Modified nucleoside compound, and oligonucleotide prepared therefrom

Abstract

A nucleoside phosphoramidite compound, which is a 2'-phosphoramidite group and 4'-modified TNA structure compound. The nucleoside phosphoramidite compound can be incorporated into the end and / or middle of an oligonucleotide, so that the modified oligonucleotide is hybridized with a portion of a target mRNA, resulting in the loss or down-regulation of the normal function of the target mRNA.

Description

A modified nucleoside compound and oligonucleotide prepared therefrom Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a modified nucleoside compound and an oligonucleotide prepared therefrom. Background Art

[0002] Oligonucleotides are polymers of nucleotides. As nucleic acid inhibitors, oligonucleotides can modulate mRNA levels in cells and have shown early promise in treating genetic diseases, metabolic disorders, cancer, and viral infections. Nucleic acid inhibitors can regulate mRNA expression via a variety of mechanisms, including RNA interference (RNAi).

[0003] RNAi is a conservative pathway found in most eukaryotes, in which double-stranded RNA molecules (dsRNA) inhibit the expression of target genes with complementary sequences to the dsRNA. In a typical RNAi pathway, longer dsRNAs are cut into shorter RNA duplexes known as small interfering RNAs ("siRNAs") by an endonuclease (Dicer). It has been shown that siRNAs associate with endonucleases, transactivation response RNA binding proteins (TRBPs), and Argonaute 2 ("Ago2") to form a complex, sometimes referred to as RNA-induced silencing complex ("RISC"). Ago2 is an endonuclease that uses the antisense strand (also referred to as a guide strand) of siRNA to guide the sequence specificity of target mRNA cutting to cut the target mRNA.

[0004] A variety of double-stranded RNAi inhibitor molecular structures have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules that simulate natural siRNA, wherein each chain has 19-25 nucleotides, including at least one 3' overhang with 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed, which were cut into active RNAi inhibitor molecules by endonucleases in vivo (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed extended double-stranded nucleic acid inhibitor molecules, wherein at least one end of at least one chain extends beyond the double-stranded targeting region of the molecule, and one of the chains includes a structure of a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent No. 8,513,207, U.S. Patent No. 8,927,705, WO2010 / 033225, and WO 2016 / 100401). Those structures include single-stranded extension structures (on one side or both sides of the molecule) and double-stranded extension structures.

[0005] In recent years, many modified nucleosides have been used for RNAi, such as common 2'-fluoro modification, 2'-methoxy modification, 2'-methoxyethyl modification, 2', 3'-open ring nucleotide, locked nucleic acid, ethylene glycol nucleic acid, 5'-(E)-vinyl phosphate and other nucleoside modifications. In some cases, chemical modifications are introduced into nucleic acid inhibitor molecules to introduce properties that may be desired under specific conditions, such as those experienced after in vivo administration. These modifications include being designed to, for example, antagonize nucleases or antagonize other enzymes that interfere with the structure or activity of oligonucleotides, increase the cellular uptake of oligonucleotides, or improve the pharmacokinetic properties of oligonucleotides.

[0006] For example, the introduction of long-chain lipids into nucleosides (see, for example, Patent Nos. WO2023283434 and WO2020006050) and modified nucleosides into corresponding oligonucleotide sequences can improve the pharmacokinetic properties and in vivo bioavailability of oligonucleotide drugs. Further research is needed on the structural modification of oligonucleotide drugs to obtain oligonucleotide drugs with better properties in various aspects.

[0007] Summary of the Invention

[0008] In view of the shortcomings of the prior art, the present invention aims to develop a nucleoside compound that, when applied to oligonucleotide molecules (including but not limited to siRNA, antisense nucleic acid, saRNA (small activating RNA), miRNA (micronucleic acid), nucleic acid aptamers, etc.), can effectively improve the activity of the oligonucleotide molecule, while improving the resistance of the corresponding oligonucleotide to enzymatic cleavage by phosphatases, exonucleases, endonucleases, etc. In addition, compared with ordinary nucleosides, the nucleoside compound can easily introduce various specific functional groups, such as various substituted alkyl lipids, without affecting its base complementary pairing ability, thereby improving the cell membrane penetration ability of the oligonucleotide and improving the organ targeting effect of the nucleic acid. When this type of modified nucleoside is used in siRNA molecules, it can increase the enrichment of siRNA molecules in the target organ and improve the pharmacokinetic properties of the drug.

[0009] The present invention provides an oligonucleotide, which is a single-stranded or double-stranded nucleotide molecule, wherein each nucleotide in the double-stranded nucleotide molecule is a modified or unmodified nucleotide, wherein the double-stranded nucleotide sequence has 16-30 nucleotides; the antisense strand of the double-stranded nucleotide molecule is reverse complementary to at least 14 consecutive nucleotides in the target mRNA; and in the sense strand of the double-stranded nucleotide molecule, in the direction from the 5' end to the 3' end, at least one nucleotide is replaced by this type of 2', 3'-phosphate-linked nucleotide (4'-modified TNA).

[0010] TNA, or α-L-threonucleic acid, is a genetic polymer. Unlike DNA or RNA, which are linked by 3', 5' phosphate bonds, TNA uses 2', 3' phosphate bonds, with the 3' substituent facing the opposite direction of natural DNA or RNA. Despite its unique structure, TNA can form a Watson-Crick double-stranded structure with its complementary bases. Due to its unique structural properties, TNA possesses excellent resistance to nuclease cleavage and is widely used in nucleic acid aptamers.

