Double-stranded sirna for inhibiting c3 expression and conjugate thereof

Abstract

Disclosed in the present invention are a double-stranded siRNA for inhibiting C3 expression and a conjugate thereof. The double-stranded siRNA for inhibiting C3 expression of the present invention comprises a sense strand and an antisense strand forming a reverse complementary double-stranded region. The antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in a sequence as shown in SEQ ID NO: 1. Lengths of the sense strand and the antisense strand are each independently 17-25 nucleotides. One or more nucleotide residues in the nucleotide sequence of the sense strand and / or the antisense strand are replaced with the following formula r: where X1, Y1, and Z1 are independently CH or N, and at least one of X1, Y1, and Z1 is N; R1 is H, optionally substituted C1-C10 alkyl, optionally substituted C1-C10 alkoxy, optionally substituted C2-C10 alkenyl, fluorine, chlorine, bromine, or iodine.

Description

Double-stranded siRNAs that inhibit C3 expression and their conjugates

[0001] This application claims priority to Chinese patent application 2024114547489, filed on 2024 / 10 / 17. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of RNA interference, specifically relating to a double-stranded siRNA that inhibits C3 expression and its conjugates. Background Technology

[0003] Complement is a group of proteins that exist in human serum and tissue fluid. After activation, they have various activities, such as assisting and supplementing specific antibodies, mediating immune lysis, hemolysis and cell lysis, enhancing phagocytosis and vascular permeability, neutralizing viruses, and activating inflammatory responses.

[0004] The complement system is currently known to consist of three pathways: the classical pathway, the alternative pathway, and the lectin pathway. All three pathways converge at complement component 3 (C3), which in turn activates the downstream C5 terminal pathway and related inflammatory responses.

[0005] In-depth research has revealed that the complement system is involved in the occurrence and development of a variety of diseases, including neurological diseases such as neuromyelitis optica (NMO) and myasthenia gravis (gMG), eye diseases such as age-related macular degeneration (AMD) and uveitis, kidney diseases such as atypical hemolytic uremic syndrome (aHUS), C3 glomerulonephropathy (C3G), IgA nephropathy, primary membranous nephropathy (PMN), lupus nephritis (LN), and hematological diseases such as paroxysmal nocturnal hemoglobinuria (PNH) and thrombotic microangiopathy (TMAs).

[0006] According to a 2020 report by the World Health Organization, CKD has risen to become one of the top ten causes of death globally. Current treatments primarily focus on symptom relief, failing to meet a significant clinical need. The primary nephron damage factors in CKD are diverse, and immune mechanisms mediate the progression of most kidney diseases. IgAN nephropathy, mentioned above, accounts for more than 50% of primary glomerular diseases in my country. It is known that most glomerulonephritis is related to the complement system, with rare diseases such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulonephropathy (C3G) showing clear associations.

[0007] Meanwhile, poor medication adherence among patients with renal failure is a widespread phenomenon, with over 60% of clinicians listing "improving patient adherence" as their top priority in renal failure treatment. Furthermore, while domestic nephrologists report that diagnosis is relatively mature, there is a lack of suitable and effective drugs. Existing imported drugs are expensive, and the large number of patients leaves a significant unmet clinical need. Therefore, in addition to drugs that alleviate symptoms, evidence-based treatments, drugs that prevent progression, and drugs that improve adherence are urgently needed for development.

[0008] While targeted therapies for C5 and C3 cyclic peptides and CFB inhibitors have been approved for the aforementioned indications, the efficacy and medication adherence of existing complement therapies remain insufficient. A sufficient number of effective drugs are lacking in various inflammatory and immune diseases, leaving unmet medical needs. Domestic and international competition is primarily focused on C5, while C3 complement therapy warrants further exploration.

[0009] Small RNA interference (siRNA) drugs are gradually developing into a mature treatment approach, offering advantages such as long-lasting efficacy and good long-term drug adherence. Currently, only three C3-targeting siRNA drugs are in Phase I clinical trials abroad, and there is a lack of related product development in China. Summary of the Invention

[0010] To further improve C3-targeting siRNA drugs and enhance their drug-likeness, such as further improving efficacy, reducing off-target effects, and lowering production difficulty and cost, this invention provides a double-stranded siRNA that inhibits C3 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 risks while maintaining high drug activity.

[0011] In one aspect, the present invention provides a double-stranded siRNA comprising a sense strand and an antisense strand forming an inverse complementary double-stranded region, wherein 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'-AGUAAUUGUAGAGAACGGCUCGG-3'), and the length of the sense strand and the antisense strand are independently 17 to 25 nucleotides.

[0012] One or more nucleotide residues in the nucleotide sequence of the sense strand and / or antisense strand are replaced by the following formula r:

[0013] 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;

[0014] R 1H, or optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 10 Alkoxy, optional substituted C2-C 10 Alkenyl, fluorine, chlorine, bromine, or iodine;

[0015] 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.

[0016] 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.

[0017] 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.

[0018] In one implementation, the C2-C 10 The alkenyl group is C2-C6 alkenyl, preferably vinyl or propenyl.

[0019] 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.

[0020] Preferably, the substituent is selected from one or more groups of the group consisting of methoxy and vinyl groups.

[0021] 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).

[0022] In one implementation, r is selected from any of the following structures:

[0023] In one implementation, the double-stranded siRNA satisfies one or more of the following conditions:

[0024] (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'-GAGCCGUUCUCUACAAUUACU-3');

[0025] (2) The antisense strand has a 2-nucleotide overhang at the 3' end; preferably, the 2 nucleotides are GG;

[0026] (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,

[0027] (4) The length of the reverse complementary double-stranded region is 19 to 23 bp, preferably 21 to 23 bp.

[0028] 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; one or more nucleotide residues in the nucleotide sequences of the sense strand and / or the antisense strand are replaced with a structure having the structure shown in Formula r, as defined above.

[0029] 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.

[0030] In one embodiment, the modifying group in the modified nucleotide is selected from methoxy modification, fluorination modification, thiophosphate linkage, nucleotide replacement with glycerol nucleic acid, (E)-vinyl phosphate, and 2'-deoxynucleotide; preferably, methoxy modification is 2'-O-methyl modification; fluorination modification is 2'-fluorination modification.

[0031] 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.

[0032] In one embodiment, the positive chain comprises two phosphate thioester bonds between three terminal nucleotides at the 5' end.

[0033] In one embodiment, the r is linked to an adjacent nucleotide residue via a phosphate ester or thiophosphate bond.

[0034] In one embodiment, one or more nucleotide residues selected from positions 2, 7-9, 12-13, 15, 17, and 22 in the antisense strand are substituted according to the formula r, from the 5' end to the 3' end.

[0035] In one embodiment, one or more nucleotide residues selected from positions 1-8 and 12-21 in the positive strand are substituted according to the formula r, from the 5' end to the 3' end.

