Double-stranded sirna for inhibiting pnpla3 expression and conjugate comprising same

Abstract

A double-stranded siRNA for inhibiting PNPLA3 expression and a conjugate comprising same. The double-stranded siRNA comprises a sense strand and an antisense strand, wherein one or more nucleotide residues in a nucleotide sequence of the sense strand and / or the antisense strand are replaced with r1 represented by formula I: formula (I). The introduction of a non-natural nucleotide monomer into the double-stranded siRNA improves the target selectivity and activity of the siRNA sequence, reducing the off-target risk while maintaining high drug activity. Also provided is a tetravalent GalNAc, making it easier to scale up synthesis and reduce costs.

Description

Double-stranded siRNA for inhibiting PNPLA3 expression and its conjugate

[0001] This application claims the priority benefit of the Chinese invention patent application with application date of November 8, 2023, application number: 202311483201.7, and invention name: "Double-stranded siRNA for inhibiting PNPLA3 expression and its conjugates". Technical Field

[0002] The present invention belongs to the field of RNA interference, and particularly relates to double-stranded siRNA for inhibiting PNPLA3 expression and a conjugate thereof. Background Art

[0003] Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease caused by excessive fat accumulation in the liver. This leads to chronic liver inflammation, which can progress to non-alcoholic steatohepatitis (NASH), triggering progressive liver fibrosis and cirrhosis, ultimately leading to liver failure, cancer, and death. Epidemiological studies show that the incidence of NAFLD has increased annually with changes in diet and lifestyle. NAFLD affects 25% of adults worldwide, 10-20% of NAFLD cases can progress to NASH, and 15-25% of NASH cases can progress to cirrhosis. In China alone, the number of people with NAFLD was estimated to be 240 million in 2016, and by 2030, this number is expected to increase to approximately 310 million. The number of NASH patients is expected to increase from 48 million to 62 million, making NASH a more prevalent chronic liver disease worldwide.

[0004] The human PNPLA3 gene is located on chromosome 22q13.31 and belongs to the PNPLA3 family. The proteins encoded by these genes contain a highly conserved patatin domain (Gly-X-Ser-X_Gly) at their N-termini, which confers nonspecific acyl hydrolase activity and promotes triglyceride hydrolysis. Human PNPLA3 is primarily expressed in hepatocytes, followed by skin and adipose tissue. At the subcellular level, it is primarily localized to the cell membrane and intracellular lipid droplets. PNPLA3 protein regulates intracellular lipids. Under normal physiological conditions, PNPLA3 primarily functions as a lipolytic enzyme. However, when lipid synthesis is inhibited, such as when triglyceride lipase is knocked out, PNPLA3 protein can synthesize triglycerides from triolein.

[0005] Human epidemiological and genome-wide association study (GWAS) analyses have shown that there is a significant association between liver fat content and the PNPLA3 gene (Meo et al. Nat Genet 2008; 40(12): 1461-1465); carriers of the PNPLA3-I148M variant corresponding to rs738409C>G have a significantly increased incidence of non-alcoholic liver disease, non-alcoholic hepatitis, cirrhosis, and hepatocellular carcinoma, and can increase the risk of liver-related death by 18 times.

[0006] The PNPLA3 protein functions as a hepatic triglyceride hydrolase. The I148M variant significantly reduces triglyceride hydrolase activity. In the presence of excess free fatty acids, overexpression of the PNPLA3-I148M variant slows the rate of intracellular triglyceride hydrolysis, promoting triglyceride deposition in the liver and ultimately leading to the development of fatty liver (Huang Y et al. J Biol Chem 2011;286:37085-37093). The PNPLA3-I148M variant also enhances its transacylase activity on phospholipids, thereby promoting cholesterol production. These two combined effects contribute to the PNPLA3-I148M variant's ability to promote lipid deposition, leading to the development of fatty liver (Pingitore P et al. BBA-Mol Cell Biol L 2019;1864:900-906). In animal experiments, knocking in the human PNPLA3-148M gene caused mice to overexpress the human PNPLA3-148M mutant protein, resulting in a significant increase in liver fat content and significant accumulation of the PNPLA3-148M mutant protein on lipid droplets in hepatocytes. This is because the mutation of amino acid 148 of PNPLA3 to methionine inhibits the ubiquitination and degradation pathway of PNPLA3, leading to the accumulation of the mutant protein on lipid droplets and promoting fat accumulation (Smagris et al. Hepatology 2015; 61:108-118).

[0007] Currently, treatment for NAFLD focuses on weight loss and managing secondary conditions such as insulin resistance or dyslipidemia. To date, only one drug has been approved in India for this disease. Therefore, NAFLD remains an unmet clinical need, and the development of treatments specifically for patients with NAFLD is urgently needed.

[0008] Small interfering RNA (siRNA) drugs are gradually becoming a mature therapeutic approach. Currently, five RNA drugs targeting PNPLA3 are in Phase I clinical trials, including an ASO developed by Ionis and four siRNA drugs each developed by Alnylam, Arrowhead, Amgen, and Dicerna Pharmaceuticals. Among them, Arrowhead's Phase I clinical results for ARO-PNPLA3 showed that after a single dose, patients with homozygous I148M mutations experienced a dose-dependent reduction in liver fat, with an average reduction of 40%. Regarding safety, no significant treatment-emergent increases in triglycerides or low-density lipoprotein cholesterol were observed. Reported adverse events (AEs) were primarily mild, with no AEs leading to treatment or study discontinuation.

[0009] There is still a need in the art for small interfering nucleic acid (siRNA) drugs for inhibiting PNPLA3 expression.

[0010] Summary of the Invention

[0011] In order to further improve the siRNA drug targeting PNPLA3 and increase its drugability, such as further improving the efficacy, reducing off-target effects, and reducing the difficulty and cost of production, the inventors have developed a novel double-stranded siRNA and GalNAc-siRNA conjugate drug. The double-stranded siRNA of the present invention introduces non-natural nucleotide monomers (Hu et al. Signal Transduct Target Ther 2020 Jun 19; 5(1): 101.), which increases the target selectivity and activity of the siRNA sequence, while maintaining high drug activity while reducing the risk of off-target. The inventors also developed a novel, symmetrical tetravalent GalNAc, making it easier to scale up synthesis and reduce costs.

[0012] In one aspect, a double-stranded siRNA is provided, comprising a sense strand and an antisense strand,

[0013] The antisense strand comprises the nucleotide sequence of SEQ ID NO: 12 (5'-ACAUAAGACAUUAUCCUAAUG-GG-3'), or a nucleotide sequence in which one or more nucleotides are substituted, added, deleted, or inserted into the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 12, and / or

[0014] the sense strand comprises the nucleotide sequence of SEQ ID NO: 13 (5'-CAUUAGGAUAAUGUCUUAUGU-3'), or a nucleotide sequence in which one or more nucleotides are substituted, added, deleted, or inserted into the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 13;

[0015] One or more nucleotide residues in the nucleotide sequence of the sense strand and / or antisense strand are replaced with r1 having formula I:

[0016] Wherein: X1, Y1 and Z1 are independently CH or N, and at least one of X1, Y1, Z1 is N;

[0017] R 1 is H, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkoxy, optionally substituted C2-C 10 Preferably, the alkyl group is C1-C6 alkyl; preferably, the alkoxy group is C1-C6 alkoxy; preferably, the alkenyl group is C2-C6 alkenyl; preferably, the substitution is with one or more groups selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, hydroxy, oxo, fluorine, chlorine, bromine or iodine, more preferably, R 1 is H or C1-C3 alkoxy optionally substituted by methoxy or C2 alkenyl.

[0018] In one embodiment, the antisense strand comprises a nucleotide sequence in which 1-10, 1-5, 1-3 or 1-2 nucleotides are substituted, added, deleted or inserted into the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 12, and / or the sense strand comprises a nucleotide sequence in which 1-10, 1-5, 1-3 or 1-2 nucleotides are substituted, added, deleted or inserted into the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: The nucleotide sequence of NO:13 has a nucleotide sequence (5'-CAUUAGGAUAAUGUCUUAUGU-3') that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

[0019] In one embodiment, r1 is selected from the following structures:

[0020] Preferably, r1 is

[0021] In one embodiment, one or more or all nucleotides of the antisense strand and / or one or more or all nucleotides of the sense strand are modified nucleotides.

[0022] In one embodiment, the modification is selected from a methoxy modification, a fluoro modification, a phosphorothioate linkage, replacement of a nucleotide with a glycerol nucleic acid, an (E)-vinyl phosphate or a 2'-deoxynucleotide; preferably, wherein the methoxy modification is a 2'-O-methyl modification; preferably, wherein the fluoro modification is a 2'-fluoro modification.

[0023] In one embodiment, the antisense strand comprises two phosphorothioate linkages between the three terminal nucleotides at the 3' end and two phosphorothioate linkages between the three terminal nucleotides at the 5' end.

[0024] In one embodiment, the sense strand comprises two phosphorothioate linkages between the three terminal nucleotides at the 5' end.

[0025] In one embodiment, r1 is linked to the adjacent nucleotide residue via a phosphate or phosphorothioate bond.

[0026] In one embodiment, the r1 substitution is made at nucleotide residue 7, 8, or 9 of the antisense strand in a 5' to 3' manner.

[0027] In one embodiment, the r1 substitution is made at nucleotide residue 2, 3, 16, 18, 19, or 20 of the sense strand in a 5' to 3' manner.

[0028] In one embodiment, one or more nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides, in a 5' to 3' manner.

[0029] In one embodiment, one or more nucleotides at positions 7 and 8-11 of the sense strand are fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides, in a 5' to 3' manner.

[0030] When r1 occupies the above-mentioned position, the position is not subjected to the corresponding fluorinated modification or 2'-O-methyl modification.

[0031] In one embodiment, the antisense strand comprises a nucleotide sequence selected from the group consisting of:

[0032] Antisense strand

[0033] 5'-mAs-fCs-mA-mU-mA-fA-mG-fA-fC-mA-mU-mU-mA-fU-mC-fC-mU-mA-mA-mU-mGs-mGs-mG-3';

[0034] 5'-mAs-fCs-mA-mU-mA-fA-r1-fA-fC-mA-mU-mU-mA-fU-mC-fC-mU-mA-mA-mU-mGs-mGs-mG-3';

[0035] 5'-mAs-fCs-mA-mU-mA-fA-mG-r1-fC-mA-mU-mU-mA-fU-mC-fC-mU-mA-mA-mU-mGs-mGs-mG-3';

[0036] 5'-mAs-fCs-mA-mU-mA-fA-mG-fA-r1-mA-mU-mU-mA-fU-mC-fC-mU-mA-mA-mU-mGs-mGs-mG-3'.

[0037] In one embodiment, the sense strand comprises a nucleotide sequence selected from the group consisting of:

[0038] mCs-r1s-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-m A-mU-mG-mU;

[0039] mCs-mAs-r1-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-mA-mU-mG-mU;

[0040] mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-r1-mU-mA-mU-mG-mU;

[0041] mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-r1-mU-mG-mU;

[0042] mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-mA-r1-mG-mU;

[0043] mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-mA-mU-r1-mU;

[0044] mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-mA-mU-mG-mU;

[0045] Wherein, mA, mU, mG, and mC are 2'-O-methyl-modified adenosine-3'-phosphate, uridine-3'-phosphate, guanosine-3'-phosphate, and cytidine-3'-phosphate, respectively; fA, fU, fG, and fC are 2'-fluoro-modified adenosine-3'-phosphate, uridine-3'-phosphate, guanosine-3'-phosphate, and cytidine-3'-phosphate, respectively; and s is a phosphorothioate bond.

[0046] In one embodiment, the double-stranded siRNA comprises the sense and antisense strand pairs of any one of SEQ ID NOs. 1-27:

[0047] Table 1

[0048] In one embodiment, the double-stranded siRNA comprises a sense and antisense strand pair of any one of:

[0049] In another aspect, a conjugate is provided, comprising the double-stranded siRNA described herein.

[0050] In one embodiment, the conjugate comprises a liver targeting moiety, preferably the liver targeting moiety comprises N-acetylgalactosamine; preferably wherein the liver targeting moiety comprises 4-valent N-acetylgalactosamine.

[0051] In one embodiment, the conjugate consists of a negative ion and a positive ion;

[0052] in,

[0053] X - O - or S - ;

[0054] M z+ is a pharmaceutically acceptable positive ion;

[0055] The negative ions The total valence of is equal to that of the positive ion;

[0056] RNA is a double-stranded siRNA according to any one of claims 1 to 8;

[0057] for for or, for for

[0058] for

[0059] A is or connecting keys;

[0060] for

[0061] k is 2;

[0062] When A is a connecting bond, A is located in L 2 meta;

[0063] When A is When A is located at L 2 Para or meta position;

[0064] n1, n2, n3, n4, n5, n6 and n7 are independently 1, 2, 3, 4, 5 or 6;

[0065] m1 and m2 are independently 0, 1, 2, 3, 4 or 5;

[0066] q1 and q2 are independently 1, 2, 3, 4 or 5.

[0067] In one embodiment, the conjugate satisfies one or more of the following conditions:

[0068] (1) n1 is 1, 2, 3, 4 or 5; preferably, n1 is 1, 2 or 3;

[0069] (2) n2 is 1, 2, 3 or 4; preferably, n2 is 1 or 2;

[0070] (3) n3 is 1, 2, 3, 4 or 5; preferably, n3 is 3;

[0071] (4) n4 is 1, 2, 3 or 4; preferably, n4 is 2;

[0072] (5) n5 is 1, 2, 3, 4, 5 or 6; preferably, n5 is 4;

[0073] (6) n6 is 1, 2, 3, 4 or 5; preferably, n6 is 3;

[0074] (7) n7 is 1, 2 or 3; preferably, n7 is 1;

[0075] (8) m1 is 1 or 2; preferably, m1 is 1;

[0076] (9) m2 is 0, 1, 2, 3, 4 or 5; preferably, m2 is 0, 1, 2 or 3;

[0077] (10) q1 is 1, 2, 3 or 4; preferably, q1 is 2;

[0078] (11) q2 is 1, 2, 3, 4 or 5; preferably, q2 is 3; and

[0079] (12)X - O - ;

[0080] (13)M z+ is a metal cation or an organic base cation; preferably, the metal cation is an alkali metal or alkaline earth metal cation, such as Na + , K + or Ca 2+ , the organic base cation may be an ammonium cation, for example

[0081] In one embodiment, the conjugate satisfies one or more of the following conditions:

[0082] (1) for Preferably, the for

[0083] (2) for Preferably, the for

[0084] (3) for

[0085] In one embodiment, the conjugate satisfies one or both of the following conditions:

[0086] (1) for Preferably, the for and

[0087] (2) for

[0088] In one embodiment, for

[0089] In one embodiment, the conjugate is any of the following structures:

[0090] In one embodiment, the conjugate is:

[0091] In one embodiment, the N-acetylgalactosamine ligand is

[0092] In one embodiment, the conjugate has the structure:

[0093] Wherein, X is O or S.

[0094] In another aspect, a pharmaceutical composition is provided, comprising the double-stranded siRNA and / or conjugate described herein and a pharmaceutically acceptable carrier.

[0095] In another aspect, provided is a use of a double-stranded siRNA, conjugate, or pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing a disease associated with PNPLA3 gene expression in a subject. In another aspect, provided is a method for treating and / or preventing a disease associated with PNPLA3 gene expression in a subject, comprising administering to the subject a double-stranded siRNA, conjugate, or pharmaceutical composition described herein. In another aspect, provided is a double-stranded siRNA, conjugate, or pharmaceutical composition described herein for treating and / or preventing a disease associated with PNPLA3 gene expression in a subject.

[0096] In one embodiment, the disease associated with PNPLA3 gene expression is chronic liver disease. In one embodiment, chronic liver disease is associated with the accumulation and / or expansion of lipid droplets in the liver. In one embodiment, chronic liver disease is selected from the group consisting of hepatitis, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic fatty liver disease (ALD), HBV, HCV-related cirrhosis, drug-induced liver injury, and hepatocellular necrosis. In one embodiment, the subject is a mammal, including but not limited to humans, cows, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs.

[0097] In another aspect, a cell is provided comprising a double-stranded siRNA and / or a conjugate described herein.