[0011] The present invention provides a nucleoside compound represented by formula (1), or a stereoisomer thereof:

[0012] in:

[0013] R 1 、R 2 、R 3 、R 4 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0014] R 5 is selected from hydrogen, halogen, cyclopropane, cyclobutane, C1-C30 alkyl, substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 alkynyl or substituted C2-C30 alkynyl;

[0015] Nu represents base or absence;

[0016] Z is -O-, -S-, or -NR 10 or -CR 10 R 11 , where R 10 and R 11 are each independently selected from hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, or substituted or unsubstituted cycloalkyl;

[0017] X1, X2 are each independently selected from -(CH2) n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0018] X3 is selected from -(CH2) n O-, -(CH2) n -、-(CH2)n S-, -(CH2) n NH(CO)-、-(CH2) n (CO)NH- or -(CH2) n O(CH2) n -A-, or, X3 is selected from -(CH2) n O-, -(CH2) n S-, -(CH2) n NH(CO)-、-(CH2) n (CO)NH- or -(CH2) n O(CH2) n -A-, wherein A is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted cycloalkyl group, and n is any integer from 0 to 10.

[0019] The nucleoside compound of formula (1) provided by the present invention is selected from the nucleoside compound shown in formula 1-a or 1-b (nucleoside phosphoramidite compound used to modify oligonucleotide, i.e., 4'-modified TNA backbone structure),

[0020] Where: R 1 、R 2 、R 3 、R 4 、R 5 , Nu, X1, X2 and X3 are as defined above.

[0021] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein,

[0022] R 1 、R 2 、R 3 、R 4 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0023] R 5 is selected from hydrogen, cyclopropane, cyclobutane, C1-C30 alkyl, substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 alkynyl, or substituted C2-C30 alkynyl;

[0024] Nu represents base or absence;

[0025] Z is -O-, -S- or -N(COR')-, wherein R' is a C1-C5 alkyl group; preferably, R' is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl;

[0026] X1, X2 are each independently selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 5; preferably, n is selected from 0, 1, 2, 3, 4 or 5;

[0027] X3 is selected from -(CH2) n O-, -(CH2) n S-, -(CH2) n NH(CO)-、-(CH2) n (CO)NH-, -(CH2) n -or-(CH2) n O(CH2) n -A-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclyl or a substituted or unsubstituted cycloalkyl, wherein n is any integer from 0 to 5; preferably, n is selected from 0, 1, 2, 3, 4 or 5.

[0028] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, Z is -O-, -S- or -N(COCH3)-.

[0029] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein X1 is -O-, -S- or -CH2O-.

[0030] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein X2 is -O- or -S-.

[0031] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein X3 is -O-, -CH2-, -CH2O-, -CH2S-, -CH2NH(CO)-, -(CO)NH- or -CH2OCH2-heteroaryl-; preferably, X3 is -O-, -CH2O-, -CH2S-, -CH2NH(CO)-, -(CO)NH- or -CH2OCH2-heteroaryl-, for example, X3 is -CH2OCH2-1,2,3-triazolyl-.

[0032] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein R 1 For hydrogen.

[0033] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein R2 For hydrogen.

[0034] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein R 3 For hydrogen.

[0035] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein R 4 is hydrogen or methoxy.

[0036] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein R 5 is hydrogen, methyl, halogen, or C10-C18 alkyl; preferably, R 5 is methyl or C10-C18 alkyl; more preferably, R 5 is methyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl or C18 alkyl; more preferably, R 5 is methyl, C10 straight chain alkyl, C11 straight chain alkyl, C12 straight chain alkyl, C13 straight chain alkyl, C14 straight chain alkyl, C15 straight chain alkyl, C16 straight chain alkyl, C17 straight chain alkyl or C18 straight chain alkyl.

[0037] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein R 1 is H, Nu does not exist.

[0038] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein Nu is a base, and the base is selected from:

[0039] According to the nucleoside compound represented by formula 1-a or 1-b of the present invention, wherein the nucleoside compound represented by formula 1-a or 1-b of the present invention is a nucleoside compound represented by formula 2-a or 2-b,

[0040] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0041] Nu is as defined above.

[0042] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound represented by formula 3-a, formula 5-a, formula 8-a or formula 9-a,

[0043] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0044] Nu is as defined above.

[0045] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 4-a or Formula 3-b, respectively,

[0046] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0047] Nu is as defined above.

[0048] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 6-a or Formula 4-b, respectively,

[0049] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0050] Nu is as defined above.

[0051] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 7-a or Formula 5-a, respectively,

[0052] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0053] Nu is as defined above.

[0054] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 10-a or Formula 6-b, respectively,

[0055] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0056] Nu is as defined above.

[0057] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 11-a or Formula 7-b, respectively,

[0058] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0059] Nu is as defined above;

[0060] R' is a C1-C5 alkyl group; preferably, R' is a methyl group, an ethyl group, an n-propyl group or an isopropyl group.

[0061] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 12-a or Formula 8-b, respectively,

[0062] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0063] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 13-a or Formula 9-a, respectively,

[0064] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0065] Nu is as defined above.

[0066] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 14-a or Formula 10-b, respectively,

[0067] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0068] Nu is as defined above.

[0069] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 15-a or Formula 11-b, respectively,

[0070] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0071] Nu is as defined above.

[0072] According to the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-a or Formula 1-b of the present invention is a nucleoside compound represented by Formula 16-a or Formula 12-b, respectively,

[0073] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group;

[0074] Nu is as defined above.

