI): m / z 386 [M+H] + ; TLC: Rf0.5 (dichloromethane:methanol=20:1).

[0587] Step 19d: Preparation of compound 18a-5: Borane dimethyl sulfide complex (10M, 5.97 ml, 59.7 mmol, 8.0 equiv) was added to a mixture of compound 18a-4 (2.87 g, 7.46 mmol, 1.0 equiv) dissolved in tetrahydrofuran (40 ml). The mixture was heated to 65°C for 5 hours. The mixture was quenched with water (25 ml). Sodium carbonate (2.37 g, 22.38 mmol, 3.0 equiv) and di-tert-butyl dicarbonate (4.88 g, 22.38 mmol, 3.0 equiv) were added. The mixture was stirred at room temperature for 3 hours. The mixture was diluted with water (50 ml) and then extracted with ethyl acetate (30 ml, ×2). The combined organic layer was washed with saturated brine (30 ml ×1), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (petroleum ether: ethyl acetate 8: 1 to 3: 1) to obtain compound 18a-5 (741 mg, yield: 18%) as a pale yellow oil. LCMS (ESI): m / z 558 [M+H] + ; TLC: Rf0.5 (petroleum ether:ethyl acetate=5:1).

[0588] Step 19e: Preparation of compound 18a-6: To a mixture of compound 18a-5 (741 mg, 1.33 mmol, 1.0 eq.) dissolved in methanol (2 ml) was added hydrogen chloride-dioxane solution (4 M, 5 ml). The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The residue was dried under vacuum to obtain a white solid crude compound 18a-6. LCMS (ESI): m / z 358 [M+H]+ ; TLC: Rf 0.2 (dichloromethane:methanol=10:1).

[0589] Step 19f: Preparation of compound 18a-7: To a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (1.38 g, 3.33 mmol, 2.5 eq) in dichloromethane (85 ml) was added dropwise over 30 minutes of a solution of compound 1a-10 (786 mg, 1.33 mmol, 1.0 eq), compound 18a-6 (616 mg, 1.33 mmol, 1.0 eq) and N,N-diisopropylethylamine (1.03 g, 7.98 mmol, 6.0 eq) in dichloromethane (15 ml) at 0°C. The mixture was warmed to room temperature and stirred for 20 minutes. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium carbonate solution (30 ml) and then extracted with ethyl acetate (20 ml x 3). The combined organic layer was washed with saturated brine (20 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (ethyl acetate: methanol 50: 1 to 30: 1) to obtain white solid compound 18a-7 (0.42 g, yield: 35%). LCMS (ESI): m / z 912.8 [M+H] + ; TLC: Rf0.3 (ethyl acetate:dichloromethane=1:1).

[0590] Step 19g: Preparation of compound 18a-8: Compound 18a-7 (0.42 g, 0.46 mmol, 1.0 eq) was dissolved in a mixture of hydrogen chloride-dioxane (4M, 4 ml,) and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (10 ml). Compound 1a-12 (618 mg, 1.38 mmol, 3.0 eq), N,N-diisopropylethylamine (475 mg, 3.68 mmol, 8.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (525 mg, 1.38 mmol, 3.0 eq) were added. The mixture was stirred at room temperature for 1 hour. The mixture was diluted with dichloromethane (20 ml), washed with saturated sodium bicarbonate solution (20 ml x 2) and saturated brine (20 ml x 1), dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (dichloromethane: methanol: triethylamine 30: 1: 0.3) to give white solid compound 18a-8 (778 mg, yield: 89%). LCMS (ESI): m / z 1902.5 [M+H] + ; TLC: Rf0.5 (dichloromethane:methanol=10:1).

[0591] Step 19h: Preparation of compound 18a-9: Palladium hydroxide / carbon (10%, 0.5 g) was added to a mixture of compound 18a-8 (778 mg, 0.409 mmol, 1.0 eq.) and trifluoroacetic acid (47 mg, 0.409 mmol, 1.0 eq.) in methanol (10 ml) and ethanol (5 ml). The mixture was stirred at room temperature overnight under hydrogen balloon pressure. The mixture was filtered. The filtrate was concentrated under reduced pressure to give a pale yellow solid compound 18a-9 (600 mg, crude product). LCMS (ESI): m / z 1721.5 [M+H] + ; TLC: Rf0.3 (dichloromethane:methanol=10:1).

[0592] Step 19i: Preparation of compound 18a-10 (compound 16): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (188 mg, 0.495 mmol, 1.5 eq.) was added to a mixture of compound 18a-9 (600 mg, 0.33 mmol, 1.0 eq.), compound 4a-6 (265 mg, 0.33 mmol, 1.0 eq.), N,N-diisopropylethylamine (255 mg, 1.98 mmol, 6.0 eq.) and N,N-dimethylformamide (2 ml). The mixture was stirred at room temperature for 0.5 hours. The mixture was purified by preparative liquid chromatography (A: pH = 7 phosphate buffer, B: acetonitrile, acetonitrile concentration 30%-90% within 30 minutes) to obtain white solid compound 18a-10 (180 mg, yield: 22%). LCMS(ESI): m / z1102.6[(M-302) / 2+H] + ; TLC: Rf0.5 (dichloromethane:methanol=10:1).

[0593] Step 19j: Preparation of compound 18a-11: Succinic anhydride (43 mg, 0.43 mmol, 6.0 eq.) was added to a solution of compound 18a-10 (180 mg, 0.072 mmol, 1.0 eq.), triethylamine (87 mg, 0.862 mmol, 12.0 eq.) and 4-dimethylaminopyridine (4.4 mg, 0.036 mmol, 0.5 eq.) in dichloromethane (2 ml). The mixture was stirred at room temperature for 20 hours. The mixture was diluted with dichloromethane (10 ml), then washed with saturated sodium bicarbonate solution (10 ml x 2) and saturated brine (10 ml x 1), dried over anhydrous sodium sulfate and concentrated to give white solid compound 18a-11 (155 mg, yield: 83%). LCMS (ESI): m / z 1152.8 [(M-302) / 2+H] + ; TLC: Rf 0.3 (dichloromethane:methanol=10:1).

[0594] Step 19k: Preparation of compound 18a-12: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34 mg, 0.09 mmol, 1.5 eq.) and 1-hydroxybenzotriazole (12 mg, 0.09 mmol, 1.5 eq.) were added to a mixture of compound 18a-11 (155 mg, 0.06 mmol, 1.0 eq.) and N,N-diisopropylethylamine (46 mg, 0.36 mmol, 6.0 eq.) dissolved in N,N-dimethylformamide (8 ml). The mixture was stirred at room temperature for 5 minutes. CPG-NH 2 (0.167 mmol / g, 775 mg, 0.13 mmol, 2.2 eq.). The mixture was stirred at room temperature overnight. The mixture was filtered. The solid was washed with dichloromethane (8 ml x 5). Pyridine (6 ml) and acetic anhydride (2 ml) were added. The mixture was stirred at room temperature for 3 hours. The mixture was filtered. The solid was washed with dichloromethane (8 ml x 5) and dried under vacuum to give a white solid compound 18a-12 (785 mg, 21.5 μmol / g, yield: 29%).

[0595] Step 191: Preparation of compound 18a-13-A-ssRNA-1: Compound 18a-12 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 18a-13-A-ssRNA-1 (MS m / z: [MH]-: calcd.9141.5925Found9140.7005).

[0596] Step 19m: Preparation of conjugate 74A-1: 53 μL PBS buffer (pH 8.0, 0.5 M), 259 μL DEPC water, 267 μL DMF, and 286 μL premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 18a-13-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 74A-1, which is then purified by HPLC to obtain conjugate 74A-1 (MS m / z: [MH]-: calcd.16468.5786Found16468.2769).

[0597] Step 19n: Preparation of conjugate 74A: Take an equivalent amount of conjugate 74A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 74A.

[0598] Example 20: Preparation of conjugate 44A (prepared according to Scheme 19a):

[0599]

[0600]

[0601] Step 20a: Preparation of compound 19a-2: To a mixture of compound 19a-1 (20 g, 66.00 mmol, 1.0 eq.) and N-methylmorpholine (6.68 g, 66.00 mmol, 1.0 eq.) in tetrahydrofuran (100 ml) was added dropwise isobutyl chloroformate (9.02 g, 66.00 mmol, 1.0 eq.) at -20°C. The mixture was stirred for 10 minutes. The mixture was filtered. A solution of sodium borohydride (5.02 g, 132.01 mmol, 2.0 eq.) in water (30 ml) was slowly added to the filtrate at -30°C. The mixture was warmed to 0°C and stirred for 0.5 hours. The mixture was diluted with water and then extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a colorless oily crude compound 19a-2 (20 g). LCMS (ESI): m / z = 290 [M+H] +.

[0602] Step 20b: Preparation of compound 19a-3: Under nitrogen protection, at 0°C, add iodine (26.26 g, 103.45 mmol, 1.5 eq.) to a mixture of triphenylphosphine (27.13 g, 103.45 mmol, 1.5 eq.), imidazole (7.04 g, 103.45 mmol, 1.5 eq.) and dichloromethane (200 ml). The mixture is stirred at room temperature for half an hour. Compound 19a-2 (20 g, 68.97 mmol, 1.0 eq.) is added to the mixture. The mixture is stirred at room temperature for 3 hours. The reaction solution is quenched with saturated sodium thiosulfate and extracted with dichloromethane. The combined organic layer is washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography on silica gel (petroleum ether: ethyl acetate = 50: 1 to 10: 1) to obtain a colorless oily compound 19a-3 (24.09 g, yield: 87.34%). LCMS (ESI): m / z = 422 [M+Na] +.

[0603] Step 20c: Preparation of compound 19a-4: A mixture of benzylamine (2.92 g, 27.27 mmol, 1.0 eq.), compound 19a-3 (24.09 g, 60.23 mmol, 2.2 eq.) and potassium carbonate (11.33 g, 82.13 mmol, 3.0 eq.) in acetonitrile (250 ml) was stirred at 60°C for 3 hours. The mixture was concentrated and diluted with water and extracted with ethyl acetate. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate = 50: 1 to 5: 1) to obtain yellow oily compound 19a-4 (14.14 g, yield: 36.13%). LCMS (ESI): m / z = 650 [M+H] +.

