GalNAc derivatives, conjugates, compositions, and uses thereof

Abstract

The present disclosure relates to the field of nucleic acid drugs, in particular to GalNAc derivatives, conjugates, compositions and uses thereof. The GalNAc derivatives have the structure shown in Formula (I). The conjugates provided by the present disclosure are formed by conjugating the GalNAc derivatives provided by the present disclosure to oligonucleotides. The conjugates provided by the present disclosure can be highly effective in targeting to the liver and effectively inhibiting the expression of target genes in the liver, and can be used for treating and / or preventing liver-derived diseases, have higher in vivo activity than control vectors, more stable and persistent pharmacodynamic effects, and are expected to have excellent safety and low animal level toxicity.

Description

Technical Field

[0001] This disclosure relates to the pharmaceutical field, and more specifically, to GalNAc derivatives, conjugates, compositions, and uses thereof. Background Technology

[0002] Small molecule nucleic acid drugs, represented by small interfering RNA (siRNA), antisense oligodeoxynucleotides (ASODN), and nucleic acid stimulating motifs (CpG), are playing an increasingly important role in gene therapy. Some of these drugs have already been approved by the FDA, and many more are currently in preclinical and clinical trials. Nucleic acid drugs are nucleic acid sequences that specifically target pathogenic genes or proteins through binding or cleavage, thereby inhibiting or promoting the expression of certain genes / proteins. These include all normal human genes that can replace defective genes, antisense nucleic acids that block gene expression, and single-stranded nucleic acids that promote triple-strand formation, such as siRNA, DNA, microRNA, or CpG.

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

[0004] This disclosure aims to address, at least to some extent, one of the technical problems existing in the prior art.

[0005] Therefore, this disclosure provides GalNAc derivatives, conjugates, compositions, and uses thereof. The conjugates provided in this disclosure are formed by conjugating the GalNAc derivatives to oligonucleotides. The conjugates provided in this disclosure can highly effectively target the liver and effectively inhibit the expression of liver target genes, and can be used to treat and / or prevent liver-related diseases. They exhibit higher in vivo activity, more stable and sustained efficacy than control vectors, and are expected to have excellent safety and low animal-level toxicity.

[0006] In a first aspect of this disclosure, this disclosure provides GalNAc derivatives or stereoisomers thereof, pharmaceutically acceptable salts, or prodrugs thereof as shown in formula (I):

[0007]

[0008] Wherein, A is selected from substituted or unsubstituted 5-7 membered saturated nitrogen heterocycles;

[0009] n is selected from 0 or 1, m is selected from 0 or 1, and n = m;

[0010] Each L1 is independently selected from substituted or unsubstituted C1-C5 alkylene groups;

[0011] Each L2 is independently selected from substituted or unsubstituted C2-C. 10 Alkylene;

[0012] Each Y is independently selected from O, S, or NH;

[0013] Each R3 is independently selected from H, substituted or unsubstituted C1-C4 alkyl acyl or substituted or unsubstituted C5-C7 aryl acyl;

[0014] p and q are each independently selected from 0, 1, 2 or 3;

[0015] R1 is selected from H, a hydroxyl protecting group, or... * Represents a linking site used to connect the active drug molecule; R 1a It is a hydroxyl or thiol group;

[0016] R2 is selected from H or R 2b Selected from solid supports containing amino functional groups, R 2a Selected from covalently linked groups connected to the amino functional group.

[0017] In some alternative embodiments of this disclosure, A may be selected from substituted or unsubstituted 4-6 membered saturated nitrogen heterocycles, 5-7 membered saturated nitrogen heterocycles, 5-6 membered saturated nitrogen heterocycles, or 6 membered saturated nitrogen heterocycles.

[0018] In some alternative embodiments of this disclosure, A is selected from

[0019] In some specific embodiments of this disclosure, A is selected from

[0020] In some alternative embodiments of this disclosure, each L1 is independently selected from substituted or unsubstituted C1-C5 straight-chain alkylene groups; for example, C1-C4 straight-chain alkylene groups, C1-C3 straight-chain alkylene groups, C2-C5 straight-chain alkylene groups, C2-C4 straight-chain alkylene groups, C2-C3 straight-chain alkylene groups, C3-C5 straight-chain alkylene groups, C3-C4 straight-chain alkylene groups, or C3 straight-chain alkylene groups, etc.

[0021] In some alternative embodiments of this disclosure, each L1 is independently selected.

[0022] In some specific embodiments of this disclosure, each L1 is selected from

[0023] In some alternative embodiments of this disclosure, each L2 is independently selected from substituted or unsubstituted C2-C. 10 Straight-chain alkylene compounds; for example, C2-C9 straight-chain alkylene compounds, C2-C8 straight-chain alkylene compounds, C2-C7 straight-chain alkylene compounds, C2-C6 straight-chain alkylene compounds, C2-C5 straight-chain alkylene compounds, C2-C4 straight-chain alkylene compounds, C3-C 10 Straight-chain alkylene, C3-C9 straight-chain alkylene, C3-C8 straight-chain alkylene, C3-C7 straight-chain alkylene, C3-C6 straight-chain alkylene, C3-C5 straight-chain alkylene, C3-C4 straight-chain alkylene, C4-C 10 Straight-chain alkylene, C4-C9 straight-chain alkylene, C4-C8 straight-chain alkylene, C4-C7 straight-chain alkylene, C4-C6 straight-chain alkylene, C4-C5 straight-chain alkylene or C4 straight-chain alkylene C4-C 10 Straight-chain alkylene groups.

[0024] In some alternative embodiments of this disclosure, each L2 is independently selected.

[0025] In some specific embodiments of this disclosure, each L2 is selected from

[0026] In some specific embodiments of this disclosure, each Y is selected from O;

[0027] In some alternative embodiments of this disclosure, each R3 is independently selected from H, substituted or unsubstituted C1-C4 alkyl acyl groups (e.g., acetyl, propionyl, n-butyryl, isobutyryl, n-valeryl, isovaleryl, 2-methylbutyryl, neovaleryl / tervaeryl) or substituted or unsubstituted C5-C7 aryl acyl groups (e.g., benzoyl); each substituent in R3 is independently selected from halogens (e.g., F).

[0028] In some alternative embodiments of this disclosure, each R3 is independently selected from H,

[0029] In some alternative embodiments of this disclosure, each R3 is independently selected from H or

[0030] In some specific embodiments of this disclosure, each R3 is selected from

[0031] In some specific embodiments of this disclosure, each R3 is selected from H.

[0032] In some alternative embodiments of this 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.

[0033] In some specific embodiments of this disclosure, p = 1 and q = 1.

[0034] In this disclosure, the hydroxyl protecting group can be any type of hydroxyl protecting group, as long as it can protect the hydroxyl group; 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 this disclosure, the hydroxyl protecting groups that can be used include, but are not limited to, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox), triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr), and 4,4',4”-trimethoxytriphenyl (TMTr).

[0035] In some alternative embodiments of this disclosure, the hydroxyl protecting group is selected from triphenylmethyl (Tr), 4-methoxytriphenylmethyl (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr), or 4,4',4”-trimethoxytriphenyl (TMTr).

[0036] In some specific embodiments of this disclosure, the hydroxyl protecting group is selected from 4,4'-dimethoxytriphenylmethyl (DMTr).

[0037] In some alternative embodiments of this disclosure, the pharmaceutically active molecule is selected from small molecule drugs, antibodies, or oligonucleotides.

[0038] In some alternative embodiments of this disclosure, the oligonucleotide is selected from single-stranded oligonucleotides and double-stranded oligonucleotides.

[0039] In some alternative embodiments of this disclosure, the oligonucleotide is selected from single-stranded oligonucleotides.