[0036] In one embodiment, one or more nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, arranged from the 5' end to the 3' end, and the remaining nucleotides are 2'-O-methyl nucleotides.

[0037] In one embodiment, one or more nucleotides at positions 7 and 9-11 of the positive strand are fluorinated nucleotides, arranged from the 5' end to the 3' end, and the remaining nucleotides are 2'-O-methyl nucleotides.

[0038] When r occupies the position mentioned above, that position is not subject to the corresponding fluorination or 2'-O-methyl modification.

[0039] In one embodiment, the antisense strand comprises a nucleotide sequence selected from the group consisting of:

[0040] Among them, r1-r12 are as described above.

[0041] In some implementations, the positive strand comprises a nucleotide sequence selected from the group consisting of:

[0042] Where Am, Um, Gm, and Cm 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; s is a thiophosphate bond; dA, dC, and dT are deoxyribonucleotides adenine, cytosine, and thymine, respectively. ;(s) indicates that the 3' terminal nucleotide residue of the sequence is linked to the delivery ligand via a thiophosphate bond; P indicates that a phosphate molecule is linked to the 5' terminal of the sequence; VPA indicates adenine nucleotide modified with 5'(E)-vinyl phosphate; gn indicates glycerol nucleotide (e.g., Tgn indicates thymine glycerol nucleotide, Cgn indicates cytosine glycerol nucleotide); 2p indicates 2'-phosphoamide nucleotide (e.g., U2p indicates uracil 2'-phosphoamide nucleotide); r1-r12 are as described above, r1 and r3-r11 are all in the category of non-natural nucleotides r as defined above, and r1, r3-r11 are linked to other nucleotide residues via phosphate esters or thiophosphate esters.

[0043] In one embodiment, the double-stranded siRNA comprises a sense strand and an antisense strand pair of any of the following sequence numbers:

[0044] Among them, r1-r12 are as described above.

[0045] In some embodiments, the 5' end of the antisense strand in the double strand is a phosphate ester nucleotide or a vinyl phosphate ester nucleotide.

[0046] In another aspect, the present invention also provides a conjugate (GalNAc conjugate) comprising the double-stranded siRNA described herein.

[0047] In one embodiment, the conjugate comprises a liver-targeting moiety, preferably comprising N-acetylgalactosamine; more preferably, the liver-targeting moiety comprises tetravalent N-acetylgalactosamine.

[0048] In one embodiment, the conjugate is shown in Formula I;

[0049] in,

[0050] X - For O - or S - ;

[0051] M z+ It is a pharmaceutically acceptable positive ion;

[0052] The negative ions mentioned The total valence is equal to the total valence of the positive ion;

[0053] The RNA is the double-stranded siRNA described in this invention;

[0054] for (The left end of the segment is connected to Gal, and the right end is connected to N);

[0055] 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

[0056] 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)-;

[0057] A is Or connect key;

[0058] for (The left end of the fragment is connected to the carbonyl group, and the right end is connected to N);

[0059] k is 2;

[0060] When A is the link key, A is located in L. 2 Interposition;

[0061] When A is When A is located at L 2 Opposite or intermediate;

[0062] n1, n2, n3, n4, n5, n6 and n7 are independently 1, 2, 3, 4, 5 or 6;

[0063] m1 and m2 are independently 0, 1, 2, 3, 4 or 5;

[0064] q1 and q2 are independently 1, 2, 3, 4 or 5.

[0065] In one implementation, n1 is 1, 2, 3, 4 or 5; preferably, n1 is 1, 2 or 3.

[0066] In one implementation, n2 is 1, 2, 3 or 4; preferably, n2 is 1 or 2.

[0067] In one implementation, n3 is 1, 2, 3, 4 or 5; preferably, n3 is 3.

[0068] In one implementation, n4 is 1, 2, 3 or 4; preferably, n4 is 2.

[0069] In one implementation, n5 is 1, 2, 3, 4, 5 or 6; preferably, n5 is 4.

[0070] In one implementation, n6 is 1, 2, 3, 4 or 5; preferably, n6 is 3.

[0071] In one implementation, n7 is 1, 2, or 3; preferably, n7 is 1.

[0072] In one implementation, m1 is 1 or 2; preferably, m1 is 1.

[0073] In one implementation, m2 is 0, 1, 2, 3, 4 or 5; preferably, m2 is 0, 1, 2 or 3.

[0074] In one implementation, q1 is 1, 2, 3 or 4; preferably, q1 is 2.

[0075] In one implementation, q2 is 1, 2, 3, 4 or 5; preferably, q2 is 3.

[0076] In one implementation, X - For O - .

[0077] 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, ...

[0078] 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.

[0079] 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.

[0080] In one implementation scheme -(CH2)q and L 3 The carbonyl group is connected to the nitrogen phase in the parent compound.

[0081] 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.

[0082] 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.

[0083] 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).

[0084] In one implementation scheme for

[0085] In one implementation scheme for

[0086] In one embodiment, the conjugate has any of the following structures:

[0087] In one embodiment, the conjugate is:

[0088] Among them, X - For O - or S - The definition of RNA is the same as that described in any embodiment of this invention.

[0089] In one embodiment, the liver-targeting portion is attached to the 3' end of the justice chain.

[0090] In another respect, the present invention provides a conjugate as shown in Formula II:

[0091] Among them, A and X - M z+ L 1 L 2 L 3 and L 4 The definitions are the same as those described above;

[0092] The double-stranded siRNA' comprises a sense strand and an antisense strand forming an inverse complementary double-stranded region, wherein the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in the sequence shown in SEQ ID NO:1 (5'-AGUAAUUGUAGAGAACGGCUCGG-3'), and the sense strand and antisense strand are each independently 17 to 25 nucleotides in length.

[0093] In one implementation, the double-stranded siRNA' satisfies one or more of the following conditions:

[0094] (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'-GAGCCGUUCUCUACAAUUACU-3');

[0095] (2) The antisense strand has a 2-nucleotide overhang at the 3' end; preferably, the 2 nucleotides are GG;

[0096] (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,

[0097] (4) The length of the reverse complementary double-stranded region is 19 to 23 bp, preferably 21 to 23 bp.

[0098] In one embodiment, the antisense chain comprises a sequence as shown in SEQ ID NO:1, and the justice chain comprises a sequence as shown in SEQ ID NO:2.

[0099] 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.

[0100] In one embodiment, the modification is selected from methoxy modification, fluorination modification, thiophosphate linkage, replacement of the nucleotide with glycerol nucleic acid, (E)-vinyl phosphate or 2'-deoxynucleotide; preferably, the methoxy modification is a 2'-O-methyl modification; preferably, the fluorination modification is a 2'-fluoro modification.

[0101] 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.

[0102] In one implementation, the positive chain comprises two phosphate thioester bonds between three terminal nucleotides at the 5' end.