[0604] Step 20d: Preparation of compound 19a-5: To a mixture of compound 19a-4 (14.14 g, 21.75 mmol, 1.0 eq.) and N,N-diisopropylethylamine (560 mg, 4.35 mmol, 0.2 eq.) in acetonitrile (110 ml) was added dropwise 1-chloroethyl chloroformate (4.67 g, 32.63 mmol, 1.0 eq.) at 0°C. The mixture was warmed to room temperature and stirred for 2 hours. The solvent was removed under reduced pressure. The residue was diluted with tetrahydrofuran / methanol / water (45 ml / 9 ml / 6.6 ml). The mixture was heated to 65°C and stirred for 1.5 hours. The solvent was removed under reduced pressure and the residue was diluted with dichloromethane. The organic layer was washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was subjected to column chromatography on silica gel (dichloromethane: methanol = 200: 1 to 20: 1) to obtain yellow oily compound 19a-5 (8.39 g, yield: 68.88%). LCMS (ESI): m / z = 560 [M+H] +.

[0605] Step 20e: Preparation of compound 19a-6: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (6.84 g, 17.98 mmol, 1.2 eq.) was added to a mixture of compound 19a-5 (8.39 g, 14.98 mmol, 1.00 eq.), 3-(tert-butyloxycarbonyl)amino)propionic acid (2.83 g, 14.98 mmol, 1.00 eq.) and N,N-diisopropylethylamine (3.87 g, 29.96 mmol, 2.0 eq.) in dichloromethane (80 ml). The mixture was stirred at room temperature for 2.5 hours. The solvent was removed under reduced pressure. The residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate = 10: 1 to 2: 1) to give a yellow oily product 19a-6 (8.31 g, yield: 75.89%). LCMS (ESI): m / z = 731 [M+H] +.

[0606] Step 20f: Preparation of compound 19a-7: A mixture of hydrogen chloride-dioxane (4 mol / L, 80 ml) of compound 19a-6 (8.31 g, 11.37 mmol, 1.0 eq) was stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue was dissolved in a mixed solvent of tetrahydrofuran (80 ml) and water (80 ml). Sodium carbonate (3.62 g, 34.10 mmol, 6.0 eq) and di-tert-butyl carbonate (12.39 g, 56.84 mmol, 5.0 eq) were added. The mixture was stirred at room temperature overnight. Tetrahydrofuran was removed under reduced pressure. The residue was diluted with water and then washed with dichloromethane. The pH was adjusted to 3 with a 2 mol / L dilute hydrochloric acid solution and then extracted with ethyl acetate. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain a colorless solid product 19a-7 (6.0 g, yield: 85.47%). LCMS (ESI): m / z = 619 [M+H] +.

[0607] Step 20g: Preparation of compound 19a-8: To a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (3.76 g, 9.08 mmol, 2.2 eq) in dichloromethane (500 ml) was added compound 1a-4 (1.28 g, 4.13 mmol, 1.0 eq), compound 19a-7 (2.55 g, 4.13 mmol, 1.0 eq) and N,N-diisopropylethylamine (2.66 g, 20.65 mmol, 5.0 eq) in dichloromethane (50 ml) at 0°C. The mixture was stirred at room temperature for 1 hour. The solvent was concentrated under reduced pressure. The residue was diluted with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate to ethyl acetate:methanol = 20:1) to obtain a white solid product 19a-8 (1.09 g, yield: 32.25%). LCMS (ESI): m / z = 820 [M+H] + .

[0608] Step 20h: Preparation of compound 19a-9: A solution of compound 19a-8 (1.09 g, 1.33 mmol, 1.0 eq.) in hydrogen chloride-dioxane (10 ml) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step without purification (crude product). LCMS (ESI): m / z=520 [M+H] + .

[0609] Step 20i: Preparation of compound 19a-10: A mixture of crude compound 19a-9, N,N-diisopropylethylamine (1.37 g, 10.63 mmol, 8.0 eq), compound 1a-12 (1.78 g, 3.99 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.62 g, 4.25 mmol, 3.2 eq) in dichloromethane (20 ml) was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / dichloromethane / methanol = 10 / 10 / 2 to dichloromethane / methanol / triethylamine = 30 / 1 / 0.2) to give a yellow solid product 19a-10 (2.18 g, yield: 90.83%). LCMS (ESI): m / z = 905 [M / 2+H]+.

[0610] Step 20j: Preparation of compound 19a-11: Under a hydrogen atmosphere, a mixture of 19a-10 (2.18 g, 1.21 mmol, 1.0 eq), palladium hydroxide (436 g, 20% mass fraction) and trifluoroacetic acid (137 mg, 1.21 mmol, 1.0 eq) in ethanol / methanol (10 ml / 10 ml) was stirred at room temperature overnight. The mixture was filtered through celite. The filter cake was washed with methanol and the filtrate was concentrated. The residue was used directly in the next step without further purification (2.18 g, crude product). LCMS (ESI): m / z=838 [M / 2+H] + .

[0611] Step 20k: Preparation of compound 19a-12 (compound 20): A mixture of compound 19a-11 (1.0 g, 0.56 mmol, 1.0 eq), compound 4a-6 (450 mg, 0.56 mmol, 1.0 eq), N,N-diisopropylethylamine (217 mg, 1.68 mmol, 3.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (255 mg, 0.67 mmol, 1.2 eq) in dichloromethane (10 ml) was stirred for 1 hour. The mixture was separated and purified using HPLC (chromatographic column: UniSil 10-120C18, 30×250 mm) to obtain a white solid product 19a-12 (470 g, yield: 34.31%). LCMS(ESI): m / z=1079[(M-302) / 2+H] + .

[0612] Step 201: Preparation of compound 19a-13: A mixture of compound 19a-12 (470 mg, 0.19 mmol, 1.0 eq), succinic anhydride (115 mg, 1.15 mmol, 6.0 eq), triethylamine (232 mg, 2.29 mmol, 12.0 eq) and 4-dimethylaminopyridine (2.3 mg, 0.019 mmol, 0.1 eq) in dichloromethane (5 ml) was stirred at room temperature overnight. The mixture was washed with 10% sodium bicarbonate solution and extracted with dichloromethane. Acetonitrile was added to the organic phase and then washed with 10% sodium bicarbonate solution. The organic phase was concentrated to give 19a-13 as a white solid (457 mg, crude product). LCMS (ESI): m / z=1129[(M-302) / 2+H] + .

[0613] Step 20m: Preparation of compound 19a-14: To a solution of compound 19a-13 (200 mg, 0.078 mmol, 1.0 eq) in N,N-dimethylformamide (8 ml) were added 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (36 mg, 0.094 mmol, 1.2 eq), 1-hydroxybenzotriazole (15 mg, 0.11 mmol, 1.4 eq) and N,N-diisopropylethylamine (40 mg, 0.31 mmol, 4.0 eq), and the mixture was stirred at room temperature for 5 minutes. CPG-NH 2 (900 mg) and stirred at room temperature overnight. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane in turn and dried under vacuum for 1 hour. Pyridine / acetic anhydride (6 ml / 2 ml) was added to the residue and stirred at room temperature for 3 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane in turn and dried under vacuum for 1 hour to obtain a light yellow solid product 19a-14 (910 mg, loading rate: 22.27 μmol / g).

[0614] Step 20n: Preparation of compound 19a-15-A-ssRNA-1: Compound 19a-14 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 19a-15-A-ssRNA-1 (MS m / z: [MH]-: calcd.9095.6115Found9093.7784).

[0615] Step 20o: Preparation of conjugate 44A-1: 53 μL PBS buffer (pH 8.0, 0.5 M), 259 μL DEPC water, 267 μL DMF, and 286 μL premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 19a-15-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 44A-1, which is then purified by HPLC to obtain conjugate 44A-1 (MS m / z: [MH]-: calcd.16422.5536Found16421.2100).

[0616] Step 20p: Preparation of conjugate 44A: Take an equivalent amount of conjugate 44A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 44A.

[0617] Example 21: Preparation of conjugate 80A (prepared according to Scheme 20a):

[0618]

[0619]

[0620] Step 21a: Preparation of compound 20a-1: Di-tert-butyl diaminoformate (5.0 g, 16.48 mmol, 1.00 eq), ethyl 2-bromoacetate (3.1 g, 18.13 mmol, 1.1 eq) and potassium carbonate (4.5 g, 32.96 mmol, 2.0 eq) were added to N,N-dimethylformamide (40 ml) and reacted overnight at room temperature. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a yellow oily product 20a-1 (6.4 g, crude product). LCMS (ESI): m / z=390 [M+H] + .

[0621] Step 21b: Preparation of compound 20a-2: Compound 20a-1 (6.2 g, 15.94 mmol, 1.0 eq.) and lithium hydroxide monohydrate (1.6 g, 39.85 mmol, 2.5 eq.) were added to a mixed solvent of tetrahydrofuran, methanol and water (40 ml + 10 ml + 10 ml), and stirred at 40°C for 2.5 hours. Water was added to the mixture, and 2 molar dilute hydrochloric acid was added to adjust the pH to 5, and the aqueous phase was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give compound 20a-2 (5.1 g, crude product). LCMS (ESI): m / z=362[M+H] + .

[0622] Step 21c: Preparation of compound 20a-3: Compound 2b-2 (13.6 g, 22.05 mmol, 1.00 eq) was dissolved in trifluoroacetic acid / dichloromethane solution (50 ml / 100 ml) and reacted at room temperature for 1 hour. The mixture was spin-dried. The residue was added with water and sodium carbonate aqueous solution to adjust the pH to 8. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a white solid product 20a-3 (6.1 g, yield: 53.6%). LCMS (ESI): m / z=517 [M+H] + .

[0623] Step 21d: Preparation of compound 20a-4: Compound 20a-2 (4.3 g, 11.82 mmol, 1.0 eq), compound 20a-3 (6.1 g, 11.82 mmol, 1.0 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.4 g, 14.18 mmol, 1.2 eq), N,N-diisopropylethylamine (3.1 g, 23.04 mmol, 2.0 eq) were added to N,N-dimethylformamide (50 ml) and stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was stirred with dichloromethane / petroleum ether = 1:1. The mixture was filtered to obtain yellow solid compound 20a-4 (10 g, crude). LCMS (ESI): m / z=861 [M+H] + .