[0098] In another aspect, provided is a method for inhibiting PNPLA3 gene expression in a cell in vitro, comprising contacting the cell with a double-stranded siRNA and / or conjugate described herein.

[0099] Advantages of the present invention include:

[0100] (1) The siRNA of the present invention has good in vitro or in vivo inhibitory activity against PNPLA3 mRNA; and

[0101] (2) The tetravalent GalNAc group of the present invention has the same level of endocytosis efficiency as the reference compound L96, showing good binding affinity with ASGPR. DETAILED DESCRIPTION

[0102] Definition and Description

[0103] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. The absence of a specific definition for a particular term or phrase should not be construed as undefined or ambiguous, but rather should be understood as meaning understood by one of ordinary skill in the art. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0104] The term "and / or" should be interpreted as inclusive, ie, including at least one of a number or list of elements, but also including more than one, and optionally, additional unlisted items.

[0105] The term "PNPLA3" refers to patatin-like phospholipase domain-containing protein 3 (PNPLA3). It refers to genes encoding patatin-like phospholipase domain-containing protein 3 from any mammalian source, including, but not limited to, humans, cows, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs, unless specifically excluded. The term also refers to fragments and variants of native PNPLA3 that retain at least one in vivo or in vitro activity of native PNPLA3. The term encompasses the full-length, unprocessed precursor form of PNPLA3, as well as forms derived from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing.

[0106] The term "nucleic acid" includes any oligonucleotide or polynucleotide or polynucleotide, wherein the fragment containing up to 60 nucleotides is generally referred to as an oligonucleotide, and the longer fragment is referred to as a polynucleotide. Deoxyribonucleotides are composed of a 5-carbon sugar called deoxyribose, which is covalently linked to phosphate on the 5' and 3' carbons of this sugar to form an alternating unbranched polymer. DNA can be, for example, an antisense molecule, plasmid DNA, pre-condensed DNA, PCR product, vector, expression cassette, chimeric sequence, chromosomal DNA or a derivative and combination of these groups. Ribonucleotides are composed of similar repeating structures, in which the 5-carbon sugar is ribose. RNA can be, for example, small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, viral RNA (vRNA), self-amplification RNA (saRNA) and the form of a combination thereof. Therefore, in the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotides or nucleoside monomers composed of naturally occurring bases, sugars and intersugar (backbone) links. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers comprising non-natural monomers or parts thereof with similar functions. Such modified or substituted oligonucleotides are generally superior to natural forms due to their properties, such as enhanced cellular uptake, reduced immunogenicity and increased stability in the presence of nucleases. The term "single-stranded oligonucleotide" described in the present disclosure refers to a single-stranded oligonucleotide having a sequence that is at least partially complementary to a target mRNA, which can hybridize to the target mRNA by hydrogen bonds under mammalian physiological conditions (or a comparable in vitro environment). In some embodiments of the present disclosure, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide (ASO). Contemplated herein are nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any specific nucleotide sequence described herein (e.g., any nucleotide sequence in Table 1, or SEQ ID NO: 12 or 13), or nucleotide sequences in which 1-10, 1-5, 1-3, or 1-2 nucleotides are substituted, added, deleted, or inserted. In the context of two or more nucleic acids, the term "identity" percentage refers to two or more identical sequences or subsequences or sequences or subsequences having a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Examples of algorithms suitable for determining sequence identity percentages and sequence similarity are BLAST and BLAST 2.0 algorithms.

[0107] The term “one or more” can refer to any suitable number, such as 1-30, 1-20, or 1-10, or any number in between (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

[0108] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues. In certain embodiments, a trivalent or tetravalent targeting ligand as described herein can be conjugated to a nucleic acid. In certain embodiments, a nucleic acid is a nucleic acid as described herein. For example, the nucleic acid used herein can be a single-stranded DNA or RNA, or a double-stranded DNA or RNA, or a DNA-RNA hybrid. Examples of double-stranded RNA are described herein, and include, for example, siRNA and other siRNAs, such as aiRNA and precursor miRNA. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides.

[0109] In certain embodiments, nucleic acid is an oligonucleotide. In specific embodiments, the length of the oligonucleotide is in the scope of about 10 to about 100 nucleotide. In various related embodiments, the length of strand, double-stranded and triple-stranded oligonucleotide can be in the scope of about 10 to about 60 nucleotide, about 15 to about 60 nucleotide, about 20 to about 50 nucleotide, about 15 to about 30 nucleotide or about 20 to about 30 nucleotide.

[0110] In certain embodiments, the nucleic acid is an antisense molecule. In certain embodiments, the nucleic acid is a miRNA molecule. In certain embodiments, the nucleic acid is a siRNA.

[0111] As used interchangeably herein, the terms "siRNA," "RNAi," "siRNA agent," and "RNA interfering agent" refer to RNA containing, as those terms are defined herein, that mediates targeted cleavage of mRNA transcripts through the RNA-induced silencing complex (RISC) pathway. siRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). siRNA modulates (e.g., inhibits) expression of the PNPLA3 gene in cells (e.g., cells of a subject, such as a mammalian subject).

[0112] The short interfering RNA (siRNA) described herein is a type of double-stranded RNA molecule, 20 to 25 base pairs in length, similar to miRNA. It operates within the RNA interference (siRNA) pathway, interfering with the translation of mRNA of a specific gene with a complementary or proximal nucleotide sequence, leading to mRNA degradation. The short interfering RNA (siRNA) described herein includes double-stranded siRNA (including both sense and antisense strands) and single-stranded siRNA (antisense strand only).

[0113] The term "antisense strand" refers to the strand of an iRNA (e.g., an siRNA) that includes a region that is substantially complementary to a target sequence (e.g., a PNPLA3 mRNA). The term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence. Where the region of complementarity is not completely complementary to the target sequence, mismatches may occur within the interior or terminal regions of the molecule. Generally, the most tolerated mismatches occur within the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end of the siRNA. Typically, siRNAs are chemically synthesized. The term "sense strand" means an iRNA that includes a region that is substantially complementary to a region of the antisense strand as defined herein. The antisense and sense strands of an siRNA can be of the same or different lengths, as is known in the art.

[0114] Oligonucleotides comprising the siRNA molecules of the present invention can be synthesized using any of a variety of techniques known in the art. The synthesis of oligonucleotides utilizes common nucleic acid protection and coupling groups, such as a p-dimethoxytrityl group at the 5' end and a phosphoramidite at the 3' end. Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those skilled in the art.

[0115] siRNA molecules can be assembled from two different oligonucleotides, wherein one oligonucleotide comprises the sense strand of the siRNA and the other comprises the antisense strand of the siRNA. For example, each can be synthesized separately and linked together by hybridization or ligation after synthesis and / or deprotection.

[0116] As used herein, a "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides described using the sequence letters of the standard nucleotide nomenclature. When 5' and 3' appear at the ends of a sequence, they indicate the direction of the sequence. Sequences are typically written in a 5' to 3' direction; single-stranded RNA is synthesized in a 3' to 5' direction.

[0117] The “modification” of nucleotides described in the present disclosure includes but is not limited to alkoxy / methoxy modification, fluorination modification, phosphorothioate linkage, and the like.

[0118] In this disclosure, unless otherwise specified, the capital letters A, U, G, and C represent adenosine-3'-phosphate, uridine-3'-phosphate, guanosine-3'-phosphate, and cytidine-3'-phosphate. The capital letters fC, fG, fU, and fA represent fluorinated modifications at the 2' position of the nucleotide represented by the corresponding capital letter. The letters mA, mG, mC, and mU respectively represent methoxy modifications at the 2' position of the nucleotide represented by the corresponding capital letter; the "s" after the letter in the sequence indicates that the two adjacent nucleotide residues of the "s" are linked by a phosphorothioate group. For example, "As-G" indicates that the A and G residues are linked by a phosphorothioate group.

[0119] The fluorinated modified nucleotides disclosed herein refer to nucleotides in which the 2'-hydroxyl group of the ribose group is replaced by fluorine, and the methoxy-modified nucleotides refer to nucleotides in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0120] In the present disclosure, "complementary" has the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary or near-complementary manner. The purine base adenine (A) is always paired with the pyrimidine base uracil (U); the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair includes a purine and a pyrimidine. When adenine on one strand is always paired with uracil on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. The present invention utilizes non-natural bases with special advantages in complementarity or near-complementarity, such as improved selectivity and thus safety advantages.

[0121] The compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. This disclosure contemplates all such compounds, including (R)- and (S)-enantiomers, diastereomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of this disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are included within the scope of this disclosure.

[0122] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0123] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0124] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0125] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0126] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0127] The term "salt" refers to salts of compounds of the present disclosure, which are prepared by reacting the compounds of the present disclosure with relatively nontoxic acids or bases, having specific substituents. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds of the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds of the present disclosure contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0128] The term "pharmaceutical excipients" refers to excipients and additives used in the production of medicines and the preparation of prescriptions. It is all substances contained in pharmaceutical preparations in addition to the active ingredients.

[0129] Unless otherwise specified, when a group has one or more linkable sites, any one or more sites of the group can be linked to other groups via chemical bonds. The chemical bonds linking the sites to other groups can be represented by wavy lines. The wavy lines in the brackets indicate that the phenyl group is connected to other groups through the carbon atoms at positions 1 and 2 in the phenyl group.

[0130] The salts of the present disclosure can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0131] r1 structural unit

[0132] The r1 described herein represents the following structural unit:

[0133] Wherein: X1, Y1 and Z1 are independently CH or N, and at least one of X1, Y1, Z1 is N;

[0134] R 1 is H, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkoxy, optionally substituted C2-C 10 Preferably, the alkyl group is C1-C6 alkyl; preferably, the alkoxy group is C1-C6 alkoxy; preferably, the alkenyl group is C2-C6 alkenyl; preferably, the substitution is with one or more groups selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, hydroxy, oxo, fluorine, chlorine, bromine or iodine, more preferably, R 1 is H or C1-C3 alkoxy optionally substituted by methoxy or C2 alkenyl.

[0135] Preferably, r1 represents the following structural unit

[0136] The r1 described herein is a non-natural nucleotide, which is different from any natural nucleotide base disclosed in any patent, and its introduction into the nucleic acid sequence brings unexpected activity and selectivity advantages.

[0137] r1 and other nucleotide residues can be linked to each other through phosphate or phosphorothioate groups. For example, "mA*r1" indicates that mA and r1 nucleotide monomers are linked through a phosphorothioate group, and "mAr1" indicates that mA and r1 nucleotide monomers are linked through a phosphate group. The connection methods are as follows:

[0138] Wherein X is O or S.

[0139] In the present disclosure, double-stranded siRNA analogs comprise a sense strand or an r1-embedded sense strand and / or an antisense strand or an r1-embedded antisense strand. The sense strand, antisense strand, r1-embedded sense strand, and r1-embedded antisense strand all contain nucleotide groups as basic structural units. It is well known to those skilled in the art that a nucleotide group contains a phosphate group, a ribose group, and a base, which will not be described in detail here.

[0140] The r1-embedded sequence disclosed herein refers to a sequence in which at least one nucleotide residue is connected to r1. The r1-embedded sequence disclosed herein includes, but is not limited to, r1-embedded double-stranded siRNA, r1-embedded sense strand, and r1-embedded antisense strand. For example, 5'-mA-fG-fU-r1-r1-fAs-fC-3', 5'-r1-fG-mG-fA-fA-fC-3', and 5'-fA-fGs-fU-r1-fA-fA-mC-fC-mU-fC-r1-3' all fall under the r1-embedded sequence.

[0141] The double-stranded siRNA with r1 embedded in the present disclosure refers to a double-stranded siRNA in which at least one nucleotide residue is connected to r1, including a double-stranded siRNA in which a nucleotide residue is replaced by r1 in the double-stranded siRNA sequence. The sense strand with r1 embedded in the present disclosure refers to a sense strand in which at least one nucleotide residue is connected to r1, including a sense strand in which one or more nucleotides are replaced by r1. The antisense strand with r1 embedded in the present disclosure refers to an antisense strand in which at least one nucleotide residue is connected to r1, including a sense strand in which one or more nucleotides are replaced by r1.

[0142] siRNA and modified nucleotides

[0143] Provided herein are siRNAs for inhibiting PNPLA3 gene expression. Each siRNA comprises a sense strand and an antisense strand. The sense and antisense strands can each be 17-25 (e.g., 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In some embodiments, the sense and antisense strands of the siRNA are each independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length. In some embodiments, the siRNA duplex has approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In some embodiments, the sense and antisense strands are complementary to each other for at least 15, 16, 17, 18, 19, 20, or 21 nucleotides in length.

[0144] The antisense strand comprises the nucleotide sequence of SEQ ID NO:12 or the nucleotide sequence of SEQ ID NO:12, which replaces, adds, deletes or inserts one or more (e.g., 2-9, e.g., 2, 3, 4, 5, 6, 7) nucleotides. The positive strand comprises the nucleotide sequence of SEQ ID NO:13 or the nucleotide sequence of SEQ ID NO:13, which replaces, adds, deletes or inserts one or more (e.g., 2-9, e.g., 2, 3, 4, 5, 6, 7) nucleotides. One or more (e.g., 1-3, e.g., 1, 2, or 3) nucleotide residues in the nucleotide sequence of the positive strand and / or the antisense strand have r1 substitutions. One or more or all nucleotides of the antisense strand and / or one or more or all nucleotides of the positive strand can be modified nucleotides. Modification can be selected from methoxy modification, fluoro modification, thiophosphate linkage, replacement of nucleotides with glycerol nucleic acid, (E)-vinyl phosphate or 2'-deoxynucleotides. Preferably, the methoxy modification is a 2'-O-methyl modification; preferably, wherein the fluoro modification is a 2'-fluoro modification.

[0145] In one embodiment, the antisense strand comprises two phosphorothioate bonds between the three terminal nucleotides at the 3' end and two phosphorothioate bonds between the three terminal nucleotides at the 5' end. In one embodiment, the sense strand comprises two phosphorothioate bonds between the three terminal nucleotides at the 5' end. In one embodiment, R1 is connected to the adjacent nucleotide residues by a phosphate or phosphorothioate bond. In one embodiment, R1 is substituted at the 7th, 8th or 9th nucleotide residue of the antisense strand in a manner from the 5' end to the 3' end. In one embodiment, R1 is substituted at the 2nd, 3rd, 16th, 18th, 19th or 20th nucleotide residue of the sense strand in a manner from the 5' end to the 3' end. In one embodiment, one or more nucleotides at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in a manner from the 5' end to the 3' end are fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides. In one embodiment, in a 5' to 3' manner, one or more nucleotides at positions 7 and 8-11 of the sense strand are fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides. When r1 occupies a position mentioned above, that position is not fluoro-modified or 2'-O-methyl modified accordingly.

[0146] Preferably, the siRNA described herein has any pair of sense and antisense strands listed in Table 1.

[0147] Conjugate

[0148] The conjugates herein comprise the siRNA described herein and a liver targeting moiety. The type of liver targeting moiety is not particularly limited. Preferably, the liver targeting moiety comprises an N-acetylgalactosamine ligand. More preferably, the liver targeting moiety comprises a trivalent or tetravalent N-acetylgalactosamine ligand. The liver targeting moiety can be attached to the 3' end of the sense strand. The tetravalent N-acetylgalactosamine ligand described herein can be a tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugate group. The trivalent N-acetylgalactosamine ligand described herein can be Alnylam's L96.

[0149] The tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugated groups or trivalent GalNAc conjugated groups of the present disclosure can enhance the delivery of therapeutic agents to specific target locations (e.g., specific organs or tissues) within a subject such as a human or animal. In some embodiments of the present disclosure, the conjugated groups can enhance the targeted delivery of expression-inhibitory oligonucleotides. In some embodiments of the present disclosure, the tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugated groups or trivalent GalNAc conjugated groups can enhance the delivery of expression-inhibitory oligonucleotides (e.g., siRNA) to hepatocytes.

