Galnac compound, conjugate, composition, and use thereof

Abstract

The present invention relates to the field of drugs, and in particular, to a compound, a conjugate, a composition, and use thereof. The oligonucleotide conjugate provided is synthesized by means of the provided compound. The oligonucleotide conjugate can target parenchymal hepatic cells with high efficiency and can effectively inhibit target gene expression in the parenchymal hepatic cells. The oligonucleotide conjugate can be used for treating and / or preventing liver-derived diseases and has the advantages of high in-vivo activity, stable and lasting pharmacodynamic action, excellent safety, and low animal-level toxicity.

Description

GalNAc compounds, conjugates, compositions, and uses thereof

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on May 24, 2023, with application number 2023105965471 and invention name “Compounds, conjugates, compositions and their uses”, the entire contents of which are incorporated by reference into this application.

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on May 24, 2023, with application number 2023105978448 and invention name “Compounds, Preparation Methods and Uses Thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of nucleic acid drugs, and in particular to GalNAc compounds, conjugates, compositions and uses thereof. Background Art

[0004] The asialoglycoprotein receptor (ASGPR), also known as liver lectin, is a transmembrane protein that is prominently and specifically expressed on the cell surface of the sinusoids and basolateral surfaces of the liver. ASGPR is mainly composed of two subunits, H1 and H2, which together have an endocytic effect. A normal mature hepatocyte has approximately 500,000 receptor molecules, and about 10% of ASGPR are expressed on the cell membrane. They have the ability to specifically recognize, bind to, and internalize some glycoproteins with galactosyl and N-acetylgalactosyl terminals in the blood circulation, allowing them to be metabolized in hepatocytes. By conjugating oligonucleotides with targeting ligands and using the targeting ligands to bind to ASGPR, liver-targeted delivery of oligonucleotides can be achieved.

[0005] Summary of the Invention

[0006] In view of this, the present disclosure provides compounds, conjugates, compositions and uses thereof.

[0007] The oligonucleotide conjugates provided by the present disclosure can be highly effectively targeted to the liver and effectively inhibit the expression of liver target genes, and can be used to treat and / or prevent liver-related diseases. They have higher in vivo activity than control vectors and more stable and long-lasting pharmacodynamic effects.

[0008] In order to achieve the above-mentioned object of the invention, the present disclosure provides the following technical solutions:

[0009] In the first aspect of the present disclosure, the present disclosure provides a compound having a structure represented by formula (Ia) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0010] In formula (Ia), R1 is selected from H, a hydroxyl protecting group or The * represents the connection site for connecting the active drug molecule;

[0011] The R2 is selected from H, a reactive phosphorus group or Among them, R 2b Selected from a solid phase support containing an amino functional group, R 2a A covalent linking group selected from the group connected to the amino functional group;

[0012] Said j is selected from 0, 1, 2 or 3;

[0013] Each M is independently selected from hydroxyl or thiol;

[0014] Each p is independently selected from 0, 1, 2, 3 or 4;

[0015] Each q is independently selected from 0, 1, 2, 3 or 4;

[0016] Each Cy is independently selected from wherein A is selected from a 5- to 6-membered oxygen-containing heterocycle; R3 is selected from H or a C1-C6 alkoxy group; n is selected from 1 or 2; each X is independently selected from O, S, or NH; each R4 is independently selected from H, a C1-C6 alkyl group, or a C1-C6 alkoxy group; each L1 is independently selected from a substituted or unsubstituted triazole group, or an amide group; m is selected from 1 or 2; each L2 is independently selected from a substituted or unsubstituted C1-C30 alkylene group or Each r is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, each Z is independently selected from O, S or NH, each R L2 Each is independently selected from substituted or unsubstituted C1-C10 alkylene; each Y is independently selected from O, S or NH; each R5 is independently selected from H, substituted or unsubstituted C1-C4 alkylacyl or substituted or unsubstituted C6-C12 arylacyl.

[0017] In some optional embodiments of the present disclosure, A is selected from a 5- to 6-membered saturated oxygen-containing heterocycle.

[0018] In some optional embodiments of the present disclosure, A is selected from a 5- to 6-membered saturated heterocyclic ring containing one oxygen atom.

[0019] In some optional embodiments of the present disclosure, the A is selected from

[0020] In some specific embodiments of the present disclosure, the A is selected from

[0021] In some specific embodiments of the present disclosure, the A is selected from

[0022] In some specific embodiments of the present disclosure, m is selected from 1.

[0023] In some specific embodiments of the present disclosure, X is selected from O.

[0024] In some specific embodiments of the present disclosure, each R4 is selected from H.

[0025] In some optional embodiments of the present disclosure, each L1 is independently selected from

[0026] In some specific embodiments of the present disclosure, each L1 is selected from

[0027] In some specific embodiments of the present disclosure, each L1 is selected from

[0028] In some optional embodiments of the present disclosure, the R3 is selected from H or C1-C3 alkoxy.

[0029] In some specific embodiments of the present disclosure, said R3 is selected from H or methoxy.

[0030] In some specific embodiments of the present disclosure, R3 is selected from H.

[0031] In some specific embodiments of the present disclosure, said R3 is selected from methoxy.

[0032] In some optional embodiments of the present disclosure, each L2 is independently selected from Each r is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, each Z is independently selected from O, S or NH, each R L2 Each is independently selected from substituted or unsubstituted C1-C10 alkylene groups.

[0033] In some optional embodiments of the present disclosure, each L L2 Each is independently selected from substituted or unsubstituted C1-C5 alkylene groups.

[0034] In some optional embodiments of the present disclosure, each L L2 Each is independently selected from substituted or unsubstituted C1-C3 alkylene.

[0035] In some optional embodiments of the present disclosure, each L L2 Each is independently selected from substituted or unsubstituted C2 alkylene.

[0036] In some optional embodiments of the present disclosure, each r is independently selected from 2 or 3.

[0037] In some optional embodiments of the present disclosure, each L2 is independently selected from The r is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0038] In some specific embodiments of the present disclosure, each L2 is independently selected from The r is selected from 2 or 3.

[0039] In some specific embodiments of the present disclosure, each L2 is selected from

[0040] In some specific embodiments of the present disclosure, each L2 is selected from

[0041] In some specific embodiments of the present disclosure, each Y is selected from O.

[0042] In some embodiments of the present disclosure, each R5 is independently selected from H,

[0043] In some specific embodiments of the present disclosure, each R5 is selected from

[0044] In some specific embodiments of the present disclosure, each R5 is selected from H.

[0045] In some embodiments of the present disclosure, said p and q are each independently selected from 0 or 1.

[0046] In some specific embodiments of the present disclosure, p=1 and q=1.

[0047] In some specific embodiments of the present disclosure, p=1 and q=0.

[0048] In some specific embodiments of the present disclosure, p=0 and q=1.

[0049] In some specific embodiments of the present disclosure, p=0 and q=0.

[0050] In the disclosure, the hydroxyl protecting group can be various hydroxyl protecting groups, and the specific type is not limited as long as it can protect the hydroxyl group. In some embodiments, the hydroxyl protecting group is stable under alkaline conditions, but can be removed under acidic conditions.

[0051] In some optional embodiments of the present disclosure, the hydroxyl protecting groups that can be used in the present disclosure include, but are not limited to, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox), trityl (Tr group), 4-methoxytrityl (MMTr group), 4,4'-dimethoxytrityl (DMTr group) and 4,4',4"-trimethoxytriphenyl (TMTr group).

[0052] In some optional embodiments of the present disclosure, the hydroxy protecting group is selected from trityl (Tr group), 4-methoxytrityl (MMTr group), 4,4'-dimethoxytrityl (DMTr group) or 4,4',4"-trimethoxytriphenyl (TMTr group).

[0053] In some specific embodiments of the present disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl (DMTr group).

[0054] In the disclosure, "reactive phosphorus group" refers to a phosphorus-containing group that can react with other compounds to be removed.

[0055] In some optional embodiments of the present disclosure, the reactive phosphorus group is selected from

[0056] In some specific embodiments of the present disclosure, the reactive phosphorus group is selected from

[0057] In some optional embodiments of the present disclosure, the pharmaceutically active molecule is selected from small molecule drugs, antibodies or oligonucleotides.

[0058] In some optional embodiments of the present disclosure, the oligonucleotide is selected from a single-stranded oligonucleotide and a double-stranded oligonucleotide.

[0059] In some optional embodiments of the present disclosure, the pharmaceutically active molecule is selected from single-stranded oligonucleotides;

[0060] In some optional embodiments of the present disclosure, the single-stranded oligonucleotide is selected from antisense oligonucleotides (ASOs).

[0061] In some specific embodiments of the present disclosure, the pharmaceutically active molecule is selected from double-stranded oligonucleotides.

[0062] In some alternative embodiments of the present disclosure, the double-stranded oligonucleotide is selected from small interfering RNA (siRNA).

[0063] In some specific embodiments of the present disclosure, the pharmaceutically active molecule is selected from siRNA.

[0064] In some specific embodiments of the present disclosure, the siRNA is conjugated to one compound, and one compound is conjugated to the 3' end of the sense strand of the siRNA.

[0065] In some optional embodiments of the present disclosure, the siRNA is conjugated to one compound, and one compound is conjugated to the 5' end of the sense strand of the siRNA.

[0066] In some specific embodiments of the present disclosure, the siRNA is conjugated to two of the compounds, and the two compounds are conjugated to the 3' end and the 5' end of the sense strand of the siRNA, respectively.

[0067] In some optional embodiments of the present disclosure, the solid phase support is selected from a resin containing an amino functional group or a controlled pore glass bead containing an amino functional group.

[0068] In some optional embodiments of the present disclosure, R 2b Selected from represents resin (eg, polystyrene resin PS) or controlled pore glass beads (CPG).

[0069] In some specific embodiments of the present disclosure, R 2a Selected from

[0070] In some specific embodiments of the present disclosure, Selected from

[0071] In some optional embodiments of the present disclosure, j is selected from 0, 1 or 2.

[0072] In some specific embodiments of the present disclosure, j is selected from 0.

[0073] In some specific embodiments of the present disclosure, j is selected from 1.

[0074] In some specific embodiments of the present disclosure, j is selected from 2.

[0075] In some optional embodiments of the present disclosure, in formula (Ia), each Cy is independently selected from any one of the following structures:

[0076] In some specific embodiments of the present disclosure, the compound is selected from any one of the following structures:

[0077] [Corrected 31.12.2024 according to Rule 91] In some specific embodiments of the present disclosure, the compound is selected from any one of the following compounds:

[0078] In the second aspect of the present disclosure, the present disclosure further provides a conjugate having a structure represented by formula (Ib) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

[0079] In formula (Ib), Nu represents an oligonucleotide;

[0080] The k is selected from 1 or 2;

[0081] The structural formula of Q is wherein said j' is selected from 1, 2, 3 or 4;

[0082] The *, M, p, q, and Cy are as defined above.

[0083] in, k Qs are connected to Nu respectively. If k is selected from 2, k Qs are connected to different positions of Nu respectively.

[0084] In some optional embodiments of the present disclosure, said j' is selected from 1, 2 or 3.

[0085] In some specific embodiments of the present disclosure, said j' is selected from 1.

[0086] In some specific embodiments of the present disclosure, said j' is selected from 2.

[0087] In some specific embodiments of the present disclosure, said j' is selected from 3.

[0088] In some specific embodiments of the present disclosure, k is selected from 1.

[0089] In some specific embodiments of the present disclosure, k is selected from 2.

[0090] In some optional embodiments of the present disclosure, the oligonucleotide is selected from a single-stranded oligonucleotide or a double-stranded oligonucleotide.

[0091] In some optional embodiments of the present disclosure, the oligonucleotide is selected from single-stranded oligonucleotides. The single-stranded oligonucleotides include, but are not limited to, antisense oligonucleotides (ASOs), aptamers, ribozymes, deoxyribozymes, circular RNA (circRNA), and the like.

[0092] In some optional embodiments of the present disclosure, the single-stranded oligonucleotide is selected from ASO.

[0093] In some specific embodiments of the present disclosure, the oligonucleotide is selected from a double-stranded oligonucleotide. The double-stranded oligonucleotide includes but is not limited to: small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small guide RNA (sgRNA), small activating RNA (saRNA) and short hairpin RNA (shRNA).

[0094] In some specific embodiments of the present disclosure, the double-stranded oligonucleotide is selected from siRNA.

[0095] In some specific embodiments of the present disclosure, the Nu represents a double-stranded oligonucleotide.

[0096] In some specific embodiments of the present disclosure, the Nu represents siRNA.

[0097] In some specific embodiments of the present disclosure, the k is selected from 1, and one Q is conjugated to the 3' end of the siRNA sense strand.

[0098] In some optional embodiments of the present disclosure, the k is selected from 1, and one Q is conjugated to the 5' end of the siRNA sense strand.

[0099] In some specific embodiments of the present disclosure, the k is selected from 2, and the two Qs are conjugated to the 3' end and the 5' end of the siRNA sense strand, respectively.

[0100] In some optional embodiments of the present disclosure, the conjugate is selected from any one of the following structures:

[0101] [Corrected 31.12.2024 according to Rule 91] In some specific embodiments of the present disclosure, the conjugate is selected from any one of the following compounds:

[0102] In the third aspect of the present disclosure, the present disclosure further provides a composition comprising the conjugate according to the second aspect or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof.

[0103] In some optional embodiments of the present disclosure, the composition further comprises optionally one or more pharmaceutically acceptable carriers or excipients.

[0104] In a fourth aspect of the present disclosure, the present disclosure further provides use of any of the following in the preparation of a medicament for preventing and / or treating a disease:

[0105] (I) the compound of the first aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0106] (II), the conjugate of the second aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0107] (III) The composition described in the third aspect.

[0108] In some embodiments of the present disclosure, the disease is selected from pathological conditions or diseases caused by abnormal expression of specific genes in target cells.

[0109] In some embodiments of the present disclosure, the disease is selected from a liver-related disease.

[0110] In a fifth aspect of the present disclosure, the present disclosure further provides use of any of the following items in the preparation of a medicament for reducing target gene expression or activity.

[0111] (I) the compound of the first aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0112] (II), the conjugate of the second aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0113] (III) The composition described in the third aspect.

[0114] In a sixth aspect of the present disclosure, the present disclosure further provides a drug comprising any of the following items and a pharmaceutically acceptable excipient or adjuvant:

[0115] (I) the compound of the first aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0116] (II), the conjugate of the second aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0117] (III) The composition described in the third aspect.

[0118] In the seventh aspect of the present disclosure, the present disclosure further provides a drug combination comprising the drug described in the sixth aspect and any other active ingredients.

[0119] In an eighth aspect of the present disclosure, the present disclosure further provides a method for treating and / or preventing a disease in a subject, comprising administering to the subject a pharmaceutically acceptable amount of any of the following:

[0120] (I) the conjugate of the second aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0121] (II), the composition of the third aspect; and / or

[0122] (III) the drug according to the sixth aspect; and / or

[0123] (IV) The drug combination described in the seventh aspect.

[0124] In some specific embodiments of the present disclosure, the disease is selected from pathological conditions or diseases caused by abnormal expression of specific genes in target cells.

[0125] In a ninth aspect of the present disclosure, the present disclosure further provides a method for reducing the expression or activity of a target gene, comprising contacting a cell with any of the following:

[0126] (I) the conjugate of the second aspect or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; and / or

[0127] (II), the composition of the third aspect; and / or

[0128] (III) the drug according to the sixth aspect; and / or

[0129] (IV) The drug combination described in the seventh aspect.

[0130] In the tenth aspect of the present disclosure, the present disclosure further provides a compound represented by formula (Ic) or a stereoisomer thereof:

[0131] In formula (Ic), p Ic and q Ic Each independently selected from 0, 1, 2 or 3;

[0132] Each n Ic Each independently selected from 1, 2, 3, 4 or 5;

[0133] R Ic-1 Selected from H or R Ic-1 ', the R Ic-1 ' is selected from hydroxyl protecting groups;

[0134] R Ic-2 Selected from H or R Ic-2 ', the R Ic-2 'Selected Among them, R Ic-2a Selected from secondary amino groups, R Ic-2b C1-C3 alkoxy group selected from secondary amino or cyano substituted;

[0135] R Ic-3 Selected from H or C1-C3 alkoxy;

[0136] Each R Ic-4 Each independently selected from

[0137] In some embodiments of the present disclosure, each R Ic-4 All selected

[0138] In some embodiments of the present disclosure, each R Ic-4 All selected

[0139] In some embodiments of the present disclosure, p Ic and q Ic Each is independently selected from 0 or 1; for example, p Ic =1 and q Ic =1, or p Ic =1 and q Ic =0, or p Ic =0 and q Ic =1, or p Ic =0 and q Ic =0.

[0140] In some embodiments of the present disclosure, p Ic =1 and q Ic =0.

[0141] In some embodiments of the present disclosure, each n is selected from 1, or each n is selected from 2, or each n is selected from 3, or each n is selected from 4, or each n is selected from 5.

[0142] In some embodiments of the present disclosure, each n Ic All are selected from 1.

[0143] For example, when p=1, q=0, and n=1, the structural formula of the compound of formula (Ic) is

[0144] In some embodiments of the present disclosure, the structural formula of the hydroxyl protecting group is Among them, each R Ic-1a Each is independently selected from H, C1-C3 alkyl or C1-C3 alkoxy.

