Double-stranded sirna which inhibits HSD17b13 expression, and conjugate thereof

Abstract

Provided in the present invention are a double-stranded siRNA which inhibits HSD17B13 expression, and a conjugate thereof. The double-stranded siRNA includes a sense strand and an antisense strand, and one or more nucleotide residues in the nucleotide sequence of the sense strand and / or the antisense strand are replaced with r which has the formula as shown by formula I.

Description

Double-stranded siRNAs and their conjugates that inhibit HSD17B13 expression

[0001] This application claims priority to Chinese patent application 2024114547493, filed on 2024 / 10 / 17, and Chinese patent application 202510575824X, filed on 2025 / 05 / 06. The full text of both of these Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of RNA interference, specifically relating to double-stranded siRNA that inhibits HSD17B13 expression and its conjugates. Background Technology

[0003] Nonalcoholic fatty liver disease (NAFLD), also known as metabolic associated fatty liver disease (MAFLD), is a complex chronic liver disease closely related to overweight, obesity, insulin resistance (IR), and metabolic dysfunction. Nonalcoholic steatohepatitis (NASH) is a progressive form of MAFLD, characterized histologically by at least 5% steatosis, ballooning degeneration, and lobular inflammation found on liver biopsy, with or without fibrosis. This disease easily progresses to end-stage liver-related diseases such as cirrhosis and liver cancer, and also increases the risk of cardiovascular events. It is estimated that there are over 100 million NASH patients worldwide, and this number is projected to exceed 350 million by 2030. However, due to the complexity of the disease, there are currently no widely used and effective treatments in clinical practice. NASH treatment research mainly focuses on reducing or reversing steatosis and inflammation, and improving fibrosis. Methodologically, besides lifestyle interventions, the main treatments are surgery and medication. While lifestyle interventions can alleviate NASH to some extent, their efficacy is limited, but they remain the cornerstone of NASH treatment. Due to potential health risks, high costs, and limited application, surgical treatment will be difficult to widely adopt for a considerable period. Medication will be the ultimate effective way to resolve NASH. Currently, most drug research is in the preclinical model stage, with some entering Phase II or even Phase III clinical trials, but these have been ultimately eliminated due to poor efficacy or significant adverse reactions, indicating that drug development for NASH treatment remains fraught with difficulties.

[0004] HSD17B13 (hydroxysteroid 17-β dehydrogenase 13) belongs to the HSD17B (17β-hydroxysteroid dehydrogenase) family and possesses NAD(P)H / dependent NAD(P)+ oxidoreductase activity. It is primarily responsible for catalyzing the interconversion between 17-ketosteroids and 17-hydroxysteroids to maintain the balance between ineffective (17-keto) and potent (17β-hydroxy) forms of estrogen and androgen, thereby participating in the activation and inactivation of sex hormones. In the liver, HSD17B13 participates in fatty acid metabolism, cholesterol biosynthesis, and bile acid production. It also acts as a retinoic acid dehydrogenase, converting retinol to retinoic acid (RA).

[0005] Recent reports describe a loss-of-function HSD17B13 mutant that offers remarkable protection against liver diseases, significantly reducing ALT and AST levels and all types of liver disease, including alcoholic liver disease (42-53%), non-alcoholic liver disease (13-43%), alcoholic cirrhosis (42-73%), non-alcoholic cirrhosis (26-49%), and hepatocellular carcinoma. In patients with fatty liver, this variant reduces the likelihood of progression to non-alcoholic fatty liver disease (13-52%) and liver fibrosis (13-61%).

[0006] HSD17B13 is one of the most abundant lipid droplet (LD)-related proteins expressed in the liver. LDs are the main storage sites for intracellular neutral lipids, composed of internal neutral lipids and cholesterol esters and an outer monolayer of phospholipids. They are bioactive organelles involved in lipid metabolism, membrane transport, and signal transduction. HSD17B13 expression is upregulated in the livers of patients with NAFLD and mice. Increased numbers and sizes of excess HSD17B13 LDs significantly increase hepatic lipogenesis and triglyceride (TG) levels, leading to a fatty liver phenotype. Increased LD accumulation is associated with various metabolic diseases and chronic fibrotic liver diseases such as liver fibrosis, NASH, and NAFLD. Numerous preclinical animal studies and human genetic data contribute to a clearer understanding of NAFLD and support the possibility that HSD17B13 may serve as a potential therapeutic target for NAFLD / NASH and chronic liver diseases.

[0007] RNA silencing (RNAi) therapy is one of the earlier developed treatments that has shown promising clinical results. RNA interference (RNAi) refers to the highly conserved, evolutionarily specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). The non-coding double-stranded RNA that causes this phenomenon is named small interfering RNA (siRNA), and the phenomenon itself is named RNAi.

[0008] siRNA contains a sense strand and an antisense strand, with the antisense strand complementary to the target mRNA. siRNA enters the cytoplasm via endocytosis and then interacts with Dicer (RNase III endonuclease), Argonaute (RNase), and the RNA-binding cofactor, a transactivation effector element, to form the RISC (RNA-Induced Silencing Complex) complex (RLC). This further forms the RISC, which can bind to the target mRNA with a complementary sequence to the siRNA guide strand. After binding to the target mRNA, the RISC induces gene silencing through various mechanisms.

[0009] siRNA drugs have gradually developed into a mature treatment approach. In the field of RNAi therapy for NASH, there are several pharmaceutical companies, including Arrowhead, Ionis Pharmaceuticals, Alnylam Pharmaceuticals, and Ribobio, among which those with faster progress have entered Phase II clinical trials. Arrowhead's Phase 1 / 2 clinical study of ARO-HSD showed that ARO-HSD is the first investigational treatment to achieve a significant reduction in mRNA and liver HSD17B13 protein levels, leading to a reduction in alanine aminotransferase (ALT).

[0010] Five patients with suspected NASH showed strong pharmacodynamic effects by liver biopsy on day 71. HSD17B13 mRNA decreased by an average of 84%, ranging from 62% to 96%. HSD17B13 protein decreased by 83% or more. Two patients showed protein reductions of 92% and 97%, while the other three patients' day 71 measurements decreased below the lower limit of quantitation. Mean ALT decreased by 46% from baseline, with all patients showing a reduction of 26%–53%. ARO-HSD was well tolerated in healthy volunteers, with no associated grade 3 or 4 laboratory abnormalities, no drug-related serious adverse events, and no discontinuation of treatment.

[0011] Numerous preclinical animal studies and human genetic data support the possibility that HSD17B13 is a potential target for the treatment of NAFLD / NASH and chronic liver disease. Summary of the Invention

[0012] To further improve the drug-likeness of siRNA drugs targeting HSD17B13, such as increasing efficacy, reducing off-target effects, and lowering production difficulty and cost, this invention provides a double-stranded siRNA that inhibits HSD17B13 expression and its conjugates. The double-stranded siRNA and its conjugates of this invention are novel double-stranded siRNAs and GalNAc-siRNA-conjugated drugs. The double-stranded siRNA of this invention incorporates non-natural nucleotide monomers (Hu et al. Signal Transduct Target Ther 2020 Jun 19; 5(1):101), increasing the target selectivity and activity of the siRNA sequence, and reducing off-target risk while maintaining high drug activity.

[0013] On one hand, a double-stranded siRNA is provided, comprising a sense strand and an antisense strand forming a reverse complementary double-stranded region, wherein:

[0014] The antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in the sequence shown in SEQ ID NO:1 (5'-AACAAGAUUAGUCUUGAUGUAGU-3'), wherein the length of the sense and antisense strands is independently 17 to 25 nucleotides; or

[0015] The antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the sequence shown in SEQ ID NO:3 (5'-AGAUAGTCCAUGCAAAAGCAUUC-3'), wherein the length of the sense strand and the antisense strand are independently 17 to 25 nucleotides.

[0016] One or more nucleotide residues in the nucleotide sequence of the sense strand and / or antisense strand are replaced by the following formula r:

[0017] Where: X 1 Y 1 and Z 1 Independently CH or N, and X 1 Y 1 and Z 1 At least one of them is N;

[0018] R 1 H, or optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkoxy, optional substituted C2-C 10 Alkenyl, fluorine, chlorine, bromine, or iodine;

[0019] The optional substituted C1-C 10 Alkyl groups, the optionally substituted C1-C groups 10 Alkoxy groups and the optional substituted C2-C 10 The substituents in the alkenyl group are selected from one or more groups from the group consisting of: C1-C6 alkyl, C-C2 alkenyl, C1-C6 alkoxy, hydroxyl, oxo, fluorine, chlorine, bromine and iodine.

[0020] In one implementation, the C1-C 10 The alkyl group is a C1-C6 alkyl group, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, such as methyl or ethyl.

[0021] In one implementation, the C1-C 10 The alkoxy group is a C1-C6 alkoxy group, preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, a sec-butoxy group, or a tert-butoxy group, such as a methoxy group.

[0022] In one implementation, the C2-C 10 The alkenyl group is C2-C6 alkenyl, preferably vinyl or propenyl.

[0023] In one embodiment, the substituent is selected from one or more groups of the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, hydroxyl, oxo, fluorine, chlorine, bromine or iodine.

[0024] Preferably, the substituent is selected from one or more groups of the group consisting of methoxy and vinyl groups.

[0025] In one implementation, R 1 C1-C is an optional substitute 10 Alkoxy, wherein the substituent is selected from one or more groups of the group consisting of: C2-C6 alkenyl, C1-C6 alkoxy, fluorine, chlorine, bromine or iodine (e.g. methoxy and vinyl).

[0026] In one implementation, R 1 It is H or a C1-C3 alkoxy group optionally substituted with a methoxy or C2 alkenyl group.

[0027] In one implementation, r is selected from any of the following structures:

[0028] In one implementation, the double-stranded siRNA satisfies one or more of the following conditions:

[0029] (1) The positive strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in SEQ ID NO:2 (5'-UACAUCAAGACUAAUCUUGUU-3') or SEQ ID NO:4 (5'-AUGCUUUUGCAUGGACUAUCU-3');

[0030] (2) The antisense strand has a 2-nucleotide overhang at the 3' end; preferably, the 2 nucleotides are GU or UC;

[0031] (3) The lengths of the sense strand and the antisense strand are each independently 19–25 nucleotides; preferably each independently 19–23 nucleotides; more preferably the sense strand is 21 nucleotides and the antisense strand is 23 nucleotides; and,

[0032] (4) The lengths of the reverse complementary double-stranded regions of the justice chain and the antisense chain are independently 19 to 23 bp, preferably 21 to 23 bp.

[0033] In one embodiment, the antisense strand comprises the sequence shown in SEQ ID NO:1 and the sense strand comprises the sequence shown in SEQ ID NO:2; or, the antisense strand comprises the sequence shown in SEQ ID NO:3 and the sense strand comprises the sequence shown in SEQ ID NO:4; one or more nucleotide residues in the nucleotide sequences of the sense strand and / or the antisense strand are replaced with r having the formula I shown, as defined above.

[0034] In one implementation, 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.

[0035] In one embodiment, the modification is selected from methoxy modification, fluorination modification, thiophosphate linkage, replacement of nucleotides with glycerol nucleic acids, (E)-vinyl phosphate, and 2'-deoxynucleotides; preferably, the methoxy modification is a 2'-O-methyl modification; preferably, the fluorination modification is a 2'-fluorination modification.

[0036] In one embodiment, the antisense strand comprises two phosphate thioester bonds between the three terminal nucleotides at the 3' end and two phosphate thioester bonds between the three terminal nucleotides at the 5' end.

[0037] In one implementation, the positive chain comprises two phosphate thioester bonds between three terminal nucleotides at the 5' end.

[0038] In one implementation, r is linked to an adjacent nucleotide residue via a phosphate ester or thiophosphate bond.

[0039] In one embodiment, r-substitution is performed at one or more nucleotide residues at positions 6, 7, 8, 10, 12, 13, 14, 15, 16, 18, and 20 of the antisense strand, in the direction from the 5' end to the 3' end.

[0040] In one embodiment, r-substitution is performed at one or more nucleotide residues at positions 2, 3, 9, 11, 13, 14, 15, 16, 18, 19, and 20 of the positive strand, in a direction from the 5' end to the 3' end.

[0041] In one embodiment, one or more nucleotides at positions 2, 14, and 16 of the antisense strand are fluorinated nucleotides, with the remaining nucleotides being 2'-O-methyl nucleotides, following the direction from the 5' end to the 3' end.

[0042] In one embodiment, one or more nucleotides at positions 7 and 9-11 of the positive strand are fluorinated nucleotides, with the remaining nucleotides being 2'-O-methyl nucleotides, oriented from the 5' end to the 3' end.

[0043] In one embodiment, one or more nucleotides at positions 2, 14, and 16 of the antisense strand are deoxynucleotides, with the remaining nucleotides being 2'-O-methyl-modified or fluorinated nucleotides.

[0044] In one embodiment, one or more nucleotides at positions 7 and 9-11 of the positive strand are deoxyribonucleotides, with the remaining nucleotides being 2'-O-methyl modified or fluorinated modified nucleotides, oriented from the 5' end to the 3' end.

[0045] In one embodiment, the nucleotide at position 7 of the antisense strand, in the direction from the 5' end to the 3' end, is a glycerol nucleic acid, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides, fluorinated modified nucleotides, or deoxynucleotides.

[0046] When r occupies the position mentioned above, no additional modifications are applied to that position.

[0047] In one implementation, the antisense strand comprises a nucleotide sequence selected from the group consisting of:

[0048] asAfscaagruuagucUfuGfauguasgsu; (SEQ ID NO:52)

[0049] asGfsauagTgnccaugcAfaAfagcaususc;

[0050] asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0051] asGfsauagrccaugcdAaAfagcaususc; (SEQ ID NO:54)

[0052] asGfsauaguccaugcAfaAfagcaususc;

[0053] asGfsauaguccrugcAfaAfagcaususc; (SEQ ID NO:55)

[0054] asGfsauagTgnccaugcdAaAfagcaususc;

[0055] asGfsauagTgnccaugcAfadAagcaususc;

[0056] asGfsauagTgnccrugcAfaAfagcaususc; (SEQ ID NO:56)

[0057] asGfsauagTgnccaurcAfaAfagcaususc; (SEQ ID NO:57)

[0058] asGfsauagTgnccaugcraAfagcaususc; (SEQ ID NO:58)

[0059] asGfsauagTgnccaugcAfaragcaususc; (SEQ ID NO:59)

[0060] asGfsauagTgnccaugcAfaAfarcaususc; (SEQ ID NO:60)

[0061] asGfsauagTgnccaugcAfaAfagcrususc; (SEQ ID NO:61)

[0062] asGfsauagrccaugcAfadAagcaususc; (SEQ ID NO:62)

[0063] asGfsauagTgnccaugcAfrAfagcaususc; (SEQ ID NO:63) and,

[0064] asGfsauagTgnccaugrAfaAfagcaususc (SEQ ID NO: 64).

[0065] In one implementation, the positive strand comprises a nucleotide sequence selected from the group consisting of:

[0066] usascaucAfaGfAfCfuaaucuuguu;

[0067] usrscaucAfaGfAfCfuaaucuuguu(SEQ ID NO:65);

[0068] usascaucAfaGfAfCfuaaucuuruu(SEQ ID NO:66);

[0069] usascaucAfaGfAfCfuaaucuugru (SEQ ID NO: 67);

[0070] asusgcuuUfuGfCfAfuggacuaucu;

[0071] asrsgcuuUfuGfCfAfuggacuaucu(SEQ ID NO:68);

[0072] asusgcuuUfuGfCfAfuggacuarcu(SEQ ID NO:69);

[0073] asusgcuuUfuGfCfAfuggacuauru(SEQ ID NO:70);

[0074] asrsgcuuUfuGfCfAfuggacuauru(SEQ ID NO:71);

[0075] asusgcuuUfuGfCfAfuggaruauru(SEQ ID NO:72);

[0076] asusgcuuUfuGfCfAfuggacuarru(SEQ ID NO:73);

[0077] asusgcuudUfuGfCfAfuggacuauru(SEQ ID NO:74);

[0078] asusgcuuUfuGfCfdAuggacuauru(SEQ ID NO:75);

[0079] asusgcuuUfuGfCfdAuggacuaucu;

[0080] asusgcuuUfuGfCfdAuggacurucu(SEQ ID NO:76);

[0081] asusgcuudUfuGfCfAfuggacuaucu;

[0082] asusgcuuUfudGCfAfuggacuaucu;

[0083] asusgcuuUfuGfdCAfuggacuaucu;

[0084] asusrcuuUfuGfCfAfuggacuaucu(SEQ ID NO:77);

[0085] asusgcuuUfurCfAfuggacuaucu(SEQ ID NO:78);

[0086] asusgcuuUfuGfCfruggacuaucu(SEQ ID NO:79);

[0087] asusgcuuUfuGfCfAfurgacuaucu(SEQ ID NO:80);

[0088] asusgcuuUfuGfCfAfugracuaucu(SEQ ID NO:81);

[0089] asusgcuuUfuGfCfAfuggrcuaucu(SEQ ID NO:82);

[0090] asusgcuuUfuGfdCAfuggrcuaucu(SEQ ID NO:83);

[0091] asusgcuuUfuGfdCAfuggacuauru(SEQ ID NO:84); and,

[0092] asusgcuudUfuGfCfAfuggrcuaucu(SEQ ID NO:85)。

[0093] The lowercase letters a, u, g, and c represent 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; Af, Uf, Gf, and Cf represent 2'-fluorine modified adenosine-3'-phosphate, 2'-fluorine modified uridine-3'-phosphate, 2'-fluorine modified guanosine-3'-phosphate, and 2'-fluorine modified cytidine-3'-phosphate, respectively; Tgn represents thymidine glycerol phosphate; s represents a thiophosphate bond; d indicates that the nucleotide adjacent to the right of the letter d is a 2'-deoxyribonucleotide; r, as defined above, is linked to other nucleotide residues via phosphate or thiophosphate.