[0040] In some alternative embodiments of this disclosure, the active pharmaceutical molecule is selected from ASO.

[0041] In some specific embodiments of this disclosure, the active pharmaceutical molecule is selected from double-stranded oligonucleotides.

[0042] In some specific embodiments of this disclosure, the active pharmaceutical molecule is selected from siRNA.

[0043] In some alternative embodiments of this disclosure, the solid support is selected from resins containing hydroxyl and / or amino functional groups (e.g., polystyrene, abbreviated PS) or glass spheres containing controlled pores (CPG).

[0044] In some specific embodiments of this disclosure, R 2a Selected from

[0045] In some specific embodiments of this disclosure, R 2b Selected from Selected from resin or glass spheres with controllable aperture.

[0046] In some specific implementation schemes disclosed herein, Selected from

[0047] In some alternative embodiments of this disclosure, Selected from

[0048] In some alternative embodiments of this disclosure, the GalNAc derivative has a structure as shown in formula (II):

[0049]

[0050] In some alternative embodiments of this disclosure, the GalNAc derivative is selected from any of the following compounds:

[0051]

[0052] In a second aspect of this disclosure, this disclosure provides conjugates or stereoisomers thereof as shown in formula (III), pharmaceutically acceptable salts or prodrugs thereof:

[0053]

[0054] Nu represents oligonucleotide;

[0055] k is selected from 1, 2, 3, or 4;

[0056] The structural formula of Q is selected from A, n, m, L1, L2, Y, p, q, R 1a *As defined above.

[0057] In some alternative embodiments of this disclosure, the structure of Q is selected from...

[0058]

[0059] In some alternative embodiments of this disclosure, the oligonucleotide is selected from single-stranded oligonucleotides or double-stranded oligonucleotides.

[0060] In some alternative embodiments of this disclosure, the single-stranded oligonucleotide is selected from antisense oligonucleotides, nucleic acid aptamers, ribozymes, deoxyribozymes, circular RNA, the sense strand of siRNA, or the antisense strand of siRNA.

[0061] In some alternative embodiments of this disclosure, the double-stranded oligonucleotide is selected from small interfering RNA, double-stranded RNA, microRNA, small guide RNA, small activating RNA, or short hairpin RNA.

[0062] In some specific embodiments of this disclosure, the oligonucleotide is selected from siRNA.

[0063] In some specific embodiments of this disclosure, k is selected from 1 or 2.

[0064] In some specific embodiments of this disclosure, k is selected from 1.

[0065] In some specific embodiments of this disclosure, k is selected from 1, the oligonucleotide is selected from siRNA, and one of the Q is conjugated to the 3' end of the positive strand of the siRNA.

[0066] In some specific embodiments of this disclosure, k is selected from 2, the oligonucleotide is selected from siRNA, and the two Qs are respectively conjugated to the 3' end and the 5' end of the positive strand of the siRNA.

[0067] In some alternative embodiments of this disclosure, the conjugate is selected from any of the following compounds:

[0068]

[0069] In a third aspect of this disclosure, a composition is provided comprising the conjugate described in the second aspect.

[0070] In some alternative embodiments of this disclosure, the composition further comprises, optionally, one or more, pharmaceutically acceptable carriers.

[0071] In a fourth aspect of this disclosure, the following are provided for use in the preparation of medicaments for the prevention and / or treatment of diseases:

[0072] (I) the compounds described in the first aspect; and / or

[0073] (II) The conjugates described in the second aspect; and / or

[0074] (III) The composition described in the third aspect.

[0075] In some alternative embodiments of this disclosure, the disease is selected from pathological conditions or diseases caused by abnormal expression of target genes in liver cells. Exemplarily, 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.

[0076] In some specific embodiments of this disclosure, the target gene is selected from SOD1, ANGPTL3, or CC3.

[0077] In a fifth aspect, this disclosure provides a method for reducing the expression or activity of a target gene, the method comprising contacting liver cells with any of the following:

[0078] (I) the conjugates described in the second aspect; and / or

[0079] (II) The composition described in the third aspect.

[0080] In some alternative embodiments of this disclosure, 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.

[0081] In some specific embodiments of this disclosure, the target gene is selected from SOD1, ANGPTL3, or CC3.

[0082] Additional aspects and advantages of this 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 this disclosure. Attached Figure Description

[0083] Figure 1 This refers to the relative expression level of the target gene in mice after administration of the siRNA conjugate described in Example 2.1;

[0084] Figure 2 This refers to the relative expression level of the target gene in mice after administration of the siRNA conjugate described in Example 2.2;

[0085] Figure 3 This refers to the relative expression level of the target gene in mice after administration of the siRNA conjugate described in Example 2.3;

[0086] Figure 4This refers to the relative expression level of the target gene in mice after administration of the siRNA conjugate described in Example 2.4;

[0087] Figure 5 This refers to the relative expression level of the target gene in mice after administration of the siRNA conjugate described in Example 2.5. Detailed Implementation

[0088] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0089] Terminology Explanation

[0090] In the context of this disclosure, the term "alkyl" refers to the general formula […]. The alkyl group can be a straight-chain alkyl group or a branched-chain alkyl group. The term "C1-C2 alkyl" refers to an alkyl group having one or two carbon atoms, such as methyl or ethyl.

[0091] In the context of this disclosure, the term "alkylene" refers to a general formula of The alkylene group can be a straight-chain alkylene or a branched alkylene. The term "C2-C" is used in this context. 10 "Alkylene" refers to an alkylene chain having 1 to 10 carbon atoms.

[0092] In the context of this disclosure, the term "alkyl" refers to the general formula […]. The alkyl group can be a straight-chain alkyl group or a branched-chain alkyl group. The term "C1-C2 alkyl" refers to an alkyl group having one or two carbon atoms, such as methyl or ethyl.

[0093] In the context of this disclosure, the term "alkylene" refers to a general formula of The alkylene group can be a straight-chain alkylene or a branched alkylene. The term "C2-C" is used in this context. 10 "Alkylene" refers to an alkylene chain having 1 to 10 carbon atoms.

[0094] In the context of this disclosure, the structural formula of the term "aromatic acyl" is: Here, Ar refers to the aryl group. In organic chemistry, Ar refers to any functional group or substituent derived from a simple aromatic ring. The simplest aryl group is phenyl, which is derived from benzene.

[0095] In the context of this disclosure, the structural formula of the term "alkoxy" is: Among them, "C1-C2 alkoxy" can be methoxy or ethoxy.

[0096] In the context of this disclosure, "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0097] In the context of this disclosure, a "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0098] In the context of this disclosure, a "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that retains the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts, with sodium salts being the most preferred. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0099] In the context of this disclosure, "oligonucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically composed of 10-50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes, including ribonuclease-mediated target degradation, splicing regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

[0100] the term This indicates the site where groups are connected by covalent bonds.

[0101] In the structural formulas of the compounds described in this disclosure, the "—" sign indicates that the configuration is not specified. If chiral isomerism exists in the chemical structure, the "—" sign can be... Or simultaneously include and Two configurations. Although all the above structural formulas are shown in some isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0102] In the structural formula of the compound described in this disclosure, the bond... This indicates that the configuration is not specified. If cis-trans isomerism exists in the chemical structure, the bond... The configuration can be E-type, Z-type, or both E-type and Z-type.

[0103] Unless otherwise stated, the following definitions as used herein shall apply. For the purposes of this disclosure, chemical elements are consistent with the CAS edition of the periodic table and the Handbook of Chemistry and Physics, 75th edition, 1994. Furthermore, general principles of organic chemistry can 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. Unless otherwise stated or there is a clear conflict in the context, the articles “a,” “an,” and “described” as used herein are intended to include “at least one” or “one or more.” Therefore, these articles as used herein refer to articles for one or more (i.e., at least one) objects. For example, “a component” refers to one or more components, meaning that more than one component may be considered for use or employed in the embodiments described.