[0103] In one embodiment, one or more nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, arranged from the 5' end to the 3' end, and the remaining nucleotides are 2'-O-methyl nucleotides; and / or

[0104] In one embodiment, one or more nucleotides at positions 7 and 9-11 of the positive strand are fluorinated nucleotides, arranged from the 5' end to the 3' end, and the remaining nucleotides are 2'-O-methyl nucleotides.

[0105] In one implementation, the antisense strand comprises a nucleotide sequence selected from the group consisting of:

[0106] The positive strand contains nucleotide sequences selected from the following group:

[0107] Where Am, Um, Gm, and Cm 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; s represents a thiophosphate bond; dA, dC, and dT represent deoxyribonucleotides adenine, cytosine, and thymine, respectively; and gn represents glycerol nucleotides (e.g., Tgn represents thymine glycerol nucleotide, and Cgn represents cytosine glycerol nucleotide).

[0108] In one implementation, the double-stranded siRNA' comprises a sense strand and an antisense strand pair of any of the following:

[0109] The conjugates provided by this invention are preferably any of the conjugates in Table 1.

[0110] In another aspect, the present invention provides a pharmaceutical composition comprising the double-stranded siRNA described herein and / or the conjugates described above and a pharmaceutically acceptable carrier.

[0111] In another aspect, the present invention provides the use of the described double-stranded siRNA, the described conjugate, or the described pharmaceutical composition in the preparation of a medicament for treating and / or preventing diseases associated with C3 gene expression in a subject.

[0112] In one embodiment, the C3 gene expression-related diseases include kidney-related C3 glomerulonephropathy (C3G); immune complex-mediated glomerulonephritis (IC-mediated GN); post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus, ischemic-perfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and other organ-related C3 diseases.

[0113] In another aspect, the present invention provides a cell comprising the double-stranded siRNA and / or conjugates described herein; said cell is a non-animal or non-plant variety.

[0114] In another aspect, the present invention provides a method for inhibiting C3 gene expression in cells in vitro, comprising contacting the cells with the double-stranded siRNA and / or conjugates described herein.

[0115] The positive and beneficial effects of this invention include:

[0116] (1) The siRNA of the present invention exhibits good in vitro or in vivo inhibitory activity against C3 mRNA; and

[0117] (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. Attached Figure Description

[0118] Figure 1 shows the remaining mRNA expression levels in the liver (n=4).

[0119] Figure 2 shows the changes in AST in each group of transgenic mice treated with the drug.

[0120] Figure 3 shows the changes in ALT levels in each group of transgenic mice treated with the drug.

[0121] Figure 4 shows the changes in serum hC3 protein levels in transgenic mice in each drug administration group. Detailed Implementation

[0122] Definitions and Explanations

[0123] 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.

[0124] 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.

[0125] The term "C3" refers to complement C3, the most abundant complement component in serum, primarily synthesized by hepatocytes and macrophages. It mediates the complement cascade by participating in both the classical and alternative complement activation pathways through activation and cleavage. Unless explicitly excluded, this term includes, but is 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 natural C3 that maintain at least one in vivo or in vitro activity of natural C3. The term encompasses C3 in its full-length, unprocessed precursor form, as well as forms obtained from cleavage after translation of the signal peptide and forms obtained from protein degradation.

[0126] 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).

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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 C3 gene in cells (such as cells of an individual, or cells of a mammalian individual).

[0131] 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 mRNA from specific genes whose nucleotide sequences are complementary to or close to theirs, 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).

[0132] The term "antisense strand" refers to the strand of an iRNA (such as a siRNA) that includes a region substantially complementary to a target sequence (e.g., a C3 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 at the 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. Typically, siRNAs are 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.

[0133] 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.

[0134] 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.

[0135] 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'.

[0136] The “modification” of nucleotides described in this disclosure includes, but is not limited to, alkoxy / methoxy modifications, fluorination modifications, and thiophosphate linkages.

[0137] In this disclosure, unless otherwise specified, the capital letters A, U, G, and C represent adenosine-3'-phosphate, uridine-3'-phosphate, guanosine-3'-phosphate, and cytidine-3'-phosphate. The capital letters Cf, Gf, Uf, and Af indicate a fluorinated modification at the 2' position of the nucleotide represented by the corresponding capital letter. The letters Am, Gm, Cm, and Um indicate a methoxy modification at the 2' position of the nucleotide represented by the corresponding capital letter; the "s" following a letter in the sequence indicates that the two nucleotide residues adjacent to "s" are linked by a thiophosphate group. For example, "As-G" indicates that the A and G residues are linked by a thiophosphate group. "d" indicates that the nucleotide adjacent to the right of the letter d is a 2'-deoxyribonucleotide.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0142] 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.

[0143] 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%.

[0144] 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%.

[0145] 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).

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] r-structure unit

[0151] In this article, 'r' represents the following structural unit:

[0152] The definitions of each group are the same as those described above.

[0153] Preferably, r1 represents the following structural unit.

[0154] 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.

[0155] r1 and other nucleotide residues can be linked together via phosphate esters or thiophosphate esters. For example, "mA*r1" indicates that mA and r1 nucleotide monomers are linked via a thiophosphate ester group, and "mAr1" indicates that mA and r1 nucleotide monomers are linked via a phosphate ester group. The linkage methods are shown below:

[0156] X - For O - or S - ;

[0157] In this disclosure, the double-stranded siRNA comprises a sense strand or an r-intercalated sense strand and / or an antisense strand or an r-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.

[0158] The r-intercalated sequence described in this disclosure refers to a sequence in which at least one nucleotide residue is linked to r. The r-intercalated sequences described in this disclosure include, but are not limited to, r-intercalated double-stranded siRNAs, r-intercalated sense strands, and r-intercalated antisense strands. For example, 5'-mAGUr1r1A*C-3' and 5'-r1GmGAAC-3' are both examples of r-intercalation.

[0159] 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 r; this includes double-stranded siRNA in which r replaces one nucleotide residue 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 where one or more nucleotides in the sense strand are 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 where one or more nucleotides in the antisense strand are replaced by r.

[0160] siRNA and modified nucleotides

[0161] This document provides siRNAs for inhibiting the expression of the Complement C3 gene. 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.

[0162] The antisense strand comprises the nucleotide sequence of SEQ ID NO:1 or 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:1. The sense strand comprises the nucleotide sequence of SEQ ID NO:2 or 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:2. One or more (e.g., 1-3, e.g., 1, 2, or 3) nucleotide residues in the nucleotide sequences of the sense strand and / or the antisense strand 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. The modification may be selected from methoxy modification, fluorination modification, thiophosphate linkage, replacement of the nucleotide with glycerol nucleic acid, (E)-vinyl phosphate, or 2'-deoxynucleotide. Preferably, the methoxy modification is a 2'-O-methyl modification; preferably, the fluorination modification is a 2'-fluoro modification.