[0094] In one implementation, the double-stranded siRNA comprises a sense strand and an antisense strand pair of any of the following:

[0095] (1) Chain of Justice: usascaucAfaGfAfCfuaaucuuguu

[0096] Antonym chain: asAfscaagruuagucUfuGfauguasgsu; (SEQ ID NO:52)

[0097] (2) Chain of Justice: usrscaucAfaGfAfCfuaaucuuguu(SEQ ID NO:65)

[0098] Antonym chain: asAfscaagruuagucUfuGfauguasgsu; (SEQ ID NO:52)

[0099] (3) Chain of Justice: usascaucAfaGfAfCfuaaucuuruu(SEQ ID NO:66)

[0100] Antonym chain: asAfscaagruuagucUfuGfauguasgsu; (SEQ ID NO:52)

[0101] (4) Chain of Justice: usascaucAfaGfAfCfuaaucuugru(SEQ ID NO:67)

[0102] Antonym chain: asAfscaagruuagucUfuGfauguasgsu; (SEQ ID NO:52)

[0103] (5) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0104] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0105] (6) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0106] Antonyms: asGfsauagTgnccrugcAfaAfagcaususc; (SEQ ID NO:56)

[0107] (7) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0108] Antonym chain: asGfsauagTgnccaurcAfaAfagcaususc; (SEQ ID NO:57)

[0109] (8) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0110] Antonym chain: asGfsauagTgnccaugcraAfagcaususc; (SEQ ID NO:58)

[0111] (9) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0112] Antonym chain: asGfsauagTgnccaugcAfaragcaususc; (SEQ ID NO:59)

[0113] (10) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0114] Antonym chain: asGfsauagTgnccaugcAfaAfarcaususc; (SEQ ID NO:60)

[0115] (11) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0116] Antonym chain: asGfsauagTgnccaugcAfaAfagcrususc; (SEQ ID NO:61)

[0117] (12) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0118] Antonym chain: asGfsauagTgnccaugcAfrAfagcaususc; (SEQ ID NO:63)

[0119] (13) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu

[0120] Antonym chain: asGfsauagTgnccaugrAfaAfagcaususc(SEQ ID NO:64);

[0121] (14) Chain of Justice: asrsgcuuUfuGfCfAfuggacuaucu(SEQ ID NO:68)

[0122] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0123] (15) Chain of Justice: asrsgcuuUfuGfCfAfuggacuaucu(SEQ ID NO:68)

[0124] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0125] (16) Chain of Justice: asusgcuuUfuGfCfAfuggacuarcu (SEQ ID NO:69)

[0126] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0127] (17) Chain of Justice: asusgcuuUfuGfCfAfuggacuarcu (SEQ ID NO:69)

[0128] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0129] (18) Chain of Justice: asusgcuuUfuGfCfAfuggacuauru (SEQ ID NO:70)

[0130] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0131] (19) Chain of Justice: asusgcuuUfuGfCfAfuggacuauru (SEQ ID NO:70)

[0132] Antonym chain: asGfsauagrccaugcdAaAfagcaususc; (SEQ ID NO:54)

[0133] (20) Chain of Justice: asusgcuuUfuGfCfAfuggacuauru (SEQ ID NO:70)

[0134] Antonyms: asGfsauagrccaugcAfadAagcaususc; (SEQ ID NO:62)

[0135] (21) Chain of Justice: asrsgcuuUfuGfCfAfuggacuauru(SEQ ID NO:71)

[0136] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0137] (22) Chain of Justice: asusgcuuUfuGfCfAfuggaruauru (SEQ ID NO:72)

[0138] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0139] (23) Chain of Justice: asusgcuuUfuGfCfAfuggacuarru(SEQ ID NO:73)

[0140] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0141] (24) Chain of Justice: asusgcuudUfuGfCfAfuggacuauru(SEQ ID NO:74)

[0142] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0143] (25) Chain of Justice: asusgcuudUfuGfCfAfuggacuauru(SEQ ID NO:74)

[0144] Antonym chain: asGfsauagrccaugcdAaAfagcaususc; (SEQ ID NO:54)

[0145] (26) Chain of Justice: asusgcuudUfuGfCfAfuggacuauru(SEQ ID NO:74)

[0146] Antonyms: asGfsauagrccaugcAfadAagcaususc; (SEQ ID NO:62)

[0147] (27) Chain of Justice: asusgcuuUfuGfCfdAuggacuauru (SEQ ID NO:75)

[0148] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0149] (28) Chain of Justice: asusgcuuUfuGfCfdAuggacuauru (SEQ ID NO:75)

[0150] Antonym chain: asGfsauagrccaugcdAaAfagcaususc; (SEQ ID NO:54)

[0151] (29) Chain of Justice: asusgcuuUfuGfCfdAuggacuaucu

[0152] Antonyms: asGfsauaguccrugcAfaAfagcaususc; (SEQ ID NO:55)

[0153] (30) Chain of Justice: asusgcuuUfuGfCfdAuggacurucu (SEQ ID NO:76)

[0154] Antonyms: asGfsauaguccaugcAfaAfagcaususc;

[0155] (31) Chain of Justice: asusrcuuUfuGfCfAfuggacuaucu(SEQ ID NO:77)

[0156] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0157] (32) Chain of Justice: asusgcuuUfurCfAfuggacuaucu(SEQ ID NO:78)

[0158] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0159] (33) Chain of Justice: asusgcuuUfuGfCfruggacuaucu(SEQ ID NO:79)

[0160] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0161] (34) Chain of Justice: asusgcuuUfuGfCfAfurgacuaucu(SEQ ID NO:80)

[0162] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0163] (35) Chain of Justice: asusgcuuUfuGfCfAfugracuaucu(SEQ ID NO:81)

[0164] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0165] (36) Chain of Justice: asusgcuuUfuGfCfAfuggrcuaucu(SEQ ID NO:82)

[0166] Antonyms: asGfsauagTgnccaugcAfaAfagcaususc;

[0167] (37) Chain of Justice: asusgcuuUfuGfdCAfuggrcuaucu(SEQ ID NO:83)

[0168] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0169] (38) Chain of Justice: asusgcuuUfuGfdCAfuggrcuaucu(SEQ ID NO:83)

[0170] Antonym chain: asGfsauagrccaugcdAaAfagcaususc; (SEQ ID NO:54)

[0171] (39) Chain of Justice: asusgcuuUfuGfdCAfuggrcuaucu(SEQ ID NO:83)

[0172] Antonyms: asGfsauagrccaugcAfadAagcaususc; (SEQ ID NO:62)

[0173] (40) Chain of Justice: asusgcuuUfuGfdCAfuggacuauru(SEQ ID NO:84)

[0174] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0175] (41) Chain of Justice: asusgcuuUfuGfdCAfuggacuauru(SEQ ID NO:84)

[0176] Antonym chain: asGfsauagrccaugcdAaAfagcaususc; (SEQ ID NO:54)

[0177] (42) Chain of Justice: asusgcuuUfuGfdCAfuggacuauru(SEQ ID NO:84)

[0178] Antonyms: asGfsauagrccaugcAfadAagcaususc; (SEQ ID NO:62)

[0179] (43) Chain of Justice: asusgcuudUfuGfCfAfuggrcuaucu(SEQ ID NO:85)

[0180] Antonym chain: asGfsauagrccaugcAfaAfagcaususc; (SEQ ID NO:53)

[0181] (44) Chain of Justice: asusgcuudUfuGfCfAfuggrcuaucu(SEQ ID NO:85)

[0182] Antonym chain: asGfsauagrccaugcdAaAfagcaususc; (SEQ ID NO:54)

[0183] (45) Chain of Justice: asusgcuudUfuGfCfAfuggrcuaucu(SEQ ID NO:85)

[0184] Antonym chain: asGfsauagrccaugcAfadAagcaususc (SEQ ID NO:62).

[0185] In one embodiment, the double-stranded siRNA comprises a sense strand and an antisense strand pair of any of the following:

[0186] r1 to r13 are the same as described above.

[0187] In another aspect, a conjugate comprising the double-stranded siRNA described herein is provided.

[0188] In one embodiment, the conjugate comprises a liver-targeting moiety, preferably comprising N-acetylgalactosamine; preferably wherein the liver-targeting moiety comprises trivalent or tetravalent N-acetylgalactosamine.

[0189] In one implementation, the liver-targeting portion is connected to the 3' end of the justice chain.

[0190] In one embodiment, the conjugate is composed of negative ions and positive ions;

[0191] X - For O - or S - ;

[0192] M z+ It is a pharmaceutically acceptable positive ion;

[0193] The negative ions mentioned The total valence is equal to the total valence of the positive ion;

[0194] The RNA is the double-stranded siRNA described in this invention;

[0195] for (The left end of the segment is connected to Gal, and the right end is connected to N);

[0196] for (The left end of the fragment is connected to a phenyl group, and the right end is connected to an L group) 3 (connected) for or, for for

[0197] The left end of the segment is -(CH2)qC(=O)-(L 3 (connected, with the right end connected to N), q is 5, 6, 7, 8, 9 or 10, and 1, 2 or 3 of -(CH2)q- can be optionally replaced by 1, 2 or 3 O and / or -NHC(=O)-;

[0198] A is Or connect key;

[0199] for (The left end of the fragment is connected to the carbonyl group, and the right end is connected to N);

[0200] k is 2;

[0201] When A is the link key, A is located in L. 2 Interposition;

[0202] When A is When A is located at L 2 Opposite or intermediate;

[0203] n1, n2, n3, n4, n5, n6 and n7 are independently 1, 2, 3, 4, 5 or 6;

[0204] m1 and m2 are independently 0, 1, 2, 3, 4 or 5;

[0205] q1 and q2 are independently 1, 2, 3, 4 or 5.

[0206] In one implementation, n1 is 1, 2, 3, 4 or 5; preferably, n1 is 1, 2 or 3.

[0207] In one implementation, n2 is 1, 2, 3 or 4; preferably, n2 is 1 or 2.

[0208] In one implementation, n3 is 1, 2, 3, 4 or 5; preferably, n3 is 3.

[0209] In one implementation, n4 is 1, 2, 3 or 4; preferably, n4 is 2.

[0210] In one implementation, n5 is 1, 2, 3, 4, 5 or 6; preferably, n5 is 4.

[0211] In one implementation, n6 is 1, 2, 3, 4 or 5; preferably, n6 is 3.

[0212] In one implementation, n7 is 1, 2, or 3; preferably, n7 is 1.

[0213] In one implementation, m1 is 1 or 2; preferably, m1 is 1.

[0214] In one implementation, m2 is 0, 1, 2, 3, 4 or 5; preferably, m2 is 0, 1, 2 or 3.

[0215] In one implementation, q1 is 1, 2, 3 or 4; preferably, q1 is 2.

[0216] In one implementation, q2 is 1, 2, 3, 4 or 5; preferably, q2 is 3.

[0217] In one implementation, X- For O - .

[0218] In one implementation scheme, M z+ It 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 can be an ammonium cation, for example, ...

[0219] In one implementation scheme, the aforementioned for Preferably, the for The left end of the above segment is connected to Gal, and the right end is connected to N.

[0220] In one implementation scheme, the aforementioned for Preferably, the for The left end of the above fragment is connected to a phenyl group, and the right end is connected to an L group. 3 Connected.

[0221] In one implementation scheme -(CH2)q and L 3 The carbonyl group is connected to the nitrogen phase in the parent compound.

[0222] In one implementation scheme for The left end of the above segment is related to L. 3 The carbonyl group is connected to the nitrogen phase in the parent compound.

[0223] In one implementation scheme, the aforementioned for The left end of the fragment is connected to a carbonyl group, and the right end is connected to a nitrogen group.

[0224] In one implementation scheme for Preferably, the for (The left end of the above segment is connected to a phenyl group, and the right end is connected to an N group).

[0225] In one implementation scheme for

[0226] In one implementation scheme for

[0227] In one embodiment, the conjugate has any of the following structures:

[0228] In one embodiment, the conjugate is:

[0229] Among them, X - For O - or S - The definition of RNA is the same as that described in any embodiment of this invention.

[0230] In one embodiment, the conjugate is any of the conjugates listed in Table 1.

[0231] In another aspect, a pharmaceutical composition is provided comprising the double-stranded siRNA and / or conjugates described herein, and a pharmaceutically acceptable carrier.

[0232] On the other hand, the use of the double-stranded siRNA, conjugate, or pharmaceutical composition described herein in the preparation of a medicament for the treatment and / or prevention of diseases associated with HSD17B13 gene expression is provided.

[0233] In one implementation, the disease associated with HSD17B13 gene expression is chronic liver disease.

[0234] In one implementation, chronic liver disease is associated with the accumulation and / or expansion of lipid droplets in the liver.

[0235] In one implementation scheme, 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.

[0236] In another aspect, a method for inhibiting HSD17B13 gene expression in cells in vitro is provided, comprising contacting the cells with the double-stranded siRNA and / or conjugates described herein.

[0237] Preferably, the cells are animal cells;

[0238] More preferably, the cells are human cells and / or hepatocytes, such as human primary hepatocytes.

[0239] The positive and beneficial effects of this invention include:

[0240] (1) The siRNA of the present invention exhibits good in vitro or in vivo inhibitory activity against HSD17B13 mRNA; and

[0241] (2) The tetravalent GalNAc group of the present invention has the same level of endocytosis efficiency as the reference compound L96 and exhibits good binding affinity with ASGPR. Detailed Implementation

[0242] Definitions and Explanations

[0243] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with the meaning as understood by one of ordinary skill in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0244] The term “and / or” should be interpreted as inclusive, that is, including at least one of the quantities or elements in the list, but also including more than one, and optionally, additional unlisted items.

[0245] The term "HSD17B13" refers to hydroxysteroid 17-β dehydrogenase 13 (HSD17B13). It is used to refer to the gene encoding hydroxysteroid 17-β dehydrogenase 13 from any mammalian source, unless explicitly excluded, including but not limited to humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs. The term also refers to fragments and variants of native HSD17B13 that maintain at least one in vivo or in vitro activity of native HSD17B13. The term encompasses HSD17B13 in its full-length, unprocessed precursor form, as well as forms obtained from post-translational cleavage of the signal peptide and forms obtained from proteolytic processing.

[0246] The term "nucleic acid" includes any oligonucleotide, polynucleotide, or polynucleotide, wherein a segment containing up to 60 nucleotides is generally called an oligonucleotide, and longer segments are called polynucleotides. Deoxyribose oligonucleotides consist of a 5-carbon sugar called deoxyribose, which is covalently linked to phosphate groups at the 5' and 3' carbons to form alternating unbranched polymers. DNA can be, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. Ribosose oligonucleotides consist of similar repetitive 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-amplifying RNA (saRNA), and combinations thereof. Therefore, in the context of this invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and inter-sugar (backbone) links. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers comprising non-naturally occurring monomers or portions thereof having similar functions. Such modified or substituted oligonucleotides are generally superior to their 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" as used in this disclosure refers to a single-stranded oligonucleotide having a sequence at least partially complementary to the target mRNA, capable of hybridizing with the target mRNA via hydrogen bonds under mammalian physiological conditions (or equivalent in vitro environments). In some embodiments of this disclosure, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide (ASO).

[0247] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by producing a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues. In some embodiments, the trivalent or tetravalent targeting ligands described herein can be conjugated to the nucleic acid. In some embodiments, the nucleic acid is the nucleic acid described herein. For example, the nucleic acid used herein can be single-stranded DNA or RNA, or 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 miRNAs, and oligonucleotides that form triplet strands.