[0624] Step 21e: Preparation of compound 20a-5: A solution of compound 20a-4 (9.5 g, 11.05 mmol, 1.0 eq.) in diethylamine / acetonitrile (20 / 80 ml) was stirred at room temperature for 1 hour. The mixture was purified by silica gel column chromatography (eluent: dichloromethane to dichloromethane / methanol = 10 / 1) to give a yellow solid product 20a-5 (5.1 g, yield: 72.5%). LCMS (ESI): m / z = 638 [M+H]+ .

[0625] Step 21f: Preparation of compound 20a-6: To a solution of (tert-butyloxycarbonyl)-L-homoserine benzyl ester (2.3 g, 7.44 mmol, 1.0 eq.) in tetrahydrofuran (30 ml) was added Dess-Martin reagent (4.7 g, 11.17 mmol, 1.5 eq.) at 0°C. The mixture was stirred at room temperature for 2 hours. The reaction solution was quenched with saturated sodium thiosulfate solution and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a yellow oily product 20a-6 (3.5 g, crude product). LCMS (ESI): m / z=308 [M+H] + .

[0626] Step 21g: Preparation of compound 20a-7: A solution of compound 20a-5 (3.65 g, 5.72 mmol, 1.0 eq), compound 20a-6 (1.8 g, 6.01 mmol, 1.05 eq) and acetic acid (343 mg, 5.72 mmol, 1.0 eq) in methanol (50 ml) was stirred at room temperature for 30 minutes. The temperature was lowered to 0°C and sodium cyanoborohydride was added. The mixture was stirred at room temperature for 1.5 hours. The mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated sodium carbonate solution and saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane to dichloromethane / methanol = 100 / 1 to 30 / 1) to give a white solid product 20a-7 (2.65 g, yield: 49.9%). LCMS (ESI): m / z = 930 [M+H] + .

[0627] Step 21h: Preparation of compound 20a-8: Compound 20a-7 (3 g, 3.23 mmol, 1.0 eq), 3-(tert-butyloxycarbonyl)amino)propionic acid (611 mg, 3.23 mmol, 1.0 eq), N,N-diisopropylethylamine (835 mg, 6.46 mmol, 2.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 mg, 3.88 mmol, 1.2 eq) were added to dichloromethane (36 ml), and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 20 / 1, 1% triethylamine was added) to give white solid compound 20a-8 (1.5 g, yield: 42.1%). LCMS (ESI): m / z = 1101 [M+H] + .

[0628] Step 21i: Preparation of compound 20a-9: Compound 20a-8 (1.5 g, 1.36 mmol, 1.0 eq) was added to 20 ml of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain a yellow solid product 20a-9 (1.1 g, crude product). LCMS (ESI): m / z=700 [M+H] + .

[0629] Step 21j: Preparation of compound 20a-10: Compound 20a-9 (1.1 g, 1.27 mmol, 1.0 eq), compound 1a-12 (2.3 g, 5.21 mmol, 4.1 eq), N,N-diisopropylethylamine (2 g, 15.24 mmol, 12.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2 g, 5.33 mmol, 4.2 eq) were added to 15 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 10 / 1, triethylamine was added) to give white solid compound 20a-10 (1.8 g, yield: 58.6%). LCMS (ESI): m / z = 1210 [M / 2+H] + .

[0630] Step 21k: Preparation of compound 20a-11: Compound 20a-10 (800 mg, 0.331 mmol, 1.0 eq) was dissolved in 8 ml of ethanol, and trifluoroacetic acid (38 mg, 0.331 mmol, 1.0 eq) and 80 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature for two days under a hydrogen atmosphere. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a white solid compound 20a-11 (890 mg, crude product). LCMS (ESI): m / z=1098 [M / 2+H] + .

[0631] Step 211: Preparation of compound 20a-12 (compound 63): Compound 20a-11 (760 mg, 0.346 mmol, 1.0 eq), N,N-diisopropylethylamine (134 mg, 1.038 mmol, 3.0 eq) and compound 2b-10 (97 mg, 0.381 mmol, 1.1 eq) were added to 10 ml of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then, N,N-diisopropylethylamine (134 mg, 1.038 mmol, 3.0 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (171 mg, 0.415 mmol, 1.2 eq) and intermediate M (249 mg, 0.415 mmol, 1.2 eq) were added, and the mixture was stirred at room temperature for 1 hour. The solvent was removed from the mixture under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to give yellow solid compound 20a-12 (272 mg, yield: 27.2%). LCMS (ESI): m / z=1306[(M-302) / 2+H] + .

[0632] Step 21m: Preparation of compound 20a-13: Compound 20a-12 (172 mg, 0.0589 mmol, 1.0 eq), triethylamine (71 mg, 0.707 mmol, 12.0 eq), succinic anhydride (35 mg, 0.354 mmol, 6.0 eq) and p-dimethylaminopyridine (1 mg, 0.00589 mmol, 0.1 eq) were added to 1 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give yellow solid compound 20a-13 (167 mg, crude product). LCMS (ESI): m / z=1359[(M-302) / 2+H]+.

[0633] Step 21n: Preparation of compound 20a-14: Compound 20a-13 (167 mg, 0.0554 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (29 mg, 0.0775 mmol, 1.2 eq), 1-hydroxybenzotriazole (12 mg, 0.0886 mmol, 1.6 eq) and N,N-diisopropylethylamine (29 mg, 0.222 mmol, 4.0 eq) were added to 2 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 501 mg of CPG-NH 2The mixture was shaken at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 6 ml of pyridine and 2 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to obtain yellow solid compound 20a-14 (532 mg, loading: 28.6 μmol / g).

[0634] Step 21o: Preparation of compound 20a-15-A-ssRNA-1: Compound 20a-14 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 20a-15-A-ssRNA-1 (MS m / z: [MH]-: calcd.9428.0085Found9427.849).

[0635] Step 21p: Preparation of conjugate 80A-1: 53 μL PBS buffer (pH 8.0, 0.5 M), 259 μL DEPC water, 267 μL DMF, and 286 μL premixed CuSO were added to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 eq.) and 20a-15-A-ssRNA-1 (200 nmol, 1.1 eq.) in sequence. 4 -THPTA (1:1) solution (conc. 14 nmol / μl), after mixing, add 1.6 mg sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction is complete, add 15 μl of ammonia water and 16 mg EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain about 2.3 mg of the crude product of conjugate 80A-1, which is then purified by HPLC to obtain conjugate 80A-1 (MS m / z: [MH]-: calcd.16754.9946Found16753.2000).

[0636] Step 21q: Preparation of conjugate 80A: Take an equivalent amount of conjugate 80A-1 and two complementary antisense strands (1167AM25 and 11040AM5) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 80A.

[0637] Example 22: Preparation of conjugate 88A (prepared according to Scheme 21a):

[0638]

[0639]

[0640] Step 22a: Preparation of compound 21a-1: 40% aqueous methylamine solution (1.5 g, 19.23 mmol, 1.1 eq) was added to a mixture of (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)butanoic acid (7.7 g, 17.48 mmol, 1.0 eq), N,N-diisopropylethylamine (3.39 g, 26.28 mmol, 1.5 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (7.95 g, 19.23 mmol, 1.1 eq) and 85 ml of dimethylformamide, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and the solid was collected by filtration. The solid was dried under reduced pressure to obtain yellow solid compound 21a-1 (6.91 g, yield: 87.2%). LCMS (ESI): m / z=454 (M+H) + .

[0641] Step 22b: Preparation of compound 21a-2: Compound 21a-1 (6.91 g, 15.24 mmol, 1.0 eq.) and 1,8-diazabicyclo[5.4.0]undec-7-ene (4.63 g, 30.48 mmol, 2.0 eq.) were added to 125 ml of acetonitrile and 50 ml of dichloromethane. The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 to 10 / 1) to obtain yellow solid compound 21a-2 (6.2 g, crude product). LCMS (ESI): m / z = 232 (M+H) + .

[0642] Step 22c: Preparation of compound 21a-3: At 0°C, compound 21a-2 (6.2 g, 15.24 mmol, 1.0 eq.) and N,N-diisopropylethylamine (3.9 g, 30.48 mmol, 2.0 eq.) were added to 50 ml of tetrahydrofuran, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 15 / 1) to obtain yellow solid compound 21a-3 (1.1 g, yield: 15.6%). LCMS (ESI): m / z = 462 (M+H) + .

[0643] Step 22d: Preparation of compound 21a-4: Under nitrogen protection, a toluene solution of diethyl azodicarboxylate (1.13 g, 6.48 mmol, 3.0 eq) was added dropwise to a toluene (30 ml) solution of compound 21a-3 (1.0 g, 2.16 mmol, 1.0 eq), tert-butyl (2-hydroxyethyl)carbamate (975 mg, 6.05 mmol, 2.8 eq) and triphenylphosphine (1.7 g, 6.48 mmol, 3.0 eq) at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction solution was filtered. The filter cake was dissolved in dichloromethane, petroleum ether was added, filtered, and the filter cake was washed with petroleum ether. The solid was dried under reduced pressure to obtain a yellow solid compound 21a-4 (1.34 g, yield: 93.0%). LCMS (ESI): m / z=605 (M+H) + .

[0644] Step 22e: Preparation of compound 21a-5: 2-Mercaptoacetic acid (408 mg, 4.43 mmol, 2.0 eq) was added to a mixture of compound 21a-4 (1.34 g, 2.21 mmol, 1.0 eq), triethylamine (670 mg, 6.63 mmol, 3.0 eq) and 15 ml of dichloromethane, and the mixture was stirred at room temperature for 10 minutes. The mixture was diluted with ethyl acetate, washed with saturated sodium carbonate solution and saturated brine, the organic phase was concentrated under reduced pressure, and the residue was dried under reduced pressure to obtain yellow solid compound 21a-5 (1.07 g, crude product). LCMS (ESI): m / z=375 (M+H) + .

[0645] Step 22f: Preparation of compound 21a-6: Compound 21a-5 (1.07 g, 2.21 mmol, 1.0 eq), compound 2b-5 (746 mg, 2.21 mmol, 1.0 eq), N,N-diisopropylethylamine (570 mg, 4.42 mmol, 2.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (924 mg, 2.43 mmol, 1.1 eq) were added to 10 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added, and the organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 20 / 1, triethylamine was added) to obtain yellow solid compound 21a-6 (940 mg, yield: 61.4%). LCMS (ESI): m / z = 694 (M+H) + .

