Compounds, conjugates, compositions and their uses

Abstract

The present invention relates to compounds, conjugates, compositions, and uses thereof, and more particularly to a compound having a structure represented by a specific formula (Ia) or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a prodrug thereof. The compound can be used to prevent and / or treat the expression or activity of a specific target gene.

Description

[Technical Field]

[0001] The present invention relates to the pharmaceutical field, specifically to compounds, conjugates, compositions and their uses. [Background technology]

[0002] Small molecule nucleic acid drugs, such as small interfering RNA (siRNA), antisense oligodeoxynucleotides (ASODN), and nucleic acid stimulatory motifs (CpG), are playing an increasingly important role in gene therapy. Some of these drugs have been approved for sale by the U.S. Food and Drug Administration (FDA), and many more are currently undergoing preclinical research and clinical trials. Nucleic acid drugs are nucleic acid sequences that specifically target disease-causing genes or proteins through binding or cleavage, inhibiting or promoting the expression of specific genes or proteins. Nucleic acid drugs include normal human genes that can replace defective gene segments, antisense nucleic acids that block gene expression, and single-stranded nucleic acids that promote triplex formation, such as siRNA, DNA, microRNA, and CpG.

[0003] Delivery systems are considered to be one of the key technologies in the development of small molecule nucleic acid drugs. Currently, the most widely studied delivery system for small molecule nucleic acids worldwide is targeted delivery via conjugation. Therefore, there is a strong need in the field to develop new drug conjugates that have high in vivo drug delivery efficiency, low toxicity, and high activity. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. [Means for solving the problem]

[0005] According to a first aspect of the present invention, there is provided a compound represented by formula (Ia), or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a prodrug thereof:

[0006] [ka]

[0007] [In formula (Ia), R1 represents a hydroxy protecting group; R2 is

[0008] [ka]

[0009] n is 0, 1, 2, or 3; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each A is independently an unsubstituted or substituted 4- to 10-membered aliphatic ring; each X is independently NH, O, or S; Each L1 is independently

[0010] [ka]

[0011] where j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0012] [ka]

[0013] and (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each Y is independently NH, O, or S; Each R4 is independently

[0014] [ka]

[0015] is] For example, when n is 0, the structural formula of the compound is as represented by formula [Ia-0].

[0016] [ka]

[0017] In some embodiments of the present invention, in formula (Ia), R1 is a trityl group (Tr group), a 4-methoxytrityl group (MMTr group), a 4,4'-dimethoxytrityl group (DMTr group), or a 4,4',4''-trimethoxytrityl group (TMTr group). In some specific embodiments of the present invention, in formula (Ia), R1 is a 4,4'-dimethoxytrityl group (DMTr group). In some specific embodiments of the present invention, in formula (Ia), R2 is

[0018] [ka]

[0019] is. In some specific embodiments of the present invention, in formula (Ia):

[0020] [ka]

[0021] In some specific embodiments of the present invention, in formula (Ia), any Z is a hydroxy group. In some embodiments of the present invention, in Formula (Ia), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (Ia), each p is 1. In some embodiments of the present invention, in Formula (Ia), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (Ia), each q is 1. In some specific embodiments of the present invention, in formula (Ia), each p is 1 and each q is 1. In some embodiments of the present invention, in formula (Ia), each A is independently an unsubstituted or substituted 4- to 10-membered cycloalkane group or an unsubstituted or substituted 4- to 10-membered cycloolefin group. In some embodiments of the present invention, in formula (Ia), each A is independently an unsubstituted or substituted 4- to 10-membered cycloalkane group. In some embodiments of the present invention, in formula (Ia), each A is independently a 4- to 10-membered cycloalkane group, for example, a monocyclic, spirocyclic, or bridged ring. In some embodiments of the present invention, in formula (Ia), each A is independently

[0022] [ka]

[0023] is. In some specific embodiments of the present invention, in formula (Ia), any A is

[0024] [ka]

[0025] is. In some specific embodiments of the present invention, in formula (Ia), any X is NH. In some specific embodiments of the present invention, in formula (Ia), any L1 is

[0026] [ka]

[0027] is. In some specific embodiments of the present invention, in formula (Ia), any R3 is H. In some embodiments of the present invention, in Formula (Ia), each L2 is independently C1-C 10 an alkylene group or

[0028] [ka]

[0029] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in formula (Ia), each L2 is independently

[0030] [ka]

[0031] is. In some specific embodiments of the present invention, in formula (Ia), any Y is O. In some specific embodiments of the present invention, in formula (Ia), any R4

[0032] [ka]

[0033] is. In some embodiments of the invention, the compound has a structure according to Formula (IIa).

[0034] [ka]

[0035] [In formula (IIa), R1 represents a hydroxy protecting group;

[0036] [ka]

[0037] n is 0, 1, 2, or 3; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each X is independently NH, O, or S; Each L1 is independently

[0038] [ka]

[0039] where j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0040] [ka]

[0041] (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each Y is independently NH, O, or S;

[0042] [ka]

[0043] Illustratively, when n is 0, the structural formula of the compound is:

[0044] [ka]

[0045] In some embodiments of the present invention, in formula (IIa), R1 is a trityl group (Tr group), a 4-methoxytrityl group (MMTr group), a 4,4'-dimethoxytrityl group (DMTr group), or a 4,4',4''-trimethoxytrityl group (TMTr group).

[0046] In some specific embodiments of the present disclosure, in formula (IIa), R 1 is a 4,4′-dimethoxytrityl group (DMTr group).

[0047] In some specific embodiments of the present invention, in formula (IIa), R2 is

[0048] [ka]

[0049] is. In some specific embodiments of the present invention, in formula (IIa), R2 is

[0050] [ka]

[0051] [ka] Illustratively, the solid support may be, for example, controlled pore glass (abbreviated as CPG), a solid polymer support (abbreviated as SPS, for example, polystyrene (PS) resin), or the like. In some specific embodiments of the present invention, in formula (IIa), any Z is a hydroxy group. In some specific embodiments of the present invention, in formula (IIa), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIa), each p is 1. In some embodiments of the present invention, in Formula (IIa), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIa), each q is 1. In some specific embodiments of the present invention, in formula (IIa), each p is 1 and each q is 1. In some specific embodiments of the present invention, in formula (IIa), any X is NH. In some specific embodiments of the present invention, in formula (IIa), any L1 is

[0052] [ka]

[0053] is. In some specific embodiments of the present invention, in formula (IIa), any R3 is H.

[0054] In some embodiments of the present invention, in Formula (IIa), each L2 is independently C1-C 10 an alkylene group or

[0055] [ka]

[0056] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0057] In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in Formula (IIa), each L2 is independently

[0058] [ka]

[0059] is. In some specific embodiments of the present invention, in formula (IIa), any Y is O. In some specific embodiments of the present invention, in formula (IIa), any R

[0060] [ka]

[0061] is. In some embodiments of the invention, the compound has a structure according to Formula (IIIa).

[0062] [ka]

[0063] [In formula (IIIa), R1 represents a hydroxy protecting group;

[0064] [ka]

[0065] n is 0, 1, 2, or 3; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0066] [ka]

[0067] (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each Y is independently NH, O, or S; Each R4 is independently

[0068] [ka]

[0069] Illustratively, when n is 0, the structural formula of the compound is as follows:

[0070] [ka]

[0071] In some embodiments of the present invention, in formula (IIIa), R1 is a trityl group (Tr group), a 4-methoxytrityl group (MMTr group), a 4,4'-dimethoxytrityl group (DMTr group), or a 4,4',4''-trimethoxytrityl group (TMTr group). In some specific embodiments of the present invention, in formula (IIIa), R 1 is a 4,4′-dimethoxytrityl group (DMTr group). In some specific embodiments of the present invention, in formula (IIIa), R2 is

[0072] [ka]

[0073] is. In some specific embodiments of the present invention, in formula (IIIa), R2 is

[0074] [ka]

[0075] In some specific embodiments of the present invention, in formula (IIIa), any Z is a hydroxy group. In some embodiments of the present invention, in Formula (IIIa), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIIa), each p is 1. In some embodiments of the present invention, in Formula (IIIa), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIIa), each q is 1. In some specific embodiments of the present invention, in formula (IIIa), each p is 1 and each q is 1. In some specific embodiments of the present invention, in formula (IIIa), any R3 is H. In some embodiments of the present invention, in Formula (IIIa), each L2 is independently C1-C 10 an alkylene group or

[0076] [ka]

[0077] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in Formula (IIIa), each L2 is independently

[0078] [ka]

[0079] is. In some specific embodiments of the present invention, in formula (IIIa), any Y is O. In some specific embodiments of the present invention, in formula (IIIa), any R

[0080] [ka]

[0081] is. In some embodiments of the invention, the compound has a structure according to Formula (IVa).

[0082] [ka]

[0083] [In formula (IVa), R1 represents a hydroxy protecting group;

[0084] [ka]

[0085] n is 0, 1, 2, or 3; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0086] [ka]

[0087] (where each R L2a are C1-C 10 is an alkylene group, and each R L2bare each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each Y is independently NH, O, or S.

[0088] Illustratively, when n is 0, the structural formula of the compound is:

[0089] [ka]

[0090] In some embodiments of the present invention, in formula (IVa), R1 is a trityl group (Tr group), a 4-methoxytrityl group (MMTr group), a 4,4'-dimethoxytrityl group (DMTr group), or a 4,4',4''-trimethoxytrityl group (TMTr group). In some specific embodiments of the present invention, in formula (IVa), R1 is a 4,4'-dimethoxytrityl group (DMTr group).

[0091] In some specific embodiments of the present invention, in formula (IVa), R2 is

[0092] [ka]

[0093] is. In some specific embodiments of the present invention, in formula (IVa), R2 is

[0094] [ka]

[0095] In some specific embodiments of the present invention, in formula (IVa), any Z is a hydroxy group. In some embodiments of the present invention, in formula (IVa), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (IVa), each p is 1. In some embodiments of the present invention, in formula (IVa), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (IVa), each q is 1. In some specific embodiments of the present invention, in formula (IVa), each p is 1 and each q is 1. In some specific embodiments of the present invention, in formula (IVa), any R3 is H. In some embodiments of the present invention, in formula (IVa), each L2 is independently C1-C 10 an alkylene group or

[0096] [ka]

[0097] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in formula (IVa), each L2 is independently

[0098] [ka]

[0099] is. In some specific embodiments of the present invention, in formula (IVa), any Y is O. In some specific embodiments of the invention, the compound has any one of the following structures:

[0100] [ka]

[0101] [ka]

[0102] [ka]

[0103] According to a second aspect of the present invention, there is provided a compound represented by formula (Ib), or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a prodrug thereof:

[0104] [ka]

[0105] [In formula (Ib), * represents a linking site for linking a pharmacoactive molecule; m is 1, 2, 3, or 4; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each A is independently an unsubstituted or substituted 4- to 10-membered aliphatic ring; each X is independently NH, O, or S; Each L1 is independently

[0106] [ka]

[0107] where j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0108] [ka]