[0145] In some embodiments of the present disclosure, each R Ic-1a Each is independently selected from H or C1-C3 alkoxy.

[0146] In some embodiments of the present disclosure, each R Ic-1a are each independently selected from H or methoxy.

[0147] In some embodiments of the present disclosure, the hydroxy protecting group is selected from trityl (Tr group), 4-methoxytrityl (MMTr group), 4,4'-dimethoxytrityl (DMTr group) or 4,4',4"-trimethoxytrityl (TMTr group).

[0148] In some embodiments of the present disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytrityl (DMTr group).

[0149] In some embodiments of the present disclosure, R Ic-2a Selected from

[0150] In some embodiments of the present disclosure, R Ic-2b Selected from

[0151] In some embodiments of the present disclosure, R Ic-2b Selected from

[0152] In some embodiments of the present disclosure, Selected from

[0153] In some embodiments of the present disclosure, Selected from

[0154] In some embodiments of the present disclosure, R Ic-2 Selected from H,

[0155] In some embodiments of the present disclosure, R Ic-2 Select H or

[0156] In some embodiments of the present disclosure, R Ic-2 Selected from H.

[0157] In some embodiments of the present disclosure, R Ic-2 Selected from

[0158] In some embodiments of the present disclosure, the compound is selected from any one of the following structures:

[0159] In some embodiments of the present disclosure, the compound is selected from any one of the following structures:

[0160] In the eleventh aspect of the present disclosure, the present disclosure further provides use of the compound or stereoisomer thereof according to the tenth aspect of the present disclosure in preparing a targeting ligand capable of binding to a cell surface receptor.

[0161] In some embodiments of the present disclosure, the receptor is selected from the group consisting of asialoglycoprotein receptors.

[0162] [Corrected 31.12.2024 according to Rule 91] Exemplarily, the targeting ligand is selected from any of the following compounds:

[0163] In the twelfth aspect of the present disclosure, the present disclosure further provides use of the compound or stereoisomer thereof according to the tenth aspect of the present disclosure in the preparation of a drug for reducing the expression or activity of a specific gene in a target cell.

[0164] In some embodiments of the present disclosure, the target cells are selected from hepatocytes.

[0165] In some embodiments of the present disclosure, the drug is selected from RNAi drugs.

[0166] In some embodiments of the present disclosure, the drug is selected from siRNA drugs.

[0167] [Corrected 31.12.2024 according to Rule 91] Exemplarily, the siRNA drug is selected from any of the following compounds:

[0168] in, Represents siRNA.

[0169] Wherein, when there is one targeting ligand in the siRNA drug, one targeting ligand is conjugated to the 3' end of the sense strand. When there are two targeting ligands in the siRNA drug, the two targeting ligands are conjugated to the 3' end and the 5' end of the sense strand, respectively.

[0170] In the thirteenth aspect of the present disclosure, the present disclosure further provides a method for preparing a compound represented by formula (Ic-1a), formula (Ic-1b), or formula (Ic-1c) or a stereoisomer thereof,

[0171] Among them, R Ic-1 '、R Ic-2 '、R Ic-3 、p Ic ,q Ic 、n Ic As defined in the tenth aspect of the present disclosure;

[0172] The preparation method comprises the following steps:

[0173] S1-1, the compound of formula (1) and the compound of formula (2) undergo acetalization reaction in the presence of an acidic catalyst to obtain a compound of formula (3); wherein R Ic-5 selected from phenyl (Ph group);

[0174] S1-2, the compound of formula (3), the hydrogenated salt and the compound of formula (4) undergo electrophilic substitution reaction to obtain a compound of formula (5); wherein, X Ic-1 represents halogen;

[0175] S1-3 and the compound of formula (5) undergo a dehydroxylation reaction under acidic conditions to obtain a compound of formula (Ic-1a);

[0176] S1-4, the compound of formula (Ic-1a) and the compound of formula (6) undergo nucleophilic substitution reaction to obtain a compound of formula (Ic-1b); wherein, X Ic-2 represents halogen;

[0177] S1-5, the compound of formula (Ic-1b) and the compound of formula (7) undergo condensation reaction to obtain the compound of formula (Ic-1c); wherein R Ic-2c is selected from halogen or secondary amino.

[0178] In some embodiments of the present disclosure, in step S1-1, the molar ratio of the compound of formula (1), the compound of formula (2) and the acidic catalyst is 1:(2-4):(0.01-0.2); for example, 1:(2.5-3.5):(0.01-0.2), 1:(2.8-3.2):(0.01-0.2), 1:3:(0.01-0.2), 1:(2-4):(0.05-0.15), 1:(2-4):(0.08-0.12), 1:(2-4):0.1 or 1:3:0.1, etc.

[0179] In some embodiments of the present disclosure, in step S1-1, the molar ratio of the compound of formula (1), the compound of formula (2), and the acidic catalyst is 1:3:0.1.

[0180] In some embodiments of the present disclosure, in step S1-1, the acidic catalyst is selected from sulfonic acid catalysts.

[0181] In some embodiments of the present disclosure, in step S1-1, the acidic catalyst is selected from DL-10-camphorsulfonic acid (CAS No. 5872-08-2).

[0182] In some embodiments of the present disclosure, in step S1-1, the reaction temperature of the acetalization reaction is selected from 20 to 40°C; for example, 25°C, 30°C, 35°C, etc.

[0183] In some embodiments of the present disclosure, in step S1-1, the reaction time of the acetal reaction is 4 to 10 h, for example, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, etc.

[0184] In some embodiments of the present disclosure, in step S1-1, the reaction solvent of the acetalization reaction is a polar aprotic solvent, such as acetonitrile.

[0185] In some embodiments of the present disclosure, in step S1-1, R Ic-5 Selected from phenyl (Ph group).

[0186] In some embodiments of the present disclosure, in step S1-2, the molar ratio of the compound of formula (3), the compound of formula (4) and the catalyst is 1:(3-5):(3-5), for example, 1:(3.5-4.5):(3-5), 1:(3.8-4.2):(3-5), 1:4:(3-5), 1:(3-5):(3.5-4.5), 1:(3-5):(3.8-4.2), 1:(3-5):4 or 1:4:4, etc.

[0187] In some embodiments of the present disclosure, in step S1-2, the molar ratio of the compound of formula (3), the compound of formula (4), and the catalyst is 1:4:4.

[0188] In some embodiments of the present disclosure, in step S1-2, the hydride salt is selected from sodium hydride, potassium hydride or magnesium hydride.

[0189] In some embodiments of the present disclosure, in step S1-2, the hydride salt is selected from sodium hydride.

[0190] In some embodiments of the present disclosure, in step S1-2, the conditions of the electrophilic substitution reaction are: the compound of formula (3) and the hydrogenated salt are first reacted at -10 to 0°C (e.g., -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, etc.) for 25 to 35 minutes (e.g., 30 minutes, etc.), and then the compound of formula (4) is added and reacted at 20 to 40°C (e.g., 25°C, 30°C, 35°C, etc.) for 1 to 5 hours (e.g., 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, etc.).

[0191] In some embodiments of the present disclosure, in step S1-2, the reaction solvent of the electrophilic substitution reaction is a polar aprotic solvent, such as N,N-dimethylformamide (DMF).

[0192] In some embodiments of the present disclosure, in step S1-2, X Ic-1 Selected from bromine (Br).

[0193] In some embodiments of the present disclosure, in step S1-3, the acidic condition is an organic acid, wherein the organic acid can be selected from formic acid (CAS No. 64-18-6), acetic acid (CAS No. 64-19-7), propionic acid (CAS No. 79-09-4), butyric acid (CAS No. 107-92-6), and valeric acid (CAS No. 109-52-4).

[0194] In some embodiments of the present disclosure, in step S1-3, the acidic condition is acetic acid, specifically 50-80% by mass acetic acid aqueous solution, more specifically 70% by mass acetic acid aqueous solution.

[0195] In some embodiments of the present disclosure, in step S1-3, the reaction temperature of the dehydroxylation protection reaction is selected from 60 to 80°C; for example, 65°C, 70°C or 75°C.

[0196] In some embodiments of the present disclosure, in step S1-3, the reaction solvent of the deprotection reaction is a polar aprotic solvent, such as dichloromethane (DCM).

[0197] In some embodiments of the present disclosure, in step S1-4, the molar ratio of the compound of formula (I-1a) to the compound of formula (6) is 1:(1.1-1.5); for example, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4 or 1:1.45, etc.

[0198] In some embodiments of the present disclosure, in step S1-4, the compound of formula (6) is selected from trityl chloride (also known as triphenylmethane chloride, abbreviated as Trt-Cl, CAS No. 76-83-5), trityl bromide (CAS No. 596-43-0), 4-methyltriphenylmethyl chloride (also known as 4-methyltriphenylmethane chloride, CAS No. 23429-44-9), 4-methoxytrityl chloride (also known as 4-methoxytriphenylmethane chloride, MMT-Cl, CAS No. 14470-28-1), 4,4'-dimethoxytrityl chloride (also known as 4,4'-dimethoxytriphenylmethane chloride, abbreviated as DMT-Cl, CAS No. 40615-36-9) and 4,4',4"-trimethoxytrityl chloride (CAS No. 49757-42-8).

[0199] In some embodiments of the present disclosure, in step S1-4, the compound of formula (6) is selected from 4,4'-dimethoxytrityl chloride.

[0200] In some embodiments of the present disclosure, in step S1-4, the nucleophilic substitution reaction is carried out in an inert atmosphere, such as nitrogen or helium.

[0201] In some embodiments of the present disclosure, in step S1-4, the reaction temperature of the nucleophilic substitution reaction is selected from 20 to 40°C; for example, 25°C, 30°C or 35°C.

[0202] In some embodiments of the present disclosure, in step S1-4, the reaction time of the nucleophilic substitution reaction is 2 to 5 h; for example, 2.5 h, 3 h, 3.5 h, 4 h or 4.5 h, etc.

[0203] In some embodiments of the present disclosure, in step S1-4, X Ic-2 Selected from bromine (Br).

[0204] In some embodiments of the present disclosure, in step S1-5, the molar ratio of the compound of formula (I-1b) to the compound of formula (7) is 1:(0.6-0.9); for example, 1:0.65, 1:0.7, 1:0.71, 1:0.72, 1:0.73, 1:0.74, 1:0.75, 1:0.76, 1:0.77, 1:0.78, 1:0.79, 1:0.8 or 1:0.85, etc.

[0205] In some embodiments of the present disclosure, in step S1-5, the molar ratio of the compound of formula (I-1b) to the compound of formula (7) is 1.5:1.1 (approximately 1:0.73).

[0206] In some embodiments of the present disclosure, in step S1-5, the compound of formula (7) is selected from 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (CAS No. 89992-70-1), bis(diisopropylamino)chlorophosphine and bis(diisopropylamino)(2-cyanoethoxy)phosphine (CAS No. 102691-36-1).

[0207] In some embodiments of the present disclosure, in step S1-5, the compound of formula (7) is selected from 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.

[0208] In some embodiments of the present disclosure, in step S1-5, R Ic-2c Selected from halogen.

[0209] In some embodiments of the present disclosure, in step S1-5, R Ic-2c Selected from chlorine (Cl).

[0210] In some embodiments of the present disclosure, in step S1-5, the condensation reaction is carried out in an inert atmosphere.

[0211] In some embodiments of the present disclosure, in step S1-5, the reaction solvent of the condensation reaction is a polar aprotic solvent, for example, dichloromethane.

[0212] In R Ic-3 When selected from H, the structural formula of the compound of formula (1) is Wherein, the compound of formula (1) (R Ic-3 H) can be purchased on the market, but the price is relatively high. In order to save costs, the compound of formula (1) (R Ic-3 H) can be obtained by the following preparation method. Ic-3 The preparation method selected from H) comprises the following steps:

[0213] Step 1: The compound of formula (1-a) undergoes a hydrodehalogenation reaction in the presence of a reducing agent and a free radical initiator to obtain a compound of formula (1-b); wherein, X Ic-4 represents a halogen (e.g., F, Cl, Br, I);

[0214] Step 2: The compound of formula (1-b) undergoes hydrolysis to obtain a compound of formula (1) (R Ic-3 Selected from H).

[0215] In some embodiments of the present disclosure, in step one, the molar ratio of the compound of formula (1-a), the reducing agent and the free radical initiator is 1:(0.9-1.2):(0.01-0.2), for example, 1:(0.95-1.2):(0.01-0.2), 1:(0.95-1.1):(0.01-0.2), 1:(0.98-1.05):(0.01-0.2), 1:(0.98-1.02):(0.01-0.2), 1:1:(0.01-0.2), 1:(0.9-1.2):(0.05-0.15), 1:(0.9-1.2):(0.08-0.12), 1:(0.9-1.2):0.1, 1:1:0.1, etc.

[0216] In some embodiments of the present disclosure, in step 1, the reducing agent is tributyltin hydride.

[0217] In some embodiments of the present disclosure, in step 1, the free radical initiator is an azo initiator, wherein the azo initiator may be azobisisobutyronitrile.

[0218] In some embodiments of the present disclosure, in step 1, the reaction temperature of the hydrodehalogenation reaction is 100-120°C, for example, 105°C, 110°C, 115°C, etc.

[0219] In some embodiments of the present disclosure, in step 1, the reaction solvent of the hydrodehalogenation reaction is an aprotic solvent, such as toluene.

[0220] In some embodiments of the present disclosure, in step 1, the reaction time of the hydrodehalogenation reaction is 1-5 h, for example, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, etc.

[0221] In some embodiments of the present disclosure, in step 2, the hydrolysis reaction is an alkaline hydrolysis reaction.

[0222] In some embodiments of the present disclosure, in step 2, the conditions for the alkaline hydrolysis reaction are: C1-C3 alkoxide and C1-C3 alcohol.

[0223] In some embodiments of the present disclosure, the C1-C3 alkoxide is selected from lithium methoxide, sodium methoxide, potassium methoxide, rubidium methoxide, cesium methoxide, lithium ethoxide, sodium ethoxide, potassium ethoxide, sodium n-propoxide, potassium n-propoxide, sodium isopropoxide, and potassium isopropoxide.

[0224] In some embodiments of the present disclosure, the C1-C3 alkoxide is selected from lithium methoxide.

[0225] In some embodiments of the present disclosure, the C1-C3 alcohol is selected from methanol, ethanol, n-propanol, and isopropanol.

[0226] In some embodiments of the present disclosure, the C1-C3 alcohol is selected from methanol.

[0227] In some embodiments of the present disclosure, in step 2, the molar ratio of the compound of formula (1-b) to the alkoxide is 1:(0.05-0.2), for example, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, 1:0.16, 1:0.17, 1:0.18, or 1:0.19.

[0228] In the fourteenth aspect of the present disclosure, the present disclosure further provides a method for preparing the compound represented by formula (Ic-2) or a stereoisomer thereof,

[0229] Among them, R Ic-1 '、R Ic-3 、p Ic ,q Ic 、n Ic As defined in the tenth aspect of the present disclosure;

[0230] The preparation method comprises the following steps:

[0231] S2-1, the compound of formula (3), the hydride salt, the compound of formula (8) and the iodide salt undergo electrophilic substitution reaction to obtain a compound of formula (9); wherein R5 is selected from substituted or unsubstituted C6-C12 aryl, X Ic-3 Represents halogen, R Ic-6 is selected from substituted or unsubstituted C1-C5 alkyl;

[0232] S2-2, the compound of formula (9) undergoes a dehydroxylation reaction under acidic conditions to obtain a compound of formula (10);

[0233] S2-3, the compound of formula (10) and the compound of formula (6) undergo a nucleophilic substitution reaction to obtain a compound of formula (11);

[0234] S2-4 and the compound represented by formula (11) undergo hydrolysis reaction to obtain the compound represented by formula (Ic-2);

[0235] In some embodiments of the present disclosure, in step S2-1, the molar ratio of the compound of formula (3), the hydrogenated salt, the compound of formula (8) and the iodized salt is 1:(3-5):(1.5-2.5):(1-2); for example, 1:(3.5-4.5):(1.5-2.5):(1-2), 1:(3.8-4.2):(1.5-2.5):(1-2), 1:4:(1.5-2.5):(1-2), 1 :(3~5):(1.8~2.2):(1~2), 1:(3~5):(1.9~2.1):(1~2), 1:(3~5):2:(1~2), 1:(3~5):(1.5~2.5):(1.3~1.7), 1:(3~5):(1.5~2.5):(1.4~1.6), 1:(3~5):(1.5~2.5):1.5 or 1:4:2:1.5, etc.

[0236] In some embodiments of the present disclosure, in step S2-1, the molar ratio of the compound of formula (3), the hydrogenated salt, the compound of formula (8), and the iodinated salt is 1:4:2:1.5.

[0237] In some embodiments of the present disclosure, in step S2-1, the conditions of the electrophilic substitution reaction are: the compound of formula (3) and the hydrogenated salt are first reacted at -10 to 0°C (e.g., -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C or -1°C, etc.) for 0.5 to 1.5 h (e.g., 0.75 h, 1 h, 1.25 h, etc.), and then the compound of formula (8) and the iodide salt are added and reacted at 20 to 40°C (e.g., 25°C, 30°C, 35°C, etc.) for 3 to 5 h (e.g., 3.5 h, 4 h, 4.5 h, etc.).