[0646] Step 22g: Preparation of compound 21a-7: Compound 21a-6 (940 mg, 1.35 mmol, 1.0 eq) was added to 10 ml of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain white solid compound 21a-7 (1.01 g, crude product). LCMS (ESI): m / z=394 (M+H) + .

[0647] Step 22h: Preparation of compound 21a-8: Compound 21a-7 (690 mg, 1.35 mmol, 1.0 eq), compound 1a-12 (1.94 g, 4.33 mmol, 3.2 eq), N,N-diisopropylethylamine (1.74 g, 13.5 mmol, 10.0 eq) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.65 g, 4.33 mmol, 3.2 eq) were added to 30 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 80 / 1 to 15 / 1, triethylamine was added) to obtain yellow solid compound 21a-8 (1.79 g, yield: 79.1%). LCMS (ESI): m / z = 1682 (M+H) + .

[0648] Step 22i: Preparation of compound 21a-9: Compound 21a-8 (1.79 g, 1.07 mmol, 1.0 eq) was dissolved in 640 ml of ethanol, and trifluoroacetic acid (216 mg, 1.89 mmol, 1.8 eq) and 270 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give a white solid compound 21a-9 (2.02 g, crude product). LCMS (ESI): m / z=1592.4 (M+H) + .

[0649] Step 22j: Preparation of compound 21a-10 (compound 66): A solution of intermediate M (709 mg, 1.18 mmol, 1.1 eq) in acetonitrile was added to a mixture of compound 21a-9 (2.02 g, 1.07 mmol, 1.0 eq), N,N-diisopropylethylamine (276 mg, 2.14 mmol, 2.0 eq), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (488 mg, 1.18 mmol, 1.1 eq) and 20 ml of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer solution / acetonitrile) to give a white solid compound 21a-10 (1.38 g, yield: 59.2%). LCMS(ESI): m / z=938((M-302) / 2+H) + .

[0650] Step 22k: Preparation of compound 21a-11: Under nitrogen protection, compound 21a-10 (500 mg, 0.230 mmol, 1.0 eq), triethylamine (326 mg, 3.22 mmol, 14.0 eq), succinic anhydride (161 mg, 1.61 mmol, 7.0 eq) and p-dimethylaminopyridine (3 mg, 0.023 mmol, 0.1 eq) were added to 3 ml of dichloromethane, and the mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane, washed with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give compound 21a-11 (536 mg, crude) as a white solid. LCMS (ESI): m / z=1975.7 ((M-302)+H) + .

[0651] Step 221: Preparation of compound 21a-12: Compound 21a-11 (536 mg, 0.23 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (122 mg, 0.32 mmol, 1.4 eq), 1-hydroxybenzotriazole (50 mg, 0.37 mmol, 1.6 eq) and N,N-diisopropylethylamine (137 mg, 1.06 mmol, 4.6 eq) were added to 20 ml of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 1.91 g of CPG-NH 2 The mixture was shaken at room temperature overnight. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure. 15 ml of pyridine and 5 ml of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to obtain yellow solid compound 21a-12.

[0652] Step 21m: Preparation of conjugate 88A-1: Compound 21a-12 was synthesized by solid phase oligonucleotide, aminolyzed and purified by HPLC to obtain 88A-1 (MS m / z: [MH]-: calcd.8195.9620 Found 8194.6426).

[0653] Step 21n: Preparation of conjugate 88A: Take equivalent amounts of conjugate 88A-1 and complementary antisense chain (1167AM25) solution, mix them evenly, heat them in a 95°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain a double-stranded conjugate 88A.

[0654] The oligonucleotide sequence information connected to each conjugate synthesized in the above examples is shown in Tables 1 to 3.

[0655] Table 1 Sequence composition information of each conjugate

[0656]

[0657]

[0658] Table 2 Summary of mass spectrometry information of positive chain of each conjugate

[0659]

[0660]

[0661] Table 3 Summary of sequences used in each conjugate

[0662]

[0663] The unmodified sequences corresponding to Table 3 are as follows:

[0664]

[0665]

[0666] Example 23: Testing the Effect of siRNA-Conjugates on AGT and PCSK9 Expression in Primary Hepatocytes of hAGT / hPCSK9 Mice

[0667] Integrate the human AGT (hAGT) gene or the human PCSK9 (hPCSK9) gene into the liver-targeted adeno-associated virus type 8 (AAV8) expression vector, and order the preparation of ultra-purified recombinant AAV8-hAGT and AAV8-hPCSK9 viruses from a vector manufacturer. Mix the AAV8-hAGT and AAV8-hPCSK9 viruses and dilute them with PBS to a virus titer of 7.5*10 11 / mL of virus solution was intravenously injected into 6-8 week old female mice (C57BL / 6), 0.2mL per mouse, that is, 1.5*10 11 Titer the virus. After 14 days, transgenic mice stably expressing hAGT / hPCSK9 were obtained.

[0668] Double humanized AGT / PCSK9 mice were purchased from Cynosure Biotechnology.

[0669] Extraction of mouse primary hepatocytes: Mouse hepatocytes were extracted by collagenase digestion through the inferior vena cava perfusion method. After filtering through a tissue cell filter (BIOLOGIX, 15-1070), mouse primary hepatocytes with good activity were obtained and resuspended in DMEM medium (containing 10% FBS, 1X penicillin-streptomycin). The cell density was determined using a Scepter automatic cell counter (Millipore, #PHCC00000). At the same time, 2 μL of siRNA-conjugate, 68 μL of Opti-MEM, and 5 μL of INTERFERin transfection reagent were mixed in a 96-well plate and incubated at room temperature for 10 minutes (no need to add INTERFERin for free uptake), and then 125 μL of complete medium containing mouse primary hepatocytes was added to each well.

[0670] RNA extraction and reverse transcription in 96-well plates: Oligo d(T)25 Magnetic Beads reagent (NEB) was used to extract mRNA from cells in 96-well plates. The medium in the 96-well plates was aspirated, and the plates were washed once with DPBS. 100 μl of cell lysis solution was added to each well, and then 20 μl of beads were added. The plates were shaken on an oscillator. The lysate in the wells was aspirated. 100 μl of washing buffer A was added to each well, and the plates were placed on a magnetic separation rack after being blown. Washing buffer A was aspirated, and then 100 μl of washing buffer B was used to blow up the beads, and the beads were transferred to a new 96-well plate, placed on a magnetic separation rack, and washing buffer B was aspirated. Then 100 μl of low-salt buffer was used to blow up the beads and transferred to a 96-well PCR plate. The 96-well PCR plate was placed on a magnetic separation rack, and the low-salt buffer was aspirated. 10 μL of elution buffer was added to each well to blow up the beads, and the plates were incubated at 50°C for 2 minutes to elute the mRNA from the beads. The reverse transcription system was prepared by using StarScriptPro one-tube de-genomic reverse transcription premix (genstar), and 5 μL was dispensed into each well of the 96-well PCR, and 5 μL of the mRNA solution obtained in the previous step was added and mixed, and the plate was sealed with a sealing film. Incubate at 37°C for 3 minutes, 50°C for 50 minutes, and then 85°C for 2 minutes on the PCR instrument, and cool to 4°C to end the reverse transcription.

[0671] Real-time fluorescence quantitative PCR: After reverse transcription, place the 96-well plate on a magnetic separation rack until the magnetic beads are adsorbed to the bottom, aspirate the reverse reagent, add the prepared QPCR system to the 96-well PCR plate, seal the plate with a sealing film, and perform PCR on the StepOnePlus real-time PCR system (applied biosystems).

[0672] Data were analyzed using the ΔΔCt method and normalized using cells transfected with 1 nM of the negative control sequence.

[0673] The negative control AD-1955 sequence is:

[0674] CUUACGCUGAGUACUUCGAdTdT(SEQ ID NO:13)

[0675] UCGAAGUACUCAGCGUAAGdTdT (SEQ ID NO: 14).

[0676] The primer sequences for detecting AGT are:

[0677]

[0678] The primer sequences for detecting PCSK9 are:

[0679]

[0680] The experimental results are shown in Tables 4 to 7.

[0681] Table 4 Results of single-target siRNA inhibition experiment of AGT in primary hepatocytes of dual humanized AGT / PCSK9 mice

[0682] siRNA-conjugate Compound No. IC50 value (nM) Conjugate 1A 31 0.05995 Conjugate 1B 31 0.03662

[0683] Conjugate 1A and Conjugate 1B use compound 31 to deliver siRNA targeting AGT. The results in Table 4 show that compound 31 can efficiently deliver siRNA in vitro.

[0684] Table 5 Experimental results of AGT single-target siRNA in primary hepatocytes of AAV8-hAGT / hPCSK9 mice at different concentrations by free uptake

[0685]

[0686] The results in Table 5 show that compounds 31 and 66 can efficiently deliver siRNA in vitro.

[0687] Table 6 Results of dual-targeting siRNA inhibition experiment of AGT / PCSK9 in primary hepatocytes of dual-humanized AGT / PCSK9 mice

[0688]

[0689] The experimental results in Table 6 show that in the primary hepatocytes of dual humanized AGT / PCSK9 mice, when the same sequence combination was delivered in vitro, both conjugate 2A and conjugate 3A could knock down both AGT and PCSK9 targets at the same time, and conjugate 2A had a better knockdown effect than conjugate 3A. This indicates that compound 54 has a slightly better delivery effect on dual-target siRNA than compound 55.

[0690] Table 7 Results of dual-targeting siRNA inhibition experiment of AGT / PCSK9 in primary hepatocytes of AAV8-hAGT / hPCSK9 mice

[0691]

[0692] The experimental results in Table 7 show that when the same sequence combination is delivered in vitro, different conjugates can inhibit both AGT and PCSK9 targets with high activity. Among them, conjugates 2A, 4A and 9A have the highest activity, indicating that compound 54 and compound 53 have better delivery effects on dual-target siRNA than compound 51. At the same time, by comparing the data of conjugates 2A, 7A, 8A and 9A, it is found that connecting the carrier compound and siRNA through different connecting chains also affects the delivery effect, among which the connection through Q13 and Q20 has a more obvious improvement in siRNA activity.

[0693] Example 24: Testing the Effects of Different siRNA-Conjugates on AGT Expression in AAV8-hAGT Mice (1 mg / kg)

[0694] The effects of 1167.25-19 delivered by different compounds on hAGT expression in the liver were evaluated in mice expressing AAV8-hAGT.

