Compounds, conjugates and their applications for targeted delivery of oligonucleotide drugs

Abstract

The present invention provides a novel compound for targeted delivery of oligonucleotide drugs and its conjugates thereof, which are capable of delivering one or more different oligonucleotide drugs, the compound having the structure shown in formula (I), formula (II) or formula (III).

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to compounds, conjugates, and their applications for targeted delivery of oligonucleotide drugs. Background Technology

[0002] Oligonucleotide drugs (such as siRNA, antisense oligonucleotides, and plasmid DNA) are emerging and promising therapies that have shown remarkable efficacy in regulating gene expression and treating a variety of diseases. However, their high molecular weight and polyanionic properties result in poor cell membrane permeability, and factors such as nucleases further limit their practical application.

[0003] Currently, utilizing receptor-mediated endocytosis to conjugate target ligands with oligonucleotide drugs to enhance cellular uptake and improve tissue-specific delivery in vivo has become a hot topic in oligonucleotide drug development. Notably, multivalent ligand clusters may sometimes outperform single ligands in promoting targeted tissue delivery, such as the desialyl glycoprotein receptor (ASGPR). Furthermore, studies have found that the ASGPR ligand N-acetylgalactosamine (GalNAc) can effectively enhance the entry of oligonucleotide drugs into hepatocytes. Further experiments have confirmed that multivalent GalNAc ligand clusters have a stronger affinity for ASGPR than single GalNAc ligands, thereby improving the efficiency of oligonucleotide delivery to hepatocytes (Chem. Soc. Rev., 2023, 52, 1273; Bioconjugate Chem. 2017, 28, 2, 283-295).

[0004] Several patent documents have reported research in this area, such as US8106022B2, CN116832169A, WO2023045995A1, CN110959011B, CN117858948A, and WO2024061157, all involving the delivery of oligonucleotide drugs using GalNAc ligands. However, most of these documents disclose the use of multivalent ligands GalNAc to deliver a single oligonucleotide drug targeting a single site. Since some single genes in organisms have multiple transcripts with sequence differences, and disease often involves the interaction of multiple genes, it is necessary to develop compounds with novel scaffolds for targeted delivery of oligonucleotide drugs, capable of simultaneously delivering two nucleotide drugs targeting a single site or dual-target nucleotide drugs. This has broad application prospects. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a novel compound for targeted delivery of oligonucleotide drugs and its conjugates with oligonucleotide drugs, which can deliver one or more different oligonucleotide drugs.

[0006] A first aspect of the invention provides a compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, said compound having the structure shown in formula (I), formula (II) or formula (III):

[0007]

[0008] Wherein: the targeting binding portion T is a group containing a ligand, which can specifically bind to a cell membrane protein receptor with endocytosis function;

[0009] W1 is selected from: groups that can bind to oligonucleotide drugs via covalent bonds;

[0010] W2 is selected from: groups capable of undergoing click chemical reactions or hydrogen;

[0011] W3 is selected from either W1 or W2, and W3 is not hydrogen;

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

[0013] L1, L2, L3, L4, L5, L6, L7, and L8 are each independently selected from one or more of the following groups and linked together without violating the principles 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, -NR C(=O)-, -NR C(=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, wherein the linking unit may be substituted with one or more R;

[0014] R is selected from: H, C1-C6 alkyl, halogen, hydroxyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0015] R1 is selected from: H, C1-C6 alkyl groups, and amino acid side chains;

[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] The R in the Y1 and Y2 structures are independently selected from: H, C1-C3 alkyl, and halogen-substituted C1-C3 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, W2 is selected from: 5-20 membered heterocyclic groups containing an alkynyl group, -SR 26 Alkyne, azide, alkenyl, 5-20 membered heterocyclic groups containing alkenyl groups, hydrogen;

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

[0024] In some embodiments, W2 is selected from:

[0025] -SR 26 , azide group, hydrogen.

[0026] In some embodiments, L1, L2, L3, L4, L5, L6, L7, and L8 are each independently selected from one or more of the following groups to form a connecting unit that does not violate 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)CRR1NR-, -OC(=O)-

[0029] , -SC(=O)-, -NRS(=O)2-, -P(=O)2O-, -P(=O)(=S)O-, C6-C 10 Aryl, 3-8 member heteromonocyclic, C3-C8 cycloalkyl, 5-10 member heterobridged cycloalkyl, 5-10 member heterospirocyclic, 5-20 member heterofused cycloalkyl, C5-C 10 The linking unit may be replaced by one or more Rs, including cycloalkyl and 5-6-membered heteroaryl groups.

[0030] R is selected from: H, C1-C3 alkyl, halogen, hydroxyl, hydroxyl-substituted C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0031] R1 is selected from: H, C1-C3 alkyl groups, and amino acid side chains.

[0032] In some embodiments, L1, L2, L3, L4, L5, L6, L7, and L8 are each independently selected from one or more of the following groups to form a connecting unit that does not violate the principle of chemical bonding:

[0033]

[0034] Or their chiral isomers;

[0035] Where j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0036] R is selected from: H, C1-C3 alkyl, halogen-substituted C1-C3 alkyl;

[0037] R1 is selected from: H, C1-C3 alkyl groups, and amino acid side chains.

[0038] In some embodiments, L1, L2, L3, L4, L5, L6, L7, and L8 are each independently selected from one or more of the following groups to form a connecting unit that does not violate the principle of chemical bonding:

[0039] Or their chiral isomers;

[0040] Where j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

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

[0042] R1 is selected from: H, methyl, ethyl, propyl, and amino acid side chains.

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

[0044]

[0045] In L1, each A is independently selected from: -C(R2)2-, -NR2-, -C(=O)NR2-, -O-, -S-, where the connection of a A does not violate the principle of chemical bonding;

[0046] Each R2 is independently selected from: H, C1-C3 alkyl groups;

[0047] 'a' is selected from: integers between 0 and 20;

[0048] b is selected from: integers between 0 and 10.

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

[0050]

[0051] Where '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 of these embodiments, 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)-, -NH(CH2)4C(=O)NH(CH2)4C(=O)-, -(CH2)2NHC(=O)(CH2)5C(=O)-, -C(=O)(CH2)5C(=O)-, -C(=O)(CH2)9C(=O)-,

[0054] -C(=O)(CH2) 10 C(=O)-, -NH(CH2)2OCH2C(=O)-,

[0055] -C(=O)(CH2)2NH(CH2)2OCH2C(=O)-.

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

[0057]

[0058] Each A in L2 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-, where the connection of c A does not violate the principle of chemical bonding;

[0059] Each B in L2 is independently selected from: -NR2-, -O-, -S-;

[0060] Each R2 is independently selected from: H, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

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

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

[0063] e is selected from: integers between 0 and 10.

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

[0065] Each R2 is independently selected from: H, methyl, and 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(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-,

[0070] -(CH2)2O(CH2)5-, -CH2(=O)NH(CH2)2NHC(=O)(CH2)5-,

[0071] In some embodiments, L3 and L4 are independently selected from: -NR6-R7-, -NR6-R7-O-R7-, and -C(=O)-R7-, respectively;

[0072] Each R6 is independently selected from: hydrogen, C1-C6 alkyl;

[0073] Each R7 is independently selected from C1-C8 alkylene groups.

[0074] In some of these embodiments, each R6 is independently selected from: hydrogen, C1-C3 alkyl;

[0075] Each R7 is independently selected from C1-C4 alkylene groups.

[0076] In some embodiments, L3 and L4 are independently selected from: -NHCH2CH2-, -NHCH2CH2OCH2-, -NHCH2CH2OCH2CH2-, and -C(=O)CH2CH2CH2-.

[0077] In some embodiments, L5 is selected from: -R8-(CR9R) 10 ) n1 -、-R8-(CR9R 10 ) n2 -R8-(CR9R 10 ) n3 -、-(CR9R 10 ) n2 -R8-(CR9R 10 ) n3 -;

[0078] Among them, R8 is selected from: -NR9-, -O-, -S-, -C (=O), -NR9C (=O)-,

[0079] R9, R 10 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0080] Each n1 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0081] Each n2 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8;

[0082] Each n3 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8.

[0083] In some embodiments, L5 is selected from: -C(=O)(CH2) n1 -、-C(=O)(CH2) n2 -NHC(=O)(CH2) n3 -、

[0084] -C(=O)(CH2) n2 -C(=O)NH(CH2) n3 -、-(CH2) n2 -NHC(=O)(CH2) n3 -、

[0085] -(CH2) n2 -O(CH2) n3 -、-C(=O)(CH2) n2 -C(=O)NH-、

[0086] Each n1 is independently selected from: 1, 2, 3, 4, 5;

[0087] Each n² is independently selected from: 1, 2, 3, 4, 5;

[0088] Each n3 is independently selected from: 1, 2, 3, 4, 5, 6.

[0089] In some embodiments, 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-,

[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 Selected independently from: -C(R 14 )2-、O、S、-NR 13 Where n4 or n5 R 12 The connection does not violate the chemical bonding principle;

[0094] Each R 13 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0095] Each R 14 The side chains are independently selected from: hydrogen, C1-C6 alkyl groups, and amino acids, preferably from: hydrogen, C1-C3 alkyl groups, and amino acids;

[0096] Each n4 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0097] Each n5 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0098] Each n6 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

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

[0100] -C(=O)NH(CH2) n4 C(=O)-、-C(=O)NH(CH2) n4 C(=O)-、

[0101] -C(=O)NH(CH2) n5 O(CH2) n7 C(=O)-、-C(=O)NH(CH2) n5 O(CH2) n7 C(=O)-、

[0102] -C(=O)NH(CH2) n4 -、-C(=O)NH(CH2) n4 NHC(=O)-、

[0103] -C(=O)(CH2) n5 -C(=O)NH(CH2) n6 C(=O)-、

[0104] Each n4 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0105] Each n5 is independently selected from: 1, 2, 3, 4, 5, 6;

[0106] Each n6 is independently selected from: 1, 2, 3, 4, 5, 6;

[0107] Each n7 is independently selected from: 1, 2, 3, 4, 5, 6.

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

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

[0110] -C(=O)NH(CH2)2OCH2C(=O)-, -C(=O)NH(CH2)2O(CH2)2C(=O)-,

[0111] -C(=O)NH(CH2)4-, -C(=O)NH(CH2)3NHC(=O)-,

[0112] -C(=O)(CH2)2-C(=O)NH(CH2)5C(=O)-,

[0113] In some embodiments, L7 is selected from: -(CH2) n’8 -R 15 -(R 16 ) n8 -;

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

[0115] Each R 16 Selected independently from: -C(R 17 )2-、O、S, where n8 are R 16 The connection does not violate the principles of chemical bonding;

[0116] Each R 13 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0117] Each R 17 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0118] Each n8 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

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

[0120] In some embodiments, 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 -;

[0121] Each n8 is independently selected from: 1, 2, 3, 4, 5, 6;

[0122] Each n9 is independently selected from: 1, 2, 3, 4, 5.

[0123] In some embodiments, L7 is selected from: -C(=O)(CH2)5-, -C(=O)(CH2)4-,

[0124] -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-.

[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 Selected independently from: -C(R 14 )2-、O、S、-NR 13 C6-C 10 Aryl, 5-6 member heteromonocyclic, C5-C6 cycloalkyl, 6-8 member heterobridged cycloalkyl, 6-8 member heterospirocyclic, 6-10 member heterocyclic cycloalkyl, C6-C 10 cycloalkyl, 5-6-membered heteroaryl, ethynyl, vinyl, wherein n 10 R 19 The connection does not violate the principles of chemical bonding;

[0128] Each R 13 Each is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0129] Each R 14 The side chains are independently selected from: hydrogen, C1-C6 alkyl groups, and amino acids, preferably from: hydrogen, C1-C3 alkyl groups, and amino acids;

[0130] each n 10 Each integer is independently selected from the range 1-20;

[0131] Alternatively, L8 can be selected from L7.

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

[0133] Each R 19Each of the following groups is independently selected from: -CH2-, -O-, -S-, -NH-, phenyl, 5-6 membered heteromonocyclic, C5-C6 cycloalkyl, 5-6 membered heteroaryl, ethynyl, and vinyl;

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

[0135] In some embodiments, L8 is selected from: -C(=O)(CH2)n 10 C(=O)-、

[0136] -C(=O)(CH2)n 11 -N(H)-(CH2CH2O)n 12 (CH2)n 13 C(=O)-、

[0137] R 19 Selected from: phenyl, 5-6 membered heteromonocyclic, C5-C6 cycloalkyl, 5-6 membered azaaryl, ethynyl, vinyl; R 19 Preferred options are:

[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)(CH2) 10 C(=O)-、

[0144] -C(=O)(CH2)2-N(H)-(CH2CH2O)2CH2C(=O)-,

[0145] In some embodiments, W1 is selected from groups that can be linked to oligonucleotides via phosphodiester bonds or thiophosphate diester bonds.

[0146] In some embodiments, W1 is selected from hydroxyl groups, protected hydroxyl groups, or groups having the following structure:

[0147]

[0148] Or its chiral isomers;

[0149] R' is a hydrogen or 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 transport protein receptors, and enzyme protein receptors.

[0151] In some embodiments, the cell membrane protein receptor is selected from: sialic acid glycoprotein receptor (ASGPR), folic acid receptor, integrin receptor, transferrin receptor, and G protein-coupled receptor (GPCR).

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

[0153]

[0154] Among them: D, E, J, and M are each independently selected from:

[0155] Or their chiral isomers;

[0156] G, V, W, and U are each independently selected from:

[0157] Or their chiral isomers;

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

[0159] X1, X'1, X2, X3, X4, and X5 are each independently selected from: single bonds, C1-C6 alkylene groups, -C(=O)-C1-C6 alkylene groups, -C(=O)-NH-C1-C6 alkylene groups, -NH-C(=O)-C1-C6 alkylene groups, -NH-C1-C6 alkylene groups, -C(=O)-NH-C1-C6 alkylene groups-O-C1-C6 alkylene groups, -NH-C(=O)-C1-C6 alkylene groups-O-C1-C6 alkylene groups, or their chiral isomers;

[0160] Connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are connection units independently selected from one or more of the following groups and linked together 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, -NR C(=O)-, -NR C(=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, wherein the linking unit may be substituted with one or more R;

[0161] R is selected from: H, C1-C6 alkyl, halogen, hydroxyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0162] R1 is selected from: H, C1-C6 alkyl groups, and amino acid side chains.

[0163] In some embodiments, the connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are each independently selected from one or more of the following groups to form a connecting unit that does not violate 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, -NR C(=O)-, -NR C(=O)NR-, -C(=O)CRR1NR-, -OC(=O)-, -SC(=O)-, -NRS(=O)2-, C6-C 10 Aryl, 3-8 member heteromonocyclic, C3-C8 cycloalkyl, 5-10 member heterobridged cycloalkyl, 5-10 member heterospirocyclic, 5-20 member heterofused cycloalkyl, C5-C 10 The linking unit may be replaced by one or more Rs, including cycloalkyl and 5-6-membered heteroaryl groups.

[0164] R is selected from: H, C1-C3 alkyl, halogen, hydroxyl, hydroxyl-substituted C1-C3 alkyl, C1-C3 alkoxy, halogen-substituted C1-C3 alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0165] R1 is selected from: H, C1-C3 alkyl groups, and amino acid side chains.

[0166] In some embodiments, the connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are each independently selected from one or more of the following groups to form a connecting unit that does not violate the principle of chemical bonding:

[0167]

[0168] Or their chiral isomers;

[0169] Where j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0170] R is selected from: H, C1-C3 alkyl, halogen-substituted C1-C3 alkyl;

[0171] R1 is selected from: H, C1-C3 alkyl groups, and amino acid side chains.

[0172] In some embodiments, the connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are each independently selected from one or more of the following groups to form a connecting unit that does not violate the principle of chemical bonding:

[0173]

[0174] Or their chiral isomers;

[0175] Where j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

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

[0177] R1 is selected from: H, methyl, ethyl, propyl, and amino acid side chains.

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

[0179] Among them, each R 20 Each of the following is independently selected from: -CH2-, -O-, and -S-, where n 15 or n16 R20 The connection does not violate the principles of chemical bonding;

[0180] R 21 Selected from: -C(=O)NH-, -C(=O)-, C6-C 10 Aryl, 3-8 member heteromonocyclic, C3-C8 cycloalkyl, 5-10 member heterobridged cycloalkyl, 5-10 member heterospirocyclic, 5-20 member heterofused cycloalkyl, C5-C 10 Cycloalkyl, 5-6-membered heteroaryl, ethynyl, vinyl, -C(=O)-3-8-membered heteromonocyclic, -C(=O)-C3-C8 cycloalkyl, or none;

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

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

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

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

[0185]

[0186] Or not.

[0187] In some embodiments, connectors K1, K3, K6, and K7 are each 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 -;

[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 of the following is independently selected from: 1, 2, 3, 4, 5, and 6;

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

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

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

[0194] Among them, each R 23 Each of the following is independently selected from: -CH2-, -O-, and -S-, where n 18 R 23 The connection does not violate the principles of chemical bonding;

[0195] R 24 Selected from: -C(=O)NH-, -C(=O)-, C6-C 10 Aryl, 3-8 member heteromonocyclic, C3-C8 cycloalkyl, 5-10 member heterobridged cycloalkyl, 5-10 member heterospirocyclic, 5-20 member heterofused cycloalkyl, C5-C 10 Cycloalkyl, 5-6-membered heteroaryl, ethynyl, vinyl, -C(=O)-3-8-membered heteromonocyclic, -C(=O)-C3-C8 cycloalkyl, or none;

[0196] R 25 Selected from: -C(=O)NH-(CH2) n19 -C(=O)-, -NHC(=O)-(CH2) n19 -C(=O)-, -C(=O)NH-(CH2)n19 -

[0197] -NHC(=O)-(CH2) n19 -、-C(=O)-(CH2) n19 - or none;

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

[0199] each n 18 Each of the following numbers is independently selected: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0200] each n 19 Each of the following is selected independently: 1, 2, and 3.

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

[0202]

[0203] Or not.

[0204] In some embodiments, connectors K2, K4, K5, K8, K9, K10, K11, K12, K13, and K14 are each 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 -;

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

[0206] 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-;

[0207] each n 17 Each of the following is independently selected from: 1, 2, 3, 4, 5, and 6;

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

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

[0210] In some of these embodiments, X1, X'1, X2, X3, X4, and X5 are each independently selected from: single bonds, C1-C3 alkylene groups, -C(=O)-C1-C3 alkylene groups, -C(=O)-NH-C1-C3 alkylene groups, -NH-C(=O)-C1-C3 alkylene groups, -NH-C1-C3 alkylene groups, -C(=O)-NH-C1-C3 alkylene groups-O-C1-C3 alkylene groups, -NH-C(=O)-C1-C3 alkylene groups-O-C1-C3 alkylene groups, or their chiral isomers.

[0211] In some embodiments, X1, X'1, X2, X3, X4, and X5 are each independently selected from: 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-mannose pyranoyl, L-mannose pyranoyl, D-arabinose, D-xylfuranose, L-xylfuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannose pyranoyl, β-D-mannose pyranoyl, α-D-mannose pyranoyl, β-D-mannose pyranoyl, α-D-glucose pyranoyl, β-D-glucose pyranoyl, α-D-glucose pyranoyl, β-D-glucose pyranoyl, α-D-glucose pyranoyl, β-D-glucose pyranoyl, α-D-glucose pyranoyl, β-D-glucose pyranoyl α-D-fructose furanose, α-D-fructose pyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactopyranose, β-D-galactopyranose, 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-sulfonamido-D-glucopyranose, N-ethanolyl-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-dehydrated-D-alosulfonyl, 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 acids and cholesterol.

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

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

[0215]

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

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

[0218]

[0219]

[0220]

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

[0222] Secondly, the present invention provides a conjugate obtained by linking the compound of the present invention with an oligonucleotide drug, wherein W1 in the compound is covalently linked to the oligonucleotide drug; and W2 in the compound is linked to the oligonucleotide drug by reacting with Q; Q is selected from: L9-L10-L11-Z2, and Z2 is a residue of the oligonucleotide drug after removing a hydroxyl group.

[0223] L9 is selected from: active groups capable of undergoing click chemistry reactions with W2 groups;

[0224] L10 is a linking 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, -NR C(=O)-, -NR C(=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, wherein the linking unit may be substituted with one or more R;

[0225] R is selected from: H, C1-C6 alkyl, halogen, hydroxyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, cyano, carboxyl, amino, aminocarbonyl;

[0226] R1 is selected from: H, C1-C6 alkyl groups, and amino acid side chains;

[0227] L11 is selected from: W1 after removing one hydroxyl hydrogen or one hydroxyl protecting group.

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

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

[0230] Azide group, -SR 26 .

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

[0232]

[0233] Or their chiral isomers.

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

[0235]

[0236] In L10, each A is independently selected from: -C(R2)2-, -NR2-, -O-, and -S-.

[0237] Each R2 is independently selected from: H, C1-C3 alkyl groups;

[0238] Each R3 is selected independently from: R5, or none;

[0239] Each R4 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-, -C(=O), -C(=S)-, where the connection of f4 R4s does not violate the principle of chemical bonding;

[0240] Each R5 is independently selected from: C6-C 10 Aryl, 3-8 member heteromonocyclic, C3-C8 cycloalkyl, 5-10 member heterobridged cycloalkyl, 5-10 member heterospirocyclic, 5-20 member heterofused cycloalkyl, C5-C 10 cycloalkyl, 5-6-membered heteroaryl, ethynyl, vinyl;

[0241] Each f1 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0242] Each f2 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0243] Each f3 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0244] Each f4 is independently selected from an integer between 0 and 20.