[0163] 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, r is linked to adjacent nucleotide residues via phosphate or phosphate bonds. In one embodiment, r substitution is performed at the 7th, 8th, or 9th nucleotide residue of the antisense strand in a 5'-to-3' manner. In one embodiment, r 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, following a 5'-to-3' end configuration, while the remaining nucleotides are 2'-O-methylated nucleotides. When r occupies a position mentioned above, that position is not subject to the corresponding fluorinated or 2'-O-methylated modification.

[0164] Preferably, the siRNA described herein has any of the pairs of sense and antisense strands listed in Table 1.

[0165] Conjugate

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] The solvents used in this disclosure are commercially available.

[0171] Unless otherwise specified, all solvent ratios used in column chromatography and preparative thin-layer silica gel chromatography in this disclosure are volume ratios.

[0172] Example

[0173] 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.

[0174] Example 1: Synthesis of phosphoramide monomer

[0175] Example 1-1: Synthesis of r1-M

[0176] Synthesis of intermediates 1-3 in step one

[0177] 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%).

[0178] LCMS(ESI) m / z: 386.1 [M+H] + ;

[0179] 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).

[0180] Synthesis of intermediates 1-4 in step two

[0181] 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%).

[0182] LCMS(ESI) m / z: 260.1 [M+H] + ;

[0183] 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).

[0184] Synthesis of intermediates 1-5 in step three

[0185] 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%).

[0186] LCMS(ESI) m / z: 502.2 [M+H] + ;

[0187] 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).

[0188] Synthesis of intermediates 1-6 in step four

[0189] 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%).

[0190] 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,2TM), 3,96-4.06(m,5H), 3,68(s,3H), 0.99-1.13(m,2H).

[0191] Synthesis of intermediates 1-7 in step five

[0192] 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).

[0193] LCMS(ESI) m / z: 274.1 [M+H] + ;

[0194] 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).

[0195] Step 6 Synthesis of intermediates 1-8

[0196] 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).

[0197] LCMS(ESI) m / z: 574.2 [MH] + .

[0198] Step 7: Synthesis of the final product r1-M

[0199] Compounds 1-8 (100 mg) were dissolved in dichloromethane (2.00 mL), and acetocyanodiisopropylphosphonoyl chloride (61.6 mg) and N,N-diisopropylethylamine (89.9 mg) were added at 0 °C. The reaction mixture was stirred at 20 °C for 2 hours. LCMS (RT = 2.367, 2.424 min) showed that the starting materials were 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).

[0200] LCMS(ESI) m / z: 776.2 [M+H] + ;

[0201] 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).

[0202] Examples 1-2: Synthesis of phosphorus amide monomer r2-M

[0203] Synthesis of intermediate 2-1 in step one

[0204] 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%).

[0205] LCMS(ESI) m / z: 542.3 [M+H]+;

[0206] Synthesis of intermediate 2-2 in step two

[0207] 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%).

[0208] Synthesis of intermediates 2-3 in step three

[0209] 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%).

[0210] LCMS(ESI) m / z: 602.2[M+H]+;

[0211] Step 4: Synthesis of the final product r2-M

[0212] 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%).

[0213] LCMS(ESI) m / z: 802.3[M+H]+;

[0214] 1H NMR (400MHz, CDCl3) δppm 0.98-1.11(m,6H)1.17(d,J=6.80Hz,6H)1.44(s,1H)2.57-2.75(m,2H)3.21-3.31(m,1H)3.44(dd,J=12.00,2.80Hz,1H)3.58(dt, J=12.00,6.80Hz,2H)3.78-3.81(m,1H)3.82-3.93(m,1H)3.91(br.d,J=7.60Hz,1H)3.96(s,3H)4.03(s,1H)4.11(br.d,J=5.60Hz ,1H)4.21(br.d,J=5.60Hz,1H)4.35(br.d,J=3.20Hz,1H)4.48(br.d,J=12.00Hz,1H)4.60(t,J=4.40Hz,1H)5.13-5.28(m,2H)5.8 0-5.93(m,1H)5.99(d,J=4.40Hz,1H)6.81(d,J=8.40Hz,4H)7.20-7.25(m,2H)7.28-7.33(m,5H)7.41(d,J=7.20Hz,2H)8.40(s,1H)

[0215] Examples 1-3: Synthesis of phosphorusamide monomer r3-M

[0216] Synthesis of final product 3-1 in step one

[0217] 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).

[0218] Step 2: Synthesis of final product 3-2

[0219] 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).

[0220] Step 3: Synthesis of final product 3-3

[0221] 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%).

[0222] LCMS(ESI) m / z: 560.3 [M+H]+;

[0223] Step 4: Synthesis of final product 3-4

[0224] 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.

[0225] LCMS(ESI)m / z:318.2[M+H]+;

[0226] Step 5: Synthesis of final product 3-5

[0227] 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).

[0228] LCMS(ESI) m / z: 620.3[M+H]+;

[0229] Step Six: Synthesis of the final product r3-M

[0230] 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%).

[0231] LCMS(ESI) m / z: 820.3[M+H]+;

[0232] 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)

[0233] Using a similar method, Examples 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 were synthesized.

[0234] Examples 1-4 Synthesis of phosphoramide monomer r4-M

[0235] LCMS(ESI) m / z: 804.3[M+H]+;

[0236] 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,1 H)6.80-6.85(m,4H)7.18-7.26(m,8H)7.31-7.37(m,2H)8.93-9.01(m,1H)

[0237] Examples 1-5 Synthesis of phosphorus amide monomer r5-M

[0238] LCMS(ESI) m / z: 776.4 [M+H]+;

[0239] 1H NMR (400MHz, DMSO-d6) δppm 1.12(d,J=6.40Hz,12H)2.60(td,J=6.00,2.31Hz,2H)3.01(dd,J=10.0,4.75 Hz,1H)3.39(s,3H)3.51-3.60(m,4H)3.72(s,7H)3.84-3.87(m,3H)4.18-4.28 (m,1H)4.45-4.52(m,1H)4.74-4.86(m,1H)6.21(d,J=3.20Hz,1H)6.79-6.83( m,4H)7.15-7.20(m,6H)7.21-7.25(m,3H)7.28-7.31(m,2H)8.43-8.45(m,1H)

[0240] Examples 1-6 Synthesis of phosphorusamide monomer r6-M

[0241] LCMS(ESI) m / z: 802.3[M+H]+;

[0242] 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(d dd,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) Examples 1-7 Synthesis of phosphorous amide monomer r7-M

[0243] LCMS(ESI) m / z: 802.3[M+H]+;

[0244] 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)

[0245] Examples 1-8 Synthesis of phosphorusamide monomer r8-M

[0246] LCMS(ESI)m / z:801.3[M+H]+;

[0247] 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)

[0248] Examples 1-9: Synthesis of phosphorous amide monomer r9-M

[0249] LCMS(ESI) m / z: 820.3[M+H]+;

[0250] 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)

[0251] Examples 1-10 Synthesis of phosphorus amide monomer r10-M

[0252] LCMS(ESI) m / z: 820.3[M+H]+;

[0253] 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)

[0254] Example 2: Compound Synthesis

[0255] Example 2-1: Synthesis of compound GAL-01-M

[0256] Step 1

[0257] 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%).