[0695] 1. Construction of hAGT transgenic mice

[0696] AAV8-hAGT virus was prepared with PBS to a virus titer of 5*10 11 / mL of virus solution was intravenously injected into 6-8 week old female mice (C57BL / 6), 0.2mL per mouse, that is, 1*10 11 The virus was titered and transgenic mice stably expressing hAGT were obtained 14 days later.

[0697] 2. Drug administration

[0698] 14 days after the mice were injected with the virus, the mice were evenly divided into groups, with 5 mice in each group. The siRNA-conjugate was dissolved in physiological saline and injected subcutaneously at a dose of 1 mg / kg.

[0699] 3.ELISA test

[0700] 14 days after the mice were injected with the virus, the tails were cut off and blood was collected. The samples were allowed to stand at room temperature for 30 minutes and centrifuged at 1000×g for 10 minutes. The supernatant serum was taken and stored in a -80°C refrigerator. The serum samples were diluted 1000 times, and the expression of hAGT in mouse serum was detected by Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Lianke Bio, Cat. No.: EK1202-96). Based on the expression of hAGT, the mice were divided into groups with 5 mice in each group. The siRNA-conjugate was dissolved in PBS and the concentration was adjusted to 0.2 mg / kg. The mice were subcutaneously injected with siRNA solution at a dose of 1 mg / kg.

[0701] 17 and 45 days after siRNA injection, a small amount of blood was collected by tail cutting, and the blood was allowed to stand at room temperature for 30 minutes and centrifuged at 1000×g for 10 minutes. The supernatant serum was collected and diluted 1000 times before ELISA to detect the expression of hAGT.

[0702] The reference compound 1A is a conjugate of L96* and 1167.25-19, obtained by conventional solid phase synthesis method. *: Structure of L96 (compound 117 (L96-DMTr protection) in US8106022B2):

[0703]

[0704] Table 8 Results of AGT targeted modified siRNA inhibition experiment in AAV8-hAGT mice

[0705]

[0706] The experimental results are shown in Table 8: In the in vivo experiment, the delivery effect of conjugate 1A is better than that of L96, indicating that the delivery effect of compound 31 is better than that of L96.

[0707] Example 25 Testing the Effect of Conjugate 2A on the Expression of AGT and PCSK9 in AAV8-hAGT / hPCSK9 Mice

[0708] The effect of conjugate 2A on hAGT / hPCSK9 expression was tested at a dose of 180 nmol / kg in mice expressing AAV8-hAGT / hPCSK9. The expression levels of hAGT and hPCSK9 in mouse serum after 14 days, 30 days and 44 days of administration were detected by Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Lianke Bio, Catalog No.: EK1202-96) and Human PCSK9 ELISA Kit (Proteintech, Catalog No.: KE00278). The experimental method refers to Examples 23 and 24.

[0709] Table 9 Results of dual-targeting siRNA inhibition experiment of AGT / PCSK9 at a dose of 180 nmol / kg in AAV8-hAGT / hPCSK9 mice

[0710]

[0711] As shown in Table 9, the experimental results show that the conjugate 2A targeting AGT and PCSK9 linked to compound 54 has high activity in vivo and can significantly inhibit the expression of hAGT and hPCSK9 in the serum of AAV8-hAGT / hPCSK9 mice. By day 44, the remaining amount of hPCSK9 was only 21.7% of that before administration, and the remaining amount of hAGT was only 10.8% of that before administration, indicating that the siRNA linked to compound 54 can persistently inhibit the expression of the two targets in vivo.

[0712] Example 26 Testing the Effects of Different siRNA-Conjugates on the Expression of AGT and PCSK9 in AAV8-hAGT / hPCSK9 Mice

[0713] The effects of different conjugates on the expression of AGT and PCSK9 were tested in mice expressing AAV8-hAGT / hPCSK9. Conjugate 2B was a conjugate that simultaneously delivered two siRNAs targeting AGT, and the rest were conjugates that simultaneously delivered siRNAs targeting AGT and PCSK9. The expression of hAGT and hPCSK9 in mouse serum after administration was detected by HumanAngiotensinogen / AGT / SerpinA8 ELISA Kit (Lianke Bio, Catalog No.: EK1202-96) and Human PCSK9 ELISA Kit (Proteintech, Catalog No.: KE00278). The experimental method was referred to Examples 23 and 24.

[0714] Table 10 Results of AGT single-targeting and AGT / PCSK9 dual-targeting siRNA inhibition experiments at a dose of 60 nmol / kg in AAV8-hAGT / hPCSK9 mice

[0715]

[0716] As shown in Table 10: At a dosage of 60 nmol / kg, each conjugate has a good knockdown effect on the hAGT target and the hPCSK9 target in vivo. At the same time, conjugate 2B is different from the other dual-target conjugates in that it uses a dual-target structure to deliver two identical sequences targeting AGT. It can be clearly seen that the simultaneous delivery of two sequences targeting AGT has a better knockdown effect on the AGT target. This shows that the use of conjugate 2B can not only deliver two target siRNAs, but also further increase the efficacy by simultaneously delivering two sequences with the same target.

[0717] Table 11 Results of dual-targeting siRNA inhibition experiment of AGT / PCSK9 at a dose of 180 nmol / kg in AAV8-hAGT / hPCSK9 mice

[0718]

[0719] As shown in Table 11, the activities of the two conjugates 9A and 10A were very good. The inhibition rates of both targets were above 80-90% on day 40.

[0720] Example 27: Testing the Effect of siRNA-Conjugates on AGT and PCSK9 Expression in Primary Hepatocytes of AAV8-hAGT / hPCSK9 Mice

[0721] The delivery effects of dual-targeted siRNA-conjugates with different structures were tested for in vitro activity by referring to the method of Example 23. Among them, 1000PM-L96 and 11000PM-L96 are Zilebesiran, an investigational drug targeting AGT in a Phase II clinical trial of Alnylam, and Inclisiran, a drug targeting PCSK9 that has been marketed, respectively, and are used for positive drug comparison.

[0722] Table 12 Results of dual-targeted siRNA inhibition experiments of AGT / PCSK9 in primary hepatocytes of AAV8-hAGT / hPCSK9 mice at different concentrations and different delivery methods

[0723]

[0724]

[0725] As shown in Table 12, each conjugate can efficiently deliver siRNA, and the inhibitory effects on AGT and PCSK9 targets are much better than the efficacy of Zilebesiran mixed with Inclisiran.

[0726] Example 28: Stability and in vivo activity of conjugate 9A

[0727] In this example, the compound AM26 with good activity in patent US11053495B2 and the compound A19a with good in vivo activity in patent WO2024061157A1 were used to synthesize conjugates DT03000 and DT03098, respectively, and the stability and in vivo activity were compared with the conjugate 9A of the present invention. The siRNAs linked to the three conjugates are 1167.25-19 and 11040.5-10; the sequence composition of the DT03000 linker is shown in Table 3.

[0728] The structures of conjugates DT03000 and DT03098 are as follows:

[0729]

[0730]

[0731] The stability of the three conjugates was tested in mouse serum. The experimental method is as follows:

[0732] The test conjugate was accurately diluted with DEPC water to a working concentration of 10 μM.

[0733] Preparation of 3h samples: Take 12μL of 10μM test conjugate, mix it with 108μL of blank mouse serum, and vortex mix for 60s to prepare a mixed solution of test conjugate and mouse serum (hereinafter referred to as mixed solution); take 50μL of the mixed solution in a centrifuge tube, the number of parallel samples is 2, and incubate it in a 37℃ incubator for 3h.

[0734] Preparation of 0h samples: Take 45μL blank mouse serum in a centrifuge tube, the number of parallel samples is 2, and incubate in a 37℃ incubator for 3h. After incubation, add 5μL 10μM test conjugate working solution and vortex mix for 60s.

[0735] After incubation, 100 μL of lysis buffer (containing 2.5 μg / ml internal standard) was added to each tube of the sample, vortexed for 30 s, and allowed to stand for 20 min.

[0736] All samples were treated with solid phase extraction, concentrated by nitrogen blowing, reconstituted with ultrapure water and centrifuged, the supernatant was taken for instrumental analysis and the relative residual rate of the sequence was calculated according to the following formula: relative residual rate (%) = (3h compound content / 3h internal standard content) / (0h compound content / 0h internal standard content) × 100%.

[0737] Table 13 Results of double-stranded stability of AGT / PCSK9 dual-targeted modified siRNA after 3h incubation in mouse serum

[0738] siRNA-conjugate Remaining ratio of antisense strand 1 (%) Remaining proportion of antisense strand 2 (%) Conjugate 9A 121.02 118.02 DT03098 101.90 98.04 DT03000 3.89 1.47

[0739] It is obvious from the data in Table 13 that after incubation in mouse serum for 3 hours, the stability of conjugates 9A and DT03098 was good and there was basically no degradation; while the remaining proportion of the two antisense chains of compound DT03000 was less than 5%. After analyzing its metabolites, it was found that multiple nucleotides at the 3' end of the two antisense chains were cut, and it was no longer possible to connect the two target siRNA duplexes together into a complete duplex through base complementary pairing, indicating that the conjugate will be degraded in the blood before being delivered into the liver, and its stability is very poor.

[0740] According to the stability test results, conjugate 9A and conjugate DT03098 were selected for internal activity test. The experimental method was similar to that of Examples 23 and 24.

[0741] Table 14 Results of AGT / PCSK9 dual-targeting siRNA inhibition experiment at a dose of 60 nmol / kg in AAV8-hAGT / hPCSK9 mice

[0742]

[0743] From the data in Table 14, it can be seen that in the case of delivering the same two sequences, the in vivo activity of conjugate 9A is significantly better than that of DT03098 in both targets, especially the inhibitory activity against PCSK9 target is significantly better than that of DT03098. This indicates that the conjugate 9A of the present invention has a better effect on the delivery of siRNA than DT03098.

[0744] Example 29: Testing the Effect of Conjugate 9A on the Expression of hAGT and hPCSK9 in AAV8-hAGT / hPCSK9 Mice

[0745] In mice expressing AAV8-hAGT / hPCSK9, the effect of conjugate 9A on the expression of hAGT and hPCSK9 was tested at a dose of 60 nmol / kg, where 1000PM-L96 and 11000PM-L96 were respectively Zilebesiran, an investigational drug targeting AGT in a Phase II clinical trial of Alnylam, and Inclisiran, a drug targeting PCSK9 that has been marketed, for positive drug comparison. The expression of hAGT and hPCSK9 in the serum of mice after administration was detected by Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Lianke Bio, Catalog No.: EK1202-96) and Human PCSK9 ELISA Kit (Proteintech, Catalog No.: KE00278), and the experimental method was referred to Examples 23 and 24.