[0245] In some embodiments, each A in L10 is independently selected from: -CH2-, -NH-, -O-, -S-;

[0246] Each R3 is selected independently from: R5, or none;

[0247] Each R4 group is independently selected from: -CH-, -NH-, -O-, -S-, -C (=O);

[0248] Each R5 is independently selected from: C6-C 10 Aryl, 5-6 member heteromonocyclic, C5-C6 cycloalkyl, 6-8 member heterobridged cycloalkyl, 6-8 member heterospirocyclic, 6-10 member heterocyclic cycloalkyl, C6-C 10 cycloalkyl, 5-6-membered heteroaryl, ethynyl, vinyl;

[0249] Each f1 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0250] Each f2 is independently selected from: 0, 1, 2, 3, 4;

[0251] Each f3 is independently selected from: 0, 1, 2, 3, 4;

[0252] Each f4 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0253] In some embodiments, each R5 is 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 ester bond or a thiophosphate ester bond with the phosphate group or thiophosphate group at the 3' and / or 5' end of the positive chain of the oligonucleotide drug via a hydroxyl group in W1.

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

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

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

[0265]

[0266] Among them, the target binding portions T, L1, L2, L3, L4, L6, L5, L7, and L8 are as described above, and L10, L11, and Z2 are as described above;

[0267] Z1 is the residue remaining after the oligonucleotide drug has lost a hydroxyl group;

[0268] W4 is the group obtained by removing a hydroxyl hydrogen or a hydroxyl protecting group from W1;

[0269] W5 is selected from: the group formed after the reaction of W2 and L9;

[0270] W6 is selected from: W4 or -W5-L10-L11-.

[0271] In some embodiments, W4 is selected from: -O-, or groups having the following structure:

[0272]

[0273] Or their chiral isomers.

[0274] In some of these embodiments, each W5 is independently selected from: 3-20 membered heterocyclic group, 5-20 membered heteroaromatic group, 3-20 membered heterocyclic thio group, -SS-.

[0275] In some embodiments, each W5 is independently selected from: 5-20 membered heterocyclic groups, 5-6 membered heteroaromatic groups, 5-6 membered heterocyclic thio groups, -SS-;

[0276] In some embodiments, each W5 is independently selected from:

[0277] -SS-.

[0278] Thirdly, the present invention also provides a carrier compound whose molecular structure is the same as that of the conjugate described in the present invention after the oligonucleotide drug is removed.

[0279] A fourth aspect of the present invention provides applications of the aforementioned compounds and conjugates, including the following technical solutions.

[0280] The use of the compounds described in this invention, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of carriers for delivering one or more oligonucleotide drugs.

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

[0282] The application of the conjugates described in this invention as active ingredients in the preparation of drugs that regulate the expression of one or more target genes.

[0283] The use of the compounds described in this invention, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, as carriers in the preparation of drugs that regulate the expression of one or more target genes.

[0284] The application of the carrier compound described in this invention as a carrier in the preparation of drugs that regulate the expression of one or more target genes.

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

[0286] This invention provides a novel scaffold compound and its conjugate with an oligonucleotide drug, which can be used for targeted delivery of oligonucleotide drugs. It can simultaneously deliver two nucleotide drugs targeting a single site or two nucleotide drugs targeting two sites, thereby enhancing the therapeutic effect of oligonucleotide drugs on corresponding diseases and has broad application prospects. Attached Figure Description

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

[0288] Figure 2 The curve showing the time-dependent inhibitory effect of conjugate 9A on the PCSK9 target in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg. Detailed Implementation

[0289] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0290] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0291] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0292] In the compounds of this invention, when any variable (e.g., R1, etc.) appears more than once in any component, the definition of each occurrence is independent of the definitions of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. It will be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be easily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is itself substituted by more than one group, it should be understood that these groups can be on the same carbon atom or on different carbon atoms, as long as structural stability is achieved.

[0293] To facilitate understanding of the technology of this invention, some terms and phrases are defined below.

[0294] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

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

[0296] The term "alkoxy" as used in this article refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0297] As used herein, the term "heterocyclic alkyl" or "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic cyclic substituent in which one or more ring atoms are selected from heteroatoms of N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Bicyclic or polycyclic groups include spirocyclic, fused, and bridged rings. Examples include: oxoheterobutyl, azaheterobutyl, morpholino, piperidinyl, tetrahydropyrrolyl, pyrrolylalkyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazoleyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiopheneyl, dihydrotriazolyl, dihydroazacyclobutane, tetrahydrofuranyl, tetrahydrothiopheneyl. And so on, and their N-oxides. The connection of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms.

[0298] As used herein, the term "heteroaryl" refers to an aromatic ring containing one or more heteroatoms selected from O, N, or S. This aromatic ring can be monocyclic, bicyclic, or polycyclic, and includes, but is not limited to: quinolinyl, pyrazolyl, pyrroloyl, thiophenyl, furanyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, etc. "Heteroaryl" is also understood to include any N-oxide derivative of a nitrogen-containing heteroaryl group. The linkage of heteroaryl groups can be achieved through carbon atoms or through heteroatoms.

[0299] As will be understood by those skilled in the art, the term “halo” or “halogen” as used herein refers to chlorine, fluorine, bromine, and iodine.

[0300] This invention includes compounds of formula (I) in their free form, as well as their pharmaceutically acceptable salts and stereoisomers. The stereoisomers described in this invention are (depending on their structure) enantiomers, diastereomers, syn- / anti-isomers, cis- / trans-isomers, epimers, and (E)- / (Z)-isomers. Compounds of formula (A) may be used in the context of this invention as pure stereoisomers or as any mixture of stereoisomers, preferably racemic in the latter case.

[0301] The abbreviations and structures of the nucleotide monomers used in the nucleic acid sequence listing of this invention are as follows:

[0302]

[0303]

[0304]

[0305] Those skilled in the art, based on existing technology, would recognize that the pharmaceutically acceptable salt of the siRNA could be a sodium or potassium salt, for example, a sodium salt of the siRNA produced during purification.

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

[0307] Oligonucleotides were synthesized using the conventional phosphoramide method, briefly described below: On a 12-channel nucleic acid synthesizer from Beijing Qingke Biotechnology Co., Ltd., 0.2-1 μmol of oligonucleotides were synthesized using a solid-phase oligonucleotide synthesis protocol. siRNA sequences were synthesized on a CPG-pre-packed column. Ammonolysis reagent was added to the synthesized oligonucleotides, and the mixture was incubated at 45-80°C to separate the oligonucleotides from the solid-phase support, freeing them. The crude oligonucleotides were then precipitated with ethanol, centrifuged at high speed to discard the supernatant, and this process was repeated twice to obtain crude oligonucleotides. The precipitate was then resuspended in DEPC-treated water. The crude oligonucleotides were purified using ion-pairing HPLC, and the collected product was dried to powder using a vacuum centrifuge. The purified product was dissolved in DEPC-treated water and analyzed by TOF LC-MS. The oligonucleotide concentration was determined, and the required volumes of equimolar amounts of the sense and antisense strands were calculated. Equimolar amounts of the sense and antisense strands were mixed thoroughly, and double-stranded oligonucleotides were prepared by annealing at 95°C for 5 minutes followed by natural 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 equivalence) and triethylamine (2.5 g, 24.8 mmol, 1.0 equivalence) in methanol (50 mL), ethyl 2,2,2-trifluoroacetate (4.4 g, 31.0 mmol, 1.25 equivalence) was added. The mixture was stirred overnight at room temperature. Methanol was removed under vacuum, the residue was dissolved in water, and the pH was adjusted to 1 to 2 with 1 mol / L hydrochloric acid solution. Extraction with ethyl acetate, washing of the organic phase with saturated brine, and concentration under vacuum gave 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 equivalence), (3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-3-ol (8.55 g, 20.4 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (7.8 g, 61.2 mmol, 3.0 equivalence) in N,N-dimethylformamide (60 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.1 g, 21.4 mmol, 1.05 equivalence) was added. 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 (elution buffer: petroleum ether / ethyl acetate = 2 / 1 to 3 / 1, with addition of 0.5% triethylamine) to give a white solid product N-(11-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)-11-oxoundecyl)-2,2,2-trifluoroacetamide (intermediate M-2, 11.6 g, yield: 80.7%). LCMS (ESI): m / z = 699 [M+H] + .

[0312] A mixture of N-(11-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)-11-oxoundecyl)-2,2,2-trifluoroacetamide (11.6 g, 16.5 mmol, 1.0 equivalence) and potassium hydroxide (9.3 g, 165 mmol, 10.0 equivalence) in methanol (110 mL) was stirred overnight at room temperature. 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: dichloromethane / methanol = 50 / 1, with 0.5% triethylamine) to give intermediate M (9.24 g, yield: 92.4%), a yellow oily product. 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)pyrrolidine-3-ol (10 g, 23.84 mmol, 1.0 equivalence), 12-methoxy-12-oxodecanoic acid (5.8 g, 23.84 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (12.3 g, 95.36 mmol, 4.0 equivalence) in dichloromethane (100 mL) was stirred at room temperature for 2.0 h. 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 oil product 1a-1 (16.7 g, yield: 79.3%).

[0317] Step 1b: Preparation of compound 1a-2: Lithium hydroxide monohydrate (3.1 g, 64.73 mmol, 2.5 equivalents) was added to a mixture of tetrahydrofuran / methanol / water (80 mL / 16 mL / 16 mL) of compound 1a-1 (16.7 g, 25.89 mmol, 1.0 equivalent). The mixture was reacted at 40 °C for 4 h. The solvent was removed from the reaction solution under reduced pressure. The residue was lyophilized in aqueous solution. The residue was dissolved in dichloromethane and filtered. The filtrate was 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: Benzyl chloroformate (2.03 g, 11.88 mmol, 1.2 equivalence) was added to a mixture of tetrahydrofuran (20 mL) and water (10 mL) of compound (azadiylbis(ethane-2,1-diyl)) tert-butyl carbamate (3.0 g, 9.9 mmol, 1.0 equivalence) and sodium carbonate (1.57 g, 14.85 mmol, 1.5 equivalence). The mixture was stirred overnight at room temperature. The mixture was diluted with water (50 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by 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: A hydrogen chloride-dioxane solution (4 mol / L, 15 mL) was added to a methanol (10 mL) mixture of compound 1a-3 (3.74 g, 8.55 mmol, 1.0 equivalence). The mixture was stirred at room temperature for 1.5 h. 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: At -20°C, isobutyl chloroformate (20.17 mL, 155.54 mmol, 1.0 equivalence) was added dropwise to a mixture of (S)-4-(tert-butoxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (45 g, 155.54 mmol, 1.0 equivalence) and N-methylmorpholine (15.7 g, 155.54 mmol, 1.0 equivalence) in tetrahydrofuran (220 mL). After stirring the mixture for 10 minutes, the mixture was filtered. At -30°C, a solution of sodium borohydride (11.8 g, 311.08 mmol, 2.0 equivalence) in water (70 mL) was slowly added to the filtrate. The mixture was brought 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 × 3). The combined organic layers were washed with saturated brine (100 ml × 1), dried over anhydrous sodium sulfate and concentrated to give a colorless oily substance 1a-5 (43.67 g, crude product).

[0321] Step 1f: Preparation of compound 1a-6: At 0°C, a mixture of compound 1a-5 (42.8 g, 155.54 mmol, 1.0 equivalent) and sodium bicarbonate (39.2 g, 466.62 mmol, 3.0 equivalent) in dichloromethane (300 mL) was added with Desmartin reagent (79.2 g, 186.65 mmol, 1.2 equivalent). The mixture was brought 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 × 1), saturated sodium bicarbonate solution (100 mL × 1), and saturated brine (100 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a pale yellow oily substance 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 equivalent), compound 1a-6 (38 g, 139.2 mmol, 2.44 equivalent), and acetic acid (3.26 mL, 57.01 mmol, 1.0 equivalent) in methanol (200 mL) was stirred at room temperature for 10 minutes. The mixture was cooled to 0°C, and sodium cyanoborohydride (12.53 g, 199.54 mmol, 3.5 equivalent) was slowly added. The mixture was brought to room temperature and stirred overnight. The solvent was removed under reduced pressure, and the residue was diluted with water (150 mL). The pH was adjusted to 9 with sodium bicarbonate solids, and the aqueous layer was extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with saturated brine (50 mL × 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 give 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: At 0°C, 1-chloroethyl chloroformate (7.16 mL, 66.34 mmol, 1.0 equivalence) was added dropwise to a mixture of compound 1a-7 (27.5 g, 44.23 mmol, 1.0 equivalence) and N,N-diisopropylethylamine (1.54 mL, 8.85 mmol, 0.2 equivalence) in acetonitrile (125 mL). The mixture was brought 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 × 1) and saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane:methanol = 200:1 to 20:1) to give a pale 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-tetramethylurea hexafluorophosphate (9.84 g, 23.78 mmol, 1.1 equivalent) was added to a mixture of compound 1a-8 (4.3 g, 22.7 mmol, 1.05 equivalent) and 3-(tert-butoxycarbonyl)amino)propionic acid (4.3 g, 22.7 mmol, 1.05 equivalent) in dichloromethane (70 mL). The mixture was stirred at room temperature for 2.5 h. The solvent was removed under reduced pressure. The residue was separated by column chromatography on silica gel (petroleum ether:ethyl acetate = 4:1 to 2:1) to give a pale 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 equivalence) and dioxane (4 mol / L, 90 mL) was stirred overnight at room temperature. 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 equivalence) and di-tert-butyl carbonate (24.36 mL, 105.95 mmol, 5.0 equivalence) were added. The mixture was stirred at room temperature for 48 hours. The tetrahydrofuran was removed under reduced pressure. The residue was diluted with water (200 mL) and washed with dichloromethane (60 mL × 4). The pH of the aqueous layer was adjusted to 3 by adding 2 mol / L dilute hydrochloric acid solution, and then extracted with ethyl acetate (60 mL × 4). The combined organic layers were washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a pale 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: At 0 °C, over 30 minutes, dropwise added a solution of Compound 1a-10 (3.76 g, 6.36 mmol, 1.0 equivalence), Compound 1a-4 (1.97 g, 6.36 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (4.92 g, 38.16 mmol, 6.0 equivalence) in dichloromethane (60 mL). The mixture was brought to room temperature and stirred for 20 minutes. 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 × 3). The combined organic layers were washed with saturated brine (50 mL × 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 give 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 equivalence) and dioxane (4 mol / L, 13 mL) was stirred at room temperature for 1.5 h. 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 equivalence), N,N-diisopropylethylamine (2.57 g, 19.92 mmol, 8.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.03 g, 7.97 mmol, 3.2 equivalence) were added. The mixture was stirred at room temperature for 1.5 h. The mixture was washed with saturated sodium bicarbonate solution (30 mL × 2) and saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane:methanol = 30:1, with the addition of 1% triethylamine) to give a pale 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 equivalence) and trifluoroacetic acid (0.15 mL, 1.95 mmol, 1.0 equivalence) 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). 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'-tetramethylurea hexafluorophosphate (0.26 g, 0.68 mmol, 1.2 equivalent) was added to a mixture of compound 1a-14 (1.0 g, 0.57 mmol, 1.0 equivalent), compound 1a-2 (0.396 g, 0.63 mmol, 1.1 equivalent), and N,N-diisopropylethylamine (0.441 g, 3.42 mmol, 6.0 equivalent) in dichloromethane (15 mL). The mixture was stirred at room temperature for 2.5 h. The solvent was removed under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (A: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 50%-70% over 30 minutes) to give 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 equivalent) was added to a mixture of compound 1a-15 (130 mg, 0.058 mmol, 1.0 equivalent), triethylamine (70 mg, 0.696 mmol, 12.0 equivalent), and 4-dimethylaminopyridine (3.5 mg, 0.029 mmol, 0.5 equivalent) in dichloromethane (1 mL). The mixture was stirred at room temperature for 2 days. The mixture was diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (5 mL × 2) and saturated brine (5 mL × 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-3O2) / 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: To a mixture of compound 1a-16 (111 mg, 0.047 mmol, 1.0 equivalence) and N,N-diisopropylethylamine (36 mg, 0.282 mmol, 6.0 equivalence) in N,N-dimethylformamide (5.5 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (21 mg, 0.056 mmol, 1.2 equivalence) and 1-hydroxybenzotriazole (9 mg, 0.066 mmol, 1.4 equivalence) were added. The mixture was shaken at room temperature for 5 minutes. CPG-NH2 (0.167 mmol / g, 550 mg, 0.092 mmol, 1.95 equivalence) was added, and the mixture was shaken at room temperature for 22 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (3 mL / 1 mL) and shaken at room temperature for 3 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to give 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 via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain conjugate 1A-1 (MS m / z: [MH]). - :calcd.8279.0520Found 8278.826).

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

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

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

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

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

[0338]

[0339]

[0340]

[0341] Step 3a: Preparation of compound 2a-2: A mixture of 6-heptanynic acid (3.00 g, 23.78 mmol, 1.0 equivalent), N,N-diisopropylethylamine (6.15 g, 47.56 mmol, 2.0 equivalent), compound 2a-1 (8.03 g, 24.97 mmol, 1.05 equivalent), and benzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (9.92 g, 26.16 mmol, 1.1 equivalent) in acetonitrile (30 mL) was stirred at room temperature for 2 hours. The mixture was added to 60.0 mL of saturated ammonium chloride aqueous solution and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1) to give 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: Trifluoroacetic acid (12 mL) was added to dichloromethane (60 mL) containing 7.82 g (18.20 mmol, 1.0 equivalent) of compound 2a-2, and the mixture was stirred at room temperature for 30 min. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 15:1) to give a pale 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 equivalent), N,N-diisopropylethylamine (6.70 g, 52.00 mmol, 2.0 equivalent), compound (3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-3-ol (11.40 g, 27.30 mmol, 1.05 equivalent), and benzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (10.80 g, 28.6 mmol, 1.1 equivalent) in acetonitrile (120.0 mL) was stirred at room temperature for 2 hours. The mixture was added to 200 mL of saturated ammonium chloride aqueous solution and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane to dichloromethane / methanol = 100 / 1, with the addition of 2% triethylamine) to give 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 equivalent) in acetonitrile (75.0 mL), and the mixture was stirred at room temperature for 15 min. The mixture was concentrated under vacuum, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1 to 10:1, with the addition of 1% triethylamine) to give a white, foamy 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 equivalent), N,N-diisopropylethylamine (1.73 g, 13.39 mmol, 2.0 equivalent), 2a-6 (3.97 g, 7.03 mmol, 1.05 equivalent), and benzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (3.05 g, 8.03 mmol, 1.2 equivalent) in acetonitrile (25.0 mL) was stirred at room temperature for 2 hours. The mixture was added to 60.0 mL of saturated ammonium chloride aqueous solution and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 to 50 / 1, 1% triethylamine) to give 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, succinic anhydride (1.83 g, 18.26 mmol, 3.0 equivalent) was added to a mixture of compound 2a-7 (2.50 g, 6.69 mmol, 1.0 equivalent), 4-dimethylaminopyridine (74.36 mg, 608.62 μmol, 0.1 equivalent), and triethylamine (3.70 g, 36.52 mmol, 6.0 equivalent) 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 mixture was separated, dried over anhydrous sodium sulfate, and concentrated under vacuum to give a pale purple bubbly 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 equivalent), benzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (2.14 g, 5.63 mmol, 1.5 equivalent), and N,N-diisopropylethylamine (0.97 g, 7.50 mmol, 2.0 equivalent) in anhydrous acetonitrile (42.0 mL) was shaken at room temperature for 10 min. CPG-NH2 (13.50 g, 500 μmol / g, 1.8 equivalent) was added, and the mixture was shaken at room temperature for 5 h. The mixture was filtered, and the filter cake was washed with acetonitrile and dichloromethane, respectively, and dried under vacuum for 1 h. Add 30 mL of anhydrous pyridine and 10 mL of acetic anhydride to the solid, shake at room temperature for 2 hours, filter the mixture, wash the filter cake with dichloromethane, acetonitrile, and dichloromethane, and dry 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 via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification 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 equivalent), (2-aminoethyl)carbamate (9.71 g, 50.00 mmol, 1.0 equivalent), N,N-diisopropylethylamine (12.90 g, 10.00 mmol, 2.0 equivalent), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (21.72 g, 52.50 mmol, 1.05 equivalent) 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 piperidine / acetonitrile (50 mL / 200 mL) of compound 2b-2 (crude product) 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 (elution: dichloromethane to dichloromethane / methanol = 20 / 1) to give 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 equivalences), tert-butyl (2-bromoethyl)carbamate (2.96 g, 13.20 mmol, 1.0 equivalences), and potassium carbonate (3.64 g, 26.40 mmol, 2.0 equivalences) in N,N-dimethylformamide (80 mL) was stirred 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. 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 equivalent), compound 2b-5 (2.39 g, 7.08 mmol, 0.95 equivalent), N,N-diisopropylethylamine (1.92 g, 14.90 mmol, 2.0 equivalent), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (3.08 g, 7.45 mmol, 1.0 equivalent) 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 equivalent) in 40 mL of dioxane-hydrochloride was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step (crude product) without purification. 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 equivalent), compound 1a-12 (7.10 g, 15.87 mmol, 3.1 equivalent), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (6.20 g, 16.31 mmol, 3.2 equivalent) 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 equivalence), palladium hydroxide / carbon (1.5 g, 25% by mass), and trifluoroacetic acid (371 mg, 3.25 mmol, 1.0 equivalence) in ethanol (100 mL) was stirred overnight at room temperature. The mixture was filtered through diatomaceous earth. The filter cake was washed with ethanol, and the filtrate was concentrated. The residue was used directly for 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 equivalence) was added to a mixture of 6-azidohexanoic acid (30 g, 190.8 mmol, 1.0 equivalence) and 1-hydroxypyrrolidine-2,5-dione (24 g, 210 mmol, 1.1 equivalence) in dichloromethane / N,N-dimethylformamide (270 / 30 mL). The mixture was stirred overnight at room temperature. Dichloromethane was removed under vacuum, and 1 mol / L hydrochloric acid solution and methyl tert-butyl ether were added. The mixture was separated, and the organic layer was washed with sodium bicarbonate solution and saturated brine. The organic phase was dried and concentrated under vacuum to give 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 equivalence), compound 2b-10 (824 mg, 3.24 mmol, 1.05 equivalence), and N,N-diisopropylethylamine (1.19 g, 9.22 mmol, 3.0 equivalence) in acetonitrile (60 mL) was stirred at room temperature for 1 hour. The reaction proceeded directly 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 equivalence), N,N-diisopropylethylamine (796 mg, 6.17 mmol, 2.0 equivalence), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (1.4 g, 3.39 mmol, 1.1 equivalence) in acetonitrile (60 mL) was stirred for 1 hour. The mixture was then purified directly by high performance liquid chromatography (column: UniSil 10-120C18, 30 × 250 mm) to give 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 equivalence), succinic anhydride (793 mg, 7.93 mmol, 6.0 equivalence), triethylamine (1.60 g, 15.84 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (16 mg, 0.13 mmol, 0.1 equivalence) 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). LCMS (ESI): m / z = 1072 [(M-302) / 2+H] + .

