Compounds, conjugates, compositions and uses thereof

Abstract

The invention relates to a compound, a conjugate, a composition and application thereof, in particular to a compound with a structure as shown in a specific formula (Ia) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof. The compounds can be used for preventing and / or treating expression or activity of specific target genes.

Description

Compounds, conjugates, compositions and their uses Technical Field

[0001] The present disclosure relates to the field of medicine, and in particular, to a compound, a conjugate, a composition and uses thereof. Background Art

[0002] Small-molecule nucleic acid drugs, represented by small interfering RNA (siRNA), antisense oligodeoxynucleotides (ASODN), and chromatin stimulatory motifs (CpGs), are playing an increasingly important role in gene therapy. Some drugs have been approved for marketing by the FDA, and a number are currently in preclinical and clinical trials. Nucleic acid drugs are nucleic acid sequences that specifically target disease-causing genes or proteins through binding or cleavage, thereby inhibiting or promoting the expression of certain genes or proteins. These include all normal human genes that can replace defective ones, antisense nucleic acids that block gene expression, and single-stranded nucleic acids that promote triplex formation, such as siRNA, DNA, microRNA, or CpGs.

[0003] Delivery systems are one of the core technologies in the development of small nucleic acid drugs. Currently, the most widely studied small nucleic acid delivery system globally is targeted conjugation delivery technology. There is an urgent need in this field to develop new drug conjugates with higher in vivo delivery efficiency, lower toxicity, and higher activity.

[0004] Summary of the Invention

[0005] The present disclosure aims to solve at least one of the technical problems existing in the prior art, at least to a certain extent.

[0006] To this end, in the first aspect of the present disclosure, the present disclosure provides a compound represented by formula (Ia) or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof:

[0007] In formula (Ia), R1 represents a hydroxyl protecting group;

[0008] R2 is in, represents a solid phase support, and R2' is a covalent linking group connected to the solid phase support;

[0009] n is 0, 1, 2, or 3;

[0010] Each Z is independently hydroxyl or thiol;

[0011] Each p is independently 1, 2 or 3;

[0012] Each q is independently 1, 2 or 3;

[0013] Each A is independently an unsubstituted or substituted 4-10 membered aliphatic ring;

[0014] Each X is independently NH, O or S;

[0015] Each L1 is independently Wherein, j is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0016] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0017] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0018] Each Y is independently NH, O or S;

[0019] Each R4 is independently

[0020] For example, when n is 0, the structural formula of the compound is as shown in formula (Ia-0):

[0021] In some embodiments of the present disclosure, 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"-trimethoxytriphenyl group (TMTr group).

[0022] In some specific embodiments of the present disclosure, in formula (Ia), R1 is 4,4'-dimethoxytrityl (DMTr group).

[0023] In some specific embodiments of the present disclosure, in formula (Ia), R2 is

[0024] In some specific embodiments of the present disclosure, in formula (Ia), R2 is in, represents a solid phase carrier, R2' is That is: in formula (Ia), R2 is

[0025] In some specific embodiments of the present disclosure, in Formula (Ia), each Z is hydroxyl.

[0026] In some embodiments of the present disclosure, in Formula (Ia), each p is independently 1 or 2.

[0027] In some specific embodiments of the present disclosure, in Formula (Ia), each p is 1.

[0028] In some embodiments of the present disclosure, in Formula (Ia), each q is independently 1 or 2.

[0029] In some specific embodiments of the present disclosure, in Formula (Ia), each q is 1.

[0030] In some specific embodiments of the present disclosure, in Formula (Ia), each p is 1, and each q is 1.

[0031] In some embodiments of the present disclosure, in Formula (Ia), each A is independently a substituted or substituted 4-10 membered cycloalkane group or a substituted or substituted 4-10 membered cycloalkene group.

[0032] In some embodiments of the present disclosure, in Formula (Ia), each A is independently a substituted or unsubstituted 4-10 membered cycloalkane group.

[0033] In some embodiments of the present disclosure, in formula (Ia), each A is independently a 4-10 membered cycloalkane group, such as a monocyclic, spirocyclic or bridged ring.

[0034] In some embodiments of the present disclosure, in formula (Ia), each A is independently

[0035] In some specific embodiments of the present disclosure, in formula (Ia), each A is

[0036] In some specific embodiments of the present disclosure, in Formula (Ia), each X is NH.

[0037] In some specific embodiments of the present disclosure, in formula (Ia), each L1 is

[0038] In some specific embodiments of the present disclosure, in Formula (Ia), each R3 is H.

[0039] In some embodiments of the present disclosure, in formula (Ia), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0040] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0041] In some specific embodiments of the present disclosure, k is 1.

[0042] In some embodiments of the present disclosure, in formula (Ia), each L2 is independently

[0043] In some specific embodiments of the present disclosure, in Formula (Ia), each Y is O.

[0044] In some specific embodiments of the present disclosure, in formula (Ia), each R4 is

[0045] In some embodiments of the present disclosure, the compound has a structure represented by formula (IIa):

[0046] In formula (IIa), R1 represents a hydroxyl protecting group;

[0047] R2 is in, represents a solid phase support, and R2' is a covalent linking group connected to the solid phase support;

[0048] n is 0, 1, 2, or 3;

[0049] Each Z is independently hydroxyl or thiol;

[0050] Each p is independently 1, 2 or 3;

[0051] Each q is independently 1, 2 or 3;

[0052] Each X is independently NH, O or S;

[0053] Each L1 is independently Wherein, j is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0054] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0055] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0056] Each Y is independently NH, O or S;

[0057] Each R4 is independently

[0058] For example, when n is 0, the structural formula of the compound is:

[0059] In some embodiments of the present disclosure, 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"-trimethoxytriphenyl group (TMTr group).

[0060] In some specific embodiments of the present disclosure, in formula (IIa), R1 is 4,4'-dimethoxytrityl (DMTr group).

[0061] In some specific embodiments of the present disclosure, in formula (IIa), R2 is

[0062] In some specific embodiments of the present disclosure, in formula (IIa), R2 is in, represents a solid phase carrier, R2' is That is: in formula (IIa), R2 is

[0063] Wherein, in the context of the present disclosure, the solid phase carrier It can also be expressed as R-SC. The solid phase carrier has the same meaning as R-SC. For example, the solid phase carrier can be a controlled pore glass (CPG) solid phase carrier, a solid phase polymer carrier (SPS, for example, polystyrene (PS) resin, etc.).

[0064] In some specific embodiments of the present disclosure, in formula (IIa), each Z is a hydroxyl group.

[0065] In some embodiments of the present disclosure, in Formula (IIa), each p is independently 1 or 2.

[0066] In some specific embodiments of the present disclosure, in Formula (IIa), each p is 1.

[0067] In some embodiments of the present disclosure, in Formula (IIa), each q is independently 1 or 2.

[0068] In some specific embodiments of the present disclosure, in Formula (IIa), each q is 1.

[0069] In some specific embodiments of the present disclosure, in Formula (IIa), each p is 1, and each q is 1.

[0070] In some specific embodiments of the present disclosure, in Formula (IIa), each X is NH.

[0071] In some specific embodiments of the present disclosure, in formula (IIa), each L1 is

[0072] In some specific embodiments of the present disclosure, in formula (IIa), each R3 is H.

[0073] In some embodiments of the present disclosure, in formula (IIa), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0074] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0075] In some specific embodiments of the present disclosure, k is 1.

[0076] In some embodiments of the present disclosure, in formula (IIa), each L2 is independently

[0077] In some specific embodiments of the present disclosure, in formula (IIa), each Y is O.

[0078] In some specific embodiments of the present disclosure, in formula (IIa), each R4 is In some embodiments of the present disclosure, the compound has a structure represented by formula (IIIa):

[0079] In formula (IIIa), R1 represents a hydroxyl protecting group;

[0080] R2 is in, represents a solid phase support, and R2' is a covalent linking group connected to the solid phase support;

[0081] n is 0, 1, 2, or 3;

[0082] Each Z is independently hydroxyl or thiol;

[0083] Each p is independently 1, 2 or 3;

[0084] Each q is independently 1, 2 or 3;

[0085] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0086] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0087] Each Y is independently NH, O or S;

[0088] Each R4 is independently

[0089] For example, when n is 0, the structural formula of the compound is:

[0090] In some embodiments of the present disclosure, 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"-trimethoxytriphenyl group (TMTr group).

[0091] In some specific embodiments of the present disclosure, in formula (IIIa), R1 is 4,4'-dimethoxytrityl (DMTr group).

[0092] In some specific embodiments of the present disclosure, in formula (IIIa), R2 is

[0093] In some specific embodiments of the present disclosure, in formula (IIIa), R2 is in, represents a solid phase carrier, R2' is That is: in formula (IIIa), R2 is

[0094] In some specific embodiments of the present disclosure, in formula (IIIa), each Z is a hydroxyl group.

[0095] In some embodiments of the present disclosure, in Formula (IIIa), each p is independently 1 or 2.

[0096] In some specific embodiments of the present disclosure, in Formula (IIIa), each p is 1.

[0097] In some embodiments of the present disclosure, in Formula (IIIa), each q is independently 1 or 2.