[0109] (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each Y is independently NH, O, or S. In some specific embodiments of the invention, m is 3. In some embodiments of the present invention, in formula (Ib), any Z is a hydroxy group. In some embodiments of the present invention, in formula (Ib), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (Ib), each p is 1. In some embodiments of the present invention, in Formula (Ib), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (Ib), each q is 1. In some specific embodiments of the present invention, in formula (Ib), each p is 1 and each q is 1. In some embodiments of the present invention, in formula (Ib), each A is independently an unsubstituted or substituted 4- to 10-membered cycloalkane group or an unsubstituted or substituted 4- to 10-membered cycloolefin group. In some embodiments of the present invention, in formula (Ib), each A is independently an unsubstituted or substituted 4- to 10-membered cycloalkane group. In some embodiments of the present invention, in formula (Ib), each A is independently a 4- to 10-membered cycloalkane group, for example, a monocyclic, spirocyclic, or bridged ring. In some embodiments of the present invention, in formula (Ib), each A is independently

[0110] [ka]

[0111] is. In some specific embodiments of the present invention, in formula (Ib), any A is

[0112] [ka]

[0113] is. In some specific embodiments of the present invention, in formula (Ib), any X is NH. In some specific embodiments of the present invention, in formula (Ib), any L1 is

[0114] [ka]

[0115] is. In some specific embodiments of the present invention, in formula (Ib), any R3 is H. In some embodiments of the present invention, in formula (Ib), each L2 is independently C1-C 10 an alkylene group or

[0116] [ka]

[0117] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in formula (Ib), each L2 is independently

[0118] [ka]

[0119] is. In some specific embodiments of the present invention, in formula (Ib), any Y is O. In some embodiments of the present invention, the pharmaceutically active molecule is a small molecule drug (eg, entecavir (ETV), small molecule chemical statins, febuxostat, allopurinol, etc.), an antibody, or an oligonucleotide. In some specific embodiments of the present invention, the pharmaceutically active molecule is an oligonucleotide. In some embodiments of the invention, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide. In some embodiments of the invention, the compound has a structure according to Formula (IIb).

[0120] [ka]

[0121] [In formula (IIb), * represents a linking site for linking a pharmacoactive molecule; m is 1, 2, 3, or 4; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each X is independently NH, O, or S; Each L1 is independently

[0122] [ka]

[0123] where j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0124] [ka]

[0125] (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each Y is independently NH, O, or S. In some specific embodiments of the present invention, in formula (IIb), m is 3. In some embodiments of the present invention, in formula (IIb), any Z is a hydroxy group. In some embodiments of the present invention, in formula (IIb), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIb), each p is 1. In some embodiments of the present invention, in formula (IIb), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIb), each q is 1. In some specific embodiments of the present invention, in formula (IIb), each p is 1 and each q is 1. In some embodiments of the present invention, in formula (IIb), any X is NH. In some specific embodiments of the present invention, in formula (IIb), any L

[0126] [ka]

[0127] is. In some specific embodiments of the present invention, in formula (IIb), any R3 is H. In some embodiments of the present invention, in formula (IIb), each L2 is independently C1-C 10 an alkylene group or

[0128] [ka]

[0129] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in Formula (IIb), each L2 is independently:

[0130] [ka]

[0131] is. In some specific embodiments of the present invention, in formula (IIb), any Y is O. In some embodiments of the invention, the compound has a structure according to Formula (IIIb).

[0132] [ka]

[0133] [In formula (IIIb), * represents a linking site for linking a pharmacoactive molecule; m is 1, 2, 3, or 4; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0134] [ka]

[0135] (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each Y is independently NH, O, or S. In some specific embodiments of the present invention, in formula (IIIb), m is 3. In some embodiments of the present invention, in formula (IIIb), any Z is a hydroxy group. In some embodiments of the present invention, in formula (IIIb), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIIb), each p is 1. In some embodiments of the present invention, in formula (IIIb), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIIb), each q is 1. In some specific embodiments of the present invention, in formula (IIIb), each p is 1 and each q is 1. In some embodiments of the present invention, in formula (IIIb), any R3 is H. In some embodiments of the present invention, in formula (IIIb), each L2 is independently C1-C 10 an alkylene group or

[0136] [ka]

[0137] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in Formula (IIIb), each L2 is independently:

[0138] [ka]

[0139] is. In some specific embodiments of the present invention, in formula (IIIb), any Y is O. In some specific embodiments of the invention, the compound has any one of the following structures:

[0140] [ka]

[0141] (where * represents a linking site for linking a pharmacoactive molecule) It should be noted that the compounds according to the second aspect of the present invention may exist in the form of a ligand.

[0142] According to a third aspect of the present invention, there is provided a conjugate represented by formula (Ic), or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a prodrug thereof:

[0143] [ka]

[0144] [In formula (Ic), Nu represents an oligonucleotide, m is 1, 2, 3, or 4; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each A is independently an unsubstituted or substituted 4- to 10-membered aliphatic ring; each X is independently NH, O, or S; Each L1 is independently

[0145] [ka]

[0146] where j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0147] [ka]

[0148] (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each Y is independently NH, O, or S.

[0149] In some embodiments of the invention, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide. In some embodiments of the present invention, the single-stranded oligonucleotide is selected from the group consisting of a modified or unmodified antisense oligonucleotide, a modified or unmodified nucleic acid aptamer, a modified or unmodified nuclease, a modified or unmodified deoxyribonuclease, a circular RNA, a sense strand of an siRNA, and an antisense strand of an siRNA. In some embodiments of the invention, the double-stranded oligonucleotide is selected from the group consisting of modified or unmodified small interfering RNA, modified or unmodified double-stranded RNA, modified or unmodified microRNA, modified or unmodified single guide RNA, modified or unmodified small activator RNA, and modified or unmodified short hairpin RNA. In some specific embodiments of the invention, in formula (Ic), Nu represents a small interfering RNA. In some specific embodiments of the present invention, in formula (Ic), m is 3. In some embodiments of the present invention, in formula (Ic), any Z is a hydroxy group. In some embodiments of the present invention, in Formula (Ic), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (Ic), each p is 1. In some embodiments of the present invention, in Formula (Ic), each q is independently 1 or 2. In some specific embodiments of the present disclosure, in formula (Ic), each q is 1. In some specific embodiments of the present disclosure, in formula (Ic), each p is 1 and each q is 1. In some embodiments of the present invention, in formula (Ic), each A is independently an unsubstituted or substituted 4- to 10-membered cycloalkane group or an unsubstituted or substituted 4- to 10-membered cycloolefin group. In some embodiments of the present invention, in formula (Ic), each A is independently an unsubstituted or substituted 4- to 10-membered cycloalkane group. In some embodiments of the present invention, in formula (Ic), each A is independently a 4- to 10-membered cycloalkane group, for example, a monocyclic, spirocyclic, or bridged ring. In some embodiments of the present invention, in formula (Ic), each A is independently

[0150] [ka]

[0151] is. In some specific embodiments of the present invention, in formula (Ic), any A is

[0152] [ka]

[0153] is. In some specific embodiments of the present invention, in formula (Ic), any X is NH. In some specific embodiments of the present invention, in formula (Ic), any L1 is

[0154] [ka]

[0155] is. In some specific embodiments of the present invention, in formula (Ic), any R3 is H. In some embodiments of the present invention, in Formula (Ic), each L2 is independently C1-C 10 an alkylene group or

[0156] [ka]

[0157] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in Formula (Ic), each L2 is independently:

[0158] [ka]

[0159] is. In some specific embodiments of the present invention, in formula (Ic), any Y is O.

[0160] In some embodiments of the invention, the conjugate has a structure according to formula (IIc):

[0161] [ka]

[0162] [In formula (IIc), Nu represents an oligonucleotide, m is 1, 2, 3, or 4; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each X is independently NH, O, or S; Each L1 is independently

[0163] [ka]

[0164] where j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0165] [ka]

[0166] (where each R L2a are C1-C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each Y is independently NH, O, or S. In some specific embodiments of the invention, in formula (IIc), Nu represents a small interfering RNA. In some specific embodiments of the present invention, in formula (IIc), m is 3. In some embodiments of the present invention, in formula (IIc), any Z is a hydroxy group. In some embodiments of the present invention, in formula (IIc), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIc), each p is 1. In some embodiments of the present invention, in Formula (IIc), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIc), each q is 1. In some specific embodiments of the present invention, in formula (IIc), each p is 1 and each q is 1. In some specific embodiments of the present invention, in formula (IIc), any R3 is H. In some embodiments of the present invention, in formula (IIc), each L2 is independently C1-C 10 an alkylene group or

[0167] [ka]

[0168] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in formula (IIc), each L2 is independently

[0169] [ka]

[0170] is. In some specific embodiments of the present invention, in formula (IIc), any Y is O.

[0171] In some embodiments of the invention, the conjugate has a structure according to formula (IIIc):

[0172] [ka]

[0173] [In formula (IIIc), Nu represents an oligonucleotide, m is 1, 2, 3, or 4; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each R3 is independently H, a C1-C6 alkyl group, a C1-C6 haloalkyl group, or a C1-C6 alkoxy group; Each L2 is independently connected to C1-C 30 an alkylene group or

[0174] [ka]

[0175] (where each R L2a are C1-C 10 is an alkylene group, and each R L2bare each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each Y is independently NH, O, or S. In some specific embodiments of the present invention, in formula (IIIc), m is 3. In some embodiments of the present invention, in formula (IIIc), any Z is a hydroxy group. In some embodiments of the present invention, in formula (IIIc), each p is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIIc), each p is 1. In some embodiments of the present invention, in Formula (IIIc), each q is independently 1 or 2. In some specific embodiments of the present invention, in formula (IIIc), each q is 1. In some specific embodiments of the present invention, in formula (IIIc), each p is 1 and each q is 1. In some embodiments of the present invention, in formula (IIIc), any R3 is H. In some embodiments of the present invention, in formula (IIIc), each L2 is independently C1-C 10 an alkylene group or

[0176] [ka]

[0177] (where each R L2a are each independently a C1-C5 alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some specific embodiments of the present invention, any R L2b is -NH-C(O)-. In some specific embodiments of the invention, k is 1. In some embodiments of the present invention, in Formula (IIIc), each L2 is independently:

[0178] [ka]

[0179] is. In some specific embodiments of the present invention, in formula (IIIc), any Y is O.

[0180] According to a fourth aspect of the present invention there is provided a composition comprising a conjugate according to the third aspect. In some embodiments of the present invention, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0181] According to a fifth aspect of the present invention there is provided the use of a compound according to the first aspect, a ligand according to the second aspect, a conjugate according to the third aspect or a composition according to the fourth aspect in the manufacture of a medicament for the prevention and / or treatment of a disease. In some embodiments of the present invention, the disease is a liver-derived disease.Exemplary liver-derived diseases include but are not limited to chronic non-alcoholic fatty liver disease, chronic alcoholic liver disease, autoimmune hepatitis, primary biliary cirrhosis, liver cirrhosis, primary liver cancer, hepatic encephalopathy, and viral hepatitis.

[0182] According to a sixth aspect of the present invention there is provided a method for treating and / or preventing a disease, comprising administering to a subject a pharmaceutically acceptable amount of a conjugate according to the third aspect or a composition according to the fourth aspect. In some embodiments of the present invention, the disease is a liver-derived disease.Exemplary liver-derived related diseases include but are not limited to chronic non-alcoholic fatty liver disease, chronic alcoholic liver disease, autoimmune hepatitis, primary biliary cirrhosis, liver cirrhosis, primary liver cancer, hepatic encephalopathy, and viral hepatitis.