[0150] The tetravalent N-acetylgalactosamine substituted aromatic amide GalNAc conjugated groups or trivalent GalNAc conjugated groups described herein can be directly or indirectly linked to a compound, such as a therapeutic agent, for example, an expression inhibitory oligonucleotide ASO or siRNA, linked to the 3' or 5' end of the expression inhibitory oligonucleotide. In some embodiments of the present disclosure, the expression inhibitory oligonucleotide includes one or more modified natural or non-natural nucleotides. In some embodiments of the present disclosure, the expression inhibitory oligonucleotide is an siRNA agent, such as a double-stranded siRNA agent comprising a sense strand and an antisense strand. In some embodiments of the present disclosure, the tetravalent N-acetylgalactosamine substituted aromatic amide GalNAc conjugated groups disclosed herein are linked to the 5' end of the sense strand of the double-stranded siRNA agent. In some embodiments, the conjugated groups disclosed herein are linked to the expression inhibitory oligonucleotide agent at the 3' end of the sense strand of the double-stranded siRNA agent via a phosphate, phosphorothioate or phosphate group. The term "link" as used herein, when referring to a connection between two molecules, refers to two molecular fragments being linked by a covalent bond or two molecules being associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). The compounds and conjugated groups of the present invention may include one or more linking groups. The structure of each linking group may vary, provided that the conjugated group functions as described herein. For example, the structure of each linking group may vary in length and atomic composition, and each linking group may be branched, unbranched, cyclic, or a combination thereof. The linking group may also regulate the solubility, stability, or aggregation properties of the conjugate.

[0151] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0152] The solvents used in the present disclosure are commercially available.

[0153] Unless otherwise specified, the solvent ratios used in the column chromatography and preparative thin-layer silica gel chromatography disclosed herein are all volume ratios.

[0154] Example

[0155] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than for limiting the claims of the invention. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0156] Example 1: Synthesis of phosphoramidite monomer

[0157] Example 1-1: Synthesis of phosphoramidite monomer r1-M

[0158] Step 1 Synthesis of Intermediate 1-3

[0159] Compound 1-1 (30.0 g) and compound 1-2 (13.1 g) were added to 4-methylbenzenesulfonic acid (649 mg). The mixture was stirred at 100°C for 4 hours. LCMS (RT = 0.616 min) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to afford compound 1-3 (20.0 g, 55.0% yield) as a yellow solid.

[0160] LCMS (ESI) m / z: 386.1 [M+H] + ;

[0161] 1 HNMR (400MHz, CDCl3): δ8.40 (s, 1H), 6.04 (d, J = 3.42Hz, 1H), 5.69-5.8l (m, 1H), 5,54 (t ,J=5.38Hz,1H),4,42-4,5l(m,2H),4.16-4.30(m,IH),3,98(s,3H),2,05-2,18(m,9H).

[0162] Step 2 Synthesis of Intermediate 1-4

[0163] Compound 1-3 (15.0 g) was dissolved in methanol (100 mL), and triethylamine (11.8 g) was added. The reaction mixture was stirred at 70°C for 12 hours. LCMS (RT = 0.284 min) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford compound 1-4 (10.0 g, 89.1% yield) as a colorless oil.

[0164] LCMS (ESI) m / z: 260.1 [M+H] + ;

[0165] 1 HNMR (400MHz, CDCl3): δ8.87 (s, 1H), 5.93 (d, J = 3.42Hz, 1H), 4.48 (dd, J = 3.48, 4.83Hz, 1H), 4.33 (t, J = 5.2 6Hz, 1H), 4.10-4.16 (m, 1H), 3.95 (s, 3H), 3.84 (dd, J = 3.24, 12.29Hz, 1H), 3.70 (dd, J = 4.46, 12.29Hz, 1H).

[0166] Step 3 Synthesis of Intermediate 1-5

[0167] Compound 1-4 (15.0 g) was dissolved in N,N-dimethylformamide (100 mL), and imidazole (11.8 g) and 1,3-dichloro-1,1,3,3-tetraisopropyldimethylsilyl ether (20.0 g) were added. The reaction mixture was stirred at 25°C for 12 hours. LCMS (RT = 1.008 min) showed complete consumption of the starting material. The reaction mixture was added with water (100 mL) and ethyl acetate (80.0 mL, 50.0 mL) for extraction. The organic phase was washed with saturated sodium chloride solution (60.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to obtain compound 1-6 (15.0 g, 51.6% yield) as a colorless oil.

[0168] LCMS (ESI) m / z: 502.2 [M+H] + ;

[0169] 1 HNMR (400MHz, CDCl3): δ8,43(s,1H),5.95(s,1H),4.73(dd,,J=4.75,8.00Hz,1H),4.41(d,J=4.75Hz,1H),4.09-4 19(m,2H),3.94-4.03(m,4H),2.71-3.34(m,1H),1.01-1.15(m,28H).

[0170] Step 4 Synthesis of Intermediate 1-6

[0171] Compound 1-5 (13.0 g) was dissolved in N,N-dimethylformamide (90.0 mL), and silver oxide (16.0 g) and iodomethane (18.3 g) were added. After the addition, the reaction solution was stirred at 25°C for 3 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.43, raw material: Rf = 0.24) showed that the starting material was completely consumed. The reaction solution was added with water (100 mL) and ethyl acetate (100 mL) for extraction. The organic phase was washed with saturated sodium chloride solution (50.0 mL), added with anhydrous sodium sulfate, dried, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to obtain compound 1-6 (1.80 g, yield 13.4%) as a colorless oil.

[0172] 1 HNMR (400MHz, CDCl3): δ8.58(s,1H),5.91(s,1H), 4,46(dd,J=4.22,9,35Hz, 1H), 4.17-4.28(m,2′H), 3,96-4.06(m,5H), 3,68(s,3H), 0.99-1.13(m,2H).

[0173] Step 5 Synthesis of Intermediate 1-7

[0174] Compound 1-6 (200 mg) was dissolved in tetrahydrofuran (2.00 mL) and triethylamine trihydrofluoride (125 mg) was added at 0°C. The reaction mixture was stirred at 25°C for 12 hours. LCMS (RT = 0.253 min) indicated complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 1-7 (105 mg, crude) as a yellow oil.

[0175] LCMS (ESI) m / z: 274.1 [M+H] + ;

[0176] 1 HNMR (400MHz, CDCl3): δ8.88(s,1H),6.04(d,Jr=3.26Hz,1H),4.44(t,J=5.33Hz,1H),4.20(dd,J=333,4.8 3Hz,1H),4.07-4.14(m,1H),3.96(s,3H),3.84(dd,J=3.20,12.36Hz,1H),3.69(dd,J=4.39,12.30Hz,1H), 3.52(s,3H).

[0177] Step 6 Synthesis of Intermediate 1-8

[0178] Compound 1-7 (100 mg) was dissolved in pyridine (2.00 mL), and 4,4-dimethoxytrityl chloride (186 mg) was added at 0°C. The reaction mixture was stirred at 25°C for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.43, starting material: Rf = 0.05) showed complete consumption of the starting material. After cooling, the reaction mixture was extracted with water (5.00 mL) and ethyl acetate (5.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 1-8 (120 mg, crude) as a colorless oil.

[0179] LCMS (ESI) m / z: 574.2 [MH] + .

[0180] Step 7 Synthesis of the final product r1-M

[0181] Compound 1-8 (100 mg) was dissolved in dichloromethane (2.00 mL). Acetonitrile diisopropylphosphonic acid chloride (61.6 mg) and N,N-diisopropylethylamine (89.9 mg) were added at 0°C. The reaction mixture was stirred at 20°C for 2 hours. LCMS (RT = 2.367, 2.424 min) showed complete consumption of the starting material. The reaction mixture was purified by HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 50%-80%, 8 min) to afford r1-M (39.8 mg, 100% purity) as a white solid.

[0182] LCMS (ESI) m / z: 776.2 [M+H] + ;

[0183] 1H NMR(400MHz,DMSO-d6)δppm 1.00(br.d,J=6.40Hz,5H)1.10-1.16(m,7H)2.81(br.t,J=5.60Hz,2H)3.10-3.15(m,1H)3 .23-3.27(m,1H)3.43(s,3H)3.48-3.59(m,2H)3.74(s,6H)3.81(br.dd,J=7.20,6.40Hz,2H )3.85(s,3H)4.15-4.25(m,1H)4.37(br.d,J=3.20Hz,1H)4.60-4.72(m,1H)6.25(d,J=2.80 Hz, 1H) 6.83 (br.t, J = 8.40Hz, 4H) 7.17-7.27 (m, 7H) 7.33 (br.d, J = 6.40Hz, 2H) 9.01 (s, 1H).

[0184] Example 1-2: Synthesis of phosphoramidite monomer r2-M

[0185] Step 1 Synthesis of Intermediate 2-1

[0186] Compound 1-5 (2.00 g) was dissolved in tetrahydrofuran (20.0 mL), and ethyl propylene carbonate (1.42 g), 1,4-bis(diphenylphosphino)butane (153 mg), and tris(dibenzylideneacetone)palladium(0) (48.3 mg) were added. The reaction mixture was stirred at 65°C for 1 hour. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.43, starting material: Rf = 0.24) showed complete consumption of the starting material. The reaction mixture was extracted with water (10.0 mL) and ethyl acetate (10.0 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to obtain compound 2-1 (1.40 g, yield 64.8%) as a colorless oil.

[0187] LCMS (ESI) m / z: 542.3 [M+H]+;

[0188] Step 2 Synthesis of Intermediate 2-2

[0189] Compound 2-1 (1.40 g) was dissolved in tetrahydrofuran (20.0 mL), and triethylamine trihydrofluoride (832 mg) was added at 0°C. The reaction mixture was stirred at 20°C for 12 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.02, starting material: Rf = 0.43) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford compound 2-2 (770 mg, 99.5% yield) as a yellow oil.

[0190] Step 3 Synthesis of Intermediate 2-3

[0191] Compound 2-2 (500 mg) was dissolved in pyridine (5.00 mL), and 4,4-dimethoxytrityl chloride (849 mg) was added at 0°C. The reaction mixture was stirred at 25°C for 12 hours. LCMS (RT = 0.800 min) showed complete consumption of the starting material. After cooling, the reaction mixture was extracted with water (10.0 mL) and ethyl acetate (8.00 mL, 5.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 2-3 (700 mg, 69.6% yield) as a colorless oil.

[0192] LCMS (ESI) m / z: 602.2 [M+H]+;

[0193] Step 4 Synthesis of the final product r2-M

[0194] Compound 2-3 (700 mg) was dissolved in dichloromethane (5.00 mL). Tetraisopropyl acetonitrile diammonium phosphate (420 mg) and 4,5-dicyanoimidazole (137 mg) were added at 0°C. The reaction mixture was stirred at 20°C for 2 hours. LCMS (RT = 2.522, 2.568 min) showed complete consumption of the starting material. The reaction mixture was purified by HPLC (column: Waters Xbridge BEH C18 250*50mm*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 70%-90%, 10 min) to afford r2 (24.0 mg, 95.2% purity) as a white solid.

[0195] LCMS (ESI) m / z: 802.3 [M+H]+;

[0196] 1H NMR (400MHz, CDCl3) δppm 0.98-1.11(m,6H)1.17(d,J=6.80Hz,6H)1.44(s,1H)2.57-2.75(m,2H)3.21-3.31(m,1H)3.44(dd,J=12.00,2.80Hz,1H)3.58(dt, J=12.00,6.80Hz,2H)3.78-3.81(m,1H)3.82-3.93(m,1H)3.91(br.d,J=7.60Hz,1H)3.96(s,3H)4.03(s,1H)4.11(br.d,J=5.60Hz ,1H)4.21(br.d,J=5.60Hz,1H)4.35(br.d,J=3.20Hz,1H)4.48(br.d,J=12.00Hz,1H)4.60(t,J=4.40Hz,1H)5.13-5.28(m,2H)5.8 0-5.93(m,1H)5.99(d,J=4.40Hz,1H)6.81(d,J=8.40Hz,4H)7.20-7.25(m,2H)7.28-7.33(m,5H)7.41(d,J=7.20Hz,2H)8.40(s,1H)

[0197] Example 1-3: Synthesis of phosphoramidite monomer r3-M

[0198] Step 1 Synthesis of final product 3-1

[0199] Compound 2-1 (10.0 g) was dissolved in a mixed solution of dichloromethane (66.0 mL) and methanol (33.0 mL). Ozone was introduced at -78°C. After the addition, the reaction solution was stirred at -78°C for 5 minutes. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.60, raw material: Rf = 0.75) showed that the raw material was completely consumed. Oxygen was introduced for 15 minutes to remove excess ozone. Dimethyl sulfide (1.38 g) was then added and stirred at 25°C for 15 minutes. The reaction solution was concentrated under reduced pressure to obtain compound 3-1 (5.20 g, crude product) as a yellow oil.

[0200] Step 2 Synthesis of final product 3-2

[0201] Compound 3-1 (5.20 g) was dissolved in a mixture of dichloromethane (35.0 mL) and methanol (16.0 mL). Sodium borohydride (1.04 g) was added at -78°C. The reaction mixture was stirred at 25°C for 1 hour. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.45, starting material: Rf = 0.60) indicated complete consumption of the starting material. After cooling, the reaction mixture was extracted with aqueous ammonium chloride (30.0 mL, 20%) and ethyl acetate (50.0 mL). The organic phase was washed with saturated sodium chloride solution (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 3-2 (4.00 g, crude product) as a colorless oil.

[0202] Step 3 Synthesis of final product 3-3

[0203] Compound 3-2 (4.00 g) was dissolved in toluene (40.0 mL), and silver oxide (8.49 g) and iodomethane (5.20 g) were added. The reaction was stirred in a liquid-stifling vessel at 80°C for 12 hours. LCMS (RT = 1.041 minutes) showed that the starting material was completely consumed. The reaction solution was added with water (20.0 mL) and ethyl acetate (40.0 mL) for extraction. The organic phase was washed with saturated sodium chloride solution (20.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to obtain compound 3-3 (1.50 g, 36.6% yield) as a colorless oil.

[0204] LCMS (ESI) m / z: 560.3 [M+H]+;

[0205] Step 4 Synthesis of final product 3-4

[0206] Compound 3-3 (1.50 g) was dissolved in tetrahydrofuran (10.0 mL), and triethylamine trihydrofluoride (864 mg) was added at 0°C. The reaction mixture was stirred at 25°C for 12 hours. LCMS (RT = 0.457 min) indicated complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 3-4 (800 mg, crude) as a yellow oil.

[0207] LCMS (ESI) m / z: 318.2 [M+H]+;

[0208] Step 5 Synthesis of final product 3-5

[0209] Compound 3-4 (800 mg) was dissolved in pyridine (8.00 mL), and 4,4-dimethoxytrityl chloride (288 mg) was added at 0°C. The reaction mixture was stirred at 25°C for 1 hour. LCMS (RT = 1.824 minutes) indicated complete consumption of the starting material. After cooling, the reaction mixture was extracted with water (5.00 mL) and ethyl acetate (8.00 mL, 5.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 3-5 (1.10 g, crude) as a colorless oil.

[0210] LCMS (ESI) m / z: 620.3 [M+H]+;

[0211] Step 6 Synthesis of the final product r3-M

[0212] Compound 3-5 (1.10 g) was dissolved in dichloromethane (10.0 mL). 4,5-Dicyanoimidazole (210 mg) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (642 mg) were added at 0°C. The reaction mixture was stirred at 20°C for 2 hours. LCMS (RT = 0.803 min, 0.829 min) indicated complete consumption of the starting material. The reaction mixture was purified by HPLC (column: Waters Xbridge BEH C18 150*40 mm*10 μm; mobile phase: [water(ammonium bicarbonate)-acetonitrile]; B%: 55%-85% over 8 minutes) to afford r3 (574 mg, 99.6% purity) as a white solid.

[0213] LCMS (ESI) m / z: 820.3 [M+H]+;

[0214] 1H NMR(400MHz,DMSO-d6)δppm 0.97(d,J=6.80Hz,3H),1.04-1.24(m,8H),1.20-1.20(m,1H),2.57-2.64(m,1H),2.74-2.83(m,1H),2.92-3.04 (m,1H),3.18(d,J=5.60Hz,2H),3.23-3.28(m,1H),3.31(s,2H),3.42-3.48(m,2H),3.50-3.63(m,2H),3.65-3. 69(m,1H),3.71(d,J=2.00Hz,7H),3.75-3.82(m,1H),3.85(s,3H),4.22(dt,J=7.60,5.60Hz,1H),4.64(dd,J=4 .00,2.13Hz,1H),4.67-4.81(m,1H),6.14-6.28(m,1H),6.72-6.88(m,4H),7.11-7.34(m,9H),8.35-8.50(m,1H)

[0215] Using a similar method, Examples 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 were synthesized.