[0104] The term “comprising” is an open-ended expression, meaning that it includes the contents specified in this disclosure, but does not exclude other contents.

[0105] "Stereoisomers" are compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers, etc.

[0106] "Chirality" refers to molecules that have the property that they cannot be superimposed on their mirror image; while "chirality" refers to molecules that can be superimposed on their mirror image.

[0107] "Enantiomers" refer to two non-overlapping but mirror-image isomers of a compound.

[0108] A diastereomer is a stereoisomer that has 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. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0109] The stereochemical definitions and rules used in this disclosure generally follow those of 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.

[0110] As described in this disclosure, the compounds of this disclosure may optionally be substituted with one or more substituents, such as the general formula compounds above, or as specific examples, subclasses, and a class of compounds included in this disclosure.

[0111] Generally, the term "substituted" means that one or more hydrogen atoms in a given structure are replaced by a specific substituent. Unless otherwise indicated, a substituted group may have one substituent at each substituted position of the group. When more than one position in a given structural formula can be replaced by one or more substituents selected from a specific group, then the substituents may be substituted at each substituted position in the same or different ways.

[0112] The term "unsubstituted" means that the specified group does not have substituents.

[0113] The term "optionally substituted with" may be used interchangeably with the term "unsubstituted or substituted with," meaning that 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, alkylamino, cycloalkyl, heterocyclic, aryl, heteroaryl, etc.

[0114] Furthermore, it should be noted that, unless otherwise explicitly stated, the descriptive phrases "each...independently is" and "...each independently is" and "...independently is" used in this disclosure are interchangeable and should be interpreted broadly. They can mean either that the specific options expressed by the same symbol in different groups do not affect each other, or that the specific options expressed by the same symbol in the same group do not affect each other. For example, regarding R3, the specific options of R3 in the structural formulas "optionally substituted C1-C50 alkylene" and "optionally substituted -C(O)-NH-C1-50 alkylene" do not affect each other.

[0115] The term "small interfering RNA (siRNA)" refers to a class of double-stranded RNAs 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 RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNAs direct the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and its conversion into proteins.

[0116] In the context of this disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence. "Sense strand (or follower strand)" refers to an iRNA strand containing an iRNA strand substantially complementary to the antisense strand. The term "substantially complementary" means fully complementary or at least partially complementary, for example, the antisense strand being fully complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can be present within the molecule or in terminal regions, wherein the most tolerant mismatches are present in terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3'-terminus of the iRNA.

[0117] It should be noted that "at least partially complementary" to mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest.

[0118] In the context of this disclosure, "oligonucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically composed of 10 to 50 nucleotides. Oligonucleotides can regulate gene expression through a series of processes, including ribonuclease-mediated target degradation, splicing regulation, non-coding RNA inhibition, gene activation, and programmed gene editing.

[0119] In the context of this disclosure, "antisense oligonucleotides (ASOs)" are single-stranded oligonucleotide molecules, typically composed of 10 to 50 nucleotides. After entering the cell, ASOs bind to their complementary target mRNAs via base pairing under the action of ribonuclease H1, thereby inhibiting the expression of the target genes.

[0120] In the context of this disclosure, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or combination thereof, all of which are known to those skilled in the art (as described in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0121] In the context of this disclosure, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0122] In the context of this disclosure, "subject" refers to any animal, such as a mammal or marsupial. Subjects of this disclosure include, but are not limited to, humans, non-human primates (e.g., monkeys), rats, pigs, horses, donkeys, cattle, sheep, and any kind of poultry.

[0123] In the context of this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, therapeutic benefits. A “therapeutic benefit” means the eradication or improvement of the underlying disorder being treated. Here, a therapeutic benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0124] In the context of this disclosure, “prevention” and “avoidance” are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” a conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0125] In the context of this disclosure, unless otherwise stated, the reagent ratios described in the embodiments of this disclosure are calculated on a volume ratio (v / v).

[0126] Unless otherwise stated, all raw materials and reagents used in the preparation of the compounds disclosed herein were purchased from Beijing Coupling Technology Co., Ltd. Details of some of the reagents used in this disclosure are shown in Table 1.

[0127] Table 1. Details of some reagents

[0128]

[0129]

[0130] Unless otherwise stated, the reagents and consumables (Table 2) and instruments and equipment (Table 3) used in this disclosure are all commercially available products from the following manufacturers.

[0131] Table 2 Main Reagents and Consumables

[0132] 1×PBS Zhongke Maichen (Beijing) Technology Co., Ltd. Hanwei RNA Extraction Kit Zhejiang Hanwei Technology Co., Ltd. Reverse Transcription System Promega Corporation SYBR Select Master Mix ABI RNALater Thermo Fisher Scientific

[0133] Table 3 Main Instruments and Equipment

[0134] Fully automated nucleic acid extractor Zhejiang Hanwei Technology Co., Ltd. High-speed refrigerated centrifuge Eppendorf NANODROP OneC Thermo Fisher Scientific Gradient PCR Amplification Instrument Eppendorf Real-time PCR instrument ABI StepOne Plus Gel Imaging Shanghai Tianneng Life Science Co., Ltd. Electrophoresis apparatus Beijing Liuyi Instrument Factory Tissuelyser II Fully Automated Tissue Homogenizer Shanghai Jingxin Industrial Development Co., Ltd.

[0135] Preparation Example 1: Synthesis of Compound CR01004Z

[0136] In this preparation example, the synthetic route of compound CR01004Z is shown below:

[0137]

[0138] (1-1) Synthesis of compound CR01004-2

[0139] Compound CR01004-1 (400 mg, 1.11 mmol, 1.0 eq), tert-butyl (3-oxopropyl)carbamate (578 mg, 3.33 mmol, 3 eq), and triethylamine (230 mg, 2.22 mmol, 2.0 eq) were dissolved in 10 mL of dichloromethane and stirred at 25 °C for 2 hours. Then, sodium triacetoxyborohydride (1.42 g, 6.66 mmol, 6 eq) was added, and the reaction mixture was stirred at 25 °C for 12 hours. After the reaction was complete, 30 mL of dichloromethane was added to the reaction system, and the mixture was washed once with 20 mL of saturated sodium bicarbonate solution and once with 20 mL of distilled water. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent: methanol / dichloromethane = 5 / 95, v / v) to give a pale yellow solid, compound CR01004-2 (600 mg, yield 84.5%). MS ESI(m / z) = 637.2 [M+H] + .

[0140] (1-2) Synthesis of compound CR01004-3

[0141] Compound CR01004-2 (600 mg, 0.94 mmol, 1.0 eq) was dissolved in 10 mL of a 4 mol / L dioxane solution of hydrogen chloride. The reaction was carried out at 25 °C for 2 hours. After the reaction was completed, the reaction solution was directly concentrated to obtain a pale yellow solid, compound CR01004-3 (7.6 g), which was used directly in the next step without purification. MS ESI (m / z) = 337.2 [M+H] + .

[0142] (1-3) Synthesis of compound CR01004-4

[0143] Compound CR01004-3 (500 mg, 1.13 mmol, 1.0 eq) was dissolved in 15 mL of N,N-dimethylformamide, and compounds Gal-5 (1.5 g, 3.39 mmol, 3.0 eq), HATU (2.15 g, 5.65 mmol, 5.0 eq), and DIEA (1.75 g, 13.56 mmol, 12.0 eq) were added. The mixture was stirred at 25 °C for 5 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (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] + .

