CN116854771A

CN116854771A - GalNAc derivatives, conjugates, compositions and uses thereof

Info

Publication number: CN116854771A
Application number: CN202310707776.6A
Authority: CN
Inventors: 黄渊余; 高永鑫
Original assignee: Beijing Xuanjingrui Pharmaceutical Technology Co ltd
Current assignee: Beijing Xuanjingrui Pharmaceutical Technology Co ltd
Priority date: 2023-06-14
Filing date: 2023-06-14
Publication date: 2023-10-10

Abstract

The present disclosure relates to the field of nucleic acid pharmaceuticals, and in particular to GalNAc derivatives, conjugates, compositions and uses thereof. The GalNAc derivative has a structure shown in a formula (I). Conjugates provided herein are formed by conjugating GalNAc derivatives provided herein to an oligonucleotide. The conjugates provided by the present disclosure are highly effective in targeting the liver and producing effective inhibition of expression of liver target genes, and are useful for treating and/or preventing liver-derived diseases, with ratios to controlThe vector has higher in vivo activity, more stable and durable pharmacodynamic action and is expected to have excellent safety and low animal level toxicity.

Description

GalNAc derivatives, conjugates, compositions and uses thereof

Technical Field

The present disclosure relates to the field of medicine, in particular, the disclosure relates to GalNAc derivatives, conjugates, compositions and uses thereof.

Background

Small molecule nucleic acid drugs represented by small interfering RNAs (small interference RNA, siRNA), antisense oligonucleotides (antisense oligodeoxynucleotide, ASODN) and nucleic acid stimulatory motifs (CpG) are increasingly important in gene therapy, some of which have been approved by the FDA for marketing, and many more are currently in preclinical and clinical trials. Nucleic acid drugs refer to nucleic acid sequences that specifically target pathogenic genes or proteins by binding or cleavage, thereby inhibiting/promoting expression of certain genes/proteins, including all human normal genes that can replace defective genes, antisense nucleic acids that block gene expression, or single-stranded nucleic acids that promote triplex formation, etc., such as siRNA, DNA, microRNA or CpG, etc.

Delivery systems are one of the key technologies in the development of small nucleic acid drugs, and the most widely studied class of delivery systems for small nucleic acid delivery systems worldwide is currently targeted conjugated delivery technology. There remains a pressing need in the art to develop a new drug conjugate with higher efficacy of active drug delivery in vivo, lower toxicity, higher activity.

Disclosure of Invention

The present disclosure is directed to solving, at least in part, at least one of the technical problems existing in the prior art.

To this end, the present disclosure provides GalNAc derivatives, conjugates, compositions, and uses thereof. Conjugates provided herein are formed by conjugating GalNAc derivatives provided herein to an oligonucleotide. The conjugates provided by the present disclosure are highly effective in targeting the liver and producing effective inhibition of expression of liver target genes, are useful for the treatment and/or prevention of liver-derived diseases, have higher in vivo activity than control vectors, have more stable and durable pharmacodynamic effects, and are expected to have excellent safety and low animal level toxicity.

In a first aspect of the present disclosure, the present disclosure provides GalNAc derivatives as shown in formula (I), or stereoisomers, pharmaceutically acceptable salts, or prodrugs thereof:

Wherein A is selected from a substituted or unsubstituted 5-7 membered saturated nitrogen heterocycle;

n is selected from 0 or 1, m is selected from 0 or 1, and n=m;

each L ₁ Each independently selected from substituted or unsubstituted C ₁ -C ₅ An alkylene group;

each L ₂ Each independently selected from substituted or unsubstituted C ₂ -C ₁₀ An alkylene group;

each Y is independently selected from O, S or NH;

each R ₃ Each independently selected from H, substituted or unsubstituted C ₁ -C ₄ Alkylacyl or substituted or unsubstituted C ₅ -C ₇ An aryl acyl group;

p and q are each independently selected from 0, 1, 2 or 3;

R ₁ selected from H, hydroxy protecting groups or* Represents a linking site for linking a pharmaceutically active molecule, R _1a Is hydroxyl or sulfhydryl;

R ₂ selected from H orR _2b Selected from solid supports containing amino functions, R _2a Selected from covalent linking groups attached to the amino functional groups.

In some alternative embodiments of the present disclosure, a may be selected from a substituted or unsubstituted 4-6 membered saturated nitrogen heterocycle, 5-7 membered saturated nitrogen heterocycle, 5-6 membered saturated nitrogen heterocycle, or 6 membered saturated nitrogen heterocycle.

In some alternative embodiments of the present disclosure, a is selected from

In some embodiments of the present disclosure, A is selected from

In some alternative embodiments of the present disclosure, each L ₁ Each independently selected from substituted or unsubstituted C ₁ -C ₅ A linear alkylene group; for example C ₁ -C ₄ Straight chain alkylene, C ₁ -C ₃ Straight chain alkylene, C ₂ -C ₅ Straight chain alkylene, C ₂ -C ₄ Straight chain alkylene, C ₂ -C ₃ Straight chain alkylene, C ₃ -C ₅ Straight chain alkylene, C ₃ -C ₄ Straight chain alkylene or C ₃ Linear alkylene groups, and the like.

In some alternative embodiments of the present disclosure, each L ₁ Each independently selected from

In some embodiments of the present disclosure, each L ₁ Are all selected from

In some alternative embodiments of the present disclosure, each L ₂ Each independently selected from substituted or unsubstituted C ₂ -C ₁₀ A linear alkylene group; for example C ₂ -C ₉ Straight chain alkylene, C ₂ -C ₈ Straight chain alkylene, C ₂ -C ₇ Straight chain alkylene, C ₂ -C ₆ Straight chain alkylene, C ₂ -C ₅ Straight chain alkylene, C ₂ -C ₄ Straight chain alkylene, C ₃ -C ₁₀ Straight chain alkylene, C ₃ -C ₉ Straight chain alkylene, C ₃ -C ₈ Straight chain alkylene, C ₃ -C ₇ Straight chain alkylene, C ₃ -C ₆ Straight chain alkylene, C ₃ -C ₅ Straight chain alkylene, C ₃ -C ₄ Straight chain alkylene, C ₄ -C ₁₀ Straight chain alkylene, C ₄ -C ₉ Straight chain alkylene, C ₄ -C ₈ Straight chain alkylene, C ₄ -C ₇ Straight chain alkylene, C ₄ -C ₆ Straight chain alkylene, C ₄ -C ₅ Straight chain alkylene or C ₄ Straight chain alkylene C ₄ -C ₁₀ A linear alkylene group.

In some alternative embodiments of the present disclosure, each L ₂ Each independently selected from

In some embodiments of the present disclosure, each L ₂ Are all selected from

In some embodiments of the disclosure, each Y is selected from O;

In some alternative embodiments of the present disclosure, each R ₃ Each independently selected from H, substituted or unsubstituted C ₁ -C ₄ Alkanoyl (e.g. acetyl, propionyl, n-butyryl, isobutyryl, n-pentanoyl, isopentanoyl, 2-methylbutanoyl, pivaloyl/pivaloyl) or substituted or unsubstituted C ₅ -C ₇ Aryl acyl (e.g., benzoyl); r is R ₃ Each of which is independently selected from halogen (e.g., F).

In some alternative embodiments of the present disclosure, each R ₃ Each independently selected from H,

In some alternative embodiments of the present disclosure, each R ₃ Each independently selected from H or

In some embodiments of the disclosure, each R ₃ Are all selected from

In some embodiments of the disclosure, each R ₃ Are all selected from H.

In some alternative embodiments of the present disclosure, p and q are each independently selected from 0 or 1; for example, p=0 and q=0, or p=0 and q=1, or p=1 and q=0, or p=1 and q=1.

In some embodiments of the present disclosure, p=1 and q=1.

In the disclosure, the hydroxyl protecting group may be various hydroxyl protecting groups, as long as the hydroxyl group can be protected, and the specific type is not limited. In some alternative embodiments, the hydroxyl protecting group is stable under basic conditions, but can be removed under acidic conditions. In some alternative embodiments of the present disclosure, hydroxyl protecting groups that may be used in the present disclosure include, but are not limited to, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and 9- (p-methoxyphenyl) xanthin-9-yl (Mox), trityl (Tr-yl), 4-methoxytrityl (MMTr-yl), 4 '-dimethoxytrityl (DMTr-yl) and 4,4',4 "-trimethoxytrityl (TMTr-yl).

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

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

In some alternative embodiments of the present disclosure, the pharmaceutically active molecule is selected from a small molecule drug, an antibody, or an oligonucleotide.