[0183] According to a seventh aspect of the present invention there is provided the use of a compound according to the first aspect, a ligand according to the second aspect, a conjugate according to the third aspect or a composition according to the fourth aspect in the manufacture of a medicament for reducing the expression or activity of a target gene. In some embodiments of the present invention, the agent is used to reduce the expression or activity of a target gene in liver cells, illustratively including, but not limited to, at least one of Apoa, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, FVII, p53, C3, C4, C5, AGT, CFB, USP20, ASGR1, FTO, INHBE, HBV, and HCV.

[0184] According to an eighth aspect of the present invention there is provided a method of reducing the expression or activity of a target gene, comprising contacting a cell with a conjugate according to the third aspect or a composition according to the fourth aspect. In some embodiments of the present invention, the cells are hepatocytes. Illustratively, the target genes include, but are not limited to, at least one of Apoa, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, FVII, p53, C3, C4, C5, AGT, CFB, USP20, ASGR1, FTO, INHBE, HBV, and HCV.

[0185] Additional aspects and advantages of the present invention will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of the present disclosure. [Brief explanation of the drawings]

[0186] The above and / or additional aspects and advantages of the present invention will be apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings.

[0187] [Figure 1]1 shows the relative expression levels of target genes in mice after administration of a trimeric CR01008 conjugate (RZ899015) and an L96 conjugate (RZ599001) in an example of the present disclosure. [Figure 2] 1 shows the relative expression levels of target genes in mice after administration of a trimeric CR01008 conjugate (RZ899015), a trimeric CR01013 conjugate (RZ899026), a trimeric CR01014 conjugate (RZ899027), and an L96 conjugate (RZ599001) in examples of the present disclosure. [Figure 3] 1 shows the relative expression levels of target genes in mice after administration of a trimeric CR01013 conjugate (RZ897001), a trimeric CR01014 conjugate (RZ897002), and an L96 conjugate (RZ597002) in an example of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0188] Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described herein are used only to illustrate and explain the present invention, and are not intended to limit the present invention. Interpretation of terms

[0189] In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The term "NH" refers to a compound having the structural formula

[0190] [ka]

[0191] It refers to the imino group. The term "CO" refers to a compound having the formula

[0192] [ka]

[0193] It refers to a carbonyl group. The term "trityl group" refers to a group having the structural formula

[0194] [ka]

[0195] is. The term "4-methoxytrityl group" refers to a group having the structural formula

[0196] [ka]

[0197] is. The term "4,4'-dimethoxytrityl group" refers to a group having the structural formula

[0198] [ka]

[0199] is. The term "4,4',4''-trimethoxytrityl group" refers to a group having the structural formula

[0200] [ka]

[0201] is. term

[0202] [ka]

[0203] represents a bonding site that covalently links groups. In the structural formula of the compounds or ligands described herein,

[0204] [ka]

[0205] For the sake of brevity, all of the above structural formulas are depicted as specific isomers, but the present disclosure can include all isomers, such as tautomers, rotamers, geometric isomers, diastereomers, racemates and enantiomers. In the structural formulae of the compounds or ligands described herein, the bond

[0206] [ka]

[0207] represents an unspecified configuration. If a chemical structure has cis-trans isomerism, the bond

[0208] [ka]

[0209] The configuration may be a Z configuration or an E configuration, or may include both E and Z configurations simultaneously.

[0210] As used herein, the following definitions apply unless otherwise stated. For purposes of this invention, chemical elements are designated by CAS number in accordance with the Periodic Table of Chemical Elements and the "Handbook of Chemistry and Physics" (75th ed., 1994). Also, for general principles of organic chemistry, see "Organic Chemistry" by Thomas Sorrell (University Science Books, Sausalito: 1999) and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March (John Wiley & Sons, New York: 2007), the entire contents of which are incorporated herein by reference.

[0211] Unless otherwise stated or clearly contradicted by context, the articles "a," "an," "one," and "the," as used herein, are intended to include "at least one" or "one or more." Thus, as used herein, these articles refer to one or more than one (i.e., at least one) of the object article. For example, "a component" refers to one or more than one component, i.e., more than one component may be applied to or used in an embodiment of the method.

[0212] The term "comprises" is open-ended, i.e., includes the subject matter expressly set forth in the disclosure, but does not exclude subject matter relating to other aspects.

[0213] "Stereoisomers" refer to compounds that have identical chemical constitution but differ with regard to the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0214] "Chiral" refers to a molecule that is not superimposable on its mirror image, while "achiral" refers to a molecule that is superimposable on its mirror image.

[0215] "Enantiomers" refer to two isomers of a compound that are non-superimposable but mirror images of one another.

[0216] "Diastereomers" refer to stereoisomers that have two or more chiral centers and are not mirror images of one another. Diastereomers differ in physical properties such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high-resolution analytical procedures, for example, electrophoresis or chromatography, such as HPLC.

[0217] The definitions and rules of stereochemistry used herein generally follow those in "McGraw-Hill Dictionary of Chemical Terms (1984) by S.P. Parker, Ed., McGraw-Hill Book Company (New York)" and "Stereochemistry of Organic Compounds (John Wiley & Sons, Inc., New York, 1994)" by Eliel, E. and Wilen, S.

[0218] As used herein, the term "pharmaceutically acceptable salts" refers to organic and inorganic salts of the compounds of the present disclosure. Pharmaceutically acceptable salts are well known in the art, as described in S. M. Berge et al., J. Pharmaceutical Sciences (66, 1-19, 1977). Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerol phosphate, gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, malonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenpropionate, picrate, pivalate, propionate, stearate, thiocyanate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C 1-4 The present disclosure is also intended to contemplate quaternary ammonium salts formed by any compound containing an N group. Water- or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable non-toxic ammonium / quaternary ammonium salts, as well as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8Included are amine cations formed with counterions such as sulfonates and aromatic sulfonates.

[0219] Most pharmaceutically acceptable salts are well known to those skilled in the art and include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituent moieties present on the compounds described herein. When the compounds disclosed in the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base under solvent-free (pure) conditions or in a suitable inert solvent, or by ion exchange (whereby one base counterion (base) in the ionic complex is replaced by another). Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, magnesium salts, or similar salts.

[0220] The term "prodrug" as used in this disclosure refers to a compound that can be converted in vivo to a compound of formula (X). Such conversion can be effected by hydrolysis of the prodrug in the blood or by enzymatic conversion of the prodrug to the parent structure in the blood or tissue. The prodrug compounds in this disclosure can be esters, and existing inventions have identified esters that can be used as prodrugs, including phenyl esters, aliphatic (C1-C 24) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, the compounds of the present disclosure contain a hydroxyl group, which can be acylated to form a prodrug form of the compound. Other forms of prodrugs include phosphate esters, such as those obtained by phosphorylation of the hydroxyl group of the parent structure. For a detailed description of prodrugs, see the following documents: T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, J. Rautio et al, Pro-drugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270, and SJ Hecker. et al., Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry, 2008, 51, 2328-2345.

[0221] In the context of this specification, unless otherwise specified, "conjugation" refers to the covalent bonding of two or more chemical moieties, each having a specific function. Correspondingly, "conjugate" refers to a compound in which individual chemical moieties are covalently bonded. Furthermore, "drug conjugate" refers to a compound in which one or more chemical moieties having a specific function are covalently bonded to an active drug. Hereinafter, particularly in the Examples, the drug conjugate of the present disclosure may be abbreviated as "conjugate." In the context of this disclosure, drug conjugate should be understood as a general term for drug conjugates or a specific drug conjugate represented by a specific structural formula.

[0222] The term "aliphatic ring" refers to a ring having a cyclic carbon skeleton structure, such as a monocyclic ring, a spiro ring, a bridged ring, etc. The term "4- to 10-membered aliphatic ring" refers to a ring having 4 to 10 carbon atoms in the cyclic carbon skeleton structure.

[0223] The term "monocyclic" refers to a cycloalkyl group that contains only one ring and is called a monocyclic ring.

[0224] The term "spirocycle" refers to a compound in which two rings share one atom.

[0225] The term "bridged ring" refers to a structure in which two or more ring structures share two non-adjacent ring atoms with each other.

[0226] The term "cycloalkane group" refers to a monovalent or polyvalent saturated monocyclic, bicyclic, or tricyclic ring system containing from 3 to 12 ring carbon atoms. In one embodiment, a cycloalkyl group contains from 7 to 12 ring carbon atoms. In another embodiment, a cycloalkyl group contains from 3 to 8 ring carbon atoms. In yet another embodiment, a cycloalkyl group contains from 3 to 6 ring carbon atoms. The cycloalkyl groups can be independently unsubstituted or substituted with one or more substituents described herein.

[0227] The term "cycloolefin group" refers to a cyclized alkenyl. 4-6 Cycloalkenyl is intended to include cycloalkenyl groups containing 4, 5, or 6 carbon atoms. Exemplary cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, and cyclohexenyl groups.

[0228] The term "cycloalkyne group" refers to a monocyclic alicyclic hydrocarbon containing one or more carbon-carbon triple bonds.

[0229] The term "alkyl group" refers to a saturated, straight- or branched-chain monovalent hydrocarbon group having 1 to 50 carbon atoms, or 1 to 40 carbon atoms, or 1 to 20 carbon atoms, or 1 to 10 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms, where the alkyl group can be independently and optionally substituted with one or more substituents described herein. Substituents include, but are not limited to, deuterium, amino, hydroxy, cyano, F, Cl, Br, I, mercapto, nitro, oxo (=O), and the like. Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (iBu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -CH(CH3)CH2CH3), and tert-butyl (t-Bu, -CH(CH3)CH2CH3). -C(CH3)3), n-pentyl group (-CH2CH2CH2CH2CH3), 2-pentyl group (-CH(CH3)CH2CH2CH3), 3-pentyl group (-CH(CH2CH3)2), 2-methyl-2-butyl group (-C(CH3)2CH2CH3), 3-methyl-2-butyl group (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl group (-CH2CH2CH(CH3)2), 2-methyl-1-butyl group (-CH2CH(CH3)CH2CH3), n-hexyl group (-CH2CH2CH2CH2CH2CH2CH3), 2-hexyl group (-CH(CH3)CH2CH2CH2CH2CH3), 3-hexyl group (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl group (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl group (-CH(CH3)CH(CH3)CH2CH3), 4-methyl- Examples include 2-pentyl group (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl group (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl group (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl group (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl group (-CH(CH3)C(CH3)3), n-heptyl group, and n-octyl group.As used herein, the term "alkyl group" and its prefix "alk-" are inclusive of all straight and branched saturated carbon chains.

[0230] The term "alkylene group" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a straight- or branched-chain saturated hydrocarbon. Unless otherwise specified, alkylene groups contain 1 to 50 carbon atoms. In some embodiments, alkylene groups contain 1 to 40 carbon atoms. In other embodiments, alkylene groups contain 1 to 20 carbon atoms. In still other embodiments, alkylene groups contain 1 to 10 carbon atoms. In still other embodiments, alkylene groups contain 1 to 6 carbon atoms. Examples include, but are not limited to, methylene (-CH-), ethylene (-CHCH-), and isopropylidene (-CH(CH)CH-).