[0144] (1-4) Synthesis of compound CR01004-5

[0145] Compound CR01004-4 (800 mg, 0.49 mmol, 1.0 eq) was dissolved in 15 mL of methanol, and wet palladium on carbon (80 mg, 10% by mass) was added. The mixture was purged three times with hydrogen, and then stirred at 25 °C for 3 hours. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated to give a white solid, compound CR01004-5 (700 mg, 92.6% yield), which was used directly in the next step without purification. MS ESI (m / z) = 1534.4 [M+H] + .

[0146] (1-5) Synthesis of compound CR01004-6

[0147] Compound CR01004-5 (770 mg, 0.50 mmol, 1.0 eq) was dissolved in 15 mL of DMF. Compounds N-SH (278 mg, 0.55 mmol, 1.1 eq), HATU (286 mg, 0.75 mmol, 1.5 eq), and DIEA (162 mg, 1.25 mmol, 2.5 eq) were added, and the mixture was stirred at 25 °C for 5 hours. After the reaction was complete, the system was directly purified by reversed-phase column chromatography (C18 column, 58%–70% ACN in H2O). The fraction was concentrated to give a white solid, compound CR01004-6 (850 mg, yield 83.3%). MS ESI (m / z) = 2021.2 [M+H] + .

[0148] (1-6) Synthesis of compound CR01004Z

[0149] Compound CR01004-6 (160 mg, 0.1 mmol, 1.0 eq.), dichloromethane (3.2 mL), 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 mixed and stirred at 25 °C for 12 hours. After the reaction was complete, the mixture was purged with nitrogen three times and stirred at 25 °C for 16 hours. The mixture was then purified by reverse-phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to give an intermediate product (100 mg).

[0150] The above intermediate product (100 mg, 0.058 mmol, 1.0 eq.), aminoCPG (1.44 g, 0.115 mmol, 80 μmol / g), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea 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. After the reaction was complete, the reaction solution was filtered, and the filter cake was washed once with 10 ml of acetonitrile and then dried under vacuum. The dried filter 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 filter cake was washed once with 10 ml of acetonitrile and then dried under vacuum to obtain compound CR01004Z (1.367 g, loading 20-30 μmol / g).

[0151] Cap1 and Cap2 are capping reagents. Cap1 is a 20 vol% N-methylimidazolium pyridine / acetonitrile mixed solution with a pyridine to acetonitrile volume ratio of 3:5. Cap2 is a 20 vol% acetic anhydride acetonitrile solution.

[0152] Preparation Example 2: Synthesis of Compound CR01005Z

[0153] In this preparation example, the synthetic route of compound CR01005Z is as follows:

[0154]

[0155] (2-1) Synthesis of compound CR01005-2

[0156] Compound CR01005-1 (19.5 g, 53.4 mmol, 1.0 eq) was dissolved in 50 mL of ultra-dry DMF, and K2CO3 (18.56 g, 134.4 mmol, 2.5 eq) and benzyl bromide (10.95 g, 64.1 mmol, 1.2 eq) were added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, 200 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (200 mL each time). The organic phases were combined, washed 10 times with saturated sodium chloride aqueous 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] + .

[0157] (2-2) Synthesis of compound CR01005-3

[0158] Compound CR01005-2 (22 g, 48.1 mmol, 1.0 eq) was dissolved in 90 mL of dichloromethane (DCM), and a solution of 1,4-dioxane in hydrogen chloride (45 mL, 4 mol / L) was added. The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, methyl tert-butyl ether was added, the mixture was slurried, filtered, and dried to give a white solid, compound CR01005-3 (24 g, 100% yield). MS ESI (m / z) = 357.2 [M+H] + .

[0159] (2-3) Synthesis of compound CR01005-5

[0160] Compounds CR01005-3 (5.2 g, 13.28 mmol, 1.1 eq) and CR01005-4 (2.5 g, 12.07 mmol, 1.0 eq) were dissolved in 70 mL of tetrahydrofuran. A solution of triethylamine (2.8 g, 27.76 mmol, 2.3 eq) was added, and the mixture was stirred at 25 °C for 2 hours. NaBH(OAc)3 (5.1 g, 24.14 mmol, 2.0 eq) was then added, and the reaction was carried out at 25 °C for 12 hours under nitrogen protection. After the reaction was complete, 30 mL of water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (50 mL each time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent: methanol / dichloromethane = 4 / 96, v / v) to give a white oily compound CR01005-5 (5 g, yield 69.0%). MS ESI (m / z) = 548.2 [M+H] + .

[0161] (2-4) Synthesis of compound CR01005-6

[0162] Compounds CR01005-5 (2.5 g, 4.56 mmol, 1.0 eq), Gal-5 (4.07 g, 9.12 mmol, 2.0 eq), and HATU (3.5 g, 9.12 mmol, 2.0 eq) were dissolved in 25 mL of N,N-dimethylformamide. DIEA (1.2 g, 9.12 mmol, 2.0 eq) was added under ice bath conditions, and the mixture was stirred at 25 °C for 12 hours. After the reaction was complete, 30 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (50 mL each time). 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 give a pale yellow solid, compound CR01005-6 (2.4 g, yield 53.8%). MS ESI (m / z) = 990 [M+Na] + .

[0163] (2-5) Synthesis of compound CR01005-7

[0164] Compound CR01005-6 (2.7 g, 2.76 mmol, 1.0 eq) was dissolved in 20 mL of methanol, and wet palladium on carbon (270 mg) and 0.1 mL of trifluoroacetic acid were added. The mixture was purged with hydrogen three times, and stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give compound CR01005-7 (1.7 g, 100% yield) as a white solid. MS ESI (m / z) = 619 [M+H] + .

[0165] (2-6) Synthesis of compound CR01005-8

[0166] Compounds Gal-5 (2.65 g, 5.9 mmol, 2.5 eq), DIEA (5 (1.8 g, 14.16 mmol, 6.0 eq)), and HATU (2.1 g, 5.43 mmol, 2.3 eq) were dissolved in 25 mL of N,N-dimethylformamide. Then, CR01005-7 (2 g, 2.36 mmol, 1.0 eq), which was freed from DIEA, was added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (C18 column, eluent: acetonitrile / water = 23 / 77, v / v) to give a white solid, compound CR01005-8 (2.4 g, yield 38%). MS ESI (m / z) = 1477 [M+H] + .

[0167] (2-6) Synthesis of compound CR01005-9

[0168] Compound CR01005-8 (1.5 g, 1.0 mmol, 1.0 eq), compound N-SH (768 mg, 1.5 mmol, 1.5 eq), and DIEA (524 mg, 4.0 mmol, 4.0 eq) were dissolved in 20 mL of N,N-dimethylformamide, and HATU (580 mg, 1.5 mmol, 1.5 eq) was added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (C18 column, eluent: acetonitrile-water = 54 / 46, v / v) to give compound CR01005-9 (1.8 g, 90% yield) as a white solid. MS ESI (m / z) = 1963 [M+H] + .

[0169] (2-7) Synthesis of compound CR01005-10

[0170] Compound CR01005-9 (200 mg, 0.1 mmol, 1.0 eq) was dissolved in 10 mL of dichloromethane, and succinic anhydride (12 mg, 0.11 mmol, 1.1 eq) and triethylamine (21 mg, 0.2 mmol, 2.0 eq) were added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain a white solid compound CR01005-10 (200 mg, 95% yield), which was used directly in the next step without purification. MS ESI (m / z) = 2065 [M+H] + .

[0171] (2-8) Synthesis of compound CR01005Z

[0172] (2-1) Synthesis of compound CR01005-2

[0173] Compound CR01005-1 (19.5 g, 53.4 mmol, 1.0 eq) was dissolved in 50 mL of ultra-dry DMF, and K2CO3 (18.56 g, 134.4 mmol, 2.5 eq) and benzyl bromide (10.95 g, 64.1 mmol, 1.2 eq) were added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, 200 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (200 mL each time). The organic phases were combined, washed 10 times with saturated sodium chloride aqueous 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] + .