[0238] In some embodiments of the present disclosure, in step S2-1, R Ic-5 selected from phenyl (Ph group);

[0239] In some embodiments of the present disclosure, in step S2-1, R Ic-6 is selected from substituted or unsubstituted C1-C3 alkyl groups.

[0240] In some embodiments of the present disclosure, in step S2-1, R Ic-6 Selected from ethyl.

[0241] In some embodiments of the present disclosure, in step S2-2, the acidic condition is an organic acid, wherein the organic acid can be selected from formic acid (CAS No. 64-18-6), acetic acid (CAS No. 64-19-7), propionic acid (CAS No. 79-09-4), butyric acid (CAS No. 107-92-6), and valeric acid (CAS No. 109-52-4).

[0242] In some embodiments of the present disclosure, in step S2-2, the acidic condition is acetic acid, specifically 50-80% by mass acetic acid aqueous solution, more specifically 70% by mass acetic acid aqueous solution.

[0243] In some embodiments of the present disclosure, in step S2-2, the reaction temperature of the dehydroxylation protection reaction is selected from 40 to 60°C; for example, 45°C, 50°C, 55°C, etc.

[0244] In some embodiments of the present disclosure, in step S2-3, the molar ratio of the compound of formula (10) to the compound of formula (6) is 1:(1.1-1.5), for example, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, etc.

[0245] In some embodiments of the present disclosure, in step S2-3, the molar ratio of the compound of formula (10) to the compound of formula (6) is 1:1.3.

[0246] In some embodiments of the present disclosure, in step S2-3, the reaction temperature of the nucleophilic substitution reaction is selected from 20 to 40°C; for example, 25°C, 30°C, 35°C, etc.

[0247] In some embodiments of the present disclosure, in step S2-4, the hydrolysis reaction is selected from an alkaline hydrolysis reaction.

[0248] In some embodiments of the present disclosure, in step S2-4, the hydrolysis reaction conditions are selected from 0.5 to 1.5 mol / L NaOH aqueous solution, such as 0.8 mol / L NaOH aqueous solution, 1 mol / L NaOH aqueous solution, 1.2 mol / L NaOH aqueous solution, etc.

[0249] In some embodiments of the present disclosure, in step S2-4, the temperature of the hydrolysis reaction is 20-40°C; for example, 25°C, 30°C, 35°C, etc. Beneficial effects of the present disclosure:

[0250] The present disclosure provides compounds, conjugates, compositions, and uses thereof. The present disclosure provides oligonucleotide conjugates provided by the present disclosure, obtained by conjugating the compounds provided by the present disclosure to specific oligonucleotides (e.g., siRNA). The compounds can highly effectively target the oligonucleotide conjugates to liver cells and effectively inhibit the expression of target genes in liver cells. These compounds can be used to treat and / or prevent pathological conditions or diseases caused by abnormal expression of target genes in liver cells, have higher in vivo activity than control vectors, more stable and long-lasting pharmacodynamic effects, and are expected to have excellent safety and low animal-level toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0251] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0252] FIG1 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 1;

[0253] FIG2 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 2;

[0254] FIG3 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 3;

[0255] FIG4 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 4;

[0256] FIG5 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 5;

[0257] FIG6 shows the relative expression levels of target genes in mice after administration of the siRNA conjugates described in Example 6;

[0258] FIG7 shows the relative expression levels of target genes in mice after administration of the siRNA conjugates described in Example 7;

[0259] FIG8 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 8;

[0260] FIG9 shows the relative expression levels of target genes in mice after administration of the siRNA conjugates described in Example 9;

[0261] FIG10 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 10;

[0262] FIG11 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 11;

[0263] FIG12 shows the relative expression levels of target genes in mice after administration of the siRNA conjugates described in Example 12;

[0264] FIG13 shows the relative expression levels of target genes in mice after administration of the siRNA conjugates described in Example 13;

[0265] FIG14 shows the relative expression levels of target genes in mice after administration of the siRNA conjugates described in Example 14;

[0266] FIG15 shows the relative expression levels of target genes in mice after administration of the siRNA conjugate described in Example 15;

[0267] FIG16a shows the detection of AST in the blood of animals in each group after administration of the siRNA conjugate described in Example 16;

[0268] FIG16 b shows the detection of ALT in the blood of animals in each group after administration of the siRNA conjugate described in Example 16;

[0269] FIG16c shows the detection of TBIL in the blood of each group of animals after administration of the siRNA conjugate described in Example 16;

[0270] FIG16d shows the detection of Urea in the blood of each group of animals after administration of the siRNA conjugate described in Example 16;

[0271] FIG16e shows the detection of Crea in the blood of each group of animals after administration of the siRNA conjugate described in Example 16;

[0272] FIG16f shows the detection of TC in the blood of each group of animals after administration of the siRNA conjugate described in Example 16;

[0273] FIG16g shows the detection of TG in the blood of animals in each group after administration of the siRNA conjugate described in Example 16;

[0274] FIG16h shows the detection of WBC in the blood of each group of animals after administration of the siRNA conjugate described in Example 16;

[0275] FIG16i shows the detection of NEUT in the blood of animals in each group after administration of the siRNA conjugate described in Example 16;

[0276] FIG16j shows the detection of LYMPH in the blood of each group of animals after administration of the siRNA conjugate described in Example 16;

[0277] FIG16k shows the histopathological examination of the liver tissues of the animals in each group after administration of the siRNA conjugates described in Example 16. DETAILED DESCRIPTION

[0278] The present disclosure discloses compounds, conjugates, compositions and their uses. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve them. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this disclosure. The methods and applications of the present disclosure have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this disclosure to implement and apply the disclosed technology.

[0279] The affinity of ASGPR to its ligand is mainly affected by the following factors:

[0280] a. Ligand terminal sugar type. Ligands with either Gal or GalNAc termini can be recognized by ASGPR. The affinity of GalNAc for binding to ASGPR is 10-50 times higher than that of Gal. Furthermore, GalNAc ligands are more likely to escape Kupffer cell recognition and target hepatocytes.

[0281] b. Sugar molecule substitution sites: 1-OH, 2-OH (or 2-amino), 3-OH, 4-OH and 5-CH2- on Gal (or GalNAc) are all involved in binding to the ASGPR receptor; when 5-CH2- is connected to a negatively charged group or 4-OH, 3-OH, 2-OH (or 2-amino) is replaced, the affinity with the receptor decreases sharply; when 1-OH undergoes glycosylation substitution, when the glycosidic bond is in the α conformation, the affinity with the receptor is weakened, and when it is in the β conformation and the substituent is a linear structure, the affinity is not affected; only 6-OH does not participate in receptor binding and points to the solvent region, making it suitable as a connection site for connection with carriers, drugs, etc.

[0282] c. Spatial distance between the terminal sugar group and the carrier: ASGPR can effectively recognize the sugar group when there is at least 6 CH2 units between the sugar group and the carrier.

[0283] In addition, different molecular cluster structures of ligands and different connection methods with oligonucleotides will significantly affect the activity of oligonucleotides in the body. Higher activity means better therapeutic effects or lower dosages. Under the same efficacy, lower dosages also mean lower toxic reactions.

[0284] Based on the affinity characteristics of the above-mentioned ASGPR receptor, we designed a ligand with high affinity for ASGPR to achieve liver-targeted delivery of drugs or genes to regulate target gene expression.

[0285] Explanation of terms

[0286] The term "aliphatic ring" refers to a structure with a cyclic carbon backbone. The term "5- to 8-membered aliphatic ring" refers to a monocyclic structure with 5 to 8 carbon atoms in the cyclic carbon backbone. The term "saturated aliphatic ring" refers to a structure with a cyclic carbon backbone composed entirely of carbon-carbon single bonds.

[0287] The term "5- to 8-membered heterocyclic aliphatic ring" refers to a ring in which one or more carbon atoms are replaced by heteroatoms. The term "saturated heterocyclic aliphatic ring" refers to a ring in which one or more carbon atoms are replaced by heteroatoms.

[0288] The term "alkane" refers to a group of the formula The term "C1-C6 alkyl" refers to a alkyl group having 1 to 6 carbon atoms.

[0289] The term "alkylene" refers to a group of the formula The chain alkylene group may be a straight chain alkylene group or a branched chain alkylene group.

[0290] The term "CH2CH2" refers to a C2 straight chain alkyl group (ie, ethylene), having the structural formula:

[0291] The term "alkoxy" refers to

[0292] The term "alkanoyl" refers to For example, "C1-C6 chain alkyl acyl" may be formyl, acetyl, propionyl, n-butyryl or isobutyryl.

[0293] The term "NH" refers to an imino group, which has the formula

[0294] The term "CO" refers to a carbonyl group, which has the structural formula

[0295] The term "CN" refers to a cyano group, which has the structural formula

[0296] The structural formula of the term "triazole" is

[0297] In the context of the present disclosure, "pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0298] In the context of this disclosure, "pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalene disulfonate, and the like. These salts can be prepared by methods known in the art.

[0299] In the context of this disclosure, "pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, following salts: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.

[0300] In the context of this disclosure, an "oligonucleotide" is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically consisting of 10-50 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.

[0301] the term Indicates the site where a group is attached via a covalent bond.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] The term "comprising" is an open expression, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0307] "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.

[0308] "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.

[0309] "Enantiomers" refer to two non-superimposable isomers of a compound that are mirror images of each other.

[0310] "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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] The term "unsubstituted" means that the designated group bears no substituents.

[0315] 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.

[0316] In addition, it should be noted that, unless otherwise expressly stated, the descriptions used in this disclosure as “each…independently is” and “…each independently is” and “…independently is” are interchangeable and should be broadly understood to mean that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other. Taking R3 as an example, the specific options of R3 in the structural formula “C1-C50 alkylene optionally substituted by R3” and the structural formula “-C(O)-NH-C1-50 alkylene optionally substituted by R3” do not affect each other.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] In the context of the present disclosure, unless otherwise stated, the ratios of reagents described in the examples of the present disclosure are calculated on a volume basis (v / v).

[0329] 2. Preparation of compounds

[0330] The reagents and sources of the reagents used in the preparation of the compounds disclosed herein are as follows:

[0331] 1) Tributyltin hydride, CAS No. 688-73-3, purchased from Beijing Coupling Technology Co., Ltd.

[0332] 2) Benzaldehyde dimethyl acetal, CAS No. 125-88-8, purchased from Beijing Coupling Technology Co., Ltd.;

[0333] 3) DL-10-camphorsulfonic acid, CAS No. 5872-08-2, purchased from Beijing Coupling Technology Co., Ltd.;

[0334] 4) 3-bromopropyne, CAS No. 106-96-7, purchased from Beijing Coupling Technology Co., Ltd.;

[0335] 5) 4,4'-bis(methoxytrityl) chloride (abbreviated as DMTrCl), CAS No. 40615-36-9, purchased from Beijing Coupling Technology Co., Ltd.;

[0336] 6) 4,5-Dicyanoimidazole (abbreviated as DCI), CAS No. 1122-28-7, purchased from Beijing Coupling Technology Co., Ltd.;

[0337] 7) Bis(diisopropylamino)(2-cyanoethoxy)phosphine, CAS No. 102691-36-1, purchased from Beijing Coupling Technology Co., Ltd.;

[0338] 8) 1-O-acetyl-2,3,5-O-tribenzoyl-BETA-L-ribofuranose, CAS No. 3080-30-6, purchased from Beijing Coupling Technology Co., Ltd.;

[0339] 9) Triethylsilane, CAS No. 617-86-7, purchased from Beijing Coupling Technology Co., Ltd.;

[0340] 10) Trimethylsilyl trifluoromethanesulfonate (abbreviated as TMSOTf), CAS No. 27607-77-8, purchased from Beijing Coupling Technology Co., Ltd.;

[0341] 11) Cesium carbonate, CAS No. 534-17-8, purchased from Beijing Coupling Technology Co., Ltd.;

[0342] 12) Silver oxide (Ag2O), CAS No. 20667-12-3, purchased from Beijing Coupling Technology Co., Ltd.

[0343] 13) Potassium iodide (KI), CAS No. 7681-11-0, purchased from Shanghai Bidex Pharmaceutical Technology Co., Ltd.

[0344] 14) p-Toluenesulfonyl chloride (TsCl), CAS No. 98-59-9, purchased from Beijing Coupling Technology Co., Ltd.;

[0345] 15) Trimethylsilyl azide (TMSN3), CAS No. 4648-54-8, purchased from Beijing Coupling Technology Co., Ltd.

[0346] 16) Potassium fluoride (KF), CAS No. 7789-23-3, purchased from Beijing Coupling Technology Co., Ltd.;

[0347] 17) N,N-dimethylformamide (DMF), CAS No. 68-12-2, purchased from Beijing Coupling Technology Co., Ltd.;

[0348] 18) D-galactosamine pentaacetate, CAS No. 76375-60-5, purchased from Beijing Coupling Technology Co., Ltd.;

[0349] 19) Scandium trifluoromethanesulfonate, CAS No. 144026-79-9, purchased from Beijing Coupling Technology Co., Ltd.

[0350] 20) (S)-(+)-Glycerol acetone acetal, CAS No. 22323-82-6, purchased from Beijing Coupling Technology Co., Ltd.;

[0351] 21) Sodium hydride (NaH), CAS No. 7646-69-7, purchased from Beijing Coupling Technology Co., Ltd.;

[0352] 21) Amberlyst-15 (Chinese name: polymer of vinylbenzenesulfonic acid and divinylbenzene, English name: amberlyst 15 ion-exchange resin), CAS No. 39389-20-3, purchased from Beijing Coupling Technology Co., Ltd.;

[0353] 22) Aminoalkyl-CPG (model C3006-1000), purchased from Beijing Coupling Technology Co., Ltd.;

[0354] 23) Succinic anhydride, CAS No. 108-30-5, purchased from Beijing Coupling Technology Co., Ltd.

[0355] 24) Reference compound L96-PS

[0356] Compound L96-PS was purchased from Agilent Pharmaceuticals (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC).

[0357] The structural formula of the reference compound L96-PS is as follows:

[0358] Wherein, PS represents polystyrene resin solid phase carrier.

[0359] Unless otherwise stated, the reagents and consumables (Table 1) and instruments and equipment (Table 2) used in this disclosure were all commercially available products from the following manufacturers.

[0360] Table 1 Main reagents and consumables

[0361] Table 2 Main instruments and equipment

[0362] The compounds, conjugates, compositions and their uses provided by the present disclosure can be purchased from the market.

[0363] The present disclosure is further described below with reference to the following embodiments:

[0364] Preparation Example 1: Synthesis of Compound NM041

[0365] In this preparation example, the synthesis process of compound NM041 is as follows:

[0366] (1-1) Synthesis of Compound NM041-2

[0367] Compound NM041-1 (25.0 g, 1.0 eq) was dissolved in 250 ml of toluene, heated to 110°C, and tributyltin hydride (17.7 g, 1.0 eq) and azobisisobutyronitrile (1 g, 0.1 eq) were added. The mixture was refluxed at 110°C for 2 hours. After completion of the reaction, the reaction solution was cooled to 25°C, and ethyl acetate (100 ml) and potassium fluoride (10.59 g in 30 ml of water, 3.0 eq) were added. The mixture was stirred at 25°C for 2 hours, filtered, and the organic phase was separated. The aqueous phase was washed twice with ethyl acetate (50 ml x 2). The organic phases were combined and washed once with 50 ml of saturated sodium chloride solution. The mixture was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound NM041-2 (18.7 g, 87.9% yield) as a light yellow oil. MS ESI (m / z) = 333.1 [M+H] + .

[0368] (1-2) Synthesis of Compound NM041-3

[0369] Compound NM041-2 (18.7 g, 1 eq) was dissolved in 50 ml of anhydrous methanol, and sodium methoxide (0.288 g, 0.1 eq) was added. The mixture was stirred at 25°C for 1 hour. After completion of the reaction, the pH of the reaction solution was adjusted to 6-7 with a 4 mol / L solution of hydrogen chloride in 1,4-dioxane under an ice bath. The solution was concentrated, eluted twice with acetonitrile, and dried under vacuum to obtain compound NM041-3 (9.4 g, 100% yield) as a white solid.

[0370] (1-3) Synthesis of Compound NM041-4

[0371] Compound NM041-3 (9.4 g, 1.0 eq) was dissolved in 150 ml of acetonitrile, and benzaldehyde dimethyl acetal (30 ml, 3 eq) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added, respectively. The mixture was stirred at 25°C for 5 hours, and 3 ml of triethylamine was added, followed by further stirring at 25°C for 30 minutes. After completion of the reaction, the reaction solution was concentrated, 100 ml of water was added, and the mixture was extracted twice with ethyl acetate (100 ml x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM041-4 (8.6 g, 59.5% yield) as a white solid. MS ESI (m / z) = 252.2 [M+H] + .