In some alternative embodiments of the present disclosure, the oligonucleotide is selected from the group consisting of a single stranded oligonucleotide and a double stranded oligonucleotide.

In some alternative embodiments of the present disclosure, the oligonucleotide is selected from single stranded oligonucleotides.

In some alternative embodiments of the present disclosure, the pharmaceutically active molecule is selected from ASO.

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

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

In some alternative embodiments of the present disclosure, the solid support is selected from resins comprising hydroxyl and/or amino functional groups (e.g., polystyrene, abbreviated PS) or glass spheres (Controlled Pore Glass, abbreviated CPG) comprising controlled pore sizes.

In some embodiments of the disclosure, R _2a Selected from the group consisting of

In some embodiments of the disclosure, R _2b Selected from the group consisting ofSelected from resin or controllable pore size glass spheres.

In some embodiments of the present disclosure,selected from->

In some alternative embodiments of the present disclosure,selected from->

In some alternative embodiments of the present disclosure, the GalNAc derivative has a structure as shown in formula (II):

in some alternative embodiments of the present disclosure, the GalNAc derivative is selected from any one of the following compounds:

in a second aspect of the present disclosure, the present disclosure provides a conjugate as shown in formula (III):

wherein Nu represents an oligonucleotide;

k is selected from 1, 2, 3 or 4;

q is selected from the group consisting ofA、n、m、L ₁ 、L ₂ 、Y、p、q、R _1a As defined above.

In some alternative embodiments of the present disclosure, Q is selected from the group consisting of

In some alternative embodiments of the present disclosure, the oligonucleotide is selected from a single-stranded oligonucleotide or a double-stranded oligonucleotide.

In some alternative embodiments of the present disclosure, the single stranded oligonucleotide is selected from the group consisting of an antisense oligonucleotide, a nucleic acid aptamer, a ribozyme, a deoxyribozyme, a circular RNA, a sense strand of an siRNA, or an antisense strand of an siRNA.

In some alternative embodiments of the present disclosure, the double-stranded oligonucleotide is selected from the group consisting of a small interfering RNA, a double-stranded RNA, a microrna, a small guide RNA, a small activating RNA, or a short hairpin RNA.

In some embodiments of the disclosure, the oligonucleotide is selected from siRNA.

In some embodiments of the disclosure, k is selected from 1 or 2.

In some embodiments of the present disclosure, k is selected from 1.

In some embodiments of the disclosure, k is selected from 1, the oligonucleotide is selected from an siRNA, and one of the Q is conjugated to the 3' end of the sense strand of the siRNA.

In some embodiments of the disclosure, k is selected from 2, the oligonucleotide is selected from an siRNA, and two of the Q are conjugated to the 3 'end of the sense strand and the 5' end of the sense strand, respectively, of the siRNA.

In some alternative embodiments of the present disclosure, the conjugate is selected from any one of the following compounds:

in a third aspect of the present disclosure, the present disclosure provides a composition comprising a conjugate of the second aspect.

In some alternative embodiments of the present disclosure, the composition further comprises optionally one or more pharmaceutically acceptable carriers.

In a fourth aspect of the present disclosure, the present disclosure provides the use of any of the following for the manufacture of a medicament for the prevention and/or treatment of a disease:

(I) The compound of the first aspect; and/or

(II) a conjugate according to the second aspect; and/or

The composition of the third aspect (III).

In some alternative embodiments of the present disclosure, the disease is selected from a pathological condition or disease caused by abnormal expression of a target gene in liver cells. Illustratively, the target genes include, but are not limited to, at least one of Apoa, apoB, apoC, ANGPTL3, PCSK9, SOD1, FVII, p53, CC3, AGT, CFB, USP20, ASGR1, FTO, INHBE, HBV, and HCV.

In some specific embodiments of the disclosure, the target gene is selected from SOD1, ANGPTL3, or CC3.

In a fifth aspect of the disclosure, the disclosure provides a method of reducing expression or activity of a target gene, the method comprising contacting a liver cell with any of:

(I) The conjugate of the second aspect; and/or

(II) the composition of the third aspect.

In some alternative embodiments of the present disclosure, the target gene includes, but is not limited to, at least one of Apoa, apoB, apoC, ANGPTL3, PCSK9, SOD1, FVII, p53, CC3, AGT, CFB, USP, ASGR1, FTO, INHBE, HBV, and HCV.

Additional aspects and advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure.

Drawings

FIG. 1 is a graph showing the relative expression levels of target genes of interest in mice following administration of siRNA conjugates described in example 2.1;

FIG. 2 is a graph showing the relative expression levels of target genes of interest in mice following administration of siRNA conjugates described in example 2.2;

FIG. 3 is a graph showing the relative expression levels of target genes of interest in mice following administration of siRNA conjugates described in example 2.3;

FIG. 4 is a graph showing the relative expression levels of target genes of interest in mice following administration of siRNA conjugates described in example 2.4;

FIG. 5 is a graph showing the relative expression levels of target genes of interest in mice following administration of siRNA conjugates described in example 2.5.

Detailed Description

The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

Interpretation of the terms

In the context of the present disclosure, the term "alkyl" refers to a compound of the formulaThe alkanyl radical of (2) may be a straight-chain alkyl radical or a branched-chain alkyl radical. Wherein the term "C ₁ -C ₂ Alkyl "refers to an alkanyl radical having 1 to 2 carbon atoms, such as methyl or ethyl.

In the context of the present disclosure, the term "alkylene" refers to a compound of the general formulaThe chain alkylene group of (a) may be a straight chain alkylene group or a branched chain alkylene group. Wherein the term "C ₂ -C ₁₀ Alkylene "refers to an alkylene chain having 1 to 10 carbon atoms.

In the context of the present disclosure, the term "aroyl" has the structural formulaWherein Ar refers to an aryl group, ar refers in organic chemistry to any functional group or substituent derived from a simple aromatic ring. The simplest aryl group is Phenyl (Phenyl), which is derived from benzene.

In the context of the present disclosure, the term "alkoxy" has the formula Wherein "C ₁ -C ₂ Alkoxy "may be methoxy or ethoxy.

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

In the context of the present disclosure, a "pharmaceutically acceptable acid addition salt" refers to a salt with an inorganic or organic acid that is capable of retaining the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include, but are not limited to, formate, acetate, 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, naphthalenedisulfonate, and the like. These salts can be prepared by methods known in the art.

In the context of the present disclosure, a "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that is capable of maintaining the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts, preferably sodium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary 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 and the like. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

In the context of the present disclosure, an "oligonucleotide" is a deoxyribonucleic acid (deoxyribonucleic acid, DNA) or ribonucleic acid (RNA), typically consisting of 10-50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes such as ribonucleic acid interference, ribonuclease-mediated target degradation, splice regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

TerminologyRepresenting the site of attachment of the group by covalent bonds.

The present disclosure describesIn the structural formula of the compound, bond "-represents an unspecified configuration. If chiral isomerism exists in the chemical structure, the bond may beOr at the same time contain->And->Two configurations. Although all of the above structural formulae are drawn as certain isomeric forms for simplicity, the present disclosure may include all isomers, for example: tautomers, rotamers, geometric isomers, diastereomers, racemates and enantiomers.

In the structural formula of the compound disclosed in the disclosure, a bondIndicating an unspecified configuration. If cis-trans isomerism exists in the chemical structure, bond +.>The configuration of (a) may be E-type, Z-type, or both E and Z-type.

The following definitions as used herein should be applied unless otherwise indicated. For the purposes of this disclosure, chemical elements are consistent with CAS version of the periodic Table of the elements, and handbook of chemistry and physics, 75 th edition, 1994. In addition, general principles of organic chemistry may be referenced to the descriptions in "Organic Chemistry", thomas Sorrell, university Science Books, sausalato: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. The articles "a," "an," and "the" are intended to include "at least one" or "one or more" unless the context clearly dictates otherwise or otherwise. Thus, as used herein, these articles refer to one or to more than one (i.e., to at least one) object. For example, "a component" refers to one or more components, i.e., more than one component is contemplated as being employed or used in embodiments of the described embodiments.

The term "comprising" is an open-ended expression, i.e., including what is indicated in the disclosure, but not excluding other aspects.

"stereoisomers" refer to compounds having the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.

"chiral" is a molecule that has properties that do not overlap with its mirror image; and "achiral" refers to a molecule that may overlap with its mirror image.

"enantiomer" refers to two isomers of a compound that do not overlap but are in mirror image relationship to each other.

"diastereoisomers" refers to stereoisomers which have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. The diastereomeric mixture may be separated by high resolution analytical procedures such as electrophoresis and chromatography, e.g., HPLC.