[0258] Step Two

[0259] 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%).

[0260] LCMS(ESI)m / z:420.5[M+H]+;

[0261] Step 3

[0262] 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%).

[0263] Step Four

[0264] 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%).

[0265] Step 5

[0266] 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%).

[0267] Step Six

[0268] 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%).

[0269] Step Seven

[0270] 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).

[0271] LCMS(ESI) m / z: 225.1 [M+H]+;

[0272] Step 8

[0273] 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).

[0274] Step Nine

[0275] 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%).

[0276] LCMS(ESI) m / z: 851.4 [M+H]+;

[0277] Step 10

[0278] 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).

[0279] Step Eleven

[0280] 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%).

[0281] LCMS(ESI) m / z: 927.4 [M+H]+;

[0282] Step Twelve

[0283] 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.

[0284] LCMS(ESI) m / z: 527.3 [M+H]+;

[0285] Step Thirteen

[0286] 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%).

[0287] Step Fourteen

[0288] 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%).

[0289] LCMS(ESI)m / z:1077.9[M+2H] / 2+;

[0290] Step Fifteen

[0291] 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%).

[0292] TOF MS ES + m / z:2711[M+H] / +;

[0293] 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)

[0294] Example 2-2: Synthesis of compound GAL-02-M

[0295] Step 1

[0296] 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).

[0297] Step Two

[0298] 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.

[0299] LCMS(ESI)m / z:223[M+H]+;

[0300] Step 3

[0301] 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).

[0302] LCMS(ESI)m / z:323[M+H]+;

[0303] Step Four

[0304] 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).

[0305] LCMS(ESI)m / z:724[M+H]+;

[0306] Step Six

[0307] 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%).

[0308] LCMS(ESI)m / z:590[M+H]+;

[0309] Step Seven

[0310] 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%).

[0311] LCMS(ESI)m / z:1212[M-DMT] / 2+;

[0312] 1 H NMR (400MHz, CHLOROFORM-d)

[0313] 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)

[0314] Examples 2-3: Synthesis of ligand GAL-03-M

[0315] Step 1

[0316] 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%).

[0317] Step Two

[0318] 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).

[0319] Step 3

[0320] 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).

[0321] LCMS(ESI) m / z: 267 [M+H] + ;

[0322] Step Four

[0323] 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%).

[0324] LCMS(ESI) m / z: 432 [M+H] + ;

[0325] Step 5

[0326] 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%).

[0327] LCMS(ESI) m / z: 1093 [M+H] + ;

[0328] Step Six

[0329] 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%).

[0330] LCMS(ESI) m / z: 958 [M+H] + ;

[0331] Step Seven

[0332] 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).

[0333] LCMS(ESI)m / z:1121[M+H] + ;

[0334] Step 8

[0335] 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.

[0336] LCMS(ESI) m / z: 958 [M+H] + ;

[0337] Step Nine

[0338] 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%).

[0339] LCMS(ESI) m / z: 1197 [M+H] + ;

[0340] Step 10

[0341] 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%).

[0342] LCMS(ESI) m / z: 1197 [M+H]+ ;

[0343] Step Eleven

[0344] 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%).

[0345] LCMS(ESI) m / z: 1257 [M+2H] 2+ / 2;

[0346] Step Twelve

[0347] 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%).

[0348] LCMS(ESI) m / z: 1212 [M+2H] + / 2;

[0349] Step Thirteen

[0350] 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%).

[0351] LCMS(ESI)m / z:1347[M-DMT] / 2 + ;

[0352] 1 H NMR (400MHz, CDCl3)

[0353] 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).

[0354] Examples 2-4: Synthesis of GAL-04-M

[0355] Step 1

[0356] 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%).

[0357] Step Two

[0358] 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%).

[0359] Step 3

[0360] 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%).

[0361] LCMS(ESI)m / z:2714[M+H]+;

[0362] 1 H NMR (400MHz, CDCl3)

[0363] 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).

[0364] Examples 2-5: Synthesis of compound GAL-05-M

[0365] Step 1

[0366] 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%).

[0367] Step Two

[0368] 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%).

[0369] 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)

[0370] Step 3

[0371] 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%).

[0372] Step Four

[0373] 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%).

[0374] Step 5

[0375] 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%).

[0376] LCMS(ESI) m / z: 618.4 [M+H]+;

[0377] 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)

[0378] Step Six

[0379] 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%).

[0380] LCMS(ESI) m / z: 604.3[M+H]+;

[0381] Step Seven

[0382] 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).

[0383] LCMS(ESI) m / z: 237.1 [MH]+;

[0384] Step 8

[0385] 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.

[0386] LCMS(ESI) m / z: 223.2[MH]+;

[0387] Step Nine

[0388] 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%).

[0389] LCMS(ESI)m / z:243.1[M+H]+;

[0390] Step 10

[0391] 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%).

[0392] LCMS(ESI) m / z: 250.1 [M+H]+;

[0393] Step Eleven

[0394] 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%).

[0395] LCMS(ESI)m / z:224.2[M+H]+;

[0396] Step Twelve

[0397] 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.

[0398] LCMS(ESI) m / z: 715.2[2M+H]+;

[0399] 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)

[0400] Step Thirteen

[0401] 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%).

[0402] LCMS(ESI) m / z: 659.2 [2M+H]+;

[0403] Step Fourteen

[0404] 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%).

[0405] Step Fifteen

[0406] 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.

[0407] LCMS(ESI) m / z: 956.3 [M+H]+;

[0408] Step Sixteen

[0409] 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%).

[0410] Step Seventeen

[0411] 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%).

[0412] LCMS(ESI)m / z:1137.4[M+2H] / 2+;

[0413] Step 18

[0414] 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%).

[0415] LCMS(ESI)m / z:1070.4[M+2H] / 2+;

[0416] Step Nineteen

[0417] 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).

[0418] LCMS(ESI)m / z:1363[M+2H] / 2+;

[0419] 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)

[0420] Synthesis of compound GAL-01-D in Examples 2-6

[0421] 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%).