[0372] (1-4) Synthesis of Compound NM041-5

[0373] Compound NM041-4 (4.3 g, 1.0 eq) was dissolved in 40 ml of N,N-dimethylformamide (DMF). Sodium hydride (2.7 g, 68 mmol, 4 eq) was added under ice-cooling and allowed to react for 30 minutes. 3-Bromopropyne (8.1 g, 4 eq) was added and stirred at 25°C for 2 hours. The mixture was quenched by adding 20 ml of water. After completion of the reaction, the reaction solution was extracted three times with ethyl acetate (50 ml x 3). The organic phases were combined and washed five times with saturated sodium chloride solution (20 ml x 5). The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound NM041-5 as a brown oil (yield 100%). MS ESI (m / z) = 329.2 [M+H] + .

[0374] (1-5) Synthesis of Compound NM041-6

[0375] Compound NM041-5 (5.6 g, 1.0 eq) was dissolved in 30 ml of dichloromethane (DCM), and 300 ml of 70% aqueous acetic acid was added. The mixture was reacted at 70°C for 1 hour. After the reaction, the reaction solution was directly concentrated to obtain compound NM041-6 as a yellow oil (yield 100%). MS ESI (m / z) = 241 [M+H] + .

[0376] (1-6) Synthesis of Compound NM041-7

[0377] Compound NM041-6 (5.6 g, 1.0 eq) was dissolved in 50 ml of pyridine. 4,4'-dimethoxytriphenylmethane (10.2 g, 1.3 eq, abbreviated as DMTrCl) was added under ice-cooling. The atmosphere was replaced with nitrogen three times, and the mixture was stirred at 25°C for 3 hours. The mixture was quenched by adding 50 ml of methanol. After completion of the reaction, the reaction solution was concentrated, 50 ml of water was added, and the mixture was extracted three times with ethyl acetate (50 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain compound NM041-7 (5 g, yield 39.6%) as a light yellow solid. MS ESI (m / z) = 543.1 [M+H] + .

[0378] (1-7) Synthesis of Compound NM041

[0379] Compound NM041-7 (2.0 g, 1.5 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (347.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.22 g, 1.1 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 sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v). The mixture was then dried in vacuo for 12 hours to obtain compound NM041 (2 g, yield 73.09%) as a white powder. MS ESI (m / z) = 743.2 [M+H] + .

[0380] 1 H NMR(400MHz, Acetonitrile-d3)δ7.53-7.47(m,2H),7.36(tt,J=9.6,3.4Hz,6H),7.28-7.22(m,1H),6.94-6 .86(m,4H),4.45(d,J=2.4Hz,1H),4.35(dt,J=6.1,1.9Hz,2H),4.18(ddd,J=11.2,5.4,2.6Hz,1H),3.81(s,7 H),3.79-3.20(m,8H),3.07(dt,J=10.5,7.0Hz,1H),2.79(q,J=2.6Hz,1H),2.72(dt,J=5.1,2.4Hz,1H),2.69 -2.63(m,1H),2.48-2.34(m,1H),2.17(d,J=1.0Hz,2H),1.08(dd,J=6.8,4.1Hz,10H),0.90(d,J=6.8Hz,2H).

[0381] Preparation Example 2: Synthesis of Reference Compound NM042

[0382] In this preparation example, the synthesis process of compound NM042 is as follows:

[0383] (2-1) Synthesis of Compound NM042-2

[0384] Compound NM042-1 (5.0 g, 1.0 eq.) and sodium hydride (3.78 g, 2.5 eq.) were added to 60 ml of DMF, cooled to 0°C, and stirred at 0°C for 0.5 hours. Propylene bromide (9.0 g, 2.0 eq.) was slowly added during stirring, and the mixture was stirred at 25°C for 4 hours. After completion of the reaction, 100 ml of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (3 x 100 ml). The organic phases were combined, washed once with 100 ml of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound NM042-2 (9.0 g) as an oil. ESI-MS (m / z) = 171 [M+H] + .

[0385] (2-2) Synthesis of Compound NM042-3

[0386] Compound NM042-2 (9.0 g, 37.8 mmol) and Amberlyst-15 resin (13.5 g, 150% w / w) were added to 180 ml of methanol, the atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 6 hours. After completion of the reaction, the reaction mixture was filtered and concentrated to obtain compound NM042-3 (9.1 g) as an oil. ESI-MS (m / z) = 131.2 [M+H] + .

[0387] (2-3) Synthesis of Compound NM042-4

[0388] Compound NM042-3 (5 g, 1.0 eq.) was added to 75 ml of pyridine solution, cooled to 0°C, and DMTrCl (16.9 g, 1.69 eq.) was added portionwise at 0°C. The mixture was stirred at 25°C for 3 hours. After completion of the reaction, the reaction solution was concentrated and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM042-4 (15 g, yield 90.4%). ESI-MS (m / z) = 433.2 [M+H] + .

[0389] (2-4) Synthesis of Compound NM042

[0390] Compound NM042-4 (2.5 g, 1.0 eq.), bis(diisopropylamino)(2-cyanoethoxy)phosphine (3.5 g, 2.0 eq.), and DCI (0.567 g, 0.8 eq.) were added to 50 ml of DCM and stirred under nitrogen for 3 hours. After completion of the reaction, the reaction solution was washed once with 50 ml of saturated sodium bicarbonate solution. The organic phase was separated and washed once with 50 ml of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by reverse-phase HPLC to obtain compound NM042 (2.0 g, 56% yield) as a white oil. ESI-MS (m / z): 619.3 [M+H] + .

[0391] 1 HNMR(400MHz,DMSO-d6)δ0.97-1.04(d,J=6.7Hz,3H),1.06-1.20(m,9H),3.05-3.14(dd,J=5.5,10.7Hz,2H),3.46-3.69(m,6H),3.70- 3.75(d,J=2.5Hz,7H),3.98-4.09(dt,J=4.9,10.3Hz,1H),4.09-4.17(m,2H),6.81-6.93(m,4H),7.15-7.34(m,7H),7.35-7.46(m,2H).

[0392] Preparation Example 3: Synthesis of Compound NM014 and Compound NM014A

[0393] In this preparation example, the synthesis process of compound NM014 and compound NM014A is as follows:

[0394] (3-1) Synthesis of Compound NM014-2

[0395] Compound NM014-1 (50 g, 1.0 eq.) was added to 100 ml of ACN, the atmosphere was replaced with nitrogen, and the temperature was lowered to 0°C. TMSOTf (44.0 g, 2.0 eq.) was added dropwise at 0°C, followed by triethylsilane (34.5 g, 3.0 eq.). The temperature was slowly raised to 25°C, and the reaction was continued at 25°C for 12 hours. After completion of the reaction, 200 ml of purified water was added to the reaction solution, and the mixture was extracted twice with 200 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: n-heptane / ethyl acetate = 4 / 1, v / v) to obtain compound NM014-2 (41 g, yield 93.1%). ESI-MS (m / z) = 445.3 [M+H] + .

[0396] (3-2) Synthesis of Compound NM014-3

[0397] Compound NM014-2 (41 g, 1.0 eq.) was added to 410 ml of monomethylamine methanol and allowed to react at 25°C for 12 hours. After completion of the reaction, the reaction solution was concentrated and purified by normal phase column chromatography (eluent: first with dichloromethane / methanol = 10 / 1 to remove impurities; then with dichloromethane / methanol = 5 / 1, v / v) to obtain compound NM014-3 (7.5 g, yield 60.6%). ESI-MS (m / z) = 135.2 [M+H] + .

[0398] (3-3) Synthesis of Compound NM014-4

[0399] Compound NM014-3 (5.16 g, 1.0 eq.) was added to 30 ml of pyridine, cooled to 0°C, and DMTrCl (14.32 g, 1.1 eq.) was added portionwise at 0°C. The mixture was stirred at 25°C for 6 hours and quenched with 10 ml of methanol. After completion of the reaction, the reaction solution was concentrated, diluted with 50 ml of ethyl acetate, washed once with 30 ml of saturated sodium bicarbonate, and twice with 30 ml of purified water. The organic phase was separated, concentrated, and purified by normal phase column chromatography (gradient elution from n-heptane / ethyl acetate = 5 / 1 to n-heptane / ethyl acetate = 1 / 1, v / v) to obtain compound NM014-4 (9.0 g, 53.6% yield). ESI-MS (m / z) = 437.3 [M+H] + .

[0400] (3-4) Synthesis of Compounds NM014-5 and NM014-5A

[0401] Compound NM014-4 (9.0 g, 1.0 eq.) was dissolved in 90 ml of DMF, cesium carbonate (7.4 g, 1.1 eq.) was added, and the mixture was stirred at 25°C for 10 min. The temperature was lowered to 0°C, and propargyl bromide (2.5 g, 1.0 eq.) was added dropwise at 0°C. After the addition was complete, the temperature was slowly raised to 25°C, and the mixture was reacted at 25°C for 16 hours. After the reaction was completed, 100 ml of ethyl acetate was added to the reaction solution, and the mixture was washed three times with 10 ml of purified water (10 ml x 3). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: n-heptane / ethyl acetate = 5 / 1, v / v, first remove impurities, then maintain polarity unchanged) to obtain the front peak compound NM014-5 (2.7 g, yield 27.6%) and the back peak compound NM014-5A (2.3 g, yield 23.7%). Among them, the ESI-MS (m / z) of the front peak product compound NM014-5 is 475.4 [M+H] +, ESI-MS (m / z) of the latter peak compound NM014-5A = 475.4 [M+H] + .

[0402] (3-5) Synthesis of Compound NM014

[0403] The pre-peak compound NM014-5 (2.57 g, 1.0 eq.) was added to 26 ml of dichloromethane with continuous stirring. DCI (0.83 g, 1.3 eq.) was added and the atmosphere was purged with nitrogen. The reaction system was now white and turbid. The temperature was cooled to 0°C and bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.44 g, 1.5 eq.) was added dropwise at 0°C. After the addition was complete, the temperature was slowly raised to 25°C and stirred at 25°C for 3 hours. After completion of the reaction, the reaction system was diluted with 50 ml of dichloromethane, and the organic phase was washed once with 20 ml of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated and purified by normal phase column chromatography (eluent: n-heptane / 0.1% triethylamine in ethyl acetate = 4 / 1, v / v) to obtain compound NM014 (907 mg). ESI-MS (m / z) = 698.2 [M+Na] + .

[0404] 1 HNMR(400MHz, DMSO-d6)δ0.88-0.94(d,J=6.8Hz,4H),1.05-1.14(m,8H),1.11-1.22(m,1H),1.18-1.26(m,1H),2.52-2.60( t,J=5.9Hz,1H),2.72-2.80(m,1H),2.89-3.00(td,J=5.1,10.7Hz,1H),3.15-3.28(ddd,J=2.8,10.2,23.1Hz,1H),3.41-3.5 9(m,4H),3.71-3.78(d,J=2.0Hz,6H),3.75-3.84(m,1H),3.88-3.99(dq,J=2.7,18.2Hz,1H),3.99-4.07(m,1H),4.14-4.21( m,1H),4.18-4.30(m,1H),4.27-4.38(m,1H),6.84-6.93(m,4H),7.17-7.35(m,7H),7.36-7.44(ddd,J=1.3,4.0,8.2Hz,2H).

[0405] (3-6) Synthesis of Compound NM014A

[0406] The late peak compound NM014-5A (2.3 g, 1.0 eq.) was added to 23 ml of dichloromethane with continuous stirring. DCI (0.74 g, 1.3 eq.) was added and the atmosphere was purged with nitrogen. The reaction system was now white and turbid. The temperature was cooled to 0°C, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.19 g, 1.5 eq.) was added dropwise at 0°C. After the addition was complete, the temperature was slowly raised to 25°C and stirred at 25°C for 3 hours. After the reaction was complete, the reaction system was diluted with 50 ml of dichloromethane, and the organic phase was washed once with 20 ml of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and filtered. The organic phase was concentrated and purified by normal phase column chromatography (eluent: n-heptane / 0.1% triethylamine in ethyl acetate = 4 / 1, v / v) to obtain compound NM014A (520 mg). ESI-MS (m / z) = 698.2 [M+Na] + .

[0407] 1 H NMR (400MHz, DMSO-d6) δ1.10-1.20 (dd, J=6.7, 8.6Hz, 12H), 2.74-2.82 (m, 2H), 2.96-3.04 (dd, J=4.4, 10. 2Hz,1H),3.11-3.19(dd,J=3.3,10.2Hz,1H),3.38-3.43(q,J=2.3Hz,1H),3.55-3.66(m,2H),3.71-3.75( s,6H),3.75-3.85(m,3H),3.85-3.94(m,1H),4.01-4.07(dd,J=4.4,9.4Hz,1H),4.09-4.20(m,2H),4.22- 4.31(m,1H),4.43-4.52(dq,J=4.1,12.0Hz,1H),6.86-6.93(m,4H),7.19-7.35(m,7H),7.38-7.43(m,2H).

[0408] Preparation Example 4: Synthesis of Compound NM043

[0409] In this preparation example, the synthesis process of compound NM043 is as follows:

[0410] (4-1) Synthesis of Compound NM043-2

[0411] Compound NM043-1 (10 g, 1.0 eq.), silver oxide (23.16 g, 1.5 eq.), and potassium iodide (12.16 g, 1.1 eq.) were added to 100 ml of dichloromethane, cooled to 0°C, and a solution of TsCl (12.7 g, 1.0 eq.) in dichloromethane (100 ml) was slowly added. The mixture was stirred at 25°C for 2 hours. After completion of the reaction, the reaction solution was filtered, and the organic phase was washed twice with 100 ml of water and once with 100 ml of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound NM043-2 (12.0 g) as an oil. ESI-MS (m / z) = 305.1 [M+H] + .

[0412] (4-2) Synthesis of Compound NM043-3

[0413] Compound NM043-2 (12.0 g, 1.0 eq), TMSN3 (azidotrimethylsilane, CAS No. 4648-54-8, 13.62 g, 3.0 eq.), and potassium fluoride (6.9 g, 3.0 eq.) were added to 72 ml of DMF, the atmosphere was purged with nitrogen three times, the temperature was raised to 60°C, and the mixture was stirred at 60°C for 1.5 hours. After completion of the reaction, the temperature was lowered to 25°C, 100 ml of water was added to the reaction solution, and the mixture was extracted three times with 60 ml of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM043-3 (3 g) as an oil. ESI-MS (m / z) = 176.3 [M+H] + .

[0414] (4-3) Synthesis of Compound NM043

[0415] Compound NM043-3 (3.0 g, 1.01 eq.) and D-galactosamine pentaacetate (6.0 g, 1.0 eq.) were added to 75 ml of dichloroethane and heated to 85°C. Scandium trifluoromethanesulfonate (0.38 g, 0.05 eq.) was added portionwise at 85°C. The mixture was stirred at 85°C for 3 hours and quenched by the addition of 16 ml of triethylamine. After completion of the reaction, the reaction system was cooled to 25°C. 60 ml of water and 60 ml of dichloromethane were added to the reaction solution, which was filtered and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM043 (4.5 g) as an oil. ESI-MS (m / z) = 505.3 [M+H] + .

[0416] Preparation Example 5: Synthesis of Compound NM015

[0417] In this preparation example, the synthesis process of compound NM015 is as follows:

[0418] Compound NM015-1 (5 g, 1.1 eq.) and D-galactosamine pentaacetate (8.07 g, 1.0 eq.) were added to 96 ml of dichloroethane and heated to 85°C. Scandium trifluoromethanesulfonate (0.38 g, 0.77 mmol) was added portionwise at 85°C. The mixture was stirred at 85°C for 3 hours and quenched by the addition of 16 ml of triethylamine. After completion of the reaction, the reaction system was cooled to 25°C. 100 ml of water and 100 ml of dichloromethane were added to the reaction solution, which was filtered and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM015 (6.6 g) as an oil. ESI-MS (m / z) = 549 [M+H] + .

[0419] Preparation Example 6: Synthesis of Compound NM064

[0420] In this preparation example, the synthesis process of compound NM064 is as follows:

[0421] (6-1) Synthesis of Compound NM064-2

[0422] Compound NM064-1 (13 g, 1.0 eq) was dissolved in 150 ml of acetonitrile, and benzaldehyde dimethyl acetal (30 ml, 3.0 eq) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added, respectively. The mixture was stirred at 25°C for 5 hours, and 3 ml of triethylamine was added, followed by further stirring at 25°C for 30 minutes. After completion of the reaction, the reaction solution was concentrated, 100 ml of water was added, and the mixture was extracted twice with ethyl acetate (100 ml x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified using a normal phase column (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM064-2 (11.5 g, 60.8% yield) as a white solid. MS ESI (m / z) = 283 [M+H] + .

[0423] (6-2) Synthesis of Compound NM064-3

[0424] Compound NM064-2 (5 g, 1.0 eq) was dissolved in 40 ml of DMF. Sodium hydride (2.7 g, 4 eq) was added under ice-cooling and the mixture was allowed to react for 30 minutes. 3-Bromopropyne (8.1 g, 4 eq) was added and stirred at 25°C for 2 hours. The mixture was quenched with 20 ml of water. After completion of the reaction, the reaction solution was extracted three times with 50 ml of ethyl acetate (50 ml x 3). The organic phases were combined and washed five times with 20 ml of saturated sodium chloride solution (20 ml x 5). The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain NM064-3 (6.3 g, 100% yield) as a brown oil. MS ESI (m / z) = 359 [M+H] + .

