A nucleic acid ligand and its conjugate, preparation method and use thereof

Abstract

This disclosure provides a nucleic acid ligand and its conjugate, a method for their preparation, and their uses. Specifically, this disclosure provides a ligand having the structure shown in Formula (I), which achieves excellent expression inhibition effects by conjugating with nucleic acids.

Description

Technical Field

[0001] This disclosure relates to a ligand and its preparation method. It also relates to nucleic acid ligand conjugates formed by covalently linking the ligand with nucleic acids, enabling the resulting nucleic acid ligand conjugates to be targeted and delivered into hepatocytes to exert RNA interference effects. Prior Technology

[0002] RNA interference (RNAi) is an effective way to silence gene expression. Statistics show that over 80% of disease-related proteins in the human body cannot be targeted by conventional small-molecule drugs and large-molecule biological agents, thus becoming untreatable proteins. Using RNAi technology, suitable siRNAs can be designed based on the mRNA encoding these proteins, specifically targeting and degrading the target mRNA, thereby inhibiting the production of the related proteins. Therefore, siRNAs hold significant promise for drug development. However, to achieve therapeutic RNAi effects in vivo, siRNA molecules need to be delivered to specific cells.

[0003] Using targeted ligand-conjugated siRNA, the targeted ligand binds to receptor molecules on the cell membrane surface, thereby endocytosis into the cell, which is an effective drug delivery method. For example, the desialylate glycoprotein receptor (ASGPR) is a receptor specifically expressed by hepatocytes, characterized by high abundance on the hepatocyte surface and rapid intracellular-extracellular conversion. Monosaccharides and polysaccharides such as galactose, galactosamine, and N-acetylglucosamine have high affinity for ASGPR. Literature reports (Yuanyu H, LIANG Zicai L, Asialoglycoprotein Receptor and Its Application in Liver-targeted Drug Delivery, Prog. Biochem. Biophys. 2015; 42(6)) that aminogalactose molecular clusters (GalNAc) can effectively deliver RNA to hepatocytes. GalNAc molecules are designed as trivalent or tetravalent molecular clusters, which can significantly improve the ability of monovalent or divalent GalNAc molecules to target hepatocytes.

[0004] Different molecular cluster structures and varying linkages with RNA significantly affect the activity of siRNA in vivo. Higher activity translates to better therapeutic effects or lower dosages, and at the same efficacy, lower dosages also imply lower toxicity. Therefore, the rational design of the covalent linkage between the targeting ligand and siRNA is of great importance.

[0005] This disclosure provides a novel molecular structure for linking GalNAc molecules to RNA, which has a simpler synthetic structure, superior delivery activity, and better RNA interference activity. Summary of the Invention

[0006] Firstly, this disclosure provides a ligand having a structure as shown in formula (I'). Wherein, L1 is a C1-C30 alkyl chain, or a C1-C30 alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O; R1 and R2 are independently chemical bonds, -NR6-, -C(=O)-, or -OC(=O)-; Q is or ; It is a single bond or a double bond, and when When it is a single bond, R3 can independently be CR7R8, NR6, O, or S. When it is a double bond, R3 can be CR9 or N independently; R4 is independently CR9 or N;

[0007] Ring A is absent, or is a cycloalkyl, heterocyclic, aryl, or heteroaryl group, and when ring A is present, R5 is independently CR9 or N, and when ring A is absent, R5 is independently CR7R8, NR6, or O;

[0008] R6 and R9 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, or heteroaryl group is selected as desired by one or more halogens. The group substituted by the following groups: hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C3-7 cycloalkyl, 3-12 member heterocyclic, 5-12 member aryl, 5-12 member heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', and C(=O)NR'(R");

[0009] R7 and R8 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, or heteroaryl group is selected as desired by one or more halogens. The group substituted by the following groups: hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C3-7 cycloalkyl, 3-12 member heterocyclic, 5-12 member aryl, 5-12 member heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', and C(=O)NR'(R");

[0010] R' and R" are independently hydrogen, deuterium, hydroxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, or heteroaryl, which may be substituted as desired by one or more substituents selected from halogen, hydroxyl, oxy, nitro, and cyano.

[0011] m, n, p, and q are independently 0, 1, 2, 3, or 4; B is or ; Rb1, Rb2, Rb3, Rb4, Rb5, Rb6, and Rb7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH2)z8-O-, or -NHC(=O)-(CH2)z9-O-; z1, z2, z3, z4, z5, z6, z7, z8, and z9 are independent integers from 0 to 10; L2 is a C1-C30 alkyl chain, or a C1-C30 alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O; G represents the targeting portion that binds to cell receptors; r is an integer from 1 to 10.

[0012] In some embodiments, certain groups are defined as follows, and undefined groups are as described in any of the preceding embodiments (hereinafter referred to as "in some embodiments"). L1 may be L3 or L3-R10-R11-L3, wherein L3 is independently a C1-C12 alkyl chain, -(CH2)j1-C(=O)-(CH2)j2- or -(CH2)j3-(CH2CH2O)1-4-(CH2)j4-, R10 and R11 are independently chemical bonds, -NR12-, -C(=O)- or -OC(=O)-, R12 is hydrogen or C1-C12 alkyl, and j1, j2, j3 and j4 are independently integers from 0 to 10, preferably integers from 0 to 2 or 4 to 10, more preferably 0, 1, 2, 6, 7, 8, 9 or 10.

[0013] In some implementations, L1 can be -(CH2)j1-C(=O)-(CH2)j2-, where j1 and j2 are defined as described in any of the previous implementations.

[0014] In some implementations, L1 can be The definitions of j1 and j2 are the same as those in the previous scheme, wherein end a1 is connected to B and end b1 is connected to R1.

[0015] In some implementations, L1 can be , , , or In this case, end a1 is connected to B, and end b1 is connected to R1.

[0016] In some implementations, R1 can be a chemical bond and R2 can be C=O.

[0017] In some implementations, R1 may be a chemical bond and R2 may be NR6, with R6 defined as described in any of the previous implementations.

[0018] In some implementations, R1 can be a chemical bond and R2 can be -OC (=O)-.

[0019] In some implementations, R1 can be NR6 and R2 can be C=O, with R6 defined as described in any of the previous implementations.

[0020] In some implementations, R1 can be NR6 and R2 can be -OC (=O)-, with R6 defined as described in any of the previous implementations.

[0021] In some implementations, R2 can be NR6 and R1 can be C=O, with R6 defined as described in any of the previous implementations.

[0022] In some implementations, R2 can be NR6 and R1 can be -OC (=O)-, with R6 defined as described in any of the previous implementations.

[0023] In some implementations, R6 may be hydrogen or a C1-6 alkyl group.

[0024] In some implementations, R6 may be hydrogen, methyl, ethyl, propyl, or isopropyl.

[0025] In some implementations, R6 may be hydrogen.

[0026] In some implementations, R7 and R8 may be hydrogen.

[0027] In some implementations, R9 may be hydrogen.

[0028] In some embodiments, when ring A is present, ring A may be C6-10 aryl, preferably phenyl.

[0029] In some implementations, m can be 0 or 1.

[0030] In some implementations, m can be 3.

[0031] In some implementations, n can be 0 or 1.

[0032] In some implementations, p and q are independently 0 or 1.

[0033] In some implementations, p=1 and q=1.

[0034] In some implementations, p=1 and q=0.

[0035] In some implementations, p=0 and q=1.

[0036] In some implementations, p=0 and q=0.

[0037] In some implementations, z1, z2, z3, z4, z5, z6, z7, z8, and z9 can be independently integers from 0 to 4, preferably 0, 1, or 2.

[0038] In some implementation schemes, B can be Rb1, Rb2, Rb3 and Rb4 are independently -C(=O)- or -NHC(=O)-, with the N atom connected to L1, and z1, z2, z3 and z4 are defined as described in any of the previous schemes.

[0039] In some implementation schemes, B can be Rb1, Rb2, Rb3, and Rb4 are independently -C(=O)- or -NHC(=O)-, with the N atom bonded to L1. Rb1, Rb3, and Rb4 are identical, and z1, z2, z3, and z4 are defined as described in any of the previous schemes.

[0040] In some implementation schemes, B can be .

[0041] In some implementation schemes, B can be .

[0042] In some implementation schemes, B can be Rb5, Rb6, and Rb7 are independently -C(=O)-(CH2)z8-O- or -NHC(=O)-(CH2)z9-O-, with the N atom bonded to L1. The definitions of z5, z6, z7, z8, and z9 are the same as described in any of the previous schemes.

[0043] In some implementation schemes, B can be Rb5, Rb6, and Rb7 are independently -C(=O)-(CH2)z8-O- or -NHC(=O)-(CH2)z9-O-, with the N atom bonded to L1. Rb5, Rb6, and Rb7 are identical, and the definitions of z5, z6, z7, z8, and z9 are the same as described in any of the previous schemes.

[0044] In some implementation schemes, B can be .

[0045] In some embodiments, L2 may be L4 or L4-R13-R14-L4, wherein L4 is independently a C1-C12 alkyl chain or -(CH2)j5-(OCH2CH2)1-4-(CH2)j6-, R13 and R14 are independently chemical bonds, -NR15-, -C(=O)- or -OC(=O)-, R15 is independently hydrogen or C1-C12 alkyl, and j5 and j6 are independently integers from 0 to 10, preferably integers from 0 to 6, more preferably 0, 1, 2, 3 or 4.

[0046] In some implementations, L2 can be -(CH2)j5-(OCH2CH2)1-4-(CH2)j6-, where j5 and j6 are defined as described in any of the previous implementations.

[0047] In some implementations, L2 can be , , , or In this configuration, the O atom is bonded to the G atom, and the C atom is bonded to the B atom. The preferred L2 is... or

[0048] In some implementations, L2 can be In this context, j6 is defined as described in the previous scheme, with the O atom connected to G and the C atom connected to B.

[0049] In some implementations, L2 can be In this case, the O atom is connected to the G atom, and the C atom is connected to the B atom.

[0050] In some implementations, G may be the desialyl glycoprotein receptor targeting region.

[0051] In some embodiments, G may be galactose, galactosamine, N-methoxygalactosamine, N-acetylglucosamine, N-propylgalactosamine, N-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0052] In some implementations, G can be .

[0053] In some implementations, r can be 3, 4, 5 or 6, for example 3.

[0054] In some implementations, Q can be or better The definitions of R3, R4, R5 and n are the same as those described in any previous scheme.

[0055] In some implementation schemes, Can be The definitions of R3, R4, R5, p, and q are the same as those described in any of the previous schemes.

[0056] In some implementation schemes, Can be , , or The definitions of R3, R4, R5, p, and q are the same as those described in any of the previous schemes.

[0057] In some implementation schemes, Can be , or better or Better The definitions of p and q are the same as described in any of the previous schemes.

[0058] In some implementation schemes, Can be , , , or better , , , , , , or Better , , or The definitions of p and q are the same as described in any of the previous schemes.

[0059] In some implementation schemes, Can be The definitions of R3, R4, n, p, and q are the same as those described in any previous scheme.

[0060] In some implementation schemes, Can be or The definitions of R3, R4, R5, n, p, and q are the same as those described in any of the previous schemes.

[0061] In some implementation schemes, Can be The definitions of n, p, and q are the same as those described in any of the previous schemes.

[0062] In some implementation schemes, Can be or The definitions of n, p, and q are the same as those described in any of the previous schemes.

[0063] This disclosure provides a ligand having a structure as shown in formula (I), Wherein, L1 is a C1-C30 alkyl chain, or a C1-C30 alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O; R1 and R2 are independently chemical bonds, -NR6-, -C(=O)-, or -OC(=O)-; Q is or ; It is a single bond or a double bond, and when When it is a single bond, R3 can independently be CR7R8, NR6, O, or S. When it is a double bond, R3 can be CR9 or N independently; R4 is independently CR9 or N;

[0064] Ring A is absent, or is a cycloalkyl, heterocyclic, aryl, or heteroaryl group, and when ring A is present, R5 is independently CR9 or N, and when ring A is absent, R5 is independently CR7R8, NR6, or O;

[0065] R6 and R9 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, or heteroaryl group is selected as desired by one or more halogens. The group substituted by the following groups: hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C3-7 cycloalkyl, 3-12 member heterocyclic, 5-12 member aryl, 5-12 member heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', and C(=O)NR'(R");

[0066] R7 and R8 are independently hydrogen, deuterium, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', or C(=O)NR'(R"), wherein the alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, or heteroaryl group is selected as desired by one or more halogens. The group substituted by the following groups: hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C3-7 cycloalkyl, 3-12 member heterocyclic, 5-12 member aryl, 5-12 member heteroaryl, SR', S(=O)R', S(=O)2R', S(=O)2NR'(R"), NR'(R"), C(=O)R', C(=O)OR', and C(=O)NR'(R");

[0067] R' and R" are independently hydrogen, deuterium, hydroxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, or heteroaryl, which may be substituted as desired by one or more substituents selected from halogen, hydroxyl, oxy, nitro, and cyano.

[0068] m, n, p, and q are independently 0, 1, 2, 3, or 4; B is or ; Rb1, Rb2, Rb3, Rb4, Rb5, Rb6, and Rb7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH2)z8-O-, or -NHC(=O)-(CH2)z9-O-; z1, z2, z3, z4, z5, z6, z7, z8, and z9 are independent integers from 0 to 10; L2 is a C1-C30 alkyl chain, or a C1-C30 alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O; r is an integer from 1 to 10.

[0069] In some embodiments, L1 may be L3 or L3-R10-R11-L3, wherein L3 is independently a C1-C12 alkyl chain, -(CH2)j1-C(=O)-(CH2)j2- or -(CH2)j3-(CH2CH2O)1-4-(CH2)j4-; R10 and R11 are independently chemical bonds, -NR12-, -C(=O)- or -OC(=O)-; R12 is hydrogen or C1-C12 alkyl; j1, j2, j3 and j4 are independently integers from 0 to 10, preferably integers from 0 to 2 or 4 to 10, more preferably 0, 1, 2, 6, 7, 8, 9 or 10.

[0070] In some implementations, L1 can be -(CH2)j1-C(=O)-(CH2)j2-, where j1 and j2 are defined as described in any of the previous implementations.

[0071] In some implementations, L1 can be The definitions of j1 and j2 are the same as those in the previous scheme, wherein end a1 is connected to B and end b1 is connected to R1.

[0072] In some implementations, L1 can be , , , or In this case, end a1 is connected to B, and end b1 is connected to R1.

[0073] In some implementations, R1 can be a chemical bond and R2 can be C=O.

[0074] In some implementations, R1 may be a chemical bond and R2 may be NR6, with R6 defined as described in any of the previous implementations.

[0075] In some implementations, R1 can be a chemical bond and R2 can be -OC (=O)-.

[0076] In some implementations, R1 can be NR6 and R2 can be C=O, with R6 defined as described in any of the previous implementations.

[0077] In some implementations, R1 can be NR6 and R2 can be -OC (=O)-, with R6 defined as described in any of the previous implementations.