[0746] The experimental results are shown in Figure 1 , Figure 2 .

[0747] As can be seen from the figure, at a dosage of 60nmol / kg, the knockdown effect of conjugate 9A on the AGT target and PCSK9 target in vivo is very good, and is much better than the efficacy of Zilebesiran mixed with Inclisiran. And from the duration of the mixed administration of the two positive drugs, it can be seen that the inhibition rate of 1000PM is still about 50% on the 56th day, while the inhibition rate of 11000PM on PCSK9 has been reduced to about 50% on the 27th day, and the duration of the inhibition of the two sequences on the target is inconsistent. The inhibitory level of the conjugate 9A of the present invention in the two targets has a basically consistent trend over time, so it is more controllable in clinical practice for the administration time and frequency.

[0748] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0749] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that: The compound has a structure shown in formula (I), formula (II) or formula (III): Wherein: the targeting binding part T is a group containing a ligand, and the ligand can specifically bind to a cell membrane protein receptor having endocytosis; W1 is selected from: a group capable of binding to an oligonucleotide drug via a covalent bond; W2 is selected from: a group capable of undergoing click chemistry reaction or hydrogen; W3 is selected from: W1 or W2, and W3 is not hydrogen; Y1 and Y2 are independently selected from: or their chiral isomers; n is an integer from 1 to 4; L1, L2, L3, L4, L5, L6, L7, L8 are independently selected from one or more of the following groups connected without violating the principle of chemical bonding: C1-C 15 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O)2-, -S(=O)-, -CH=CH-, B Alkynyl, -NRC(=O)-, -NRC(=O)NR-, -C(=O)CRR1NR-, -OC(=O)-, -SC(=O)-, -NRS(=O)2-, C6-C 10 Aryl, 3-20 membered heterocyclic group, C3-C 10 Cycloalkyl, 5-10 membered heteroaryl, the linking unit may be substituted by one or more R; R is selected from the group consisting of: H, C1-C6 alkyl, halogen, hydroxy, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, cyano, carboxyl, amino, and aminocarbonyl; R1 is selected from: H, C1-C6 alkyl, amino acid side chain; X is selected from: -O-, -S-, -NR-, -C(=O)-, 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: Y1 is selected from: or their chiral isomers; n is: 1, 2, 3 or 4; Y2 is selected from: or their chiral isomers; R in the structures of Y1 and Y2 are independently selected from: H, C1-C3 alkyl, and halogen-substituted C1-C3 alkyl.

3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: Y1 is selected from: or their chiral isomers; Y2 is selected from: or their chiral isomers.

4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: W2 is selected from: 5-20 membered heterocyclic group containing alkynyl, -SR 26 , alkynyl, azido, alkenyl, 5-20 membered heterocyclic group containing alkenyl, hydrogen; R 26 Selected from: hydrogen, a thiol protecting group.

5. The compound according to claim 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: W2 is selected from: -SR 26 , azido, hydrogen.

6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L1, L2, L3, L4, L5, L6, L7, L8 are independently selected from one or more of the following groups connected without violating the principle of chemical bonding: C1-C 12 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O)2-, -S(=O)-, -CH=CH-, ethynyl, -NRC(=O)-, -NRC(=O)NR-, -C(=O)CRR1NR-, -OC(=O)-, -SC(=O)-, -NRS(=O)2-, -P(=O)2O-, -P(=O)(=S)O-, C6-C 10 Aryl, 3-8 membered heteromonocyclic group, C3-C8 cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospirocyclic group, 5-20 membered heterocyclic group, C5-C 10 Cycloalkyl, 5-6 membered heteroaryl, the linking unit may be substituted by one or more R; R is selected from the group consisting of: H, C1-C3 alkyl, halogen, hydroxy, hydroxy-substituted C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, cyano, carboxyl, amino, and aminocarbonyl; R1 is selected from: H, C1-C3 alkyl, amino acid side chain.

7. The compound according to claim 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: Wherein L1, L2, L3, L4, L5, L6, L7, and L8 are independently selected from one or more of the following groups connected to form a connecting unit that does not violate the principle of chemical bonding: or their chiral isomers; Wherein, j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R is selected from: H, C1-C3 alkyl, halogen-substituted C1-C3 alkyl; R1 is selected from: H, C1-C3 alkyl, amino acid side chain.

8. The compound according to claim 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: Wherein L1, L2, L3, L4, L5, L6, L7, and L8 are independently selected from one or more of the following groups connected to form a connecting unit that does not violate the principle of chemical bonding: or their chiral isomers; Wherein, j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R is selected from: H, methyl, ethyl, propyl; R1 is selected from: H, methyl, ethyl, propyl, amino acid side chain.

9. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L1 is selected from: wherein each A in L1 is independently selected from: -C(R2)2-, -NR2-, -C(=O)NR2-, -O-, -S-, wherein the connection of a A does not violate the principle of chemical bonding; Each R2 is independently selected from: H, C1-C3 alkyl; a is selected from: an integer between 0 and 20; b is selected from: an integer between 0-10.

10. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L1 is selected from: wherein a is selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; b is selected from: 0, 1, 2, 3, 4, 5, 6; Preferably, L1 is selected from: -NH(CH2) 10 C(=O)-, -NH(CH2)4C(=O)-, -NH(CH2)5C(=O)-, -NH(CH2)6C(=O)-, -(CH2)2NHC(=O)(CH2) 10 C(=O)-, -(CH2)2NHC(=O)(CH2)5C(=O)-, -NH(CH2)4C(=O)NH(CH2)4C(=O)-, -C(=O)(CH2)5C(=O)-, -C(=O)(CH2)9C(=O)-, -C(=O)(CH2) 10 C(=O)-、-NH(CH2)2OCH2C(=O)-、 -C(=O)(CH2)2NH(CH2)2OCH2C(=O)-.

11. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L2 is selected from: Each A in L2 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-, wherein the connection of c A does not violate the principle of chemical bonding; Each B in L2 is independently selected from: -NR2-, -O-, -S-; Each R2 is independently selected from: H, C1-C6 alkyl, preferably selected from: hydrogen, C1-C3 alkyl; c is selected from: an integer between 0 and 20; d is selected from: an integer between 0 and 10; e is selected from: an integer between 0-10.

12. The compound according to claim 11, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L2 is selected from: wherein each R2 is independently selected from: H, methyl, ethyl; c is selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; d is selected from: 0, 1, 2, 3, 4, 5, 6; e is selected from: 0, 1, 2, 3, 4, 5, 6; Preferably, L2 is selected from: -NH(CH2)2NHC(=O)(CH2)5-, -NH(CH2)2NHC(=O)(CH2)3-, -(CH2)2NH(CH2)2-, -(CH2)2N(CH3)(CH2)2-, -C(=O)(CH2)5-, -C(=O)(CH2)4-, -NH(CH2)6-, -NH(CH2)5-, -NH(CH2)-, -NH(CH2)2-, <h2 style=";text-align:left;direction:ltr">-(CH2)2O(CH2)5-、-CH2(=O)NH(CH2)2NHC(=O)(CH2)5-、<h2 style=";text-align:left;direction:ltr"> 13. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L3 and L4 are independently selected from: -NR6-R7-, -NR6-R7-O-R7-, -C(=O)-R7-; Each R6 is independently selected from: hydrogen, C1-C6 alkyl, preferably selected from: hydrogen, C1-C3 alkyl; Each R7 is independently selected from: C1-C8 alkylene, preferably selected from: C1-C4 alkylene.

14. The compound according to claim 13, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L3 and L4 are independently selected from: -NHCH2CH2-, -NHCH2CH2OCH2-, -NHCH2CH2OCH2CH2-, and -C(=O)CH2CH2CH2-.

15. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L5 is selected from: -R8-(CR9R 10 ) n1 -、-R8-(CR9R 10 ) n2 -R8-(CR9R 10 ) n3 -、-(CR9R 10 ) n2 -R8-(CR9R 10 ) n3 -; Wherein, R8 is selected from: -NR9-, -O-, -S-, -C(=O), -NR9C(=O)-, R9, R 10 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably selected from: hydrogen, C1-C3 alkyl; Each n1 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n2 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8; Each n3 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8.

16. The compound according to claim 15, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L5 is selected from: -C(=O)(CH2) n1 -, -C(=O)(CH2) n2 -NHC(=O)(CH2) n3 -, -C(=O)(CH2) n2 -C(=O)NH(CH2) n3 -、-(CH2) n2 -NHC(=O)(CH2) n3 -、-(CH2) n2 -O(CH2) n3 -, -C(=O)(CH2) n2 -C(=O)NH-, Each n1 is independently selected from: 1, 2, 3, 4, 5; Each n2 is independently selected from: 1, 2, 3, 4, 5; Each n3 is independently selected from: 1, 2, 3, 4, 5, 6; Preferably, L5 is selected from: -C(=O)CH2-, -C(=O)CH2CH2-, -C(=O)CH2CH2CH2-, -C(=O)CH2CH2NHC(=O)CH2CH2-, -CH2CH2NHC(=O)CH2CH2-, -CH2CH2OCH2CH2-, -CH2CH2OCH2-, -C(=O)CH2CH2C(=O)NH(CH2)4-, -C(=O)CH2CH2C(=O)NH-, 17. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L6 is selected from: -R 11 -(R 12 ) n4 -、-R 11 -(R 12 ) n4 -R 11 -、-R 11 -(R 12 ) n5 -R 11 -(R 12 ) n6 -R 11 -; Among them, R 11 Selected from: -C(=O), -NR 13 C(=O)-; Each R 12 Each independently selected from: -C(R 14 )2-、O、S、-NR 13 , where n4 or n5 R 12 The connection does not violate the principle of chemical bonding; Each R 13 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably selected from: hydrogen, C1-C3 alkyl; Each R 14 Each of the following is independently selected from: hydrogen, C1-C6 alkyl, and the side chain of an amino acid, preferably selected from: hydrogen, C1-C3 alkyl, and the side chain of an amino acid; Each n4 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; Each n5 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n6 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