[0248] In some embodiments, the nucleic acid is an oligonucleotide. In a particular embodiment, the length of the oligonucleotide is in the range of about 10 to about 100 nucleotides. In various related embodiments, the length of single-stranded, double-stranded, and triple-stranded oligonucleotides can be in the range of about 10 to about 60 nucleotides, about 15 to about 60 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides.

[0249] In some implementations, the nucleic acid is an antisense molecule. In some implementations, the nucleic acid is a miRNA molecule. In some implementations, the nucleic acid is a siRNA.

[0250] As used interchangeably herein, the terms “siRNA,” “RNAi,” “siRNA agent,” and “RNA interfering agent” refer to RNA containing the terms defined herein and which mediates the targeted cleavage of mRNA transcripts via 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) the expression of the HSD17B13 gene in cells (such as cells of an individual, or cells of a mammalian individual).

[0251] The short interfering RNAs (siRNAs) described in this disclosure are a class of double-stranded RNA molecules, 20-25 base pairs in length, similar to miRNAs, and operate within the RNA interference (siRNA) pathway. They interfere with the translation of mRNAs of specific genes that are complementary to or close to their nucleotide sequences, leading to mRNA degradation. The short interfering RNAs (siRNAs) described in this disclosure include double-stranded siRNAs (containing both sense and antisense strands) and single-stranded siRNAs (antense strand only).

[0252] The term "antisense strand" refers to a strand of iRNA (such as siRNA) that includes a region substantially complementary to a target sequence (e.g., HSD17B13 mRNA). The term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence. In cases where the complementary region is not perfectly complementary to the target sequence, mismatches may occur within the molecule or in terminal regions. Typically, the most tolerant mismatches are found in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of the siRNA. siRNA is typically chemically synthesized. The term "sense strand" means an iRNA containing a region substantially complementary to the region of the antisense strand as defined herein. The antisense and sense strands of siRNA can have the same or different lengths, as is known in the art.

[0253] 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 protecting and coupling groups, such as p-dimethoxytriphenylmethyl at the 5′ end and 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.

[0254] siRNA molecules can be assembled from two distinct oligonucleotides, one containing the sense strand of the siRNA and the other containing the antisense strand. For example, each oligonucleotide can be synthesized separately and linked together by hybridization or conjugation after synthesis and / or deprotection.

[0255] The term "sequence" or "nucleotide sequence" as used in this disclosure refers to the order or sequence of nucleobases or nucleotides described using sequence letters in standard nucleotide naming conventions. When 5' and 3' appear at the ends of a sequence, they indicate the direction of the sequence; sequences are typically written in the direction of 5' to 3'; the direction of synthesis for single-stranded RNA nucleotide chains is 3' to 5'.

[0256] The “modification” of nucleotides described in this disclosure includes, but is not limited to, alkoxy / methoxy modifications, fluorination modifications, and thiophosphate linkages.

[0257] The lowercase letters a, u, g, and c represent 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; Af, Uf, Gf, and Cf represent 2'-fluorine modified adenosine-3'-phosphate, 2'-fluorine modified uridine-3'-phosphate, 2'-fluorine modified guanosine-3'-phosphate, and 2'-fluorine modified cytidine-3'-phosphate, respectively; Tgn represents thymidine glycerol phosphate; s represents a thiophosphate bond; and d indicates that the nucleotide adjacent to the right of the letter d is a 2'-deoxyribonucleotide.

[0258] The fluorinated nucleotides described in this disclosure refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, and the methoxylated nucleotides refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group.

[0259] In this disclosure, "complementary" has the meaning known to those skilled in the art, namely, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary or near-complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises one purine and one pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. This invention utilizes the unique advantages of non-natural bases in complementarity or near-complementarity, such as improved selectivity, and consequently, safety advantages.

[0260] The compounds disclosed herein may exist in specific geometric or stereoisomeric forms. This disclosure envisions all such compounds, including (R)- and (S)-enantiomers, diastereomers, racemic mixtures, and other mixtures, such as mixtures enriched with enantiomers or diastereomers, 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 mixtures thereof are included within the scope of this disclosure.

[0261] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0262] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0263] Unless otherwise stated, the terms "rich in one isomer," "isomer enrichment," "rich in one enantiomer," or "enantiomer enrichment" mean that the content of one isomer or enantiomer is less than 100%, and the content of the 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%.

[0264] Unless otherwise stated, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomer excess (ee value) is 80%.

[0265] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound disclosed herein, 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 desired enantiomer in pure form. 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 salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to carbamates).

[0266] The term "salt" refers to the salt of the compounds disclosed herein, prepared by reacting a compound with a relatively non-toxic acid or base, as discovered in this disclosure, with a specific substituent. When the compounds of this disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds of this disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds of this disclosure contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0267] The term "pharmaceutical excipients" refers to the excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. They are all substances contained in pharmaceutical preparations, excluding the active ingredient.

[0268] Unless otherwise specified, when a group has one or more connectable sites, any one or more of these sites can be chemically bonded to other groups. The chemical bonds connecting these sites to other groups can be represented by a wavy line. The wavy line in parentheses indicates that the phenyl group is bonded to other groups through the carbon atoms at positions 1 and 2.

[0269] The salts disclosed herein can be synthesized from parent compounds containing anions or bases using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both.

[0270] r-structure unit

[0271] In this article, 'r' represents the following structural unit:

[0272] The definitions of each group are the same as those described above.

[0273] Preferably, r1 represents the following structural unit.

[0274] The r1 mentioned in this article is a non-natural nucleotide, unlike any publicly patented natural nucleotide base, which brings unexpected activity and selectivity advantages when introduced into the nucleic acid sequence.

[0275] R1 and other nucleotide residues can be linked together via phosphate esters or thiophosphate esters. For example, "asr1" indicates that the A and R1 nucleotide monomers are linked via a thiophosphate ester group, and "ar1" indicates that the A and R1 nucleotide monomers are linked via a phosphate ester group. The linkage methods are shown below:

[0276] Where X is O or S.

[0277] In this disclosure, the double-stranded siRNA comprises a sense strand or an r1-intercalated sense strand and / or an antisense strand or an r1-intercalated antisense strand. The sense strand, antisense strand, r-intercalated sense strand, and r-intercalated antisense strand all contain nucleotide groups as basic structural units. As is known to those skilled in the art, nucleotide groups contain phosphate groups, ribose groups, and bases, which will not be elaborated further here.

[0278] The r-intercalated sequence described in this disclosure refers to a sequence in which at least one nucleotide residue is linked to r1. The r-intercalated sequences described in this disclosure include, but are not limited to, r1-intercalated double-stranded siRNAs, r1-intercalated sense strands, and r1-intercalated antisense strands. For example, 5'-aGfUfr1r1asc-3' and 5'-r1gGfAfAfc-3' are both examples of r-intercalation.

[0279] The r-embedded double-stranded siRNA described in this disclosure refers to a double-stranded siRNA in which at least one nucleotide residue is linked to r1; this includes double-stranded siRNA in which one nucleotide residue is replaced by r in the sequence of the double-stranded siRNA. The r-embedded sense strand described in this disclosure refers to a sense strand in which at least one nucleotide residue is linked to r, including one or more nucleotides in the sense strand being replaced by r. The r-embedded antisense strand described in this disclosure refers to an antisense strand in which at least one nucleotide residue is linked to r, including one or more nucleotides in the antisense strand being replaced by r.

[0280] siRNA and modified nucleotides

[0281] This document provides siRNAs for inhibiting HSD17B13 gene expression. Each siRNA comprises a sense strand and an antisense strand. The length of each sense strand and antisense strand can be 17-25 (e.g., 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides. In some embodiments, the lengths of the sense and antisense strands of the siRNA are independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides. In some embodiments, the siRNA duplex has about 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some embodiments, the sense and antisense strands are complementary with a length of at least 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0282] An escape chain includes:

[0283] A: The nucleotide sequence of SEQ ID NO:1 or the nucleotide sequence of SEQ ID NO:1 in which one or more (e.g., 2-9, e.g., 2, 3, 4, 5, 6, 7) nucleotides are substituted, added, deleted or inserted.

[0284] B: The nucleotide sequence of SEQ ID NO:3 or the nucleotide sequence of SEQ ID NO:3 in which one or more (e.g., 2-9, e.g., 2, 3, 4, 5, 6, 7) nucleotides are substituted, added, deleted or inserted.

[0285] The Justice Chain includes

[0286] A: The nucleotide sequence of SEQ ID NO:2 or the nucleotide sequence of SEQ ID NO:2 in which one or more (e.g., 2-9, e.g., 2, 3, 4, 5, 6, 7) nucleotides are substituted, added, deleted or inserted.

[0287] B: A nucleotide sequence in which one or more (e.g., 2-9, e.g., 2, 3, 4, 5, 6, 7) nucleotides are substituted, added, deleted, or inserted in the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:2.

[0288] One or more (e.g., 1-3, e.g., 1, 2, or 3) nucleotide residues in the nucleotide sequence of the sense and / or antisense strands have r-substitution. One or more or all nucleotides of the antisense strand and / or one or more or all nucleotides of the sense strand may be modified nucleotides. Modifications may be selected from methoxy modifications, fluorinated modifications, thiophosphate linkages, replacement of the nucleotide with a glycerol nucleic acid, (E)-vinyl phosphate, or 2'-deoxynucleotides. Preferably, the methoxy modification is a 2'-O-methyl modification; preferably, the fluorinated modification is a 2'-fluoro modification.

[0289] In one embodiment, the antisense strand comprises two phosphate thioester bonds between the three terminal nucleotides at the 3' end and two phosphate thioester bonds between the three terminal nucleotides at the 5' end. In one embodiment, the sense strand comprises two phosphate thioester bonds between the three terminal nucleotides at the 5' end. In one embodiment, r1 is linked to adjacent nucleotide residues via phosphate or phosphate thioester bonds. In one embodiment, r1 substitution is performed at the 7th, 8th, or 9th nucleotide residue of the antisense strand in a 5'-to-3' manner. In one embodiment, r1 substitution is performed at the 2nd, 3rd, 16th, 18th, 19th, or 20th nucleotide residue of the sense strand in a 5'-to-3' manner. In one embodiment, one or more nucleotides at positions 2, 6th, 8th, 9th, 14th, and 16th of the antisense strand in a 5'-to-3' manner are fluorinated nucleotides, and the remaining nucleotides are 2'-O-methyl nucleotides. In one embodiment, one or more nucleotides at positions 7 and 8-11 of the positive strand are fluorinated nucleotides, arranged from the 5' end to the 3' end, while the remaining nucleotides are 2'-O-methylated nucleotides. When r1 occupies a position mentioned above, that position is not subject to the corresponding fluorinated or 2'-O-methylated modification.

[0290] Preferably, the siRNA described herein has any of the pairs of sense and antisense strands listed in Table 1.

[0291] Conjugate

[0292] The conjugate described herein comprises the siRNA described herein and includes 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 may be attached to the 3' end of the positive strand. The tetravalent N-acetylgalactosamine ligand described herein may be a tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugate group. The trivalent N-acetylgalactosamine ligand described herein may be L96 from Alnylam.

[0293] The tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugates or trivalent GalNAc conjugates described in this disclosure can enhance the delivery of therapeutic agents to specific target sites (e.g., specific organs or tissues) within a subject, such as a human or animal. In some embodiments of this disclosure, the conjugates can enhance the targeted delivery of expressed repressive oligonucleotides. In some embodiments of this disclosure, the tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugates or trivalent GalNAc conjugates can enhance the delivery of expressed repressive oligonucleotides (e.g., siRNA) to hepatocytes.

[0294] The tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugate group or trivalent GalNAc conjugate group described herein can be directly or indirectly linked to compounds, such as therapeutic agents, for example, expression repressive oligonucleotides (ASOs) or siRNAs, linked to the 3' or 5' end of the expression repressive oligonucleotide. In some embodiments of this disclosure, the expression repressive oligonucleotide comprises one or more modified natural or non-natural nucleotides. In some embodiments of this disclosure, the expression repressive oligonucleotide is an siRNA reagent, such as a double-stranded siRNA reagent comprising a sense strand and an antisense strand. In some embodiments of this disclosure, the tetravalent N-acetylgalactosamine-substituted aromatic amide GalNAc conjugate group disclosed herein is linked to the 5' end of the sense strand of the double-stranded siRNA reagent. In some embodiments, the conjugate group disclosed herein is linked to the expression repressive oligonucleotide reagent at the 3' end of the sense strand of the double-stranded siRNA reagent via a phosphate ester, thiophosphate ester, or phosphate ester group. The term "link" as used herein, when referring to a connection between two molecules, means that two molecular segments are linked by a covalent bond or that two molecules are linked by a non-covalent bond (e.g., a hydrogen bond or an ionic bond). The compounds and conjugation groups of the present invention may include one or more linking groups. The structure of each linking group may vary, provided that the conjugation 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. Linking groups may also modulate the solubility, stability, or aggregation properties of the conjugate.

[0295] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0296] The solvents used in this disclosure are commercially available.

[0297] Unless otherwise specified, all solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this disclosure are volume ratios.

[0298] Example

[0299] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to 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.

[0300] Example 1: Synthesis of phosphoramide monomer

[0301] Example 1-1: Synthesis of r1-M

[0302] Synthesis of intermediates 1-3 in step one

[0303] Compound 1-1 (30.0 g) and compound 1-2 (13.1 g) were added to 4-methylbenzenesulfonic acid (649 mg). After addition, the mixture was stirred at 100 °C for 4 hours. LCMS (RT = 0.616 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1-1 / 1) to give a yellow solid compound 1-3 (20.0 g, yield 55.0%).

[0304] LCMS(ESI) m / z: 386.1 [M+H] + ;

[0305] 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).

[0306] Synthesis of intermediates 1-4 in step two

[0307] Compounds 1-3 (15.0 g) were dissolved in methanol (100 mL), and triethylamine (11.8 g) was added. After the addition was complete, the reaction mixture was stirred at 70 °C for 12 hours. LCMS (RT = 0.284 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a colorless oily compound 1-4 (10.0 g, yield 89.1%).

[0308] LCMS(ESI) m / z: 260.1 [M+H] + ;

[0309] 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).

[0310] Synthesis of intermediates 1-5 in step three

[0311] Compounds 1-4 (15.0 g) were dissolved in N,N-dimethylformamide (100 mL), and imidazole (11.8 g) and 1,3-dichloro-1,1,3,3-tetraisopropyl dimethylsilyl ether (20.0 g) were added. The reaction mixture was stirred at 25 °C for 12 hours. LC-MS (RT = 1.008 min) showed that the starting material was completely consumed. The reaction mixture was extracted with water (100 mL) and ethyl acetate (80.0 mL, 50.0 mL). 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 / 1-1 / 1) to give colorless oily compounds 1-6 (15.0 g, yield 51.6%).

[0312] LCMS(ESI) m / z: 502.2 [M+H] + ;

[0313] 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).

[0314] Synthesis of intermediates 1-6 in step four

[0315] Compounds 1-5 (13.0 g) were dissolved in N,N-dimethylformamide (90.0 mL), and silver oxide (16.0 g) and iodomethane (18.3 g) were added. The reaction mixture was stirred at 25 °C for 3 hours after the addition was complete. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.43, starting material: Rf = 0.24) showed that the starting material was completely consumed. The reaction mixture was extracted with water (100 mL) and ethyl acetate (100 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1-0 / 1) to give colorless oily compounds 1-6 (1.80 g, yield 13.4%).

[0316] 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,2H),3,96-4.06(m,5H),3,68(s,3H),0.99-1.13(m,2H).

[0317] Synthesis of intermediates 1-7 in step five

[0318] Compounds 1-6 (200 mg) were 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 after the addition was complete. LCMS (RT = 0.253 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a yellow oily compound 1-7 (105 mg, crude product).

[0319] LCMS(ESI) m / z: 274.1 [M+H] + ;

[0320] 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=333,4.83Hz,1 H), 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).

[0321] Step 6 Synthesis of intermediates 1-8

[0322] Compounds 1-7 (100 mg) were dissolved in pyridine (2.00 mL), and 4,4-dimethoxytriphenylmethyl chloride (186 mg) was added at 0 °C. After addition, 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 that the starting material was completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a colorless oily compound 1-8 (120 mg, crude product).

[0323] LCMS(ESI) m / z: 574.2 [MH] + .