[0098] In some specific embodiments of the present disclosure, in Formula (IIIa), each q is 1.

[0099] In some specific embodiments of the present disclosure, in Formula (IIIa), each p is 1, and each q is 1.

[0100] In some specific embodiments of the present disclosure, in formula (IIIa), each R3 is H.

[0101] In some embodiments of the present disclosure, in formula (IIIa), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0102] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0103] In some specific embodiments of the present disclosure, k is 1.

[0104] In some embodiments of the present disclosure, in formula (IIIa), each L2 is independently

[0105] In some specific embodiments of the present disclosure, in formula (IIIa), each Y is O.

[0106] In some specific embodiments of the present disclosure, in formula (IIIa), each R4 is

[0107] In some embodiments of the present disclosure, the compound has a structure represented by formula (IVa):

[0108] In formula (IVa), R1 represents a hydroxyl protecting group;

[0109] R2 is in, represents a solid phase support, and R2' is a covalent linking group connected to the solid phase support;

[0110] n is 0, 1, 2, or 3;

[0111] Each Z is independently hydroxyl or thiol;

[0112] Each p is independently 1, 2 or 3;

[0113] Each q is independently 1, 2 or 3;

[0114] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0115] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0116] Each Y is independently NH, O or S.

[0117] For example, when n is 0, the structural formula of the compound is:

[0118] In some embodiments of the present disclosure, 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"-trimethoxytriphenyl group (TMTr group).

[0119] In some specific embodiments of the present disclosure, in formula (IVa), R1 is 4,4'-dimethoxytrityl (DMTr group).

[0120] In some specific embodiments of the present disclosure, in formula (IVa), R2 is

[0121] In some specific embodiments of the present disclosure, in formula (IVa), R2 is in, represents a solid phase carrier, R2' is That is: in formula (IVa), R2 is

[0122] In some specific embodiments of the present disclosure, in formula (IVa), each Z is hydroxyl.

[0123] In some embodiments of the present disclosure, in Formula (IVa), each p is independently 1 or 2.

[0124] In some specific embodiments of the present disclosure, in Formula (IVa), each p is 1.

[0125] In some embodiments of the present disclosure, in Formula (IVa), each q is independently 1 or 2.

[0126] In some specific embodiments of the present disclosure, in Formula (IVa), each q is 1.

[0127] In some specific embodiments of the present disclosure, in Formula (IVa), each p is 1, and each q is 1.

[0128] In some specific embodiments of the present disclosure, in formula (IVa), each R3 is H.

[0129] In some embodiments of the present disclosure, in formula (IVa), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0130] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0131] In some specific embodiments of the present disclosure, k is 1.

[0132] In some embodiments of the present disclosure, in formula (IVa), each L2 is independently

[0133] In some specific embodiments of the present disclosure, in formula (IVa), each Y is O.

[0134] In some specific embodiments of the present disclosure, the compound is any of the following structures:

[0135] in, Stands for solid phase support.

[0136] In a second aspect of the present disclosure, the present disclosure provides a compound represented by formula (Ib) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0137] In formula (Ib), * represents the linking site for connecting the active pharmaceutical molecule;

[0138] m is 1, 2, 3 or 4;

[0139] Each Z is independently hydroxyl or thiol;

[0140] Each p is independently 1, 2 or 3;

[0141] Each q is independently 1, 2 or 3;

[0142] Each A is independently an unsubstituted or substituted 4-10 membered aliphatic ring;

[0143] Each X is independently NH, O or S;

[0144] Each L1 is independently Wherein, j is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0145] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0146] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2aEach independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0147] Each Y is independently NH, O or S.

[0148] In some specific embodiments of the present disclosure, m is 3.

[0149] In some embodiments of the present disclosure, in Formula (Ib), each Z is hydroxyl.

[0150] In some embodiments of the present disclosure, in Formula (Ib), each p is independently 1 or 2.

[0151] In some specific embodiments of the present disclosure, in Formula (Ib), each p is 1.

[0152] In some embodiments of the present disclosure, in Formula (Ib), each q is independently 1 or 2.

[0153] In some specific embodiments of the present disclosure, in Formula (Ib), each q is 1.

[0154] In some specific embodiments of the present disclosure, in Formula (Ib), each p is 1, and each q is 1.

[0155] In some embodiments of the present disclosure, in Formula (Ib), each A is independently a substituted or substituted 4-10 membered cycloalkane group or a substituted or substituted 4-10 membered cycloalkene group.

[0156] In some embodiments of the present disclosure, in Formula (Ib), each A is independently a substituted or unsubstituted 4-10 membered cycloalkane group.

[0157] In some embodiments of the present disclosure, in formula (Ib), each A is independently a 4-10 membered cycloalkane group, such as a monocyclic, spirocyclic or bridged ring.

[0158] In some embodiments of the present disclosure, in formula (Ib), each A is independently

[0159] In some specific embodiments of the present disclosure, in formula (Ib), each A is

[0160] In some specific embodiments of the present disclosure, in Formula (Ib), each X is NH.

[0161] In some specific embodiments of the present disclosure, in formula (Ib), each L1 is

[0162] In some specific embodiments of the present disclosure, in Formula (Ib), each R3 is H.

[0163] In some embodiments of the present disclosure, in formula (Ib), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0164] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0165] In some specific embodiments of the present disclosure, k is 1.

[0166] In some embodiments of the present disclosure, in formula (Ib), each L2 is independently

[0167] In some specific embodiments of the present disclosure, in Formula (Ib), each Y is O.

[0168] In some embodiments of the present disclosure, the pharmaceutically active molecule is a small molecule drug (eg, entecavir (ETV), statin small molecule chemical drugs, febuxostat, allopurinol, etc.), an antibody, or an oligonucleotide.

[0169] In some specific embodiments of the present disclosure, the pharmaceutically active molecule is an oligonucleotide.

[0170] In some embodiments of the present disclosure, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide.

[0171] In some embodiments of the present disclosure, the compound has a structure represented by formula (IIb):

[0172] In formula (IIb), * represents the linking site for connecting the active pharmaceutical molecule;

[0173] m is 1, 2, 3 or 4;

[0174] Each Z is independently hydroxyl or thiol;

[0175] Each p is independently 1, 2 or 3;

[0176] Each q is independently 1, 2 or 3;

[0177] Each X is independently NH, O or S;

[0178] Each L1 is independently Wherein, j is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0179] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0180] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0181] Each Y is independently NH, O or S.

[0182] In some specific embodiments of the present disclosure, in formula (IIb), m is 3.

[0183] In some embodiments of the present disclosure, in Formula (IIb), each Z is hydroxyl.

[0184] In some embodiments of the present disclosure, in Formula (IIb), each p is independently 1 or 2.

[0185] In some specific embodiments of the present disclosure, in Formula (IIb), each p is 1.

[0186] In some embodiments of the present disclosure, in Formula (IIb), each q is independently 1 or 2.

[0187] In some specific embodiments of the present disclosure, in Formula (IIb), each q is 1.

[0188] In some specific embodiments of the present disclosure, in Formula (IIb), each p is 1, and each q is 1.

[0189] In some embodiments of the present disclosure, in Formula (IIb), each X is NH.

[0190] In some specific embodiments of the present disclosure, in formula (IIb), each L1 is

[0191] In some specific embodiments of the present disclosure, in formula (IIb), each R3 is H.

[0192] In some embodiments of the present disclosure, in formula (IIb), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0193] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0194] In some specific embodiments of the present disclosure, k is 1.

[0195] In some embodiments of the present disclosure, in formula (IIb), each L2 is independently any of the following structures:

[0196] In some specific embodiments of the present disclosure, in formula (IIb), each Y is O.

[0197] In some embodiments of the present disclosure, the compound has the structure shown in formula (IIIb):

[0198] In formula (IIIb), * represents the linking site for connecting the active pharmaceutical molecule;

[0199] m is 1, 2, 3 or 4;

[0200] Each Z is independently hydroxyl or thiol;

[0201] Each p is independently 1, 2 or 3;

[0202] Each q is independently 1, 2 or 3;

[0203] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0204] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2bare each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0205] Each Y is independently NH, O or S.

[0206] In some specific embodiments of the present disclosure, in formula (IIIb), m is 3.

[0207] In some embodiments of the present disclosure, in Formula (IIIb), each Z is hydroxyl.

[0208] In some embodiments of the present disclosure, in Formula (IIIb), each p is independently 1 or 2.

[0209] In some specific embodiments of the present disclosure, in Formula (IIIb), each p is 1.

[0210] In some embodiments of the present disclosure, in Formula (IIIb), each q is independently 1 or 2.

[0211] In some specific embodiments of the present disclosure, in Formula (IIIb), each q is 1.

[0212] In some specific embodiments of the present disclosure, in Formula (IIIb), each p is 1, and each q is 1.

[0213] In some embodiments of the present disclosure, in Formula (IIIb), each R3 is H.

[0214] In some embodiments of the present disclosure, in formula (IIIb), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0215] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0216] In some specific embodiments of the present disclosure, k is 1.

[0217] In some embodiments of the present disclosure, in formula (IIIb), each L2 is independently

[0218] In some specific embodiments of the present disclosure, in formula (IIIb), each Y is O.