[0425] (6-3) Synthesis of Compound NM064-4

[0426] Compound NM064-3 (6.3 g, 1.0 eq) was dissolved in 30 ml of DCM, and 300 ml of 70% aqueous acetic acid was added. The mixture was reacted at 70°C for 1 hour. After completion of the reaction, the reaction solution was directly concentrated to obtain compound NM064-4 (4.78 g, 100% yield) as a yellow oil. MS ESI (m / z) = 271 [M+H] + .

[0427] (6-4) Synthesis of Compound NM064-5

[0428] Compound NM064-4 (4.78 g, 1.0 eq) was dissolved in 50 ml of pyridine. DMTrCl (7.8 g, 1.3 eq) was added under ice-cooling, the atmosphere was replaced with nitrogen three times, and the mixture was stirred at 25°C for 3 hours. The mixture was quenched with 50 ml of methanol. After completion of the reaction, the reaction solution was concentrated, 50 ml of water was added, and the mixture was extracted three times with 50 ml of ethyl acetate (50 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain compound NM064-5 (6.7 g, yield 66.3%) as a light yellow solid. MS ESI (m / z) = 573 [M+H] + .

[0429] (6-5) Synthesis of Compound NM064

[0430] Compound NM064-5 (2.0 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (330.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.16 g, 1.1 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 (20 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v). The mixture was dried in vacuo for 12 hours to obtain compound NM064 (2 g, yield 74.07%) as a white powder. MS ESI (m / z) = 774 [M+H] + .

[0431] 1 H NMR(400MHz, DMSO-d6)δ7.45-7.39(d,J=7.8Hz,2H),7.36-7.18(tt,J=14.5,8.5Hz,7 H),6.95-6.84(d,J=7.5Hz,4H),5.01-4.96(s,1H),4.43-4.28(s,4H),3.81-3.70(s,8 H),3.65-3.35(m,12H),3.28-3.18(dt,J=14.5,7.2Hz,1H),3.06-2.97(t,J=9.5Hz,1 H),2.75-2.68(m,1H),1.07-0.95(q,J=7.4,6.8Hz,10H),0.84-0.78(d,J=6.6Hz,2H).

[0432] Preparation Example 7: Synthesis of Compound CR01022 and Compound CR01022Z (a cluster of CR01022, denoted as (CR01022)×1)

[0433] In this preparation example, the synthesis process of compound CR01022 and compound CR01022Z is ​​as follows:

[0434] (7-1) Synthesis of Compound CR01022-1

[0435] Compound NM015 (1114 mg, 2.03 mmol, 2.2 eq.), compound NM041-7 (500 mg, 0.922 mmol, 1.0 eq.), cuprous iodide (701 mg, 3.69 mmol, 4.0 eq.), and N,N-diisopropylethylamine (476 mg, 3.69 mmol, 4.0 eq.) were added to 3 ml of N,N-dimethylformamide solution and stirred at 25°C for 1.5 hours. After completion of the reaction, 5 ml of ethyl acetate and 5 ml of water were added to the reaction solution, filtered, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound CR01022-1 (1 g) as a foam. ESI-MS (m / z) = 1639 [M+H] + .

[0436] (7-2) Synthesis of Compound CR01022

[0437] Compound CR01022-1 (850 mg, 0.523 mmol, 1.0 eq.) was added to 17 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (313 mg, 1.04 mmol, 2.0 eq.) was added portionwise, followed by 4,5-dicyanoimidazole (49 mg, 0.415 mmol, 0.8 eq.). The atmosphere was purged with nitrogen three times and stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was washed once with saturated aqueous sodium bicarbonate. The organic phase was separated, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01022 (560 mg). MS ESI (m / z) = 1839 [M+H] + .

[0438] (7-3) Synthesis of Compound CR01022Z

[0439] To a 25 ml reaction vessel, compound CR01022-1 (160 mg, 0.1 mmol, 1.0 eq.) was added to 3.2 ml of dichloromethane. Succinic anhydride (18 mg, 0.18 mmol, 1.8 eq.), 4-dimethylaminopyridine (1.2 mg, 0.01 mmol, 0.01 eq.), and triethylamine (19.7 mg, 0.2 mmol, 2.0 eq.) were also added. The atmosphere was purged with nitrogen three times and stirred at 25°C for 16 hours. After completion of the reaction, the product was purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain 100 mg of the product.

[0440] The above product (100 mg, 0.058 mmol, 1.0 eq.), aminoCPG (1.44 g, 0.115 mmol, 80 umol / g), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (32.67 mg, 0.086 mmol, 1.5 eq.) and N,N-diisopropylethylamine (14.8 mg, 0.115 mmol, 2.0 eq.) were mixed and shaken for 16 hours. The reaction mixture was filtered, the solid was washed once with 10 ml of acetonitrile and dried in vacuo. The solid was mixed with 4-dimethylaminopyridine (2 mg, 0.017 mmol), Cap1 (15 ml) and Cap2 (1.5 ml) and shaken for 6 hours. The reaction mixture was filtered, the filter cake was washed once with 10 ml of acetonitrile and dried in vacuo to obtain compound CR01022Z (1.366 g, loading 20-30 umol / g).

[0441] Cap1 and Cap2 are capping reagent solutions. 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.

[0442] Preparation Example 8: Synthesis of Compound CR01027 and Compound CR01027Z (a cluster of CR01027, denoted as (CR01027)×1)

[0443] In this preparation example, the synthesis process of compound CR01027 and compound CR01027Z is as follows:

[0444] (8-1) Synthesis of Compound CR01027-1

[0445] Compound NM015 (1125 mg, 2.03 mmol, 2.2 eq.), compound NM064-5 (500 mg, 0.922 mmol, 1.0 eq.), cuprous iodide (701 mg, 3.69 mmol, 4.0 eq.), and N,N-diisopropylethylamine (476 mg, 3.69 mmol, 4.0 eq.) were added to 3 ml of N,N-dimethylformamide and stirred at 25°C for 1.5 hours. After completion of the reaction, 5 ml of ethyl acetate and 5 ml of water were added to the reaction solution, which was filtered and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01027-1 (1 g, yield 68.5%) as a foam. ESI-MS (m / z): 1669.7 [M+H] + .

[0446] (8-2) Synthesis of Compound CR01027

[0447] Compound CR01027-1 (870 mg, 0.530 mmol, 1.0 eq.) was added to 20 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (330 mg, 1.04 mmol, 2.0 eq.) was added portionwise, followed by 4,5-dicyanoimidazole (52 mg, 0.415 mmol, 0.8 eq.). The atmosphere was purged with nitrogen three times and stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed once with aqueous sodium bicarbonate solution. The organic phase was separated, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01027 (560 mg, 60.8% yield). MS ESI (m / z) = 1869.8 [M+H] + .

[0448] (8-3) Synthesis of Compound CR01027Z

[0449] Compound CR01027Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 9: Synthesis of Compound CR01020 and Compound CR01020Z

[0450] In this preparation example, the synthesis process of compound CR01020 and compound CR01020Z is as follows:

[0451] (9-1) Synthesis of Compound CR01020-1

[0452] Compound NM015 (2.55 g, 4.64 mmol, 2.2 eq.), compound NM014-5 (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide and stirred at 25°C for 1.5 hours. After completion of the reaction, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, which was filtered and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01020-1 (1.6 g, yield 74.1%) as a foam. ESI-MS (m / z) = 1023.2 [M+H] + .

[0453] (9-2) Synthesis of Compound CR01020

[0454] Compound CR01020-1 (1.0 g, 0.98 mmol, 1.0 eq.) was added to 20 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.59 g, 1.96 mmol, 2.0 eq.) was added portionwise, followed by 4,5-dicyanoimidazole (92 mg, 0.78 mmol, 0.8 eq.). The atmosphere was purged with nitrogen three times and stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was washed once with aqueous sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01020 (780 mg, 65.0% yield). MS ESI (m / z) = 1223.5 [M+H] + .

[0455] (9-3) Synthesis of Compound CR01020Z

[0456] Compound CR01020Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 10: Synthesis of compound CR01021 and compound CR01021Z (a cluster of CR01021, denoted as (CR01021)×1)

[0457] In this preparation example, the synthesis process of compound CR01021 and compound CR01021Z is as follows:

[0458] (10-1) Synthesis of Compound CR01021-1

[0459] Compound NM015 (2.55 g, 4.64 mmol, 2.2 eq.), compound NM014-5A (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide and stirred at 25° C. for 1.5 hours. After completion of the reaction, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, which was filtered and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01021-1 (1.7 g, 78.7% yield) as a foam. ESI-MS (m / z): 1023.2 [M+H] + .

[0460] (10-2) Synthesis of Compound CR01021

[0461] Compound CR01020-1 (1.0 g, 0.98 mmol, 1.0 eq.) was added to 20 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.59 g, 1.96 mmol, 2.0 eq.) was added portionwise, followed by 4,5-dicyanoimidazole (92 mg, 0.78 mmol, 0.8 eq.). The atmosphere was purged with nitrogen three times and stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed once with aqueous sodium bicarbonate solution. The organic phase was separated, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28) to obtain compound CR01021 (880 mg, 73.3% yield). MS ESI (m / z) = 1223.5 [M+H] + .

[0462] (10-3) Synthesis of Compound CR01021Z

[0463] Compound CR01021Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 11: Synthesis of compound CR01023 and compound CR01023Z (a cluster of CR01023, denoted as (CR01023)×1)

[0464] In this preparation example, the synthesis process of compound CR01023 and compound CR01023Z is as follows:

[0465] (11-1) Synthesis of Compound CR01023-1

[0466] Compound NM015 (2.79 g, 5.08 mmol, 2.2 eq.), compound NM042-4 (1.0 g, 2.31 mmol, 1.0 eq.), cuprous iodide (1.76 g, 9.24 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.19 g, 9.24 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide, stirred at 25°C for 1.5 hours, and then 15 ml of ethyl acetate and 15 ml of water were added. The mixture was filtered and separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01023-1 (1.6 g, yield 70.5%) as a foam. ESI-MS (m / z) = 980.2 [M+H] + .

[0467] (11-2) Synthesis of Compound CR01023

[0468] Compound CR01023-1 (1.0 g, 1.02 mmol, 1.0 eq.) was added to 20 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.61 g, 2.04 mmol, 2.0 eq.) was added portionwise, followed by 4,5-dicyanoimidazole (96 mg, 0.82 mmol, 0.8 eq.). The atmosphere was purged with nitrogen three times and stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was washed once with aqueous sodium bicarbonate. The organic phase was separated, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01023 (880 mg, 73.3% yield). MS ESI (m / z) = 1181.5 [M+H] + .

[0469] (11-3) Synthesis of Compound CR01023Z

[0470] Compound CR01023Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 12: Synthesis of compound CR01024 and compound CR01024Z (a cluster of CR01024, denoted as (CR01024)×1)

[0471] In this preparation example, the synthesis process of compound CR01024 and compound CR01024Z is as follows:

[0472] (12-1) Synthesis of Compound CR01024-1

[0473] Compound NM043 (2.34 g, 4.64 mmol, 2.2 eq.), compound NM014-5 (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide and stirred at 25°C for 1.5 hours. After completion of the reaction, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, filtered, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound CR01024-1 (1.7 g, 80% yield) as a foam. ESI-MS (m / z): 979.2 [M+H] + .

[0474] (12-2) Synthesis of Compound CR01024

[0475] To a 25 ml reaction vessel was added compound CR01024-1 (1.0 g, 1.02 mmol, 1.0 eq.) and dichloromethane (20 ml). Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.61 g, 2.04 mmol, 2.0 eq.) and 4,5-dicyanoimidazole (96 mg, 0.82 mmol, 0.8 eq.) were added portionwise. The atmosphere was purged with nitrogen three times and stirred at room temperature for 1 hour. The mixture was washed once with aqueous sodium bicarbonate solution, separated, and the organic phase was concentrated and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01024 (770 mg, 64.2% yield). MS ESI (m / z) = 1179.5 [M+H] + .

[0476] (12-3) Synthesis of Compound CR01024Z

[0477] Compound CR01024Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 13: Synthesis of compound CR01025 and compound CR01025Z (a cluster of CR01025, denoted as (CR01025)×1)

[0478] In this preparation example, the synthesis process of compound CR01025 and compound CR01025Z is as follows:

[0479] (13-1) Synthesis of Compound CR01025-1

[0480] Compound NM043 (2.34 g, 4.64 mmol, 2.2 eq.), compound NM14-5A (1.0 g, 2.11 mmol, 1.0 eq.), cuprous iodide (1.61 g, 8.44 mmol, 4.0 eq.), and N,N-diisopropylethylamine (1.09 g, 8.44 mmol, 4.0 eq.) were added to 10 ml of N,N-dimethylformamide and stirred at 25°C for 1.5 hours. After completion of the reaction, 15 ml of ethyl acetate and 15 ml of water were added to the reaction solution, filtered, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01025-1 (1.7 g, 80% yield) as a foam. ESI-MS (m / z) = 979.2 [M+H] + .

[0481] (13-2) Synthesis of Compound CR01025

[0482] Compound CR01025-1 (1.0 g, 1.02 mmol, 1.0 eq.) was added to 20 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.61 g, 2.04 mmol, 2.0 eq.) was added portionwise, followed by 4,5-dicyanoimidazole (96 mg, 0.82 mmol, 0.8 eq.). The atmosphere was purged with nitrogen three times and stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was washed once with aqueous sodium bicarbonate. The organic phase was separated, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to afford compound CR01025 (775 mg, 64.3% yield). MS ESI (m / z) = 1179.5 [M+H] + .

[0483] (13-3) Synthesis of Compound CR01025Z

[0484] Compound CR01025Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 14: Synthesis of compound CR01026 and compound CR01026Z (a cluster of CR01026, denoted as (CR01026)×1)

[0485] In this preparation example, the synthesis process of compound CR01026 and compound CR01026Z is as follows:

[0486] (14-1) Synthesis of Compound CR01026-1

[0487] Compound NM043 (4.08 g, 8.10 mmol, 4.4 eq.), compound NM041-7 (1.0 g, 1.84 mmol, 1.0 eq.), cuprous iodide (2.10 g, 11.04 mmol, 6.0 eq.), and N,N-diisopropylethylamine (1.43 g, 11.04 mmol, 6.0 eq.) were added to 20 ml of N,N-dimethylformamide and stirred at 25° C. for 1.5 hours. After the reaction, 25 ml of ethyl acetate and 25 ml of water were added to the reaction solution, which was filtered and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01026-1 (2.5 g, yield 87.4%) as a foam. ESI-MS (m / z) = 1551.2 [M+H] + .

[0488] (14-2) Synthesis of Compound CR01026

[0489] Compound CR01026-1 (1.0 g, 0.64 mmol, 1.0 eq.) was added to 20 ml of dichloromethane. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.39 g, 1.28 mmol, 2.0 eq.) was added portionwise, followed by 4,5-dicyanoimidazole (60 mg, 0.51 mmol, 0.8 eq.). The atmosphere was purged with nitrogen three times and stirred at 25°C for 1 hour. After completion of the reaction, the reaction mixture was washed once with aqueous sodium bicarbonate. The organic phase was separated, concentrated, and purified by reverse phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to obtain compound CR01026 (670 mg, 55.8% yield). MS ESI (m / z) = 1751.5 [M+H] + .

[0490] (14-3) Synthesis of Compound CR01026Z

[0491] Compound CR01026Z was prepared according to the synthesis method of compound CR01022Z (7-3) in Preparation Example 7. Preparation Example 15: Synthesis of compound CR01017 and compound CR01017Z (a cluster of CR01017, denoted as (CR01017)×1)

[0492] In this preparation example, the synthesis process of compound CR01017 and compound CR01017Z is as follows:

[0493] (15-1) Synthesis of Compound CR01017-1

[0494] Compound NM041-4 (4.3 g, 17.0 mmol, 1.0 eq) was dissolved in 50 ml of ultra-dry DMF. NaH (2.7 g, 68 mmol, 4.0 eq) was added under ice-cooling and stirred for 1 hour. Ethyl bromoacetate (5.7 g, 34 mmol, 2.0 eq, CAS No. 105-36-2) and KI (4.3 g, 25.9 mmol, 1.5 eq) were then added and allowed to react at 25°C for 3 hours. The mixture was then quenched by addition of saturated aqueous ammonium chloride. After completion of the reaction, 20 ml of water was added to the reaction solution, and the mixture was extracted three times with 50 ml of ethyl acetate (50 ml x 3). The organic phases were combined and washed ten times with 20 ml of saturated aqueous sodium chloride (20 ml x 10), dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound CR01017-1 as a dark brown oil, which was used directly in the next step. MS ESI (m / z) = 425.2 [M+H] + .

[0495] (15-2) Synthesis of Compound CR01017-2

[0496] CR01017-1 (5 g, 1.0 eq) was dissolved in 30 ml of dichloromethane (DCM), and 300 ml of 70% glacial acetic acid was added. The mixture was stirred at 50°C for 2 hours. After the reaction, the reaction solution was concentrated to obtain compound CR01017-2 as a dark brown oil, which was used directly in the next step. MS ESI (m / z) = 337 [M+H] + .