[0231] The term "alkenyl group" means an alkenyl group having at least one carbon-carbon sp 2 Alkenyl refers to a linear or branched monovalent hydrocarbon group having a double bond, including "cis" and "trans" configurations, or "E" and "Z" configurations, where the alkenyl group may be optionally substituted with one or more substituents described herein. In some embodiments, alkenyl groups contain 2 to 50 carbon atoms. In other embodiments, alkenyl groups contain 3 to 50 carbon atoms. In still other embodiments, alkenyl groups contain 2 to 40 carbon atoms. In still other embodiments, alkenyl groups contain 2 to 20 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), and the like.

[0232] The term "alkenylene group" means an alkenylene group having at least one carbon-carbon sp 2 It refers to a straight-chain or branched-chain divalent hydrocarbon group containing a double bond.

[0233] The term "alkynyl group" refers to a linear or branched monovalent hydrocarbon group containing 2 to 50 carbon atoms and at least one carbon-carbon sp triple bond, wherein the alkynyl group is optionally substituted with one or more substituents described herein. In one embodiment, an alkyne group contains 3 to 12 carbon atoms. In another embodiment, an alkyne group contains 2 to 6 carbon atoms. In yet another embodiment, an alkyne group contains 2 to 4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propynyl (-CHC≡CH), 1-propynyl (-C≡C-CH), and the like.

[0234] The term "alkylene group" refers to a straight- or branched-chain divalent alkynyl group having at least one carbon-carbon sp triple bond.

[0235] The term "alkoxy group" refers to an alkyl group attached to the remainder of the molecule by an oxygen atom, wherein alkyl group has the meaning described in this disclosure. Unless otherwise specified, the alkoxy group contains 1 to 50 carbon atoms. In some embodiments, the alkoxy group contains 1 to 40 carbon atoms. In other embodiments, the alkoxy group contains 1 to 20 carbon atoms. In still other embodiments, the alkoxy group contains 1 to 10 carbon atoms. The alkoxy group may be optionally substituted with one or more substituents described in this disclosure. Examples of alkoxy groups include methoxy (MeO, -OCH3), ethoxy (EtO, -OCH2CH3), 1-propoxy (n-PrO, -OCH2CH2CH3), 2-propoxy (i-PrO, -OCH(CH3)2), 1-butoxy (n-BuO, -OCH2CH2CH2CH3), 2-methyl-1-propoxy (i-BuO, -OCH2CH(CH3)2), 2-butoxy (s-BuO, -OCH(CH3)CH2CH3), 2-methyl-2-propoxy t-Butoxy group (t-BuO, t-butoxy, -OC(CH3)3), 1-pentyloxy group (n-pentyloxy group, -OCH2CH2CH2CH3), 2-pentyloxy group (-OCH(CH3)CH2CH2CH3), 3-pentyloxy group (-OCH(CH2CH3)2), 2-methyl-2-butoxy group (-OC(CH3)2CH2CH3), 3-methyl-2-butoxy group (-OCH(CH3)CH(CH3)2), 3-methyl-l-butoxy group (-OCH2CH2CH(CH3)2), 2-methyl-l-butoxy group (-OCH2CH(CH3)CH2CH 3) These include, but are not limited to:

[0236] The term "alkylamino group" includes "N-alkylamino group" and "N,N-dialkylamino group," wherein each amino group is independently substituted with one or two alkyl groups, the alkyl groups having the meanings described herein. In some embodiments, the alkylamino group is a lower alkylamino group formed by one or two C1-C6 alkyl groups bound to the nitrogen atom. In other embodiments, the alkylamino group is an alkylamino group formed by one or two C1-C4 lower alkyl groups bound to the nitrogen atom. Suitable alkylamino groups are monoalkylamino groups or dialkylamino groups. Examples include, but are not limited to, N-methylamino group, N-ethylamino group, N,N-dimethylamino group, N,N-diethylamino group, and the like.

[0237] The term "haloalkyl group," "haloalkenyl group," "haloalkoxy group," or "haloalkylamino group" refers to an alkyl group, alkenyl group, alkoxy group, or alkylamino group substituted with one or more halogen atoms, wherein the alkyl group, alkenyl group, alkoxy group, or alkylamino group has the meaning described herein. Examples of such groups include, but are not limited to, trifluoromethyl, 2,2,3,3-tetrafluoropropyl, trifluoromethoxy, trifluoromethylamino, and the like.

[0238] The terms "cycloalkyl group" or "heterocycloalkyl group," independently or in combination with other terms, refer to cyclic versions of "alkyl groups" and "heteroalkyl groups," respectively. Additionally, for heterocycloalkyl groups, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl groups include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms "cycloalkenyl" and "heterocycloalkenyl" refer to the divalent derivatives of cycloalkyl and heterocycloalkyl groups, respectively.

[0239] As described herein, the compounds of the present invention, such as those of the general formula above, or the specific examples, subclasses, and classes of compounds included within the embodiments, may be optionally substituted with one or more substituents.

[0240] Generally speaking, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced with a specified substituent. Unless otherwise specified, a substituent may have one substituent at each substitutable position of the group. When more than one position in a given structural formula is substituted with one or more substituents selected from a specified group, the substituents at each substitutable position may be the same or different.

[0241] The term "unsubstituted" means that the given group bears no substituents.

[0242] The term "optionally substituted with" is used interchangeably with the term "unsubstituted or substituted with." That is, the structure is either unsubstituted or substituted with one or more substituents described herein. The substituents described herein include, but are not limited to, D, F, Cl, Br, I, N, CN, NO, OH, SH, NH, alkyl groups, haloalkyl groups, haloalkoxy groups, haloalkylamino groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylamino groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, and the like.

[0243] In addition, unless otherwise clearly stated, the expressions "each ... is independently ...", "each ... is each independently ..." and "... is independently ..." used in this specification are used interchangeably, but should be understood in a broad sense, and may mean that specific options represented by the same symbols in different groups do not affect each other, or that specific options represented by the same symbols in the same group do not affect each other. Taking R3 as an example, the structural formula "C1-C optionally substituted with R3" is 50 Alkylene group" and structural formula "-C(O)-NH-C optionally substituted with R3 1-50 The specific choices of R3 in both "alkylene groups" do not affect each other.

[0244] The term "small interfering RNA (siRNA)" refers to a type of double-stranded RNA consisting of a sense strand and an antisense strand, each of which is 17 to 30 nucleotides in length. siRNA mediates targeted cleavage of RNA transcripts by the RNA-induced silencing complex (RISC) pathway by forming the RISC. Specifically, siRNA inhibits the translation of mRNA into amino acids and protein by inducing specific degradation of mRNA sequences through the known RNA interference (RNAi) process.

[0245] In the context of the present invention, the term "antisense strand (also called guide strand)" includes a region that is substantially complementary to a target sequence. The term "sense strand (or passenger strand)" refers to an iRNA strand that is substantially complementary to the antisense strand. The term "substantially complementary" means fully complementary or at least partially complementary. For example, the antisense strand is fully complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can occur within the internal or terminal regions of the molecule, with mismatches most tolerated within the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the iRNA.

[0246] The antisense strand being "at least partially substantially complementary" to an mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest.

[0247] In the context of the present invention, "oligonucleotide" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) consisting of 10 to 50 nucleotides. Oligonucleotides can regulate gene expression through several processes, including ribonucleic acid interference, ribonuclease-mediated target degradation, splicing regulation, non-coding RNA suppression, gene activation, and programmed genome editing.

[0248] In the context of this invention, "antisense oligonucleotides (ASOs)" are single-stranded oligonucleotide molecules typically consisting of 10 to 50 nucleotides. After entering cells, ASOs bind to complementary target mRNA through base pairing under the action of RNase H1, thereby inhibiting target gene expression.

[0249] In the context of the present invention, unless otherwise specified, capital letters A, U, G, C, and T represent the base composition of a nucleotide, lowercase letter m represents that one nucleotide adjacent to the left of the letter m is a nucleotide modified with a 2'-methoxy group, lowercase letter f represents that one nucleotide adjacent to the left of the letter f is a nucleotide modified with a 2'-fluoro group, and lowercase letter s represents that the two nucleotides adjacent to both sides of the letter s are linked by a phosphorothioate bond between them.

[0250] In the context of the present invention, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (antibacterial, antifungal, etc.), isotonic agent, salt, drug stabilizer, binder, excipient, dispersing agent, lubricant, sweetener, flavoring agent, coloring agent, or combination thereof. These carriers are known to those skilled in the art (see Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is encompassed.

[0251] In the context of the present invention, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, other liquid excipient, etc., that is suitable for a particular intended dosage form. To the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, e.g., to the extent that it produces adverse biological effects or interacts in an adverse manner with other components of the pharmaceutically acceptable composition, its use is also encompassed within the scope of the present invention.

[0252] In the context of the present invention, a "subject" refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (such as monkeys), mice, pigs, horses, donkeys, cattle, sheep, and any type of poultry.

[0253] In the context of the present invention, "treatment," "alleviation," or "amelioration" are used interchangeably herein. These terms refer to an approach to obtaining a beneficial or desired result, including, but not limited to, therapeutic benefit. "Therapeutic benefit" refers to the eradication or amelioration of the underlying disorder being treated. Therapeutic benefit may also be achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, resulting in an observed improvement in the subject, although the subject may still be afflicted with the underlying disorder.

[0254] In the context of the present invention, "prevention" and "prevention" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi reagent, or composition can be administered to a subject at risk of developing a particular disease, or to a subject who may not yet have been diagnosed but who has reported one or more physiological symptoms of the disease. General Experiment

[0255] The present disclosure will be described in detail below with reference to examples. All reagents and media used in the following examples were commercially available, and nucleic acid electrophoresis, real-time PCR, and other procedures were performed according to techniques known to those skilled in the art.

[0256] Unless otherwise specified, the siRNA sequences used herein were synthesized by Suzhou Biosyntech Biotechnology Co., Ltd. (Biosyntech). The PCR primers used herein were synthesized by Beijing Tsingke Biotechnology Co., Ltd. (Tsingke). The experimental animals used herein, C57BL / 6J mice, were purchased from SPF (Beijing) Biotechnology Co., Ltd. (SPF).

[0257] Unless otherwise specified, the meanings of base compositions and modifications described in various examples of the present disclosure are as follows: capital letters A, U, G, C, and T represent nucleotide base compositions, lowercase letter m represents a methoxy-modified nucleotide represented by the preceding letter, lowercase letter f represents a fluorine-modified nucleotide, and lowercase letter s represents a nucleotide represented by the preceding and following two letters that is linked by a phosphorothioate bond.

[0258] Unless otherwise specified, the in vivo activity experimental data were expressed as X ± SD. All experimental data were compiled and analyzed using GraphPad Prism 8.0 software.

[0259] Unless otherwise stated, the reagent ratios set forth in each example of the present invention were calculated on a volume to volume (v / v) basis.

[0260] The reagents and reagent sources used in the examples of the present invention are detailed in Table 1-1 and Table 1-2 below.

[0261] [Table 1-1]

[0262] All reagents in Table 1-1 were purchased from Beijing Ouhe Technology Co., Ltd. (Ouhe Company).

[0263] [Table 1-2]

[0264] The sources of equipment used in the examples of the present invention are detailed in Table 2 below.

[0265] [Table 2]

[0266] Example 1: Preparation of compounds (1) Synthesis of compound CR01008

[0267] [ka]

[0268] The synthetic route of compound CR01008 is as follows, where "Molecular Weight" represents the molecular weight.