[0078] In some implementations, R2 can be NR6 and R1 can be C=O, with R6 defined as described in any of the previous implementations.

[0079] In some implementations, R2 can be NR6 and R1 can be -OC (=O)-, with R6 defined as described in any of the previous implementations.

[0080] In some implementations, R6 may be hydrogen or a C1-6 alkyl group.

[0081] In some implementations, R6 may be hydrogen, methyl, ethyl, propyl, or isopropyl.

[0082] In some implementations, R6 may be hydrogen.

[0083] In some implementations, R7 and R8 may be hydrogen.

[0084] In some implementations, R9 may be hydrogen.

[0085] In some embodiments, when ring A is present, ring A may be C6-10 aryl, preferably phenyl.

[0086] In some implementations, m can be 0 or 1.

[0087] In some implementations, m can be 3.

[0088] In some implementations, n can be 0 or 1.

[0089] In some implementations, p and q are independently 0 or 1.

[0090] In some implementations, p=1 and q=1.

[0091] In some implementations, p=1 and q=0.

[0092] In some implementations, p=0 and q=1.

[0093] In some implementations, p=0 and q=0.

[0094] In some implementations, z1, z2, z3, z4, z5, z6, z7, z8, and z9 can be independently integers from 0 to 4, preferably 0, 1, or 2.

[0095] In some implementation schemes, B can be Rb1, Rb2, Rb3, and Rb4 are independently -C(=O)- or -NHC(=O)-, with the N atom bonded to L1. The definitions of z1, z2, z3, and z4 are the same as described in any of the previous schemes (e.g., The N atom at the a2 end is connected to L1.

[0096] In some implementation schemes, B can be Rb1, Rb2, Rb3, and Rb4 are independently -C(=O)- or -NHC(=O)-, with the N atom bonded to L1. Rb1, Rb3, and Rb4 are identical, and z1, z2, z3, and z4 are defined as described in any of the previous schemes (e.g., The N atom at the a2 end is connected to L1.

[0097] In some implementation schemes, B can be (For example (where the N atom at the a2 end is connected to L1).

[0098] In some implementation schemes, B can be (For example (where the N atom at the a2 end is connected to L1).

[0099] In some implementation schemes, B can be Rb5, Rb6, and Rb7 are independently -C(=O)-(CH2)z8-O- or -NHC(=O)-(CH2)z9-O-, with the N atom bonded to L1. The definitions of z5, z6, z7, z8, and z9 are the same as described in any of the previous schemes.

[0100] In some implementation schemes, B can be Rb5, Rb6, and Rb7 are independently -C(=O)-(CH2)z8-O- or -NHC(=O)-(CH2)z9-O-, with the N atom bonded to L1. Rb5, Rb6, and Rb7 are identical, and the definitions of z5, z6, z7, z8, and z9 are the same as described in any of the previous schemes.

[0101] In some implementation schemes, B can be Rb5, Rb6, and Rb7 are independently -C(=O)-(CH2)z8-O- or -NHC(=O)-(CH2)z9-O-, with the N atom at the a2 end connected to L1. The definitions of z5, z6, z7, z8, and z9 are the same as described in any of the previous schemes.

[0102] In some implementation schemes, B can be Rb5, Rb6, and Rb7 are independently -C(=O)-(CH2)z8-O- or -NHC(=O)-(CH2)z9-O-, with the N atom at the a2 end connected to L1. Rb5, Rb6, and Rb7 are the same, and the definitions of z5, z6, z7, z8, and z9 are the same as described in any of the previous schemes.

[0103] In some implementation schemes, B can be (For example (where the N atom at the a2 end is connected to L1).

[0104] In some embodiments, L2 may be L4 or L4-R13-R14-L4, wherein L4 is independently a C1-C12 alkyl chain or -(CH2)j5-(OCH2CH2)1-4-(CH2)j6-, R13 and R14 are independently chemical bonds, -NR15-, -C(=O)- or -OC(=O)-, R15 is independently hydrogen or C1-C12 alkyl, and j5 and j6 are independently integers from 0 to 10, preferably integers from 0 to 6, more preferably 0, 1, 2, 3 or 4.

[0105] In some implementations, L2 can be -(CH2)j5-(OCH2CH2)1-4-(CH2)j6-, where j5 and j6 are defined as described in any of the previous implementations.

[0106] In some implementations, L2 can be , , , or better or In this case, the O atom is connected to the G atom, and the C atom is connected to the B atom.

[0107] In some implementations, L2 may be a C1-C12 alkyl chain.

[0108] In some implementations, L2 can be , ,

[0109] In some implementations, L2 can be better Better In this case, terminal a3 is connected to O, and terminal b3 is connected to B.

[0110] In some implementations, L2 can be In this case, terminal a3 is connected to O, and terminal b3 is connected to B.

[0111] In some implementations, L2 can be .

[0112] In some implementations, r can be 3, 4, 5 or 6, with 3 being preferred.

[0113] In some implementations, Q can be The definitions of R3, R4, and R5 are the same as those described in any of the previous schemes.

[0114] In some implementation schemes, Can be The definitions of R3, R4, R5, p, and q are the same as those described in any of the previous schemes.

[0115] In some implementation schemes, Can be , , or The definitions of R3, R4, R5, p, and q are the same as those described in any of the previous schemes.

[0116] In some implementation schemes, Can be , or better or Better The definitions of p and q are the same as described in any of the previous schemes.

[0117] In some implementation schemes, Can be , , , or better , , , , , , or Better , , or The definitions of p and q are the same as described in any of the previous schemes.

[0118] In some implementations, -R1-R2- can be -C(=O)-NR6-, wherein the C atom is preferably connected to L1, and R6 is defined as described in any of the previous implementations.

[0119] In some implementations, m can be 1.

[0120] In some implementations, m can be 1; Can be better The definitions of p and q are the same as described in any of the previous schemes.

[0121] In some implementation schemes, Can be better

[0122] In some implementations, the ligand may be any of the following structures:

[0123] In some implementations, the ligand may be any of the following structures:

[0124] In some embodiments, the N-acetylglucosamine portion of the above ligands can be replaced by N-trifluoroacetylglucosamine, N-propylgalactosamine, N-n-butylgalactosamine, or N-isobutylgalactosamine.

[0125] Secondly, this disclosure provides a compound as shown in formula (II'), Where X is a hydroxyl protecting group; Y is hydrogen, deuterium, or hydrogen protecting group. or ; j7 is 1, 2, 3, or 4; W represents a polymer compound; G represents the targeting portion that binds to cell receptors; The definitions of L1, R1, R2, Q, B, L2, m, p, q, and r are the same as those for any of the ligands having the structure shown in equation (I').

[0126] In some embodiments, the hydroxyl protecting group may be an ester protecting group, an alkoxymethyl protecting group, an alkyl protecting group, a silyl protecting group, or an aryl protecting group, preferably an aryl protecting group, more preferably MMTr and DMTr, and even more preferably DMTr.

[0127] In some implementations, j7 can be 2.

[0128] In some implementations, G may be the desialyl glycoprotein receptor targeting region.

[0129] In some embodiments, G may be N-acetylglucosamine triacetate, N-trifluoroacetylglucosamine triacetate, N-propionic acid galactosamine triacetate, N-n-butyl galactosamine triacetate or N-isobutyl galactosamine triacetate, with N-acetylglucosamine triacetate being preferred.

[0130] In some embodiments, the polymer compound may be a resin, preferably a macroporous resin, and more preferably a macroporous aminomethyl resin.

[0131] In some implementation schemes, Can be The definitions of R3, R4, R5, X, Y, p, and q are the same as those described in any of the previous schemes.

[0132] In some implementation schemes, Can be , , or The definitions of R3, R4, R5, X, Y, p, and q are the same as those described in any of the previous schemes.

[0133] In some implementation schemes, Can be , or better or Better The definitions of X, Y, p, and q are the same as those described in any of the previous schemes.

[0134] In some implementation schemes, Can be , , , or better , , , , , , or Better , , or The definitions of X, Y, p, and q are the same as those described in any of the previous schemes.

[0135] In some implementation schemes, Can be The definitions of R3, R4, X, Y, n, p, and q are the same as those described in any previous scheme.

[0136] In some implementation schemes, Can be or The definitions of R3, R4, X, Y, n, p, and q are the same as those described in any previous scheme.

[0137] In some implementation schemes, Can be The definitions of X, Y, n, p, and q are the same as described in any of the previous schemes.

[0138] In some implementation schemes, Can be or The definitions of X, Y, n, p, and q are the same as described in any of the previous schemes.

[0139] In some embodiments, the compound may be a compound represented by formula (II'-1), (II'-2), or (II'-3). in, The resin is preferred, a macroporous resin, and more preferably a macroporous aminomethyl resin; L1, R1, R2, Q, B, L2, G, m, p, q, and r are defined as in any of the compounds shown in formula (II').

[0140] This disclosure provides a compound as shown in formula (II), Where X is a hydroxyl protecting group; Y is hydrogen, deuterium, or hydrogen protecting group. or ; j7 is 1, 2, 3, or 4; W represents a polymer compound; The definitions of L1, R1, R2, Q, B, L2, m, p, q, and r are the same as those for any of the ligands having the structure shown in equation (I).

[0141] In some embodiments, the hydroxyl protecting group may be an ester protecting group, an alkoxymethyl protecting group, an alkyl protecting group, a silyl protecting group, or an aryl protecting group, preferably an aryl protecting group, more preferably MMTr and DMTr, and even more preferably DMTr.

[0142] In some implementations, j7 can be 2.

[0143] In some embodiments, the polymer compound may be a resin, preferably a macroporous resin, and more preferably a macroporous aminomethyl resin.

[0144] In some implementation schemes, Can be The definitions of R3, R4, R5, X, Y, p, and q are the same as those described in any of the previous schemes.

[0145] In some implementation schemes, Can be , , or The definitions of R3, R4, R5, X, Y, p, and q are the same as those described in any of the previous schemes.

[0146] In some implementation schemes, Can be , or better or Better The definitions of X, Y, p, and q are the same as those described in any of the previous schemes.

[0147] In some implementation schemes, Can be , , , or better , , , , , , or Better , , or The definitions of X, Y, p, and q are the same as those described in any of the previous schemes.

[0148] In some implementation schemes, Can be The definitions of R3, R4, X, Y, n, p, and q are the same as those described in any previous scheme.

[0149] In some implementation schemes, Can be or The definitions of R3, R4, X, Y, n, p, and q are the same as those described in any previous scheme.

[0150] In some implementation schemes, Can be The definitions of X, Y, n, p, and q are the same as described in any of the previous schemes.

[0151] In some implementation schemes, Can be or The definitions of X, Y, n, p, and q are the same as described in any of the previous schemes.

[0152] In some embodiments, the compound may be a compound represented by formula (II-1), (II-2), or (II-3). in, The resin is preferred, a macroporous resin, and more preferably a macroporous amine methyl resin; L1, R1, R2, Q, B, L2, m, p, q, and r are defined as described in any of the compounds shown in formula (II).

[0153] In some embodiments, the compound may have any of the following structures: Where Y is hydrogen or ; Or, Y is , For resins, macroporous resins are preferred, and macroporous aminomethyl resins are even better.

[0154] In some embodiments, the compound may have any of the following structures: Where Y is hydrogen or ; Or, Y is , It is a resin, preferably a macroporous resin, and even better, a macroporous aminomethyl resin.

[0155] In some embodiments, the N-acetylated galactosamine triacetate portion of the above compounds may be replaced by N-trifluoroacetylgalactosamine triacetate, N-propionic acid galactosamine triacetate, N-n-butyl galactosamine triacetate, or N-isobutyl galactosamine triacetate.

[0156] This disclosure provides a nucleic acid ligand conjugate comprising a nucleic acid and one or more of the aforementioned ligands, which are conjugated to the ends of the nucleic acid, and the ligands may be the same or different.

[0157] In some implementations, the nucleic acid and ligand can be linked by a phosphate ester group, a thiophosphate ester group, or a phosphonic acid group.

[0158] In some implementations, the nucleic acid may include, but is not limited to: oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribonuclease, interfering RNA molecules, and Dicer enzyme receptors.

[0159] In some implementations, the 3' end of the nucleic acid can be conjugated to a ligand.

[0160] In some implementations, the nucleic acid may be a single-stranded nucleic acid.

[0161] In some implementations, the single-stranded nucleic acid may be the positive strand of siRNA.

[0162] In some implementations, the single-stranded nucleic acid may be the antisense strand of siRNA.

[0163] In some implementations, the nucleic acid may be a double-stranded nucleic acid. The double-stranded nucleic acid may include at least one double-stranded region, wherein the first-stranded nucleic acid and the second-stranded nucleic acid within the double-stranded region are at least partially complementary. (1) The two ends of the first-strand nucleic acid are not conjugated to the ligand; and the 5' end of the second-strand nucleic acid is conjugated to the ligand, but the 3' end is not conjugated to the ligand, or the 3' end of the second-strand nucleic acid is conjugated to the ligand, but the 5' end is not conjugated to the ligand, or both the 3' and 5' ends of the second-strand nucleic acid are conjugated to the ligand; (2) The two ends of the second-strand nucleic acid are not conjugated to the ligand; and the 5' end of the first-strand nucleic acid is conjugated to the ligand, but the 3' end is not conjugated to the ligand, or the 3' end of the first-strand nucleic acid is conjugated to the ligand, but the 5' end is not conjugated to the ligand, or both the 3' and 5' ends of the first-strand nucleic acid are conjugated to the ligand; (3) Both the 5' ends of the first-strand nucleic acid and the second-strand nucleic acid are conjugated to the ligand, while the 3' ends are not conjugated to the ligand; or both the 3' ends of the first-strand nucleic acid and the second-strand nucleic acid are conjugated to the ligand, while the 5' ends are not conjugated to the ligand; or both the 3' and 5' ends of the first-strand nucleic acid and the second-strand nucleic acid are conjugated to the ligand; or both the 3' and 5' ends of the first-strand nucleic acid and the second-strand nucleic acid are conjugated to the ligand, while neither the 5' end of the first-strand nucleic acid nor the 3' end of the second-strand nucleic acid is conjugated to the ligand; or both the 5' end of the first-strand nucleic acid and the 3' end of the second-strand nucleic acid are conjugated to the ligand, while neither the 3' end of the first-strand nucleic acid nor the 5' end of the second-strand nucleic acid is conjugated to the ligand; (4) Both the 3' and 5' ends of the first-strand nucleic acid are conjugated to the ligand, and either the 5' or 3' end of the second-strand nucleic acid is conjugated to the ligand; or both the 3' and 5' ends of the second-strand nucleic acid are conjugated to the ligand, and either the 5' or 3' end of the first-strand nucleic acid is conjugated to the ligand.

[0164] In some implementations, the double-stranded nucleic acid may be siRNA.

[0165] In some implementations, the nucleic acid may include one or more modified nucleotides.