Stereochemical definitions and rules as used in the present disclosure generally follow s.p. 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.

As described in the present disclosure, 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 specific examples within the examples, subclasses, and classes of compounds encompassed by the present disclosure.

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

The term "unsubstituted" means that the specified group does not carry a substituent.

The term "optionally substituted with … …" may be used interchangeably with the term "unsubstituted or substituted with … …," i.e., the structure is unsubstituted or substituted with one or more substituents described in this disclosure. Substituents described in this disclosure 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.

In addition, unless explicitly indicated otherwise, the descriptions used in this disclosure of the manner in which each … is independently "and" … is independently "and" … is independently "are to be construed broadly as meaning that particular items expressed between the same symbols in different groups do not affect each other, or that particular items expressed between the same symbols in the same groups do not affect each other. Taking R3 as an example, the specific options of R3 between the structural formula "C1-C50 alkylene optionally substituted with R3" and the structural formula "C (O) -NH-C1-50 alkylene optionally substituted with R3" are not affected by each other.

The term "small interfering RNA (Small interfering RNA; siRNA)" is a type of double-stranded RNA comprising a sense strand and an antisense strand, each strand being 17 to 30 nucleotides in length. siRNA mediates RNA transcript targeted cleavage of the RISC pathway by forming silencing complexes (RNA-induced silencing complex, RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting translation of mRNA into amino acids and conversion to proteins.

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" follower strand) "means that it contains an iRNA strand that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, e.g., the antisense strand is complete complementarity or at least partial complementarity to a target sequence. In the case of partial complementarity, the mismatch may be present within the interior or terminal region of the molecule, wherein the most tolerated mismatch is present within the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5 '-and/or 3' -end of the iRNA.

It is noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.

In the context of the present disclosure, an "oligonucleotide" is a deoxyribonucleic acid (deoxyribonucleic acid, DNA) or ribonucleic acid (RNA), typically consisting of 10 to 50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes such as ribonucleic acid interference, ribonuclease-mediated target degradation, splice regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

In the context of the present disclosure, an "antisense oligonucleotide (antisense oligonucleotides, ASO)" is a single stranded oligonucleotide molecule, typically consisting of 10 to 50 nucleotides. ASO enters cells and then is combined with complementary target mRNA through the base complementary pairing principle under the action of ribonuclease H1, so that the expression of target genes is inhibited.

In the context of the present disclosure, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring, coloring agent, or combination thereof, all of which are known to those of skill in the art (as described in Remington's Pharmaceutical Sciences,18th Ed.Mack Printing Company,1990,pp.1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

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

In the context of the present disclosure, "subject" refers to any animal, such as a mammal or a pouched animal. 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 variety of poultry.

In the context of the present disclosure, "treatment," "alleviation," or "improvement" may be used interchangeably herein. These terms refer to methods of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. Here, therapeutic benefit is obtained by eradicating or ameliorating one or more of the 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.

In the context of the present disclosure, "prevent" and "prevent" are used interchangeably. These terms refer to methods of achieving a beneficial or desired result, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," the conjugate, RNAi agent, or composition may be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though a diagnosis of the disease may not have been made.

In the context of the present disclosure, the ratio of reagents described in the various embodiments of the present disclosure are calculated as volume ratio (v/v) unless otherwise indicated.

Unless otherwise indicated, the starting materials and reagents used in the preparation of the compounds provided by the present disclosure were purchased from Beijing coupling technologies Inc. Details of some of the reagents used in the present disclosure are shown in table 1.

TABLE 1 details of reagents in part

The reagent consumables (Table 2) and instrumentation (Table 3) used in the present disclosure were all derived from commercial products from the following manufacturers, unless otherwise specified.

Table 2 Primary reagent consumable

Name of the name	Manufacturer' s
		1×PBS	Midkine technology Co.Ltd
Van-Vibrio RNA extraction kit	Zhejiang Hanwei science and technology Limited liability company
		Reverse Transcription System	Promega Corporation
SYBR Select Master Mix	ABI
		RNALater	Thermo Fisher Scientific

TABLE 3 Main instrumentation

Name of the name	Manufacturing factories
		Full-automatic nucleic acid extractor	Zhejiang Hanwei science and technology Limited liability company
High-speed refrigerated centrifuge	Eppendorf
		NANODROP OneC	Thermo Fisher Scientific
Gradient PCR amplification instrument	Eppendorf
		Fluorescent quantitative PCR instrument	ABI StepOne Plus
Gel imaging instrument	Shanghai Tianneng Life Science Co., Ltd.
		Electrophoresis apparatus	Beijing Liuyi Instrument Factory
Tissuelyser II type full-automatic tissue homogenate instrument	SHANGHAI JINGXIN INDUSTRIAL DEVELOPMENT Co.,Ltd.

Preparation example 1: synthesis of Compound CR01004Z

In this preparation, the synthetic route for compound CR01004Z is shown below:

(1-1) Synthesis of Compound CR01004-2

Compound CR01004-1 (400 mg,1.11mmol,1.0 eq), (3-oxopropyl) carbamic acid tert-butyl ester (578 mg,3.33mmol,3 eq) and triethylamine (230 mg,2.22mmol,2.0 eq) were dissolved in 10ml dichloromethane and stirred at 25℃for 2 hours, sodium triacetoxyborohydride (1.42 g,6.66mmol,6 eq) was added and reacted at 25℃for 12 hours. After completion of the reaction, 30ml of methylene chloride was added to the reaction system, washed once with 20ml of saturated sodium hydrogencarbonate solution, washed once with 20ml of distilled water, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound CR01004-2 (600 mg, yield 84.5%) as a pale yellow solid, which was purified by column chromatography as a normal phase (eluent: methanol/methylene chloride=5/95, v/v). MS ESI (m/z) =637.2 [ m+h ] ⁺ .

(1-2) Synthesis of Compound CR01004-3

Compound CR01004-2 (600 mg,0.94mmol,1.0 eq) was dissolved in 10ml of a 4mol/L dioxane solution of hydrogen chloride, reacted at 25℃for 2 hours, and after the reaction was completed, the reaction solution was directly concentrated to give compound CR01004-3 (7.6 g) as a pale yellow solid, which was used in the next step without purification. MS ESI (m/z) =337.2 [ m+h ]] ⁺ .

(1-3) Synthesis of Compound CR01004-4

Compound CR01004-3 (500 mg,1.13mmol,1.0 eq) was dissolved in 15ml of N, N-dimethylformamide, and compound Gal-5 (1.5 g,3.39mmol,3.0 eq), HATU (2.15 g,5.65mmol,5.0 eq) and DIEA (1.75 g, 13) were added56mmol,12.0 eq) and stirred for 5 hours at 25 ℃. After the completion of the reaction, the reaction mixture was directly subjected to column chromatography reverse phase purification (C18 column, elution gradient: acetonitrile/water=48/52 to acetonitrile/water=56/44 can, v/v) to give compound CR01004-4 (800 mg, yield 44.4%) as a white solid. MS ESI (m/z) =1657.2 [ m+h] ⁺ .

(1-4) Synthesis of Compound CR01004-5

Compound CR01004-4 (800 mg,0.49mmol,1.0 eq) was dissolved in 15ml of methanol, wet palladium on carbon (80 mg, loading 10 mass%) was added, hydrogen was replaced three times, and after replacement, the mixture was stirred at 25℃for 3 hours. After completion of the reaction, the reaction solution was filtered, and the filtrate was concentrated to give compound CR01004-5 (700 mg, yield 92.6%) as a white solid, which was used in the next step without purification. MS ESI (m/z) =1534.4 [ m+h ] ⁺ .

(1-5) Synthesis of Compound CR01004-6

Compound CR01004-5 (770 mg,0.50mmol,1.0 eq) was dissolved in 15ml DMF, and compound N-SH (278 mg,0.55mmol,1.1 eq), HATU (284 mg,0.75mmol,1.5 eq), DIEA (162 mg,1.25mmol,2.5 eq) was added and stirred at 25℃for 5 hours. After the reaction is finished, the system directly performs column chromatography reverse phase purification (C18 chromatographic column, 58% -70% ACN in H) ₂ O), the fraction was concentrated to give compound CR01004-6 (850 mg, yield 83.3%) as a white solid. MS ESI (m/z) =2021.2 [ M+H ]] ⁺ .

(1-6) Synthesis of Compound CR01004Z

Compound CR01004-6 (160 mg,0.1mmol,1.0 eq.) in methylene chloride (3.2 ml), succinic anhydride (18 mg,0.18mmol,1.8 eq.), 4-dimethylaminopyridine (1.2 mg,0.01mmol,0.01 eq.), triethylamine (19.7 mg,0.2mmol,2.0 eq.) are mixed and stirred at 25℃for 12 hours. After the completion of the reaction, nitrogen was substituted 3 times, and stirred at 25℃for 16 hours, followed by column chromatography reversed phase purification (C18 column, acetonitrile/water=72/28, v/v) to give an intermediate (100 mg).