[0360] Step 3t: Preparation of compound 2b-14: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (56 mg, 0.15 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (23 mg, 0.17 mmol, 1.4 equivalence), and N,N-diisopropylethylamine (63 mg, 0.49 mmol, 4.0 equivalence) were added to an N,N-dimethylformamide solution of compound 2b-13 (300 mg, 0.12 mmol, 1.4 equivalence). The mixture was shaken at room temperature for 5 minutes. 1.5 g of CPG-NH2 was added to the mixture, and the mixture was shaken overnight at room temperature. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (12 mL / 4 mL), and the mixture was 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 give 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 via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification 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 added sequentially 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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added, and the mixture was shaken for 5-10 minutes. The crude product of conjugate 2A-1 (approximately 2.3 mg) was obtained by ethanol precipitation, and 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 equal amounts of conjugate 2A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 2A.

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

[0365] Compound 2a-10-B-ssRNA-2 (182 nmol, 1 equivalent) and 2b-15-B-ssRNA-1 (200 nmol, 1.1 equivalent) were sequentially added 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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added, and the mixture was shaken for 5-10 minutes. Ethanol precipitation yielded approximately 2.0 mg of crude conjugate 2B-1, which was then purified by HPLC to obtain conjugate 2B-1 (MS m / z: [MH]). - :calcd.15682.1370Found 15682.1011).

[0366] Take equal amounts of conjugate 2B-1 and two complementary antisense chains (1167AM25 and 1167AM25) solutions, mix them thoroughly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain 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: 6.04 g benzyl glycine hydrochloride (29.96 mmol, 2.0 equivalence), (9H-fluorene-9-yl)methyl(2-oxoethyl)carbamate (4.21 g, 14.98 mmol, 1.0 equivalence), and acetic acid (90 mg, 1.5 mmol, 0.1 equivalence) were added to 80 mL of methanol and stirred at room temperature for 30 min. Sodium cyanoborohydride (1.4 g, 22.47 mmol, 1.5 equivalence) was added, and the mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure, and extracted with ethyl acetate after adding a saturated aqueous sodium carbonate solution. 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 give a 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: 18 mL of an aqueous solution of sodium carbonate (3.16 g, 29.77 mmol, 4.0 equivalence) was added to 70 mL of a tetrahydrofuran solution of compound 3a-1 (3.22 g, 7.44 mmol, 1.0 equivalence). Di-tert-butyl dicarbonate (3.24 g, 14.88 mmol, 2.0 equivalence) was added, and the mixture was stirred at room temperature for 2 hours. Extraction was performed 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 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 equivalent) was dissolved in 90 mL of methanol, and 341 mg of 10% palladium on carbon was added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily substance 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 equivalence), (2-aminoethyl)carbamate (1.17 g, 6.04 mmol, 1.0 equivalence), N,N-diisopropylethylamine (1.56 g, 12.08 mmol, 2.0 equivalence), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (2.5 g, 6.04 mmol, 1.0 equivalence) were added to 20 mL of N,N-dimethylformamide. 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 give a colorless syrupy compound 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 equivalent) and 1.5 mL piperidine were added to 24 mL acetonitrile. The mixture was stirred at room temperature for 30 min. 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 the addition of triethylamine) to give 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 equivalence), tert-butyl (2-oxoethyl)carbamate (768 mg, 4.82 mmol, 1.0 equivalence), acetic acid (29 mg, 0.48 mmol, 0.1 equivalence), and sodium cyanoborohydride (337 mg, 5.2 mmol, 1.1 equivalence) were added to 20 mL of methanol, and the mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure, and extracted with ethyl acetate by adding sodium carbonate solution. 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 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 equivalence), compound 2b-5 (631 mg, 1.87 mmol, 1.1 equivalence), N,N-diisopropylethylamine (439 mg, 3.4 mmol, 2.0 equivalence), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (774 mg, 1.87 mmol, 1.7 equivalence) were added to 15 mL of N,N-dimethylformamide. 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 give a 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 equivalent) was added to 40 mL of a 4 mol / L solution of hydrogen chloride-dioxane. 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 equivalent), compound 1a-12 (2.05 g, 4.58 mmol, 3.2 equivalent), N,N-diisopropylethylamine (1.85 g, 14.3 mmol, 10.0 equivalent), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.74 g, 4.58 mmol, 3.2 equivalent) were added to 30 mL of acetonitrile. 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 a 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 equivalent) was dissolved in 60 mL of ethanol, and trifluoroacetic acid (95 mg, 0.84 mmol, 1.0 equivalent) and 200 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 3a-10 (1.42 g, crude product) as a white solid. 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 equivalence), N,N-diisopropylethylamine (162 mg, 1.25 mmol, 1.5 equivalence), and compound 2b-10 (234 mg, 0.92 mmol, 1.1 equivalence) were added to 15 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h. N,N-diisopropylethylamine (162 mg, 1.25 mmol, 1.5 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (414 mg, 1.0 mmol, 1.2 equivalence), and intermediate M were added to the mixture, and the mixture was stirred at room temperature for 1 h. 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 high-performance liquid chromatography (HPLC) (eluent: potassium dihydrogen phosphate-potassium hydroxide buffer / acetonitrile, pH 7.0) to give a white solid compound 3a-11 (compound 55) (550 mg, yield: 27.9%). LCMS (ESI): m / z = 1022 [(M-302) / 2+H] + .

[0383] Step 4l: Preparation of compound 3a-12: Compound 3a-11 (550 mg, 0.235 mmol, 1.0 equivalence), N,N-diisopropylethylamine (285 mg, 2.82 mmol, 12.0 equivalence), succinic anhydride (141 mg, 1.41 mmol, 6.0 equivalence), and 4-dimethylaminopyridine (3 mg, 0.024 mmol, 0.1 equivalence) were added to 3 mL of dichloromethane, and the mixture 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 under reduced pressure to give compound 3a-12 (550 mg, crude) as a white solid. 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 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (117 mg, 0.308 mmol, 1.4 equivalence), 1-hydroxybenzotriazole (49 mg, 0.36 mmol, 1.6 equivalence), and N,N-diisopropylethylamine (133 mg, 1.03 mmol, 4.6 equivalence) 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 overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and dried under reduced pressure. 18 mL of pyridine and 6 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 give 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 via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification 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: Take 109 nmol of 2a-10-A-ssRNA-2 (109 nmol, 1 equivalent) and 120 nmol of 3a-14-A-ssRNA-1 (1.1 equivalent), and add 32 μL of PBS buffer (pH 8.0, 0.5 M), 186 μL of DEPC water, 160 μL of DMF, and 171 μL of premixed CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL). After mixing, add 1 mg of sodium ascorbate, purge with nitrogen, and react at 35 °C for 2 h. After the reaction is complete, add 9 μL of ammonia 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). Then purify conjugate 3A-1 by HPLC (MS m / z: [MH]). - :calcd.16299.4826Found 16299.417).

[0387] Step 4p: Preparation of conjugate 3A: Take equal amounts of conjugate 3A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 3A.

[0388] Example 5: Preparation of conjugate 4A (prepared according to scheme 4a):

[0389]

[0390]

[0391] Step 5a: Preparation of compound 4a-2: Under nitrogen protection, a mixture of compound 4a-1 (10 g, 62 mmol, 1.0 equivalent), compound 2b-10 (17 g, 68 mmol, 1.1 equivalent), and N,N-diisopropylethylamine (18 g, 136 mmol, 2.2 equivalent) in acetonitrile (100 mL) was stirred overnight at room temperature. The acetonitrile was removed under vacuum, and a 1 mol / L hydrochloric acid solution and methyl tert-butyl ether were added. The mixture was separated, and the organic layer was washed with water and saturated brine. The organic phase was dried and concentrated under vacuum to give a yellow oily product 4a-2 (18.5 g, yield: 99.4%). LCMS (ESI): m / z = 301 [M+H] + .

[0392] Step 5b: Preparation of compound 4a-3: To a mixture of compound 4a-2 (18.5 g, 61.5 mmol, 1.0 equivalent), tert-butyl 3-(2-aminoethoxy)propionate (11.6 g, 61.5 mmol, 1.0 equivalent), and N,N-diisopropylethylamine (17.4 g, 135.2 mmol, 2.0 equivalent) in N,N-dimethylformamide (180 mL), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (28 g, 67.6 mmol, 1.1 equivalent) was added. 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 (elution buffer: petroleum ether / ethyl acetate = 2 / 1 to dichloromethane / methanol = 20 / 1) to give product 4a-3 as a white solid (14.8 g, yield: 51.4%). LCMS (ESI): m / z = 472 [M+H] + .

[0393] Step 5c: Preparation of compound 4a-4: A mixture of compound 4a-3 (8.86 g, 20.7 mmol, 1.0 equivalent) and trifluoroacetic acid / dichloromethane (10 / 50 mL) was stirred at room temperature for 5.0 h. The mixture was concentrated under vacuum to give a yellow oily product 4a-4 (8 g, crude). LCMS (ESI): m / z = 416 [M+H] + .

[0394] Step 5d: Preparation of compound 4a-5: To a mixture of compound 4a-4 (8.86 g, 18.7 mmol, 1.0 equivalence), (3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-3-ol (7.8 g, 18.7 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (9.6 g, 75 mmol, 4.0 equivalence) in N,N-dimethylformamide (90 mL), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (8.1 g, 19.7 mmol, 1.1 equivalence) was added. 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 (elution buffer: petroleum ether / dichloromethane / methanol = 50 / 50 / 1 to 30 / 30 / 1) to give product 4a-5 as a white solid (7.5 g, yield: 49%). LCMS (ESI): m / z = 817 [M+H] + .

[0395] Step 5e: Preparation of compound 4a-6: A mixture of compound 4a-5 (7.5 g, 9.19 mmol, 1.0 equivalent) and lithium hydroxide monohydrate (1.1 g, 27.5 mmol, 3.0 equivalent) in tetrahydrofuran / methanol / water (60 / 30 / 30 mL) was stirred overnight at room temperature. 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] - .

[0396] Step 5f: Preparation of compound 4a-7 (compound 53): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (148 mg, 0.39 mmol, 1.2 equivalence) was added to a mixture of compound 1a-14 (570 mg, 0.32 mmol, 1.0 equivalence), compound 4a-6 (260 mg, 0.32 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (146 mg, 1.13 mmol, 3.5 equivalence) in N,N-dimethylformamide (2.5 mL). The mixture was stirred overnight at room temperature. The mixture was purified by preparative high-performance liquid chromatography (A: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%-90% over 30 minutes) to give a white solid product 4a-7 (compound 53) (245 mg, yield: 31%). LCMS(ESI): m / z=1065.4[(M-302) / 2+H] + .

[0397] Step 5g: Preparation of compound 4a-8: Succinic anhydride (60.5 mg, 0.60 mmol, 6.0 equivalence) was added to a mixture of compound 4a-7 (245 mg, 0.10 mmol, 1.0 equivalence), triethylamine (121 mg, 1.2 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (12 mg, 0.10 mmol, 1.0 equivalence) in dichloromethane (2 mL). The mixture was stirred at room temperature for 20 hours. The mixture was diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (10 mL × 2) and saturated brine (10 mL × 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] + .

[0398] Step 5h: Preparation of compound 4a-9: To a mixture of compound 4a-8 (253 mg, 0.10 mmol, 1.0 equivalence) and N,N-diisopropylethylamine (77 mg, 0.60 mmol, 6.0 equivalence) in N,N-dimethylformamide (10 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 equivalence) and 1-hydroxybenzotriazole (20 mg, 0.15 mmol, 1.5 equivalence) were added. The mixture was shaken at room temperature for 5 minutes. CPG-NH2 (0.167 mmol / g, 1.27 g, 0.21 mmol, 2.1 equivalence) was added, and the mixture was shaken at room temperature for 22 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (12 mL / 4 mL) and shaken at room temperature for 3 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to give a white solid 4a-9 (1.36 g, loading: 30 μmol / g).

[0399] Step 5i: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 4a-10-A-ssRNA-1 (MS m / z: [MH]). - :calcd.9067.5135Found9066.8616).

[0400] 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 sequentially to 86 μL of PBS buffer (pH 8.0, 0.5 M), 189 μL of DEPC water, 429 μL of DMF, and 161 μL of premixed CuSO4-THPTA (1:1) solution (conc. 40 nmol / μL). After mixing, 2.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 25 μL of ammonia and 26 mg of EDTA were added, and the mixture was shaken for 5-10 minutes. The crude product of conjugate 4A-1 (approximately 2.6 mg) was obtained by ethanol precipitation. This crude product was then purified by HPLC to obtain conjugate 4A-1 (MS m / z: [MH]). - :calcd.16385.4886Found16385.4753).

[0401] Step 5k: Preparation of conjugate 4A: Take equal amounts of conjugate 4A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 4A.

[0402] Example 6: Preparation of conjugate 6A (prepared according to scheme 5a):

[0403]

[0404]

[0405] 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 equivalent), benzylamine (1.80 g, 15.99 mmol, 0.5 equivalent), and potassium carbonate (13.9 g, 100.75 mmol, 3.0 equivalent) in acetonitrile (60 mL) was stirred overnight at 75 °C. 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 (elution: 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] + .

[0406] 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 equivalent) and palladium hydroxide / carbon (1.26 g, 20% mass fraction) in ethanol (50 mL) was stirred overnight at room temperature. The mixture was filtered, and the filtrate was concentrated to give product 5a-2 (crude product). LCMS (ESI): m / z = 392 [M+H] + .

[0407] 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 equivalents), and sodium carbonate (2.07 g, 19.54 mmol, 1.5 equivalents) 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] + .

[0408] Step 6d: Preparation of compound 5a-4: A mixture of compound 5a-3 (5.4 g, 10.27 mmol, 1.0 equivalence) and dioxane (40 mL) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly for the next step (crude product) without purification. LCMS (ESI): m / z = 326 [M+H] + .

[0409] Step 6e: Preparation of compound 5a-5: At 0 °C, over 30 minutes, a mixture of dichloromethane containing compounds 5a-4 (2.8 g, 7.05 mmol, 1.0 equivalence), 1a-10 (4.17 g, 7.05 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (4.55 g, 35.26 mmol, 5.0 equivalence) was added dropwise to a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (5.90 g, 15.52 mmol, 2.2 equivalence). 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 (elution: ethyl acetate / methanol = 20 / 1) to give a white solid product 5a-5 (1.54 g, yield: 24.84%). LCMS (ESI): m / z = 880 [M+H] + .

[0410] Step 6f: Preparation of compound 5a-6: A mixture of compound 5a-5 (1.54 g, 1.75 mmol, 1.0 equivalence) and dioxane (10 mL) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly for the next step (crude product) without purification. LCMS (ESI): m / z = 580 [M+H] + .

[0411] 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 equivalence), compound 1a-12 (2.35 g, 5.25 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.13 g, 5.60 mmol, 3.2 equivalence) 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 (elution buffer: 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] + .

[0412] 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 equivalent), palladium hydroxide / carbon (380 g, 20% by mass), and trifluoroacetic acid (116 mg, 1.02 mmol, 1.0 equivalent) in isopropanol (20 mL) was stirred overnight at room temperature. The mixture was filtered through diatomaceous earth. The filter cake was washed with ethanol, and the filtrate was concentrated. The residue (2.08 g, crude product) was used directly for the next step without further purification. LCMS (ESI): m / z = 868 [M / 2+H] + .

[0413] Step 6i: Preparation of compound 5a-9 (compound 51): A mixture of compound 5a-8 (792 mg, 0.46 mmol, 1.0 equivalence), compound 4a-6 (367 mg, 0.46 mmol, 1.0 equivalence), N,N-diisopropylethylamine (177 mg, 1.37 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (209 mg, 0.55 mmol, 1.2 equivalence) 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: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%-90% over 30 minutes) to give a white solid product 5a-9 (compound 51) (210 mg, yield: 18.26%). LCMS(ESI): m / z=1109[(M-302) / 2+H] + .

[0414] Step 6j: Preparation of compound 5a-10: A mixture of compound 5a-9 (210 mg, 0.083 mmol, 1.0 equivalence), succinic anhydride (51 mg, 0.50 mmol, 6.0 equivalence), triethylamine (101 mg, 1.00 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (2 mg, 0.008 mmol, 0.1 equivalence) in dichloromethane (5 mL) was stirred overnight at room temperature. 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 under vacuum to give a white solid product 5a-10 (210 mg, crude). LCMS (ESI): m / z = 1159 [(M-302) / 2+H] + .

[0415] Step 6k: Preparation of compound 5a-11: To an N,N-dimethylformamide solution (8 mL) of compound 5a-10 (210 mg, 0.08 mmol, 1.0 equivalence), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (36 mg, 0.096 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (16 mg, 0.11 mmol, 1.4 equivalence), and N,N-diisopropylethylamine (42 mg, 0.32 mmol, 4.0 equivalence) were added, and the mixture was shaken at room temperature for 5 min. CPG-NH2 (850 mg) was added to the mixture, and the mixture was shaken overnight at room temperature. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 h. The residue was added to pyridine / acetic anhydride (6 mL / 2 mL), and the mixture was shaken at room temperature for 3 h. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to give a light yellow solid product 5a-11 (990 mg, loading: 40 μmol / g).

[0416] Step 61: Preparation of compound 5a-12-A-ssRNA-1: Compound 5a-11 was synthesized via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 5a-12-A-ssRNA-1 (MS m / z: [MH]). - :calcd.9155.6195Found9155.8439).

[0417] 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 sequentially to 59 μL of PBS buffer (pH 8.0, 0.5 M), 257 μL of DEPC water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL). After mixing, 1.7 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 17 μL of ammonia and 18 mg of EDTA were added, and the mixture was shaken for 5-10 minutes. The crude conjugate 6A-1 was obtained by ethanol precipitation, yielding approximately 2.2 mg of crude product. This crude product was then purified by HPLC to obtain conjugate 6A-1 (MS m / z: [MH]). - :calcd.16473.5946Found16471.1600).

[0418] Step 6n: Preparation of conjugate 6A: Take equal amounts of conjugate 6A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 6A.

[0419] Example 7: Preparation of conjugate 7A (prepared according to scheme 6a):

[0420]

[0421]

[0422] Step 7a: Preparation of compound 6a-1: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (6.11 g, 16.07 mmol, 1.15 equivalent) was added to a mixture of methyl piperidine-4-carboxylate (2.0 g, 13.97 mmol, 1.0 equivalent), 3-(tert-butoxycarbonyl)amino)propionic acid (2.77 g, 14.67 mmol, 1.05 equivalent) and N,N-diisopropylethylamine (3.6 g, 27.94 mmol, 2.0 equivalent) in dichloromethane (15 mL). 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 × 2). The combined organic layers were washed with saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a brown oily substance 6a-1 (8.64 g, crude product). LCmS (ESI): m / z = 315 [m + H] + .

[0423] Step 7b: Preparation of compound 6a-2: A mixture of compound 6a-1 (4.39 g, 13.97 mmol, 1.0 equivalence) and dioxane (4 m, 17 mL) was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (40 mL). Heptan-6-acetylic acid (1.76 g, 13.97 mmol, 1.0 equivalence), N,N-diisopropylethylamine (5.4 g, 41.91 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.84 g, 15.37 mmol, 1.1 equivalence) were added. The mixture was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The mixture was diluted with a saturated sodium carbonate solution (50 mL) and then extracted with ethyl acetate (30 mL × 2). The combined organic layers were washed with saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane:methanol = 60:1) to give a pale yellow oil 6a-2 (4.5 g, yield: 100%). LCMS (ESI): m / z = 323 [M+H] + .

[0424] Step 7c: Preparation of compound 6a-3: Sodium hydroxide (0.84 g, 20.96 mmol, 1.5 equivalence) was added to a mixture of compound 6a-2 (4.5 g, 13.97 mmol, 1.0 equivalence) in methanol (7.5 mL) and water (7.5 mL). The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The pH was adjusted to 3 with 2 mol / L dilute hydrochloric acid solution, and then extracted with ethyl acetate (20 mL × 5). The combined organic layers were washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a pale yellow oil, 6a-3 (4.32 g, crude). LCMS (ESI): m / z = 309 [M+H] + .