[0166] In some implementations, the nucleic acid ligand conjugate may be the following structure or a pharmaceutically acceptable salt thereof: Wherein, T represents the aforementioned ligands, and each T may be the same or different; L5 is independently a C1-C30 alkyl chain, or a C1-C30 alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O; M can be either O or S independently; g is an independent integer between 0 and 4; Represents nucleic acid.

[0167] In some implementations, L5 can be ,

[0168] In some implementations, g can be 0 or 1.

[0169] In some implementations, g can be 0.

[0170] In some implementations, M can be S.

[0171] In some implementations, the nucleic acid ligand conjugate may be any of the following structures or a pharmaceutically acceptable salt thereof.

[0172] In some implementations, the nucleic acid ligand conjugate may be any of the following structures or a pharmaceutically acceptable salt thereof.

[0173] In some implementations, the pharmaceutically acceptable salt may be a salt conventional in the art, including but not limited to: sodium salts, potassium salts, ammonium salts, amine salts, etc.

[0174] On the other hand, this disclosure provides an RNAi reagent comprising the above-mentioned nucleic acid ligand conjugate.

[0175] In some implementations, the RNAi reagent may include, but is not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer receptors.

[0176] In some implementations, the RNAi reagent may be siRNA.

[0177] On the other hand, this disclosure provides a composition comprising the above-described nucleic acid ligand conjugate or the above-described RNAi reagent, and one or more pharmaceutically acceptable excipients.

[0178] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, transporter, diluent, and / or delivery polymer.

[0179] In some implementations, the nucleic acid ligand conjugate or RNAi reagent may be a therapeutically effective amount.

[0180] In some implementations, the unit dose of the component may be 0.001 mg to 1000 mg.

[0181] In some implementations, the content of the nucleic acid ligand conjugate or RNAi reagent may be 0.01-99.99%, or 0.1-99.9%, or 0.5%-99.5%, or even 1%-99%, or even 2%-98%, depending on the total weight of the components.

[0182] In some implementations, the content of the pharmaceutically acceptable excipient may be 0.01-99.99%, or 0.1-99.9%, or 0.5%-99.5%, or even 1%-99%, or even 2%-98%, depending on the total weight of the composition.

[0183] In some embodiments, the nucleic acid ligand conjugates or RNAi reagents or compositions disclosed herein, when contacted with cells expressing a target gene, inhibit the expression of the target gene by, for example: psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence assays, such as Western blotting or flow cytometry, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0184] In some embodiments, when the above-mentioned conjugate or RNAi reagent or composition comes into contact with cells expressing the target gene, the percentage of residual expression of the target gene mRNA induced by the above-mentioned siRNA is determined by, for example: psiCHECK activity screening and luciferase reporter gene detection, other methods such as PCR or branched DNA (bDNA) based methods, or protein-based methods such as immunofluorescence analysis, such as Western Blot or flow cytometry, to be no higher than 99%, no higher than 95%, no higher than 90%, no higher than 85%, no higher than 80%, no higher than 75%, no higher than 70%, no higher than 65%, no higher than 60%, no higher than 55%, no higher than 50%, no higher than 45%, no higher than 40%, no higher than 35%, no higher than 30%, no higher than 25%, no higher than 20%, no higher than 15%, or no higher than 10%.

[0185] On the other hand, this disclosure provides the use of the above-mentioned nucleic acid ligand conjugate, the above-mentioned RNAi reagent, or the above-mentioned composition in the preparation of a medicament for treating a patient's disease. The disease is preferably a hepatogenic disease.

[0186] This disclosure provides a nucleic acid ligand conjugate or RNAi reagent or composition for treating a patient's disease, as previously described. The disease is preferably a hepatogenic disease.

[0187] This disclosure provides a nucleic acid ligand conjugate or RNAi reagent or composition for inhibiting mRNA expression in patients, as described above.

[0188] This disclosure provides a ligand, nucleic acid ligand conjugate, or RNAi reagent or composition for in vivo delivery of an expression-inhibiting oligomeric compound to the liver, the ligand, nucleic acid ligand conjugate, or RNAi reagent or composition being as previously described.

[0189] On the other hand, this disclosure provides a method for treating a patient's disease, comprising administering the aforementioned nucleic acid ligand conjugate or RNAi reagent or composition to the patient. The disease is preferably a hepatogenic disease. The nucleic acid ligand conjugate or RNAi reagent or composition may be a therapeutically effective amount.

[0190] On the other hand, this disclosure provides a method for inhibiting mRNA expression in a patient, comprising administering the aforementioned nucleic acid ligand conjugate or RNAi reagent or composition to the patient. The nucleic acid ligand conjugate or RNAi reagent or composition may be in a therapeutically effective amount.

[0191] On the other hand, this disclosure provides a method for in vivo delivery of an expression-inhibiting oligomeric compound to the liver, comprising administering the aforementioned nucleic acid ligand conjugate or RNAi reagent or composition to a patient. The nucleic acid ligand conjugate or RNAi reagent or composition may be in a therapeutically effective amount.

[0192] The nucleic acid ligand conjugates or RNAi reagents or compositions and methods disclosed herein can reduce the level of target mRNA in cells, cell populations, tissues or subjects, including: administering a therapeutically effective amount of the expression-inhibiting oligomer described herein to the subject, the expression-inhibiting oligomer being linked to a ligand thereby inhibiting the expression of target mRNA in the subject, the ligand being as previously described.

[0193] In some implementations, the subject has been previously identified as having pathogenic upregulation of the target gene in the targeted cells or tissues.

[0194] The patients described in this disclosure are subjects suffering from diseases or conditions that would benefit from a reduction or inhibition of target mRNA expression.

[0195] Delivery can be made by local administration (e.g., direct injection or implantation) or systemic administration, or by oral, rectal or parenteral routes, including but not limited to subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, inhalation administration (e.g., aerosol), mucosal administration (e.g., sublingual or intranasal administration), intracranial administration, etc.

[0196] In some implementations, the nucleic acid ligand conjugates or RNAi reagents or compositions provided herein can be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, duodenal, or intraperitoneal injection.

[0197] In some implementations, the nucleic acid ligand conjugates or RNAi reagents or compositions provided in this disclosure may be packaged in a kit.

[0198] In some embodiments, this disclosure also provides a cell comprising the above-described nucleic acid ligand conjugate or RNAi reagent.

[0199] This disclosure provides a method for preparing nucleic acid ligand conjugates, which includes the following steps: starting with the above formula (II-3) or formula (II'-3), linking nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement sequence.

[0200] In some implementation schemes, each monomer linkage involves four steps: deprotection, coupling, capping, oxidation, or sulfidation. After the last monomer linkage is completed, the nucleic acid sequence linked on the solid-phase support is sequentially cleaved, deprotected, purified, and desalted, and then freeze-dried to obtain the nucleic acid ligand conjugate.

[0201] This disclosure provides a method for preparing the compound shown in formula (II-3), comprising the following steps: conjugating the compound shown in formula (II-2) with a polymeric compound to obtain the compound shown in formula (II-3). Among them, L1, R1, R2, Q, B, L2, m, p, q, r and The definition is the same as described above.

[0202] In some implementations, the conditions and operations of the coupling may be those conventional in the art.

[0203] In some embodiments, the compound shown in formula (II-2) can be prepared by the following steps: in a solvent, under the action of a base, the compound shown in formula (II-1) is reacted with succinic anhydride as shown below to obtain the compound shown in formula (II-2); The definitions of L1, R1, R2, Q, B, L2, m, p, q, and r are as described above.

[0204] In some implementations, the conditions and operation of the reaction may be those conventional for this type of reaction in the art.

[0205] In some embodiments, the compound shown in formula (II-1) can be prepared by the following steps: reacting the compound shown in formula (III) with the compound shown in formula (IV) as shown below to obtain the compound shown in formula (II-1); The definitions of L1, R1, R2, Q, B, L2, m, p, q, and r are as described above.

[0206] In some implementations, the conditions and operation of the reaction may be those conventional for this type of reaction in the art.

[0207] This disclosure provides a method for preparing the compound shown in formula (II-1), comprising the following steps: reacting the compound shown in formula (III) with the compound shown in formula (IV) as shown below to obtain the compound shown in formula (II-1); The definitions of L1, R1, R2, Q, B, L2, m, p, q, and r are as described above.

[0208] In some implementations, the conditions and operation of the reaction may be those conventional for this type of reaction in the art. Terminology Definition

[0209] On the other hand, without limiting specific configurations in this disclosure, the disclosed compounds may exist in specific geometric or stereoisomeric forms. This disclosure envisions all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and their racemic mixtures and other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure.

[0210] Furthermore, the compounds and intermediates disclosed herein may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton transfer, such as keto-enol and imine-enamine, and endo-imine isomerization. An example of an endo-imine equilibrium is between A and B as shown below.

[0211] All compounds disclosed herein may be classified as type A or type B. All tautomers are within the scope of this disclosure. The nomenclature of compounds does not exclude any tautomers.

[0212] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds containing asymmetric carbon atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0213] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound disclosed herein, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated, and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amine) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished using chromatography employing a chiral stationary phase and, as needed, combined with chemical derivatization (e.g., from amines to form carbamates).

[0214] This disclosure also includes compounds identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 123I, 125I, and 36Cl, respectively.

[0215] Unless otherwise stated, when a position is specifically designated as deuterium (D), that position should be understood as having a deuterium abundance of at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium doping). The natural abundance of deuterium in the example compounds can be at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even higher. This disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) by referring to relevant literature. When preparing the deuterated form of compound (I), commercially available deuterated starting materials can be used, or conventional techniques can be used to synthesize it with deuterated reagents, including but not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.

[0216] "As needed" or "as needed" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "C1-6 alkyl group substituted with halogen or cyano group as needed" means that halogen or cyano group may but does not have to be present, and the description includes cases where the alkyl group is substituted with halogen or cyano group and cases where the alkyl group is not substituted with halogen or cyano group.

[0217] In the chemical structure of the compound disclosed herein, the bond " "" indicates that the configuration is not specified, meaning that if a chiral isomer exists in the chemical structure, the bond " "can be " "or" ", or both contain " "and" "Two configurations. Although for simplicity all the above structural formulas are drawn in some isomer forms, this disclosure can include all isomers, such as tautomers, rotatimers, geometric isomers, diastereomers, racemates, and enantiomers. In the chemical structure of the compounds described in this disclosure, the bond " "No configuration specified, i.e., key" The configuration of “” can be E-type or Z-type, or it can contain both E-type and Z-type configurations at the same time.

[0218] The term "composition" refers to a mixture of a drug containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a composition is to facilitate drug delivery to a living organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity.

[0219] The terms “pharmaceutical-grade excipient” or “pharmaceutical-acceptable excipient” include, but are not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0220] Unless otherwise specified, the terms "compound," "ligand," "nucleic acid ligand conjugate," and "nucleic acid" disclosed herein may exist independently as salts, mixed salts, or non-salts (e.g., free acids or free bases). When present as salts or mixed salts, they may be pharmaceutically acceptable salts.

[0221] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0222] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylates, and naphthalenedisulfonates. These salts can be prepared by methods known in the art.

[0223] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium being the most preferred. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as amines, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0224] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 30 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and even more preferably an alkyl group containing 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, the alkyl group contains 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, syloxy, carboxyl, or carboxylic acid ester group.

[0225] The term "alkylene" refers to the portion of an alkane molecule remaining after the removal of two hydrogen atoms, comprising straight-chain and branched subgroups with 1 to 20 carbon atoms. Alkylenes containing 1 to 6 carbon atoms, in non-limiting examples, include methylene (-CH2-), ethylene (e.g., -CH2CH2- or -CH(CH3)-), propylene (e.g., -CH2CH2CH2- or -CH(CH2CH3)-), and butylene (e.g., -CH2CH2CH2CH2-). Unless otherwise specified, alkylenes can be substituted or unsubstituted; when substituted, the substituent can be substituted at any usable junction, preferably one or more of the following groups, independently selected from deuterium, aryl, heteroaryl, and halogen-substituted groups.

[0226] The terms "cycloalkyl" or "carbocyclic" refer to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. The cycloalkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any usable connection point. Preferably, one or more of the following groups are independently selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, wherein the C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl groups are substituted as needed by one or more groups selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano.

[0227] The cycloalkyl ring can be fused to an aryl or heteroaryl ring, wherein the ring connected to the parent structure is a cycloalkyl ring, and non-limiting examples include indenyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl group may be substituted or unsubstituted as desired. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, wherein the C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl is substituted as desired by one or more groups selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano.

[0228] The term "heterocyclic alkyl" or "heterocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, wherein 1 to 4 are heteroatoms; more preferably, it comprises 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocyclic alkyl groups include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic alkyl groups include spirocyclic, fused-ring, and bridged-ring heterocyclic alkyl groups. Non-limiting examples of "heterocyclic alkyl" include: and ,etc.

[0229] The heterocyclic alkyl ring can be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocyclic alkyl ring, and non-limiting examples include: and wait.

[0230] Heterocyclic alkyl groups may be substituted or unsubstituted as desired. When substituted, the substituents are preferably one or more of the following groups, independently selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocyclic alkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, wherein the C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocyclic alkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl groups are substituted as desired by one or more groups selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano.

[0231] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6 to 12-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic alkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, and non-limiting examples include:

[0232] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, wherein the C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl is substituted as desired by one or more groups selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano.

[0233] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl groups are preferably 5 to 12 members, more preferably 5 or 6 members. For example, non-limiting examples include: imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrole, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazole, benzimidazolyl, etc. , , wait.

[0234] The heteroaryl ring can be fused to an aryl, heterocyclic alkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0235] The heteroaryl group may be substituted or unsubstituted as desired. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl, wherein the C1-6 alkyl, C1-6 alkoxy, C2-6 alkenoxy, C2-6 alkynoxy, C3-6 cycloalkoxy, 3 to 6-membered heterocycloalkoxy, C3-8 cycloalkenoxy, 5 to 6-membered aryl or heteroaryl group is substituted as desired by one or more groups selected from halogen, deuterium, hydroxyl, lateral oxygen, nitro, cyano.

[0236] The term "spiroring" refers to a compound in which two rings share a single atom.

[0237] The term "spirocycloalkyl" refers to a polycyclic group consisting of 5 to 20 rings sharing a single carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Spirocycloalkyl groups are classified as monospirocycloalkyl, bispirocycloalkyl, or polyspirocycloalkyl groups based on the number of shared spiro atoms between the rings, with monospirocycloalkyl and bispirocycloalkyl groups being preferred. More preferably, they are 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 monospirocycloalkyl groups. "Spirocarbon ring" refers to the ring system within the spirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups include:

[0238] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or multispirocyclic groups based on the number of shared spiro atoms between rings, with monospirocyclic and bispirocyclic groups being more preferred. More preferably, it consists of 4 / 4, 4 / 5, 4 / 6, 5 / 5, or 5 / 6 monospirocyclic groups. "Spiroheterocyclic" refers to the ring system within the spirocyclic group. Non-limiting examples of spirocyclic groups include:

[0239] The term "fused ring" refers to a compound in which two or more rings are fused together by sharing two adjacent atoms.