[0075] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is the nucleoside compound shown in Table A in the specific embodiment.

[0076] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound as shown below:

[0077] According to the nucleoside compound represented by formula 1-b of the present invention, wherein the nucleoside compound represented by formula 1-b is the nucleoside compound shown in Table B in the specific embodiment.

[0078] According to the nucleoside compound represented by formula 1-b of the present invention, wherein the nucleoside compound represented by formula 1-b is a nucleoside compound as shown below:

[0079] Another object of the present invention is to provide an oligonucleotide, wherein the oligonucleotide is prepared from at least one nucleoside compound represented by Formula 1-a or Formula 1-b as an intermediate; preferably, the oligonucleotide is siRNA, antisense nucleic acid, saRNA, miRNA, or nucleic acid aptamer.

[0080] According to the oligonucleotide of the present invention, the nucleoside compound has the structure of the following formula 17-a or formula 18-a in the oligonucleotide, and ligated to the rest of the oligonucleotide,

[0081] where R 1 、R 2 、R 3 、R 4 、R 5 , Z, X1, X2, X3 and Nu are as defined above;

[0082] Preferably,

[0083] The oligonucleotide is siRNA;

[0084] The nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA sense strand; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; even further preferably, the structure of Formula 17-a or Formula 18-a is the 1st nucleotide at the 5' end of the siRNA sense strand;

[0085] Alternatively, the nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA antisense chain; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

[0086] According to the oligonucleotide of the present invention, the nucleoside compound has the structure of the following formula 13-b or formula 14-b in the oligonucleotide, and ligated to the rest of the oligonucleotide,

[0087] where R1 、R 2 、R 3 、R 4 、R 5 , Z, X1, X2, X3 and Nu are as defined in claim 1;

[0088] Preferably,

[0089] The oligonucleotide is siRNA;

[0090] The nucleoside compound has a structure of Formula 13-b or Formula 14-b in the siRNA sense strand; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; even further preferably, the structure of Formula 13-b or Formula 14-b is the 1st nucleotide at the 5' end of the siRNA sense strand;

[0091] Alternatively, the nucleoside compound has a structure such as Formula 13-b or Formula 14-b in the siRNA antisense chain; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

[0092] Another object of the present invention is to provide the use of the nucleoside compound represented by Formula 1-a or 1-b for preparing oligonucleotides.

[0093] According to the use of the present invention, the nucleoside compound has the structure of the following formula 17-a or formula 18-a in the oligonucleotide, and ligated to the rest of the oligonucleotide,

[0094] where R 1 、R 2 、R 3 、R 4 、R 5 , Z, X1, X2, X3 and Nu are as defined above;

[0095] Preferably,

[0096] The oligonucleotide is siRNA;

[0097] The nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA sense strand; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; even further preferably, the structure of Formula 17-a or Formula 18-a is the 1st nucleotide at the 5' end of the siRNA sense strand;

[0098] Alternatively, the nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA antisense chain; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

[0099] According to the use of the present invention, the nucleoside compound has the structure of the following formula 13-b or formula 14-b in the oligonucleotide, and ligated to the rest of the oligonucleotide,

[0100] where R 1 、R 2 、R 3 、R 4 、R 5 , Z, X1, X2, X3 and Nu are as defined in claim 1;

[0101] Preferably,

[0102] The oligonucleotide is siRNA;

[0103] The nucleoside compound has a structure of Formula 13-b or Formula 14-b in the siRNA sense strand; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; even further preferably, the structure of Formula 13-b or Formula 14-b is the 1st nucleotide at the 5' end of the siRNA sense strand;

[0104] Alternatively, the nucleoside compound has a structure such as Formula 13-b or Formula 14-b in the siRNA antisense chain; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

[0105] In the present invention, when the structures of Formula 17-a, Formula 18-a, Formula 13-b, and Formula 14-b are the first nucleotide at the 5' end of the oligonucleotide, X1 is Connected to hydrogen.

[0106] In the present invention, when the structures of Formula 17-a, Formula 18-a, Formula 13-b, and Formula 14-b are the first nucleotide at the 3' end of the oligonucleotide, X2 is not connected to the phosphate group but to hydrogen. Alternatively, when the structures of Formula 17-a, Formula 18-a, Formula 13-b, and Formula 14-b are the first nucleotide at the 3' end of the oligonucleotide, X2 is connected to hydrogen through the phosphate group. Attached to a targeting ligand.

[0107] One of the purposes of the present invention is to provide a nucleoside compound represented by formula 1-a (a nucleoside phosphoramidite compound used to modify an oligonucleotide, i.e., a 4'-modified TNA backbone structure),

[0108] in:

[0109] R 1 、R 2 、R 3 、R 4 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0110] R 5 is selected from hydrogen, cyclopropane, cyclobutane, C1-C30 alkyl, substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 alkynyl, or substituted C2-C30 alkynyl;

[0111] Nu represents base or absence;

[0112] Z is -O-, -S-, or -NR 10 or -CR 10 R 11 , where R 10 and R 11Each is independently hydrogen, halogen, acyl (acyl is -COR', wherein R' is C1-C5 alkyl), substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle or substituted or unsubstituted cycloalkyl;

[0113] X1, X2 are each independently selected from -(CH2) n O- or -(CH2) n S-, where n is any integer from 0 to 10;

[0114] X3 is selected from -(CH2) n O-, -(CH2) n S-, -(CH2) n NH(CO)- or -(CH2) n O(CH2) n -A-, wherein A is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted cycloalkyl group, and n is any integer from 0 to 10.