[0425] Step 7d: Preparation of compound 6a-4: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (400 mg, 1.05 mmol, 1.2 equivalence) was added to a mixture of compound 2a-6 (415 mg, 1.35 mmol, 1.52 equivalence), compound 6a-3 (500 mg, 0.886 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (286 mg, 2.22 mmol, 2.5 equivalence) in N,N-dimethylformamide (3 mL). The mixture was stirred at room temperature for 1 hour. The mixture was purified by preparative high-performance liquid chromatography (A: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%-90% over 30 minutes) to give compound 6a-4 (363 mg, yield: 48%) as a white solid. LCMS(ESI): m / z = 877.7 [M + Na] + .

[0426] Step 7e: Preparation of compound 6a-5: Succinic anhydride (255 mg, 2.55 mmol, 6.0 equivalent) was added to a mixture of compound 6a-4 (363 mg, 0.43 mmol, 1.0 equivalent), triethylamine (515 mg, 5.1 mmol, 12.0 equivalent), and 4-dimethylaminopyridine (26 mg, 0.21 mmol, 0.5 equivalent) in dichloromethane (3 mL). The mixture was stirred overnight at room temperature. The mixture was diluted with dichloromethane (15 mL), washed with saturated sodium bicarbonate solution (15 mL × 2) and saturated brine (15 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a brown solid product 6a-5 (448 mg, crude). LCMS (ESI): m / z = 978 [M + Na] + .

[0427] Step 7f: Preparation of compound 6a-6: To a mixture of compound 6a-5 (210 mg, 0.22 mmol, 1.0 equivalence) and N,N-diisopropylethylamine (170 mg, 1.32 mmol, 6.0 equivalence) in N,N-dimethylformamide (20 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (125 mg, 0.33 mmol, 1.5 equivalence) and 1-hydroxybenzotriazole (45 mg, 0.33 mmol, 1.5 equivalence) were added. The mixture was shaken at room temperature for 5 minutes. CPG-NH2 (2.37 g, 0.40 mmol, 1.8 equivalence) was added. The mixture was shaken at room temperature overnight. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (20 mL / 6 mL) and shaken at room temperature for 3 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to give a pale yellow solid product 6a-6 (2.48 g, loading: 67 μmol / g).

[0428] Step 7g: Preparation of compound 6a-7-A-ssRNA-2: Compound 6a-6 was synthesized via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 6a-7-A-ssRNA-2 (MS m / z: [MH]). - :calcd.7252.9070Found7252.1811).

[0429] Step 7h: Preparation of conjugate 7A-1: Take 200 nmol (1 equivalent) of 6a-7-A-ssRNA-2 and 2b-15-A-ssRNA-1 (220 nmol (1.1 equivalent)), and add 59 μL of PBS buffer (pH 8.0, 0.5 M), 397 μL of DEPC water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL). After mixing, add 1.7 mg of sodium ascorbate, purge with nitrogen, and react at 35 °C for 2 h. After the reaction is complete, add 17 μL of ammonia and 18 mg of EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain approximately 2.0 mg of crude conjugate 7A-1. Then, purify by HPLC to obtain conjugate 7A-1 (MS m / z: [MH]). - :calcd.16234.4146Found16234.1400).

[0430] Step 7i: Preparation of conjugate 7A: Take equal amounts of conjugate 7A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, 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 double-chain conjugate 7A.

[0431] Example 8: Preparation of conjugate 8A (prepared according to scheme 7a):

[0432]

[0433]

[0434] Step 8a: Preparation of compound 7a-1: Methyl 4-chloro-4-oxobutyrate (1.38 mL, 11.28 mmol, 1.0 equivalence) was added dropwise to a mixture of piperazine-1-carboxylate (2.0 g, 10.74 mmol, 1.0 equivalence) and N,N-diisopropylethylamine (2.08 g, 16.11 mmol, 1.5 equivalence) in dichloromethane (15 mL) at 0 °C. The mixture was stirred at 0 °C for 20 min. 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 × 2). The combined organic layers were washed with saturated brine (30 mL × 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] + .

[0435] Step 8b: Preparation of compound 7a-2: A mixture of compound 7a-1 (3.19 g, 10.60 mmol, 1.0 equivalence) and dioxane (4 mol / L, 12 mL) was stirred at room temperature for 1.5 h. 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 equivalence), N,N-diisopropylethylamine (4.31 g, 33.39 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.64 g, 12.19 mmol, 1.15 equivalence) were added. The mixture was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The mixture was diluted with a saturated sodium carbonate solution (50 mL) and then extracted with ethyl acetate (30 mL × 2). The combined organic layers were 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] + .

[0436] Step 8c: Preparation of compound 7a-3: A mixture of compound 7a-2 (4.73 g, 10.60 mmol, 1.0 equivalence) and dioxane (4 mol / L, 12 mL) was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (30 mL). Heptan-6-acetylic acid (1.34 g, 10.60 mmol, 1.0 equivalence), N,N-diisopropylethylamine (4.1 g, 31.8 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.43 g, 11.66 mmol, 1.1 equivalence) were added. The mixture was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The mixture was diluted with a saturated sodium carbonate solution (50 mL) and then extracted with ethyl acetate (30 mL × 2). The combined organic layers were washed with saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane:methanol = 40:1) to give a brown oily product 7a-3 (3.72 g, yield: 77%). LCMS (ESI): m / z = 455 [M+H] + .

[0437] Step 8d: Preparation of compound 7a-4: Sodium hydroxide (0.49 g, 12.29 mmol, 1.5 equivalence) was added to a mixture of compound 7a-3 (3.72 g, 8.19 mmol, 1.0 equivalence) in methanol (7.5 mL) and water (7.5 mL). The mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The pH was adjusted to 3 with 2 mol / L dilute hydrochloric acid solution, and then extracted with ethyl acetate (20 mL × 5). The combined organic layers were washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a pale yellow oil 7a-4 (3.65 g, crude). LCMS (ESI): m / z = 441 [M+H] + .

[0438] Step 8e: Preparation of compound 7a-5: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (411 mg, 1.08 mmol, 1.2 equivalence) was added to a mixture of compound 7a-4 (600 mg, 1.36 mmol, 1.52 equivalence), compound 2a-6 (510 mg, 0.90 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (290 mg, 2.25 mmol, 2.5 equivalence) in N,N-dimethylformamide (3 mL). The mixture was stirred at room temperature for 1 hour. The mixture was purified by preparative high-performance liquid chromatography (A: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%-90% over 30 minutes) to give a white solid product 7a-5 (201 mg, yield: 23%). LCMS(ESI): m / z=684.5[M-302+H] + .

[0439] Step 8f: Preparation of compound 7a-6: Succinic anhydride (123 mg, 1.22 mmol, 6.0 equivalent) was added to a mixture of compound 7a-5 (201 mg, 0.204 mmol, 1.0 equivalent), triethylamine (247 mg, 2.45 mmol, 12.0 equivalent), and 4-dimethylaminopyridine (12 mg, 0.102 mmol, 0.5 equivalent) in dichloromethane (2 mL). The mixture was stirred at room temperature for 4 hours. The mixture was diluted with dichloromethane (15 mL), washed with saturated sodium bicarbonate solution (15 mL × 2) and saturated brine (15 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a white solid 7a-6 (243 mg, crude). LCMS (ESI): m / z = 784.6 [M-302+H] + .

[0440] Step 8g: Preparation of compound 7a-7: To a mixture of compound 7a-6 (222 mg, 0.20 mmol, 1.0 equivalence) and N,N-diisopropylethylamine (158 mg, 1.22 mmol, 6.0 equivalence) in N,N-dimethylformamide (20 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (116 mg, 0.31 mmol, 1.5 equivalence) and 1-hydroxybenzotriazole (41 mg, 0.31 mmol, 1.5 equivalence) were added. The mixture was shaken at room temperature for 5 minutes. CPG-NH2 (2.20 g, 0.37 mmol, 1.8 equivalence) was added. The mixture was shaken at room temperature overnight. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (20 mL / 6 mL) and shaken at room temperature for 3 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to give a pale yellow solid product 7a-7 (2.34 g, loading: 62 μmol / g).

[0441] Step 8h: Preparation of compound 7a-8-A-ssRNA-2: Compound 7a-7 was synthesized via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 7a-8-A-ssRNA-2 (MS m / z: [MH]). - :calcd.7384.0380Found7383.3414).

[0442] 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 sequentially to 59 μL of PBS buffer (pH 8.0, 0.5 M), 290 μL of DEPC water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL). After mixing, 1.7 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 17 μL of ammonia and 18 mg of EDTA were added, and the mixture was shaken for 5-10 minutes. The crude product of conjugate 8A-1 was obtained by ethanol precipitation, yielding approximately 2.1 mg of crude product. This crude product was then purified by HPLC to obtain conjugate 8A-1 (MS m / z: [MH]). - :calcd.16365.5456Found16365.2100).

[0443] Step 8j: Preparation of conjugate 8A: Take equal amounts of conjugate 8A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 8A.

[0444] Example 9: Preparation of conjugate 9A (prepared according to scheme 8a):

[0445]

[0446]

[0447] Step 9a: Preparation of compound 8a-2: A mixture of compound 8a-1 (2.72 g, 18.99 mmol, 1.0 equivalent), methyl piperidine 4-carboxylate (2.72 g, 18.99 mmol, 1.0 equivalent), N,N-diisopropylethylamine (4.90 g, 37.98 mmol, 2.0 equivalent), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.47 g, 22.28 mmol, 1.2 equivalent) 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). LCMS (ESI): m / z = 389 [M+H] + .

[0448] 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 (crude) without purification. LCMS (ESI): m / z = 289 [M+H] + .

[0449] Step 9c: Preparation of compound 8a-4: A mixture of compound 8a-3 (crude), hepta-6-acetylic acid (2.35 g, 18.69 mmol, 1.0 equivalent), N,N-diisopropylethylamine (7.23 g, 56.06 mmol, 3.0 equivalent), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.52 g, 22.42 mmol, 1.2 equivalent) in dichloromethane (50 mL) was stirred at room temperature for 30 min. 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 (elution: 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] + .

[0450] Step 9d: Preparation of compound 8a-5: A mixture of compound 8a-4 (8.19 g, 18.69 mmol, 1.0 equivalent) and sodium hydroxide (1.24 g, 30.94 mmol, 1.5 equivalent) 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 a yellow oily product 8a-5 (6.0 g, crude). LCMS (ESI): m / z = 383 [M+H] + .

[0451] Step 9e: Preparation of compound 8a-6: A mixture of compound 8a-5 (1.1 g, 2.87 mmol, 1.0 equivalence), compound 2a-6 (1.22 g, 2.15 mmol, 0.75 equivalence), N,N-diisopropylethylamine (1.11 g, 8.62 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.31 g, 3.45 mmol, 1.2 equivalence) 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: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%-90% over 30 minutes) to give a white solid product 8a-6 (320 mg, yield: 16%). LCMS(ESI): m / z=627[(M-302)+H] + .

[0452] Step 9f: Preparation of compound 8a-7: A mixture of compound 8a-6 (320 mg, 0.34 mmol, 1.0 equivalence), succinic anhydride (207 mg, 2.07 mmol, 6.0 equivalence), triethylamine (418 mg, 4.14 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (5 mg, 0.034 mmol, 0.1 equivalence) 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 under vacuum to give a yellow solid product 8a-7 (380 mg, crude). LCMS (ESI): m / z = 727 [(M-302)+H] + . 1 H 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).

[0453] Step 9g: Preparation of compound 8a-8: To a solution of compound 8a-7 (380 mg, 0.37 mmol, 1.0 equivalence) in N,N-dimethylformamide (10 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (169 mg, 0.44 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (70 mg, 0.52 mmol, 1.4 equivalence), and N,N-diisopropylethylamine (191 mg, 1.48 mmol, 4.0 equivalence) were added, and the mixture was shaken at room temperature for 5 min. Then, CPG-NH2 (1.9 g) was added to the reaction mixture, and the mixture was shaken overnight at room temperature. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane and dried under vacuum for 1 h. The residue was added to pyridine / acetic anhydride (7.5 mL / 2.5 mL), and the mixture was shaken at room temperature for 3 h. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to give a light yellow solid product 8a-8 (2.08 g, loading: 101 μmol / g).

[0454] Step 9h: Preparation of compound 8a-9-A-ssRNA-2: Compound 8a-8 was synthesized via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 8a-9-A-ssRNA-2 (MS m / z: [MH]). - :calcd.7326.9860Found7326.2315).

[0455] Step 9i: Preparation of conjugate 9A-1: Take 200 nmol (1 equivalent) of 8a-9-A-ssRNA-2 and 2b-15-A-ssRNA-1 (220 nmol (1.1 equivalent)), and add sequentially 59 μL of PBS buffer (pH 8.0, 0.5 M), 590 μL of DEPC water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL). After mixing, add 1.7 mg of sodium ascorbate, purge with nitrogen, and react at 35 °C for 2 h. After the reaction is complete, add 17 μL of ammonia and 18 mg of EDTA, shake for 5-10 minutes, and precipitate with ethanol to obtain approximately 2.1 mg of crude conjugate 9A-1. Further purification by HPLC yields conjugate 9A-1 (MS m / z: [MH]). - :calcd.16308.4936Found 16308.1200).

[0456] Step 9j: Preparation of conjugate 9A: Take equal amounts of conjugate 9A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 9A.

[0457] Example 10: Preparation of conjugate 5A (prepared according to scheme 9a):

[0458]

[0459]

[0460] Step 10a: Preparation of compound 9a-1: A mixture of compound 1a-8 (8.15 g, 15.32 mmol, 1.0 equivalent) and dioxane (4 M, 60 mL) was stirred at room temperature for 2 days. The solvent was removed under reduced pressure. The residue was dissolved in a mixture of tetrahydrofuran (100 mL) and water (100 mL). Sodium carbonate (9.7 g, 91.92 mmol, 6.0 equivalent) and di-tert-butyl carbonate (20.06 g, 91.92 mmol, 6.0 equivalent) were added. The mixture was stirred at room temperature for 2 days. The tetrahydrofuran was removed under reduced pressure. The residue was diluted with water (200 mL) and then washed with dichloromethane (60 mL × 4). The pH of the aqueous layer was adjusted to 3 by adding 2N dilute hydrochloric acid solution, and then extracted with ethyl acetate (60 mL × 4). The combined organic layers were washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a white solid, compound 9a-1 (7.08 g, yield: 89%). LCMS (ESI): m / z 520 [M+H] + TLC: Rf0.4 (dichloromethane:methanol = 10:1).

[0461] Step 10b: Preparation of compound 9a-2: At 0°C, a solution of compound 9a-1 (4.0 g, 6.76 mmol, 1.0 equivalence), compound 1a-4 (2.1 g, 6.76 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (5.23 g, 40.56 mmol, 6.0 equivalence) in dichloromethane (70 mL) was added dropwise over 30 minutes to a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (6.99 g, 16.9 mmol, 2.5 equivalence) and dichloromethane (600 mL). The mixture was brought to room temperature and stirred for 20 minutes. 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 × 3). The combined organic layers were washed with saturated brine (30 mL × 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 give a pale 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).

[0462] Step 10c: Preparation of compound 9a-3: A mixture of compound 9a-2 (4.88 g, 6.76 mmol, 1.0 equivalence) and dioxane (4 M, 25 mL) was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (50 mL). 3-(tert-Butoxycarbonyl)amino)propionic acid (3.84 g, 20.28 mmol, 3.0 equivalence), N,N-diisopropylethylamine (6.98 g, 54.08 mmol, 8.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (7.7 g, 20.28 mmol, 3.0 equivalence) were added. The mixture was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The mixture was diluted with a saturated sodium carbonate solution (60 mL) and then extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane:methanol 40:1) to give a pale 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).

[0463] Step 10d: Preparation of compound 9a-4: A mixture of compound 9a-3 (2.19 g, 2.34 mmol, 1.0 equivalent) and dioxane (4 M, 20 mL) was stirred at room temperature for 1.5 h. 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 equivalent), N,N-diisopropylethylamine (2.41 g, 18.72 mmol, 8.0 equivalent), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.85 g, 7.49 mmol, 3.2 equivalent) were added. The mixture was stirred at room temperature for 1 h. The mixture was washed with saturated sodium bicarbonate solution (40 mL × 2) and saturated brine (50 mL × 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 give 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).

[0464] 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 equivalence), trifluoroacetic acid (0.14 mL, 1.82 mmol, 1.0 equivalence), 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 compound 9a-5 (3.48 g, crude) as a white solid. LCMS (ESI): m / z 1790.4 [M+H] + TLC: Rf0.4 (dichloromethane:methanol = 10:1).

[0465] Step 10f: Preparation of compound 9a-6 (compound 52): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (178 mg, 0.47 mmol, 1.5 equivalence) was added to a mixture of compound 9a-5 (735 mg, 0.39 mmol, 1.25 equivalence), compound 4a-6 (250 mg, 0.312 mmol, 1.0 equivalence), N,N-diisopropylethylamine (240 mg, 1.86 mmol, 6.0 equivalence), and N,N-dimethylformamide (2 mL). The mixture was stirred overnight at room temperature. The mixture was purified by preparative liquid chromatography (A: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%–90% over 30 minutes) to give compound 9a-6 (199 mg, yield: 25%) as a white solid. LCMS(ESI): m / z 1136.5[(M-302) / 2+H] + TLC: Rf0.5 (dichloromethane:methanol = 10:1).

[0466] Step 10g: Preparation of compound 9a-7: Succinic anhydride (46 mg, 0.462 mmol, 6.0 equivalent) was added to a mixture of compound 9a-6 (199 mg, 0.077 mmol, 1.0 equivalent), triethylamine (93 mg, 0.92 mmol, 12.0 equivalent), 4-dimethylaminopyridine (9 mg, 0.077 mmol, 1.0 equivalent), and dichloromethane (2 mL). The mixture was stirred overnight at room temperature. The mixture was diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (10 mL × 2) and saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a white solid compound 9a-7 (212 mg, crude). LCMS (ESI): m / z 1186.5 [(M-302) / 2+H] + TLC: Rf0.4 (dichloromethane:methanol = 10:1).

[0467] Step 10h: Preparation of compound 9a-8: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (44 mg, 0.116 mmol, 1.5 equivalence) and 1-hydroxybenzotriazole (16 mg, 0.116 mmol, 1.5 equivalence) were added to a mixture of compound 9a-7 (206 mg, 0.077 mmol, 1.0 equivalence), N,N-diisopropylethylamine (60 mg, 0.462 mmol, 6.0 equivalence), and N,N-dimethylformamide (8 mL). The mixture was stirred at room temperature for 5 minutes. CPG-NH2 (0.167 mmol / g, 1.0 g, 0.167 mmol, 2.2 equivalence) was added. The mixture was stirred at room temperature for 22 hours. The mixture was filtered. The solid was washed with dichloromethane (8 mL × 5). Add pyridine (6 mL) and acetic anhydride (2 mL). Stir the mixture at room temperature for 3 hours. Filter the mixture. Wash the solid with dichloromethane (8 mL × 5) and dry under vacuum to give a white solid compound 9a-8 (1.0 g, loading: 35 μmol / g, yield: 43%).

[0468] Step 10i: Preparation of compound 9a-9-A-ssRNA-1: Compound 9a-8 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 9a-9-A-ssRNA-1 (MS m / z: [MH]-: calcd 9209.6715. Found 9208.8616).

[0469] 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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 9a-9-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, crude conjugate 5A-1 was obtained by ethanol precipitation, about 2.3 mg. Conjugate 5A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16536.6576 Found 16536.1842).

[0470] Step 10k: Preparation of conjugate 5A: Take equal amounts of conjugate 5A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 5A.

[0471] Example 11: Preparation of conjugate 12A (prepared according to scheme 10a):

[0472]

[0473]

[0474] Step 11a: Preparation of compound 10a-1: Under nitrogen protection, ethyl chloroformate (11.26 g, 103.74 mmol, 3.5 equivalence) was added dropwise to a mixture of compound 2b-5 (10.0 g, 29.64 mmol, 1.0 equivalence), triethylamine (10.8 g, 106.7 mmol, 3.6 equivalence), 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, followed by the addition of sodium borohydride (2.25 g, 59.2 mmol, 2.0 equivalence) in portions at 0 °C. 80 mL of methanol was added, and the mixture was stirred at room temperature for 30 minutes. Sodium cyanoborohydride (1.4 g, 22.47 mmol, 1.5 equivalence) 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 give a yellow oily compound 10a-1 (5.53 g, yield: 57.6%). LCMS (ESI): m / z = 324 (M+H) + .

[0475] Step 11b: Preparation of compound 10a-2: At room temperature, 2-iodobenzoic acid (5.73 g, 20.47 mmol, 1.2 equivalence) was added to a solution of compound 10a-1 (5.1 g, 17.1 mmol, 1.0 equivalence) in dimethyl sulfoxide (50 mL). 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 give compound 10a-2 (5.11 g, crude product) as a yellow oil. LCMS (ESI): m / z = 322 (M+H) + .

[0476] Step 11c: Preparation of compound 10a-4: A solution of (2-aminoethyl)carbamate (10.16 g, 52.3 mmol, 0.95 equivalents) in N,N-dimethylformamide was added to a solution of compound 10a-3 (25 g, 55.0 mmol, 1.0 equivalent), N,N-diisopropylethylamine (10.64 g, 82.5 mmol, 1.5 equivalent), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (27.3 g, 66.0 mmol, 1.2 equivalent) in N,N-dimethylformamide (160 mL). 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). LCMS (ESI): m / z = 631 (M+H) + .

[0477] Step 11d: Preparation of compound 10a-5: Compound 10a-4 (38 g, 60.23 mmol, 1.0 equivalent) 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, with the addition of triethylamine) to give a yellow oily compound 10a-5 (16.7 g, yield: 67.8%). LCMS (ESI): m / z = 409 (M+H) + .