[0240] The term "fused-ring alkyl" refers to a 5- to 20-membered polycyclic carbon group in which each ring in the system shares an adjacent pair of carbon atoms with the other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused-ring alkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused-ring alkyl groups include:

[0241] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. "Fused ring" refers to the ring system in the fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0242] The term "fused aryl" can refer to an unsaturated aromatic fused ring structure containing 5-14 ring atoms (including at least one heteroatom), formed by two or more ring structures sharing two adjacent atoms, and also includes cases where carbon, nitrogen, and sulfur atoms can be oxidized. Preferred terms include "5-12 member fused aryl", "7-12 member fused aryl", "9-12 member fused aryl", etc. Examples include benzofuranyl, benzoisofuranyl, benzothiopheneyl, indolyl, isoindolyl, benzoxazolyl, benzoimidazolyl, indazole, benzotriazolyl, quinolinyl, 2-quinolinoneyl, 4-quinolinoneyl, 1-isoquinolinoneyl, isoquinolinyl, acridineyl, phenanthidineyl, benzopyridinyl, phthalazinyl, quinazolinyl, quinoxalazinyl, quinoxalazinyl, phenazinyl, pteridineyl, purineyl, naphthidineyl, phenazinyl, phenothiazinyl, etc. "Dense aromatic rings" refers to the ring system in dense aromatic groups.

[0243] The fused heteroaryl group can be substituted or unsubstituted as needed. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamine, halogen, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0244] The term "bridged ring" refers to a structure formed by two or more ring structures sharing two non-adjacent ring atoms.

[0245] The term "bridged cycloalkyl" refers to a polycyclic aromatic hydrocarbon group with 5 to 20 members, in which any two rings share two non-directly bonded carbon atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it has 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0246] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0247] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be substituted or unsubstituted as desired. When substituted, the substituent is preferably one or more of the following groups, independently selected from halogens, deuterium, hydroxyl groups, oxy groups, nitro groups, cyano groups, C1-6 alkyl groups, C1-6 alkoxy groups, C2-6 alkenoxy groups, C2-6 alkynoxy groups, C3-6 cycloalkoxy groups, 3 to 6-membered heterocyclic alkoxy groups, C3-8 cycloalkenoxy groups, and 5 to 6-membered aryl or heteroaryl groups, wherein the C1-6 alkyl group, C1-6 alkoxy group, C2-6 alkenoxy group, C2-6 alkynoxy group, C3-6 cycloalkoxy group, 3 to 6-membered heterocyclic alkoxy group, C3-8 cycloalkenoxy group, and 5 to 6-membered aryl or heteroaryl group are substituted as desired by one or more groups selected from halogens, deuterium, hydroxyl groups, oxy groups, nitro groups, and cyano groups. Similarly, the definitions of "alkynoxy group," "alkenoxy group," "cycloalkoxy group," "heterocyclic alkoxy group," and "cycloalkenoxy group" are the same as those for "alkoxy group" as described above.

[0248] The term "substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions without much effort (by experiment or theory).

[0249] "Being replaced by one or more substituents" means that it can be replaced by one or more substituents. When replaced by multiple substituents, it can be a plurality of identical substituents or a combination of one or a plurality of different substituents.

[0250] The term "connection," when referring to the link between two molecules, means that the two molecules are linked by a covalent bond or by a non-covalent bond (e.g., hydrogen bond or ionic bond), including direct and indirect connections.

[0251] The term "direct link" refers to the connection between a first compound or group and a second compound or group without any intercalating atoms or groups. The term "indirect link" refers to the connection between a first compound or group and a second compound or group via an intermediate group, compound, or molecule (e.g., a linking group).

[0252] The term "hydroxyl group" refers to -OH.

[0253] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0254] The term "haloalkyl" refers to an alkyl group that has been substituted with a halogen, wherein the alkyl group is as defined above.

[0255] The term "cyano" refers to -CN.

[0256] The term "nitro" refers to -NO2.

[0257] The term "side oxygen" refers to the =O group. For example, a carbon atom and an oxygen atom are connected by a double bond, forming a ketone or aldehyde group.

[0258] The term "amine" refers to -NH2.

[0259] The term "cyano" refers to -CN.

[0260] The term "carboxyl group" refers to -C(O)OH.

[0261] The term "aldehyde group" refers to -CHO.

[0262] In this disclosure, the terms “comprising” and “including” can be replaced with “consisting of”.

[0263] In this disclosure, the terms "phosphate ester group," "phosphate ester group," and "phosphate ester bond" are used interchangeably, including monophosphate, diephosphate, or triphosphate. The term "phosphate ester group" in "thiophosphate ester group" has the same meaning. Unless otherwise specified, the internucleotide phosphate ester group in natural nucleotides is a diephosphate group.

[0264] In this disclosure, the thiophosphate group refers to a phosphate diester group modified by replacing a non-bridging oxygen atom with a sulfur atom. , (M represents S atoms) can be used interchangeably.

[0265] As used in this article, in the case of RNA-mediated gene silencing, the sense strand of siRNA (also known as SS, SS chain, or sense strand) refers to a strand containing a sequence that is identical or substantially identical to the target mRNA sequence; the antisense strand of siRNA (also known as AS or AS chain) refers to a strand containing a sequence that is complementary to the target mRNA sequence.

[0266] In this disclosure, the "5' region," also known as the "5' end" or "5' terminus," of the sense or antisense strand can be substituted. For example, the nucleotides at positions 2 to 8 of the 5' region of the antisense strand can be replaced with the nucleotides at positions 2 to 8 of the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand can also be substituted.

[0267] As used herein, the terms “complementary” or “reverse complementary” are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired in a complementary manner with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, “mismatch” in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired in a complementary manner.

[0268] As used herein, “chemical modification” or “modification” means a structure that is chemically different from its naturally occurring counterpart, including all alterations made by chemical means, such as the addition or removal of a chemical part, or the substitution of one chemical part for another.

[0269] In the context of this disclosure, Bz represents benzoyl; MMTr represents methoxyphenyl diphenylmethyl; and DMTr represents dimethoxytriphenylmethyl.

[0270] As used herein, the term "base" includes any known DNA and RNA base, base analogues such as purines or pyrimidines, and also includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, hypoxanthin, and natural analogues.

[0271] Unless otherwise specified, in the context of this disclosure, uppercase letters C, G, U, A, and T represent the base composition of nucleotides; lowercase letter d indicates that the nucleotide adjacent to the right of d is a deoxyribonucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of f is a fluorinated nucleotide; and lowercase letter s indicates that the two nucleotides adjacent to s are linked by thiophosphate groups.

[0272] As used herein, the term "fluorine-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribosome is replaced by fluorine; the term "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribosome is replaced by a methoxy group.

[0273] As used herein, the term “inhibition” may be used interchangeably with “reduction,” “silencing,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in one or more of these variables at an absolute or relative level compared to a control level. This control level can be any type of control level used in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only or inert control) subjects, cells, or samples. For example, the degree of inhibition of target gene expression by siRNA can be characterized by residual mRNA expression levels, such as residual mRNA expression levels not exceeding 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%. The inhibition rate of target gene expression can be detected using the Dual-Glo® Luciferase Assay System. The chemiluminescence values ​​of firefly and Renilla are read separately, and the relative value Ratio = Ren / Fir is calculated. In this disclosure, the proportion of remaining mRNA expression (or remaining activity %) = Ratio (siRNA treatment group) / Ratio (no siRNA control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0274] "Effective amount," "effective dose," "effective therapeutic amount," or "therapeutic effective amount" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition. For therapeutic applications, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various conditions related to the target gene, target mRNA, or target protein of this disclosure, or improving one or more symptoms of the condition, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the condition related to the target gene, target mRNA, or target protein in a patient. An effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0275] As used herein, the terms “subject,” “patient,” “subject,” or “individual” are used interchangeably and include human or non-human animals, such as mammals, such as humans or monkeys.

[0276] In some embodiments, after delivery of the oligomeric compound to cells expressing a gene, the oligomeric compound is able to inhibit the expression of the underlying gene and is referred to herein as an "expression-inhibiting oligomeric compound," which can inhibit gene expression in vitro or in vivo. "Oligomeric compounds" include, but are not limited to: oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribonucleotides, interfering RNA molecules, and Dicer enzyme receptors.

[0277] Unless otherwise specified, the symbols used in this article are as follows: This indicates that it can be connected with one or more groups according to the scope of disclosure described herein.

[0278] As used in this article, the first chain may be referred to as the antisense chain, and the second chain may be referred to as the justice chain. The terms first chain and antisense chain, or second chain and justice chain, should be considered interchangeable.

[0279] The “RNAi reagent” described in this disclosure refers to a reagent containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules capable of degrading or inhibiting the transcription and translation of target messenger RNA (mRNA) in a sequence-specific manner. The RNAi reagents described in this disclosure can be manipulated via RNA interference mechanisms (i.e., by inducing RNA interference through interaction with the RNA interference pathway-forming mechanisms of mammalian cells (RNA-induced silencing complex or RISC)) or through any other mechanism or pathway. RNAi reagents include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer receptors.

[0280] The RNAi reagents described herein include oligonucleotides having a strand that is at least partially complementary to the targeted mRNA. Simple Explanation of the Diagram

[0281] Figure 1 shows the expression levels of conjugates TRD002218, TRD007203, TRD007204, and TRD007205 in TTR 7 days after drug administration. Figure 2 shows the expression levels of conjugates TRD002218, TRD007203, TRD007204, and TRD007205 in the TTR 28 days after drug administration. Figure 3 shows the inhibitory activity of GalNAc conjugated siRNA on mTTR gene expression in mouse primary hepatocytes in Experiment 6. Figure 4 shows the in vivo inhibitory activity of GalNAc conjugated siRNA on the mouse mTTR gene in Experiment 7. Implementation

[0282] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified can be obtained from any supplier of molecular biology reagents at the quality / purity required for molecular biology applications.

[0283] compound [NAG0024]、 [NAG0026] was purchased from Tianjin WuXi AppTec New Drug Development Co., Ltd. Unless otherwise specified, all reagents used in the following examples are commercially available products.

[0284] [Preparation Example 1: Synthesis of Compound NAG0039]

[0285] [Compound 2] At room temperature, under a nitrogen atmosphere, in the compound [1] DIEA (469 mg, 3.63 mmol), 3A molecular sieve, monomethyl succinate (342 mg, 1.82 mmol), DCC (487 mg, 2.36 mmol), and HOBt (319 mg, 2.36 mmol) were added sequentially to a 20 mL solution of dried THF (1.00 g, 1.82 mmol). The reaction mixture was reacted overnight at 40 °C. The reaction mixture was filtered and concentrated, purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O), and lyophilized to give an intermediate (1.14 g, 1.58 mmol, yield 87%).

[0286] The intermediate (1.14 g, 1.58 mmol) was dissolved in THF (3 mL) and MeOH (3 mL), and then a solution of NaOH (126 mg, 3.16 mmol) in water (3 mL) was added dropwise. The reaction mixture was stirred at room temperature for 4 h. Most of the organic solvent was removed under reduced pressure, and then purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [2](851 mg, 1.17 mmol, sodium salt, yield 74%).

[0287] [Compound 3] At room temperature, under a nitrogen atmosphere, in the compound [NAG0026] (863 mg, 0.566 mmol) was added sequentially to a dry THF (15 mL) solution with DIEA (146 mg, 1.13 mmol), 3A molecular sieve, compound 2 (400 mg, 0.566 mmol), DCC (140 mg, 0.679 mmol), and HOBt (92 mg, 0.679 mmol). The reaction mixture was reacted overnight at 45 °C. The reaction solution was filtered and concentrated, purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O), and lyophilized to obtain the compound. [3](918mg, 0.415mmol, yield 73%).

[0288] [Compound NAG0039] Room temperature, towards the compound [3] A 3A molecular sieve, succinic anhydride (36 mg, 0.361 mmol), and DMAP (22 mg, 0.181 mmol) were added to a pyridine solution (400 mg, 0.181 mmol). The reaction was carried out overnight at 45 °C under a nitrogen atmosphere. The reaction solution was filtered, concentrated under reduced pressure, and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain 248 mg of crude product. The two batches of this crude product were combined and separated by HPLC preparative chromatography (column: Xbridge 150x50 mm, 5 μm; mobile phase: A: 0.1% NH3H2O ​​+ 0.005% FA aqueous solution; B: MeCN; gradient: 20% B-95% B within 9 min) to obtain the compound. [NAG0039] (240 mg, 0.104 mmol, yield 33%). MS(ESI)m / z=2312.3[M-1]-, Theoretical: 2313.0. 1H NMR(400MHz, Acetonitrile-d 3)δ 7.63-6.59(m,27H),5.40-5.22(m,4H),5.16-5.02(m,3H),4.74-4.57(m,3H),4.45-3.05(m,47H),2.70-1.96(m,57H),1.63-1.27(m,11H).

[0289] [Preparation Example 2: Synthesis of Compound NAG0046]

[0290] [Compound 3] Under a nitrogen atmosphere, the compound was added to the solvent DCM (5 mL). [2] (203 mg, 0.830 mmol), DIEA (0.206 mL, 1.246 mmol), HATU (237 mg, 0.623 mmol). The mixture was stirred at 20 °C, and then the compounds were... [1] (200 mg, 0.415 mmol) was added to the above system. After addition, the reaction was carried out at 20 °C for 1 h under a nitrogen atmosphere. H2O (10 mL) was added to the reaction solution and extracted with DCM (3 x 20 mL). The mixture was dried over anhydrous Na2SO4, filtered, concentrated, and the residue was purified by column chromatography (DCM:MeOH (100:0-90:10)) to obtain the compound. [3](250mg, yield 82%). LCMS: Chromatographic conditions 10-80 AB 2 min, retention time 1.534 min; MS (ESI) m / z = 682.6 [M+Na]+. H NMR(400MHz, CDCl3)δ 7.42(d,J=7.0Hz,1H),7.34-7.29(m,6H),7.21-7.17(m,2H),6.86(d,J=9.0Hz,4H),4.44(br d,J=7.5Hz,1H),4.31-4.08(m,1H),4.01-3.84(m,1H),3.82(d,J=2.0Hz,6H),3.69(d,J=2.3Hz,3H),3.1 7-3.06(m,1H),2.36-2.25(m,3H),2.19-1.97(m,4H),1.57-1.54(m,3H),1.47(d,J=6.8Hz,4H),1.28(br d, J = 6.8 Hz, 10 Hz.