[0269] [ka]

[0270] (1-1) Synthesis of Compound 2

[0271] [ka]

[0272] Compound 1 (trans-4-(Boc-amino)cyclohexanecarbaldehyde, 10.0 g, 1.0 equivalent) and aqueous formaldehyde solution (8.9 g, 37% by weight, 2.4 equivalents) were dissolved in 33 mL of methanol, and 13 mL of 45.3% by weight aqueous KOH solution was added dropwise. After the addition, the reaction mixture was stirred at 25°C for 30 minutes, then heated to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain the crude product as a white solid. Next, a small amount of water was added to the crude product to form a slurry, which was then filtered to obtain compound 2 as a white solid (9 g, 78.9% yield). MS-ESI (m / z) = 260 [M + H] + .

[0273] (1-2) Synthesis of Compound 3

[0274] [ka]

[0275] Compound 2 (9 g, 1 equivalent) prepared according to step (1-1) was dissolved in 70 ml of 1,4-dioxane, and a 1,4-dioxane solution of hydrogen chloride (45 ml, 4 M) was added and reacted with stirring at 25° C. for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 as a white solid (6.8 g, 100% yield).

[0276] (1-3) Synthesis of Compound 5

[0277] [ka]

[0278] Compound 3 (1.8 g, 2.0 equiv.) prepared according to step (1-2), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 equiv.), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 equiv.) were dissolved in 15 mL of DMF, and HBTU (1.9 g, 1.1 equiv.) was added. The mixture was stirred at 25 °C under a N atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse-phase chromatography (22% acetonitrile in water) to give compound 5 as a white solid (1.78 g, 64.4% yield). MS-ESI (m / z) = 589 [M + H] + .

[0279] (1-4) Synthesis of Compound 6

[0280] [ka]

[0281] Compound 5 (1.54 g, 1.0 equiv.) prepared according to step (1-3) was dissolved in 15 mL of pyridine. The reaction mixture was cooled to 0 °C in an ice-water bath. DMTrCl (chloro-4,4'-dimethoxytriphenylmethane, 1.32 g, 1.5 equiv.) was added at 0 °C and the mixture was allowed to react at 25 °C for 3 hours. The reaction was quenched by adding 15 mL of methanol. After completion of the reaction, the reaction mixture was evaporated to dryness under reduced pressure and purified by reverse-phase chromatography (60% acetonitrile by volume) to give compound 6 as a yellow solid (1 g, 42.7% yield). MS-ESI (m / z) = 891 [M + H] + .

[0282] (1-5) Synthesis of Compound CR01008

[0283] [ka]

[0284] Compound 6 (1.08 g, 1.0 equiv.) prepared according to step (1-4) was dissolved in 20 mL of anhydrous dichloromethane, and DCI (115 mg, 0.8 equiv.) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 equiv.) were added separately. The reaction mixture was purged with nitrogen gas three times and stirred at 25 °C for 2 hours. After the reaction was completed, 20 mL of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 mL of dichloromethane (3 × 20 mL). The organic phases were combined, evaporated to dryness under reduced pressure, purified by reverse-phase chromatography (72% acetonitrile by volume), and then dried under vacuum for 12 hours to obtain compound CR01008 as a white powder (1 g, 76.0% yield). MS-ESI (m / z) = 1091 [M + Na] + .

[0285] 1H NMR (400 MHz, DMSO-d6) δ 1.05 (d, J = 6.7 Hz, 6H).1.14 (d, J = 6.7 Hz, 6H), 1.37 - 1.17 (m, 5H), 1.60 - 1.40 (m, 6H),1.68 - 1.62 (m, 1H),1.80 (s, 3H),1.80 (s, 3H),1.92 (s, 3H), 2.02 (s, 5H),2.13 (s, 3H),2.71 (t, J = 5.9 Hz, 2H), 2.79 (d, J = 8.4 Hz, 1H), 2.87 (d, J = 8.4 Hz, 1H),3.36 (s, 1H), 3.58 - 3.39 (m, 3H), 3.69 - 3.60 (m, 2H), 3.75 (s, 7H), 3.90 (dt, J = 11.2, 8.8 Hz, 1H), 4.05 (s, 3H),4.51 (d, J = 8.4 Hz, 1H), 4.99 (dd, J = 11.3, 3.4 Hz, 1H), 5.24 (d, J = 3.4 Hz, 1H), 5.78 (s, 1H), 6.93 - 6.87 (m, 4H),7.35 - 7.21 (m, 7H), 7.44 - 7.37 (m, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H).

[0286] (2) Synthesis of compound CR01008Z

[0287] [ka]

[0288] Compound CR01008Z was prepared by coupling compound 6, which was used to synthesize compound CR01008, to the solid support CPG.

[0289] The synthetic route of compound CR01008Z is as follows.

[0290] [ka]

[0291] (2-1) Synthesis of Compound 9

[0292] [ka]

[0293] Compound 6 (500 mg) prepared according to step (1-4) was dissolved in 10 mL of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at 25°C for 16 hours. The reaction solution was purified by flash chromatography to give compound 9 (300 mg, 53.6% yield). MS-ESI (m / z) = 1013 [M + Na] + .

[0294] (2-2) Synthesis of compound CR01008Z

[0295] [ka]

[0296] Compound 9 (50 mg), prepared according to step (2-1), amino CPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 mL vial and reacted for 16 hours on a shaker. After the reaction was completed, the reaction solution was filtered to obtain a filter cake. The filter cake was then washed once with 10 mL (1 × 10 mL) of acetonitrile and dried under vacuum. The dried filter cake, DMAP (3 mg), Cap1 (10 mL, 200 V), and Cap2 (1 mL, 20 V) were added to a 20 mL vial and reacted for 6 hours on a shaker. After the reaction was completed, the reaction solution was filtered to obtain a filter cake. The filter cake was then washed once with 10 mL (1 × 10 mL) of acetonitrile and dried under vacuum to obtain compound CR01008Z (1.03 g, loading: 20-30 μmol / g).

[0297] Here, Cap1 and Cap2 are capping agents. Cap1 is a 20% by volume solution of N-methylimidazole in a pyridine / acetonitrile mixture, where the volume ratio of pyridine to acetonitrile is 3:5. Cap2 is a 20% by volume solution of acetic anhydride in acetonitrile.

[0298] (3) Synthesis of compound CR01013

[0299] [ka]

[0300] The synthetic route of compound CR01013 is as follows.

[0301] [ka]

[0302] (3-1) Synthesis of Compound 2

[0303] [ka]

[0304] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 4.9 g) was dissolved in 17 ml of methanol, and aqueous formaldehyde solution (4.21 g, concentration 37% by mass) and aqueous sodium hydroxide solution (6.5 ml, concentration 45.3% by mass) were added dropwise. After the addition was complete, the reaction system was heated to 60°C and allowed to react at 60°C for 2 hours with stirring. After the reaction was complete, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude product as a white solid. Next, a small amount of water was added to the crude product to form a slurry, which was then filtered and dried to obtain compound 2 as a white solid (4.8 g, yield 85.9%). ESI-MS (m / z) = 260.2 [M+H] + .

[0305] (3-2) Synthesis of Compound 3

[0306] [ka]

[0307] Compound 2 (4.8 g) prepared in step (3-1) was dissolved in 25 ml of 1,4-dioxane, and a 1,4-dioxane solution of hydrochloric acid (25 ml, 4 M, dioxane) was added. The reaction mixture was stirred at 25° C. for 2 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 as a white solid (3.6 g, yield 99.4%).

[0308] (3-3) Synthesis of Compound 11

[0309] [ka]

[0310] Compound 3 (3.6 g) prepared according to step (3-2) was dissolved in 36 mL of DMF, followed by the addition of TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g). The reaction mixture was stirred at 25 °C for 16 hours. After completion of the reaction, the reaction mixture was added to 200 mL of saturated aqueous sodium bicarbonate solution and extracted three times with 100 mL of ethyl acetate (3 × 100 mL). The combined organic phases were washed with 50 mL of saturated aqueous sodium chloride solution (1 × 50 mL), dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and purified by column chromatography (elution gradient: dichloromethane:methanol = 10:1) to give compound 11 as a white solid (2.3 g, 33.0% yield). ESI MS (m / z) = 379.5 [M+H]. + .

[0311] (3-4) Synthesis of Compound 12

[0312] [ka]

[0313] Compound 11 (2.3 g) prepared in step (3-3) was dissolved in 23 mL of methanol, and wet palladium-carbon (230 mg, 10% by weight loading) was added. The atmosphere was purged with hydrogen gas three times, and the reaction system was stirred under a hydrogen atmosphere (15 psi) at 25° C. for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filtrate, which was then evaporated to dryness under reduced pressure to obtain compound 12 as a yellow oil (1.48 g, 99.8% yield).

[0314] (3-5) Synthesis of Compound 13

[0315] [ka]

[0316] Compound 12 (1.48 g) prepared in step (3-4) was dissolved in 15 mL of DMF, and triethylamine (TEA, 1.22 g), compound 4 (1.35 g), and HBTU (3.45 g) were added. The reaction mixture was stirred at 25 °C for 16 hours. After completion of the reaction, the reaction mixture was added to 150 mL of saturated aqueous sodium bicarbonate solution and extracted three times with 50 mL of ethyl acetate (3 × 50 mL). The combined organic phases were washed with 30 mL of saturated aqueous sodium chloride solution (1 × 30 mL), dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and purified by column chromatography (elution gradient: water:acetonitrile = 5:1) to obtain compound 13 as a white solid (1.3 g, 31.8% yield). ESI-MS (m / z): 674.3 [M+H] + .

[0317] (3-6) Synthesis of Compound 14

[0318] [ka]

[0319] Compound 13 (1.1 g) prepared in step (3-5) was dissolved in 11 mL of pyridine, the reaction mixture was cooled to 0 °C in an ice-water bath, and DMTrCl (813 mg) was added batchwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour. After completion of the reaction, methanol was added to the reaction mixture to quench the reaction. The reaction mixture was evaporated to remove the solvent, and the mixture was purified by column chromatography (elution gradient: water:acetonitrile = 1:4) to obtain compound 14 as a white solid (800 mg, 50.3% yield). ESI-MS (m / z): 976.5 [M+H] + .

[0320] (3-7) Synthesis of compound CR01013

[0321] [ka]

[0322] Compound 14 (550 mg) was dissolved in 5 mL of dichloromethane (DCM) at room temperature, and 4,5-dicyanoimidazole (DCl, 53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 255.4 mg) were added. The atmosphere was purged with nitrogen three times, and the reaction mixture was stirred under a nitrogen atmosphere at 25 °C for 1 hour. After completion of the reaction, the reaction solution was washed twice with 5 mL of saturated aqueous sodium bicarbonate (2 × 5 mL) and once with 30 mL of saturated aqueous sodium chloride (1 × 30 mL). The organic phase was separated and dried over anhydrous sodium sulfate. The organic layer was evaporated to dryness under reduced pressure, and the mixture was purified by column chromatography (elution gradient: dichloromethane:methanol = 20:1) to obtain compound CR01013 as a white solid (532 mg, 80.4% yield). ESI-MS (m / z): 1176.7[M+H] + .