[0174] (2-2) Synthesis of compound CR01005-3

[0175] Compound CR01005-2 (22 g, 48.1 mmol, 1.0 eq) was dissolved in 90 mL of dichloromethane (DCM), and a solution of 1,4-dioxane in hydrogen chloride (45 mL, 4 mol / L) was added. The mixture was stirred at 25 °C for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure, methyl tert-butyl ether was added, the mixture was slurried, filtered, and dried to give a white solid, compound CR01005-3 (24 g, 100% yield). MS ESI (m / z) = 357.2 [M+H] + .

[0176] (2-3) Synthesis of compound CR01005-5

[0177] Compounds CR01005-3 (5.2 g, 13.28 mmol, 1.1 eq) and CR01005-4 (2.5 g, 12.07 mmol, 1.0 eq) were dissolved in 70 mL of tetrahydrofuran. A solution of triethylamine (2.8 g, 27.76 mmol, 2.3 eq) was added, and the mixture was stirred at 25 °C for 2 hours. NaBH(OAc)3 (5.1 g, 24.14 mmol, 2.0 eq) was then added, and the reaction was carried out at 25 °C for 12 hours under nitrogen protection. After the reaction was complete, 30 mL of water was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (50 mL each time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent: methanol / dichloromethane = 4 / 96, v / v) to give a white oily compound CR01005-5 (5 g, yield 69.0%). MS ESI (m / z) = 548.2 [M+H] + .

[0178] (2-4) Synthesis of compound CR01005-6

[0179] Compounds CR01005-5 (2.5 g, 4.56 mmol, 1.0 eq), Gal-5 (4.07 g, 9.12 mmol, 2.0 eq), and HATU (3.5 g, 9.12 mmol, 2.0 eq) were dissolved in 25 mL of N,N-dimethylformamide. DIEA (1.2 g, 9.12 mmol, 2.0 eq) was added under ice bath conditions, and the mixture was stirred at 25 °C for 12 hours. After the reaction was complete, 30 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (50 mL each time). 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 give a pale yellow solid, compound CR01005-6 (2.4 g, yield 53.8%). MS ESI (m / z) = 990 [M+Na] + .

[0180] (2-5) Synthesis of compound CR01005-7

[0181] Compound CR01005-6 (2.7 g, 2.76 mmol, 1.0 eq) was dissolved in 20 mL of methanol, and wet palladium on carbon (270 mg) and 0.1 mL of trifluoroacetic acid were added. The mixture was purged with hydrogen three times, and stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give compound CR01005-7 (1.7 g, 100% yield) as a white solid. MS ESI (m / z) = 619 [M+H] + .

[0182] (2-6) Synthesis of compound CR01005-8

[0183] Compounds Gal-5 (2.65 g, 5.9 mmol, 2.5 eq), DIEA (5 (1.8 g, 14.16 mmol, 6.0 eq)), and HATU (2.1 g, 5.43 mmol, 2.3 eq) were dissolved in 25 mL of N,N-dimethylformamide. Then, CR01005-7 (2 g, 2.36 mmol, 1.0 eq), which was freed from DIEA, was added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (C18 column, eluent: acetonitrile / water = 23 / 77, v / v) to give a white solid, compound CR01005-8 (2.4 g, yield 38%). MS ESI (m / z) = 1477 [M+H] + .

[0184] (2-6) Synthesis of compound CR01005-9

[0185] Compound CR01005-8 (1.5 g, 1.0 mmol, 1.0 eq), compound N-SH (768 mg, 1.5 mmol, 1.5 eq), and DIEA (524 mg, 4.0 mmol, 4.0 eq) were dissolved in 20 mL of N,N-dimethylformamide, and HATU (580 mg, 1.5 mmol, 1.5 eq) was added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (C18 column, eluent: acetonitrile-water = 54 / 46, v / v) to give compound CR01005-9 (1.8 g, 90% yield) as a white solid. MS ESI (m / z) = 1963 [M+H] + .

[0186] (2-7) Synthesis of compound CR01005-10

[0187] Compound CR01005-9 (200 mg, 0.1 mmol, 1.0 eq) was dissolved in 10 mL of dichloromethane, and succinic anhydride (12 mg, 0.11 mmol, 1.1 eq) and triethylamine (21 mg, 0.2 mmol, 2.0 eq) were added. The mixture was stirred at 25 °C for 12 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain a white solid compound CR01005-10 (200 mg, 95% yield), which was used directly in the next step without purification. MS ESI (m / z) = 2065 [M+H] + .

[0188] (2-8) Synthesis of compound CR01005Z

[0189] Compounds CR01005-10 (100 mg, 0.048 mmol, 1.0 eq), HBTU (27 mg, 0.073 mmol, 1.5 eq), and DIEA (12 mg, 0.096 mmol, 2.0 eq) were dissolved in 10 mL of acetonitrile solution and stirred for 5 minutes. Amino CPG (80 μmol / g, 1.17 g, 2 eq) was added, and the mixture was reacted in a shaker at 25 °C for 18 hours. The mixture was filtered, and the filter cake was washed twice with 10 mL of dichloromethane (10 mL each time) and then twice with acetonitrile (10 mL each time). The mixture was then dried under vacuum. The dried filter cake, 4-dimethylaminopyridine (2 mg, 0.017 mmol), Cap1 (8 ml) and Cap2 (8 ml) were mixed and reacted in a shaker at 25 °C for 5 hours. After filtration, the filter cake was washed three times with acetonitrile (10 ml each time) and dried under vacuum to obtain compound CR01005Z (1.3 g, loading 20-30 μmol / g).

[0190] Preparation Example 3: Synthesis of Reference Compound CR01007Z

[0191] In this preparation example, the synthetic route of the reference compound CR01005Z is shown below:

[0192]

[0193] (3-1) Synthesis of compound CR01007-3

[0194] Compounds CR01007-1 (5.0 g, 15.5 mmol, 1.0 eq), CR01007-2 (2.8 g, 17.05 mmol, 1.1 eq), HATU (8.8 g, 23.25 mmol, 1.5 eq), and DIEA (8.0 g, 62.0 mmol, 4.0 eq) were dissolved in 50 mL of dichloromethane and stirred at 25 °C for 4 hours. After the reaction was complete, 30 mL of dichloromethane was added to the reaction solution, and the mixture was washed once with 20 mL of saturated sodium bicarbonate solution and once with 20 mL of distilled water. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent: ethyl acetate / petroleum ether = 30 / 70, v / v) to give a yellow solid, compound CR01007-3 (5.0 g, yield 74.0%). MS ESI (m / z) = 437.2 [M+H] + .

[0195] (3-2) Synthesis of compound CR01007-4

[0196] Compound CR01007-3 (5.0 g, 11.47 mmol, 1.0 eq) was dissolved in 50 mL of methanol, and 0.5 g of wet palladium on carbon was added. The mixture was purged three times with hydrogen gas, and then stirred at 25 °C for 3 hours. After the reaction was complete, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a white solid, compound CR01007-4 (3.0 g, yield 88.2%), which was used directly in the next step without purification. MS ESI (m / z) = 303.4 [M+H] + .