[0291] [Compound 4] compound [3] (250 mg, 0.348 mmol) was dissolved in MeOH (1 mL) and H2O (0.5 mL), and then LiOH (146 mg, 3.48 mmol) was added. The reaction was carried out at room temperature for 12 h. The reaction solution was passed through a reverse-phase column to obtain the compound. [4](166mg, yield 71%). LCMS: Chromatographic conditions 10-80 CD_3 min, retention time 1.353 min; MS (ESI) m / z = 668.6 [M+Na]+. HPLC: Chromatographic conditions 10-80 CD_6 min, retention time 1.897 min. H NMR (400MHz, CD3OD) δ ppm 7.45(d,J=7.2Hz,2H),7.34-7.27(m,6H),7.24-7.19(m,1H),6.92-6.83(m ,4H),4.32(q,J=8.0Hz,1H),4.13-4.02(m,1H),3.80(s,6H),3.21-3.14(m, 1H),3.13-3.06(m,1H),2.37-2.28(m,1H),2.14(td,J=7.5,15.6Hz,5H),1. 99-1.89(m,1H),1.72-1.70(m,1H),1.59-1.50(m,4H),1.36-1.23(m,13H).

[0292] [Compound 5] compound [NAG0026] (120 mg, 0.079 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (2 mL), 3A molecular sieve (50 mg) was added, and then the compounds were added sequentially. [4] (54 mg, 0.084 mmol), HOBt (28 mg, 0.205 mmol), DCC (39 mg, 0.189 mmol), and DIEA (0.08 mL, 0.472 mmol). The reaction solution was reacted at 40 °C for 20 h. After the reaction was confirmed to be complete by LC-MS, water was added to quench the reaction, and the mixture was filtered. The filtrate was concentrated under reduced pressure and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [5] (90 mg, yield 53%).

[0293] [Compound NAG0046] compound [5] (90 mg, 0.042 mmol) was dissolved in anhydrous pyridine (3 mL), and 3A molecular sieve (50 mg), DMAP (26 mg, 0.209 mmol), and succinic anhydride (42 mg, 0.364 mmol) were added. The reaction solution was stirred at 50 °C for 48 h. LC-MS showed that the reactant had reacted to approximately 50%. The reaction solution was filtered, concentrated, and purified by reversed-phase column chromatography (Boston C18 column, H2O / MeCN, extraction from 5% to 70%) to obtain [NAG0046] (30mg). MS(ESI)m / z=2251.4[M-1]-, Theoretical: 2252.0. 1H NMR(400MHz, Acetonitrile-d 3)δ 7.50-7.14(m,14H),6.89(d,J=8.6Hz,7H),6.56(d,J=7.8Hz,1H),5.32(d,J=3. 4Hz,3H),5.09(t,J=12.5Hz,4H),4.65(dd,J=8.6,4.8Hz,3H),4.32-3.11(m,52 H),2.54(t,J=6.8Hz,3H),2.32(d,J=7.6Hz,16H),2.14-2.04(m,17H),1.89(d, J=2.9Hz,10H),1.75(dt,J=14.6,7.9Hz,4H),1.62-1.49(m,5H),1.29(s,13H).

[0294] [Preparation Example 3: Synthesis of Compound NAG0047]

[0295] [Compound 3] At 0°C and under a nitrogen atmosphere, the compound was... [2] SOCl2 (0.581 mL, 8.009 mmol) was added dropwise to a 10 mL solution of MeOH (300 mg, 1.60 mmol). The mixture was stirred at 60 °C for 3 hours. The reaction solution was then concentrated to obtain the compound. [3](0.32g, 1.510mmol, yield 95%). 1H NMR: (400MHz, CD3OD) δ ppm 3.75-3.59(m,3H),2.93(t,J=7.2Hz,2H),2.33(t,J=7.4Hz,2H),1.64(td,J=7.0,14.4Hz,4H),1.48-1.27(m,10H).

[0296] [Compound 4] At 0°C and under a nitrogen atmosphere, the compound was... [1] DIEA (0.267 mL, 1.61 mmol), 4A molecular sieve (500 mg), and compound were added to a 25 mL THF (250 mg, 0.538 mmol) solution. [3] (271 mg, 1.08 mmol), DCC (333 mg, 1.61 mmol), and HOBt (218 mg, 1.61 mmol). The reaction solution was stirred at 50 °C for 5 hours. After the reaction was completed, it was quenched with H2O (50 mL) and extracted with EtOAc (100 mL x 3). The combined organic phases were washed with saturated brine (50 mL), dried over Na2SO4, filtered, concentrated, and then obtained by silicone column chromatography (0-80% EtOAc / PE) to obtain the compound. [4](420mg, 0.486mmol, yield 90%). LCMS: Chromatographic conditions 10-80 CD_7 min, retention time 5.586 min; MS (ESI) m / z = 670.3 [M+Na]+. 1H NMR: (400MHz, CD3OD) δ ppm 7.44(d,J=7.6Hz,2H),7.36-7.29(m,6H),7.28-7.22(m,1H),6.89(d,J=8.8Hz,4H),4.53(t,J=7.8Hz,1H),4.3 5-4.27(m,1H),4.02(q,J=4.0Hz,1H),3.86-3.74(m,6H),3.66(s,3H),3.52-3.42(m,1H),3.33-3.30(m,1H),3. 29-3.23(m,1H),3.18(td,J=7.0,13.5Hz,1H),3.00(td,J=7.0,13.5Hz,1H),2.33-2.22(m,3H),2.14(ddd,J=5 .8,7.8,13.3Hz,1H),1.92-1.82(m,2H),1.73(td,J=3.6,13.2Hz,2H),1.66-1.53​​(m,3H),1.43-1.26(m,7H).

[0297] [Compound 5] At 20°C, the compound was subjected to... [4] LiOH (82 mg, 1.95 mmol) was added to a solution of 420 mg (0.486 mmol) THF (5 mL) and H2O (2 mL). The reaction solution was stirred at 20 °C for 15 hours. After the reaction was completed, the solution was concentrated and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [5](236 mg, 0.155 mmol, 32% yield, lithium salt). LCMS: Chromatographic conditions 0-60 CD_7 min, retention time 3.950 min; MS (ESI) m / z = 656.4 [M+Na]+.

[0298] [Compound 6] compound [NAG0026] (236 mg, 0.155 mmol) was dissolved in anhydrous THF (3 mL), and 3A molecular sieve (100 mg) was added, followed by the addition of compounds sequentially. [5] (98 mg, 0.155 mmol), HOBt (25 mg, 0.186 mmol), DCC (41 mg, 0.201 mmol), and DIEA (0.08 mL, 0.464 mmol). The reaction solution was reacted at 40 °C for 16 h. After the reaction was confirmed to be complete by LC-MS, water was added to quench the reaction, and the mixture was filtered. The filtrate was concentrated and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [6](160mg, 0.075mmol, yield 48%).

[0299] [Compound NAG0047] compound [6] (160 mg, 0.075 mmol) was dissolved in anhydrous pyridine (3 mL), and 3A molecular sieve (100 mg), DMAP (46 mg, 0.374 mmol), and succinic anhydride (75 mg, 0.747 mmol) were added sequentially. The reaction solution was stirred at 50 °C for 48 h, filtered, concentrated, and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain [NAG0047] (80mg). MS(ESI)m / z=2239.1[M-1]-, Theoretical: 2240.0. 1H NMR(400MHz, Acetonitrile-d 3)δ 7.53-7.09(m,14H),6.89(dd,J=7.2,5.1Hz,8H),5.31(dt,J=3.2,1.5Hz, 3H),5.22-5.05(m,4H),4.70-4.61(m,3H),4.44(dd,J=9.7,6.7Hz,1H),4 .29(dq,J=14.6,7.6Hz,2H),4.18-3.84(m,16H),3.80(s,6H),3.69(dd,J =11.2,4.3Hz,3H),3.64-3.03(m,24H),2.57(h,J=2.3Hz,5H),2.36-2.27 (m,7H),2.24-2.16(m,4H),2.11(s,3H),2.01-1.96(m,22H),1.88(q,J=2.9,2.2Hz,8H),1.56(s,2H),1.24(d,J=17.7Hz,13H).

[0300] [Preparation Example 4: Synthesis of Compound NAG0048]

[0301] [Compound 3] At 0°C and under a nitrogen atmosphere, the compound was... [2] SOCl2 (0.968 mL, 13.3 mmol) was added dropwise to a 10 mL solution of MeOH (500 mg, 2.67 mmol). The mixture was stirred at 60 °C for 12 hours. The reaction solution was then concentrated to obtain the compound. [3](550mg, 2.46mmol, 92%). 1H NMR: (400MHz, CD3OD) δ ppm 3.69-3.62(m,3H),2.94(s,2H),2.33(t,J=7.2Hz,2H),1.73-1.57(m,4H),1.46-1.31(m,10H).

[0302] [Compound 4] At 0°C and under a nitrogen atmosphere, the compound was... [1] DIEA (0.267 mL, 1.614 mmol), 4A molecular sieve (500 mg), and compound were added to a 25 mL THF (250 mg, 0.538 mmol) solution. [3] (271 mg, 1.08 mmol), DCC (333 mg, 1.61 mmol), and HOBt (218 mg, 1.61 mmol). The reaction solution was stirred at 50 °C for 5 hours. After the reaction was completed, it was quenched with H2O (50 mL) and extracted with EtOAc (100 mL x 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous Na2SO4, filtered, concentrated, and purified by silicone column chromatography (0-80% EtOAc / PE) to obtain the compound. [4](350mg, 0.486mmol, yield 90%). LCMS: Chromatographic conditions 10-80 CD_7 min, retention time 5.670 min; MS (ESI) m / z = 670.3 [M+Na]+. 1H NMR: (400MHz, CD3OD) δ ppm 7.45(d,J=7.4Hz,2H),7.36-7.27(m,6H),7.25-7.19(m,1H),6.90-6.85(m,4H),4.50(dd,J=4.6,9.0Hz,1H),4 .27(td,J=3.0,5.6Hz,1H),4.20-4.14(m,1H),3.80(s,6H),3.68-3.62(m,3H),3.53-3.43(m,1H),3.27-3.18( m,3H),3.16-3.09(m,1H),2.54(ddd,J=5.8,8.8,13.2Hz,1H),2.31(t,J=7.4Hz,2H),2.08(td,J=4.0,13.2Hz, 1H),1.91-1.82(m,2H),1.73(td,J=3.6,13.2Hz,2H),1.67-1.50(m,5H),1.39-1.32(m,2H),1.27-1.12(m,3H).

[0303] [Compound 5] At 20°C, the compound was subjected to... [4] LiOH (82 mg, 1.95 mmol) was added to a solution of 350 mg (0.486 mmol) THF (5 mL) and H2O (2 mL). The reaction solution was stirred at 20 °C for 15 hours. After the reaction was completed, the compound was directly concentrated to obtain the compound. [5](300mg, 0.469mmol, yield 96%). LCMS: Chromatographic conditions 0-60 CD_7 min, retention time 4.081 min; MS (ESI) m / z = 656.4 [M+Na]+.

[0304] [Compound 6] compound [NAG0026] (300 mg, 0.197 mmol) was dissolved in anhydrous THF (5 mL), 3A molecular sieve (100 mg) was added, and then the compounds were added sequentially. [5] (125 mg, 0.197 mmol), HOBt (32 mg, 0.236 mmol), DCC (53 mg, 0.256 mmol), and DIEA (0.10 mL, 0.590 mmol). The reaction solution was reacted at 40 °C for 16 h. After the reaction was confirmed to be complete by LC-MS, water was added to quench the reaction, and the mixture was filtered. The filtrate was concentrated under reduced pressure and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [6](260mg, yield 62%).

[0305] [Compound NAG0048] compound [6] (260 mg, 0.121 mmol) was dissolved in anhydrous pyridine (4 mL), and 3A molecular sieve (100 mg), DMAP (30 mg, 0.242 mmol), and succinic anhydride (73 mg, 0.726 mmol) were added sequentially. The reaction solution was stirred at 50 °C for 48 h. LC-MS showed that the reactant had reacted to approximately 70%. The reaction solution was filtered, concentrated, and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain [NAG0048] (170 mg, yield 62%). MS(ESI)m / z=2239.1[M-1]-, Theoretical: 2240.0. 1H NMR(400MHz, Acetonitrile-d 3)δ 7.68(s,3H),7.51-7.17(m,12H),7.11-6.85(m,7H),5.31(dt,J=2.5,1.3Hz,3H),5.18-5.04(m,4H),4.74- 4.53(m,4H),4.30(d,J=4.9Hz,3H),4.21-3.93(m,12H),3.79(s,9H),3.69(dt,J=10.4,4.7Hz,3H),3.62-3 .14(m,22H),3.09(dd,J=10.1,4.4Hz,2H),2.91(d,J=7.2Hz,1H),2.46(s,6H),2.28(ddt,J=28.8,20.0,7. 2Hz,8H),2.11(s,3H),2.01-1.96(m,18H),1.90-1.87(m,8H),1.54(d,J=25.2Hz,4H),1.37-1.18(m,16H).

[0306] [Preparation Example 5: Synthesis of Compound NAG0049]

[0307] [Compound 3] compound [2] (293 mg, 1.20 mmol) was dissolved in DCM (3 mL), DIEA (0.298 mL, 1.80 mmol) and HATU (457 mg, 1.20 mmol) were added, and then the compound was added. [1] (300 mg, 0.601 mmol, derived from Preparation Example 7), reacted at 20 °C for 1 hour. The reaction solution was extracted with dichloromethane (60 mL) and water (60 mL), the organic phase was washed three times with water (60 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography (PE:EtOAc = 0:1) to give the compound. [3](300mg, yield 90%). LCMS: Chromatographic conditions 30-90 CD_3 min, retention time 2.225 min; MS (ESI) m / z = 698.4 [M+Na]+.

[0308] [Compound 4] compound [3] (300 mg, 0.444 mmol) was dissolved in THF (3 mL) and H2O (1 mL), and LiOH·H2O (75 mg, 1.78 mmol) was added. The reaction mixture was reacted at 20 °C for 12 hours. The reaction solution was concentrated under reduced pressure, dissolved in water (5 mL) and methanol (5 mL), and purified by reverse-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [4](212mg, yield 100%). LCMS: Chromatographic conditions 10-80 CD_3 min, retention time 1.333 min; MS (ESI) m / z = 684.3 [M+Na]+. HPLC: Chromatographic conditions 10-80 CD_6 min, retention time 1.853 min. 1H NMR: (400MHz, CD3OD) δ ppm 7.47-7.38(m,2H),7.35-7.25(m,6H),7.24-7.17(m,1H),6.86(d,J=8.8Hz,4H),4.30-4.17(m,2H),3.99-3.88(m,1H),3.78 (s,6H),3.42-3.33(m,2H),3.16-3.04(m,2H),2.18-2.05(m,4H),1.92-1.70(m,2H),1.65-1.46(m,4H),1.36-1.17(m,12H).