[0422] LCMS(ESI)m / z:1903[M-DMT]+;

[0423] 1 H NMR (400MHz, DMSO-d6)

[0424] δppm 1.27(br,s,6H)1.32-1.55(m,22H)1.56-1.63(m,4H)1.67-1.75(m,4H)1.75-1.82(m,12H)1.82-1.88(m,2H)1.93(br,s,5H)2.01-2.11(m,5 H)2.20(br,t,J=7.60Hz,2H)2.58(br,d,J=9.60Hz,4H)2.80-2.91(m,4H)2.98(br,d,J=5.60Hz,2H)3.07-3.19(m,9H)3.40(br,dd,J=6.00,2 .63Hz,12H)3.46-3.57(m,8H)3.60-3.77(m,18H)4.06-4.31(m,6H)4.34-4.42(m,1H)4.47(d,J=4.00Hz,4H)4.52-4.64(m,8H)4.85-4.99(m, 1H)6.83-6.92(m,4H)7.16-7.23(m,5H)7.25-7.37(m,4H)7.43(s,1H)7.62(br,d,J=8.00Hz,6H)7.77-7.90(m,4H)8.64(br,d,J=1.20Hz,1H)

[0425] Using the same synthesis method as in Examples 2-6, Examples 2-7, 2-8, and 2-9 were synthesized:

[0426] Examples 2-7: Compound GAL-02-D

[0427] LCMS(ESI)m / z:1919.8[M-DMT]+;

[0428] 1 H NMR (400MHz, DMSO-d6)

[0429] 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)

[0430] Examples 2 - 8: Compound GAL - 03 - D

[0431] LCMS(ESI) m / z: 1095 [M - DMT] / 2+;

[0432] 1 H NMR (400 MHz, MeOD - d4)

[0433] 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) �.37 - 3.46 (m, 10H) 3.48 - 3.55 (m, 9H) 3.61 (dt, J = 10.4, 3.20 Hz, 6H) 3.74 - 3.82 (m, 14H) <3.85 (d, J = 2.80 Hz, 4H) 3.89 - 3.99 (m, 8H) 4.25 - 4.31 (m, 1H) 4.37 (dd, J = 8.40, 4.40 Hz, 4H) 4.57 - 4.63 (m, 1H) 6.84 - 6.90 (m, 4H) 7.19 - 7.33 (m, 7H) 7.38 (br, d, J = 7.20 Hz, 2H) 7.49 (br, d, J = 8.40 Hz, 2H) 7.94 (d, J = 8.00 Hz, 2H)

[0434] Examples 2-9: Compound GAL-04-D

[0435] LCMS(ESI)m / z:1908[M-DMT] + .

[0436] Example 3: Synthesis of double-stranded siRNA reagent

[0437] 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.

[0438] 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:

[0439] Where X - For O - or S - ;

[0440] Step 1: Preparation of GAL-03 succinate

[0441] 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%).

[0442] Step 2: General process for synthesizing GAL-03-CPG

[0443] 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).

[0444] 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)

[0445] 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).

[0446] Phosphoramide monomer or r monomer was prepared into a 0.06 M acetonitrile solution, and anhydrous dimethylformamide and molecular sieve were added.

[0447] 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.

[0448] 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 (r monomer).

[0449] 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.

[0450] 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.

[0451] 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.

[0452] 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.

[0453] 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.

[0454] 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.

[0455] Step 4: Synthesize the antisense chain of the conjugate

[0456] 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.

[0457] 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.

[0458] Step 5: Synthesis of double-stranded conjugates

[0459] The sense and antisense strands were separately dissolved 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 an annealed product. The 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 were consistent with the theoretical values, indicating that the synthesized product is a double-stranded siRNA. Quantification was performed using ultraviolet spectrophotometry.

[0460] *The monomers containing the r sequence are selected from the corresponding rM in the examples. The ester groups (methyl ester, ethyl ester) contained in their structures react with ammonia to generate amides during the cleavage and deprotection processes of sequence synthesis. Taking r1 as an example, it is shown below:

[0461] Using the above synthesis methods, the representative novel GalNAc-siRNAs of this invention are summarized in Table 1.

[0462] Table 1: Novel GalNAc-siRNA sequences of the sense and antisense strands that inhibit human gene C3 expression, and their conjugates.

[0463] Table 1

[0464] In the table, dA, dC, and dT represent adenine, cytosine, and thymine, respectively; (s) indicates that the 3' terminal nucleotide residue is linked to the delivery ligand via a thiophosphate bond; P indicates that a phosphate molecule is linked to the 5' terminal of the sequence; VPA indicates adenine nucleotide modified with 5'(E)-vinyl phosphate; r1-r12 are the ranges of non-natural nucleotides r as defined above.

[0465] In the table, Gal01, Gal02, and Gal03 are all novel tetravalent GalNAc groups that bind to nucleic acids via covalent bonds, as shown in the following formula:

[0466] Where X - For O - or S - ;

[0467] In the sense / antisense chain sequence, the marker(s) refers to the X in the ligand. - For S - Unmarked refers to the X in the ligand. - For O - .

[0468] Example 4: Evaluation of the endocytosis efficiency of GalNAc compounds in primary cynomolgus monkey hepatocytes (PCMH) using flow cytometry

[0469] Experimental Objective

[0470] This invention uses flow cytometry to evaluate the endocytosis efficiency of tested GalNAc compounds in primary cynomolgus monkey hepatocytes (PCMH). Both the tested GalNAc compound and GalNAc3-Cy5 can bind to the ASGPR receptor on the surface of PCMH and be internalized into the cells. The competing compound GalNAc3-Cy5 is a fluorescently labeled compound; its fluorescence signal can be detected by flow cytometry after internalization. The competition between the tested GalNAc compound and GalNAc3-Cy5 leads to a decrease in the detected fluorescence signal, and the degree of decrease in fluorescence signal reflects the endocytosis efficiency of the tested compound.

[0471] Experimental materials and instruments:

[0472] 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.

[0473] 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).

[0474] Experimental steps and methods:

[0475] Flow cytometry was used to evaluate the endocytosis efficiency of compounds in PCMH.

[0476] On day 0, the revived PCMH suspension was adjusted to a suitable density and then inoculated into 48-well plates.

[0477] 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.

[0478] Data Analysis

[0479] The inhibition rate of the compound at each concentration point on GalNAc3-Cy5 reflects the endocytosis efficiency of the compound at each concentration point. Using GraphPad Prism software, the endocytosis efficiency EC50 value of the test compound was calculated by fitting the curve of the inhibition rate of the compound at each concentration point on GalNAc3-Cy5 using the equation "log(agonist) vs. response–variable slope".

[0480] The inhibition rate of the compound against GalNAc3-Cy5 = (1-(MFI) Sample -MFI Neg ) / (MFI Pos -MFI Neg ))*100%

[0481] MFI Sample It is the median fluorescence intensity of the compound in the APC channel.

[0482] MFI Neg Background fluorescence value

[0483] MFI Pos The maximum uptake fluorescence intensity of GalNAc3-Cy5

[0484] Experimental Results and Conclusions:

[0485] Under conditions of competition with 0.5 μM GalNAc3-Cy5, the endocytosis efficiency of the test compound in PCMH was detected by flow cytometry. The EC50 value of the reference compound L96 was 0.181 μM, and the EC50 value of the test compound GAL-03-D was [missing value]. 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.

