A liver-targeting compound and its use in drug conjugates

Abstract

A compound, or a racemic mixture, stereoisomer, isotope label, or salt thereof, and its uses are provided. An oligonucleotide conjugate and its uses are also provided. The compound structure contains a novel ASGPR ligand and a phosphoramidite structure, enabling it to participate in solid-phase nucleic acid synthesis in any type, quantity, and insertion site, thereby forming an oligonucleotide conjugate containing the ASGPR ligand. This conjugate exhibits excellent in vivo liver-targeting effects, enabling long-term in vivo knockout of target genes.

Description

A liver-targeted compound and its use in drug conjugates Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a liver-targeting compound and its use in delivering active drugs (such as oligonucleotides) by forming a conjugate with the compound. The present invention also relates to a conjugate formed from the compound and its use. Background Art

[0002] The asialoglycoprotein receptor (ASGPR), also known as the hepatic lectin, is an abundant endocytic receptor primarily found on the sinusoidal surface of liver parenchymal cells. It has specificity for sugar recognition and endocytosis. ASGPR is a type I transmembrane receptor located on the plasma membrane and is divided into four functional domains: the cytoplasmic domain, the transmembrane domain, the pedicle domain, and the carbohydrate recognition domain (CRD). ASGPR is abundant on the plasma membrane of hepatocytes and is rapidly internalized, a property that gives it significant potential for clearing circulating glycoproteins, thereby regulating the homeostasis of asialoseromucoid (ASOR) (Progress in Biochemistry and Biophysics 2015, 42, 501-510). Glycoproteins with non-reducing galactose (Gal) or N-acetylgalactosamine (GalNAc) residues at the end or other similar structures can be recognized by ASGPR and then internalized into the cell (Iobst ST et al, J Biol Chem, 1996, 271(12):6686-6693).

[0003] Liver-targeted oligonucleotides (siRNAs, antisense oligonucleotides, and other drugs) mediated by ASGPR proteins are a hot topic in the field of innovative nucleic acid drug research. In 2019, givosiran, the first GalNAc-siRNA conjugate developed by Alnylam, was approved by the US FDA. In 2014, IONIS Pharmaceuticals (USA) achieved liver-targeted drug delivery in animals by covalently linking triantennary GalNAc to antisense nucleic acids (Prakash, T et al., Nucleic Acids Res. 42, 8796-807).

[0004] The targeting and actual efficacy of ASGPR-based oligonucleotide drug conjugates are closely related to their delivery structure. The design, modification, and application of ASGPR ligands are of great significance for the actual in vivo application of oligonucleotide drugs. Through in-depth interpretation and research on the characteristics of ASGPR ligands, it is possible to enhance the efficacy, pharmacokinetic (PK), metabolic stability, and long-term efficacy of liver-targeted drug conjugates and drug combinations. Summary of the Invention

[0005] To overcome the deficiencies in the prior art, one object of the present invention is to provide a phosphoramidite derivative comprising a novel ASGPR ligand and its use, which can be used to form oligonucleotide conjugates or other types of drug conjugates with good liver targeting.

[0006] Another object of the present invention is to provide an oligonucleotide conjugate and use thereof.

[0007] The first aspect of the present invention provides a compound as shown in formula (I), or its racemate, stereoisomer, isotope-labeled substance or salt thereof,

[0008] Wherein, Y1 represents a hydroxyl protecting group PG1;

[0009] Y2 represents the structure shown in formula (I-1):

[0010] In formula (I-1), R 1 represents a straight or branched alkylene group of -C1 to C8, -CN, R 2 、R 3 Each independently represents a C1 to C8 straight or branched chain alkyl group;

[0011] A represents a structure represented by formula (I-2), formula (I-2'), formula (I-3), formula (I-4) or formula (I-5):

[0012] In formula (I-2), formula (I-2') and formula (I-3), R 4 represents hydrogen or a C1-C8 straight-chain or branched alkyl group, L A1 , L A2 Each independently represents absence or represents a C1-C8 straight or branched alkylene group, ring A0 represents a 4-10 membered (e.g., 4, 5, 6, 7, 8, 9 or 10 membered) saturated heterocyclic ring containing at least one N atom, R A5represents -C(O)NH-, -OC(O)NH-, -NHC(O)-, -NHC(O)O-, -O-, -S-, -NH-, -C(O)-, or -S(O)-, and s represents an integer of 0 to 4 (e.g., 0, 1, 2, 3, or 4);

[0013] In formula (I-4) and formula (I-5), R A1 represents the absence or represents a C1-C8 straight or branched alkylene group, one or more carbon atoms of which are optionally replaced by one or more -O- or -C(O)-, R A2 Indicates absence or p represents an integer of 0 to 5 (for example, 0, 1, 2, 3, 4 or 5), R A3 express R A4 represents -C(O)NH-, -NHC(O)-, -O-, -S-, -NH-, -C(O)- or -S(O)-, L A3 represents the absence or a C1-C8 straight-chain or branched alkylene group, Base represents a natural base A, T, C, G, or U, wherein the amino group is protected by a protecting group; 1 represents the connection site with B, 2 represents the connection site with Y1, and 3 represents the connection site with Y2;

[0014] B represents absence or represents a C1-C30 straight or branched alkylene group, wherein one or more carbon atoms are optionally replaced by one or more of the following groups: -C(O)NH-, -O-, -S-, -NH-, -C(O)-, -S(O)-, -S(O2)-, a 4-10 membered (e.g., 4, 5, 6, 7, 8, 9 or 10 membered) saturated or unsaturated carbocyclic ring, a 4-10 membered (e.g., 4, 5, 6, 7, 8, 9 or 10 membered) saturated or unsaturated heterocyclic ring containing at least one heteroatom selected from N, O, S;

[0015] Preferably, B is absent or represents a C25-C30, C1-C25, C1-C20, C1-C15, C1-C10 or C1-C5 straight or branched alkylene group, wherein one or more carbon atoms are optionally replaced by one or more of the following groups: -C(O)NH-, -O-, -S-, -NH-, -C(O)-, -S(O)-, -S(O2)-, a 4-10 membered (e.g., 4, 5, 6, 7, 8, 9 or 10 membered) saturated or unsaturated carbocyclic ring, a 4-10 membered (e.g., 4, 5, 6, 7, 8, 9 or 10 membered) saturated or unsaturated heterocyclic ring containing at least one heteroatom selected from N, O or S;

[0016] G represents the structure shown in formula (I-6):

[0017] In formula (I-6), R G1 represents hydrogen, hydroxyl, C1-C20 straight chain or branched alkyl, C2-C20 straight chain or branched alkenyl, -O-C1-C20 straight chain or branched alkyl, -S-C1-C20 straight chain or branched alkyl, -NH-C1-C20 straight chain or branched alkyl, -N-(C1-C20 straight chain or branched alkyl)2, -O-C0-C8 straight chain or branched alkylene, C6-C20 aryl, -S-C0 -C0-C8 straight chain or branched alkylene-C6-C20 aryl, -O-C0-C8 straight chain or branched alkylene-C3-C10 cycloalkyl, -S-C0-C8 straight chain or branched alkylene-C3-C10 cycloalkyl, -O-C0-C8 straight chain or branched alkylene-5-12 membered heterocyclic group, -S-C0-C8 straight chain or branched alkylene-5-12 membered heterocyclic group, galactosyl or galactosylamide group, or q represents an integer of 0 to 16 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16), wherein the aryl, cycloalkyl or heterocyclic group is optionally substituted with one or more C1 to C8 linear or branched alkyl groups, and the heterocyclic group contains at least one heteroatom selected from N, O and S;

[0018] Preferably, R G1 represents hydrogen, hydroxyl, C1-C16 straight chain or branched alkyl, C2-C16 straight chain or branched alkenyl, -O-C1-C16 straight chain or branched alkyl, -S-C1-C16 straight chain or branched alkyl, -NH-C1-C16 straight chain or branched alkyl, -N-(C1-C16 straight chain or branched alkyl)2, -O-C0-C6 straight chain or branched alkylene-C6-C16 aryl, -S-C -O-C0-C6 straight chain or branched chain alkylene-C6-C16 aryl, -O-C0-C6 straight chain or branched chain alkylene-C3-C8 cycloalkyl, -S-C0-C6 straight chain or branched chain alkylene-C3-C8 cycloalkyl, -O-C0-C6 straight chain or branched chain alkylene-5-10 membered heterocyclic group, -S-C0-C8 straight chain or branched chain alkylene-5-10 membered heterocyclic group, galactosyl or galactosylamide group, or q represents an integer from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), wherein the aryl, cycloalkyl or heterocyclic group is optionally substituted with one or more C1-C6 linear or branched alkyl groups, and the heterocyclic group contains at least one heteroatom selected from N, O and S;

[0019] More preferably, R G1represents hydrogen, hydroxyl, C1-C12 straight chain or branched alkyl, C2-C12 straight chain or branched alkenyl, -O-C1-C12 straight chain or branched alkyl, -S-C1-C12 straight chain or branched alkyl, -NH-C1-C12 straight chain or branched alkyl, -N-(C1-C12 straight chain or branched alkyl)2, -O-C0-C4 straight chain or branched alkylene-C6-C12 aryl, -S- C0~C4 straight chain or branched alkylene-C6~C12 aryl, -O-C0~C4 straight chain or branched alkylene-C3~C6 cycloalkyl, -S-C0~C4 straight chain or branched alkylene-C3~C6 cycloalkyl, -O-C0~C4 straight chain or branched alkylene-5~8 membered heterocyclic group, -S-C0~C4 straight chain or branched alkylene-5~8 membered heterocyclic group, galactosyl or galactosylamide group, or q represents an integer of 0 to 6 (for example, 0, 1, 2, 3, 4, 5 or 6), wherein the aryl, cycloalkyl or heterocyclic group is optionally substituted with one or more C1 to C4 linear or branched alkyl groups, and the heterocyclic group contains at least one heteroatom selected from N, O and S;

[0020] More preferably, R G1 represents hydrogen, hydroxyl, C1~C8 straight chain or branched alkyl, C2~C8 straight chain or branched alkenyl, -O-C1~C8 straight chain or branched alkyl, -S-C1~C8 straight chain or branched alkyl, -NH-C1~C8 straight chain or branched alkyl, -N-(C1~C8 straight chain or branched alkyl)2, -O-C0~C4 straight chain or branched alkylene, -C6~C10 aryl, -S-C0~ C4 straight chain or branched alkylene-C6~C10 aryl, -O-C0~C4 straight chain or branched alkylene-C3~C5 cycloalkyl, -S-C0~C4 straight chain or branched alkylene-C3~C5 cycloalkyl, -O-C0~C4 straight chain or branched alkylene-5~6 membered heterocyclic group, -S-C0~C4 straight chain or branched alkylene-5~6 membered heterocyclic group, galactosyl or galactosylamide group, or q represents an integer of 0 to 4 (for example, 0, 1, 2, 3 or 4), wherein the aryl, cycloalkyl or heterocyclic group is optionally substituted with one or more C1 to C4 linear or branched alkyl groups, and the heterocyclic group contains at least one heteroatom selected from O;

[0021] R G2 Represents -C(O)NR 5 R 6, -C(O)-C1~C8 straight or branched alkyl, -C(O)-C3~C10 cycloalkyl, -S(O2)-C1~C8 straight or branched alkyl, -S(O2)-C3~C10 cycloalkyl, -C(O)-C0~C8 straight or branched alkylene-C6~C20 aryl, -S(O2)-C0~C8 straight or branched alkylene-C6~C20 aryl, -C(O)-C1~C8 straight or branched haloalkyl or -S(O2)-C1~C8 straight or branched haloalkyl, wherein the aryl is optionally substituted with one or more C1~C8 straight or branched alkyl or C1~C8 straight or branched haloalkyl; R 5 、R 6 Each is independently selected from hydrogen or a C1-C8 straight or branched chain alkyl group;

[0022] Preferably, R G2 Represents -C(O)NR 5 R 6 , -C(O)-C1~C6 straight chain or branched alkyl, -C(O)-C3~C8 cycloalkyl, -S(O2)-C1~C6 straight chain or branched alkyl, -S(O2)-C3~C8 cycloalkyl, -C(O)-C0~C6 straight chain or branched alkylene-C6~C16 aryl, -S(O2)-C0~C6 straight chain or branched alkylene-C6~C16 aryl, -C(O)-C1~C6 straight chain or branched haloalkyl or -S(O2)-C1~C6 straight chain or branched haloalkyl, wherein the aryl is optionally substituted with one or more C1~C6 straight chain or branched alkyl or C1~C6 straight chain or branched haloalkyl; R 5 、R 6 Each is independently selected from hydrogen or a C1-C6 straight or branched chain alkyl group;

[0023] More preferably, R G2 Represents -C(O)NR 5 R 6 , -C(O)-C1~C4 straight chain or branched alkyl, -C(O)-C3~C6 cycloalkyl, -S(O2)-C1~C4 straight chain or branched alkyl, -S(O2)-C3~C6 cycloalkyl, -C(O)-C0~C4 straight chain or branched alkylene-C6~C12 aryl, -S(O2)-C0~C4 straight chain or branched alkylene-C6~C12 aryl, -C(O)-C1~C4 straight chain or branched haloalkyl or -S(O2)-C1~C4 straight chain or branched haloalkyl, wherein the aryl is optionally substituted with one or more C1~C6 straight chain or branched alkyl or C1~C6 straight chain or branched haloalkyl; R 5 、R 6Each is independently selected from hydrogen or a C1-C6 straight or branched chain alkyl group;

[0024] R G3 and R G4 Each independently represents hydrogen or a hydroxyl protecting group PG2;

[0025] Z represents -C(O)-, -O-, -S-, -NH-, -C(O)NH-, -OC(O)NH-, -OP(O)(OH)- or -R 8 -(CH2) n -R 7 -, where R 8 represents a triazole subunit, for example R 7 represents -C(O)-, -O-, -S- or -NH-, and n represents an integer of 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0026] In some preferred embodiments, in the formula (I-6), the R G1 represents hydrogen, hydroxyl, C1-C6 straight-chain or branched alkyl, C2-C6 straight-chain or branched alkenyl, -O-C1-C16 straight-chain or branched alkyl (for example, -O-C1-C6 straight-chain or branched alkyl), -S-C1-C6 straight-chain or branched alkyl, -NH-C1-C6 straight-chain or branched alkyl, -N-(C1-C6 straight-chain or branched alkyl)2, -O-C0-C4 straight-chain or branched alkylene-phenyl, -O-C0-C4 straight-chain or branched alkylene-C3-C6 cycloalkyl or -O-C0-C4 straight-chain or branched alkylene-5- to 6-membered heterocyclic group, wherein the phenyl, cycloalkyl or heterocyclic group is optionally substituted with one or more C1-C4 straight-chain or branched alkyl groups, and the heterocyclic group contains at least one heteroatom selected from O.

[0027] In some more preferred embodiments, the R G1 Represents one of the following structures:

[0028] OH.

[0029] In some preferred embodiments, the R G2It represents -C(O)-C1~C6 straight chain or branched alkyl, -C(O)-C3~C6 cycloalkyl, -S(O2)-C1~C6 straight chain or branched alkyl, -C(O)-C0~C4 straight chain or branched alkylene-phenyl, -S(O2)-C0~C4 straight chain or branched alkylene-phenyl, -C(O)-halogenated C1~C4 straight chain or branched alkyl or -S(O2)-halogenated C1~C4 straight chain or branched alkyl, wherein the aryl group is optionally substituted by one or more C1~C4 straight chain or branched alkyl or C1~C4 straight chain or branched halogenated alkyl.

[0030] In some more preferred embodiments, the R G2 Represents one of the following structures:

[0031] In some preferred embodiments, Z represents one of the following structures:

[0032] In some preferred embodiments, when A represents formula (I-2), formula (I-2') or formula (I-3), it may represent one of the following structures:

[0033] When A represents formula (I-4) or formula (I-5), Base represents a natural base A, T, C, G or U, wherein the amino group is protected by a protecting group, which can be an amino protecting group commonly used in the art, including but not limited to one or more of acetyl (Ac), benzoyl (Bz) or isobutyryl (iBu).

[0034] In some more preferred embodiments, R A3 Represents one of the following structures:

[0035] 1 represents the binding site with B, 2 represents the binding site with Y1, and 3 represents the binding site with Y2.

[0036] In some preferred embodiments, the B represents a structure represented by formula (I-7):

[0037] In formula (I-7), ring B0 is absent or represents a 4- to 10-membered (e.g., 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered) saturated carbocyclic or heterocyclic ring containing W, W represents CH or N, and R 9 Indicates absence or one or more of -C(O)-, -O-, -S-, and -NH-, L B1represents the absence or represents a C1-C20 straight or branched alkylene group, one or more carbon atoms of which are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, -S-, -SS-, R 10 represents absence or represents -C(O)-, -O-, -S- or -NH-, and ring B0, R 9 , L B1 、R 10 Different indicates absence; 1 indicates the connection site with A, and 2 indicates the connection site with G;

[0038] In some more preferred embodiments, Ring B0 represents one of the following ring structures:

[0039] In some further preferred embodiments, B represents a structure represented by formula (I-8):

[0040] In formula (I-8), ring B0 represents a 4- to 6-membered (for example, a 4-, 5-, or 6-membered) saturated heterocyclic ring containing a nitrogen atom, and L B1 represents the absence or represents a C1-C20 straight or branched alkylene group, one or more carbon atoms of which are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, and -SS-, and R 10 indicates absence or indicates -C(O)-, -O- or -NH-; 1 indicates the linking site with A, and 2 indicates the linking site with G.

[0041] In some most preferred embodiments, the B is absent or represents one of the following structures:

[0042] 1 indicates the binding site with A, and 2 indicates the binding site with G.

[0043] In some preferred embodiments, the PG1 represents one or more of dimethoxytrityl (DMTr), p-methoxytrityl (MMTr), and trityl (TRT), for example, DMTr.

[0044] In some preferred embodiments, Y2 represents the following structure:

[0045] In some preferred embodiments, the PG2 represents one or more of methoxymethyl (MOM), benzyl (Bn), p-methoxybenzyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), acetyl (Ac), and benzoyl (Bz).

[0046] In some preferred embodiments, the compounds provided by the present invention may have structures represented by formula (I-10) and formula (I-11):

[0047] In formula (I-10) and formula (I-11), R G1 Represents the following structure:

[0048] In other preferred embodiments, the compounds provided by the present invention may also have structures represented by formula (I-12) and formula (I-13):

[0049] In formula (I-12) and formula (I-13), R G2 Represents the following structure:

[0050] The compounds provided by the present invention, or their racemates, stereoisomers, isotope-labeled substances or salts thereof include but are not limited to the following:

[0051] The second aspect of the present invention provides a compound as shown in formula (III), or its racemate, stereoisomer, isotope-labeled substance or salt thereof,

[0052] Wherein, Y1, Y2, A, and G are each independently defined as in any one of the above technical solutions, for example, each independently defined as in the first aspect of the present invention;

[0053] B' represents a C2-C60 straight or branched alkylene group, wherein one or more carbon atoms are optionally replaced by one or more of the following groups: -C(O)NH-, -OC(O)NH-, -NHC(O)-, -NHC(O)O-, -O-, -S-, -NH-, -C(O)-, -S(O)-, -S(O2)-, a 4-10 membered (e.g., 4, 5, 6, 7, 8, 9 or 10 membered) saturated or unsaturated carbocyclic ring, a 4-10 membered (e.g., 4, 5, 6, 7, 8, 9 or 10 membered) saturated or unsaturated heterocyclic ring containing at least one heteroatom selected from N, O, and S;

[0054] m represents 2 to 5 (for example, 2, 3, 4, or 5).

[0055] In some preferred embodiments, B' represents a structure represented by formula (III-1) or formula (III-2):

[0056] In formula (III-1) or formula (III-2), Lb1 represents absence or represents a C1 to C20 straight chain or branched alkylene group, one or more carbon atoms of which are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, and -S-; R11 represents absence or represents -C(O)-, -O-, -S-, or -NH-; Lb2 are the same or different and each independently represents a C1 to C20 straight chain or branched alkylene group, one or more carbon atoms of which are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, and -S-; R12 are the same or different and each independently represents absence or represents -C(O)-, -O-, -S-, or -NH-; 1 represents the connection site with A, and 2 represents the connection site with G.

[0057] In some more preferred embodiments, B' represents a structure represented by formula (III-3), formula (III-4), formula (III-5) or formula (III-6):

[0058] In formula (III-3), formula (III-4), formula (III-5) or formula (III-6), Lb1 is absent or represents a C1 to C12 straight chain or branched alkylene group; R11 is absent or represents -C(O)-, -O-, -S- or -NH-; Lb3 are the same or different and each independently represents a C1 to C10 straight chain or branched alkylene group; Lb4 are the same or different and each independently represents a C1 to C10 straight chain or branched alkylene group; Lb5 are the same or different and each independently represents a C1 to C6 straight chain or branched alkylene group; Lb6 are the same or different and each independently represents a C1 to C6 straight chain or branched alkylene group; Lb7 are the same or different and each independently represents a straight chain or branched alkylene group of C1 to C6; R12 are the same or different and each independently represents a straight chain or branched alkylene group of C1 to C6; R12 are the same or different and each independently represents -C(O)-, -O-, -S- or -NH-; m1 are the same or different and each independently represents 1 to 8 (for example, 1, 2, 3, 4, 5, 6, 7 or 8); m2 are the same or different and each independently represents 1 to 5 (for example, 1, 2, 3, 4 or 5); 1 represents the connection site with A, and 2 represents the connection site with G.

[0059] In some further preferred embodiments, B' represents one of the following structures:

[0060] In some preferred embodiments, the compound represented by formula (III) comprises:

[0061] The third aspect of the present invention provides the use of the compound described in any one of the above technical solutions, or its racemate, stereoisomer, isotope-labeled product or salt thereof in the preparation of liver-targeted drugs (such as liver-targeted oligonucleotide conjugates).

[0062] In some preferred embodiments, the use described in the present invention is the use of the compound described in any one of the above technical solutions, or its racemate, stereoisomer, isotope label or salt as a preparation raw material (i.e., synthetic monomer) in the preparation of liver-targeted drugs (such as liver-targeted oligonucleotide conjugates).

[0063] The fourth aspect of the present invention provides an oligonucleotide conjugate, wherein the oligonucleotide conjugate includes one or more compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof.

[0064] In some preferred embodiments, at least one oligonucleotide chain in the oligonucleotide conjugate comprises one or more compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof.

[0065] In some more preferred embodiments, one or more of the compounds, or their racemates, stereoisomers, isotope labels, or salts thereof can be used as synthetic monomers when preparing at least one oligonucleotide chain in the oligonucleotide conjugate by a preparation method commonly used in the art, such as nucleic acid solid-phase chemical synthesis, and inserted into the oligonucleotide chain in any type, any number, and any position, and corresponding deprotection treatments can be performed as needed.

[0066] In some preferred embodiments, the number of the compound, or its racemate, stereoisomer, isotope-labeled substance, or salt thereof is 1 to 12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some more preferred embodiments, the number may be 1 to 6, and further may be 1 to 4.

[0067] In some preferred embodiments, at least one oligonucleotide chain in the oligonucleotide conjugate comprises one or more compounds described in any of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof at the 3'-end, 5'-end or mid-chain position.

[0068] In some preferred embodiments, the 5'-end of one oligonucleotide chain in the oligonucleotide conjugate includes 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts.

[0069] In some preferred embodiments, the 3'-end and the 5'-end of one oligonucleotide chain in the oligonucleotide conjugate simultaneously include 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof.

[0070] In some preferred embodiments, the 3'-end and 5'-end of one oligonucleotide chain in the oligonucleotide conjugate simultaneously include 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof, and the 3'-end of the other oligonucleotide chain in the oligonucleotide conjugate includes 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof.

[0071] The fifth aspect of the present invention provides an oligonucleotide conjugate, wherein one or more of the synthetic monomers for preparing the oligonucleotide conjugate is a compound described in any one of the above technical solutions, or a racemate, stereoisomer, isotope-labeled substance or a salt thereof.

[0072] In some preferred embodiments, when preparing the oligonucleotide conjugate, one or more compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof are used as synthetic monomers in at least one oligonucleotide chain.

[0073] In some more preferred embodiments, one or more of the compounds, or their racemates, stereoisomers, isotope labels, or salts thereof can be used as synthetic monomers when preparing at least one oligonucleotide chain in the oligonucleotide conjugate by a preparation method commonly used in the art, such as nucleic acid solid-phase chemical synthesis, and inserted into the oligonucleotide chain in any type, any number, and any position, and corresponding deprotection treatments can be performed as needed.

[0074] In some preferred embodiments, the number of the compound, or its racemate, stereoisomer, isotope-labeled substance, or salt thereof is 1 to 12, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some more preferred embodiments, the number may be 1 to 6, and further may be 1 to 4.

[0075] In some preferred embodiments, when preparing the oligonucleotide conjugate, one or more compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels or salts thereof are used as synthetic monomers at the 3'-end, 5'-end or mid-chain position of at least one oligonucleotide chain.

[0076] In some preferred embodiments, when preparing the oligonucleotide conjugate, 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels, or salts thereof are used as synthetic monomers at the end of the 5'-end of one of the oligonucleotide chains.

[0077] In some preferred embodiments, when preparing the oligonucleotide conjugate, 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels, or salts thereof are used simultaneously as synthetic monomers at the 3'-end and 5'-end of one of the oligonucleotide chains.

[0078] In some preferred embodiments, when preparing the oligonucleotide conjugate, 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels, or salts thereof are used simultaneously as synthetic monomers at the 3'-end and 5'-end of one of the oligonucleotide chains, and 1 to 6 (e.g., 1 to 3) compounds described in any one of the above technical solutions, or their racemates, stereoisomers, isotope labels, or salts thereof are used as synthetic monomers at the 3'-end of the other oligonucleotide chain.

[0079] In some preferred embodiments, at least one oligonucleotide chain in the oligonucleotide conjugate may have one of the following structures:

[0080] wherein X represents O or S, k1 and k2 each independently represent an integer from 0 to 6 (e.g., each independently represents 0, 1, 2, 3, 4, 5, or 6), k1 and k2 do not simultaneously represent 0. k3 represents an integer from 1 to 6 (e.g., represents 1, 2, 3, 4, 5, or 6), ONS represents an oligonucleotide chain, A, B, B', G, and m are each independently defined as in any one of the above technical solutions, and wherein R G3 、R G4 For H.

[0081] In some more preferred embodiments, at least one oligonucleotide chain in the oligonucleotide conjugate may have one of the following structures:

[0082] Among them, R G1 , Base are each independently defined as any one of the above technical solutions.

[0083] The sixth aspect of the present invention provides an oligonucleotide conjugate comprising at least one G structure as shown in formula (I-6):

[0084] In formula (I-6), R G1 、R G2 、R G3 、R G4 and Z are each independently defined as in any one of the above technical solutions, for example, as defined in the first aspect of the present invention.

[0085] In some preferred embodiments, the oligonucleotide conjugate comprises 1, 2, 3 or 4 G structures as shown in formula (I-6).

[0086] In the oligonucleotide conjugates provided by the present invention, the oligonucleotide can be any type commonly used in the art, including but not limited to one or more of antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), aptamer, immunostimulatory substance, G-quadrupole, alternative spliceosome, single-stranded RNA, ribozyme, and decoy.

[0087] The oligonucleotide conjugates provided by the present invention can be prepared by at least two preparation methods:

[0088] 1) First, prepare the compound described in the above technical solution, or its racemate, stereoisomer, isotope-labeled substance or salt thereof. Based on the phosphoramidite structure therein, use one or more of the compounds, or its racemate, stereoisomer, isotope-labeled substance or salt thereof as synthetic monomers when preparing an oligonucleotide chain by methods commonly used in the art, such as nucleic acid solid-phase chemical synthesis, and insert them into the oligonucleotide chain in any type, any number, and any position.

[0089] 2) First, prepare an intermediate containing a phosphoramidite structure and an active linker (such as -NH2, -C≡CH, -SH, -N3, etc.), including but not limited to the following:

[0090] Based on the phosphoramidite structure in the active intermediate, one or more of the intermediates are used as synthetic monomers when preparing an oligonucleotide chain through methods commonly used in the art, such as nucleic acid solid-phase chemical synthesis, and are inserted into the oligonucleotide chain in any type, any number, and any position, and then connected to the ASGPR ligand portion through an active linker to obtain an oligonucleotide conjugate.

[0091] For example, the ASGPR ligand portion can have the structure shown in Formula (II),

[0092] In formula (II), R G1 、R G2 、R G3 、R G4 Each is independently defined as in any one of the above technical solutions; Q represents a group or linker capable of coupling with the active linker in the above intermediate, including but not limited to the following:

[0093] The seventh aspect of the present invention provides a pharmaceutical composition comprising the oligonucleotide conjugate described in any one of the above technical solutions and a pharmaceutically acceptable carrier.

[0094] In some preferred embodiments, the pharmaceutical composition may further comprise any other active ingredients for use in combination with the oligonucleotide conjugate, including but not limited to chemical drugs, biological drugs, drug conjugates, etc.

[0095] The eighth aspect of the present invention provides the use of the compound described in any one of the above technical solutions, or its racemate, stereoisomer, isotope-labeled product or salt thereof, or the oligonucleotide conjugate described in any one of the above technical solutions, or the pharmaceutical composition described in the above technical solution in the preparation of a drug for inhibiting mRNA expression in vivo, or a drug for detecting, preventing and / or treating hepatic diseases.

[0096] The ninth aspect of the present invention provides the use of the compound described in any one of the above technical solutions, or its racemate, stereoisomer, isotope-labeled product or salt thereof, or the oligonucleotide conjugate described in any one of the above technical solutions, or the pharmaceutical composition described in the above technical solutions as a drug (i.e., for therapeutic use).

[0097] The tenth aspect of the present invention provides a method for preventing and / or treating hepatic diseases, comprising administering to a patient a therapeutically effective dose of a compound described in any one of the above technical solutions, or its racemate, stereoisomer, isotope-labeled substance or salt thereof, or the oligonucleotide conjugate described in any one of the above technical solutions, or the pharmaceutical composition described in the above technical solutions.