The above intermediate (100 mg,0.058mmol,1.0 eq.) amino CPG (1.44 g,0.115mmol,80 umol/g) and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (32.67 mg,0.086mmol,1.5 eq.) N, N-diisopropylethylamine (14.8 mg,0.115mmol,2.0 eq.) were mixed together and the shaking table was shaking table for 16 hours. After the reaction was completed, the reaction solution was filtered, and the cake was washed once with 10ml of acetonitrile and dried under vacuum. The dried cake, 4-dimethylaminopyridine (2 mg,0.017 mmol), cap1 (15 ml) and Cap2 (1.5 ml) were mixed and shaken for 6 hours. After the reaction was completed, the reaction solution was filtered, and the cake was washed once with 10ml of acetonitrile and then dried under vacuum to obtain compound CR01004Z (1.367 g, loading 20-30 umol/g).

Cap1 and Cap2 are capping reagents, cap1 is a pyridine/acetonitrile mixed solution of 20 volume percent of N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; cap2 is a 20% by volume acetic anhydride in acetonitrile.

Preparation example 2: synthesis of Compound CR01005Z

In this preparation, the synthetic route for compound CR01005Z is shown below:

(2-1) Synthesis of Compound CR01005-2

Compound CR01005-1 (19.5 g,53.4mmol,1.0 eq) is dissolved in 50ml super-dry DMF and K is added ₂ CO ₃ (18.56 g,134.4mmol,2.5 eq) benzyl bromide (10.95 g,64.1mmol,1.2 eq) and stirred at 25℃for 12 hours. After completion of the reaction, 200ml of water was added to the reaction mixture, which was extracted 3 times with ethyl acetate (200 ml each time), and the organic phases were combined, and the organic phase was washed 10 times with saturated aqueous sodium chloride solution (50 ml each time), dried over anhydrous sodium sulfate, filtered, and concentrated to give compound CR01005-2 (22 g, yield 91.7%) as a white solid. MS ESI (m/z) =457 [ M+H ]] ⁺ .

(2-2) Synthesis of Compound CR01005-3

Compound CR01005-2 (22 g,48.1mmol,1.0 eq) was dissolved in 90mL dichloromethane DCM and a solution of 1, 4-dioxane (45 mL,4 mol/L) of hydrogen chloride was added and stirred at 25℃for 2 hours. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, slurried with methyl t-butyl ether, filtered, and dried to give Compound CR01005-3 (24 g, yield 100%) as a white solid )。MS ESI(m/z)＝357.2[M+H] ⁺ .

(2-3) Synthesis of Compound CR01005-5

Compound CR01005-3 (5.2 g,13.28mmol,1.1 eq) and compound CR01005-4 (2.5 g,12.07mmol,1.0 eq) are dissolved in 70ml tetrahydrofuran, a solution of triethylamine (2.8 g,27.76mmol,2.3 eq) is added and stirred at 25℃for 2 hours; naBH (OAc) is added ₃ (5.1 g,24.14mmol,2.0 eq) under nitrogen at 25℃for 12 hours. After completion of the reaction, 30mL of water was added to the reaction mixture, extracted three times with ethyl acetate (50 mL each time), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give the compound CR01005-5 (5 g, yield 69.0%) as a white oil. MS ESI (m/z) =548.2 [ m+h] ⁺ .

(2-4) Synthesis of Compound CR01005-6

Compound CR01005-5 (2.5 g,4.56mmol,1.0 eq), compound Gal-5 (4.07 g,9.12mmol,2.0 eq), HATU (3.5 g,9.12mmol,2.0 eq) were dissolved in 25ml of N, N-dimethylformamide and DIEA (1.2 g,9.12mmol,2.0 eq) was added under ice-bath and stirred for 12 hours at 25 ℃. After completion of the reaction, 30ml of water was added to the reaction mixture, which was extracted three times with ethyl acetate (50 ml each time), and the organic phases were combined, dried over anhydrous sodium sulfate, suction-filtered, concentrated, and purified by column chromatography in reverse phase (C18 column, eluent: acetonitrile/water=72/28, v/v) to give compound CR01005-6 (2.4 g, yield 53.8%) as a pale yellow solid. MS ESI (m/z) =990 [ M+Na ] ] ⁺ .

(2-5) Synthesis of Compound CR01005-7

Compound CR01005-6 (2.7 g,2.76mmol,1.0 eq) was dissolved in 20ml methanol, wet palladium on carbon (270 mg) and 0.1ml trifluoroacetic acid were added, replaced three times with hydrogen, and stirred at 25℃for 12 hours. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound CR01005-7 (1.7 g, yield 100%) as a white solid. MS ESI (m/z) =619 [ M+H ]] ⁺ .

(2-6) Synthesis of Compound CR01005-8

Compounds Gal-5 (2.65 g,5.9mmol,2.5 eq), DIEA (5(1.8 g,14.16mmol,6.0 eq) and HATU (2.1 g,5.43mmol,2.3 eq) were dissolved in 25ml of N, N-dimethylformamide and compound CR01005-7 (2 g,2.36mmol,1.0 eq) free in DIEA was added thereto and stirred at 25℃for 12 hours. After the completion of the reaction, the reaction mixture was directly purified by column chromatography in reverse phase (C18 column, eluent: acetonitrile water=23/77, v/v) to give compound CR01005-8 (2.4 g, yield 38%) as a white solid. MS ESI (m/z) =1477 [ M+H ]] ⁺ .

(2-6) Synthesis of Compound CR01005-9

Compound CR01005-8 (1.5 g,1.0mmol,1.0 eq), compound N-SH (768 mg,1.5mmol,1.5 eq), DIEA (524 mg,4.0mmol,4.0 eq) were dissolved in 20ml of N, N-dimethylformamide, HATU (580 mg,1.5mmol,1.5 eq) was added and stirred at 25℃for 12 hours. After the completion of the reaction, the reaction mixture was directly purified by column chromatography in reverse phase (C18 column, eluent: acetonitrile water=54/46, v/v) to give compound CR01005-9 (1.8 g, yield 90%) as a white solid. MS ESI (m/z) =1963 [ m+h ] ] ⁺ .

(2-7) Synthesis of Compound CR01005-10

Compound CR01005-9 (200 mg,0.1mmol,1.0 eq) was dissolved in 10ml of dichloromethane, succinic anhydride (12 mg,0.11mmol,1.1 eq) and triethylamine (21 mg,0.2mmol,2.0 eq) were added and stirred at 25℃for 12 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give compound CR01005-10 (200 mg, yield 95%) as a white solid, which was used in the next step without purification. MS ESI (m/z) =2065 [ m+h ]] ⁺ .

(2-8) Synthesis of Compound CR01005Z

(2-1) Synthesis of Compound CR01005-2

Compound CR01005-1 (19.5 g,53.4mmol,1.0 eq) is dissolved in 50ml super-dry DMF and K is added ₂ CO ₃ (18.56 g,134.4mmol,2.5 eq) benzyl bromide (10.95 g,64.1mmol,1.2 eq) and stirred at 25℃for 12 hours. After completion of the reaction, 200ml of water was added to the reaction mixture, followed by extraction with ethyl acetate 3 times (200 ml each time), the organic phase was combined, the organic phase was washed with saturated aqueous sodium chloride solution 10 times (50 ml each time), dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid-like productCompound CR01005-2 (22 g, 91.7% yield). MS ESI (m/z) =457 [ M+H ]] ⁺ .

(2-2) Synthesis of Compound CR01005-3

Compound CR01005-2 (22 g,48.1mmol,1.0 eq) was dissolved in 90mL dichloromethane DCM and a solution of 1, 4-dioxane (45 mL,4 mol/L) of hydrogen chloride was added and stirred at 25℃for 2 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, slurried with methyl t-butyl ether, filtered, and dried to give compound CR01005-3 (24 g, yield 100%) as a white solid. MS ESI (m/z) =357.2 [ m+h ] ] ⁺ .