[0309] [Compound 5] compound [NAG0026] (300 mg, 0.197 mmol) was dissolved in anhydrous DMF (3 mL), 3A molecular sieve (100 mg) was added, and then the compounds were added sequentially. [4] (143 mg, 0.216 mmol), HOBt (32 mg, 0.236 mmol), DCC (53 mg, 0.256 mmol), and DIEA (0.1 mL, 0.590 mmol). The reaction solution was reacted at 40 °C for 16 h. After the reaction was confirmed to be complete by LC-MS, water was added to quench the reaction, and the mixture was filtered. The filtrate was concentrated and purified by reversed-phase column chromatography (Boston C18 column, H2O / MeCN, extraction from 5% to 80%) to obtain the compound. [5](110mg, yield 28%).

[0310] [Compound NAG0049] compound [5] (110 mg, 0.051 mmol) was dissolved in anhydrous pyridine (3 mL), and 3A molecular sieve (100 mg), DMAP (31 mg, 0.255 mmol), and succinic anhydride (51 mg, 0.510 mmol) were added sequentially. The reaction solution was stirred at 50 °C for 48 h. LC-MS showed that the reactant had reacted to approximately 50%. The reaction solution was filtered, concentrated, and purified by reversed-phase column chromatography (Boston C18 column, H2O / MeCN, extraction from 5% to 70%) to obtain [NAG0049] (45mg, yield 39%). MS(ESI)m / z=2267.0[M-1]-, Theoretical: 2268.0. 1H NMR(400MHz,CH3CN-d 3)δ 7.62-6.40(m,22H),5.31(s,3H),5.21-5.02(m,4H),4.64(dd,J=8.7,6.2Hz,3H),4.16(s,2H),4.13-3.94(m,14H),3.79(s,10H),3. 72-3.38(m,24H),3.32-3.11(m,5H),2.56(t,J=3.4Hz,4H),2.34-2.20(m,16H),1.99(d,J=11.3Hz,24H),1.41(d,J=127.6Hz,21H).

[0311] [Preparation Example 6: Synthesis of Compound NAG0050]

[0312] [Compound 3] compound [2] (435 mg, 1.780 mmol) was dissolved in DCM (10 mL), DIEA (0.441 mL, 2.67 mmol) and HATU (677 mg, 1.78 mmol) were added, and then the compound was added. [1] (400 mg, 0.890 mmol, derived from Preparation Example 8), reacted at 20 °C for 1 hour. The reaction solution was extracted with dichloromethane (60 mL) and water (60 mL), the organic phase was washed three times with water (60 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (PE:EtOAc = 0:1) to give the compound. [3](600mg, yield 90%). LCMS: Chromatographic conditions 30-90 CD_3 min, retention time 2.745 min; MS (ESI) m / z = 698.4 [M+Na]+. 1H NMR: (400MHz, CD3OD) δ ppm 7.46-7.38(m,2H),7.35-7.24(m,6H),7.22-7.16(m,1H),6.90-6.78(m,4H),4.29-4.21(m,2H),4.02-3.95(m,1H),3.7 7(s,6H),3.66-3.62(m,3H),3.41(s,1H),3.18-3.04(m,2H),2.36-2.17(m,5H),1.71-1.50(m,5H),1.39-1.25(m,14H).

[0313] [Compound 4] compound [3] (600 mg, 0.799 mmol) was dissolved in THF (3 mL) and H2O (1 mL), and LiOH·H2O (134 mg, 3.20 mmol) was added. The reaction mixture was reacted at 20 °C for 12 hours. The reaction solution was concentrated under reduced pressure, dissolved in water (5 mL) and methanol (5 mL), and purified by reverse-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [4](460 mg, 100% yield, lithium salt). LCMS: Chromatographic conditions 10-80 CD_3 min, retention time 1.346 min; MS (ESI) m / z = 684.3 [M+Na]+. HPLC: Chromatographic conditions 10-80 CD_6 min, retention time 1.879 min. 1H NMR: (400MHz, CD3OD) δ ppm 7.47-7.39(m,2H),7.35-7.24(m,6H),7.22-7.15(m,1H),6.91-6.79(m,4H),4.31-4.18(m,2H),4.02-3.95(m,1H),3.78(s ,6H),3.44-3.33(m,2H),3.18-3.04(m,2H),2.35-2.27(m,1H),2.24-2.10(m,4H),1.70-1.51(m,5H),1.31-1.23(m,12H).

[0314] [Compound 5] compound [NAG0026] (300 mg, 0.197 mmol) was dissolved in anhydrous DMF (3 mL), 3A molecular sieve (100 mg) was added, and then the compounds were added sequentially. [4] (143 mg, 0.216 mmol), HOBt (32 mg, 0.236 mmol), DCC (53 mg, 0.256 mmol), and DIEA (0.1 mL, 0.590 mmol). The reaction solution was reacted at 40 °C for 16 h. After the reaction was confirmed to be complete by LC-MS, water was added to quench the reaction, and the mixture was filtered. The filtrate was concentrated and purified by reversed-phase column chromatography (Boston C18 column, H2O / MeCN, extraction from 5% to 80%) to obtain the compound. [5](300mg, yield 73%).

[0315] [Compound NAG0050] compound [5] (310 mg, 0.143 mmol) was dissolved in anhydrous pyridine (5 mL), and 3A molecular sieve (100 mg), DMAP (87 mg, 0.714 mmol), and succinic anhydride (143 mg, 1.429 mmol) were added sequentially. The reaction mixture was stirred at 50 °C for 48 h. LC-MS showed that the reactant had reacted to approximately 50%. The reaction mixture was filtered, concentrated, and purified by reversed-phase column chromatography (Boston C18 column, H2O / MeCN, extraction from 5% to 70%) to obtain [NAG0050] (140 mg, yield 43%). MS(ESI)m / z=2267.1[M-1]-, Theoretical: 2268.0. 1H NMR(400MHz,CH3CN-d 3)δ 7.54-7.14(m,14H),6.97-6.64(m,8H),5.32(d,J=3.4Hz,3H),5.21-5.04(m,4H),4.71-4.60(m ,3H),4.30(d,J=6.8Hz,3H),4.17-3.95(m,14H),3.93-3.84(m,3H),3.79(s,7H),3.74-3.65(m ,3H),3.63-3.08(m,26H),2.57(d,J=2.0Hz,6H),2.46-2.28(m,8H),2.20(dt,J=15.1,7.4Hz,5 H),2.11(s,2H),2.01-1.97(m,16H),1.90-1.88(m,9H),1.65-1.55(m,4H),1.39-1.20(m,14H).

[0316] [Preparation Example 7: Compound NAG0051]

[0317] [Compound 2] Add the compound to the solvent dichloromethane (100 mL) [1] (5.00 g, 21.4 mmol) and TEA (8.04 mL, 57.9 mmol) [。] The mixture was stirred at 0°C, and DMTrCl (9.08 g, 26.8 mmol) was slowly added to the above system. After the addition was complete, the temperature was raised to 25°C and the reaction was carried out for 12 h. A saturated sodium bicarbonate solution (100 mL) was added to the reaction solution, and the mixture was extracted with DCM (100 x 3). The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (PE:EtOAc 100:0-70:30) to obtain the compound. [2](12g, yield 94%). LCMS: Chromatographic conditions 5-95AB_1.5min, retention time 1.166min; MS(ESI) m / z=558.1[M+Na]+. 1H NMR: (400MHz, CDCl3) δ ppm 7.52-7.47(m,2H),7.42-7.35(m,8H),7.34-7.27(m,3H),7.26-7.19(m,1 H),6.89-6.81(m,4H),5.68(d,J=5.6Hz,1H),5.18-5.08(m,3H),4.83(br d,J=9.2Hz,1H),4.55-4.42(m,2H),3.82(s,6H),2.43-2.29(m,1H),1.39-1.32(m,2H).

[0318] [Compound 3] Add the compound to the solvent THF (100 mL) [2] (6.00 g, 10.1 mmol), BH3THF (1 M, 20.2 mL, 20.2 mmol) was added to the above system at 0 °C. After the addition was complete, the reaction was carried out at 20 °C for 12 h. Then the temperature was lowered to 0 °C, and EtOH (6 mL) and NaOH (20 mL, 60.0 mmol) were slowly added at 0 °C. Finally, H2O2 (20 mL, 33.4 mmol) was slowly added dropwise to the reaction solution at 0 °C, and then the temperature was slowly raised to 20 °C for 12 h. The reaction solution was filtered, and H2O (200 mL) and EtOAc (200 mL) were added. The solution was extracted with EtOAc (3 x 200 mL), washed with saturated Na2SO3 (100 mL) solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (DCM / MeOH) to obtain 3.5 g of crude product. The product was then separated by alkaline preparative chromatography (column: Xbridge 150x50 mm, 5 μm; mobile phase: A: 0.1% NH3H2O ​​+ 0.005% FA aqueous solution; B: MeCN; gradient: 20% B - 95% B in 9 min) to obtain the compound. [3] (850 mg, yield 15%) and compounds [4] (800 mg, yield 14%).

[0319] [Compound 3] LCMS: Chromatographic conditions 10-80AB_7min, retention time 4.214min; MS(ESI) m / z=576.3[M+Na]+. 1H NMR: (400MHz, CDCl3) δ ppm 7.31-7.23(m,6H),7.23-7.19(m,5H),7.12-7.08(m,3H),6.79-6.73(m,4H),5.12(s,1H),5.01(s,2H ),4.13(s,2H),4.01-3.95(m,1H),3.75-3.71(m,6H),2.48-2.27(m,1H),2.12-1.84(m,2H),1.57(br d,J=14.6Hz,1H).

[0320] [Compound 4] LCMS: Chromatographic conditions 10-80AB_7min, retention time 4.305min; MS(ESI) m / z=576.3[M+Na]+. 1H NMR: (400MHz, CDCl3) δ ppm 7.37-7.33(m,1H),7.31-7.19(m,11H),7.15-7.08(m,2H),6.78-6.69(m,4H),5.08-4.98(m,3H),4.39- 4.05(m,2H),3.71(m,6H),3.54-3.36(m,1H),2.37-1.97(m,1H),1.87-1.65(m,2H),1.14-0.99(m,1H).

[0321] [Compound 5] Pd / C 5% (300 mg, 0.141 mmol) was added to the compound. [3] 600 mg (1.030 mmol) of EtOAc (10 mL) solution was reacted in a hydrogen atmosphere (15 psi) at 20 °C for 2 h. The mixture was filtered and concentrated to obtain a crude product, which was then separated by alkaline preparative chromatography (column: Xbridge 150 x 50 mm, 5 μm; mobile phase: A: 0.1% NH3H2O ​​+ 0.005% FA aqueous solution; B: MeCN; gradient: 20% B - 95% B in 9 min) to obtain the compound. [5](301mg, yield 70%). LCMS: Chromatographic conditions 10-80 CD_3 min, retention time 2.444 min; MS (ESI) m / z = 839.5 [2M+H]+. HPLC: Chromatographic conditions 10-80 CD_7 min, retention time 4-100 min. 1H NMR: (400MHz, CD3OD) δ ppm 7.49-7.44(m,2H),7.34(d,J=8.8Hz,4H),7.32-7.26(m,2H),7.24-7.18(m,1H),6.91-6.82(m,4H),4.22-4.11(m,1H),3.85 -3.81(m,1H),3.79(s,6H),2.78-2.68(m,1H),1.90-1.83(m,1H),1.73-1.65(m,1H),1.41-1.33(m,1H),1.29-1.25(m,1H).

[0322] [Compound 7] compound [6] (233 mg, 0.953 mmol), HATU (272 mg, 0.715 mmol), and DIEA (0.236 mL, 1.430 mmol) were dissolved in DCM (10 mL), and the compound was added. [5] (200 mg, 0.477 mmol), reacted at 25 °C for 1 hour. H₂O (20 mL) was added, and the mixture was extracted with DCM (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by column chromatography (PE / EtOAc = 1 / 1~1 / 2) to obtain the compound. [7](290mg). LCMS: Chromatographic conditions 10-80 CD_3 min, retention time 2.834 min; MS (ESI) m / z = 668.4 [M+Na]+. 1H NMR: (400MHz, CDCl3) δ ppm 7.49-7.44(m,2H),7.41-7.30(m,6H),7.25-7.18(m,1H),6.89-6.79(m ,4H),4.30-4.07(m,2H),3.87-3.79(m,7H),3.73-3.64(m,4H),2.8-2. 80(m,7H),2.32(t,J=7.6Hz,2H),2.24-2.13(m,2H),2.01-1.89(m,1H) ,1.80-1.75(m,1H),1.65-1.60(m,5H),1.58(s,4H),1.20-1.10(m,1H).

[0323] [Compound 8] compound [7] (290 mg, 0.404 mmol) was dissolved in THF (10 mL) and H2O (5 mL), and LiOH (51 mg, 1.212 mmol) was added. The reaction was carried out at 25 °C for 3 hours. TLC (DCM / MeOH = 10 / 1) showed that about 20% of the starting material had not reacted completely. LiOH (50 mg) was added, and the reaction was carried out at 25 °C for 12 hours. TLC (DCM / MeOH = 10 / 1) showed that the starting material had reacted completely. The reaction solution was concentrated, and the residue was separated by reversed-phase chromatography (H2O / CH3CN = 5 / 1~3 / 1) to obtain the compound. [8](217mg). LCMS: Chromatographic conditions 5-95 CDN_1.5 min, retention time 0.879 min; MS (ESI) m / z = 630.3 [MH]+. HPLC: Chromatographic conditions 10-80 CD_7 min, retention time 2.677 min. 1H NMR: (400MHz, CD3OD) δ ppm 7.50-7.44(m,2H),7.38-7.32(m,4H),7.32-7.27(m,2H),7.24-7.19(m ,1H),6.91-6.85(m,4H),4.25-4.14(m,1H),4.02-3.96(m,1H),3.80(s, 6H),3.73-3.65(m,1H),2.20-2.10(m,4H),1.97-1.86(m,1H),1.75-1. 70(m,1H),1.66-1.53​​(m,4H),1.51-1.41(m,1H),1.37-1.26(m,13H).

[0324] [Compound 9] compound [NAG0026] (300 mg, 0.197 mmol) was dissolved in anhydrous DMF (4 mL), 3A molecular sieve (100 mg) was added, and then the compounds were added sequentially. [8] (137 mg, 0.217 mmol), HOBt (32 mg, 0.236 mmol), DCC (53 mg, 0.256 mmol), and DIEA (0.10 mL, 0.591 mmol). The reaction solution was reacted at 40 °C for 16 h. The reaction was quenched with water and filtered. The filtrate was concentrated and purified by reversed-phase column chromatography (Boston C18 column, H2O / MeCN, extraction from 5% to 80%) to obtain the compound. [9](330mg).

