Compound for targeted delivery of oligonucleotide drug, conjugate, and use

Abstract

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

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511426773.0 (22) Application Date 2024.09.03 (66) Domestic Priority Data 202410763028.4 2024.06.13 CN (62) Divisional Application Data 202411229259.3 2024.09.03 (71) Applicant Guangzhou Bibet Pharmaceutical Co., Ltd. Address 510660 Room 802, 8th Floor, Building A-3, No. 25, Yayingshi Road, Science City, High-tech Industrial Development Zone, Guangzhou, Guangdong Province (72) Inventors Weng Yunwo, Liu Xinjian, Fan Fushun, Qing Yuanhui, Wang Binjie, Cai Xiong, Qian Changgeng (74) Patent Agency Guangzhou Guangdian Intellectual Property Agency (General Partnership) 44365 Patent Attorney Zeng Yinfeng (51) Int.Cl. C07H 15 / 04(2006.01) A61K 47 / 54(2017.01) C07D 519 / 00(2006.01) A61K 31 / 713(2006.01) C07H 21 / 02(2006.01) C07H 1 / 00(2006.01) A61P 43 / 00(2006.01) (54) Invention Title Compounds, Conjugates and Applications for Targeted Delivery of Oligonucleotide Drugs (57) Abstract This invention provides a novel compound for targeted delivery of oligonucleotide drugs and conjugates thereof bound to an oligonucleotide drug, which are capable of delivering one or more different oligonucleotide drugs, said compound having the structure shown in formula (I), formula (II) or formula (III). Claims 12 pages, Specification 110 pages, Sequence List (electronic publication), Drawings 1 page, CN 121270626 A 2026.01.06 CN 1 21 27 06 26 A 1. A compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that the compound has the structure shown in formula (II) or formula (III): wherein: the targeting binding portion T is a group containing a ligand having the structure shown in formula (IV), formula (V) or formula (VI), the ligand being capable of specifically binding to a cell membrane protein receptor with endocytosis activity, the cell membrane protein receptor with endocytosis activity being selected from: sialic acid glycoprotein receptor; D and E are each independently selected from: or their chiral isomers; each r is each independently selected from: 0, 1, 2, 3; X1 and X'1 are independently selected from: single bond, C1-C6 alkylene, -C(=O)-C1-C6 alkylene, -C(=O)-NH-C1-C6 alkylene, -NH-C(=O)-C1-C6 alkylene, and -NH-C1-C6 alkylene.Or their chiral isomers; Connectors K1 and K3 are independently selected from: -(CH2)n15-R22-, -(CH2)n15-R21-(CH2)n16-R22-, -(CH2)n15-O-R21-CH2O-(CH2)n16-R22-, -(CH2)n15-R21-CH2CH2O-(CH2)n16-R22-, -(CH2)n15-R21-(CH2CH2O)2-, -(CH2)n15-R21-(CH2)n16-; R21 is selected from: -C(=O)NH-, -C(=O)-, or none; R22 is selected from: -C(=O)NH-, -NH-, -C(=O)-; Each n15 is independently selected from: 1, 2, 3, 4, 5, 6; Each n16 is independently selected from: 0, 1, 2, 3, 4; the connector K2 is selected from: -(CH2)n17-R25-, -(CH2)n17-R24-(CH2)n18-R25-, -(CH2)n17-OCH2-R24-CH2O- (CH2)n18-R25-, -(CH2)n17-R24-(CH2)n18-, -(CH2)n17-R24-CH2CH2O-(CH2)n18-R25-, -(CH2)n17-R24- (CH2CH2O)2-(CH2)n18-, -(CH2)n17-; Claims 1 / 12 page 2 CN 121270626 A R24 is selected from: -C(=O)NH-, -C(=O)-, or none; R25 is selected from: -C(=O)NH-(CH2) 2-C(=O)-, -NHC(=O)-(CH2) 2-C(=O)-, -C(=O)NH- (CH2)2-, -NHC(=O)-(CH2)2-, -C(=O)-(CH2)2-; Each n17 is independently selected from: 1, 2, 3, 4, 5, 6; Each n18 is independently selected from: 0, 1, 2, 3, 4; W1 is selected from: groups that can bind to oligonucleotide drugs through phosphodiester bonds or thiophosphodiester bonds; W2 is a group that can undergo click chemistry or hydrogen; selected from: 5-20 membered heterocyclic groups containing alkyne, -S-R26, alkyne, azide, alkenyl, 5-20 membered heterocyclic groups containing alkenyl, hydrogen; R26 is selected from: hydrogen, mercapto protecting group; W3 is selected from: W1 or W2.And W3 is not hydrogen; Y1 and Y2 are independently selected from: or their chiral isomers; R in the structures of Y1 and Y2 are independently selected from: H, C1-C6 alkyl; L3 and L4 are independently selected from: -NR6-R7-, -NR6-R7-O-R7-, -C(=O)-R7-; each R6 is independently selected from: hydrogen, C1-C6 alkyl; each R7 is independently selected from: C1-C8 alkylene; L5 is selected from: -R8-(CR9R10)n1-; in L5, R8 is selected from: -C(=O); R9 and R10 are independently selected from: hydrogen, C1-C6 alkyl; each n1 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; L6 is selected from: -C(=O)NH(CH2)n4C(=O)-, -C(=O)NH(CH2)n5O(CH2)n7C(=O)-; In L6, each n4 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; each n5 is independently selected from: 1, 2, 3, 4, 5, 6; each n7 is independently selected from: 1, 2, 3, 4, 5, 6; L7 is selected from: -(CH2)n'8-R15-(R16)n8-; In L7, R15 is selected from: -C(=O); each R16 is independently selected from: -C(R17)2-; each R17 is independently selected from: hydrogen, C1-C6 alkyl; each n8 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; each n'8 is independently selected from: 0; L8 is selected from: -R18-(R19)n10-R18- or L7; in L8, R18 is selected from: -C (=O); each R19 is independently selected from: -C(R14)2-, O, S, wherein the connection of n10 R19s does not violate the principle of chemical bonding; each R14 is independently selected from: hydrogen, C1-C6 alkyl; each n10 is independently selected from: integers between 1 and 20. 2. The compound according to claim 1 or its pharmaceutically acceptable salt or its stereoisomer, characterized in that Y1 is selected from: or their chiral isomers; Y2 is selected from: or their chiral isomers; R in the structures of Y1 and Y2 is independently selected from: H, C1-C3 alkyl; preferably, Y1 is selected from: or their chiral isomers; preferably, Y2 is selected from: or their chiral isomers. 3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that W2 is selected from: (Claims 2 / 12, page 3, CN 121270626 A-S-R26, azide, hydrogen). 4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that each R6 in L3 and L4 is independently selected from: hydrogen, C1-C3 alkyl; each R7 is independently selected from: C1-C4 alkylene; preferably,L3 and L4 are each independently selected from: -NHCH2CH2-, -NHCH2CH2OCH2-, -NHCH2CH2OCH2CH2-, -C(=O)CH2CH2CH2-. 5. The compound according to claim 1, or its pharmaceutically acceptable salt or its stereoisomer, is characterized in that R9 and R10 in L5 are each independently selected from: hydrogen and C1-C3 alkyl groups; preferably, L5 is selected from: -C(=O)(CH2)n1-, and each n1 is independently selected from: 1, 2, 3, 4, 5; more preferably, L5 is selected from: -C(=O)CH2-, -C(=O)CH2CH2-, -C(=O)CH2CH2CH2-. 6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that L6 is selected from: -C(=O)NH(CH2)5C(=O)-, -C(=O)NH(CH2)4C(=O)-, -C(=O)NH(CH2)2OCH2C(=O)-, -C(=O)NH(CH2)2O(CH2)2C(=O)-. 7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that each R17 in L7 is independently selected from: hydrogen, C1-C3 alkyl; preferably, L7 is selected from: -C(=O)(CH2)n8-, and each n8 is independently selected from: 1, 2, 3, 4, 5, 6; more preferably, L7 is selected from: -C(=O)(CH2)5-, -C(=O)(CH2)4-. 8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that each R14 in L8 is independently selected from: hydrogen, C1-C3 alkyl; preferably, L8 is selected from: -C(=O)(CH2)n10C(=O)-, n10 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; more preferably, L8 is selected from: -C(=O)(CH2)10C(=O)-. 9. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that W1 is selected from: hydroxyl, protected hydroxyl, or a group having the following structure: or a chiral isomer thereof; wherein R' is a hydrogen or hydroxyl protecting group. 10. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that the connectors K1 and K3 are each independently selected from: Claims 3 / 12, page 4, CN 121270626 A 11. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that the connector K2 is selected from: 12. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that,X1 and X'1 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, or their chiral isomers. Preferably, X1 and X'1 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, -NH-C(=O)-propylene. 13. The compound according to any one of claims 1-8, or its pharmaceutically acceptable salt or its stereoisomer, characterized in that the ligand is selected from: N-acetylgalactosamine, N-propionylgalactosamine. 14. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that the ligand is selected from: or or where the hydrogen on the hydroxyl group is replaced by an acetyl group. 15. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that the targeting binding moiety T is selected from: (Claims 4 / 12, page 5, CN 121270626 A) or or where the hydrogen on the hydroxyl group is replaced by an acetyl group. 16. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that the compound is selected from: Claims 5 / 12 page 6 CN 121270626 A 17. A conjugate, characterized in that it is obtained by linking the compound according to any one of claims 1-16 with an oligonucleotide drug, wherein W1 in the compound is covalently linked to the oligonucleotide drug; W2 in the compound is linked to the oligonucleotide drug by reacting with Q Claims 6 / 12 page 7 CN 121270626 A; Q is selected from: L9-L10-L11-Z2, Z2 is a residue of the oligonucleotide drug after the removal of a hydroxyl group; L9 is an active group capable of undergoing a click chemical reaction with the W2 group, selected from: a 5-20 membered heterocyclic group containing an alkyne, -S-R26, alkyne, azide; R26 is selected from: H or a thiol protecting group; L10 is selected from: In L10, each A is independently selected from: -C(R2)2-, -NR2-, -O-, -S-; each R2 is independently selected from: H, C1-C3 alkyl; each R3 is independently selected from: or none; each R4 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-, -C(=O), -C(=S)-.The connection of R4 in f4 does not violate the principle of chemical bonding; each R5 is independently selected from: 3-8 member heteromonocyclic group, C3-C8 cycloalkyl group, 5-10 member heterobridged cycloalkyl group, 5-10 member heterospirocyclic group, 5-20 member heterocyclic cycloalkyl group, C5-C10 cycloalkyl group; each f1 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; each f2 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; each f3 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; each f4 is independently selected from: integers between 0 and 20; L11 is selected from: the group obtained by removing one hydroxyl hydrogen or one hydroxyl protecting group from W1. 18. The conjugate according to claim 17, wherein L9 is selected from: azide group, -S-R26. 19. The conjugate according to claim 17, wherein L11 is selected from: -O-, or groups having the following structure: or their chiral isomers. 20. The conjugate according to claim 17, characterized in that each A in L10 is independently selected from: -CH2-, -NH-, -O-, -S-; each R3 is independently selected from: R5, or none; each R4 is independently selected from: -CH-, -NH-, -O-, -S-, -C(=O); each R5 is independently selected from: 5-6 member heteromonocyclic group, C5-C6 cycloalkyl group, 6-8 member heterobridged cycloalkyl group, 6-8 member heterospirocyclic group, 6-10 member heterocyclic cycloalkyl group, C6-C10 cycloalkyl group; each f1 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8; each f2 is independently selected from: 0, 1, 2, 3, 4; each f3 is independently selected from: 0, 1, 2, 3, 4; Each f4 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; preferably, each R5 in L10 is independently selected from: or their chiral isomers; preferably, L10 is selected from: or their chiral isomers. 21. The conjugate according to claim 17, wherein Q is selected from: Claims 8 / 12 Page 9 CN 121270626 A 22. The conjugate according to any one of claims 17-21, wherein the compound forms a phosphate ester bond or a thiophosphate bond with the phosphate group or thiophosphate group at the 3' and / or 5' end of the positive chain of the oligonucleotide drug through the hydroxyl group in W1. 23. The conjugate according to any one of claims 17-21, wherein L11 is connected to the 3' and / or 5' end of the positive chain of the oligonucleotide drug through a phosphate ester bond or a thiophosphate bond. 24. The conjugate according to any one of claims 17-21, wherein...The oligonucleotide drug includes siRNA, ASO, saRNA, miRNA, PNA, and aptamers. 25. The conjugate according to any one of claims 17-21, characterized in that it has the structure shown in formula (A), formula (B), or formula (C): wherein the targeting binding portions T, L3, L4, L5, L6, L7, and L8 are as described in any one of claims 1-16, and L10, L11, and Z2 are as described in any one of claims 17-21; Z1 is a residue of the oligonucleotide drug after the removal of a hydroxyl group; W4 is a group of W1 as described in any one of claims 1-16 after the removal of a hydroxyl hydrogen or a hydroxyl protecting group; W5 is selected from: the group formed after the reaction of W2 as described in any one of claims 1-16 and L9 as described in any one of claims 17-21; W6 is selected from: W4 or -W5-L10-L11-. 26. The conjugate according to claim 25, wherein W4 is selected from: -O-, or groups having the following structure: or their chiral isomers. 27. The conjugate according to claim 25, wherein each W5 is independently selected from: 3-20-membered heterocyclic groups, 5-20-membered heteroaromatic groups, 3-20-membered heterocyclic thio groups, -S-S-; preferably, each W5 is independently selected from: 5-20-membered heterocyclic groups, 5-6-membered heteroaromatic groups, 5-6-membered heterocyclic thio groups, -S-S-; preferably, each W5 is independently selected from: -S-S-. 28. The conjugate according to claim 17, characterized in that the conjugate is selected from: Claims 10 / 12 pages 11 CN 121270626 A Claims 11 / 12 pages 12 CN 121270626 A 29. A carrier compound, characterized in that its molecular structure is identical to the molecular structure of the conjugate according to any one of claims 17-28 after the removal of the oligonucleotide drug. 30. Use of the compound according to any one of claims 1-16 or its pharmaceutically acceptable salt or its stereoisomer in the preparation of a carrier for delivering one or more oligonucleotide drugs; preferably, the oligonucleotide drug comprises siRNA, ASO, saRNA, miRNA, PNA, aptamer. 31. Use of the conjugate according to any one of claims 17-28 as an active ingredient in the preparation of a drug for regulating the expression of one or more target genes. 32. Use of the compound according to any one of claims 1-16 or its pharmaceutically acceptable salt or its stereoisomer, and / or the carrier compound according to claim 29 as a carrier in the preparation of a drug for regulating the expression of one or more target genes. 33. A pharmaceutical formulation for regulating the expression of one or more target genes, characterized in that,Prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the conjugate as described in any one of claims 17-28. Claims 12 / 12 pages 13 CN 121270626 A Compounds, conjugates and their applications for targeted delivery of oligonucleotide drugs

[0001] This invention is a divisional application of Chinese patent application filed on September 3, 2024, with application number 2024112292593 and application title "Compounds, conjugates and their applications for targeted delivery of oligonucleotide drugs". Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to compounds, conjugates and their applications for targeted delivery of oligonucleotide drugs. Background Art

[0003] Oligonucleotide drugs (such as siRNA, antisense oligonucleotides and plasmid DNA, etc.), as an emerging and promising therapy, have shown outstanding performance in regulating gene expression and treating many diseases. However, their high molecular weight and polyanionic properties result in poor cell membrane permeability, and the influence of factors such as nucleases further limits the practical application of oligonucleotide drugs.

[0004] Currently, utilizing receptor-mediated endocytosis to conjugate target ligands with oligonucleotide drugs to enhance cellular uptake and improve tissue specificity for in vivo delivery has become a hot topic in the field of oligonucleotide drug development. It is worth noting that sometimes multivalent ligand clusters may outperform single ligands in promoting targeted tissue delivery, such as the desialyl glycoprotein receptor (ASGPR). In addition, studies have found that the ASGPR ligand N-acetylgalactosamine (GalNAc) can effectively enhance the entry of oligonucleotide drugs into hepatocytes. Further experiments confirmed that, compared with single GalNAc ligands, multivalent GalNAc ligand clusters have a stronger affinity for ASGPR binding, thereby improving the delivery efficiency of oligonucleotides targeting hepatocytes (Chem. Soc. Rev., 2023, 52, 1273; Bioconjugate Chem. 2017, 28, 2, 283–295).

[0005] Several patent documents have reported research in this area, such as US8106022B2, CN116832169A, WO2023045995A1, CN110959011B, CN117858948A, and WO2024061157, all of which involve the delivery of oligonucleotide drugs using GalNAc ligands. However, most of the documents disclosed in these documents utilize multivalent GalNAc ligands to deliver single-target single oligonucleotide drugs. Some single genes in organisms have multiple transcripts with sequence differences, and disease development often involves the interaction of multiple genes. Therefore, 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.

[0006] Based on this, the object 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.

[0007] In a first aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, the compound having the structure shown in formula (I), formula (II) or formula (III): Specification 1 / 110 page 14 CN 121270626 A

[0008]

[0009] wherein: the targeting binding portion T is a group containing a ligand that can specifically bind to a cell membrane protein receptor with endocytosis activity;

[0010] W1 is selected from: a group capable of binding to an oligonucleotide drug via a covalent bond;

[0011] W2 is selected from: a group capable of undergoing a click chemistry reaction or hydrogen;

[0012] W3 is selected from: W1 or W2, and W3 is not hydrogen;

[0013] Y1 and Y2 are each independently selected from: or their chiral isomers; n is an integer from 1 to 4;

[0014] 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-C15 alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -S-S-, -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-C10 aryl, 3-20 membered heterocyclic, C3-C10 cycloalkyl, 5-10 membered heteroaryl. The linking unit can be substituted with one or more R groups. (Specification 2 / 110, page 15, CN 121270626 A)

[0015] 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;

[0016] R1 is selected from: H, C1-C6 alkyl, amino acid side chain;

[0017] X is selected from: -O-, -S-, -NR-, -C(=O)-,

[0018] In some embodiments, Y1 is selected from: or their chiral isomers; n is: 1, 2, 3 or 4;

[0019] Y2 is selected from: or their chiral isomers;

[0020] R in the Y1 and Y2 structures is independently selected from: H, C1-C3 alkyl, halogen-substituted C1-C3 alkyl.

[0021] In some embodiments,Y1 is selected from: or their chiral isomers;

[0022] Y2 is selected from: or their chiral isomers.

[0023] In some embodiments, W2 is selected from: 5-20 membered heterocyclic groups containing an alkynyl group, -S-R26, alkynyl, azide, alkenyl, 5-20 membered heterocyclic groups containing an alkenyl group, hydrogen;

[0024] R26 is selected from: hydrogen, mercapto protecting group.

[0025] In some embodiments, W2 is selected from:

[0026] -S-R26, azide, hydrogen.

[0027] 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 linking unit that does not violate the principle of chemical bonding:

[0028] C1-C12 alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -S-S-, -CR=N-, -S(=O)2-, -

[0029] 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-C10 aryl、3-8 member heteromonocyclic、C3-C8 cycloalkyl、5-10 member heterobridged cycloalkyl、5-10 member heterospirocyclic、5-20 member heterocyclic cycloalkyl、C5-C10 cycloalkyl、5-6 member heteroaryl、the linking unit can be substituted by one or more R;

[0030] R is selected from: H、C1-C3 alkyl、halogen、hydroxyl、hydroxy-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、amino acid side chain.

[0032] In some embodiments, L1, L2, L3, L4, L5, L6, L7, and L8 are each independently selected from one or more linking units formed by connecting groups without violating the principle of chemical bonding, as described on page 3 / 110 of the specification, CN 121270626 A:

[0033] or their chiral isomers;

[0034] wherein, j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0035] R is selected from: H, C1-C3 alkyl, halogen-substituted C1-C3 alkyl;

[0036] R1 is selected from: H, C1-C3 alkyl, amino acid side chain.

[0037] 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 linking unit that does not violate the principle of chemical bonding: or their chiral isomers;

[0038] wherein,j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12; Specification 4 / 110 page 17 CN 121270626 A

[0039] R is selected from: H, methyl, ethyl, propyl;

[0040] R1 is selected from: H, methyl, ethyl, propyl, amino acid side chain.

[0041] In some embodiments, L1 is selected from:

[0042]

[0043] Wherein, each A in L1 is independently selected from: -C(R2)2-, -NR2-, -C(=O)NR2-, -O-, -S-, wherein the connection of a A does not violate the principle of chemical bonding;

[0044] Each R2 is independently selected from: H, C1-C3 alkyl;

[0045] a is selected from: an integer between 0 and 20;

[0046] b is selected from: an integer between 0 and 10.

[0047] In some embodiments, L1 is selected from:

[0048]

[0049] where a is selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0050] b is selected from: 0, 1, 2, 3, 4, 5, 6.