[0323] 1H NMR (400 MHz, DMSO-d6) δ 0.95 - 1.05 (d, J = 6.7 Hz, 5H), 1.06 - 1.15 (q, J = 7.6 Hz, 8H), 1.15 - 1.21 (t, J = 7.2 Hz, 14H), 1.72 - 1.80 (s, 3H), 1.84 - 1.92 (s, 3H), 1.94 - 2.07 (d, J = 16.0 Hz, 7H), 2.07 - 2.14 (s, 3H), 2.64 - 2.72 (q, J = 5.8 Hz, 2H), 2.74 - 2.89 (d, J = 8.5 Hz, 2H), 3.35 - 3.56 (m, 4H), 3.57 - 3.70 (m, 4H), 3.71 - 3.77 (s, 6H), 3.81 - 3.93 (m, 1H), 3.96 - 4.09 (d, J = 6.4 Hz, 3H), 6.82 - 6.97 (d, J = 8.7 Hz, 4H), 7.17 - 7.27 (t, J = 8.7 Hz, 5H), 7.27 - 7.34 (t, J = 7.6 Hz, 2H), 7.34 - 7.43 (d, J = 7.5 Hz, 2H).

[0324] (4) Synthesis of compound CR01013Z

[0325] [ka]

[0326] Compound CR01013Z was prepared by coupling compound 14, which was used to synthesize compound CR01013, to the solid support CPG.

[0327] The synthetic route of compound CR01013Z is as follows.

[0328] [ka]

[0329] (4-1) Synthesis of Compound 15

[0330] [ka]

[0331] Compound 14 (100 mg, 0.10 mmol) was dissolved in 2 mL of dichloromethane at room temperature, and triethylamine (25.9 mg, 0.25 mmol), DMAP (1.25 mg, 0.01 mmol), and compound 8 (succinic anhydride, 15.4 mg, 0.15 mmol) were added. The reaction mixture was stirred at 25 °C for 16 hours. After completion of the reaction, the solvent was removed by evaporation. The mixture was purified by column chromatography (elution gradient: water:acetonitrile = 2:1) to give compound 15 as a yellow oil (110 mg, 0.10 mmol, 100% yield). ESI-MS (m / z) = 1099.3 [M+Na] + .

[0332] (4-2) Synthesis of compound CR01013Z

[0333] [ka]

[0334] Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 mL of acetonitrile, and HBTU (24.2 mg, 0.06 mmol), DIEA (11.0 mg, 0.08 mmol), and amino CPG (1.06 g, loading: 80 μmol / g) were added. The reaction mixture was stirred at 25 °C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filter cake. The filter cake was washed twice with 50 mL of dichloromethane (2 × 50 mL), three times with 50 mL of acetonitrile (3 × 50 mL), and once with 50 mL of ethyl acetate (1 × 50 mL), and then vacuum dried. Cap1 (4.8 mL), Cap2 (0.54 mL), and DMAP (2.59 mg) were added to the dried filter cake, and the reaction mixture was stirred at 25 °C for 5 hours. After the reaction was completed, the reaction solution was filtered to obtain a filter cake, which was washed three times with 50 ml of acetonitrile (3 x 50 ml) and dried in vacuo to obtain compound CR01013Z (900 mg, loading: 20-30 μmol / g).

[0335] Here, Cap1 and Cap2 are capping agents. Cap1 was a 20% by volume solution of N-methylimidazole in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5, and Cap2 was a 20% by volume solution of acetic anhydride in acetonitrile.

[0336] (5) Synthesis of reference compound CR01014

[0337] [ka]

[0338] The synthetic route of the reference compound CR01014 is as follows.

[0339] [ka]

[0340] (5-1) Synthesis of Compound 18

[0341] [ka]

[0342] Compound 16 (maleic anhydride, 5.0 g), compound 17 (N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine, 12.1 g), and TFA (trifluoroacetic acid, 0.58 g) were dissolved in 35 mL of dichloromethane. The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at 25°C for 3 hours. After completion of the reaction, the reaction solution was washed once with 10 mL of purified water (1 x 10 mL) and once with 10 mL of saturated aqueous sodium chloride (1 x 10 mL). The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to give compound 18 (11 g), which was used directly in the next reaction without further purification. MS-ESI (m / z) = 232.0 [M + H] + .

[0343] (5-2) Synthesis of Compound 19

[0344] [ka]

[0345] Compound 18 (4.1 g) prepared in step (5-1) was dissolved in 20 mL of tetrahydrofuran and purged with nitrogen gas three times. A 1.0 M solution of LiAlH4 in tetrahydrofuran (17.7 mL) was added dropwise at 0 °C. The reaction mixture was cooled to 0 °C in an ice-water bath and stirred at 0 °C for 1 hour. After completion of the reaction, 32 mL of purified water and 24 mL of 1.0 M aqueous sodium hydroxide solution were added dropwise. The reaction mixture was filtered to obtain the filtrate. The filtrate was evaporated to dryness under reduced pressure to obtain compound 19 (6.0 g), which was used directly in the next reaction without further purification. MS-ESI (m / z) = 222.3 [M + H] + .

[0346] (5-3) Synthesis of Compound 20

[0347] [ka]

[0348] Compound 19 (5.0 g) prepared in step (5-2) was dissolved in 50 mL of methanol, and wet palladium-carbon (0.5 g, 10% by weight loading) and palladium-carbon hydroxide (0.5 g, 10% by weight loading) were added separately. The atmosphere was purged with hydrogen gas three times, and the reaction system was heated to 40°C and stirred at 40°C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain the filtrate, which was then evaporated to dryness under reduced pressure to obtain compound 20 (2.9 g). MS-ESI (m / z) = 132.18 [M + H] + .

[0349] (5-4) Synthesis of Compound 22

[0350] [ka]

[0351] Compound 20 (4.0 g) prepared in step (5-3), compound 21 (N-benzyloxycarbonyl-4-aminobutyric acid, 2.8 g), and DIEA (N,N-diisopropylethylamine, 15.6 g) were dissolved in 20 mL of N,N-dimethylformamide (DMF). HBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 17.2 g) was added. The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse-phase chromatography to give compound 22 (1.46 g, 35.6% yield). MS-ESI (m / z) = 367.41 [M + H]. + .

[0352] (5-5) Synthesis of Compound 23

[0353] [ka]

[0354] Compound 22 (1.46 g) prepared in step (5-4) was dissolved in 10 mL of methanol, and wet palladium-carbon (0.15 g, 10% by weight) was added. The mixture was purged with hydrogen gas three times and stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain the filtrate, which was then evaporated to dryness under reduced pressure to obtain crude product 23 (1.06 g), which was used directly in the next reaction without further purification. MS-ESI (m / z) = 233.28 [M + H] + .

[0355] (5-6) Synthesis of Compound 24

[0356] [ka]

[0357] Compound 23 (1.02 g) prepared in step (5-5) and compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetyloxy-6-(acetyloxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 1.41 g) were dissolved in 14 mL of N,N-dimethylformamide, and DIEA (N,N-diisopropylethylamine, 0.81 g) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.8 g) were added. The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by flash chromatography to give compound 24 (1.28 g, 63.5% yield). MS-ESI (m / z) = 648.68 [M + H] + .

[0358] (5-7) Synthesis of Compound 25

[0359] [ka]

[0360] Compound 24 (1.18 g) prepared in step (5-6) was dissolved in 12 mL of pyridine, and DMTrCl (4,4'-bismethoxytriphenylmethyl chloride, 0.98 g) was added batchwise. The mixture was purged with nitrogen gas three times and stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by flash chromatography to give compound 25 (1.0 g, 61.1% yield). MS-ESI (m / z) = 951.0 [M + H] + .

[0361] (5-8) Synthesis of compound CR01014

[0362] [ka]

[0363] Compound 25 (300 mg) prepared in step (5-7) was dissolved in 6 mL of dichloromethane, and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 152 mg) and 4,5-dicyanoimidazole (DCI, 30 mg) were added in batches. The reaction mixture was purged with nitrogen gas three times and stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by flash chromatography to give compound CR01014 (270 mg, 74.4% yield). MS-ESI (m / z) = 1151.27 [M + H] + .

[0364] 1H NMR (400 MHz, DMSO-d6) δ: 0.92 - 1.03 (d, J = 6.7 Hz, 8H), 1.04 - 1.13 (d, J = 6.8 Hz, 7H), 1.16 - 1.29 (m, 4H), 1.38 - 1.53 (dq, J = 7.2, 14.6 Hz, 4H), 1.55 - 1.68 (dt, J = 9.0, 15.1 Hz, 2H), 1.75 - 1.82 (s, 3H), 1.86 - 1.94 (s, 3H), 1.97 - 2.07 (s, 6H), 2.09 - 2.15 (s, 3H), 2.15 - 2.29 (m, 2H), 2.64 - 2.76 (d, J = 5.5 Hz, 3H), 2.99 - 3.16 (dt, J = 6.7, 14.4 Hz, 4H), 3.20 - 3.30 (d, J = 14.8 Hz, 1H), 3.37 - 3.53 (tt, J = 7.1, 14.6 Hz, 6H), 3.53 - 3.66 (dt, J = 8.4, 17.5 Hz, 3H), 3.74 - 3.78 (s, 6H), 4.00 - 4.09 (s, 3H), 6.84 - 6.96 (d, J = 8.3 Hz, 4H), 7.19 - 7.28 (t, J = 7.6 Hz, 5H), 7.28 - 7.41 (dt, J = 7.8, 22.9 Hz, 4H), 7.70 - 7.88 (d, J = 5.9 Hz, 2H).

[0365] (6) Synthesis of reference compound CR01014Z

[0366] [ka]

[0367] Compound CR01014Z was prepared by coupling the compound used to synthesize compound CR01014 to the solid support CPG.

[0368] The synthetic route of compound CR01014Z is as follows.

[0369] [ka]

[0370] (6-1) Synthesis of Compound 26

[0371] [ka]

[0372] Compound 25 (100 mg) prepared in step (5-7) was dissolved in 2 mL of dichloromethane, and compound 8 (succinic anhydride, 15.7 mg), 4-dimethylaminopyridine (DMAP, 1.2 mg), and triethylamine (TEA, 19.7 mg) were added. The atmosphere was purged with nitrogen gas three times, and the reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by flash chromatography to give compound 26 (73 mg, 66.7% yield). MS-ESI (m / z) = [M + H] + .

[0373] (6-2) Synthesis of compound CR01014Z

[0374] [ka]

[0375] Compound 26 (50 mg) prepared in step (6-1), amino CPG (1.19 g), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (27 mg), and N,N-diisopropylethylamine (12 mg) were added to a 20 mL vial and reacted for 16 hours on a shaker. After the reaction was completed, the reaction solution was filtered to obtain a filter cake. The filter cake was washed once with 10 mL (1 x 10 mL) of acetonitrile and then vacuum dried. The dried filter cake, 4-dimethylaminopyridine (DMAP, 3 mg), Cap1 (10 mL), and Cap2 (1 mL) were added to a 20 mL vial and reacted for 6 hours on a shaker. After the reaction was completed, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml (1×10 ml) of acetonitrile and dried in vacuo to obtain compound CR01014Z (1.03 g, loading: 20-30 μmol / g).

[0376] Here, Cap1 and Cap2 are capping agents. Cap1 is a 20% by volume solution of N-methylimidazole in a pyridine / acetonitrile mixture, where the volume ratio of pyridine to acetonitrile is 3:5. Cap2 is a 20% by volume solution of acetic anhydride in acetonitrile.

[0377] (7) Synthesis of reference compound L96-PS Compound L96-PS was purchased from Asymchem Laboratories (Tianjin) Co., Ltd., and the loading was 120±12 μmol / g as determined by UV / HPLC.