[0115] According to the nucleoside compound represented by formula 1-a of the present invention, wherein,

[0116] R 1 、R 2 、R 3 、R 4 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0117] R 5 is selected from hydrogen, cyclopropane, cyclobutane, C1-C30 alkyl, substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 alkynyl, or substituted C2-C30 alkynyl;

[0118] Nu represents base or absence;

[0119] Z is -O-, -S- or -N(COR')-, wherein R' is a C1-C5 alkyl group;

[0120] X1, X2 are each independently selected from -(CH2) n O- or -(CH2) n S-, where n is any integer from 0 to 5;

[0121] X3 is selected from -(CH2) nO-, -(CH2) n S-, -(CH2) n NH(CO)- or -(CH2) n O(CH2) n -A-, wherein A is a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted cycloalkyl group, and n is any integer from 0 to 5.

[0122] According to the nucleoside compound represented by formula 1-a of the present invention, Z is -O-, -S- or -N(COCH3)-.

[0123] According to the nucleoside compound represented by formula 1-a of the present invention, X1 is -O-, -S- or -CH2O-.

[0124] According to the nucleoside compound represented by formula 1-a of the present invention, wherein X2 is -O- or -S-.

[0125] According to the nucleoside compound represented by formula 1-a of the present invention, wherein X3 is -O-, -CH2O-, -CH2S-, -CH2NH(CO)-, -(CO)NH- or -CH2OCH2-heteroaryl-, for example, X3 is -CH2OCH2-triazolyl-.

[0126] According to the nucleoside compound represented by formula 1-a of the present invention, wherein R 1 For H.

[0127] According to the nucleoside compound represented by formula 1-a of the present invention, wherein R 2 For H.

[0128] According to the nucleoside compound represented by formula 1-a of the present invention, wherein R 3 For H.

[0129] According to the nucleoside compound represented by formula 1-a of the present invention, wherein R 4 is H or methoxy.

[0130] According to the nucleoside compound represented by formula 1-a of the present invention, wherein R 5 It is methyl or C10-C18 alkyl.

[0131] According to the nucleoside compound represented by formula 1-a of the present invention, wherein R 1 is H, Nu does not exist.

[0132] According to the nucleoside compound represented by formula 1-a of the present invention, wherein Nu is a base, and the base is selected from:

[0133] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound represented by formula 2-a,

[0134] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group;

[0135] Nu is as defined above.

[0136] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound represented by formula 3-a,

[0137] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group;

[0138] Nu is as defined above.

[0139] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound represented by formula 4-a,

[0140] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group;

[0141] Nu is as defined above.

[0142] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound represented by formula 7-a,

[0143] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group;

[0144] Nu is as defined above.

[0145] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is the nucleoside compound represented by formula 8-a,

[0146] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group;

[0147] Nu is as defined above.

[0148] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound represented by formula 10-a,

[0149] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group;

[0150] Nu is as defined above.

[0151] According to the nucleoside compound represented by formula 1-a of the present invention, wherein the nucleoside compound represented by formula 1-a of the present invention is a nucleoside compound represented by formula 12-a,

[0152] R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group.

[0153] Another object of the present invention is to provide an oligonucleotide, wherein the oligonucleotide comprises at least one nucleoside compound represented by formula 1-a.

[0154] According to the oligonucleotide of the present invention, the nucleoside compound has the following structure in the oligonucleotide:

[0155] where R 1 、R 2 、R 3 、R 4 、R 5 , Z, X1, X2, X3 and Nu are as defined above.

[0156] Another object of the present invention is to provide the use of the nucleoside compound represented by formula 1-a for preparing oligonucleotides.

[0157] According to the use of the present invention, the nucleoside compound has the following structure in the oligonucleotide:

[0158] where R 1 、R 2 、R 3 、R 4 、R 5 , Z, X1, X2, X3 and Nu are as defined above.

[0159] In the present invention, the oligonucleotide can be siRNA, antisense nucleic acid, saRNA, miRNA, or nucleic acid aptamer.

[0160] In the present invention, the siRNA has a double-stranded structure, comprising a sense strand and an antisense strand; wherein the antisense strand is 17 to 30 nucleotides in length; and the sense strand is 17 to 30 nucleotides in length and is at least partially complementary to the antisense strand. Preferably, the sense strand and the antisense strand are complementary for at least 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0161] In some embodiments of the present invention, the antisense strand is 21 to 23 nucleotides in length; and the sense strand is 19 to 21 nucleotides in length.

[0162] In some embodiments of the present invention, the siRNA comprises one or more single-stranded nucleotide overhangs. For example, an overhang of 1, 2, 3, or 4 nucleotides. In some embodiments of the present invention, the overhang can be on the sense strand, the antisense strand, or any combination thereof. In some embodiments of the present invention, the overhang is present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the siRNA. Preferably, the 3' end of the antisense strand of the siRNA has an overhang of 2 nucleotides.

[0163] In some embodiments of the present invention, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length; or the antisense strand is 22 nucleotides in length and the sense strand is 20 nucleotides in length; or the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length; or the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length; or the antisense strand is 19 nucleotides in length and the sense strand is 19 nucleotides in length.

[0164] In some embodiments of the present invention, the siRNA contains at least one modified nucleotide.

[0165] In some embodiments of the present invention, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides or nucleotide analogs.