[0098] In some preferred embodiments, the liver-derived diseases include, but are not limited to, hypercholesterolemia, type II hyperlipoproteinemia, primary hyperlipidemia, atherosclerosis, heterozygous familial hypercholesterolemia, dyslipidemia, primary hypercholesterolemia, atherosclerotic plaques, homozygous familial hypercholesterolemia, preeclampsia, hypertension, cardiovascular disease, diabetes, homozygous familial hypercholesterolemia, hypertriglyceridemia, familial hyperchylomicronemia, chronic hepatitis D, hepatitis D, hepatitis B, chronic hepatitis B, transthyretin amyloidosis , transthyretin amyloid neuropathy, transthyretin amyloid cardiomyopathy, familial amyloidosis, Stargardt disease, nephrolithiasis, hepatic porphyria, acute hepatic porphyria, hereditary porphyrias, acute intermittent porphyria, multiforme porphyria, paroxysmal nocturnal hemoglobinuria, myasthenia gravis, C3 glomerulopathy, immunoglobulin A kidney / liver disease, bleeding, hemophilia B, hemophilia A, nonalcoholic steatohepatitis, liver cancer, alpha-1 antitrypsin deficiency, primary hyperoxaluria type 1, primary hyperoxaluria type 2, end-stage renal disease.

[0099] The technical solution provided by the present invention has the following beneficial effects:

[0100] On the one hand, compared with the prior art, the present invention has the following advantages:

[0101] 1) Based on common GalNAc, the present invention synthesized a series of new ASGPR ligand substrates (as shown in formula (I-6)), which have improved stability against glycoside hydrolases and higher activity.

[0102] 2) The compounds and isomers provided by the present invention have more stable chemical connection modes compared to common ASGPR protein ligands. Through more stable chemical connection modes, the types of ASGPR ligands and their connection sites with linkers are expanded.

[0103] 3) The compounds provided by the present invention are based on phosphoramidite structures, and thus the monovalent ASGPR protein ligand compounds described in the present invention can participate in nucleic acid solid-phase chemical synthesis in different types and quantities and different insertion sites, thereby forming oligonucleotide conjugates containing ASGPR ligands. Thus, one or more ligand-modified oligonucleotide conjugates can be obtained, and various forms of oligonucleotide conjugates can be formed in combination. For example, the monovalent ASGPR ligand (i.e., the compound described in the present invention) can be connected to the 3' end or 5' end of the oligonucleotide chain in different quantities, or the 3' end and the 5' end can be connected at the same time, or inserted at any position in the oligonucleotide chain. The oligonucleotide conjugates formed have good pharmacokinetic and pharmacodynamic properties.

[0104] On the other hand, the delivery structure design and ligand number of oligonucleotide drugs also have a significant impact on the cost of drugs. The present invention starts from the design of a single ASGPR ligand, simplifies the currently used trivalent or multivalent antennae branched delivery design, and reduces the cost of chemical synthesis. At the same time, the present invention also introduces the structure-activity relationship (SAR) design of small molecule drugs, optimizes the binding affinity of the delivery ligand to the ASGPR receptor protein, and realizes a significant increase in the activity of the ligand structure. The oligonucleotide conjugate drug or its pharmaceutical composition comprising the compound of the present invention has an excellent in vivo liver targeting effect, and can achieve a long-term knockout effect of the target gene in vivo through mRNA expression inhibition mechanisms such as RNA interference. Further, by applying the ligand compound provided by the present invention, the active effect of the obtained divalent oligonucleotide conjugate is equivalent to or better than the active effect of the trivalent antennae delivery structure, breaking through the conclusion limitation of the prior art on the number of ligands and delivery activity efficiency (Nucleic Acid Ther. 2018 Jun; 28 (3): 109-118.). BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG1 is a schematic diagram of the preparation process of the oligonucleotide conjugate described in Example 51;

[0106] FIG2 is a graph showing gene suppression efficiency of some conjugates of Test Example 1;

[0107] FIG3 is a graph showing gene suppression efficiency of some conjugates of Test Example 1;

[0108] FIG4 is a graph showing gene suppression efficiency of some conjugates of Test Example 1;

[0109] FIG5 is a graph showing gene suppression efficiency of some conjugates of Test Example 1;

[0110] FIG6 is a graph showing gene suppression efficiency of some conjugates of Test Example 1;

[0111] FIG7 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 2;

[0112] FIG8 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 2;

[0113] FIG9 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 2;

[0114] FIG10 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on the FXII target gene;

[0115] FIG11 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on the PCSK9 target gene;

[0116] FIG12 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on the APOC3 target gene;

[0117] FIG13 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on the ANGPTL3 target gene;

[0118] FIG14 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on HBsAg protein of HBV virus;

[0119] FIG15 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on the FVII target gene;

[0120] FIG16 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on AGT angiotensinogen protein;

[0121] FIG17 is a graph showing the in vivo inhibitory effect of the conjugate of Test Example 3 on mTTR protein. DETAILED DESCRIPTION

[0122] the term

[0123] The term "absent" as used herein, alone or in combination, means that the indicated group does not exist in the chemical structure and the other groups to which the group is connected are directly connected via a chemical bond.

[0124] The term "C0" as used herein alone or in combination also means that the group is not present and the other groups to which the group is connected are directly connected via a chemical bond.

[0125] When a numerical range is listed herein, each value and sub-range within the range is intended to be included. For example, the "C1-C6" group means that there are 1 to 6 carbon atoms in the moiety, that is, the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms. Specifically, "C1-C6" includes C1, C2, C3, C4, C5, C6, C1-C2, C1-C3, C1-C4, C1-C5, C1-C6, C2-C3, C2-C4, C2-C5, C2-C6, C3-C4, C3-C5, C3-C6, C4-C5, C4-C6, and C5-C6. Thus, for example, "C1-C4 alkyl" refers to an alkyl group containing 1 to 4 carbon atoms, i.e., the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Numeric ranges herein, such as "1 to 30," "1 to 12," etc., refer to each integer in the given range.

[0126] The term "alkyl" as used herein, alone or in combination, refers to an optionally substituted straight-chain or optionally substituted branched saturated aliphatic hydrocarbon. The "alkyl" herein preferably has 1 to 6 carbon atoms, for example, 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1-3 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, and the like. When a group defined herein, such as "alkyl", appears in a numerical range, for example, "C1-C6 alkyl" refers to an alkyl group that can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms. The alkyl group herein also includes the case where no numerical range is specified.

[0127] As used herein, "alkyl" refers to an alkyl group attached to another group, for example, an alkyl group in an alkoxy group or a haloalkyl group, and has the same definition as when used alone.

[0128] The term "haloalkyl" as used herein, alone or in combination, refers to an alkyl group in which one or more, or even all, hydrogen atoms are replaced by halogen. Non-limiting examples of haloalkyl groups include trifluoromethyl, trichloromethyl, and the like.

[0129] The term "alkenyl" as used herein, alone or in combination, refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, non-limiting examples of which include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2- or 3-butenyl, etc. Alkenyl groups may be optionally substituted or unsubstituted.

[0130] The terms "cycloalkyl" or "carbocycle" used herein, alone or in combination, are used interchangeably herein and refer to saturated or partially saturated monocyclic, condensed, bridged, and spirocyclic carbocycles. Preferably, a C3-C10 cycloalkyl, more preferably a C3-C8 cycloalkyl, most preferably a C3-C6 cycloalkyl. Non-limiting examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc., preferably cyclopropyl, cyclohexenyl. Cycloalkyl can be optionally substituted or unsubstituted.

[0131] The terms "heterocyclyl" or "heterocycle" as used herein, alone or in combination, are used interchangeably herein and refer to non-aromatic heterocyclic groups in which one or more of the atoms forming the ring are heteroatoms, such as oxygen, nitrogen, sulfur atoms, and the like, including monocyclic, polycyclic, fused, bridged, and spirocyclic rings. Preferably, the heterocyclic group has a 4- to 10-membered monocyclic ring or a 7- to 10-membered bi- or tricyclic ring, which may contain 1, 2, or 3 atoms selected from nitrogen, oxygen, and / or sulfur. Non-limiting examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetane, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazinyl, and the like. The heterocyclic group may be substituted or unsubstituted.

[0132] The term "aryl" as used herein, alone or in combination, refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be fused together. The term "aryl" includes monocyclic or bicyclic aromatic groups, such as phenyl, naphthyl, and tetrahydronaphthyl aromatic groups. Preferred aromatic groups are phenyl and naphthyl, with phenyl being most preferred. Aryl groups may be substituted or unsubstituted.

[0133] The term "hydroxy," as used herein, alone or in combination, refers to an -OH group.

[0134] The term "halogen," as used herein, alone or in combination, refers to fluorine, chlorine, bromine, and iodine.

[0135] The term "amino," as used herein, alone or in combination, refers to -NH2.

[0136] The term "benzyl," as used herein, alone or in combination, refers to -CH2-phenyl.

[0137] As used herein, a group may be connected to other groups in any order unless the connection site is indicated. For example, the group "-C(O)NH-" connected to Q may be represented as "QC(O)NH-" or "Q-NHC(O)-".

[0138] In the structural fragments used herein, if there are numbers such as "1", "2", "3" (e.g. ), representing that the structural fragment is connected to the groups at both ends in a specific order, and the numerical limitation indicates the connection direction; if there is no numerical limitation (e.g. ), it means that the structural fragment can be connected to the groups at both ends in different orders.

[0139] The term "pharmaceutically acceptable," as used herein, alone or in combination, refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compound or conjugate of the invention and is relatively non-toxic, i.e., the substance can be administered to a subject without causing an adverse biological response or interacting in an adverse manner with any components contained in the composition.

[0140] The term "pharmaceutical composition" as used herein, alone or in combination, refers to a biologically active compound or conjugate optionally mixed with at least one pharmaceutically acceptable chemical component, including but not limited to carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, excipients, and the like.

[0141] The term "salt" or "pharmaceutically acceptable salt" as used herein, alone or in combination, refers to a compound or conjugate of the present invention that retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with an inorganic base or an organic base, or the free base is obtained by reacting with a non-toxic inorganic acid or organic acid. Salts can be obtained using standard procedures well known in the art. Suitable salts are listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).

[0142] The term "isotopically labeled" as used herein, alone or in combination, refers to a compound or conjugate having the same structure as the compound or conjugate of the present invention, but with one or more atoms in the structure replaced by an atom having the same number of protons but a different number of neutrons. Examples of isotopes that may be incorporated into the compound or conjugate of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, and iodine, such as2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F. 36 Cl and 131 I etc.

[0143] The compounds or conjugates of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds or conjugates of the present invention, including but not limited to diastereomers, enantiomers, steric isomers and geometric (conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention. Unless otherwise indicated, the structures described herein also include all isomers of the structure (e.g., diastereomers, enantiomers, steric isomers and geometric (conformation) isomer forms; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, steric isomers of biphenyl structures (see "Basic Organic Chemistry" (Second Edition), Volume 1, Xing Qiyi et al., p104-105); PAC, 1996, 68, 2193. (Basic terminology of stereochemistry (IUPAC Recommendations 1996, on page 2201)), (Z) and (E) conformational isomers. Therefore, single stereoisomers of the compounds and conjugates of the present invention, as well as mixtures of enantiomers, diastereomers, steric isomers and geometric (conformation) isomers are all within the scope of the present invention.

[0144] For example, R G1 The connection site can be connected by a racemate, α-stereoconfiguration or β-stereoconfiguration chemical bond (for example, by a β-stereoconfiguration chemical bond). The α and β configurations are related to the cyclic structure of the monosaccharide. When the monosaccharide changes from a linear structure to a cyclic structure, the carbonyl carbon atom becomes a new chiral center, resulting in C1 (carbonyl carbon) epimerization to produce two diastereomers. The anomeric carbon hydroxyl group (i.e., R G1 ) and the 6-carbon atom have the same orientation, it is β type, otherwise it is α type.

[0145] As used herein, the terms "treat," ...

[0146] The technical solution of the present invention is further described in detail below with reference to specific embodiments.

[0147] Unless otherwise specified, the raw materials or reagents used in the examples and comparative examples of the present invention are commercially available products.

[0148] The reversed-phase Flash rapid purification instrument used in the examples and comparative examples of the present invention was purchased from Biotage. The Selekt instrument used a reversed-phase preparative column, a C18 column purchased from Agela Technology Co., Ltd., in sizes of 40 g, 80 g, 120 g, and 330 g. The elution system consisted of 5%-95% acetonitrile / water (volume ratio), with the aqueous phase consisting of 0.01% ammonium bicarbonate solution (mass ratio). Unless otherwise specified, the column chromatography used was silica gel column chromatography.

[0149] The ratio of the eluent used in the examples and comparative examples of the present invention is by volume, and other percentages are by mass unless otherwise specified.

[0150] The meanings of the abbreviations used in the examples and comparative examples of the present invention are shown in the following table:

[0151] 2-Cyanoethyl N,N-diisopropylphosphoramidite is abbreviated as CEP, and 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite is abbreviated as CEP-Cl, as shown below:

[0152] The targeting ligand L96 has the following structure; it is prepared according to methods known in the art (such as the method described in patent application WO2014025805A1) and linked to the siRNA molecule.

[0153] Example 1 Synthesis of Compound 3

[0154] Compound 1 (50 g, 0.55 mol, 1.0 eq) was dissolved in 500 mL of acetonitrile. After nitrogen replacement, 167 g (1.65 mol, 3.0 eq) of triethylamine and 160 g (1.1 mol, 2.0 eq) of ethyl trifluoroacetate were added and allowed to react overnight at room temperature. After completion of the reaction, the solvent was removed by direct concentration under reduced pressure, as monitored by TLC, to afford a crude oil.

[0155] The crude oil was dissolved in 400 mL of pyridine and replaced with nitrogen. The mixture was cooled to 0°C in an ice-water bath and 186 g (0.55 mol, 1.0 eq) of 4,4'-bis(methoxytrityl) chloride was added portionwise. After the addition, the mixture was allowed to react at room temperature overnight. After completion of the reaction, the mixture was quenched with water and extracted twice with ethyl acetate. The organic phases were combined, dried, concentrated, and purified by column chromatography to afford 233 g of compound 2, with a two-step yield of 86.9%.

[0156] Compound 2 (233 g, 0.47 mol, 1.0 eq) was dissolved in 1.5 L of methanol, and 53.4 g (0.95 mol, 2.0 eq) of potassium hydroxide (prepare a 3.0 mol / L aqueous solution of potassium hydroxide) was added. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction, the solvent was removed by TLC monitoring and concentrated under reduced pressure. The mixture was then purified by flash column chromatography to obtain 174 g of compound 3 in a yield of 92.8%. 1 H NMR (400MHz, DMSO-d6) δ7.40(d,J=7.5Hz,2H),7.33–7.18(m,7H),6.88(d,J=8.5Hz,4H),4.75(s,1H),3.73(s,6H),3.56(p,J=6.1Hz,1H),2.9 4(dd,J=9.1,5.4Hz,1H),2.83(dd,J=9.0,6.1Hz,1H),2.69(dd,J=12.7,4.0Hz,1H),2.49–2.42(m,1H),1.97(d,J=81.8Hz,1.7H).MS(ESI):m / z calcd for C 24 H 27 NO4[MH] - :392.19,found:392.16.

[0157] The R-configuration and S-configuration compounds 3 were synthesized from the corresponding R-configuration and S-configuration starting materials 1, respectively, using the same operation as the racemic compound, and the NMR results were consistent.

[0158] Example 2 Synthesis of Compound 8

[0159] Compound 4 (5.0 g, 49.5 mmol, 1.0 eq) was dissolved in 50 mL of acetonitrile. After nitrogen purge, triethylamine (15.0 g, 148 mmol, 3.0 eq) and 14.1 g (99.0 mmol, 2.0 eq) of ethyl trifluoroacetate were added and allowed to react overnight at room temperature. After completion of the reaction, the mixture was directly concentrated under reduced pressure to obtain a crude oil (which was directly reacted in the next step with a 100% yield), as monitored by TLC.

[0160] 2.0 g (10.0 mmol, 1.0 eq) of the crude oil and compound 3 (3.93 g, 10.0 mmol, 1.0 eq) were dissolved in 20 mL of N,N-dimethylformamide. 2.5 g (20.0 mmol, 2.0 eq) of N,N-diisopropylethylamine and 4.5 g (12.0 mmol, 1.2 eq) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, extracted with ethyl acetate, and the organic phase washed with saturated brine, separated, dried, concentrated, and purified by reverse phase preparative column chromatography (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to afford 2.3 g of compound 5 as a white solid in a yield of 39.6%.

[0161] Compound 5 (2.3 g, 4.0 mmol, 1.0 eq) was dissolved in 20 mL of methanol, and 0.45 g (8.0 mmol, 2.0 eq) of potassium hydroxide (prepared to a 3.0 mol / L aqueous solution) was added. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction, the solvent was removed by TLC monitoring and concentrated under reduced pressure. Compound 6 was then purified by flash column chromatography to obtain 1.75 g of a light yellow oil in a 91.6% yield.

[0162] Compound 6 (1.06 g, 2.2 mmol, 1.0 eq) and compound 7 (1.0 g, 2.2 mmol, 1.0 eq, prepared in Example 4) were dissolved in 10 mL of N,N-dimethylformamide. Under nitrogen, 0.57 g (4.4 mmol, 2.0 eq) of N,N-diisopropylethylamine and 1.01 g (2.6 mmol, 1.2 eq) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were added. The reaction was allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, and concentrated. The product was purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 1.2 g of compound 8 as a white solid in a 63.2% yield. MS (ESI): m / z calculated for C 47 H 59 N3O 15 [MH] -:904.39,found:904.37.

[0163] Example 3 Synthesis of Compound 9

[0164] Compound 8 (1.2 g, 1.3 mmol, 1.0 eq) and 4-dimethylaminopyridine (32 mg, 0.26 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 20 mL of anhydrous dichloromethane and N,N-diisopropylethylamine (0.34 g, 2.6 mmol, 2.0 eq) were added. 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.46 g, 1.9 mmol, 1.5 eq) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified by reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 1.1 g of compound 9 as a white solid in a 75.3% yield. 1 H NMR(400MHz, DMSO-d6)δ7.96(dt,J=21.1,5.9Hz,1H),7.81(d,J=9.2Hz,1H),7.43–7.3 8(m,2H),7.26(tdd,J=12.0,7.9,4.3Hz,7H),6.87(dd,J=8.6,6.0Hz,4H),5.22(d,J=3. 4Hz,1H),4.97(dt,J=11.3,3.8Hz,1H),4.50(dd,J=8.5,3.5Hz,1H),4.11(dt,J=9.2,2 .6Hz,1H),4.02(d,J=4.6Hz,4H),3.87(ddt,J=16.0,11.4,5.5Hz,2H),3.74(d,J=2.9Hz ,6H),3.71–3.66(m,2H),3.59(dt,J=10.7,6.9Hz,1H),3.51(dt,J=10.1,6.8Hz,1H),3 .44–3.39(m,1H),3.32–3.16(m,3H),3.10–2.99(m,1H),2.90(dd,J=9.9,5.4Hz,1H),2. 77(t,J=5.9Hz,1H),2.67–2.59(m,1H),2.10(s,3H),2.07(s,5H),1.99(s,5H),1.89(s, 3H),1.78(s,3H),1.47(q,J=4.6,3.3Hz,4H),1.26–1.07(m,8H),1.00(d,J=6.7Hz,3H). 31P NMR(162MHz,DMSO)δ148.40,147.96,147.91.MS(ESI):m / z calcd for C 56 H 76 N5O 16 P[MH] - :1104.50,found:1104.52.

[0165] Example 4 Synthesis of Compound 7

[0166] In a 1L round-bottom flask, δ-valerolactone 10 (100g, 1mol), sodium hydroxide (40g, 1mol), and 400mL of deionized water were mixed and reacted at 70°C for 6 hours. The reaction was monitored by TLC for completion. The reaction solution was spin-dried, 200mL of toluene was added, and the mixture was spin-dried to obtain 140g of a white solid. In a 1L round-bottom flask, compound 10 (140g, 1mol), 500mL of anhydrous acetone, benzyl bromide (205.2g, 1.2mol), and catalyst tetrabutylammonium bromide (16.2g, 0.05mol) were added and heated to reflux. The reaction was monitored by TLC and completed after 24 hours. After the reaction solution was cooled to room temperature, the acetone was removed under reduced pressure. The residue was dissolved in 500mL of ethyl acetate and washed sequentially with 200mL of saturated sodium bisulfate, 200mL of saturated sodium bicarbonate, and 200mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column (petroleum ether:ethyl acetate V:V=1:1) to obtain a transparent oily liquid compound 11, 175 g, with a yield of 84%.

[0167] To a 1L round-bottom flask, D-galactose hydrochloride compound 12 (100g, 0.46mol) and 450mL of anhydrous pyridine were added. Acetic anhydride (325mL), triethylamine (64.5mL, 0.46mol), and 4-dimethylaminopyridine (2g, 0.016mol) were slowly added under an ice bath. The reaction was allowed to proceed overnight at room temperature, resulting in the precipitation of a large amount of solid. The filter cake was filtered and rinsed with 200mL of 0.5N HCl solution to afford 162.5g of compound 13 as a white solid in a 90% yield.

[0168] Compound 13 (10 g, 25.7 mmol) and 100 mL of anhydrous dichloromethane were added to a 250 mL round-bottom flask. After stirring for 10 minutes, trimethylsilyl trifluoromethanesulfonate (7 mL, 38.7 mmol) was added. The reaction was then allowed to react overnight at room temperature. The reaction solution was slowly poured into an aqueous solution (200 mL) of sodium bicarbonate (7 g, 79.5 mmol) and stirred for 0.5 hours. The organic phase was separated and dried over anhydrous sodium sulfate. After concentration under reduced pressure, a light yellow colloid was obtained. In a 100 mL round-bottom flask, the above crude product (5 g, 15.2 mmol) and compound 11 (3.8 g, 18.25 mmol) were dissolved in 50 mL of anhydrous 1,2-dichloroethane. After stirring for 10 minutes, trimethylsilyl trifluoromethanesulfonate (0.55 mL, 3 mmol) was added. The reaction was allowed to react overnight at room temperature. The reaction solution was extracted with dichloromethane. The obtained organic phase was washed twice with 50 mL of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The product was separated on a silica gel column (petroleum ether:ethyl acetate V:V=3:2) to obtain a transparent oily liquid compound 14 (6.94 g, yield 85%).

[0169] In a 50 mL round-bottom flask, compound 14 (3.3 g, 6.1 mmol) and Pd / C (0.33 g, 10%) were dissolved in 5 mL of methanol and 20 mL of ethyl acetate. A hydrogen balloon was then added and the reaction was allowed to proceed overnight at room temperature. The reaction solution was filtered through celite, which was then rinsed with methanol. The filtrate was concentrated to dryness under reduced pressure and purified by flash silica gel column chromatography (petroleum ether:ethyl acetate, v:v = 3:1) to afford compound 7 as a white solid (2.8 g, 95.5% yield).

[0170] 1 H NMR (400MHz, DMSO-d6) δ7.82(d,J=9.2Hz,1H),5.21(d,J=3.4Hz,1H),4.96(dd,J=11.2, 3.4Hz,1H),4.49(d,J=8.4Hz,1H),4.03(d,J=5.4Hz,4H),3.88(dt,J=11.2,8.9Hz,1H), 3.71(dt,J=9.7,5.2Hz,2H),2.19(t,J=7.0Hz,2H),2.10(s,3H),1.99(d,J=4.2Hz,4H), 1.89(s,3H),1.77(s,3H),1.50(dq,J=8.2,4.6Hz,4H),1.26–1.14(m,1H).MS(ESI):m / z calcd for C 19 H 29 NO 11 [MH] - :446.17,found:446.14.

[0171] Example 5 Synthesis of Compound 19

[0172] 5.0 g (38.1 mmol, 1.0 eq) of compound 15 was weighed and dissolved in 50 mL of acetonitrile. After nitrogen purge, 11.5 g (114.5 mmol, 3.0 eq) of triethylamine and 10.8 g (76.3 mmol, 2.0 eq) of ethyl trifluoroacetate were added. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was directly concentrated under reduced pressure to obtain a crude oil, compound 16, as monitored by TLC.

[0173] Compound 16 (1.3 g, 6.1 mmol, 1.2 eq) and compound 3 (2.0 g, 5.1 mmol, 1.0 eq) were weighed and dissolved in 20 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (1.3 g, 10.1 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (2.3 g, 6.1 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 2.3 g of an oil (yield: 79.3%).

[0174] Dissolve 2.3 g of the oil in 20 mL of methanol, add 0.45 g (8.0 mmol, 2.0 eq) of potassium hydroxide (prepare a 3.0 mol / L aqueous solution), and allow to react at room temperature for 2 h. After completion of the reaction, monitor the reaction by TLC, concentrate under reduced pressure to remove the solvent, and then purify the mixture via flash column chromatography to obtain 1.75 g of compound 17 (yield: 91.6%).

[0175] Compound 17 (1.3 g, 2.6 mmol, 1.2 eq) and compound 7 (1.0 g, 2.2 mmol, 1.0 eq) were weighed and dissolved in 10 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (0.57 g, 4.4 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1.01 g, 2.6 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 1.5 g of compound 18 in a yield of 72.8%.

[0176] Compound 18 (1.5 g, 1.6 mmol, 1.0 eq) and 4-dimethylaminopyridine (39 mg, 0.32 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and 420 mg (3.2 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. Then, 460 mg (1.9 mmol, 1.2 eq) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified on a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 1.2 g of compound 19 in a 65.9% yield. 1 H NMR(400MHz,DMSO-d6)δ7.82(d,J=9.2Hz,1H),7.68(d,J=6.1Hz,1H),7.67–7.56(m ,1H),7.44–7.37(m,2H),7.32–7.17(m,7H),6.87(dd,J=8.6,6.1Hz,4H),5.22(d,J= 3.3Hz,1H),4.97(dd,J=11.3,3.3Hz,1H),4.49(d,J=8.5Hz,1H),4.03(s,2H),4.01( d,J=5.3Hz,2H),3.93–3.76(m,2H),3.68(dt,J=7.5,3.7Hz,2H),3.56(ddt,J=30.9, 9.5,6.6Hz,2H),3.41(dt,J=9.5,6.1Hz,1H),3.33(s,2H),3.18(q,J=7.6Hz,1H),2 .99(p,J=6.5,5.6Hz,3H),2.95–2.87(m,1H),2.77(t,J=6.1Hz,1H),2.67–2.59(m,1 H),2.10(s,3H),2.07(s,2H),2.03(t,J=6.9Hz,2H),1.99(s,3H),1.89(s,3H),1.77 (s,3H),1.53–1.28(m,9H),1.18–1.07(m,10H),1.01(d,J=6.7Hz,3H).MS(ESI):m / z calcd for C 58 H 82 N5O 16 P[MH] - :1134.54,found:1134.49.

[0177] Example 6 Synthesis of Compound 20

[0178] Compound 20 was synthesized with compound 16 and R-configuration compound 3 as raw materials and with reference to compound 19. 1 H NMR (400MHz, DMSO-d6) δ7.81(d,J=9.2Hz,1H),7.69(d,J=5.7Hz,1H),7.62(d,J=23.3Hz, 1H),7.40(d,J=8.5Hz,2H),7.26(dq,J=14.6,11.6,9.1Hz,7H),6.93–6.83(m,4H),5.21( d,J=3.3Hz,1H),4.96(dd,J=11.3,3.3Hz,1H),4.48(d,J=8.5Hz,1H),4.02(s,3H),3.93– 3.84(m,1H),3.73(d,J=2.6Hz,6H),3.71–3.64(m,2H),3.52(q,J=7.7,7.0Hz,1H),3.40( d,J=8.5Hz,1H),3.19(d,J=16.3Hz,1H),2.98(d,J=7.2Hz,2H),2.94–2.87(m,1H),2.80– 2.73(m,1H),2.63(dt,J=7.3,3.5Hz,1H),2.10(s,3H),2.07(s,2H),2.06–2.01(m,2H),1 .99(s,4H),1.95(d,J=8.6Hz,1H),1.89(s,3H),1.77(s,3H),1.42(dt,J=45.1,10.6Hz,8 H),1.24(s,2H),1.15(ddd,J=12.7,9.9,5.9Hz,10H),1.00(d,J=6.7Hz,3H).MS(ESI):m / z calcd for C 58 H 82 N5O 16 P[MH] - :1134.54,found:1134.50.

[0179] Example 7 Synthesis of Compound 21

[0180] Compound 21 was synthesized with compound 16 and S-configuration compound 3 as raw materials and with reference to compound 19. 1H NMR(400MHz,DMSO-d6)δ7.82(d,J=9.2Hz,1H),7.73–7.56(m,2H),7.40(d,J=7.6 Hz,2H),7.33–7.18(m,7H),6.87(dd,J=8.5,6.2Hz,4H),5.21(d,J=3.4Hz,1H),4. 97(dd,J=11.3,3.3Hz,1H),4.48(d,J=8.5Hz,1H),4.02(s,3H),3.93–3.76(m,2H ),3.73(s,6H),3.55(ddt,J=23.3,9.8,7.0Hz,2H),3.46–3.34(m,1H),3.32(s,2H ),3.18(dq,J=9.8,5.4Hz,1H),3.00(dd,J=11.8,5.9Hz,2H),2.97–2.83(m,2H), 2.77(t,J=6.0Hz,1H),2.63(t,J=6.1Hz,1H),2.10(s,3H),2.08–2.01(m,3H),1.9 9(s,3H),1.97–1.92(m,1H),1.89(s,3H),1.77(s,3H),1.40(ddq,J=34.8,13.6,7 .2,6.4Hz,8H),1.14(dt,J=12.6,5.0Hz,9H),1.01(d,J=6.7Hz,3H).MS(ESI):m / z calcd for C 58 H 82 N5O 16 P[MH] - :1134.54,found:1134.51.

[0181] Example 8 Synthesis of Compound 26

[0182] 5.0 g (43.5 mmol, 1.0 eq) of compound 22 was dissolved in 50 mL of acetonitrile. After nitrogen purge, 15.0 g (130.5 mmol, 3.0 eq) of triethylamine and 14.1 g (87.0 mmol, 2.0 eq) of ethyl trifluoroacetate were added and allowed to react overnight at room temperature. After completion of the reaction, the mixture was directly concentrated under reduced pressure to obtain a crude oil (which was directly used in the next step in a 100% yield), as monitored by TLC.