[0219] In some specific embodiments of the present disclosure, the compound is any of the following structures:

[0220] Wherein, * represents the connection site for connecting the active drug molecule.

[0221] It should be noted that the compound described in the second aspect of the present application may exist in the form of a ligand.

[0222] In the third aspect of the present disclosure, the present disclosure provides a conjugate represented by formula (Ic) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0223] In formula (Ic), Nu represents an oligonucleotide;

[0224] m is 1, 2, 3 or 4;

[0225] Each Z is independently hydroxyl or thiol;

[0226] Each p is independently 1, 2 or 3;

[0227] Each q is independently 1, 2 or 3;

[0228] Each A is independently an unsubstituted or substituted 4-10 membered aliphatic ring;

[0229] Each X is independently NH, O or S;

[0230] Each L1 is independently Wherein, j is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0231] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0232] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0233] Each Y is independently NH, O or S.

[0234] In some embodiments of the present disclosure, the oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide.

[0235] In some embodiments of the present disclosure, the single-stranded oligonucleotide is selected from a modified or unmodified antisense oligonucleotide, a modified or unmodified nucleic acid aptamer, a modified or unmodified ribozyme, a modified or unmodified deoxyribozyme, a circular RNA, a sense strand of siRNA, or an antisense strand of siRNA.

[0236] In some embodiments of the present disclosure, the double-stranded oligonucleotide is selected from modified or unmodified small interfering RNA, modified or unmodified double-stranded RNA, modified or unmodified microRNA, modified or unmodified small guide RNA, modified or unmodified small activating RNA and modified or unmodified short hairpin RNA.

[0237] In some specific embodiments of the present disclosure, in formula (Ic), Nu represents small interfering RNA

[0238] In some specific embodiments of the present disclosure, in formula (Ic), m is 3.

[0239] In some embodiments of the present disclosure, in Formula (Ic), each Z is hydroxyl.

[0240] In some embodiments of the present disclosure, in Formula (Ic), each p is independently 1 or 2.

[0241] In some specific embodiments of the present disclosure, in Formula (Ic), each p is 1.

[0242] In some embodiments of the present disclosure, in Formula (Ic), each q is independently 1 or 2.

[0243] In some specific embodiments of the present disclosure, in Formula (Ic), each q is 1.

[0244] In some specific embodiments of the present disclosure, in Formula (Ic), each p is 1, and each q is 1.

[0245] In some embodiments of the present disclosure, in Formula (Ic), each A is independently a substituted or substituted 4-10 membered cycloalkane group or a substituted or substituted 4-10 membered cycloalkene group.

[0246] In some embodiments of the present disclosure, in formula (Ic), each A is independently a substituted or unsubstituted 4-10 membered cycloalkane group.

[0247] In some embodiments of the present disclosure, in formula (Ic), each A is independently a 4-10 membered cycloalkane group, such as a monocyclic, spirocyclic or bridged ring.

[0248] In some embodiments of the present disclosure, in formula (Ic), each A is independently

[0249] In some specific embodiments of the present disclosure, in formula (Ic), each A is

[0250] In some specific embodiments of the present disclosure, in Formula (Ic), each X is NH.

[0251] In some specific embodiments of the present disclosure, in formula (Ic), each L1 is

[0252] In some specific embodiments of the present disclosure, in formula (Ic), each R3 is H.

[0253] In some embodiments of the present disclosure, in formula (Ic), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0254] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0255] In some specific embodiments of the present disclosure, k is 1.

[0256] In some embodiments of the present disclosure, in formula (Ic), each L2 is independently

[0257] In some specific embodiments of the present disclosure, in formula (Ic), each Y is O. In some embodiments of the present disclosure, the conjugate has a structure represented by formula (IIc):

[0258] In formula (IIc), Nu represents an oligonucleotide;

[0259] m is 1, 2, 3 or 4;

[0260] Each Z is independently hydroxyl or thiol;

[0261] Each p is independently 1, 2 or 3;

[0262] Each q is independently 1, 2 or 3;

[0263] Each X is independently NH, O or S;

[0264] Each L1 is independently Wherein, j is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0265] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0266] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0267] Each Y is independently NH, O or S.

[0268] In some specific embodiments of the present disclosure, in formula (IIc), Nu represents small interfering RNA

[0269] In some specific embodiments of the present disclosure, in formula (IIc), m is 3.

[0270] In some embodiments of the present disclosure, in Formula (IIc), each Z is hydroxyl.

[0271] In some embodiments of the present disclosure, in Formula (IIc), each p is independently 1 or 2.

[0272] In some specific embodiments of the present disclosure, in Formula (IIc), each p is 1.

[0273] In some embodiments of the present disclosure, in Formula (IIc), each q is independently 1 or 2.

[0274] In some specific embodiments of the present disclosure, in Formula (IIc), each q is 1.

[0275] In some specific embodiments of the present disclosure, in Formula (IIc), each p is 1, and each q is 1.

[0276] In some specific embodiments of the present disclosure, in formula (IIc), each R3 is H.

[0277] In some embodiments of the present disclosure, in formula (IIc), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents a C1-C5 alkylene group, and each RL2b are each independently O, S, NH or -NH-C(O)-, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0278] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0279] In some specific embodiments of the present disclosure, k is 1.

[0280] In some embodiments of the present disclosure, in formula (IIc), each L2 is independently

[0281] In some specific embodiments of the present disclosure, in formula (IIc), each Y is O.

[0282] In some embodiments of the present disclosure, the conjugate has the structure shown in formula (IIIc):

[0283] In formula (IIIc), Nu represents an oligonucleotide;

[0284] m is 1, 2, 3 or 4;

[0285] Each Z is independently hydroxyl or thiol;

[0286] Each p is independently 1, 2 or 3;

[0287] Each q is independently 1, 2 or 3;

[0288] Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy;

[0289] Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, each R L2b are each independently O, S, NH or -NH-C(O)-, k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0290] Each Y is independently NH, O or S.

[0291] In some specific embodiments of the present disclosure, in formula (IIIc), m is 3.

[0292] In some embodiments of the present disclosure, in Formula (IIIc), each Z is hydroxyl.

[0293] In some embodiments of the present disclosure, in Formula (IIIc), each p is independently 1 or 2.

[0294] In some specific embodiments of the present disclosure, in Formula (IIIc), each p is 1.

[0295] In some embodiments of the present disclosure, in Formula (IIIc), each q is independently 1 or 2.

[0296] In some specific embodiments of the present disclosure, in Formula (IIIc), each q is 1.

[0297] In some specific embodiments of the present disclosure, in Formula (IIIc), each p is 1, and each q is 1.

[0298] In some embodiments of the present disclosure, in Formula (IIIc), each R3 is H.

[0299] In some embodiments of the present disclosure, in formula (IIIc), each L2 is independently C1-C 10 Alkylene or Among them, each R L2a Each independently represents 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.

[0300] In some specific embodiments of the present disclosure, each R L2b They are all -NH-C(O)-.

[0301] In some specific embodiments of the present disclosure, k is 1.

[0302] In some embodiments of the present disclosure, in formula (IIIc), each L2 is independently

[0303] In some specific embodiments of the present disclosure, in formula (IIIc), each Y is O.

[0304] In a fourth aspect of the present disclosure, the present disclosure provides a composition comprising the conjugate according to the third aspect.

[0305] In some embodiments of the present disclosure, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0306] In the fifth aspect of the present disclosure, the present disclosure provides use of the compound described in the first aspect, the ligand described in the second aspect, the conjugate described in the third aspect, or the composition described in the fourth aspect in the preparation of a medicament for preventing and / or treating a disease.

[0307] In some embodiments of the present disclosure, the disease is a liver-related disease. Exemplarily, the liver-related disease includes but is not limited to chronic non-alcoholic fatty liver disease, chronic alcoholic liver disease, autoimmune hepatitis, primary biliary cirrhosis, cirrhosis, primary liver cancer, hepatic encephalopathy, and viral hepatitis.

[0308] In a sixth aspect of the present disclosure, the present disclosure provides a method for treating and / or preventing a disease, the method comprising: administering a pharmaceutically acceptable amount of the conjugate of the third aspect or the composition of the fourth aspect to a subject.

[0309] In some embodiments of the present disclosure, the disease is a liver-related disease. Exemplarily, the liver-related disease includes but is not limited to chronic non-alcoholic fatty liver disease, chronic alcoholic liver disease, autoimmune hepatitis, primary biliary cirrhosis, cirrhosis, primary liver cancer, hepatic encephalopathy, and viral hepatitis.

[0310] In the seventh aspect of the present disclosure, the present disclosure provides a use of the compound of the first aspect, the ligand of the second aspect, the conjugate of the third aspect or the composition of the fourth aspect in the preparation of a drug for reducing the expression or activity of a target gene.

[0311] In some embodiments of the present disclosure, the drug is used to reduce the expression or activity of a target gene in liver cells. Exemplarily, the target gene includes but is 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.

[0312] In an eighth aspect of the present disclosure, the present disclosure provides a method for reducing the expression or activity of a target gene, the method comprising contacting the conjugate of the third aspect or the composition of the fourth aspect with a cell.

[0313] In some embodiments of the present disclosure, the cell is a liver cell. Exemplarily, the target gene includes but is 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.