[0166] In some embodiments of the present invention, the modified nucleotide is selected from 2'-methoxy nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-split nucleotide analogs, 2'-fluoroarabinonucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 3'-methoxy nucleotides, 2'-allyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides (LNA), unlocked nucleotides (UNA), glycerol nucleotides (GNA) or nucleotides having the structure represented by formula (1) of the present invention, but the present invention is not limited thereto.

[0167] In some embodiments of the present invention, the siRNA modified nucleotide sequence is selected from:

[0168] 1) The nucleotides at positions 7, 9, 10, and 11 at the 5′ end of the sense strand are 2′-fluoro nucleotides; and / or

[0169] 2) the nucleotides at positions 9, 10, and 11 at the 5′ end of the sense strand are 2′-fluoro nucleotides; and / or

[0170] 3) the nucleotides at positions 2, 6, 8, 9, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides; and / or

[0171] 4) The nucleotides at positions 2, 6, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides.

[0172] In some embodiments of the present invention, the siRNA modified nucleotide sequence is selected from:

[0173] 1) The nucleotides at positions 7, 9, 10, and 11 at the 5′ end of the sense strand are 2′-fluoro nucleotides, and the remaining positions are 2′-methoxy nucleotides or nucleotides represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention; and / or

[0174] 2) the nucleotides at positions 9, 10, and 11 at the 5′ end of the sense strand are 2′-fluoro nucleotides, and the remaining positions are 2′-methoxy nucleotides or nucleotides represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention; and / or

[0175] 3) the nucleotides at positions 2, 6, 8, 9, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides, and the remaining positions are 2′-methoxy nucleotides or nucleotides represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention; and / or

[0176] 4) The nucleotides at positions 2, 6, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides, and the remaining positions are 2′-methoxy nucleotides or nucleotides represented by Formula 17-a, Formula 18-a, Formula 13-b, and Formula 14-b of the present invention.

[0177] In some embodiments of the present invention, the siRNA modified nucleotide sequence is selected from:

[0178] 1) the nucleotides at positions 7, 9, 10, and 11 at the 5′ end of the sense strand are 2′-fluoro nucleotides, the nucleotide at position 1 at the 5′ end of the sense strand is a nucleotide represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention, and the remaining positions are 2′-methoxy nucleotides; and / or

[0179] 2) the nucleotides at positions 9, 10, and 11 at the 5′ end of the sense strand are 2′-fluoro nucleotides, the nucleotide at position 1 at the 5′ end of the sense strand is a nucleotide represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention, and the remaining positions are 2′-methoxy nucleotides; and / or

[0180] 3) the nucleotides at positions 2, 6, 8, 9, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides, the nucleotide at position 1 at the 3′ end of the antisense strand is a nucleotide represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention, and the remaining positions are 2′-methoxy nucleotides; and / or

[0181] 4) the nucleotides at positions 2, 6, 8, 9, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides, the nucleotide at position 2 at the 3′ end of the antisense strand is a nucleotide represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention, and the remaining positions are 2′-methoxy nucleotides; and / or

[0182] 5) the nucleotides at positions 2, 6, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides, the nucleotide at position 1 at the 3′ end of the antisense strand is a nucleotide represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention, and the remaining positions are 2′-methoxy nucleotides; and / or

[0183] 6) The nucleotides at positions 2, 6, 14, and 16 at the 5′ end of the antisense strand are 2′-fluoro nucleotides, the nucleotide at position 2 at the 3′ end of the antisense strand is a nucleotide represented by Formula 17-a, Formula 18-a, Formula 13-b, or Formula 14-b of the present invention, and the remaining positions are 2′-methoxy nucleotides.

[0184] In some embodiments of the present invention, the 3' end of the siRNA sense strand is further connected to a targeting ligand.

[0185] In some embodiments of the present invention, the targeting ligand is L96, having the following structure:

[0186] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0187] In the structure of the compound of the present application, "DMTr" is 4,4'-dimethoxytrityl.

[0188] In the present application, when Nu represents "absent", it means that the nucleotide compound does not have a base, that is, the base is replaced by hydrogen.

[0189] The term "stereoisomer" as used herein refers to compounds that have the same chemical structure but differ in the way the atoms or groups are arranged in space. The compounds of the present invention may contain asymmetric centers or chiral centers, and therefore exist as different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc., and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D, L or R, S are used to indicate the absolute configuration of the chiral center of the molecule. These stereoisomers have the same chemical structure, but their three-dimensional structures are different. "Stereoisomers" include enantiomers or diastereomers. A specific stereoisomer can be an enantiomer, and a mixture of isomers is often called an enantiomeric mixture.

[0190] The carbon, hydrogen, oxygen, sulfur, nitrogen or F, Cl, Br, I involved in the groups and compounds described in this application include their isotopes, and the carbon, hydrogen, oxygen, sulfur or nitrogen involved in the groups and compounds described in this application are optionally further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C. 13 C and 14 C, hydrogen isotopes include protium (H), deuterium (D, also called heavy hydrogen), tritium (T, also called super tritium), oxygen isotopes include 16 O. 17 O and 18 O, sulfur isotopes include 32 S. 33 S. 34 S and 36 S, nitrogen isotopes include 14 N and 15N, fluorine isotopes include 17 F and 19 F, chlorine isotopes include 35 Cl and 37 Isotopes of Cl, bromine include 79 Br and 81 Br, Iodine isotopes include 127 iodine, 129 Iodine and 131 iodine.