[0324] Step 7: Synthesis of the final product r1-M

[0325] Compounds 1-8 (100 mg) were dissolved in dichloromethane (2.00 mL), and N,N-diisopropylphosphonamide (2-cyanoethyl) ester (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 that the starting material was completely consumed. The reaction mixture was purified by high performance liquid chromatography (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 50%-80%, 8 min) to obtain a white solid r1-M (39.8 mg, 100% purity).

[0326] LCMS(ESI) m / z: 776.2 [M+H] + ;

[0327] 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).

[0328] Examples 1-2: Synthesis of phosphorus amide monomer r2-M

[0329] Synthesis of intermediate 2-1 in step one

[0330] Compound 1-5 (2.00 g) was dissolved in tetrahydrofuran (20.0 mL), and ethyl propylene carbonate (1.42 g), 1,4-bis(diphenylphosphine)butane (153 mg), and tris(dibenzylacetone)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 that the starting material was completely consumed. 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 / 1-0 / 1) to give a colorless oily compound 2-1 (1.40 g, yield 64.8%).

[0331] LCMS(ESI) m / z: 542.3 [M+H]+;

[0332] Synthesis of intermediate 2-2 in step two

[0333] 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 after the addition. 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 give a yellow oily compound 2-2 (770 mg, yield 99.5%).

[0334] Synthesis of intermediates 2-3 in step three

[0335] Compound 2-2 (500 mg) was dissolved in pyridine (5.00 mL), and 4,4-dimethoxytriphenylmethyl chloride (849 mg) was added at 0 °C. After the addition was complete, the reaction mixture was stirred at 25 °C for 12 hours. LCMS (RT = 0.800 min) showed that the starting material was completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a colorless oily compound 2-3 (700 mg, yield 69.6%).

[0336] LCMS(ESI) m / z: 602.2[M+H]+;

[0337] Step 4: Synthesis of final product r2

[0338] Compounds 2-3 (700 mg) were dissolved in dichloromethane (5.00 mL), and diammonium cyanotetraisopropylphosphate (420 mg) and 4,5-dicyanimidazole (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 that the starting materials were completely consumed. The reaction mixture was purified by high performance liquid chromatography (column: Waters Xbridge BEH C18250*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 70%-90%, 10 min) to obtain a white solid r2 (24.0 mg, purity 95.2%).

[0339] LCMS(ESI) m / z: 802.3[M+H]+;

[0340] 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)5.80 -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).

[0341] Examples 1-3: Synthesis of phosphorusamide monomer r3-M

[0342] Synthesis of final product 3-1 in step one

[0343] Compound 2-1 (10.0 g) was dissolved in a mixed solution of dichloromethane (66.0 mL) and methanol (33.0 mL). Ozone was bubbled through the solution at -78 °C, and the reaction mixture was stirred at -78 °C for 5 minutes. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.60, feedstock: Rf = 0.75) showed that the feedstock was completely consumed. Oxygen was bubbled through the solution for 15 minutes to remove excess ozone. Then, dimethyl sulfide (1.38 g) was added, and the mixture was stirred at 25 °C for 15 minutes. The reaction mixture was concentrated under reduced pressure to give a yellow oily compound 3-1 (5.20 g, crude product).

[0344] Step 2: Synthesis of final product 3-2

[0345] Compound 3-1 (5.20 g) was dissolved in a mixed solution of dichloromethane (35.0 mL) and methanol (16.0 mL). Sodium borohydride (1.04 g) was added at -78 °C, and the reaction mixture was stirred at 25 °C for 1 hour. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.45, feedstock: Rf = 0.60) showed that the feedstock was completely consumed. After cooling, the reaction mixture was extracted with ammonium chloride aqueous solution (30.0 mL, 20%) and ethyl acetate (50.0 mL). The organic phase was washed with saturated sodium chloride solution (30.0 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a colorless oily compound 3-2 (4.00 g, crude product).

[0346] Step 3: Synthesis of final product 3-3

[0347] 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 mixture was stirred at 80 °C for 12 hours in a liquid-sealed container. LCMS (RT = 1.041 min) showed that the starting material was completely consumed. The reaction mixture was extracted with water (20.0 mL) and ethyl acetate (40.0 mL). 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 / 1-0 / 1) to give a colorless oily compound 3-3 (1.50 g, yield 36.6%).

[0348] LCMS(ESI) m / z: 560.3 [M+H]+;

[0349] Step 4: Synthesis of final product 3-4

[0350] Compound 3-3 (1.50 g) was dissolved in tetrahydrofuran (10.0 mL), and triethylamine trihydrofluoride (864 mg) was added at 0 °C. After the addition was complete, the reaction mixture was stirred at 25 °C for 12 hours. LCMS (RT = 0.457 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give compound 3-4 (800 mg, crude product), a yellow oil.

[0351] LCMS(ESI)m / z:318.2[M+H]+;

[0352] Step 5: Synthesis of final product 3-5

[0353] Compounds 3-4 (800 mg) were dissolved in pyridine (8.00 mL), and 4,4-dimethoxytriphenylmethyl chloride (288 mg) was added at 0 °C. After the addition was complete, the reaction mixture was stirred at 25 °C for 1 hour. LCMS (RT = 1.824 min) showed that the starting material was completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a colorless oily compound 3-5 (1.10 g, crude product).

[0354] LCMS(ESI) m / z: 620.3[M+H]+;

[0355] Step Six: Synthesis of Final Product r3

[0356] Compounds 3-5 (1.10 g) were dissolved in dichloromethane (10.0 mL), and 4,5-dicyanimidazolium (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) showed that the starting material was completely consumed. The reaction mixture was purified by high performance liquid chromatography (column: Waters Xbridge BEH C18 150*40 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 55%-85%, 8 min) to obtain a white solid r3 (574 mg, purity 99.6%).

[0357] LCMS(ESI) m / z: 820.3[M+H]+;

[0358] 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)

[0359] Using a similar method, Examples 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 and 1-13 were synthesized.

[0360] Examples 1-4 Synthesis of phosphoridamide projectile r4-M

[0361] LCMS(ESI) m / z: 804.3[M+H]+;

[0362] 1H NMR (400MHz, DMSO-d6) δppm 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,1H )6.80-6.85(m,4H)7.18-7.26(m,8H)7.31-7.37(m,2H)8.93-9.01(m,1H).

[0363] Examples 1-5 Synthesis of phosphorus amide monomer r5-M

[0364] LCMS(ESI) m / z: 776.4 [M+H]+;

[0365] 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=10.0,4.75H z,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).

[0366] Examples 1-6 Synthesis of phosphorusamide monomer r6-M

[0367] LCMS(ESI) m / z: 775.3 [M+H]+;

[0368] 1H NMR (400MHz, DMSO-d6) δppm 1.03-1.17 (m, 12H) 2.60 (br.t, J = 5.60Hz, 2H) 3.11 (br.dd, J = 12.00, 5.44Hz, 1H) 3.36 (s, 3H) 3.54-3.63 (m, 4H) 3.73 (s, 9H) 4.13-4.25 (m, 1H) 4.27-4.36 (m, 1H) 4.58-4.73 (m, 1H) 5.92-6.02 (m, 1H) 6.81-6.87 (m, 4H) 7.20-7.29 (m, 8H) 7.34-7.40 (m, 2H) 7.99 (s, 1H) 8.62 (s, 1H).

[0369] Examples 1-7 Synthesis of phosphorus amide monomer r7-M

[0370] LCMS(ESI) m / z: 802.3[M+H]+;

[0371] 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).

[0372] Examples 1-8 Synthesis of phosphorusamide monomer r8-M

[0373] LCMS(ESI) m / z: 802.3[M+H]+;

[0374] 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).

[0375] Examples 1-9: Synthesis of phosphorous amide monomer r9-M

[0376] LCMS(ESI)m / z:801.3[M+H]+;

[0377] 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)3.04-3.1 4(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=18.00,3.60H z,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).

[0378] Examples 1-10 Synthesis of phosphorus amide monomer r10-M

[0379] LCMS(ESI) m / z: 820.3[M+H]+;

[0380] 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),3.65-3.6 9(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).

[0381] Example 1-11 Synthesis of phosphorusamide monomer r11-M

[0382] LCMS(ESI) m / z: 820.3[M+H]+;

[0383] 1H NMR(400MHz,DMSO-d6)δppm 0.12-0.20(m,6H), 0.25-0.35(m,6H), 1.84-1.90(m,1H), 1.96(t,J=6.00Hz,2H), 2.25-2.33(m,2H), 2.38(s,3H), 2.64 (t,J=4.40Hz,2H), 2.67-2.74(m,2H), 2.90(s,6H), 2.92(s,1H), 2.94-2.98(m,2H), 3.01(s,2H), 3.03(s,1H), 3.42(br d,J=2.80Hz,1H), 3.73-3.81(m,1H), 3.82-3.87(m,1H), 5.45(d,J=3.20Hz ,1H)6.00(dd,J=8.80,5.25Hz,4H), 6.35-6.52(m,9H), 7.93-8.25(m,1H).

[0384] Example 1-12 Synthesis of phosphorusamide monomer r12-M

[0385] LCMS(ESI) m / z: 819.3 [M+H]+;

[0386] 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).

[0387] Examples 1-13 Synthesis of phosphorusamide monomer r13-M

[0388] Synthesis of intermediate 4-1 in step one

[0389] Compounds 1-5 (14.0 g, 27.9 mmol) were dissolved in dichloromethane (140 mL), and 4-dimethylaminopyridine (6.8 g, 55.8 mmol) was added. The reaction mixture was cooled to 0 °C and stirred under nitrogen protection. Then, phenyl thiochloroformate (5.1 g, 29.3 mmol) was slowly added. After the addition was complete, the reaction mixture was heated to 25 °C and stirred for 12 hours. The reaction progress was monitored by liquid chromatography-mass spectrometry (LC-MS) (retention time RT = 1.008 min) to confirm that the starting material was completely consumed. After the reaction was complete, water (100 mL) was added to the reaction mixture for extraction, the organic phase was separated, and washed with saturated sodium chloride solution (60.0 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 100 / 1-1 / 1) to finally give a colorless oily compound 4-1 (6.2 g, yield 35%). LCMS (ESI) m / z: 622.9 [M+H]+;

[0390] Synthesis of intermediate 4-2 in step four

[0391] Compound 4-1 (6.2 g, 9.97 mmol) was dissolved in toluene (62.0 mL), and azobisisobutyronitrile (1.64 g, 9.97 mmol) and tributyltin hydride (11.6 g, 39.88 mmol) were added sequentially. After the addition was complete, the reaction solution was heated to 90 °C and stirred for 2 hours. The reaction progress was monitored by thin-layer chromatography (TLC, eluent: petroleum ether / ethyl acetate = 2 / 1). The results showed that the starting material spot (Rf = 0.54) disappeared and the product spot (Rf = 0.23) formed, indicating that the starting material was completely consumed. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was directly purified by wet column chromatography (eluent: petroleum ether / ethyl acetate = 100 / 1-1 / 1) to give a colorless oily compound 4-2 (4.3 g, yield 88%).

[0392] LCMS(ESI) m / z: 486.7 [M+H]+;

[0393] Synthesis of intermediate 4-3 in step five

[0394] Compound 4-2 (4.3 g, 8.85 mmol) was dissolved in tetrahydrofuran (43.0 mL), and triethylamine trihydrofluoride (2.85 g, 17.71 mmol) was slowly added at 0 °C. After the addition was complete, the reaction mixture was heated to 25 °C and stirred for 12 hours. The reaction progress was monitored by thin-layer chromatography (TLC, developing solvent: petroleum ether / ethyl acetate = 1 / 1). The results showed that the starting material spot (Rf = 0.75) disappeared and the product spot (Rf = 0.05) formed, indicating that the starting material was completely consumed. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain crude yellow oily compound 4-3, which could be used directly in the next reaction without further purification.

[0395] Synthesis of intermediate 4-4 in step six

[0396] Compound 4-3 (3.0 g, 12.33 mmol) was dissolved in pyridine (15.0 mL), and 4,4-dimethoxytriphenylmethyl chloride (4.6 g, 13.57 mmol) was slowly added at 0 °C. After the addition was complete, the reaction mixture was heated to 25 °C and stirred for 12 hours. The reaction progress was monitored by thin-layer chromatography (TLC, developing solvent: petroleum ether / ethyl acetate = 1 / 1). The results showed that the starting material spot (Rf = 0.05) disappeared and the product spot (Rf = 0.43) formed, indicating that the starting material was completely consumed. After the reaction was completed, the reaction mixture was cooled, and water (50.0 mL) and ethyl acetate (50.0 mL) were added for extraction. The organic phase was separated and washed with saturated sodium chloride solution (50.0 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a colorless oily compound 4-4 (1.3 g, yield 19%). LCMS(ESI) m / z: 546.6 [MH]+.

[0397] Step 7: Synthesis of the final product r13-M

[0398] Compound 4-4 (1.10 g, 2.02 mmol) was dissolved in dichloromethane (11.00 mL), and N,N-diisopropylphosphonamide (2-cyanoethyl) ester (669 mg, 2.82 mmol) and N,N-diisopropylethylamine (391 mg, 3.02 mmol) were added sequentially at 0 °C. After the addition was complete, the reaction mixture was heated to 20 °C and stirred for 2 hours. The reaction progress was monitored by thin-layer chromatography (TLC, developing solvent: petroleum ether / ethyl acetate = 1 / 1). The results showed that the starting material spot (Rf = 0.43) disappeared and the product spot (Rf = 0.53) formed, indicating that the starting material was completely consumed. After the reaction was completed, the reaction solution was purified by high performance liquid chromatography (column: Waters Xbridge Prep OBD C18 150*40mm*10μm; mobile phase: [water (containing ammonium bicarbonate)-acetonitrile]; gradient: B% 50%-80%, 8min) to obtain white solid r13-M (680.0mg, yield 45%).

[0399] LCMS(ESI) m / z: 746.2 [M+H] + 768.2 [M+Na] +

[0400] 1 H NMR(400MHz,DMSO-d6)δppm 1.10-1.73(m,12H)2.46-2.49(t,J=12.0Hz,1H)2.61-2.74(m,2H)2.76-2.84(m,1H)3.30-3.38(m,2H)3.54-3.89(m,10H)3.99(s, 3H)4.30-4.35(m,1H)4.62-4.70(m,1H)6.24-6.28(m,1H)6.79-6.84(m,4H)7.19-7.31(m,7H)7.38-7.41(m,2H)8.39-8.41(m,1H).

[0401] Example 2: Compound Synthesis

[0402] Example 2-1: Synthesis of compound GAL-01-M

[0403] Step 1

[0404] Compound 01-1 (500 mg) was dissolved in pyridine (5.00 mL), and 4,4-dimethoxytriphenylmethyl chloride (741 mg) was added at 0 °C. After the addition was complete, 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 = 0.24) showed that the starting material was completely consumed. The reaction mixture was extracted by slowly adding water (10.0 mL) and ethyl acetate (8.00 mL, 6.00 mL). The organic phase was washed with saturated sodium chloride solution (6.00 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow solid compound 01-2 (300 mg, yield 27.2%).

[0405] Step Two

[0406] Compound 01-2 (300 mg) was dissolved in methanol (3.00 mL), and palladium on carbon (100 mg, 10%) was added. After the addition was complete, the reaction mixture was stirred at 30 °C and 30 Psi under a hydrogen atmosphere for 12 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.15, feed: Rf = 0.54) showed that the feed was completely consumed. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a colorless oily compound 01-3 (200 mg, yield 88.0%).

[0407] LCMS(ESI)m / z:420.5[M+H]+;

[0408] Step 3

[0409] Compound 01-4 (700 mg) was dissolved in a mixed solution of tetrahydrofuran (14.0 mL) and water (7.00 mL), sodium bicarbonate (769 mg) was added, and benzyl chloroformate (780 mg) was added at 0 °C. After the addition was complete, the reaction mixture was stirred at 25 °C for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.72, feed: Rf = 0.24) showed that the feed was completely consumed. The reaction mixture was extracted by slowly adding water (10.0 mL) and ethyl acetate (8.00 mL, 6.00 mL). The organic phase was washed with saturated sodium chloride solution (6.00 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a colorless oily compound 01-5 (500 mg, yield 45.1%).

[0410] Step Four

[0411] 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, feedstock: Rf = 0.72) showed that the feedstock was completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a colorless oily compound 01-6 (400 mg, yield 94.6%).

[0412] Step 5

[0413] Compounds 01-6 (200 mg) and 01-3 (400 mg) were dissolved in N,N-dimethylformamide (6.00 mL), and N,N-diisopropylethylamine (219 mg, 295 μL) and O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethylurea hexafluorophosphine salt (322 mg) were added. The reaction mixture was stirred at 20 °C for 2 hours after the addition was complete. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.43, feed: Rf = 0.1) showed that the feed was completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow oily compound 01-7 (130 mg, yield 43.4%).