[0478] Step 11e: Preparation of compound 10a-6: Compound 10a-5 (6.4 g, 15.58 mmol, 1.0 equivalence), compound 10a-2 (5.0 mg, 15.58 mmol, 1.0 equivalence), acetic acid (935 mg, 15.58 mmol, 1.0 equivalence), and sodium cyanoborohydride (2.94 g, 46.74 mmol, 3.0 equivalence) were added to 75 mL of methanol. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and extracted with ethyl acetate after adding sodium carbonate solution. 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, with the addition of triethylamine) to give compound 10a-6 (2.35 g, yield: 20.8%) as a white solid. LCMS(ESI): m / z = 714.5(M+H) + .

[0479] Step 11f: Preparation of compound 10a-7: Compound 10a-6 (2.3 g, 3.22 mmol, 1.0 equivalence), 3-(tert-butoxycarbonyl)amino)propionic acid (700 mg, 3.70 mmol, 1.15 equivalence), N,N-diisopropylethylamine (831 mg, 6.44 mmol, 2.0 equivalence), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (1.53 g, 3.70 mmol, 1.15 equivalence) were added to 20 mL of N,N-dimethylformamide. 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, with the addition of triethylamine) to give a yellow oil solid compound 10a-7 (2.2 g, yield: 77.2%). LCMS (ESI): m / z = 885.5 (M+H) + .

[0480] Step 11g: Preparation of compound 10a-8: Compound 10a-7 (2.2 g, 2.26 mmol, 1.0 equivalent) was added to 25 mL of 4 M 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 compound 10a-8 (2.7 g, crude). LCMS (ESI): m / z = 585.5 (M+H) + .

[0481] Step 11h: Preparation of compound 10a-9: Compound 10a-8 (2.7 g, 2.26 mmol, 1.0 equivalence), compound 1a-12 (3.24 g, 7.23 mmol, 3.2 equivalence), N,N-diisopropylethylamine (2.92 g, 22.6 mmol, 10.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.75 g, 7.23 mmol, 3.2 equivalence) were added to 40 mL of acetonitrile. 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, with the addition of triethylamine) to give a white solid compound 10a-9 (2.46 g, yield: 58.1%). LCMS (ESI): m / z = 937.7 (M / 2+H). + .

[0482] Step 11i: Preparation of compound 10a-10: Compound 10a-9 (2.46 g, 1.31 mmol, 1.0 equivalent) was dissolved in 50 mL of ethanol, and trifluoroacetic acid (150 mg, 1.31 mmol, 1.0 equivalent) and 300 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 10a-10 (2.31 g, crude product) as a white solid. LCMS (ESI): m / z = 825 (M / 2+H) + .

[0483] Step 11j: Preparation of compound 10a-11 (compound 60): Compound 10a-10 (2.31 g, 1.31 mmol, 1.0 equivalence), N,N-diisopropylethylamine (507 mg, 3.93 mmol, 3.0 equivalence), and compound 2b-10 (383 mg, 1.51 mmol, 1.15 equivalence) were added to 20 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h. Then, N,N-diisopropylethylamine (254 mg, 1.96 mmol, 1.5 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (662 mg, 1.60 mmol, 1.22 equivalence), and intermediate M (963 mg, 1.60 mmol, 1.22 equivalence) were added, and the mixture was stirred at room temperature for 1 h. 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 / acetonitrile) to give a white solid compound 10a-11 (1.81 g, yield: 58.1%). LCMS (ESI): m / z = 1036((M-302) / 2+H) + .

[0484] Step 11k: Preparation of compound 10a-12: Under nitrogen protection, compound 10a-11 (300 mg, 0.126 mmol, 1.0 equivalence), triethylamine (154 mg, 1.52 mmol, 12.0 equivalence), succinic anhydride (76 mg, 0.76 mmol, 6.0 equivalence), and p-dimethylaminopyridine (1.5 mg, 0.0126 mmol, 0.1 equivalence) were added to 1.5 mL of dichloromethane. The mixture was stirred overnight at room temperature. The mixture was diluted with dichloromethane / acetonitrile and 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) + .

[0485] Step 11l: Preparation of compound 10a-13: Compound 10a-9 (350 mg, 0.126 mmol, 1.0 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (64.8 mg, 0.170 mmol, 1.35 equivalence), 1-hydroxybenzotriazole (27 mg, 0.20 mmol, 1.6 equivalence), and N,N-diisopropylethylamine (74 mg, 0.57 mmol, 4.5 equivalence) 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 overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and 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 give a yellow solid compound 10a-13 (32.1 μmol / g).

[0486] Step 11m: Preparation of compound 10a-14-A-ssRNA-1: Compound 10a-13 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 10a-14-A-ssRNA-1 (MS m / z: [MH]-: calcd.9009.5615 Found9008.8216).

[0487] Step 11n: Preparation of conjugate 12A-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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compounds 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 10a-14-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, about 2.3 mg of crude conjugate 12A-1 was obtained by ethanol precipitation. Conjugate 12A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16336.5476 Found 16336.1821).

[0488] Step 11o: Preparation of conjugate 12A: Take equal amounts of conjugate 12A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 12A.

[0489] Example 12: Preparation of conjugate 11A (prepared according to scheme 11a):

[0490]

[0491]

[0492] Step 12a: Preparation of compound 11a-1: Compound 10a-6 (3.03 g, 5.49 mmol, 1.0 equivalent), compound 2b-5 (2.01 g, 6.04 mmol, 1.1 equivalent), N,N-diisopropylethylamine (1.42 g, 10.98 mmol, 2.0 equivalent), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (2.5 g, 6.04 mmol, 1.1 equivalent) were added to 15 mL of N,N-dimethylformamide. 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, with the addition of triethylamine) to give a white solid compound 11a-1 (4.75 g, yield: 99.37%). LCMS (ESI): m / z = 871 (M+H) + .

[0493] Step 12b: Preparation of compound 11a-2: Compound 11a-1 (4.75 g, 5.49 mmol, 1.0 equivalent) was added to 20 mL of 4 M 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 compound 11a-2 (4.85 g, crude). LCMS (ESI): m / z = 571 (M+H) + .

[0494] Step 12c: Preparation of compound 11a-3: Compound 11a-2 (4.85 g, 5.49 mmol, 1.0 equivalence), compound 1a-12 (7.6 g, 17.02 mmol, 3.1 equivalence), N,N-diisopropylethylamine (7.08 g, 54.9 mmol, 10.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (6.67 g, 17.57 mmol, 3.2 equivalence) were added to 30 mL of acetonitrile. 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, with the addition of triethylamine) to give a white solid compound 11a-3 (6.92 g, yield: 67.84%). LCMS (ESI): m / z = 930.6 (M / 2 + H). + .

[0495] Step 12d: Preparation of compound 11a-4: Compound 11a-3 (2 g, 1.07 mmol, 1.0 equivalent) was dissolved in 10 mL of ethanol, and trifluoroacetic acid (123 mg, 1.07 mmol, 1.0 equivalent) and 200 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 11a-4 (1.77 g, crude) as a yellow solid. LCMS (ESI): m / z = 1635 (M+H) + .

[0496] Step 12e: Preparation of compound 11a-5 (compound 59): Compound 11a-4 (1.77 g, 1.07 mmol, 1.0 equivalence), N,N-diisopropylethylamine (417 mg, 3.23 mmol, 3 equivalence), and compound 2b-10 (302 mg, 1.18 mmol, 1.1 equivalence) were added to 10 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h. Then, N,N-diisopropylethylamine (417 mg, 3.23 mmol, 3 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (535 mg, 1.29 mmol, 1.2 equivalence), and intermediate M (778 mg, 1.29 mmol, 1.2 equivalence) were added, and the mixture was stirred at room temperature for 1 h. Extraction was performed 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 preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer / acetonitrile) to give a 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).

[0497] Step 12f: Preparation of compound 11a-6: Compound 11a-5 (200 mg, 0.09 mmol, 1.0 equivalent), triethylamine (108 mg, 1.08 mmol, 12.0 equivalent), succinic anhydride (54 mg, 0.54 mmol, 6.0 equivalent), and p-dimethylaminopyridine (1 mg, 0.009 mmol, 0.1 equivalent) were added to 2 mL of dichloromethane. The mixture 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 under reduced pressure to give compound 11a-6 (229 mg, crude) as a white solid. LCMS (ESI): m / z = 1079((M-302) / 2+H) + .

[0498] Step 12g: Preparation of compound 11a-7: Compound 11a-6 (229 mg, 0.09 mmol, 1.0 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (41 mg, 0.11 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (17 mg, 0.13 mmol, 1.4 equivalence), and N,N-diisopropylethylamine (46 mg, 0.36 mmol, 4.0 equivalence) 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 overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and 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 solid phase was filtered, washed with dichloromethane and acetonitrile, and dried under reduced pressure to give a yellow solid compound 11a-7 (1.2 g, loading: 41.7 μmol / g).

[0499] Step 12m: Preparation of compound 11A-8-A-ssRNA-1: Compound 11a-7 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 11A-8-A-ssRNA-1 (MS m / z: [MH]-: calcd. 8995.5345 Found 8997.8616).

[0500] Step 12n: Preparation of conjugate 11A-1: To a mixture of compound 8a-9-A-ssRNA-2 (182 nanomoles, 1 equivalent) and 11A-8-A-ssRNA-1 (200 nanomoles, 1.1 equivalent), 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 CuSO4-THPTA (1:1) solution (conc. 14 nanomoles / μL) were added sequentially. After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 11A-1 was obtained by ethanol precipitation. The conjugate 11A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16322.5206 Found 16322.1033).

[0501] Step 12o: Preparation of conjugate 11A: Take equal amounts of conjugate 11A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 11A.

[0502] Example 13: Preparation of conjugate 10A (prepared according to scheme 12a):

[0503]

[0504]

[0505] Step 13a: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized via solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 4a-10-A-ssRNA-1 (MS m / z: [MH]). - :calcd.Found).

[0506] Step 13b: Preparation of conjugate 10A-1: To a mixture of compound 8a-9-A-ssRNA-2 (182 nanomoles, 1 equivalent) and 4a-10-A-ssRNA-1 (200 nanomoles, 1.1 equivalent), 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 CuSO4-THPTA (1:1) solution (conc. 14 nanomoles / μL) were added sequentially. After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 10A-1 was obtained by ethanol precipitation. Conjugate 10A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16394.4996 Found 16394.0447).

[0507] Step 13c: Preparation of conjugate 10A: Take equal amounts of conjugate 10A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 10A.

[0508] Example 14: Preparation of conjugate 13A (prepared according to scheme 13a):

[0509]

[0510]

[0511] Step 14a: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 4a-10-A-ssRNA-1 (MS m / z: [MH]-: calcd.Found).

[0512] Step 14b: Preparation of conjugate 13A-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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 6a-7-A-ssRNA-2 (182 nmol, 1 equivalent) and 4a-10-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 13A-1 was obtained by ethanol precipitation. Conjugate 13A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16320.4206 Found 16319.8622).

[0513] Step 14c: Preparation of conjugate 13A: Take equal amounts of conjugate 13A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 13A.

[0514] Example 15: Preparation of conjugate 79A (prepared according to scheme 14a):

[0515]

[0516]

[0517] Step 15a: Preparation of compound 14a-1: A mixture of compound 2b-7 (2.6 g, 3.0 mmol, 1.0 equivalence), 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid (2 g, 9.4 mmol, 3.1 equivalence), N,N-diisopropylethylamine (4 g, 30.4 mmol, 10.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.7 g, 9.7 mmol, 3.2 equivalence) 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 with silica gel (elution buffer: petroleum ether / ethyl acetate = 1 / 2, 0.5% triethylamine) to give a white solid compound 14a-1 (2.08 g, yield: 59.4%). LCMS (ESI): m / z = 1161.5 [M+H] + .

[0518] Step 15b: Preparation of compound 14a-2: A solution of compound 14a-1 (2.08 g, 1.79 mmol, 1.0 equivalence) in hydrogen chloride-dioxane (5 mL) was stirred at room temperature for 1 hour. The mixture was concentrated to give crude compound 14a-2. LCMS (ESI): m / z = 860.7 [M+H] + .

[0519] Step 15c: Preparation of compound 14a-3: A mixture of compound 14a-2 (2.08 g, 1.79 mmol, 1.0 equivalence), compound 1a-12 (2.5 g, 5.5 mmol, 3.1 equivalence), N,N-diisopropylethylamine (2.3 g, 17.9 mmol, 10.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.2 g, 5.7 mmol, 3.2 equivalence) in acetonitrile (20 mL) was stirred at room temperature for 1 hour. The mixture was diluted with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified with silica gel (elution: dichloromethane / methanol = 50 / 1 to 20 / 1) to give compound 14a-3 (1.46 g, yield: 38.4%) as a white solid. LCMS(ESI): m / z=1075.4[M / 2+H] + .

[0520] 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 equivalent), palladium hydroxide / carbon (146 mg, 25% by mass), and trifluoroacetic acid (77.5 mg, 0.64 mmol, 1.0 equivalent) in ethanol (60 mL) was stirred overnight at room temperature. The mixture was filtered through diatomaceous earth. The filter cake was washed with ethanol, and the filtrate was concentrated. The residue was used directly for the next step without purification (1.23 g, yield: 94.61%). LCMS (ESI): m / z = 963 [M / 2+H] + .

[0521] Step 15e: Preparation of compound 14a-5: A mixture of compound 14a-4 (1.23 g, 0.6 mmol, 1.0 equivalence), compound 2b-10 (186.8 mg, 0.7 mmol, 1.15 equivalence), and N,N-diisopropylethylamine (247.5 mg, 1.9 mmol, 3.0 equivalence) in acetonitrile (10 mL) was stirred at room temperature for 1 hour. The reaction proceeded directly to the next step without post-treatment. LCMS (ESI): m / z = 1032.9 [M / 2+H]+.

[0522] 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 equivalences), N,N-diisopropylethylamine (123.7 mg, 0.96 mmol, 1.5 equivalences), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (322.9 mg, 0.78 mmol, 1.22 equivalences) in acetonitrile (60 mL) was stirred at room temperature for 1 hour. The mixture was then purified directly by high performance liquid chromatography (column: UniSil 10-120C18, 30 × 250 mm) to give a white solid product 14a-6 (360 mg, yield: 21.3%). LCMS (ESI): m / z = 1174 [(M-302) / 2+H] + .

[0523] Step 15g: Preparation of compound 14a-7: A mixture of compound 14a-6 (330 mg, 0.12 mmol, 1.0 equivalence), succinic anhydride (74.8 mg, 0.75 mmol, 6.0 equivalence), triethylamine (151.1 mg, 1.5 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (1.5 mg, 0.012 mmol, 0.1 equivalence) 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). LCMS (ESI): m / z = 1224 [(M-302) / 2+H] + .

[0524] Step 15h: Preparation of compound 14a-8: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (57.8 mg, 0.14 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (22.7 mg, 0.17 mmol, 1.4 equivalence), and N,N-diisopropylethylamine (62 mg, 0.49 mmol, 4.0 equivalence) were added to an N,N-dimethylformamide solution of compound 14a-7 (330 mg, 0.12 mmol, 1.4 equivalence), and the mixture was shaken at room temperature for 5 minutes. 1.5 g of CPG-NH2 was added to the mixture, and the mixture was shaken overnight at room temperature. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (12 mL / 4 mL), and the mixture was 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 give a light yellow solid product 14a-8 (1.7 g, loading: 23.1 μmol / g).

[0525] Step 15i: Preparation of compound 14a-9-A-ssRNA-1: Compound 14a-8 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 14a-9-A-ssRNA-1 (MS m / z: [MH]-: calcd.9284.8225 Found 9284.8267).

[0526] Step 15j: Preparation of conjugate 79A-1: To a mixture of compound 8a-9-A-ssRNA-2 (182 nanomoles, 1 equivalent) and 14a-9-A-ssRNA-1 (200 nanomoles, 1.1 equivalent), 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 CuSO4-THPTA (1:1) solution (conc. 14 nanomoles / μL) were added sequentially. After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 79A-1 was obtained by ethanol precipitation. Conjugate 79A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16611.8086 Found 16611.1957).

[0527] Step 15k: Preparation of conjugate 79A: Take equal amounts of conjugate 79A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 79A.

[0528] Example 16: Preparation of conjugate 82A (prepared according to scheme 15a):

[0529]

[0530]

[0531] Step 16a: Preparation of compound 15a-1: Potassium carbonate (5.07 g, 36.75 mmol, 2.5 equivalence) was added to a mixture of compound 10a-5 (6.0 g, 14.7 mmol, 1.5 equivalence) and tert-butyl (2-bromoethyl)carbamate (2.2 g, 9.8 mmol, 1.0 equivalence) and 30 mL of N,N-dimethylformamide 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) to give compound 15a-1 (3.29 g, yield: 60.8%) as a white solid. LCMS (ESI): m / z = 552 (M+H) + .

[0532] Step 16b: Preparation of compound 15a-2: Compound 15a-1 (1.7 g, 3.08 mmol, 1.0 equivalence), compound 10a-2 (3.2 g, 9.97 mmol, 3.2 equivalence), acetic acid (185 mg, 3.08 mmol, 1.0 equivalence), and sodium cyanoborohydride (626 mg, 9.97 mmol, 3.2 equivalence) were added to 24 mL of methanol. The mixture was stirred at room temperature for 96 hours, concentrated under reduced pressure, and extracted with ethyl acetate after adding sodium carbonate solution. 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, with the addition of ammonia) to give compound 15a-2 (1.72 g, yield: 54.0%) as a white solid. LCMS (ESI): m / z = 857.5 (M+H) + .

[0533] Step 16c: Preparation of compound 15a-3: Compound 15a-2 (1.72 g, 2.01 mmol, 1.0 equivalent) was added to 20 mL of 4 M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give compound 15a-3 (1.57 g, crude product). LCMS (ESI): m / z = 557.7 (M+H) + .

[0534] Step 16d: Preparation of compound 15a-4: Crude compound 15a-3 (1.57 g, 2.01 mmol, 1.0 equivalent), compound 1a-12 (2.87 g, 6.41 mmol, 3.2 equivalent), N,N-diisopropylethylamine (2.58 g, 20.1 mmol, 10.0 equivalent), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.87 g, 6.41 mmol, 3.2 equivalent) were added to 35 mL of acetonitrile. 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, with the addition of triethylamine) to give a white solid compound 15a-4 (2.01 g, yield: 54.1%). LCMS (ESI): m / z = 923.7 (M / 2+H). + .

[0535] Step 16e: Preparation of compound 15a-5: Compound 15a-4 (2.0 g, 1.08 mmol, 1.0 equivalent) was dissolved in 45 mL of ethanol, and trifluoroacetic acid (160 mg, 1.41 mmol, 1.3 equivalent) and 300 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 15a-5 (2.0 g, crude) as a white solid. LCMS (ESI): m / z = 1621.5 (M+H) + .

[0536] Step 16f: Preparation of compound 15a-6 (compound 61): Compound 15a-5 (2.0 g, 1.08 mmol, 1.0 equivalence), N,N-diisopropylethylamine (418 mg, 3.24 mmol, 3.0 equivalence), and compound 2b-10 (307 mg, 1.21 mmol, 1.12 equivalence) were added to 15 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h. Then, N,N-diisopropylethylamine (209 mg, 1.62 mmol, 1.5 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (538 mg, 1.30 mmol, 1.2 equivalence), and intermediate M (783 mg, 1.30 mmol, 1.2 equivalence) were added, and the mixture was stirred at room temperature for 1 h. 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 / acetonitrile) to give a white solid compound 15a-6 (1.81 g, yield: 37.1%). LCMS (ESI): m / z = 1022.5 ((M-3O2) / 2+H) + .

[0537] Step 16g: Preparation of compound 15a-7: Under nitrogen protection, compound 15a-6 (300 mg, 0.128 mmol, 1.0 equivalence), triethylamine (155 mg, 1.54 mmol, 12.0 equivalence), succinic anhydride (77 mg, 0.768 mmol, 6.0 equivalence), and p-dimethylaminopyridine (1.6 mg, 0.013 mmol, 0.1 equivalence) were added to 1.5 mL of dichloromethane. The mixture was stirred overnight at room temperature. The mixture was diluted with dichloromethane / acetonitrile and 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 product) as a white solid. LCMS (ESI): m / z = 1072((M-302) / 2+H) + .

[0538] Step 16h: Preparation of compound 15a-8: Compound 15a-7 (375 mg, 0.128 mmol, 1.0 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (68 mg, 0.179 mmol, 1.4 equivalence), 1-hydroxybenzotriazole (28 mg, 0.205 mmol, 1.6 equivalence), and N,N-diisopropylethylamine (76 mg, 0.59 mmol, 4.6 equivalence) 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-NH2 was added, and the mixture was shaken overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and 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 solid phase was filtered, washed with dichloromethane and acetonitrile, and dried under reduced pressure to give a yellow solid compound 15a-8 (1.39 g, 37.5 μmol / g).

[0539] Step 16i: Preparation of compound 15a-9-A-ssRNA-1: Compound 15a-8 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 15a-9-A-ssRNA-1 (MS m / z: [MH]-: calcd.8981.5515Found8980.7234).

[0540] Step 16j: Preparation of conjugate 82A-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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 15a-9-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 82A-1 was obtained by ethanol precipitation. Conjugate 82A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16308.5376 Found 16307.9277).

[0541] Step 16k: Preparation of conjugate 82A: Take equal amounts of conjugate 82A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 82A.