[0197] (3-3) Synthesis of compound CR01007-5

[0198] Compounds CR01007-4 (3.7 g, 12.2 mmol, 1.0 eq), CR01007-1 (4.34 g, 13.42 mmol, 1.1 eq), HATU (6.96 g, 18.3 mmol, 1.5 eq), and DIEA (3.94 g, 30.5 mmol, 2.5 eq) were dissolved in 50 mL of dichloromethane and stirred at 25 °C for 4 hours. After the reaction was complete, 30 mL of dichloromethane was added to the reaction solution, and the mixture was washed once with 20 mL of saturated sodium bicarbonate solution and once with 20 mL of distilled water. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (eluent: ethyl acetate / petroleum ether = 50 / 50, v / v) to give a yellow solid, compound CR01007-5 (6.0 g, yield 81.1%). MS ESI (m / z) = 608.2 [M+H] + .

[0199] (3-4) Synthesis of compound CR01007-6

[0200] Compound CR01007-5 (6.0 g, 9.88 mmol, 1.0 eq) was dissolved in 20 mL of dichloromethane. 50 mL of formic acid was added at 25 °C. After the formic acid addition was complete, the reaction mixture was heated to 40 °C and reacted at 40 °C for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain a yellow solid, compound CR01007-6 (4.0 g), which was used directly in the next step without purification. MS ESI (m / z) = 440.2 [M+H] + .

[0201] (3-5) Synthesis of compound CR01007-7

[0202] Compounds CR01007-6 (1.0 g, 2.27 mmol, 1.0 eq), GAL-6 (3.2 g, 7.49 mmol, 3.3 eq), HATU (3.45 g, 9.08 mmol, 4.0 eq), and DIEA (2.64 g, 20.43 mmol, 9.0 eq) were dissolved in 30 mL of DMF and stirred at 25 °C for 4 hours. After the reaction was complete, 40 mL of ethyl acetate was added to the reaction system, and the mixture was washed four times with 20 mL of saturated sodium chloride each time, followed by one wash with 20 mL of distilled water. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal-phase column chromatography (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] + .

[0203] (3-6) Synthesis of compound CR01007-8

[0204] Compound CR01007-7 (2.0 g, 1.19 mmol, 1.0 eq) was dissolved in 15 mL of methanol, and 200 mg of wet palladium on carbon was added. The mixture was purged three times with hydrogen gas, and then stirred at 25 °C for 6 hours. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a white solid, compound CR01007-8 (1.5 g, yield 82.6%), which was used directly in the next reaction without purification. MSESI (m / z) = 1564.4 [M+H] + .

[0205] (3-7) Synthesis of compound CR01007-9

[0206] Compound CR01007-8 (2.0 g, 1.29 mmol, 1.0 eq) was dissolved in 20 mL of dichloromethane, and triethylamine (0.26 g, 2.58 mmol, 2.0 eq) and succinic anhydride (0.14 g, 1.42 mmol, 1.1 eq) were added. The mixture was stirred at 25 °C for 5 hours. After the reaction was complete, the reaction solution was directly purified by reverse-phase column chromatography (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] + .

[0207] (3-8) Synthesis of compound CR01007-10

[0208] Compound CR01007-9 (1.6 g, 0.97 mmol, 1.0 eq) was dissolved in 15 mL of DMF. Compounds N-SH (726 mg, 1.44 mmol, 1.5 eq), HATU (547 mg, 1.44 mmol, 1.5 eq), and DIEA (375 mg, 2.91 mmol, 3.0 eq) were added, and the mixture was stirred at 25 °C for 5 hours. After the reaction was complete, the reaction solution was directly purified by reversed-phase column chromatography (C18 column, elution gradient: acetonitrile / water = 55 / 45 to acetonitrile / water = 70 / 30, v / v) to give a white solid, compound CR01007-10 (1.6 g, yield 76.3%). MS ESI (m / z) = 2064.2 [M+H] + .

[0209] (3-8) Synthesis of compound CR01007Z

[0210] Compound CR01007-10 (210 mg, 0.1 mmol, 1.0 eq.) was dissolved in 3.0 mL of dichloromethane, and 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 added. The reaction system was stirred at 25 °C for 12 hours, purged with nitrogen three times, and stirred at 25 °C for 16 hours. The mixture was purified by reverse-phase column chromatography (C18 column, acetonitrile / water = 72 / 28, v / v) to give 100 mg of intermediate product.

[0211] The above intermediate product (100 mg, 0.045 mmol, 1.0 eq.), aminoCPG (1.44 g, 0.115 mmol, 80 μmol / g), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (25.67 mg, 0.068 mmol, 1.5 eq.), and N,N-diisopropylethylamine (11.6 mg, 0.09 mmol, 2.0 eq.) were mixed and shaken in a shaker for 16 hours. The mixture was filtered, and the filter cake was washed twice with acetonitrile (10 ml each time) and dried under vacuum. The dried filter cake, 4-dimethylaminopyridine (2 mg, 0.017 mmol), cap1 (15 ml), and cap2 (1.5 ml) were mixed and shaken for 6 hours. The mixture was then filtered, and the filter cake was washed twice with acetonitrile (10 ml each time). The mixture was then dried under vacuum to obtain compound CR01007Z (1.31 g, loading 20-30 μmol / g).

[0212] Preparation Example 4: Preparation of siRNA conjugates

[0213] (4-1) Synthesis of the Chain of Justice (SS)

[0214] The phosphoramidite-based solid-phase nucleic acid synthesis method utilizes the aforementioned compounds (CR01004Z, CR01005Z, and CR01007Z) linked to a solid support as the starting cycle, and sequentially links nucleoside monomers along the 3'-5' direction according to the nucleotide sequence. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below:

[0215] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0216] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0217] The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2mol / L acetonitrile / pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

[0218] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; the capping reagent solution was a 1:1 molar ratio of Cap1 and Cap2, where Cap1 was a 20% (v / v) N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 was a 20% (v / v) acetic anhydride acetonitrile solution; the molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

[0219] The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water / pyridine mixed solvent (water to pyridine volume ratio 1:9).

[0220] After the last nucleoside monomer was ligated, the nucleic acid sequence ligated on the solid-phase support was sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the positive strand, wherein:

[0221] The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25% (w / w) ammonia solution at a concentration of 0.5 mL / μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL / μmol triethylamine and 0.6 mL / μmol triethylamine trifluoride were added to deprotect the 2'-O-TBDMS protection on the ribose.

[0222] Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio of 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixture (water to acetonitrile volume ratio of 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reverse chromatographic purification column. Desalting conditions included using a dextran gel column (g25 dextran gel) and elution with deionized water.

[0223] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the compound was conjugated to the 3' end of the positive strand of the siRNA.

[0224] (4-2) Synthesis of antisense strand (AS)

[0225] The antisense chain was synthesized using a general solid-phase support. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting conditions of the solid-phase synthesis method of the antisense chain are the same as those of the synthesis of the sense chain in step (4-1).

[0226] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the siRNA antisense strand had been obtained.

[0227] (4-3) Synthesis of siRNA conjugates

[0228] 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°C. The mixture was then slowly cooled to room temperature and kept at room temperature for 10 minutes to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonds, thereby obtaining the siRNA conjugate with the sense and antisense strands shown in Table 4.

[0229] UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmCmUmAmAmAm, as shown in SEQ ID NO:1;

[0230] UmsUfsUmAmGmAfGmUmGmAmGmGmAmUfUmAfAmAmAmsUmsGm, as shown in SEQ ID NO:2;

[0231] CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm, as shown in SEQ ID NO:3;

[0232] UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsUmsUm, as shown in SEQ ID NO:4;

[0233] CmsAmsGmAmCmAmGfAfCfAmAmGmAmCmCmAmUmCmUm, as shown in SEQ ID NO:5;

[0234] AmsGfsAmUmGmGfUmCmUmUmGmUmCmUfGmUfCmUmGmsGmsAm, as shown in SEQ ID NO:6.