18. The compound according to claim 17, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L6 is selected from: -C(=O)NH(CH2) n4 C(=O)-、-C(=O)NH(CH2) n4 C(=O)-、 -C(=O)NH(CH2) n5 O(CH2) n7 C(=O)-、-C(=O)NH(CH2) n5 O(CH2) n7 C(=O)-、 -C(=O)NH(CH2) n4 -、-C(=O)NH(CH2) n4 NHC(=O)-、 -C(=O)(CH2) n5 -C(=O)NH(CH2) n6 C(=O)-、 Each n4 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n5 is independently selected from: 1, 2, 3, 4, 5, 6; Each n6 is independently selected from: 1, 2, 3, 4, 5, 6; Each n7 is independently selected from: 1, 2, 3, 4, 5, 6; Preferably, L6 is selected from: -C(=O)NH(CH2)5C(=O)-, -C(=O)NH(CH2)4C(=O)-, -C(=O)NH(CH2)2OCH2C(=O)-, -C(=O)NH(CH2)2O(CH2)2C(=O)-, -C(=O)NH(CH2)4-, -C(=O)NH(CH2)3NHC(=O)-, -C(=O)(CH2)2-C(=O)NH(CH2)5C(=O)-、 19. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L7 is selected from: -(CH2) n’8 -R 15 -(R 16 ) n8 -; Among them, R 15 Selected from: -C(=O), -NR 13 C(=O)-; Each R 16 Each independently selected from: -C(R 17 )2-, O, S, n8 R 16 The connection does not violate the principle of chemical bonding; Each R 13 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably selected from: hydrogen, C1-C3 alkyl; Each R 17 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably selected from: hydrogen, C1-C3 alkyl; Each n8 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n'8 is independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, and 6.

20. The compound according to claim 19, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L7 is selected from: -C(=O)(CH2) n8 -、-C(=O)NH(CH2) n8 -, -NHC(=O)(CH2) n8 -、-C(=O)CH2O(CH2) n9 -、-(CH2)4NHC(=O)(CH2) n8 -; Each n8 is independently selected from: 1, 2, 3, 4, 5, 6; Each n9 is independently selected from: 1, 2, 3, 4, 5; Preferably, L7 is selected from: -C(=O)(CH2)5-, -C(=O)(CH2)4-, -C(=O)CH2O(CH2)3-, -C(=O)NH(CH2)5-, -C(=O)NH(CH2)4-, -NHC(=O)(CH2)5-, -NHC(=O)(CH2)4-, -(CH2)4NHC(=O)(CH2)5-.

21. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L8 is selected from: -R 18 -(R 19 ) n10 -R 18 -; Among them, R 18 Selected from: -C(=O), -NR 13 C(=O)-; Each R 19 Each independently selected from: -C(R 14 )2-、O、S、-NR 13 、C6-C 10 Aryl, 5-6 membered heteromonocyclic group, C5-C6 cycloalkyl, 6-8 membered heterobridged ring group, 6-8 membered heterospirocyclic group, 6-10 membered heterocyclic group, C6-C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl, wherein n 10 R 19 The connection does not violate the principle of chemical bonding; Each R 13 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably selected from: hydrogen, C1-C3 alkyl; Each R 14 Each of the following is independently selected from: hydrogen, C1-C6 alkyl, and the side chain of an amino acid, preferably selected from: hydrogen, C1-C3 alkyl, and the side chain of an amino acid; Each n 10 are independently selected from: an integer between 1 and 20; Or L8 is selected from L7 described in claim 19 or 20.

22. The compound according to claim 21, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: L8 is selected from: -C(=O)(CH2)n 10 C(=O)-, -C(=O)(CH2)n 11 -N(H)-(CH2CH2O)n 12 (CH2)n 13 C(=O)-、 R 19 Selected from: phenyl, 5-6 membered heteromonocyclic group, C5-C6 cycloalkyl, 5-6 membered nitrogen heteroaryl, ethynyl, vinyl; R 19 Preferably: n 10 Selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; n 11 Selected from: 1, 2, 3, 4, 5, 6; n 12 Selected from: 1, 2, 3, 4; n 13 Selected from: 1, 2, 3, 4; n 14 Selected from: 1, 2, 3, 4, 5, 6, 7, 8; Preferably, L8 is selected from: -C(=O)(CH2) 10 C(=O)-, -C(=O)(CH2)2-N(H)-(CH2CH2O)2CH2C(=O)-、 23. The compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: W1 is selected from the group consisting of: a group capable of being linked to an oligonucleotide via a phosphodiester bond or a phosphorothioate diester bond.

24. The compound according to claim 23, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: W1 is selected from: hydroxyl, protected hydroxyl or a group having the following structure: or a chiral isomer thereof; Wherein, R' is hydrogen or a hydroxyl protecting group.

25. The compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The cell membrane protein receptor with endocytosis is selected from: cell protein receptors, cell channel receptors, cell transporter receptors and enzyme protein receptors; Preferably, the cell membrane protein receptor is selected from the group consisting of: sialoglycoprotein receptor, folate receptor, integrin receptor, transferrin receptor, and G protein-coupled receptor.

26. The compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The targeting binding moiety T has a structure as shown in the following formula (IV), formula (V), formula (VI) or formula (VII): Wherein: D, E, J, M are independently selected from: or their chiral isomers; G, V, W, and U are independently selected from: or their chiral isomers; r, t are independently selected from: 0, 1, 2, 3, 4, 5, 6; X1, X'1, X2, X3, X4, and X5 are independently selected from the group consisting of a single bond, C1-C6 alkylene, -C(=O)-C1-C6 alkylene, -C(=O)-NH-C1-C6 alkylene, -NH-C(=O)-C1-C6 alkylene, -NH-C1-C6 alkylene, -C(=O)-NH-C1-C6 alkylene-O-C1-C6 alkylene, -NH-C(=O)-C1-C6 alkylene-O-C1-C6 alkylene, or chiral isomers thereof; The connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from one or more of the following groups, and are connected to form a connection unit that does not violate the principle of chemical bonding: C1-C 15 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O)2-, -S(=O)-, -CH=CH-, B Alkynyl, -NRC(=O)-, -NRC(=O)NR-, -C(=O)CRR1NR-, -OC(=O)-, -SC(=O)-, -NRS(=O)2-, C6-C 10 Aryl, 3-20 membered heterocyclic group, C3-C 10 Cycloalkyl, 5-10 membered heteroaryl, the linking unit may be substituted by one or more R; R is selected from the group consisting of: H, C1-C6 alkyl, halogen, hydroxy, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, cyano, carboxyl, amino, and aminocarbonyl; R1 is selected from: H, C1-C6 alkyl, amino acid side chain.

27. The compound according to claim 26, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from the following connecting units formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C 12 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O)2-, -S(=O)-, -CH=CH-, B Alkynyl, -NRC(=O)-, -NRC(=O)NR-, -C(=O)CRR1NR-, -OC(=O)-, -SC(=O)-, -NRS(=O)2-, C6-C 10 Aryl, 3-8 membered heteromonocyclic group, C3-C8 cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospirocyclic group, 5-20 membered heterocyclic group, C5-C 10 Cycloalkyl, 5-6 membered heteroaryl, the linking unit may be substituted by one or more R; R is selected from the group consisting of: H, C1-C3 alkyl, halogen, hydroxy, hydroxy-substituted C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, cyano, carboxyl, amino, and aminocarbonyl; R1 is selected from: H, C1-C3 alkyl, amino acid side chain.

28. The compound according to claim 27, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from connecting units formed by connecting one or more of the following groups without violating the principle of chemical bonding: or their chiral isomers; Wherein, j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R is selected from: H, C1-C3 alkyl, halogen-substituted C1-C3 alkyl; R1 is selected from: H, C1-C3 alkyl, amino acid side chain.

29. The compound according to claim 28, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independently selected from connecting units formed by connecting one or more of the following groups without violating the principle of chemical bonding: or their chiral isomers; Wherein, j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; R is selected from: H, methyl, ethyl, propyl; R1 is selected from: H, methyl, ethyl, propyl, amino acid side chain.

30. The compound according to claim 29, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The connectors K1, K3, K6, and K7 are independently selected from: -(R 20 ) n15 -R 21 -(R 20 ) n16 -R 22 -; Each R 20 Each is independently selected from: -CH2-, -O-, -S-, wherein n 15 or n16 R 20 The connection does not violate the principle of chemical bonding; R 21 Selected from: -C(=O)NH-, -C(=O)-, C6-C 10 Aryl, 3-8 membered heteromonocyclic group, C3-C8 cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospirocyclic group, 5-20 membered heterocyclic group, C5-C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl, -C(=O)-3-8 membered heteromonocyclic group, -C(=O)-C3-C8 cycloalkyl, or none; R 22 Selected from: -C(=O)NH-, -NH-, -C(=O)-, or none; Each n 15 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n 16 Each of the following is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

31. The compound according to claim 30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 21 Selected from: -C(=O)NH-, -C(=O)-, phenyl, Or not.

32. The compound according to claim 30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: Linkers K1, K3, K6, K7 are independently selected from: -(CH2) n15 -R 22 -、-(CH2) n15 -R 21 -(CH2) n16 -R 22 -、-(CH2) n15 -OR 21 -CH2O-(CH2) n16 -R 22 -、-(CH2) n15 -R 21 -CH2CH2O-(CH2) n16 -R 22 -、-(CH2) n15 -R 21 -(CH2CH2O)2-, -(CH2) n15 -R 21 -(CH2) n16 -; R 21 Selected from: -C(=O)NH-, -C(=O)-, phenyl, R 22 Selected from: -C(=O)NH-, -NH-, -C(=O)-; Each n 15 Each independently selected from: 1, 2, 3, 4, 5, 6; Each n 16 Each is independently selected from: 0, 1, 2, 3, 4; Preferably, the connectors K1, K3, K6, and K7 are independently selected from:

33. The compound according to claim 29, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The connectors K2, K4, K5, K8, K9, K10, K11, K12, K13, and K14 are independently selected from: -(R 23 ) n17 -R 24 -(R 23 ) n18 -R 25 -; Among them, each R 23 Each is independently selected from: -CH2-, -O-, -S-, wherein n 18 R 23 The connection does not violate the principle of chemical bonding; R 24 Selected from: -C(=O)NH-, -C(=O)-, C6-C 10 Aryl, 3-8 membered heteromonocyclic group, C3-C8 cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospirocyclic group, 5-20 membered heterocyclic group, C5-C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl, -C(=O)-3-8 membered heteromonocyclic group, -C(=O)-C3-C8 cycloalkyl, or none; R 25 Selected from: -C(=O)NH-(CH2) n19 -C(=O)-, -NHC(=O)-(CH2) n19 -C(=O)-, -C(=O)NH-(CH2) n19 -、-NHC(=O)-(CH2) n19 -、-C(=O)-(CH2) n19 -, or not; Each n 17 Each independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n 18 Each is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n 19 Independently selected from: 1, 2, 3.