[0497] (15-3) Synthesis of Compound CR01017-3

[0498] Compound CR01017-2 (3.2 g, 9.52 mmol, 1.0 eq) was dissolved in 70 ml of pyridine, and DMTrCl (4.19 g, 12.4 mmol, 1.3 eq) was added under ice-cooling. The mixture was stirred at 25°C for 3 hours and quenched by the addition of 30 ml of methanol. After completion of the reaction, the reaction solution was concentrated, 20 ml of water was added, and the mixture was extracted three times with 50 ml of ethyl acetate (50 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 52 / 48, v / v) to obtain compound CR01017-3 (5 g, yield 83%) as a yellow solid. MS ESI (m / z) = 639 [M+H] + .

[0499] (15-4) Synthesis of Compound CR01017-4

[0500] Compound CR01017-3 (2 g, 3.16 mmol, 1.0 eq) was dissolved in 10 ml of methanol, and aqueous NaOH (6.3 ml, 1 mol / L) was added. The mixture was stirred at 25°C for 1 hour. After completion of the reaction, the pH of the reaction solution was adjusted to neutral with hydrochloric acid. The reaction solution was filtered, the filtrate was concentrated, and the product was purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 26 / 74, v / v) to obtain compound CR01017-4 (920 mg, 51% yield) as a light yellow solid. MS ESI (m / z) = 583 [M+H] + .

[0501] (15-5) Synthesis of Compound CR01017-5

[0502] Compound CR01017-4 (900 mg, 1.55 mmol, 1 eq), (2R,3R,4R,5R,6R)-5-acetylamino-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy) (2.02 g, 3.88 mmol, 2.5 eq), and HATU (1.35 g, 3.56 mmol, 2.3 eq) were dissolved in 25 ml of N,N-dimethylformamide. DIEA (800 mg, 6.2 mmol, 4.0 eq) was added under ice-cooling, and the mixture was stirred at 25° C. for 12 hours. After completion of the reaction, the mixture was purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 20 / 80, v / v) to obtain compound CR01017-5 (700 mg, yield 28.6%) as a white solid. MS ESI (m / z) = 1591.2 [M+H] + .

[0503] (15-6) Synthesis of Compound CR01017

[0504] Compound CR01017-5 (500 mg, 0.31 mmol, 1.0 eq), 4,5-dicyanoimidazole (30 mg, 0.25 mmol, 0.8 eq), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (138 mg, 0.46 mmol, 1.5 eq) were dissolved in 10 ml of ultra-dry dichloromethane and reacted at 25°C under a nitrogen atmosphere for 2 hours. After completion of the reaction, the reaction solution was washed twice with 10 ml of saturated sodium bicarbonate (10 ml x 2). The organic phase was concentrated and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 80 / 20, v / v) to obtain compound CR01017 (480 mg, yield 83.1%) as a white solid. MS ESI (m / z) = 1791.2 [M+H] + .

[0505] (15-7) Synthesis of Compound CR01017-6

[0506] Compound CR01017-5 (200 mg, 0.13 mmol, 1.0 eq) was dissolved in 10 ml of dichloromethane, and succinic anhydride (14 mg, 0.14 mmol, 1.1 eq) and triethylamine (26 mg, 0.26 mmol, 2.0 eq) were added. The mixture was stirred at 25°C for 12 hours. After completion of the reaction, the reaction solution was concentrated and purified by reverse phase column chromatography (C18 column, eluent: acetonitrile / water = 36 / 64, v / v) to obtain compound CR01017-6 (150 mg, yield 71.4%) as a white solid. MS ESI (m / z) = 1691.2 [M+H] + .

[0507] (15-8) Synthesis of Compound CR01017Z

[0508] Compound CR01017-6 (70 mg, 0.041 mmol, 1.0 eq), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (22 mg, 0.062 mmol, 1.5 eq, abbreviated as HBTU), and N,N-diisopropylethylamine (10 mg, 0.082 mmol, 2.0 eq) were dissolved in 10 ml of acetonitrile and stirred at 25 ° C for 5 minutes. Amino CPG (970 mg, 80 umol / g) was added and the mixture was shaken at 25 ° C for 18 hours. The mixture was filtered and the filter cake was washed twice with 10 ml of dichloromethane (10 ml × 2) and then washed twice with 10 ml of acetonitrile (10 ml × 2). The filter cake, DMAP (3 mg, 0.024 mmol), Cap1 (10 ml) and Cap2 (1 ml) were mixed and shaken at 25°C for 5 hours. The mixture was filtered and the filter cake was rinsed three times with 10 ml of acetonitrile and dried to obtain compound CR01017Z (720 mg).

[0509] Cap1 and Cap2 are capping reagent solutions. 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.

[0510] Preparation Example 16: Synthesis Method 1 of siRNA Conjugate

[0511] (16-1) Synthesis of positive chain SS

[0512] By the phosphoramidite nucleic acid solid phase synthesis method, starting with the above-mentioned compounds connected to the solid phase support (i.e., CR01017Z, CR01020Z, CR01021Z, CR01022Z, CR01023Z, CR01024Z, CR01025Z, CR01026Z, CR01027Z, L96-PS), nucleoside monomers are linked one by one in a 3'-5' direction according to the nucleotide sequence (compounds CR01017, CR01020, CR01021, CR01022, CR01023, CR01024, CR01025 and CR01026 can be regarded as a nucleoside monomer respectively).

[0513] Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given below:

[0514] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0515] 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.

[0516] 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.

[0517] 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.

[0518] 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).

[0519] 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:

[0520] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to a solid support was added to 0.5 ml / μmol of 25% 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.

[0521] 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 eluent ratio 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.

[0522] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured and theoretical values ​​were consistent, the compound was conjugated to the 3' end of the siRNA sense strand.

[0523] During the synthesis of the sense chain, the following ligands (carriers) were synthesized:

[0524] [Corrected 31.12.2024 according to Rule 91] The structural formula of the two-cluster CR01017 (denoted as (CR01017)×2) is:

[0525] The structural formula of three clusters of CR01020 (denoted as (CR01020)×3) is:

[0526] The structural formula of three clusters of CR01021 (denoted as (CR01021)×3) is:

[0527] The structural formula of the two-cluster CR01022 (denoted as (CR01022)×2) is:

[0528] The structural formula of three clusters of CR01022 (denoted as (CR01022)×3) is:

[0529] The structural formula of three clusters of CR01023 (denoted as (CR01023)×3) is:

[0530] The structural formula of three clusters of CR01024 (denoted as (CR01024)×3) is:

[0531] The structural formula of three clusters of CR01025 (denoted as (CR01025)×3) is:

[0532] The structural formula of the two-cluster CR01026 (denoted as (CR01026)×2) is:

[0533] The structural formula of the two-cluster CR01027 (denoted as (CR01027)×2) is:

[0534] (16-2) Synthesis of antisense strand (AS)

[0535] 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 (16-1) for synthesizing the sense chain.

[0536] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS, purchased from Waters, model: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured and theoretical values ​​were consistent, the siRNA antisense strand was obtained.

[0537] (16-3) Synthesis of siRNA Conjugates

[0538] The sense chain synthesized in step (16-1) and the antisense chain synthesized in step (16-2) are mixed in an equimolar ratio, dissolved in water for injection and heated to 95°C, slowly cooled to room temperature and kept at room temperature for 10 minutes, so that the sense chain and the antisense chain form a double-stranded structure through hydrogen bonds, thereby obtaining a target siRNA conjugate.

[0539] Detection: Each conjugate was diluted to a concentration of 0.2 mg / ml (based on siRNA) using ultrapure water (Milli-Q ultrapure water analyzer, resistivity 18.2 MΩ*cm (25°C)). Molecular weights were then determined using liquid chromatography-mass spectrometry (LC-MS, Waters, LCT Premier). The measured values ​​were consistent with the theoretical values, demonstrating that the synthesized conjugates contained the desired double-stranded nucleic acid sequence.

[0540] Preparation Example 17: Synthesis Method 2 of siRNA Conjugate

[0541] In this preparation example, in the siRNA conjugates provided by the present disclosure, the linking group used to connect the siRNA and the ligand group is shown in Formula A (NM014, NM014A, NM041 and NM042):

[0542] The ligand groups are shown in Formula B (NM015 and NM043):

[0543] Taking the linker group NM041 and the ligand group NM015 as an example, the siRNA synthesis process includes the following steps:

[0544] (17-1) Synthesis of the Sense Chain

[0545] The synthetic route of the sense chain is as follows:

[0546] Specifically, the preparation method of the sense chain includes the following steps:

[0547] S1. Compound NM041 is treated as a nucleoside monomer and attached to a solid phase support via a phosphoramidite solid-phase synthesis method. Nucleoside monomers are then attached one by one in the 3' to 5' direction according to the nucleotide types and sequence of the sense strand of the siRNA sequence. The attachment of each nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The deprotection, coupling, capping, oxidation, or sulfurization reaction conditions in the synthesis of the sense strand are the same as those for the synthesis of the sense strand in step (16-1).

[0548] The sense strand hemiconjugate containing the linker was isolated (denoted as SS-(NM041)×2), wherein the linker NM041 was connected to the 3' end of the sense strand of the siRNA.

[0549] The terminal alkyne group in S2 and SS-(NM041)×2 was conjugated with the ligand group NM015 via a click reaction to obtain a positive chain conjugate.

[0550] S2-1. Mix 150 μL of H2O, 70 μL of 0.2 mol / L carbonate buffer solution (pH = 9.2), and 70 μL of N,N-dimethylformamide (DMF) to obtain a mixed solvent; dissolve SS-(NM041)×2 in the mixed solvent to obtain a 1.0 eq SS-(NM041)×2 solution.

[0551] S2-2, take 6.0eq of ligand compound NM015 and dissolve it in 70μL of DMF to obtain NM015 solution

[0552] S2-3. Mix the SS-(NM041)×2 solution obtained in step S2-1 and the NM015 solution obtained in step S2-2 to obtain a reactant mixture.

[0553] S2-4. Take 10.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 3.0 eq of CuSO4·5H2O in a volume ratio of THPTA:CuSO4·5H2O=10:3, shake at 40°C for 5 minutes, take 37 μL and add it to the reactant mixture obtained in the above step S2-3, and vortex to obtain an intermediate product mixture. The pH of the intermediate product mixture is measured to be 8.

[0554] S3. Take 25.0 eq of sodium ascorbate and quickly add it to the intermediate product mixture obtained in step 2-4, and vortex and shake it. React at 40° C. for 1 h to obtain a product mixture.

[0555] S4. Purification: Take 3 μL of the product mixture obtained in step S3, dilute it with a mixture of DMF and HO (DMF:HO volume ratio of 1:5), and then separate and purify it using HPLC. The HPLC treatment uses a C18 column, an ammonium bicarbonate buffer solution as the mobile phase, and a gradient elution method. The product after HPLC treatment is lyophilized.

[0556] (17-2) Synthesis of antisense strand

[0557] Prepared according to the synthesis method of the antisense chain (AS) (16-2) in Preparation Example 16.

[0558] (17-3) Synthesis of siRNA Conjugates

[0559] Prepared according to the synthesis method of siRNA conjugate (16-3) in Preparation Example 16.

[0560] In the two methods for synthesizing siRNA conjugates provided in the present disclosure, the first method for synthesizing the siRNA conjugate is to first synthesize the carrier, then synthesize the sense chain conjugate, and finally synthesize the siRNA conjugate; the second method for synthesizing the siRNA conjugate is to first synthesize the sense chain half conjugate, then synthesize the sense chain conjugate, and finally synthesize the siRNA conjugate.

[0561] Among them, when synthesizing the same amount of siRNA conjugates, compared with synthesis method 2, synthesis method 1 uses less raw materials in synthesizing the carrier, but takes longer to synthesize the siRNA conjugate; compared with synthesis method 1, synthesis method 2 takes less time to synthesize the siRNA conjugate, but uses more raw materials in synthesizing the carrier.

[0562] [Corrected 31.12.2024 according to Rule 91] When the carrier is a cluster of CR01017, the structural formula of the siRNA conjugate is as follows:

[0563] [Corrected 31.12.2024 according to Rule 91] When the carrier is a two-cluster CR01017, the structural formula of the siRNA conjugate is as follows:

[0564] When the vector is three clusters of CR01020, the structural formula of the siRNA conjugate is as follows:

[0565] When the vector is three clusters of CR01021, the structural formula of the siRNA conjugate is as follows:

[0566] When the carrier is a cluster of CR01022, the structural formula of the siRNA conjugate is:

[0567] When the carrier is two clusters of CR01022, the structural formula of the siRNA conjugate is:

[0568] When the vector is three clusters of CR01022, the structural formula of the siRNA conjugate is:

[0569] When the vector is three clusters of CR01023, the structural formula of the siRNA conjugate is:

[0570] When the vector is three clusters of CR01024, the structural formula of the siRNA conjugate is:

[0571] When the vector is three clusters of CR01025, the structural formula of the siRNA conjugate is:

[0572] When the carrier is two clusters of CR01026, the structural formula of the siRNA conjugate is:

[0573] When the carrier is two clusters of CR01027, the structural formula of the siRNA conjugate is:

[0574] When the vector is L96, the structural formula of the siRNA conjugate is:

[0575] The present disclosure synthesized the siRNA conjugates in Table 3 according to the methods of Preparation Example 16 and Preparation Example 17, respectively.

[0576] In the present disclosure, the conjugates RZ502031, RZ599001, RZ597002, RZ899038, RZ899041, and RZ899042 can only be synthesized using the synthesis method 1 of the siRNA conjugate described in Preparation Example 16.

[0577] Conjugates RZ802008, RZ802009, RZ899007, RZ802014, RZ899022, RZ899023, RZ899024, RZ802010, RZ899009, RZ899011, RZ899040, RZ899025, RZ802011, RZ899010, RZ899048, and RZ897013 can be synthesized using either the first method for synthesizing the siRNA conjugate described in Preparation Example 16 or the second method for synthesizing the siRNA conjugate described in Preparation Example 17. Specifically, in the present disclosure, conjugates RZ802010, RZ899009, RZ899011, and RZ899040 were synthesized using the first method for synthesizing the siRNA conjugate described in Preparation Example 16; conjugates RZ802008, RZ802009, RZ899007, RZ802014, RZ899022, RZ899023, RZ899024, RZ899025, RZ802011, RZ899010, RZ899048, and RZ897013 were synthesized using the second method for synthesizing the siRNA conjugate described in Preparation Example 17.

[0578] UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm, as shown in SEQ ID No. 1;

[0579] UmsUfsUmAmGmAfGmUmGmAmGmGmAmUfUmAfAmAmAmsUmsGm, as shown in SEQ ID No. 2;

[0580] CmsAmsGmAmCmAmGfAfCfAmAmGmAmCmCmAmUmCm, as shown in SEQ ID No. 3;

[0581] AmsGfsAmUmGmGfUmCmUmUmGmUmCmUfGmUfCmUmGmsGmsAm, as shown in SEQ ID No.4;

[0582] CmsAmsGmAmCmAmGfAfCfAmAmGmAmCmCmAmUmCmUm, as shown in SEQ ID No.5;

[0583] CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm, as shown in SEQ ID No.6;

[0584] UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsUmsUm, as shown in SEQ ID No.7.

[0585] Table 3 siRNA conjugates

[0586] 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.

[0587] Unless otherwise specified, the siRNA sequences used in this application and this disclosure were commissioned to Suzhou Beixin Biotechnology Co., Ltd. for synthesis; the PCR primers used in this application and this disclosure were commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis; and the experimental animals C57BL / 6J mice used in this application and this disclosure were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0588] Table 4 Detection results of siRNA conjugates

[0589] It can be seen from the data in Table 4 that the sense strand (SS) and the antisense strand (AS) can be well attached to the ligand while maintaining a high purity.

[0590] General experiments

[0591] Method for evaluating target gene inhibitory activity in mice

[0592] 6-8 week old C57BL / 6J mice were randomly divided into groups (all female) by body weight. The mice in each group were dosed according to body weight, and the siRNA conjugates were configured into corresponding concentrations (calculated as siRNA) solutions for administration with a dosing volume of 5ml / kg (calculated as mouse body weight) using PBS solution. The PBS control group was given PBS solution (without drug conjugates) and the volume was 5ml / kg (calculated as mouse body weight). The administration was recorded as the 1st day (referred to as D1) on that day, and 5 mice were killed in each group at the preset time after administration. The mice that were killed were subjected to gross dissection and the liver tissue of each mouse that was killed was collected, and the liver tissue was cut into about 2mm 3 Small pieces were preserved with RNALater.

[0593] Liver tissue samples were collected from the RNAlater at different time points in different experimental groups and 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.

[0594] Take 1 μg of total RNA and use a reverse transcription kit (Promega, Reverse Transcription System, A3500) and select Oligo (dT) 15 reverse transcription primer. Prepare 20 μL reverse transcription system according to the instructions of the reverse transcription kit and complete the reverse transcription reaction. After the reaction is completed, add 80 μL RNase-Free water to the reverse transcription system to obtain cDNA solution. Then use 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, 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 above reverse transcription reaction, 10μL SYBR TMSelect 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 ) were used for real-time PCR amplification using a three-step method: 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 expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method. The calculation method is as follows:

[0595] ΔCt(test group) = Ct(test group target gene) - Ct(test group reference gene)

[0596] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0597] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0598] ΔΔCt(control group) = ΔCt(control group) - ΔCt(average of control group)

[0599] 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.