[0414] Step Six

[0415] Compound 01-7 (110 mg) was dissolved in methanol (2.00 mL), and palladium on carbon (200 mg, 10%) was added. After addition, the reaction mixture was stirred at 20°C for 12 hours under a hydrogen atmosphere at a pressure of 15 Psi. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.1, feed: Rf = 0.43) showed that the feed was completely consumed. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily compound 01-8 (90.5 mg, yield 82.2%).

[0416] Step Seven

[0417] Compound 01-9 (30.0 g) was dissolved in methanol (210 mL), and sodium hydroxide aqueous solution (107 mL, 1 M) was added. After the addition was complete, the reaction mixture was stirred at 70 °C for 3 hours. LCMS (RT = 0.542 min) showed that the starting material was completely consumed. After the reaction mixture was cooled, dilute hydrochloric acid was added to adjust the pH to 4, and the solution was concentrated under reduced pressure to obtain a white solid compound 01-10 (28.0 g, crude product).

[0418] LCMS(ESI) m / z: 225.1 [M+H]+;

[0419] Step 8

[0420] Compound 01-10 (200 mg) was dissolved in dichloromethane (2.00 mL), and thionyl chloride (510 mg) was added. After the addition was complete, 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 that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a yellow oily compound 01-11 (150 mg, crude product).

[0421] Step Nine

[0422] Compound 01-11 (150 mg) was dissolved in dichloromethane (2.00 mL) and added dropwise at 0 °C to a solution of 1,9-bis-tert-butoxycarbonyl-1,5,9-triazanonane in dichloromethane (2.00 mL). N,N-diisopropylethylamine (371 mg) was then added, and the reaction mixture was stirred at 25 °C for 12 hours. LC-MS (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 / 1-1 / 1) to give a yellow solid compound 01-12 (480 mg, yield 98.2%).

[0423] LCMS(ESI) m / z: 851.4 [M+H]+;

[0424] Step 10

[0425] Compound 01-12 (480 mg) was dissolved in tetrahydrofuran (2.00 mL) and water (600 μL), and lithium hydroxide (11.3 mg) was added. After the addition was complete, the reaction mixture was stirred at 20 °C for 2 hours. TLC (dichloromethane / methanol = 10 / 1, product: Rf = 0.24, raw material: Rf = 0.43) showed that the raw material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a yellow solid compound 01-13 (150 mg).

[0426] Step Eleven

[0427] 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 after the addition was complete. LCMS (RT = 0.851 min) showed that the starting material was completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 50 / 1-1 / 1) to give a white solid compound 01-14 (110 mg, yield 83.3%).

[0428] LCMS(ESI) m / z: 927.4 [M+H]+;

[0429] Step Twelve

[0430] Compound 01-14 (130 mg) was dissolved in ethyl acetate (1.00 mL), and hydrochloric acid / ethyl acetate (300 μL) was added. After the addition was complete, the reaction mixture was stirred at 20 °C for 1 hour. LCMS (RT = 0.346 min) showed that the starting material was completely consumed. The organic phase was concentrated under reduced pressure to give compound 01-15 (70.0 mg, crude product) as a white solid.

[0431] LCMS(ESI) m / z: 527.3 [M+H]+;

[0432] Step Thirteen

[0433] Compounds 01-15 (70.0 mg) and 01-16 (238 mg) were dissolved in N,N-dimethylformamide (2.00 mL), and N,N-diisopropylethylamine (137 mg, 185 μL) and O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethylurea hexafluorophosphine salt (303 mg) were added. The reaction mixture was stirred at 20 °C for 2 hours after the addition was complete. TLC (dichloromethane / methanol = 10 / 1, product: Rf = 0.43, starting material: Rf = 0.15) showed that the starting material was completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow solid compound 01-17 (100 mg, yield 33.5%).

[0434] Step Fourteen

[0435] 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 was complete, the reaction mixture was stirred at 20 °C for 12 hours under a hydrogen atmosphere at a pressure of 15 Psi. LCMS (RT = 1.127 min) showed that the starting material was completely consumed. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a white solid 01-18 (50.0 mg, yield 74.4%).

[0436] LCMS(ESI)m / z:1077.9[M+2H] / 2+;

[0437] Step Fifteen

[0438] Compounds 01-18 (50.0 mg) and 01-8 (20.0 mg) were dissolved in N,N-dimethylformamide (1.00 mL), and N,N-diisopropylethylamine (9.00 mg) and tri-n-propylcyclic phosphoric 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) showed that the starting materials were completely consumed. 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (column: Waters Xbridge Prep OBDC18 150*40mm*10um; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 35%-55%, 8min) to obtain a white solid compound GAL-01 (11.3mg, purity 100%).

[0439] TOF MS ES + m / z:2711[M+H] / +;

[0440] 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).

[0441] Example 2-2: Synthesis of compound GAL-02-M

[0442] Step 1

[0443] Compound 02-1 (20.0 g) was dissolved in a mixture of anhydrous tetrahydrofuran (400 mL) and water (120 mL). Sodium bicarbonate (26.8 g) was added, followed by benzyl chloroformate (27.2 g) at 0 °C. The reaction mixture was stirred at 25 °C for 2 hours after the addition was complete. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.43, feedstock: Rf = 0.1) showed that the feedstock was completely consumed. The reaction mixture was extracted by slowly adding water (100 mL) and ethyl acetate (80.0 mL, 60.0 mL). The organic phase was washed with saturated sodium chloride solution (60.0 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a white solid compound 02-2 (30.0 g, crude product).

[0444] Step Two

[0445] Compound 02-2 (30.0 g) was dissolved in ethyl acetate (100 mL), and hydrochloric acid / ethyl acetate (100 mL) was added. After the addition was complete, the reaction mixture was stirred at 25 °C for 12 hours. LCMS (Rt = 0.413 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give compound 02-3 (8.00 g, crude product), a white solid.

[0446] LCMS(ESI)m / z:223[M+H]+;

[0447] Step 3

[0448] Compound 02-3 (8.00 g) was dissolved in tetrahydrofuran (160 mL), and succinic anhydride (3.60 g) was added. After the addition was complete, the reaction mixture was stirred at 25 °C for 1 hour. LCMS (Rt = 1.904 min) showed that the starting material was completely consumed, and a main peak meeting the requirements was detected. The reaction mixture was filtered to obtain a white solid compound 02-4 (11.0 g, crude product).

[0449] LCMS(ESI)m / z:323[M+H]+;

[0450] Step Four

[0451] 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-tetramethylurea hexafluorophosphine salt (11.8 g) was added at 0 °C. LCMS (Rt = 1.886 min) showed complete consumption of the starting material, and a main peak meeting the requirements was detected. The reaction solution 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a colorless oily compound 02-5 (9.00 g, crude product).

[0452] LCMS(ESI)m / z:724[M+H]+;

[0453] Step Six

[0454] Compound 02-5 (9.00 g) was dissolved in methanol (100 mL), and palladium on carbon (2.85 g, 10%) was added. After addition, the reaction mixture was stirred at 20 °C for 12 hours under a hydrogen atmosphere at a pressure of 15 Psi. LCMS (Rt = 1.440 min) showed that the starting material was completely consumed, and a main peak meeting the requirements was detected. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a yellow oily compound 02-6 (4.00 g, yield 54.5%).

[0455] LCMS(ESI)m / z:590[M+H]+;

[0456] Step Seven

[0457] Compounds 1-18 (4.00 g) and 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-tetramethylurea hexafluorophosphine salt (1.41 g) was added at 0 °C. LCMS (Rt = 2.885 min) showed complete consumption of the starting material, and a main peak meeting the requirements was detected. The reaction solution was purified by high performance liquid chromatography (HPLC) (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonia)-acetonitrile]; B%: 35%-55%, 8 min) to obtain a white solid compound GAL-02-M (2.04 g, purity 93.2%).

[0458] LCMS(ESI)m / z:1212[M-DMT] / 2+;

[0459] 1 H NMR (400MHz, CHLOROFORM-d)

[0460] 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).

[0461] Examples 2-3: Synthesis of ligand GAL-03-M

[0462] Step 1

[0463] 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, feedstock: Rf = 0.88) showed that the feedstock was completely consumed. The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was then slurried with petroleum ether at 25 °C for 10 minutes to give a white solid compound 03-2 (110 g, yield 87.1%).

[0464] Step Two

[0465] 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, feedstock: Rf = 0.2) showed complete consumption of the feedstock. The reaction mixture was extracted by slowly adding water (100 mL) and ethyl acetate (80.0 mL, 60.0 mL). The organic phase was washed with saturated sodium chloride solution (60.0 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a white oily compound 03-3 (10.4 g, crude product).

[0466] Step 3

[0467] Compound 03-3 (10.4 g) was dissolved in ethyl acetate (100 mL), and hydrochloric acid / ethyl acetate (100 mL) was added. After the addition was complete, the reaction mixture was stirred at 25 °C for 12 hours. LCMS (Rt = 0.714 min) showed that the starting material was completely consumed, and a main peak meeting the requirements was detected. The reaction mixture was concentrated under reduced pressure to give a white solid compound 03-4 (4.20 g, crude product).

[0468] LCMS(ESI) m / z: 267 [M+H] + ;

[0469] Step Four

[0470] Compound 03-2 (10.0 g) was dissolved in tetrahydrofuran (200 mL), and succinic anhydride (3.02 g) was added. After the addition was complete, the reaction mixture was stirred at 25 °C for 1 hour. LCMS (Rt = 1.081 min) showed that the starting material was completely consumed, and a main peak meeting the requirements was detected. The reaction mixture was filtered to obtain a colorless oily compound 03-6 (13.0 g, yield 99.8%).

[0471] LCMS(ESI) m / z: 432 [M+H] + ;

[0472] Step 5

[0473] Compounds 03-6 (6.63 g) and 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-tetramethylurea hexafluorophosphine salt (5.84 g) was then added at 0 °C. LCMS (Rt = 1.919 min) showed complete consumption of the starting material, and a main peak meeting the required mass spectrometry 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by reversed-phase high-performance liquid chromatography (under 0.1% ammonium bicarbonate conditions) to obtain a colorless oily compound 03-7 (6.40 g, yield 80.1%).

[0474] LCMS(ESI) m / z: 1093 [M+H] + ;

[0475] Step Six

[0476] Compound 03-7 (6.00 g) was dissolved in methanol (60.0 mL), and palladium on carbon (5.85 g, 10%) was added. After the addition was complete, the reaction mixture was stirred at 20 °C for 12 hours under a hydrogen atmosphere at a pressure of 15 Psi. LCMS (Rt = 2.225 min) showed that the starting material was completely consumed, and a main peak meeting the requirements was detected. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a yellow oily compound 03-8 (5.00 g, yield 88.5%).

[0477] LCMS(ESI) m / z: 958 [M+H] + ;

[0478] Step Seven

[0479] 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-tetramethylurea hexafluorophosphine salt (4.15 g) was added at 0 °C. LCMS (Rt = 1.814 min) showed complete consumption of the starting material, and a main peak meeting the requirements 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a colorless oily compound 03-9 (5.50 g, crude product).

[0480] LCMS(ESI)m / z:1121[M+H] + ;

[0481] Step 8

[0482] Compound 03-9 (5.50 g) was dissolved in a mixed solution 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 after the addition. LCMS (Rt = 1.472 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give compound 03-10 (5.00 g, yield 94.9%) as a white solid.

[0483] LCMS(ESI) m / z: 958 [M+H] + ;

[0484] Step Nine

[0485] 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) showed complete consumption of the starting material, and a main peak meeting the requirements was 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by high performance liquid chromatography (HPLC) (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonia)-acetonitrile]; B%: 30%-60%, 8 min) to obtain a white solid compound 03-11 (5.00 g, yield 84.6%).

[0486] LCMS(ESI) m / z: 1197 [M+H] + ;

[0487] Step 10

[0488] Compound 03-11 (5.00 g) was dissolved in ethyl acetate (50.0 mL), and hydrochloric acid / ethyl acetate (50.0 mL) was added. After the addition was complete, the reaction mixture was stirred at 25 °C for 12 hours. LCMS (Rt = 0.885 min) showed that the starting material was completely consumed, and a main peak meeting the requirements was detected. The reaction mixture was concentrated under reduced pressure to give a white solid compound 03-12 (4.00 g, yield 93.6%).

[0489] LCMS(ESI) m / z: 1197 [M+H]+ ;

[0490] Step Eleven

[0491] Compounds 03-12 (4.00 g) and 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-tetramethylurea hexafluorophosphine salt (9.69 g) was then added at 0 °C. LCMS (Rt = 1.430 min) showed complete consumption of the starting material, and a main peak meeting the required mass spectrometry 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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by reversed-phase high-performance liquid chromatography (under 0.1% ammonium bicarbonate conditions) to obtain a colorless oily compound 03-13 (8.00 g, yield 79.6%).

[0492] LCMS(ESI) m / z: 1257 [M+2H] 2+ / 2;

[0493] Step Twelve

[0494] Compound 03-13 (8.00 g) was dissolved in methanol (160 mL), and palladium on carbon (4.85 g, 10%) was added. After addition, the reaction mixture was stirred at 20 °C for 12 hours under a hydrogen atmosphere at a pressure of 15 Psi. LCMS (Rt = 1.085 min) showed that the starting material was completely consumed, and a main peak meeting the requirements was detected. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a yellow oily compound 03-14 (7.00 g, yield 89.5%).

[0495] LCMS(ESI) m / z: 1212 [M+2H] + / 2;

[0496] Step Thirteen

[0497] Compounds 03-14 (4.00 g) and 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-tetramethylurea hexafluorophosphine salt (1.26 g) was added at 0 °C. LCMS (Rt = 1.696 min) showed complete consumption of the starting material, and a main peak meeting the requirements was detected. The reaction solution was purified by high performance liquid chromatography (HPLC) (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonia)-acetonitrile]; B%: 35%-55%, 8 min) to obtain a white solid compound GAL-03-M (1.13 g, purity 93.28%).

[0498] LCMS(ESI)m / z:1347[M-DMT] / 2 + ;

[0499] 1 H NMR (400MHz, CDCl3)

[0500] 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).

[0501] Examples 2-4: Synthesis of GAL-04-M

[0502] Step 1

[0503] 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-tetramethylurea hexafluorophosphine salt (1.22 g) was added at 0 °C, and 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. The reaction mixture was extracted by slowly adding water (10.0 mL) and ethyl acetate (8.00 mL, 6.00 mL). The organic phase was washed with saturated sodium chloride solution (6.00 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a white solid, compound 04-2 (200 mg, yield 17.5%).

[0504] Step Two

[0505] Compound 04-2 (200 mg) was dissolved in methanol (1.00 mL), and palladium on carbon (299 mg, 10%) was added. After the addition was complete, the reaction mixture was stirred at 30 Psi under a hydrogen atmosphere at 25 °C for 1 hour. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.15, feed: Rf = 0.54) showed that the feed was completely consumed. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a white oily compound 04-3 (110 mg, yield 67.5%).

[0506] Step 3

[0507] Compound 01-18 (250 mg) and compound 04-3 (110 mg) were dissolved in N,N-dimethylformamide (20.0 mL), followed by the addition of N,N-diisopropylethylamine (60.0 mg) and 50% ethyl acetate solution of tri-n-propyl cyclophosphine (148 mg). The reaction mixture was stirred at 25 °C for 1 hour. LCMS (ET61818-330-P1A1, Rt = 2.744) showed complete consumption of the starting materials. 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 (HPLC) (column: Waters Xbridge BEHC18 250*30mm*10um; mobile; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 33%-2%, 8 min) to obtain a white solid compound GAL-04-M (37.2 mg, yield 11.8%).

[0508] LCMS(ESI)m / z:2714[M+H]+;

[0509] 1 H NMR (400MHz, CDCl3)

[0510] 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).

[0511] Examples 3-5: Synthesis of compound GAL-05-M

[0512] Step 1

[0513] Compound 01-1 (12.0 g) and imidazole (13.0 g) were dissolved in N,N-dimethylformamide (100 mL), and tert-butyldimethylchlorosilane (18.0 g) was added at 0 °C. After addition, the reaction mixture was stirred at 25 °C for 6 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, product: Rf = 0.72, feed: Rf = 0.24) showed that the feed was completely consumed. The reaction mixture was extracted by slowly adding water (100 mL) and ethyl acetate (80.0 mL, 60.0 mL). The organic phase was washed with saturated sodium chloride solution (60.0 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow solid compound 05-1 (20.0 g, yield 87.2%).