[0314] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0315] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0316] FIG1 shows the relative expression levels of target genes in mice after administration of three clusters of CR01008 conjugates (RZ899015) and L96 conjugates (RZ599001) according to an embodiment of the present disclosure;

[0317] FIG2 shows the relative expression levels of target genes in mice after administration of three clusters of CR01008 conjugates (RZ899015), three clusters of CR01013 conjugates (RZ899026), three clusters of CR01014 conjugates (RZ899027), and L96 conjugate (RZ599001) according to an embodiment of the present disclosure;

[0318] 3 shows the relative expression levels of target genes in mice after administration of three clusters of CR01013 conjugates (RZ897001), three clusters of CR01014 conjugates (RZ897002), and L96 conjugates (RZ597002) according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0319] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not intended to limit the present disclosure.

[0320] Explanation of terms

[0321] To facilitate understanding of the present disclosure, some technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. The term "NH" refers to an imino group, the structural formula of which is

[0322] The term "CO" refers to a carbonyl group, which has the structural formula

[0323] The structural formula of the term "trityl" is:

[0324] The structural formula of the term "4-methoxytrityl" is

[0325] The structural formula of the term "4,4'-dimethoxytrityl" is

[0326] The structural formula of the term "4,4',4"-trimethoxytriphenyl" is

[0327] the term Indicates the site where a group is attached via a covalent bond.

[0328] In the structural formula of the compound or ligand disclosed herein, the bond Indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond Can be or include both Although all of the above structural formulae are drawn as certain isomers for simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0329] In the structural formula of the compound or ligand disclosed herein, the bond Indicates that the configuration is not specified. If there are cis-trans isomers in the chemical structure, the bond The configuration can be E-type, Z-type, or both E and Z configurations.

[0330] Unless otherwise indicated, the following definitions used herein shall apply. For the purposes of this disclosure, the chemical elements are referred to in accordance with the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry may be found in "Organic Chemistry," 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.

[0331] Unless otherwise specified or clearly contradicted by context, the articles "a," "an," 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 objects. For example, "a component" refers to one or more components, i.e., more than one component may be contemplated for use or use in implementing the described embodiments.

[0332] The term "comprising" is an open expression, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0333] "Stereoisomers" are compounds that have identical chemical constitutions but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0334] "Chiral" refers to a molecule that is non-superimposable on its mirror image; "achiral" refers to a molecule that is superimposable on its mirror image.

[0335] "Enantiomers" refer to two non-superimposable isomers of a compound that are mirror images of each other.

[0336] "Diastereoisomers" refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of one another. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Diastereomeric mixtures can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0337] Stereochemical definitions and conventions used in this disclosure generally follow SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

[0338] As used herein, "pharmaceutically acceptable salts" refer 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. Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups, such as hydrochlorides, hydrobromides, phosphates, sulfates, and perchlorates, and organic acid salts, such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates, or these salts may be obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C 1-4 The present disclosure also contemplates quaternary ammonium salts formed by compounds containing any N group. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium / quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0339] Pharmaceutically acceptable salts are generally known to those of ordinary skill in the art and include salts of active compounds prepared with relatively non-toxic acids or bases based on the specific substituents present on the compounds described herein. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of the compounds with a sufficient amount of the desired base under neat conditions or in a suitable inert solvent or by ion exchange (whereby one basic counterion (base) in an ionic complex is replaced by another). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts.

[0340] The term "prodrug" as used in this disclosure refers to a compound that is converted into a compound represented by formula (X) in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or by the conversion of the prodrug into the parent structure by enzymes in the blood or tissues. The prodrug compounds disclosed in this disclosure may be esters. In the prior art, esters that can be used as prodrugs include phenyl esters, aliphatic (C1-C 24 ) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, a compound of the present disclosure containing a hydroxyl group can be acylated to produce a prodrug form of the compound. Other prodrug forms include phosphate esters, such as these phosphate ester compounds, which are obtained by phosphorylating a hydroxyl group on the parent compound. For a complete discussion of prodrugs, see 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., Prodrugs: 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.

[0341] In the context of this disclosure, unless otherwise specified, "conjugation" refers to the covalent attachment of two or more chemical moieties, each with a specific function, to one another; accordingly, a "conjugate" refers to a compound formed by covalent attachment of these chemical moieties. Furthermore, a "drug conjugate" refers to a compound formed by covalently attaching one or more chemical moieties with specific functions to an active drug. Hereinafter, and particularly in the Examples, the drug conjugates of the present disclosure may sometimes be referred to simply as "conjugates." Depending on the context, a drug conjugate should be understood as a general term for drug conjugates or a specific drug conjugate represented by a specific structural formula.

[0342] The term "aliphatic ring" refers to a ring having a carbon skeleton structure, such as a monocyclic ring, a spirocyclic 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 carbon skeleton structure.

[0343] The term "monocyclic" refers to a cycloalkyl group containing only one ring.

[0344] The term "spirocyclic" refers to a compound in which two rings share one atom.

[0345] The term "bridged ring" refers to a structure formed by two or more cyclic structures sharing two non-adjacent ring atoms.

[0346] The term "cycloalkyl" refers to a monovalent or multivalent saturated monocyclic, bicyclic, or tricyclic ring system containing 3-12 ring carbon atoms. In one embodiment, the cycloalkyl group contains 7-12 ring carbon atoms; in another embodiment, the cycloalkyl group contains 3-8 ring carbon atoms; in another embodiment, the cycloalkyl group contains 3-6 ring carbon atoms. The cycloalkyl groups may independently be unsubstituted or substituted with one or more substituents described herein.

[0347] The term "cycloalkenyl" refers to a cyclized alkenyl group. 4-6 Cycloalkenyl is intended to include C4, C5 and C6 cycloalkenyls. Exemplary cycloalkenyls include, but are not limited to, cyclobutenyl, cyclopentenyl and cyclohexenyl.

[0348] The term "cycloalkynyl" refers to a monocyclic alicyclic hydrocarbon containing one or more carbon-carbon triple bonds.

[0349] The term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon group of 1-50 carbon atoms, or 1-40 carbon atoms, or 1-20 carbon atoms, or 1-10 carbon atoms, or 1-6 carbon atoms, or 1-4 carbon atoms, wherein the alkyl group may be independently and optionally substituted with one or more substituents described herein, including but not limited to deuterium, amino, hydroxyl, cyano, F, Cl, Br, I, mercapto, nitro, oxo (=O), and the like. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -CH(CH3)CH2CH3), u, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2- Methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2 CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, and the like. The term "alkyl" and its prefix "alkane" as used herein include both straight and branched saturated carbon chains.

[0350] The term "alkylene" refers to a saturated divalent hydrocarbon radical derived by removing two hydrogen atoms from a saturated straight-chain or branched hydrocarbon radical. Unless otherwise specified, an alkylene group contains 1-50 carbon atoms. In some embodiments, an alkylene group contains 1-40 carbon atoms; in other embodiments, an alkylene group contains 1-20 carbon atoms; in yet other embodiments, an alkylene group contains 1-10 carbon atoms; and in still other embodiments, an alkylene group contains 1-6 carbon atoms. Examples include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), isopropylene (-CH(CH3)CH2-), and the like.

[0351] The term "alkenyl" refers to a straight or branched monovalent hydrocarbon radical having at least one carbon-carbon sp 2 Double bonds, including "cis" and "trans" positioning, or "E" and "Z" positioning. Wherein, the alkenyl group may be optionally substituted with one or more substituents described in the present disclosure. In some embodiments, the alkenyl group contains 2-50 carbon atoms; in other embodiments, the alkenyl group contains 3-50 carbon atoms; in other embodiments, the alkenyl group contains 2-40 carbon atoms; in yet another embodiment, the alkenyl group contains 2-20 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), and the like.

[0352] The term "alkenylene" refers to a straight or branched chain divalent hydrocarbon radical having at least one carbon-carbon sp 2 Double bond.

[0353] The term "alkynyl" refers to a straight or branched monovalent hydrocarbon radical containing 2 to 50 carbon atoms, wherein at least one carbon-carbon sp triple bond is present, wherein the alkynyl group may be optionally substituted with one or more substituents described herein. In one embodiment, the alkynyl group contains 3 to 12 carbon atoms; in another embodiment, the alkynyl group contains 2 to 6 carbon atoms; in yet another embodiment, the alkynyl group contains 2 to 4 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propargyl (-CH2C≡CH), 1-propynyl (-C≡C-CH3), and the like.

[0354] The term "alkynylene" refers to a straight or branched divalent alkynyl group having at least one carbon-carbon sp triple bond.