[0183] The crude oil and 17.1 g (43.5 mmol, 1.0 eq) of compound 3 were dissolved in 100 mL of N,N-dimethylformamide. Under nitrogen, 11.2 g (87.0 mmol, 2.0 eq) of N,N-diisopropylethylamine and 19.8 g (52.2 mmol, 1.2 eq) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were added. The reaction was allowed to react at room temperature for 2 h. The reaction was monitored by TLC. The reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified by column chromatography to afford 17.5 g of compound 23, with a two-step yield of 68.2%.

[0184] Dissolve 17.5 g (29.8 mmol, 1.0 eq) of compound 23 in 200 mL of methanol, add 3.4 g (59.7 mmol, 2.0 eq) of potassium hydroxide (prepare a 3.0 mol / L aqueous solution), and allow to react at room temperature for 2 h. After completion of the reaction, monitor the reaction by TLC, concentrate under reduced pressure to remove the solvent, and then purify the mixture via flash column chromatography to obtain 13.8 g of compound 24 in a 95.1% yield.

[0185] Compound 24 (1.3 g, 2.6 mmol, 1.2 eq) and compound 7 (1.0 g, 2.2 mmol, 1.0 eq) were weighed and dissolved in 10 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (0.57 g, 4.4 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1.01 g, 2.6 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 415 mg of compound 25 in a yield of 20.1%.

[0186] 410 mg (0.45 mmol, 1.0 eq) of compound 25 and 11 mg (0.09 mmol, 0.2 eq) of 4-dimethylaminopyridine were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and 115 mg (0.9 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. 158 mg (0.67 mmol, 1.5 eq) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite were then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified by reverse-phase preparative column to obtain 270 mg of compound 26 in a yield of 54.5%. 1H NMR(400MHz,DMSO-d6)δ7.82(dt,J=15.6,6.2Hz,1H),7.75–7.57(m,1H),7.45–7.36(m, 2H),7.36–7.17(m,7H),6.87(t,J=7.1Hz,4H),5.21(d,J=2.6Hz,1H),4.96(dt,J=9.1,4 .4Hz,1H),4.48(t,J=9.6Hz,1H),4.26–4.12(m,1H),4.02(d,J=3.5Hz,4H),3.94–3.77( m,2H),3.73(d,J=2.3Hz,8H),3.67–3.58(m,1H),3.59–3.48(m,1H),3.39(dd,J=17.2,9. 9Hz,3H),3.27(d,J=5.8Hz,1H),3.25–3.09(m,1H),3.06–2.99(m,1H),2.93–2.82(m,1H) ),2.78(t,J=5.8Hz,1H),2.69–2.60(m,1H),2.22(s,1H),2.09(d,J=10.4Hz,4H),1.99( d,J=3.0Hz,3H),1.89(s,4H),1.85–1.78(m,1H),1.76(t,J=2.6Hz,4H),1.64(dd,J=27. 4,7.1Hz,1H),1.48(s,3H),1.38(d,J=5.5Hz,1H),1.28–1.10(m,9H),1.08–0.98(m,3H). 31 P NMR(162MHz,DMSO)δ148.43,148.34,148.24,148.22,148.12,148.04,147.99,147.84,147.82.MS(ESI):m / z calcd for C 57 H 78 N5O 16 P[MH] - :1118.51,found:1118.56.

[0187] Example 9 Synthesis of Compound 31

[0188] 5.0 g (31.8 mmol, 1.0 eq) of compound 27 was dissolved in 50 mL of 1,4-dioxane and 5 mL of water. After nitrogen replacement, 6.7 g (63.6 mmol, 2.0 eq) of sodium carbonate and 9.8 g (38.1 mmol, 1.2 eq) of 9-fluorenylmethylcarbamoyl chloride were added and allowed to react at room temperature for 4 h. After completion of the reaction, the pH was adjusted to 2-3 by TLC, and the mixture was filtered. The filter cake was washed with water and air-dried (50°C) to afford 11.2 g of a white solid (yield: 93.3%).

[0189] 5.0 g (13.2 mmol, 1.0 eq) of the white solid and 5.2 g (13.2 mmol, 1.0 eq) of compound 3 were dissolved in 100 mL of N,N-dimethylformamide. 3.4 g (26.4 mmol, 2.0 eq) of N,N-diisopropylethylamine and 6.0 g (15.8 mmol, 1.2 eq) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, and concentrated to afford 10.0 g of crude compound 28 as an oil.

[0190] 2 g of crude compound 28 was dissolved in 20 mL of dichloromethane, and 4 mL of piperidine was added. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction, the solvent was removed by TLC monitoring, and the mixture was concentrated under reduced pressure. The mixture was then purified by flash column chromatography to obtain 1.2 g of compound 29.

[0191] Compound 29 (1.2 g, 2.2 mmol, 1.0 eq) and compound 7 (1.0 g, 2.2 mmol, 1.0 eq) were weighed and dissolved in 10 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (0.57 g, 4.4 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1.01 g, 2.6 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 1.4 g of compound 30 in a yield of 63.6%.

[0192] Compound 30 (1.4 g, 1.0 eq) and 4-dimethylaminopyridine (36 mg, 0.3 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 20 mL of anhydrous dichloromethane and 0.38 g (2.95 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.52 g, 2.21 mmol, 1.5 eq) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 700 mg of compound 31 in a yield of 42.1%. 1 H NMR (400MHz, DMSO-d6) δ7.82(d,J=9.2Hz,1H),7.70(t,J=5.8Hz,1H),7.55(dt,J=28.0,5. 8Hz,1H),7.41(d,J=7.8Hz,2H),7.31–7.16(m,7H),6.86(dd,J=8.7,6.7Hz,4H),5.22(d,J =3.4Hz,1H),4.97(dd,J=11.3,3.4Hz,1H),4.49(d,J=8.5Hz,1H),4.03(s,3H),3.94–3.77 (m,2H),3.73(d,J=2.6Hz,6H),3.70(d,J=3.5Hz,1H),3.58(ddt,J=26.0,10.3,6.7Hz,2H), 3.46–3.38(m,1H),3.29(d,J=18.1Hz,1H),3.16(dt,J=21.4,6.2Hz,1H),3.08–2.93(m,1H ),2.88(q,J=5.8,5.1Hz,3H),2.78(t,J=6.3Hz,1H),2.68–2.60(m,1H),2.10(s,3H),2.07 (s,6H),1.99(s,3H),1.89(s,3H),1.77(s,3H),1.67(d,J=13.0Hz,2H),1.56–1.41(m,5H) ,1.26(d,J=18.1Hz,2H),1.18–1.11(m,8H),1.05(d,J=6.7Hz,3H),0.81(d,J=13.0Hz,2H). 31 P NMR(162MHz,DMSO)δ148.28,148.01.MS(ESI):m / z calcd for C 60 H 84 N5O 16 P[MH] -:1160.56,found:1160.49.

[0193] Example 10 Synthesis of Compound 36

[0194] Compound 32 (11.4 g, 0.1 mol) and compound 33 (5.5 g, 0.1 mol) were weighed and dissolved in 100 mL of dichloromethane. The atmosphere was replaced with nitrogen and stirred at room temperature. After the reaction was completed as monitored by TLC, the system was directly concentrated to obtain 17.0 g of product, with a yield of 100.6%.

[0195] Compound 34 (4.7 g, 27.9 mmol) and compound 3 (10.0 g, 25.4 mmol) were dissolved in 100 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (6.6 g, 50.8 mmol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.6 g, 30.5 mmol) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified by column chromatography to obtain 7.8 g of compound 35 in a yield of 56.5%.

[0196] Compound 35 (3.3 g, 6.0 mmol) and 4-dimethylaminopyridine (148 mg, 1.2 mmol) were weighed into a 100 mL single-necked flask. After nitrogen purge, 30 mL of anhydrous dichloromethane and N,N-diisopropylethylamine (1.56 g, 12.1 mmol) were added. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (2.1 g, 9.1 mmol) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified on a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 2.4 g of compound 36 in a 54.3% yield. 1H NMR(400MHz, DMSO-d6)δ8.21(d,J=5.8Hz,1H),7.67(dt,J=20.8,5.8Hz,1H),7.44–7.36(m,2H),7.32–7.20(m,7H),6. 87(dd,J=8.7,6.1Hz,4H),4.02(dt,J=11.5,5.6Hz,1H),3.87–3.76(m,3H),3.74(s,6H),3.69(s,1H),3.57–3.44(m,1 H),3.33(s,1H),3.26–3.14(m,2H),3.13–2.96(m,3H),2.95–2.83(m,1H),2.77(t,J=6.1Hz,1H),2.63(t,J=5.3Hz,1H ),2.01(ddd,J=29.8,14.4,7.4Hz,4H),1.63(h,J=7.6Hz,2H),1.25–1.09(m,8H),1.00(d,J=6.7Hz,4H).MS(ESI):m / z calcd for C 41 H 53 N4O7P[MH] - :743.36,found:743.33.

[0197] Example 11 Synthesis of Compound 44

[0198] 5.2 g (50.0 mol, 1.0 eq) of compound 37 was dissolved in 100 mL of acetonitrile. After nitrogen purge, 12.0 g (118.8 mmol, 2.4 eq) of triethylamine and 17.6 g (123.8 mmol, 2.5 eq) of ethyl trifluoroacetate were added. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was concentrated under reduced pressure, monitoring by TLC, to yield 14.0 g of a crude oil.

[0199] Dissolve 14.0 g of the crude oil in 200 mL of pyridine, replace the atmosphere with nitrogen, cool to 0°C in an ice-water bath, and add 18.6 g (55.0 mol, 1.1 eq) of 4,4'-bis(methoxytrityl) chloride portionwise. Return the mixture to room temperature and allow to react overnight. After TLC monitoring, quench the reaction with water and extract twice with ethyl acetate. The organic phases are combined, dried, concentrated, and purified by column chromatography to afford 20.2 g of compound 38, with a two-step yield of 80.2%.

[0200] Dissolve 20.2 g (40.0 mmol, 1.0 eq) of compound 38 in 200 mL of methanol, add 4.5 g (80.0 mol, 2.0 eq) of potassium hydroxide (prepare a 3.0 mol / L aqueous solution), and allow to react at room temperature for 2 h. After completion of the reaction, monitor the reaction by TLC, concentrate under reduced pressure to remove the solvent, and then purify the mixture via flash column chromatography to obtain 15.5 g of compound 39 in a 95.1% yield.

[0201] Compound 27 (5.0 g, 31.8 mmol, 1.0 eq) was dissolved in 50 mL of 1,4-dioxane and 5 mL of water. After nitrogen replacement, 6.7 g of sodium carbonate (63.6 mmol, 2.0 eq) and 9.8 g of 9-fluorenylmethylcarbamoyl chloride (38.1 mmol, 1.2 eq) were added and allowed to react at room temperature for 4 h. After completion of the reaction, the pH was adjusted to 2-3 by TLC, and the mixture was filtered. The filter cake was washed with water and air-dried (50°C) to obtain 11.2 g of compound 40 as a white solid in a yield of 93.3%.

[0202] Compound 40 (5.0 g, 13.2 mmol, 1.0 eq) and compound 39 (5.4 g, 13.2 mmol, 1.0 eq) were dissolved in 100 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (3.4 g, 26.4 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (6.0 g, 15.8 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, and concentrated to afford 10.0 g of crude compound 41 as an oil.

[0203] 4 g of crude compound 41 was dissolved in 20 mL of dichloromethane, and 4 mL of piperidine was added. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction, the solvent was removed by TLC monitoring, and the mixture was concentrated under reduced pressure. The mixture was then purified on a silica gel flash column to obtain 2.4 g of compound 42.

[0204] Compound 42 (1.91 g, 3.5 mmol, 1.0 eq) and valeric acid (0.4 g, 4.2 mmol, 1.2 eq) were dissolved in 10 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (0.9 g, 7.0 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1.6 g, 4.2 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified on a reverse-phase C18 preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford compound 43 as a white solid (1.32 g, yield: 60.9%).

[0205] Compound 43 (320 mg, 0.51 mmol, 1.0 eq) and 4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and 132 mg (1.02 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. 181 mg (0.77 mmol, 1.5 eq) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified by reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 274 mg of compound 44 as a white solid in a 65.2% yield. 1 H NMR (400MHz, DMSO-d6) δ7.84(t,J=5.8Hz,1H),7.37(d,J=7.9Hz,3H),7.32–7.19(m,7H),6.90–6.82(m,4H),4.12(d,J=8. 9Hz,1H),3.73(s,6H),3.48(tdd,J=14.0,9.5,6.5Hz,3H),3.32(s,2H),3.05(ddd,J=21.1,9.1,6.0Hz,2H),2.94(dd,J=14 .4,6.3Hz,2H),2.90(s,1H),2.77–2.67(m,2H),2.61(t,J=5.9Hz,1H),2.35(td,J=7.7,6.7,2.3Hz,2H),2.30–2.16(m,3H ),1.75(t,J=10.4Hz,4H),1.36(tdd,J=16.3,12.5,11.5,7.1Hz,3H),1.14–0.96(m,14H),0.96–0.83(m,3H).MS(ESI):m / z calcd for C47 H 63 N4O7P[MH] - :825.44,found:825.38.

[0206] Example 12 Synthesis of Compound 47

[0207] Synthesis of compound 46

[0208] Compound 45 (5 g, 54.88 mmol) was dissolved in anhydrous acetonitrile (100 mL), and triethylamine (19 mL, 137.20 mmol, 2.5 eq.) was added. After stirring, ethyl trifluoroacetate (13 mL, 109.76 mmol, 2.0 eq.) was added dropwise and allowed to react at room temperature. The reaction was allowed to react for approximately 1 h, monitored by TLC and LC-MS. After completion of the reaction, 4,4'-bismethoxytrityl chloride (16.7 g, 49.39 mmol, 0.9 eq.) was added in small portions. The reaction was then allowed to react at room temperature for approximately 1 h, monitored by TLC and LC-MS. After completion of the reaction, the reaction was quenched by addition of saturated sodium bicarbonate solution, extracted three times with ethyl acetate, and the organic phases were combined, dried, and concentrated to obtain the crude product, which was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 2:1) to afford compound 46 (12.5 g, 47% yield) as a white solid.

[0209] Synthesis of compound 47

[0210] Compound 46 (10.5 g, 20.4 mmol) was dissolved in methanol (100 mL) and stirred. Aqueous potassium hydroxide (2.3 g in 50 mL of water, 40.8 mmol, 2.0 eq.) was added, and the reaction was allowed to proceed at room temperature. After approximately 1 h, the reaction was monitored by TLC and LC-MS. Upon completion of the reaction, the methanol was removed by distillation under reduced pressure. Saturated aqueous sodium chloride solution was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated to obtain a crude product, which was used directly without further purification. 1 H NMR (400MHz, DMSO-d6) δ7.40(d,J=7.5Hz,2H),7.33–7.18(m,7H),6.88(d,J=8.5Hz,4H),4.75(s,1H),3.73(s,6H),3.56( p,J=6.1Hz,1H),3.44(dd,J=9.1,5.4Hz,1H),3.31(m,2H),2.69(dd,J=12.7,4.0Hz,1H),2.31–2.42(m,2H).MS(ESI):m / z calcd for C 24 H27 NO4[MH] - :392.19,found:392.12.

[0211] Example 13 Synthesis of Compound 50

[0212] 5.8 g (50.0 mol, 1.0 eq) of compound 48 was dissolved in 100 mL of acetonitrile. After nitrogen purge, 12.0 g (118.8 mmol, 2.4 eq) of triethylamine and 17.6 g (123.8 mmol, 2.5 eq) of ethyl trifluoroacetate were added. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was directly concentrated under reduced pressure, monitoring by TLC, to yield 13.0 g of a crude oil.

[0213] Dissolve 13.0 g of the crude oil in 200 mL of pyridine, replace the atmosphere with nitrogen, and cool to 0°C in an ice-water bath. Add 4,4'-bismethoxytrityl chloride (18.6 g, 55.0 mol, 1.1 eq) portionwise. Return to room temperature and allow to react overnight. After TLC monitoring, quench the reaction with water and extract twice with ethyl acetate. The organic phases are combined, dried, concentrated, and purified by column chromatography to afford 21.6 g of compound 49, with a two-step yield of 84.2%.

[0214] Compound 49 (21.6 g, 42.0 mmol, 1.0 eq) was dissolved in 200 mL of methanol, and 4.7 g (84.0 mol, 2.0 eq) of potassium hydroxide (prepared into a 3.0 mol / L aqueous solution) was added. The mixture was allowed to react at room temperature for 2 h. After completion of the reaction, the solvent was removed by TLC monitoring, and the mixture was concentrated under reduced pressure. The mixture was then purified by flash column chromatography to obtain 16.9 g of compound 50 (96.1% yield). 1 H NMR(400MHz,DMSO-d6)δ7.40(d,J=7.4Hz,2H),7.33–7.16(m,7H),6.88(d,J=8.9Hz ,4H),4.59(s,1H),4.13(s,1H),3.73(s,6H),3.46–3.40(m,1H),3.17(s,1H),2.89( ddd,J=16.5,9.9,5.5Hz,2H),2.78(dd,J=8.8,5.8Hz,1H),2.64(dd,J=11.2,2.7Hz, 1H),1.70(dd,J=13.1,6.9Hz,1H),1.40(ddd,J=12.9,8.5,5.9Hz,1H).MS(ESI):m / z calcd for C 26 H 29 NO4[MH] - :418.20,found:418.11.

[0215] Example 14 Synthesis of Compound 56

[0216] Synthesis of compound 52

[0217] Compound 13 (31 g, 79.66 mmol) was dissolved in anhydrous methanol (310 mL), and sodium methoxide (3.44 g, 63.73 mmol, 0.8 eq.) was added. The system was stirred at room temperature under nitrogen. After approximately 1 h of reaction, the reaction was monitored by TLC and LC-MS. After completion of the reaction, a cation exchange resin was added until the system became neutral. The mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 10:1 to 5:1) to obtain Compound 52 (15 g, yield: 81%) as a white solid. MS (ESI): m / z calculated for C8H 15 NO6[M+H] + :222.09,found:222.07.

[0218] Synthesis of compound 53

[0219] Compound 52 (10.5 g, 47.49 mmol) was dissolved in dry pyridine (210 mL), and TBDPSCl (13.5 mL, 52.24 mmol, 1.1 eq.) and 4-dimethylaminopyridine (1.16 g, 9.50 mmol, 0.2 eq.) were added. The reaction system was stirred at room temperature under nitrogen. After 4 h of reaction, TLC and LC-MS monitoring revealed that the reaction of the starting compound 52 was complete and the subsequent steps were carried out in one pot without post-treatment. MS (ESI): m / z calculated for C 24 H 33 NO6Si[M+H] + :460.21,found:460.22.

[0220] Synthesis of compound 54

[0221] The reaction system was placed at 0°C, and benzoyl chloride (22 mL, 189.89 mmol, 4 eq.) and 4-dimethylaminopyridine (3.48 g, 28.48 mmol, 0.6 eq.) were added dropwise. After the addition was complete, the reaction system was stirred at room temperature under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After approximately 12 hours of reaction at room temperature, the reaction was complete. The reaction system was diluted with ethyl acetate and washed sequentially with water, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain Compound 54 (24.7 g, 68% yield over two steps) as a white solid. MS (ESI): m / z calcd for C 45 H 45 NO9Si[M+H] + :772.29,found:772.26.

[0222] Synthesis of compound 55

[0223] Compound 54 (24.7 g, 32 mmol) was dissolved in anhydrous tetrahydrofuran (247 mL), and triethylamine trihydrofluoride (26 mL, 160 mmol, 5 eq.) was added. The reaction system was stirred at 50°C under nitrogen protection, and the reaction was monitored by TLC and LC-MS. After about 12 hours of reaction at room temperature, the reaction was complete. The reaction system was directly concentrated under reduced pressure to remove most of the solvent, diluted with ethyl acetate, and extracted with water, saturated sodium bicarbonate solution, and saturated brine in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:1) to obtain compound 55 (11.6 g, yield: 68%) as a white solid. MS (ESI): m / z calcd for C 29 H 27 NO9[M+H] + :554.17,found:554.15.

[0224] Synthesis of compound 56

[0225] Compound 55 (5.0 g, 9.37 mmol) was dissolved in anhydrous tetrahydrofuran (75 mL) and the mixture was placed at 0°C. PPh3 (4.93 g, 18.74 mmol, 2 eq.) and DPPA (4.0 mL, 18.74 mmol, 2 eq.) were added, and DIAD (3.7 mL, 18.74 mmol, 2 eq.) was slowly added dropwise. The mixture was stirred at 0°C under nitrogen for 4 h, then allowed to react at room temperature for approximately 12 h. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1 to 3:1) to obtain Compound 56 (3.0 g, 60% yield) as a white solid. 1 H NMR (400MHz, DMSO) δ8.24–8.20(m,2H),7.96(d,J=7.2Hz,2H),7.78–7.70(m ,6H),7.65–7.56(m,5H),7.43(t,J=7.8Hz,5H),6.48(d,J=3.5Hz,1H),5.87 (d,J=3.1Hz,1H),5.69(dd,J=11.7,3.1Hz,1H),4.90–4.82(m,1H),4.76–4. 64(m,1H),3.58–3.50(m,1H),3.49–3.40(m,1H),1.72(s,3H).MS(ESI):m / z calcd for C 29 H 26 N4O8[M+H] + :559.18,found:559.17.

[0226] Example 15 Synthesis of Compounds 57-64

[0227] Synthesis of compound 57

[0228] Compound 56 (800 mg, 1.432 mmol) was dissolved in dry 1,2-DCE (15 mL). The reaction mixture was heated to 0°C and trimethylsilyl trifluoromethanesulfonate (0.4 mL, 2.148 mmol, 1.5 eq.) was added. The reaction mixture was stirred at 0°C under nitrogen for 10 min and then placed in a 50°C oil bath for 12 h. TLC and LC-MS confirmed the complete reaction. The mixture was then heated to room temperature over 4A molecular sieves and stirred for 30 min. Anhydrous 3-pentanol (0.24 mL, 2.148 mmol, 1.5 eq.) was then added. The reaction mixture was stirred at room temperature under nitrogen for 12 h. TLC and LC-MS confirmed the complete reaction. After completion of the reaction, triethylamine was added until the mixture was neutral. The mixture was then extracted sequentially with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain compound 57 (400 mg, yield: 53%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ8.01–7.91(m,3H),7.75–7.68(m,3H),7.63–7.53(m,3H) ),7.40(t,J=7.8Hz,2H),5.62(d,J=3.4Hz,1H),5.34(dd,J=11.1,3.4Hz,1H),4 .77(d,J=8.5Hz,1H),4.26–4.15(m,2H),3.60–3.49(m,2H),3.32(d,J=3.7Hz,1 H),1.70(s,3H),1.61–1.42(m,4H),0.87(dt,J=17.7,7.4Hz,6H).MS(ESI):m / z calcd for C 27 H 32 N4O7[M+H] + :525.23,found:525.25.

[0229] Synthesis of compound 58

[0230] The synthesis of compound 58 was carried out by referring to compound 57, using compound 56 (1.5 g, 2.96 mmol), trimethylsilyl trifluoromethanesulfonate (730 μL, 4.03 mmol, 1.5 eq.), 1,2-DCE (50 mL) and anhydrous isopropanol (610 μL, 8.88 mmol, 3 eq.) as raw materials to obtain compound 58 (900 mg, yield: 68%). 1H NMR (400MHz, DMSO-d6) δ8.11–7.93(m,3H),7.78–7.63(m,3H),7.55–7.43(m,3H),7.38(t,J=7.3Hz,2H),5.62(d,J=3.4Hz,1H),5.34(dd,J=1 1.1,3.4Hz,1H),4.85(d,J=8.5Hz,1H),4.16–4.07(m,2H),3.61–3.50(m,2H),3.27(d,J=3.7Hz,1H),1.72(s,3H),0.83(m,6H).MS(ESI):m / z calcd for C 25 H 28 N4O7[M+H] + :497.21,found:497.20.

[0231] Synthesis of compound 59

[0232] The synthesis of compound 59 was carried out by referring to compound 57, using compound 56 (1500 mg, 2.96 mmol), trimethylsilyl trifluoromethanesulfonate (730 μL, 4.03 mmol, 1.5 eq.), 1,2-DCE (50 mL) and anhydrous ethanol (370 mg, 8.88 mmol, 3 eq.) as raw materials to obtain compound 59 (1000 mg, yield: 70%). 1 H NMR (400MHz, CDCl3) δ8.03(d,J=7.3Hz,2H),7.75(d,J=7.5Hz,2H),7.56(t,J=7.4Hz,1H),7.42(t,J =7.5Hz,3H),7.23(t,J=7.8Hz,2H),5.67–5.57(m,2H),5.49(d,J=8.7Hz,1H),4.85(d,J=8.3Hz,1H) ,4.18(dd,J=19.0,8.6Hz,1H),3.98(tt,J=13.9,6.9Hz,2H),3.61(dd,J=9.6,7.1Hz,1H),3.55(dd, J=13.0,8.4Hz,1H),3.18(dd,J=13.0,3.8Hz,1H),1.83(s,3H),1.21(t,J=7.1Hz,3H).MS(ESI):m / z calcd for C 24 H 26 N4O7[M+H] + :483.18,found:483.19.

[0233] Synthesis of compound 60

[0234] The synthesis of compound 60 was carried out by referring to compound 57, using compound 56 (1500 mg, 2.96 mmol), trimethylsilyl trifluoromethanesulfonate (730 μL, 4.03 mmol, 1.5 eq.), 1,2-DCE (50 mL) and anhydrous methanol (280 mg, 8.88 mmol, 3 eq.) as raw materials to obtain compound 60 (1040 mg, yield: 75%). 1 H NMR (400MHz, CDCl3) δ8.10(d,J=7.3Hz,2H),7.82(d,J=7.5Hz,2H),7.63(t,J=7.4Hz,1H),7 .49(t,J=7.5Hz,3H),7.30(t,J=7.7Hz,2H),5.70(d,J=2.9Hz,1H),5.61(dd,J=11.1,3.3Hz, 1H), 5.49 (d, J=8.7Hz, 1H), 4.77 (d, J=8.3Hz, 1H), 4.33 (dd, J=19.7, 8.8Hz, 1H), 4.05 (dd, J= 8.3,3.5Hz,1H),3.67–3.54(m,4H),3.24(dd,J=13.0,3.8Hz,1H),1.90(s,3H).MS(ESI):m / z calcd for C 23 H 24 N4O7[M+H] + :469.16,found:469.17.

[0235] Synthesis of compound 61

[0236] The synthesis of compound 61 was carried out by referring to compound 57, using compound 56 (1500 mg, 2.96 mmol), trimethylsilyl trifluoromethanesulfonate (730 μL, 4.03 mmol, 1.5 eq.), 1,2-DCE (50 mL) and ethanethiol (550 mg, 8.88 mmol, 3 eq.) as raw materials to obtain compound 61 (930 mg, yield: 63%). 1H NMR (400MHz, DMSO) δ8.00(d,J=9.5Hz,1H),7.93(d,J=7.2Hz,2H),7.72(t,J=7.4Hz,3H),7.59(t,J=7 .6Hz, 3H), 7.40 (t, J = 7.8Hz, 2H), 5.67 (d, J = 3.1Hz, 1H), 5.32 (dd, J = 10.7, 3.2Hz, 1H), 4.86 (d, J = 10. 4Hz, 1H), 4.40 (q, J=10.3Hz, 1H), 4.26 (dd, J=8.2, 3.9Hz, 1H), 3.55 (dd, J=13.0, 8.2Hz, 1H), 3.36 (d, J=3.9Hz,1H),2.88–2.75(m,1H),2.74–2.64(m,1H),1.70(s,3H),1.28(t,J=7.4Hz,3H).MS(ESI):m / z calcd for C 24 H 26 N4O6S[M+H] + :499.16,found:499.15.

[0237] Synthesis of compound 62

[0238] The synthesis of compound 62 was carried out by referring to compound 57, using compound 56 (150 mg, 0.296 mmol), trimethylsilyl trifluoromethanesulfonate (73 μL, 0.403 mmol, 1.5 eq.), 1,2-DCE (5.0 mL) and n-pentanol (96 μL, 0.888 mmol, 3 eq.) as raw materials to obtain compound 62 (119 mg, yield: 74%). 1 H NMR (400MHz, CDCl3) δ8.03(d,J=7.5Hz,2H),7.74(d,J=7.4Hz,2H),7.55(t,J=7.4Hz,1H),7.41(t, J=7.5Hz,3H),7.22(t,J=7.7Hz,2H),5.62(dd,J=17.5,5.9Hz,3H),4.80(d,J=8.3Hz,1H),4.21(dd, J=18.7,8.7Hz,1H),4.01(dd,J=8.2,3.6Hz,1H),3.96–3.85(m,1H),3.54(dd,J=13.1,8.6Hz,1H), 3.48(dd,J=16.7,7.4Hz,1H),3.17(dd,J=13.1,3.7Hz,1H),1.81(s,3H),1.18(s,9H).MS(ESI):m / z calcd for C 27 H32 N4O7[M+H] + :524.22,found:524.30.

[0239] Synthesis of compound 63

[0240] The synthesis of compound 63 was carried out by referring to compound 57. Compound 63 (90 mg, yield: 68%) was obtained using compound 56 (150 mg, 0.296 mmol), trimethylsilyl trifluoromethanesulfonate (73 μL, 0.403 mmol, 1.5 eq.), 1,2-DCE (5.0 mL) and benzyl alcohol (61 μL, 0.807 mmol, 3 eq.) as raw materials. 1 H NMR (400MHz, CDCl3) δ8.14(d,J=7.6Hz,2H),7.83(d,J=7.8Hz,2H),7.66(t,J=7.4Hz,1H),7.52(t,J=7.8Hz,3H) ,7.46–7.35(m,5H),7.32(t,J=7.7Hz,2H),5.71(d,J=3.1Hz,1H),5.55(dd,J=11.2,3.3Hz,1H),5.38(d,J=8.8H z,1H),5.05(d,J=12.2Hz,1H),4.83(d,J=8.4Hz,1H),4.75(d,J=12.1Hz,1H),4.46(dd,J=19.7,8.7Hz,1H),4.0 5(dd,J=8.5,3.7Hz,1H),3.68(dd,J=13.1,8.5Hz,1H),3.28(dd,J=13.0,3.7Hz,1H),1.89(s,3H).MS(ESI):m / z calcd for C 29 H 28 N4O7[M+H] + :545.20,found:545.22.