[0514] Step Two

[0515] Compound 05-1 (15.0 g) was dissolved in methanol (100 mL), and palladium on carbon (1.00 g, 10%) was added. After the addition was complete, the reaction mixture was stirred at 30 °C for 12 hours under a hydrogen atmosphere at a pressure of 30 Psi. TLC (petroleum ether / ethyl acetate = 2 / 1, product: Rf = 0.15, feed: Rf = 0.54) showed that the feed was completely consumed. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a yellow oily compound 05-2 (10.0 g, yield 92.5%).

[0516] 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).

[0517] Step 3

[0518] 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-tetramethylurea hexafluorophosphine salt (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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow oily compound 05-3 (6.50 g, yield 41.3%).

[0519] Step Four

[0520] Compound 05-3 (8.50 g) was dissolved in tetrahydrofuran (56.0 mL), and tetrabutylammonium fluoride (15.6 mL, 1 M) was added. After the addition was complete, 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 that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a yellow solid compound 05-4 (5.00 g, yield 91.3%).

[0521] Step 5

[0522] Compound 05-4 (5.00 g) was dissolved in pyridine (35.0 mL), and 4,4-dimethoxytriphenylchloromethane (8.06 g) was added. After 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 was extracted with saturated sodium bicarbonate aqueous solution (50.0 mL) and ethyl acetate (50.0 mL, 30.0 mL). The organic phase was washed with saturated sodium chloride solution (50.0 mL), dried with 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 give a yellow oily compound 05-5 (4.00 mg, yield 40.8%).

[0523] LCMS(ESI) m / z: 618.4 [M+H]+;

[0524] 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).

[0525] Step Six

[0526] Compound 05-5 (800 mg) was dissolved in dioxane (8.00 mL), and lithium hydroxide aqueous solution (1.29 mL, 2 M) was added. After the addition was complete, the reaction mixture was stirred at 20 °C for 3 hours. LCMS (RT = 1.750 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a yellow solid compound 05-6 (500 mg, yield 63.9%).

[0527] LCMS(ESI) m / z: 604.3[M+H]+;

[0528] Step Seven

[0529] Compound 01-9 (30.0 g) was dissolved in methanol (210 mL), and sodium hydroxide aqueous solution (107 mL, 1 M) was added. After the addition was complete, the reaction mixture was stirred at 20 °C for 12 hours. LCMS (RT = 0.572 min) showed that the starting material was completely consumed. After the reaction mixture was cooled, dilute hydrochloric acid was added to adjust the pH to 4, and the solution was concentrated under reduced pressure to obtain a white solid compound 05-7 (28.0 g, crude product).

[0530] LCMS(ESI) m / z: 237.1 [MH]+;

[0531] Step 8

[0532] 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. After the addition was complete, the reaction mixture was stirred at 20 °C for 12 hours. LC-MS (RT = 0.775 min) showed that the starting material was completely consumed. The reaction mixture was cooled to 0 °C, and methanol (50.0 mL) was slowly added, followed by 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 give compound 05-8 (8.00 g, yield 30.3%) as a white solid.

[0533] LCMS(ESI) m / z: 223.2[MH]+;

[0534] Step Nine

[0535] Compound 05-8 (8.00 g) was dissolved in dichloromethane (55.0 mL), and N,N-dimethylformamide (137 μL) and thionyl chloride (5.18 mL) were added at 0 °C. After addition, the reaction mixture was stirred at 20 °C for 2 hours. LCMS (RT = 0.741 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a yellow solid compound 05-9 (8.00 g, yield 92.4%).

[0536] LCMS(ESI)m / z:243.1[M+H]+;

[0537] Step 10

[0538] 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. After the addition was complete, the reaction mixture was stirred at 60 °C for 12 hours. LCMS (RT = 0.589 min) showed that the starting material was completely consumed. 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 give a white solid compound 05-10 (8.00 g, yield 97.3%).

[0539] LCMS(ESI) m / z: 250.1 [M+H]+;

[0540] Step Eleven

[0541] Compound 05-10 (8.00 g) was dissolved in methanol (55.0 mL), and palladium on carbon (800 mg, 10%) was added. After the addition was complete, the reaction mixture was stirred at 25 °C for 12 hours under a hydrogen atmosphere at a pressure of 15 Psi. LCMS (RT = 0.589 min) showed that the starting material was completely consumed. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a white solid compound 05-11 (8.00 g, yield 89.3%).

[0542] LCMS(ESI)m / z:224.2[M+H]+;

[0543] Step Twelve

[0544] Compound 05-11 (8.00 g) was dissolved in tetrahydrofuran (28.0 mL) and water (28.0 mL), followed by the addition of benzyl chloroformate (6.11 g) and sodium bicarbonate (9.03 g). The reaction mixture was stirred at 25 °C for 12 hours after the addition was complete. LCMS (RT = 0.619 min) showed complete consumption of the starting material. Extraction was performed with water (20.0 mL) and ethyl acetate (30.0 mL, 20.0 mL). 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 (silica, petroleum ether / ethyl acetate = 100 / 1-1 / 1) to give compound 05-12 (5.00 g, yield 39.0%) as a white solid.

[0545] LCMS(ESI) m / z: 715.2[2M+H]+;

[0546] 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).

[0547] Step Thirteen

[0548] Compound 05-12 (4.00 g) was dissolved in tetrahydrofuran (30.0 mL) and water (6.00 mL), and an aqueous solution of lithium hydroxide (536 mg) was added. After the addition was complete, the reaction mixture was stirred at 20 °C for 12 hours. LCMS (RT = 0.52 min) showed that the starting material was completely consumed. After cooling the reaction mixture, dilute hydrochloric acid was added to adjust the pH to 4, and the solution was concentrated under reduced pressure to give a white solid compound 05-13 (3.50 g, yield 94.9%).

[0549] LCMS(ESI) m / z: 659.2 [2M+H]+;

[0550] Step Fourteen

[0551] Compound 05-13 (1.00 g) was dissolved in dichloromethane (10.0 mL), and N,N-dimethylformamide (140 μL) and oxaloyl chloride (797 μL) were added at 0 °C. After addition, 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 that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to give a yellow oily compound 05-14 (1.00 g, yield 89.9%).

[0552] Step Fifteen

[0553] 1,9-bis-Boc-1,5,9-triazanonane (905 mg) and N,N-diisopropylethylamine (705 mg, 951 μL) were dissolved in dichloromethane (5.00 mL). Compound 05-14 (500 mg) was slowly added to dichloromethane (5.00 mL) at 0 °C. After addition, the reaction mixture was stirred at 20 °C for 3 hours. LC-MS (RT = 0.788 min) showed complete consumption of the starting material. Extraction was performed by adding water (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. The crude product was purified by column chromatography (silica, petroleum ether / ethyl acetate = 50 / 1-0 / 1) to give compound 05-15 (800 mg, yield 61.4%) as a white solid.

[0554] LCMS(ESI) m / z: 956.3 [M+H]+;

[0555] Step Sixteen

[0556] Compound 05-15 (600 mg) was dissolved in dichloromethane (6.00 mL), and trifluoroacetic acid (280 μL) was slowly added. After the addition was complete, the reaction mixture was stirred at 20 °C for 3 hours. TLC (dichloromethane / methanol = 10 / 1, product: Rf = 0.0, feed: Rf = 0.24) showed that the feed was completely consumed. The reaction mixture was extracted with saturated sodium bicarbonate aqueous solution (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 with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow oily compound 05-16 (450 mg, yield 81.6%).

[0557] Step Seventeen

[0558] Compound 05-16 (50.0 mg) was dissolved in N,N-dimethylformamide (1.00 mL), and N,N-diisopropylethylamine (73.6 mg, 749 μL), compound 01-16 (127 mg), and O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethylurea hexafluorophosphine salt (162 mg) were added. The reaction mixture was stirred at 20 °C for 3 hours. LCMS (RT = 1.529 min) showed that the starting material was completely consumed. The crude product was purified by high performance liquid chromatography (HPLC) (column: Waters Xbridge BEHC18250*50 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 40%-60%, 10 min) to obtain a white solid compound 05-17 (100 mg, yield 61.7%).

[0559] LCMS(ESI)m / z:1137.4[M+2H] / 2+;

[0560] Step 18

[0561] 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 was complete, the reaction mixture was stirred at 20 °C for 3 hours under a hydrogen atmosphere at a pressure of 15 Psi. LCMS (RT = 1.229 min) showed that the starting material was completely consumed. After cooling, the reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give a white solid compound 05-18 (30.0 mg, yield 63.7%).

[0562] LCMS(ESI)m / z:1070.4[M+2H] / 2+;

[0563] Step Nineteen

[0564] Compound 05-18 (30.0 mg) and compound 05-6 (8.47 mg) were dissolved in tetrahydrofuran (500 μL). N,N-diisopropylethylamine (3.62 mg) and O-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethylurea hexafluorophosphine salt (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 materials. The reaction mixture was purified by high-performance liquid chromatography (HPLC) (column: Waters Xbridge Prep OBDC18 150*40 mm*10 μm; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 30%-60%, 8 min) to obtain a white solid compound GAL-05 (1.90 mg, 100% purity).

[0565] LCMS(ESI)m / z:1363[M+2H] / 2+;

[0566] 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).

[0567] Synthesis of compound GAL-01-D in Examples 2-6

[0568] Compound GAL-01 (100 mg) was dissolved in methanol (2.00 mL), and 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*50 mm*10 μm; mobile; mobile phase: [water (ammonium bicarbonate)-acetonitrile]; B%: 35%-55%, 10 min) to obtain a white solid compound GAL-01-D (23.0 mg, yield 28.3%).

[0569] LCMS(ESI)m / z:1903[M-DMT]+;

[0570] 1 H NMR (400MHz, DMSO-d6)

[0571] 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,5H )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,1 H)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).

[0572] Using the same synthesis method as in Examples 2-6, Examples 2-7, 2-8, and 2-9 were synthesized:

[0573] Examples 2-7: Compound GAL-02-D

[0574] LCMS(ESI)m / z:1919.8[M-DMT]+;

[0575] 1 H NMR (400MHz, DMSO-d6)

[0576] 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)

[0577] Examples 2 - 8: Compound GAL - 03 - D

[0578] LCMS(ESI) m / z: 1095 [M - DMT] / 2+;

[0579] 1 1H NMR (400 MHz, MeOD - d4)

[0580] 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 =

[0581] Examples 2-9: Compound GAL-04-D

[0582] LCMS(ESI)m / z:1908[M-DMT] + .

[0583] Example 3: Synthesis of double-stranded siRNA reagent

[0584] The sense and antisense strands of the RNAi reagent are synthesized using a solid-phase phosphoramidite triester technique employed in oligonucleotide synthesis. Such standard synthesis is generally known in the art. Depending on scale, a 12-channel nucleic acid synthesizer or an OP 100 nucleic acid synthesizer is used. Synthesis is performed on controlled-pore glass beads (CPG). or The synthesis of single-stranded oligonucleotides containing targeting ligands is performed on a solid support made of controllable microporous glass spheres covalently linked to the targeting ligand molecules. Monomers positioned at the 3' end of the corresponding strand are attached to the solid support as the starting point for synthesis. Each nucleotide monomer attachment involves a four-step reaction: deprotection, coupling, oxidation or sulfidation, and capping. The required 2'-modified RNA phosphoramidite monomers (2'-methoxy and fluorinated modified RNA phosphoramidite monomers) and auxiliary reagents are commercially available.

[0585] The following description uses the synthesis of siRNA linked by the GAL-03-M conjugate group as an example. The synthesis of other siRNA reagents (shown in Table 1) is carried out in the same way:

[0586] Step 1: Preparation of GAL-03 succinate

[0587] Under a nitrogen atmosphere, 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). After the additions were complete, the mixture was stirred at room temperature for 12 hours. After the reaction was confirmed to be complete 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 removed by vacuum evaporation below 30 °C to obtain 400 mg of a grayish-white solid product (yield: 77%).

[0588] Step 2: General process for synthesizing GAL-03-CPG

[0589] The GAL-03 succinate (400 mg, 128 μmol) solution obtained in the previous step was mixed with acetonitrile (10 mL). HBTU (73 mg, 193 μmol) and DIEA (50 mg, 387 μmol) were added to the mixture, and the reaction mixture was stirred for 5 minutes to obtain a clear solution. Amino-controlled microporous glass beads (NH2-CPG, NH2-controlled pore glass) were then added to the solution. The amino loading was 180 μmol / g (2 g). The reaction was carried out at 25 °C on a shaker at 150 rpm. After 24 hours of reaction, the mixture was filtered, and the filter cake was thoroughly washed with dichloromethane and acetonitrile, then dried under vacuum. The dried solid support was then capped with a 25% acetic anhydride / pyridine capping reagent and reacted for 3 hours. After filtration, the filter cake was thoroughly washed with acetonitrile and dried to obtain the desired GAL-03-CPG solid support (2.1 g, measured loading: 20 μmol / g).

[0590] Step 3: Synthesize the positive chain of the conjugate (the synthetic method is a general method applicable to all mentioned monomers, including r, 2-O-methyl, and fluorinated nucleotides)

[0591] Using the solid-phase phosphoramide method, the GAL-03-CPG solid support prepared in step two above was used to start the cycle, and nucleoside monomers were linked one by one from the 3'-5' direction according to the nucleotide arrangement sequence of the positive strand (as shown in Table 1).

[0592] Phosphoramide monomer or R1 monomer was prepared into a 0.06 M acetonitrile solution, and anhydrous dimethylformamide and molecular sieve were added.

[0593] The deprotection reaction conditions were the same for each step, including a reaction temperature of 25°C, a dichloromethane solution of dichloroacetic acid (3% W / v) as the deprotection reagent, a molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support of 5:1, and a reaction time of 70 seconds.

[0594] The coupling reaction conditions for each step included a temperature of 25°C, the use of 5-benzylthio-1H-tetrazole (BTT, 0.3M, acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 0.25M acetonitrile solution) as the activator, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:8, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, and coupling times of 3 minutes (modified RNA monomer, fluoroRNA monomer, or 2'-O-methyl modified RNA monomer) and 5 minutes (r1 monomer).

[0595] Each capping step was performed under the following conditions: a temperature of 25°C, a capping reagent solution consisting of a 1:1 molar ratio of CapA (10% acetic anhydride, acetonitrile) and CapB (pyridine / N-methylimidazole / acetonitrile, 10:14:76, v / v / v), and a reaction time of 15 seconds.

[0596] The oxidation reaction conditions for each step included a temperature of 25°C, an oxidizing agent of iodine solution (0.05M, THF / pyridine / water, 70:20:10), a molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step of 30:1, and a reaction time of 15 seconds.

[0597] The conditions for each sulfurization reaction step included a temperature of 25°C, a sulfurizing agent of PADS (diphenylacetyl disulfide) (0.2M, dissolved in pyridine / acetonitrile = 7:3), a molar ratio of sulfurizing agent to the nucleic acid sequence linked on the solid-phase support in the coupling step of 120:1, and a reaction time of 300 seconds.

[0598] The cleavage and deprotection conditions of the oligomer bound to CPG are as follows: The synthesized nucleotide sequence linked to the carrier is added to ammonia water with a concentration of 25 wt% (0.5 mL / μmol), and reacted at 55 °C for 6 hours. The liquid is removed, and the residue is concentrated to dryness under vacuum.

[0599] Purification and Desalting: Nucleic acids were purified using a preparative ion chromatography column with a gradient elution of NaCl. Specifically: Eluent A: 0.05M NaOH aqueous solution; Eluent B: 1M NaCl, 0.05M NaOH aqueous solution; Elution gradient: Eluent A: Eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a dextran gel column (Sephadex G25 packing material) with water for injection.

[0600] 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 molecule is the positive chain of a conjugated molecule with GAL-03 at the 3' end.

[0601] Step 4: Synthesize the antisense chain of the conjugate

[0602] The solid-phase phosphoramidite triester method was employed, using universally controllable glass microspheres (CPG) to initiate the cycle. Nucleoside monomers were sequentially linked from the 3'-5' direction according to the antisense nucleotide arrangement sequence (as shown in Table 1). The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense strand.

[0603] Testing: 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 material was an antisense chain.

[0604] Step 5: Synthesize the double-stranded conjugate

[0605] The sense and antisense strands were dissolved separately in water for injection to obtain a 40 mg / mL solution. These solutions were mixed in an equimolar ratio, heated at 95°C for 10 min, and slowly cooled to obtain the annealed product. The lyophilized product was then lyophilized to obtain a lyophilized powder. Molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured values ​​in this example are consistent with the theoretical values, indicating that the synthesized product is a double-stranded siRNA. Quantification was performed using ultraviolet spectrophotometry.

[0606] Using the above synthesis methods, the representative novel GalNAc-siRNAs of this invention are summarized in Table 1.