34. The compound according to claim 33, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 24 Selected from: -C(=O)NH-, -C(=O)-, phenyl, Or not.

35. The compound according to claim 33, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: Linkers K2, K4, K5, K8, K9, K10, K11, K12, K13, K14 are independently selected from: -(CH2) n17 -R 25 -、-(CH2) n17 -R 24 -(CH2) n18 -R 25 -、-(CH2) n17 -OCH2-R 24 -CH2O-(CH2) n18 -R 25 -、-(CH2) n17 -R 24 -(CH2) n18 -、-(CH2) n17 -R 24 -CH2CH2O-(CH2) n18 -R 25 -、-(CH2) n17 -R 24 -(CH2CH2O)2-(CH2) n18 -、-(CH2) n17 -; R 24 Selected from: -C(=O)NH-, -C(=O)-, phenyl, R 25 Selected from: -C(=O)NH-(CH2)2-C(=O)-, -NHC(=O)-(CH2)2-C(=O)-, -C(=O)NH-(CH2)2-, -NHC(=O)-(CH2)2-, -C(=O)-(CH2)2-; Each n 17 Each independently selected from: 1, 2, 3, 4, 5, 6; Each n 18 Each is independently selected from: 0, 1, 2, 3, 4; Preferably, connectors K2, K4, K5, K8, K9, K10, K11, K12, K13, K14 are independently selected from:

36. The compound according to claim 26, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: X1, X'1, X2, X3, X4, and X5 are independently selected from the group consisting of a single bond, C1-C3 alkylene, -C(=O)-C1-C3 alkylene, -C(=O)-NH-C1-C3 alkylene, -NH-C(=O)-C1-C3 alkylene, -NH-C1-C3 alkylene, -C(=O)-NH-C1-C3 alkylene-O-C1-C3 alkylene, -NH-C(=O)-C1-C3 alkylene-O-C1-C3 alkylene, or chiral isomers thereof; Preferably, X1, X'1, X2, X3, X4, and X5 are independently selected from the group consisting of methylene, ethylene, propylene, -C(=O)-methylene, -C(=O)-ethylene, -C(=O)-propylene, -NH-C(=O)-methylene, -NH-C(=O)-ethylene, and -NH-C(=O)-propylene.

37. The compound according to claim 26, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The ligand is selected from the group consisting of: D-pyranose, L-pyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-pyranose, β-D-mannopyranose, α-D-glucose, β-D-pyranose, α-D-glucose, β-D-glucose, α-D-glucose, β-D-glucose, α-D-furanose, α-D-glucose Sugar, α-D-fructose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, 4-fluorodeoxy-N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-difluoropropionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-triphenylmethyl methyl-α-D-glucopyranose, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranose heptanoside ethyl ester, 2,5-anhydro-D-allose nitrile, D-ribose, D-4-thioribose, L-ribose and L-4-thioribose, RGD peptide, folic acid molecule, TfR1 peptide, integrin ligand small molecule, lipid, fatty acid, bile acid and cholesterol; Preferably, the ligand is selected from: N-acetylgalactosamine, 4-fluorodeoxy-N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-difluoropropionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, folic acid molecules, integrin ligand small molecules.

38. The compound according to claim 37, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The ligand is selected from: , or the hydrogen on its hydroxyl group is replaced by an acetyl group.

39. The compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The targeting binding moiety T is selected from: or the hydrogen on its hydroxyl group is replaced by an acetyl group.

40. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The compound is selected from:

41. A conjugate, characterized in that The compound according to any one of claims 1 to 40 is connected to an oligonucleotide drug, wherein W1 in the compound is connected to the oligonucleotide drug through a covalent bond; W2 in the compound is connected to the oligonucleotide drug by reacting with Q; Q is selected from: L9-L10-L11-Z2, and Z2 is the residue after removing a hydroxyl group from the oligonucleotide drug; L9 is selected from: an active group capable of undergoing click chemistry reaction with the W2 group; L10 is selected from a connecting unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C 15 Alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -SS-, -CR=N-, -S(=O)2-, -S(=O)-, -CH=CH-, ethynyl, -NRC(=O)-, -NRC(=O)NR-, -C(=O)CRR1NR-, -OC(=O)-, -SC(=O)-, -NRS(=O)2-, -P(=O)2O-, -P(=O)(=S)O-, C6-C 10 Aryl, 3-20 membered heterocyclic group, C3-C 10 Cycloalkyl, 5-10 membered heteroaryl, the linking unit may be substituted by one or more R; R is selected from the group consisting of: H, C1-C6 alkyl, halogen, hydroxy, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, cyano, carboxyl, amino, and aminocarbonyl; R1 is selected from: H, C1-C6 alkyl, amino acid side chain; L11 is selected from: W1 without a hydroxyl hydrogen or a hydroxyl protecting group.

42. The conjugate according to claim 41, characterized in that L9 is selected from: 5-20 membered heterocyclic group containing alkynyl, -SR 26 , alkynyl, azido; R 26 Selected from: H or a thiol protecting group.

43. The conjugate according to claim 42, characterized in that L9 is selected from: Azide, -SR 26 .

44. The conjugate according to claim 41, characterized in that L11 is selected from: -O-, or a group having the following structure: or their chiral isomers.

45. The conjugate according to claim 41, characterized in that L10 is selected from: wherein each A in L10 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-; Each R2 is independently selected from: H, C1-C3 alkyl; Each R3 is independently selected from: R5, or none; Each R4 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-, -C(=O), -C(=S)-, wherein the connection of f4 R4 does not violate the principle of chemical bonding; Each R5 is independently selected from: C6-C 10 Aryl, 3-8 membered heteromonocyclic group, C3-C8 cycloalkyl, 5-10 membered heterobridged ring group, 5-10 membered heterospirocyclic group, 5-20 membered heterocyclic group, C5-C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl; Each f1 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; Each f2 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; Each f3 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; Each f4 is independently selected from an integer between 0-20.

46. ​​The conjugate according to claim 45, characterized in that Each A in L10 is independently selected from: -CH2-, -NH-, -O-, -S-; Each R3 is independently selected from: R5, or none; Each R4 is independently selected from: -CH-, -NH-, -O-, -S-, -C(=O); Each R5 is independently selected from: C6-C 10 Aryl, 5-6 membered heteromonocyclic group, C5-C6 cycloalkyl, 6-8 membered heterobridged ring group, 6-8 membered heterospirocyclic group, 6-10 membered heterocyclic group, C6-C 10 Cycloalkyl, 5-6 membered heteroaryl, ethynyl, vinyl; Each f1 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; Each f2 is independently selected from: 0, 1, 2, 3, 4; Each f3 is independently selected from: 0, 1, 2, 3, 4; Each f4 is independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

47. The conjugate according to claim 46, characterized in that Each R5 is independently selected from: or their chiral isomers.

48. The conjugate according to claim 45, characterized in that L10 is selected from: or their chiral isomers.

49. The conjugate according to claim 41, characterized in that Q is selected from:

50. The conjugate according to any one of claims 41 to 49, characterized in that The compound forms a phosphate bond or a phosphorothioate bond with the phosphate group or the phosphorothioate group at the 3' and / or 5' end of the sense strand in the oligonucleotide drug via the hydroxyl group in W1.

51. The conjugate according to any one of claims 41 to 49, characterized in that L11 is linked to the 3' and / or 5' end of the sense strand of the oligonucleotide drug via a phosphate bond or a phosphorothioate bond.

52. The conjugate according to any one of claims 41 to 49, characterized in that The oligonucleotide drugs include siRNA, ASO, saRNA, miRNA, PNA, and aptamers.

53. The conjugate according to any one of claims 41 to 49, characterized in that It has a structure shown in the following formula (A), formula (B) or formula (C): Wherein, the targeting binding moieties T, L1, L2, L3, L4, L5, L6, L7, and L8 are as described in any one of claims 1 to 40, and L10, L11, and Z2 are as described in any one of claims 41 to 49; Z1 is the residue after removing one hydroxyl group from the oligonucleotide drug; W4 is the group obtained by removing a hydroxyl hydrogen or a hydroxyl protecting group from W1 as described in any one of claims 1 to 40; W5 is selected from: a group formed by reacting W2 described in any one of claims 1 to 40 and L9 described in any one of claims 41 to 49; W6 is selected from: W4 or -W5-L10-L11-.

54. The conjugate according to claim 53, characterized in that W4 is selected from: -O-, or a group having the following structure: or their chiral isomers.

55. The conjugate according to claim 53, characterized in that Each W5 is independently selected from: a 3-20 membered heterocyclic group, a 5-20 membered heteroaromatic ring group, a 3-20 membered heterocyclic thio group, -SS-; Preferred is: 5-20 membered heterocyclic group, 5-6 membered heteroaromatic ring group, 5-6 membered heterocyclic thio group, -SS-; More preferably: -S-S-。 56. The conjugate according to claim 41, characterized in that The conjugate is selected from:

57. A carrier compound, characterized in that Its molecular structure is the same as the molecular structure of the conjugate described in any one of claims 41-56 after the oligonucleotide drug is removed.

58. Use of a compound according to any one of claims 1 to 40 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a carrier for delivering one or more oligonucleotide drugs; Preferably, the oligonucleotide drug includes siRNA, ASO, saRNA, miRNA, PNA, and aptamer.

59. Use of the conjugate according to any one of claims 41 to 56 as an active ingredient in the preparation of a medicament for regulating the expression of one or more target genes.

60. Use of the compound according to any one of claims 1 to 40 or a pharmaceutically acceptable salt or a stereoisomer thereof, and / or the carrier compound according to claim 57 as a carrier in the preparation of a drug for regulating the expression of one or more target genes.

61. A pharmaceutical preparation for regulating the expression of one or more target genes, characterized in that The composition is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient comprises the conjugate according to any one of claims 41 to 56.

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