[0241] Synthesis of compound 64

[0242] The synthesis of compound 64 was carried out by referring to compound 57, using compound 56 (150 mg, 0.296 mmol), trimethylsilyl trifluoromethanesulfonate (73 μL, 0.403 mmol, 1.5 eq.), 1,2-DCE (5.0 mL) and 1-hexadecanol (216 mg, 0.888 mmol, 3 eq.) as raw materials to obtain compound 64 (130 mg, yield: 65%). 1H NMR (400MHz, CDCl3) δ8.10(d,J=7.5Hz,2H),7.81(d,J=7.4Hz,2H),7.62(t,J=7.4Hz,1H),7.48(t,J= 7.5Hz,3H),7.29(t,J=7.7Hz,2H),5.70(s,1H),4.87(d,J=8.3Hz,1H),4.28(dd,J=18.7,8.7Hz,1H), 4.08(dd,J=8.2,3.6Hz,1H),4.04–3.94(m,1H),3.61(dd,J=13.1,8.6Hz,1H),3.55(dd,J=16.7,7.4H z,1H),3.24(dd,J=13.1,3.7Hz,1H),1.88(s,3H),1.25(s,30H),0.87(t,J=6.7Hz,3H).MS(ESI):m / z calcd for C 38 H 54 N4O7[M+H] + :679.40,found:679.45.

[0243] Example 16 Synthesis of Compound 65

[0244] Referring to the synthesis of compound 57, compound 13 (116.8 mg, 0.296 mmol), trimethylsilyl trifluoromethanesulfonate (73 μL, 0.403 mmol, 1.5 eq.), 1,2-DCE (5.0 mL), and 6-azido-1-hexanol (127 mg, 0.888 mmol, 3 eq.) were used as starting materials to obtain compound 65 (123 mg, yield: 72%). MS (ESI): m / z calcd for C 28 H 33 N7O7[M+H] + :580.24,found:580.26.

[0245] Example 17 Synthesis of Compound 73

[0246] Synthesis of compound 66

[0247] A 100 mL three-necked flask was charged with 3.9 g (10 mmol, 1.0 eq) of compound 13. A reflux condenser was installed and the atmosphere was purged with nitrogen three times. Subsequently, 20 mL of dichloromethane (water ≤ 50 ppm) was added. 1.1 mL (12 mmol, 1.2 eq) of TiCl₄ was slowly added dropwise at 0°C for 30 minutes. The mixture was then refluxed at 50°C for 16 hours. The endpoint was determined by TLC and LC-MS. After completion of the reaction, saturated aqueous sodium chloride solution was added to quench the reaction. The aqueous phase was extracted with dichloromethane, and the organic phases were combined. The organic phase was dried and concentrated under reduced pressure to obtain the desired product 66, which was directly carried out in the next step.

[0248] Synthesis of compound 67

[0249] A 100 mL three-necked flask was charged with 0.37 g (1.0 mmol, 1.0 eq) of compound 66. A reflux condenser was installed and the atmosphere was purged with nitrogen three times. Subsequently, 20 mL of toluene (water ≤ 50 ppm) was added, followed by the dropwise addition of 0.27 mL (1.2 mmol, 1.2 eq) of BuSnH and 0.03 g (0.2 mmol, 0.2 eq) of azobisisobutyronitrile (dissolved in 2 mL of toluene). The reaction was then refluxed at 100°C for 3 hours. The endpoint was determined by TLC and LC-MS. After completion of the reaction, the system was directly concentrated under reduced pressure to remove most of the toluene. Petroleum ether and acetonitrile were then added for extraction, and the acetonitrile phase was combined and collected. The product was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate = 4:1 to 1:1) to obtain the desired product as a white solid (254.9 mg, 77% yield).

[0250] Synthesis of compound 68

[0251] To a 100 mL round-bottom flask was added 0.33 g (1.0 mmol, 1.0 eq) of compound 67, followed by 20 mL of methanol and 0.05 g (1.0 mmol, 1.0 eq) of sodium methoxide. The mixture was allowed to react at room temperature for 3 hours, and the endpoint was determined by TLC and LC-MS. After the reaction, HCl (in MeOH) was added dropwise in an ice bath until the reaction was neutral. The system was concentrated under reduced pressure and directly proceeded to the next step.

[0252] Synthesis of compound 69

[0253] A 100 mL round-bottom flask was charged with 0.21 g (1.0 mmol, 1.0 eq) of compound 68, followed by 25 mL of N,N-dimethylformamide and 0.13 g (2.0 mmol, 2.0 eq) of imidazole. The atmosphere was then purged with nitrogen three times, and 0.26 mL (1.0 mmol, 1.0 eq) of TBDPSCl was slowly added dropwise at 0°C. The reaction was allowed to react at room temperature for 3 hours, and the endpoint was determined by TLC and LC-MS. After completion of the reaction, the reaction was quenched with saturated brine and extracted with ethyl acetate. The organic phases were combined, dried, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 4:1 to 1:1) to obtain the desired product as a white solid (430.3 mg, 97% yield).

[0254] Synthesis of compound 70

[0255] To a 100 mL round-bottom flask was added 0.44 g (1.0 mmol, 1.0 eq) of compound 69, followed by 20 mL of pyridine, 0.06 g (0.5 mmol, 0.5 eq) of 4-dimethylaminopyridine, and 0.76 g (3.5 mmol, 3.5 eq) of benzoic anhydride. The mixture was allowed to react at room temperature for 16 hours, and the endpoint was determined by TLC and LC-MS. After completion of the reaction, the reaction was quenched with saturated brine and extracted with ethyl acetate. The organic phases were combined, dried, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to afford the desired product as a white solid (547.5 mg, 84% yield). 1 H NMR (400MHz, DMSO) δ8.03(d,J=8.2Hz,1H),7.90(d,J=7.5Hz,2H),7.77(d,J=7.8Hz,2H),7.72(d,J= 7.3Hz,1H),7.59(dd,J=12.1,6.9Hz,5H),7.43(dt,J=11.9,6.8Hz,7H),7.31(t,J=7.4Hz,1H),7.10( t,J=7.4Hz,2H),5.95(s,1H),5.31(dd,J=11.0,2.4Hz,1H),4.55–4.28(m,1H),4.16(t,J=6.8Hz,1H) ,3.95(dd,J=10.9,4.9Hz,1H),3.65–3.54(m,2H),3.40(t,J=11.1Hz,1H),1.73(s,3H),0.90(s,9H).

[0256] Synthesis of compound 71

[0257] To a 100 mL round-bottom flask, 0.65 g (1.0 mmol, 1.0 eq) of compound 70 was added, followed by 10 mL of tetrahydrofuran. Once dissolved, 0.81 mL (5.0 mmol, 5.0 eq) of triethylamine hydrofluoride was slowly added dropwise. The reaction was allowed to proceed at 50°C for 16 hours, and the endpoint was determined by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure, dissolved in ethyl acetate, and washed three times with saturated brine. The organic phases were combined. The organic phases were dried, concentrated, and purified by column chromatography to yield the desired product as a white solid (334.8 mg, 81% yield). 1 H NMR (400MHz, DMSO) δ7.65(dd,J=7.0,5.7Hz,5H),7.51–7.31(m,6H),4.67(d,J=6.2Hz,1H),4.56(d,J=4.6Hz,1H),3.96–3.83(m,1 H),3.83–3.76(m,2H),3.69(ddd,J=16.6,10.4,5.7Hz,2H),3.44–3.36(m,2H),2.88(t,J=10.8Hz,1H),1.81(s,3H),0.99(s,9H).

[0258] Synthesis of compound 72

[0259] To a 100 mL round-bottom flask, 0.41 g (1.0 mmol, 1.0 eq) of compound 71 was added, followed by 20 mL of dichloromethane. After dissolution, 0.42 mL (3.0 mmol, 3.0 eq) of triethylamine was added. The mixture was reacted at 0°C and 0.33 g (1.5 mmol, 1.5 eq) of p-nitrobenzenesulfonyl chloride was added portionwise. The reaction was allowed to react at room temperature for 18 hours. The endpoint was determined by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain the desired product as a white solid (586.6 mg, 98% yield). 1H NMR (400MHz, DMSO) δ8.31(d,J=8.6Hz,2H),8.10(d,J=8.7Hz,2H),7.97(d,J=8.2Hz,1H),7.77(d ,J=7.5Hz,2H),7.69(d,J=7.5Hz,2H),7.65(d,J=7.4Hz,1H),7.59(t,J=7.3Hz,1H),7.50(t,J=7 .6Hz,2H),7.40(t,J=7.6Hz,2H),5.66(s,1H),5.21(d,J=11.1Hz,1H),4.40–4.30(m,1H),4.27( d,J=5.6Hz,1H),4.22(s,2H),3.94(dd,J=11.1,5.0Hz,1H),3.36(d,J=11.3Hz,1H),1.71(s,3H).

[0260] Synthesis of compound 73

[0261] To a 100 mL round-bottom flask, 0.60 g (1.0 mmol, 1.0 eq) of compound 72 was added, followed by 20 mL of acetonitrile (water ≤ 30 ppm). After dissolution, 0.39 mL (3.0 mmol, 3.0 eq) of trimethylsilyl azide was added, followed by the dropwise addition of 3 mL (3.0 mmol, 3.0 eq) of tetrabutylammonium fluoride (1 M in tetrahydrofuran). The reaction was allowed to react at room temperature for 18 hours, and endpoint was determined by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure, dissolved in ethyl acetate, and washed three times with aqueous citric acid. The organic phases were combined, dried, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 4:1 to 1:1) to afford the desired product as a white solid (0.33 g, 75% yield). 1 H NMR (400MHz, DMSO) δ8.00(d,J=8.2Hz,1H),7.93(d,J=7.9Hz,2H),7.71(t,J=8. 2Hz,3H),7.58(q,J=7.5Hz,3H),7.41(t,J=7.6Hz,2H),5.69(d,J=2.1Hz,1H),5. 25(dd,J=11.0,2.7Hz,1H),4.50–4.39(m,1H),4.12–4.07(m,1H),4.02(dd,J=11 .1,5.0Hz,1H),3.52–3.43(m,1H),3.42–3.33(m,2H),1.73(s,3H).MS(ESI):m / z calcd for C 22 H 22 N4O6[M+H] + :439.16,found:439.11.

[0262] Example 18 Synthesis of Compound 78

[0263] Compound 13 (20.0 g, 51.2 mmol, 1.0 eq) was weighed and dissolved in 200 mL of ultra-dry dichloromethane. The mixture was cooled to 5°C in an ice-water bath under nitrogen atmosphere. TiCl4 (11.6 g, 61.5 mmol, 1.2 eq) was added dropwise and the mixture was heated to reflux. After completion of the reaction as monitored by TLC, aqueous sodium bicarbonate was added to quench the reaction. The mixture was extracted with dichloromethane, separated, dried, and concentrated to yield 18.8 g of an oil. The oil was dissolved in 200 mL of tetrahydrofuran, followed by the addition of allyltributyltin (50.8 g, 153.6 mmol, 3.0 eq) and azobisisobutyronitrile (2.5 g, 15.4 mmol, 0.3 eq). The reaction was allowed to react at 50°C. After completion of the reaction as monitored by TLC, the solvent was removed by concentration and purified by column chromatography (dichloromethane:methanol = 10:1 to 5:1) to yield 5.0 g of compound 74, with a two-step yield of 26.3%.

[0264] Compound 74 (2.5 g, 6.7 mmol, 1.0 eq) was weighed and dissolved in 50 mL of dichloromethane. 50 mL of 20% acetic acid was added, followed by 4.1 g of trioctylmethylammonium chloride (10.1 mmol, 1.5 eq). The mixture was cooled in an ice-water bath and portionwise added with 3.9 g of potassium permanganate (10.1 mmol, 3.7 eq). After the addition, the mixture was allowed to react at room temperature overnight. After completion of the reaction, as monitored by TLC, sodium sulfite was added to quench the reaction. The mixture was extracted with dichloromethane, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to afford 2.1 g of compound 75 in a yield of 79.6%.

[0265] 1.0 g of compound 75 (2.56 mmol, 1.0 eq) and 1.26 g of compound 16 (2.56 mmol, 1.0 eq) were dissolved in 10 mL of N,N-dimethylformamide. Under nitrogen, 0.66 g of N,N-diisopropylethylamine (5.12 mmol, 2.0 eq) and 1.16 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (3.07 mmol, 1.2 eq) were added. The mixture was allowed to react at room temperature for 2 h. The reaction was monitored by TLC. The mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 1.0 g of compound 76 in a yield of 77.3%.

[0266] 1.0 g of compound 76 (2 mmol, 1 eq) and 1.26 g of compound 3 (2 mmol, 1.0 eq) were dissolved in 10 mL of N,N-dimethylformamide. Under nitrogen, 0.51 g (4 mmol, 2.0 eq) of N,N-diisopropylethylamine and 0.93 g (2.4 mmol, 1.2 eq) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were added. The mixture was allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 1.33 g of compound 77 in a yield of 75.5%.

[0267] Compound 77 (0.88 g, 1.0 mmol, 1.0 eq) and 4-dimethylaminopyridine (24 mg, 0.2 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 20 mL of anhydrous dichloromethane and 0.26 g (2.0 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.28 g, 1.2 mmol, 1.2 eq) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 0.46 g of compound 78 in a yield of 42.8%. The isomer ratio:dr = 4:1. 1H NMR(400MHz, DMSO-d6)δ8.03(d,J=7.8Hz,0.83H),7.90(d,J=9.1Hz,0.2H),7.76(t,J=5.5Hz,0.91H) ,7.65(t,J=5.8Hz,0.24H),7.59(t,J=5.7Hz,1H),7.40(dd,J=7.5,1.8Hz,2H),7.33–7.18(m,9H),6. 87(dd,J=8.8,6.5Hz,5H),5.75(s,1H),5.32–5.25(m,1H),4.94(td,J=13.8,12.6,5.4Hz,1H),4.42( dt,J=11.3,4.6Hz,1H),4.34–4.21(m,2H),4.02(td,J=11.6,5.9Hz,2H),3.95–3.85(m,1H),3.84–3. 76(m,1H),3.73(d,J=2.8Hz,8H),3.51(dq,J=10.0,6.6Hz,2H),3.38–3.29(m,1H),3.21(dd,J=12.9, 6.4Hz,2H),3.07–2.94(m,4H),2.91(dd,J=9.6,5.0Hz,1H),2.77(t,J=6.1Hz,2H),2.68–2.61(m,1H) ,2.21(d,J=6.1Hz,0.2H),2.14(dd,J=14.3,3.8Hz,1H),2.08(d,J=4.6Hz,3H),2.01–1.92(m,8H),1. 79(d,J=14.2Hz,4H),1.37(ddd,J=24.3,13.1,7.3Hz,5H),1.24–1.10(m,11H),1.01(d,J=6.7Hz,5H). 31 P NMR(162MHz,DMSO)δ148.42,147.92.MS(ESI):m / zcalcd for C 55 H 76 N5O 15 P[M-DMTr] + :774.37,found:774.34.

[0268] Example 19 Synthesis of Compound 81

[0269] 2.0 g (7.7 mmol, 1.0 eq) of compound 79 was dissolved in 20 mL of pyridine and the atmosphere was replaced with nitrogen. The mixture was cooled to 0°C in an ice-water bath and 2.6 g (7.7 mmol, 1.0 eq) of 4,4'-bis(methoxytrityl) chloride was added portionwise. After the addition, the mixture was allowed to react at room temperature overnight. After TLC monitoring, the reaction was quenched with water and extracted twice with ethyl acetate. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain 3.3 g of compound 80 in a yield of 75.6%.

[0270] Compound 80 (2.0 g, 3.6 mmol, 1.0 eq) and 4-dimethylaminopyridine (87 mg, 0.7 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 20 mL of anhydrous dichloromethane and 0.93 g (7.2 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.0 g, 4.3 mmol, 1.5 eq) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 1.6 g of compound 81 in a yield of 59.3%. 1 H NMR(400MHz,DMSO-d6)δ7.41(d,J=7.8Hz,2H),7.36–7.15(m,7H),6.87(dd,J=8.8,6.7Hz ,4H),3.90(tt,J=10.7,5.4Hz,1H),3.73(d,J=2.7Hz,6H),3.68–3.47(m,3H),3.03(ddd,J =30.6,9.5,5.3Hz,2H),2.73(t,J=5.9Hz,1H),2.59(dt,J=10.0,4.5Hz,1H),1.7–1.43(m, 2H),1.35–1.17(m,24H),1.13(t,J=6.7Hz,9H),1.02(d,J=6.7Hz,3H),0.88–0.80(m,3H). 31 P NMR (162MHz, DMSO) δ147.57,147.25.

[0271] Example 20 Synthesis of Compound 85

[0272] Synthesis of compound 82

[0273] Compound 57 (1.0 g, 1.90 mmol) was dissolved in dry dichloromethane / ethyl acetate (v:v = 1:1, 100 mL) and Pd / C (300 mg) was added. The system was replaced with H2 three times and stirred at room temperature under hydrogen for 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was filtered and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 82 (420 mg, 44%). MS (ESI): m / z calculated for C 27 H 34 N2O7[M+H] + :499.24,found:499.25.

[0274] Synthesis of compound 83

[0275] Compound 24 (1.4 g, 2.86 mmol) and monomethyl dodecanedioate (0.698 g, 2.86 mmol) were dissolved in dry dichloromethane (60 mL). Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1.19 g, 3.14 mmol, 1.1 eq.) and N,N-diisopropylethylamine (1.11 g, 6.57 mmol, 3 eq.) were added. The reaction was stirred at room temperature under nitrogen for 2 h and monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:5 to 1:10) to obtain the intermediate (1.9 g, 95%). This was dissolved in methanol (50 mL), and a solution of NaOH (318 mg, 7.96 mmol, 3 eq.) in water (10 mL) was added. The reaction was stirred at room temperature for 2 h and monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (dichloromethane:methanol = 20:5 to 10:1) to afford compound 83 (1.7 g, 91%) as a colorless syrup. MS (ESI): m / z calculated for C 41 H 54 N2O8[M+H] + :703.39,found:703.33.

[0276] Synthesis of compound 84

[0277] Compound 83 (710 mg, 1.012 mmol) and compound 82 (420 g, 0.842 mmol) were dissolved in dry dichloromethane (20 mL). Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (799 mg, 2.105 mmol, 2.5 eq.) and N,N-diisopropylethylamine (0.34 mL, 2.021 mmol, 2.4 eq.) were added. The reaction was stirred at room temperature under nitrogen for 12 h and monitored by TLC and LC-MS. After completion of the reaction, the system was directly concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified on a C18 reverse-phase column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to obtain compound 84 (480 mg, 48%). MS (ESI): m / z calculated for C 68 H 86 N4O 14 [M+H] + :1183.62,found:1183.61.

[0278] Synthesis of compound 85

[0279] Compound 84 (685 mg, 0.579 mmol) was dissolved in dry dichloromethane (12 mL), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.2 mL, 0.869 mmol, 1.5 eq.), 4-dimethylaminopyridine (14 mg, 0.116 mmol, 0.2 eq.), and N,N-diisopropylethylamine (0.2 mL, 1.158 mmol, 2 eq.) were added. The reaction was stirred at room temperature under nitrogen for 0.5 h and monitored by TLC and LC-MS. After completion of the reaction, the system was directly concentrated under reduced pressure to obtain the crude product, which was then purified on a C18 reverse-phase column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to yield compound 85 (634 mg, 79%). 1H NMR (400MHz, DMSO) δ7.94–7.90(m,3H),7.74–7.66(m,3H),7.61–7.54(m,3H),7.42–7.37(m,4H),7.32(t,J=7.6H z,2H),7.28–7.22(m,6H),6.90(d,J=8.3Hz,4H),5.54(d,1H),5.29(dd,J=11.1,3.0Hz,1H),4.66(d,J=8.4Hz,1H) ,4.64–4.53(m,1H),4.29–4.11(m,5H),4.03(t,1H),3.53–3.17(m,15H),2.95–2.87(m,3H),2.28–2.17(m,2H),2 .09–1.99(m,4H),1.95–1.74(m,3H),1.62–1.39(m,10H),1.22(t,J=7.8Hz,17H),0.91–0.79(m,6H); MS(ESI):m / z calcd for C 77 H 103 N6O 15 P[M+H] + :1382.72,found:1382.74.

[0280] Example 21 Synthesis of Compound 90

[0281] The synthesis procedure of compound 88 refers to the synthesis of compound 43.

[0282] Compound 88 (612 mg, 1.0 mmol, 1.0 eq) and compound 57 (524 mg, 1.0 mmol, 1.0 eq) were weighed and dissolved in 10 mL of methanol. 500 mg of copper sulfate pentahydrate (2.0 mmol, 2.0 eq) and 400 mg of sodium ascorbate (2.0 mmol, 2.0 eq) were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC. After concentration, the mixture was purified on a reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to afford 910 mg of compound 89 as a white solid in an 80% yield.

[0283] Compound 89 (569 g, 0.5 mmol, 1.0 eq) and 4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and N,N-diisopropylethylamine (132 mg, 1.02 mmol, 2.0 eq) were added. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (181 mg, 0.77 mmol, 1.5 eq) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified by reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 430 mg of compound 90 as a white solid in a 65.2% yield. 1 H NMR(400MHz,DMSO-d6)δ7.96–7.89(m,3H),7.91–7.80(m,1H),7.78–7.65(m,4 H),7.55(t,J=7.5Hz,3H),7.46–7.36(m,4H),7.36–7.16(m,7H),6.93–6.82(m, 4H),5.63(d,J=3.5Hz,1H),5.34(dt,J=11.3,3.7Hz,1H),4.56(dt,J=9.8,6.3 Hz,2H),4.52–4.41(m,2H),4.22(tq,J=16.2,7.4,6.9Hz,2H),3.83(dq,J=12.9 ,6.3Hz,1H),3.75(m,J=2.4Hz,7H),3.57(ddt,J=23.6,10.2,6.3Hz,2H),3.27 (dt,J=16.0,5.8Hz,3H),3.09–2.98(m,2H),2.85–2.68(m,3H),2.68–2.51(m,3 H),2.12-2.07(m,3H),1.90–1.79(m,3H),1.68–1.51(m,J=1.7Hz,4H),1.46–1. 27(m,4H),1.21–1.11(m,9H),1.05(dd,J=6.8,4.4Hz,3H),0.83–0.65(m,12H). 31 P NMR(162MHz,DMSO)δ148.32,148.11.MS(ESI):m / z calcd for C 73 H 93 N8O 14 P[MH] - :1335.64,found:1335.51.

[0284] Example 22 Synthesis of Compound 94

[0285] Compound 24 (1.7 g, 3.5 mmol, 1.0 eq) and valeric acid (0.4 g, 4.2 mmol, 1.2 eq) were dissolved in 10 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (0.9 g, 7.0 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.6 g, 4.2 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford compound 92 as a white solid (1.2 g, yield: 60.9%).

[0286] Compound 92 (570 mg, 1.0 mmol, 1.0 eq) and compound 57 (524 mg, 1.0 mmol, 1.0 eq) were weighed and dissolved in 10 mL of methanol. 500 mg of copper sulfate pentahydrate (2.0 mmol, 2.0 eq) and 400 mg of sodium ascorbate (2.0 mmol, 2.0 eq) were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC. After concentration, the mixture was purified on a reverse-phase preparative column (5%-95% acetonitrile / water, with the aqueous phase containing 0.01% ammonium bicarbonate) to afford 560 mg of compound 93 in a yield of 51.2%.

[0287] Compound 93 (560 g, 0.51 mmol, 1.0 eq) and 4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and 132 mg (1.02 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. 181 mg (0.77 mmol, 1.5 eq) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified by reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 430 mg of compound 94 as a white solid in a 65.2% yield. 1H NMR(400MHz,DMSO-d6)δ7.98–7.91(m,3H),7.91–7.80(m,1H),7.78–7.63(m,4H), 7.59(t,J=7.5Hz,3H),7.46–7.36(m,4H),7.36–7.16(m,7H),6.93–6.82(m,4H),5. 66(d,J=3.5Hz,1H),5.34(dt,J=11.3,3.7Hz,1H),4.59(dt,J=9.8,6.3Hz,2H),4.5 2–4.41(m,2H),4.22(tq,J=16.2,7.4,6.9Hz,2H),4.05(dt,J=10.3,5.2Hz,1H),3. 83(dq,J=12.9,6.3Hz,1H),3.73(d,J=2.4Hz,7H),3.57(ddt,J=23.6,10.2,6.3Hz ,2H),3.38(s,1H),3.27(dt,J=16.0,5.8Hz,3H),3.09–2.98(m,1H),2.92–2.68(m, 4H),2.68–2.51(m,2H),2.07(s,3H),1.90–1.77(m,1H),1.68(d,J=1.7Hz,5H),1.4 8–1.22(m,5H),1.21–1.10(m,8H),1.03(dd,J=6.8,4.4Hz,3H),0.83–0.62(m,6H). 31 P NMR(162MHz,DMSO)δ148.36,148.32,148.22,148.16,148.05,147.87.MS(ESI):m / z calcd for C 70 H 86 N8O 14 P[MH] - :1293.60,found:1293.63.

[0288] Example 23 Synthesis of Compound 97

[0289] 10.0 g (102 mmol, 1.0 eq) of valeric acid was weighed and dissolved in 20 mL of acetonitrile. 21.1 g (102 mmol, 1.0 eq) of N,N'-dicyclohexylcarbodiimide was dissolved in acetonitrile and added dropwise to the system. The mixture was allowed to react overnight at room temperature. The reaction was filtered and dried under reduced pressure to yield 20.0 g of a white solid. The 20.0 g white solid was dissolved in 150 mL of tetrahydrofuran and compound 15 (13.3 g, 102 mmol, 1.0 eq) was added. The mixture was allowed to react at room temperature. Mass spectrometry analysis was performed. After completion of the reaction, the mixture was filtered and dried under reduced pressure to yield compound 95 (19.75 g, 92% yield).

[0290] Compound 24 (1.9 g, 3.9 mmol, 1.0 eq) and compound 95 (1.0 g, 4.7 mmol, 1.2 eq) were weighed and dissolved in 10 mL of dichloromethane. Under nitrogen, 1.0 g (7.7 mmol, 2.0 eq) of N,N-diisopropylethylamine and 1.8 g (4.7 mmol, 1.2 eq) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were added. The mixture was allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 1.5 g of compound 96 (yield: 46.8%).

[0291] 846 mg (1.24 mmol, 1.0 eq) of compound 96 and 650 mg (1.24 mmol, 1.0 eq) of compound 57 were dissolved in 10 mL of methanol. 620 mg (2.5 mmol, 2.0 eq) of copper sulfate pentahydrate and 496 mg (2.5 mmol, 2.0 eq) of sodium ascorbate were dissolved in 10 mL of water and added to the system. The reaction was monitored for completion by TLC. After concentration, the product was purified on a reverse-phase preparative column (5%-95% acetonitrile / water, with the aqueous phase containing 0.01% ammonium bicarbonate) to yield 590 mg of the product.

[0292] 590 mg (0.49 mmol, 1.0 eq) of product and 12 mg (0.1 mmol, 0.2 eq) of 4-dimethylaminopyridine were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and 132 mg (1.02 mmol, 2.0 eq) of N,N-diisopropylethylamine were added. 181 mg (0.77 mmol, 1.5 eq) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. Completion of the reaction was monitored by TLC. The product was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 380 mg of compound 97 in a 56.7% yield. 1H NMR (400MHz, DMSO) δ7.95(d,J=7.1Hz,3H),7.86–7.80(m,1H),7.79(t,J=4.5Hz,1H),7.72(t,J=7.9Hz, 3H),7.59(t,J=7.7Hz,3H),7.39(t,J=7.8Hz,4H),7.33–7.18(m,7H),6.87(t,J=7.2Hz,4H),5.66(d,J=3 .2Hz,1H),5.34(dd,J=11.1,3.2Hz,1H),4.59(dd,J=12.4,8.2Hz,2H),4.51–4.38(m,2H),4.28–4.12(m ,2H),4.09–3.96(m,1H),3.87–3.78(m,1H),3.73(d,J=2.1Hz,7H),3.66–3.58(m,1H),3.58–3.50(m,1H) ,3.46–3.36(m,2H),3.25(dd,J=11.4,5.8Hz,2H),3.02(dd,J=12.4,6.2Hz,3H),2.91–2.84(m,1H),2.8 3–2.74(m,3H),2.64(t,J=4.9Hz,1H),2.38(t,J=7.3Hz,2H),2.20(dd,J=11.7,6.5Hz,1H),2.13–1.98(m ,1H),1.84(dt,J=18.1,8.0Hz,1H),1.77–1.55(m,6H),1.52–1.27(m,9H),1.25(d,J=7.0Hz,3H),1.15(d d,J=12.7,6.7Hz,9H),1.04(t,J=6.8Hz,3H),0.79(t,J=7.3Hz,3H),0.73(t,J=7.4Hz,3H).MS(ESI):m / z calcd for C 76 H 98 N9O 15 P[MH] - :1406.68,found:1406.69.

[0293] Example 24 Synthesis of Compound 101

[0294] Compound 24 (1.5 g, 3.1 mmol, 1.0 eq) and compound 98 (0.67 g, 3.7 mmol, 1.2 eq) were weighed and dissolved in 10 mL of dichloromethane. N,N-diisopropylethylamine (0.8 g, 6.1 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (1.4 g, 3.7 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 1.6 g of compound 99 (80.0% yield).

[0295] Compound 99 (655 mg, 1.0 mmol, 1.0 eq) and Compound 57 (524 mg, 1.0 mmol, 1.0 eq) were weighed and dissolved in 10 mL of methanol. Copper sulfate pentahydrate (500 mg, 2.0 eq) and sodium ascorbate (400 mg, 2.0 eq) were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC. After concentration, the product was purified using a reverse-phase preparative column (5%-95% acetonitrile / water, with the aqueous phase containing 0.01% ammonium bicarbonate) to afford 700 mg of Compound 100 (yield: 59.0%).