[0355] The term "alkoxy" refers to an alkyl group attached to the rest of the molecule through an oxygen atom, wherein the alkyl group has the meaning as described in the present disclosure. Unless otherwise specified, the alkoxy group contains 1-50 carbon atoms. In some embodiments, the alkoxy group contains 1-40 carbon atoms; in other embodiments, the alkoxy group contains 1-20 carbon atoms; in yet other embodiments, the alkoxy group contains 1-10 carbon atoms. The alkoxy group may be optionally substituted with one or more substituents described in the present disclosure. Examples of alkoxy groups include, but are not limited to, methoxy (MeO, -OCH3), ethoxy (EtO, -OCH2CH3), 1-propoxy (n-PrO, n-propoxy, -OCH2CH2CH3), 2-propoxy (i-PrO, i-propoxy, -OCH(CH3)2), 1-butoxy (n-BuO, n-butoxy, -OCH2CH2CH2CH3), 2-methyl-1-propoxy (i-BuO, i-butoxy, -OCH2CH(CH3)2), 2-butoxy (s-BuO, s-butoxy, -OCH(CH3)CH2CH3), 2-methyl-2-propoxy (t-BuO, t-butoxy, -OC(CH3)3), 1-pentoxy (n-pentoxy, -OCH2CH2CH2CH2CH3), 2-pentyloxy (-OCH(CH3)CH2CH2CH3), 3-pentyloxy (-OCH(CH2CH3)2), 2-methyl-2-butoxy (-OC(CH3)2CH2CH3), 3-methyl-2-butoxy (-OCH(CH3)CH(CH3)2), 3-methyl-l-butoxy (-OCH2CH2CH(CH3)2), 2-methyl-l-butoxy (-OCH2CH(CH3)CH2CH3) and the like.

[0356] The term "alkylamino" includes "N-alkylamino" and "N,N-dialkylamino", wherein the amino group is independently substituted with one or two alkyl groups; the alkyl group has the meaning described in the present disclosure. In some embodiments, the alkylamino group is a lower alkylamino group formed by one or two C1-C6 alkyl groups attached to a nitrogen atom. In other embodiments, the alkylamino group is an alkylamino group formed by one or two C1-C4 lower alkyl groups attached to a nitrogen atom. Suitable alkylamino groups can be monoalkylamino or dialkylamino, and such examples include but are not limited to: N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino, and the like.

[0357] The term "haloalkyl", "haloalkenyl", "haloalkoxy" or "haloalkylamino" means that an alkyl, alkenyl, alkoxy or alkylamino group is substituted with one or more halogen atoms, wherein the alkyl, alkenyl, alkoxy or alkylamino group has the meaning described in the present disclosure, and such examples include but are not limited to: trifluoromethyl, 2,2,3,3-tetrafluoropropyl, trifluoromethoxy, trifluoromethylamino, etc.

[0358] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, refer to cyclic forms of "alkyl" and "heteroalkyl," respectively. Additionally, for heterocycloalkyl, a heteroatom may occupy the position at which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, and 2-piperazinyl. The terms "cycloalkenyl" and "heterocycloalkenyl" refer to divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0359] As described herein, the compounds of the present disclosure may be optionally substituted with one or more substituents, such as the compounds of the general formula above, or as described in the specific examples, subclasses, and classes of compounds encompassed by the present disclosure.

[0360] In general, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced with a specified substituent. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structure can be substituted with one or more substituents selected from a specified group, the substituents may be the same or different at each substitutable position.

[0361] The term "unsubstituted" means that the designated group bears no substituents.

[0362] The term "optionally substituted with" can be used interchangeably with the term "unsubstituted or substituted with," meaning that the structure is unsubstituted or substituted with one or more substituents disclosed herein. Substituents disclosed herein include, but are not limited to, D, F, Cl, Br, I, N3, CN, NO2, OH, SH, NH2, alkyl, haloalkyl, haloalkoxy, haloalkylamino, alkenyl, alkynyl, alkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like.

[0363] In addition, it should be noted that, unless otherwise explicitly stated, the descriptions used in this disclosure as “each ... is independently” and “... are independently” and “... are independently” can be interchanged and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or in the same group, the specific options expressed by the same symbols do not affect each other. Taking R3 as an example, the structural formula “C1-C 50 Alkylene" and the structural formula "-C(O)-NH-C optionally substituted by R3 1-50 The specific options of R3 between the two "alkylene" are not affected by each other.

[0364] The term "small interfering RNA (siRNA)" refers to a class of double-stranded RNA molecules consisting of a sense strand and an antisense strand, each 17 to 30 nucleotides in length. siRNAs mediate the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway by forming a silencing complex. Specifically, siRNAs direct the specific degradation of mRNA sequences through a process known as RNA interference (RNAi), inhibiting the translation of mRNA into amino acids and protein.

[0365] In the context of the present disclosure, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence. "Sense strand (or trailing strand)" refers to an iRNA strand that contains a region that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, for example, the antisense strand is completely complementary to the target sequence or at least partially complementary. In the case of partial complementarity, mismatches can exist within the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, for example, within 5, 4, 3 or 2 nucleotides of the 5'- and / or 3'-end of the iRNA.

[0366] It should be noted that the "at least part of the antisense strand being substantially complementary to the mRNA" means that the antisense strand has a polynucleotide that is substantially complementary to a continuous part of the mRNA of interest.

[0367] In the context of this disclosure, an "oligonucleotide" is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically consisting of 10 to 50 nucleotides. Oligonucleotides can regulate gene expression through a range of processes, including RNA interference, ribonuclease-mediated target degradation, splicing regulation, noncoding RNA inhibition, gene activation, and programmed gene editing.

[0368] In the context of this disclosure, "antisense oligonucleotides (ASOs)" are single-stranded oligonucleotide molecules, typically consisting of 10 to 50 nucleotides. After entering cells, ASOs bind to their complementary target mRNA through base pairing under the action of RNase H1, inhibiting the expression of the target gene.

[0369] In the context of the present disclosure, unless otherwise specified, capital letters A, U, G, C, and T represent the base composition of a nucleotide; a lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; a lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; and a lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0370] In the context of the present disclosure, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, drug stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or a combination thereof, which are known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except for the case where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical composition is encompassed.

[0371] In the context of the present disclosure, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. Except to the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0372] In the context of the present disclosure, "subject" refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., monkeys), mice, pigs, horses, donkeys, cattle, sheep, and any kind of poultry.

[0373] In the context of this disclosure, "treating," "alleviating," or "amortization" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Here, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0374] In the context of this disclosure, "prevention" and "prevent" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0375] General experiments

[0376] The present disclosure is described in detail below through examples. The reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed according to protocols well known to those skilled in the art.

[0377] Unless otherwise specified, the siRNA sequences used in this disclosure were commissioned to Suzhou Beixin Biotechnology Co., Ltd. for synthesis; the PCR primers used in this disclosure were commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis; and the experimental animals C57BL / 6J mice used in this disclosure were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0378] Unless otherwise specified, the base composition and modification meanings described in the embodiments of the present disclosure are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; the lowercase letter m indicates that the nucleotide represented by the previous letter is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide represented by the previous letter is a fluorinated-modified nucleotide; the lowercase letter s indicates that the nucleotides represented by the two letters before and after it are connected by a phosphorothioate bond.

[0379] Unless otherwise specified, the in vivo activity data are based on The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0380] Unless otherwise specified, the reagent ratios described in the embodiments of the present disclosure are calculated based on volume ratio (v / v).

[0381] The reagents and their sources used in the embodiments of the present disclosure are shown in Table 1-1 and Table 1-2:

[0382] Table 1-1

[0383] The reagents listed in Table 1-1 were purchased from Beijing Coupling Technology Co., Ltd.

[0384] Table 1-2

[0385] The sources of the instruments and equipment used in the embodiments of this disclosure are shown in Table 2:

[0386] Table 2

[0387] Example 1: Preparation of compounds

[0388] (1) Synthesis of compound CR01008

[0389] The synthetic route of compound CR01008 is as follows:

[0390] (1-1) Synthesis of Compound 2

[0391] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3% by mass KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25 ° C for 30 minutes, 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 a crude white solid. A small amount of water was added to the crude product to slurry, and filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M + H] +.

[0392] (1-2) Synthesis of Compound 3

[0393] Compound 2 (9 g, 1 eq) prepared in step (1-1) was dissolved in 70 ml of 1,4-dioxane. A 4 M solution of hydrogen chloride in 1,4-dioxane (45 ml) was added, and the mixture was stirred 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 (6.8 g, 100% yield) as a white solid.

[0394] (1-3) Synthesis of Compound 5

[0395] Compound 3 (1.8 g, 2.0 eq) prepared in step (1-2), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and 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 reverse-phase purified (22% acetonitrile in water) to obtain compound 5 (1.78 g, 64.4% yield) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0396] (1-4) Synthesis of Compound 6

[0397] Compound 5 (1.54 g, 1.0 eq) prepared in step (1-3) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (60% acetonitrile in water) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0398] (1-5) Synthesis of Compound CR01008

[0399] Compound 6 (1.08 g, 1.0 eq) prepared according to step (1-4) was dissolved in 20 ml of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 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 and evaporated to dryness under reduced pressure. After reverse purification (72 vol% acetonitrile aqueous solution), the mixture was dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0400] 1H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,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=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,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=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,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=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0401] (2) Synthesis of compound CR01008Z

[0402] Compound CR01008Z was obtained by linking compound 6, which was used to synthesize compound CR01008, to a solid phase support CPG.