[0191] "Alkyl" refers to a straight or branched saturated aliphatic hydrocarbon group of 1 to 30 carbon atoms, preferably an alkyl group of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms, more preferably an alkyl group of 1 to 6 carbon atoms or an alkyl group of 10 to 18 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, a straight or branched C 10-18 The alkyl group may be optionally further substituted with one or more substituents.

[0192] "Alkoxy" refers to a group formed by linking an alkyl group with an oxygen atom. The definition of the alkyl group is the same as that of "alkyl" described above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexyloxy, cyclopropyloxy, cyclobutyloxy, linear or branched C 10-18 Alkoxy. The alkoxy may be further substituted with one or more substituents.

[0193] "Cycloalkyl" refers to a saturated cyclic hydrocarbon group, which can be a 3-10-membered (e.g., 3, 4, 5, 6, 7, 8, 9, 10-membered) monocyclic ring, a 4-12-membered (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10-20-membered (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-membered) polycyclic ring system, preferably 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The cycloalkyl group may optionally be further substituted with one or more substituents.

[0194] "Alkenyl" refers to a straight-chain or branched unsaturated aliphatic hydrocarbon group consisting of 2 to 30 carbon atoms and containing 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) carbon-carbon double bonds, preferably an alkenyl group of 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms, more preferably an alkenyl group of 2 to 8 carbon atoms or an alkenyl group of 10 to 18 carbon atoms. Non-limiting examples of alkenyl groups include ethenyl, propen-2-yl, buten-2-yl, penten-2-yl, penten-4-yl, hexen-2-yl, hexen-3-yl, hepten-2-yl, hepten-3-yl, hepten-4-yl, octen-3-yl, nonen-3-yl, decen-4-yl, undecen-3-yl, straight or branched C 12-18 The alkenyl group may be optionally further substituted by one or more substituents.

[0195] "Alkynyl" refers to a straight-chain or branched unsaturated aliphatic hydrocarbon group consisting of 2 to 30 carbon atoms and containing 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) carbon-carbon triple bonds, preferably an alkynyl group of 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms, more preferably an alkynyl group of 2 to 8 carbon atoms or an alkynyl group of 10 to 18 carbon atoms. Non-limiting examples of alkynyl groups include ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, butyn-3-yl, 3,3-dimethylbutyn-2-yl, pentyn-1-yl, pentyn-2-yl, hexyn-1-yl, 1-heptyn-1-yl, heptyn-3-yl, heptyn-4-yl, octyn-3-yl, nonyn-3-yl, decyn-4-yl, undecyn-3-yl, dodecyn-4-yl, a straight or branched C 13-18 The alkynyl group may be optionally further substituted by one or more substituents.

[0196] Halogens include F, Cl, Br and I.

[0197] An acyl group is a structure consisting of a carbonyl-containing functional group connected to a hydrogen atom or a substituent group, represented by -COR'. In the present invention, R' represents an alkyl group, an alkenyl group, or an alkynyl group. The definitions of these alkyl groups are the same as those for "alkyl," "alkenyl," and "alkynyl" described above. Non-limiting examples of acyl groups include formyl, acetyl, propionyl, and butyryl.

[0198] "Aryl" refers to an aromatic ring group having a conjugated planar ring system, which can be a 5-8 membered (e.g., 5, 6, 7, 8 membered) monocyclic ring, a 5-12 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12 membered) bicyclic ring, or a 10-15 membered (e.g., 10, 11, 12, 13, 14, 15 membered) tricyclic ring system, which can be a bridged ring or a spirocyclic ring. Non-limiting examples of aryl include phenyl and naphthyl. The aryl group can optionally be further substituted with one or more substituents.

[0199] "Heteroaryl" refers to an aromatic ring group having a conjugated planar ring system and containing heteroatoms, which can be a 3-8 membered (e.g., 3, 4, 5, 6, 7, 8 membered) monocyclic ring, a 5-12 membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12 membered) bicyclic ring, or a 10-15 membered (e.g., 10, 11, 12, 13, 14, 15 membered) tricyclic ring system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O, or S. Non-limiting examples of heteroaryl include triazolyl, pyridyl, furyl, thienyl, pyranyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinylbenzimidazolyl, benzopyridinyl, pyrrolopyridinyl. The heteroaryl group may be optionally further substituted with one or more substituents.

[0200] "Heterocyclyl" or "heterocycle" refers to a saturated or unsaturated aromatic heterocycle or non-aromatic heterocycle. When it is an aromatic heterocycle, its definition is the same as the above "heteroaryl"; when it is a non-aromatic heterocycle, it can be a 3-10 membered (e.g., 3, 4, 5, 6, 7, 8, 9, 10 membered) monocyclic ring, a 4-12 membered (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12 membered) bicyclic ring or a 10-15 membered (e.g., 10, 11, 12, 13, 14, 15 membered) tricyclic ring system, and contains 1 to 4 (e.g., 1, 2, 3, 4) heteroatoms selected from N, O or S, and is preferably a 3- to 8-membered heterocyclyl. Non-limiting examples of "heterocyclyl" or "heterocycle" include oxetanyl, azetidinyl, thietanyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxanyl, azepanyl, oxepinyl, thiepanyl, triazolyl, pyridinyl, piperidinyl, furanyl, thienyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, 1,3-dithianyl, dihydrofuranyl, dithiopentyl, thiazolyl, 1,3-dithianyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, 1,3-dithianyl, dihydrofuranyl, dithiopentyl, thioxanyl, 1,3-dithian ... The "heterocyclyl" or "heterocycle" may be optionally substituted with one or more substituents.