[0296] Compound 100 (700 mg, 0.6 mmol, 1.0 eq) and 4-dimethylaminopyridine (15 mg, 0.12 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and 153 mg of N,N-diisopropylethylamine (1.2 mmol, 2.0 eq) were added. 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (210 mg, 0.9 mmol, 1.5 eq) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and purified on a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 600 mg of compound 101 in a yield of 73.5%. 1H NMR (400MHz, DMSO-d6) δ7.95 (dd, J=8.7, 4.0Hz, 3H), 7.78 (d, J=8.0Hz, 1H), 7.76–7.69 (m, 3H), 7.59 (t, J=7. 7Hz,3H),7.40(dd,J=9.9,5.6Hz,4H),7.33–7.18(m,7H),6.86(dt,J=9.1,4.6Hz,4H),5.76(s,1H),5.67(d,J =3.3Hz,1H),5.34(dd,J=11.1,3.4Hz,1H),4.60(dd,J=9.5,6.1Hz,2H),4.46(d,J=10.8Hz,2H),4.20(q,J=10 .2,9.8Hz,2H),4.11–3.95(m,1H),3.88–3.75(m,1H),3.73(d,J=2.5Hz,7H),3.67–3.50(m,2H),3.46–3.34(m ,2H),3.29(s,1H),3.27–3.14(m,2H),3.02(dq,J=22.7,8.2Hz,2H),2.87(dp,J=12.2,6.6Hz,1H),2.78(t,J =6.0Hz,1H),2.64(td,J=6.8,6.0,2.9Hz,1H),2.56(q,J=8.5,8.1Hz,3H),2.21(q,J=6.5Hz,1H),2.06(dt,J= 15.5,8.4Hz,1H),1.83(ddd,J=13.9,11.0,6.1Hz,1H),1.74(dd,J=11.8,5.8Hz,1H),1.68(s,3H),1.55(q,J= 7.3Hz,2H),1.46–1.34(m,4H),1.34–1.22(m,8H),1.20–1.10(m,10H),1.07–0.99(m,3H),0.82–0.68(m,6H). 31 P NMR(162MHz,DMSO)δ148.48,148.35,148.23,148.21,148.04,147.98,147.83.MS(ESI):m / z calcd for C 76 H 99 N8O 14 P[MH] - :1377.69,found:1377.66.

[0297] Example 25 Synthesis of Compound 104

[0298] Compound 6 (3.8 g, 8.0 mmol, 1.0 eq) and valeric acid (0.94 g, 9.6 mmol, 1.2 eq) were weighed and dissolved in 30 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (2.1 g, 16.0 mmol, 2.0 eq) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (3.6 g, 9.6 mmol, 1.2 eq) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 3.2 g of compound 102 in a yield of 72.3%.

[0299] 500 mg (0.9 mmol, 1.0 eq) of compound 102 and 490 mg (0.9 mmol, 1.0 eq) of compound 63 were dissolved in 10 mL of methanol. 450 mg (1.8 mmol, 2.0 eq) of copper sulfate pentahydrate and 360 mg (1.8 mmol, 2.0 eq) of sodium ascorbate were dissolved in 10 mL of water and added to the system. The reaction was monitored by TLC. After concentration, the mixture was purified on a reverse-phase preparative column (5%-95% acetonitrile / water, with the aqueous phase containing 0.01% ammonium bicarbonate) to afford 780 mg of compound 103 in a yield of 78.8%.

[0300] Compound 103 (400 mg, 0.36 mmol, 1.0 eq) and 4-dimethylaminopyridine (9 mg, 0.07 mmol, 0.2 eq) were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and 98 mg of N,N-diisopropylethylamine (0.7 mmol, 2.0 eq) were added. 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (128 mg, 0.54 mmol, 1.5 eq) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. Completion of the reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 310 mg of compound 104 in a yield of 65.6%. 1H NMR (400MHz, DMSO-d6) δ7.95 (h, J=8.6Hz, 4H), 7.87 (d, J=1.5Hz, 1H), 7.76–7. 67(m,3H),7.59(dq,J=7.9,3.3Hz,3H),7.40(dt,J=7.8,3.4Hz,4H),7.34(t,J= 7.2Hz,2H),7.31–7.20(m,9H),6.87(dd,J=8.6,5.8Hz,4H),5.34(dd,J=11.1, 3.3Hz,1H),4.76–4.62(m,3H),4.52(dd,J=27.4,10.3Hz,3H),4.33(q,J=9.5Hz ,1H),4.12–4.00(m,2H),4.00–3.75(m,3H),3.72(d,J=2.4Hz,7H),3.69–3.63 (m,1H),3.54(ddt,J=30.4,10.0,6.6Hz,2H),3.20(d,J=22.3Hz,3H),3.14–2.9 4(m,2H),2.94–2.69(m,4H),2.67–2.59(m,1H),2.36(q,J=5.4,3.0Hz,2H),2. 07(s,1H),1.71(s,2H),1.24–1.07(m,9H),0.99(d,J=6.7Hz,3H).MS(ESI):m / z calcd for C 71 H 81 N8O 14 P[MH] - :1299.55,found:1299.51.

[0301] Example 26 Synthesis of Compound 57

[0302] Synthesis Route 2

[0303] Compound 13 (30 g, 77.09 mmol) was dissolved in dry 1,2-DCE (300 mL). The system was heated to 0°C, and trimethylsilyl trifluoromethanesulfonate (21 mL, 115.64 mmol, 1.5 eq.) was added. The reaction system was stirred at 0°C under nitrogen for 10 min and then placed in a 50°C oil bath for 12 h. TLC and LC-MS confirmed the complete reaction. The system was then heated to room temperature over 4A molecular sieves and stirred for 30 min. Anhydrous 3-pentanol (12.7 mL, 115.64 mmol, 1.5 eq.) was then added. The system was stirred at room temperature under nitrogen for 12 h. TLC and LC-MS monitored the reaction. After completion, triethylamine was added until the system was neutral. The mixture was then extracted sequentially with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1 to 1:2) to obtain compound 105 as a white solid (19 g, two-step yield: 60%). MS (ESI): m / z calculated for C 19 H 31 NO9[M+H] + :418.20,found:418.21.

[0304] Compound 105 (58 g, 139.568 mmol) was dissolved in dry NH3 / MeOH solution (300 mL). The reaction was stirred at room temperature under nitrogen for 12 h. TLC and LC-MS were used to monitor the reaction. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product. The crude product was used directly in the subsequent reaction without further purification. MS (ESI): m / z calculated for C 13 H 25 NO6[M+H] + :292.17,found:292.19.

[0305] Compound 106 (40.6 g, 139.57 mmol) was dissolved in dry pyridine (800 mL), and TBDPSCl (47 mL, 181.44 mmol, 2 eq.) and 4-dimethylaminopyridine (5.12 g, 41.87 mmol, 0.3 eq.) were added. The system was stirred at room temperature under nitrogen for 12 h and monitored by TLC and LC-MS. After completion of the reaction, the product was directly used for the next reaction without post-treatment. MS (ESI): m / z calculated for C 29 H 43 NO6Si[M+H] + :530.29,found:530.26.

[0306] The reaction system of compound 107 was placed at 0°C, and benzoyl chloride (48 mL, 418.8 mmol, 2 eq.) and 4-dimethylaminopyridine (6.8 g, 55.8 mmol, 0.4 eq.) were added dropwise. The system was stirred at room temperature under nitrogen for 12 h, and the reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction system was diluted with ethyl acetate and extracted sequentially with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by flash silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain compound 108 as a white solid, which was directly carried to the next step. MS (ESI): m / z calculated for C 43 H 51 NO8Si[M+H] + :738.34,found:738.33.

[0307] Compound 108 (103 g, 139.57 mmol) was dissolved in anhydrous tetrahydrofuran (800 mL), and triethylamine trihydrofluoride (114 mL, 697.85 mmol, 5.0 eq.) was added. The reaction system was stirred at 50°C under nitrogen protection and monitored by TLC and LC-MS. After approximately 12 h of reaction at room temperature, the reaction was complete. The reaction system was directly concentrated under reduced pressure to remove most of the solvent, diluted with ethyl acetate, and extracted sequentially with water, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 1:1) to obtain Compound 109 (147 g, 65% yield over four steps starting from Compound 105) as a white solid. MS (ESI): m / z calculated for C 27 H 33 NO8[M+H] + :500.22,found:500.23.

[0308] Compound 109 (10 g, 20.031 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL). The mixture was heated to 0°C, and PPh3 (10.5 g, 40.062 mmol, 2 eq.) and DPPA (8.6 mL, 40.062 mmol, 2 eq.) were added. DIAD (7.9 mL, 40.062 mmol, 2 eq.) was slowly added dropwise. The reaction was stirred at 0°C under nitrogen for 4 h, and then allowed to react at room temperature for approximately 12 h. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 8:1 to 3:1) to obtain Compound 57 (4.7 g, 44%) as a white solid. NMR spectra were the same as above. MS (ESI): m / z calculated for C 27 H 32 N4O7[M+H] + :525.23,found:525.24.

[0309] Example 27 Synthesis of Compound 113

[0310] Compound 57 (1.0 g, 1.90 mmol) was dissolved in dry dichloromethane / ethyl acetate (v:v = 1:1, 100 mL) and Pd / C (300 mg) was added. The system was replaced with H2 three times and stirred at room temperature under hydrogen for 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was filtered and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 110 (420 mg, 44%). LC-MS (ESI): m / z calculated for C 27 H 34 N2O7[M+H] + :499.24,found:499.25.

[0311] Compound 110 (100 mg, 0.201 mmol) was dissolved in dry dichloromethane (5 mL), and glutaric anhydride (25 mg, 0.211 mmol, 1.1 eq.) was added. The system was stirred at room temperature for 0.5 h and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 111 (103 mg, 84%). LC-MS (ESI): m / z calculated for C 32 H 40 N2O 10 [M+H] +:613.28,found:613.21.

[0312] Compound 111 (100 mg, 0.163 mmol) was dissolved in dry dichloromethane (5 mL), and compound 24 (80 mg, 0.163 mmol, 1.0 eq.), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (155 mg, 0.408 mmol, 2.5 eq.), and N,N-diisopropylethylamine (54 μL, 0.326 mmol, 2.0 eq.) were added. The reaction was stirred at room temperature for 2 h and monitored by TLC and LC-MS. After completion of the reaction, the mixture was diluted with dichloromethane and extracted sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 112 (113 mg, 64%). LC-MS (ESI): m / z calculated for C 61 H 72 N4O 14 [M+H] + :1085.26,found:1085.27.

[0313] Compound 112 (680 mg, 0.627 mmol) was dissolved in dry dichloromethane (10 mL). 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (222 mg, 0.941 mmol, 1.5 eq.), 4-dimethylaminopyridine (16 mg, 0.125 mmol, 0.2 eq.), and N,N-diisopropylethylamine (137 mg, 1.254 mmol, 2 eq.) were added. The reaction was stirred at room temperature under nitrogen for 0.5 h and monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reverse-phase column (water / acetonitrile system) to obtain compound 113 (508 mg, 63%). 1H NMR (400MHz, DMSO) δ7.90(d,J=7.2Hz,4H),7.71(d,J=7.5Hz,3H),7.58(d,J=7.6Hz,3H),7.40(d,J=7.3Hz,4H),7.33–7.16( m,9H),6.87(d,J=8.5Hz,5H),5.53(s,1H),5.28(d,J=11.4Hz,1H),5.11–4.91(m,1H),4.66(d,J=8.4Hz,1H),4.27–3.98(m, 3H),3.73(s,9H),3.54–3.37(m,2H),3.34(d,J=9.8Hz,4H),3.30–3.05(m,4H),3.05–2.74(m,4H),2.20(s,1H),2.08(d,J=1 0.0Hz, 4H), 2.01 (s, 1H), 1.94–1.78 (m, 2H), 1.77–1.35 (m, 13H), 1.23 (s, 1H), 0.85 (dt, J = 18.5, 7.3Hz, 6H); LC-MS (ESI): m / z calcd for C 70 H 89 N6O 15 P[M+H] + :1285.61,found:1285.66.

[0314] Example 28 Synthesis of Compound 120

[0315] Compound 109 (1.6 g, 3.205 mmol) was dissolved in dry dichloromethane (20 mL), and N,N'-disuccinimidyl carbonate (980 mg, 3.846 mmol, 1.2 eq.) and triethylamine (1.62 g, 16.025 mmol, 5 eq.) were added. The system was stirred at room temperature for 0.5 h and the reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 114 (1.88 g, 91%). LC-MS (ESI): m / z calculated for C 32 H 36 N2O 12 [M+H] + :641.23,found:641.26.

[0316] Compound 114 (1.6 g, 2.499 mmol) was dissolved in dry dichloromethane (15 mL), and compound 115 (522 mg, 2.999 mmol, 1.2 eq.) and triethylamine (1.26 g, 12.495 mmol, 5 eq.) were added. The system was stirred at room temperature for 0.5 h and the reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 116 (1.56 g, 89%). LC-MS (ESI): m / z calculated for C 36 H 49 N3O 11 [M+H] + :700.34,found:700.36.

[0317] Compound 116 (1.56 g, 2.231 mmol) was dissolved in acetonitrile (5 mL), and trifluoroacetic acid (5 mL) was added. The system was stirred at room temperature for 3 h and monitored by TLC and LC-MS. After completion, the reaction was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified on a C18 reverse-phase column (water / acetonitrile system) to obtain compound 117 (860 mg, 64%). LC-MS (ESI): m / z calculated for C 31 H 41 N3O9[M+H] + :600.28,found:600.25.

[0318] Compound 117 (198 mg, 0.331 mmol) was dissolved in dry N,N-dimethylformamide (4 mL). Compound 118 (200 mg, 0.331 mmol, 1.0 eq.), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (314 mg, 0.828 mmol, 2.5 eq.), and N,N-diisopropylethylamine (140 μL, 0.828 mmol, 2.0 eq.) were added. The reaction was stirred at room temperature for 2 h and monitored by TLC and LC-MS. After completion, the reaction was diluted with dichloromethane and extracted sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 119 (313 mg, 79%). LC-MS(ESI):m / z calcd for C 65 H 79 N5O 16 [M+H] + :1185.37,found:1185.39.

[0319] Compound 119 (500 mg, 0.421 mmol) was dissolved in dry dichloromethane (5 mL). 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (150 mg, 0.632 mmol, 1.5 eq.), 4-dimethylaminopyridine (11 mg, 0.084 mmol, 0.2 eq.), and N,N-diisopropylethylamine (0.12 mL, 0.842 mmol, 2 eq.) were added. The reaction was stirred at room temperature under nitrogen for 0.5 h and monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reverse-phase column (water / acetonitrile system) to obtain compound 120 (251 mg, 43%). 1 H NMR (400MHz, DMSO) δ7.92 (dd, J=13.1, 8.4Hz, 3H), 7.70 (t, J=7.7Hz, 4H), 7.58 (q,J=7.4Hz,3H),7.40(t,J=7.7Hz,4H),7.31–7.14(m,8H),6.87(dd,J=8.1,6 .3Hz,4H),5.62(d,J=3.1Hz,1H),5.31(dd,J=11.1,3.0Hz,1H),4.71(d,J=8.4 Hz,1H),4.22–4.07(m,4H),3.99(dd,J=15.0,8.9Hz,2H),3.71(t,J=9.9Hz,8H) ,3.56–3.43(m,2H),3.07–2.85(m,6H),2.81–2.75(m,1H),2.64(dd,J=9.8,5. 6Hz,1H),2.19(d,J=7.2Hz,2H),2.09–1.94(m,5H),1.84(dd,J=18.7,11.7Hz, 1H),1.71–1.60(m,6H),1.49(tt,J=23.4,7.2Hz,7H),1.24(s,2H),1.19–1.11 (m,8H),1.03(t,J=6.8Hz,3H),0.84(dd,J=14.7,7.4Hz,6H).;LC-MS(ESI):m / z calcd for C 74 H 96 N7O 17 P[M+H] + :1385.66,found:1385.67.

[0320] Example 29 Synthesis of Compound 127

[0321] Compound 121 (5 g, 30.643 mmol) was dissolved in dry acetonitrile (30 mL). Ethyl trifluoroacetate (7.3 mL, 61.286 mmol, 2.0 eq.) and triethylamine (13 mL, 16.025 mmol, 3.0 eq.) were added. The system was stirred at room temperature for 3 h and monitored by TLC and LC-MS. Upon completion of the reaction, the product was concentrated under reduced pressure to obtain the crude product, which was carried on to the next step without further treatment.

[0322] Compound 122 (600 mg, 2.286 mmol) was dissolved in dry N,N-dimethylformamide (10 mL). Compound 24 (1.12 g, 2.286 mmol, 1.0 eq.), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.2 g, 5.715 mmol, 2.5 eq.), and N,N-diisopropylethylamine (0.76 mL, 4.572 mmol, 2.0 eq.) were added. The reaction was stirred at room temperature for 12 h and monitored by TLC and LC-MS. After completion of the reaction, the system was diluted with dichloromethane and extracted sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 123 (333 mg, 64%). LC-MS(ESI):m / z calcd for C 37 H 44 N3O9[M+H] + :732.30,found:732.33.

[0323] Compound 123 (1.67 g, 2.286 mmol) was dissolved in methanol / water (v / v, 2:1, 15 mL) and potassium carbonate (790 mg, 5.715 mmol, 2.5 eq.) was added. The system was stirred at room temperature for 10 h and the reaction was monitored by TLC and LC-MS. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The product was then separated and purified on a C18 reverse-phase column (water / acetonitrile system) to obtain compound 124 (609 mg). LC-MS (ESI): m / z calculated for C 35 H 45 N3O8[M+H] + :636.76,found:636.77.

[0324] Compound 124 (609 mg, 0.959 mmol) was dissolved in dry N,N-dimethylformamide (5 mL). Pentacene acid (103 mg, 1.054 mmol, 1.1 eq.), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (909 mg, 2.398 mmol, 2.5 eq.), and N,N-diisopropylethylamine (0.32 mL, 1.918 mmol, 2.0 eq.) were added. The reaction was stirred at room temperature for 4 h and monitored by TLC and LC-MS. After completion of the reaction, the system was diluted with dichloromethane and extracted sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reverse-phase column (water / acetonitrile system) to obtain compound 125 (510 mg, 74%). LC-MS (ESI): m / z calculated for C 40 H 49 N3O9[MH] - :714.35,found:714.43.

[0325] Compound 125 (510 mg, 0.713 mmol) and compound 57 (374 mg, 0.713 mmol, 1.0 eq.) were dissolved in methanol / water (v / v, 3:1, 8 mL). Copper sulfate pentahydrate (89 mg, 0.357 mmol, 0.5 eq.) and sodium ascorbate (71 mg, 0.357 mmol, 0.5 eq.) were added. The reaction was stirred at room temperature for 0.5 h and monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated to remove the methanol, diluted with dichloromethane, and extracted sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reverse-phase column (water / acetonitrile system) to obtain compound 126 (644 mg, 73%). LC-MS (ESI): m / z calculated for C 67 H 81 N7O 16 [MH] - :1238.57,found:1238.74.

[0326] Compound 126 (630 mg, 0.508 mmol) was dissolved in dry dichloromethane (5 mL). 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (0.17 mL, 0.762 mmol, 1.5 eq.), 4-dimethylaminopyridine (13 mg, 0.102 mmol, 0.2 eq.), and N,N-diisopropylethylamine (0.14 mL, 1.016 mmol, 2 eq.) were added. The reaction was stirred at room temperature under nitrogen for 0.5 h and monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reverse-phase column (water / acetonitrile system) to obtain compound 127 (459 mg, 63%). 1 H NMR (400MHz, DMSO) δ7.95(d,J=7.2Hz,4H),7.79(s,1H),7.72(t,J=7.8Hz,3H),7.59(t,J =7.8Hz,3H),7.43–7.36(m,4H),7.32–7.18(m,7H),7.07–7.03(m,1H),6.87(dd,J=8.4,6 .0Hz,4H),5.66(d,J=3.2Hz,1H),5.34(dd,J=11.1,3.3Hz,1H),4.67–4.54(m,2H),4.51– 4.40(m,2H),4.27–4.13(m,2H),4.11–3.99(m,2H),3.81–3.65(m,8H),3.65–3.47(m,5H) ,3.40(ddd,J=17.9,14.4,3.9Hz,5H),3.25(ddd,J=20.7,13.4,5.5Hz,4H),2.98(ddd,J= 46.1,19.1,10.4Hz,2H),2.83–2.74(m,3H),2.64(t,J=5.2Hz,1H),2.40(t,J=7.7Hz,2H) ,2.07(s,2H),1.74–1.64(m,4H),1.35(tdd,J=28.1,17.8,11.1Hz,6H),1.21–1.06(m,10 H), 1.03 (t, J = 7.7Hz, 3H), 0.79 (t, J = 7.3Hz, 3H), 0.72 (t, J = 7.4Hz, 3H); LC-MS (ESI): m / z calcd for C 76 H 98 N9O 17 P[M+H] + :1440.57,found:1440.63.

[0327] Example 30 Synthesis of Compound 131

[0328] The synthesis of compound 128 followed the synthetic procedure of compound 24.

[0329] Compound 128 (20.0 g, 40.8 mmol) and valeric acid (4.4 g, 44.9 mmol) were dissolved in 100 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (10.5 g, 81.60 mmol) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (18.6 g, 49.0 mmol) were added under nitrogen and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, quenched with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine, separated, dried, concentrated, and purified by column chromatography to afford 19.7 g of compound 129 in an 85.4% yield.

[0330] Compound 129 (4.1 g, 7.2 mmol) and compound 57 (3.8 g, 7.2 mmol) were weighed and dissolved in 40 mL of methanol. Copper sulfate pentahydrate (2.7 g, 10.8 mmol) and sodium ascorbate (2.1 g, 10.8 mmol) were dissolved in 20 mL of water and added to the system. The reaction was monitored by TLC. After concentration, the product was purified on a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 4.4 g of compound 130 (yield: 56.0%).

[0331] Compound 130 (4.2 g, 3.5 mmol) and 4-dimethylaminopyridine (85 mg, 0.7 mmol) were weighed into a 100 mL single-necked flask. After nitrogen purge, 30 mL of anhydrous dichloromethane and N,N-diisopropylethylamine (0.9 g, 6.9 mmol) were added. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (1.2 g, 5.2 mmol) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. Completion of the reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 3.5 g of compound 131 in a 68% yield. 1H NMR(400MHz,DMSO-d6)δ7.99–7.91(m,3H),7.91–7.80(m,1H),7.77–7.63(m,4H),7.59(t ,J=7.8Hz,3H),7.46–7.36(m,4H),7.36–7.17(m,7H),6.87(ddd,J=8.7,6.1,2.2Hz,4H), 5.66(d,J=3.4Hz,1H),5.34(dt,J=11.1,3.7Hz,1H),4.65–4.52(m,2H),4.46(d,J=12.1H z,2H),4.28–4.13(m,2H),4.13–3.96(m,1H),3.87–3.75(m,1H),3.73(d,J=2.3Hz,7H),3 .65–3.49(m,2H),3.40(d,J=7.4Hz,2H),3.31–3.23(m,2H),3.04(dddd,J=27.0,20.7,13 .5,5.3Hz,2H),2.91–2.75(m,3H),2.64(td,J=5.7,2.2Hz,1H),2.57(d,J=8.2Hz,1H),2. 07(s,2H),1.92–1.80(m,1H),1.79–1.70(m,1H),1.68(s,3H),1.65–1.56(m,1H),1.47–1 .20(m,5H),1.15(dd,J=12.7,6.7Hz,8H),1.03(dd,J=6.8,4.6Hz,3H),0.83–0.65(m,6H). 31 P NMR (162MHz, DMSO) δ148.32,148.22,148.04,147.86.

[0332] Example 31 Synthesis of Compound 136

[0333] Compound 109 (1.6 g, 3.2 mmol) was dissolved in dry dichloromethane (20 mL), and N,N'-disuccinimidyl carbonate (980 mg, 3.8 mmol, 1.2 eq.) and triethylamine (1.62 g, 16.0 mmol, 5 eq.) were added. The system was stirred at room temperature for 0.5 h and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 114 (1.88 g, 91%). Compound 114: LC-MS (ESI): m / z calculated for C 32 H 36 N2O12 [M+H] + :641.23,found:641.26.

[0334] Compound 114 (1.6 g, 2.5 mmol) was dissolved in dry dichloromethane (15 mL), and compound 132 (664 mg, 3 mmol, 1.2 eq.) and triethylamine (1.26 g, 12.5 mmol, 5 eq.) were added. The system was stirred at room temperature for 0.5 h and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 133 (1.12 g, 60%). Compound 133: LC-MS (ESI): m / z calculated for C 41 H 50 N2O 11 [M+H] + :747.35,found:747.39.

[0335] Compound 133 (1.12 g, 1.5 mmol) was dissolved in an equal volume mixture of ethyl acetate and methanol, and Pd / C (0.22 g, 20%) was added. The system was stirred at room temperature for 3 h and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the mixture was filtered through a fritted funnel and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified on a C18 reverse phase column (water / acetonitrile system) to obtain compound 134 (936 mg, 95%). Compound 134: LC-MS (ESI): m / z calculated for C 34 H 44 N2O 11 [M+H] + :657.29,found:657.25.

[0336] Compound 134 (920 mg, 1.4 mmol, 1.0 eq) was dissolved in dry N,N-dimethylformamide (4 mL). Compound 24 (687 mg, 1.4 mmol, 1.0 eq.), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.33 g, 3.5 mmol, 2.5 eq.), and N,N-diisopropylethylamine (0.5 mL, 2.8 mmol, 2.1 eq.) were added. The reaction was stirred at room temperature for 2 h and monitored by TLC and LC-MS. After completion of the reaction, the mixture was diluted with dichloromethane and extracted sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by silica gel column chromatography (dichloromethane:methanol = 20:1 to 10:1) to obtain compound 135 (1.1 g, 70%). Compound 135: LC-MS (ESI): m / z calculated for C 63 H 76 N4O 15 [M+H] + :1130.53,found:1130.48.

[0337] Compound 135 (1.0 g, 0.88 mmol) was dissolved in dry dichloromethane (5 mL), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (312 mg, 1.32 mmol, 1.5 eq.), 4-dimethylaminopyridine (21.5 mg, 0.176 mmol, 0.2 eq.), and N,N-diisopropylethylamine (0.25 mL, 0.842 mmol, 2 eq.) were added. The system was stirred at room temperature under nitrogen for 0.5 h and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by C18 reverse phase column (water, acetonitrile system) to obtain compound 136 (737 mg, 63%). Compound 136: LC-MS (ESI): m / z calculated for C 72 H 93 N6O 16 P[M+H] + :1329.64,found[M-DMTr] + :1025.50. 1H NMR (400MHz, DMSO) δ7.93 (dd, J=13.7, 8.6Hz, 3H), 7.71 (dd, J=12.2, 7.5Hz, 3H ),7.60(dt,J=14.5,6.7Hz,3H),7.40(dd,J=9.7,5.9Hz,4H),7.33–7.12(m,8H) ,6.91–6.83(m,4H),5.63(d,J=2.2Hz,1H),5.32(dd,J=11.0,2.8Hz,1H),4.72 (d,J=8.4Hz,1H),4.26–4.02(m,5H),4.02–3.92(m,1H),3.87–3.77(m,1H),3.7 7–3.66(m,8H),3.65–3.39(m,4H),3.38–3.22(m,4H),3.08–2.97(m,2H),2.91 (t,J=12.0Hz,3H),2.78(t,J=6.0Hz,1H),2.64(dd,J=9.1,3.9Hz,1H),2.19(dd ,J=13.3,6.5Hz,1H),1.90–1.77(m,1H),1.67(d,J=19.8Hz,4H),1.62–1.27(m, 10H),1.24–1.10(m,11H),1.03(d,J=6.6Hz,3H),0.86(dt,J=21.1,7.4Hz,6H).

[0338] Example 32 Synthesis of Compound 137

[0339] Compound 129 (1.14 g, 2.0 mmol) was dissolved in dry dichloromethane (10 mL), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (710 mg, 3 mmol, 1.5 eq.) and 4-dimethylaminopyridine (48.8 mg, 0.4 mmol, 0.2 eq.) and N,N-diisopropylethylamine (1.2 mL, 4.0 mmol, 2 eq.) were added. The system was stirred at room temperature under nitrogen for 0.5 h, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by C18 reverse phase column (water, acetonitrile system) to obtain compound 137 (1.0 g, 65%). Compound 137: 1H NMR(400MHz,DMSO-d6)δ7.93(d,J=34.4Hz,0.38H),7.63(dt,J=22.0,5.9H z,0.7H),7.44–7.36(m,2H),7.33–7.19(m,8H),6.86(dt,J=11.3,5.6Hz,4H ),4.30(td,J=7.4,6.5,2.4Hz,0.37H),4.17(ddd,J=20.2,8.6,3.0Hz,0.6 8H),4.05(qd,J=10.8,9.6,4.3Hz,1H),3.88–3.76(m,1H),3.73(d,J=2.5Hz ,7H),3.72–3.68(m,1H),3.57(dddd,J=27.6,13.5,10.4,6.8Hz,2H),3.48 –3.34(m,2H),3.27(t,J=6.1Hz,1H),3.13–2.93(m,2H),2.90–2.73(m,3H), 2.72–2.61(m,1H),2.48–2.42(m,1H),2.37–2.25(m,2H),2.07(s,2H),2.04 –1.94(m,1H),1.91–1.45(m,4H),1.20–1.08(m,9H),1.03(t,J=6.4Hz,3H). 31 P NMR(162MHz,DMSO)δ148.42,148.25,148.14,147.84.LC-MS(ESI):m / z calcd for C 43 H 55 N4O7P[MH] - :769.37,found[MH] - :769.31.

[0340] Example 33 Synthesis of Compound 141

[0341] Compound 57 (1.5 g, 2.860 mmol) and valeric acid (310 g, 3.150 mmol) were dissolved in tetrahydrofuran (15 mL). A 7.5 mL aqueous solution of copper sulfate pentahydrate (210 mg, 0.860 mmol, 0.3 eq.) and a 7.5 mL aqueous solution of sodium ascorbate (280 mg, 1.430 mmol, 0.5 eq.) were added, and the system was stirred at room temperature. After approximately 0.5 h of reaction, the reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure, diluted with dichloromethane, washed with water and saturated brine, and the organic phase dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by reverse phase preparative column chromatography (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to yield Compound 139 (1.24 g, 70%) as a white solid. Compound 139: MS (ESI): m / z calculated for C 32 H 38 N4O9[M+H] + :623.68,found:623.26.