[0403] The synthetic route of compound CR01008Z is as follows:

[0404] (2-1) Synthesis of Compound 9

[0405] Compound 6 (500 mg) prepared in 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 three times, and the mixture was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0406] (2-2) Synthesis of Compound CR01008Z

[0407] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (2-1) were added to a 20 ml sample vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0408] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0409] (3) Synthesis of compound CR01013

[0410] The synthetic route of compound CR01013 is as follows:

[0411] (3-1) Synthesis of Compound 2

[0412] 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 of 37% by mass) and aqueous sodium hydroxide solution (6.5 ml, concentration of 45.3% by mass) were added dropwise. After the addition was complete, the temperature was raised to 60°C and stirred 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 a crude white solid. A small amount of water was added to the crude product to make a pulp, filtered and dried to obtain compound 2 (4.8 g, yield 85.9%) as a white solid. ESI-MS (m / z) = 260.2 [M+H] + .

[0413] (3-2) Synthesis of Compound 3

[0414] Compound 2 (4.8 g) prepared in step (3-1) was dissolved in 25 ml of 1,4-dioxane. A 1,4-dioxane solution of hydrochloric acid (25 ml, 4 M) was added, and the 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 (3.6 g, 99.4% yield) as a white solid.

[0415] (3-3) Synthesis of Compound 11

[0416] Compound 3 (3.6 g) prepared in step (3-2) was dissolved in 36 ml of DMF, and TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g) were added, and the mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution 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 organic phases were combined, washed once with 50 ml of saturated aqueous sodium chloride solution (1×50 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by column chromatography (elution gradient: dichloromethane:methanol = 10:1) to obtain compound 11 (2.3 g, yield 33.0%) as a white solid. ESI MS (m / z) = 379.5 [M+H] + .

[0417] (3-4) Synthesis of Compound 12

[0418] Compound 11 (2.3 g) prepared in step (3-3) was dissolved in 23 ml of methanol, and wet palladium on carbon (230 mg, 10% loading) was added. The atmosphere was replaced with hydrogen three times, and the reaction system was stirred at 25°C under a hydrogen atmosphere (15 psi) for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filtrate, which was evaporated to dryness under reduced pressure to obtain compound 12 (1.48 g, 99.8% yield) as a yellow oil.

[0419] (3-5) Synthesis of Compound 13

[0420] 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 mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution 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 organic phases were combined, washed once with 30 ml of saturated aqueous sodium chloride solution (1 × 30 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by column chromatography (elution gradient: water:acetonitrile = 5:1) to obtain compound 13 (1.3 g, yield 31.8%) as a white solid. ESI-MS (m / z): 674.3 [M+H] + .

[0421] (3-6) Synthesis of Compound 14

[0422] Compound 13 (1.1 g) prepared in step (3-5) was dissolved in 11 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath, and DMTrCl (813 mg) was added portionwise at 0°C. The reaction system was stirred at 0°C for 1 hour. After the reaction, methanol was added to the reaction solution to quench the reaction. The solvent was evaporated, and the solution was purified by column chromatography (elution gradient: water:acetonitrile = 1:4) to obtain compound 14 (800 mg, yield 50.3%) as a white solid. ESI-MS (m / z): 976.5 [M+H] + .

[0423] (3-7) Synthesis of Compound CR01013

[0424] At room temperature, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM), 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 system was stirred at 25°C for 1 hour under a nitrogen atmosphere. After completion of the reaction, the reaction solution was washed twice with 5 ml of saturated sodium bicarbonate solution (2 × 5 ml) and once with 30 ml of saturated sodium chloride solution (1 × 30 ml). The organic phase was separated and dried over anhydrous sodium sulfate. The organic phase solvent was evaporated to dryness under reduced pressure and purified by column chromatography (elution gradient: dichloromethane:methanol = 20:1) to obtain compound CR01013 (532 mg, yield 80.4%) as a white solid. ESI-MS (m / z): 1176.7 [M+H] + .

[0425] 1H NMR(400MHz, DMSO-d6)δ0.95–1.05(d,J=6.7Hz,5H),1.06–1.15(q,J=7.6Hz,8H),1.15–1.21(t,J=7.2Hz,14H),1.72–1.80(s,3H),1.84–1.92(s, 3H),1.94–2.07(d,J=16.0Hz,7H),2.07–2.14(s,3H),2.64–2.72(q,J=5 .8Hz,2H),2.74–2.89(d,J=8.5Hz,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=6.4Hz,3H),6.82–6.97(d,J=8.7H z, 4H), 7.17–7.27 (t, J = 8.7Hz, 5H), 7.27–7.34 (t, J = 7.6Hz, 2H), 7.34–7.43 (d, J = 7.5Hz, 2H).

[0426] (4) Synthesis of compound CR01013Z

[0427] Compound CR01013Z was obtained by linking compound 14, which was used to synthesize compound CR01013, to a solid phase support CPG.

[0428] The synthetic route of compound CR01013Z is as follows:

[0429] (4-1) Synthesis of Compound 15

[0430] At room temperature, compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane. 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 system was stirred at 25°C for 16 hours. After completion of the reaction, the solvent was evaporated and the reaction solution was purified by column chromatography (elution gradient: water:acetonitrile = 2:1) to obtain compound 15 (110 mg, 0.10 mmol, yield 100%) as a yellow oil. ESI-MS (m / z) = 1099.3 [M+Na] + .

[0431] (4-2) Synthesis of Compound CR01013Z

[0432] 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 system was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filter cake, which 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. Cap 1 (4.8 ml), Cap 2 (0.54 ml), and DMAP (2.59 mg) were added to the dried filter cake, and the reaction system 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 (3×50 ml) and dried in vacuo to obtain compound CR01013Z (900 mg, loading amount of 20-30 μmol / g).

[0433] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0434] (5) Synthesis of reference compound CR01014

[0435] The synthetic route of reference compound CR01014 is as follows:

[0436] (5-1) Synthesis of Compound 18

[0437] 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 replaced with nitrogen three times, and the 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 sodium chloride solution (1 x 10 ml). The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure to obtain compound 18 (11 g), which was directly used in the next step without purification. MS-ESI (m / z) = 232.0 [M+H] + .

[0438] (5-2) Synthesis of Compound 19

[0439] Compound 18 (4.1 g) prepared in step (5-1) was dissolved in 20 ml of tetrahydrofuran, and the atmosphere was replaced with nitrogen three times. A 1.0 M solution of LiAlH4 in tetrahydrofuran (17.7 ml) was added dropwise at 0°C. The reaction system was cooled to 0°C in an ice-water bath and stirred at 0°C for 1 hour. After the reaction, 32 ml of purified water was added dropwise to the reaction solution, followed by 24 ml of a 1.0 M aqueous sodium hydroxide solution. The filtrate was filtered and evaporated to dryness under reduced pressure to obtain compound 19 (6.0 g), which was directly used in the next step without purification. MS-ESI (m / z) = 222.3 [M+H] + .

[0440] (5-3) Synthesis of Compound 20

[0441] 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% loading) and palladium hydroxide carbon (0.5 g, 10% loading) were added, respectively. The atmosphere was purged with hydrogen three times, and the reaction system was heated to 40°C and stirred at 40°C for 16 hours. After the reaction, the reaction solution was filtered to obtain a filtrate, which was evaporated to dryness under reduced pressure to obtain compound 20 (2.9 g). MS-ESI (m / z) = 132.18 [M+H] + .

[0442] (5-4) Synthesis of Compound 22

[0443] Compound 20 (4.0 g), compound 21 (N-benzyloxycarbonyl-4-aminobutyric acid, 2.8 g), and DIEA (N,N-diisopropylethylamine, 15.6 g) prepared in step (5-3) were dissolved in 20 ml of N,N-dimethylformamide (DMF). HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 17.2 g) was added. The atmosphere was purged with nitrogen 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 subjected to reverse purification to obtain compound 22 (1.46 g, 35.6% yield). MS-ESI (m / z) = 367.41 [M+H] + .

[0444] (5-5) Synthesis of Compound 23

[0445] 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 mass) was added. The atmosphere was replaced with hydrogen three times, and the mixture was stirred at 25°C for 16 hours. After the reaction, the reaction solution was filtered to obtain a filtrate, which was evaporated to dryness under reduced pressure to obtain a crude product of compound 23 (1.06 g). The crude product was directly used in the next step without purification. MS-ESI (m / z) = 233.28 [M+H] + .

[0446] (5-6) Synthesis of Compound 24

[0447] Compound 23 (1.02 g) prepared in step (5-5) and compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 1.41 g) were dissolved in 14 ml of N,N-dimethylformamide. DIEA (N,N-diisopropylethylamine, 0.81 g) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.8 g) were added. The atmosphere was purged with nitrogen three times and the 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 flash purified to obtain compound 24 (1.28 g, yield 63.5%). MS-ESI (m / z) = 648.68 [M+H] + .

[0448] (5-7) Synthesis of Compound 25

[0449] Compound 24 (1.18 g) prepared in step (5-6) was dissolved in 12 ml of pyridine, and DMTrCl (4,4'-bis(methoxytrityl) chloride, 0.98 g) was added in batches. The atmosphere was purged with nitrogen three times, and the 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 flash purified to obtain compound 25 (1.0 g, yield 61.1%). MS-ESI (m / z) = 951.0 [M+H] + .