[0600] 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%.

[0601] Relative expression level of target gene mRNA in the test group = 2-ΔΔCt (test group) × 100%

[0602] Test group target gene mRNA inhibition rate = (1-test group target gene mRNA relative expression level) × 100%

[0603] Unless otherwise stated, the in vivo activity data are based on The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0604] Example 1: In vivo activity evaluation of three clusters of CR01020 vector-conjugated complement component 3 (CC3) siRNA

[0605] This example uses the in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the target gene CC3 in mice by using siRNA conjugates RZ802008 and RZ802009, which are conjugated to three clusters of CR01020 vectors at the 3' end of the siRNA sense strand at the same CC3 target site, and siRNA conjugate RZ502031, which is conjugated to the L96 vector.

[0606] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 15 mice in each group. Each group of mice was given PBS solution and the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen, wherein the dose given to each mouse in the PBS control group was 5 ml / kg; the dose given to each mouse in the siRNA conjugate experimental group was 3 mg / kg (in terms of siRNA), and the administration volume was 5 ml / kg. The day of administration was recorded as the first day (D1). After administration, 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 15th day (D15), and 5 mice were killed in each group on the 29th day (D29). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0607] The primer sequences of this example are shown in Table 5.

[0608] Table 5 Primer sequence list

[0609] The results of Example 1 show that the in vivo activity and duration of the conjugate RZ802008, in which the 3' end of the siRNA sense chain is conjugated with three clusters of CR01020, are basically equivalent to those of the conjugate RZ502031, in which the 3' end of the siRNA sense chain is conjugated with L96; the three-cluster CR01020 conjugate RZ802009 has better in vivo activity and comparable sustained pharmacodynamics than the L96 conjugate RZ502031 ( Figure 1 , Table 6 ).

[0610] Table 6 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0611] Example 2: In vivo activity evaluation of three clusters of CR01020 vectors conjugated with superoxide dismutase 1 (SOD1) siRNA

[0612] This example uses the in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of the target gene SOD1 in mice by using the siRNA conjugate RZ899007, which is conjugated to three clusters of CR01020 vectors at the 3' end of the siRNA sense strand at the same SOD1 target site, and the conjugate RZ599001, which is conjugated to the L96 vector.

[0613] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups according to body weight, with 20 mice in each group. Each group of mice was administered PBS solution and the siRNA conjugates mentioned in the present embodiment by subcutaneous administration in the abdomen, wherein the dosage of each mouse in the PBS control group was 5 ml / kg, and the dosage of each mouse in the siRNA conjugate experimental group was 1 mg / kg (in terms of siRNA), and the administration volume was 5 ml / kg. The administration day was recorded as the first day (D1), and after administration, 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 15th day (D15), 5 mice were killed in each group on the 29th day (D29), and 5 mice were killed in each group on the 43rd day (D43). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the aforementioned ΔΔCt method.

[0614] The primer sequences of this example are shown in Table 7.

[0615] Table 7 Primer sequence list

[0616] The results of Example 2 showed that the conjugate RZ899007, in which the 3' end of the siRNA sense strand was conjugated with three clusters of CR01020, had comparable maximum inhibitory activity and better sustained efficacy than the conjugate RZ599001, in which the 3' end of the siRNA sense strand was conjugated with L96 ( Figure 2 , Table 8 ).

[0617] Table 8 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0618] Example 3: In vivo activity evaluation of three clusters of CR01021 vectors and three clusters of CR01020 vectors conjugated with CC3 target siRNA

[0619] This example used an in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of three siRNA conjugates, RZ802014, conjugated to the 3' end of the sense strand of the CC3 target siRNA, three siRNA conjugates, RZ802009, conjugated to the 3' end of the CR01020 vector, and three siRNA conjugates, RZ502031, conjugated to the L96 vector. The difference between the three CR01021 and three CR01020 vectors is that the circular cores are isomers of each other.

[0620] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 10 mice in each group. Each group of mice was given PBS solution and the siRNA conjugates mentioned in the present embodiment by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA), and the administration volume was 5 ml / kg. The day of administration was recorded as the first day (D1), and after administration, 5 mice were killed in each group on the 8th day (D8) and 5 mice were killed in each group on the 22nd day (D22). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0621] The primer sequences of this example are shown in Table 5 in Example 1.

[0622] The results of Example 3 showed that the in vivo activity of the three-cluster CR01021 conjugate RZ802014 was superior to that of the L96 conjugate RZ502031; the pharmacodynamic activity of the three-cluster CR01020 conjugate RZ802009 was basically equivalent to that of the L96 conjugate RZ502031 ( Figure 3 , Table 9 ).

[0623] Table 9 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0624] Example 4: In vivo activity evaluation of three clusters of CR01021 vectors and three clusters of CR01020 vectors conjugated with SOD1 target siRNA

[0625] This example used an in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of three siRNA conjugates, RZ899022, conjugated to the 3' end of three CR01021 vectors; three siRNA conjugates, RZ899007, conjugated to three CR01020 vectors; and three siRNA conjugates, RZ599001, conjugated to the L96 vector, against the target gene SOD1 in mice. The difference between the three CR01021 and three CR01020 vectors is that the circular cores are isomers of each other.

[0626] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 5 mice in each group. Each group of mice was given PBS solution and the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA) with a dosing volume of 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice were killed in each group on the 8th day (D8) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0627] The primer sequences of this example are shown in Table 7 in Example 2.

[0628] The results of Example 4 showed that the in vivo activities of siRNA conjugates RZ899022, in which the 3' end of the siRNA sense strand was conjugated with three clusters of CR01021, and RZ899007, in which the siRNA conjugate was conjugated with three clusters of CR01020, were superior to those of L96 conjugate RZ599001 ( FIG. 4 , Table 10).

[0629] Table 10 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0630] Example 5: In vivo activity evaluation of three clusters of CR01024 vectors and three clusters of CR01025 vectors conjugated with SOD1 target siRNA

[0631] This example used an in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of three siRNA conjugates, RZ899023, conjugated to the 3' end of three CR01024 vectors, three siRNA conjugates, RZ899024, conjugated to three CR01025 vectors, and RZ599001, conjugated to L96, against the target gene SOD1 in mice. The three CR01024 and three CR01020 siRNAs share the same circular core structure but utilize linkers of different lengths. CR01025 and CR01021 share the same circular core structure but utilize linkers of different lengths.

[0632] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 5 mice in each group. Each group of mice was given PBS solution and the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA), and the administration volume was 5 ml / kg. The day of administration was recorded as the first day (D1), and the mice were killed on the 8th day (D8) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0633] The primer sequences of this example are shown in Table 7 in Example 2.

[0634] The results of Example 5 showed that the in vivo activities of the conjugates RZ899023, in which the siRNA sense strand 3' end was conjugated with three clusters of CR01024, and RZ899024, in which the siRNA sense strand 3' end was conjugated with three clusters of CR01025, were both better than those of the L96 conjugate RZ599001 ( FIG. 5 , Table 11 ).

[0635] The results of Example 4 and Example 5 show that different linker lengths have little effect on the activity of the vector.

[0636] Table 11 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0637] Example 6: In vivo activity evaluation of three clusters of CR01022 vector-conjugated CC3 target siRNA

[0638] This example uses the in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of the same CC3 target siRNA, the siRNA conjugate RZ802010, which is conjugated to the 3' end of the sense strand of the three clusters of CR01022, and the L96 conjugate RZ502031, on the target gene CC3 in mice.

[0639] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups according to body weight, with 15 mice in each group. Each group of mice was given PBS solution and the siRNA conjugates mentioned in the present embodiment by subcutaneous administration in the abdomen, wherein the dosage of each mouse in the PBS control group was 5 ml / kg, and the dosage of each mouse in the siRNA conjugate experimental group was 3 mg / kg (in terms of siRNA), and the administration volume was 5 ml / kg. The administration day was recorded as the first day (D1), and after administration, 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 15th day (D15), and 5 mice were killed in each group on the 29th day (D29). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0640] The primer sequences of this example are shown in Table 5 in Example 1.

[0641] The results of Example 6 showed that the conjugate RZ802010, in which three clusters of CR01022 were conjugated to the 3' end of the siRNA sense strand, had comparable highest inhibitory activity and better sustained efficacy than the L96 conjugate RZ502031 ( FIG. 6 , Table 12).

[0642] Table 12 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0643] Example 7: In vivo activity evaluation of two clusters of CR01022 vectors and three clusters of CR01022 vectors conjugated with SOD1 target siRNA

[0644] In this example, the in vivo target gene inhibitory activity evaluation method was used to evaluate the inhibitory activity of the same SOD1 target siRNA in vivo, including three CR01022 siRNA sequences RZ899009 conjugated to the 3' end of the positive strand, two CR01022 siRNA sequences RZ899011 conjugated to the L96 conjugate RZ599001, and the L96 conjugate RZ599001.

[0645] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 20 mice in each group. Each group of mice was given PBS solution and the siRNA conjugates mentioned in the present embodiment by subcutaneous administration in the abdomen, wherein the dosage of each mouse in the PBS control group was 5 ml / kg, and the dosage of each mouse in the siRNA conjugate experimental group was 1 mg / kg (in terms of siRNA), and the administration volume was 5 ml / kg. The administration day was recorded as the first day (D1), and after administration, 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 15th day (D15), 5 mice were killed in each group on the 29th day (D29), and 5 mice were killed in each group on the 43rd day (D43). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0646] The primer sequences of this example are shown in Table 7 in Example 2.

[0647] The results of Example 7 showed that the siRNA sequence RZ899009, which was conjugated with three clusters of CR01022 at the 3' end of the siRNA sense chain, and the siRNA sequence RZ899011, which was conjugated with two clusters of CR01022, had similar maximum inhibitory activity and sustained efficacy against the target gene SOD1, and both were superior to the L96 conjugated sequence RZ599001 ( Figure 7 , Table 13 ).

[0648] Table 13 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0649] Example 8: In vivo activity evaluation of one cluster of CR01022 vectors and two clusters of CR01022 vectors conjugated with SOD1 target siRNA

[0650] In this example, the in vivo target gene inhibitory activity evaluation method was used to evaluate the inhibitory activity of the same SOD1 target siRNA in mice, including the siRNA sequence RZ899011 conjugated to two CR01022 vectors at the 3' end of the positive strand, the siRNA sequence RZ899040 conjugated to one CR01022 vector, and the L96 conjugate RZ599001.

[0651] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 5 mice in each group. Each group of mice was given PBS solution and the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen, wherein the dose of each mouse in the PBS control group was 5 ml / kg, and the dose of each mouse in the siRNA conjugate experimental group was 1 mg / kg (in terms of siRNA), and the administration volume was 5 ml / kg. The day of administration was recorded as the first day (D1), and the mice were killed on the 8th day (D8) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0652] The primer sequences of this example are shown in Table 7 in Example 2.

[0653] The results of Example 8 showed that the siRNA sequence RZ899011 conjugated with two clusters of CR01022 at the 3' end of the siRNA sense strand and the siRNA sequence RZ899040 conjugated with one cluster of CR01022 were both able to produce a highly effective inhibitory effect on the target gene SOD1 at D8, with basically equivalent inhibitory activities, and both were slightly better than the L96 conjugate RZ599001 ( Figure 8 , Table 14 ).

[0654] Table 14 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0655] Example 9: In vivo activity evaluation of two clusters of CR01026 vectors and two clusters of CR01022 conjugated SOD1 target siRNA

[0656] This example used an in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of the same SOD1 target gene in mice using two CR01026 vector-based siRNA sequences, RZ899025, two CR01022 vector-based siRNA sequences, and the L96 conjugate RZ599001, conjugated to the 3' end of the sense strand of the same SOD1 target siRNA. The two CR01026 and two CR01022 vector-based siRNA sequences had the same circular core structure but employed linkers of varying lengths.

[0657] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 20 mice in each group. Each group of mice was given PBS solution and the siRNA conjugates mentioned in this example by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA) with a dose of 5 ml / kg. The day of administration was recorded as the first day (D1), and 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 29th day (D29), 5 mice were killed in each group on the 43rd day (D43), and 5 mice were killed in each group on the 57th day (D57). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0658] The primer sequences of this example are shown in Table 7 in Example 2.

[0659] The results of Example 8 showed that the siRNA sense chain 3' end conjugated with the siRNA sequence RZ899025 of the two clusters CR01026 and the siRNA sequence RZ899011 of the two clusters CR01022 had in vivo activities comparable to those of the L96 conjugated sequence RZ599001 at D8 and D29, and significantly better than that of the L96 conjugated sequence RZ599001 at D43, indicating that the two clusters CR01026 conjugate RZ899025 and the two clusters CR01022 conjugate RZ899011 had better sustained efficacy than the L96 conjugate RZ599001, and that different linker lengths had little effect on the activity of the CR01026 and CR01022 vectors ( Figure 9 , Table 15 ).

[0660] Table 15 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0661] Example 10: In vivo activity evaluation of three clusters of CR01023 vector-conjugated CC3 target siRNA

[0662] This example uses a method for evaluating target gene inhibition activity in mice to evaluate the inhibitory activity of the siRNA sequence RZ802011, which is conjugated to three clusters of CR01023 vectors at the 3' end of the sense strand, and the L96 conjugate RZ502031, on the target gene CC3 in mice.

[0663] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups according to body weight, with 15 mice in each group. Each group of mice was given PBS solution and the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA) with a dose of 5 ml / kg. The day of administration was recorded as the first day (D1), and after administration, 5 mice were killed in each group on D8, 5 mice were killed in each group on D15, and 5 mice were killed in each group on D29. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0664] The primer sequences of this example are shown in Table 5 in Example 1.

[0665] The results of Example 10 showed that the three-cluster CR01023 conjugate RZ802011 and the L96 conjugate RZ502031 had comparable overall activity at D8 and D15, but the L96 conjugate RZ502031 had a relatively higher target gene inhibition effect at D29 ( Figure 10 , Table 16 ).

[0666] Table 16 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0667] Example 11: In vivo activity evaluation of three clusters of CR01023 vectors conjugated to SOD1 target siRNA

[0668] This example uses a method for evaluating target gene inhibition activity in mice to evaluate the inhibitory activity of siRNA sequence RZ899010, which is conjugated with three clusters of CR01023 at the 3' end of the siRNA sense strand, and L96 conjugate RZ599001, on the target gene SOD1 in mice.

[0669] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups according to body weight, with 15 mice in each group. Each group of mice was given PBS solution and the siRNA conjugates mentioned in the present embodiment by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 1 mg / kg (in terms of siRNA) with a dose of 5 ml / kg. The administration day was recorded as the first day (D1), and after administration, 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 15th day (D15), and 5 mice were killed in each group on the 29th day (D29). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0670] The primer sequences of this example are shown in Table 7 in Example 2.

[0671] The results of Example 11 showed that compared with the three-cluster CR01023 conjugate RZ899010 and the L96 conjugate RZ599001, the two groups of test substances had comparable overall activities at D8 and D15, but at D29, the L96 conjugate RZ599001 had a relatively higher target gene inhibition effect ( Figure 11 , Table 17 ).

[0672] Table 17 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0673] Example 12: In vivo activity evaluation of two clusters of CR01027 vectors and two clusters of CR01022 vectors conjugated with SOD1 target siRNA

[0674] This example used an in vivo target gene inhibition activity assessment method to evaluate the inhibitory activity of the sense strand of siRNAs conjugated to the 3' end of two CR01027 vectors containing siRNA sequence RZ899048, two CR01022 vectors containing siRNA sequence RZ899011, and L96 conjugate RZ599001 against the target gene SOD1 in mice. The difference between CR01027 and CR01022 is that CR01027 adds a methoxy group to the 1-position of the CR01022 core structure.

[0675] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 5 mice in each group. Each group of mice was given the siRNA conjugate mentioned in this embodiment by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg, with a dose volume of 5 mL / kg. The day of administration was recorded as the first day (D1), and 5 mice were killed in each group on the 8th day (D8) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0676] The primer sequences of this example are shown in Table 7 in Example 2.

[0677] The results of Example 12 showed that the conjugate RZ899048 of cluster 2 CR01027 had a higher target gene inhibition effect at D8 than the conjugate RZ599001 of L96, and the effect was comparable to the conjugate (RZ899011) of cluster 2 CR01022 ( FIG. 12 , Table 18).

[0678] Table 18 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0679] Example 13: In vivo activity evaluation of two clusters of CR01027 vectors conjugated to angiopoietin-like 3 (ANGPTL3) target siRNA

[0680] This example uses the in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequence RZ897013, which is conjugated to the two-cluster CR01027 vector at the 3' end of the positive strand, and the conjugate RZ597002, which is conjugated to L96, against the target gene ANGPTL3 in mice.

[0681] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 5 mice in each group. Each group of mice was given the siRNA conjugate mentioned in this example by subcutaneous administration in the abdomen. Each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg, with a dose volume of 5 mL / kg. The day of administration was recorded as the first day (D1), and 5 mice were killed in each group on the 8th day (D8) after administration. Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0682] The primer sequences of this example are shown in Table 19.

[0683] Table 19 Primer sequence table

[0684] The results of Example 13 showed that the second cluster CR01027 conjugate RZ897013 had comparable inhibitory effects compared to the L96 conjugate RZ597002 ( FIG. 13 , Table 20 ).