(2-3) Synthesis of Compound CR01005-5

(2-4) Synthesis of Compound CR01005-6

(2-5) Synthesis of Compound CR01005-7

(2-6) Synthesis of Compound CR01005-8

Compound Gal-5 (2.65 g,5.9mmol,2.5 eq), DIEA (5 (1.8 g,14.16mmol,6.0 eq) and HATU (2.1 g,5.43mmol,2.3 eq) were dissolved in 25ml of N, N-dimethylformamide, then compound CR01005-7 (2 g,2.36mmol,1.0 eq) free of DIEA was added thereto, and stirred at 25℃for 12 hours after completion of the reaction, the reaction mixture was directly subjected to column chromatography reverse phase purification (C18 column, eluent: acetonitrile water=23/77, v/v) to give compound CR01005-8 (2.4 g, yield 38%) as a white solid, MS ESI (m/z) =1477 [ M+H ]] ⁺ .

(2-6) Synthesis of Compound CR01005-9

(2-7) Synthesis of Compound CR01005-10

(2-8) Synthesis of Compound CR01005Z

Compounds CR01005-10 (100 mg,0.048mmol,1.0 eq), HBTU (27 mg,0.073mmol,1.5 eq), DIEA (12 mg,0.096mmol,2.0 eq) were dissolved in 10ml acetonitrile, stirred for 5 min, amino CPG (80 umol/g,1.17g,2 eq) was added and reacted on a shaker at 25℃for 18 hours, filtered, the filter cake washed twice with 10ml dichloromethane (10 ml each time), twice with acetonitrile (10 ml each time) and dried in vacuo. The dried cake, 4-dimethylaminopyridine (2 mg,0.017 mmol), cap1 (8 ml) and Cap2 (8 ml) were mixed and reacted on a shaker at 25℃for 5 hours, filtered, and the cake was washed 3 times with acetonitrile (10 ml each time) and dried in vacuo to give the compound CR01005Z (1.3 g, loading 20 to 30 umol/g).

Preparation example 3: synthesis of reference Compound CR01007Z

In this preparation, the synthetic route for the reference compound CR01005Z is shown below:

(3-1) Synthesis of Compound CR01007-3

Compound CR01007-1 (5.0 g,15.5mmol,1.0 eq), compound CR01007-2 (2.8 g,17.05mmol,1.1 eq), HATU (8.8 g,23.25mmol,1.5 eq), DIEA (8.0 g,62.0mmol,4.0 eq) were dissolved in 50ml dichloromethane and stirred at 25℃for 4 hours. After completion of the reaction, 30ml of methylene chloride was added to the reaction mixture, which was washed once with 20ml of a saturated sodium hydrogencarbonate solution and once with 20ml of distilled water, and an organic phase was separated, dried over anhydrous sodium sulfate, suction-filtered, concentrated, and purified by column chromatography as a normal phase (eluent: ethyl acetate/petroleum ether=30/70, v/v) to give compound CR01007-3 (5.0 g, yield 74.0%) as a yellow solid. MS ESI (m/z) =437.2 [ m+h ]] ⁺ .

(3-2) Synthesis of Compound CR01007-4

Compound CR01007-3 (5.0 g,11.47mmol,1.0 eq) was dissolved in 50ml of methanol, and 0.5g of wet palladium on carbon was added thereto, replaced with hydrogen three times, and stirred at 25℃for 3 hours after the replacement. After the completion of the reaction, the reaction mixture was suction-filtered, and the filtrate was concentrated under reduced pressure to give Compound CR01007-4 (3.0 g, yield 88.2%) as a white solid, which was used as such without purification for the next reaction And (3) step (c). MS ESI (m/z) =303.4 [ m+h ]] ⁺ .

(3-3) Synthesis of Compound CR01007-5

Compound CR01007-4 (3.7 g,12.2mmol,1.0 eq), compound CR01007-1 (4.34 g,13.42mmol,1.1 eq), HATU (6.96 g,18.3mmol,1.5 eq), DIEA (3.94 g,30.5mmol,2.5 eq) were dissolved in 50ml dichloromethane and stirred at 25℃for 4 hours. After completion of the reaction, 30ml of methylene chloride was added to the reaction mixture, which was washed once with 20ml of a saturated sodium hydrogencarbonate solution and once with 20ml of distilled water, and an organic phase was separated, dried over anhydrous sodium sulfate, suction-filtered, concentrated, and purified by column chromatography as a normal phase (eluent: ethyl acetate/petroleum ether=50/50, v/v) to give compound CR01007-5 (6.0 g, yield 81.1%) as a yellow solid. MS ESI (m/z) =608.2 [ m+h] ⁺ .

(3-4) Synthesis of Compound CR01007-6

Compound CR01007-5 (6.0 g,9.88mmol,1.0 eq) was dissolved in 20ml of methylene chloride, 50ml of formic acid was added at 25℃and after the addition of formic acid, the reaction system was warmed to 40℃and reacted at 40℃for 3 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give compound CR01007-6 (4.0 g) as a yellow solid, which was used in the next step without purification. MS ESI (m/z) =440.2 [ m+h] ⁺ .

(3-5) Synthesis of Compound CR01007-7

Compound CR01007-6 (1.0 g,2.27mmol,1.0 eq), compound GAL-6 (3.2 g,7.49mmol,3.3 eq), HATU (3.45 g,9.08mmol,4.0 eq), DIEA (2.64 g,20.43mmol,9.0 eq) were dissolved in 30ml DMF and stirred at 25℃for 4 hours. After completion of the reaction, 40ml of ethyl acetate was added to the reaction system, washed four times with saturated sodium chloride (20 ml each time), washed once with 20ml of distilled water, and the organic phase was separated, dried over anhydrous sodium sulfate, suction-filtered, concentrated, and purified by column chromatography as a normal phase (eluent: methanol/dichloromethane=5/95, v/v) to give compound CR01007-7 (2.0 g, yield 52.6%) as a white solid. MS ESI (m/z) =1698.2 [ m+h] ⁺ .

(3-6) Synthesis of Compound CR01007-8

Compound CR01007-7 (2.0 g,1.19mmol,1.0 eq) is dissolved in 15ml methanol200mg of wet palladium on carbon was added, the mixture was replaced with hydrogen three times, and the mixture was stirred at 25℃for 6 hours after the replacement. After the completion of the reaction, the reaction mixture was filtered under suction, and the filtrate was concentrated under reduced pressure to give compound CR01007-8 (1.5 g, yield 82.6%) as a white solid, which was used in the next reaction without purification. MS ESI (m/z) =1564.4 [ m+h] ⁺ .

(3-7) Synthesis of Compound CR01007-9

Compound CR01007-8 (2.0 g,1.29mmol,1.0 eq) was dissolved in 20ml of dichloromethane, and triethylamine (0.26 g,2.58mmol,2.0 eq) and succinic anhydride (0.14 g,1.42mmol,1.1 eq) were added and stirred at 25℃for 5 hours. After the completion of the reaction, the reaction mixture was directly subjected to column chromatography reverse phase purification (C18 column, elution gradient: acetonitrile/water=48/52 to acetonitrile/water=56/44, v/v) to give compound CR01007-9 (1.5 g, yield 70.4%) as a white solid. MS ESI (m/z) =1650.2 [ m+h ] ⁺ .

(3-8) Synthesis of Compound CR01007-10

Compound CR01007-9 (1.6 g,0.97mmol,1.0 eq) was dissolved in 15ml DMF, and compound N-SH (726 mg,1.44mmol,1.5 eq), HATU (547 mg,1.44mmol,1.5 eq), DIEA (375 mg,2.91mmol,3.0 eq) was added and stirred at 25℃for 5 hours. After the completion of the reaction, the reaction mixture was directly subjected to column chromatography reverse phase purification (C18 column, elution gradient: acetonitrile/water=55/45 to acetonitrile/water=70/30, v/v) to give compound CR01007-10 (1.6 g, yield 76.3%) as a white solid. MS ESI (m/z) =2064.2 [ m+h] ⁺ .

(3-8) Synthesis of Compound CR01007Z

Compound CR01007-10 (210 mg,0.1mmol,1.0 eq.) is dissolved in 3.0ml of dichloromethane, succinic anhydride (18 mg,0.18mmol,1.8 eq.) 4-dimethylaminopyridine (1.2 mg,0.01mmol,0.01 eq.) triethylamine (19.7 mg,0.2mmol,2.0 eq.) is added, the reaction system is stirred at 25℃for 12 hours, nitrogen is displaced 3 times, stirred at 25℃for 16 hours, and column chromatography is reversed phase purified (C18 column, acetonitrile/water=72/28, v/v) to give 100mg of intermediate.