[0607] The structure of r1-r13 in this invention is as follows:

[0608] Table 1: Sensitive and antisense strand sequences of GalNAc-siRNA that inhibit human HSD17B13 mRNA expression, and their combinations.

[0609] The r structure and connection method in the table are as described above. The amide in the r structure is derived from the ester group in rM. The monomers used in its synthesis process are selected from the rM corresponding to the numbers in the examples. The ester groups (methyl ester, ethyl ester) contained in its structure react with ammonia water to generate amides during the cleavage and deprotection process of sequence synthesis, as shown below:

[0610] r1 is derived from r1-M structure, r2 is derived from r2-M structure, r3 is derived from r3-M structure, r4 is derived from r2-M structure, r5 is derived from r5-M structure, r6 is derived from r6-M structure, r7 is derived from r7-M structure, r8 is derived from r8-M structure, r9 is derived from r9-M structure, r10 is derived from r10-M structure, r11 is derived from r11-M structure, r12 is derived from r12-M structure, and r13 is derived from r13-M structure;

[0611] In the table, GAL-01 is a novel tetravalent GalNAc group that binds to nucleic acids via covalent bonds, as shown below:

[0612] In the table, GAL-02 is a novel tetravalent GalNAc group that binds to nucleic acids via covalent bonds, as shown in the following formula:

[0613] In the table, GAL-03 is a novel tetravalent GalNAc group that binds to nucleic acids via covalent bonds, as shown in the following formula:

[0614] Where: M + for Na + ;X - For O - .

[0615] Example 4: Evaluation of the endocytosis efficiency of GalNAc compounds in primary cynomolgus monkey hepatocytes (PCMH) using flow cytometry

[0616] Experimental Objective

[0617] This study used flow cytometry to evaluate the endocytosis efficiency of tested GalNAc compounds in primary cynomolgus monkey hepatocytes (PCMH). Both the tested GalNAc compounds and GalNAc3-Cy5 could bind to the ASGPR receptor on the surface of PCMH and be internalized into the cells. The competing compound GalNAc3-Cy5 was a fluorescently labeled compound, and its fluorescence signal could be detected by flow cytometry after internalization. The competition between the tested GalNAc compounds and GalNAc3-Cy5 led to a decrease in the detected fluorescence signal, and the degree of decrease in fluorescence signal reflected the endocytosis efficiency of the tested compounds.

[0618] Experimental materials and instruments:

[0619] 1. Cell lines and compounds: Primary monkey hepatocytes PCMH (batch number: GSYG202102) were provided by Shanghai WuXi AppTec Co., Ltd., and the competing compound GalNAc3-Cy5 (batch number: ET45235-14-P1) and the reference compound L96 (batch number: ET63057-7-P1) were provided by Shanghai WuXi AppTec Co., Ltd.

[0620] 2. Major 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).

[0621] Experimental steps and methods:

[0622] Flow cytometry was used to evaluate the endocytosis efficiency of compounds in PCMH.

[0623] On day 0, the revived PCMH suspension was adjusted to a suitable density and then inoculated into 48-well plates.

[0624] On day 1, pre-mixed and diluted test compounds were added to cells in a mixture with GalNAc3-Cy5. Test compounds GAL-01-D, GAL-02-D, GAL-03-D, GAL-04-D, and reference compound L96 were added at 100 μM, diluted 3-fold, with 11 concentration points per well. The final concentration of the competing compound GalNAc3-Cy5 was 0.5 μM. The final concentration of DMSO 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 detected by flow cytometry.

[0625] Data Analysis

[0626] The inhibition rates of compounds at various concentrations against GalNAc3-Cy5 reflect their endocytosis efficiency at those concentrations. Using GraphPad Prism software, the endocytosis efficiency (EC) of the tested compounds was calculated by fitting curves to the inhibition rates of compounds at various concentrations against GalNAc3-Cy5 using the equation "log(agonist) vs. response–variable slope". 50 value.

[0627] The inhibition rate of the compound against GalNAc3-Cy5 = (1-(MFI) Sample -MFI Neg ) / (MFI Pos -MFI Neg))*100%

[0628] MFI Sample It is the median fluorescence intensity of the compound in the APC channel.

[0629] MFI Neg Background fluorescence value

[0630] MFI Pos The maximum uptake fluorescence intensity of GalNAc3-Cy5

[0631] Experimental Results and Conclusions:

[0632] The endocytosis efficiency of the test compounds in PCMH was determined by flow cytometry in the presence of 0.5 μM GalNAc3-Cy5 competition, with reference to the EC50 of compound L96. 50 The EC value was 0.181 μM, indicating that the test compound GAL-03-D had an EC value of 0.181 μM. 50 The concentration was 0.189 μM, which was at the same level as the reference compound L96, both showing good binding affinity to ASGPR.

[0633] Table 2: Internalization efficiency of compounds in PCMH

[0634] L96 is a delivery group developed by Alnylm that binds to nucleic acids via covalent bonds, and its linkage is shown in the following formula: (where L96 succinate is a commercially available product, and its sequence synthesis method is the same as described in Example 3).

[0635] Example 5: Assay of the activity of the compound in inhibiting HSD17B13 mRNA in human primary hepatocytes (PHH)

[0636] Experimental materials and instruments:

[0637] Cells: PHH (batch number: HDS) were provided by Shanghai WuXi AppTec Co., Ltd. PHH cells were cultured in InvitroGRO™ CP Medium (BIOIVT, catalog number S03316) containing 10% fetal bovine serum (ExCellBio, catalog number FSP500) and 1% penicillin-streptomycin (HyClone, catalog number SV30010). Reagent: Lipofectamine TMiRNAiMAX transfection reagent (Invitrogen, catalog number 13778150), SensiFAST SYBR Hi Rox kit Mix (Meridian Bioscience, catalog number CSA-01135), RNA extraction kit (Qiagen, catalog number 74182), FastKing cDNA first-strand synthesis kit (TianGen, catalog number KR116-02), 96-well plates (Costar 3599). Instruments: Fluorescence qPCR instrument (Applied Biosystems, model QuantStudio 7Flex), cell counter (Countstar Rigel2).

[0638] Experimental steps and methods:

[0639] The test compound was serially diluted 8 times with PBS. Frozen PHH cells (batch number: HDS) were revived, counted using Countstar Rigel 2, and adjusted to 6 × 10⁶ cells / year. 5 / mL density. Free-access dosing: Add the diluted compound directly to a 96-well cell culture plate (10 μL / well), followed by adding 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.

[0640] Cells were collected, and RNA was extracted according to the Qiagen-74182 RNA Extraction Kit instructions. RNA was then reverse transcribed into cDNA according to the FastKing cDNA First-Strand Synthesis Kit instructions. The target gene cDNA was detected using qPCR. GAPDH was used as an internal control gene, and qPCR was performed in 384-well plates. Primers for amplification were included in the kit and provided by the supplier. The qPCR reaction program was as follows: first, heat at 50°C for 2 minutes, then at 95°C for 2 minutes, followed by cycling at 95°C for 5 seconds, then at 60°C for 30 seconds, for a total of 40 cycles; finally, heat at 95°C for 15 seconds, then at 60°C for 1 minute.

[0641] Data Analysis

[0642] The expression of the target gene in each sample was analyzed using the ΔΔCt method, a relative quantification method. This method measures the Ct difference (ΔCt) between the target gene (Human HSD17B13) and the internal reference gene (GAPDH), and compares the ΔCt values ​​of the compound-treated samples with those of the control group.

[0643] The formula is:

[0644] ΔCt = Average Ct of the target gene - Average Ct of the reference gene.

[0645] ΔCt = ΔCt of the sample after compound treatment – ​​average ΔCt of the control group.

[0646] Gene expression level = 2 - ΔΔCT

[0647] Experimental Results and Conclusions:

[0648] In experiments using primary human hepatocytes, the inhibitory activity of the test compounds on HSD17B13 mRNA after incubation in PHH cells for 2 days is shown in Table 3. AD-288917 and AD-288996 are selected from preferred sequences in Alnylam's related patent WO 2019 / 183164, and both test compounds significantly reduced the relative expression level of mRNA. VB13010 exhibited superior activity compared to *Codonopsis pilosula* at all three tested concentrations.

[0649] Table 3: Inhibition rate of the test compounds on HSD17B13 mRNA in PHH cells

[0650] Example 6: Assay on the activity and persistence of the compound in inhibiting HSD17B13 mRNA in human primary hepatocytes (PHH).

[0651] Experimental steps and methods:

[0652] (1) Take out the PHH (human primary hepatocytes) frozen in liquid nitrogen, revive them in a 37°C water bath, and centrifuge at 100g for 10 minutes.

[0653] (2) After resuspension counting, add 4-5 x 10⁴ / 100 μL / well to the 96-well collagen plate.

[0654] (3) After standing in the incubator for 4 to 6 hours to adhere to the wall, change the medium and add the siRNA mixture prepared with OptiMEM (11058021, GIBCO).

[0655] (4) After processing for the specified time, discard the supernatant and extract mRNA using Dynabeads (61012, Thermo) and reverse it (R222-01, Vyzame).

[0656] (5) SYBR Green qPCR (Q111-02, Vyzame) was performed on a Roche LightCycler 480II.

[0657] Data Analysis

[0658] The expression of the target gene in each sample was analyzed using the ΔΔCt method, a relative quantification method. This method measures the Ct difference (ΔCt) between the target gene (Human HSD17B13) and the internal reference gene (Human GAPDH), and compares the ΔCt values ​​of the compound-treated samples with those of the control group.

[0659] The formula is:

[0660] ΔCt = average Ct of target gene - average Ct of internal reference gene.

[0661] ΔCt = ΔCt of the sample after compound treatment – ​​average ΔCt of the control group.

[0662] Gene expression level = 2 - ΔΔCT

[0663] Experimental results

[0664] In experiments with human primary hepatocytes, the percentage of HSD17B13 mRNA remaining in PHH cells after incubation of the test compounds for 2-7 days is shown in Tables 4 and 5. All test compounds reduced the relative expression level of mRNA to varying degrees. Among them, VB13020 and VB13046 showed higher inhibitory activity than AD-288996 after 7 days of incubation in PHH, demonstrating excellent persistence.

[0665] Table 4: Inhibitory activity of the test compounds against HSD17B13 mRNA in PHH cells

[0666] Table 5: Inhibitory activity of the test compounds against HSD17B13 mRNA in PHH cells

[0667] Example 7: Assay of the activity of the compound in inhibiting HSD17B13 mRNA in an HDI mouse model

[0668] Experimental Objective: Hyperbaric tail vein injection (HDI) is an in vivo gene transduction method that involves the rapid injection of a large volume of plasmid DNA solution into mouse hepatocytes via the tail vein over a short period, thereby mediating the transient transfection of exogenous plasmid DNA into mouse hepatocytes. Utilizing the liver-targeting nature of this method, a single hyperbaric tail vein injection of the HSD17B13 replicon plasmid can mediate the short-term, efficient replication of HSD17B13 DNA in the mouse liver. This experiment used a mouse model of hyperbaric tail vein injection of HSD17B13 to evaluate the in vivo activity of a compound inhibiting HSD17B13 mRNA.

[0669] Experimental materials and instruments

[0670] 1. Animals: BALB / c mice, purchased from Shanghai Lingchang Biotechnology Co., Ltd., female, 6-8 weeks old.

[0671] 2. Solvent: Phosphate buffer, provided by WuXi AppTec. Filtered on the day of administration for drug preparation.

[0672] 3. Plasmid DNA: pcDNA-HSD17B13 I148M (I148M mutant) plasmid, provided by Shanghai WuXi AppTec Co., Ltd., batch number 880-1095686535_R2 / PA30592-2 / V1506362, concentration 2.4 μg / μL, diluted with normal physiological saline for later use. Reagents and consumables: Trizol (Invitrogen, 15596018); FastKing cDNA First Strand Synthesis Kit (Genomic De-generated) (Qiagen-Kr116-02); MagMAX TM mirVana TM Total RNA extraction kit (Thermo-A27828); HSD17B13 gene expression kit (Thermo-4351368); Stainless steel beads (Qiagen-69989); KingFisher 96-well standard plate (200 μL) (Thermo-97002540B); KingFisher 96-well magnetic rod sleeve (Thermo-97002534B); KingFisher 96-well plate (Thermo-95040450B).

[0673] 4. Instruments: Centrifuge (Beckman-Allegra X-15R); Tissue homogenizer (Qiagen-Tissue Lyser II); PCR instrument (Bio-RAD-T100TM Thermal cycer); Real-time quantitative PCR instrument (Applied Biosystems-QuantStudio 7Flex); Spectrophotometer (Thermo-Nanodrop ONE); Nucleic acid extractor KingFisher Apex (Thermo-APEX96DW714);

[0674] Experimental steps and methods:

[0675] 1. In vivo experiments

[0676] The day of drug administration to mice was defined as day 0 of the experiment, the day before was day -1, the day after was day 1, and so on. On day 0, mice were subcutaneously injected with a solvent or test compound at a volume of 5 mL / kg. On day 3, mice were hyperbarically injected with HSD17B13 plasmid DNA solution via tail vein. The plasmid DNA was prepared in advance with physiological saline and stored at 4°C until use. All mice were injected via tail vein within 5 seconds with 8% of their body weight of the plasmid DNA solution (injection volume (mL) = mouse body weight (g) × 8%), and the mass of the plasmid injected by each mouse was 10 μg. On day 4, 24 hours after the hyperbaric tail vein injection of HSD17B13 plasmid, all mice were euthanized by CO2 inhalation. After euthanasia, blood was collected from the heart, and liver samples were collected, approximately 70 mg of liver tissue was immersed in CO2. After incubating overnight at 4°C, the supernatant was discarded and transferred to a -80°C freezer for HSD17B13 mRNA detection.

[0677] 2. Sample Analysis

[0678] RNA was extracted from the liver using Trizol. The method is briefly described below: 50-70 mg of liver tissue and 1.2 mL of Trizol were homogenized with Qiagen Tissue Lyser II at 26 Hz for 2 min; lysis was performed at room temperature for 5 min; 1 mL of supernatant was collected, 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 s. After standing for 3 min, the supernatant was centrifuged and collected; an equal volume of isopropanol was added, and the mixture was incubated overnight at -20°C to precipitate the RNA; the sample was washed twice with 70% ethanol and dissolved in RNase-free water. The RNA concentration was determined using Nanodrop ONE, and the sample was diluted to 400 ng / μL with RNase-free water for reverse transcription.

[0679] The reverse transcription procedure was performed according to the FastKing cDNA First-Strand Synthesis Kit (Genomic Removal) 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 plate, incubate at 42°C for 3 min, and then 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 plate for reverse transcription. Reaction conditions: 42°C, 15 min; 95°C, 3 min. Store the cDNA at 4°C for further analysis.

[0680] The method for quantitative PCR detection of HSD17B13 and NEO gene expression levels in mouse liver is briefly described below: Prepare a qPCR reaction mixture, add 2 μL of sample, and perform the PCR reaction. Primers for amplification are included in the kit and provided by the supplier. Reaction conditions: 95℃, 10 min; 95℃, 15 s, 60℃, 1 min, 40 cycles.

[0681] Data Analysis

[0682] The expression of the target gene in each sample was analyzed using the ΔΔCt method, a relative quantitative method. This method measures the Ct difference (ΔCt) between the target gene (HSD17B13) and the internal reference gene (Neo), and compares the ΔCt values ​​of the compound-treated samples with those of the control group. The formulas are: ΔCt = average Ct of the target gene - average Ct of the reference gene; ΔΔCt = ΔCt of the compound-treated sample – average ΔCt of the control group; Gene expression level = 2 - ΔΔCt.

[0683] Experimental Results and Conclusions:

[0684] Compared with the PBS group, the following compounds all significantly reduced the relative expression levels of mRNA in liver tissue (see Tables 6 and 7). Among them, VB13005 and VB13006 were superior to Yangshen AD-288917.

[0685] Table 6: Inhibition of HSD17B13 mRNA expression levels by the compounds in the HDI mouse model

[0686] Table 7: Inhibition of HSD17B13 mRNA expression levels by the compounds in the HDI mouse model

[0687] In summary, to develop superior GalNAc-siRNA conjugates targeting HSD17B13, this invention optimized both the siRNA sequence and GalNAc. In experiments with primary human hepatocytes, to compare the effect of the siRNA sequence on activity, this invention designed a series of compounds, introducing non-natural nucleotide monomers into the siRNA sequence. Results showed that compounds VB13005 and VB13006 exhibited superior in vivo activity compared to *Gynostemma pentaphyllum*, and the introduction of non-natural nucleotide monomers simultaneously reduced off-target risk. To further optimize activity and enhance novelty, this invention developed a novel symmetrical tetravalent GalNAc, whose delivery efficiency can be maintained while facilitating CMC-scaled synthesis and reducing costs. Conjugating the optimized siRNA sequence with the new tetravalent GalNAc resulted in GalNAc-siRNA conjugates with superior selectivity and activity.