[0216] Example 1-4 Synthesis of phosphoramidite monomer r4-M

[0217] LCMS (ESI) m / z: 804.3 [M+H]+;

[0218] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.04-1.17(m,15H)1.17-1.23(m,3H)2.61(td,J=6.40,2.13Hz,1H)3.09- 3.15(m,1H)3.49-3.64(m,6H)3.68-3.70(m,1H)3.72(s,6H)3.73-3.75(m, 1H)4.16-4.33(m,3H)4.41-4.50(m,1H)4.53-4.62(m,1H)6.09-6.18(m,1 H)6.80-6.85(m,4H)7.18-7.26(m,8H)7.31-7.37(m,2H)8.93-9.01(m,1H)

[0219] Example 1-5 Synthesis of phosphoramidite monomer r5-M

[0220] LCMS (ESI) m / z: 776.4 [M+H]+;

[0221] 1H NMR (400MHz, DMSO-d6) δppm 1.12(d,J=6.40Hz,12H)2.60(td,J=6.00,2.31Hz,2H)3.01(dd,J=10.0,4.75 Hz,1H)3.39(s,3H)3.51-3.60(m,4H)3.72(s,7H)3.84-3.87(m,3H)4.18-4.28 (m,1H)4.45-4.52(m,1H)4.74-4.86(m,1H)6.21(d,J=3.20Hz,1H)6.79-6.83( m,4H)7.15-7.20(m,6H)7.21-7.25(m,3H)7.28-7.31(m,2H)8.43-8.45(m,1H)

[0222] Example 1-6 Synthesis of Phosphoramidite Monomer r6-M

[0223] LCMS (ESI) m / z: 802.3 [M+H]+;

[0224] 1H NMR (400MHz, CDCl3-d) δppm 1.04 (d, J = 6.80Hz, 3H) 1.09-1.23 (m,9H)2.38(t,J=6.40Hz,1H)2.65(br,d,J=10.80Hz,1H)3.08-3.19(m,1H)3.43(ddd,J=17.20,10.40 ,3.20Hz,1H)3.52-3.72(m,3H)3.79(d,J=3.60Hz,6H)3.82-3.91(m,1H)3.93(s,3H)4.03-4.19(m,2H)4 .30-4.42(m,1H)4.63-4.77(m,2H)5.07-5.26(m,2H)5.74-5.93(m,1H)6.24(t,J=3.60Hz,1H)6.79(t, J=8.40Hz,4H)7.16-7.26(m,3H)7.32(dq,J=7.20,4.25Hz,4H)7.39-7.48(m,2H)8.11(d,J=5.20Hz,1H)

[0225] Example 1-7 Synthesis of phosphoramidite monomer r7-M

[0226] LCMS (ESI) m / z: 802.3 [M+H]+;

[0227] 1H NMR (400MHz, CDCl3-d) δppm 1.01-1.09(m,2H)1.13-1.24(m,10H)2.41(t,J=6.40Hz,1H)2.53-2.73(m,1H)3.02-3.15(m,1H)3.25-3.36(m,1H)3.38 -3.52(m,1H)3.54-3.67(m,3H)3.67-3.77(m,1H)3.78-3.83(m,6H)3.83-3.89(m,3H)3.89-3.99(m,2H)4.02-4.18(m,2 H)4.19-4.32(m,1H)4.34-4.60(m,2H)4.65-4.76(m,1H)5.09-5.31(m,2H)5.63-5.78(m,1H)5.87(td,J=11.20,5.32Hz ,1H)6.09-6.16(m,1H)6.77-6.90(m,4H)7.28-7.35(m,5H)7.36-7.47(m,2H)8.26-8.42(m,1H)8.74(d,J=16.80Hz,1H)

[0228] Example 1-8 Synthesis of Phosphoramidite Monomer r8-M

[0229] LCMS (ESI) m / z: 801.3 [M+H]+;

[0230] 1H NMR(400MHz, DMSO-d6)δppm 0.96(d,J=6.80Hz,3H)1.10(br,d,J=7.20Hz,9H)2.55-2.61(m,1H)2.73-2.79(m,1H)3.04-3.14(m,1H) 3.16-3.30(m,1H)3.46-3.63(m,3H)3.67-3.82(m,11H)4.05-4.25(m,3H)4.47(br,d,J=4.40Hz,1H)4.60(br,d,J=5.60Hz,1H)5.05-5.27( m,2H)5.76(s,1H)6.02(dd,J=18.00,3.60Hz,1H)6.78-6.89(m,4H)7.16-7.28(m,7H)7.33-7.40(m,2H)7.98(s,1H)8.63(d,J=4.40Hz,1H)

[0231] Example 1-9 Synthesis of Phosphoramidite Monomer r9-M

[0232] LCMS (ESI) m / z: 820.3 [M+H]+;

[0233] 1H NMR(400MHz,DMSO-d6)δppm 0.97(d,J=6.80Hz,3H),1.04-1.24(m,8H),1.20-1.20(m,1H),2.57-2.64(m,1H),2.74-2.83(m,1H),2.92-3.04 (m,1H),3.18(d,J=5.60Hz,2H),3.23-3.28(m,1H),3.31(s,2H),3.42-3.48(m,2H),3.50-3.63(m,2H),3.65-3. 69(m,1H),3.71(d,J=2.00Hz,7H),3.75-3.82(m,1H),3.85(s,3H),4.22(dt,J=7.60,5.60Hz,1H),4.64(dd,J=4 .00,2.13Hz,1H),4.67-4.81(m,1H),6.14-6.28(m,1H),6.72-6.88(m,4H),7.11-7.34(m,9H),8.35-8.50(m,1H)

[0234] Example 1-10 Synthesis of phosphoramidite monomer r10-M

[0235] LCMS (ESI) m / z: 820.3 [M+H]+;

[0236] 1H NMR (400MHz, CDCl3-d6) δppm 1.03(d,J=6.80Hz,3H)1.11-1.24(m,9H)2.38(t,J=6.40Hz,1H)2.65(d,J=6.40Hz,1H )3.17-3.27(m,1H)3.31(s,3H)3.34-3.45(m,1H)3.48-3.71(m,6H)3.73-4.00(m,11H) 4.28-4.41(m,1H)4.46-4.63(m,2H)5.75-5.94(m,1H)6.80(br t,J=7.60Hz,4H)7.15-7.26(m,3H)7.30-7.38(m,4H)7.41-7.48(m,2H)7.96(d,J=4.40Hz,1H)8.18(d,J=10.20Hz,1H)

[0237] Example 2: Compound Synthesis

[0238] Example 2-1: Synthesis of compound GAL-01-M

[0239] Step 1

[0240] Compound 01-1 (500 mg) was dissolved in pyridine (5.00 mL) and 4,4-dimethoxytrityl chloride (741 mg) was added at 0°C. After addition, the reaction solution was stirred at 25°C for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.43, starting material: Rf = 0.24) showed complete consumption of the starting material. Water (10.0 mL) and ethyl acetate (8.00 mL, 6.00 mL) were slowly added to the reaction solution for extraction. The organic phase was washed with saturated sodium chloride solution (6.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 01-2 (300 mg, 27.2% yield) as a yellow solid.

[0241] Step 2

[0242] Compound 01-2 (300 mg) was dissolved in methanol (3.00 mL), and palladium on carbon (100 mg, 10%) was added. The reaction mixture was stirred at 30°C for 12 hours under a hydrogen atmosphere at 30 psi. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.15, starting material: Rf = 0.54) showed complete consumption of the starting material. The reaction mixture was cooled and filtered through celite. The filtrate was concentrated under reduced pressure to afford compound 01-3 (200 mg, 88.0% yield) as a colorless oil.

[0243] LCMS (ESI) m / z: 420.5 [M+H]+;

[0244] Step 3

[0245] Compound 01-4 (700 mg) was dissolved in a mixed solution of tetrahydrofuran (14.0 mL) and water (7.00 mL), and sodium bicarbonate (769 mg) was added. Benzyl chloroformate (780 mg) was added at 0°C, and the reaction mixture was stirred at 25°C for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.72, raw material: Rf = 0.24) showed complete consumption of the starting material. Water (10.0 mL) and ethyl acetate (8.00 mL, 6.00 mL) were slowly added to the reaction mixture for extraction. The organic phase was washed with saturated sodium chloride solution (6.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 01-5 (500 mg, 45.1% yield) as a colorless oil.

[0246] Step 4

[0247] Compound 01-5 (500 mg) was dissolved in dichloromethane (7.00 mL), and trifluoroacetic acid (2.68 g) was added. The reaction mixture was stirred at 25°C for 5 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.2, starting material: Rf = 0.72) showed complete consumption of the starting material. The reaction mixture was extracted with water (5.00 mL) and dichloromethane (5.00 mL, 3.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford Compound 01-6 (400 mg, 94.6% yield) as a colorless oil.

[0248] Step 5

[0249] Compounds 01-6 (200 mg) and 01-3 (400 mg) were dissolved in N,N-dimethylformamide (6.00 mL). N,N-diisopropylethylamine (219 mg, 295 μL) and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (322 mg) were added. The reaction mixture was stirred at 20°C for 2 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.43, starting material: Rf = 0.1) showed complete consumption of the starting material. The reaction mixture was extracted with water (5.00 mL) and ethyl acetate (4.00 mL, 3.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 01-7 (130 mg, 43.4% yield) as a yellow oil.

[0250] Step 6

[0251] Compound 01-7 (110 mg) was dissolved in methanol (2.00 mL), and palladium on carbon (200 mg, 10%) was added. The reaction mixture was stirred at 20°C for 12 hours under a hydrogen atmosphere at 15 psi. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.1, starting material: Rf = 0.43) showed complete consumption of the starting material. The reaction mixture was cooled and filtered through celite. The filtrate was concentrated under reduced pressure to afford compound 01-8 (90.5 mg, 82.2% yield) as a yellow oil.

[0252] Step 7

[0253] Compound 01-9 (30.0 g) was dissolved in methanol (210 mL) and aqueous sodium hydroxide (107 mL, 1 M) was added. The reaction mixture was stirred at 70°C for 3 hours. LCMS (RT = 0.542 min) showed complete consumption of the starting material. After cooling, dilute hydrochloric acid was added to the reaction mixture, and the pH was adjusted to 4. The mixture was then concentrated under reduced pressure to afford Compound 01-10 (28.0 g, crude) as a white solid.

[0254] LCMS (ESI) m / z: 225.1 [M+H]+;

[0255] Step 8

[0256] Compound 01-10 (200 mg) was dissolved in dichloromethane (2.00 mL), and thionyl chloride (510 mg) was added. After the addition, the reaction mixture was stirred at 20°C for 1.5 hours. TLC (dichloromethane / methanol = 10 / 1, product (with methanol): Rf = 0.6, starting material: Rf = 0.24) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 01-11 (150 mg, crude) as a yellow oil.

[0257] Step 9

[0258] Compound 01-11 (150 mg) was dissolved in dichloromethane (2.00 mL) and added dropwise to a solution of 1,9-bis-tert-butyloxycarbonyl-1,5,9-triazanonane in dichloromethane (2.00 mL) at 0°C. N,N-diisopropylethylamine (371 mg) was added, and the reaction mixture was stirred at 25°C for 12 hours. LCMS (RT = 0.857 min) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 50 / 1 to 1 / 1) to afford Compound 01-12 (480 mg, 98.2% yield) as a yellow solid.

[0259] LCMS (ESI) m / z: 851.4 [M+H]+;

[0260] Step 10

[0261] Compound 01-12 (480 mg) was dissolved in tetrahydrofuran (2.00 mL) and water (600 uL). Lithium hydroxide (11.3 mg) was added, and the reaction mixture was stirred at 20°C for 2 hours. TLC (dichloromethane / methanol = 10 / 1, product: Rf = 0.24, starting material: Rf = 0.43) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 01-13 (150 mg) as a yellow solid.

[0262] Step 11

[0263] Compound 01-13 (150 mg) and benzyl bromide (29.4 mg) were dissolved in N,N-dimethylformamide (1.00 mL). Potassium carbonate (29.7 mg) was added at 0°C, and the reaction mixture was stirred at 20°C for 6 hours. LCMS (RT = 0.851 min) showed complete consumption of the starting material. The reaction mixture was extracted with water (5.00 mL) and ethyl acetate (5.00 mL, 4.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 50 / 1 to 1 / 1) to afford Compound 01-14 (110 mg, 83.3% yield) as a white solid.

[0264] LCMS (ESI) m / z: 927.4 [M+H]+;

[0265] Step 12

[0266] Compound 01-14 (130 mg) was dissolved in ethyl acetate (1.00 mL). Hydrochloric acid / ethyl acetate (300 μL) was added, and the reaction mixture was stirred at 20°C for 1 hour. LCMS (RT = 0.346 min) showed complete consumption of the starting material. The organic phase was concentrated under reduced pressure to afford Compound 01-15 (70.0 mg, crude) as a white solid.

[0267] LCMS (ESI) m / z: 527.3 [M+H]+;

[0268] Step 13

[0269] Compound 01-15 (70.0 mg) and Compound 01-16 (238 mg) were dissolved in N,N-dimethylformamide (2.00 mL). N,N-diisopropylethylamine (137 mg, 185 μL) and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (303 mg) were added. The reaction mixture was stirred at 20°C for 2 hours. TLC (dichloromethane / methanol = 10 / 1, product: Rf = 0.43, starting material: Rf = 0.15) showed complete consumption of the starting material. The reaction mixture was extracted with water (8.00 mL) and ethyl acetate (6.00 mL, 4.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford Compound 01-17 (100 mg, 33.5% yield) as a yellow solid.

[0270] Step 14

[0271] Compound 01-17 (100 mg) was dissolved in methanol (1.00 mL), and palladium on carbon (50.0 mg, 10%) was added. After the addition, the reaction mixture was stirred at 20°C for 12 hours under a hydrogen atmosphere at 15 psi. LCMS (RT = 1.127 min) showed complete consumption of the starting material. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to afford 01-18 (50.0 mg, 74.4% yield) as a white solid.

[0272] LCMS(ESI)m / z:1077.9[M+2H] / 2+;

[0273] Step 15

[0274] Compounds 01-18 (50.0 mg) and 01-8 (20.0 mg) were dissolved in N,N-dimethylformamide (1.00 mL). N,N-diisopropylethylamine (9.00 mg) and tri-n-propyl cyclophosphoric anhydride solution (29.5 mg, 50% ethyl acetate solution) were added. The reaction mixture was stirred at 25°C for 1 hour. LCMS (RT = 1.790 min) indicated complete consumption of the starting material. After cooling, the reaction mixture was extracted with water (3.00 mL) and ethyl acetate (5.00 mL, 3.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (chromatographic column: Waters Xbridge Prep OBDC18150*40mm*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 35%-55%, 8 min) to obtain a white solid compound GAL-01 (11.3 mg, purity 100%).

[0275] TOF MS ES + m / z:2711[M+H] / +;

[0276] 1H NMR (400MHz, CDCl3) δppm 1.23-1.45(m,21H)1.75-1.90(m,25H)1.91-1.95(m,12H)1.95-2.01(m,16H)2.01-2.13(m,13H)2.14 -2.28(m,6H)2.98-3.63(m,29H)3.73(s,6H)3.75-3.92(m,9H)3.96-4.16(m,12H)4.21-4.30(m,1H)4 .33-4.40(m,1H)4.40-4.60(m,4H)5.01-5.20(m,4H)5.24-5.33(m,4H)6.52-6.61(m,1H)6.75-6.79( m,4H)6.84-6.92(m,2H)7.10(d,J=8.80Hz,4H)7.21-7.27(m,4H)7.41-7.51(m,1H)7.81-7.85(m,1H)

[0277] Example 2-2: Synthesis of compound GAL-02-M

[0278] Step 1

[0279] Compound 02-1 (20.0 g) was dissolved in a mixture of anhydrous tetrahydrofuran (400 mL) and water (120 mL), and sodium bicarbonate (26.8 g) was added. Benzyl chloroformate (27.2 g) was added at 0°C, and the reaction mixture was stirred at 25°C for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.43, raw material: Rf = 0.1) showed complete consumption of the starting material. Water (100 mL) and ethyl acetate (80.0 mL, 60.0 mL) were slowly added to the reaction mixture for extraction. The organic phase was washed with saturated sodium chloride solution (60.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 02-2 (30.0 g, crude product) as a white solid.