[0342] Compound 139 (1.4 g, 2.250 mmol) and compound 49 (943 mg, 2.250 mmol, 1.0 eq.) were dissolved in dry dichloromethane (28 mL). Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (943 mg, 5.625 mmol, 2.5 eq.) and N,N-diisopropylethylamine (0.75 mL, 4.500 mmol, 2.0 eq.) were added. The reaction system was stirred at room temperature under nitrogen. After 4 h of reaction at room temperature, the reaction was monitored by TLC and LC-MS. The reaction was complete. The system was diluted with dichloromethane, washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by reverse phase preparative column chromatography (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to obtain Compound 140 (1.96 g, 75%) as a white solid. Compound 140: 1H NMR (400MHz, DMSO) δ7.98–7.92(m,3H),7.83(s,1H),7.75–7.69(m,3H),7.62–7.54(m,3H),7.43–7.36(m,2H),7.34–7.24(m,5H) ,7.23–7.14(m,5H),6.90–6.81(m,4H),5.71–5.66(m,1H),5.39–5.32(m,1H),5.02(d,J=4.1Hz,1H),4.64–4.55(m,2H),4.50–4.3 6(m,3H),4.27–4.12(m,2H),3.75–3.70(m,6H),3.60(dd,J=10.5,5.1Hz,1H),3.33–3.17(m,2H),3.08–2.94(m,1H),2.89–2.72( m,2H),2.65–2.52(m,1H),2.10–1.98(m,1H),1.95–1.81(m,1H),1.68(s,3H),1.44–1.24(m,4H),0.81–0.63(m,6H).MS(ESI):m / z calcd for C 58 H 65 N5O 12 [M+H] + :1024.43,found:1024.42.

[0343] Compound 140 (1.96 g, 1.915 mmol) was dissolved in dry dichloromethane (20 mL), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (0.65 mL, 2.873 mmol, 1.5 eq.) and 4-dimethylaminopyridine (47 mg, 0.383 mmol, 0.2 eq.) and N,N-diisopropylethylamine (0.53 mL, 3.830 mmol, 2 eq.) were added. The system was stirred at room temperature under nitrogen for 0.5 h, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by a C18 reverse phase column (5%-95% acetonitrile / water, the aqueous phase was 0.01% ammonium bicarbonate) to obtain compound 141 (1.63 g, 70%). Compound 141: 1H NMR (400MHz, DMSO) δ7.95(d,J=7.9Hz,3H),7.85–7.79(m,1H),7.75–7.68(m,3H),7.58(t,J=7.5Hz,3H),7.39(t,J=7.7Hz,2H), 7.35–7.24(m,4H),7.23–7.14(m,5H),6.89–6.81(m,4H),5.73–5.64(m,1H),5.35(dd,J=11.1,2.9Hz,1H),4.72–4.54(m,3H),4. 50–4.35(m,2H),4.25–4.12(m,2H),3.80–3.63(m,9H),3.60–3.40(m,3H),3.29–3.18(m,2H),3.08–2.94(m,1H),2.89–2.69(m, 4H),2.64–2.54(m,2H),2.17–2.09(m,1H),2.06–1.96(m,1H),1.68(s,3H),1.42–1.23(m,4H),1.12(m,12H),0.81–0.62(m,6H); 31 P NMR (162MHz, DMSO) δ146.92, 146.83, 146.64, 146.42; MS (ESI): m / z calcd for C 67 H 82 N7O 13 P[M+H] + :1224.57,found:1224.60.LC-MS(ESI):m / z calcd for C 67 H 82 N7O 13 P[MH] - :1222.57,found[MH] - :1222.51.

[0344] Example 34. Synthesis of Compound 143

[0345] Compound 129 (2 g, 3.5 mmol) and compound 73 (1.53 g, 3.5 mmol) were weighed and dissolved in 40 mL of methanol. Copper sulfate pentahydrate (174.8 mg, 0.7 mmol) and sodium ascorbate (277 mg, 1.4 mmol) were dissolved in 20 mL of water and added to the system. The reaction was monitored by TLC. The mixture was concentrated and purified on a reverse-phase C18 preparative column to afford 3.5 g of compound 142 (yield: 96.0%).

[0346] Compound 142 (3.5 g, 3.4 mmol) and 4-dimethylaminopyridine (85 mg, 0.7 mmol) were weighed into a 100 mL single-necked flask. After nitrogen purge, 30 mL of anhydrous dichloromethane and N,N-diisopropylethylamine (0.9 g, 6.9 mmol) were added. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (1.2 g, 5.2 mmol) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. Completion of the reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified on a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 2.8 g of compound 143 in a 68% yield. 1 H NMR (400MHz, DMSO-d6) δ7.99(d,J=8.4Hz,1H),7.97–7.90(m,2H),7.85(d,J=1.7Hz,1H),7.79–7.64(m,4H),7.59(q,J=7.4Hz,3H),7.40(t,J=7. 7Hz,4H),7.34–7.17(m,7H),6.93–6.79(m,4H),5.70(d,J=3.2Hz,1H),5 .27(dd,J=11.0,3.3Hz,1H),4.57–4.39(m,4H),4.32–4.14(m,1H),4.01( m,2H),3.87–3.66(m,8H),3.66–3.49(m,2H),3.37(s,2H),3.27(d,J=16 .8Hz,1H),3.18–2.93(m,2H),2.89–2.72(m,3H),2.67–2.54(m,2H),2.0 7(s,6H),1.86(dt,J=19.5,9.1Hz,1H),1.72(s,4H),1.61(d,J=10.7Hz, 1H),1.50(s,1H),1.20–1.10(m,7H),1.07–0.97(m,3H).LC-MS(ESI):m / z calcd for C 65 H 77 N8O 13 P[MH] - :1207.53,found[MH] - :1207.41.

[0347] Example 35 Synthesis of Compound 144

[0348] Compound 109 (1.70 g, 3.4 mmol) and 4-dimethylaminopyridine (85 mg, 0.7 mmol) were weighed into a 100 mL single-necked flask. After nitrogen purge, 30 mL of anhydrous dichloromethane and N,N-diisopropylethylamine (0.9 g, 6.9 mmol) were added. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (1.2 g, 5.2 mmol) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified by reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 1.43 g of compound 144 in a 60% yield. 1 H NMR (400MHz, DMSO-d6) δ7.93(pd,J=11.1,9.5,3.5Hz,5H),7.77–7.68(m,3H),7.68–7.52(m,4H),7.52–7.37(m,3H),5.70(d ,J=3.3Hz,1H),5.42(d,J=5.8Hz,0H),5.35(s,0H),5.30(dd,J=11.1,3.4Hz,1H),4.89(dd,J=6.2,4.4Hz,1H),4.70(d,J=8.4 Hz,1H),4.18(dt,J=11.1,8.9Hz,1H),3.93(t,J=6.9Hz,1H),3.98(dd,J=11.3,5.3Hz,1H),3.86–3.76(m,1H),3.60–3.38(m, 3H),3.18–2.92(m,2H),2.07(m,2H),1.70(s,3H),1.65–1.34(m,4H),1.22–1.08(m,9H),1.02–0.77(m,9H).LC-MS(ESI):m / z calcd for C 36 H 50 N3O9P[MH] - :698.77,found[MH] - :698.73.

[0349] Example 36 Synthesis of Compound 147

[0350] Compound 57 (8 g, 15.267 mmol) was dissolved in a methanol solution of NH3 (80 mL), and the system was stirred at room temperature for 12 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the crude product was concentrated under reduced pressure to obtain a crude product. The crude product was rotary evaporated three times with anhydrous pyridine, dissolved in pyridine (50 mL), placed in an ice-water bath, and acetyl chloride (3.3 mL, 45.802 mmol) was added dropwise. After the addition was complete, the system was stirred at room temperature under nitrogen protection for 5 h. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was diluted with dichloromethane, washed with water, saturated sodium bicarbonate solution and saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography to obtain compound 145 (4.1 g, two-step yield: 85%) as a white solid.

[0351] Compound 129 (2 g, 3.5 mmol) and compound 145 (1.40 g, 3.5 mmol) were weighed and dissolved in 40 mL of methanol. Copper sulfate pentahydrate (174.8 mg, 0.7 mmol) and sodium ascorbate (277 mg, 1.4 mmol) were dissolved in 20 mL of water and added to the system. The reaction was monitored by TLC. After concentration, the mixture was purified on a reverse-phase C18 preparative column to afford 3.13 g of compound 146 (yield: 92.0%).

[0352] Compound 146 (340 mg, 0.35 mmol) and 4-dimethylaminopyridine (8.5 mg, 0.07 mmol) were weighed into a 100 mL single-necked flask. After nitrogen purge, 10 mL of anhydrous dichloromethane and N,N-diisopropylethylamine (0.1 g, 0.7 mmol) were added. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (120 mg, 0.52 mmol) was then added dropwise via syringe. The mixture was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. The mixture was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified on a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate as the aqueous phase) to afford 266 mg of compound 147 in a 65% yield. 1H NMR (400MHz, DMSO-d6) δ7.91–7.74(m,2H),7.74–7.64(m,1H),7.46–7.36(m,2H),7. 36–7.17(m,7H),6.93–6.79(m,4H),5.22(q,J=3.4Hz,1H),5.01(dt,J=11.5,4.0Hz,1 H),4.49–4.33(m,3H),4.29–4.13(m,2H),4.06(dq,J=11.4,6.2Hz,1H),3.82(dt,J= 12.1,9.0Hz,2H),3.73(d,J=2.4Hz,6H),3.69–3.50(m,2H),3.47–3.33(m,2H),3.29( t,J=6.2Hz,1H),3.16(p,J=5.6,5.2Hz,1H),3.12–2.93(m,2H),2.91–2.74(m,3H),2 .64(td,J=6.0,2.1Hz,1H),2.60–2.53(m,1H),2.19–2.12(m,3H),2.07(s,5H),1.89( s,4H),1.75(d,J=1.7Hz,4H),1.67–1.59(m,1H),1.51(dd,J=10.9,5.5Hz,1H),1.40– 1.19(m,5H),1.15(dd,J=14.2,6.6Hz,7H),1.04(t,J=6.1Hz,3H),0.79–0.60(m,6H). 31 P NMR(162MHz,DMSO)δ148.35,148.25,148.04,147.86.LC-MS(ESI):m / z calcd for C 60 H 83 N8O 14 P[MH] - :1169.57,found[MH] - :1169.53.

[0353] Example 37 Synthesis route of compound 151

[0354] Example 38 Synthesis route of compound 159

[0355] Example 39 Synthesis of Compound 167

[0356] Compound 160 (5 g, 13.465 mmol) was dissolved in dry pyridine (50 mL), and diisopropyldichlorosilane (5.2 mL, 16.158 mmol, 1.2 eq.) was added. The system was stirred at room temperature under nitrogen for 5 h. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the system was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product, which was directly used for subsequent reactions.

[0357] Compound 161 (5 g, 8.145 mmol) was dissolved in dry dichloromethane (50 mL). Triethylamine (1.75 mL, 16.290 mmol, 2 eq.) and 4-nitrophenyl chloroformate (2.46 g, 12.220 mmol, 1.2 eq.) were added. The system was stirred at room temperature under nitrogen for 0.5 h. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the subsequent reaction was carried out directly.

[0358] Compound 148 (1.75 g, 12.220 mmol, 1.2 eq.) was added to the reaction system of compound 162. The system was stirred at room temperature under nitrogen for 0.5 h and the reaction was monitored by TLC and LC-MS. After the reaction of compound 162 was complete, the system was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by silica gel column chromatography (dichloromethane / methanol) to obtain compound 163 (4 g, yield: 60%).

[0359] Compound 163 (2.2 g, 2.810 mmol) was dissolved in dry tetrahydrofuran (22 mL), and TBAF tetrahydrofuran solution (1.0 mL) was added. The system was stirred at room temperature under nitrogen for 0.5 h, and the reaction was monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain a crude product, which was then separated and purified by silica gel column chromatography (dichloromethane / methanol) to obtain compound 164 (1 g, yield: 66%).

[0360] Compound 164 (1 g, 1.851 mmol) was dissolved in dry pyridine (10 mL), and 4,4'-bismethoxytrityl chloride (760 mg, 2.222 mmol, 1.2 eq.) was added. The system was stirred at room temperature under nitrogen for 4 h. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the system was diluted with dichloromethane and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product, Compound 165, which was directly used for subsequent reactions.

[0361] Compound 165 (1.5 g, 1.780 mmol) and compound 57 (932 mg, 1.780 mmol) were dissolved in tetrahydrofuran (15 mL). A 7.5 mL aqueous solution of copper sulfate pentahydrate (131 mg, 0.534 mmol, 0.3 eq.) and a 7.5 mL aqueous solution of sodium ascorbate (175 mg, 0.890 mmol, 0.5 eq.) were added, and the mixture was stirred at room temperature. After approximately 0.5 h, the reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure, diluted with dichloromethane, washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude product. The crude product was purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to yield Compound 166 (1.8 g, 75%) as a white solid.

[0362] Compound 166 (1.8 g, 1.316 mmol) was dissolved in dry dichloromethane (15 mL). 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (0.45 mL, 1.974 mmol, 1.5 eq.), 4-dimethylaminopyridine (32 mg, 0.263 mmol, 0.2 eq.), and N,N-diisopropylethylamine (0.36 mL, 2.630 mmol, 2 eq.) were added. The reaction was stirred at room temperature under nitrogen for 0.5 h and monitored by TLC and LC-MS. After completion of the reaction, the system was concentrated under reduced pressure to obtain the crude product. The crude product was purified by C18 reverse-phase column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to obtain compound 167 (1.55 g, yield: 71%). 1H NMR (400MHz, DMSO) δ8.34–8.25(m,1H),8.10–8.04(m,2H),7.99–7.91(m,3H),7.77–7.67(m,3H),7.58(t,J=7.6Hz,3H),7.43–7.3 0(m,4H),7.25–7.11(m,11H),6.85–6.77(m,4H),6.10–5.99(m,1H),5.68(d,J=3.1Hz,1H),5.34(dd,J=11.1,3.2Hz,1H),5.21–5. 02(m,1H),4.67–4.59(m,2H),4.56–4.47(m,4H),4.31–4.15(m,2H),3.93–3.83(m,1H),3.81–3.66(m,9H),3.26–3.14(m,2H),2.8 0–2.68(m,2H),2.62–2.52(m,2H),1.67(s,3H),1.44–1.22(m,6H),1.18–1.09(m,9H),1.08–0.93(m,12H),0.81–0.58(m,9H); 31P NMR (162MHz, DMSO) δ147.57,147.25; MS (ESI): m / z calcd for C 82 H 95 N 12 O 18 P[M+H] + :1567.66,found:1567.72.

[0363] Example 40 Synthesis of Compound 168

[0364] The synthetic route of compound 168 is as follows:

[0365] Example 41 Synthesis of Compounds 170-172

[0366] Compound 57 (1.86 g, 3.548 mmol) was dissolved in ethanol (15 mL), and a KOH solution (5.940 g) in water (15 mL) was added. The reaction system was allowed to react at 90°C overnight. Completion of the reaction was monitored by TLC and LC-MS. The reaction was concentrated under reduced pressure at room temperature to remove ethanol and most of the water, and then purified on a reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to afford compound 169 (660 mg, 68%).

[0367] Compound 169 (100 mg, 0.365 mmol) was dissolved in dry dichloromethane (2 mL) and placed in an ice bath. DIPEA (0.12 mL, 0.730 mmol) and p-TosCl (70 mg, 0.365 mmol) were added. The reaction was stirred in an ice bath. Completion of the reaction was monitored by TLC and LC-MS. The product was concentrated under reduced pressure at room temperature to remove dichloromethane, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 50 mg of compound 170.

[0368] Compound 169 (100 mg, 0.365 mmol) was dissolved in dry dichloromethane (2 mL) and placed in an ice bath. DIPEA (0.12 mL, 0.730 mmol) and MsCl (0.032 mL, 0.365 mmol) were added. The reaction was stirred in an ice bath. Completion of the reaction was monitored by TLC and LC-MS. The product was concentrated under reduced pressure at room temperature to remove dichloromethane, and then purified using a reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford 30 mg of compound 171.

[0369] Compound 169 (110 mg, 0.401 mmol) was dissolved in dichloromethane (3 mL) and the system was placed at -70°C. Triethylamine (0.11 mL, 0.802 mmol), TFAA (0.055 mL, 0.4015 mmol), and DMAP (3 mg, 0.020 mmol) were added. The reaction system was stirred at -70°C. Completion of the reaction was monitored by TLC and LC-MS. The product was concentrated under reduced pressure at room temperature to remove dichloromethane, and then purified using a reverse phase preparative column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to afford 15 mg of compound 172.

[0370] Example 42 Solid phase carrier loading example

[0371] Compound 130 (420 mg, 0.35 mmol) and 4-dimethylaminopyridine (8.5 mg, 0.07 mmol) were added to a 100 mL single-necked flask. After nitrogen purge, 30 mL of anhydrous dichloromethane and succinic anhydride (70 mg, 0.7 mmol) were added and allowed to react overnight. Completion of the reaction was monitored by TLC. The product was concentrated under reduced pressure at room temperature to remove dichloromethane and then purified using a reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to afford 350 mg of compound 173.

[0372] Compound 173 is coupled with an amino support (polystyrene resin (PS) or CPG powder) through a coupling reaction and a capping reaction to prepare a solid-phase synthesis support preloaded with BT-021. After analysis and testing to determine the support loading, it is used as a standby support for solid-phase synthesis and subsequent base monomer coupling. This synthetic strategy can also produce conjugates containing the structures described in this invention, which can serve as a supplement to the preparation method of conjugates synthesized from phosphoramidite monomers.

[0373] Example 43 Synthesis of Compound 175

[0374] Compound 43 (400 mg, 0.64 mmol) and compound 57 (351 mg, 0.64 mmol) were dissolved in tetrahydrofuran (15 mL). A 7.5 mL aqueous solution of copper sulfate pentahydrate (32 mg, 0.13 mmol, 0.2 eq.) and a 7.5 mL aqueous solution of sodium ascorbate (38 mg, 0.19 mmol, 0.3 eq.) were added. The mixture was stirred at room temperature. After approximately 0.5 h of reaction, the reaction was monitored by TLC and LC-MS. After completion of the reaction, the mixture was concentrated under reduced pressure, diluted with dichloromethane, washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude product. The crude product was purified by reverse phase separation to yield Compound 174 (600 mg, 82%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ7.98–7.91(m,3H),7.81(d,J=16.8Hz,2H),7.76–7.67(m,3H),7.59(td,J=7.5,1.5Hz,3H),7.44–7.34(m,5H),7.32–7.16(m ,7H),6.90–6.82(m,4H),5.66(d,J=3.4Hz,1H),5.33(dd,J=11.1,3.4Hz,1 H),4.65–4.54(m,2H),4.52–4.41(m,3H),4.20(dt,J=11.1,8.9Hz,1H),3. 89(dp,J=9.2,3.3Hz,2H),3.73(s,6H),3.25(p,J=5.9Hz,1H),3.05(dd,J =8.8,5.3Hz,1H),2.94–2.76(m,5H),2.40(dd,J=8.9,6.6Hz,2H),2.19(t, J=12.0Hz,1H),1.72(d,J=12.7Hz,4H),1.68(s,3H),1.48–1.24(m,7H),0. 93(d,J=6.1Hz,3H),0.87(d,J=12.1Hz,2H),0.76(dt,J=21.1,7.4Hz,6H).

[0375] Compound 174 (600 mg, 0.52 mmol) was dissolved in dry dichloromethane (10 mL), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (CEP-Cl) (0.18 mL, 0.78 mmol, 1.5 eq.), 4-dimethylaminopyridine (13 mg, 0.10 mmol, 0.2 eq.), and N,N-diisopropylethylamine (0.2 mL, 1.04 mmol, 2 eq.) were added. The system was stirred at room temperature under nitrogen for 0.5 h, and the reaction was monitored by TLC and LC-MS. After the reaction was complete, the system was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to C 18 Reverse phase (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) separation and purification gave Compound 175 (500 mg, 70%). 1H NMR (400MHz, DMSO-d6) δ7.95 (dd, J=8.2, 1.4Hz, 3H), 7.84 (t, J=5.8Hz, 1H), 7.80 (s, 1H), 7.77–7.68 ( m,3H),7.59(td,J=7.6,1.7Hz,3H),7.44–7.35(m,5H),7.24(tdt,J=9.5,7.3,4.7Hz,7H),6.86(dd,J= 8.8,6.9Hz,4H),5.66(d,J=3.4Hz,1H),5.34(dd,J=11.1,3.4Hz,1H),4.65–4.54(m,2H),4.52–4.41(m ,2H),4.20(dt,J=11.2,8.9Hz,1H),4.15–4.05(m,2H),3.73(d,J=2.2Hz,6H),3.55–3.50(m,1H),3.50 –3.38(m,2H),3.33–3.20(m,2H),3.06(ddd,J=14.4,8.3,3.7Hz,1H),3.02–2.93(m,1H),2.92(d,J=7 .4Hz,2H),2.81(dd,J=8.9,6.6Hz,2H),2.71(t,J=5.8Hz,1H),2.61(t,J=5.9Hz,1H),2.41(dd,J=8.9, 6.7Hz,2H),2.08(s,2H),1.84–1.70(m,4H),1.68(s,3H),1.48–1.23(m,8H),1.23–1.14(m,1H),1.14– 1.05(m,8H),1.02(d,J=7.5Hz,4H),0.96(d,J=6.7Hz,3H),0.90(s,1H),0.76(dt,J=21.1,7.3Hz,7H).

[0376] Example 44 Synthesis of Compound 180

[0377] The synthetic route of compound 180 is as follows:

[0378] Example 45 Synthesis of Compound 185

[0379] Compound 181 (5.22 g, 11.918 mmol) was added to a 500 mL round-bottom flask, followed by dichloromethane (200 mL). Once the solution was substantially dissolved, HBTU (5.42 g, 14.302 mmol) and DIEA (3.08 g, 23.837 mmol) were added. Once the solution was homogeneous, compound 51 (5 g, 11.918 mmol) was added. The reaction was allowed to react at room temperature and monitored by TLC and LCMS. Upon completion of the reaction, the reaction system was stripped of most of the dichloromethane, diluted with ethyl acetate, washed sequentially with water and saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield the crude product. The crude product was dissolved in DCM (200 mL), and the reaction reagent, piperidine (11.787 mL, 119.185 mmol), was added. The reaction system was stirred at room temperature under N2 protection. After the reaction was completed, most of the dichloromethane was removed from the reaction system and the mixture was slurried three times with petroleum ether (300 mL) to obtain a crude product which was used directly in the next step without separation. MS (ESI): m / z calcd for C 38 H 52 N2O5[M+H] + :617.39,found:617.45.

[0380] Fmoc-6-Aminohexanoic Acid (4.20 g, 11.888 mmol) was added to a 500 mL round-bottom flask and dissolved in DCM (230 mL). DIEA (3.07 g, 23.776 mmol) and HBTU (5.41 g, 14.266 mmol) were then added and allowed to react for half an hour. Compound 182 (7.33 g, 11.888 mmol) was then added. The reaction was allowed to proceed at room temperature and monitored by TLC and LCMS. After the reaction was completed, the reaction system was stripped of most of the dichloromethane, diluted with ethyl acetate, washed sequentially with water and saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in DCM (200 mL), and the reaction reagent, piperidine (11.787 mL, 119.185 mmol), was added. The reaction system was stirred at room temperature under N2 protection. After the reaction was completed, most of the dichloromethane was removed from the reaction system and the mixture was slurried three times with petroleum ether (300 mL) to obtain a crude product which was used directly in the next step without separation. MS (ESI): m / z calcd for C 44 H 63 N3O6[M+H] + :730.47,found:730.54.

[0381] 4-Pentynoic acid (0.20 g, 2.055 mmol) was added to a 50 mL round-bottom flask and dissolved in DCM (20 mL). HBTU (0.94 g, 2.466 mmol) and DIEA (0.53 g, 4.110 mmol) were then added. After reacting at room temperature for half an hour, compound 183 (1.5 g, 2.055 mmol) was added. The reaction system was stirred at room temperature under N2 protection and monitored by TLC and LCMS. After the reaction was completed, the reaction system was stripped of most of the dichloromethane and diluted with ethyl acetate. The product was washed sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Compound 184 (0.50 g, 28%) was purified by reverse phase preparative column chromatography (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase). MS (ESI): m / z calculated for C 49 H 67 N3O7[M+H] + :810.50,found:810.60.

[0382] Compound 184 (0.37 g, 0.457 mmol) and DMAP (0.01 g, 0.091 mmol) were added to a 50 mL round-bottom flask. After purging the nitrogen atmosphere three times, DCM (20 mL) was added to dissolve the mixture. DIEA (0.12 g, 0.913 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.12 g, 0.502 mmol) were then added dropwise. The reaction system was stirred at room temperature under N2 protection and monitored by TLC and LCMS. After the reaction was completed, the reaction system was stripped of most of the dichloromethane and diluted with ethyl acetate. The mixture was washed sequentially with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Compound 185 (0.31 g, 67%) was purified by reverse-phase preparative column chromatography (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford compound 185. 1H NMR (400MHz, DMSO) δ7.83(t,J=5.3Hz,1H),7.69(t,J=5.3Hz,1H),7.37–7.26(m,4H),7.25–7.14(m,5H), 6.90–6.82(m,4H),4.70–4.46(m,1H),4.22–4.09(m,1H),3.77–3.64(m,9H),3.62–3.44(m,3H),3.26–3.1 5(m,1H),3.10–2.91(m,5H),2.78–2.68(m,3H),2.37–2.29(m,2H),2.27–2.18(m,3H),2.18–2.05(m,2H), 2.02(t,J=7.4Hz,3H),1.51–1.42(m,4H),1.41–1.32(m,4H),1.29–1.16(m,17H),1.16–1.06(m,13H); 31P NMR(162MHz,DMSO)δ146.98,146.75,146.55,146.20.MS(ESI):m / z calcd for C 58 H 84 N5O8P[M+H] + :1010.61,found:1010.68.

[0383] Example 46 Synthesis of Compound 190

[0384] Reactant 186 (4.90 g, 22.049 mmol) and reactant 51 (7.4 g, 17.639 mmol) were added to a 50 mL round-bottom flask and dissolved in dry dichloromethane (150 mL). Reagents DIEA (11.522 mL, 66.147 mmol) and HBTU (9.32 g, 24.582 mmol) were added. The reaction system was stirred at 25°C under nitrogen for 1.5 h. The reaction was monitored by TLC and LC-MS. Upon completion of the reaction, the reaction system was diluted with dichloromethane, washed sequentially with water and saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified using a reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford compound 187 (4.49 g, 32%). MS (ESI): m / z calculated for C 34 H 41 NO 10 [M+H] + :624.27,found:624.35.

[0385] Reactant 187 (4.2 g, 6.734 mmol) and compound 188 (2.08 g, 13.468 mmol) were added to a 100 mL round-bottom flask and dissolved in dry dichloromethane (60 mL). DIEA (2.346 mL, 13.468 mmol) and HBTU (2.81 g, 7.407 mmol) were added, and the reaction system was stirred at room temperature under N2 protection. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction system was diluted with dichloromethane, washed sequentially with water and saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified using a reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to obtain compound 189 (4.49 g, 32%). MS (ESI): m / z calcd for C 42 H 53 N3O 10 [M+H] + :760.37,found:760.45.

[0386] Reactant 189 (420 mg, 0.553 mmol) was added to a 50 mL round-bottom flask and dissolved in dry dichloromethane (3 mL). Reagents DMAP (14 mg, 0.111 mmol), DIEA (0.193 mL, 1.105 mmol), and CEPCl (0.185 mL, 0.829 mmol) were then added. The reaction system was stirred at 25°C under nitrogen for 30 min. After completion of the reaction, the system was concentrated under reduced pressure under nitrogen to afford the crude product, which was then purified on a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford product 190 (395 mg, 74%). 1 H NMR (400MHz, DMSO) δ7.87(t,J=5.5Hz,1H),7.70(t,J=5.9Hz,1H),7.36–7.25(m,4H),7.25–7.16(m, 5H),6.93–6.80(m,4H),4.70–4.58(m,1H),4.27–4.16(m,1H),4.15–4.02(m,2H),3.85(s,2H),3.79 –3.68(m,9H),3.62–3.39(m,12H),3.25–3.00(m,6H),2.79–2.69(m,3H),2.35(td,J=6.6,2.2Hz,2H ),2.29–2.21(m,2H),2.16–2.08(m,1H),2.05–1.95(m,1H),1.57–1.48(m,2H),1.18–1.06(m,12H); 31P NMR (162MHz, DMSO) δ148.42,147.92; MS (ESI): m / z calcd for C 51 H 70 N5O 11 P[M+H] + :960.48,found:960.52.

[0387] Example 47 Synthesis of Compound 199

[0388] Synthesis of compound 192

[0389] Reactant 51 (12.3 g, 29.319 mmol) and reagent 191 (10.36 g, 29.319 mmol) were added to a 500 mL round-bottom flask and dissolved in dry dichloromethane (200 mL). HBTU reagent O-(IH-benzotriazole-1-yl)-N,N,N',N'-tetramethylisourea phosphorus hexafluoride (13.34 g, 35.183 mmol) and DIEA (10.214 mL, 58.638 mmol) were added. The reaction system was stirred at room temperature under nitrogen protection. The reaction was monitored by TLC and LC-MS. After the reaction was completed, the reaction system was washed with water and saturated brine in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Compound 192 was directly used for subsequent reactions without isolation and purification. MS (ESI): m / z calcd for C 47 H 50 N2O7[M+H] + :755.36,found:755.39.

[0390] Synthesis of compound 193

[0391] Reactant 192 (22 g, 29.142 mmol) was added to a 250 mL round-bottom flask, dissolved in dichloromethane (88 mL), and the reaction reagent piperidine (17.6 mL) was added. The reaction system was stirred at 25°C under N2 protection. The reaction was monitored by TLC and LC-MS. After the reaction was complete, the reaction system was directly concentrated under reduced pressure to obtain the crude product. The crude product was slurried with a large amount of petroleum ether to obtain the product, which was used directly in the subsequent reaction without subsequent separation and purification. MS (ESI): m / z calcd for C 32 H 40 N2O5[M+H] + :533.29,found:533.32.