[0450] (5-8) Synthesis of Compound CR01014

[0451] 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 atmosphere was purged with nitrogen three times, and the 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 flash purified to obtain compound CR01014 (270 mg, yield 74.4%). MS-ESI (m / z) = 1151.27 [M+H] + .

[0452] 1 H NMR(400MHz, DMSO-d6)δ:0.92–1.03(d,J=6.7Hz,8H),1.04–1.13(d,J=6.8Hz,7H),1.16–1.29(m,4H),1.38–1.53(dq,J=7.2,14.6Hz,4H),1.55–1.68 (dt,J=9.0,15.1Hz,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=5.5Hz,3H), 2.99–3.16(dt,J=6.7,14.4Hz,4H),3.20–3.30(d,J=14.8Hz,1H),3.37–3. 53(tt,J=7.1,14.6Hz,6H),3.53–3.66(dt,J=8.4,17.5Hz,3H),3.74–3.78 (s,6H),4.00–4.09(s,3H),6.84–6.96(d,J=8.3Hz,4H),7.19–7.28(t,J=7 .6Hz,5H),7.28–7.41(dt,J=7.8,22.9Hz,4H),7.70–7.88(d,J=5.9Hz,2H).

[0453] (6) Synthesis of reference compound CR01014Z

[0454] The reference compound CR01014Z was obtained by linking the compound synthesized from the compound CR01014 to the solid phase support CPG.

[0455] The synthetic route of reference compound CR01014Z is as follows:

[0456] (6-1) Synthesis of Compound 26

[0457] Compound 25 (100 mg) prepared in step (5-7) was dissolved in 2 ml of dichloromethane. 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 three times and stirred at room temperature for 16 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and flash purified to obtain compound 26 (73 mg, yield 66.7%). MS-ESI (m / z) = [M+H] + .

[0458] (6-2) Synthesis of Compound CR01014Z

[0459] To a 20 ml vial, compound 26 (50 mg) prepared in step (6-1), aminoCPG (1.19 g), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (27 mg), and N,N-diisopropylethylamine (12 mg) were added and shaken for 16 hours. After the reaction, the reaction mixture was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1 x 10 ml) and then dried under vacuum. To a 20 ml vial, the dried filter cake, 4-dimethylaminopyridine (DMAP, 3 mg), 10 ml of Cap 1, and 1 ml of Cap 2 were added and shaken for 6 hours. After the reaction, the reaction mixture was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1 x 10 ml) and then dried under vacuum to obtain compound CR01014Z (1.03 g, loading 20-30 μmol / g).

[0460] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0461] (7) Reference compound L96-PS

[0462] Compound L96-PS was purchased from Agilent Pharmaceuticals (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC).

[0463] The structural formula of compound L96-PS is as follows:

[0464] Wherein, PS represents polystyrene resin solid phase carrier.

[0465] Example 2 Synthesis of siRNA Conjugates

[0466] (1) Polymerization of compounds

[0467] By phosphoramidite nucleic acid solid phase synthesis, the aforementioned compounds attached to a solid support (i.e., CR01008Z, CR01013Z, and CR01014Z) were used as the starting cycles to ligate the compounds not attached to a solid support (i.e., CR01008, CR01013, and CR01014). Specifically, CR01008 was ligate one by one in the cycles starting with CR01008Z, CR01013Z was ligate one by one in the cycles starting with CR01013Z, and CR01014Z was ligate one by one in the cycles starting with CR01014Z.

[0468] Each compound connection involves four steps: deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given below:

[0469] The compounds not linked to the solid phase support (ie, CR01008, CR01013, and CR01014) were prepared into 0.1 M solutions using acetonitrile.

[0470] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0471] The conditions for each coupling reaction were the same. The coupling reaction conditions were: a temperature of 25°C, a molar ratio of the compound attached to the solid support to the compound not attached to the solid support of 1:10, a molar ratio of the compound attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0472] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the compound attached to the solid support was 1:1:1.

[0473] The conditions for each oxidation or sulfidation reaction were identical. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; sulfidation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of sulfidation agent to the compound attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9).

[0474] Through the above method, three clusters CR01008 (denoted as (CR01008)×3), three clusters CR01013 (denoted as (CR01013)×3), and three clusters CR01014 (denoted as (CR01014)×3) were obtained respectively.

[0475] The structural formula of the three-cluster CR01008 is as follows:

[0476] The structural formula of the three-cluster CR01013 is as follows:

[0477] The structural formula of the three-cluster CR01014 is as follows:

[0478] (2) Synthetic sense strand (SS)

[0479] By using the phosphoramidite nucleic acid solid phase synthesis method, the above compounds connected to the solid phase support are used to start the cycle (i.e., three clusters of CR01008, three clusters of CR01013, three clusters of CR01014, and L96), and the nucleoside monomers are connected one by one in the 3'-5' direction according to the nucleotide sequence. Each connection of a nucleoside monomer includes four steps: deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0480] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0481] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0482] The conditions for each coupling reaction were the same. The coupling reaction conditions were: temperature 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0483] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0484] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0485] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0486] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to the solid phase support was added to 0.5 ml / μmol of 25% by mass ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0487] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the elution gradient was eluent 1:eluent 2 = (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0488] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC). Molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value ≈ the theoretical value, the compound was conjugated to the 3' end of the siRNA sense strand.

[0489] Taking the three-cluster CR01008 as an example, the structural formula of the sense chain is as follows:

[0490] Taking the three-cluster CR01013 as an example, the structural formula of the sense chain is as follows:

[0491] (2) Synthesis of antisense strand (AS)

[0492] The antisense chain was synthesized using a universal solid phase support. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis method of the antisense chain are the same as those in step (1) for synthesizing the sense chain.

[0493] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured molecular weight with the theoretical value. If the measured value ≈ the theoretical value, it indicates that the siRNA antisense chain is obtained. (3) Synthesis of siRNA conjugates

[0494] 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, and heated to 95° C. The mixture was slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense strand and the antisense strand to form a double-stranded structure through hydrogen bonding, thereby obtaining siRNA conjugates having the sense strand and the antisense strand shown in Table 3. As can be seen from the data in Table 4, the sense strand (SS) and the antisense strand (AS) can be well attached to the ligand with high purity.

[0495] Table 3 Sense and antisense strands of siRNA conjugates

[0496] Table 4 Detection results of siRNA conjugates

[0497] Example 3 In vivo toxicity experiment of siRNA conjugates

[0498] C57BL / 6J mice were randomly divided into two groups, each consisting of two mice, half male and half female. Each experimental group received a single subcutaneous injection of 300 mg / kg of siRNA conjugate per mouse body weight (calculated as siRNA). No animal mortality or clinical symptoms related to adverse drug reactions were observed for 14 consecutive days. Following the observation period, gross autopsies of the mice were performed, revealing no abnormalities. Therefore, these results demonstrate that the siRNA conjugates disclosed herein are safe and have low animal-level toxicity.

[0499] Example 4 Method for inhibiting target gene mRNA expression in mice using siRNA conjugates

[0500] 6-8 week old C57BL / 6J mice are randomly divided into groups (all female) by body weight. The mice in each group are dosed according to body weight, and the siRNA conjugates are configured into corresponding concentration (calculated as siRNA) solution for administration with PBS solution, and the administration volume is 5ml / kg mouse body weight (calculated as siRNA). The PBS control group is given the PBS solution (not containing drug conjugate) of the same volume. The administration is recorded as the 1st day (recorded as D1) on the same day. At the preset time after administration, such as the 8th day (recorded as D8), the 15th day (recorded as D15) and the 29th day (recorded as D29), 5 mice are killed in each group. The mice killed are respectively subjected to gross dissection and the liver tissue of each mouse killed is collected, and the liver tissue is cut into about 2mm 3 Small pieces were stored with RNA Later.

[0501] Liver tissue samples were obtained from the above RNA later at different time points in different experimental groups, and the liver tissue samples were disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Total RNA was then extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd.) according to the standard operating procedures for total RNA extraction.

[0502] Take 1 μg of total RNA and use a reverse transcription kit (Promega, Reverse Transcription System, A3500) and select Oligo (dT) 15Reverse transcription primers were prepared according to the instructions of the reverse transcription kit to prepare 20 μL of reverse transcription system and complete the reverse transcription reaction. After the reaction was completed, 80 μL of RNase-free water was added to the reverse transcription system to obtain cDNA solution. Then, a real-time fluorescence quantitative PCR kit (ABI, SYBR TM Select Master Mix, Catalog number: 4472908) to detect the expression level of target gene mRNA in liver tissue. In this real-time fluorescence quantitative PCR method, primers for the target gene and primers for the internal reference gene are used to detect the target gene and the internal reference gene respectively. According to the method described in the instructions of the real-time fluorescence quantitative PCR kit, a 20μL real-time PCR reaction system is configured for each PCR detection well. Each reaction system contains 5μL of the cDNA solution obtained by the reverse transcription reaction above, 10μL of SYBR TM Select Master Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 4 μL RNase-Free H2O. The prepared reaction system was placed in a real-time fluorescence quantitative PCR instrument (ABI, StepOnePlus TM ), a three-step method was used for Real-time PCR amplification. The amplification program was pre-denaturation at 95°C for 10 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The denaturation, annealing, and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the ΔΔCt method was used to perform relative quantitative calculations of the expression level and inhibition rate of the target gene mRNA in each test group. The calculation method is as follows: ΔCt(test group) = Ct(target gene in the test group) – Ct(reference gene in the test group) ΔCt(control group) = Ct(target gene in the control group) – Ct(reference gene in the control group) ΔΔCt(test group) = ΔCt(test group) – ΔCt(average of the control group) ΔΔCt(control group) = ΔCt(control group) – ΔCt(average of the control group)

[0503] Here, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) values ​​of five mice sacrificed at the same time point in the control group. Therefore, each mouse in the test group and the control group corresponds to a ΔΔCt value.