[0280] Step 2

[0281] Compound 02-2 (30.0 g) was dissolved in ethyl acetate (100 mL), and hydrochloric acid / ethyl acetate (100 mL) was added. The reaction mixture was stirred at 25°C for 12 hours. LCMS (Rt = 0.413 min) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford compound 02-3 (8.00 g, crude) as a white solid.

[0282] LCMS (ESI) m / z: 223 [M+H]+;

[0283] Step 3

[0284] Compound 02-3 (8.00 g) was dissolved in tetrahydrofuran (160 mL), and succinic anhydride (3.60 g) was added. The reaction mixture was stirred at 25°C for 1 hour. LCMS (Rt = 1.904 min) showed complete consumption of the starting material, and a major peak meeting the mass spectrometry requirements was detected. The reaction mixture was filtered to yield Compound 02-4 (11.0 g, crude) as a white solid.

[0285] LCMS (ESI) m / z: 323 [M+H]+;

[0286] Step 4

[0287] Compound 02-4 (5.00 g) and compound 3A (7.81 g) were dissolved in N,N-dimethylformamide (100 mL), and N,N-diisopropylethylamine (8.02 g, 10.8 mL) was added. O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphonate (11.8 g) was added at 0°C. LCMS (Rt = 1.886 min) indicated complete consumption of the starting material, with a major peak meeting the mass spectrometry requirements. The reaction mixture was extracted with water (200 mL) and ethyl acetate (100 mL, 100 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 02-5 (9.00 g, crude) as a colorless oil.

[0288] LCMS (ESI) m / z: 724 [M+H]+;

[0289] Step 6

[0290] Compound 02-5 (9.00 g) was dissolved in methanol (100 mL), and palladium on carbon (2.85 g, 10%) was added. The reaction mixture was stirred at 20°C for 12 hours under a hydrogen atmosphere at 15 psi. LCMS (Rt = 1.440 min) indicated complete consumption of the starting material, with a single, consistent main peak detected in the mass spectrum. After cooling, the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to afford compound 02-6 (4.00 g, 54.5% yield) as a yellow oil.

[0291] LCMS (ESI) m / z: 590 [M+H]+;

[0292] Step 7

[0293] Compound 1-18 (4.00 g) and compound 02-6 (1.10 g) were dissolved in N,N-dimethylformamide (40.0 mL), and N,N-diisopropylethylamine (959 mg, 1.29 mL) was added. O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphonate (1.41 g) was then added at 0°C. LCMS (Rt = 2.885 min) indicated complete consumption of the starting material, and a major peak meeting the mass spectra was detected. The reaction mixture was purified by HPLC (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonia)-acetonitrile]; B%: 35%-55% over 8 min) to afford compound GAL-02-M (2.04 g, 93.2% purity) as a white solid.

[0294] LCMS(ESI)m / z:1212[M-DMT] / 2+;

[0295] 1 H NMR (400 MHz, CHLOROFORM-d)

[0296] δppm 1.61(br,d,J=4.40Hz,20H), 1.80-1.90(m,16H), 1.94(br,s,6H), 1.96-2.09(m,28H), 2.12-2.18(m,1 3H), 2.18-2.34(m,6H), 3.00-3.67(m,30H), 3.79(d,J=3.20Hz,7H), 3.83-3.99(m,8H), 4.02-4.23(m,1 3H), 4.30-4.47(m,2H), 4.52-4.65(m,4H), 5.11-5.27(m,4H), 5.30-5.41(m,4H), 6.59-6.67(m,1H), 6 .80-6.85(m,5H), 6.94-7.06(m,3H), 7.16-7.26(m,6H), 7.33-7.38(m,2H), 7.50-7.57(m,1H), 7.95(br d,J=8.40Hz,1H)

[0297] Example 2-3: Synthesis of ligand GAL-03-M

[0298] Step 1

[0299] Compound 03-1 (50.0 g) and compound 1a (128 g) were dissolved in anhydrous toluene (500 mL), and the reaction mixture was stirred at 60°C for 12 hours. TLC (dichloroethane / methanol = 10 / 1, product: Rf = 0.43, starting material: Rf = 0.88) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was then slurried with petroleum ether at 25°C for 10 minutes to afford compound 03-2 (110 g, 87.1% yield) as a white solid.

[0300] Step 2

[0301] Compound 03-2 (10.0 g) was dissolved in a mixture of tetrahydrofuran (200 mL) and water (60.0 mL), and sodium bicarbonate (7.60 g) was added. Benzyl chloroformate (7.72 g) was added at 0°C, and the reaction mixture was stirred at 25°C for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.53, raw material: Rf = 0.2) showed complete consumption of the starting material. Water (100 mL) and ethyl acetate (80.0 mL, 60.0 mL) were slowly added to the reaction mixture for extraction. The organic phase was washed with saturated sodium chloride solution (60.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 03-3 (10.4 g, crude product) as a white oil.

[0302] Step 3

[0303] Compound 03-3 (10.4 g) was dissolved in ethyl acetate (100 mL), and hydrochloric acid / ethyl acetate (100 mL) was added. The reaction mixture was stirred at 25°C for 12 hours. LCMS (Rt = 0.714 min) showed complete consumption of the starting material, and a major peak meeting the mass spectrometry requirements was detected. The reaction mixture was concentrated under reduced pressure to afford Compound 03-4 (4.20 g, crude) as a white solid.

[0304] LCMS (ESI) m / z: 267 [M+H] + ;

[0305] Step 4

[0306] Compound 03-2 (10.0 g) was dissolved in tetrahydrofuran (200 mL), and succinic anhydride (3.02 g) was added. The reaction mixture was stirred at 25°C for 1 hour. LCMS (Rt = 1.081 min) indicated complete consumption of the starting material, with a single, consistent main peak detected in the mass spectrum. The reaction mixture was filtered to afford Compound 03-6 (13.0 g, 99.8% yield) as a colorless oil.

[0307] LCMS (ESI) m / z: 432 [M+H] + ;

[0308] Step 5

[0309] Compound 03-6 (6.63 g) and compound 03-4 (2.60 g) were dissolved in N,N-dimethylformamide (80.0 mL), and N,N-diisopropylethylamine (3.97 g, 5.36 mL) was added. O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphonate (5.84 g) was added at 0° C. LCMS (Rt = 1.919 min) showed complete consumption of the starting material, and a major peak meeting the requirements of the mass spectrum was detected. The reaction solution was extracted with water (150 mL) and ethyl acetate (80.0 mL, 80.0 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by reverse-phase high-performance liquid chromatography (under 0.1% ammonium bicarbonate) to give compound 03-7 (6.40 g, yield 80.1%) as a colorless oil.

[0310] LCMS (ESI) m / z: 1093 [M+H] + ;

[0311] Step 6

[0312] Compound 03-7 (6.00 g) was dissolved in methanol (60.0 mL), and palladium on carbon (5.85 g, 10%) was added. The reaction mixture was stirred at 20°C for 12 hours under a hydrogen atmosphere at 15 psi. LCMS (Rt = 2.225 min) indicated complete consumption of the starting material, with a single, consistent main peak detected in the mass spectrum. After cooling, the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to afford Compound 03-8 (5.00 g, 88.5% yield) as a yellow oil.

[0313] LCMS (ESI) m / z: 958 [M+H] + ;

[0314] Step 7

[0315] Compound 03-8 (4.90 g) and monomethyl terephthalate (1.04 g) were dissolved in N,N-dimethylformamide (100 mL), and N,N-diisopropylethylamine (2.67 g, 3.66 mL) was added. O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphonate (4.15 g) was added at 0°C. LCMS (Rt = 1.814 min) indicated complete consumption of the starting material, with a major peak meeting the mass spectrum. The reaction mixture was extracted with water (150 mL) and ethyl acetate (80.0 mL, 80.0 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford Compound 03-9 (5.50 g, crude) as a colorless oil.

[0316] LCMS (ESI) m / z: 1121 [M+H] + ;

[0317] Step 8

[0318] Compound 03-9 (5.50 g) was dissolved in a mixture of tetrahydrofuran (36.0 mL), water (12.0 mL), and methanol (12.0 mL). Lithium hydroxide monohydrate (898 mg) was added, and the reaction mixture was stirred at 25°C for 2 hours. LCMS (Rt = 1.472 min) indicated complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 03-10 (5.00 g, 94.9% yield) as a white solid.

[0319] LCMS (ESI) m / z: 958 [M+H] + ;

[0320] Step 9

[0321] Compound 03-10 (5.00 g) and potassium carbonate (1.03 g) were dissolved in N,N-dimethylformamide (100 mL). Benzyl bromide (1.45 g) was added at 0°C, and the reaction mixture was stirred at 20°C for 12 hours. LCMS (Rt = 2.022 min) indicated complete consumption of the starting material, with a major peak meeting the mass spectrometry requirements detected. The reaction mixture was extracted with water (200 mL) and ethyl acetate (100 mL, 80.0 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford the crude product. The crude product was purified by HPLC (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonia)-acetonitrile]; B%: 30%-60% over 8 minutes) to afford Compound 03-11 (5.00 g, 84.6% yield) as a white solid.

[0322] LCMS (ESI) m / z: 1197 [M+H] + ;

[0323] Step 10

[0324] Compound 03-11 (5.00 g) was dissolved in ethyl acetate (50.0 mL), and hydrochloric acid / ethyl acetate (50.0 mL) was added. The reaction mixture was stirred at 25°C for 12 hours. LCMS (Rt = 0.885 min) showed complete consumption of the starting material, with a single, consistent main peak detected in the mass spectrum. The reaction mixture was concentrated under reduced pressure to afford Compound 03-12 (4.00 g, 93.6% yield) as a white solid.

[0325] LCMS (ESI) m / z: 1197 [M+H]+ ;

[0326] Step 11

[0327] Compound 03-12 (4.00 g) and compound 01-18 (7.60 g) were dissolved in N,N-dimethylformamide (90.0 mL), and N,N-diisopropylethylamine (4.39 g, 5.92 mL) was added. O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphonate (9.69 g) was added at 0° C. LCMS (Rt = 1.430 min) showed complete consumption of the starting material, and a main peak meeting the requirements of the mass spectrum was detected. The reaction solution was extracted with water (150 mL) and ethyl acetate (80.0 mL, 80.0 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by reverse-phase high-performance liquid chromatography (under 0.1% ammonium bicarbonate) to give compound 03-13 (8.00 g, yield 79.6%) as a colorless oil.

[0328] LCMS (ESI) m / z: 1257 [M+2H] 2+ / 2;

[0329] Step 12

[0330] Compound 03-13 (8.00 g) was dissolved in methanol (160 mL), and palladium on carbon (4.85 g, 10%) was added. The reaction mixture was stirred at 20°C for 12 hours under a hydrogen atmosphere at 15 psi. LCMS (Rt = 1.085 min) indicated complete consumption of the starting material, with a single, consistent main peak detected in the mass spectrum. After cooling, the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to afford Compound 03-14 (7.00 g, 89.5% yield) as a yellow oil.

[0331] LCMS (ESI) m / z: 1212 [M+2H] + / 2;

[0332] Step 13

[0333] Compound 03-14 (4.00 g) and compound 02-6 (1.07 g) were dissolved in N,N-dimethylformamide (40.0 mL), and N,N-diisopropylethylamine (853 mg, 1.15 mL) was added. O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphonate (1.26 g) was then added at 0°C. LCMS (Rt = 1.696 min) indicated complete consumption of the starting material, with a major peak meeting the mass spectra. The reaction mixture was purified by HPLC (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonia)-acetonitrile]; B%: 35%-55% over 8 min) to afford compound GAL-03-M (1.13 g, 93.28% purity) as a white solid.

[0334] LCMS (ESI) m / z: 1347 [M-DMT] / 2 + ;

[0335] 1 H NMR (400 MHz, CDCl3)

[0336] δppm 1.54-1.77(m,38H), 1.81-1.89(m,6H), 1.89-1.95(m,10H), 2.00(s,11H), 2.05(s,11H), 2.08-2.11(m,2H ), 2.13-2.19(m,15H), 2.53-2.71(m,6H), 3.00-3.58(m,35H), 3.65(br,s,2H)3.80(d,J=2.80Hz,6H), 3.9 0(br,s,7H), 4.06-4.24(m,12H), 4.54-4.67(m,4H), 5.11-5.27(m,4H), 5.35(br,d,J=2.40Hz,4H), 6.82( br,t,J=8.40Hz,7H), 6.91-6.97(m,2H), 6.99-7.06(m,1H), 7.20-7.26(m,6H), 7.30-7.43(m,5H), 7.89(br d,J=8.40Hz,2H).

[0337] Example 2-4: Synthesis of GAL-04-M

[0338] Step 1

[0339] Compound 04-1 (500 mg) and compound 01-3 (674 mg) were dissolved in N,N-dimethylformamide (5.00 mL), and N,N-diisopropylethylamine (830 mg) was added. O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (1.22 g) was added at 0°C. The reaction mixture was stirred at 25°C for 1 hour. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.72, starting material: Rf = 0.24) showed complete consumption of the starting material. Water (10.0 mL) and ethyl acetate (8.00 mL, 6.00 mL) were slowly added to the reaction mixture for extraction. The organic phase was washed with saturated sodium chloride solution (6.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 04-2 (200 mg, 17.5% yield) as a white solid.

[0340] Step 2

[0341] Compound 04-2 (200 mg) was dissolved in methanol (1.00 mL), and palladium on carbon (299 mg, 10%) was added. The reaction mixture was stirred at 25°C for 1 hour under a hydrogen atmosphere at 30 psi. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.15, starting material: Rf = 0.54) showed complete consumption of the starting material. The reaction mixture was cooled and filtered through celite. The filtrate was concentrated under reduced pressure to afford compound 04-3 (110 mg, 67.5% yield) as a white oil.

[0342] Step 3

[0343] Compound 01-18 (250 mg) and compound 04-3 (110 mg) were dissolved in N,N-dimethylformamide (20.0 mL). N,N-diisopropylethylamine (60.0 mg) and a 50% ethyl acetate solution of tri-n-propyl cyclic phosphoric anhydride (148 mg) were added. The reaction mixture was stirred at 25°C for 1 hour. LCMS (ET61818-330-P1A1, Rt = 2.744) indicated complete consumption of the starting material. After cooling, the reaction mixture was extracted with water (1.00 mL) and ethyl acetate (2.00 mL, 1.00 mL). The organic phase was washed with saturated sodium chloride solution (5.00 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (chromatographic column: Waters Xbridge BEHC18250*30mm*10um; mobile; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 33%-2%, 8 minutes) to obtain a white solid compound GAL-04-M (37.2 mg, yield 11.8%).

[0344] LCMS (ESI) m / z: 2714 [M+H]+;

[0345] 1 H NMR (400 MHz, CDCl3)

[0346] δppm 1.36-1.53(m,8H)1.64(br,s,18H)1.79-1.91(m,14H)1.95(br,s,8H)2.00(s,12H) 2.02-2.11(m,17H)2.12-2.34(m,19H)2.41-2.71(m,2H)3.07-3.36(m,15H)3.37-3 .45(m,3H)3.47-3.73(m,20H)3.75-3.82(m,10H)3.84-3.98(m,8H)4.00-4.23(m,1 2H)4.24-4.47(m,3H)4.47-4.71(m,5H)5.12-5.29(m,3H)5.16-5.17(m,1H)5.35(br s,4H)6.46-6.66(m,2H)6.82(br,t,J=8.40Hz,6H)7.01(s,3H)7.27(s,5H )7.33-7.40(m,3H)7.49-7.59(m,1H)7.89-8.06(m,2H)8.33-8.46(m,1H)

[0347] Example 2-5: Synthesis of compound GAL-05-M

[0348] Step 1

[0349] Compound 01-1 (12.0 g) and imidazole (13.0 g) were dissolved in N,N-dimethylformamide (100 mL). Tert-butyldimethylsilyl chloride (18.0 g) was added at 0°C. The reaction mixture was stirred at 25°C for 6 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.72, raw material: Rf = 0.24) showed complete consumption of the starting material. Water (100 mL) and ethyl acetate (80.0 mL, 60.0 mL) were slowly added to the reaction mixture for extraction. The organic phase was washed with saturated sodium chloride solution (60.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 05-1 (20.0 g, 87.2% yield) as a yellow solid.