The above intermediate (100 mg,0.045mmol,1.0 eq.) amino CPG (1.44 g,0.115mmol,80 umol/g), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (25.67 mg,0.068mmol,1.5 eq.) N, N-diisopropylethylamine (11.6 mg,0.09mmol,2.0 eq.) were mixed, shaking bed for 16 hours, filtered, the filter cake washed twice with acetonitrile (10 ml each) and dried in vacuo. The dried cake, 4-dimethylaminopyridine (2 mg,0.017 mmol), cap1 (15 ml), cap2 (1.5 ml) were mixed, shaken for 6 hours, filtered, and the cake washed twice with acetonitrile (10 ml each time) and dried in vacuo to give compound CR01007Z (1.31 g, load 20-30 umol/g).

Preparation example 4: preparation of siRNA conjugates

(4-1) Synthesis of Sense Strand (SS)

By phosphoramidite nucleic acid solid phase synthesis method, the above-mentioned compound (CR 01004Z, CR01005Z, CR 01007Z) attached to a solid phase carrier is used to initiate a cycle, and nucleoside monomers are attached one by one in the 3'-5' direction according to the nucleotide sequence. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The synthesis conditions were given as follows:

nucleoside monomers were formulated as an acetonitrile solution of nucleoside monomers at a concentration of 0.1M.

The deprotection conditions are the same for each step. Conditions for deprotection reaction: the temperature is 25 ℃, the reaction time is 70 seconds, the deprotection reagent is dichloromethane solution (3 vol%) of dichloroacetic acid, and the molar ratio of the dichloroacetic acid to the 4,4' -dimethoxytrityl protecting group on the solid carrier is 5:1.

The conditions for each coupling reaction were identical. The conditions of the coupling reaction are: the temperature is 25 ℃, the mole ratio of the nucleic acid sequence connected on the solid carrier to the nucleoside monomer is 1:10, the mole ratio of the nucleic acid sequence connected on the solid carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with the concentration of 0.5M, and the thioagent is an acetonitrile/pyridine mixed solution of hydrogenation Huang Yuansu with the concentration of 0.2mol/L (the volume ratio of acetonitrile to pyridine is 1:1).

The conditions for the capping reaction were the same for each step. The conditions of the capping reaction are: the temperature is 25 ℃; the reaction time was 2 minutes; the Cap reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, wherein Cap1 is a pyridine/acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume percent, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is an acetonitrile solution of acetic anhydride with a annual attack rate of 20 volume percent; the molar ratio of the N-methylimidazole in the Cap1 capping reagent to the acetic anhydride in the Cap2 capping reagent to the nucleic acid sequence connected to the solid carrier is 1:1:1.

The conditions for each oxidation reaction are the same. The oxidation reaction conditions were: the temperature is 25 ℃; the reaction time was 3 seconds; iodine water with the concentration of 0.05M of the oxidizing reagent, wherein the molar ratio of iodine to the nucleic acid sequence connected to the solid carrier in the coupling reaction is 30:1; the oxidation reaction was carried out in a water/pyridine mixed solvent (volume ratio of water to pyridine: 1:9). The conditions of the vulcanization reaction are as follows: the temperature is 25 ℃; the reaction time was 360 seconds; a pyridine solution with a concentration of 0.2M hydrogenation Huang Yuansu of the thio reagent, wherein the molar ratio of the thio reagent to the nucleic acid sequence connected to the solid carrier in the coupling reaction is 4:1; the thio-reaction was carried out in a water/pyridine mixed solvent (volume ratio of water to pyridine: 1:9).

After the last nucleoside monomer is connected, the nucleic acid sequence connected on the solid phase carrier is sequentially cut, deprotected, purified and desalted, and then freeze-dried to obtain the sense strand, wherein:

the cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence to which the solid phase carrier was attached was added to aqueous ammonia having a concentration of 25% by mass, the amount of aqueous ammonia was 0.5 ml/. Mu.mol, reacted at 55℃for 16 hours, the solvent was removed, and concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved with 0.4 ml/. Mu.mol of N-methylpyrrolidone, followed by the addition of 0.3 ml/. Mu.mol of triethylamine and 0.6 ml/. Mu.mol of triethylamine-tricofluoride, relative to the amount of single-stranded nucleic acid, and the 2' -O-TBDMS protection on ribose was removed.

Conditions for purification and desalination: purification of nucleic acids was accomplished by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent 1 is 20mM sodium phosphate (pH=8.1), solvent is water/acetonitrile mixed solution (volume ratio of water and acetonitrile is 9:1); eluent 2 is 1.5M sodium chloride, 20mM sodium phosphate (pH=8.1), solvent is water/acetonitrile mixed solution (volume ratio of water and acetonitrile is 9:1); the elution gradient is eluent 1, eluent 2= (100:0) - (50:50). Collecting and combining product eluents, desalting by using a reverse chromatography purification column, wherein the desalting conditions comprise desalting by using a sephadex column, eluting with deionized water, wherein the filler is sephadex G25.

And (3) detection: purity detection using ion exchange chromatography (IEX-HPLC); and (3) detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the measured value and the theoretical value of the molecular weight, wherein if the measured value and the theoretical value are consistent, the obtained compound is conjugated at the 3' end of the siRNA sense strand.

(4-2) Synthesis of Antisense Strand (AS)

Antisense strands were synthesized using a universal solid support. The conditions of deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting in the solid phase synthesis method of antisense strand are the same as those of step (4-1) for synthesizing sense strand.

And (3) detection: purity detection using ion exchange chromatography (IEX-HPLC); molecular weight detection is performed by using liquid chromatography-mass spectrometry (LC-MS), and the measured value and the theoretical value of the molecular weight are compared, and if the measured value and the theoretical value are consistent, the siRNA antisense strand is obtained.

(4-3) Synthesis of siRNA conjugates

The sense strand synthesized in step (4-1) and the antisense strand synthesized in step (4-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃, slowly cooled to room temperature and maintained at room temperature for 10 minutes, so that the sense strand and the antisense strand formed a double-stranded structure through hydrogen bonds, thereby obtaining siRNA conjugates having the sense strand and the antisense strand shown in table 4.

UmsUmsUmAmUfCfCfUmCmAmCmUmCmUmAmAm, as shown in SEQ ID NO 1;

UmsUfsUmGlumGlumGlumGlumGlumGlufAmAmAmAmAmAmsUmsGm, as shown in SEQ ID NO. 2;

CmsCmsAMAMAMmGmfCfAfCmAmAmAmAmAmAmAmAmAmCmUmAm, as shown in SEQ ID NO 3;

UmsAfsGmUmCfUmUmGmUmGmUmCmUfCmUmGmGmGmGmMmSmSmUmsum, as shown in SEQ ID NO. 4;

CmsAmsMGmAmGfAfCfAmAmGmAmAmCmCmCmCmAmUmCmUm, as shown in SEQ ID NO. 5;

AmsGfsAmmUmGfUmCmUmUmUmUmCmUfGmUmGmMmGmSam as shown in SEQ ID NO. 6.

Wherein, when the carrier conjugated to the 3' -end of the sense strand of the siRNA is CR01004, the structural formula of the siRNA conjugate is:

when the carrier conjugated to the 3' -end of the sense strand of the siRNA is CR01005, the siRNA conjugate has the structural formula:

when the carrier conjugated to the 3' -end of the sense strand of the siRNA is CR01007, the siRNA conjugate has the structural formula:

TABLE 4 sequence information for siRNA conjugates

Unless otherwise indicated, the base compositions and modifications described in the examples of the present disclosure are as follows: capital A, U, G, C, T indicates the base composition of the nucleotide, and lowercase m indicates that the nucleotide indicated by the preceding letter is a methoxy-modified nucleotide; the lower case letter f indicates that the nucleotide indicated by the preceding letter is a fluoro-modified nucleotide; lower case letter s indicates that phosphorothioate linkages are between the nucleotides indicated by the two letters before and after.

Unless otherwise indicated, the siRNA sequences used in the present disclosure were all delegated to su Bei Xin biotechnology limited; the PCR primer synthesis used in the present disclosure is completed by Beijing qingke biotechnology Co., ltd; experimental animals C57BL/6J mice used in the present disclosure were purchased from St Bei Fu (Beijing) Biotechnology Co., ltd.

TABLE 5 detection results of siRNA conjugates

From the 5 data, it can be seen that the Sense Strand (SS) and the Antisense Strand (AS) can be attached to the ligand better and with higher purity.

General experiment

Experimental example 1 in vivo toxicity experiment of siRNA conjugate

C57BL/6J mice were randomly divided into 3 groups (i.e., RZ899039, RZ899017, RZ899021, RZ897011, RZ897005, RZ897006, RZ802012, RZ 802013) of 2 mice each, each half of which were individually administered 300mg/kg of siRNA conjugate of mouse body weight (as siRNA) by subcutaneous injection, and the mice of each group were continuously observed for 14 days without death of the animals or clinical symptoms related to adverse drug reactions, and after the completion of the observation, the mice were subjected to gross dissection and no abnormalities were observed. Thus, the above results indicate that the siRNA conjugates of the present disclosure are safe and have low animal level toxicity.