[0051] In some embodiments, L1 is selected from: -NH(CH2)10C(=O)-, -NH(CH2)4C(=O)-, -NH(CH2)5C(=O)-, -NH(CH2)6C(=O)-, -(CH2)2NHC(=O)(CH2)10C(=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)-,

[0052] -C(=O)(CH2)10C(=O)-, -NH(CH2)2OCH2C(=O)-,

[0053] -C(=O)(CH2)2NH(CH2)2OCH2C(=O)-.

[0054] In some embodiments, L2 is selected from:

[0055]

[0056] Each A in L2 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-, wherein the connection of c A does not violate the principle of chemical bonding;

[0057] Each B in L2 is independently selected from: -NR2-, -O-, -S-;

[0058] Each R2 is independently selected from: H, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0059] c is selected from: an integer between 0 and 20;

[0060] d is selected from: an integer between 0 and 10;

[0061] e is selected from: an integer between 0 and 10.

[0062] In some embodiments,L2 is selected from: Page 5 / 110 of the specification, CN 121270626 A

[0063] wherein each R2 is independently selected from: H, methyl, ethyl;

[0064] c is selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0065] d is selected from: 0, 1, 2, 3, 4, 5, 6;

[0066] e is selected from: 0, 1, 2, 3, 4, 5, 6.

[0067] 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-,

[0068] -(CH2)2O(CH2)5-, -CH2(=O)NH(CH2)2NHC(=O)(CH2)5-,

[0069] In some embodiments, L3 and L4 are independently selected from: -NR6-R7-, -NR6-R7-O-R7-, -C(=O)-R7-;

[0070] Each R6 is independently selected from: hydrogen, C1-C6 alkyl;

[0071] Each R7 is independently selected from: C1-C8 alkylene.

[0072] In some embodiments, each R6 is independently selected from: hydrogen, C1-C3 alkyl;

[0073] Each R7 is independently selected from: C1-C4 alkylene.

[0074] In some embodiments, L3 and L4 are independently selected from: -NHCH2CH2-, -NHCH2CH2OCH2-, -NHCH2CH2OCH2CH2-, -C(=O)CH2CH2CH2-.

[0075] In some embodiments, L5 is selected from: -R8-(CR9R10)n1-, -R8-(CR9R10)n2-R8-(CR9R10)n3-, -(CR9R10)n2-R8-(CR9R10)n3-;

[0076] Wherein, R8 is selected from: -NR9-, -O-, -S-, -C(=O), -NR9C(=O)-,

[0077] R9 and R10 are each independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0078] Each n1 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0079] Each n2 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8;

[0080] Each n3 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8.

[0081] In some embodiments,L5 is selected from: -C(=O)(CH2)n1-, -C(=O)(CH2)n2-NHC(=O)(CH2)n3-,

[0082] -C(=O)(CH2)n2-C(=O)NH(CH2)n3-, -(CH2)n2-NHC(=O)(CH2)n3-,

[0083] -(CH2)n2-O(CH2)n3-, -C(=O)(CH2)n2-C(=O)NH-,

[0084] Each n1 is independently selected from: 1, 2, 3, 4, 5;

[0085] Each n2 is independently selected from: 1, 2, 3, 4, 5;

[0086] Each n3 is independently selected from: 1, 2, 3, 4, 5, 6.

[0087] 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-,

[0088] Specification 6 / 110 pages 19 CN 121270626 A

[0089] In some embodiments, L6 is selected from: -R11-(R12)n4-, -R11-(R12)n4-R11-, -R11-(R12)n5-R11- (R12)n6-R11-;

[0090] Wherein, R11 is selected from: -C(=O), -NR13C(=O)-;

[0091] Each R12 is independently selected from: -C(R14)2-, O, S, -NR13, wherein the connection of n4 or n5 R12 does not violate the chemical bonding principle;

[0092] Each R13 is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0093] Each R14 is independently selected from: hydrogen, C1-C6 alkyl, side chain of amino acid, preferably from: hydrogen, C1-C3 alkyl, side chain of amino acid;

[0094] Each n4 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0095] Each n5 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0096] Each n6 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0097] In some embodiments,L6 is selected from:

[0098] -C(=O)NH(CH2)n4C(=O)-, -C(=O)NH(CH2)n4C(=O)-,

[0099] -C(=O)NH(CH2)n5O(CH2)n7C(=O)-, -C(=O)NH(CH2)n5O(CH2)n7C(=O)-,

[0100] -C(=O)NH(CH2)n4-, -C(=O)NH(CH2)n4NHC(=O)-,

[0101] -C(=O)(CH2)n5-C(=O)NH(CH2)n6C(=O)-,

[0102] Each n4 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0103] Each n5 is independently selected from: 1, 2, 3, 4, 5, 6;

[0104] Each n6 is independently selected from: 1, 2, 3, 4, 5, 6;

[0105] Each n7 is independently selected from: 1, 2, 3, 4, 5, 6.

[0106] In some embodiments, L6 is selected from:

[0107] -C(=O)NH(CH2)5C(=O)-, -C(=O)NH(CH2)4C(=O)-,

[0108] -C(=O)NH(CH2)2OCH2C(=O)-, -C(=O)NH(CH2)2O(CH2)2C(=O)-,

[0109] -C(=O)NH(CH2)4-, -C(=O)NH(CH2)3NHC(=O)-,

[0110] -C(=O)(CH2)2-C(=O)NH(CH2)5C(=O)-,

[0111] In some embodiments, L7 is selected from: -(CH2)n'8-R15-(R16)n8-;

[0112] Wherein, R15 is selected from: -C(=O), -NR13C(=O)-;

[0113] Each R16 is independently selected from: -C(R17)2-, O, S, wherein the connection of n8 R16 does not violate the principle of chemical bonding;

[0114] Each R13 is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0115] Each R17 is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0116] Each n8 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Specification 7 / 110 pages 20 CN 121270626 A

[0117] Each n'8 is independently selected from: 0, 1, 2, 3, 4, 5, 6.

[0118] 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-;

[0119] Each n8 is independently selected from: 1, 2, 3, 4, 5, 6;

[0120] Each n9 is independently selected from: 1, 2, 3, 4, 5.

[0121] In some embodiments, L7 is selected from: -C(=O)(CH2)5-, -C(=O)(CH2)4-,

[0122] -C(=O)CH2O(CH2)3-, -C(=O)NH(CH2)5-, -C(=O)NH(CH2)4-, -NHC(=O)(CH2)5-, -NHHC(=O)(CH2)4-, -(CH2)4NHC(=O)(CH2)5-.

[0123] In some embodiments, L8 is selected from: -R18-(R19)n10-R18-;

[0124] wherein, R18 is selected from: -C(=O), -NR13C(=O)-;

[0125] each R19 is independently selected from: -C(R14)2-, O, S, -NR13, C6-C10 aryl, 5-6 member heteromonocyclic, C5-C6 cycloalkyl, 6-8 member heterobridged cycloalkyl, 6-8 member heterospirocyclic, 6-10 member heterocyclocyclic, C6-C10 cycloalkyl, 5-6 member heteroaryl, ethynyl, vinyl, wherein the connection of n10 R19s does not violate the principle of chemical bonding;

[0126] each R13 is independently selected from: hydrogen, C1-C6 alkyl, preferably from: hydrogen, C1-C3 alkyl;

[0127] Each R14 is independently selected from: hydrogen, C1-C6 alkyl, and the side chain of an amino acid, preferably from: hydrogen, C1-C3 alkyl, and the side chain of an amino acid;

[0128] Each n10 is independently selected from: an integer between 1 and 20;

[0129] Or L8 is selected from L7.

[0130] In some embodiments, R18 is selected from: -C(=O), -NHC(=O)-;

[0131] Each R19 is independently selected from: -CH2-, -O-, -S-, -NH-, phenyl, 5-6-membered heteromonocyclic, C5-C6 cycloalkyl, 5-6-membered heteroaryl, ethynyl, vinyl;

[0132] Each n10 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

[0133] In some embodiments,L8 is selected from: -C(=O)(CH2)n10C(=O)-,

[0134] -C(=O)(CH2)n11-N(H)-(CH2CH2O)n12(CH2)n13C(=O)-,

[0135] R19 is selected from: phenyl, 5-6-membered heterocyclic, C5-C6 cycloalkyl, 5-6-membered azaaryl, ethynyl, vinyl; R19 is preferably:

[0136] n10 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15;

[0137] n11 is selected from: 1, 2, 3, 4, 5, 6;

[0138] n12 is selected from: 1, 2, 3, 4;

[0139] n13 is selected from: 1, 2, 3, 4;

[0140] n14 is selected from: 1, 2, 3, 4, 5, 6, 7, 8.

[0141] In some embodiments, L8 is selected from: -C(=O)(CH2)10C(=O)-,

[0142] -C(=O)(CH2)2-N(H)-(CH2CH2O)2CH2C(=O)-,

[0143] In some embodiments, W1 is selected from: groups that can be linked to oligonucleotides via phosphodiester bonds or thiophosphate diester bonds.

[0144] In some embodiments, W1 is selected from hydroxyl groups, protected hydroxyl groups, or groups having the following structure: Specification 8 / 110 page 21 CN 121270626 A

[0145] or their chiral isomers;

[0146] wherein R' is a hydrogen or hydroxyl protecting group.

[0147] 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.

[0148] 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).

[0149] In some embodiments, the targeting binding portion T has the structure shown in formula (IV), formula (V), formula (VI), or formula (VII):

[0150]

[0151] wherein: D, E, J, and M are each independently selected from:

[0152] or their chiral isomers;

[0153] G, V, W, and U are each independently selected from: or their chiral isomers;

[0154] r,t is independently selected from: 0, 1, 2, 3, 4, 5, 6;

[0155] X1, X'1, X2, X3, X4, X5 are independently selected from: single bond, C1-C6 alkylene, -C(=O)-C1-C6 alkylene, -C(=O)-NH-C1-C6 alkylene, -NH-C(=O)-C1-C6 alkylene, -NH-C1-C6 alkylene, -C(=O)-NH-C1-C6 alkylene, or their chiral isomers;

[0156] Connectors K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, and K14 are independent linking units formed by selecting one or more of the following groups without violating the principle of chemical bonding: C1-C15 alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -S-S-, -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-C10 aryl, 3-20 membered heterocyclic, C3-C10 cycloalkyl, 5-10 membered heteroaryl. These linking units can be substituted with one or more R groups.

[0157] 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, amino carbonyl;

[0158] R1 is selected from: H, C1-C6 alkyl, amino acid side chain.

[0159] 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-C12 alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -S-S-, -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-C10 aryl, 3-8 membered heteromonocyclic, C3-C8 cycloalkyl, 5-10 membered heterobridged cycloalkyl, 5-10 membered heterospirocyclic, 5-20 membered heterofused cycloalkyl, C5-C10 fused cycloalkyl, 5-6 membered heteroaryl,The connecting unit may be replaced by one or more R;

[0160] 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, amino carbonyl;

[0161] R1 is selected from: H, C1-C3 alkyl, amino acid side chain.

[0162] 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: Specification 10 / 110 page 23 CN 121270626 A

[0163] or their chiral isomers;

[0164] wherein, j1 is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12;

[0165] R is selected from: H, C1-C3 alkyl, halogen-substituted C1-C3 alkyl;

[0166] R1 is selected from: H, C1-C3 alkyl, amino acid side chain.

[0167] 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:

[0168] or their chiral isomers as specified on page 11 / 110 of CN 121270626 A;

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

[0170] R is selected from: H, methyl, ethyl, propyl;

[0171] R1 is selected from: H, methyl, ethyl, propyl, amino acid side chain.

[0172] In some embodiments, the connectors K1, K3, K6, and K7 are each independently selected from: -(R20)n15-R21-(R20)n16-R22-;

[0173] wherein each R20 is independently selected from: -CH2-, -O-, -S-, wherein the connection of n15 or n16 R20s does not violate the principle of chemical bonding;

[0174] R21 is selected from: -C(=O)NH-, -C(=O)-, C6-C10 aryl, 3-8 member heteromonocyclic, C3-C8 cycloalkyl, 5-10 member heterobridged cycloalkyl, 5-10 member heterospirocyclic, 5-20 member heterocycloalkyl, C5-C10 cycloalkyl, 5-6 member heteroaryl, ethynyl, vinyl, -C(=O)-3-8 member heteromonocyclic, -C(=O)-C3-C8 cycloalkyl, or none;

[0175] R22 is selected from: -C(=O)NH-, -NH-, -C(=O)-,Or none;

[0176] Each n15 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0177] Each n16 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0178] In some embodiments, R21 is selected from: -C(=O)NH-, -C(=O)-, phenyl,

[0179] or none.

[0180] In some embodiments, connectors K1, K3, K6, and K7 are each independently selected from: -(CH2)n15-R22-, -(CH2)n15-R21-(CH2)n16-R22-, -(CH2)n15-O-R21-CH2O-(CH2)n16-R22-, -(CH2)n15-R21-CH2CH2O-(CH2)n16-R22-, -(CH2)n15-R21-(CH2CH2O)2-, -(CH2)n15-R21-(CH2)n16-;

[0181] R21 is selected from: -C(=O)NH-, -C(=O)-, phenyl;

[0182] R22 is selected from: -C(=O)NH-, -NH-, -C(=O)-;

[0183] Each n15 is independently selected from: 1, 2, 3, 4, 5, 6;

[0184] Each n16 is independently selected from: 0, 1, 2, 3, 4.

[0185] In some embodiments, connectors K1, K3, K6, K7 are independently selected from: Specification 12 / 110 page 25 CN 121270626 A

[0186] In some embodiments, connectors K2, K4, K5, K8, K9, K10, K11, K12, K13, K14 are independently selected from: -(R23)n17-R24-(R23)n18-R25-;

[0187] Wherein, each R23 is independently selected from: -CH2-, -O-, -S-, wherein the connection of n18 R23s does not violate the principle of chemical bonding;

[0188] R24 is selected from: -C(=O)NH-, -C(=O)-, C6-C10 aryl, 3-8 heteromonocyclic, C3-C8 cycloalkyl, 5-10 heterobridged cycloalkyl, 5-10 heterospirocyclic, 5-20 heterocyclic, C5-C10 cycloalkyl, 5-6 heteroaryl, ethynyl, vinyl, -C(=O)-3-8 heteromonocyclic, -C(=O)-C3-C8 cycloalkyl, or none;

[0189] R25 is selected from: -C(=O)NH-(CH2)n19-C(=O)-, -NHC(=O)-(CH2)n19-C(=O)-, -C(=O)NH-(CH2)n19-, -NHC(=O)-(CH2)n19-, -C(=O)-(CH2)n19-,Or none;

[0190] Each n17 is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0191] Each n18 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0192] Each n19 is independently selected from: 1, 2, 3.

[0193] In some embodiments, R24 is selected from: -C(=O)NH-, -C(=O)-, phenyl,

[0194] or none.

[0195] In some embodiments, connectors K2, K4, K5, K8, K9, K10, K11, K12, K13, and K14 are each independently selected from: -(CH2)n17-R25-, -(CH2)n17-R24-(CH2)n18-R25-, -(CH2)n17-OCH2-R24-CH2O-(CH2)n18-R25-, -(CH2)n17-R24-(CH2)n18-, -(CH2)n17-R24-CH2CH2O-(CH2)n18-R25-, -(CH2)n17-R24-(CH2CH2O)2-(CH2)n18-, -(CH2)n17-;

[0196] R24 is selected from: -C(=O)NH-, -C(=O)-, phenyl;

[0197] R25 is 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-;

[0198] Each n17 is independently selected from: 1, 2, 3, 4, 5, 6; Specification 13 / 110 page 26 CN 121270626 A

[0199] Each n18 is independently selected from: 0, 1, 2, 3, 4.

[0200] In some embodiments, the connectors K2, K4, K5, K8, K9, K10, K11, K12, K13, and K14 are each independently selected from:

[0201] In some 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.

[0202] 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, -NH-C(=O)-propylene.

[0203] In some embodiments, the ligand is selected from: D-mannylpyranose, L-mannylpyranose, D-arabinose, D-xylfuranose, L-xylfuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannylfuranose, β-D- Furanomannose, α-D-Mannopyranose, β-D-Mannopyranose, α-D-Glucose Pyranopyranose, β-D-Glucose Pyranopyranose, α-D-Fructose Pyranopyranose, α-D-Fructose Pyranopyranose, 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-carbamate-2,3-di-O-methyl-D-mannose pyranose, 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-triphenylmethyl-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-alokonitrile, D-ribose, D-4-thioribose, L-ribose and L-4-thioribose, RGD polypeptide, folic acid molecule, TfR1 polypeptide, integrin ligand small molecule, lipids, fatty acids, bile acids and cholesterol.

[0204] 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 molecule, integrin ligand small molecule.

[0205] In some embodiments, the ligand is selected from:

[0206] , or the hydrogen on the hydroxyl group is replaced by an acetyl group. Specification 14 / 110 pages 27 CN 121270626 A

[0207] In some embodiments,The targeted binding portion T is selected from:

[0208] Specification 15 / 110 page 28 CN 121270626 A

[0209] Specification 16 / 110 page 29 CN 121270626 A

[0210] Specification 17 / 110 page 30 CN 121270626 A

[0211] or the hydrogen on the hydroxyl group is replaced by an acetyl group.

[0212] In a second aspect, 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 linked to the oligonucleotide drug via a covalent bond; 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;

[0213] L9 is selected from: an active group capable of undergoing a click chemical reaction with the W2 group;

[0214] L10 is selected from one or more of the following groups linked together without violating the principle of chemical bonding to form a linking unit: C1-C15 alkylene, -C(=O)-, -C(=S)-, -NR-, -O-, -S-, -S-S-, -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-C10 aryl, 3-20 heterocyclic, C3-C10 cycloalkyl, 5-10 heteroaryl, wherein the linking unit may be substituted by one or more R;

[0215] 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;

[0216] R1 is selected from: H, C1-C6 alkyl, amino acid side chain;

[0217] L11 is selected from: the group after removing one hydroxyl hydrogen or removing one hydroxyl protecting group from W1.

[0218] In some embodiments, L9 is selected from: 5-20 membered heterocyclic groups containing an alkynyl group, -S-R26, alkynyl, azide; R26 is selected from: H or a mercapto protecting group.

[0219] In some embodiments, L9 is selected from:

[0220] azide, -S-R26.

[0221] In some embodiments, L11 is selected from: -O-, or groups having the following structure:

[0222] or their chiral isomers.

[0223] In some embodiments, L10 is selected from:

[0224]

[0225] wherein,Each A in L10 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-; Specification 18 / 110 page 31 CN 121270626 A

[0226] Each R2 is independently selected from: H, C1-C3 alkyl;

[0227] Each R3 is independently selected from: R5, or none;

[0228] Each R4 is independently selected from: -C(R2)2-, -NR2-, -O-, -S-, -C(=O), -C(=S)-, wherein the connection of f4 R4s does not violate the principle of chemical bonding;

[0229] Each R5 is independently selected from: C6-C10 aryl, 3-8 member heteromonocyclic, C3-C8 cycloalkyl, 5-10 member heterobridged cycloalkyl, 5- 10-membered heterospirocyclic group, 5-20-membered heterocyclic group, C5-C10 cycloalkyl group, 5-6-membered heteroaryl group, ethynyl group, vinyl group;

[0230] Each f1 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0231] Each f2 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0232] Each f3 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0233] Each f4 is independently selected from: integers between 0 and 20.

[0234] In some embodiments, each A in L10 is independently selected from: -CH2-, -NH-, -O-, -S-;

[0235] Each R3 is independently selected from: R5, or none;

[0236] Each R4 is independently selected from: -CH-, -NH-, -O-, -S-, -C(=O);

[0237] Each R5 is independently selected from: C6-C10 aryl, 5-6 heteromonocyclic, C5-C6 cycloalkyl, 6-8 heterobridged cycloalkyl, 6-8 heterospirocyclic, 6-10 heterocycloalkyl, C6-C10 cycloalkyl, 5-6 heteroaryl, ethynyl, vinyl;

[0238] Each f1 is independently selected from: O, 1, 2, 3, 4, 5, 6, 7, 8;

[0239] Each f2 is independently selected from: O, 1, 2, 3, 4;

[0240] Each f3 is independently selected from: 0, 1, 2, 3, 4;

[0241] Each f4 is independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0242] In some embodiments, each R5 is independently selected from: or their chiral isomers.

[0243] In some embodiments, L10 is selected from: Specification 19 / 110 page 32 CN 121270626 A

[0244] or their chiral isomers.

[0245] In some embodiments,Q is selected from:

[0246] Specification 20 / 110 page 33 CN 121270626 A

[0247]

[0248]

[0249] In some embodiments, the compound forms a phosphate ester bond or a thiophosphate bond with the phosphate group or thiophosphate group at the 3' and / or 5' end of the sense strand of the oligonucleotide drug via the hydroxyl group in W1.

[0250] In some embodiments, L11 is linked to the 3' and / or 5' end of the sense strand of the oligonucleotide drug via a phosphate ester bond or a thiophosphate bond.

[0251] 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.