[0350] Step 2

[0351] Compound 05-1 (15.0 g) was dissolved in methanol (100 mL), and palladium on carbon (1.00 g, 10%) was added. The reaction mixture was stirred at 30°C for 12 hours under a hydrogen atmosphere at 30 psi. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.15, starting material: Rf = 0.54) showed complete consumption of the starting material. The reaction mixture was cooled and filtered through celite. The filtrate was concentrated under reduced pressure to afford compound 05-2 (10.0 g, 92.5% yield) as a yellow oil.

[0352] 1 H NMR (400MHz, CDCl3) δppm 0.05 (s, 12H) 0.89 (d, J = 4.80Hz, 18H) 1.60-1.80 (m, 2H) 2.72-2.83 (m, 1H) 3.07 ( dd, J=12.00, 5.20Hz, 1H) 3.33-3.46 (m, 1H) 3.51-3.64 (m, 2H) 4.30-4.36 (m, 1H)

[0353] Step 3

[0354] Compound 05-2 (10.0 g) and monomethyl sebacate (6.26 g) were dissolved in N,N-dimethylformamide (70.0 mL). N,N-diisopropylethylamine (11.2 g, 15.1 mL) and O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphonate (22.0 g) were added. The reaction mixture was stirred at 20°C for 2 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.43, starting material: Rf = 0.15) showed complete consumption of the starting material. The reaction mixture was extracted with water (80.0 mL) and ethyl acetate (60.0 mL, 40.0 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford compound 05-3 (6.50 g, 41.3% yield) as a yellow oil.

[0355] Step 4

[0356] Compound 05-3 (8.50 g) was dissolved in tetrahydrofuran (56.0 mL), and tetrabutylammonium fluoride (15.6 mL, 1 M) was added. The reaction mixture was stirred at 20°C for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.05, starting material: Rf = 0.65) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford compound 05-4 (5.00 g, 91.3% yield) as a yellow solid.

[0357] Step 5

[0358] Compound 05-4 (5.00 g) was dissolved in pyridine (35.0 mL), and 4,4-dimethoxytriphenylmethane (8.06 g) was added. After the addition, the reaction mixture was stirred at 20°C for 3 hours. LCMS (RT = 2.287 min) showed that the starting material was completely consumed. After cooling the reaction mixture, saturated aqueous sodium bicarbonate solution (50.0 mL) and ethyl acetate (50.0 mL, 30.0 mL) were added for extraction. The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, petroleum ether / ethyl acetate = 100 / 1-1 / 1) to obtain compound 05-5 (4.00 mg, 40.8% yield) as a yellow oil.

[0359] LCMS (ESI) m / z: 618.4 [M+H]+;

[0360] 1 H NMR (400MHz, CDCl3) δppm 1.32(br.s,8H)1.57-1.72(m,6H)2.27-2.37(m,4H)3.52-3.64(m,3H)3.66-3.69(m,3H)3.69-3.75(m,1H)3.7 8-3.84 (m, 6H) 4.34-4.43 (m, 1H) 4.45-4.52 (m, 1H) 6.79-6.89 (m, 4H) 7.18 (d, J = 8.80Hz, 4H) 7.27-7.35 (m, 5H)

[0361] Step 6

[0362] Compound 05-5 (800 mg) was dissolved in dioxane (8.00 mL), and aqueous lithium hydroxide (1.29 mL, 2 M) was added. The reaction mixture was stirred at 20°C for 3 hours. LCMS (RT = 1.750 min) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 05-6 (500 mg, 63.9% yield) as a yellow solid.

[0363] LCMS (ESI) m / z: 604.3 [M+H]+;

[0364] Step 7

[0365] Compound 01-9 (30.0 g) was dissolved in methanol (210 mL) and aqueous sodium hydroxide (107 mL, 1 M) was added. The reaction mixture was stirred at 20°C for 12 hours. LCMS (RT = 0.572 min) showed complete consumption of the starting material. After cooling, dilute hydrochloric acid was added to the reaction mixture, and the pH was adjusted to 4. The mixture was then concentrated under reduced pressure to afford Compound 05-7 (28.0 g, crude) as a white solid.

[0366] LCMS (ESI) m / z: 237.1 [MH]+;

[0367] Step 8

[0368] Compound 05-7 (28.0 g) was dissolved in tetrahydrofuran (180 mL), and borane dimethyl sulfide (23.5 mL, 10 M) was added at 0°C. The reaction mixture was stirred at 20°C for 12 hours. LCMS (RT = 0.775 min) showed complete consumption of the starting material. The reaction mixture was cooled to 0°C, and methanol (50.0 mL) was slowly added, followed by further stirring at room temperature for 30 minutes. The organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (silica, petroleum ether / ethyl acetate = 100 / 1-1 / 1) to afford compound 05-8 (8.00 g, 30.3% yield) as a white solid.

[0369] LCMS (ESI) m / z: 223.2 [MH]+;

[0370] Step 9

[0371] Compound 05-8 (8.00 g) was dissolved in dichloromethane (55.0 mL). N,N-dimethylformamide (137 μL) and thionyl chloride (5.18 mL) were added at 0°C. The reaction mixture was stirred at 20°C for 2 hours. LCMS (RT = 0.741 min) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 05-9 (8.00 g, 92.4% yield) as a yellow solid.

[0372] LCMS (ESI) m / z: 243.1 [M+H]+;

[0373] Step 10

[0374] Compound 05-9 (8.00 g) was dissolved in acetone (45.0 mL) and water (15.0 mL), and sodium azide (4.29 g) was added. The reaction mixture was stirred at 60°C for 12 hours. LCMS (RT = 0.589 min) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure, dissolved in chloroform (50.0 mL), and then washed with water (50.0 mL) and saturated brine (40.0 mL). The organic phase was separated, dried, filtered, and concentrated under reduced pressure to afford Compound 05-10 (8.00 g, 97.3% yield) as a white solid.

[0375] LCMS (ESI) m / z: 250.1 [M+H]+;

[0376] Step 11

[0377] Compound 05-10 (8.00 g) was dissolved in methanol (55.0 mL), and palladium on carbon (800 mg, 10%) was added. The reaction mixture was stirred at 25°C for 12 hours under a hydrogen atmosphere at 15 psi. LCMS (RT = 0.589 min) showed complete consumption of the starting material. The reaction mixture was cooled and filtered through celite. The filtrate was concentrated under reduced pressure to afford Compound 05-11 (8.00 g, 89.3% yield) as a white solid.

[0378] LCMS (ESI) m / z: 224.2 [M+H]+;

[0379] Step 12

[0380] Compound 05-11 (8.00 g) was dissolved in tetrahydrofuran (28.0 mL) and water (28.0 mL), and benzyl chloroformate (6.11 g) and sodium bicarbonate (9.03 g) were added. After the addition, the reaction solution was stirred at 25° C. for 12 hours. LCMS (RT = 0.619 min) showed that the starting material was completely consumed. Water (20.0 mL) and ethyl acetate (30.0 mL, 20.0 mL) were added for extraction. The organic phase was washed with saturated sodium chloride solution (20.0 mL), added with anhydrous sodium sulfate, dried, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, petroleum ether / ethyl acetate = 100 / 1-1 / 1) to give compound 05-12 (5.00 g, yield 39.0%) as a white solid.

[0381] LCMS (ESI) m / z: 715.2 [2M+H]+;

[0382] 1 H NMR (400MHz, DMSO-d6) δppm 3.85-3.92 (m, 6H) 4.34 (d, J=6.40Hz, 2H) 5.06 (s, 2H) 7.28-7.41 (m, 5H) 7.95-8.05 (m, 1H) 8.12 (s, 2H) 8.34-8.42 (m, 1H)

[0383] Step 13

[0384] Compound 05-12 (4.00 g) was dissolved in tetrahydrofuran (30.0 mL) and water (6.00 mL). Aqueous lithium hydroxide (536 mg) was added, and the reaction mixture was stirred at 20°C for 12 hours. LCMS (RT = 0.52 min) showed complete consumption of the starting material. After cooling, the reaction mixture was added with dilute hydrochloric acid, the pH was adjusted to 4, and the mixture was concentrated under reduced pressure to afford Compound 05-13 (3.50 g, 94.9% yield) as a white solid.

[0385] LCMS (ESI) m / z: 659.2 [2M+H]+;

[0386] Step 14

[0387] Compound 05-13 (1.00 g) was dissolved in dichloromethane (10.0 mL). N,N-dimethylformamide (140 μL) and oxalyl chloride (797 μL) were added at 0°C. The reaction mixture was stirred at 20°C for 2 hours. TLC (dichloromethane / methanol = 10 / 1, product (methanol solution): Rf = 0.43, starting material: Rf = 0.05) showed complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to afford Compound 05-14 (1.00 g, 89.9% yield) as a yellow oil.

[0388] Step 15

[0389] 1,9-Bis-Boc-1,5,9-triazanonane (905 mg) and N,N-diisopropylethylamine (705 mg, 951 uL) were dissolved in dichloromethane (5.00 mL). Compound 05-14 (500 mg) in dichloromethane (5.00 mL) was slowly added at 0°C. The reaction mixture was stirred at 20°C for 3 hours. LCMS (RT = 0.788 min) showed complete consumption of the starting material. Water (10.0 mL) and dichloromethane (10.0 mL, 5.00 mL) were added for extraction. The organic phase was washed with saturated sodium chloride solution (10.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, petroleum ether / ethyl acetate = 50 / 1 to 0 / 1) to afford compound 05-15 (800 mg, 61.4% yield) as a white solid.

[0390] LCMS (ESI) m / z: 956.3 [M+H]+;

[0391] Step 16

[0392] Compound 05-15 (600 mg) was dissolved in dichloromethane (6.00 mL), and trifluoroacetic acid (280 μL) was slowly added. The reaction mixture was stirred at 20°C for 3 hours. TLC (dichloromethane / methanol = 10 / 1, product: Rf = 0.0, starting material: Rf = 0.24) showed complete consumption of the starting material. The reaction mixture was extracted with saturated aqueous sodium bicarbonate (10.0 mL) and dichloromethane (10.0 mL, 5.00 mL). The organic phase was washed with saturated sodium chloride solution (10.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford Compound 05-16 (450 mg, 81.6% yield) as a yellow oil.

[0393] Step 17

[0394] Compound 05-16 (50.0 mg) was dissolved in N,N-dimethylformamide (1.00 mL), and N,N-diisopropylethylamine (73.6 mg, 749 uL), compound 01-16 (127 mg), and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (162 mg) were added. The reaction mixture was stirred at 20°C for 3 hours. LCMS (RT = 1.529 min) showed complete consumption of the starting material. The crude reaction mixture was purified by HPLC (column: Waters Xbridge BEHC18 250*50mm*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 40%-60%, 10 min) to afford compound 05-17 (100 mg, 61.7% yield) as a white solid.

[0395] LCMS(ESI)m / z:1137.4[M+2H] / 2+;

[0396] Step 18

[0397] Compound 05-17 (50.0 mg) was dissolved in tetrahydrofuran (1.00 mL), and palladium on carbon (10.0 mg, 10%) was added. After the addition, the reaction mixture was stirred at 20°C under a hydrogen atmosphere at 15 psi for 3 hours. LCMS (RT = 1.229 min) showed complete consumption of the starting material. The reaction mixture was cooled and filtered through celite. The filtrate was concentrated under reduced pressure to afford Compound 05-18 (30.0 mg, 63.7% yield) as a white solid.

[0398] LCMS(ESI)m / z:1070.4[M+2H] / 2+;

[0399] Step 19

[0400] Compound 05-18 (30.0 mg) and compound 05-6 (8.47 mg) were dissolved in tetrahydrofuran (500 μL), and N,N-diisopropylethylamine (3.62 mg) and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (10.6 mg) were added. The reaction mixture was stirred at 20°C for 12 hours. LCMS (RT = 1.780 min) showed complete consumption of the starting material. The reaction mixture was purified by HPLC (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 30%-60% over 8 min) to afford compound GAL-05 (1.90 mg, 100% purity) as a white solid.

[0401] LCMS (ESI) m / z: 1363 [M+2H] / 2+;

[0402] 1 H NMR (400MHz, CDCl3) δppm 1.27-1.41(m,16H)1.82-1.89(m,9H)1.91-2.11(m,49H)2.13-2.20(m,15H)2 .24-2.40(m,8H)3.03-3.26(m,9H)3.26-3.39(m,5H)3.40-3.48(m,3H)3.50( s, 7H) 3.52-3.63 (m, 7H) 3.81 (s, 6H) 3.87-3.99 (m, 9H) 4.06-4.23 (m, 13H) 4.3 0-4.78(m,11H)5.13-5.25(m,4H)5.34-5.40(m,4H)6.82-6.87(m,5H)7.18(br d, J=8.80Hz, 7H) 7.29 (br d, J=4.00Hz, 3H) 7.31-7.36 (m, 2H)

[0403] Example 2-6 Synthesis of Compound GAL-01-D

[0404] Compound GAL-01 (100 mg) was dissolved in methanol (2.00 mL), and aqueous ammonia (1.29 mg, 100%) was added. The reaction mixture was stirred at 70°C for 2 hours. The reaction mixture was purified by high-performance liquid chromatography (HPLC column: Waters Xbridge BEHC18 250*50mm*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 35%-55%, 10 minutes) to obtain compound GAL-01-D (23.0 mg, 28.3% yield) as a white solid.

[0405] LCMS (ESI) m / z: 1903 [M-DMT]+;

[0406] 1 H NMR (400 MHz, DMSO-d6)

[0407] δppm 1.27(br,s,6H)1.32-1.55(m,22H)1.56-1.63(m,4H)1.67-1.75(m,4H)1.75-1.82(m,12H)1.82-1.88(m,2H)1.93(br,s,5H)2.01-2.11(m,5 H)2.20(br,t,J=7.60Hz,2H)2.58(br,d,J=9.60Hz,4H)2.80-2.91(m,4H)2.98(br,d,J=5.60Hz,2H)3.07-3.19(m,9H)3.40(br,dd,J=6.00,2 .63Hz,12H)3.46-3.57(m,8H)3.60-3.77(m,18H)4.06-4.31(m,6H)4.34-4.42(m,1H)4.47(d,J=4.00Hz,4H)4.52-4.64(m,8H)4.85-4.99(m, 1H)6.83-6.92(m,4H)7.16-7.23(m,5H)7.25-7.37(m,4H)7.43(s,1H)7.62(br,d,J=8.00Hz,6H)7.77-7.90(m,4H)8.64(br,d,J=1.20Hz,1H)

[0408] Using the same synthesis method as Example 2-6, Examples 2-7, 2-8, and 2-9 were synthesized:

[0409] Example 2-7: Compound GAL-02-D

[0410] LCMS(ESI)m / z:1919.8[M-DMT]+;

[0411] 1 H NMR (400 MHz, DMSO-d6)

[0412] δ ppm 1.25 - 1.65 (m, 26H), 1.65 - 1.88 (m, 18H), 1.90 - 1.99 (m, 4H), 2.03 - 2.14 (m, 4H), 2.24 - 2.47 (m, 4H), 2.87 (br, d, J = 2.40 Hz, 4H), 2.99 - 3.21 (m, 12H), 3.22 - 3.31 (m, 9H), 3.39 - 3.46 (m, 8H), 3.51 (dt, J = 11.20, 5.50 Hz, 8H), 3.61 - 3.79 (m, 19H), 4.06 - 4.27 (m, 5H), 4.37 - 4.44 (m, 1H), 4.47 (d, J = 4.00 Hz, 4H), 4.52 - 4.64 (m, 8H), 4.86 - 5.04 (m, 1H), 6.88 (d, J = 8.40 Hz, 4H), 7.15 - 7.25 (m, 5H), 7.27 - 7.36 (m, 4H), 7.44 (s, 1H), 7.56 - 7.73 (m, 6H), 7.77 - 7.91 (m, 5H), 8.67 (br, s, 1H)

[0413] Examples 2 - 8: Compound GAL - 03 - D

[0414] LCMS(ESI) m / z: 1095 [M - DMT] / 2+;

[0415] 1 H NMR (400 MHz, MeOD - d4)

[0416] δ ppm 1.56 - 1.62 (m, 9H) 1.62 - 1.77 (m, 17H) 1.82 - 1.98 (m, 8H) 1.99 (s, 11H) 2.09 (s, 1H) 2.23 (q, J = 7.20 Hz, 10H) 2.37 (br, d, J = 5.63 Hz, 3H) 2.47 - 2.75 (m, 10H) 3.13 - 3.28 (m, 14H) 3.37 - 3.46 (m, 10H) 3.48 - 3.55 (m, 9H) 3.61 (dt, J = 10.4, 3.20 Hz, 6H) 3.74 - 3.82 (m, 14H) 3.85 (d, J = 2.80 Hz, 4H) 3.89 - 3.99 (m, 8H) 4.25 - 4.31 (m, 1H) 4.37 (dd, J = 8.40, 4.40 Hz, 4H) 4.57 - 4.63 (m, 1H) 6.84 - 6.90 (m, 4H) 7.19 - 7.33 (m, 7H) 7.38 (br, d, J = 7.20 Hz, 2H) 7.49 (br, d, J = 8.40 Hz, 2H) 7.94 (d, J = 8.00 Hz, 2H)

[0417] Example 2-9: Compound GAL-04-D

[0418] LCMS (ESI) m / z: 1908 [M-DMT] + .