[0201] When the above-mentioned "alkyl", "alkoxy", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl" and "heterocyclyl" are substituted, they may be further substituted by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents selected from F, Cl, Br, I, hydroxyl, thiol, nitro, cyano, amino, C1-6 alkylamino, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 heterocycloalkyl, C6-10 aryl and C5-10 heteroaryl.

[0202] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "heterocyclyl optionally substituted with alkyl" means that the alkyl group may but need not be present, and that the description includes instances where the heterocyclyl group is substituted with alkyl and instances where the heterocyclyl group is not substituted with alkyl. Beneficial effects

[0203] The present invention provides a modified nucleoside compound that can be incorporated into the oligonucleotide head end and / or the middle, so that the modified oligonucleotide is hybridized with a part of the target RNA, thereby causing the loss or reduction of the normal function of the target RNA. Such oligonucleotides may also be included in a double-stranded composition. Specifically, the nucleoside compound shown in Formula 1-a and 1-b of the present invention can be applied to oligonucleotides (including but not limited to siRNA, antisense nucleic acids, saRNA, miRNA, nucleic acid aptamers, etc.), can effectively improve the activity of oligonucleotides, and simultaneously improve the enzymatic resistance of its corresponding oligonucleotide to phosphatases, exonucleases, endonucleases, etc. In addition, the nucleoside compound shown in Formula 1-a of the present invention is relatively ordinary nucleoside, and can be easily introduced into various functional groups of specific effects, such as various substituted alkyl lipids, without affecting its base complementary pairing ability, to improve the cell membrane penetration ability of the oligonucleotide sequence, and improve the organ targeting effect of the oligonucleotide (for example, when such modified nucleosides are used in siRNA molecules, the enrichment degree of siRNA molecules in target organs can be increased, etc.). Through cell and mouse experiments, the in vitro and in vivo activities, pharmacokinetic properties and bioavailability of the oligonucleotide drugs prepared from the nucleoside compounds represented by formula 1-a and 1-b of the present invention are greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0204] FIG1 shows the remaining amount of the test sample at each time point in the 3′-exonuclease stability test of single-stranded molecules in Experiment 4.

[0205] FIG2 shows the TTR protein inhibition rate of wild-type mice on Day 6 of the experiment.

[0206] FIG3 shows the liver exposure of mice in Experiment 7 for 14 days.

[0207] FIG4 shows the TTR protein inhibition rate of wild-type mice on Day 21 of Experiment 13.

[0208] FIG5 shows the investigation of TTR protein inhibitory activity in wild-type mice in Experiment 13. DETAILED DESCRIPTION

[0209] Unless otherwise specified, the instruments used in the present invention are conventional instruments, and the reagents used are conventional reagents.

[0210] The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0211] NMR shifts (δ) are given in units of 10-6 (ppm). NMR measurements were performed using a Broker Avance III 400 nuclear magnetic resonance spectrometer. The solvents used were tritiated dimethyl sulfoxide (DMSO-d6), tritiated chloroform (CDCl3), and tritiated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0212] MS was determined using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0213] Thin layer chromatography silica gel plates used were Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications of the Guijiao plates used in thin layer chromatography (TLC) were 0.15 mm to 0.20 mm, and the specifications used for thin layer chromatography separation and purification products were 0.4 mm to 0.5 mm.

[0214] Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0215] Oligo solid-phase synthesis was performed on an LK-48E synthesizer (Lingkun).

[0216] Description of the abbreviations of materials used in this invention:

[0217] The present invention will be described in detail below through examples. Where specific conditions are not specified in the examples, the experimental methods are carried out according to conventional conditions. The examples are provided to better illustrate the present invention, but it should not be understood that the present invention is limited to the examples. Any non-essential improvements and adjustments made by those skilled in the art to the embodiments based on the above invention are still within the scope of protection of the present invention.

[0218] Examples

[0219] The following Examples 1-4 are preparation examples of the nucleoside compound represented by Formula 2-a of the present invention, wherein the nucleoside compound represented by Formula 2-a is a compound having a 2'-phosphoramidite group, a 3'-ODMTr, and a 4'-modified TNA structure.

[0220] Example 1: Synthesis of Compound 11

[0221] Synthesis of compound 2:

[0222] Dried compound 1 (20.0 g, 105.15 mmol, 1.0 eq.) was dissolved in DCM (200 mL). Imidazole (17.9 g, 262.88 mmol, 2.5 eq.) was added to the reaction system, and the reaction mixture was cooled to 0°C and stirred for 30 minutes. Tert-butyldiphenylsilyl chloride (TBDPSCl, 31.79 g, 115.67 mmol, 1.1 eq.) was then slowly added dropwise to the reaction system. After the addition, the ice bath was removed, and the reaction mixture was gradually allowed to return to room temperature. The reaction mixture was allowed to react overnight at room temperature. TLC analysis of the reaction indicated that compound 1 had reacted completely. Water was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=100 / 15) to give compound 2 (35.24 g, 82.29 mmol, 78.3% yield). ESI-MS: m / z 451.2 [M+Na] +

[0223] Synthesis of compound 3:

[0224] Compound 2 (38.2 g, 89.13 mmol, 1.0 eq.) was dissolved in DMF (300 mL) and stirred until completely dissolved. The reaction system was cooled to approximately 0°C. NaH (5.35 g, 133.70 mmol, 1.5 eq.) was slowly added portionwise to the reaction. After addition, the reaction system was stirred at 0°C for 30 minutes. BnBr (22.87 g, 133.70 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, maintaining the reaction temperature at approximately 0°C. After addition, the reaction system was returned to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 2. The reaction system was slowly poured into a saturated aqueous ammonium chloride solution at approximately 0°C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield crude product 3 (68.2 g), which was used directly in the next step. ESI-MS: m / z 519.3[M+H] +