Claims

1. A double-stranded siRNA, characterized in that, It includes a justice chain and an antithesis chain, in which: The antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in the sequence shown in SEQ ID NO:1, wherein the length of the sense and antisense strands is independently 17 to 25 nucleotides; or The antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the sequence shown in SEQ ID NO:3, wherein the length of the sense strand and the antisense strand are independently 17 to 25 nucleotides. one or more of the nucleotide residues in the nucleotide sequence of the sense strand and / or antisense strand is replaced with r, having the 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 H, optionally substituted C1-C 10 alkyl, optionally substituted C1-C 10 alkoxy, optionally substituted C2-C 10 alkenyl, fluoro, chloro, bromo, or iodo; The optional substituted C1-C 10 Alkyl groups, the optionally substituted C1-C groups 10 Alkoxy groups and the optional substituted C2-C 10 The substituents in the alkenyl group are selected from one or more groups from the group consisting of: C1-C6 alkyl, C-C2 alkenyl, C1-C6 alkoxy, hydroxyl, oxo, fluorine, chlorine, bromine and iodine.

2. The double stranded siRNA of claim 1, wherein r is selected from any one of the following structures:

3. The double-stranded siRNA of claim 1 or 2, wherein The double-stranded siRNA satisfies one or more of the following conditions: (1) The positive strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in SEQ ID NO:2 or SEQ ID NO:4; (2) The antisense strand has a 2-nucleotide overhang at the 3' end; preferably, the 2 nucleotides are GU or UC; (3) The lengths of the sense strand and the antisense strand are each independently 19–25 nucleotides; preferably 19–23 nucleotides; more preferably 21 nucleotides for the sense strand and 23 nucleotides for the antisense strand; and, (4) The lengths of the reverse complementary double-stranded regions of the justice chain and the antisense chain are independently 19 to 23 bp, preferably 21 to 23 bp; Preferably, the antisense chain comprises the sequence shown in SEQ ID NO:1, and the justice chain comprises the sequence shown in SEQ ID NO:2; or, the antisense chain comprises the sequence shown in SEQ ID NO:3, and the justice chain comprises the sequence shown in SEQ ID NO:

4.

4. The double-stranded siRNA of any one of claims 1-3, wherein, One or more nucleotides of the antisense strand and / or one or more nucleotides of the sense strand are modified nucleotides.

5. The double stranded siRNA of claim 4, wherein The modification is selected from methoxy modification, fluorination modification, thiophosphate linkage, glycerol nucleic acid, (E)-vinyl phosphate and 2'-deoxynucleotide substitution; preferably, the methoxy modification is 2'-O-methyl modification, and / or the fluorination modification is 2'-fluorination modification.

6. The double-stranded siRNA of any one of claims 1-5, wherein, The double-stranded siRNA satisfies one or more of the following conditions: (1) The antisense strand contains two thiophosphate bonds between the three terminal nucleotides at the 3' end and two thiophosphate bonds between the three terminal nucleotides at the 5' end; (2) The positive strand contains two phosphate thioester bonds between the three terminal nucleotides at the 5' end; (3) r is linked to adjacent nucleotide residues via phosphate ester or thiophosphate bonds. (4) r substitutions were made at one or more nucleotide residues at positions 6, 7, 8, 10, 12, 13, 14, 15, 16, 18 and 20 of the antisense strand, in the direction from the 5' end to the 3' end; (5) r substitutions were made at one or more nucleotide residues at positions 2, 3, 9, 11, 13, 14, 15, 16, 18, 19 and 20 of the positive strand, in the direction from the 5' end to the 3' end; (6) In the direction from the 5' end to the 3' end, one or more nucleotides at positions 2, 14 and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl nucleotides. (7) In the direction from the 5' end to the 3' end, one or more nucleotides at positions 7 and 9-11 of the positive strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl nucleotides. (8) In the direction from the 5' end to the 3' end, one or more nucleotides at positions 2, 14 and 16 of the antisense strand are deoxynucleotides, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl or fluorinated. (9) Following the direction from the 5' end to the 3' end, one or more nucleotides at positions 7 and 9-11 of the positive strand are deoxyribonucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides or fluorinated modified nucleotides; and, (10) Following the direction from the 5' end to the 3' end, the nucleotide at the 7th position of the antisense strand is a glycerol nucleic acid, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl, fluorinated, or deoxynucleotides.

7. The double-stranded siRNA of any one of claims 1-6, wherein, The antisense strand contains nucleotide sequences selected from the following group: asAfscaagruuagucUfuGfauguasgsu; asGfsauagTgnccaugcAfaAfagcaususc; asGfsauagrccaugcAfaAfagcaususc; asGfsauagrccaugcdAaAfagcaususc; asGfsauaguccaugcAfaAfagcaususc; asGfsauaguccrugcAfaAfagcaususc; asGfsauagTgnccaugcdAaAfagcaususc; asGfsauagTgnccaugcAfadAagcaususc; asGfsauagTgnccrugcAfaAfagcaususc; asGfsauagTgnccaurcAfaAfagcaususc; asGfsauagTgnccaugcraAfagcaususc; asGfsauagTgnccaugcAfaragcaususc; asGfsauagTgnccaugcAfaAfarcaususc; asGfsauagTgnccaugcAfaAfagcrususc; asGfsauagrccaugcAfadAagcaususc; asGfsauagTgnccaugcAfrAfagcaususc; and, asGfsauagTgnccaugrAfaAfagcaususc; And / or, The positive strand contains nucleotide sequences selected from the following group: usascaucAfaGfAfCfuaaucuuguu; usrscaucAfaGfAfCfuaaucuuguu; usascaucAfaGfAfCfuaaucuuruu; usascaucAfaGfAfCfuaaucuugru; asusgcuuUfuGfCfAfuggacuaucu; asrsgcuuUfuGfCfAfuggacuaucu; asusgcuuUfuGfCfAfuggacuarcu; asusgcuuUfuGfCfAfuggacuauru; asrsgcuuUfuGfCfAfuggacuauru; asusgcuuUfuGfCfAfuggaruauru; asusgcuuUfuGfCfAfuggacuarru; asusgcuudUfuGfCfAfuggacuauru; asusgcuuUfuGfCfdAuggacuauru; asusgcuuUfuGfCfdAuggacuaucu; asusgcuuUfuGfCfdAuggacurucu; asusgcuudUfuGfCfAfuggacuaucu; asusgcuuUfudGCfAfuggacuaucu; asusgcuuUfuGfdCAfuggacuaucu; asusrcuuUfuGfCfAfuggacuaucu; asusgcuuUfurCfAfuggacuaucu; asusgcuuUfuGfCfruggacuaucu; asusgcuuUfuGfCfAfurgacuaucu; asusgcuuUfuGfCfAfugracuaucu; asusgcuuUfuGfCfAfuggrcuaucu; asusgcuuUfuGfdCAfuggrcuaucu; asusgcuuUfuGfdCAfuggacuauru;and, asusgcuudUfuGfCfAfuggrcuaucu; Wherein lowercase letters a, u, g, and c 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; Af, Uf, Gf, and Cf are 2'-fluorine modified adenosine-3'-phosphate, 2'-fluorine modified uridine-3'-phosphate, 2'-fluorine modified guanosine-3'-phosphate, and 2'-fluorine modified cytidine-3'-phosphate, respectively; Tgn is thymidine glycerol phosphate; s is a thiophosphate bond; d indicates that the nucleotide adjacent to the right of the letter d is a 2'-deoxyribonucleotide; r is as defined in claim 1 or 2, and is interconnected with other nucleotide residues via phosphate or thiophosphate.

8. The double-stranded siRNA of any one of claims 1-7, wherein, It contains a justice chain and an antithesis chain pair of any of the following: (1) Chain of Justice: usascaucAfaGfAfCfuaaucuuguu Antonyms: asAfscaagruuagucUfuGfauguasgsu; (2) Chain of Justice: usrscaucAfaGfAfCfuaaucuuguu Antonyms: asAfscaagruuagucUfuGfauguasgsu; (3) Chain of Justice: usascaucAfaGfAfCfuaaucuuruu Antonyms: asAfscaagruuagucUfuGfauguasgsu; (4) Chain of Justice: usascaucAfaGfAfCfuaaucuugru Antonyms: asAfscaagruuagucUfuGfauguasgsu; (5) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagrccaugcAfaAfagcaususc; (6) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccrugcAfaAfagcaususc; (7) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaurcAfaAfagcaususc; (8) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcraAfagcaususc; (9) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcAfaragcaususc; (10) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcAfaAfarcaususc; (11) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcrususc; (12) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcAfrAfagcaususc; (13) Chain of Justice: asusgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugrAfaAfagcaususc; (14) Chain of Justice: asrsgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagrccaugcAfaAfagcaususc; (15) Chain of Justice: asrsgcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (16) Chain of Justice: asusgcuuUfuGfCfAfuggacuarcu Antonyms: asGfsauagrccaugcAfaAfagcaususc; (17) Chain of Justice: asusgcuuUfuGfCfAfuggacuarcu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (18) Chain of Justice: asusgcuuUfuGfCfAfuggacuauru Antonyms: asGfsauagrccaugcAfaAfagcaususc; (19) Chain of Justice: asusgcuuUfuGfCfAfuggacuauru Antonyms: asGfsauagrccaugcdAaAfagcaususc; (20) Chain of Justice: asusgcuuUfuGfCfAfuggacuauru Antonyms: asGfsauagrccaugcAfadAagcaususc; (21) Chain of Justice: asrsgcuuUfuGfCfAfuggacuauru Antonyms: asGfsauagrccaugcAfaAfagcaususc; (22) Chain of Justice: asusgcuuUfuGfCfAfuggaruauru Antonyms: asGfsauagrccaugcAfaAfagcaususc; (23) Chain of Justice: asusgcuuUfuGfCfAfuggacuarru Antonyms: asGfsauagrccaugcAfaAfagcaususc; (24) Chain of Justice: asusgcuudUfuGfCfAfuggacuauru Antonyms: asGfsauagrccaugcAfaAfagcaususc; (25) Chain of Justice: asusgcuudUfuGfCfAfuggacuauru Antonyms: asGfsauagrccaugcdAaAfagcaususc; (26) Chain of Justice: asusgcuudUfuGfCfAfuggacuauru Antonyms: asGfsauagrccaugcAfadAagcaususc; (27) Chain of Justice: asusgcuuUfuGfCfdAuggacuauru Antonyms: asGfsauagrccaugcAfaAfagcaususc; (28) Chain of Justice: asusgcuuUfuGfCfdAuggacuauru Antonyms: asGfsauagrccaugcdAaAfagcaususc; (29) Chain of Justice: asusgcuuUfuGfCfdAuggacuaucu Antonyms: asGfsauaguccrugcAfaAfagcaususc; (30) Chain of Justice: asusgcuuUfuGfCfdAuggacurucu Antonyms: asGfsauaguccaugcAfaAfagcaususc; (31) Chain of Justice: asusrcuuUfuGfCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (32) Chain of Justice: asusgcuuUfurCfAfuggacuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (33) Chain of Justice: asusgcuuUfuGfCfruggacuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (34) Chain of Justice: asusgcuuUfuGfCfAfurgacuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (35) Chain of Justice: asusgcuuUfuGfCfAfugracuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (36) Chain of Justice: asusgcuuUfuGfCfAfuggrcuaucu Antonyms: asGfsauagTgnccaugcAfaAfagcaususc; (37) Chain of Justice: asusgcuuUfuGfdCAfuggrcuaucu Antonyms: asGfsauagrccaugcAfaAfagcaususc; (38) Chain of Justice: asusgcuuUfuGfdCAfuggrcuaucu Antonyms: asGfsauagrccaugcdAaAfagcaususc; (39) Chain of Justice: asusgcuuUfuGfdCAfuggrcuaucu Antonyms: asGfsauagrccaugcAfadAagcaususc; (40) Chain of Justice: asusgcuuUfuGfdCAfuggacuauru Antonyms: asGfsauagrccaugcAfaAfagcaususc; (41) Chain of Justice: asusgcuuUfuGfdCAfuggacuauru Antonyms: asGfsauagrccaugcdAaAfagcaususc; (42) Chain of Justice: asusgcuuUfuGfdCAfuggacuauru Antonyms: asGfsauagrccaugcAfadAagcaususc; (43) Chain of Justice: asusgcuudUfuGfCfAfuggrcuaucu Antonyms: asGfsauagrccaugcAfaAfagcaususc; (44) Chain of Justice: asusgcuudUfuGfCfAfuggrcuaucu Antonyms: asGfsauagrccaugcdAaAfagcaususc; (45) Chain of Justice: asusgcuudUfuGfCfAfuggrcuaucu Antonyms: asGfsauagrccaugcAfadAagcaususc; Preferably, the double-stranded siRNA comprises a sense strand and an antisense strand pair of any one of the following:

9. A conjugate, characterized in that, It contains the double-stranded siRNA as described in any one of claims 1-8.

10. The conjugate according to claim 9, characterized in that, It includes a liver-targeting component, and preferably the liver-targeting component includes N-acetylgalactosamine; Preferably, the liver-targeting portion comprises trivalent or tetravalent N-acetylgalactosamine; preferably, the liver-targeting portion is attached to the 3' end of the positive chain.

11. The conjugate according to claim 9 or 10, characterized in that, It consists of negative and positive ions; in, X - is O - or S - ; M z+ are pharmaceutically acceptable positive ions; The negative ions among them The total valence is equal to the total valence of the positive ion; The RNA is the double-stranded siRNA as described in any one of claims 1-8; For For For or For For The value is -(CH2)qC(=O)-, where q is 5, 6, 7, 8, 9 or 10, and 1, 2 or 3 of -(CH2)q- can be optionally replaced by 1, 2 or 3 O and / or -NHC(=O)-; A is Or connect key; For k is 2; When A is a bond, A is located between L 2 meta position; When A is At time A, A is located at L 2 para or meta; 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 of claim 11, wherein, The conjugates thereon satisfy 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 - is 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+ , and the organic base cation can be an ammonium cation, such as 13. The conjugate of claim 12, wherein, The conjugates thereon satisfy one or more of the following conditions: (1) the For Preferably, said For (2) the For Preferably, said For (3) -(CH2)q and L 3 The carbonyl group is connected to the nitrogen phase in the parent compound. (4) For (5) the For and (6) the For 14. The conjugate of claim 13, wherein, The conjugate satisfies one or two of the following conditions: (1) the For Preferably, said For 15. The conjugate of claim 14, wherein, The For 16. The conjugate of claim 15, wherein, The conjugate is any of the following structures: Preferably, the conjugate is:

17. The conjugate of any one of claims 9-16, wherein The conjugate is any one of: wherein, GAL-01 is a tetravalent symmetric GalNAc group, which is covalently bound to the nucleic acid, and the connection mode is as shown in the following formula: GAL-02 is a tetravalent symmetric GalNAc group, which is covalently bound to the nucleic acid, and the connection mode is as shown in the following formula: GAL-03 is a tetravalent symmetric GalNAc group, which is covalently bound to the nucleic acid, and the connection mode is as shown in the following formula: wherein: M + is Na + ; X - is O - .

18. A pharmaceutical composition, characterized by, It comprises a double-stranded siRNA of any one of claims 1-8 and / or a conjugate of any one of claims 9-17, and a pharmaceutically acceptable carrier.

19. Use of the double-stranded siRNA of any one of claims 1-8, the conjugate of any one of claims 9-17, or the pharmaceutical composition of claim 18 in the preparation of a medicament for the treatment and / or prevention of diseases associated with HSD17B13 gene expression.

20. Use according to claim 19, characterized in that, The disease associated with HSD17B13 gene expression is chronic liver disease. Preferably, the chronic liver disease is associated with the accumulation and / or expansion of lipid droplets in the liver; More preferably, the chronic liver disease is selected from the group consisting of: hepatitis, liver fibrosis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, cirrhosis, alcoholic steatohepatitis, alcoholic fatty liver disease, cirrhosis caused by HBV or HCV, drug-induced liver injury, and hepatocellular necrosis.

21. A method of inhibiting expression of a HSD17B13 gene in a cell in vitro, comprising contacting the cell with a compound of any one of claims 1-20. 21 It includes contacting cells with the double-stranded siRNA of any one of claims 1-8 and / or the conjugate of any one of claims 9-17; Preferably, the cells are animal cells; More preferably, the cells are human cells and / or hepatocytes, such as human primary hepatocytes.

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