[0325] [Compound NAG0051] compound [9] (330 mg, 0.154 mmol) was dissolved in anhydrous pyridine (5 mL), and 3A molecular sieve (100 mg), DMAP (94 mg, 0.771 mmol), and succinic anhydride (154 mg, 1.543 mmol) were added sequentially. The reaction mixture was stirred at 50 °C for 48 h. LC-MS showed that the reactant had reacted to approximately 60%. The reaction mixture was filtered, concentrated, and purified by reversed-phase column chromatography (Boston C18 column, H2O / MeCN, extraction from 5% to 70%) to obtain [NAG0051] (190mg). MS(ESI)m / z=2237.3[M-1]-, Theoretical: 2238.0. 1H NMR(400MHz, Acetonitrile-d 3)δ 7.54-7.16(m,14H),7.01-6.53(m,8H),5.32(d,J=5.1Hz,3H),5.20-4.89(m,4H),4.65(q,J=7.2Hz,3H ),4.38-3.20(m,52H),2.51-2.44(m,4H),2.36-2.20(m,20H),2.02-1.97(m,20H),1.64-1.20(m,22H).

[0326] [Preparation Example 8: Synthesis of NAG0052]

[0327] [Compound 3] At room temperature and under a nitrogen atmosphere, the compound was... [NAG0024] (271 mg, 0.151 mmol) was dissolved in anhydrous THF (2 mL) and anhydrous DMF (4 mL), 3A molecular sieve was added, and then the compounds were added sequentially. [2] (100 mg, 0.151 mmol), HOBt (25 mg, 0.181 mmol), DCC (38 mg, 0.181 mmol), and DIEA (39 mg, 0.302 mmol). The reaction solution was reacted at 45 °C for 16 h. After the reaction was confirmed to be complete by LC-MS, water was added to quench the reaction, and the mixture was filtered. The filtrate was concentrated and purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O) to obtain the compound. [3](210mg, yield 57%).

[0328] [Compound NAG0052] Room temperature, compound [3] (230 mg, 0.094 mmol) was dissolved in pyridine (5 mL), and molecular sieves were added. DMAP (12 mg, 0.283 mmol) and succinic anhydride (28 mg, 0.283 mmol) were added. The mixture was stirred at 50 °C for 16 hours under a nitrogen atmosphere. The reaction was detected by LCMS to be complete. The mixture was filtered and concentrated under reduced pressure. The residue was purified by reversed-phase column chromatography (Boston C18 column, 0-100% MeCN / H2O), and then purified again by preparative HPLC chromatography (column: Xbridge 150x50 mm, 5 μm; mobile phase: A: 0.1% NH3H2O ​​+ 0.005% FA aqueous solution B: MeCN; gradient: 20% B-95% B in 9 min) to obtain the compound. [NAG0052] (123 mg, 0.048 mmol, yield 51%). MS(ESI)m / z=2535.3[M-1]-, Theoretical: 2536.2. 1H NMR(400MHz,CH3CN-d 3) δ7.48-7.43(m,2H),7.37-7.12(m,11H),7.00-6.85(m,10H),6.66(s,1H),5.31(dd,J=3.4,1.1Hz,3H),5.20- 5.13(m,1H),5.05(dd,J=11.3,3.4Hz,3H),4.56(d,J=8.5Hz,3H),4.30(dd,J=7.7,5.3Hz,1H),4.18-3.93(m,14H ),3.79(s,10H),3.65(q,J=4.7,3.6Hz,13H),3.56-3.07(m,24H),2.56(s,6H),2.37(t,J=5.8Hz,10H),2.17(t, J=7.5Hz,9H),2.02-1.96(m,20H),1.88(s,8H),1.82-1.73(m,2H),1.60(dt,J=15.0,7.3Hz,16H),1.27(s,13H).

[0329] [Preparation Example 9: Synthesis of L96 and NAG1]

[0330] L96 was prepared according to the method described in patent application WO2014025805A1, and NAG1 was prepared according to the method described in patent application WO2021254360A1. The above patent applications (in full) are incorporated herein by reference.

[0331] [Preparation Example 10: Synthesis of Nucleic Acid Ligand Conjugates] 1. Preparation of resin with carrier Compounds containing carboxylic acid groups [NAG0050] (140 mg, 0.062 mmol) was dissolved in anhydrous DMF (3 mL). After the reactant was completely dissolved, anhydrous acetonitrile (4 mL), DIEA (0.03 mL, 0.154 mmol, 2.5 eq), and HBTU (35 mg, 0.093 mmol, 1.5 eq) were added sequentially. After the reaction solution was mixed thoroughly, macroporous amine methyl resin (476 mg, blank loading 0.41 mmol / g, target loading 0.1 mmol / g) was added. The reaction solution was placed on a shaker (temperature: 25 °C, speed: 200 rpm) and shaken overnight. The reaction solution was filtered, and the filter cake was washed sequentially with DCM and anhydrous acetonitrile. The solid was collected and dried under vacuum overnight.

[0332] The solid from the previous step was dispersed in anhydrous acetonitrile (5 mL), and pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL), and CapB1 (2.68 mL) were added sequentially. The reaction mixture was placed on a shaker (temperature: 25℃, speed: 200 rpm) and shaken for 2 h. The reaction mixture was filtered, the filter cake was washed with anhydrous acetonitrile, the solid was collected, and vacuum dried overnight to obtain the resin with the support. The loading was determined to be 0.1 mmol / g.

[0333] compound [NAG0051] and [NAG0052] A resin with a carrier was obtained using the same reaction conditions.

[0334] 2. For those already bonded to the resin [NAG0050-NAG0052] uses this resin as a starting material to sequentially link nucleoside monomers from the 3'-5' direction according to the nucleotide arrangement sequence. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. Instrument models: Biolytic Dr. Oligo 48 solid phase synthesizer, Comma BioEmbed™ CPG Frits universal synthesis column DS0200, Comma BioEmbed™ 96-well plate desalting column DC189650 (80mg).

[0335] Table 1. Reagents used for synthesizing siRNA conjugates

[0336] The synthesis conditions are as follows: Nucleoside monomers were provided in 0.05 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 3 min reaction time, DCA as the deprotection reagent, and 180 μL injection volume.

[0337] The coupling reaction conditions were identical for each step, including a temperature of 25°C and a reaction time of 3 minutes. The injection volume of the nucleoside monomer was 90 μL, and the injection volume of the ACT catalyst was 110 μL.

[0338] Each capping step was conducted under identical conditions, including a temperature of 25°C and a reaction time of 2 minutes. The capping reagent solution was a 1:1 molar ratio mixture of CapA and CapB (CapB1:CapB2 = 1:1). The injection volume of the capping reagent was 180 μL.

[0339] The oxidation reaction conditions were the same for each step, including a temperature of 25°C, a reaction time of 3 minutes, and an injection volume of 180 μL for the oxidizing reagent OXD.

[0340] The vulcanization reaction conditions were identical for each step, including a temperature of 25°C, a reaction time of 4 minutes, and a 0.05 M PADS pyridine acetonitrile solution as the vulcanizing agent. The injection volume of the vulcanizing agent was 180 μL.

[0341] 3. After the last nucleoside monomer is ligated, the nucleic acid sequence ligated on the solid-phase support is sequentially cut, deprotected, purified, and desalted, then freeze-dried to obtain the sense and antisense strands. 3-1 The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked with the vector was added to a mixture of ammonia and ethanol in a ratio of 3:1 to a volume of 0.8 mL. The reaction was carried out at 50 °C for 15 h. The remaining vector was removed by filtration, and the supernatant was concentrated to dryness under vacuum.

[0342] 3-2 Purification and desalting conditions are as follows: Desalting was performed using a comma-based 96-well plate desalting column DC189650 (80 mg). Specific conditions included: 3-2-1 Sample Preparation Add 0.1M TEAA (triethylamine acetate) to the oligonucleotide sample to a volume of 0.8mL.

[0343] 3-2-2 Activation of 96-well plates Activation: 0.8 mL of acetonitrile was used to activate the plate in each well of a 96-well plate. Equilibration: Equilibrate the 96-well plate with 0.8 mL of TEAA (pH 7.0) solution.

[0344] 3-2-3 The purification process shall be carried out in the following order: A 0.8 mL solution containing oligonucleotides was passed through a desalting column; the 96-well plate was washed twice with 0.8 mL of 6.5% ammonia to remove failed sequences; the 96-well plate was rinsed twice with 0.8 mL of deionized water to remove salt; the 96-well plate was rinsed three times with 0.8 mL of 3% trifluoroacetic acid to remove DMT, and the adsorbed layer turned orange-red; the 96-well plate was rinsed with 0.8 mL of 0.1 M TEAA; the 96-well plate was rinsed twice with 0.8 mL of deionized water to remove trifluoroacetic acid and residual salt; the plate was extracted with 0.6 mL of 20% acetonitrile and collected and lyophilized.

[0345] The detection method is as follows: The purity of the above-mentioned sense and antisense chains was detected and the molecular weight was analyzed using a Waters Acquity UPLC-SQD2 LCMS (column: ACQUITY UPLC BEH C18). The measured values ​​are consistent with the theoretical values, indicating that the synthesized sense and antisense chains are 3'-terminally conjugated with conjugated molecules.

[0346] 4. The annealing process is as follows: The sense and antisense strands synthesized in step 3 were dissolved separately in water for injection to obtain a 1000 ng / μL solution. These solutions were mixed in an equal molar ratio using a qPCR instrument (Applied Biosystems QuantStudio 6 & 7 Pro), heated at 90°C for 10 minutes, maintained for 3 minutes at 5°C increments, and finally maintained at 25°C for 10 minutes, allowing them to form a double-stranded structure via hydrogen bonding. The measured values ​​matched the theoretical values, indicating that the synthesized siRNA conjugate is the target-designed double-stranded nucleic acid sequence with the conjugated molecule. This siRNA possesses the sense and antisense strands shown in Tables 2 and 3.

[0347] Table 2

[0348] Table 3 Nucleic acid sequences of the positive and negative strands.

[0349] The structures of the above conjugates are as follows: Among them, the conjugate TRD002218 was used as the reference positive compound.

[0350] [Experimental Example 1] This experiment investigates the efficiency of the siRNA conjugates with different structures disclosed in this study in inhibiting the expression of target gene mRNA in vivo.

[0351] Male C57BL / 6 mice aged 6-8 weeks were randomly divided into groups of 6 mice each, with 3 mice in each group at each time point. Each group of mice was given the conjugates disclosed herein (3 conjugates: TRD007203, TRD007204, and TRD007205), the reference positive nucleic acid ligand conjugate TRD002218, and PBS.

[0352] All animals were given a single subcutaneous injection based on their body weight. The dosage of the siRNA conjugate (based on the amount of siRNA) was 1 mg / kg, and the administration volume was 5 mL / kg. Mice were sacrificed 7 and 28 days after administration, and their livers were collected and preserved using RNA later (Sigma Aldrich). The liver tissue was then homogenized using a tissue homogenizer, and total RNA was extracted from the liver tissue using a tissue RNA extraction kit (Fanzhi Medical Technology, FG0412) according to the instructions. The total RNA was reverse transcribed into cDNA, and the expression level of TTR mRNA in the liver tissue was detected using real-time quantitative PCR. In this quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene, and the expression levels of TTR and GAPDH mRNA were detected using TaqMan probe primers targeting TTR and GAPDH, respectively.

[0353] Table 4 Grouping information of compounds tested in mice

[0354] Table 5 shows the sequence of the detection primer.

[0355] TTR mRNA expression levels are calculated according to the following equation: TTR mRNA expression level = [(TTR mRNA expression level in test group / GAPDH mRNA expression level in test group) / (TTR mRNA expression level in control group / GAPDH mRNA expression level in control group)] x 100%.

[0356] After 7 and 28 days of drug administration, the inhibitory efficiencies of the siRNA conjugates with different structures disclosed in this study on the expression of target gene mRNA in vivo are shown in Figures 1 and 2, respectively. As shown in Figure 1, both the NAG0050 conjugate TRD007203 and the NAG0052 conjugate TRD007205 showed good inhibitory effects on TTR mRNA expression 7 days after drug administration. Among them, the NAG0050 conjugate TRD007203 was significantly superior to the L96 conjugate TRD002218, indicating that it can mediate more efficient siRNA delivery. As shown in Figure 2, after 28 days of administration, the inhibitory effects of the NAG0050 conjugate TRD007203, the NAG0051 conjugate TRD007204, and the NAG0052 conjugate TRD007205 on the mRNA expression of the target gene were all superior to those of the L96 conjugate TRD002218. Among them, the NAG0050 conjugate TRD007203 showed the most significant advantage.

[0357] [Preparation Example 11: Synthesis of Nucleic Acid Ligand Conjugates] The following siRNA conjugate was synthesized following the steps of Preparation Example 10. This siRNA has the sense and antisense strands shown in Tables 6 and 7.

[0358] Table 6 List of Nucleic Acid Compounds Among them, conjugates TRD6233, TRD6238, and TRD6253 were used as reference positive compounds.

[0359] Table 7 Nucleic acid sequences of the positive and negative strands. Wherein, 036 represents the (-)hmpNA(A) modification disclosed in WO2022028462A1 (the base changes with the sequence).

[0360] [Experimental Example 2: siRNA sequence psiCHECK 11 concentration at target activity] In HEK293A cells (Nanjing Kebai), 11 concentration gradients were used to screen the siRNA sequences in Table 6 for in vitro molecular-level target activity simulation.

[0361] The target sequences corresponding to human FXI, ANGPTL3, and MARC1 genes were constructed and inserted into the psiCHECK-2 plasmid (Sangon Biotech Co., Ltd.). This plasmid contains the *Rhizopus luciferase* gene and the *Firefly luciferase* gene. As a dual reporter gene system, the target sequence of the siRNA is inserted into the 3'UTR region of the *Rhizopus luciferase* gene. The activity of the siRNA for the target sequence can be reflected by detecting the expression of *Rhizopus luciferase* after calibration with *Firefly luciferase*, using the Dual-Luciferase Reporter Assay System (Promega, E2940).

[0362] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before transfection, HEK293A cells were seeded into 96-well plates at a density of 8 × 10³ cells per well with 100 μL of medium per well.

[0363] Cells were co-transfected with siRNA and corresponding plasmids using Lipofectamine 2000 (ThermoFisher, 11668019), with 0.2 μL of Lipofectamine 2000 and 20 ng of plasmid per well. For the target sequence plasmid, 11 concentration points were set for siRNA, with the highest concentration point ending at 20 nM, using a 3-fold serial dilution. Target levels were detected 24 h post-transfection using the Dual-Luciferase Repoter Assay System (Promega, E2940). Data were analyzed using GraphPad Prism5, and the results are shown in Table 8.

[0364] Table 8. Results of siRNA sequence psi-CHECK target activity screening.

[0365] The results show that the siRNAs conjugated with different structural sequences by the ligand NAG0052 disclosed in this study can effectively inhibit the expression of target gene mRNAs on cells.

[0366] [Experimental Example 3: Inhibition of human FXI, ANGPTL3, and MARC1 by siRNA in primary human hepatocytes (PHH)] The activity of the siRNAs listed in Table 6 was screened in human primary hepatocytes (PHH, Novabiosis) using 5 or 7 concentration gradients.