[0252] In some embodiments, the conjugate has the structure shown in formula (A), formula (B), or formula (C):

[0253]

[0254] wherein the targeting 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;

[0255] Z1 is a residue of the oligonucleotide drug after removing a hydroxyl group;

[0256] W4 is a group of W1 after removing a hydroxyl hydrogen or a hydroxyl protecting group;

[0257] W5 is selected from: the group formed after the reaction of W2 and L9;

[0258] W6 is selected from: W4 or -W5-L10-L11-.

[0259] In some embodiments, W4 is selected from: -O-, or a group having the following structure:

[0260] or their chiral isomers.

[0261] In some embodiments, each W5 is independently selected from: 3-20 membered heterocyclic group, 5-20 membered heteroaromatic group, 3-20 membered heterocyclic thio group, -S-S-.

[0262] In some embodiments, each W5 is independently selected from: 5-20 membered heterocyclic group, 5-6 membered heteroaromatic group, 5-6 membered heterocyclic thio group, -S-S-;

[0263] In some embodiments, each W5 is independently selected from:

[0264] -S-S-.

[0265] In a third aspect, the present invention also provides a carrier compound whose molecular structure is the same as the molecular structure of the conjugate of the present invention after the oligonucleotide drug is removed.

[0266] In a fourth aspect, the present invention provides the application of the compound and the conjugate,The following technical solutions are included.

[0267] The use of the compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer in the preparation of a carrier for delivering one or more oligonucleotide drugs.

[0268] 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.

[0269] The use of the conjugate of the present invention as an active ingredient in the preparation of a drug for regulating the expression of one or more target genes.

[0270] The use of the compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer as a carrier in the preparation of a drug for regulating the expression of one or more target genes.

[0271] The use of the carrier compound of the present invention as a carrier in the preparation of a drug for regulating the expression of one or more target genes.

[0272] 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 a pharmaceutically acceptable excipient, wherein the active ingredient comprises the conjugate of the present invention.

[0273] This invention provides a novel scaffold compound and its conjugates with oligonucleotide drugs, which can be used for targeted delivery of oligonucleotide drugs. It can simultaneously deliver two nucleotide drugs targeting a single site or a dual-target nucleotide drug, enhancing the therapeutic effect of oligonucleotide drugs on corresponding diseases and has broad application prospects. Brief Description of the Drawings

[0274] Figure 1 shows the time-varying effect of conjugate 9A on the AGT target in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg.

[0275] Figure 2 shows the time-varying effect of conjugate 9A on the PCSK9 target in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg. Detailed Description of the Embodiments

[0276] To facilitate understanding of this invention, a more comprehensive description is provided below. This 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 this invention.

[0277] Experimental methods in the following examples, unless otherwise specified, were performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples were commercially available products, page 23 / 110, 36 CN 121270626 A.

[0278] 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.

[0279] 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 definition of other occurrences. Similarly, combinations of substituents and variables are permitted, provided that 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 raw materials by means of the art and the methods presented below. If a substituent is substituted by more than one group, it should be understood that these groups may be on the same carbon atom or on different carbon atoms, as long as the structure is stable.

[0280] To facilitate understanding of the technology of this invention, some terms and phrases are defined below.

[0281] The term “alkyl” as used herein means a saturated aliphatic hydrocarbon group comprising branched and straight chains 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.

[0282] 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.

[0283] As used herein, the term "alkoxy" refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0284] As used herein, the terms "heterocyclic alkyl" or "heterocyclic group" refer to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic cyclic substituent, wherein 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 compounds include spirocyclic, fused-ring, and bridged-ring compounds. Examples include: oxo-heterocyclic butyl, aza-heterocyclic butyl, morpholino, piperidinyl, tetrahydropyrrolyl, pyrrolylalkyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrrolyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophene, dihydrotriazolyl, dihydroaza-heterocyclic butylalkyl, tetrahydrofuranyl, tetrahydrothiophene, etc., and their N-oxides. The linkage of heterocyclic substituents can be achieved through carbon atoms or through heteroatoms.

[0285] As used herein, the term “heteroaryl” refers to an aromatic ring containing one or more heteroatoms selected from O, N, or S. The 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 via carbon atoms or via heteroatoms.

[0286] As will be understood by those skilled in the art, as used herein, “halogen” or “halogen” means chlorine, fluorine, bromine, and iodine.

[0287] This invention includes the free form of compounds of formula (I), as well as their pharmaceutically acceptable salts and stereoisomers. The stereoisomers described in this invention are (depending on their structure) enantiomers, diastereomers, cis / trans isomers (syn- / anti-isomer), cis / trans isomers (cis- / trans-isomer), epimers, and (E)- / (Z)-isomers. Compounds of formula (A) can be used in the context of this invention as pure stereoisomers or as any mixture of stereoisomers, preferably racemic in the latter case.

[0288] The abbreviations and structures of the nucleotide monomers used in the nucleic acid sequence listing of this invention are as follows:

[0289]

[0290] Specification 25 / 110 pages 38 CN 121270626 A

[0291]

[0292] Those skilled in the art, based on existing technology, will recognize that the pharmaceutically acceptable salt of the siRNA can be a sodium or potassium salt, for example, a sodium salt of siRNA is produced during purification.

[0293] The invention will be further described in detail below with reference to specific embodiments.

[0294] The oligonucleotides were synthesized using the conventional phosphoramide method, briefly described below: 0.2–1 μmol of oligonucleotides were synthesized using a solid-phase oligonucleotide synthesis protocol on a 12-channel nucleic acid synthesizer from Beijing Qingke Biotechnology Co., Ltd.siRNA sequences need to be synthesized on a CPG-pre-packed column. Ammonolysis reagent is added to the synthesized oligonucleotides, and the mixture is incubated at 45–80 °C to separate the oligonucleotides from the solid-phase support, freeing them. The crude oligonucleotides are then precipitated with ethanol, centrifuged at high speed to discard the supernatant, and this process is repeated twice to obtain crude oligonucleotides. The precipitate is then resuspended in DEPC-treated water. The crude oligonucleotides are purified by ion-paired HPLC, and the collected product is dried to powder using a vacuum centrifuge. The purified product is dissolved in DEPC-treated water and analyzed by TOF LC-MS. The oligonucleotide concentration is determined, and the required volumes of equimolar amounts of the sense and antisense strands are calculated. Equimolar amounts of the sense and antisense strands are mixed thoroughly, and double strands are prepared by annealing at 95 °C for 5 minutes followed by natural cooling to room temperature.

[0295] Preparation of intermediate M:

[0296]

[0297] To a mixture of 11-aminoundecanoic acid (5 g, 24.8 mmol, 1.0 equivalent) and triethylamine (2.5 g, 24.8 mmol, 1.0 equivalent) in methanol (50 mL), ethyl 2,2,2-trifluoroacetate (4.4 g, 31.0 mmol, 1.25 equivalent) 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]+.

[0298] To a mixture of 11-(2,2,2-trifluoroacetamide)undecanoic acid (6.06 g, 20.4 mmol, 1.0 equivalent), (3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-3-ol (8.55 g, 20.4 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (7.8 g, 61.2 mmol, 3.0 equivalent) in N,N-dimethylformamide (60 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.1 g, 21.4 mmol, 1.05 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 3 / 1, with the addition of 0.5% triethylamine) to give a white solid product N-(11-((2S,4S, 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]+.

[0299] 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 equivalent) and potassium hydroxide (9.3 g, 165 mmol, 10.0 equivalent) 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 (elution: dichloromethane / methanol = 50 / 1, with 0.5% triethylamine) to give a yellow oily intermediate M (9.24 g, yield: 92.4%). LCMS (ESI): m / z = 625 [M + Na] +.

[0300] Example 1: Preparation of conjugate 1A: (prepared according to scheme 1a): Specification 27 / 110 pages 40 CN 121270626 A

[0301] Specification 28 / 110 pages 41 CN 121270626 A

[0302]

[0303] 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%).

[0304] Step 1b: Preparation of compound 1a-2: Lithium hydroxide monohydrate (3.1 g, 64.73 mmol) was added to a mixture of compound 1a-1 (16.7 g, 25.89 mmol, 1.0 equivalent) in tetrahydrofuran / methanol / water (80 mL / 16 mL / 16 mL).(2.5 equivalents). The mixture was reacted at 40°C for 4 hours. The solvent was removed from the reaction solution under reduced pressure. The aqueous solution of the residue was lyophilized. 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 [M-H]-.

[0305] Step 1c: Preparation of compound 1a-3: Benzyl chloroformate (2.03 g, 11.88 mmol, 1.2 equivalent) was added to a mixture of tetrahydrofuran (20 mL) and water (10 mL) of butyl ester (3.0 g, 9.9 mmol, 1.0 equivalent) and sodium carbonate (1.57 g, 14.85 mmol, 1.5 equivalent). The mixture was stirred overnight at room temperature. The mixture was diluted with water (50 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 (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]+.

[0306] 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 equivalent). 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 product). LCMS (ESI): m / z = 238 [M+H]+.

[0307] Step 1e: Preparation of compound 1a-5: At -20°C, isobutyl chloroformate (20.17 mL, 155.54 mmol, 1.0 equivalent) 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 equivalent) and N-methylmorpholine (15.7 g, 155.54 mmol, 1.0 equivalent) 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 equivalent) 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 oil, 1a-5 (43.67 g).Crude product).

[0308] 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 obtain a pale yellow oily substance 1a-6 (40 g, crude product).

[0309] 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). Sodium bicarbonate solid was added to adjust the pH to 9, 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 oily substance 1a-7 (24.1 g, yield: 68%). LCMS (ESI): m / z 622 [M+H]+.

[0310] Step 1h: Preparation of compound 1a-8: At 0°C, 1-chloroethyl chloroformate (7.16 mL, 66.34 mmol, 1.0 equivalent) was added dropwise to a mixture of compound 1a-7 (27.5 g, 44.23 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (1.54 mL, 8.85 mmol, 0.2 equivalent) 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]+. Specification 30 / 110 pages 43 CN 121270626 A

[0311] 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) and N,N-diisopropylethylamine (5.58 g, 43.24 mmol, 2.0 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]+.

[0312] Step 1j: Preparation of compound 1a-10: A mixture of compound 1a-9 (14.9 g, 21.19 mmol, 1.0 equivalent) 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 equivalent) and di-tert-butyl carbonate (24.36 mL, 105.95 mmol, 5.0 equivalent) were added. The mixture was stirred at room temperature for 48 hours. Tetrahydrofuran was removed under reduced pressure. The residue was diluted with water (200 mL) and then washed with dichloromethane (60 mL × 4). The aqueous layer was adjusted to pH 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]+.

[0313] Step 1k: Preparation of compound 1a-11: At 0°C, over 30 minutes, 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) were added dropwise to a mixture of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (6.58 g, 15.9 mmol, 2.5 equivalence) and dichloromethane (570 mL).A solution of 6.0 equivalents of 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]+.

[0314] Step 11: Preparation of compound 1a-13: A mixture of 1a-11 (1.97 g, 2.49 mmol, 1.0 equivalents) and hydrogen chloride-dioxane (4 mol / L, 13 mL) was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue was diluted with dichloromethane (60 mL). Compound 1a-12 (3.34 g, 7.47 mmol, 3.0 equivalence) was added, along with 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). 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 1% triethylamine) to give a pale yellow solid, 1a-13 (3.43 g, yield: 77%). LCMS (ESI): m / z = 1782.2 [M+H]+.

[0315] 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 product). LCMS (ESI): m / z = 1646.9 [M+H]+.

[0316] Step 1n: Preparation of compound 1a-15 (compound 31): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.26 g, 1.0 g) 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).0.68 mmol (1.2 equivalents). 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, concentration 50%-70% within 30 minutes, according to the acetonitrile specification 31 / 110 page 44 CN 121270626 A) 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]+.

[0317] 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‑302) / 2+H]+.1H NMR (500MHz, DMSO) δ8.42– 8.17(m ,2H) ,8.06(t,J=25.8Hz,2H) ,7 .89–7 .70(m ,4H) ,7 .30(dd ,J=14.4 ,6.9Hz,4H) , 7.20(dd ,J=10.1 ,6.9Hz,5H) ,6.93–6.85(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).

[0318] Step 1p: Preparation of compound 1a-17: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (21 mg,0.056 mmol, 1.0 equivalence) was added 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). 1.2 equivalents) and 1-hydroxybenzotriazole (9 mg, 0.066 mmol, 1.4 equivalents). The mixture was shaken at room temperature for 5 minutes. CPG-NH2 (0.167 mmol / g, 550 mg, 0.092 mmol, 1.95 equivalents) 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 with 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, 1.2 equivalents).Loading capacity: 35 μmol / g).

[0319] Step 1q: Preparation of conjugate 1A-1: Compound 1a-17 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain conjugate 1A-1 (MS m / z: [M-H]-: calcd. 8279.0520 Found 8278.826).

[0320] Step 1r: Preparation of conjugate 1A: Equivalent amounts of conjugate 1A-1 and complementary antisense chain (1167AM25) solution were taken, mixed evenly, heated in a 95℃ water bath for 5-10 minutes, and then naturally cooled to room temperature to obtain double-stranded conjugate 1A.

[0321] Example 2: Preparation of conjugate 1B (preparation method is the same as that of conjugate 1A):

[0322] Step 2a: Conjugate 1B-1: Compound 1a-17 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain conjugate 1B-1 (MS m / z: [M-H]-: calcd. 9027.6065 Found 9026.9963).

[0323] 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-stranded conjugate 1B.

[0324] Example 3: Preparation of conjugates 2A and 2B (prepared according to schemes 2a and 2b): Specification 32 / 110 pages 45 CN 121270626 A

[0325] Specification 33 / 110 pages 46 CN 121270626 A

[0326]

[0327]

[0328] Step 3a: Preparation of compound 2a-2: 6-Heptynic 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... A mixture of acetonitrile (30 mL) and benzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (9.92 g, 26.16 mmol, 1.1 equivalent) 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 /

[0329] Step 3b: Preparation of compound 2a-3: Compound 2a-3 was prepared by adding acetonitrile (30 mL) of benzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (9.92 g, 26.16 mmol, 1.1 equivalent) to ...1.0 equivalent of dichloromethane (60 mL) was added to trifluoroacetic acid (12 mL), and the mixture was stirred at room temperature for 30 minutes. 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]+.

[0330] 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]+.

[0331] 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 minutes. 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]+.

[0332] 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]+.

[0333] 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 organic phase 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]+.

[0334] 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 minutes. CPG-NH2 (13.50 g, 500 μmol / g, 1.8 equivalent) was added, and the mixture was shaken at room temperature for 5 hours. The mixture was filtered, and the filter cake was washed with acetonitrile and dichloromethane, and dried under vacuum for 1 hour. 30 mL of anhydrous pyridine and 10 mL of acetic anhydride were added to the solid, and the mixture was shaken at room temperature for 2 hours. The mixture was then filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to obtain 2a-9 (17.8 g, loading of 182.5 μmol / g).

[0335] Step 3h: Preparation of compound 2a-10-A-ssRNA-2: Compound 2a-9 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 2a-10-A-ssRNA-2 (MS m / z: [M-H]-: calcd .7317 .9750 Found 7317.2356).

[0336] Step 3i: Preparation of compound 2b-2: 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, ... (Note: The last part of the text appears to be a fragment and requires further context for accurate translation.)A mixture of 1.05 equivalents of 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]+.1H 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=7.2Hz,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, 6.6 Hz, 1H), 1.37 (s, 9H).

[0337] Step 3j: Preparation of compound 2b-3: The piperidine / acetonitrile (50 mL / 200 mL) mixture 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 yellow solid product 2b-3 (15.0 g, yield: 76.14%). LCMS (ESI): m / z = 395 [M+H]+.

[0338] Step 3k: Preparation of compound 2b-4: A mixture of compound 2b-3 (7.80 g, 19.80 mmol, 1.5 equivalence), tert-butyl (2-bromoethyl)carbamate (2.96 g, 13.20 mmol, 1.0 equivalence), and potassium carbonate (3.64 g, 26.40 mmol, 2.0 equivalence) 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 (elution: 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]+.1H 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).

[0339] 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 (elution buffer: 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).

[0340] Step 3m: Preparation of compound 2b-7: Compound 2b-6 (4.4 g, 5.13 mmol,A solution of 1.0 equivalent of hydrogen chloride-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 = 557 [M+H]+.

[0341] Step 3n: Preparation of compound 2b-8: A mixture of 2b-7 (crude product), 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 with 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%), as described on page 49 of the specification 36 / 110 of CN 121270626 A. LCMS (ESI): m / z = 923 [M / 2+H]+.

[0342] 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 equivalent), palladium hydroxide / carbon (1.5 g, 25% mass fraction), and trifluoroacetic acid (371 mg, 3.25 mmol, 1.0 equivalent) 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 in the next step without purification (5.0 g, yield: 94.93%).

[0343] LCMS (ESI): m / z = 811[M / 2+H]+.

[0344] Step 3p: Preparation of compound 2b-10: Under nitrogen protection, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (40.2 g, 210 mmol, 1.1 equivalent) was added to a mixture of 6-azidohexanoic acid (30 g, 190.8 mmol, 1.0 equivalent) and 1-hydroxypyrrolidine-2,5-dione (24 g, 210 mmol, 1.1 equivalent) 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]+.

[0345] Step 3q: Preparation of compound 2b-11: Compound 2b-9 (5.0 g,A mixture of acetonitrile (60 mL) of compound 2b-10 (824 mg, 3.24 mmol, 1.05 equivalent) and N,N-diisopropylethylamine (1.19 g, 9.22 mmol, 3.0 equivalent) 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] +.

[0346] Step 3r: Preparation of compound 2b-12 (compound 54): A mixture of compound 2b-11 (crude product), intermediate M (2.04 g, 3.39 mmol, 1.1 equivalent), N,N-diisopropylethylamine (796 mg, 6.17 mmol, 2.0 equivalent) and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (1.4 g, 3.39 mmol, 1.1 equivalent) in acetonitrile (60 mL) was stirred for 1 hour. The mixture was directly purified 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=6.8Hz,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,10H) ,1.89(s,9H) ,1.84(d ,J=13.3Hz,3H) ,1.77(s,9H) ,1.68(d ,J=35.4Hz,2H) , 1.50 (dd, J = 14.8, 7.4 Hz, 19H), 1.35 (s, 3H), 1.30–1.18 (m, 13H).

[0347] 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]+.

[0348] Step 3t: Preparation of compound 2b-14: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (56 mg, 0.15 mmol, 1.2 equivalent), 1-hydroxybenzotriazole (23 mg, 0.17 mmol, 1.4 equivalent) and N,N-diisopropylethylamine (63 mg,0.49 mmol (4.0 equivalents) was added to the mixture and shaken at room temperature for 5 minutes. 1.5 g of CPG-NH2 was added to the mixture and 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 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 2b-14 (1.61 g, loading: 41.1 μmol / g).

[0349] Step 3u: Preparation of compound 2b-15-A-ssRNA-1: Compound 2b-14 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 2b-15-A-ssRNA-1 (MS m / z: [M-H]-: calcd.8981.5075Found8980.9595).

[0350] Step 3v: Preparation of conjugate 2A-1: Compound 2a-10-A-ssRNA-2 (182 nanomoles, 1 equivalent) and 2b-15-A-ssRNA-1 (200 nanomoles, 1.1 equivalent) were added sequentially to 53 μL of PBS buffer (pH 8.0, 0.5M), 259 μL of DEPC water, 267 μL of DMF, and 286 μL of premixed CuSO4-THPTA (1:1) solution (conc. 14 nanomoles / μL). After mixing, 1.6 mg of sodium ascorbate was added, and after nitrogen purging, the reaction was carried out 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 2A-1 was obtained by ethanol precipitation. Then, conjugate 2A-1 was purified by HPLC (MS m / z: [M-H]-: calcd. 16299.4826 Found 16299.3828).

[0351] Step 3w: Preparation of conjugate 2A: Take equal amounts of conjugate 2A-1 and two complementary antisense chains (1167AM25 and 11040AM5) 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 2A.

[0352] Step 3x: Preparation of conjugate 2B (preparation method is the same as conjugate 2A):

[0353] Compound 2a-10-B-ssRNA-2 (182 nanomoles, 1 equivalent) and 2b-15-B-ssRNA-1 (200 nanomoles, 1.1 equivalent) were added sequentially to 53 μL of PBS buffer (pH 8.0, 0.5M), 259 μL of DEPC water, and 267 μL of DMF.And a premixed 286 μL CuSO4-THPTA (1:1) solution (conc. 14 nanomoles / μL) was added, and 1.6 mg of sodium ascorbate was added. After nitrogen purging, the mixture was 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 2.0 mg of crude conjugate 2B-1 was obtained by ethanol precipitation. The crude product was then purified by HPLC to obtain conjugate 2B-1 (MS m / z: [M-H]-: calcd. 15682.1370 Found 15682.1011).

[0354] Equivalent amounts of conjugate 2B-1 and two complementary antisense chains (1167AM25 and 1167AM25) solutions were mixed evenly and heated in a 95 °C water bath for 5-10 minutes. Then, the mixture was naturally cooled to room temperature to obtain double-chain conjugate 2B.