[0542] Example 17: Preparation of conjugate 87A (prepared according to scheme 16a):

[0543]

[0544]

[0545]

[0546] Step 17a: Preparation of compound 16a-1: Under nitrogen protection and ice bath conditions, a solution of 2,4-dinitrobenzenesulfonyl chloride (2.6 g, 9.7 mmol, 1.2 equivalent) in tetrahydrofuran (10 mL) was added dropwise to a mixture of compound 2b-3 (3.2 g, 8.1 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (2.0 g, 16.2 mmol, 2.0 equivalent) in tetrahydrofuran (40 mL). The mixture was stirred in an ice bath for 1 hour. Ethyl acetate was added to the mixture, followed by washing with water, dilute hydrochloric acid, sodium bicarbonate solution, and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a brown solid 16a-1 (6.0 g, crude product). LCMS (ESI): m / z = 582 [M+H] + .

[0547] Step 17b: Preparation of compound 16a-2: Compound 16a-1 (1 g, 1.6 mmol, 1.0 equivalence), (tert-butoxycarbonyl)-L-homoserine benzyl ester (1.24 g, 4.0 mmol, 2.5 equivalence), and triphenylphosphine (1.26 g, 4.8 mmol, 3.0 equivalence) were added to 30 mL of toluene and stirred at room temperature for 5 min. Diethyl azodicarbonate (0.835 g, 4.8 mmol, 3.0 equivalence) was added dropwise under ice bath, and the mixture was stirred at room temperature for 2 h. 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 h. The mixture was filtered. The residue was washed with dichloromethane / petroleum ether (1 / 1). The residue was dried to give compound 16a-2 (1.42 g, yield: 96.59%) as a yellow solid. LCMS(ESI): m / z = 916(M+H) + .

[0548] Step 17c: Preparation of compound 16a-3: Compound 16a-2 (1.6 g, 1.75 mmol, 1.0 equivalence) and triethylamine (530 mg, 5.25 mmol, 3.0 equivalence) were added to 10 mL of dichloromethane. 2-Mercaptoacetic acid (322 mg, 3.5 mmol, 2.0 equivalence) was added, and the mixture was stirred at room temperature for 10 min. Extraction was performed with ethyl acetate. The organic phase was washed with aqueous sodium carbonate solution and saturated brine. The organic phase was concentrated under reduced pressure to give a yellow oily compound 16a-3 (1.2 g, crude). LCMS (ESI): m / z = 686 (M+H) + .

[0549] Step 17d: Preparation of compound 16a-4: Compound 16a-3 (1.2 g, 1.75 mmol, 1.0 equivalence), 3-(tert-butoxycarbonyl)amino)propionic acid (331 mg, 1.75 mmol, 1.0 equivalence), N,N-diisopropylethylamine (451 mg, 3.5 mmol, 2.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (798 mg, 2.1 mmol, 1.2 equivalence) were added to 30 mL of dichloromethane. The mixture was stirred at room temperature for 1 hour. Extraction was performed 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 = 25 / 1 to 10 / 1, with the addition of triethylamine) to give a white solid compound 16a-4 (740 mg, yield: 49.33%). LCMS (ESI): m / z = 858 (M+H) + .

[0550] Step 17e: Preparation of compound 16a-5: Compound 16a-4 (740 mg, 0.86 mmol, 1.0 equivalence) was added to 5 mL of 4 M 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, compound 16a-5 (482 mg, crude). LCMS (ESI): m / z = 557 (M+H) + .

[0551] Step 17f: Preparation of compound 16a-6: Compound 16a-5 (482 mg, 0.86 mmol, 1.0 equivalence), compound 1a-12 (1.2 g, 2.68 mmol, 3.1 equivalence), N,N-diisopropylethylamine (1.12 g, 8.64 mmol, 10.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.05 g, 2.76 mmol, 3.2 equivalence) were added to 10 mL of acetonitrile. 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, with the addition of triethylamine) to give a white solid compound 16a-6 (879 mg, yield: 55.28%). LCMS (ESI): m / z = 1844 (M+H) + .

[0552] Step 17g: Preparation of compound 16a-7: Compound 16a-6 (879 mg, 0.476 mmol, 1.0 equivalent) was dissolved in 5 mL of ethanol, and trifluoroacetic acid (54 mg, 0.476 mmol, 1.0 equivalent) and 88 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 16a-7 (800 mg, crude) as a white solid. LCMS (ESI): m / z = 1621 (M+H) + .

[0553] Step 17h: Preparation of compound 16a-8 (compound 65): Compound 16a-7 (800 mg, 0.476 mmol, 1.0 equivalence), N,N-diisopropylethylamine (184 mg, 1.428 mmol, 3 equivalence), and compound 2b-10 (133 mg, 0.523 mmol, 1.1 equivalence) were added to 10 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h. Then, N,N-diisopropylethylamine (184 mg, 1.428 mmol, 3 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (236 mg, 0.57 mmol, 1.2 equivalence), and intermediate M (343 mg, 0.57 mmol, 1.2 equivalence) were added, and the mixture was stirred at room temperature for 1 h. Extraction was performed 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 preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer / acetonitrile) to give a yellow solid compound 16a-8 (343 mg, yield: 30.90%).

[0554] Step 17i: Preparation of compound 16a-9: Compound 16a-8 (343 mg, 0.146 mmol, 1.0 equivalence), triethylamine (177 mg, 1.752 mmol, 12.0 equivalence), succinic anhydride (88 mg, 0.878 mmol, 6.0 equivalence), and p-dimethylaminopyridine (2 mg, 0.014 mmol, 0.1 equivalence) were added to 5 mL of dichloromethane. The mixture 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 under reduced pressure to give a yellow solid, compound 16a-9 (396 mg, crude). LCMS (ESI): m / z = 1072((M-302) / 2+H) + .

[0555] Step 17j: Preparation of compound 16a-10: Compound 16a-9 (396 mg, 0.162 mmol, 1.0 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (74 mg, 0.194 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (30 mg, 0.226 mmol, 1.4 equivalence), and N,N-diisopropylethylamine (84 mg, 0.648 mmol, 4.0 equivalence) 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-NH2 was added, and the mixture was shaken overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and 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 solid phase was filtered, washed with dichloromethane and acetonitrile, and dried under reduced pressure to give a yellow solid compound 16a-10 (1.68 g, loading: 45.7 μmol / g).

[0556] Step 17k: Preparation of compound 16a-11-A-ssRNA-1: Compound 16a-10 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 16a-11-A-ssRNA-1 (MS m / z: [MH]-: calcd.8981.5075 Found8983.7126).

[0557] Step 17l: Preparation of conjugate 87A-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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 16a-11-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 87A-1 was obtained by ethanol precipitation. Conjugate 87A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16308.4936 Found 16307.8157).

[0558] Step 17m: Preparation of conjugate 87A: Take equal amounts of conjugate 87A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 87A.

[0559] Example 18: Preparation of conjugate 81A (prepared according to scheme 17a):

[0560]

[0561]

[0562]

[0563] Step 18a: Preparation of compound 17a-2: Compound 17a-1 (3.03 g, 10.0 mmol, 1.0 equivalence) was added to an 80 mL mixture of acetonitrile containing 3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)propionic acid (3.43 g, 11.0 mmol, 1.1 equivalence), N,N-diisopropylethylamine (1.94 g, 15.0 mmol, 1.5 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.56 g, 12.0 mmol, 1.2 equivalence). 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 give a yellow solid compound 17a-2 (6.88 g, crude). LCMS (ESI): m / z = 597 (M+H) + .

[0564] Step 18b: Preparation of compound 17a-3: Compound 17a-2 (3.44 g, 5.76 mmol, 1.0 equivalent) and 5 mL piperidine were added to 35 mL 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, with the addition of ammonia) to give compound 17a-3 (1.9 g, yield: 87.9%) as a white solid. LCMS (ESI): m / z = 375 (M+H) + .

[0565] Step 18c: Preparation of compound 17a-5: A mixture of compound 17a-4 (5.0 g, 19.1 mmol, 1.0 equivalence), (2-aminoethyl)carbamate (3.9 g, 20 mmol, 1.05 equivalence), N,N-diisopropylethylamine (4.9 g, 38.2 mmol, 2.0 equivalence), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (8.3 g, 20 mmol, 1.05 equivalence) 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 under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 50 / 1) to give compound 17a-5 (7.1 g, yield: 85.5%) as a white solid. LCMS(ESI): m / z = 438[M+H] + .

[0566] Step 18d: Preparation of compound 17a-6: A mixture of compound 17a-5 (7.4 g, 17 mmol, 1.0 equivalent), lithium bromide (7.4 g, 85 mmol, 5.0 equivalent), and N,N-diisopropylethylamine (6.6 g, 51 mmol, 3.0 equivalent) 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. Extraction was performed with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to give a white solid, compound 17a-6 (6.0 g, yield: 83.8%). LCMS (ESI): m / z = 424 [M+H] + .

[0567] Step 18e: Preparation of compound 17a-7: A mixture of 17a-6 (1.6 g, 3.8 mmol, 1.0 equivalence), 1-hydroxypyrrolidine-2,5-dione (0.7 g, 5.7 mmol, 1.5 equivalence), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.1 g, 5.7 mmol, 1.5 equivalence) in dichloromethane (30 mL) was stirred overnight at room temperature. The mixture was washed successively with water, aqueous sodium bicarbonate solution, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to give compound 17a-7 (1.9 g, crude) as a white solid. LCMS (ESI): m / z = 521 [M+H] + .

[0568] Step 18f: Preparation of compound 17a-8: A mixture of compound 17a-7 (1.8 g, 3.46 mmol, 1.0 equivalence), (S)-2-amino-5-(benzyloxy)-5-oxovaleric acid (0.8 g, 3.46 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (1.3 g, 10.38 mmol, 3.0 equivalence) in acetonitrile (30 mL) was stirred at room temperature for 2 hours. Ethyl acetate and water were added, and the pH was adjusted to 5-6 with dilute hydrochloric acid. The mixture was separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to give a white solid, compound 17a-8 (2.0 g, yield: 90.9%). LCMS (ESI): m / z = 643 [M+H] + .

[0569] Step 18g: Preparation of compound 17a-9: 6-Chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (823 mg, 1.99 mmol, 1.1 equivalence) was added to a mixture of 15 mL acetonitrile containing compound 17a-3 (678 mg, 1.81 mmol, 1.0 equivalence), compound 17a-8 (1.28 g, 1.99 mmol, 1.1 equivalence), and N-methylimidazole (298 mg, 3.63 mmol, 2.0 equivalence). 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, with the addition of ammonia) to give compound 17a-9 (1.12 g, yield: 61.8%) as a yellow solid. LCMS(ESI): m / z = 999.5(M+H) + .

[0570] Step 18h: Preparation of compound 17a-10: Compound 17a-9 (1.12 g, 1.12 mmol, 1.0 equivalent) was added to 10 mL of 4 M 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 compound 17a-10 (854 mg, crude). LCMS (ESI): m / z = 699 (M+H) + .

[0571] Step 18i: Preparation of compound 17a-11: Compound 17a-10 (854 mg, 1.12 mmol, 1.0 equivalence), compound 1a-12 (1.65 g, 3.70 mmol, 3.3 equivalence), N,N-diisopropylethylamine (1.45 g, 11.2 mmol, 10.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.41 g, 3.70 mmol, 3.3 equivalence) were added to 13 mL of acetonitrile. 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, with the addition of triethylamine) to give a white solid compound 17a-11 (1.59 g, yield: 70.0%). LCMS (ESI): m / z = 994.7 (M / 2 + H). + .

[0572] Step 18j: Preparation of compound 17a-12: Compound 17a-11 (1.59 g, 0.8 mmol, 1.0 equivalent) was dissolved in 60 mL of ethanol, and trifluoroacetic acid (100 mg, 0.88 mmol, 1.1 equivalent) and 230 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 17a-12 (2.31 g, crude product), a white solid. LCMS (ESI): m / z = 882.7 (M / 2+H) + .

[0573] Step 18k: Preparation of compound 17a-13 (compound 64): Compound 17a-12 (1.52 g, 0.8 mmol, 1.0 equivalence), N,N-diisopropylethylamine (413 mg, 3.2 mmol, 4.0 equivalence), and compound 2b-10 (227 mg, 0.90 mmol, 1.12 equivalence) were added to 10 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h. Then, N,N-diisopropylethylamine (155 mg, 1.2 mmol, 1.5 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (397 mg, 0.96 mmol, 1.2 equivalence), and intermediate M (578 mg, 0.96 mmol, 1.2 equivalence) were added, and the mixture was stirred at room temperature for 1 h. 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 / acetonitrile) to give a white solid compound 17a-13 (886 mg, yield: 44.6%). LCMS (ESI): m / z = 1093((M-302) / 2+H) + .

[0574] Step 18l: Preparation of compound 17a-14: Under nitrogen protection, compound 17a-13 (300 mg, 0.121 mmol, 1.0 equivalence), triethylamine (171 mg, 1.69 mmol, 14.0 equivalence), succinic anhydride (85 mg, 0.844 mmol, 7.0 equivalence), and p-dimethylaminopyridine (1.5 mg, 0.0121 mmol, 0.1 equivalence) were added to 2.5 mL of dichloromethane. The mixture was stirred overnight at room temperature. The mixture was diluted with dichloromethane / acetonitrile and 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 17a-14 (269 mg, yield: 85.9%) as a white solid. LCMS (ESI): m / z = 1143((M-302) / 2+H) + .

[0575] Step 18m: Preparation of compound 17a-15: Compound 17a-14 (269 mg, 0.104 mmol, 1.0 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (56 mg, 0.146 mmol, 1.4 equivalence), 1-hydroxybenzotriazole (23 mg, 0.166 mmol, 1.6 equivalence), and N,N-diisopropylethylamine (62 mg, 0.478 mmol, 4.6 equivalence) were added to 10 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 807 mg of CPG-NH2 was added, and the mixture was shaken overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and 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 solid phase was filtered, washed with dichloromethane and acetonitrile, and dried under reduced pressure to give a yellow solid compound 17a-15 (770 mg, 43.71 μmol / g).

[0576] Step 18n: Preparation of compound 17a-16-A-ssRNA-1: Compound 17a-15 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 17a-16-A-ssRNA-1 (MS m / z: [MH]-: calcd. 9123.6655 Found 9122.7623).

[0577] 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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 17a-16-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 81A-1 was obtained by ethanol precipitation. Conjugate 81A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16450.6516 Found 16450.1231).

[0578] Step 18p: Preparation of conjugate 81A: Take equal amounts of conjugate 81A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 81A.

[0579] Example 19: Preparation of conjugate 74A (prepared according to scheme 18a):

[0580]

[0581]

[0582] Step 19a: Preparation of compound 18a-2: Benzyl bromide (13.1 mL, 109.89 mL, 2.5 equivalences) was added to a mixture of compound 18a-1 (4.0 g, 43.96 mmol, 1.0 equivalences), potassium carbonate (18.2 g, 131.88 mmol, 3.0 equivalences), 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 × 2). The combined organic layers were washed with saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane:methanol 80:1) to give compound 18a-2 (9.89 g, yield: 83%) as a white solid. LCMS (ESI): m / z 272 [M+H] + TLC: Rf0.5 (dichloromethane:methanol ester = 50:1).

[0583] Step 19b: Preparation of compound 18a-3: Sodium hydroxide (15 g, 375 mmol, 15 equivalents) was added to a mixture of compound 18a-2 (6.7 g, 24.72 mmol, 1.0 equivalent), tert-butyl bromoacetate (28.78 g, 147.55 mmol, 6.0 equivalent), tetrabutylammonium hydrogen sulfate (1.26 g, 3.71 mmol, 0.15 equivalent), 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 × 2). The combined organic layers were washed with saturated brine (50 mL × 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 give a 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).

[0584] Step 19c: Preparation of compound 18a-4: A mixture of compound 18a-3 (3.73 g, 7.46 mmol, 1.0 equivalence) and dioxane (4 M, 20 mL) was stirred overnight at room temperature. 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 equivalence), N,N-diisopropylethylamine (7.7 g, 59.68 mmol, 8.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.51 g, 22.38 mmol, 3.0 equivalence) were added. The mixture was stirred at room temperature for 7 hours. The solvent was removed under reduced pressure. The residue was diluted with a saturated sodium carbonate solution (50 mL) and then extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a pale yellow oily compound 18a-4 (3.5 g, crude). LCMS (ESI): m / z 386 [M+H] + TLC: Rf0.5 (dichloromethane:methanol = 20:1).

[0585] Step 19d: Preparation of compound 18a-5: Borane dimethyl sulfide complex (10M, 5.97 mL, 59.7 mmol, 8.0 equivalence) was added to a mixture of compound 18a-4 (2.87 g, 7.46 mmol, 1.0 equivalence) dissolved in tetrahydrofuran (40 mL). The mixture was heated to 65 °C and reacted for 5 hours. The mixture was quenched with water (25 mL). Sodium carbonate (2.37 g, 22.38 mmol, 3.0 equivalence) and di-tert-butyl dicarbonate (4.88 g, 22.38 mmol, 3.0 equivalence) 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 layers were 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 give a pale yellow oily compound 18a-5 (741 mg, yield: 18%). LCMS (ESI): m / z 558 [M+H] + TLC: Rf0.5 (petroleum ether: ethyl acetate = 5:1).

[0586] Step 19e: Preparation of compound 18a-6: A solution of hydrogen chloride-dioxane (4 M, 5 mL) was added to a mixture of compound 18a-5 (741 mg, 1.33 mmol, 1.0 equivalence) dissolved in methanol (2 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 give crude compound 18a-6 as a white solid. LCMS (ESI): m / z 358 [M+H] + TLC: Rf0.2 (dichloromethane:methanol = 10:1).

[0587] Step 19f: Preparation of compound 18a-7: At 0 °C, a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (1.38 g, 3.33 mmol, 2.5 equivalences) in dichloromethane (85 mL) was added dropwise over 30 minutes to a mixture of compound 1a-10 (786 mg, 1.33 mmol, 1.0 equivalences), compound 18a-6 (616 mg, 1.33 mmol, 1.0 equivalences), and N,N-diisopropylethylamine (1.03 g, 7.98 mmol, 6.0 equivalences) in dichloromethane (15 mL). The mixture was brought 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 × 3). The combined organic layers were washed with saturated brine (20 mL × 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 give a 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).

[0588] Step 19g: Preparation of compound 18a-8: Compound 18a-7 (0.42 g, 0.46 mmol, 1.0 equivalence) was dissolved in a mixture of hydrogen chloride-dioxane (4 M, 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 equivalence), N,N-diisopropylethylamine (475 mg, 3.68 mmol, 8.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (525 mg, 1.38 mmol, 3.0 equivalence) 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 × 2) and saturated brine (20 mL × 1), dried over anhydrous sodium sulfate, and concentrated. The residue was separated by column chromatography on silica gel (dichloromethane:methanol:triethylamine 30:1:0.3) to give a white solid compound 18a-8 (778 mg, yield: 89%). LCMS (ESI): m / z 1902.5 [M+H] + TLC: Rf0.5 (dichloromethane:methanol = 10:1).

[0589] 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 equivalence) and trifluoroacetic acid (47 mg, 0.409 mmol, 1.0 equivalence) in methanol (10 mL) and ethanol (5 mL). The mixture was stirred overnight at room temperature under hydrogen balloon pressure. The mixture was filtered. The filtrate was concentrated under reduced pressure to give compound 18a-9 (600 mg, crude) as a pale yellow solid. LCMS (ESI): m / z 1721.5 [M+H] + TLC: Rf0.3 (dichloromethane:methanol = 10:1).

[0590] Step 19i: Preparation of compound 18a-10 (compound 16): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (188 mg, 0.495 mmol, 1.5 equivalence) was added to a mixture of compound 18a-9 (600 mg, 0.33 mmol, 1.0 equivalence), compound 4a-6 (265 mg, 0.33 mmol, 1.0 equivalence), N,N-diisopropylethylamine (255 mg, 1.98 mmol, 6.0 equivalence), and N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 0.5 h. The mixture was purified by preparative liquid chromatography (A: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%-90% over 30 min) to give compound 18a-10 (180 mg, yield: 22%) as a white solid. LCMS(ESI): m / z1102.6[(M-302) / 2+H] + TLC: Rf0.5 (dichloromethane:methanol = 10:1).

[0591] Step 19j: Preparation of compound 18a-11: Succinic anhydride (43 mg, 0.43 mmol, 6.0 equivalence) was added to a solution of compound 18a-10 (180 mg, 0.072 mmol, 1.0 equivalence), triethylamine (87 mg, 0.862 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (4.4 mg, 0.036 mmol, 0.5 equivalence) in dichloromethane (2 mL). The mixture was stirred at room temperature for 20 hours. The mixture was diluted with dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (10 mL × 2) and saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a 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).

[0592] Step 19k: Preparation of Compound 18a-12: To a mixture of compound 18a-11 (155 mg, 0.06 mmol, 1.0 equivalence) and N,N-diisopropylethylamine (46 mg, 0.36 mmol, 6.0 equivalence) dissolved in N,N-dimethylformamide (8 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (34 mg, 0.09 mmol, 1.5 equivalence) and 1-hydroxybenzotriazole (12 mg, 0.09 mmol, 1.5 equivalence) were added. The mixture was stirred at room temperature for 5 minutes. CPG-NH2 (0.167 mmol / g, 775 mg, 0.13 mmol, 2.2 equivalence) was added. The mixture was stirred at room temperature overnight. The mixture was filtered. The solid was washed with dichloromethane (8 mL × 5). Add pyridine (6 mL) and acetic anhydride (2 mL). Stir the mixture at room temperature for 3 hours. Filter the mixture. Wash the solid with dichloromethane (8 mL × 5) and dry under vacuum to give a white solid compound 18a-12 (785 mg, 21.5 μmol / g, yield: 29%).

[0593] Step 19l: Preparation of compound 18a-13-A-ssRNA-1: Compound 18a-12 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 18a-13-A-ssRNA-1 (MS m / z: [MH]-: calcd.9141.5925 Found9140.7005).