[0486] Table 2: Internalization efficiency of compounds in PCMH

[0487] Example 5: Activity test of the conjugate in inhibiting C3 protein expression in human primary hepatocytes (PHH)

[0488] This invention uses adherent human primary hepatocytes to evaluate the in vitro persistent (7-14 days) interference effect of compounds on inhibiting Human C3 protein expression (dose unit: nmol).

[0489] Experimental methods and procedures:

[0490] (1) Take out the PHH frozen in liquid nitrogen, revive it in a water bath at 37°C, and centrifuge it at 100g for 10 minutes.

[0491] (2) After re-suspension counting, count 4-5 x 10. 4 100 μL / well was added to a 96-well collagen plate.

[0492] (3) After standing in the incubator for 4 to 6 hours to adhere to the wall, change the medium and add the specified concentration of siRNA mixture prepared with OptiMEM (11058021, GIBCO).

[0493] (4) After processing for a specified time, the supernatant was collected by swiping the plate and the C3 protein level was detected using the Human C3 ELISA kit (CSB-E08665h, Huamei Biotechnology).

[0494] Data Analysis

[0495] Each assay was performed in duplicate or triplet wells. After sample collection, pooled ELISA was used for detection, and the relative change in protein expression was calculated with the blank control as 100%.

[0496] Tables 3-1 to 3-4 show the inhibition rates of different conjugates on C3 protein expression (dosage unit: nmol). Table 3-1

[0497] Table 3-2

[0498] Table 3-3

[0499] Table 3-4

[0500] The siRNA in *Codonopsis pilosula* is numbered AD-570714 (sequence information is shown in Table 4):

[0501] Table 4

[0502] Example 6: Assay of the compound's activity in inhibiting C3 mRNA in an HDI mouse model

[0503] This experiment used a mouse model of high-pressure tail vein injection of Human C3 to evaluate the in vivo activity of the compound in inhibiting Human C3 mRNA.

[0504] Experimental materials and instruments

[0505] 1. Animals: BALB / c mice, female, 6-8 weeks old.

[0506] 2. Solvent: Phosphate buffer solution, filtered and used for drug preparation on the day of administration.

[0507] 3. Plasmid: pcDNA-CMV-C3, provided by Shanghai WuXi AppTec New Drug Development Co., Ltd., prepared in advance with physiological saline before injection and stored at 4℃ until use.

[0508] Experimental steps and methods:

[0509] 1. In vivo experiments

[0510] 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 -14 or -21, mice were subcutaneously injected with the solvent or test compound at 3 mpk. On day 0, mice were injected with hC3 plasmid solution via high-pressure tail vein. On day 1, 24 hours after the high-pressure tail vein injection of the plasmid, all mice were euthanized by CO2 inhalation, and liver samples were collected for subsequent mRNA detection.

[0511] 2. Sample Analysis

[0512] RNA was extracted from the liver using Trizol.

[0513] After reverse transcription, quantitative PCR was used to detect the expression levels of C3 and NEO genes in mouse liver.

[0514] Data Analysis

[0515] 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 (C3) and the internal reference gene (Neo), and compares the ΔCt values ​​of the compound-treated samples with those of the control group. The formula is:

[0516] ΔCt = Average Ct of the target gene - Average Ct of the reference gene.

[0517] ΔCt = ΔCt of the sample after compound treatment – ​​average ΔCt of the control group.

[0518] Gene expression level = 2 - ΔΔCT

[0519] Experimental results:

[0520] As shown in Figure 1, the conjugate of the present invention exhibits good residual mRNA expression levels in the liver, wherein VB20100 in Figure 1 is identical to the ginseng number AD-570714.

[0521] Example 7: Activity test of the compound in inhibiting C3 protein expression and its downstream functions in a Human C3 transgenic mouse model

[0522] Experimental materials and instruments

[0523] 1. Animals: Human C3 homozygous transgenic mice (NM-HU-2000079, Shanghai Southern Model Animal Center), female, 18 weeks old.

[0524] 2. Solvent: Phosphate buffer solution, filtered and used for drug preparation on the day of administration.

[0525] Experimental steps and methods:

[0526] 1. In vivo experiments

[0527] Transgenic mice were housed in an environment that met IACUC requirements until 18 weeks of age. They were then given a single subcutaneous administration of 10 mpk siRNA solution and observed until 24 weeks of age (6 weeks after administration) before being euthanized and sampled.

[0528] 2. Sample Collection and Analysis

[0529] (1) Baseline blood was collected before administration, and blood was collected once a week in the first and second weeks after administration, and then once every two weeks thereafter. Blood was collected from the heart after euthanasia.

[0530] (2) AST / ALT levels were detected in blood samples and hC3 protein levels in serum were detected by ELISA.

[0531] Data Analysis

[0532] Changes in serum hC3 protein levels relative to baseline (100%) in each group.

[0533] Experimental results:

[0534] See Figure 2-4 for details.

[0535] As shown in Figures 2 and 3, there were no significant changes in liver enzymes in the transgenic mice compared to the solvent group, indicating that no hepatotoxicity was observed at a drug efficacy dose of 10 mpk.

[0536] As shown in Figure 4, a single dose of transgenic mice resulted in a sustained and significant reduction in serum hC3 compared to baseline. The VB20115 / VB20118 administration group showed similar or better reduction efficiency compared to the same dose of Yangshen.

[0537] In summary, to develop superior GalNAc-siRNA conjugates targeting Complement Component 3, this invention optimized both the siRNA sequence and GalNAc. In experiments using human primary hepatocytes, to compare the effect of the siRNA sequence on activity, this invention used a series of compounds in which GalNAc remained unchanged, but non-natural nucleotide monomers were introduced into the siRNA sequence. The results showed that the compounds exhibited superior activity compared to *Gynostemma pentaphyllum*, and the introduction of non-natural nucleotide monomers simultaneously reduced the off-target risk.

[0538] To further optimize activity and enhance novelty, this invention provides a novel, symmetrical tetravalent GalNAc, wherein the delivery efficiency of GalNAc can be maintained while facilitating easier CMC-scaled synthesis and reducing costs. Conjugating the optimized siRNA sequence with the novel tetravalent GalNAc ultimately yields a GalNAc-siRNA conjugate drug with superior selectivity and activity.