[0355] Example 4: Preparation of conjugate 3A (prepared according to scheme 3a):

[0356] Specification 38 / 110 pages 51 CN 121270626 A

[0357]

[0358] Specification 39 / 110 pages 52 CN 121270626 A

[0359] Step 4a: Preparation of compound 3a-1: glycine benzyl ester hydrochloride (6.04 g, 29.96 mmol, 2.0 equivalent), (9H-fluorene-9-yl)methyl(2-oxoethyl)carbamate (4.21 g, 14.98 mmol, 1.0 equivalent) and acetic acid (90 mg, 1.5 mmol, 0.1 equivalent) were added to 80 mL of methanol and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (1.4 g, 22.47 mmol, 1.5 equivalence) was added, and the mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure, and a saturated aqueous sodium carbonate solution was added, followed by extraction with ethyl acetate. The organic phase was washed with water and saturated brine, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1 to 1 / 2) to give a colorless oil 3a-1 (3.22 g, yield: 49.9%). LCMS (ESI): m / z = 431 [M+H]+.

[0360] 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 equivalent) was added, and the mixture was stirred at room temperature for 2 hours. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1 to 1 / 3) to give a yellow oil 3a-2 (3.41 g,Yield: 86.4%. LCMS (ESI): m / z = 531 [M+H]+.

[0361] 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]+.

[0362] Step 4d: Preparation of compound 3a-4: Compound 3a-3 (2.65 g, 6.04 mmol, 1.0 equivalent), (2-aminoethyl)carbamate benzyl ester (1.17 g, 6.04 mmol, 1.0 equivalent), N,N-diisopropylethylamine (1.56 g, 12.08 mmol, 2.0 equivalent) and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (2.5 g, 6.04 mmol, 1.0 equivalent) were added to 20 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1 to 1 / 3) to give a colorless syrup 3a-4 (2.80 g, yield: 75.2%). LCMS (ESI): m / z = 617 [M+H]+.

[0363] 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 minutes. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 to 10 / 1, with addition of triethylamine) to give a colorless oil 3a-5 (1.25 g, yield: 69.8%). LCMS (ESI): m / z = 395 [M+H]+.

[0364] Step 4f: Preparation of compound 3a-6: Compound 3a-5 (1.9 g, 4.82 mmol, 1.0 equivalent), tert-butyl (2-oxoethyl)carbamate (768 mg, 4.82 mmol, 1.0 equivalent), acetic acid (29 mg, 0.48 mmol, 0.1 equivalent) and sodium cyanoborohydride (337 mg, 5.2 mmol, 1.1 equivalent) were added to 20 mL of methanol, and the mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure, and sodium carbonate solution was added, followed by extraction 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 the addition of triethylamine) to give a yellow oily substance 3a-6 (914 mg,Yield: 35.2%. LCMS (ESI): m / z = 538 [M+H]+.

[0365] 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, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, plus triethylamine) to give a yellow oily substance 3a-7 (1.23 g, yield: 84.2%). LCMS (ESI): m / z = 857.5 [M+H]+.

[0366] 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 4 mol / L hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give a yellow solid 3a-8 (1.01 g, crude product). LCMS (ESI): m / z = 557 [M+H]+.

[0367] 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, and the mixture was stirred at room temperature for 1 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, plus triethylamine) to give a white solid compound 3a-9 (1.54 g, yield: 58.3%). LCMS (ESI): m / z = 923.7 [M / 2+H]+.

[0368] 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, ...) was added.1.0 equivalent) and 200 mg of 10% palladium hydroxide / carbon. 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 white solid compound 3a-10 (1.42 g, crude). LCMS (ESI): m / z = 1622 [M+H]+.

[0369] Step 4k: Preparation of compound 3a-11 (compound 55): Compound 3a-10 (1.42 g, 0.84 mmol, 1.0 equivalent), N,N-diisopropylethylamine (162 mg, 1.25 mmol, 1.5 equivalent) and compound 2b-10 (234 mg, 0.92 mmol, 1.1 equivalent) were added to 15 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 h.

[0370] 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 hour. The mixture was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (eluent: potassium dihydrogen phosphate-potassium hydroxide buffer solution / 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]+.

[0371] Step 41: Preparation of compound 3a-12: Compound 3a-11 (550 mg, 0.235 mmol, 1.0 equivalent), N,N-diisopropylethylamine (285 mg, 2.82 mmol, 12.0 equivalent), succinic anhydride (141 mg, 1.41 mmol, 6.0 equivalent) and 4-dimethylaminopyridine (3 mg, 0.024 mmol, 0.1 equivalent) 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 a white solid compound 3a-12 (550 mg, crude product). LCMS (ESI): m / z = 1072[(M-302) / 2+H]+.

[0372] 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 equivalents) were added to 25 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 5 minutes. 2.75 g of

[0373] 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 mixture was filtered, the solid phase was 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). Instructions 41 / 110 pages 54 CN 121270626 A

[0374] Step 4n: Preparation of compound 3a-14-A-ssRNA-1: Compound 3a-13 was synthesized by solid-phase oligonucleotide synthesis, ammonolyzed and purified by HPLC to obtain 3a-14-A-ssRNA-1 (MS m / z: [M-H]-: calcd .8981 .5075 Found 8980 .9588).

[0375] Step 4o: Preparation of conjugate 3A-1: 2a-10-A-ssRNA-2 (109 nanomoles, 1 equivalent) and 3a-14-A-ssRNA-1 (120 nanomoles, 1.1 equivalent) were added sequentially to 32 μL of PBS buffer (pH 8.0, 0.5M) and 186 μL of DEPC. Water, 160 μL of DMF, and 171 μL of premixed CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were mixed, and 1 mg of sodium ascorbate was added. After nitrogen purging, the mixture was reacted at 35 °C for 2 h. After the reaction was complete, 9 μL of ammonia and 10 mg of EDTA were added. After shaking for 5–10 minutes, conjugate 3A-1 (crude product, approximately 1.7 mg) was obtained by ethanol precipitation. Conjugate 3A-1 was then purified by HPLC (MS m / z: [M-H]-: calcd. 16299.4826 Found 16299.417).

[0376] 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°C water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 3A.

[0377] Example 5: Preparation of conjugate 4A (prepared according to scheme 4a):

[0378] Specification 42 / 110 pages 55 CN 121270626 A

[0379]

[0380] Step 5a: Preparation of compound 4a-2: Under nitrogen protection, 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, ...A mixture of 2.2 equivalents of 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]+.

[0381] 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: 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]+.

[0382] Step 5c: Preparation of compound 4a-4: A mixture of compound 4a-3 (8.86 g, 20.7 mmol, 1.0 equivalent) in 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]+.

[0383] 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 (7.5 g) as a white solid.Yield: 49%. LCMS (ESI): m / z = 817 [M+H]+. Specification 43 / 110 pages 56 CN 121270626 A

[0384] Step 5e: Preparation of compound 4a-6: A mixture of tetrahydrofuran / methanol / water (60 / 30 / 30 mL) 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) 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 [M-H]-.

[0385] 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 equivalent) was added to a mixture of compound 1a-14 (570 mg, 0.32 mmol, 1.0 equivalent), compound 4a-6 (260 mg, 0.32 mmol, 1.0 equivalent), and N,N-diisopropylethylamine (146 mg, 1.13 mmol, 3.5 equivalent) 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]+.

[0386] Step 5g: Preparation of compound 4a-8: Succinic anhydride (60.5 mg, 0.60 mmol, 6.0 equivalent) was added to a mixture of compound 4a-7 (245 mg, 0.10 mmol, 1.0 equivalent), triethylamine (121 mg, 1.2 mmol, 12.0 equivalent) and 4-dimethylaminopyridine (12 mg, 0.10 mmol, 1.0 equivalent) in dichloromethane (2 mL). The mixture was stirred at room temperature for 20 hours. The mixture was diluted with dichloromethane (10 mL), then washed with saturated sodium bicarbonate solution (10 mL × 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-3O2) / 2+H]+.

[0387] Step 5h: Preparation of compound 4a-9: Compound 4a-8 (253 mg, 0.10 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (77 mg, 0.60 mmol,To a mixture of N,N-dimethylformamide (6.0 equivalents) (10 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (57 mg, 0.15 mmol, 1.5 equivalents) and 1-hydroxybenzotriazole (20 mg, 0.15 mmol, 1.5 equivalents) 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 equivalents) 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 obtain a white solid 4a-9 (1.36 g, loading: 30 μmol / g).

[0388] Step 5i: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 4a-10-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9067.5135Found9066.8616).

[0389] Step 5j: Preparation of conjugate 4A-1: Take 2a-10-A-ssRNA-2 (293 nanomoles, 1 equivalent) and 4a-10-A-ssRNA-1 (322 nanomoles, 1.1 equivalent), and add 86 μL of PBS buffer (pH 8.0, 0.5M), 189 μL of DEPC water, 429 μL of DMF, and 161 μL of premixed CuSO4-THPTA (1:1) solution (conc. 40 nanomoles / μL) in sequence. After mixing, add 2.6 mg of sodium ascorbate, replace with nitrogen, and react at 35°C for 2 h. After the reaction was complete, 25 μL of ammonia and 26 mg of EDTA were added. After shaking for 5-10 minutes, about 2.6 mg of crude conjugate 4A-1 was obtained by ethanol precipitation. Then, conjugate 4A-1 was purified by HPLC (MS m / z: [M-H]-: calcd. 16385.4886 Found 16385.4753).

[0390] Step 5k: Preparation of conjugate 4A: Take equal amounts of conjugate 4A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solution, mix them evenly, heat in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature.The double-chain conjugate 4A was obtained.

[0391] Example 6: Preparation of conjugate 6A (prepared according to scheme 5a): Specification 44 / 110 page 57 CN 121270626 A

[0392] Specification 45 / 110 page 58 CN 121270626 A

[0393]

[0394] 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 (eluent: petroleum ether / dichloromethane / acetone = 10 / 10 / 1) to give a yellow oily substance 5a-1 (6.28 g, yield: 77.43%). LCMS (ESI): m / z = 482 [M+H]+. Specification 46 / 110 pages 59 CN 121270626 A

[0395] 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]+.

[0396] 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]+.

[0397] Step 6d: Preparation of compound 5a-4: Compound 5a-3 (5.4 g, 10.27 mmol,A 1.0 equivalent (40 mL) mixture of hydrogen chloride and dioxane 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 further purification. LCMS (ESI): m / z = 326 [M+H]+.

[0398] Step 6e: Preparation of compound 5a-5: At 0°C, over 30 minutes, a mixture of dichloromethane containing compound 5a-4 (2.8 g, 7.05 mmol, 1.0 equivalent), compound 1a-10 (4.17 g, 7.05 mmol, 1.0 equivalent), and N,N-diisopropylethylamine (4.55 g, 35.26 mmol, 5.0 equivalent) was added dropwise to a mixture of dichloromethane containing N,N-diisopropylethylamine (4.55 g, 35.26 mmol, 5.0 equivalent). 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]+.

[0399] Step 6f: Preparation of compound 5a-6: A mixture of compound 5a-5 (1.54 g, 1.75 mmol, 1.0 equivalent) and 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 = 580 [M+H]+.

[0400] 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]+.

[0401] Step 6h: Preparation of compound 5a-8: Compound 5a-7 (1.9 g, 1.02 mmol, 1.0 equivalent) was subjected to hydrogen balloon pressure.A mixture of palladium hydroxide / carbon (380 g, 20% by mass) and trifluoroacetic acid (116 mg, 1.02 mmol, 1.0 equivalence) 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]+.

[0402] 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 at pH 7, B: acetonitrile, acetonitrile concentration 30%-90% over 30 minutes) to give white solid products 5a-9 (compound 51) (210 mg, yield: 18.26%). (See manual page 47 / 110, 60 CN 121270626 A). LCMS (ESI): m / z = 1109[(M-302) / 2+ H]+.

[0403] Step 6j: Preparation of compound 5a-10: A mixture of compound 5a-9 (210 mg, 0.083 mmol, 1.0 equivalent), succinic anhydride (51 mg, 0.50 mmol, 6.0 equivalent), triethylamine (101 mg, 1.00 mmol, 12.0 equivalent) and 4-dimethylaminopyridine (2 mg, 0.008 mmol, 0.1 equivalent) 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 product). LCMS (ESI): m / z = 1159[(M-302) / 2 +H]+.

[0404] Step 6k: Preparation of compound 5a-11: 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 to an N,N-dimethylformamide solution (8 mL) of compound 5a-10 (210 mg, 0.08 mmol, 1.0 equivalence).The mixture was shaken at room temperature for 5 minutes. 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 hour. The residue was added to pyridine / acetic anhydride (6 mL / 2 mL), and the mixture was 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 5a-11 (990 mg, loading: 40 μmol / g).

[0405] Step 61: Preparation of compound 5a-12-A-ssRNA-1: Compound 5a-11 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 5a-12-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9155.6195Found9155.8439).

[0406] Step 6m: Preparation of conjugate 6A-1: 2a-10-A-ssRNA-2 (200 nanomoles, 1 equivalent) and 5a-12-A-ssRNA-1 (220 nanomoles, 1.1 equivalent) were added sequentially to 59 μL of PBS buffer (pH 8.0, 0.5M) and 257 μL of DEPC. Water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL) were mixed, and 1.7 mg of sodium ascorbate was added. After nitrogen purging, the mixture was 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 min. The crude product of conjugate 6A-1 was obtained by ethanol precipitation, which was then purified by HPLC to obtain conjugate 6A-1 (MS m / z: [M-H]-: calcd. 16473.5946 Found 16471.1600).

[0407] 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°C water bath for 5-10 minutes, and then cool them naturally to room temperature to obtain double-chain conjugate 6A.

[0408] Example 7: Preparation of conjugate 7A (prepared according to scheme 6a): Specification 48 / 110 pages 61 CN 121270626 A

[0409]

[0410]

[0411] Step 7a: Preparation of compound 6a-1: 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,Instructions for Use, pages 49 / 110, 62 CN 121270626 A: 27.94 mmol (2.0 equivalent) of dichloromethane (15 mL) was mixed with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (6.11 g, 16.07 mmol, 1.15 equivalent). 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). LCmS(ESI): m / z = 315[m+H]+.

[0412] Step 7b: Preparation of compound 6a-2: A mixture of compound 6a-1 (4.39 g, 13.97 mmol, 1.0 equivalent) 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 equivalent), N,N-diisopropylethylamine (5.4 g, 41.91 mmol, 3.0 equivalent) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.84 g, 15.37 mmol, 1.1 equivalent) 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 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]+.

[0413] Step 7c: Preparation of compound 6a-3: Sodium hydroxide (0.84 g, 20.96 mmol, 1.5 equivalent) was added to a mixture of compound 6a-2 (4.5 g, 13.97 mmol, 1.0 equivalent) 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 by adding 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 product). LCMS (ESI): m / z = 309 [M+H]+.

[0414] Step 7d: Preparation of compound 6a-4: Compound 2a-6 (415 mg, 1.35 mmol, 1.52 equivalences) was added.A mixture of 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) was mixed with 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (400 mg, 1.05 mmol, 1.2 equivalence). 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]+.

[0415] 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 product). LCMS (ESI): m / z = 978 [M+Na]+.

[0416] 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 again, 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 capacity: 67 μmol / g). Instructions 50 / 110 pages 63 CN 121270626 A

[0417] Step 7g: Preparation of compound 6a-7-A-ssRNA-2: Compound 6a-6 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 6a-7-A-ssRNA-2 (MS m / z: [M-H]-: calcd .7252 .9070Found 7252 .1811).

[0418] Step 7h: Preparation of conjugate 7A-1: Take 6a-7-A-ssRNA-2 (200 nanomoles, 1 equivalent) and 2b-15-A-ssRNA-1 (220 nanomoles, 1.1 equivalent), and add 59 μL of PBS buffer (pH 8.0, 0.5M) and 397 μL of DEPC in sequence. Water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL) were mixed, and 1.7 mg of sodium ascorbate was added. After nitrogen purging, the mixture was 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 min. The crude product of conjugate 7A-1 was obtained by ethanol precipitation, and then purified by HPLC to obtain conjugate 7A-1 (MS m / z: [M-H]-: calcd. 16234.4146 Found 16234.1400).

[0419] Step 7i: Preparation of conjugate 7A: Take equal amounts of conjugate 7A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solution, mix them evenly, heat in a 95°C water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 7A.

[0420] Example 8: Preparation of conjugate 8A (prepared according to scheme 7a):

[0421] Specification 51 / 110 pages 64 CN 121270626 A

[0422]

[0423] Step 8a: Preparation of compound 7a-1: At 0°C, 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). The mixture was stirred at 0°C for 20 minutes. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium carbonate solution (30 mL) and then extracted with ethyl acetate (30 mL × 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]+.

[0424] Step 8b: Preparation of compound 7a-2: A mixture of compound 7a-1 (3.19 g, 10.60 mmol, 1.0 equivalent) 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). Add 2,2-dimethyl-4-oxo-3,8,11-trioxo-5-azatridecane-13-acid (2.93 g, 11.13 mmol, 1.05 equivalents), N,N-diisopropylethylamine (4.31 g, 33.39 mmol, 3.0 equivalents), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.64 g, 12.19 mmol, 1.15 equivalents). Stir the mixture at room temperature for 1.5 hours. Remove the solvent under reduced pressure. Dilute the mixture with saturated sodium carbonate solution (50 mL) and extract with ethyl acetate (30 mL × 2). Wash the combined organic layers with saturated brine (30 mL × 1), dry over anhydrous sodium sulfate, and concentrate to give a brown oily product 7a-2 (7.57 g, crude). LCMS (ESI): m / z = 447 [M+H]+.

[0425] Step 8c: Preparation of compound 7a-3: A mixture of compound 7a-2 (4.73 g, 10.60 mmol, 1.0 equivalent) 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 equivalent), N,N-diisopropylethylamine (4.1 g, 31.8 mmol, 3.0 equivalent) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (4.43 g, 11.66 mmol, 1.1 equivalent) 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 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]+.

[0426] Step 8d: Preparation of compound 7a-4: Sodium hydroxide (0.49 g, 12.29 mmol, 1.0 equivalent) was added to a mixture of compound 7a-3 (3.72 g, 8.19 mmol, 1.0 equivalent) in methanol (7.5 mL) and water (7.5 mL).(1.5 equivalents). Mixing instructions 52 / 110 pages 65 CN 121270626 A. Stir the mixture at room temperature for 1 hour. Remove the solvent under reduced pressure. Adjust the pH to 3 with 2 mol / L dilute hydrochloric acid solution, and then extract with ethyl acetate (20 mL × 5). Wash the combined organic layers with saturated brine (50 mL × 1), dry over anhydrous sodium sulfate and concentrate to give a pale yellow oil 7a-4 (3.65 g, crude product). LCMS (ESI): m / z = 441 [M+H]+.

[0427] Step 8e: Preparation of compound 7a-5: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (411 mg, 1.08 mmol, 1.2 equivalent) was added to a mixture of N,N-dimethylformamide (3 mL) of compound 7a-4 (600 mg, 1.36 mmol, 1.52 equivalent), compound 2a-6 (510 mg, 0.90 mmol, 1.0 equivalent), and N,N-diisopropylethylamine (290 mg, 2.25 mmol, 2.5 equivalent). 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]+.

[0428] 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), then 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 product). LCMS (ESI): m / z = 784.6 [M-302+H]+.

[0429] Step 8g: Preparation of compound 7a-7: To a mixture of compound 7a-6 (222 mg, 0.20 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (158 mg, 1.22 mmol, 6.0 equivalent) in N,N-dimethylformamide (20 mL), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (116 mg, 0.31 mmol, 1.5 equivalent) and 1-hydroxybenzotriazole (41 mg, 0.31 mmol, ...1.5 equivalents). The mixture was shaken at room temperature for 5 minutes. CPG-NH2 (2.20 g, 0.37 mmol, 1.8 equivalents) was added. 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 (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).

[0430] Step 8h: Preparation of compound 7a-8-A-ssRNA-2: Compound 7a-7 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 7a-8-A-ssRNA-2 (MS m / z: [M-H]-: calcd .7384 .0380 Found 7383 .3414).

[0431] Step 8i: Preparation of conjugate 8A-1: 7a-8-A-ssRNA-2 (200 nanomoles, 1 equivalent) and 2b-15-a-ssRNA-1 (220 nanomoles, 1.1 equivalent) were added sequentially to 59 μL of PBS buffer (pH 8.0, 0.5M) and 290 μL of DEPC. Water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL) were mixed, and 1.7 mg of sodium ascorbate was added. After nitrogen purging, the mixture was 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, which was then purified by HPLC to obtain conjugate 8A-1 (MS m / z: [M-H]-: calcd. 16365.5456 Found 16365.2100).

[0432] Step 8j: Preparation of conjugate 8A: Take equal amounts of conjugate 8A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solution, mix them evenly, heat in a 95°C water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 8A.

[0433] Example 9: Preparation of conjugate 9A (prepared according to scheme 8a): Specification 53 / 110 pages 66 CN 121270626 A

[0434]

[0435]

[0436] Step 9a: Preparation of compound 8a-2: 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, ...A mixture of 2.0 equivalents) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (8.47 g, 22.28 mmol, 1.2 equivalents) 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]+.