[0419] Example 3: Synthesis of double-stranded siRNA reagent

[0420] The sense and antisense strands of the RNAi reagents were synthesized using the solid phase phosphoramidite triplysate method used in oligonucleotide synthesis. Such standard syntheses are generally known in the art. Depending on the scale, a 12-channel nucleic acid synthesizer or an OP 100 nucleic acid synthesizer was used. The synthesis was performed on a controlled pore glass (CPG) sphere. or ) is carried out on a solid support made of a single-stranded oligoribonucleotide containing a targeting ligand, and is synthesized on a solid support formed by covalently linking a controllable microporous glass ball and a targeting ligand molecule. The monomer placed at the 3' end of the corresponding chain is attached to the solid support as the starting point for synthesis. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and capping. The 2'-modified RNA phosphoramidite monomers (2'-methoxy and fluoro-modified RNA phosphoramidite monomers) and auxiliary reagents required for the synthesis are commercially available.

[0421] The synthesis of siRNA linked to the GAL-03-M conjugate group is described below as an example. The synthesis of other siRNA reagents (shown in Table 1) follows the same method:

[0422] Step 1: Preparation of GAL-03 succinate

[0423] Under nitrogen, GAL-03-M (500 mg, 167 μmol) was dissolved in 10 mL of dichloromethane. Triethylamine (85 mg, 834 μmol) was added, followed by succinic anhydride (67 mg, 667 μmol) and DMAP (40 mg, 334 μmol). The mixture was stirred at room temperature for 12 hours. After completion of the reaction as determined by LCMS, the reaction solution was diluted with 50 mL of dichloromethane, washed with 5% NaCl solution (20 mL x 3), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure below 30°C to obtain 400 mg of an off-white solid product (yield: 77%).

[0424] Step 2: General process for synthesizing GAL-03-CPG

[0425] The GAL-03 succinate solution (400 mg, 128 μmol) obtained in the previous step was dissolved in acetonitrile (10 mL). HBTU (73 mg, 193 μmol) and DIEA (50 mg, 387 μmol) were added to the solution. The reaction mixture was mixed for 5 minutes to obtain a clear solution. NH2-controlled pore glass beads (NH2-CPG, NH2-controlled pore glass, The reaction was carried out on a shaker at 25°C and 150 rpm (amino loading 180 μmol / g, 2 g). After 24 hours of reaction, the mixture was filtered, the filter cake was washed thoroughly with dichloromethane and acetonitrile, and vacuum dried. The dried solid phase support was capped with 25% acetic anhydride / pyridine capping reagent for 3 hours, filtered, the filter cake was washed thoroughly with acetonitrile, and dried to obtain the desired GAL-03-CPG solid phase support (2.1 g, measured loading: 20 μmol / g).

[0426] Step 3: Synthesis of the conjugate sense strand (the synthesis method is general and applicable to all monomers mentioned, including R1, 2-O-methyl and fluorinated modified nucleotides)

[0427] The solid phase phosphoramidite method was used to initiate the cycle using the GAL-03-CPG solid phase support prepared in step 2 above. Nucleoside monomers were linked one by one from the 3'-5' direction according to the arrangement order of the positive chain nucleotides (sequence shown in Table 1).

[0428] Phosphoramidite monomer or r1 monomer is prepared into 0.06M acetonitrile solution, and anhydrous dimethylformamide and molecular sieves are added.

[0429] The deprotection reaction conditions for each step were the same, including a reaction temperature of 25° C., a deprotection reagent of a dichloroacetic acid solution in dichloromethane (3% w / v), a molar ratio of dichloroacetic acid to the 4,4′-dimethoxytrityl protecting group on the solid support of 5:1, and a reaction time of 70 seconds.

[0430] The coupling reaction conditions for each step included a temperature of 25°C, the use of 5-benzylthio-1H-tetrazole (BTT, 0.3 M, acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 0.25 M acetonitrile solution) as the activator solution, a molar ratio of the nucleic acid sequence connected to the solid support to the nucleoside monomer of 1:8, a molar ratio of the nucleic acid sequence connected to the solid support to the coupling reagent of 1:65, and a coupling time of 3 minutes (modified RNA monomer, fluorinated RNA monomer or 2'-O-methyl modified RNA monomer) and 5 minutes (r1 monomer).

[0431] The capping conditions for each step included a temperature of 25°C, a capping reagent solution of a mixed solution of CapA (10% acetic anhydride, acetonitrile) and CapB (pyridine / N-methylimidazole / acetonitrile, 10:14:76, v / v / v) in a molar ratio of 1:1, and a reaction time of 15 seconds.

[0432] The oxidation reaction conditions for each step included a temperature of 25° C., an iodine solution (0.05 M, THF / pyridine / water, 70:20:10) as the oxidizing reagent, a molar ratio of iodine to the nucleic acid sequence attached to the solid phase support in the coupling step of 30:1, and a reaction time of 15 seconds.

[0433] The sulfurization reaction conditions for each step included a temperature of 25°C, a sulfurization reagent of PADS (phenylacetyl disulfide) (0.2 M, dissolved in pyridine / acetonitrile = 7:3), a molar ratio of the sulfurization reagent to the nucleic acid sequence attached to the solid phase support in the coupling step of 120:1, and a reaction time of 300 seconds.

[0434] The cleavage and deprotection conditions for oligomers bound to CPG are as follows: the synthesized nucleotide sequence connected to the carrier is added to 25 wt% ammonia water at a concentration of 0.5 mL / μmol, and the reaction is carried out at 55°C for 6 hours. The liquid is removed and the residue is concentrated to dryness in vacuo.

[0435] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column with a NaCl gradient elution. Specifically, the following were used: Eluent A: 0.05M NaOH aqueous solution; Eluent B: 1M NaCl, 0.05M NaOH aqueous solution; the elution gradient was: Eluent A:Eluent B = 100:0 to 50:50. The product eluates were collected and combined, and desalted using a Sephadex G25 column, using water for injection.

[0436] Detection: Purity was determined using high performance liquid chromatography (HPLC); molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS). The measured values ​​in the examples were consistent with the theoretical values, indicating that the synthesized molecules were the positive-sense strands of the conjugated molecules with GAL-03 conjugated to the 3' end.

[0437] Note: The monomer used to comprise the r1 sequence is selected from the corresponding numbered r1-M in the example. The ester group contained in its structure reacts with ammonia to form an amide during the cleavage and deprotection process of the sequence synthesis, as shown below:

[0438] Step 4: Synthesis of the conjugate antisense strand

[0439] Using a solid-phase phosphoramidite trysterase method, using universal controlled glass (CPG) microspheres for initiation, nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand (please confirm the sequence shown in Table 1). The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification, and desalting conditions used in the solid-phase synthesis method are the same as those used for synthesizing the sense strand.

[0440] Detection: Purity was determined using high performance liquid chromatography (HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured values ​​in the examples were consistent with the theoretical values, indicating that the synthesized strand was an antisense strand.

[0441] Step 5: Synthesis of double-stranded conjugate

[0442] The sense strand and antisense strand were dissolved separately in water for injection to obtain a 40 mg / mL solution, which was then mixed in an equimolar ratio and heated at 95°C for 10 minutes. After slow cooling, the annealed product was lyophilized to obtain a lyophilized powder. Molecular weight determination was performed using liquid chromatography-mass spectrometry (LC-MS). The measured values ​​in the examples were consistent with the theoretical values, indicating that the synthesized siRNA was a double-stranded RNA. Quantification was performed using ultraviolet spectrophotometry.

[0443] Using the above synthesis method, representative novel GalNAc-siRNAs of the present invention are summarized in Table 1.

[0444] Table 1: Sense and antisense strand sequences of novel GalNAc-siRNAs that inhibit human PNPLA3 mRNA expression, and their compositions.

[0445] *GAL-03 in the table is a novel tetravalent symmetrical GalNAc group that binds to nucleic acids through covalent bonds. The linkage is shown in the following formula:

[0446] L is the residue after chemical reaction of the small molecule fragment L96, which is bound to the nucleic acid through a covalent bond, and its linkage method is shown in the following formula: (wherein L96 succinate is a commercially available product, and the sequence synthesis method is the same as that described in Example 3).

[0447] Example 4: Evaluation of the endocytosis efficiency of GalNAc compounds in primary cynomolgus monkey hepatocytes (PCMH) using flow cytometry

[0448] Purpose of the experiment

[0449] The purpose of this study was to evaluate the endocytosis efficiency of test GalNAc compounds in primary cynomolgus macaque hepatocytes (PCMH) using flow cytometry. Both the test GalNAc compound and GalNAc3-Cy5 bind to the ASGPR receptor on the PCMH surface and are internalized into the cells. The competing compound, GalNAc3-Cy5, is a fluorescently labeled compound. After internalization, its fluorescence signal can be detected in the cells using flow cytometry. Competition between the test GalNAc compound and GalNAc3-Cy5 results in a decrease in the detected fluorescence signal, and the degree of this decrease reflects the endocytosis efficiency of the test compound.

[0450] Experimental materials and instruments:

[0451] 1. Cell lines and compounds: Monkey primary hepatocytes PCMH (Batch No. GSYG202102) were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd., and the competitive compound GalNAc3-Cy5 (Batch No. ET45235-14-P1) and the reference compound L96 (Batch No. ET63057-7-P1) were also provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.

[0452] 2. Main instruments and reagents: DMEM (Gibco, 11965-092), FBS (ExCell Bio, FSP500), InvitroGRO CP Medium (BioIVT, S03316), Staining buffer (Thermo Fisher, 00-4222-26), 1x Fixation buffer (BD, 554655); centrifuge (Beckman Allegra, X15R Centrifuge), cell counter (Alit Life Science, Rigel S2), flow cytometer (BD, FACSCanto TM Plus).

[0453] Experimental steps and methods:

[0454] Evaluation of compound internalization efficiency in PCMH by flow cytometry

[0455] On day 0, the resuscitated PCMH suspension was adjusted to an appropriate density and inoculated into a 48-well plate.

[0456] On day 1, pre-mixed and diluted test compounds and GalNAc3-Cy5 were added to the cells. Test compounds GAL-01-D, GAL-02-D, GAL-03-D, and GAL-04-D, as well as the reference compound L96, were diluted three-fold starting at 100 μM, with 11 concentration points per well. The final concentration of the competing compound, GalNAc3-Cy5, was 0.5 μM. The final DMSO concentration in the cell culture medium was 2%. After incubation with the compounds for 4 hours, the cells were digested, fixed, and the median fluorescence intensity (MFI) of PCMH in the APC channel was measured by flow cytometry.

[0457] Data Analysis

[0458] The inhibition rate of the compound at each concentration point on GalNAc3-Cy5 reflects the endocytosis efficiency of the compound at each concentration point. Using GraphPad Prism software, the inhibition rate of the compound at each concentration point on GalNAc3-Cy5 was analyzed by fitting the curve using the "log (agonist) vs. response–variable slope" equation to calculate the EC50 value of the endocytosis efficiency of the test compound.

[0459] Inhibition rate of compound on GalNAc3-Cy5 = (1-(MFI Sample -MFI Neg ) / (MFI Pos -MFI Neg ))*100%

[0460] MFI Sample is the median fluorescence intensity of the compound in the APC channel

[0461] MFI Neg is the background fluorescence value

[0462] MFI Pos is the maximum fluorescence intensity of GalNAc3-Cy5 uptake

[0463] Experimental results and conclusions:

[0464] In the presence of 0.5 μM GalNAc3-Cy5 competition, the endocytosis efficiency of the test compounds in PCMH was detected by flow cytometry. The EC50 value of the reference compound L96 was 0.181 μM, and the test compound GAL-03-MEC50 was 0.189 μM, which was at the same level as the reference compound L96, and both showed good binding affinity to ASGPR.

[0465] Table 2: Internalization efficiency of compounds in PCMH

[0466] Example 5: Activity test of the compound in inhibiting PNPLA3 mRNA in primary human hepatocytes (PHH)

[0467] Experimental materials and instruments:

[0468] 1. Cell Line

[0469] PHH (lot number: HDS) cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. PHH cells were cultured in InvitroGRO™ CP Medium (BIOIVT, catalog number S03316) supplemented with 10% fetal bovine serum (ExCellBio, catalog number FSP500) and 1% penicillin-streptomycin (HyClone catalog number SV30010).

[0470] 2. Main instruments and reagents and consumables

[0471] The main instruments used in this experiment include a fluorescence qPCR instrument (Applied Biosystems, model QuantStudio 7Flex), a cell counter (Countstar Rigel2); Lipofectamine TM iRNAiMAX transfection reagent (Invitrogen-13778150), SensiFAST SYBR Hi Rox kit Mix (Meridian Bioscience-CSA-01135), RNA extraction kit (Qiagen-74182), FastKing cDNA first-strand synthesis kit (TianGen-KR116-02), 96-well plate (Costar 3599), PNPLA3 gene expression kit (Thermo-4351368).

[0472] Experimental steps and methods:

[0473] The test compound was diluted with PBS to 8 concentrations. Frozen PHH cells (lot number: HDS) were revived and counted using Countstar Rigel2.

[0474] The cells were adjusted to 6 × 10 5 / mL density. Ad libitum dosing: Directly add diluted compound to a 96-well cell culture plate (10 μL / well). Subsequently, add 90 μL / well of cells to the 96-well plate, for a final volume of 100 μL per well. After addition, incubate in a 5% CO2, 37°C incubator for 48 hours.

[0475] Cells were collected, and RNA was extracted according to the Qiagen-74182 RNA Extraction Kit instructions. RNA was reverse transcribed into cDNA according to the FastKing cDNA First-Strand Synthesis Kit instructions. Target gene cDNA was detected by qPCR. GAPDH was used as an internal reference gene, and qPCR was performed in a 384-well plate. Amplification primers were included in the kit and provided by the supplier. The qPCR reaction program was as follows: first, heating at 50°C for 2 minutes, then 95°C for 2 minutes, followed by cycling mode: heating at 95°C for 5 seconds, followed by 60°C for 30 seconds, for a total of 40 cycles, and finally heating at 95°C for 15 seconds, followed by 60°C for 1 minute.

[0476] Data Analysis

[0477] The ΔΔCt method was used to analyze the expression of the target gene in each test sample. This method measures the Ct difference (ΔCt) between the target gene (Human PNPLA3) and the reference gene (GAPDH), and compares the ΔCt values ​​of the compound-treated samples with those of the control group.

[0478] The formula is: ΔCt = average Ct value of target gene - average Ct value of reference gene. ΔΔCt = ΔCt value of compound-treated sample - average ΔCt value of control group. Gene expression level = 2 - ΔΔCT

[0479] Experimental results and conclusions:

[0480] In experiments with primary human hepatocytes, the following compounds, all of which use the GalNAc moiety conjugated to L96, patented by Alnylam, VB16102-VB16107, significantly inhibited mRNA expression. AD-519347.1 (inferred from publicly available data from Alnylam's related project US11149276B2) had an IC50 of 10.02 nM, while VB16105 and VB16106 exhibited superior activity compared to Yangshen, with IC50 values ​​of 3.49 nM and 4.75 nM, respectively (see Table 2).