[0378] Here, the structural formula of the compound L96-PS is as follows:

[0379] [ka]

[0380] In the formula, PS represents a polystyrene resin solid phase support. Example 2: Synthesis of siRNA conjugates

[0381] (1) Polymerization of compounds According to the solid-phase nucleic acid synthesis using the phosphoramidite method, the above compounds linked to the solid support (i.e., CR01008Z, CR01013Z, CR01014Z) were used as the starting point of the cycle, and compounds not bound to the solid support (i.e., CR01008, CR01013, CR01014) were linked one by one. Specifically, CR01008Z was used as the starting point of the cycle to link CR01008 one by one, CR01013Z was used as the starting point of the cycle to link CR01013 one by one, and CR01014Z compound was used as the starting point of the cycle to link CR01014 one by one.

[0382] The connection of each compound involves four steps of reactions: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are as follows:

[0383] Each compound not bound to a solid support (ie, CR01008, CR01013, CR01014) was prepared into a solution with a concentration of 0.1 M using acetonitrile.

[0384] The deprotection reaction conditions for each step were the same: temperature 25°C, reaction time 70 seconds, deprotection agent dichloroacetic acid in dichloromethane (3% by volume), and molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support 5:1.

[0385] The coupling reaction conditions for each step were the same: temperature 25°C, molar ratio of the solid-phase-bound compound to the unbound compound 1:10, molar ratio of the solid-phase-bound compound to the coupling agent 1:65, reaction time 600 seconds, coupling agent 0.5 M 5-ethylthio-1H-tetrazole in acetonitrile, and thioagent 0.2 M xanthan hydride in acetonitrile / pyridine (volume ratio of acetonitrile to pyridine = 1:1).

[0386] The capping reaction conditions for each step were the same: temperature 25°C, reaction time 2 minutes, capping agent a mixed solution of Cap1 and Cap2 (molar ratio 1:1), Cap1 a 20% by volume N-methylimidazole solution in a pyridine / acetonitrile mixture (volume ratio of pyridine:acetonitrile = 3:5), Cap2 a 20% by volume acetic anhydride solution in acetonitrile, and the molar ratio of N-methylimidazole in the capping agent Cap1 to acetic anhydride in the capping agent Cap2 to the compound linked to the solid support was 1:1:1.

[0387] The conditions for the oxidation and sulfurization reactions were the same for each step. The oxidation reaction was performed at 25°C for 3 seconds, with 0.05 M iodine water used as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked to the solid support in the coupling reaction was 30:1. The oxidation reaction was performed in a water / pyridine mixed solvent (water:pyridine volume ratio = 1:9). The sulfurization reaction was performed at 25°C for 360 seconds, with 0.2 M xanthan hydride in pyridine as the thiol agent. The molar ratio of thiol to the compound linked to the solid support in the coupling reaction was 4:1. The sulfurization reaction was performed in a water / pyridine mixed solvent (water:pyridine volume ratio = 1:9).

[0388] Trimer CR01008 (denoted as (CR01008) × 3), trimer CR01013 (denoted as (CR01013) × 3), and trimer CR01014 (denoted as (CR01014) × 3) were separately prepared by the above method.

[0389] The structural formula of the trimer CR01008 is as follows:

[0390] [ka]

[0391] The structural formula of the trimer CR01013 is as follows:

[0392] [ka]

[0393] The structural formula of the trimer CR01014 is as follows:

[0394] [ka]

[0395] (2) Synthesis of the sense strand (SS) Following solid-phase nucleic acid synthesis using the phosphoramidite method, compounds linked to a solid support (i.e., trimer CR01008, trimer CR01013, trimer CR01014, and L96) were used as the starting point for the cycle, and nucleoside monomers were linked one by one in the 3'-5' direction according to the nucleotide sequence. Linking each nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are as follows:

[0396] The nucleoside monomer was prepared as a 0.1 M solution of the nucleoside monomer in acetonitrile.

[0397] The deprotection reaction conditions for each step were the same: temperature 25°C, reaction time 70 seconds, deprotection agent dichloroacetic acid in dichloromethane (3% by volume), and molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support 5:1.

[0398] The coupling reaction conditions for each step were the same: temperature 25°C, molar ratio of the nucleic acid sequence linked to the solid support to the nucleotide monomer 1:10, molar ratio of the nucleic acid sequence linked to the solid support to the coupling agent 1:65, reaction time 600 seconds, coupling agent 0.5 M 5-ethylthio-1H-tetrazole in acetonitrile, and thioagent 0.2 M xanthan hydride in acetonitrile / pyridine (volume ratio of acetonitrile:pyridine = 1:1).

[0399] The capping reaction conditions for each step were the same: temperature 25°C, reaction time 2 minutes, capping agent a mixed solution of Cap1 and Cap2 (molar ratio 1:1), Cap1 a 20% by volume N-methylimidazole solution in a pyridine / acetonitrile mixture (volume ratio of pyridine:acetonitrile = 3:5), Cap2 a 20% by volume acetic anhydride solution in acetonitrile, and the molar ratio of N-methylimidazole in the capping agent Cap1 to acetic anhydride in the capping agent Cap2 to the nucleic acid sequence linked to the solid phase support was 1:1:1.

[0400] The oxidation reaction conditions for each step were the same. The oxidation reaction conditions were a temperature of 25°C, a reaction time of 3 seconds, 0.05 M iodine water as the oxidizing agent, and a molar ratio of iodine to the nucleic acid sequence linked to the solid support in the coupling reaction of 30:1. The oxidation reaction was carried out in a water / pyridine mixed solvent (water:pyridine volume ratio = 1:9). The sulfurization reaction conditions were a temperature of 25°C, a reaction time of 360 seconds, 0.2 M xanthan hydride pyridine solution as the thiol agent, and a molar ratio of thiol to the compound linked to the solid support in the coupling reaction of 4:1. The sulfurization reaction was carried out in a water / pyridine mixed solvent (water:pyridine volume ratio = 1:9).

[0401] After the final nucleoside monomer was linked, the nucleic acid sequence linked to the solid support was sequentially cleaved, deprotected, purified, desalted, and lyophilized to obtain the sense strand.

[0402] The cleavage and deprotection conditions were as follows: The solid-phase support-linked synthetic nucleotide sequence was added to a 25% by weight aqueous ammonia solution (0.5 ml / μmol used) and reacted at 55°C for 16 hours. The solvent was removed and the mixture was concentrated to dryness under vacuum. After treatment with aqueous ammonia, the single-stranded nucleic acid was dissolved in 0.4 ml / μmol of N-methylpyrrolidone, and then 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride were added to remove the 2'-O-TBDMS protecting group from the ribose.

[0403] The purification and desalting conditions were as follows. Nucleic acids were purified using a preparative ion chromatography column (Source 15Q) with a gradient elution of NaCl. Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1) and the solvent was a water / acetonitrile mixture (water:acetonitrile volume ratio = 9:1). Eluent 2 was 1.5 M sodium chloride and 20 mM sodium phosphate (pH 8.1) and the solvent was a water / acetonitrile mixture (water:acetonitrile volume ratio = 9:1), with an elution gradient of eluent 1:eluent 2 = (100:0) to (50:50). The collected eluents were combined and desalted using a reverse-phase chromatography purification column. The desalting conditions included desalting using a Sephadex column packed with Sephadex G25 and elution with deionized water.

[0404] Detection: Purity was detected by ion exchange chromatography (IEX-HPLC). Molecular weight was measured by liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value was approximately equal to the theoretical value, it indicated that the compound was conjugated to the 3' end of the siRNA sense strand.

[0405] Taking the trimer CR01008 as an example, the structural formula of the sense strand is as follows:

[0406] [ka]

[0407] Taking the trimer CR01013 as an example, the structural formula of the sense strand is as follows:

[0408] [ka]

[0409] (2) Synthesis of antisense strand (AS) The antisense strand was synthesized using a standard solid support. The conditions for deprotection, coupling, capping, oxidation or sulfurization, cleavage and deprotection, and purification and desalting in the solid-phase synthesis of the antisense strand were the same as those used in the synthesis of the sense strand in step (1).

[0410] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC). Molecular weight was measured by liquid chromatography mass spectrometry (LC-MS), and the measured molecular weight was compared with the theoretical molecular weight. If the measured molecular weight was approximately equal to the theoretical value, it indicated that the antisense strand of siRNA was obtained.

[0411] (3) Synthesis of siRNA conjugates The sense strand synthesized in step (1) and the antisense strand synthesized in step (2) were mixed in an equimolar ratio, dissolved in water for injection, heated to 95°C, slowly cooled to room temperature, and left at room temperature for 10 minutes to allow the sense strand and antisense strand to form a double-stranded structure through hydrogen bonding, thereby obtaining the siRNA conjugates having the sense strand and antisense strand shown in Table 3 below. The data in Table 4 below show that the sense strand (SS) and antisense strand (AS) exhibit good binding with the ligand and maintain higher purity.

[0412] [Table 3]

[0413] [Table 4]

[0414] Example 3: In vivo toxicity testing of siRNA conjugates C57BL / 6J mice were randomly divided into two groups (one male and one female in each group). Mice in each test group received a single subcutaneous injection of 300 mg / kg (calculated as siRNA) of the siRNA conjugate. After 14 days of observation, no animal deaths or clinical symptoms related to adverse drug reactions were observed. After completion of the observation period, all mice were roughly necropsied, and no abnormalities were found. These results demonstrate the high safety of the siRNA conjugate and its low toxicity at the animal level.

[0415] Example 4: Inhibition of mRNA expression of target genes in mice by siRNA conjugates Six- to eight-week-old C57BL / 6J female mice were randomly assigned to each group based on their body weight. The drug dose for each group was calculated based on their body weight. Each siRNA conjugate was prepared in PBS at the corresponding concentration (calculated based on siRNA) and administered subcutaneously to the abdomen of each mouse at a volume of 5 ml / kg (calculated based on siRNA). The PBS control group received the same volume of PBS solution without siRNA conjugate. The day of administration was recorded as day 1 (D1). At predetermined time points after administration, such as day 8 (D8), day 15 (D15), and day 29 (D29), five mice from each group were sacrificed. The sacrificed mice were grossly dissected, and liver tissue was collected and cut into approximately 2 mm3 pieces and stored in RNALater.

[0416] Liver tissue samples obtained from different test groups at different time points were collected from the above RNALater and homogenized for 60 seconds using a Tissuelyser II automatic tissue homogenizer. Total RNA was then extracted using an automatic nucleic acid extraction device (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd.) according to the standard procedure for total RNA extraction.