[0594] Step 19m: Preparation of conjugate 74A-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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 18a-13-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 74A-1 was obtained by ethanol precipitation. Conjugate 74A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16468.5786 Found 16468.2769).

[0595] Step 19n: Preparation of conjugate 74A: Take equal amounts of conjugate 74A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 74A.

[0596] Example 20: Preparation of conjugate 44A (prepared according to scheme 19a):

[0597]

[0598]

[0599] Step 20a: Preparation of compound 19a-2: At -20°C, isobutyl chloroformate (9.02 g, 66.00 mmol, 1.0 equivalent) was added dropwise to a mixture of compound 19a-1 (20 g, 66.00 mmol, 1.0 equivalent) and N-methylmorpholine (6.68 g, 66.00 mmol, 1.0 equivalent) in tetrahydrofuran (100 mL). The mixture was stirred for 10 minutes. The mixture was filtered. At -30°C, a solution of sodium borohydride (5.02 g, 132.01 mmol, 2.0 equivalent) in water (30 mL) was slowly added to the filtrate. The mixture was brought 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]+.

[0600] Step 20b: Preparation of compound 19a-3: Under nitrogen protection, at 0°C, elemental iodine (26.26 g, 103.45 mmol, 1.5 equivalents) was added to a mixture of triphenylphosphine (27.13 g, 103.45 mmol, 1.5 equivalents), imidazole (7.04 g, 103.45 mmol, 1.5 equivalents), and dichloromethane (200 mL). The mixture was stirred at room temperature for half an hour. Compound 19a-2 (20 g, 68.97 mmol, 1.0 equivalent) was added to the mixture. The mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with saturated sodium thiosulfate and extracted with dichloromethane. The combined organic layers were 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 give a colorless oily compound 19a-3 (24.09 g, yield: 87.34%). LCMS (ESI): m / z = 422 [M+Na]+.

[0601] Step 20c: Preparation of compound 19a-4: A mixture of benzylamine (2.92 g, 27.27 mmol, 1.0 equivalent), compound 19a-3 (24.09 g, 60.23 mmol, 2.2 equivalent), and potassium carbonate (11.33 g, 82.13 mmol, 3.0 equivalent) in acetonitrile (250 mL) was stirred at 60 °C for 3 hours. The mixture was concentrated and diluted with water, then extracted with ethyl acetate. The combined organic layers were 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 5:1) to give a yellow oily compound 19a-4 (14.14 g, yield: 36.13%). LCMS (ESI): m / z = 650 [M+H]+.

[0602] Step 20d: Preparation of compound 19a-5: At 0°C, 1-chloroethyl chloroformate (4.67 g, 32.63 mmol, 1.0 equivalent) was added dropwise to a mixture of compound 19a-4 (14.14 g, 21.75 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (560 mg, 4.35 mmol, 0.2 equivalent) in acetonitrile (110 mL). The mixture was brought 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 separated by column chromatography on silica gel (dichloromethane:methanol = 200:1 to 20:1) to give a yellow oily compound 19a-5 (8.39 g, yield: 68.88%). LCMS (ESI): m / z = 560 [M+H]+.

[0603] Step 20e: Preparation of compound 19a-6: To a mixture of compound 19a-5 (8.39 g, 14.98 mmol, 1.00 equivalence), 3-(tert-butoxycarbonyl)amino)propionic acid (2.83 g, 14.98 mmol, 1.00 equivalence), and N,N-diisopropylethylamine (3.87 g, 29.96 mmol, 2.0 equivalence) in dichloromethane (80 mL), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (6.84 g, 17.98 mmol, 1.2 equivalence) was added. The mixture was stirred at room temperature for 2.5 h. The solvent was removed under reduced pressure. The residue was separated by 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]+.

[0604] Step 20f: Preparation of Compound 19a-7: A mixture of compound 19a-6 (8.31 g, 11.37 mmol, 1.0 equivalent) and dioxane (4 mol / L, 80 mL) was stirred overnight at room temperature. The solvent was removed under reduced pressure. The residue was dissolved in a mixture of tetrahydrofuran (80 mL) and water (80 mL). Sodium carbonate (3.62 g, 34.10 mmol, 6.0 equivalent) and di-tert-butyl carbonate (12.39 g, 56.84 mmol, 5.0 equivalent) were added. The mixture was stirred overnight at room temperature. The 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 solution of dilute hydrochloric acid, 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 solid product 19a-7 (6.0 g, yield: 85.47%). LCMS (ESI): m / z = 619 [M+H]+.

[0605] Step 20g: Preparation of Compound 19a-8: At 0°C, a solution of Compound 1a-4 (1.28 g, 4.13 mmol, 1.0 equivalence), Compound 19a-7 (2.55 g, 4.13 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (2.66 g, 20.65 mmol, 5.0 equivalence) in dichloromethane (50 mL) was added to a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (3.76 g, 9.08 mmol, 2.2 equivalence) and dichloromethane (50 mL). 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 (elution: ethyl acetate to ethyl acetate:methanol = 20:1) to give a white solid product 19a-8 (1.09 g, yield: 32.25%). LCMS (ESI): m / z = 820 [M+H] + .

[0606] Step 20h: Preparation of compound 19a-9: A solution of compound 19a-8 (1.09 g, 1.33 mmol, 1.0 equivalent) in 10 mL of 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 (crude product) without purification. LCMS (ESI): m / z = 520 [M+H] + .

[0607] 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 equivalence), compound 1a-12 (1.78 g, 3.99 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.62 g, 4.25 mmol, 3.2 equivalence) 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 (elution buffer: 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]+.

[0608] Step 20j: Preparation of compound 19a-11: Under a hydrogen atmosphere, a mixture of 19a-10 (2.18 g, 1.21 mmol, 1.0 equivalent), palladium hydroxide (436 g, 20% by mass), and trifluoroacetic acid (137 mg, 1.21 mmol, 1.0 equivalent) in ethanol / methanol (10 mL / 10 mL) was stirred overnight at room temperature. The mixture was filtered through diatomaceous earth. The filter cake was washed with methanol, and the filtrate was concentrated. The residue was used directly for the next step without further purification (2.18 g, crude product). LCMS (ESI): m / z = 838 [M / 2+H] + .

[0609] Step 20k: Preparation of compound 19a-12 (compound 20): A mixture of compound 19a-11 (1.0 g, 0.56 mmol, 1.0 equivalence), compound 4a-6 (450 mg, 0.56 mmol, 1.0 equivalence), N,N-diisopropylethylamine (217 mg, 1.68 mmol, 3.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (255 mg, 0.67 mmol, 1.2 equivalence) in dichloromethane (10 mL) was stirred for 1 hour. The mixture was purified by HPLC (column: UniSil 10-120C18, 30 × 250 mm) to give a white solid product 19a-12 (470 g, yield: 34.31%). LCMS(ESI): m / z=1079[(M-302) / 2+H] + .

[0610] Step 20l: Preparation of compound 19a-13: A mixture of compound 19a-12 (470 mg, 0.19 mmol, 1.0 equivalence), succinic anhydride (115 mg, 1.15 mmol, 6.0 equivalence), triethylamine (232 mg, 2.29 mmol, 12.0 equivalence), and 4-dimethylaminopyridine (2.3 mg, 0.019 mmol, 0.1 equivalence) in dichloromethane (5 mL) was stirred overnight at room temperature. The mixture was washed with 10% sodium bicarbonate solution and extracted with dichloromethane. After adding acetonitrile to the organic phase, it was washed with 10% sodium bicarbonate solution. The organic phase was concentrated to give 19a-13 as a white solid (457 mg, crude). LCMS (ESI): m / z = 1129 [(M-302) / 2+H] + .

[0611] Step 20m: Preparation of compound 19a-14: To an N,N-dimethylformamide solution (8 mL) of compound 19a-13 (200 mg, 0.078 mmol, 1.0 equivalence), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (36 mg, 0.094 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (15 mg, 0.11 mmol, 1.4 equivalence), and N,N-diisopropylethylamine (40 mg, 0.31 mmol, 4.0 equivalence) were added, and the mixture was stirred at room temperature for 5 minutes. Then, CPG-NH2 (900 mg) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. The mixture was filtered, and the filter cake was washed successively with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (6 mL / 2 mL), and the mixture was stirred at room temperature for 3 hours. The mixture was filtered, and the filter cake was washed sequentially with dichloromethane, acetonitrile, and dichloromethane, and then dried under vacuum for 1 hour to obtain a light yellow solid product 19a-14 (910 mg, loading rate: 22.27 μmol / g).

[0612] Step 20n: Preparation of compound 19a-15-A-ssRNA-1: Compound 19a-14 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 19a-15-A-ssRNA-1 (MS m / z: [MH]-: calcd.9095.6115Found9093.7784).

[0613] Step 20o: Preparation of conjugate 44A-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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 19a-15-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, about 2.3 mg of crude conjugate 44A-1 was obtained by ethanol precipitation. Conjugate 44A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16422.5536 Found 16421.2100).

[0614] Step 20p: Preparation of conjugate 44A: Take equal amounts of conjugate 44A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 44A.

[0615] Example 21: Preparation of conjugate 80A (prepared according to scheme 20a):

[0616]

[0617]

[0618] Step 21a: Preparation of compound 20a-1: Di-tert-butyl dicarboxylate (5.0 g, 16.48 mmol, 1.00 equivalent), ethyl 2-bromoacetate (3.1 g, 18.13 mmol, 1.1 equivalent), and potassium carbonate (4.5 g, 32.96 mmol, 2.0 equivalent) 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). LCMS (ESI): m / z = 390 [M+H] + .

[0619] Step 21b: Preparation of compound 20a-2: Compound 20a-1 (6.2 g, 15.94 mmol, 1.0 equivalent) and lithium hydroxide monohydrate (1.6 g, 39.85 mmol, 2.5 equivalent) 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 h. Water was added to the mixture, and the pH was adjusted to 5 with 2 mol of dilute hydrochloric acid. The aqueous phase was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound 20a-2 (5.1 g, crude product). LCMS (ESI): m / z = 362 [M+H] + .

[0620] Step 21c: Preparation of compound 20a-3: Compound 2b-2 (13.6 g, 22.05 mmol, 1.00 equivalent) was dissolved in a trifluoroacetic acid / dichloromethane solution (50 mL / 100 mL) and reacted at room temperature for 1 hour. The mixture was evaporated to dryness. The residue was adjusted to pH 8 with water and an aqueous sodium carbonate solution. 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] + .

[0621] Step 21d: Preparation of compound 20a-4: Compound 20a-2 (4.3 g, 11.82 mmol, 1.0 equivalence), compound 20a-3 (6.1 g, 11.82 mmol, 1.0 equivalence), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.4 g, 14.18 mmol, 1.2 equivalence), and N,N-diisopropylethylamine (3.1 g, 23.04 mmol, 2.0 equivalence) 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 at a ratio of 1:1. The mixture was filtered to give compound 20a-4 (10 g, crude), a yellow solid. LCMS(ESI): m / z = 861[M+H] + .

[0622] Step 21e: Preparation of compound 20a-5: A solution of compound 20a-4 (9.5 g, 11.05 mmol, 1.0 equivalent) in diethylamine / acetonitrile (20 / 80 mL) was stirred at room temperature for 1 hour. The mixture was purified by silica gel column chromatography (elution: 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]+ .

[0623] Step 21f: Preparation of compound 20a-6: At 0 °C, Desmartin reagent (4.7 g, 11.17 mmol, 1.5 equivalence) was added to a tetrahydrofuran (30 mL) solution of (tert-butyloxycarbonyl)-L-homoserine benzyl ester (2.3 g, 7.44 mmol, 1.0 equivalence). The mixture was stirred at room temperature for 2 hours. The reaction solution was quenched with saturated sodium thiosulfate solution and then 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). LCMS (ESI): m / z = 308 [M+H] + .

[0624] Step 21g: Preparation of compound 20a-7: A methanol (50 mL) solution of compound 20a-5 (3.65 g, 5.72 mmol, 1.0 equivalent), compound 20a-6 (1.8 g, 6.01 mmol, 1.05 equivalent), and acetic acid (343 mg, 5.72 mmol, 1.0 equivalent) was stirred at room temperature for 30 min. The mixture was cooled to 0 °C, and sodium cyanoborohydride was added. The mixture was stirred at room temperature for 1.5 h. 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 (elution: 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] + .

[0625] Step 21h: Preparation of compound 20a-8: Compound 20a-7 (3 g, 3.23 mmol, 1.0 equivalence), 3-(tert-butoxycarbonyl)amino)propionic acid (611 mg, 3.23 mmol, 1.0 equivalence), N,N-diisopropylethylamine (835 mg, 6.46 mmol, 2.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.5 mg, 3.88 mmol, 1.2 equivalence) 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, with the addition of 1% triethylamine) to give a white solid compound 20a-8 (1.5 g, yield: 42.1%). LCMS (ESI): m / z = 1101 [M+H] + .

[0626] Step 21i: Preparation of compound 20a-9: Compound 20a-8 (1.5 g, 1.36 mmol, 1.0 equivalent) was added to 20 mL of 4 M 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 product 20a-9 (1.1 g, crude product). LCMS (ESI): m / z = 700 [M+H] + .

[0627] Step 21j: Preparation of compound 20a-10: Compound 20a-9 (1.1 g, 1.27 mmol, 1.0 equivalence), compound 1a-12 (2.3 g, 5.21 mmol, 4.1 equivalence), N,N-diisopropylethylamine (2 g, 15.24 mmol, 12.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2 g, 5.33 mmol, 4.2 equivalence) were added to 15 mL of acetonitrile. 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, with the addition of triethylamine) to give a white solid compound 20a-10 (1.8 g, yield: 58.6%). LCMS (ESI): m / z = 1210 [M / 2 + H] + .

[0628] Step 21k: Preparation of compound 20a-11: Compound 20a-10 (800 mg, 0.331 mmol, 1.0 equivalent) was dissolved in 8 mL of ethanol, and trifluoroacetic acid (38 mg, 0.331 mmol, 1.0 equivalent) 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 compound 20a-11 (890 mg, crude product), a white solid. LCMS (ESI): m / z = 1098 [M / 2+H] + .

[0629] Step 21l: Preparation of compound 20a-12 (compound 63): Compound 20a-11 (760 mg, 0.346 mmol, 1.0 equivalence), N,N-diisopropylethylamine (134 mg, 1.038 mmol, 3.0 equivalence), and compound 2b-10 (97 mg, 0.381 mmol, 1.1 equivalence) were added to 10 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h. Then, N,N-diisopropylethylamine (134 mg, 1.038 mmol, 3.0 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (171 mg, 0.415 mmol, 1.2 equivalence), and intermediate M (249 mg, 0.415 mmol, 1.2 equivalence) were added, and the mixture was stirred at room temperature for 1 h. 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 / acetonitrile) to give a yellow solid compound 20a-12 (272 mg, yield: 27.2%). LCMS (ESI): m / z = 1306 [(M-302) / 2+H] + .

[0630] Step 21m: Preparation of compound 20a-13: Compound 20a-12 (172 mg, 0.0589 mmol, 1.0 equivalent), triethylamine (71 mg, 0.707 mmol, 12.0 equivalent), succinic anhydride (35 mg, 0.354 mmol, 6.0 equivalent), and p-dimethylaminopyridine (1 mg, 0.00589 mmol, 0.1 equivalent) were added to 1 mL of dichloromethane. The mixture 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 under reduced pressure to give compound 20a-13 (167 mg, crude) as a yellow solid. LCMS (ESI): m / z = 1359[(M-302) / 2+H]+.

[0631] Step 21n: Preparation of compound 20a-14: Compound 20a-13 (167 mg, 0.0554 mmol, 1.0 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (29 mg, 0.0775 mmol, 1.2 equivalence), 1-hydroxybenzotriazole (12 mg, 0.0886 mmol, 1.6 equivalence), and N,N-diisopropylethylamine (29 mg, 0.222 mmol, 4.0 equivalence) were added to 2 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 501 mg of CPG-NH2 was added, and the mixture was shaken overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and 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 solid phase was filtered, washed with dichloromethane and acetonitrile, and dried under reduced pressure to give a yellow solid compound 20a-14 (532 mg, loading: 28.6 μmol / g).

[0632] Step 21o: Preparation of compound 20a-15-A-ssRNA-1: Compound 20a-14 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 20a-15-A-ssRNA-1 (MS m / z: [MH]-: calcd. 9428.0085 Found 9427.849).

[0633] Step 21p: Preparation of conjugate 80A-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 CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nmol, 1 equivalent) and 20a-15-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and the mixture was purged with nitrogen and reacted at 35 °C for 2 h. After the reaction was complete, 15 μL of ammonia and 16 mg of EDTA were added. After shaking for 5-10 minutes, approximately 2.3 mg of crude conjugate 80A-1 was obtained by ethanol precipitation. Conjugate 80A-1 was then purified by HPLC (MS m / z: [MH]-: calcd. 16754.9946 Found 16753.2000).

[0634] Step 21q: Preparation of conjugate 80A: Take equal amounts of conjugate 80A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions, mix them evenly, heat them in a 95℃ water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 80A.

[0635] Example 22: Preparation of conjugate 88A (prepared according to scheme 21a):

[0636]

[0637]

[0638] Step 22a: Preparation of compound 21a-1: A 40% aqueous solution of methylamine (1.5 g, 19.23 mmol, 1.1 equivalence) was added to a mixture of (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (7.7 g, 17.48 mmol, 1.0 equivalence), N,N-diisopropylethylamine (3.39 g, 26.28 mmol, 1.5 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (7.95 g, 19.23 mmol, 1.1 equivalence), and 85 mL of dimethylformamide. 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 21a-1 (6.91 g, yield: 87.2%). LCMS (ESI): m / z = 454 (M+H) + .

[0639] Step 22b: Preparation of compound 21a-2: Compound 21a-1 (6.91 g, 15.24 mmol, 1.0 equivalent) and 1,8-diazabicyclo[5.4.0]undec-7-ene (4.63 g, 30.48 mmol, 2.0 equivalent) were added to 125 mL of acetonitrile and 50 mL of dichloromethane. The mixture was stirred at room temperature for 30 min. 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 give compound 21a-2 (6.2 g, crude) as a yellow solid. LCMS (ESI): m / z = 232 (M+H) + .

[0640] Step 22c: Preparation of compound 21a-3: Compound 21a-2 (6.2 g, 15.24 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (3.9 g, 30.48 mmol, 2.0 equivalent) were added to 50 mL of tetrahydrofuran at 0 °C, 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 give compound 21a-3 (1.1 g, yield: 15.6%) as a yellow solid. LCMS (ESI): m / z = 462 (M+H) + .

[0641] Step 22d: Preparation of compound 21a-4: Under nitrogen protection, a toluene solution of diethyl azodicarbonate (1.13 g, 6.48 mmol, 3.0 equivalent) was added dropwise to a toluene solution of compound 21a-3 (1.0 g, 2.16 mmol, 1.0 equivalent), tert-butyl (2-hydroxyethyl)carbamate (975 mg, 6.05 mmol, 2.8 equivalent), and triphenylphosphine (1.7 g, 6.48 mmol, 3.0 equivalent) for 1 hour at room temperature. The reaction mixture was filtered. The filter cake was dissolved in dichloromethane, and petroleum ether was added. The mixture was filtered again and washed with petroleum ether. The solid was dried under reduced pressure to give a yellow solid, compound 21a-4 (1.34 g, yield: 93.0%). LCMS (ESI): m / z = 605 (M+H) + .

[0642] Step 22e: Preparation of compound 21a-5: 2-Mercaptoacetic acid (408 mg, 4.43 mmol, 2.0 equivalence) was added to a mixture of compound 21a-4 (1.34 g, 2.21 mmol, 1.0 equivalence), triethylamine (670 mg, 6.63 mmol, 3.0 equivalence), and 15 mL of dichloromethane. 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, and the organic phase was concentrated under reduced pressure. The residue was dried under reduced pressure to give a yellow solid, compound 21a-5 (1.07 g, crude). LCMS (ESI): m / z = 375 (M+H) + .

[0643] Step 22f: Preparation of compound 21a-6: Compound 21a-5 (1.07 g, 2.21 mmol, 1.0 equivalence), compound 2b-5 (746 mg, 2.21 mmol, 1.0 equivalence), N,N-diisopropylethylamine (570 mg, 4.42 mmol, 2.0 equivalence), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (924 mg, 2.43 mmol, 1.1 equivalence) were added to 10 mL of N,N-dimethylformamide. 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, with the addition of triethylamine) to give a yellow solid compound 21a-6 (940 mg, yield: 61.4%). LCMS (ESI): m / z = 694 (M+H) + .

[0644] Step 22g: Preparation of compound 21a-7: Compound 21a-6 (940 mg, 1.35 mmol, 1.0 equivalence) was added to 10 mL of 4 M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give a white solid compound 21a-7 (1.01 g, crude product). LCMS (ESI): m / z = 394 (M+H) + .

[0645] Step 22h: Preparation of compound 21a-8: Compound 21a-7 (690 mg, 1.35 mmol, 1.0 equivalence), compound 1a-12 (1.94 g, 4.33 mmol, 3.2 equivalence), N,N-diisopropylethylamine (1.74 g, 13.5 mmol, 10.0 equivalence), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.65 g, 4.33 mmol, 3.2 equivalence) were added to 30 mL of acetonitrile. 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, with the addition of triethylamine) to give a yellow solid compound 21a-8 (1.79 g, yield: 79.1%). LCMS (ESI): m / z = 1682 (M+H) + .

[0646] Step 22i: Preparation of compound 21a-9: Compound 21a-8 (1.79 g, 1.07 mmol, 1.0 equivalent) was dissolved in 640 mL of ethanol, and trifluoroacetic acid (216 mg, 1.89 mmol, 1.8 equivalent) and 270 mg of 10% palladium hydroxide / carbon were added. The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give compound 21a-9 (2.02 g, crude product), a white solid. LCMS (ESI): m / z = 1592.4 (M+H) + .