[0367] Human primary hepatocytes (PHH) were cryopreserved in liquid nitrogen. Twenty-four hours before transfection, PHH cells were thawed and seeded into 96-well plates at a density of 3 × 10⁴ cells per well, with 80 μL of culture medium per well. siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150). The final concentrations of the siRNA transfection gradients for the seven concentration sites were 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM. The final concentrations of the siRNA transfection gradients for the five concentration sites were 5 nM, 0.625 nM, 0.0781 nM, 0.00977 nM, and 0.00122 nM. Twenty-four hours after treatment, total RNA was extracted from cells using a high-throughput cell RNA extraction kit (Fanzhi, FG0417), and reverse transcribed using an RNA reverse transcription kit (Takara, 6210A). The mRNA levels of human FXI, ANGPTL3, and MARC1 were measured using a Taqman probe Q-PCR kit (ThermoFisher, 4444964). The mRNA levels of human FXI, ANGPTL3, and MARC1 were corrected based on the GAPDH internal reference gene level. Taqman probe primer information is shown in Table 9. Data processing was performed using… The results were expressed as the percentage of remaining human FXI, ANGPTL3, and MACR1 mRNA expression relative to cells treated with control siRNA. The IC50 results of the inhibition rate are shown in Tables 10 and 11. △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - remaining amount of target gene expression) × 100%.

[0368] Table 9 Taqman Primer Information Table

[0369] Table 10. Inhibitory activity of siRNA in PHH cells at multiple doses

[0370] Table 11. Inhibitory activity of siRNA at multiple doses in PHH cells

[0371] The results showed that the siRNAs conjugated with different structural sequences of the ligand NAG0052 disclosed in this study could effectively inhibit the expression of target gene mRNAs in human primary hepatocytes. The NAG0052 conjugates TRD008024, TRD008025, and TRD008026 exhibited superior inhibitory activity in PHH cells compared to the NAG1 conjugates TRD6233, TRD6238, and TRD6253.

[0372] [Experiment 4: Inhibition of FXI in cynomolgus monkey primary hepatocytes (PCH) by siRNA - Inhibitory activity at 7 concentration points] In primary monkey hepatocytes (PCH, Miaoshun Biotechnology), the reverse transfection activity of two siRNAs from FXI listed in Table 6 was screened using seven concentration gradients. The initial and final concentrations of each siRNA sample were 10 nM, with 5-fold serial dilutions and seven concentration points.

[0373] Primary monkey hepatocytes (PCH) were cryopreserved in liquid nitrogen. Before transfection, PCH cells were thawed and seeded into 96-well plates at a density of 3 × 10⁴ cells per well, with 90 μL of culture medium per well. siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) at final concentrations of 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM. Twenty-four hours after treatment, total RNA was extracted from the cells using a high-throughput cell RNA extraction kit (Fanzhi, FG0417). Reverse transcription was performed using an RNA reverse transcription kit (Takara, 6210A), and the mRNA level of monkey FXI was determined using a Taqman probe Q-PCR kit (ThermoFisher, 4444964). The mRNA levels of monkey FXI were corrected based on the levels of the GAPDH internal reference gene. Taqman probe primer information is shown in Table 12. Data processing was performed using... The results were expressed as the percentage of remaining monkey FXI mRNA expression relative to cells treated with control siRNA. The IC50 results of the inhibition rate are shown in Table 13. △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - remaining amount of target gene expression) × 100%.

[0374] Table 12 Monkey Taqman Probe Primer Information Table 13. Inhibitory activity of siRNA at multiple doses in PCH

[0375] The results showed that the TRD008002 and TRD008003 conjugates of NAG0052 had good inhibitory activity against FXI in PCH cells.

[0376] [Experimental Example 5: Inhibition of Human ANGPTL3 by siRNA in Huh7 Cells - Inhibitory Activity at 7 Concentration Points] In Huh7 cells (Nanjing Kebai), the siRNAs listed in Table 6 were screened for Huh7 cell viability using seven concentration gradients. Each siRNA sample started at a concentration of 10 nM, with 5-fold serial dilutions at seven concentration points.

[0377] Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before transfection, Huh7 cells were seeded into 96-well plates at a density of 10,000 cells per well with 100 μL of medium per well. siRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150) at final concentrations of 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM. 24 hours after treatment, total RNA was extracted from the cells using a high-throughput cell RNA extraction kit (Fanzhi, FG0417), and reverse transcribed using an RNA reverse transcription kit (Takara, 6210A). Human ANGPTL3 mRNA levels were measured using a Taqman probe Q-PCR kit (ThermoFisher, 4444964). The mRNA level of human ANGPTL3 was corrected based on the level of the GAPDH internal reference gene. Data processing was performed using... The results were expressed as the percentage of residual human ANGPTL3 mRNA expression relative to cells treated with control siRNA. The IC50 results for the inhibition rate are shown in Table 14. △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - remaining amount of target gene expression) × 100%.

[0378] Table 14. Inhibitory activity of siRNA against human ANGPTL3 in Huh7 at multiple doses

[0379] The results showed that in Huh7 cells, the TRD008004, TR008005, and TRD008006 conjugates of NAG0052 exhibited effective inhibitory activity against ANGPTL3.

[0380] [Preparation Example 12: Synthesis of Nucleic Acid Ligand Conjugates] The following siRNA conjugates were synthesized according to the steps of Preparation Example 10 for Experiments 6 and 7. The siRNA has the sense and antisense strands shown in Table 15.

[0381] Table 15

[0382] [Experimental Example 6: Inhibition of mRNA expression in primary liver cells by siRNA conjugated with aminogalactose molecules] Fresh mouse primary hepatocytes were isolated using the method reported by Severgini et al. (Cytotechnology.2012;64(2):187-195.).

[0383] After primary hepatocyte isolation, they were seeded at a rate of 100,000 cells per well in 24-well plates. The target siRNA compound was added at final concentrations of 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM, respectively. The primary hepatocytes were then cultured at 37°C in 5% CO2 for 24 hours. After 24 hours, the mTTR mRNA expression level was detected using qPCR.

[0384] Figure 3 shows the mRNA inhibition rates of different concentrations of siRNA conjugates S-1 (NAG1 conjugate) and S-L96 (L96 conjugate). As shown in Figure 3, S-1 exhibited excellent mTTR gene expression inhibition efficiency. Compared with the control group S-L96 (IC50 value of 0.280 nM), S-1 had an IC50 value of 0.131 nM, indicating that the NAG1 conjugate siRNA was more efficiently taken up by primary hepatocytes in vitro than the control group, and that the NAG1 conjugate could more efficiently mediate siRNA entry into primary hepatocytes. Since the NAG0052 conjugate siRNA showed stronger inhibitory activity against target gene expression than the NAG1 conjugate siRNA in Experiment 3, the combined data indicate that the NAG0052 conjugate has superior inhibitory activity compared to the L96 conjugate.

[0385] [Experimental Example 7: Inhibition of mRNA expression in vivo by siRNA conjugated with aminogalactose molecules] Eight-week-old C57BL / 6 mice (Zhao derivative, SPF grade, female) were used to deliver the aforementioned galactosyl-conjugated siRNA subcutaneously. On day 1, 100 μl of a solution containing either 1 mg / kg (mpk) of the corresponding galactosyl-conjugated siRNA (S-L96 or S-1) in PBS (designated as the Mock group, i.e., blank control group) or 0.2 mpk dose of the siRNA prepared in PBS was injected subcutaneously into the loose skin of the neck and shoulder of the mice. Six mice were injected in each group.

[0386] Three days after administration, mice were euthanized by cervical dislocation, and the mRNA expression level of mTTR in mouse liver tissue was detected by qPCR.

[0387] Figure 4 shows the mRNA expression levels in mouse liver tissue after administration of different doses of S-1 and S-L96. As shown in Figure 4, S-1 exhibited excellent mTTR gene expression inhibition efficiency. S-1 showed superior activity levels at 1 mpk and 0.2 mpk compared to the control group S-L96. Since the NAG0052-conjugated siRNA showed stronger inhibitory activity against target gene expression than the NAG1-conjugated siRNA in Experiment 3, the combined data indicate that the NAG0052 conjugate has superior inhibitory activity compared to the L96 conjugate.

Claims

1. A ligand having the structure shown in formula (I), wherein, L1 is a C1-C30 alkyl chain, or a C1-C30 alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms, or C=O; R1 and R2 are independently chemical bonds, -NR6-, -C(=O)-, or -OC(=O)-; is or; R6 is hydrogen or C1-6 alkyl; m, p, and q are independently 0, 1, 2, 3, or 4; B is or; Rb1, Rb2, Rb3, Rb4, Rb5, Rb6, and Rb7 are independently -C(=O)-, -NHC(=O)-, -C(=O)O-, -C(=O)-(CH2)z8-O-, or -NHC(=O)-(CH2)z9-O-; z1, z2, z3, z4, z5, z6, z7, z8, and z9 are independently integers from 0 to 10; L2 is a C1-C30 alkyl chain, or a C1-C30 alkyl chain interrupted by one or more oxygen, sulfur, nitrogen atoms or C=O; r is 3.

2. The ligand as described in claim 1, wherein, L1 is L3 or L3-R10-R11-L3, wherein L3 is independently a C1-C12 alkyl chain, -(CH2)j1-C(=O)-(CH2)j2- or -(CH2)j3-(CH2CH2O)1-4-(CH2)j4-; R10 and R11 are independently chemical bonds, -NR12-, -C(=O)- or -OC(=O)-; R12 is hydrogen or a C1-C12 alkyl group; j1, j2, j3 and j4 are independently integers from 0 to 10.

3. The ligand as described in claim 2, wherein, L1 is -(CH2)j1-C(=O)-(CH2)j2-, where j1 and j2 are independent integers of 0-2 or 4-10.

4. The ligand as described in claim 3, wherein, L1 is a variable; j2 is an independent integer from 4 to 10; where a1 is connected to B and b1 is connected to R1.

5. The ligand as described in claim 4, wherein, L1 can be any combination of , , , or ; where a1 is connected to B and b1 is connected to R1.

6. The ligand as described in claim 1, wherein, R1 and R2 can be any of the following combinations: Combination 1: R1 is a chemical bond and R2 is C=O; Combination 2: R1 is a chemical bond and R2 is NR6; Combination 3: R1 is a chemical bond and R2 is -OC(=O)-; Combination 4: R1 is NR6 and R2 is C=O; Combination 5: R1 is NR6 and R2 is -OC(=O)-; Combination 6: R2 is NR6 and R1 is C=O; Combination 7: R2 is NR6 and R1 is -OC(=O)-.

7. The ligand as described in claim 1, wherein, R6 is hydrogen, deuterium, methyl, ethyl, propyl, or isopropyl.

8. The ligand as described in claim 1, wherein, m is 0 or 1.

9. The ligand as described in claim 1, wherein, p and q can be any of the following combinations: Combination 1: p=1 and q=1; Combination 2: p=1 and q=0; Combination 3: p=0 and q=1; Combination 4: p=0 and q=0.

10. The ligand as described in claim 1, wherein, B is OR, Rb1, Rb2, Rb3, Rb4, Rb5, Rb6 and Rb7 are independently -C(=O)-, -NHC(=O)-(CH2)z9-O- or -NHC(=O)-, the N atom at the a2 end is connected to L1, and z9 is an independent integer from 0 to 10.

11. The ligand as described in claim 10, wherein, B is either 0 or 1, and the N atom at the a2 end is connected to L1.

12. The ligand as described in claim 1, wherein, L2 is L4 or L4-R13-R14-L4, wherein L4 is independently a C1-C12 alkyl chain or -(CH2)j5-(OCH2CH2)1-4-(CH2)j6-, R13 and R14 are independently chemical bonds, -NR15-, -C(=O)- or -OC(=O)-, R15 is independently hydrogen or C1-C12 alkyl, and j5 and j6 are independently integers from 0 to 10.

13. The ligand as described in claim 12, wherein, L2 is -(CH2)j5-(OCH2CH2)1-4-(CH2)j6-, where j5 and j6 are independent integers from 0 to 6.

14. The ligand as described in claim 13, wherein, L2 is...

15. The ligand as described in claim 12, wherein, L2 is a, where a3 is connected to O and b3 is connected to B.

16. The ligand as described in claim 15, wherein, L2 is a, where a3 is connected to O and b3 is connected to B.

17. The ligand as described in claim 16, wherein, L2 is a, where a3 is connected to O and b3 is connected to B.

18. The ligand as described in claim 17, wherein, L2 is a, where a3 is connected to O and b3 is connected to B.

19. The ligand as claimed in claim 1, wherein, For, , , , , , or.

20. The ligand as claimed in claim 1, wherein, The ligand is any of the following structures: , , , , , , , , , , or.

21. The ligand as described in claim 1, wherein, The ligand is any of the following structures: , ...

22. A compound as shown in formula (II-1), (II-2), or (II-3), wherein, , , ; is a macroporous amine methyl resin; is or ; L1, R1, R2, B, L2, m, p, q and r are defined as described in claim 1.

23. A nucleic acid ligand conjugate comprising a nucleic acid and one or more ligands as described in claim 1, the ligands being conjugated to the ends of the nucleic acid, the ligands being the same or different.

24. The nucleic acid ligand conjugate as described in claim 23, wherein, The nucleic acid is linked to the ligand via a phosphate ester group, a thiophosphate ester group, or a phosphonic acid group; and / or, the 3' end of the nucleic acid is conjugated to the ligand; and / or, the nucleic acid is the positive strand of siRNA.

25. The nucleic acid ligand conjugate as described in claim 23, wherein, The nucleic acid ligand conjugate is any of the following structures or a pharmaceutically acceptable salt thereof, , , , , , , , , or, where represents a nucleic acid.

26. The nucleic acid ligand conjugate as described in claim 23, wherein, The nucleic acid ligand conjugate is any of the following structures or a pharmaceutically acceptable salt thereof, ...

27. An RNAi reagent comprising a nucleic acid ligand conjugate as described in any one of claims 23 to 26.

28. A composition comprising a nucleic acid ligand conjugate as described in any one of claims 23 to 26 or an RNAi reagent as described in claim 27, and one or more pharmaceutically acceptable excipients.

29. Use of a nucleic acid ligand conjugate as described in any one of claims 23 to 26, or an RNAi reagent as described in claim 27, or a composition as described in claim 28, in the preparation of a medicament for treating a patient’s disease.

30. The use as described in claim 29, wherein, This disease is of hepatic origin.

31. Use of a nucleic acid ligand conjugate as described in any one of claims 23 to 26, or an RNAi reagent as described in claim 27, or a composition as described in claim 28, in the preparation of a medicament for inhibiting mRNA expression in a patient.

32. A method for preparing a nucleic acid ligand conjugate as described in any one of claims 23 to 26, comprising the following steps: Starting with the compound described in claim 22, nucleoside monomers are linked one by one in the nucleotide sequence from 3' to 5'.

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