[0392] Synthesis of compound 196

[0393] Reactant 193 (7.5 g, 14.09 mmol) and reactant 194 (8.32 g, 14.090 mmol) were added to a 500 mL round-bottom flask and dissolved in dry dichloromethane (200 mL). The reagents DIEA (4.909 mL, 28.180 mmol) and HBTU (5.88 g, 15.499 mmol) were added. The reaction system was stirred at room temperature under nitrogen protection. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction system was washed with water and saturated brine in sequence. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Compound 195 was directly used in the subsequent reaction without isolation and purification. The crude compound 195 was dissolved in dichloromethane (200 mL) and the reagent piperidine (14 mL) was added. The reaction system was stirred at 25°C under N2 protection. The reaction was monitored by TLC and LC-MS. After completion, the reaction system was concentrated under reduced pressure to yield a solid residue. This residue was dissolved in a small amount of dichloromethane and slurried with a large amount of petroleum ether to obtain a crude product. The crude product was purified on a reverse-phase preparative column (5%-95% acetonitrile / water, aqueous phase containing 0.01% ammonium bicarbonate) to afford compound 196 (7.5 g, 80%). MS (ESI): m / z calculated for C 38 H 52 N4O6[M+H] + :660.39,found:660.37.

[0394] Synthesis of compound 198

[0395] Reactant 196 (5.5 g, 8.328 mmol) and compound 197 (4.98 g, 16.657 mmol) were added to a 500 mL round-bottom flask and dissolved in dry dichloromethane (100 mL). Reagents DIEA (7.254 mL, 41.642 mmol) and HBTU (6.95 g, 18.323 mmol) were added. The reaction system was stirred at room temperature under nitrogen. The reaction was monitored by TLC and LC-MS. After completion of the reaction, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried, concentrated, and purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford compound 198 (6.13 g, 68%). MS (ESI): m / z calculated for C 54 H 72 F6N6O 10 [M+H] + :1079.52,found:1079.62.

[0396] Synthesis of compound 199

[0397] Reactant 198 (2.5 g) and the reaction reagent DMAP (0.06 g, 0.464 mmol) were added to a 100 mL round-bottom flask and dissolved in dry dichloromethane (25 mL). DIEA (0.808 mL, 4.636 mmol) and CEPCl (0.776 mL, 3.477 mmol) were added. The reaction system was stirred at room temperature under N2 protection for 30 min. The reaction system was directly concentrated under reduced pressure under nitrogen protection to obtain the crude product, which was purified by reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to obtain the yellow product, Compound 199 (1.7 g, 57%). 1 H NMR (400MHz, DMSO) δ9.44–9.30(m,2H),7.91–7.77(m,2H),7.71(t,J=5.5Hz,1H),7.37–7.25(m,4H), 7.25–7.14(m,5H),6.92–6.81(m,4H),4.70–4.60(m,1H),4.20–4.10(m,2H),3.82–3.65(m,9H),3.62 –3.48(m,3H),3.33(s,5H),3.19–3.11(m,4H),3.08–2.93(m,5H),2.79–2.70(m,2H),2.25–2.08(m,4 H),2.05–1.96(m,3H),1.54–1.41(m,10H),1.41–1.30(m,4H),1.31–1.16(m,8H),1.17–1.06(m,12H); 31 P NMR (162MHz, DMSO) δ148.42,147.92; MS (ESI): m / z calcd for C 63 H 89 F6N8O 11 P[M+H] + :1279.63,found:1279.68.

[0398] Example 48 Synthesis of Compound 202

[0399] Synthesis of compound 201

[0400] Reactant 200 (2.5 g, 2.818 mmol) was added to a 250 mL round-bottom flask and dissolved in dry dichloromethane (15 mL). The reaction reagents DIEA (2.945 mL, 16.908 mmol), HBTU reagent O-(IH-benzotriazol-1-yl)-N,N,N',N'-tetramethylisouronium phosphorus hexafluoride (3.42 g, 9.017 mmol), and valeric acid (0.83 g, 8.454 mmol) were added. The reaction system was stirred at room temperature under N2 protection. After completion of the reaction, the reaction system was washed with water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified on a reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to obtain product 201 (1.81 g, 61%). MS (ESI): m / z calculated for C 60 H 82 N6O 10 [M+H] + :1047.61,found:1046.68.

[0401] Synthesis of compound 202

[0402] Reactant 201 (1.41 g, 1.346 mmol) was added to a 50 mL round-bottom flask and dissolved in dichloromethane (20 mL). The reagents DMAP (0.03 g, 0.269 mmol), DIEA (0.469 mL, 2.693 mmol), and CEPCl (0.48 g, 2.019 mmol) were then added. The reaction was stirred at 25°C under nitrogen for 30 min. The reaction was concentrated under reduced pressure under nitrogen to afford the crude product, which was then purified using a reverse-phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford product 202 (877 mg, 52%). 1H NMR (400MHz, DMSO) δ7.90–7.77(m,4H),7.75–7.67(m,1H),7.36–7.25(m,4H),7.24–7.13(m,5H),6.96 –6.80(m,4H),4.71–4.55(m,1H),4.23–4.08(m,2H),3.73(s,8H),3.62–3.47(m,3H),3.33(s,5H),3.09 –2.92(m,9H),2.79–2.71(m,3H),2.37–2.30(m,4H),2.28–2.20(m,5H),2.16–2.06(m,3H),2.05–1.98( m,3H),1.63–1.52(m,2H),1.52–1.41(m,7H),1.41–1.30(m,8H),1.29–1.17(m,9H),1.16–1.08(m,9H); 31 P NMR (162MHz, DMSO) δ148.42,147.92; MS (ESI): m / z calcd for C 69 H 99 N8O 11 P[M+H] + :1247.72,found:1247.68.

[0403] Example 49 Synthesis of Compound 208

[0404] Synthesis of compound 204

[0405] Reactant 203 (5.00 g, 4.846 mmol) was added to a 250 mL round-bottom flask and dissolved in dry dichloromethane (100 mL). TFA (49.97 mL, 0.653 mol) was then added. The reaction system was stirred at 25°C under nitrogen for 3 h. The reaction was monitored by TLC and LC-MS. Upon completion of the reaction, the reaction system was concentrated under reduced pressure three times with 100 mL of toluene to obtain the crude product, which was used directly in the next step without further separation. MS (ESI): m / z calculated for C 35 H 69 N7O9[M+H] + :732.52,found:732.59.

[0406] Synthesis of compound 205

[0407] Reactant 204 (3.54 g, 4.846 mmol) was added to a 250 mL round-bottom flask and dissolved in dry dichloromethane (100 mL). DIEA (5.912 mL, 33.936 mmol), HBTU (7.32 g, 19.358 mmol), and 4-pentynoic acid (1.659 g, 16.939 mmol) were added. The reaction system was stirred at room temperature under N2 protection. The reaction was monitored by TLC and LC-MS. Upon completion of the reaction, the reaction system was diluted with dichloromethane, washed sequentially with water and saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified using a reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to obtain compound 205 (3.81 g, 81%). MS (ESI): m / z calculated for C 50 H 81 N7O 12 [M+H] + :972.59,found:972.66.

[0408] Synthesis of compound 206

[0409] Reactant 205 (3.81 g, 3.921 mmol) was added to a 250 mL round-bottom flask and dissolved in methanol (100 mL). NaOH (0.47 g, 11.739 mmol) was dissolved in pure water (2 mL) and added to the system. The reaction system was stirred at room temperature under nitrogen protection. TLC and LC-MS were used to monitor the reaction. After the reaction was completed, the reaction system was neutralized with hydrochloric acid to pH = 7 and concentrated under reduced pressure to obtain the crude product, which was used directly in the next step without separation. MS (ESI): m / z calculated for C 49 H 79 N7O 12 [M+H] + :958.58,found:958.66.

[0410] Synthesis of compound 207

[0411] Reactant 206 (2 g, 2.09 mmol) was added to a 250 mL round-bottom flask and dissolved in dry dichloromethane (10 mL). DIEA (0.54 mg, 4.180 mmol), HBTU (950 mg, 2.508 mmol), and compound 3 (821 mg, 2.09 mmol) were then added. The reaction system was stirred at room temperature under N2 protection. The reaction was monitored by TLC and LC-MS. Upon completion of the reaction, the reaction system was diluted with dichloromethane, washed sequentially with water and saturated brine, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified using a reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford compound 207 (2.216 g, 80%). MS (ESI): m / z calculated for C 73 H 104 N8O 15 [M+H] + :1333.76,found:1333.72.

[0412] Synthesis of compound 208

[0413] Reactant 207 (420 mg, 0.315 mmol) was added to a 50 mL round-bottom flask and dissolved in dry dichloromethane (3 mL). Reagents DIEA (81 mg, 0.630 mmol) and succinic anhydride (32 mg, 0.315 mmol) were then added. The reaction system was stirred at 25°C under nitrogen for 30 min. After completion of the reaction, the system was concentrated under reduced pressure under nitrogen to obtain the crude product. The crude product was then purified on a reverse phase preparative column (5%-95% acetonitrile / water, 0.01% ammonium bicarbonate in the aqueous phase) to afford product 208 (343 mg, 76%). MS (ESI): m / z calculated for C 77 H 108 N8O 18 [M+H] + :1433.78,found:1433.85.

[0414] Example 50 Synthesis of Compounds 209-211

[0415] The synthesis of compound 209 was carried out by referring to compound 57, using compound 56 (150 mg, 0.269 mmol), trimethylsilyl trifluoromethanesulfonate (73 μL, 0.403 mmol, 1.5 eq.), 1,2-DCE (5 mL) and anhydrous methoxyethanol (61 mg, 0.807 mmol, 3 eq.) as raw materials to obtain compound 209 (48 mg, yield: 35%). 1H NMR (400MHz, CDCl3) δ8.09(d,J=7.5Hz,2H),7.83(d,J=7.5Hz,2H),7.61(t,J=7.4 Hz,1H),7.51–7.45(m,3H),7.32–7.26(m,2H),6.03(d,J=8.6Hz,1H),5.75–5.65( m,2H),4.98(d,J=8.4Hz,1H),4.39–4.29(m,1H),4.15–4.04(m,2H),3.87–3.78(m ,1H),3.65–3.56(m,3H),3.38(s,3H),3.26(dd,J=13.0,3.8Hz,1H),1.89(s,3H). MS(ESI):m / z calcd for C 25 H 28 N4O8[M+H] + :513.52,found:512.42.

[0416] The synthesis of compound 210 was carried out by referring to compound 57, using compound 56 (150 mg, 0.269 mmol), trimethylsilyl trifluoromethanesulfonate (73 μL, 0.403 mmol, 1.5 eq.), 1,2-DCE (5 mL) and anhydrous tetrahydrofuran-4-ol (83 mg, 0.807 mmol, 3 eq.) as raw materials to obtain compound 210 (25 mg, yield: 17%). 1 H NMR (400MHz, CDCl3) δ8.10(d,J=7.3Hz,2H),7.81(d,J=7.3Hz,2H),7.63(t,J=7.4Hz,1H),7.54–7.45(m,3H ),7.34–7.27(m,2H),5.91(d,J=8.3Hz,1H),5.80(dd,J=11.2,3.4Hz,1H),5.72(d,J=3.2Hz,1H),5.13(d,J =8.3Hz,1H),4.21–4.10(m,2H),4.03–3.86(m,3H),3.60(dd,J=13.0,8.6Hz,1H),3.51–3.42(m,2H),3.24( dd,J=13.0,3.5Hz,1H),2.03–1.94(m,1H),1.89(s,4H),1.82–1.73(m,1H),1.66–1.55(m,1H).MS(ESI):m / z calcd for C 27 H 30 N4O8[M+H] + :539.56,found:539.66.

[0417] The synthesis of compound 211 was carried out by referring to compound 57, using compound 56 (150 mg, 0.269 mmol), trimethylsilyl trifluoromethanesulfonate (73 μL, 0.403 mmol, 1.5 eq.), 1,2-DCE (5 mL) and anhydrous cyclopentanol (68 mg, 0.807 mmol, 3 eq.) as raw materials to obtain compound 211 (72 mg, yield: 52%). 1 H NMR (400MHz, CDCl3) δ8.10(d,J=7.4Hz,2H),7.81(d,J=7.5Hz,2H),7.61(t,J=7.4Hz,1H),7.47(t,J=7.5H z,4H),7.28(t,J=6.9Hz,2H),6.09(d,J=8.6Hz,1H),5.79–5.69(m,2H),4.94(d,J=8.4Hz,1H),4.46–4.36 (m,1H),4.23(dd,J=19.4,8.6Hz,1H),4.11(dd,J=8.3,3.4Hz,1H),3.61(dd,J=13.0,8.4Hz,1H),3.26(dd ,J=13.0,3.8Hz,1H),1.88(s,3H),1.86–1.80(m,2H),1.74–1.63(m,3H),1.57–1.49(m,2H).MS(ESI):m / z calcd for C 27 H 30 N4O7[M+H] + :523.56,found:523.78.

[0418] The synthesis numbers of some compounds and their corresponding exemplary compound numbers (as well as the nucleoside numbers corresponding to the compounds introduced into the oligonucleotide sequences) are as follows:

[0419] Example 51 Synthesis of oligonucleotide sequences

[0420] As shown in Figure 1, the solid phase synthesis process of oligonucleotide sequences is carried out according to the following process:

[0421] Using a universal solid phase synthesis carrier (UnyLinker TMLoaded HL Solid Supports (Kinovate Life Sciences) are used as support. Using phosphoramidite nucleoside monomers as starting materials, the phosphoramidite solid-phase synthesis method connects nucleoside monomers one by one from the 3'-5' direction according to the oligonucleotide arrangement order. Each nucleoside monomer connection includes four steps: deprotection, coupling, oxidation or sulfurization, and capping. Ammonolysis reaction produces a crude oligonucleotide. After purification, ultrafiltration, and lyophilization, the two single-stranded oligonucleotides that constitute the siRNA are obtained. These two single strands are annealed to form a double-stranded siRNA with complementary base pairing, and the final product is obtained by freeze-drying.

[0422] 1. Synthesis principle, including the following four reaction steps

[0423] a. Deprotection of dimethoxytrityl groups: The DMTr (dimethoxytrityl) protecting group on the conjugate monomer (i.e., the phosphoramidite derivative prepared in the example) / nucleotide is removed using dichloroacetic acid at room temperature (20-25°C) to obtain active hydroxyl groups available for coupling reactions. The deprotection reagent is a 3% v / v solution of dichloroacetic acid in toluene or a 3% v / v solution of dichloroacetic acid in dichloromethane.

[0424] b. Coupling Reaction: Nucleotide phosphoramidite monomers or conjugate monomers and an activator are simultaneously injected into a solid-phase synthesis column. The phosphoramidite groups are activated and undergo a coupling condensation reaction with the active hydroxyl groups to form a phosphite triester. The activator is a 0.6 M solution of 5-ethylthio-1H-tetrazole (ETT) in acetonitrile.

[0425] c1. Oxidation reaction: Under the action of an oxidizing agent, the phosphite triester generated in the previous coupling condensation reaction is converted into a stable phosphate triester, the oxidizing agent being a 0.04M iodine / water / pyridine solution, v (water): v (pyridine) = 1:9; or

[0426] c2. Thiolysis reaction: Under the action of the thiolation reagent PADS / ADTT, the phosphite triester generated in the previous coupling condensation step is converted into a stable thiophosphate triester.

[0427] d. Capping reaction: The active hydroxyl groups that did not react completely during the coupling reaction were capped to prevent them from participating in subsequent reactions. The capping reagents were Cap A (acetic anhydride / acetonitrile = 1:4, volume ratio) and Cap B (NMI:Py:acetonitrile = 2:3:5, volume ratio). When used, the capping reagents Cap A and Cap B were mixed in a volume ratio of 1:1.

[0428] The above four-step cycle is repeated, and different types of nucleoside phosphoramidite monomers are added in sequence according to the oligonucleotide sequence. After completing the four-step cycle of the desired nucleoside phosphoramidite, the 5'-DMTr group at the end of the oligonucleotide is removed, and the cyanoethyl-protected phosphate is removed using a diethylamine acetonitrile solution (20% v / v). The solid-phase reaction is completed to obtain a solid-phase support for the linked oligonucleotide.

[0429] Cleavage and Deprotection: Aminolysis of the solid support containing the oligonucleotide is performed using an aminolysis reagent (25% to 28% concentrated ammonia solution). This cleaves the oligonucleotide from the support and simultaneously removes the various protecting groups on the nucleoside bases. The resulting solution is concentrated to yield the crude oligonucleotide. The crude product is then tested using high-resolution liquid chromatography-mass spectrometry (LC-MS) to determine the target molecular weight.

[0430] Purification and desalting: Purification of crude oligonucleotides by anion exchange chromatography

[0431] 1. Anion Exchange Chromatography: The column was packed with strong anion exchange medium Nano15Q (Suzhou Nanomicro Technology) with a packing height of 120 mm. Buffer A: 10 mM NaOH aqueous solution, Buffer B: 10 mM NaOH and 2 M NaCl aqueous solution, and Buffer C: 50 mM NaH2PO4 aqueous solution. Buffer B was used as the elution phase with a gradient of 10% to 40% at a flow rate of 150 cm / h. The eluate was monitored at 290 nm. The eluate was collected and neutralized with Buffer C, and the pH was adjusted to 7.0. Separate fractions were collected and combined if they met the purity requirements.

[0432] 2. Desalting: Desalting is done using a gel column, the gel column is HiTrap TM Desalting (Si Tuo Fan), eluting with deionized water.

[0433] Annealing: The desalted sense and antisense strands are mixed in an equimolar ratio and heated to 65°C. The mixture is then cooled to room temperature naturally, allowing the two strands to form a double-stranded structure through hydrogen bonding. After annealing, purity is analyzed using ion-pair reversed-phase chromatography (IPRP-HPLC) and molecular weight is determined using high-resolution liquid chromatography-mass spectrometry (LC-MS).

[0434] Lyophilization: After annealing, the sample is packaged according to certain specifications. After packaging, the sample is freeze-dried to obtain lyophilized powder of the siRNA double-stranded sample.

[0435] The synthesized single-stranded oligonucleotides and the double-stranded conjugates formed by complementary pairing are shown in Table 1.

[0436] Table 1 Oligonucleotide conjugates

[0437] Table 1 Oligonucleotide conjugates

[0438] In the table, "G," "C," "A," "T," and "U" represent nucleotides based on guanine, cytosine, adenine, thymine, and uracil, respectively. A lowercase letter "m" indicates that the nucleotide to the left of the letter "m" is a 2'-methoxy-modified nucleotide; a lowercase letter "f" indicates that the nucleotide to the left of the letter "f" is a 2'-fluorine-modified nucleotide; a lowercase letter "s" indicates that the connection between the two nucleotides to the left and right of the letter "s," or between the phosphoramidite monomers described in the present invention, is a phosphorothioate linkage; and a lowercase letter "d" indicates that the nucleotide to the right of the letter "d" is a deoxyribonucleotide. SS indicates the sense strand, and AS indicates the antisense strand. "-" indicates that the result is the same as or equivalent to the value / information of the aforementioned sequence or conjugate with the same number.

[0439] The conjugates of the present invention were synthesized based on the following sequence information:

[0440] In the present invention, the mRNA expressed by the target gene includes but is not limited to the mRNA transcribed by the following genes: ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C3, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, X0, INSR, SREBF 1, HDV, RPTOR, TLK2, LPA, AGT.

[0441] Example 52 Post-sequence modification

[0442] Place 20.0 nmol of Seq 23 (BPR302-M01D0113-SS) in a 1.5 mL centrifuge tube and add 60 μL of ultrapure water to fully dissolve it. Dissolve 0.35 mg (800 nmol, 40.0 eq) of compound 73 in 100 μL of methanol (water ≤ 50 ppm) and add the resulting mixture dropwise to the centrifuge tube. Vortex the mixture thoroughly to homogenize. Dissolve 0.015 mg (60 nmol, 3.0 eq) of copper sulfate pentahydrate and 0.036 mg (180 nmol, 9.0 eq) of sodium ascorbate in 1.5 mL centrifuge tubes and add 20 μL of ultrapure water to each solution. Add the resulting mixture to the reaction mixture. Once the mixture is completely clarified, thoroughly vortex and centrifuge to ensure that the mixture remains at the bottom of the tube. The reaction mixture is incubated at 60°C for 15 min under microwave conditions at 50 W power. LC-MS analysis is used to determine the endpoint. After the reaction, the system was concentrated under vacuum at 40°C to remove the solvent. After purification by preparative chromatography, the resulting product was placed in a 1.5 mL centrifuge tube and concentrated under vacuum at 40°C to remove the solvent. The product was then added with 300 μL of aqueous ammonia and placed in a 55°C water bath for 1 hour for aminolysis. The endpoint and purity were determined by LC-MS. After the reaction, the system was concentrated under vacuum at 40°C to remove the solvent, yielding the sense strand (SS) compound.

[0443] Annealing procedure: Add 100 μL of ultrapure water to obtain 9.0 nmol of target product (as determined by Nano Drop assay). After complete dissolution, add an equal amount of the corresponding AS sequence and shake until homogenous. Centrifuge at 1000 rad / s for 5 seconds to ensure that the system is located at the bottom of the centrifuge tube. Then, anneal in a 55°C water bath for 20 minutes to obtain the corresponding RNA duplex. The system is determined by LC to determine the endpoint and purity.

[0444] Compounds 57, 58, 59, 60, 61, 62, 63, 64 and 73 were used as reaction substrates with different oligonucleotide sequences, and the above operation was repeated to obtain oligonucleotide conjugates. After quantification, the corresponding antisense strand (AS) with complementary base pairing was added for annealing to obtain double-stranded oligonucleotide sequences, as shown in Tables 2, 3, 4, 5 and 6.

[0445] Table 2

[0446] In the table, "-" indicates that the result is the same or equivalent to the value / information of the aforementioned sequence or conjugate with the same number. NA indicates not applicable or blank.

[0447] Table 3

[0448] In the table, "-" indicates that the result is the same or equivalent to the value / information of the aforementioned sequence or conjugate with the same number. NA indicates not applicable or blank.

[0449] Table 4

[0450] In the table, "-" indicates that the result is the same or equivalent to the value / information of the aforementioned sequence or conjugate with the same number. NA indicates not applicable or blank.

[0451] Table 5

[0452] In the table, "-" indicates that the result is the same or equivalent to the value / information of the aforementioned sequence or conjugate with the same number. NA indicates not applicable or blank.

[0453] Table 6

[0454] In the table, "-" indicates that the result is the same or equivalent to the value / information of the aforementioned sequence or conjugate with the same number. NA indicates not applicable or blank.

[0455] Test Example 1 Free uptake experiment of primary mouse hepatocytes

[0456] 1.1 Experimental operation

[0457] 1) Cell plating:

[0458] a) PMH cells (purchased from Milestone, catalog number CMH-100CBP-PQ) were revived and plated onto a collagen-coated 96-well plate at 40,000 cells / well in 90 μL / well.

[0459] b) Culture in a 37° C., 5% CO 2 incubator. After 6 hours, the culture medium was replaced with hepatocyte maintenance medium (purchased from Milestone, catalog number CMHEP064) and culture was continued overnight.

[0460] 2) Cell transfection:

[0461] a) Dilute the test substance solution to the desired concentration (20 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.315 nM).

[0462] b) Add 10 μL of transfection mixture to the corresponding wells and set up 3 replicate wells for each concentration.

[0463] 3). 48 hours after transfection, perform qPCR detection:

[0464] a) Cell lysis: Cell lysis was performed according to the instructions of Cells-to-Ct bulk lysis reagents.

[0465] b) Reverse transcription (RNA to cDNA): Perform reverse transcription according to the instructions of Cells-to-Ct Fast advanced RT reagent.

[0466] c) qPCR detection:

[0467] Prepare the qPCR reaction mixture as shown in the table below.

[0468] The qPCR procedure was performed as follows:

[0469] d) Data analysis: Ct values ​​were automatically calculated using the default settings of Quant Studio 7 software and exported to Excel files.

[0470] Control wells without GalNAc-siRNA (PBS negative control group) and internal reference gene GAPDH mRNA control wells were set up.

[0471] After exporting the Ct value, the relative expression of the gene was calculated using the following formula:

[0472] ΔCt=Ct(target gene)–Ct(GAPDH)

[0473] ΔΔCt = ΔCt (test sample) - ΔCt (PBS negative control group)

[0474] Remaining ratio of genes relative to the blank group = 2^(-ΔΔCt)

[0475] Gene inhibition rate relative to the blank group = 1 – residual ratio

[0476] 1.2 The reagents and consumables used in the test examples are commercially available products.

[0477] The instruments used in the test case include the following:

[0478] 1.3 TTR mRNA expression inhibition rate results

[0479] The results of the inhibition rates of some oligonucleotide conjugates prepared by the present invention on TTR mRNA expression in mouse C57 primary hepatocytes are shown in Figures 2-6.

[0480] As can be seen from Figure 2, almost all the conjugates tested have high target gene inhibition activity at higher concentrations, and still show good inhibitory effects at low concentrations (0.625nM). In different conjugate designs, different G structures have different effects on the TTR gene inhibition effect. Conjugates BPR302-M01D0113-G1, BPR302-M01D0113-G6, BPR302-M01D0116-G2, BPR302-M01D0113-G3, BPR302-M01D0113-G2, BPR302-M01D0113-G7, etc. showed very good activity, and at the lower concentration tested (0.625nM), they had significantly higher inhibitory activity than the conjugate connected to ordinary galnac (BPR302-M01D0111).

[0481] As can be seen from FIG3 , the inhibition rate of the tested compounds on TTR at a relatively low concentration (0.3125 nM) was higher than 60%, and even BPR302-M01D0150-G2 achieved an inhibition rate of 80%.

[0482] As shown in Figures 4 and 5 , some conjugates also exhibited significant gene knockdown efficacy in other sequences. In Figure 4 , the conjugate using the compound described herein (BPR302-M02D0104-G5) exhibited higher inhibitory activity than the unmodified, standard galnac conjugate (BPR302-M02D0101), with activity twice that of the control conjugate at the lowest concentration tested (2.5 nM). In Figure 5 , the conjugate corresponding to the partial G structure still achieved greater than 50% target gene knockdown at the lowest concentration tested (1.25 nM).

[0483] As shown in Figure 6, when the compound structure is attached to the 3'-terminus of the sense strand of an RNAi drug, or to both the 3' and 5' ends, the resulting conjugate still exhibits good target gene inhibition activity. When the compound described herein is coupled to a non-terminal end of one of the RNAi drug's strands, the conjugate, BPR302-M01D0161, exhibits inhibitory activity close to that of the terminal end.

[0484] Test Example 2 In vivo experiment in mice

[0485] 2.1 Target gene TTR

[0486] Wild-type mice C57BL / 6JGPT (Chengdu Yaokang Biotechnology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against mouse TTR gene expression. At least one week in advance, 6-8 week-old male mice C57BL / 6JGPT were brought into the room for adaptive feeding. After the adaptive feeding was completed, the mice were randomly divided into groups according to their body weight, with 4-6 animals in each group, and the day of grouping was D0. After blood collection on D0, a single dose of subcutaneous injection was performed; a negative control group (PBS) and experimental groups (conjugate BPR302-M01D0119, conjugate BPR302-M01D0127, conjugate BPR302-M01D0129, conjugate BPR302-M01D0140, conjugate BPR302-M01D0141, conjugate BPR302-M01D014 3. Conjugate BPR302-M01D0150-G2, conjugate BPR302-M01D0150-G11, conjugate BPR302-M01D0151, conjugate BPR302-M01D0152, conjugate BPR302-M02D0122, conjugate BPR302-M02D0128) were administered at a dose of 0.5 mg / kg and a dosing volume of 10 μL / g. Blood was subsequently collected from the orbit or neck at time points such as D7, D14, D21, D28, D35, D42, and D56. The blood volume was 100-300 μL (determined by the mouse's condition) and serum was obtained after separation. Finally, TTR protein was quantitatively detected using an ELISA kit (Abcam ab282297). The specific procedures were the same as those in the kit instructions.

[0487] The experimental results are shown in Figures 7, 8, and 9. The results show that some of the oligonucleotide conjugates prepared by the present invention can effectively knock out the TTR target gene in mice.

[0488] Test Example 3

[0489] This test case studies the inhibitory activity of oligonucleotide conjugates against the expression of different target genes in mice.

[0490] 3.1 PTEN gene

[0491] Wild-type mice C57BL / 6JGPT (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against mouse PTEN gene expression. At least one week in advance, 6-8 week-old male C57BL / 6JGPT mice were brought into the room for adaptive feeding. After adaptive feeding, the mice were randomly divided into groups according to body weight, with 4 animals in each group, and the day of grouping was D0. After blood collection on D0, a single dose of subcutaneous injection was performed; a negative control group (PBS or saline) and an experimental group (BPR3T01-M10D0102) were set up, with a dose of 3 mg / kg and a dosing volume of 10 μL / g. The mice were sacrificed on D7, the liver organs were isolated, and the expression of the PTEN gene was detected according to the following procedures. The activity of the oligonucleotide conjugate was evaluated by the reduction effect compared to the pre-administration effect.

[0492] 1. Preparation of experimental materials

[0493] Take 10-90 mg of mouse liver;

[0494] 2. Grinding

[0495] Add tissue lysis buffer at a ratio of 1:10, grind 5 magnetic beads, and grind on a tissue homogenizer at 6800 rpm for 5 minutes;

[0496] 3. RNA Extraction

[0497] RNA was extracted using VAZYME FastPure Universal Plant Total RNA Isolation Kit V2 (Cat. No. PC112-01, Batch No. 017E2272CB) according to the kit instructions;

[0498] 4. Reverse transcription

[0499] Reverse transcription was performed using VAZYME HiScript III 1 st Reverse transcription was performed using the Strand cDNA Synthesis Kit (+gDNA wiper) (Cat. No.: R312-02, Batch No.: 7F581J1) according to the manufacturer's instructions;

[0500] 5. cDNA dilution

[0501] Dilute the reverse transcribed cDNA in step 4 3 times;

[0502] 6. qPCR

[0503] 1) Main reagents

[0504] SYBR Green dye was Taq Pro Universal SYBR qPCR Master Mix (Cat. No.: Q712-02, Batch No.: 027E1260LB) produced by Vazyme.

[0505] 2) Add sample

[0506] Two replicate wells were added for each sample;

[0507] 3) Reaction system

[0508] 4) Reaction conditions

[0509] 50℃2min;

[0510] 95℃2min;

[0511] 95℃5sec;

[0512] 60℃1min;

[0513] read, 40 cycles in total;

[0514] The melting curve was 95°C 5 sec, 65°C 5 sec to 95°C, 0.5°C / read. The data in Table 7 demonstrate that the oligonucleotide conjugates of the present invention have good applicability to the PTEN gene. After 7 days of administration, approximately 39% of the PTEN gene remained in the mouse liver.