[0235] When the vector conjugated to the 3' end of the siRNA's positive strand is CR01004, the structural formula of the siRNA conjugate is as follows:

[0236]

[0237] When the vector conjugated to the 3' end of the siRNA's positive strand is CR01005, the structural formula of the siRNA conjugate is as follows:

[0238]

[0239] When the vector conjugated to the 3' end of the siRNA's positive strand is CR01007, the structural formula of the siRNA conjugate is:

[0240]

[0241] Table 4 Sequence information of siRNA conjugates

[0242]

[0243] Unless otherwise specified, the base composition and modifications described in the embodiments of this disclosure have the following meanings: uppercase letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide represented by the preceding letter is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide represented by the preceding letter is a fluorinated nucleotide; and lowercase letter s indicates that the nucleotides represented by the preceding and following letters are linked by a thiophosphate bond.

[0244] Unless otherwise stated, all siRNA sequences used in this disclosure were synthesized by Suzhou Beixin Biotechnology Co., Ltd.; all PCR primers used in this disclosure were synthesized by Beijing Qingke Biotechnology Co., Ltd.; and all C57BL / 6J mice used in this disclosure were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0245] Table 5. Detection results of siRNA conjugates

[0246]

[0247]

[0248] As can be seen from the data in Figure 5, the sense strand (SS) and the antisense strand (AS) can be attached to the ligand in a good manner and with high purity.

[0249] General Experiment

[0250] Experimental Example 1: In vivo toxicity test of siRNA conjugate

[0251] C57BL / 6J mice were randomly divided into three groups (RZ899039, RZ899017, RZ899021, RZ897011, RZ897005, RZ897006, RZ802012, and RZ802013), with two mice in each group (half male and half female). Each group was administered a single subcutaneous injection of 300 mg / kg body weight (based on siRNA) of the siRNA conjugate. Mice were observed for 14 consecutive days. No animal deaths or adverse drug reaction-related clinical symptoms were observed. Gross necropsy of the mice at the end of the observation period revealed no abnormalities. Therefore, these results indicate that the disclosed siRNA conjugate has good safety and low animal-level toxicity.

[0252] Experimental Example 2: Method for Assessing Target Gene Repressive Activity in Mice

[0253] Six- to eight-week-old C57BL / 6J mice (all female) were randomly divided into groups based on body weight. The drug dosage for each group was calculated based on body weight and administered via a single subcutaneous injection. Each siRNA conjugate was prepared into a solution of the appropriate concentration (based on siRNA) using PBS solution for administration, with a dosage of 5 ml / kg (based on mouse body weight). The PBS control group received 5 ml / kg (based on mouse body weight) of PBS solution (without drug conjugates). The day of administration was designated as Day 1 (D1). At a predetermined time after administration, five mice from each group were sacrificed. The sacrificed mice were grossly dissected, and liver tissue was collected from each mouse. The liver tissue was cut into pieces approximately 2 mm in size. 3 Small pieces, stored using RNA Later.

[0254] Liver tissue samples were taken from different experimental groups at different time points from the RNA later sample. The liver tissue samples were homogenized in a Tissuelyser II fully automated tissue homogenizer for 60 seconds, and then total RNA was extracted using a fully automated 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 procedure for total RNA extraction.

[0255] Take 1 μg of total RNA, and use a reverse transcription kit (Promega, Reverse Transcription System, A3500) with Oligo(dT)15 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the instructions of the reverse transcription kit and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain a cDNA solution. Then, use a real-time quantitative PCR kit (ABI, SYBR). TM Select Master Mix (Catalog number: 4472908) was used to detect the expression level of target gene mRNA in liver tissue. In this real-time quantitative PCR method, primers targeting the target gene and primers targeting the internal reference gene were used to detect the target gene and the internal reference gene, respectively. A 20 μL Real-time PCR reaction system was prepared for each PCR detection well according to the instructions of the real-time quantitative PCR kit. Each reaction system contained 5 μL of cDNA solution obtained from the above reverse transcription reaction and 10 μL of SYBR. TM Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of RNase-Free H2O. Place the prepared reaction mixture in a real-time quantitative PCR instrument (ABI, StepOnePlus).TM Real-time PCR amplification was performed using a three-step method. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated for 40 cycles. In this real-time quantitative PCR method, the ΔΔCt method was used to calculate the relative quantitative levels and inhibition rates of the target gene mRNA in each test group. The calculation method is as follows:

[0256] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0257] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0258] ΔCt(test group) = ΔCt(test group) – ΔCt(control group average)

[0259] ΔCt(control group) = ΔCt(control group) – ΔCt(control group average)

[0260] Here, ΔCt (control group mean) is the arithmetic mean of the ΔCt (control group) values ​​of the five mice sacrificed at the same time point in the control group. Therefore, each mouse in both the test group and the control group corresponds to a ΔCt value.

[0261] Using the control group as a baseline, the expression level of the target gene mRNA in the test group was normalized, and the expression level of the target gene mRNA in the control group was defined as 100%.

[0262] The relative expression level of the target gene mRNA in the test group = 2 - ΔΔCt(test group) × 100%

[0263] The inhibition rate of target gene mRNA expression in the test group = (1 – relative expression level of target gene mRNA in the test group) × 100%

[0264] Unless otherwise stated, all in vivo activity data are expressed as X±SD, and all data were plotted and analyzed using GraphPadprism 8.0 software.

[0265] Example 2.1 In vivo activity assessment of superoxide dismutase 1 (SOD1) siRNA conjugated to CR01005 and CR01007 vectors

[0266] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of siRNA sequences RZ899017 (3' end conjugated to the CR01005 vector) and RZ899021 (3' end conjugated to the CR01007 vector) on the target gene SOD1 in mice. CR01005 has a different branching structure compared to CR01007.

[0267] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of ten mice each, based on body weight. Each group was administered either PBS solution or the siRNA conjugate mentioned in this embodiment via subcutaneous abdominal administration. In the siRNA conjugate group, the dosage was 3 mg / kg (based on siRNA), with an administration volume of 5 mL / kg body weight. The PBS control group received 5 mL / kg body weight of PBS solution without the siRNA conjugate. The day of administration was designated as day 1 (D1). Five mice from each group were sacrificed on day 15 (D15) and day 29 (D29). Liver tissue was collected for RNA extraction, reverse transcription, and Real-time PCR detection. The relative quantification of target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0268] Table 6 Primer sequence listing for Example 2.1

[0269]

[0270] The results of this embodiment show that both the siRNA conjugate RZ899017 conjugated to the CR01005 vector and the siRNA conjugate RZ899021 conjugated to the CR01007 vector can effectively inhibit the target gene. At day 15, the highest inhibitory activity of RZ899017 reached 95.43%, and that of RZ899021 reached 86.41%. At day 29, the inhibitory activity of RZ899017 was 76.75%, and that of RZ899021 was 76.36%. Therefore, the siRNA conjugate RZ899017 of CR01005 is superior to the siRNA conjugate RZ899021 of CR01007. Figure 1 (Table 7).

[0271] Table 7 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 2.1.

[0272]

[0273]

[0274] Example 2.2 In vivo activity assessment of angiopoietin-like 3 (ANGPTL3) siRNA conjugated to CR01005 and CR01007 vectors

[0275] In this embodiment, the inhibitory activity of siRNA sequences RZ897005 (3' end conjugated to CR01005 vector) and RZ897006 (3' end conjugated to CR01007 vector) on the target gene ANGPTL3 in mice was evaluated using a mouse in vivo target gene inhibitory activity assessment method.