Experimental example 2 method for evaluating inhibition activity of target gene in mouse

The 6-8 week old C57BL/6J mice were randomly grouped by body weight (females). The mice in each group were dosed in a single dose by abdominal subcutaneous injection based on body weight, and each siRNA conjugate was formulated with PBS solution as a corresponding concentration (calculated as siRNA) solution for dosing, with a dosing volume of 5ml/kg (calculated as body weight of the mice). The PBS control group was given 5ml/kg (based on the weight of the mice) of PBS solution (without drug conjugate). Administration when the day 1 (noted as D1) was recorded, 5 mice were sacrificed for each group at the preset time after administration.The sacrificed mice were subjected to general dissection and liver tissue of each sacrificed mouse was collected, and the liver tissue was cut into about 2mm ³ The pellet was stored with RNA Later.

Taking liver tissue samples at different time points from the RNA later, crushing the liver tissue samples in a tissue lyser II type full-automatic tissue homogenizer for 60s, and extracting the total RNA by using a full-automatic nucleic acid extractor (purchased from Zhejiang Han Wei technology Co., ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Han Wei technology Co., ltd.) according to standard operation steps of total RNA extraction.

Using the above 1. Mu.g of total RNA, a reverse transcription kit (Promega Corp., reverse Transcription System, A3500) was used and Oligo (dT) 15 reverse transcription primer was selected, and a 20. Mu.L reverse transcription system was prepared according to the method described in the specification of the reverse transcription kit to complete the reverse transcription reaction. After completion of the reaction, 80. Mu.L of RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. Then, a real-time fluorescent quantitative PCR kit (ABI, SYBR was used ^TM Select Master Mix, catalyst number:4472908 Detecting the expression level of mRNA of the target gene in liver tissue. In the real-time fluorescent quantitative PCR method, a primer for a target gene and a primer for an internal reference gene are used to detect the target gene and the internal reference gene, respectively. 20. Mu.L Real-time PCR reaction systems were prepared for each PCR detection well according to the method described in the Real-time fluorescent quantitative PCR kit, each reaction system containing 5. Mu.L of the cDNA solution obtained by the above-mentioned reverse transcription reaction and 10. Mu.L of SYBR ^TM Select Master Mix, 0.5. Mu.L of 10. Mu.M upstream primer, 0.5. Mu.L of 10. Mu.M downstream primer, 4. Mu.L RNase-Free H2O. Placing the prepared reaction system in a real-time fluorescence quantitative PCR instrument (ABI company, stepOneGlus) ^TM ) The three-step method was used to perform Real-time PCR amplification by pre-denaturing at 95℃for 10min, then denaturing at 95℃for 30s, annealing at 60℃for 30s, and extending at 72℃for 30s, and repeating the denaturation, annealing, and extension steps for 40 cycles. In the real-time fluorescence quantitative PCR method, the delta Ct method is adopted to perform relative quantitative calculation on the expression level and the inhibition rate of the target gene mRNA in each test group, and the calculation method is as follows:

Delta Ct (test group) =ct (test group target gene) -Ct (test group reference gene)

Delta Ct (control) =ct (control target gene) -Ct (control reference gene)

ΔΔct (test group) =Δct (test group) - Δct (control group average)

ΔΔct (control) =Δct (control) - Δct (control average)

Wherein, Δct (control group average) is the arithmetic average of Δct (control group) of each of 5 mice sacrificed at the same time point in the control group. Thus, each mouse of the test and control groups corresponds to one ΔΔct value.

And normalizing the mRNA expression level of the target gene of the test group by taking the control group as a reference, and defining the mRNA expression level of the target gene of the control group as 100%.

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

Test group target gene mRNA expression inhibition ratio= (1-test group target gene mRNA relative expression level) ×100%

Unless otherwise indicated, in vivo activity assay data are all expressed as X+ -SD, and the assay data are plotted and analyzed using GraphPad prism 8.0 software.

Example 2.1CR01005, evaluation of in vivo Activity of CR01007 vector conjugated superoxide dismutase 1 (Superoxide Dismutase, SOD 1) siRNA

In this example, the inhibitory activity of the 3' -end of the sense strand of the siRNA to the target gene SOD1 of the target gene in mice was evaluated by using the method for evaluating the inhibitory activity of the target gene in mice, wherein the sequence RZ899017 of the siRNA was conjugated to the CR01005 vector, and the sequence RZ899021 of the siRNA was conjugated to the CR01007 vector. CR01005 has a different branched structure than CR 01007.

The 6-8 week old C57BL/6j mice were randomly grouped by body weight, 10 per group, 3 total groups. Each group of mice was administered PBS solution and siRNA conjugate mentioned in this example using abdominal subcutaneous administration, respectively. Wherein, each mouse in the siRNA conjugate experimental group is dosed with 3mg/kg (calculated as siRNA), and the dosage volume is 5mL/kg of the body weight of the mouse; the PBS control group was given 5mL/kg of mouse body weight in PBS solution without siRNA conjugate. Dosing when the day one (D1) was recorded, 5 mice were sacrificed on each of the 15 th (D15) and 29 th (D29) day after dosing, respectively. Liver tissue was collected for RNA extraction, reverse transcription and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the ΔΔct method described above.

TABLE 6 primer sequence listing of example 2.1

The results of this example show that both the siRNA conjugate RZ899017 conjugated to CR01005 vector and the siRNA conjugate RZ899021 conjugated to CR01007 vector are capable of producing effective inhibition of the target gene of interest, with the highest inhibitory activity of RZ899017 reaching 95.43% at D15, the highest inhibitory activity of RZ899021 reaching 86.41%, the inhibitory activity of RZ899017 at D29 being 76.75% and the inhibitory activity of RZ899021 being 76.36%. It can be seen that siRNA conjugate RZ899017 of CR01005 is superior to siRNA conjugate RZ899021 of CR01007 (fig. 1, table 7).

TABLE 7 inhibition Activity of target genes of interest in mice following administration of siRNA conjugates described in example 2.1

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Example 2.2CR01005, evaluation of in vivo Activity of CR01007 vector conjugated angiopoietin-like protein 3 (ANGPTL 3) siRNA

In this example, the inhibitory activity of the 3' -end of the sense strand of the siRNA to the target gene ANGPTL3 of the target gene in mice was evaluated by using the method for evaluating the inhibitory activity of the target gene in mice, wherein the sequence RZ897005 of the siRNA was conjugated to the CR01005 vector, and the sequence RZ897006 of the siRNA was conjugated to the CR01007 vector.

The 6-8 week old C57BL/6j mice were randomly grouped by body weight, 10 per group, 3 total groups. Each group of mice was administered with the siRNA conjugate and PBS solution described in this example, respectively, by subcutaneous administration via the abdomen. Wherein, each mouse in the siRNA conjugate experimental group is dosed with 3mg (based on siRNA)/kg (based on mouse weight) and the dosing volume is 5mL/kg (based on mouse weight); the PBS control group was given 5mL/kg (based on the weight of the mice) of PBS solution without siRNA conjugate. Dosing when the day one (D1) was recorded, 5 mice were sacrificed on each of the 15 th (D15) and 29 th (D29) day after dosing, respectively. Liver tissue was collected for RNA extraction, reverse transcription and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the ΔΔct method described above.

TABLE 8 primer sequence listing of example 2.2

The results of this example demonstrate that both the siRNA conjugate RZ897005 conjugated to CR01005 vector and the siRNA conjugate RZ897006 conjugated to CR01007 vector are capable of producing an inhibitory effect on the target gene of interest, wherein the activity of CR01005 conjugate RZ897005 is better than CR01007 conjugate RZ897006 and the highest inhibitory activity of RZ897005 at D15 is up to 84.44% (fig. 2, table 9).

TABLE 9 inhibition Activity of target genes of interest in mice following administration of siRNA conjugates described in example 2.2

Example 2.3CR01005, evaluation of in vivo Activity of CR01007 vector conjugated complement component 3 (complement component 3, CC3) siRNA

In this example, the inhibitory activity of the 3' -end of the sense strand of the siRNA to the target gene CC3 of the target gene in mice was evaluated by using the method for evaluating the inhibitory activity of the target gene in mice, wherein the 3' -end of the sense strand of the siRNA was conjugated with the sequence RZ802012 of the siRNA of the CR01005 vector, and the 3' -end of the sense strand of the siRNA was conjugated with the sequence RZ802013 of the CR01007 vector.