[0515] Table 7

[0516] 3.2 SOD1 gene

[0517] Wild-type C57BL / 6JGPT mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against mouse SOD1 gene expression. At least one week in advance, 6-8 week-old male C57BL / 6JGPT mice were housed and adapted to feeding. After adaptation, the mice were randomly divided into groups according to body weight, with 4 animals per group, and the day of grouping was designated as day 0. After blood collection on day 0, a single subcutaneous injection was performed; a negative control group (PBS or saline) and an experimental group (BPR3T01-M11007) were set up, with a dose of 6 mg / kg and a dosing volume of 10 μL / g. After 7 days, the mice were sacrificed and the liver organs were isolated. The expression of the SOD1 gene was detected using the same procedure as above. The activity of the oligonucleotide conjugate was evaluated by the reduction effect compared to the pre-administration level.

[0518] The data in Table 8 show that the oligonucleotide conjugates of the present invention have good applicability in SOD1 gene. After 7 days of administration, the SOD1 gene in the mouse liver remained at about 4%.

[0519] Table 8

[0520] 3.3 FXII gene (F12 gene)

[0521] Wild-type mice C57BL / 6JGPT (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against mouse FXII gene expression. At least one week in advance, 6-8 week-old male C57BL / 6JGPT mice were brought into the room for adaptive feeding. After adaptive feeding, the mice were randomly divided into groups according to body weight, with 4 animals per group, and the day of grouping was D0. After blood collection on D0, a single dose of subcutaneous injection was performed; a negative control group (PBS or saline) and an experimental group (BPR302-M05D0102 and BPR302-M05D0103) were set up, with a dose of 1 mg / kg and a dosing volume of 10 μL / g. Subsequently, blood was collected from the orbit or suborbital area at time points (D7, D14, D21, D28, D35, D42, and D56) in a volume of 100-300 μL (depending on the mouse's condition) and serum was separated. Finally, FXII protein was quantitatively assayed using an ELISA kit (Abcam, Cat. No. 272776) following the same procedures as the kit instructions. The activity of the oligonucleotide conjugate was assessed by reducing the concentration of FXII compared to the pre-administration level.

[0522] The results in Figure 10 indicate that the oligonucleotide conjugates BPR302-M05D0102 and BPR302-M05D0103 have excellent expression inhibition effects on the FXII target gene. BPR302-M05D0103 is superior to the control BPR302-M05D0102 in terms of maximum knockout activity.

[0523] 3.4 PCSK9 gene

[0524] Humanized hPCSK9 mice (source: Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against humanized mouse PCSK9 gene expression. At least one week in advance, male mice aged 6-8 weeks were brought into the room for adaptive feeding. After adaptive feeding, the mice were randomly divided into groups according to body weight, with 4 animals per group, and the day of grouping was designated as day 0. After blood collection on day 0, a single dose of subcutaneous injection was administered; a negative control group (PBS or saline) and an experimental group (BPR-30300105) were set up, with a dose of 3 mg / kg and a dosing volume of 10 μL / g. Subsequently, blood was collected from the orbit or neck at time points such as D7, D14, D21, D28, D35, D42, and D56. The blood volume was 100-300 μL (determined according to the mouse's condition), and serum was obtained after separation. Finally, hPCSK9 protein was quantitatively detected using an ELISA kit (Proteintech, Cat. No. KE00278) according to the manufacturer's instructions. The activity of the oligonucleotide conjugate was assessed by comparing the reduction in hPCSK9 protein levels to that before administration.

[0525] The results in FIG11 show that the oligonucleotide conjugate BPR-30300105 has an excellent expression inhibition effect on the PCSK9 target gene.

[0526] 3.5 APOC3 gene

[0527] Humanized hAPOC3 mice (source: Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were injected subcutaneously with a single dose to investigate the inhibitory activity of the oligonucleotide conjugate against APOC3 gene expression in humanized mice. At least one week prior to initiation, 6-8-week-old male mice were housed and acclimated. After completion of acclimation, mice were randomly divided into groups of four based on body weight, with grouping occurring on day 0 (D0). Following blood collection on D0, a single subcutaneous dose of 1 mg / kg (10 μL / kg) was administered to a negative control group (PBS or saline) and an experimental group (BPR-30505555). Blood was subsequently collected from the orbit or suborbital area at D7, D14, D21, D28, D35, D42, and D56, with a volume of 100-300 μL (depending on the mouse's condition) and serum was separated. Finally, hAPOC3 protein was quantitatively detected using an ELISA kit (Thermo brand, catalog number: EHAPOC3). The detection method was performed according to the corresponding instructions, and the activity of the oligonucleotide conjugate was evaluated by the reduction effect compared to the pre-administration level.

[0528] The results in FIG12 show that the oligonucleotide conjugate BPR-30505555 has an excellent expression inhibition effect on the APOC3 target gene.

[0529] 3.6 ANGPTL3 gene

[0530] Wild-type C57BL / 6JGPT mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were injected subcutaneously with a single dose to investigate the inhibitory activity of the oligonucleotide conjugate against mouse ANGPTL3 gene expression. At least one week prior to the start of the experiment, 6-8-week-old male mice were housed and acclimated. After completion of acclimation, mice were randomly divided into groups of four according to body weight, with grouping occurring on day 0 (D0). Following blood collection on D0, a single subcutaneous injection of 1 mg / kg (10 μL / g) was administered to a negative control group (PBS or saline) and an experimental group (BPR323-H06001). Blood was subsequently collected from the orbit or neck at D7, D14, D21, D28, D35, D42, and D56, with a volume of 100-300 μL (depending on the mouse's condition) and serum was obtained after separation. Finally, ANGPTL3 protein was quantitatively detected using an ELISA kit (RD brand, catalog number: MANL30) according to the corresponding instructions. The activity of the oligonucleotide conjugate was evaluated by the reduction effect compared to the pre-administration level.

[0531] The results in FIG13 indicate that the oligonucleotide conjugate BPR323-H06001 has an excellent expression inhibition effect on the ANGPTL3 target gene.

[0532] 3.7 HBV viral protein expression levels

[0533] The in vivo efficacy of the oligonucleotide conjugate was evaluated using a transgenic HBV mouse model. The transgenic mouse strain was C57B / 6N-Tg(1.28HBV) / Vst, purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were grouped according to the HBsAg level and body weight in the serum, and there was no statistical difference in HBsAg and body weight in the serum. Mice with lower HBsAg levels and lighter body weight were removed from the experiment. Each group of animals consisted of 4 males, the dosage was 3 mg / kg, and the administration method was a single subcutaneous administration. The saline group containing the drug solvent was the negative control group (NC). Before and on the 7th day after administration, blood samples were collected from the orbital venous plexus of the mice. The collected blood samples were placed at room temperature for 3 minutes, centrifuged at 5 rpm for 1 minute, and the supernatant was taken for HBV DNA and HBsAg detection. The detection reagents were the Hepatitis B Virus Nucleic Acid Assay Kit (Shengxiang) and the Hepatitis B Virus Surface Antigen Assay Kit (Mike), and the detection method was carried out according to the corresponding instructions. The serum HBV DNA and HBsAg expression values ​​on the 7th day after administration were compared with the corresponding Log10 expression values ​​before administration to evaluate the efficacy of the conjugate based on the reduction effect compared with the pre-administration value.

[0534] The results in FIG14 indicate that the oligonucleotide conjugate BPR301-6681001 has an excellent expression inhibitory effect on the HBsAg protein of the HBV virus.

[0535] 3.8 FVII gene

[0536] Wild-type mice C57BL / 6JGPT (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against mouse FVII gene expression. At least one week in advance, male C57BL / 6JGPT mice aged 6-8 weeks were brought into the room for adaptive feeding. After adaptive feeding was completed, the mice were randomly divided into groups according to body weight, with 4 animals in each group, and the day of grouping was D0. After completing blood collection on D0, a single dose of subcutaneous injection was performed; a negative control group (PBS or saline) and an experimental group (BPR323M-15001) were set up, with a dose of 1 mg / kg and a dosing volume of 10 μL / g. Subsequently, blood was collected from the orbit or neck at time points such as D7, D14, D21, D28, D35, D42, and D56, with a blood volume of 100-300 μL (determined according to the mouse state), and serum was obtained after separation. Finally, FVII protein was quantitatively detected using an ELISA kit (SAB brand, Cat. No. EK4042) following the kit instructions. The activity of the oligonucleotide conjugate was assessed by the reduction in FVII protein compared to the pre-administration level.

[0537] The results in FIG15 indicate that the oligonucleotide conjugate BPR323M-15001 has an excellent expression inhibition effect on the FVII target gene.

[0538] 3.9 AGT gene

[0539] Humanized hAGT mice (source: Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against humanized mouse hAGT gene expression. At least one week in advance, male mice aged 6-8 weeks were brought into the room for adaptive feeding. After adaptive feeding, the mice were randomly divided into groups according to body weight, with 4 animals per group, and the day of grouping was designated as day 0. After blood collection on day 0, a single dose of subcutaneous injection was administered; a negative control group (PBS or saline) and an experimental group (BPR-30900101) were set up, with a dose of 1 mg / kg and a dosing volume of 10 μL / g. Subsequently, blood was collected from the orbit or neck at time points such as D7, D14, D21, D28, D35, D42, and D56. The blood volume was 100-300 μL (determined according to the condition of the mouse), and serum was obtained after separation. Finally, hAPOC3 protein was quantitatively detected using an ELISA kit (Abcam, Cat. No. ab287170) according to the manufacturer's instructions. The activity of the oligonucleotide conjugate was assessed by comparing the reduction in protein levels before administration.

[0540] The results in FIG16 indicate that the oligonucleotide conjugate BPR30900101 has an excellent expression-inhibiting effect on angiotensinogen protein (AGT).

[0541] 3.10 TTR gene

[0542] Wild-type mice C57BL / 6JGPT (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were injected subcutaneously with a single dose to study the inhibitory activity of the oligonucleotide conjugate against mouse TTR gene expression. At least one week in advance, 6-8 week-old male C57BL / 6JGPT mice were brought into the room for adaptive feeding. After the adaptive feeding was completed, the mice were randomly divided into groups according to their body weight, with 4 animals in each group, and the day of grouping was D0. After blood collection on D0, a single dose of subcutaneous injection was performed; a negative control group (PBS or saline) and an experimental group (BPR-3T01M01196, BPR-3T01M01202, BPR302-M01D0161) were set up, with a dose of 0.15 mg / kg and a dosing volume of 10 μL / g. Subsequently, blood was collected from the orbit or neck at time points such as D7, D14, D21, D28, D35, D42, and D56. A volume of 100-300 μL (depending on the mouse's condition) was collected and serum was obtained after separation. Finally, TTR protein was quantitatively assayed using an ELISA kit (Abcam, Cat. No. ab282297) following the same procedures as the kit instructions. The activity of the oligonucleotide conjugate was assessed by reducing the concentration of the oligonucleotide conjugate compared to the pre-administration level.

[0543] The results in FIG17 show that the oligonucleotide conjugates BPR-3T01M01196, BPR-3T01M01202, and BPR302-M01D0161 still have an expression-inhibiting effect on mTTR protein at 0.15 mg / kg.

[0544] Unless otherwise defined, the terms used in the present invention have the same meanings as those commonly understood by those skilled in the art.

[0545] The embodiments described in the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various other substitutions, changes and improvements within the scope of the present invention. Therefore, the present invention is not limited to the above-mentioned embodiments, but is only limited by the claims.

Claims

1. A compound represented by formula (I), or a racemate, stereoisomer, isotope-labeled compound or salt thereof, Among them, Y1 represents a hydroxyl protecting group PG1; Y2 represents the structure shown in formula (I-1): In formula (I-1), R 1 represents a straight-chain or branched-chain C1-C8 alkylene -CN, R 2 , R 3 each independently represents a straight-chain or branched-chain C1-C8 alkyl group; A represents the structure shown by formula (I-2), formula (I-2'), formula (I-3), formula (I-4) or formula (I-5): In formula (I-5), formula (I-2), formula (I-2'), and formula (I-3), R 4 represents hydrogen or a linear or branched alkyl group having 1 to 8 carbon atoms, L A1 , L A2 each independently represents absent or a linear or branched alkylene group having 1 to 8 carbon atoms, ring A0 represents a 4- to 10-membered saturated heterocycle containing at least one N atom, R A5 represents -C(O)NH-, -OC(O)NH-, -NHC(O)-, -NHC(O)O-, -O-, -S-, -NH-, -C(O)-, or -S(O)-, and s represents an integer from 0 to 4; In Formula (I-4) and Formula (I-5), R A1 represents absent or a C1-C8 straight-chain or branched-chain alkylene group, where one or more carbon atoms are optionally replaced by one or more -O- or -C(O)-, R A2 represents absent or represents p represents an integer from 0 to 5, R A3 represents R A4 represents -C(O)NH-, -NHC(O)-, -O-, -S-, -NH-, -C(O)- or -S(O)-, L A3 represents absent or represents a C1-C8 straight-chain or branched-chain alkylene group, Base represents a natural base A, T, C, G or U, the amino group of which is protected by a protecting group; 1 represents the connection site with B, 2 represents the connection site with Y1, and 3 represents the connection site with Y2; B represents absent or represents a C1-C30 straight-chain or branched alkylene group, where one or more carbon atoms are optionally replaced by one or more of the following groups: -C(O)NH-, -O-, -S-, -NH-, -C(O)-, -S(O)-, -S(O2)-, a 4- to 10-membered saturated or unsaturated carbocyclic ring, a 4- to 10-membered saturated or unsaturated heterocyclic ring, and the heterocyclic ring contains at least one heteroatom selected from N, O, and S; G represents the structure shown in formula (I-6): In formula (I-6), R G1 represents hydrogen, a hydroxyl group, a linear or branched alkyl group having 1 to 20 carbon atoms, a linear or branched alkenyl group having 2 to 20 carbon atoms, -O-a linear or branched alkyl group having 1 to 20 carbon atoms, -S-a linear or branched alkyl group having 1 to 20 carbon atoms, -NH-a linear or branched alkyl group having 1 to 20 carbon atoms, -N-(a linear or branched alkyl group having 1 to 20 carbon atoms)2, -O-a linear or branched alkylene group having 0 to 8 carbon atoms-C6-C20 aryl, -S-a linear or branched alkylene group having 0 to 8 carbon atoms-C6-C20 aryl, -O-a linear or branched alkylene group having 0 to 8 carbon atoms-C3-C10 cycloalkyl, -S-a linear or branched alkylene group having 0 to 8 carbon atoms-C3-C10 cycloalkyl, -O-a linear or branched alkylene group having 0 to 8 carbon atoms-a 5- to 12-membered heterocyclic group, -S-a linear or branched alkylene group having 0 to 8 carbon atoms-a 5- to 12-membered heterocyclic group, a galactosyl group or a galactosylamide group, or q represents an integer from 0 to 16, where the aryl, cycloalkyl, or heterocyclic group is optionally substituted by one or more C1-C8 straight-chain or branched alkyl groups, and the heterocyclic group contains at least one heteroatom selected from N, O, and S; R G2 represents -C(O)NR 5 R 6 , -C(O)-a straight-chain or branched-chain alkyl of C1-C8, -C(O)-a cycloalkyl of C3-C10, -S(O2)-a straight-chain or branched-chain alkyl of C1-C8, -S(O2)-a cycloalkyl of C3-C10, -C(O)-a straight-chain or branched-chain alkylene of C0-C8-C6-C20 aryl, -S(O2)-a straight-chain or branched-chain alkylene of C0-C8-C6-C20 aryl, -C(O)-a straight-chain or branched-chain haloalkyl of C1-C8 or -S(O2)-a straight-chain or branched-chain haloalkyl of C1-C8, wherein the aryl is optionally substituted by one or more straight-chain or branched-chain alkyls of C1-C8 or straight-chain or branched-chain haloalkyls of C1-C8; R 5 , R 6 each independently selected from hydrogen or a straight-chain or branched-chain alkyl of C1-C8; R G3 and R G4 each independently represents hydrogen or a hydroxy protecting group PG2; Z represents -C(O)-, -O-, -S-, -NH-, -C(O)NH-, -OC(O)NH-, -OP(O)(OH)- or -R 8 -(CH2) n -R 7 -, wherein R 8 represents a triazolyl subunit, for example represents R 7 represents -C(O)-, -O-, -S- or -NH-, and n represents an integer of 0 to 10.

2. The compound according to claim 1, or a racemate, stereoisomer, isotope-labeled compound or a salt thereof, wherein, In the formula (I-6), the R G1 represents hydrogen, a hydroxyl group, a linear or branched alkyl group having 1 to 6 carbon atoms, a linear or branched alkenyl group having 2 to 6 carbon atoms, a linear or branched alkyl group having 1 to 16 carbon atoms (e.g., a linear or branched alkyl group having 1 to 6 carbon atoms) -O-, a linear or branched alkyl group having 1 to 6 carbon atoms -S-, a linear or branched alkyl group having 1 to 6 carbon atoms -NH-, -N- (a linear or branched alkyl group having 1 to 6 carbon atoms) 2, -O- a linear or branched alkylene group having 0 to 4 carbon atoms - phenyl, -O- a linear or branched alkylene group having 0 to 4 carbon atoms - C3-C6 cycloalkyl or -O- a linear or branched alkylene group having 0 to 4 carbon atoms - 5- to 6-membered heterocyclic group, wherein the phenyl group, cycloalkyl group or heterocyclic group is optionally substituted by one or more linear or branched alkyl groups having 1 to 4 carbon atoms, and the heterocyclic group contains at least one heteroatom selected from O; Preferably, the R G1 represents one of the following structures: OH; Said R G2 represents a straight-chain or branched C1-C6 alkyl group of -C(O)-, a C3-C6 cycloalkyl group of -C(O)-, a straight-chain or branched C1-C6 alkyl group of -S(O2)-, a straight-chain or branched C0-C4 alkylene-phenyl group of -C(O)-, a straight-chain or branched C0-C4 alkylene-phenyl group of -S(O2)-, a straight-chain or branched C1-C4 alkyl group of -C(O)-halo or a straight-chain or branched C1-C4 alkyl group of -S(O2)-halo, wherein the aryl group is optionally substituted by one or more straight-chain or branched C1-C4 alkyl groups or straight-chain or branched C1-C4 haloalkyl groups; Preferably, the R G2 represents one of the following structures: Preferably, Z represents one of the following structures:

3. The compound according to claim 1 or 2, or a racemate, stereoisomer, isotopically labeled compound or a salt thereof, wherein, When the said A represents Formula (I-2), Formula (I-2') or Formula (I-3), it preferably represents one of the following structures: When A represents formula (I-4) or formula (I-5), Base represents a natural base A, T, C, G, or U, where the amino group is protected by a protecting group, and the protecting group is preferably one or more of acetyl (Ac), benzoyl (Bz), or isobutyryl (iBu); R A3 preferably represents one of the following structures: 1 represents the connection site with B, 2 represents the connection site with Y1, and 3 represents the connection site with Y2.

4. A compound according to any one of claims 1-3, or a racemate, stereoisomer, isotopically labeled compound or salt thereof, wherein, Said B represents the structure shown in formula (I-7): In formula (I-7), ring B0 represents absent or a 4- to 10-membered saturated carbon or heterocyclic ring containing W, W represents CH or N, R 9 represents absent or one or more of -C(O)-, -O-, -S-, -NH-, L B1 represents absent or a C1-C20 straight or branched alkylene group, where one or more of the carbon atoms are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, -S-, -S-S-, R 10 represents absent or -C(O)-, -O-, -S- or -NH-, and ring B0, R 9 , L B1 , R 10 do not simultaneously represent absent; 1 represents the attachment site to A, and 2 represents the attachment site to G; Preferably, ring B0 represents one of the following ring structures: More preferably, B represents the structure shown in Formula (I-8): In formula (I-8), ring B0 represents a 4- to 6-membered saturated heterocycle containing an N atom, L B1 represents absent or represents a C1-C20 straight-chain or branched-chain alkylene group, one or more of the carbon atoms of which are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, -S-S-, R 10 represents absent or represents -C(O)-, -O- or -NH-; 1 represents the attachment site to A, and 2 represents the attachment site to G.

5. The compound according to claim 4, or a racemate, stereoisomer, isotopically labeled compound or a salt thereof, wherein, B represents non - existence or represents one of the following structures: 1 represents the connection site with A, and 2 represents the connection site with G.

6. The compound according to any one of claims 1-5, or a racemate, stereoisomer, isotope-labeled substance or salt thereof, wherein, PG1 represents one or more of dimethoxytrityl (DMTr), p-methoxytrityl (MMTr), or trityl (TRT), and preferably represents DMTr; The Y2 represents the following structure:

7. A compound according to any one of claims 1-6, or a racemate, stereoisomer, isotopically labeled compound or salt thereof, wherein, PG2 represents one or more of methoxymethyl (MOM), benzyl (Bn), p-methoxybenzyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butoxyphenylsilyl (TBMPS), acetyl (Ac), or benzoyl (Bz).

8. A compound according to any one of claims 1-7, or a racemate, stereoisomer, isotope-labeled compound or a salt thereof, wherein, The compound has the structure shown in formula (I-9): In formula (I-9), R G1 represents the following structure:

9. A compound according to any one of claims 1-7, or a racemate, stereoisomer, isotopically labeled compound or a salt thereof, wherein, The compound has the structure shown in formula (I-10): In formula (I-10), R G1 represents the following structure:

10. A compound according to any one of claims 1-7, or a racemate, stereoisomer, isotope-labeled compound or salt thereof, wherein, The compound has the structure shown in formula (I-11): In formula (I-11), R G1 represents the following structure:

11. A compound according to any one of claims 1-7, or a racemate, stereoisomer, isotope-labeled substance or salt thereof, wherein, The compound has the structure shown in formula (I-12): In formula (I-12), R G2 represents the following structure:

12. A compound according to any one of claims 1-7, or a racemate, stereoisomer, isotopically labeled compound or a salt thereof, wherein, The compound has the structure shown in formula (I-13): In formula (I-13), R G2 represents the following structure:

13. The compound according to any one of claims 1-7, or its racemate, stereoisomer, isotope-labeled compound or its salt, wherein, The compounds include:

14. A compound represented by formula (III), or a racemate, stereoisomer, isotope-labeled compound or salt thereof, Among them, Y1, Y2, A, and G are each independently defined as in any one of claims 1-13; B' represents a C2-C60 straight-chain or branched alkylene group, where one or more carbon atoms are optionally replaced by one or more of the following groups: -C(O)NH-, -OC(O)NH-, -NHC(O)-, -NHC(O)O-, -O-, -S-, -NH-, -C(O)-, -S(O)-, -S(O2)-, a 4- to 10-membered saturated or unsaturated carbocyclic ring, a 4- to 10-membered saturated or unsaturated heterocyclic ring, and the heterocyclic ring contains at least one heteroatom selected from N, O, and S; m represents 2 to 5; Preferably, B’ represents the structure shown in formula (III-1) or formula (III-2): In formula (Ⅲ-1) or formula (Ⅲ-2), Lb1 represents absent or a C1-C20 linear or branched alkylene group, where one or more carbon atoms are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, -S-; R11 represents absent or represents -C(O)-, -O-, -S- or -NH-; Lb2 are the same or different and each independently represents a C1-C20 linear or branched alkylene group, where one or more carbon atoms are optionally replaced by one or more of -C(O)-, -C(O)NH-, -O-, -NH-, -S-; R12 are the same or different and each independently represents absent or represents -C(O)-, -O-, -S- or -NH-; 1 represents the connection site to A, and 2 represents the connection site to G; More preferably, B’ represents the structure shown by formula (III-3), formula (III-4), formula (III-5) or formula (III-6): In formula (Ⅲ-3), formula (Ⅲ-4), formula (Ⅲ-5) or formula (Ⅲ-6), Lb1 represents absent or a C1-C12 linear or branched alkylene group; R11 represents absent or represents -C(O)-, -O-, -S- or -NH-; Lb3 are the same or different and each independently represents a C1-C10 linear or branched alkylene group; Lb4 are the same or different and each independently represents a C1-C10 linear or branched alkylene group; Lb5 are the same or different and each independently represents a C1-C6 linear or branched alkylene group; Lb6 are the same or different and each independently represents a C1-C6 linear or branched alkylene group; Lb7 are the same or different and each independently represents a C1-C6 linear or branched alkylene group; R12 are the same or different and each independently represents -C(O)-, -O-, -S- or -NH-; m1 are the same or different and each independently represents 1-8; m2 are the same or different and each independently represents 1-5; 1 represents the connection site to A, and 2 represents the connection site to G; Further preferably, B' represents one of the following structures: Most preferably, the compounds represented by formula (III) include:

15. Use of the compound according to any one of claims 1-14, or its racemate, stereoisomer, isotope-labeled compound or its salt in the preparation of a liver-targeted drug; Preferably, the drug is an oligonucleotide conjugate.

16. An oligonucleotide conjugate, wherein, The oligonucleotide conjugate includes one or more of the compounds according to any one of claims 1-13, or its racemate, stereoisomer, isotope-labeled compound or its salt; Preferably, the number of the compound, or its racemate, stereoisomer, isotope-labeled compound or its salt is 1-12, preferably 1-6, more preferably 1-4; Preferably, one or more of the compounds according to any one of claims 1-14, or its racemate, stereoisomer, isotope-labeled compound or its salt are included at the 3'-end, 5'-end or in-chain position of at least one oligonucleotide chain in the oligonucleotide conjugate.

17. The oligonucleotide conjugate according to claim 16, wherein, 1-6 (preferably 1-3) of the compounds according to any one of claims 1-14, or its racemate, stereoisomer, isotope-labeled compound or its salt are included at the 5'-end of an oligonucleotide chain in the oligonucleotide conjugate.

18. The oligonucleotide conjugate according to claim 16, wherein, The 3'-end and 5'-end of one oligonucleotide chain in the oligonucleotide conjugate simultaneously comprise 1 to 6 (preferably 1 to 3) compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof.

19. The oligonucleotide conjugate according to claim 16, wherein, The 3'-end and 5'-end of one oligonucleotide chain in the oligonucleotide conjugate simultaneously comprise 1 to 6 (preferably 1 to 3) compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof, and the 3'-end of another oligonucleotide chain in the oligonucleotide conjugate comprises 1 to 6 (preferably 1 to 3) compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof.

20. An oligonucleotide conjugate, wherein, One or more of the synthetic monomers for preparing the oligonucleotide conjugate are the compounds described in any one of claims 1-13, or their racemates, stereoisomers, isotope-labeled substances or salts thereof; Preferably, the number of the compounds, or their racemates, stereoisomers, isotope-labeled substances or salts thereof is 1 to 12, preferably 1 to 6, more preferably 1 to 4; Preferably, when preparing the oligonucleotide conjugate, one or more of the compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof are used as synthetic monomers at the 3'-end, 5'-end or in-chain position of at least one oligonucleotide chain therein; More preferably, when preparing the oligonucleotide conjugate, 1 to 6 (preferably 1 to 3) compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof are used as synthetic monomers at the 5'-end of one oligonucleotide chain therein; or when preparing the oligonucleotide conjugate, 1 to 6 (preferably 1 to 3) compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof are simultaneously used as synthetic monomers at the 3'-end and 5'-end of one oligonucleotide chain therein; or when preparing the oligonucleotide conjugate, 1 to 6 (preferably 1 to 3) compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof are simultaneously used as synthetic monomers at the 3'-end and 5'-end of one oligonucleotide chain therein, and 1 to 6 (preferably 1 to 3) compounds described in any one of claims 1-14, or their racemates, stereoisomers, isotope-labeled substances or salts thereof are used as synthetic monomers at the 3'-end of another oligonucleotide chain therein.

21. The oligonucleotide conjugate according to any one of claims 16-20, wherein, At least one oligonucleotide chain in the oligonucleotide conjugate has one of the following structures: Wherein, X represents O or S, k1 and k2 each independently represent an integer from 0 to 6, k1 and k2 do not simultaneously represent 0; k3 represents an integer from 1 to 6, ONS represents an oligonucleotide chain, A, B, and G are as defined in any one of claims 1-13, B', and m are as defined in any one of claims 14, and wherein R G3 , R G4 is H.

22. The oligonucleotide conjugate according to claim 21, wherein, At least one oligonucleotide chain in the oligonucleotide conjugate has one of the following structures: wherein, R G1 , and Base are each independently defined as in any one of claims 1-14.

23. The oligonucleotide conjugate according to any one of claims 16-22, wherein, The oligonucleotide is one or more of antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), aptamer, immunostimulant, G-quadruplex, alternative spliceosome, single-stranded RNA, ribozyme, decoys.

24. A pharmaceutical composition comprising the oligonucleotide conjugate according to any one of claims 16-23 and a pharmaceutically acceptable carrier.

25. Use of the compound according to any one of claims 1-14, or a racemate, stereoisomer, isotopically labeled compound or a salt thereof, or the oligonucleotide conjugate according to any one of claims 16-23, or the pharmaceutical composition according to claim 24, in the preparation of a medicament for inhibiting mRNA expression in vivo, or a medicament for detecting, preventing and / or treating hepatogenic diseases; Preferably, the hepatogenic diseases include hypercholesterolemia, type II hyperlipoproteinemia, primary hyperlipidemia, atherosclerosis, heterozygous familial hypercholesterolemia, dyslipidemia, primary hypercholesterolemia, atherosclerotic plaque, homozygous familial hypercholesterolemia, preeclampsia, hypertension, cardiovascular disease, diabetes, homozygous familial hypercholesterolemia, hypertriglyceridemia, familial hyperchylomicronemia, chronic hepatitis D, hepatitis D, hepatitis B, chronic hepatitis B, transthyretin amyloidosis, transthyretin amyloidotic neuropathy, transthyretin amyloid cardiomyopathy, familial amyloidosis, Stargardt disease, nephrolithiasis, hepatic porphyria, acute hepatic porphyria, hereditary coproporphyria, acute intermittent porphyria, variegate porphyria, paroxysmal nocturnal hemoglobinuria, myasthenia gravis, C3 glomerulopathy, immunoglobulin a nephropathy / liver disease, bleeding, hemophilia B, hemophilia A, non-alcoholic steatohepatitis, liver cancer, alpha-1 antitrypsin deficiency, primary hyperoxaluria type 1, primary hyperoxaluria type 2, end-stage renal disease.

Images