[0276] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of ten mice each, based on their body weight. Each group was administered the siRNA conjugate described in this embodiment via subcutaneous abdominal administration, while the control group received 5 mL / kg of PBS solution without the siRNA conjugate. The day of administration was designated as day 1 (D1). Five mice from each group were sacrificed on day 15 (D15) and day 29 (D29). Liver tissue was collected for RNA extraction, reverse transcription, and Real-time PCR detection. The relative quantification of the target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0277] Table 8 Primer sequence listing for Example 2.2

[0278]

[0279] The results of this embodiment show that both the siRNA conjugate RZ897005 conjugated to the CR01005 vector and the siRNA conjugate RZ897006 conjugated to the CR01007 vector can inhibit the target gene. The activity of the CR01005 conjugate RZ897005 is superior to that of the CR01007 conjugate RZ897006, and the highest inhibitory activity of RZ897005 at day 15 reaches 84.44%. Figure 2 (Table 9).

[0280] Table 9 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 2.2.

[0281]

[0282] Example 2.3 In vivo activity assessment of complement component 3 (CC3) siRNA conjugated to CR01005 and CR01007 vectors.

[0283] In this embodiment, the inhibitory activity of the siRNA sequence RZ802012 (3' end conjugated to the CR01005 vector) and the siRNA sequence RZ802013 (3' end conjugated to the CR01007 vector) on the target gene CC3 in mice was evaluated using a mouse in vivo target gene inhibitory activity assessment method.

[0284] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of five mice each, based on body weight. Each group was administered the siRNA conjugate described in this embodiment via subcutaneous abdominal administration, while the control group received 5 mL / kg of PBS solution without the siRNA conjugate. The day of administration was designated as day 1 (D1), and mice were sacrificed on day 8 (D8). Liver tissue was collected for RNA extraction, reverse transcription, and real-time PCR detection. The relative quantification of the target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0285] Table 10 Primer sequence listing for Example 2.3

[0286]

[0287] The results of this embodiment show that both the siRNA conjugate RZ802012 conjugated to the CR01005 vector and the siRNA conjugate RZ802013 conjugated to the CR01007 vector can effectively inhibit the target gene CC3, with RZ802012 exhibiting relatively higher inhibitory activity, reaching 95.59%. Figure 3 (Table 11).

[0288] Table 11 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 2.3.

[0289]

[0290] Example 2.4 In vivo activity assessment of SOD1 target siRNA conjugated to the CR01004 vector

[0291] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequence RZ899039, which is conjugated to the CR01004 vector at the 3' end of the siRNA positive strand, on the target gene SOD1 in mice.

[0292] Six- to eight-week-old C57BL / 6j mice were randomly divided into two groups of 15 mice each, based on body weight. Each group was administered the siRNA conjugate described in this embodiment via subcutaneous abdominal administration, while the control group received 5 mL / kg of PBS solution without the siRNA conjugate. The day of administration was designated as day 1 (D1). Five mice from each group were sacrificed on days 8 (D8), 29 (D29), and 43 (D43). Liver tissue was collected for RNA extraction, reverse transcription, and Real-time PCR detection. The relative quantification of target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0293] The primer sequences for this embodiment are shown in Table 6 of Example 2.1.

[0294] The results of this embodiment show that the CR01004 conjugate RZ899039 can produce sustained and effective inhibitory activity against the target gene, with the highest D15 inhibitory activity reaching 96.31%. Figure 4 (Table 12).

[0295] Table 12 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 2.4.

[0296]

[0297]

[0298] Example 2.5 In vivo activity assessment of ANGPTL3 target siRNA conjugated to the CR01004 vector

[0299] This embodiment uses a mouse in vivo target gene inhibitory activity assessment method to evaluate the inhibitory activity of the siRNA sequence RZ897011, which is conjugated to the CR01004 vector at the 3' end of the siRNA positive strand, on the target gene ANGPTL3 in mice.

[0300] Six- to eight-week-old C57BL / 6j mice were randomly divided into two groups of 15 mice each, based on body weight. Each group was administered the siRNA conjugate described in this embodiment via subcutaneous abdominal administration, while the control group received 5 mL / kg of PBS solution without the siRNA conjugate. The day of administration was designated as day 1 (D1). Five mice from each group were sacrificed on days 8 (D8), 29 (D29), and 43 (D43). Liver tissue was collected for RNA extraction, reverse transcription, and Real-time PCR detection. The relative quantification of target gene mRNA in each test group was performed using the aforementioned ΔΔCt method.

[0301] The primer sequences for this embodiment are shown in Table 8 of Example 2.2.

[0302] The results of this embodiment show that the CR01004 conjugate RZ897011 can produce sustained and effective inhibitory activity against the target gene, with the highest D15 inhibitory activity reaching 88.87%. Figure 5 (Table 13).

[0303] Table 13 shows the inhibitory activity of the target gene in mice after administration of the siRNA conjugate described in Example 2.5.

[0304]

[0305] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of this disclosure, and this disclosure is not limited thereto. Various modifications and improvements can be made by those skilled in the art without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A GalNAc derivative or a pharmaceutically acceptable salt thereof as shown in formula (I): in, A is selected from ; n is selected from 0 or 1, m is selected from 0 or 1, and n = m; Each L1 is independently selected from an unsubstituted C1-C5 alkylene group; Each L2 is independently selected from unsubstituted C2-C. 10 Alkylene; Y is selected from O; Each R3 is independently selected from H or C1-C4 alkyl acyl groups; Both p and q are selected from 1; R1 is selected from DMTr or ; R represents the linking site used to connect oligonucleotides. 1a It is a hydroxyl group; R2 is selected from H or , Selected from resin or glass spheres with controllable aperture.

2. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, A is selected from .

3. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Each L1 is independently selected from an unsubstituted C1-C5 straight-chain alkylene group.

4. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Each L2 is independently selected , , , , , , , or .

5. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Each L2 is selected from .

6. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Each R3 is independently selected from H, , , , , , , or .

7. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Each R3 is independently selected from H or .

8. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Each R3 is selected from H.

9. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The oligonucleotides are selected from single-stranded oligonucleotides and double-stranded oligonucleotides.

10. The GalNAc derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The oligonucleotides are selected from siRNA.

11. A GalNAc derivative or a pharmaceutically acceptable salt thereof, characterized in that, The GalNAc derivative is selected from any of the following compounds: ； This represents a linkage site for linking oligonucleotides, which are selected from single-stranded and double-stranded oligonucleotides. Selected from resin or glass spheres with controllable aperture.

12. Conjugates as shown in formula (III) or pharmaceutically acceptable salts thereof: in, Nu stands for oligonucleotide; k is selected from 1, 2, 3, or 4; The structural formula of Q is selected from ,A,n,m,L1,L2,Y,p,q,R 1a , As defined in any one of claims 1-4.

13. The conjugate or a pharmaceutically acceptable salt thereof according to claim 12, characterized in that, The structural formula of Q is selected from .

14. The conjugate or a pharmaceutically acceptable salt thereof according to claim 13, characterized in that, The oligonucleotides are selected from siRNA.

15. The conjugate or a pharmaceutically acceptable salt thereof according to claim 13, characterized in that, k is selected from 1, the oligonucleotide is selected from siRNA, and one of the Q is conjugated to the 3' end of the positive strand of the siRNA.

16. A conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The conjugate is selected from any of the following compounds: ； Nu stands for oligonucleotide.

17. A composition, characterized in that, The composition comprises the conjugate of any one of claims 12-16 or a pharmaceutically acceptable salt thereof.

18. The composition according to claim 17, characterized in that, The composition further comprises, optionally, one or more pharmaceutically acceptable carriers.

19. Use of the GalNAc derivative of any one of claims 1-11 or a pharmaceutically acceptable salt thereof, and / or the conjugate of any one of claims 12-16 or a pharmaceutically acceptable salt thereof, and / or the composition of any one of claims 17-18 in the preparation of a medicament for the prevention and / or treatment of pathological conditions or diseases caused by abnormal expression of target genes in liver cells.

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