The 6-8 week old C57BL/6j mice were randomly grouped by body weight, 5 per group, and 3 total groups. Each group of mice was administered with the siRNA conjugate and PBS solution described in this example, respectively, by subcutaneous administration via the abdomen. Wherein, each mouse in the siRNA conjugate experimental group is dosed with 9mg (based on siRNA)/kg (based on mouse weight) and the dosing volume is 5mL/kg (based on mouse weight); the PBS control group was given 5mL/kg (based on the weight of the mice) of PBS solution without siRNA conjugate. Dosing when the day one (D1) was noted, mice were sacrificed on day 8 (D8) after dosing. Liver tissue was collected for RNA extraction, reverse transcription and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the ΔΔct method described above.

Table 10 primer sequence listing of example 2.3

The results of this example demonstrate that both the siRNA conjugate RZ802012 conjugated to CR01005 vector and the siRNA conjugate RZ802013 conjugated to CR01007 vector are capable of producing an effective inhibition effect on the target gene of interest CC3, wherein the inhibition activity of RZ802012 is relatively higher, up to 95.59% (fig. 3, table 11).

Table 11 inhibitory Activity of target genes of interest in mice following administration of the siRNA conjugates described in example 2.3

Example 2.4CR01004 in vivo Activity assessment of vector conjugated SOD1 target siRNA

In this example, the inhibitory activity of the 3 '-end of the sense strand of the siRNA to the target gene SOD1 of interest in mice was evaluated by using the method for evaluating the inhibitory activity of the target gene in mice, which was used to attach the RZ899039 sequence of CR01004 vector to the 3' -end of the sense strand of the siRNA.

The 6-8 week old C57BL/6j mice were randomly grouped by body weight, 15 mice per group, and 2 total groups. Each group of mice was administered with the siRNA conjugate and PBS solution described in this example, respectively, by subcutaneous administration via the abdomen. Wherein, each mouse in the siRNA conjugate experimental group is dosed with 3mg (based on siRNA)/kg (based on mouse weight), and the dosing volume is 5mL/kg (based on mouse weight). The PBS control group was given 5mL/kg (based on the weight of the mice) of PBS solution without siRNA conjugate. Dosing when the day one (D1) was recorded, 5 mice were sacrificed on each of the 8 th (D8), 29 th (D29) and 43 th (D43) groups after dosing, respectively. Liver tissue was collected for RNA extraction, reverse transcription and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the ΔΔct method described above.

The primer sequences of this example are shown in Table 6 of example 2.1.

The results of this example demonstrate that CR01004 conjugate RZ899039 produces sustained potent inhibitory activity against the target gene of interest, with D15 inhibitory activity up to 96.31% (fig. 4, table 12).

Table 12 inhibitory Activity of target genes of interest in mice following administration of the siRNA conjugates described in example 2.4

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Example 2.5CR01004 vector conjugated ANGPTL3 target siRNA in vivo Activity assessment

In this example, the inhibitory activity of the 3' -end of the sense strand of the siRNA to the target gene ANGPTL3 of the target gene in mice was evaluated by using the method for evaluating the inhibitory activity of the target gene in mice, which was conjugated with the RZ897011 sequence of the CR01004 vector.

The 6-8 week old C57BL/6j mice were randomly grouped by body weight, 15 mice per group, and 2 total groups. Each group of mice was administered with the siRNA conjugate and PBS solution described in this example, respectively, by subcutaneous administration via the abdomen. Wherein, each mouse in the siRNA conjugate experimental group is dosed with 3mg (based on siRNA)/kg (based on mouse weight) and the dosing volume is 5mL/kg (based on mouse weight); each mouse in the PBS control group was given 5mL/kg (based on the weight of the mouse) of PBS solution without siRNA conjugate. Dosing when the day one (D1) was recorded, 5 mice were sacrificed on each of the 8 th (D8), 29 th (D29) and 43 th (D43) groups after dosing, respectively. Liver tissue was collected for RNA extraction, reverse transcription and Real-time PCR detection, and relative quantitative calculation of target gene mRNA in each test group was performed according to the ΔΔct method described above.

The primer sequences of this example are shown in Table 8 in example 2.2.

The results of this example demonstrate that CR01004 conjugate RZ897011 produces sustained potent inhibitory activity against the target gene of interest, with D15 inhibitory activity up to 88.87% (fig. 5, table 13).

TABLE 13 inhibition Activity of target genes of interest in mice following administration of siRNA conjugates described in example 2.5

It is to be understood that the above embodiments are merely exemplary embodiments employed to illustrate the principles of the present disclosure, however, the present disclosure is not limited thereto. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the disclosure, and are also considered to be within the scope of the disclosure.

Claims

1. GalNAc derivative represented by formula (I) or a stereoisomer, a pharmaceutically acceptable salt or a prodrug thereof:

wherein A is selected from substituted or unsubstituted 4-7 membered saturated nitrogen heterocycle;

n is selected from 0 or 1, m is selected from 0 or 1, and n=m;

each Y is independently selected from O, S or NH;

p and q are each independently selected from 0, 1, 2 or 3;

2. GalNAc derivative according to claim 1, characterized in that a is selected from

Optionally, A is selected from

Optionally, each L ₁ Each independently selected from substituted or unsubstituted C ₁ -C ₅ A linear alkylene group;

optionally, each L ₁ Each independently selected from

Optionally, each L ₁ Are all selected from

Optionally, each L ₂ Each independently selected from substituted or unsubstituted C ₂ -C ₁₀ A linear alkylene group;

optionally, each L ₂ Each independently selected from

Optionally, each L ₂ Are all selected from

Optionally, each Y is selected from O;

optionally, each R ₃ Each independently selected from H,

Optionally, each R ₃ Each independently selected from H or

Optionally, each R ₃ Are all selected from

Optionally, each R ₃ Are all selected from H;

optionally, p and q are each independently selected from 0 or 1;

optionally, p=1 and q=1;

Optionally, the hydroxyl protecting group is selected from trityl, 4-methoxytrityl, 4 '-dimethoxytrityl or 4,4',4 "-trimethoxytrityl;

optionally, the hydroxyl protecting group is selected from 4,4' -dimethoxytrityl;

optionally, the pharmaceutically active molecule is selected from a small molecule drug, an antibody or an oligonucleotide;

optionally, the oligonucleotide is selected from the group consisting of a single stranded oligonucleotide and a double stranded oligonucleotide;

optionally, the oligonucleotide is selected from single stranded oligonucleotides;

optionally, the pharmaceutically active molecule is selected from ASO;

optionally, the pharmaceutically active molecule is selected from double stranded oligonucleotides;

optionally, the pharmaceutically active molecule is selected from siRNA;

optionally, the solid support is selected from resins comprising hydroxyl and/or amino functional groups or glass spheres comprising a controlled pore size;

optionally R _2a Selected from the group consisting of

Optionally R _2b Selected from the group consisting of Selected from resin or controllable pore size glass spheres;

optionally, the composition may be used in combination with,selected from->

3. GalNAc derivative according to claim 1, characterized in that it has a structure as shown in formula (II):

4. GalNAc derivative according to claim 1, characterized in that it is selected from any one of the following compounds:

5. A conjugate as shown in formula (III) or a stereoisomer, pharmaceutically acceptable salt or prodrug thereof:

wherein Nu represents an oligonucleotide;

k is selected from 1, 2, 3 or 4;

q is selected from the group consisting ofA、n、m、L ₁ 、L ₂ 、Y、p、q、R _1a As defined in any one of claims 1 to 4.

6. The conjugate of claim 5, wherein Q is of a formula selected from the group consisting of

Optionally, the oligonucleotide is selected from a single-stranded oligonucleotide or a double-stranded oligonucleotide;

optionally, the oligonucleotide is selected from siRNA;

optionally, k is selected from 1;

optionally, k is selected from 1, said oligonucleotide is selected from siRNA, one of said Q is conjugated to the 3' end of the sense strand of said siRNA.

7. The conjugate of claim 5, wherein the conjugate is selected from any one of the following compounds:

8. a composition comprising the conjugate of any one of claims 5-7;

optionally, the composition further comprises optionally one or more pharmaceutically acceptable carriers.

9. Use of any of the following for the manufacture of a medicament for the prevention and/or treatment of a disease:

(I) A compound according to any one of claims 1 to 5; and/or

(II) the conjugate of any one of claims 6-7; and/or

(III) the composition of claim 8;

optionally, the disease is selected from a pathological condition or disease caused by abnormal expression of a target gene in liver cells.

10. A method of reducing expression or activity of a target gene, the method comprising contacting a liver cell with any of:

(I) The conjugate of any one of claims 6-7; and/or

(II) the composition of claim 8.