[0685] Table 20 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0686] Example 14: In vivo activity evaluation of two clusters of CR01017 vectors and two clusters of CR01022 vectors conjugated with SOD1 target siRNA

[0687] This example used an in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the SOD1 target gene in mice using the 3' end of the sense strand of SOD1-targeting siRNA conjugated to two CR01017 vectors containing the siRNA sequence RZ899038, two CR01022 vectors containing the siRNA sequence RZ899011, and the L96 conjugate RZ599001. The primary difference between the two CR01017 vectors and the two CR01022 vectors is that the linker portion of the CR01017 vectors uses an amide bond structure, while the linker portion of the CR01022 vectors uses a triazole structure.

[0688] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, with 15 mice in each group. Each group of mice was given PBS solution and the siRNA conjugates mentioned in the present embodiment by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA) with a dose of 5 ml / kg. The day of administration was recorded as the first day (D1), and after administration, 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 29th day (D29), and 5 mice were killed in each group on the 54th day (D54). Liver tissue was collected for RNA extraction, reverse transcription reaction and Real-time PCR detection, and the target gene mRNA in each test group was relatively quantitatively calculated according to the aforementioned ΔΔCt method.

[0689] The primer sequences of this example are shown in Table 7 in Example 2.

[0690] The results of Example 14 showed that the siRNA sense strand 3' end conjugated to the siRNA sequence RZ899038 of the two clusters CR01017 vectors and the siRNA sequence RZ899011 of the two clusters CR01022 vectors had better in vivo activity than the L96 conjugated sequence RZ599001, and the use of a triazole structure (CR01022) or an amide structure (CR01017) as the linker had little effect on the vector activity ( Figure 14 , Table 21).

[0691] Table 21 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0692] Example 15 In vivo activity evaluation of different amounts and locations of CR01017 conjugated to SOD1 target siRNA

[0693] In this example, the in vivo target gene inhibitory activity assessment method for mice was used to evaluate the inhibitory activity of the siRNA sequence RZ897041, in which the 3' end of the siRNA sense strand was conjugated to a cluster of CR01017 vectors; the siRNA sequence RZ897042, in which the 5' and 3' ends of the siRNA sense strand were respectively conjugated to a cluster of CR01017 vectors; the siRNA sequence RZ897038, in which the 3' end of the siRNA sense strand was conjugated to two clusters of CR01017 vectors; and the L96 conjugate RZ599001, against the target gene SOD1 in mice.

[0694] 6-8 week old C57BL / 6j mice were randomly divided into 5 groups according to body weight, with 15 mice in each group. Each group of mice was given PBS solution or the siRNA conjugates mentioned in the present embodiment by subcutaneous administration in the abdomen, wherein each mouse in the PBS control group was given a dose of 5 ml / kg, and each mouse in the siRNA conjugate experimental group was given a dose of 3 mg / kg (in terms of siRNA) with a dose of 5 ml / kg. The day of administration was recorded as the first day (D1), and after administration, 5 mice were killed in each group on the 8th day (D8), 5 mice were killed in each group on the 29th day (D29), and 5 mice were killed in each group on the 54th day (D54).

[0695] The primer sequences of this example are shown in Table 7 in Example 2.

[0696] The results of Example 15 showed that the siRNA conjugate RZ899038, in which the 3' end of the siRNA sense strand was conjugated to two CR01017 vectors, the siRNA conjugate RZ899041, in which the 3' end of the siRNA sense strand was conjugated to one CR01017 vector, and the siRNA conjugate RZ899042, in which the 5' end and 3' end of the siRNA sense strand were each conjugated to one CR01017 vector, had comparable inhibitory effects as the L96 conjugate RZ599001 at D8 and D29, and had a higher inhibitory effect on the target gene than the L96 conjugate RZ599001 at D59. This indicates that the CR01017 conjugate has a longer duration of pharmacodynamic action ( Figure 15 , Table 22).

[0697] Table 22 Inhibitory activity of target genes in mice after administration of the siRNA conjugates described in this example

[0698] Example 16: The present invention selects two clusters of CR01022 conjugated with SOD1 target siRNA sequence RZ899011 and two clusters of CR01017 vector conjugated with SOD1 target siRNA sequence RZ899038 for in vivo toxicity evaluation in mice

[0699] In this example, subcutaneous injection was used to evaluate the nature and extent of the toxicity of the siRNA sequence RZ899011 conjugated to two clusters of CR01022 and the siRNA sequence RZ899038 conjugated to two clusters of CR01017 vectors in ICR mice.

[0700] 6-8 week old ICR mice were randomly divided into 3 groups (10 mice per group, half male and half female) based on body weight. Each group of mice was subcutaneously administered with either PBS solution (without siRNA conjugate) or the siRNA conjugates described in this example. Each mouse in the PBS control group received a dose of 10 ml / kg of mouse body weight, while each mouse in the siRNA conjugate-treated group received a dose of 300 mg / kg of mouse body weight (calculated as siRNA) in a 10 ml / kg volume.

[0701] The day of administration was designated as the first day (D1). Clinical observations were performed at least once a day after the first administration during the experimental period. On the 15th day of the experiment (designated as D15), blood was collected from all animals (mice were fasted for at least 12 hours before sampling and testing, but water was not withheld) for hematological and blood biochemical tests. Blood was collected using EDTA-K2 anticoagulation and an automatic modular blood and body fluid analyzer (Sysmex XN-10B1) was used for routine blood tests. Blood was collected using sodium citrate anticoagulation and centrifuged at room temperature for 10 minutes (centrifugal force of approximately 2000g). Plasma was separated and tested for coagulation indexes using a fully automatic coagulation analyzer (Mindray C3510). Blood samples (without anticoagulation) were collected and placed at room temperature for approximately 30 minutes. After the blood coagulated, it was centrifuged at 2000g for 10 minutes at 4°C. Blood biochemical tests were performed using a fully automatic biochemical analyzer (Sysmex BX-4000). All animals were dissected on the 15th day of the experiment (denoted as D15). The mice were fasted but not watered for at least 12 h before dissection and anesthetized with Zotal 50 (Vicco, France, 8G4VA). After blood collection, the animals were euthanized by bleeding from the abdominal aorta and subjected to gross anatomical observation. The livers of all animals were subjected to histopathological examination (using hematoxylin-eosin staining). If necessary, histopathological examination was performed on organs with obvious abnormal changes in size, morphology, color, texture, etc.

[0702] The primer sequences of this example are shown in Table 7 in Example 2.

[0703] The results of Example 16 show that after the siRNA conjugates described in this example, no abnormalities were observed in the clinical observations of the PBS group, the RZ899011 group, and the RZ899038 group during the experiment; at the experimental endpoint D15, no obvious abnormalities were observed in the gross anatomy; no abnormalities were found in the hematological and blood biochemical tests of the animals in each group ( Figures 16a-j, Table 23); and no abnormalities were found in the histopathological staining of the liver tissues of the animals in each group ( Figure 16k ); this indicates that the siRNA conjugates and their carrier molecules disclosed herein are safe and have low animal-level toxicity.

[0704] Table 23 Hematological and blood biochemical test results of mice after administration of the siRNA conjugates described in this example

[0705] The compounds, conjugates, compositions, and uses thereof provided by the present invention are described in detail above. The principles and implementation methods of the present invention are described herein using specific examples. The description of the above examples is intended only to facilitate understanding of the methods and core concepts of the present invention. It should be noted that those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0706] Sequence Listing

Claims

1. A compound having a structure represented by formula (Ia) or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof: In formula (Ia), R1 is selected from H, a hydroxyl protecting group or The * represents a connection site for connecting a drug active molecule; The R2 is selected from H, a reactive phosphorus group or in, R 2b Selected from a solid phase carrier containing an amino functional group, R 2a A covalent linking group selected from the group consisting of a covalent linking group connected to the amino functional group; The j is selected from 0, 1, 2 or 3; Each M is independently selected from hydroxyl or thiol; Each p is independently selected from 0, 1, 2, 3 or 4; Each q is independently selected from 0, 1, 2, 3 or 4; Each Cy is independently selected from wherein A is selected from a 5-6 membered oxygen-containing heterocyclic ring; R3 is selected from H or C1-C6 alkoxy; n is selected from 1 or 2; each X is independently selected from O, S, or NH; each R4 is independently selected from H, C1-C6 alkyl or C1-C6 alkoxy; each L1 is independently selected from a substituted or unsubstituted triazole group, or an amide group; m is selected from 1 or 2; each L2 is independently selected from a substituted or unsubstituted C1-C30 alkylene group or Each r is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, each Z is independently selected from O, S or NH, each R L2 Each is independently selected from substituted or unsubstituted C1-C10 alkylene; each Y is independently selected from O, S or NH; each R5 is independently selected from H, substituted or unsubstituted C1-C4 alkylacyl or substituted or unsubstituted C6-C 12 Aryl acyl.

2. The compound according to claim 1, or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof, characterized in that: A is selected from a 5- to 6-membered saturated oxygen-containing heterocyclic ring; Optionally, A is selected from a 5-6 membered saturated heterocyclic ring containing one oxygen atom; Optionally, said m is selected from 1; Optionally, X is selected from O; Optionally, each R4 is H; Optionally, each L1 is independently selected from Optionally, each L1 is selected from Optionally, each L1 is selected from Optionally, the R3 is selected from H or C1-C3 alkoxy; Optionally, said R3 is selected from H or methoxy; Optionally, each L2 is independently selected from Each r is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, each Z is independently selected from O, S or NH, each R L2 Each is independently selected from substituted or unsubstituted C1-C10 alkylene; Optionally, each L L2 Each is independently selected from substituted or unsubstituted C1-C5 alkylene; Optionally, each L L2 Each is independently selected from substituted or unsubstituted C1-C3 alkylene; Optionally, each L L2 Each is independently selected from substituted or unsubstituted C2 alkylene; Optionally, each r is independently selected from 2 or 3; Optionally, each L2 is independently selected from The r is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Optionally, each L2 is independently selected from The r is selected from 2 or 3; Optionally, Y is selected from O; Optionally, each R5 is independently selected from H, Optionally, each R5 is selected from Optionally, each R5 is selected from H; Optionally, said p and q are each independently selected from 0 or 1; Optionally, p=1 and q=1, or p=1 and q=0, or p=0 and q=1, or p=0 and q=0; Optionally, p=1 and q=0; Optionally, the hydroxy protecting group is selected from trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytriphenyl; Optionally, the hydroxy protecting group is selected from 4,4'-dimethoxytrityl; Optionally, the reactive phosphorus group is selected from Optionally, the reactive phosphorus group is selected from Optionally, the pharmaceutically active molecule is selected from a small molecule drug, an antibody or an oligonucleotide; Optionally, the oligonucleotide is selected from a single-stranded oligonucleotide and a double-stranded oligonucleotide; Optionally, the pharmaceutically active molecule is selected from double-stranded oligonucleotides; Optionally, the double-stranded oligonucleotide is selected from siRNA; Optionally, the solid support is selected from a resin containing an amino functional group or a controlled pore glass bead containing an amino functional group; Optionally, R 2b Selected from represents resin or controlled pore glass beads; Optionally, R 2a Selected from Optionally, Selected from Optionally, the j is selected from 0, 1 or 2 Optionally, said j is selected from 0; Optionally, said j is selected from 1; Optionally, j is selected from 2.

3. The compound according to claim 1 or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof, characterized in that: Each Cy is independently selected from any of the following structures:

4. The compound according to claim 1, or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof, characterized in that: The compound is selected from any of the following structures:

5. [Corrected 31.12.2024 in accordance with Rule 91] A compound according to claim 1, or a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a prodrug thereof, characterized in that The compound is selected from any of the following compounds:

6. A conjugate having a structure represented by formula (Ib) or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof: In formula (Ib), Nu represents an oligonucleotide; The k is selected from 1 or 2; The structural formula of Q is in, The j' is selected from 1, 2, 3 or 4; the *, M, p, q, Cy are as defined in any one of claims 1 to 5.

7. The conjugate according to claim 6, characterized in that The j' is selected from 1, 2 or 3; Optionally, said j' is selected from 1; Optionally, said j' is selected from 2; Optionally, said j' is selected from 3; Optionally, said k is selected from 1; Optionally, said k is selected from 2; Optionally, the oligonucleotide is selected from a single-stranded oligonucleotide or a double-stranded oligonucleotide; Optionally, said Nu represents a double-stranded oligonucleotide; Optionally, the double-stranded oligonucleotide is selected from siRNA; Optionally, the k is selected from 1; and one Q is conjugated to the 3' end of the siRNA sense strand, Optionally, the k is selected from 2, and the two Qs are conjugated to the 3' end and 5' end of the siRNA sense strand, respectively.

8. The conjugate according to claim 6, or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof, characterized in that: The conjugate has any of the following structures:

9. [Corrected 31.12.2024 according to Rule 91] The conjugate according to claim 6, or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof, characterized in that The conjugate is selected from any one of the following compounds:

10. A composition, characterized in that The composition comprises the conjugate according to any one of claims 6 to 9 or its stereoisomer, pharmaceutically acceptable salt or prodrug thereof; Optionally, the composition further comprises one or more pharmaceutically acceptable carriers or excipients.

11. Use of any of the following in the preparation of a medicament for preventing and / or treating a disease: (I), a compound according to any one of claims 1 to 5, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (II), the conjugate according to any one of claims 6 to 9, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (III) The composition of claim 10; Optionally, the disease is selected from a liver-related disease.

12. Use of any of the following in the preparation of a drug for reducing target gene expression or activity: (I), a compound according to any one of claims 1 to 5, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (II), the conjugate according to any one of claims 6 to 9, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (III) The composition according to claim 10.

13. A drug, characterized in that Includes any of the following items and pharmaceutically acceptable excipients or adjuvants: (I) a compound according to any one of claims 1 to 5, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (II), the conjugate according to any one of claims 6 to 9, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (III) The composition according to claim 10.

14. A pharmaceutical combination, characterized in that The method comprises the drug according to claim 13 and any other active ingredients.

15. A method for treating and / or preventing a disease in a subject, characterized in that It comprises administering to a subject a pharmaceutically acceptable amount of any of the following: (I), the conjugate according to any one of claims 6 to 9, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (II), the composition according to claim 10; and / or (III), the medicament according to claim 13; and / or (IV) The pharmaceutical combination according to claim 14.

16. A method for reducing the expression or activity of a target gene, characterized in that: It includes contacting the cell with any of the following: (I), the conjugate according to any one of claims 6 to 9, or a stereoisomer thereof, a pharmaceutically acceptable salt or a prodrug thereof; and / or (II) the composition according to claim 10; and / or (III), the medicament according to claim 13; and / or (IV) The pharmaceutical combination according to claim 14.

17. A compound represented by formula (Ic) or a stereoisomer thereof: In formula (Ic), p Ic and q Ic Each independently selected from 0, 1, 2 or 3; Each n Ic Each independently selected from 1, 2, 3, 4 or 5; R Ic-1 Select from H or R Ic-1 ', the R Ic-1 ' is selected from hydroxyl protecting groups; R Ic-2 Select from H or R Ic-2 ', the R Ic-2 'Selected from in, R Ic-2a Selected from secondary amino groups, R Ic-2b Selected from C1-C3 alkoxy substituted with secondary amino or cyano; R Ic-3 Selected from H or C1-C3 alkoxy; Each R Ic-4 Each independently selected from 18. The compound or stereoisomer thereof according to claim 17, characterized in that: Each R Ic-4 All selected from 19. The compound or stereoisomer thereof according to claim 17, characterized in that: Each R Ic-4 All selected from 20. The compound or stereoisomer thereof according to claim 17, characterized in that: p Ic and q Ic Each independently selected from 0 or 1; Optionally, p Ic =1 and q Ic =0; Optionally, each n Ic All selected from 1; Optionally, the structural formula of the hydroxyl protecting group is Among them, each R Ic-1a Each independently selected from H, C1-C3 alkyl or C1-C3 alkoxy; Optionally, each R Ic-1a Each independently selected from H or methoxy; Optionally, the hydroxy protecting group is selected from a trityl group (Tr group), a 4-methoxytrityl group (MMTr group), a 4,4'-dimethoxytrityl group (DMTr group) or a 4,4',4"-trimethoxytrityl group (TMTr group); Optionally, the hydroxy protecting group is selected from 4,4'-dimethoxytrityl (DMTr group). Optionally, R Ic-2a Selected from Optionally, R Ic-2b Selected from Optionally, Selected from Optionally, Selected from 21. The compound or stereoisomer thereof according to claim 17, characterized in that: The compound is selected from any of the following structures:

22. The compound or stereoisomer thereof according to claim 17, characterized in that: The compound is selected from any of the following structures:

23. Use of the compound according to any one of claims 17 to 22 or its stereoisomer in the preparation of a targeting ligand capable of binding to a cell surface receptor; Optionally, the receptor is selected from asialoglycoprotein receptors.

24. Use of a compound according to any one of claims 17 to 23 or a stereoisomer thereof in the preparation of a drug for reducing the expression or activity of a specific gene in a target cell; Optionally, the target cells are selected from hepatocytes; Optionally, the drug is selected from RNAi drugs; Optionally, the drug is selected from siRNA drugs.

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