[0647] Step 22j: Preparation of compound 21a-10 (compound 66): An acetonitrile solution of intermediate M (709 mg, 1.18 mmol, 1.1 equivalence) was added to a mixture of compound 21a-9 (2.02 g, 1.07 mmol, 1.0 equivalence), N,N-diisopropylethylamine (276 mg, 2.14 mmol, 2.0 equivalence), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (488 mg, 1.18 mmol, 1.1 equivalence), and 20 mL of acetonitrile. 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 / acetonitrile) to give compound 21a-10 (1.38 g, yield: 59.2%) as a white solid. LCMS(ESI): m / z=938((M-302) / 2+H) + .

[0648] Step 22k: Preparation of compound 21a-11: Under nitrogen protection, compound 21a-10 (500 mg, 0.230 mmol, 1.0 equivalence), triethylamine (326 mg, 3.22 mmol, 14.0 equivalence), succinic anhydride (161 mg, 1.61 mmol, 7.0 equivalence), and p-dimethylaminopyridine (3 mg, 0.023 mmol, 0.1 equivalence) were added to 3 mL of dichloromethane. The mixture 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 under reduced pressure to give compound 21a-11 (536 mg, crude product) as a white solid. LCMS (ESI): m / z = 1975.7 ((M-302)+H) + .

[0649] Step 22l: Preparation of compound 21a-12: Compound 21a-11 (536 mg, 0.23 mmol, 1.0 equivalence), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (122 mg, 0.32 mmol, 1.4 equivalence), 1-hydroxybenzotriazole (50 mg, 0.37 mmol, 1.6 equivalence), and N,N-diisopropylethylamine (137 mg, 1.06 mmol, 4.6 equivalence) 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-NH2 was added, and the mixture was shaken overnight at room temperature. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and 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 solid phase was filtered, washed with dichloromethane and acetonitrile, and dried under reduced pressure to give a yellow solid compound 21a-12.

[0650] Step 21m: Preparation of conjugate 88A-1: Compound 21a-12 was synthesized by solid-phase oligonucleotide synthesis, followed by ammonolysis and purification by HPLC to obtain 88A-1 (MS m / z: [MH]-: calcd. 8195.9620 Found 8194.6426).

[0651] Step 21n: Preparation of conjugate 88A: Take equal amounts of conjugate 88A-1 and complementary antisense chain (1167AM25) solution, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 88A.

[0652] The oligonucleotide sequence information of each conjugate synthesized in the above embodiments is shown in Tables 1-3.

[0653] Table 1. Sequence Composition Information of Each Conjugate

[0654]

[0655]

[0656] Table 2 Summary of positive-strand mass spectrometry information for each conjugate

[0657]

[0658]

[0659] Table 3 Summary of sequences used for each conjugate

[0660]

[0661] The unmodified sequences corresponding to Table 3 are as follows:

[0662]

[0663]

[0664] Example 23: Testing the effect of siRNA-conjugates on the expression of AGT and PCSK9 in primary hepatocytes of hAGT / hPCSK9 mice

[0665] The human AGT (hAGT) gene or the human PCSK9 (hPCSK9) gene was integrated into a liver-targeted adeno-associated virus type 8 (AAV8) expression vector. Ultrapure recombinant AAV8-hAGT and AAV8-hPCSK9 viruses were prepared by ordering from a vector manufacturer. The AAV8-hAGT and AAV8-hPCSK9 viruses were mixed and diluted with PBS to a viral titer of 7.5 × 10⁻⁶. 11The viral fluid was administered intravenously at a concentration of 0.2 mL / mL to 6-8 week old female mice (C57BL / 6), equivalent to 1.5 x 10⁹ / mL per mouse. 11 Titer virus. Transgenic mice stably expressing hAGT / hPCSK9 were obtained after 14 days.

[0666] The dual-humanized AGT / PCSK9 mice were purchased from Cyagen Biosciences.

[0667] Primary mouse hepatocyte extraction: Mouse hepatocytes were extracted via inferior vena cava perfusion and digested with collagenase. After filtration through a tissue cell filter (BIOLOGIX, 15-1070), viable primary mouse hepatocytes were obtained and resuspended in DMEM medium (containing 10% FBS and 1X penicillin-streptomycin). Cell density was measured using a Scepter automated cell counter (Millipore, #PHCC00000). Simultaneously, 2 μL of siRNA-conjugate, 68 μL of Opti-MEM, and 5 μL of INTERFERin transfection reagent were mixed in 96-well plates and incubated at room temperature for 10 minutes (INTERFERin is not required for the free-take method). Then, 125 μL of complete culture medium containing primary mouse hepatocytes was added to each well.

[0668] 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 culture medium in the 96-well plates was aspirated, and the plates were washed once with DPBS. 100 μl of cell lysis buffer was added to each well, followed by 20 μl of beads. The plates were shaken on a vortex mixer, and the 96-well plates were placed on a magnetic separator. The lysis buffer was aspirated, and 100 μl of washing buffer A was added to each well. After pipetting, the plates were placed on a magnetic separator, and washing buffer A was aspirated. Then, 100 μl of washing buffer B was used to lift the beads, which were then transferred to a new 96-well plate. The plate was placed on a magnetic separator, and washing buffer B was aspirated. 100 μl of low-salt buffer was used to lift the beads, which were then transferred to a 96-well PCR plate. The PCR plate was placed on a magnetic separator, and the low-salt buffer was aspirated. 10 μL of elution buffer was added to each well to lift 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 using StarScriptPro one-tube genomic de-generated reverse transcription premix (Genstar). 5 μL of the mixture was dispensed into each well of a 96-well PCR instrument, and 5 μL of the mRNA solution obtained in the previous step was added and mixed. The mixture was briefly centrifuged and sealed with a sealing film. The PCR instrument was incubated at 37°C for 3 minutes, 50°C for 50 minutes, and then at 85°C for 2 minutes. The temperature was then lowered to 4°C to complete the reverse transcription.

[0669] Real-time quantitative PCR: After reverse transcription, place the 96-well plate on the magnetic separator until the magnetic beads are adsorbed to the bottom. Remove the reverse reagent, add the prepared qPCR system to the 96-well PCR plate, seal the plate with the sealing membrane, and perform PCR on the StepOnePlus real-time PCR system (applied biosystems).

[0670] Data were analyzed using the ΔΔCt method, and the test was standardized using cells transfected with a 1 nM negative control sequence.

[0671] The negative control AD-1955 sequence is as follows:

[0672] CUUACGCUGAGUACUUCGAdTdT(SEQ ID NO:13)

[0673] UCGAAGUACUCAGCGUAAGdTdT (SEQ ID NO: 14).

[0674] The primer sequences for detecting AGT are:

[0675]

[0676] The primer sequences for detecting PCSK9 are as follows:

[0677]

[0678] The experimental results are shown in Tables 4-7.

[0679] Table 4. Results of AGT single-target siRNA inhibition experiments in primary hepatocytes from dual-humanized AGT / PCSK9 mouse cells.

[0680] siRNA conjugates Compound numbering IC50 value (nM) Conjugate 1A 31 0.05995 Conjugate 1B 31 0.03662

[0681] Conjugates 1A and 1B used compound 31 to deliver siRNA targeting AGT. The results in Table 4 show that compound 31 can efficiently deliver siRNA in vitro.

[0682] Table 5. Experimental results of AGT single-target siRNA at different concentrations in primary hepatocytes of AAV8-hAGT / hPCSK9 mice via free uptake.

[0683]

[0684] Table 5 shows that compounds 31 and 66 can efficiently deliver siRNA in vitro.

[0685] Table 6. Results of AGT / PCSK9 dual-targeting siRNA inhibition experiments in primary hepatocytes of dual-humanized AGT / PCSK9 mice.

[0686]

[0687] The experimental results in Table 6 show that, in primary humanized AGT / PCSK9 mouse hepatocytes, when the same sequence combination was delivered in vitro, both conjugate 2A and conjugate 3A could simultaneously knock down both AGT and PCSK9 targets, with conjugate 2A showing better knockdown than conjugate 3A. This indicates that compound 54 is slightly more effective than compound 55 in delivering dual-target siRNA.

[0688] Table 7 Results of AGT / PCSK9 dual-targeting siRNA inhibition experiments in AAV8-hAGT / hPCSK9 mouse primary hepatocytes

[0689]

[0690] As shown in Table 7, different conjugates can simultaneously inhibit both AGT and PCSK9 targets with high activity when delivering the same sequence combination in vitro. Conjugates 2A, 4A, and 9A exhibited the highest activity, indicating that compounds 54 and 53 are more effective than compound 51 in delivering dual-target siRNA. Furthermore, comparing the data from conjugates 2A, 7A, 8A, and 9A revealed that the connection between the vector compound and siRNA via different linker strands also affected the delivery efficiency; the connections via Q13 and Q20 showed a more significant improvement in siRNA activity.

[0691] Example 24: Testing the effect of different siRNA-conjugates on AGT expression in AAV8-hAGT mice (1 mg / kg)

[0692] The effects of different compound deliverables of 1167.25-19 on hepatic hAGT expression were evaluated in mice expressing AAV8-hAGT.

[0693] 1. Construction of hAGT transgenic mice

[0694] AAV8-hAGT virus was prepared with PBS to a viral titer of 5*10. 11 The viral fluid was administered intravenously at a concentration of 0.2 mL / mL to 6-8 week old female mice (C57BL / 6), equivalent to 1*10^6 / mL per mouse. 11 Titer virus. Transgenic mice stably expressing hAGT were obtained after 14 days.

[0695] 2. Administration

[0696] Fourteen days after injection of the virus, the mice were divided into five groups. The siRNA conjugate was dissolved in physiological saline and administered subcutaneously at a dose of 1 mg / kg.

[0697] 3. ELISA testing

[0698] Fourteen days after injection of the virus, mice underwent tail amputation and blood collection. The blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 1000×g for 10 minutes. The supernatant serum was collected, aliquoted, and stored at -80℃. Serum samples were diluted 1000-fold, and hAGT expression in mouse serum was detected using the Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Link Biotech, catalog number: EK1202-96). Mice were randomly divided into groups of five based on hAGT expression levels. The siRNA conjugate was dissolved in PBS and adjusted to a concentration of 0.2 mg / kg. Mice were subcutaneously injected with the siRNA solution at a dose of 1 mg / kg.

[0699] 17 and 45 days after siRNA injection, a small amount of blood was collected from the tail, allowed to stand at room temperature for 30 minutes, centrifuged at 1000×g for 10 minutes, and the supernatant serum was collected, diluted 1000 times, and the hAGT expression level was detected by ELISA.

[0700] Reference compound 1A is a conjugate of L96* with 1167.25-19, obtained by conventional solid-phase synthesis. *: L96 structure (compound 117 (L96-DMTr protected) in US8106022B2):

[0701]

[0702] Table 8. Results of AGT-targeted siRNA inhibition experiments in AAV8-hAGT mice.

[0703]

[0704] The experimental results are shown in Table 8: In the in vivo experiment, the delivery effect of conjugate 1A was better than that of L96, indicating that the delivery effect of compound 31 on siRNA was better than that of L96.

[0705] Example 25: Testing the effect of conjugate 2A on the expression of AGT and PCSK9 in AAV8-hAGT / hPCSK9 mice.

[0706] The effect of conjugate 2A on hAGT / hPCSK9 expression was tested in mice expressing AAV8-hAGT / hPCSK9 at a dose of 180 nmol / kg. The expression levels of hAGT and hPCSK9 in mouse serum were detected 14, 30, and 44 days after drug administration using the Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Link Biotech, catalog number: EK1202-96) and the Human PCSK9 ELISA Kit (Proteintech, catalog number: KE00278), following the methods described in Examples 23 and 24.

[0707] Table 9. Results of AGT / PCSK9 dual-targeting siRNA inhibition experiments in AAV8-hAGT / hPCSK9 mice at a dose of 180 nmol / kg.

[0708]

[0709] As shown in Table 9, the experimental results indicate that the conjugate 2A targeting AGT and PCSK9 linked by compound 54 exhibits high activity in vivo, significantly inhibiting 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 the pre-drug level, and the remaining amount of hAGT was only 10.8% of the pre-drug level, indicating that the siRNA linked by compound 54 can persistently inhibit the expression of both targets in vivo.

[0710] Example 26: Testing the effects of different siRNA-conjugates on the expression of AGT and PCSK9 in AAV8-hAGT / hPCSK9 mice.

[0711] In mice expressing AAV8-hAGT / hPCSK9, the effects of different conjugates on AGT and PCSK9 expression were tested. Conjugate 2B delivered two siRNAs targeting AGT simultaneously, while the others delivered siRNAs targeting both AGT and PCSK9 simultaneously. The expression of hAGT and hPCSK9 in mouse serum after drug administration was detected using the Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Link Biotech, catalog number: EK1202-96) and the Human PCSK9 ELISA Kit (Proteintech, catalog number: KE00278), following the experimental methods described in Examples 23 and 24.

[0712] Table 10 Results of AGT single-target and AGT / PCSK9 dual-target siRNA inhibition experiments in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg

[0713]

[0714] As shown in Table 10, at a dosage of 60 nmol / kg, all conjugates exhibited excellent in vivo knockdown effects on both hAGT and hPCSK9 targets. Furthermore, conjugate 2B differs from other dual-target conjugates in that it utilizes a dual-target structure to deliver two identical sequences targeting AGT. It is evident that simultaneously delivering two AGT-targeting sequences results in superior AGT knockdown efficacy. This indicates that conjugate 2B not only delivers siRNA targeting two targets but also further enhances efficacy by simultaneously delivering two identical target sequences.

[0715] Table 11 Results of AGT / PCSK9 dual-targeting siRNA inhibition experiments in AAV8-hAGT / hPCSK9 mice at a dose of 180 nmol / kg

[0716]

[0717] As shown in Table 11, both conjugates 9A and 10A exhibited excellent activity. On day 40, the inhibition rates against both targets were above 80-90%.

[0718] Example 27: Testing the effect of siRNA-conjugates on the expression of AGT and PCSK9 in primary hepatocytes of AAV8-hAGT / hPCSK9 mice

[0719] The delivery efficacy of dual-targeting siRNA conjugates with different structures was tested in vitro, following the method described in Example 23. 1000PM-L96 and 11000PM-L96 were used for positive drug comparison: Zilebesiran, an investigational drug targeting AGT developed by alnylam in a phase II clinical trial, and Inclisiran, a marketed drug targeting PCSK9.

[0720] Table 12 Results of AGT / PCSK9 dual-targeting siRNA inhibition experiments at different concentrations and delivery methods in AAV8-hAGT / hPCSK9 mouse primary hepatocytes.

[0721]

[0722]

[0723] As shown in Table 12, each conjugate can efficiently deliver siRNA, and its inhibitory effects on AGT and PCSK9 targets are far better than those of the Zilebesiran mixed with Inclisiran.

[0724] Example 28: Stability and in vivo activity of conjugate 9A

[0725] In this embodiment, based on the highly active compound AM26 from patent US11053495B2 and the highly active compound A19a from patent WO2024061157A1, conjugates DT03000 and DT03098 were synthesized, respectively, and their stability and in vivo activity were compared with conjugate 9A of this invention. The siRNAs linked to the three conjugates were 1167.25-19 and 11040.5-10, respectively; the sequence composition of the linker portion of DT03000 is shown in Table 3.

[0726] The structures of conjugates DT03000 and DT03098 are as follows:

[0727]

[0728]

[0729] The stability of the three conjugates was tested in mouse serum. The experimental method is as follows:

[0730] The test conjugate was precisely diluted with DEPC water to a working solution with a concentration of 10 μM.

[0731] Preparation of 3h samples: Take 12μL of 10μM test conjugate and mix it with 108μL of blank mouse serum. Vortex mix for 60s to prepare a mixed solution of test conjugate and mouse serum (hereinafter referred to as mixed solution); take 50μL of mixed solution into a centrifuge tube, with 2 parallel samples, and incubate at 37℃ for 3h.

[0732] Preparation of 0h samples: Take 45 μL of blank mouse serum into a centrifuge tube, with 2 parallel samples, and incubate at 37℃ for 3h. After incubation, add 5 μL of 10 μM working solution of the test conjugate and vortex mix for 60s.

[0733] After incubation, add 100 μL of lysis buffer (containing 2.5 μg / ml internal standard) to each sample tube, vortex for 30 s, and let stand for 20 min.

[0734] 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%.

[0735] Table 13 Results of double-stranded stability of AGT / PCSK9 dual-targeted modified siRNA in mouse serum after 3 hours of incubation.

[0736] siRNA conjugates Remaining percentage of antisense chain 1 (%) Remaining percentage of antisense chain 2 (%) Conjugate 9A 121.02 118.02 DT03098 101.90 98.04 DT03000 3.89 1.47

[0737] Table 13 clearly shows that after 3 hours of incubation in mouse serum, conjugates 9A and DT03098 exhibited good stability with virtually no degradation; however, the remaining percentage of the two antisense strands in compound DT03000 was less than 5%. Analysis of its metabolites revealed that multiple nucleotides at the 3' ends of the two antisense strands were cleaved, making it impossible to link the two target siRNA duplexes together into a complete double strand through base pairing. This indicates that the conjugate degrades in the bloodstream before being delivered to the liver, demonstrating its poor stability.

[0738] Based on the stability test results, conjugate 9A and conjugate DT03098 were selected for internal activity testing. The experimental methods are the same as those in Examples 23 and 24.

[0739] Table 14 Results of AGT / PCSK9 dual-targeting siRNA inhibition experiments in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg

[0740]

[0741] As shown in Table 14, when delivering the same two sequences, conjugate 9A exhibits significantly better in vivo activity than DT03098 at both target sites, especially showing significantly better inhibitory activity against the PCSK9 target. This indicates that conjugate 9A of the present invention is more effective than DT03098 in delivering siRNA.

[0742] Example 29: Testing the effect of conjugate 9A on the expression of hAGT and hPCSK9 in AAV8-hAGT / hPCSK9 mice.

[0743] In mice expressing AAV8-hAGT / hPCSK9, the effect of conjugate 9A on hAGT and hPCSK9 expression was tested at a dose of 60 nmol / kg. 1000 PM-L96 and 11000 PM-L96 were used for positive drug comparison, representing alnylam's investigational drug Zilebesiran (targeting AGT) in a phase II clinical trial and the marketed drug Inclisiran (targeting PCSK9), respectively. The expression of hAGT and hPCSK9 in mouse serum after drug administration was detected using the Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Link Biotech, catalog number: EK1202-96) and the Human PCSK9 ELISA Kit (Proteintech, catalog number: KE00278), following the experimental methods described in Examples 23 and 24.

[0744] The experimental results are shown in Figure 1 , Figure 2 .

[0745] As shown in the figure, at a dosage of 60 nmol / kg, conjugate 9A exhibits excellent in vivo knockdown effects on both AGT and PCSK9 targets, significantly outperforming the combination of Zilebesiran and Inclisiran. Furthermore, the duration of combined administration of the two positive control drugs reveals that 1000 PM maintained approximately 50% inhibition on PCSK9 by day 56, while 11000 PM's inhibition rate on PCSK9 had already decreased to approximately 50% by day 27, indicating inconsistent durations of inhibitory effects on the targets by the two sequences. In contrast, the inhibitory levels of conjugate 9A at both targets exhibit a generally consistent trend over time, thus offering greater controllability regarding dosing time and frequency in clinical practice.

[0746] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0747] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that, The compound is selected from:

2. A conjugate, characterized in that, The compound of claim 1 is obtained by linking it with an oligonucleotide drug, wherein W1 in the compound is linked to the oligonucleotide drug via a covalent bond; and W2 in the compound is linked to the oligonucleotide drug by reacting with Q; Q is selected from: L9-L10-L11-Z2, and Z2 is a residue of the oligonucleotide drug after removing a hydroxyl group; W1 is W2 is an azide group; L9 is an active group capable of undergoing click chemistry with the W2 group, selected from: L10 is selected from: L11 is Or its chiral isomers.

3. The conjugate according to claim 2, characterized in that, L9 is selected from:

4. The conjugate according to claim 2, characterized in that, L10 is selected from:

5. The conjugate according to claim 2, characterized in that, Q is selected from:

6. The conjugate according to any one of claims 2-5, characterized in that, The compound forms a phosphate ester bond or a thiophosphate ester bond with the phosphate group or thiophosphate group at the 3' and / or 5' end of the positive chain of the oligonucleotide drug via the hydroxyl group in W1.

7. The conjugate according to any one of claims 2-4, characterized in that, L11 is linked to the 3' and / or 5' ends of the positive strand of the oligonucleotide drug via a phosphate ester bond or a thiophosphate ester bond.

8. The conjugate according to any one of claims 2-5, characterized in that, The oligonucleotide drugs include siRNA, ASO, saRNA, miRNA, PNA, and aptamers.

9. The conjugate according to claim 2, characterized in that, The conjugate is selected from:

10. A carrier compound, characterized in that, Its molecular structure is the same as that of the conjugate described in any one of claims 2-9 after the oligonucleotide drug is removed.

11. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a carrier for delivering one or more oligonucleotide drugs.

12. The application according to claim 11, characterized in that, The oligonucleotide drugs include siRNA, ASO, saRNA, miRNA, PNA, and aptamers.

13. The use of the conjugate according to any one of claims 2-9 as an active ingredient in the preparation of a medicament for regulating the expression of one or more target genes.

14. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and / or the carrier compound of claim 10 as a carrier in the preparation of a medicament for regulating the expression of one or more target genes.

15. A pharmaceutical formulation for regulating the expression of one or more target genes, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the conjugate as described in any one of claims 2-9.

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