[0504] The target gene mRNA expression level of the test group was normalized with the control group as the benchmark, and the target gene mRNA expression level of the control group was defined as 100%.

[0505] Relative expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0506] Test group target gene mRNA inhibition rate = (1 – test group target gene mRNA relative expression level) × 100%

[0507] Example 4-1 In vivo activity evaluation of three clusters of CR01008 vectors conjugated to superoxide dismutase 1 siRNA

[0508] This example evaluated the inhibitory activity of the siRNA sequence RZ899015 (RZ899015), conjugated to the CR01008 vector and the L96 conjugate RZ599001 (RZ599001), against the target gene SOD1 in mice using a method for inhibiting target gene mRNA expression in vivo. RZ899015 and RZ599001 have identical nucleic acid sequences and chemical modifications, differing only in the delivery vector structure.

[0509] Six- to eight-week-old C57BL / 6J mice were randomly divided into three groups based on body weight: a PBS control group, an RZ899015 group, and an RZ599001 group, with five mice in each group. Each group was subcutaneously administered with PBS solution, RZ899015, and RZ599001 at a dose of 1 mg / kg per mouse in a 5 mL / kg volume. The day of administration was designated Day 1 (D1), and mice were sacrificed on Day 8 (D8).

[0510] The experimental results are shown in Figure 1 and Table 6, indicating that on day 8, the three-cluster CR01008 conjugate RZ899015 group and the L96 conjugate RZ599001 group had comparable in vivo activities.

[0511] Table 5 Primer sequence list of Example 4-1

[0512] Table 6 Inhibitory activity of target genes in mice

[0513] Example 4-2 In vivo activity evaluation of three clusters of CR01008, CR01013, and CR01014 vectors conjugated to SOD1 target siRNA

[0514] This example evaluated the inhibitory activity of three siRNA sequences (RZ899015, RZ899015) conjugated to the CR01008 vector at the 3' end of the sense strand, three siRNA sequences (RZ899026, RZ899026) conjugated to the CR01013 vector, and three siRNA sequences (RZ899027, RZ899027) conjugated to the CR01014 vector, along with the L96 conjugate (RZ599001, RZ599001)) against the target gene SOD1 in mice using a method for inhibiting target gene mRNA expression in vivo. RZ899015, RZ899026, RZ899027, and RZ599001 share identical nucleic acid sequences and chemical modifications, differing only in the delivery vector structure. Among them, the difference between RZ899015 (CR01008 vector conjugated) and RZ899026 (CR01013 vector conjugated) is only the difference in the linker length of the vector.

[0515] 6-8 week old C57BL / 6J mice were randomly divided into 5 groups according to body weight, namely PBS control group, RZ899015 group, RZ899026 group, RZ899027 group and RZ599001 group, with 20 mice in each group. Each group of mice was administered PBS solution, RZ899015, RZ899026, RZ899027 and RZ599001 by subcutaneous administration in the abdomen, with a dose of 3 mg / kg per mouse and a dosing volume of 5 mL / kg. The day of administration was recorded as the first day (D1), and 5 mice were sacrificed in each group on the 25th day (D15), 29th day (D29), 43rd day (D43) and 57th day (D57) after administration.

[0516] The experimental results are shown in Figure 2 and Table 8. The three-cluster CR01008 conjugate (RZ899015) and the three-cluster CR01013 conjugate (RZ899026) have better in vivo inhibitory effects and sustained efficacy on target genes than the L96 conjugate (RZ599001). In particular, at D57, RZ899015 and RZ899026 still maintained 70.39% and 62.42% inhibitory activity on the target gene, respectively, while the L96 conjugate (RZ599001) had only 52.19% inhibitory activity.

[0517] Table 7 Primer sequence table of Example 4-2

[0518] Table 8 Inhibitory activity of target genes in mice

[0519] Example 4-3 In vivo activity evaluation of three clusters of CR01013 and CR01014 vectors conjugated with angiopoietin-like protein 3 siRNA

[0520] This example evaluated the inhibitory activity of three siRNA sequences, RZ897001 (RZ897001), conjugated to the CR01013 vector at the 3' end of the sense strand (three clusters), three siRNA sequences, RZ897002 (RZ897002), conjugated to the CR01014 vector (RZ897002), and the L96 conjugate, RZ597002 (RZ597002), against the target gene ANGPTL3 in mice using a method for inhibiting target gene mRNA expression in vivo. RZ897001, RZ897002, and RZ597002 have identical nucleic acid sequences and chemical modifications, differing only in the delivery vector structure.

[0521] 6-8 week old C57BL / 6J mice were randomly divided into 4 groups according to body weight: PBS control group, RZ897001 group, RZ897002 group, and RZ597002 group, with 20 mice in each group. Each group of mice was administered PBS solution, RZ897001, RZ897002, and RZ597002 subcutaneously in the abdomen, with a dose of 3 mg / kg per mouse and a dosing volume of 5 mL / kg. The day of administration was recorded as the first day (D1). After administration, 5 mice in each group were sacrificed on D15, D29, D43, and D57.

[0522] The experimental results, shown in Figure 3 and Table 10, demonstrate that the three-cluster CR01013 conjugate (RZ897001) exhibited superior in vivo inhibitory efficacy and sustained efficacy against target genes compared to the L96 conjugate (RZ597002). At D43, RZ897001 exhibited 74.45% inhibitory activity against the target gene, while the L96 conjugate (RZ597002) exhibited only 62.84% inhibitory activity. At D57, RZ897001 maintained 51.58% inhibitory activity against the target gene, while the L96 conjugate (RZ597002) exhibited only 37.17% inhibitory activity.

[0523] Table 9 Primer sequence list of Example 4-3

[0524] Table 10 Inhibitory activity of target genes in mice

[0525] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0526] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A compound represented by formula (Ia) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof: In formula (Ia), R1 represents a hydroxyl protecting group; R2 is in, represents a solid phase carrier, and R2' is a covalent linking group connected to the solid phase carrier; n is 0, 1, 2 or 3; Each Z is independently hydroxyl or thiol; Each p is independently 1, 2 or 3; Each q is independently 1, 2 or 3; each A is independently an unsubstituted or substituted 4-10 membered aliphatic ring; Each X is independently NH, O or S; Each L1 is independently Wherein, j is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Each R3 is independently H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; Each L2 is independently C1-C 30 Alkylene or Among them, each R L2a Each independently is C1-C 10 Alkylene, 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 2. The compound according to claim 1, characterized in that Each A is independently 3. The compound according to claim 1, characterized in that Every X is NH.

4. The compound according to claim 1, characterized in that Each L1 is 5. The compound according to claim 1, characterized in that R1 is trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytriphenyl.

6. The compound according to claim 1, characterized in that Each L2 is independently any of the following structures:

7. The compound according to claim 1, characterized in that The compound has a structure shown in formula (IIa): In formula (IIa), R1, R2, n, p, q, Z, X, L1, R3, L2, Y, and R4 are as defined in formula (Ia) of claim 1.

8. The compound according to claim 1, characterized in that The compound has a structure shown in formula (IIIa): In formula (IIIa), R1, R2, n, p, q, Z, R3, L2, Y, and R4 are as defined in formula (Ia) of claim 1.

9. The compound according to claim 8, characterized in that When n is 0, the structural formula of the compound is:

10. The compound according to claim 1, characterized in that The compound has a structure shown in formula (IVa): In formula (IVa), R1, R2, n, p, q, Z, R3, L2, and Y are as defined in formula (Ia) of claim 1.

11. The compound according to claim 10, characterized in that When n is 0, the structural formula of the compound is:

12. The compound according to claim 1, characterized in that The compound is any of the following structures: in, Stands for solid phase support.

13. A compound having a structure represented by formula (IIIb) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof: In formula (IIIb), * represents the connection site for connecting the active drug molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L2 and Y are as defined in formula (Ia) of claim 1.

14. The compound according to claim 13, characterized in that The compound is any of the following structures: Wherein, * represents the connection site for connecting the active drug molecule.

15. A conjugate, characterized in that The conjugate has a structure shown in formula (IIIc): In formula (IIIc), Nu represents an oligonucleotide; m is 1, 2, 3 or 4; Z, p, q, R3, L2 and Y are as defined in formula (Ia) of claim 1.

16. A composition, characterized in that The composition comprises the conjugate of claim 15.

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

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

19. A method for reducing the expression or activity of a target gene, characterized in that: The method comprises: The conjugate of claim 15 or the composition of claim 16 is contacted with a cell.

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