Claims

1. A double-stranded siRNA, characterized in that, It includes a sense strand and an antisense strand forming a reverse complementary double-stranded region, wherein the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the sequence shown in SEQ ID NO:1, and the sense strand and antisense strand are each independently 17 to 25 nucleotides in length; one or more of the nucleotide residues in the nucleotide sequence of the sense strand and / or the antisense strand is replaced with the following formula r: wherein: X 1 , Y 1 and Z 1 are independently CH or N, and at least one of X 1 , Y 1 and Z 1 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 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 1 is optionally substituted C1-C 10 alkoxy, the substituents being selected from one or more groups of C2-C6alkenyl, C1-C6alkoxy, fluorine, chlorine, bromine or iodine; Preferably, 3. The double-stranded siRNA of claim 1 or 2, wherein, It meets 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; (2) The antisense strand has a 2-nucleotide overhang at the 3' end; Preferably, the two nucleotides are GG; (3) The lengths of the sense strand and the antisense strand are each 19 to 25 nucleotides, respectively; Preferably, each nucleotide is independently composed of 19 to 23 nucleotides; More preferably, the sense strand is 21 nucleotides and the antisense strand is 23 nucleotides; (4) The length of the reverse complementary double-stranded region is 19 to 23 bp, preferably 21 to 23 bp; (5) 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; The modifying groups in the modified nucleotides are selected from methoxy modification, fluorination modification, thiophosphate linkage, replacing the nucleotide with glycerol nucleic acid, (E)-vinyl phosphate and 2'-deoxynucleotide; Preferably, the methoxy modification is replaced by a 2'-O-methyl modification; the fluorination modification is replaced by a 2'-fluoro modification; (6) The antisense strand comprises 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; (7) The positive strand contains two phosphate thioester bonds between the three terminal nucleotides at the 5' end; (8) Formula r is linked to adjacent nucleotide residues via phosphate ester or thiophosphate ester bonds. (9) Substitution of formula r in the antisense strand by one or more nucleotide residues selected from positions 2, 7-9, 12-13, 15, 17, and 22, in a manner from the 5' end to the 3' end; and, (10) The formula r substitution is performed on one or more nucleotide residues selected from positions 1-8 and 12-21 in the positive strand in a manner from the 5' end to the 3' end.

4. The double-stranded siRNA of claim 3, wherein, It meets one or more of the following conditions: (1) In the manner from the 5' end to the 3' end, one or more nucleotides at positions 2, 6, 8, 9, 14 and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl nucleotides. (2) Following the order 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; and, (3) When r occupies the aforementioned position, the position is not subject to the corresponding fluorination or 2'-O-methyl modification.

5. The double-stranded siRNA of claim 1, wherein It meets one or more of the following conditions: (1) the antisense strand comprises a nucleotide sequence selected from the group consisting of: The structure of r1-r12 is as described in claim 2; (2) the sense strand comprises a nucleotide sequence selected from the group consisting of: The structure of r1-r12 is as described in claim 2.

6. The double-stranded siRNA of claim 1, wherein The double-stranded siRNA comprises the following sense and antisense strand pairs of any one of the following sequences: Preferably, the 5' end of the antisense strand in the double strand is a phosphate ester nucleotide or a vinyl phosphate ester nucleotide.

7. A conjugate comprising double-stranded siRNA as described in any one of claims 1-6.

8. The conjugate of claim 7, wherein, The conjugate is shown in formula I: in, X - is O - or S - ; M z+ are pharmaceutically acceptable positive ions; The negative ions are selected from the group consisting of The total valence is equal to the total valence of the positive ion; The RNA is a double-stranded siRNA as described in any one of claims 1-6; 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 (The left end of the fragment is connected to the carbonyl group, and the right end is connected to N); k is 2; When A is a bond, A is located between L 2 meta; 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.

9. The conjugate of claim 8, wherein, It meets 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; (12) X - is O - ; (13)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, ...

10. The conjugate of claim 8, wherein, It meets 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 11. The conjugate of claim 8, wherein, It meets one or more of the following conditions: (1) For Preferably, said For (2) For 12. The conjugate of claim 8, wherein, The conjugate is any of the following structures: Preferably: Among them, X - For O - or S - The RNA is a double-stranded siRNA as described in any one of claims 1-6; Preferably, the liver-targeting portion is connected to the 3' end of the positive chain.

13. A conjugate, characterized in that, which is represented by Formula II: wherein A, X - , M z+ , L 1 , L 2 , L 3 and L 4 are each as defined in any one of claims 8-12; The double-stranded siRNA' comprises a sense strand and an antisense strand forming a reverse complementary double-stranded region, wherein the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in the sequence shown in SEQ ID NO:1, and the length of the sense strand and the antisense strand is independently 17 to 25 nucleotides.

14. The conjugate of claim 13, wherein, It meets 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; (2) The antisense strand has a 2-nucleotide overhang at the 3' end; Preferably, the two nucleotides are GG; (3) The lengths of the sense strand and the antisense strand are each 19 to 25 nucleotides, respectively; Preferably, each nucleotide is independently composed of 19 to 23 nucleotides; More preferably, the sense strand is 21 nucleotides and the antisense strand is 23 nucleotides; (4) The length of the reverse complementary double-stranded region is 19 to 23 bp, preferably 21 to 23 bp; (5) 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; The modifying groups in the modified nucleotides are selected from methoxy modification, fluorination modification, thiophosphate linkage, replacing the nucleotide with glycerol nucleic acid, (E)-vinyl phosphate and 2'-deoxynucleotide; Preferably, the methoxy modification is replaced by a 2'-O-methyl modification; the fluorination modification is replaced by a 2'-fluoro modification; (6) The antisense strand comprises 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; (7) The positive chain contains two phosphate thioester bonds between the three terminal nucleotides at the 5' end.

15. The conjugate of claim 14, wherein, It meets one or more of the following conditions: (1) Following the order from the 5' end to the 3' end, one or more nucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl nucleotides; and, (2) In the manner 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.

16. The conjugate of claim 13, wherein It meets one or more of the following conditions: (1) the antisense strand comprises a nucleotide sequence selected from the group consisting of: (2) the sense strand comprises a nucleotide sequence selected from the group consisting of:

17. The conjugate of claim 13, wherein The double-stranded siRNA comprises the following sense and antisense strand pairs of any one of the following sequences:

18. The conjugate of any one of claims 7-17, which is any one of the following conjugates: The structure of r1-r12 in the table is as described in claim 2; In the table, Gal01, Gal02 and Gal03 are covalently bound to the nucleic acid, and the linkage is as shown in the following formula: wherein X - is O - or S - .

19. A pharmaceutical composition comprising a double-stranded siRNA as described in any one of claims 1-6, a conjugate as described in any one of claims 7-17, and a pharmaceutically acceptable carrier.

20. Use of a double-stranded siRNA as described in any one of claims 1-6, a conjugate as described in any one of claims 7-17, or a pharmaceutical composition as described in claim 19 in the preparation of a medicament for treating and / or preventing a disease in a subject associated with C3 gene expression; The diseases associated with C3 gene expression include one or more of the following: kidney-related C3 glomerulonephropathy (C3G); immune complex-mediated glomerulonephritis (IC-mediated GN); post-infectious glomerulonephritis (PIGN); systemic lupus erythematosus; ischemic-perfusion injury and IgA nephropathy; paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); and other organ-related C3-related diseases.

21. A cell, comprising: It comprises double-stranded siRNA as described in any one of claims 1-6, and conjugates as described in any one of claims 7-17; the cells are non-animal or non-plant varieties.

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