[0437] 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]+. Specification 54 / 110 pages 67 CN 121270626 A

[0438] Step 9c: Preparation of compound 8a-4: A mixture of compound 8a-3 (crude), heptyl-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 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. 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]+.

[0439] 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]+.

[0440] Step 9e: Preparation of compound 8a-6: Compound 8a-5 (1.1 g, 2.87 mmol, 1.0 equivalent), compound 2a-6 (1.22 g, 2.15 mmol, 0.75 equivalent), N,N-diisopropylethylamine (1.11 g, 8.62 mmol, ...A mixture of 3.0 equivalents of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.31 g, 3.45 mmol, 1.2 equivalents) and 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]+.

[0441] Step 9f: Preparation of compound 8a-7: A mixture of compound 8a-6 (320 mg, 0.34 mmol, 1.0 equivalent), succinic anhydride (207 mg, 2.07 mmol, 6.0 equivalent), triethylamine (418 mg, 4.14 mmol, 12.0 equivalent) and 4-dimethylaminopyridine (5 mg, 0.034 mmol, 0.1 equivalent) 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]+ .1H NMR(500MHz,MeOD)δ7.42–7.12(m,9H) ,6.93–6.71(m,4H) ,5.55–5.28(m,1H) ,4.40(dd, J=44.2,7.2Hz,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=13.4,6.2Hz,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) .

[0442] Step 9g: Preparation of compound 8a-8: N, Add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (169 mg, 0.44 mmol, 1.2 equivalents) and 1-hydroxybenzotriazole (70 mg, 0.52 mmol) to an N-dimethylformamide solution (10 mL).1.4 equivalents) and N,N-diisopropylethylamine (191 mg, 1.48 mmol, 4.0 equivalents) were mixed and shaken at room temperature for 5 minutes. 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 hour. The residue was added to pyridine / acetic anhydride (7.5 mL / 2.5 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 8a-8 (2.08 g, loading: 101 μmol / g).

[0443] Step 9h: Preparation of compound 8a-9-A-ssRNA-2: Compound 8a-8 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 8a-9-A-ssRNA-2 (MS m / z: [M-H]-: calcd .7326 .9860 Found 7326 .2315). Specification 55 / 110 pages 68 CN 121270626 A

[0444] Step 9i: Preparation of conjugate 9A-1: Take 8a-9-A-ssRNA-2 (200 nanomoles, 1 equivalent) and 2b-15-A-ssRNA-1 (220 nanomoles, 1.1 equivalent), and add 59 μL of PBS buffer (pH 8.0, 0.5M) and 590 μL of DEPC sequentially. Water, 293 μL of DMF, and 73 μL of premixed CuSO4-THPTA (1:1) solution (conc. 60 nmol / μL) were mixed, and 1.7 mg of sodium ascorbate was added. After nitrogen purging, the mixture was 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 min. The crude product of conjugate 9A-1 was obtained by ethanol precipitation, and then purified by HPLC to obtain conjugate 9A-1 (MS m / z: [M-H]-: calcd. 16308.4936 Found 16308.1200).

[0445] 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 in a 95°C water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 9A.

[0446] Example 10: Preparation of conjugate 5A (prepared according to scheme 9a):

[0447] Specification 56 / 110 pages 69 CN 121270626 A

[0448]

[0449] Step 10a: Preparation of compound 9a-1: Mix compound 1a-8 (8.15 g, 15.32 mmol, 1.0 equivalent) and hydrogen chloride-dioxane (4 M, 60 mL,The mixture was stirred at room temperature for 2 days. The solvent was removed under reduced pressure. The residue was dissolved in a mixed solvent of tetrahydrofuran (100 mL) and water (100 mL). Sodium carbonate (9.7 g, 91.92 mmol, 6.0 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 washed with dichloromethane (60 mL × 4). The aqueous layer was adjusted to pH 3 with 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: Rf 0.4 (dichloromethane:methanol = 10:1).

[0450] Step 10b: Preparation of compound 9a-2: At 0°C, a solution of compound 9a-1 (4.0 g, 6.76 mmol, 1.0 equivalent), compound 1a-4 (2.1 g, 6.76 mmol, 1.0 equivalent), and N,N-diisopropylethylamine (5.23 g, 40.56 mmol, 6.0 equivalent) 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 equivalent) 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: Rf 0.5 (ethyl acetate:dichloromethane = 1:1).

[0451] Step 10c: Preparation of compound 9a-3: A mixture of compound 9a-2 (4.88 g, 6.76 mmol, 1.0 equivalent) and hydrogen chloride-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). Add 3-(tert-Butoxycarbonyl)amino)propionic acid (3.84 g, 20.28 mmol, 3.0 equivalents), N,N-diisopropylethylamine (6.98 g, 54.08 mmol),8.0 equivalents) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (7.7 g, 20.28 mmol, 3.0 equivalents). The mixture was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure. The mixture was diluted with saturated sodium carbonate solution (60 mL) and then extracted with ethyl acetate (30 mL × 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: Rf 0.5 (dichloromethane:methanol = 20:1).

[0452] 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).

[0453] 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 equivalent), trifluoroacetic acid (0.14 mL, 1.82 mmol, 1.0 equivalent), isopropanol (50 mL), and ethanol (50 mL). The mixture was stirred at room temperature for 2 days under hydrogen balloon pressure. The mixture was then filtered. The filtrate was concentrated under reduced pressure to give a white solid compound 9a-5 (3.48 g).Crude product). LCMS (ESI): m / z 1790.4 [M+H]+; TLC: Rf 0.4 (dichloromethane:methanol = 10:1).

[0454] 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 equivalent) was added to a mixture of compound 9a-5 (735 mg, 0.39 mmol, 1.25 equivalent), compound 4a-6 (250 mg, 0.312 mmol, 1.0 equivalent), N,N-diisopropylethylamine (240 mg, 1.86 mmol, 6.0 equivalent) 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 at pH 7, B: acetonitrile, acetonitrile concentration 30%-90% over 30 minutes) to give a white solid compound 9a-6 (199 mg, yield: 25%). LCMS (ESI): m / z 1136.5 [(M-302) / 2+H]+; TLC: Rf 0.5 (dichloromethane:methanol = 10:1).

[0455] 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), then washed with saturated sodium bicarbonate solution (10 mL × 2) and saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, and concentrated to give a white solid compound 9a-7 (212 mg, crude). LCMS (ESI): m / z 1186.5 [(M-3O2) / 2+H]+; TLC: Rf 0.4 (dichloromethane:methanol = 10:1).

[0456] Step 10h: Preparation of compound 9a-8: 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), 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. The mixture was stirred at room temperature for 5 minutes. CPG-NH2 (0.167 mmol / g, 1.0 g, 0.167 mmol / g) was added.2.2 equivalents). The mixture was stirred at room temperature for 22 hours. The mixture was filtered. The solid was washed with dichloromethane (8 mL × 5). Pyridine (6 mL) and acetic anhydride (2 mL) were added. The mixture was stirred at room temperature for 3 hours. The mixture was filtered. The solid was washed with dichloromethane (8 mL × 5) and dried under vacuum to give a white solid compound 9a-8 (1.0 g, loading: 35 μmol / g, yield: 43%). Specification 58 / 110 pages 71 CN 121270626 A

[0457] Step 10i: Preparation of compound 9a-9-A-ssRNA-1: Compound 9a-8 was synthesized by solid-phase oligonucleotide synthesis, ammonolyzed and purified by HPLC to give 9a-9-A-ssRNA-1 (MS m / z: [M-H]-: calcd 9209 .6715 . Found 9208 .8616).

[0458] Step 10j: Preparation of conjugate 5A-1: 53 μL of PBS buffer (pH 8.0, 0.5M), 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: [M-H]-: calcd. 16536.6576 Found 16536.1842).

[0459] Step 10k: Preparation of conjugate 5A: Equivalent amounts of conjugate 5A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solution were taken, mixed evenly, heated in a 95℃ water bath for 5-10 minutes, and then naturally cooled to room temperature to obtain double-chain conjugate 5A.

[0460] Example 11: Preparation of conjugate 12A (prepared according to scheme 10a): Specification 59 / 110 pages 72 CN 121270626 A

[0461] Specification 60 / 110 pages 73 CN 121270626 A

[0462]

[0463] Step 11a: Preparation of compound 10a-1: Under nitrogen protection, compound 2b-5 (10.0 g, 29.64 mmol, 1.0 equivalent), triethylamine (10.8 g, 106.7 mmol, ...Ethyl chloroformate (11.26 g, 103.74 mmol, 3.5 equivalents) was added dropwise to a mixture of 3.6 equivalents of tetrahydrofuran 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 equivalents) 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 equivalents) 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)+.

[0464] Step 11b: Preparation of compound 10a-2: At room temperature, 2-iodobenzoic acid (5.73 g, 20.47 mmol, 1.2 equivalent) was added to a solution of compound 10a-1 (5.1 g, 17.1 mmol, 1.0 equivalent) 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)+.

[0465] 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 equivalents), N,N-diisopropylethylamine (10.64 g, 82.5 mmol, 1.5 equivalents), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (27.3 g, 66.0 mmol, 1.2 equivalents) 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 obtain a yellow solid compound 10a-4 (37.97 g, crude product). LCMS (ESI): m / z = 631(M+H)+.

[0466] 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, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1).Adding triethylamine yielded a yellow oily compound 10a-5 (16.7 g, yield: 67.8%). LCMS (ESI): m / z = 409(M+H)+.

[0467] Step 11e: Preparation of compound 10a-6: Compound 10a-5 (6.4 g, 15.58 mmol, 1.0 equivalent), compound 10a-2 (5.0 mg, 15.58 mmol, 1.0 equivalent), acetic acid (935 mg, 15.58 mmol, 1.0 equivalent) and sodium cyanoborohydride (2.94 g, 46.74 mmol, 3.0 equivalent) 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 a white solid compound 10a-6 (2.35 g, yield: 20.8%). LCMS (ESI): m / z = 714.5(M+H)+.

[0468] 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, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 10 / 1, 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)+.

[0469] 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 4M 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 product). LCMS (ESI): m / z = 585.5(M+H)+.

[0470] Step 11h: Preparation of compound 10a-9: Compound 10a-8 (2.7 g, 2.26 mmol, 1.0 equivalent), compound 1a-12 (3.24 g, 7.23 mmol, 3.2 equivalent), N,N-diisopropylethylamine (2.92 g, 22.6 mmol, ...10.0 equivalents) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.75 g, 7.23 mmol, 3.2 equivalents) were added to 40 mL of acetonitrile, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 15 / 1, 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)+.

[0471] 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 obtain a white solid compound 10a-10 (2.31 g, crude product). LCMS (ESI): m / z = 825(M / 2+H)+.

[0472] 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 hours. Then, N,N-diisopropylethylamine (254 mg, 1.96 mmol, 1.5 equivalences), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (662 mg, 1.60 mmol, 1.22 equivalences), and intermediate M (963 mg, 1.60 mmol, 1.22 equivalences) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer / acetonitrile) to give a white solid compound 10a-11 (1.81 g, yield: 58.1%). LCMS (ESI): m / z = 1036((M-302) / 2+H)+.

[0473] Step 11k: Preparation of compound 10a-12: Under nitrogen protection, compound 10a-11 (300 mg, 0.126 mmol, 1.0 equivalent), triethylamine (154 mg, 1.52 mmol, 12.0 equivalent), succinic anhydride (76 mg, 0.76 mmol) was prepared.6.0 equivalents) and p-dimethylaminopyridine (1.5 mg, 0.0126 mmol, 0.1 equivalents) were added to 1.5 mL of dichloromethane, and 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)+.

[0474] Step 111: 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 the solid phase was dried under reduced pressure. 12 mL of pyridine and 4 mL of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to give a yellow solid compound 10a-13 (32.1 μmol / g).

[0475] Step 11m: Preparation of compound 10a-14-A-ssRNA-1: Compound 10a-13 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to give 10a-14-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9009.5615Found9008.8216).

[0476] Step 11n: Preparation of conjugate 12A-1: To a mixture of compounds 8a-9-A-ssRNA-2 (182 nanomoles, 1 equivalent), and 10a-14-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, and the mixture was shaken for 5-10 minutes. The crude product of conjugate 12A-1 was obtained by ethanol precipitation, yielding approximately 2.3 mg.The conjugate 12A-1 was then purified by HPLC (MS m / z: [M-H]-: calcd .16336 .5476 Found 16336 .1821).

[0477] 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 in a 95℃ water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 12A.

[0478] Example 12: Preparation of conjugate 11A (prepared according to scheme 11a): Specification 63 / 110 page 76 CN 121270626 A

[0479] Specification 64 / 110 page 77 CN 121270626 A

[0480]

[0481] 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)+.

[0482] 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 4M 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 product). LCMS (ESI): m / z = 571(M+H)+.

[0483] Step 12c: Preparation of compound 11a-3: Compound 11a-2 (4.85 g, 5.49 mmol, 1.0 equivalent), compound 1a-12 (7.6 g, 17.02 mmol, 3.1 equivalent), N,N-diisopropylethylamine (7.08 g, 54.9 mmol, 10.0 equivalent) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (6.67 g, 17.57 mmol, 3.2 equivalent) 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, triethylamine added) to give a white solid compound 11a-3 (6.92 g, yield: 67.84%). LCMS (ESI): m / z = 930.6(M / 2+H)+.

[0484] 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 obtain a yellow solid compound 11a-4 (1.77 g, crude product). LCMS (ESI): m / z = 1635(M+H)+.

[0485] Step 12e: Preparation of compound 11a-5 (compound 59): Compound 11a-4 (1.77 g, 1.07 mmol, 1.0 equivalent), N,N-diisopropylethylamine (417 mg, 3.23 mmol, 3 equivalent) and compound 2b-10 (302 mg, 1.18 mmol, 1.1 equivalent) were added to 10 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then N,N-diisopropylethylamine (417 mg, 3.23 mmol, 3 equivalents), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (535 mg, 1.29 mmol, 1.2 equivalents), and intermediate M (778 mg, 1.29 mmol, 1.2 equivalents) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer / 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) ,3.41(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) .

[0486] 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, 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 obtain a white solid compound 11a-6 (229 mg, crude product). LCMS (ESI): m / z = 1079((M-302) / 2+H)+.

[0487] Step 12g: Preparation of compound 11a-7: Compound 11a-6 (229 mg, 0.09 mmol, 1.0 equivalent), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (41 mg, 0.11 mmol, 1.2 equivalent), 1-hydroxybenzotriazole (17 mg, 0.13 mmol, 1.4 equivalent) and N,N-diisopropylethylamine (46 mg,0.36 mmol (4.0 equivalences) was 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 mixture was filtered, the solid phase was 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).

[0488] Step 12m: Preparation of compound 11A-8-A-ssRNA-1: Compound 11a-7 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 11A-8-A-ssRNA-1 (MS m / z: [M-H]-: calcd.8995.5345Found8997.8616).

[0489] Step 12n: Preparation of conjugate 11A-1: 53 μL of PBS buffer (pH 8.0, 0.5 M), 259 μL of DEPC water, 267 μL of DMF, and 286 μL of premixed 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 11A-8-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 11A-1 was obtained by ethanol precipitation. Then, conjugate 11A-1 was purified by HPLC (MS m / z: [M-H]-: calcd .16322 .5206 Found Specification 66 / 110 page 79 CN 121270626 A 16322.1033).

[0490] Step 12o: Preparation of conjugate 11A: Take equal amounts of conjugate 11A-1 and two complementary antisense chains (1167AM25 and 11040AM5) 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 11A.

[0491] Example 13: Preparation of conjugate 10A (prepared according to scheme 12a):

[0492]

[0493]

[0494] Step 13a: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized by solid-phase oligonucleotide synthesis, purified by HPLC after ammonolysis.4a-10-A-ssRNA-1 was obtained (MS m / z: [M-H]-: calcd.Found).

[0495] Step 13b: Preparation of conjugate 10A-1: 53 μL of PBS buffer (pH 8.0, 0.5M), 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 4a-10-A-ssRNA-1 (200 nmol, 1.1 equivalent). After mixing, 1.6 mg of sodium ascorbate was added, and after nitrogen purging, the mixture was 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 10A-1 was obtained by ethanol precipitation. Then, conjugate 10A-1 was purified by HPLC (MS m / z: [M-H]-: calcd .16394 .4996 Found 16394 .0447).

[0496] Step 13c: Preparation of conjugate 10A: Take equal amounts of conjugate 10A-1 and two complementary antisense chain solutions (1167AM25 and 11040AM5) and mix them evenly. Heat in a 95°C water bath for 5-10 minutes and then cool naturally to room temperature to obtain double-chain conjugate 10A.

[0497] Example 14: Preparation of conjugate 13A (prepared according to scheme 13a):

[0498]

[0499]

[0500] Step 14a: Preparation of compound 4a-10-A-ssRNA-1: Compound 4a-9 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 4a-10-A-ssRNA-1 (MS m / z: [M-H]-: calcd.Found).

[0501] Step 14b: Preparation of conjugate 13A-1: To a mixture of compound 6a-7-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, and the mixture was shaken for 5-10 minutes. The crude product of conjugate 13A-1 was obtained by ethanol precipitation, yielding approximately 2.3 mg.The conjugate 13A-1 was then purified by HPLC (MS m / z: [M-H]-: calcd .16320 .4206 Found 16319.8622). Specification 68 / 110 pages 81 CN 121270626 A

[0502] Step 14c: Preparation of conjugate 13A: Take equal amounts of conjugate 13A-1 and two complementary antisense chains (1167AM25 and 11040AM5) 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 13A.

[0503] Example 15: Preparation of conjugate 79A (prepared according to scheme 14a):

[0504] Specification 69 / 110 pages 82 CN 121270626 A

[0505]

[0506] Step 15a: Preparation of compound 14a-1: Compound 2b-7 (2.6 g, 3.0 mmol, 1.0 equivalent), 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid (2 g, 9.4 mmol, 3.1 equivalent), N,N-diisopropylethylamine (4 g, 30.4 mmol, 10.0 equivalent) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (3.7 g, 3.0 mmol, 1.0 equivalent) were prepared. A mixture of acetonitrile (30 mL) in 2.08 g (9.7 mmol, 3.2 equivalents) 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 (eluent: petroleum ether / ethyl acetate = 1 / 2, 0.5% triethylamine) to give compound 14a-1 (2.08 g, yield: 59.4%) as a white solid. LCMS (ESI): m / z = 1161.5 [M+H]+.

[0507] Step 15b: Preparation of compound 14a-2: A solution of compound 14a-1 (2.08 g, 1.79 mmol, 1.0 equivalents) 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]+.

[0508] Step 15c: Preparation of compound 14a-3: A mixture of compound 14a-2 (2.08 g, 1.79 mmol, 1.0 equivalent), compound 1a-12 (2.5 g, 5.5 mmol, 3.1 equivalent), N,N-diisopropylethylamine (2.3 g, 17.9 mmol, 10.0 equivalent) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.2 g, 5.7 mmol, 3.2 equivalent) in acetonitrile (20 mL) was stirred at room temperature for 1 hour. The mixture was diluted with water, extracted with ethyl acetate, and the organic phase was washed with saturated brine.The residue was dried over anhydrous sodium sulfate and concentrated. The residue was purified with silica gel (elution: dichloromethane / methanol = 50 / 1 to 20 / 1) to give a white solid, compound 14a-3 (1.46 g, yield: 38.4%). LCMS (ESI): m / z = 1075.4 [M / 2+H]+.

[0509] 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]+.

[0510] 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] +.

[0511] 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 equivalence), N,N-diisopropylethylamine (123.7 mg, 0.96 mmol, 1.5 equivalence), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (322.9 mg, 0.78 mmol, 1.22 equivalence) 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]+.

[0512] Step 15g: Preparation of compound 14a-7: 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,A mixture of 0.1 equivalents of 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 pale purple solid compound 14a-7 (330 mg, crude). LCMS (ESI): m / z = 1224[(M-302) / 2+H]+.

[0513] Step 15h: Preparation of compound 14a-8: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (57.8 mg, 0.14 mmol, 1.2 equivalent), 1-hydroxybenzotriazole (22.7 mg, 0.17 mmol, 1.4 equivalent), and N,N-diisopropylethylamine (62 mg, 0.49 mmol, 4.0 equivalent) were added to an N,N-dimethylformamide solution of compound 14a-7 (330 mg, 0.12 mmol, 1.0 equivalent), 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 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 obtain a light yellow solid product 14a-8 (1.7 g, loading: 23.1 μmol / g).

[0514] Step 15i: Preparation of compound 14a-9-A-ssRNA-1: Compound 14a-8 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 14a-9-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9284.8225Found9284.8267).

[0515] 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, 0.5M), 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, and the mixture was shaken for 5-10 minutes. The crude product of conjugate 79A-1 was obtained by ethanol precipitation, yielding approximately 2.3 mg.The conjugate 79A-1 was then purified by HPLC (MS m / z: [M-H]-: calcd .16611 .8086 Found 16611.1957).