[0417] 1 μg of the total RNA was collected and reverse-transcribed using a reverse transcription kit (Promega, Reverse Transcription System, A3500). 15Primers were selected, and 20 μL of reverse transcription system was prepared according to the instructions of the reverse transcription kit, and the reverse transcription reaction was carried out. After the reaction was completed, 80 μL of RNase-free water was added to the reverse transcription system to obtain a cDNA solution. Next, a real-time fluorescent quantitative PCR kit (ABI, SYBR TM The mRNA expression level of the target gene in liver tissue was measured using Select Master Mix (catalog number: 4472908). In the real-time fluorescent quantitative PCR assay, the target gene and the internal standard gene were detected using primers for the target gene and primers for the internal standard gene, respectively. 20 μL of real-time PCR reaction system was prepared per PCR reaction well according to the method described in the real-time fluorescent quantitative PCR kit's instructions. Each reaction system contained 5 μL of the cDNA solution obtained by the reverse transcription reaction described above, SYBR TM The reaction mixture contained 10 μL of Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of RNase-free H2O. The reaction mixture was run on a real-time fluorescent quantitative PCR system (ABI StepOnePlus). TM ) and performed real-time PCR amplification using a three-step method. The amplification procedure included pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The above denaturation, annealing, and extension process was repeated 40 times. In this real-time fluorescent quantitative PCR assay, the ΔΔCt method was used to relatively quantify the mRNA expression level and inhibition rate of the target gene in each test group, which was calculated as follows:

[0418] ΔCt(test group) = Ct(target gene of test group) - Ct(internal control gene of test group) ΔCt(control group) = Ct(target gene of control group) - Ct(internal standard gene of control group) ΔΔCt (test group) = ΔCt (test group) - ΔCt (mean value of control group) ΔΔCt(control group) = ΔCt(control group) - ΔCt(mean value of the control group) Here, ΔCt (mean value of control group) is the arithmetic mean of the ΔCt (control group) values ​​for five mice sacrificed at the same time point in the control group. Therefore, each mouse in the test and control groups corresponds to a single ΔΔCt value.

[0419] The mRNA expression levels of the target genes in the test groups were normalized to those in the control group, where the mRNA expression levels of the target genes in the control group were defined as 100%. Relative expression level of target gene mRNA in the test group = 2 - ΔΔ Ct (Test group) x 100% Inhibition rate of target gene mRNA in test group = (1 - relative expression level of target gene mRNA in test group) x 100%

[0420] Example 4-1 Evaluation of in vivo activity of superoxide dismutase 1 siRNA conjugated with trimeric CR01008 carrier In this example, we measured the inhibitory activity of the siRNA sequence RZ899015 (abbreviated as RZ899015), conjugated to the trimeric CR01008 carrier at the 3' end of the sense strand, and the L96-conjugated RZ599001 (abbreviated as RZ599001), on the target gene SOD1 in vivo, according to a method for inhibiting mRNA expression of a target gene in vivo in mice. RZ899015 and RZ599001 have the same nucleic acid sequence and chemical modifications, but differ only in their delivery carrier structures.

[0421] Six- to eight-week-old C57BL / 6J mice were randomly divided into three groups (five mice per group) based on body weight: a PBS control group, an RZ899015 group, and an RZ599001 group. Each group received PBS solution, RZ899015, or RZ599001 subcutaneously in the abdominal cavity at a dose of 1 mg / kg per mouse in a volume of 5 mL / kg. The day of administration was recorded as day 1 (D1), and the mice were sacrificed on day 8 (D8) after administration.

[0422] As shown in Figure 1 and Table 6, the experimental results revealed that on day 8, the trimeric CR01008 conjugate RZ899015 group and the L96 conjugate RZ599001 group exhibited comparable in vivo activity.

[0423] [Table 5]

[0424] [Table 6]

[0425] Example 4-2 Evaluation of in vivo activity of siRNA targeting SOD1 conjugated with trimeric CR01008 carrier, CR01013 carrier, and CR01014 carrier In this example, the inhibitory activity of the siRNA sequence RZ899015 (abbreviated as RZ899015) conjugated to a trimer CR01008 carrier at the 3' end of the sense strand, the siRNA sequence RZ899026 (abbreviated as RZ899026) conjugated to a trimer CR01013 carrier, the siRNA sequence RZ899027 (abbreviated as RZ899027) conjugated to a trimer CR01014 carrier, and the L96 conjugate RZ599001 (abbreviated as RZ599001) on the target gene SOD1 in mice was measured according to a method for inhibiting mRNA expression of a target gene in mice. RZ899015, RZ899026, RZ899027, and RZ599001 have the same nucleic acid sequence and chemical modifications, and only differ in delivery carrier structure. However, the only difference between RZ899015 (bound to the CR01008 carrier) and RZ899026 (bound to the CR01013 carrier) was the linker length.

[0426] Six- to eight-week-old C57BL / 6J mice were randomly divided into five groups (20 mice per group) based on body weight: PBS control group, RZ899015 group, RZ899026 group, RZ899027 group, and RZ599001 group. Each group received PBS solution, RZ899015, RZ899026, RZ899027, or RZ599001 subcutaneously in the abdominal region at a dose of 3 mg / kg per mouse in a volume of 5 mL / kg. The day of administration was recorded as day 1 (D1), and five mice in each group were sacrificed on days 25 (D25), 29 (D29), 43 (D43), and 57 (D57) after administration.

[0427] As shown in Figure 2 and Table 8, the experimental results revealed that the trimeric CR01008 conjugate (RZ899015) and the trimeric CR01013 conjugate (RZ899026) exhibited superior in vivo inhibitory activity and sustained efficacy against target genes compared to the L96 conjugate (RZ599001). In particular, at D57, RZ899015 and RZ899026 maintained 70.39% and 62.42% inhibitory activity against the target gene, respectively, while the L96 conjugate (RZ599001) maintained only 52.19% inhibitory activity.

[0428] [Table 7]

[0429] [Table 8]

[0430] Example 4-3 Evaluation of in vivo activity of angiopoietin-like protein 3 siRNA conjugated to trimeric CR01013 and CR01014 carriers In this example, we measured the inhibitory activity of the target gene ANGPTL3 in mice using the siRNA sequence RZ897001 (abbreviated as RZ897001) conjugated to a trimer CR01013 carrier at the 3' end of the sense strand, the siRNA sequence RZ897002 (abbreviated as RZ897002) conjugated to a trimer CR01014 carrier, and the L96-conjugated RZ597002 (abbreviated as RZ597002). RZ897001, RZ897002, and RZ597002 have the exact same nucleic acid sequence and chemical modifications, with only the delivery carrier structure differing.

[0431] Six- to eight-week-old C57BL / 6J mice were randomly divided into four groups (20 mice per group) based on body weight: PBS control group, RZ897001 group, RZ897002 group, and RZ597002 group. Each group received PBS solution, RZ897001, RZ897002, or RZ597002 subcutaneously in the abdominal cavity at a dose of 3 mg / kg per mouse in a volume of 5 mL / kg. The day of administration was recorded as day 1 (D1), and five mice from each group were sacrificed on days 15 (D15), 29 (D29), 43 (D43), and 57 (D57) after administration.

[0432] As shown in Figure 3 and Table 10, the experimental results demonstrated that the trimeric CR01013 conjugate (RZ897001) exhibited superior in vivo inhibitory activity and sustained efficacy against target genes compared to the L96 conjugate (RZ597002). At D43, RZ897001 maintained 74.45% inhibitory activity against the target gene, while the L96 conjugate (RZ597002) maintained only 62.84% inhibitory activity. At D57, RZ897001 still maintained 51.58% inhibitory activity against the target gene, while the L96 conjugate (RZ597002) maintained only 31.17% inhibitory activity.

[0433] [Table 9]

[0434] [Table 10]

[0435] As used herein, terms such as "embodiments," "some embodiments," "examples," "particular examples," and "some examples" mean that a particular feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. Furthermore, particular features, structures, materials, or characteristics described herein can be combined in any suitable manner in any one or more embodiments or examples. Furthermore, unless mutually inconsistent, those skilled in the art can combine different embodiments or examples described herein and features of different embodiments or examples.

[0436] Although the embodiments of the present invention have been described above, it should be understood that the above embodiments are merely illustrative and are not intended to limit the present invention. Those skilled in the art may make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A compound represented by the following formula (Ia), or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a prodrug thereof: 【Chemistry 1】 [In formula (Ia), R 1 represents a hydroxy protecting group, 【Chemistry 2】 n is 0, 1, 2, or 3; each Z is independently a hydroxy group or a mercapto group; each p is independently 1, 2, or 3; each q is independently 1, 2, or 3; each A is independently an unsubstituted or substituted 4- to 10-membered aliphatic ring; each X is independently NH, O, or S; Each L 1 are each independently 【Transformation 3】 where j is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each R 3 are each independently H, C 1 -C 6 Alkyl group, C 1 -C 6 haloalkyl group, or C 1 -C 6 is an alkoxy group, Each L 2 are each independently C 1 -C 30 an alkylene group or 【Chemistry 4】 (where each R L2a are each independently C 1 -C 10 is an alkylene group, and each R L2b are each independently O, S, NH, or —NH—C(O)—, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each Y is independently NH, O, or S; Each R 4 are each independently 【Transformation 5】

2. Each A is independently 【Transformation 6】 2. The compound of claim 1, wherein:

3. 2. The compound of claim 1, wherein any X is NH.

4. Either L 1 but 【Transformation 7】 2. The compound of claim 1, wherein:

5. R 1 2. The compound according to claim 1, wherein is a trityl group, a 4-methoxytrityl group, a 4,4'-dimethoxytrityl group, or a 4,4',4''-trimethoxytrityl group.

6. Each L 2 is each independently one of the following structures: 【Transformation 8】

7. 2. The compound of claim 1, having a structure represented by formula (IIa): 【Chemistry 9】 [In formula (IIa), R 1 , R 2 , n, p, q, Z, X, L 1 , R 3 , L 2 , Y and R 4 is as defined in formula (Ia) in claim 1.

8. 2. The compound of claim 1, having a structure represented by formula (IIIa): 【Chemistry 10】 [In formula (IIIa), R 1 , R 2 , n, p, q, Z, R 3 , L 2 , Y and R 4 is as defined in formula (Ia) in claim 1.

9. 9. The compound of claim 8, wherein when n is 0, the structural formula of the compound is: 【Chemistry 11】

10. 2. The compound of claim 1, wherein the compound has a structure represented by formula (IVa): 【Chemistry 12】 [In formula (IVa), R 1 , R 2 , n, p, q, Z, R 3 , L 2 and Y are as defined in formula (Ia) in claim 1.

11. 11. The compound of claim 10, wherein when n is 0, the structural formula of the compound is: 【Chemistry 13】

12. 2. The compound of claim 1, wherein the compound has one of the following structures: 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】

13. A compound represented by the following formula (IIIb), or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a prodrug thereof: 【Chemistry 17】 [In formula (IIIb), * represents a linking site for linking a pharmaceutically active molecule, m is 1, 2, 3, or 4; Z, p, q, R 3 , L 2 and Y is as defined in formula (Ia) in claim 1.

14. 14. The compound of claim 13, wherein the compound has one of the following structures: [Chemistry 18] (where * represents the linking site for linking the pharmaceutically active molecule)

15. A conjugate having a structure represented by the following formula (IIIc): 【Chemistry 19】 [In formula (IIIc), Nu represents an oligonucleotide; m is 1, 2, 3, or 4; Z, p, q, R 3 , L 2 and Y are as defined in formula (Ia) in claim 1.

16. A composition comprising the conjugate of claim 15.

17. Use of a compound according to any one of claims 1 to 14, a conjugate according to claim 15 or a composition according to claim 16 in the manufacture of a medicament for the prevention and / or treatment of a disease.

18. Use of a compound according to any one of claims 1 to 14, a conjugate according to claim 15 or a composition according to claim 16 in the manufacture of a medicament for reducing the expression or activity of a target gene.

19. 17. A method for reducing the expression or activity of a target gene, comprising contacting a cell with the conjugate of claim 15 or the composition of claim 16.

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