[0481] Table 3: Inhibition rate of PNPLA3 mRNA by different test compounds

[0482] Example 6: Activity test of the compound in inhibiting PNPLA3 mRNA in the HDI mouse model

[0483] Objective: High-pressure tail vein injection (HDI) is an in vivo gene delivery method that involves the rapid injection of a large volume of plasmid DNA solution into mouse hepatocytes within a short period of time through the tail vein. Leveraging the liver-targeting nature of this method, a single HDI injection of a PNPLA3 replicon plasmid mediated efficient and short-term replication of PNPLA3 DNA in the mouse liver. This study utilized the PNPLA3 HDI mouse model to evaluate the in vivo activity of compounds inhibiting PNPLA3 mRNA.

[0484] Experimental materials and instruments

[0485] 1. Animals: BALB / c mice, female, 6-8 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0486] 2. Solvent: Phosphate buffered saline, provided by Shanghai WuXi AppTec. Filter and use for dispensing on the day of administration.

[0487] 3. Plasmid DNA: pcDNA-PNPLA3 I148M (I148M mutation) plasmid, provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd., batch number 880-1095686535_R2 / PA30592-2 / V1506362, concentration 2.4 μg / μL, diluted with normal saline. Reagents and consumables: Trizol (Invitrogen, 15596018); FastKing cDNA First-Strand Synthesis Kit (Genomic Depleted) (Qiagen-Kr116-02); MagMAX TM mirVana TM Total RNA extraction kit (Thermo-A27828); PNPLA3 gene expression kit (Thermo-4351368); stainless steel beads (Qiagen-69989); KingFisher 96-well standard plate (200 μL) (Thermo-97002540B); KingFisher 96 deep-well magnetic rod holder (Thermo-97002534B); KingFisher 96 deep-well plate (Thermo-95040450B).

[0488] 4. Instruments: centrifuge (Beckman-Allegra X-15R); tissue disruptor (Qiagen-Tissue Lyser II); PCR instrument (Bio-RAD-T100TM Thermal cycer); real-time fluorescence quantitative PCR instrument (Applied Biosystems-QuantStudio 7Flex); spectrophotometer (Thermo-Nanodrop ONE); nucleic acid extraction instrument KingFisher Apex (Thermo-APEX96DW714);

[0489] Experimental steps and methods:

[0490] 1. In vivo experiments

[0491] The day on which the mice were dosed was defined as day 0 of the experiment, the day before that was day -1, the day after that was day 1, and so on. On day 0, the mice were subcutaneously injected with a solvent or test compound at a volume of 5 mL / kg. On day 3, the PNPLA3 plasmid DNA solution was injected into the tail vein of the mice at high pressure. The plasmid DNA was prepared with physiological saline before injection and stored at 4°C until use. All mice were injected with a plasmid DNA solution of 8% of their body weight through the tail vein within 5 seconds (injection volume (mL) = mouse body weight (g) × 8%), and the mass of the injected plasmid for each mouse was 10 μg. On day 4, 24 hours after the high-pressure tail vein injection of PNPLA3 plasmid, all mice were euthanized by CO2 inhalation. After euthanasia, blood was collected through the heart, and liver samples were collected. About 70 mg of liver tissue was immersed in After incubation at 4°C overnight, the supernatant was discarded and transferred to a -80°C refrigerator for PNPLA3 mRNA detection.

[0492] 2. Sample Analysis

[0493] RNA from the liver was extracted using Trizol. The method is briefly described as follows: 50-70 mg of liver tissue and 1.2 mL of Trizol were homogenized using a Qiagen Tissue Lyser II at 26 Hz for 2 minutes. Lysis was performed at room temperature for 5 minutes. 1 mL of the supernatant was collected, 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 seconds. After standing for 3 minutes, the supernatant was centrifuged and collected. An equal volume of isopropanol was added and the mixture was precipitated overnight at -20°C. The mixture was washed twice with 70% ethanol and dissolved in RNase-free water. The RNA sample concentration was determined using a Nanodrop ONE and diluted to 400 ng / μL with RNase-free water for reverse transcription.

[0494] The reverse transcription experimental steps refer to the FastKing cDNA First-Strand Synthesis Kit (Genomic Depletion) instructions. A brief description is as follows: Prepare the gDNA removal mixture. Add the mixture (5 μL / sample) and 400 ng / μL RNA sample to a 96-well PCR reaction plate, incubate at 42°C for 3 minutes, and cool on ice. Prepare the reverse transcription mixture, add the reverse transcription mixture (10 μL / sample) and the DNA removal product from the previous step to a 96-well PCR reaction plate, and perform reverse transcription. Reaction conditions: 42°C for 15 minutes; 95°C for 3 minutes. Store the cDNA at 4°C for further analysis.

[0495] The quantitative PCR method for detecting PNPLA3 and NEO gene expression in mouse liver is briefly described as follows: qPCR reaction mixture was prepared, 2 μL of sample was added, and PCR was performed. Amplification primers were provided by the supplier and included in the kit. Reaction conditions were: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

[0496] Data Analysis

[0497] The ΔΔCt method was used to analyze the expression of the target gene in each test sample. This method measures the Ct difference (ΔCt) between the target gene (PNPLA3) and the reference gene (Neo). The ΔCt values ​​of compound-treated samples were compared with those of the control group. The formula is: ΔCt = average Ct value of the target gene - average Ct value of the reference gene. ΔΔCt = ΔCt value of compound-treated samples - average ΔCt value of the control group. Gene expression level = 2 - ΔΔCt.

[0498] Experimental results and conclusions:

[0499] Compared with the PBS group, the following compounds can significantly reduce the relative expression levels of mRNA in liver tissue, see Table 3.

[0500] Table 4: Remaining mRNA expression levels in the liver (n=5 PBS or 3 dosing groups)

[0501] In summary, to develop a more optimal GalNAc-siRNA drug conjugate targeting PNPLA3, we optimized both the siRNA sequence and the GalNAc. In experiments with primary human hepatocytes, to compare the effect of siRNA sequence on activity, we designed a series of compounds in which the GalNAc was unchanged but non-natural nucleomonomers were introduced into the siRNA sequence. The results showed that compounds VB16105 and VB16106 exhibited superior activity compared to Yangshen (see Example 1), and the introduction of non-natural nucleomonomers also reduced the risk of off-target effects. To further optimize activity and enhance novelty, we developed a novel, symmetrical tetravalent GalNAc, which maintains the delivery efficiency of the GalNAc (see Example 2), making it easier to scale up CMC synthesis and reducing costs. The optimized siRNA sequence was conjugated to the novel tetravalent GalNAc, ultimately yielding a GalNAc-siRNA drug conjugate with improved selectivity and activity.

Claims

1. A double-stranded siRNA comprising a sense strand and an antisense strand, wherein: The antisense strand comprises the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence in which one or more nucleotides are substituted, added, deleted or inserted into the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the nucleotide sequence of SEQ ID NO: 12, and / or The sense strand comprises the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence in which one or more nucleotides are substituted, added, deleted or inserted into the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 13; One or more nucleotide residues in the nucleotide sequence of the sense strand and / or antisense strand are replaced with r1 having formula I: Wherein: X1, Y1 and Z1 are independently CH or N, and at least one of X1, Y1 and Z1 is N; R 1 is H, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkoxy, optionally substituted C2-C 10 Preferably, the alkyl group is C1-C6 alkyl; Preferably, the alkoxy group is C1-C6 alkoxy; Preferably, the alkenyl group is C2-C6 alkenyl; Preferably, the substitution is substituted with one or more groups selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, hydroxy, oxo, fluorine, chlorine, bromine or iodine, more preferably, R 1 is H or C1-C3 alkoxy optionally substituted by methoxy or C2 alkenyl.

2. The double-stranded siRNA according to claim 1, wherein the antisense strand comprises a nucleotide sequence in which 1-10, 1-5, 1-3 or 1-2 nucleotides are substituted, added, deleted or inserted into the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the nucleotide sequence of SEQ ID NO: 12, and / or the sense strand comprises a nucleotide sequence in which 1-10, 1-5, 1-3 or 1-2 nucleotides are substituted, added, deleted or inserted into the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with the nucleotide sequence of SEQ ID NO:

13. The nucleotide sequence of NO:13 has a nucleotide sequence with at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity; preferably, 15-21, 18-21 or 20-21 nucleotides in the sense strand and the antisense strand are complementary.

3. The double-stranded siRNA according to claim 1 or 2, wherein r1 is selected from the following structures: Preferably, r1 is 4 . The double-stranded siRNA according to claim 1 , wherein one or more or all nucleotides of the antisense strand and / or one or more or all nucleotides of the sense strand are modified nucleotides.

5. The double-stranded siRNA according to claim 4, wherein the modification is selected from methoxy modification, fluoro modification, phosphorothioate linkage, glycerol nucleic acid, (E)-vinyl phosphate or 2'-deoxynucleotide substitution; preferably, the methoxy modification is 2'-O-methyl modification; preferably, the fluoro modification is 2'-fluoro modification.

6. The double-stranded siRNA according to any one of claims 1 to 5, wherein the antisense strand comprises two phosphorothioate bonds between the three terminal nucleotides at the 3' end and two phosphorothioate bonds between the three terminal nucleotides at the 5' end; and / or wherein the sense strand comprises two phosphorothioate bonds between the three terminal nucleotides at the 5' end; and / or wherein r1 is linked to the adjacent nucleotide residue via a phosphate or phosphorothioate bond; and / or wherein r1 substitution is made at nucleotide residues 7, 8 and / or 9 of the antisense strand in a 5' to 3' manner; and / or wherein r1 substitution is made at nucleotide residue 2, 3, 16, 18, 19 and / or 20 of the sense strand in a 5' to 3' manner; and / or wherein one or more nucleotides at positions 2, 6, 8, 9, 14 and 16 of the antisense strand are fluorinated modified nucleotides and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides in a 5' to 3' manner; and / or In the manner from the 5' end to the 3' end, one or more nucleotides at positions 7 and 8-11 of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides.

7. The double-stranded siRNA according to any one of claims 1 to 6, wherein the antisense strand comprises a nucleotide sequence selected from the group consisting of: mAs-fCs-mA-mU-mA-fA-mG-fA-fC-mA-mU-mU-mA-fU-mC-fC-mU-mA -mA-mU-mGs-mGs-mG; mAs-fCs-mA-mU-mA-fA-r1-fA-fC-mA-mU-mU-mA-fU-mC-fC-mU-mA- mA-mU-mGs-mGs-mG; mAs-fCs-mA-mU-mA-fA-mG-r1-fC-mA-mU-mU-mA-fU-mC-fC-mU-mA- mA-mU-mGs-mGs-mG; mAs-fCs-mA-mU-mA-fA-mG-fA-r1-mA-mU-mU-mA-fU-mC-fC-mU-mA- mA-mU-mGs-mGs-mG; The sense strand comprises a nucleotide sequence selected from the group consisting of: mCs-r1s-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-m A-mU-mG-mU; mCs-mAs-r1-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-m A-mU-mG-mU; mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-r1-mU-m A-mU-mG-mU; mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU-r 1-mU-mG-mU; mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU- mA-r1-mG-mU; mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU- mA-mU-r1-mU; mCs-mAs-mU-mU-mA-mG-fG-mA-fU-fA-fA-mU-mG-mU-mC-mU-mU- mA-mU-mG-mU; Among them, mA, mU, mG, and mC are 2'-O-methyl-modified adenosine-3'-phosphate, 2'-O-methyl-modified uridine-3'-phosphate, 2'-O-methyl-modified guanosine-3'-phosphate, and 2'-O-methyl-modified cytidine-3'-phosphate, respectively; fA, fU, fG, and fC are 2'-fluoro-modified adenosine-3'-phosphate, 2'-fluoro-modified uridine-3'-phosphate, 2'-fluoro-modified guanosine-3'-phosphate, and 2'-fluoro-modified cytidine-3'-phosphate, respectively; and s is a phosphorothioate bond.

8. The double-stranded siRNA according to any one of claims 1 to 7, comprising a sense strand and an antisense strand pair of any one of sequence numbers 1 to 27: Preferably, the double-stranded siRNA comprises a sense strand and an antisense strand pair of any one of the following:

9. A conjugate comprising the double-stranded siRNA according to any one of claims 1 to 8.

10. The conjugate according to claim 9, comprising a liver targeting moiety, preferably the liver targeting moiety comprises an N-acetylgalactosamine ligand; preferably wherein the liver targeting moiety comprises a trivalent or tetravalent N-acetylgalactosamine ligand; preferably, the ligand is linked to the 3' end of the sense strand.

11. The conjugate according to claim 9 or 10, which consists of negative ions and positive ions; in, X - O - or S - ; M z+ is a pharmaceutically acceptable positive ion; The negative ions The total valence of is equal to that of the positive ion; RNA is a double-stranded siRNA according to any one of claims 1 to 8; for for for or, for for for A is or connection key; for k is 2; When A is a connecting bond, A is located in L 2 meta; When A is When A is located at L 2 Para or meta position; n1, n2, n3, n4, n5, n6 and n7 are independently 1, 2, 3, 4, 5 or 6; m1 and m2 are independently 0, 1, 2, 3, 4 or 5; q1 and q2 are independently 1, 2, 3, 4 or 5.

12. The conjugate according to claim 11, wherein the conjugate satisfies one or more of the following conditions: (1) n1 is 1, 2, 3, 4 or 5; preferably, n1 is 1, 2 or 3; (2) n2 is 1, 2, 3 or 4; preferably, n2 is 1 or 2; (3) n3 is 1, 2, 3, 4 or 5; preferably, n3 is 3; (4) n4 is 1, 2, 3 or 4; preferably, n4 is 2; (5) n5 is 1, 2, 3, 4, 5 or 6; preferably, n5 is 4; (6) n6 is 1, 2, 3, 4 or 5; preferably, n6 is 3; (7) n7 is 1, 2 or 3; preferably, n7 is 1; (8) m1 is 1 or 2; preferably, m1 is 1; (9) m2 is 0, 1, 2, 3, 4 or 5; preferably, m2 is 0, 1, 2 or 3; (10) q1 is 1, 2, 3 or 4; preferably, q1 is 2; (11) q2 is 1, 2, 3, 4 or 5; preferably, q2 is 3; and (12)X - O - ; (13)M z+ is a metal cation or an organic base cation; preferably, the metal cation is an alkali metal or alkaline earth metal cation, such as Na + , K + or Ca 2+ , the organic base cation may be an ammonium cation, for example 13. The conjugate according to claim 12, wherein the conjugate satisfies one or more of the following conditions: (1) for Preferably, the for (2) for Preferably, the for (3) for 14. The conjugate according to claim 13, wherein the conjugate satisfies one or both of the following conditions: (1) for Preferably, the for and (2) for 15. The conjugate according to claim 14, wherein the for 16. The conjugate according to claim 15, wherein the conjugate is any of the following structures: Preferably, the conjugate is:

17. The conjugate according to claim 9 or 10, wherein the N-acetylgalactosamine ligand is Preferably, the conjugate has the following structure: in, X is O or S.

18. A pharmaceutical composition comprising the double-stranded siRNA according to any one of claims 1 to 8 and / or the conjugate according to any one of claims 9 to 17 and a pharmaceutically acceptable carrier.

19. Use of the double-stranded siRNA of any one of claims 1 to 8, the conjugate of any one of claims 9 to 17, or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating and / or preventing a disease associated with PNPLA3 gene expression in a subject.

20. The use according to claim 19, wherein the disease associated with PNPLA3 gene expression is chronic liver disease; preferably, chronic liver disease is associated with accumulation and / or expansion of lipid droplets in the liver; preferably, the chronic liver is selected from the group consisting of hepatitis, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic fatty liver disease (ALD), HBV, HCV-related cirrhosis, drug-induced liver damage, and hepatocellular necrosis.

21. A cell comprising the double-stranded siRNA of any one of claims 1 to 8 and / or the conjugate of any one of claims 9 to 17.

22. A method for inhibiting PNPLA3 gene expression in cells in vitro, comprising contacting the cells with the double-stranded siRNA of any one of claims 1 to 8 and / or the conjugate of any one of claims 9 to 17.

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