[0516] Step 15k: Preparation of conjugate 79A: Take equal amounts of conjugate 79A-1 and two complementary antisense chains (1167AM25 and 11040AM5) 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 79A.

[0517] Example 16: Preparation of Conjugate 82A (prepared according to Scheme 15a): Specification 71 / 110 page 84 CN 121270626 A

[0518] Specification 72 / 110 page 85 CN 121270626 A

[0519]

[0520] Step 16a: Preparation of Compound 15a-1: Potassium carbonate (5.07 g, 36.75 mmol, 2.5 equivalent) was added to a mixture of compound 10a-5 (6.0 g, 14.7 mmol, 1.5 equivalent) and (2-bromoethyl) tert-butyl carbamate (2.2 g, 9.8 mmol, 1.0 equivalent) 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 added) to give a white solid compound 15a-1 (3.29 g, yield: 60.8%). LCMS (ESI): m / z = 552(M+H)+.

[0521] Step 16b: Preparation of compound 15a-2: Compound 15a-1 (1.7 g, 3.08 mmol, 1.0 equivalent), compound 10a-2 (3.2 g, 9.97 mmol, 3.2 equivalent), acetic acid (185 mg, 3.08 mmol, 1.0 equivalent) and sodium cyanoborohydride (626 mg, 9.97 mmol, 3.2 equivalent) 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, ammonia added) to obtain a white solid compound 15a-2 (1.72 g, yield: 54.0%). LCMS (ESI): m / z = 857.5 (M+H)+.

[0522] 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). LCMS (ESI): m / z = 557.7(M+H)+.

[0523] 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)+.

[0524] 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 a white solid compound 15a-5 (2.0 g, crude). LCMS (ESI): m / z = 1621.5(M+H)+.

[0525] 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 hours. Then N,N-diisopropylethylamine (209 mg, 1.62 mmol, 1.5 equivalences), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (538 mg, 1.30 mmol, 1.2 equivalences), and intermediate M (783 mg, 1.30 mmol, 1.2 equivalences) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine.Concentrate 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-302) / 2+H)+.

[0526] 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, and 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 a white solid compound 15a-7 (375 mg, crude). LCMS (ESI): m / z = 1072((M-302) / 2+H)+.

[0527] 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 the solid phase was dried under reduced pressure. 10 mL of pyridine and 3.4 mL of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to give a yellow solid compound 15a-8 (1.39 g, 37.5 μmol / g).

[0528] Step 16i: Preparation of compound 15a-9-A-ssRNA-1: Compound 15a-8 was synthesized by solid-phase oligonucleotide synthesis, purified by HPLC after ammonolysis.15a-9-A-ssRNA-1 was obtained (MS m / z: [M-H]-: calcd.8981.5515Found8980.7234). Instructions 74 / 110 pages 87 CN 121270626 A

[0529] Step 16j: Preparation of conjugate 82A-1: 53 μL of PBS buffer (pH 8.0, 0.5M), 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 nanomoles, 1 equivalent) and 15a-9-A-ssRNA-1 (200 nanomoles, 1.1 equivalent) and mixed well. 1.6 mg of sodium ascorbate was added, and after nitrogen purging, the mixture was 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 82A-1 was obtained by ethanol precipitation. Then, conjugate 82A-1 was purified by HPLC (MS m / z: [M-H]-: calcd .16308 .5376 Found 16307 .9277).

[0530] Step 16k: Preparation of conjugate 82A: Take equal amounts of conjugate 82A-1 and two complementary antisense chains (1167AM25 and 11040AM5) 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 82A.

[0531] Example 17: Preparation of Conjugate 87A (prepared according to Scheme 16a):

[0532] Specification 75 / 110 pages 88 CN 121270626 A

[0533]

[0534]

[0535] Step 17a: Preparation of Compound 16a-1: Under nitrogen protection, a solution of 2,4-dinitrobenzenesulfonyl chloride (2.2 g, 8.1 mmol, 1.0 equivalent) in tetrahydrofuran (40 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 (10 mL). The mixture was stirred in an ice bath for 1 hour. Specification 76 / 110 pages 89 CN 121270626 A 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]+.

[0536] 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 minutes. 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 hours. The mixture was filtered. The residue was dissolved in 30 mL of dichloromethane and 30 mL of petroleum ether was added. The mixture was stirred at room temperature for 1 hour. The mixture was filtered. The residue was washed with dichloromethane / petroleum ether (1 / 1). The product was dried to give a yellow solid compound 16a-2 (1.42 g, yield: 96.59%). LCMS (ESI): m / z = 916(M + H)+.

[0537] 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 minutes. Ethyl acetate was added for extraction. The organic phase was washed with an 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 product). LCMS (ESI): m / z = 686(M+H)+.

[0538] 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, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, triethylamine added) to give a white solid compound 16a-4 (740 mg, yield: 49.33%). LCMS (ESI): m / z = 858(M+H)+.

[0539] Step 17e: Preparation of compound 16a-5: Compound 16a-4 (740 mg, 0.86 mmol,1.0 equivalent 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)+.

[0540] 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, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 25 / 1 to 10 / 1, 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)+.

[0541] 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 a white solid compound 16a-7 (800 mg, crude). LCMS (ESI): m / z = 1621(M+H)+.

[0542] Step 17h: Preparation of compound 16a-8 (compound 65): Compound 16a-7 (800 mg, 0.476 mmol, 1.0 equivalent), N,N-diisopropylethylamine (184 mg, 1.428 mmol, 3 equivalent) and compound 2b-10 (133 mg, 0.523 mmol, 1.1 equivalent) were added to 10 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then N,N-diisopropylethylamine (184 mg, 1.428 mmol, 3 equivalents), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (236 mg, 0.57 mmol, 1.2 equivalents), and intermediate M (343 mg, 0.57 mmol, 1.2 equivalents) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine.Concentrate 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%).

[0543] 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, 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 obtain a yellow solid compound 16a-9 (396 mg, crude product). LCMS (ESI): m / z = 1072((M-302) / 2+H)+.

[0544] Step 17j: Preparation of compound 16a-10: Compound 16a-9 (396 mg, 0.162 mmol, 1.0 equivalent), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (74 mg, 0.194 mmol, 1.2 equivalent), 1-hydroxybenzotriazole (30 mg, 0.226 mmol, 1.4 equivalent) and N,N-diisopropylethylamine (84 mg, 0.648 mmol, 4.0 equivalent) 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 the solid phase was dried under reduced pressure. 9 mL of pyridine and 3 mL of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to give a yellow solid compound 16a-10 (1.68 g, loading: 45.7 μmol / g).

[0545] Step 17k: Preparation of compound 16a-11-A-ssRNA-1: Compound 16a-10 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to give 16a-11-A-ssRNA-1 (MS m / z: [M-H]-: calcd.8981.5075Found8983.7126).

[0546] Step 17l: Preparation of conjugate 87A-1: 53 μL of PBS buffer (pH 8.0, 0.5 M) was added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nanomoles, 1 equivalent) and 16a-11-A-ssRNA-1 (200 nanomoles, 1.1 equivalent).259 μL of DEPC-treated water, 267 μL of DMF, and 286 μL of premixed CuSO4-THPTA (1:1) solution (conc. 14 nmol / μL) were mixed, and 1.6 mg of sodium ascorbate was added. After nitrogen purging, the mixture was 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 87A-1 was obtained by ethanol precipitation, which was then purified by HPLC to obtain conjugate 87A-1 (MS m / z: [M-H]-: calcd .16308 .4936 Found 16307 .8157).

[0547] 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 in a 95°C water bath for 5-10 minutes, and then cool naturally to room temperature to obtain double-chain conjugate 87A.

[0548] Example 18: Preparation of conjugate 81A (prepared according to scheme 17a): Specification 78 / 110 pages 91 CN 121270626 A

[0549] Specification 79 / 110 pages 92 CN 121270626 A

[0550] Specification 80 / 110 pages 93 CN 121270626 A

[0551]

[0552] Step 18a: Preparation of compound 17a-2: Compound 17a-1 (3.03 g, 10.0 mmol, 1.0 equivalence) was added to an acetonitrile mixture of 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)+.

[0553] 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, ammonia added) to give a white solid compound 17a-3 (1.9 g, crude).Yield: 87.9%. LCMS (ESI): m / z = 375(M+H)+.

[0554] Step 18c: Preparation of compound 17a-5: A mixture of compound 17a-4 (5.0 g, 19.1 mmol, 1.0 equivalent), (2-aminoethyl)carbamate (3.9 g, 20 mmol, 1.05 equivalent), N,N-diisopropylethylamine (4.9 g, 38.2 mmol, 2.0 equivalent) and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (8.3 g, 20 mmol, 1.05 equivalent) in N,N-dimethylformamide (70 mL) was stirred at room temperature for 1 hour. The mixture was poured into water, 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 = 100 / 1 to 50 / 1) to give a white solid compound 17a-5 (7.1 g, yield: 85.5%). LCMS (ESI): m / z = 438 [M+H]+.

[0555] 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. Extracted 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]+.

[0556] 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, sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain a white solid compound 17a-7 (1.9 g, crude product). LCMS (ESI): m / z = 521 [M+H]+.

[0557] Step 18f: Preparation of compound 17a-8: Compound 17a-7 (1.8 g, 3.46 mmol, 1.0 equivalent), (S)-2-amino-5-(benzyloxy)-5-oxovaleric acid (0.8 g, 3.46 mmol, ...A mixture of acetonitrile (30 mL) and N,N-diisopropylethylamine (1.3 g, 10.38 mmol, 3.0 equivalent) 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]+.

[0558] 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 15 mL mixture of 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 a yellow solid compound 17a-9 (1.12 g, yield: 61.8%). LCMS (ESI): m / z = 999.5(M+H)+.

[0559] 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 4M 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 product). LCMS (ESI): m / z = 699(M+H)+.

[0560] 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, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1 to 15 / 1, 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)+.

[0561] 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 obtain a white solid compound 17a-12 (2.31 g, crude product). LCMS (ESI): m / z = 882.7(M / 2+H)+.

[0562] 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 equivalences), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (397 mg, 0.96 mmol, 1.2 equivalences), and intermediate M (578 mg, 0.96 mmol, 1.2 equivalences) were added, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated to dryness under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer / acetonitrile) to give a white solid compound 17a-13 (886 mg, yield: 44.6%). LCMS (ESI): m / z = 1093((M-302) / 2+H)+.

[0563] Step 18l: Preparation of compound 17a-14: Under nitrogen protection, compound 17a-13 (300 mg, 0.121 mmol, 1.0 equivalent), triethylamine (171 mg, 1.69 mmol, 14.0 equivalent), succinic anhydride (85 mg, 0.844 mmol, 7.0 equivalent) and p-dimethylaminopyridine (1.5 mg, 0.0121 mmol, 0.1 equivalent) were added to 2.5 mL of dichloromethane, and the mixture was stirred overnight at room temperature. The mixture was diluted with dichloromethane / acetonitrile and washed with 10% sodium bicarbonate solution and saturated saline solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a white solid compound 17a-14 (269 mg).Yield: 85.9%. LCMS (ESI): m / z = 1143((M-302) / 2+H)+.

[0564] 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 the solid phase was dried under reduced pressure. 7.5 mL of pyridine and 2.5 mL of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to give a yellow solid compound 17a-15 (770 mg, 43.71 μmol / g).

[0565] Step 18n: Preparation of compound 17a-16-A-ssRNA-1: Compound 17a-15 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to give 17a-16-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9123.6655Found9122.7623).

[0566] Step 18o: Preparation of conjugate 81A-1: 53 μL of PBS buffer (pH 8.0, 0.5M), 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. The crude product was then purified by HPLC to obtain conjugate 81A-1 (MS m / z: [M-H]-: calcd .16450 .6516 Found 16450.1231).

[0567] Step 18p: Preparation of conjugate 81A: Equivalent amounts of conjugate 81A-1 and two complementary antisense chains (1167AM25 and 11040AM5) solutions were mixed evenly.Heating in a 95°C water bath for 5-10 minutes, then naturally cooling to room temperature, yields double-chain conjugate 81A.

[0568] Example 19: Preparation of conjugate 74A (prepared according to scheme 18a): Specification 83 / 110 page 96 CN 121270626 A

[0569] Specification 84 / 110 page 97 CN 121270626 A

[0570]

[0571] Step 19a: Preparation of compound 18a-2: Benzyl bromide (13.1 mL, 109.89 mmol, 2.5 equivalence) was added to a mixture of compound 18a-1 (4.0 g, 43.96 mmol, 1.0 equivalence), potassium carbonate (18.2 g, 131.88 mmol, 3.0 equivalence), 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 a white solid compound 18a-2 (9.89 g, yield: 83%). LCMS (ESI): m / z 272 [M+H]+; TLC: Rf 0.5. (Note: The last line appears to be a reference to a product manual and is not translated.)

[0572] 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: Rf 0.5 (petroleum ether: ethyl acetate = 30:1).

[0573] Step 19c: Preparation of compound 18a-4: Compound 18a-3 (3.73 g, 7.46 mmol, 1.0 equivalent) was subjected to hydrochloride-dioxane (4 M, 20 mL, 4 M, 20 mL) of dioxane (4 M, 20 mL, ...The mixture 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 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: Rf 0.5 (dichloromethane:methanol = 20:1).

[0574] 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 ditert-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: Rf 0.5 (petroleum ether: ethyl acetate = 5:1).

[0575] Step 19e: Preparation of compound 18a-6: A hydrogen chloride-dioxane solution (4 M, 5 mL) was added to a mixture of compound 18a-5 (741 mg, 1.33 mmol, 1.0 equivalent) 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 a white solid crude compound 18a-6. LCMS (ESI): m / z 358 [M+H]+; TLC: Rf 0.2 (dichloromethane:methanol = 10:1).

[0576] Step 19f: Preparation of compound 18a-7: At 0°C, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (1.38 g, 3.33 mmol,A mixture of 2.5 equivalences of dichloromethane (85 mL) was added dropwise over 30 minutes to a solution of compound 1a-10 (786 mg, 1.33 mmol, 1.0 equivalence), compound 18a-6 (616 mg, 1.33 mmol, 1.0 equivalence), and N,N-diisopropylethylamine (1.03 g, 7.98 mmol, 6.0 equivalence) 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: Rf 0.3 (ethyl acetate: dichloromethane = 1:1).

[0577] Step 19g: Preparation of compound 18a-8: Compound 18a-7 (0.42 g, 0.46 mmol, 1.0 equivalent) 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 chloromethane (10 mL). Compound 1a-12 (618 mg, 1.38 mmol, 3.0 equivalence) was added, along with 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). 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: Rf 0.5 (dichloromethane:methanol = 10:1).

[0578] 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 equivalent) and trifluoroacetic acid (47 mg, 0.409 mmol, 1.0 equivalent) 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 a pale yellow solid compound 18a-9 (600 mg,Crude product). LCMS (ESI): m / z 1721.5 [M+H]+; TLC: Rf 0.3 (dichloromethane:methanol = 10:1).

[0579] 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 equivalent) was added to a mixture of compound 18a-9 (600 mg, 0.33 mmol, 1.0 equivalent), compound 4a-6 (265 mg, 0.33 mmol, 1.0 equivalent), N,N-diisopropylethylamine (255 mg, 1.98 mmol, 6.0 equivalent), and N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature for 0.5 hours. The mixture was purified by preparative liquid chromatography (A: phosphate buffer, pH 7; B: acetonitrile, acetonitrile concentration 30%–90% over 30 minutes) to give a white solid compound 18a-10 (180 mg, yield: 22%). LCMS (ESI): m / z 1102.6 [(M-302) / 2+H]+; TLC: Rf 0.5 (dichloromethane:methanol = 10:1).

[0580] Step 19j: Preparation of compound 18a-11: Succinic anhydride (43 mg, 0.43 mmol, 6.0 equivalent) was added to a dichloromethane (2 mL) solution of compound 18a-10 (180 mg, 0.072 mmol, 1.0 equivalent), triethylamine (87 mg, 0.862 mmol, 12.0 equivalent), and 4-dimethylaminopyridine (4.4 mg, 0.036 mmol, 0.5 equivalent). 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).

[0581] Step 19k: Preparation of compound 18a-12: 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 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). The mixture was stirred at room temperature for 5 minutes. Add CPG-NH2 (0.167 mmol / g, 775 mg),0.13 mmol, 2.2 equivalence). The mixture was stirred overnight at room temperature. The mixture was filtered. The solid was washed with dichloromethane (8 mL × 5). Pyridine (6 mL) and acetic anhydride (2 mL) were added. The mixture was stirred at room temperature for 3 hours. The mixture was filtered. The solid was washed with dichloromethane (8 mL × 5) and dried under vacuum to give a white solid compound 18a-12 (785 mg, 21.5 μmol / g, yield: 29%).

[0582] Step 191: Preparation of compound 18a-13-A-ssRNA-1: Compound 18a-12 was synthesized by solid-phase oligonucleotide synthesis, ammonolyzed and purified by HPLC to give 18a-13-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9141.5925Found9140.7005).

[0583] Step 19m: Preparation of conjugate 74A-1: 53 μL of PBS buffer (pH 8.0, 1 equivalent) was added sequentially to a mixture of compound 8a-9-A-ssRNA-2 (182 nanomoles, 1 equivalent) and 18a-13-A-ssRNA-1 (200 nanomoles, 1.1 equivalent), 259 μL of DEPC water, 267 μL of DMF, and 286 μL of premixed CuSO4-THPTA (1:1) solution (conc. 14 nanomoles / 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. After shaking for 5-10 minutes, about 2.3 mg of crude conjugate 74A-1 was obtained by ethanol precipitation. Then, conjugate 74A-1 was purified by HPLC (MS m / z: [M-H]-: calcd .16468 .5786 Found 16468.2769).

[0584] Step 19n: Preparation of conjugate 74A: Take equal amounts of conjugate 74A-1 and two complementary antisense chains (1167AM25 and 11040AM5) 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 74A.

[0585] Example 20: Preparation of conjugate 44A (prepared according to scheme 19a):

[0586] Specification 88 / 110 pages 101 CN 121270626 A

[0587]

[0588] Step 20a: Preparation of compound 19a-2: At -20°C, compound 19a-1 (20 g, 66.00 mmol, 1.0 equivalent) and N-methylmorpholine (6.68 g, 66.00 mmol, 1.0 equivalent) were added.Isobutyl chloroformate (9.02 g, 66.00 mmol, 1.0 equivalent) was added dropwise to a mixture of 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]+.

[0589] Step 20b: Preparation of compound 19a-3: Under nitrogen protection, at 0°C, elemental iodine (26.26 g, 103.45 mmol, 1.5 equivalent) was added to a mixture of triphenylphosphine (27.13 g, 103.45 mmol, 1.5 equivalent), imidazole (7.04 g, 103.45 mmol, 1.5 equivalent), 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 solution 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]+.

[0590] 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, diluted with water, and extracted with ethyl acetate. The combined specification, pages 89 / 110, 102 CN 121270626 A, describes the organic layer being 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]+.

[0591] Step 20d: Preparation of compound 19a-5: At 0°C, compound 19a-4 (14.14 g, 21.75 mmol, 1.0 equivalent) and N,N-diisopropylethylamine (560 mg, 4.35 mmol, ...1-Chloroethyl chloroformate (4.67 g, 32.63 mmol, 1.0 equivalent) was added dropwise to a mixture of acetonitrile (110 mL) at 0.2 equivalents. 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]+.

[0592] 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 hours. 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]+.

[0593] Step 20f: Preparation of compound 19a-7: A mixture of compound 19a-6 (8.31 g, 11.37 mmol, 1.0 equivalent) in hydrogen chloride-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 mixed solvent 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. Tetrahydrofuran was removed under reduced pressure. The residue was diluted with water and then washed with dichloromethane. The pH was adjusted to 3 with a 2 mol / L dilute hydrochloric acid solution and then extracted with ethyl acetate. The combined organic 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]+.

[0594] Step 20 g: Preparation of compound 19a-8: At 0 °C, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (3.76 g,A mixture of 9.08 mmol (2.2 equivalences) of dichloromethane (500 mL) was mixed with a solution of compound 1a-4 (1.28 g, 4.13 mmol, 1.0 equivalences), compound 19a-7 (2.55 g, 4.13 mmol, 1.0 equivalences), and N,N-diisopropylethylamine (2.66 g, 20.65 mmol, 5.0 equivalences) in 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]+.

[0595] Step 20h: Preparation of compound 19a-9: A solution of compound 19a-8 (1.09 g, 1.33 mmol, 1.0 equivalent) in hydrogen chloride-dioxane (10 mL) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step (crude product) without purification. LCMS (ESI): m / z = 520 [M+H]+.

[0596] 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 equivalent), compound 1a-12 (1.78 g, 3.99 mmol, 3.0 equivalent), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.62 g, 4.25 mmol, 3.2 equivalent) 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 with 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]+.

[0597] 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% mass fraction), 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.The filtrate was concentrated. The residue was used directly for the next step without further purification (2.18 g, crude). LCMS (ESI): m / z = 838 [M / 2 + H] +.

[0598] 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 separated and purified by HPLC (column: UniSil 10-120C18, 30×250mm) to give a white solid product 19a-12 (470 g, yield: 34.31%). LCMS (ESI): m / z = 1079[(M-302) / 2+H]+.

[0599] Step 201: 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 a 10% sodium bicarbonate solution and extracted with dichloromethane. The organic phase was then washed with a 10% sodium bicarbonate solution after the addition of acetonitrile. The organic phase was concentrated to give 19a-13 as a white solid (457 mg, crude product). LCMS (ESI): m / z = 1129[(M-302) / 2+H]+.

[0600] Step 20m: Preparation of compound 19a-14: 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 to an N,N-dimethylformamide solution (8 mL) of compound 19a-13 (200 mg, 0.078 mmol, 1.0 equivalence), 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 sequentially 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 stirred at room temperature for 3 hours. The mixture was then filtered.The filter cake was washed sequentially with dichloromethane, acetonitrile, and dichloromethane, and dried under vacuum for 1 hour to obtain a light yellow solid product 19a-14 (910 mg, loading rate: 22.27 μmol / g).

[0601] Step 20n: Preparation of compound 19a-15-A-ssRNA-1: Compound 19a-14 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to obtain 19a-15-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9095.6115Found9093.7784).

[0602] Step 20o: Preparation of conjugate 44A-1: 53 μL of PBS buffer (pH 8.0, 0.5M), 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. Then, conjugate 44A-1 was purified by HPLC (MS m / z: [M-H]-: calcd .16422 .5536 Found 16421 .2100).

[0603] Step 20p: Preparation of conjugate 44A: Take equal amounts of conjugate 44A-1 and two complementary antisense chain solutions (1167AM25 and 11040AM5) from page 91 / 110 of the specification, 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 44A.

[0604] Example 21: Preparation of conjugate 80A (prepared according to scheme 20a):

[0605] Specification 92 / 110 pages 105 CN 121270626 A

[0606]

[0607] 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.The product 20a-1 (6.4 g, crude) was dried and concentrated with anhydrous sodium sulfate. LCMS (ESI): m / z = 390 [M+H]+. Specification 93 / 110 pages 106 CN 121270626 A

[0608] 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 hours. Water was added to the mixture, and 2 mol of dilute hydrochloric acid was added to adjust the pH to 5. The aqueous phase was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried and concentrated with anhydrous sodium sulfate to obtain compound 20a-2 (5.1 g, crude). LCMS (ESI): m / z = 362 [M+H]+.

[0609] 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 with 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]+.

[0610] Step 21d: Preparation of compound 20a-4: Compound 20a-2 (4.3 g, 11.82 mmol, 1.0 equivalent), compound 20a-3 (6.1 g, 11.82 mmol, 1.0 equivalent), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.4 g, 14.18 mmol, 1.2 equivalent), and N,N-diisopropylethylamine (3.1 g, 23.04 mmol, 2.0 equivalent) were added to N,N-dimethylformamide (50 mL) and stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was stirred with dichloromethane / petroleum ether = 1:1. The mixture was filtered to give a yellow solid compound 20a-4 (10 g, crude). LCMS (ESI): m / z = 861 [M+H]+.

[0611] 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, crude).Yield: 72.5%. LCMS (ESI): m / z = 638 [M+H]+.

[0612] Step 21f: Preparation of compound 20a-6: At 0°C, Desmartin reagent (4.7 g, 11.17 mmol, 1.5 equivalent) was added to a tetrahydrofuran (30 mL) solution of (tert-butoxycarbonyl)-L-homoserine benzyl ester (2.3 g, 7.44 mmol, 1.0 equivalent). 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 with anhydrous sodium sulfate and concentrated to give a yellow oily product 20a-6 (3.5 g, crude product). LCMS (ESI): m / z = 308 [M+H]+.

[0613] 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 minutes. The mixture was cooled to 0°C and sodium cyanoborohydride was added. The mixture was stirred at room temperature for 1.5 hours. The mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated sodium carbonate solution and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (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]+.

[0614] 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]+.

[0615] Step 21i: Preparation of compound 20a-9: Compound 20a-8 (1.5 g, 1.36 mmol,Add 1.0 equivalent to the sample (page 94 / 110, CN 121270626 A) to 20 mL of 4M hydrogen chloride-dioxane solution. Stir the mixture at room temperature for 1 hour. Concentrate the mixture under reduced pressure to give a yellow solid product 20a-9 (1.1 g, crude product). LCMS (ESI): m / z = 700 [M+H]+.

[0616] Step 21j: Preparation of compound 20a-10: Compound 20a-9 (1.1 g, 1.27 mmol, 1.0 equivalent), compound 1a-12 (2.3 g, 5.21 mmol, 4.1 equivalent), N,N-diisopropylethylamine (2 g, 15.24 mmol, 12.0 equivalent) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2 g, 5.33 mmol, 4.2 equivalent) were added to 15 mL of acetonitrile, and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 10 / 1, 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]+.

[0617] 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 a white solid compound 20a-11 (890 mg, crude). LCMS (ESI): m / z = 1098 [M / 2 + H] +.

[0618] Step 211: Preparation of compound 20a-12 (compound 63): Compound 20a-11 (760 mg, 0.346 mmol, 1.0 equivalent), N,N-diisopropylethylamine (134 mg, 1.038 mmol, 3.0 equivalent) and compound 2b-10 (97 mg, 0.381 mmol, 1.1 equivalent) were added to 10 mL of acetonitrile, and the mixture was stirred at room temperature for 1.5 hours. Then N,N-diisopropylethylamine (134 mg, 1.038 mmol, 3.0 equivalents), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (171 mg, 0.415 mmol, 1.2 equivalents) and intermediate M (249 mg, 0.415 mmol, 1.2 equivalents) were added.The mixture was stirred at room temperature for 1 hour. The solvent was removed from the mixture under reduced pressure. The residue was purified by preparative liquid chromatography (eluent: pH 7.0 potassium dihydrogen phosphate-potassium hydroxide buffer / acetonitrile) to give a yellow solid compound 20a-12 (272 mg, yield: 27.2%). LCMS (ESI): m / z = 1306[(M-302) / 2+H]+.

[0619] 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, and the mixture was stirred at room temperature overnight. 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 20a-13 (167 mg, crude). LCMS (ESI): m / z = 1359 [(M-302) / 2+H]+.

[0620] 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 the solid phase was dried under reduced pressure. 6 mL of pyridine and 2 mL of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to give a yellow solid compound 20a-14 (532 mg, loading: 28.6 μmol / g).

[0621] Step 21o: Preparation of compound 20a-15-A-ssRNA-1: Compound 20a-14 was synthesized by solid-phase oligonucleotide synthesis, and purified by HPLC after ammonolysis to give 20a-15-A-ssRNA-1 (MS m / z: [M-H]-: calcd.9428.0085Found9427.849).

[0622] Step 21p: Preparation of conjugate 80A-1: In compound 8a-9-A-ssRNA-2 (182 nanomolar),Add 53 μL of PBS buffer (pH 8.0, CN 121270626 A 0.5M) to a mixture of 1 equivalent of 20a-15-A-ssRNA-1 (200 nanomoles, 1.1 equivalents), 259 μL of DEPC water, 267 μL of DMF, and 286 μL of premixed CuSO4-THPTA (1:1) solution (conc. 14 nanomoles / μL). After mixing, add 1.6 mg of sodium ascorbate, purge with nitrogen, and react 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 80A-1 was obtained by ethanol precipitation. Then, conjugate 80A-1 was purified by HPLC (MS m / z: [M-H]-: calcd .16754 .9946 Found 16753 .2000).

[0623] Step 21q: Preparation of conjugate 80A: Take equal amounts of conjugate 80A-1 and two complementary antisense chains (1167AM25 and 11040AM5) 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 80A.

[0624] Example 22: Preparation of conjugate 88A (prepared according to scheme 21a):

[0625] Specification 96 / 110 pages 109 CN 121270626 A

[0626]

[0627] Step 22a: Preparation of compound 21a-1: 40% aqueous methylamine (1.5 g, 19.23 mmol, 1.1 equivalents) was added to (S)-4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (7.7 g). The mixture was stirred at room temperature for 1 hour in a mixture of N,N-diisopropylethylamine (3.39 g, 26.28 mmol, 1.5 equivalent), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (7.95 g, 19.23 mmol, 1.1 equivalent), and 85 mL of dimethylformamide. The mixture was poured into water and filtered to collect the solid. 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)+.

[0628] 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 minutes. The mixture was concentrated under reduced pressure.The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1 to 10 / 1) to give a yellow solid compound 21a-2 (6.2 g, crude). LCMS (ESI): m / z = 232(M+H)+.

[0629] 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 a yellow solid compound 21a-3 (1.1 g, yield: 15.6%). LCMS (ESI): m / z = 462(M+H)+.

[0630] 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 (30 mL) 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). The mixture was stirred at room temperature for 1 hour. The reaction solution was filtered. The filter cake was dissolved in dichloromethane, petroleum ether was added, and 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)+.

[0631] 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, the organic phase was concentrated under reduced pressure, and the residue was dried under reduced pressure to give a yellow solid compound 21a-5 (1.07 g, crude product). LCMS (ESI): m / z = 375(M+H)+.

[0632] Step 22f: Preparation of compound 21a-6: Compound 21a-5 (1.07 g, 2.21 mmol, 1.0 equivalent), compound 2b-5 (746 mg, 2.21 mmol, 1.0 equivalent), N,N-diisopropylethylamine (570 mg, 4.42 mmol, ...2.0 equivalents) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (924 mg, 2.43 mmol, 1.1 equivalents) were added to 10 mL of N,N-dimethylformamide, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added, and the organic phase was washed with water and saturated brine, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 20 / 1, with the addition of triethylamine) to give a yellow solid compound 21a-6 (940 mg, yield: 61.4%). LCMS Specification 97 / 110 pages 110 CN 121270626 A (ESI): m / z=694(M+H)+.

[0633] Step 22g: Preparation of compound 21a-7: Compound 21a-6 (940 mg, 1.35 mmol, 1.0 equivalent) was added to 10 mL of 4M hydrogen chloride-dioxane solution. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain white solid compound 21a-7 (1.01 g, crude product). LCMS (ESI): m / z = 394(M+H)+.

[0634] 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, and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 80 / 1 to 15 / 1, 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)+.

[0635] 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 a white solid compound 21a-9 (2.02 g, crude product). LCMS (ESI): m / z = 1592.4(M+H)+.

[0636] Step 22j: Preparation of compound 21a-10 (compound 66): intermediate M (709 mg, 1.18 mmol,A solution of acetonitrile (1.1 equivalents) was added to a mixture of compound 21a-9 (2.02 g, 1.07 mmol, 1.0 equivalents), N,N-diisopropylethylamine (276 mg, 2.14 mmol, 2.0 equivalents), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (488 mg, 1.18 mmol, 1.1 equivalents), 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)+.

[0637] Step 22k: Preparation of compound 21a-11: Under nitrogen protection, compound 21a-10 (500 mg, 0.230 mmol, 1.0 equivalent), triethylamine (326 mg, 3.22 mmol, 14.0 equivalent), succinic anhydride (161 mg, 1.61 mmol, 7.0 equivalent) and p-dimethylaminopyridine (3 mg, 0.023 mmol, 0.1 equivalent) 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 obtain a white solid compound 21a-11 (536 mg, crude product). LCMS (ESI): m / z = 1975.7((M-302)+H)+.

[0638] Step 221: 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 the solid phase was dried under reduced pressure. 15 mL of pyridine and 5 mL of acetic anhydride were added, and the mixture was shaken for 3 hours. The mixture was filtered, the solid phase was washed with dichloromethane and acetonitrile, and the solid phase was dried under reduced pressure to give a yellow solid compound 21a-12.

[0639] Step 21m: Preparation of conjugate 88A-1: Compound 21a-12 was synthesized by solid-phase oligonucleotide synthesis, purified by HPLC after ammonolysis.88A-1 was obtained (MS m / z: [M-H]-: calcd. 8195.9620 Found 8194.6426).

[0640] 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-stranded conjugate 88A. Specification 98 / 110 pages 111 CN 121270626 A

[0641] The oligonucleotide sequence information of each conjugate synthesized in the above examples is shown in Tables 1-3.

[0642] Table 1 Sequence Composition Information of Each Conjugate

[0643]

[0644]

[0645] Table 2 Summary Table of Right-Side Stroke Mass Spectrometry Information of Each Conjugate (Instruction Manual 99 / 110, Page 112, CN 121270626 A)

[0646] (Instruction Manual 100 / 110, Page 113, CN 121270626 A)

[0647]

[0648] Table 3 Summary Table of Sequences Used for Each Conjugate (Instruction Manual 101 / 110, Page 114, CN 121270626 A)

[0649]

[0650] The unmodified sequences corresponding to Table 3 are as follows:

[0651] (Instruction Manual 102 / 110, Page 115, CN 121270626 A)

[0652]

[0653] Example 23: Testing the effect of siRNA-conjugates on AGT and PCSK9 expression in primary hepatocytes of hAGT / hPCSK9 mice

[0654] 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. Ultrapurified recombinant AAV8-hAGT and AAV8-hPCSK9 viruses were prepared by ordering from a vector manufacturer. After mixing the AAV8-hAGT and AAV8-hPCSK9 viruses, the mixture was diluted with PBS to a viral titer of 7.5*10¹¹ / mL and injected intravenously into 6-8 week old female mice (C57BL / 6), 0.2 mL per mouse, i.e., 1.5*10¹¹ viral titer per mouse. Transgenic mice stably expressing hAGT / hPCSK9 were obtained after 14 days.

[0655] The dual-humanized AGT / PCSK9 mice were purchased from Cyagen Biosciences.

[0656] Extraction of primary mouse hepatocytes: Mouse hepatocytes were extracted by collagenase digestion via inferior vena cava perfusion. 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 a 96-well plate and incubated at room temperature for 10 minutes (INTERFERin is not required for the free-up method). Then, 125 μL of complete culture medium containing mouse primary hepatocytes was added to each well

[0657] . 96-well plate RNA extraction and reverse transcription: mRNA was extracted from cells in the 96-well plate using Oligo d(T)25 Magnetic Beads reagent (NEB). The culture medium in the 96-well plate was aspirated, and the cells were washed once with DPBS. 100 μL of cell lysis buffer was added to each well, followed by 20 μL of beads. The plate was shaken on a shaker, and the 96-well plate was placed on a magnetic separator. The lysis buffer in the wells was aspirated, and 100 μL of lysis buffer was added to each well. Washing buffer A was used to lift the beads with 100 μl of washing buffer B and transfer them to a new 96-well plate. The plate was placed on a magnetic separator and the washing buffer B was removed. Then, 100 μl of low-salt buffer was used to lift the beads and transfer them to a new 96-well PCR plate. The 96-well PCR plate was placed on a magnetic separator and the low-salt buffer was removed. 10 μL of elution buffer was added to each well to lift the beads, and the plate was incubated at 50°C for 2 minutes to elute the mRNA from the beads. The reverse transcription system was prepared using StarScript Pro one-tube genomic de-generated reverse transcription premix (Genstar). 5 μL of the premixed mRNA solution was aliquoted into each well of the 96-well PCR plate, mixed, briefly centrifuged, and sealed with sealing film. The plate was incubated at 37°C for 3 minutes, 50°C for 50 minutes, and then at 85°C for 2 minutes. The plate was then cooled to 4°C to complete the reverse transcription.

[0658] Real-time quantitative PCR: After reverse transcription, place the 96-well plate on a magnetic separator until all magnetic beads are adsorbed to the bottom. Remove the reverse transcription reagent, add the prepared qPCR system to the 96-well PCR plate, seal the plate with a sealing membrane, and perform PCR on the StepOnePlus real-time PCR system (applied biosystems).

[0659] Data were analyzed using the ΔΔCt method.Cells transfected with a 1 nM negative control sequence were used for testing and standardization.

[0660] The negative control AD-1955 sequence is:

[0661] CUUACGCUGAGUACUUCGAdTdT (SEQ ID NO:13)

[0662] UCGAAGUACUCAGCGUAAGdTdT (SEQ ID NO:14).

[0663] The primer sequences for detecting AGT are:

[0664]

[0665] The primer sequences for detecting PCSK9 are:

[0666]

[0667] The experimental results are shown in Tables 4-7.

[0668] Table 4 Results of AGT single-target siRNA inhibition experiment in primary hepatocytes of dual-humanized AGT / PCSK9 mice

[0669] siRNA-conjugate compound number IC50 value (nM) Conjugate 1A 31 0.05995 Conjugate 1B 31 0.03662

[0670] Conjugate 1A and conjugate 1B used compound 31 to deliver siRNA targeting AGT. The results in Table 4 show that compound 31 can efficiently deliver siRNA in vitro.

[0671] Table 5 Results of AGT single-target siRNA under different concentrations in primary hepatocytes of AAV8-hAGT / hPCSK9 mice via free uptake

[0672]

[0673] The results in Table 5 show that compounds 31 and 66 can efficiently deliver siRNA in vitro.

[0674] Table 6 Results of AGT / PCSK9 dual-target siRNA inhibition experiment in dual-humanized AGT / PCSK9 mouse primary hepatocytes

[0675]

[0676] As shown in Table 6, when the same sequence combination was delivered in vitro in dual-humanized AGT / PCSK9 mouse primary hepatocytes, both conjugate 2A and conjugate 3A could simultaneously knock down both AGT and PCSK9 targets. Conjugate 2A (Instructions for Use, pages 104 / 110, CN 121270626 A) showed better knockdown effect than conjugate 3A. This indicates that compound 54 was slightly better than compound 55 in delivering dual-target siRNA.

[0677] Table 7 Results of AGT / PCSK9 dual-target siRNA inhibition experiment in AAV8-hAGT / hPCSK9 mouse primary hepatocytes

[0678]

[0679] As can be seen from the experimental results in Table 7, when delivering the same sequence combination in vitro, different conjugates can inhibit both AGT and PCSK9 targets simultaneously with high activity. Among them, conjugates 2A, 4A, and 9A have the highest activity, indicating that compounds 54 and 53 have better delivery effects on dual-target siRNA than compound 51. At the same time, comparing the data of conjugates 2A, 7A, 8A, and 9A, it was found that connecting the vector compound and siRNA with different linker strands also affects the delivery effect.The enhancement of siRNA activity is more pronounced when Q13 and Q20 are linked.

[0680] Example 24: Testing the effect of different siRNA-conjugates on AGT expression in AAV8-hAGT mice (1 mg / kg)

[0681] The effect of 1167.25-19 delivered by different compounds on hAGT expression in the liver was evaluated in mice expressing AAV8-hAGT.

[0682] 1. Construction of transgenic mice of hAGT

[0683] AAV8-hAGT virus was prepared into a viral solution with a viral titer of 5*10¹¹ / mL using PBS and injected intravenously into 6-8 week old female mice (C57BL / 6), 0.2 mL per mouse, that is, each mouse was injected with a viral titer of 1*10¹¹. Transgenic mice stably expressing hAGT were obtained after 14 days.

[0684] 2. Administration

[0685] 14 days after the mice were injected with the virus, the mice were divided into groups of 5 mice each. The siRNA-conjugate was dissolved in physiological saline and administered subcutaneously at a dose of 1 mg / kg.

[0686] 3. ELISA detection

[0687] Fourteen days after the mice were injected with the virus, blood was collected by tail amputation, allowed to stand at room temperature for 30 minutes, centrifuged at 1000×g for 10 minutes, and the supernatant serum was collected, aliquoted and frozen at -80℃. The serum sample was diluted 1000 times and the expression of hAGT in mouse serum was detected by Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Lianke Biotechnology, catalog number: EK1202–96). Based on the hAGT expression level, the mice were divided into groups of 5 mice each. The siRNA-conjugate was dissolved in PBS and the concentration was adjusted to 0.2 mg / kg. The mice were subcutaneously injected with the siRNA solution at a dose of 1 mg / kg.

[0688] 17 and 45 days after siRNA injection, a small amount of blood was collected by tail cutting, 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.

[0689] Among them, the reference compound 1A is a conjugate of L96* conjugate 1167 .25-19, which was obtained by conventional solid-phase synthesis method. *: L96 structure (compound 117 (L96-DMTr protected) in US8106022B2): Specification 105 / 110 pages 118 CN 121270626 A

[0690]

[0691] Table 8 Results of AGT-targeted modified siRNA inhibition experiment in AAV8-hAGT mice

[0692]

[0693] The experimental results are shown in Table 8: In the in vivo experiment, the delivery effect of conjugate 1A is better than that of L96.This indicates that compound 31 is superior to L96 in delivering siRNA.

[0694] Example 25: Testing the effect of conjugate 2A on the expression of AGT and PCSK9 in AAV8-hAGT / hPCSK9 mice

[0695] The effect of conjugate 2A on the expression of hAGT / hPCSK9 was tested at a dose of 180 nmol / kg in mice expressing AAV8-hAGT / hPCSK9. The expression levels of hAGT and hPCSK9 in mouse serum were detected 14, 30 and 44 days after 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). The experimental methods were the same as in Examples 23 and 24.

[0696] Table 9 Results of AGT / PCSK9 dual-targeting siRNA inhibition experiment in AAV8-hAGT / hPCSK9 mice at a dose of 180 nmol / kg

[0697]

[0698] As shown in Table 9, the experimental results show that the conjugate 2A targeting AGT and PCSK9 linked by compound 54 has high activity in vivo and can significantly inhibit the expression of hAGT and hPCSK9 in the serum of AAV8-hAGT / hPCSK9 mice. By day 44, the remaining amount of hPCSK9 was only 21.7% of that before administration, and the remaining amount of hAGT was only 10.8% of that before administration, indicating that the siRNA linked by compound 54 can persistently inhibit the expression of the two targets in vivo.

[0699] Example 26: Testing the effect of different siRNA-conjugates on the expression of AGT and PCSK9 in AAV8-hAGT / hPCSK9 mice

[0700] The effect of different conjugates on the expression of AGT and PCSK9 was tested in mice expressing AAV8-hAGT / hPCSK9. Conjugate 2B was a conjugate that simultaneously delivered two siRNAs targeting AGT, and the others were conjugates that simultaneously delivered siRNAs targeting AGT and PCSK9. The expression of hAGT and hPCSK9 in the serum of mice after drug administration was detected by Human Angiotensinogen / AGT / SerpinA8 ELISA Kit (Link Biotech, catalog number: EK1202–96) and Human PCSK9 ELISA Kit (proteintech, catalog number: KE00278).The experimental methods were as described in Examples 23 and 24.

[0701] 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

[0702]

[0703] As shown in Table 10: At a dose of 60 nmol / kg, all conjugates showed good in vivo knockdown effects on both hAGT and hPCSK9 targets. Meanwhile, conjugate 2B differs from other dual-target conjugates in that it utilizes a dual-target structure to deliver two identical AGT-targeting sequences. It is evident that simultaneously delivering two AGT-targeting sequences results in a superior knockdown effect on the AGT target. This indicates that using conjugate 2B not only delivers siRNA targeting two targets but also further enhances efficacy by simultaneously delivering two identical target sequences.

[0704] Table 11 Results of AGT / PCSK9 dual-targeting siRNA inhibition experiment in AAV8-hAGT / hPCSK9 mice at a dose of 180 nmol / kg

[0705]

[0706] As shown in Table 11, the activities of the two conjugates 9A and 10A were very good. The inhibition rates of both targets were above 80-90% on day 40.

[0707] Example 27: Testing the effect of siRNA-conjugates on the expression of AGT and PCSK9 in primary hepatocytes of AAV8-hAGT / hPCSK9 mice

[0708] The delivery effect of dual-targeting siRNA-conjugates with different structures was tested in vitro according to the method of Example 23. Among them, 1000PM-L96 and 11000PM-L96 are alnylam's investigational drug Zilebesiran, which targets AGT in a phase II clinical trial, and Inclisiran, a marketed drug targeting PCSK9, respectively, and were used for positive drug comparison.

[0709] Table 12 Results of AGT / PCSK9 dual-target siRNA inhibition experiment in AAV8-hAGT / hPCSK9 mouse primary hepatocytes at different concentrations and delivery methods. (Instructions for use, pages 107 / 110, CN 121270626 A)

[0710]

[0711]

[0712] As shown in Table 12, each conjugate can efficiently deliver siRNA, and the inhibitory effects on both AGT and PCSK9 targets are far better than those of Zilebesiran mixed with Inclisiran.

[0713] Example 28: Stability and in vivo activity of conjugate 9A

[0714] This example refers to compound AM26 with good activity in patent US11 053495 B2, and compound A19a with good in vivo activity in patent WO2024061157A1.Conjugates DT03000 and DT03098 were synthesized separately, and their stability and in vivo activity were compared with conjugate 9A of the present 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.

[0715] The structures of conjugates DT03000 and DT03098 are as follows: Specification 108 / 110 pages 121 CN 121270626 A

[0716]

[0717]

[0718] The stability of the three conjugates was tested in mouse serum. The experimental method is as follows:

[0719] The test conjugates were precisely diluted with DEPC water to a working solution with a concentration of 10 μM.

[0720] Preparation of 3h samples: Take 12 μL of 10 μM test conjugate and mix it with 108 μL of blank mouse serum. Vortex for 60 s to prepare a mixed solution of test conjugate and mouse serum (hereinafter referred to as the mixed solution). Take 50 μL of the mixed solution into a centrifuge tube, with 2 parallel samples, and incubate at 37℃ for 3 h.

[0721] Preparation of 0h samples: Take 45 μL of blank mouse serum into a centrifuge tube, with 2 parallel samples, and incubate at 37℃ for 3 h. After incubation, add 5 μL of 10 μM test conjugate working solution and vortex for 60 s.

[0722] 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.

[0723] All samples were treated with solid phase extraction, concentrated by nitrogen blowing, and then 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%.

[0724] Table 13 Results of double-stranded stability of AGT / PCSK9 dual-targeted modified siRNA in mouse serum after 3 h of incubation

[0725] siRNA-conjugate antisense strand 1 remaining percentage (%) Antisense strand 2 remaining percentage (%) Conjugate 9A 121.02 118.02 DT03098 101.90 98.04 DT03000 3.89 1.47

[0726] It is clear from the data in Table 13 that after 3 h of incubation in mouse serum, conjugate 9A and DT03098 have good stability and are basically not degraded; while the remaining percentage of the two antisense strands of compound DT03000 is 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, resulting in poor stability.

[0727] Based on the stability test results,Conjugate 9A and conjugate DT03098 were selected for in vivo activity testing. The experimental methods were as described in Examples 23 and 24.

[0728] Table 14 Results of AGT / PCSK9 dual-target siRNA inhibition experiment in AAV8-hAGT / hPCSK9 mice at a dose of 60 nmol / kg. (Instructions for use, pages 109 / 110, CN 121270626 A)

[0729]

[0730] As can be seen from the data in Table 14, when delivering the same two sequences, the in vivo activity of conjugate 9A was significantly better than that of DT03098 at both target sites, especially the inhibitory activity against the PCSK9 target was significantly better than that of DT03098. This indicates that conjugate 9A of the present invention has a better siRNA delivery effect than DT03098.

[0731] Example 29: Testing the effect of conjugate 9A on the expression of hAGT and hPCSK9 in AAV8-hAGT / hPCSK9 mice

[0732] In mice expressing AAV8-hAGT / hPCSK9, the effect of conjugate 9A on the expression of hAGT and hPCSK9 was tested at a dose of 60 nmol / kg. 1000PM-L96 and 11000PM-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), with experimental methods as described in Examples 23 and 24.

[0733] ​​The experimental results are shown in Figures 1 and 2.

[0734] As can be seen from the figures, at a drug dosage of 60 nmol / kg, conjugate 9A showed good in vivo knockdown effects on both AGT and PCSK9 targets, and was far superior to the efficacy of Zilebesiran mixed with Inclisiran. Furthermore, the duration of combined administration of the two positive drugs showed that the inhibition rate of 1000PM was still around 50% on day 56, while the inhibition rate of 11000PM on PCSK9 had already decreased to around 50% on day 27, indicating that the duration of the inhibitory effect of the two sequences on the targets was inconsistent. The inhibitory levels of the conjugate 9A of the present invention at the two targets show a basically consistent trend over time, thus providing greater control over the timing and frequency of administration in clinical practice.

[0735] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However,As long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification.

[0736] The above-described embodiments only illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those skilled in the art, several modifications and improvements can be made 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. Instruction Manual 110 / 110 Page 123 CN 121270626 A Figure 1 Figure 2 Instruction Manual Drawings 1 / 1 Page 124 CN 121270626 A ABSTRACT Provided are a compound for targeted delivery of an oligonucleotide drug, and a conjugate formed by binding the compound to the oligonucleotide drug, for use in delivering one or more different oligonucleotide drugs. The compound has a structure as shown in formula (I), formula (II), or formula (III). (I) Y1 Y2 L3 L5 L6 W1 L7 W2 Targeting binding portion T L4 (III) (III)

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that, The compound has a structure shown in formula (II) or formula (III): In the structure, the targeting binding moiety T is a group containing a ligand capable of specifically binding to a cell membrane protein receptor with endocytosis selected from the group consisting of sialoglycoprotein receptor; D, E are each independently selected from: or their chiral isomers; Each r is independently selected from 0, 1, 2, 3; X1, X'1 are independently selected from a single bond, C1-C6 alkylene, -C(=O)-C1-C6 alkylene, -C(=O)-NH-C1-C6 alkylene, -NH-C(=O)-C1-C6 alkylene, -NH-C1-C6 alkylene, or chiral isomers thereof; K1, K3 are independently selected from the group consisting of: -(CH2) n15 -R 22 -, -(CH2) n15 -R 21 -(CH2) n16 -R 22 -, -(CH2) n15 -O-R 21 -CH2O-(CH2) n16 -R 22 -, -(CH2) n15 -R 21 -CH2CH2O-(CH2) n16 -R 22 -, -(CH2) n15 -R 21 -(CH2CH2O)2-, -(CH2) n15 -R 21 -(CH2) n16 -; R 21 is selected from: -C(=O)NH-, -C(=O)-, or nothing; R 22 is selected from: -C(=O)NH-, -NH-, -C(=O)-; Each n15 is independently selected from 1, 2, 3, 4, 5, 6; Each n16 is independently selected from 0, 1, 2, 3, 4; Linker K2 is selected from: -(CH2) n17 -R 25 - -(CH2) n17 -R 24 -(CH2) n18 -R 25 - -(CH2) n17 -OCH2-R 24 -CH2O-(CH2) n18 -R 25 - -(CH2) n17 -R 24 -(CH2) n18 - -(CH2) n17 -R 24 -CH2CH2O-(CH2) n18 -R 25 - -(CH2) n17 -R 24 -(CH2CH2O)2-(CH2) n18 - -(CH2) n17 - R 24 is selected from: -C(=O)NH-, -C(=O)-, or nothing; R 25 is selected from: -C(=O)NH-(CH2)2-C(=O)-, -NHC(=O)-(CH2)2-C(=O)-, -C(=O)NH- (CH2)2-, -NHC(=O)-(CH2)2-, -C(=O)-(CH2)2-; each n 17 Each of the following is independently selected from: 1, 2, 3, 4, 5, and 6; each n 18 Each of the following can be independently selected from: 0, 1, 2, 3, and 4; W1 is selected from a group capable of binding to an oligonucleotide drug through a phosphodiester bond or a phosphorothioate bond; W2is a group capable of undergoing a click chemistry reaction or hydrogen; selected from: 5-20 membered heterocyclyl containing an alkynyl group, -S-R 26 , alkynyl, azido, alkenyl, 5-20 membered heterocyclyl containing an alkenyl group, hydrogen; R 26 is selected from: hydrogen, thiol protecting group; W3 is selected from W1 or W2, and W3 is not hydrogen; Y1, Y2are each independently selected from the group consisting of: or chiral isomers thereof; R in the structure of Y1and Y2is each independently selected from the group consisting of: H, C1-C6alkyl; L3, L4 are independently selected from -NR6-R7-, -NR6-R7-O-R7-, -C(=O)-R7-; each R6 is independently selected from hydrogen, C1-C6 alkyl; each R7 is independently selected from C1-C8 alkylene; L5is selected from: -R8-(CR9R 10 ) n1 -; In L5, R8is selected from: -C(=0); R9, R 10 each n1is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; L6is selected from: -C(=O)NH(CH2) n4 C(=O)-, -C(=O)NH(CH2) n5 O(CH2) n7 C(=O)-; In L6, each n4 is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Each n5 is independently selected from 1, 2, 3, 4, 5, 6; Each n7 is independently selected from 1, 2, 3, 4, 5, 6; L7is selected from: -(CH2) n’8 -R 15 -(R 16 ) n8 -; L7is selected from: -C(=O); each R 15 is independently selected from: -C(=O); each R 16 is independently selected from: -C(R 17 )2-; each R 17 is independently selected from: hydrogen, C1-C6alkyl; each n8is independently selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; each n’8is independently selected from: 0; L8is selected from: -R 18 -(R 19 ) n10 -R 18 - or L7; In L8, R 18 Selected from: -C (=O); each R 19 Selected independently from: -C(R 14 )2-、O、S, where n 10 R 19 The connection does not violate the principle of chemical bonding; each R 14 Each is independently selected from: hydrogen, C1-C6 alkyl; each n 10 Each integer is independently selected from the range 1 to 20.

2. The compound or its pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, characterized in that, Y1is selected from: or their chiral isomers; Y2is selected from: or their chiral isomers; R in the structure of Y1 and Y2 is independently selected from H, C1-C3 alkyl; Preferably, Y1is selected from: or their chiral isomers; Preferably, Y2is selected from: or their chiral isomers.

3. The compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, wherein W2 is selected from: -S-R 26 , azido, hydrogen.

4. The compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, wherein Each R6 in L3, L4 is independently selected from hydrogen, C1-C3 alkyl; Each R7 is independently selected from C1-C4 alkylene; Preferably, L3, L4 are independently selected from -NHCH2CH2-, -NHCH2CH2OCH2-, -NHCH2CH2OCH2CH2-, -C(=O)CH2CH2CH2-.

5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein R9 and R in L5 10 Each of the following is independently selected from: hydrogen and C1-C3 alkyl groups; Preferably, L5is selected from: -C(=0)(CH2) n1 - each n1is independently selected from: 1, 2, 3, 4, 5; More preferably, L5 is selected from -C(=O)CH2-, -C(=O)CH2CH2-, -C(=O)CH2CH2CH2-.

6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein L6 is selected from -C(=O)NH(CH2)5C(=O)-, -C(=O)NH(CH2)4C(=O)-, -C(=O)NH(CH2)2OCH2C(=O)-, -C(=O)NH(CH2)2O(CH2)2C(=O)-.

7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein each R in L7 17 are each independently selected from the group consisting of: hydrogen, C1-C3alkyl; Preferably, L7is selected from: -C(=0)(CH2) n8 - each n8is independently selected from: 1, 2, 3, 4, 5, 6; More preferably, L7 is selected from -C(=O)(CH2)5-, -C(=O)(CH2)4-.

8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein each R in L8 14 are each independently selected from the group consisting of: hydrogen, C1-C3alkyl; Preferably, L8is selected from: -C(=0)(CH2)n 10 C(=0)-, n 10 selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15; More preferably, L8is selected from: -C(=O)(CH2) 10 C(=O)-.

9. The compound or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-8, wherein W1 is selected from hydroxyl, protected hydroxyl, or a group having the following structure: or a chiral isomer thereof; In the structure, R' is hydrogen or a hydroxyl protecting group.

10. The compound or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-8, wherein Linkers K1, K3 are each independently selected from:

11. The compound or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-8, wherein Linker K2 is selected from:

12. The compound or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-8, wherein X1, X'1are independently selected from a single bond, C1-C3 alkylene, -C(=O)-C1-C3 alkylene, -C(=O)-NH-C1-C3 alkylene, -NH-C(=O)-C1-C3 alkylene, -NH-C1-C3 alkylene, or chiral isomers thereof. Preferably, X1, X'1are independently selected from methylene, ethylene, propylene, -C(=O)-methylene, -C(=O)-ethylene, -C(=O)-propylene, -NH-C(=O)-methylene, -NH-C(=O)-ethylene, -NH-C(=O)-propylene.

13. The compound or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-8, wherein The ligand is selected from N-acetylgalactosamine, N-propionylgalactosamine.

14. The compound or pharmaceutically acceptable salt or stereoisomer thereof according to any one of claims 1-8, wherein The ligand is selected from or the hydrogen on the hydroxyl group is replaced by acetyl.

15. The compound of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein, The targeting binding moiety T is selected from or the hydrogen on the hydroxyl group is replaced by an acetyl group.

16. The compound of claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein The compound is selected from 17. A conjugate, characterized in that, The compound of any one of claims 1-16 is linked to an oligonucleotide drug, wherein W1 in the compound is linked to the oligonucleotide drug via a covalent bond; W2 in the compound is linked to the oligonucleotide drug by reacting with Q; Q is selected from L9-L10-L11-Z2, Z2 is a residue of the oligonucleotide drug after removing one hydroxyl group; L9is an active group capable of click chemistry reaction with the W2group, selected from: 5-20 membered heterocyclyl containing an alkyne group, -S-R 26 , alkyne, azido; R 26 is selected from: H or a thiol protecting group; L10 is selected from In L10, each A is independently selected from the group consisting of: -C(R2)2-, -NR2-, -O-, -S-; each R2is independently selected from the group consisting of: H, C1-C3 alkyl; each R3is independently selected from the group consisting of: or none; each R4is independently selected from the group consisting of: -C(R2)2-, -NR2-, -O-, -S-, -C(=O), -C(=S)-, wherein the attachment of f4 R4s does not violate the principles of chemical bonding; each R5is independently selected from the group consisting of: 3-8 membered heteromonocyclic group, C3-C8 cycloalkyl, 5-10 membered heterobicyclic group, 5-10 membered heterospirocyclyl, 5-20 membered heteroannulated cycloalkyl, C5-C 10 cycloalkyl; each f1is independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8; each f2is independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8; each f3is independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8; each f4is independently selected from the group consisting of: an integer between 0-20; L11 is selected from a group after removing one hydroxyl hydrogen of W1 or removing one hydroxyl protecting group of W1.

18. The conjugate of claim 17, wherein, L9 is selected from azido, -S-R 26 .

19. The conjugate of claim 17, wherein, L11 is selected from -O-, or a group having the following structure: or their chiral isomers.

20. The conjugate of claim 17, wherein, Each A in L10 is independently selected from -CH2-, -NH-, -O-, -S-; each R3is independently selected from: R5, or none; Each R4 is independently selected from -CH-, -NH-, -O-, -S-, -C(=O); Each R5 is independently selected from: 5-6 member heteromonocyclic groups, C5-C6 cycloalkyl groups, 6-8 member heterobridged cycloalkyl groups, 6-8 member heterospirocyclic groups, 6-10 member heterofused cycloalkyl groups, C6-C 10 cycloalkyl; Each f1 is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8; Each f2 is independently selected from 0, 1, 2, 3, 4; Each f3 is independently selected from 0, 1, 2, 3, 4; Each f4 is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; Preferably, each R5in L10is independently selected from: or chiral isomers thereof; Preferably, L10 is selected from: or their chiral isomers.

21. The conjugate of claim 17, wherein, Q is selected from 22. The conjugate according to any one of claims 17-21, characterized in that, The compound is linked to the oligonucleotide drug via a hydroxyl group in W1 and a phosphate group or a thiophosphate group at the 3' and / or 5' end of the sense strand of the oligonucleotide drug.

23. The conjugate of any one of claims 17-21, wherein, L11 is linked to the 3' and / or 5' end of the sense strand of the oligonucleotide drug via a phosphate bond or a thiophosphate bond.

24. The conjugate of any one of claims 17-21, wherein, The oligonucleotide drug comprises siRNA, ASO, saRNA, miRNA, PNA, aptamer.

25. The conjugate of any one of claims 17-21, wherein, It has the following formula (A), formula (B) or formula (C) structure: wherein the targeting binding moiety T, L3, L4, L5, L6, L7, L8 are as described in any one of claims 1-16, L10, L11, Z2 are as described in any one of claims 17-21; Z1 is a residue of the oligonucleotide drug after removing one hydroxyl group; W4 is a group after removing one hydroxyl hydrogen or one hydroxyl protecting group of W1 described in any one of claims 1-16; W5 is selected from W2 described in any one of claims 1-16 and a group formed after the reaction of W2 described in any one of claims 1-16 and L9 described in any one of claims 17-21; W6 is selected from W4 or -W5-L10-L11-.

26. The conjugate of claim 25, wherein, W4is selected from: -O-, or a group having the following structure: or their chiral isomers.

27. The conjugate of claim 25, wherein, each W5is independently selected from: 3-20 membered heterocyclyl, 5-20 membered heteroarylyl, 3-20 membered heterocyclylthio, -S-S-; Preferably, each W5is independently selected from: 5-20 membered heterocyclyl, 5-6 membered heteroarylyl, 5-6 membered heterocyclylthio, -S-S- Preferably, each W5is independently selected from: -S-S-.

28. The conjugate of claim 17, wherein, The conjugate is selected from:

29. A carrier compound, characterized in that, The conjugate is selected from:

30. Use of a compound of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the manufacture of a carrier for delivering one or more oligonucleotide drugs. Preferably, the oligonucleotide drug comprises siRNA, ASO, saRNA, miRNA, PNA, aptamer.

31. Use of a conjugate of any one of claims 17-28 as an active ingredient in the manufacture of a medicament for modulating the expression of one or more target genes.

32. Use of a compound of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and / or a carrier compound of claim 29 as a carrier in the manufacture of a medicament for modulating the expression of one or more target genes.

33. A pharmaceutical preparation for modulating the expression of one or more target genes, comprising, prepared from an active ingredient which comprises a conjugate of any one of claims 17-28 and a pharmaceutically acceptable adjuvant.

Images