[0225] Synthesis of compound 4:

[0226] Crude compound 3 (68.2 g) was dissolved in THF (400 mL) and stirred until completely dissolved. TBAF (1 M in THF, 100 mL, 100 mmol, 1.12 eq.) was added to the reaction mixture, and stirred at room temperature overnight until reaction of compound 3 was complete. The reaction solution was concentrated under reduced pressure to remove the solvent, and the resulting crude product was purified by column chromatography (PE / EA = 10 / 3) to afford compound 4 (22.07 g, 78.73 mmol, 88.3% yield over two steps).1 H NMR (400MHz, DMSO-d6) δ7.43-7.23 (m, 5H), 5.84 (d, J=3.6Hz, 1H), 4.74 (t, J=5.2Hz, 1H), 4.67 (m, 2H), 4.5 4-4.47 (d, J=12Hz, 1H), 4.10 (m, 1H), 3.89 (d, J=3.2Hz, 1H), 3.71-3.55 (m, 2H), 1.39 (s, 3H), 1.26 (s, 3H).

[0227] Synthesis of compound 5:

[0228] Compound 4 (22.07 g, 78.73 mmol, 1.0 eq.) was dissolved in DMF (200 mL) and stirred evenly. The reaction mixture was cooled to 0°C and stirred for 30 minutes. Bromododecane (23.55 g, 94.48 mmol, 1.2 eq.) was slowly added dropwise to the reaction system. After addition, the reaction mixture was returned to room temperature and allowed to react overnight. TLC analysis indicated that compound 4 had reacted completely. The reaction mixture was slowly poured into ice water to quench the mixture completely. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 100 / 9) to obtain compound 5 (32.0 g, 71.37 mmol, 90.6% yield). 1 H NMR (400MHz, DMSO-d6) δ7.39-7.24(m, 5H), 5.85(d, J=3.8Hz, 1H), 4.73-4.62(m, 2H), 4.48 (d, J=12.0Hz, 1H), 4.17 (td, J=5.9, 3.2Hz, 1H), 3.88 (d, J=3.2Hz, 1H), 3 .62 (dd, J=10.1, 5.4Hz, 1H), 3.51 (dd, J=10.1, 6.4Hz, 1H), 3.46-3.30 (m, 2H), 1. 47(m, 2H), 1.39(s, 3H), 1.24(d, J=11.6Hz, 21H), 0.89-0.81(m, 3H)., ESI-MS: m / z 471.3[M+Na] +

[0229] Synthesis of compound 6:

[0230] Compound 5 (35.32 g, 78.73 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask, and AcOH (120 mL) and Ac2O (40.19 g, 393.65 mmol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (5 mL) was slowly added dropwise to the reaction system, keeping the temperature at 0-5°C at all times. After the addition, the reaction system was returned to room temperature and continued to stir for 8 hours until compound 5 was completely reacted. The reaction system was cooled to about -5°C and neutralized with ammonia water to a pH of about 7. Water was added to the reaction system, and the reaction was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 6. The crude product 6 was purified by column chromatography (PE / EA=10 / 2) to give compound 6 (19.0 g, 38.59 mmol, 49% yield). 1 H NMR (400MHz, DMSO-d6) δ7.36-7.25 (m, 5H), 6.24 (d, J=8.4, 1H), 5.19-5.15 (m, 1 H), 4.72-4.53(m, 2H), 4.39-4.09(m, 2H), 3.67-3.46(m, 2H), 3.42-3.32(m, 2H), 2.04(s, 3H), 2.02(s, 3H), 1.50-1.42(m, 2H), 1.31-1.19(m, 18H), 1.23(t, J=6.8, 3H).ESI-MS: m / z 493.3[M+H] +

[0231] Synthesis of compound 7:

[0232] Compound 6 (5.0 g, 10.15 mmol, 1.00 eq.) and U (2.28 g, 20.30 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (60 mL) was added and stirred to dissolve. BSA (6.19 g, 30.45 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (2.26 g, 10.15 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 6. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 7. The crude product was purified by column chromatography (PE / EA = 5 / 2) to afford compound 7 (4.71 g, 8.65 mmol, 85.2% yield). 1 H NMR (400MHz, DMSO-d6) δ11.37 (s, 1H), 7.62 (d, J = 8.1Hz, 1H), 7.35-7.25 (m, 5H), 5.91 (d, J = 2.1Hz, 1H) , 5.58 (dd, J=8.1, 2.2Hz, 1H), 5.22 (t, J=1.9Hz, 1H), 4.71 (d, J=12.0Hz, 1H), 4.54 (d, J=12.0Hz, 1H), 4. 26 (m, 1H), 4.12 (dd, J=4.1, 1.6Hz, 1H), 3.71 (dd, J=10.6, 4.5Hz, 1H), 3.64 (dd, J=10.6, 6.1Hz, 1H), 3.4 8-3.34(m, 2H), 2.08(s, 3H), 1.50-1.43(m, 2H), 1.29-1.20(m, 18H), 1.18(t, J=7.1Hz, 3H).ESI-MS: m / z 545.3[M+H] +

[0233] Synthesis of compound 8:

[0234] Compound 7 (4.71 g, 8.65 mmol) was added to a 250 ml three-necked flask. Ultra-dry DCM (50 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. BCl3 (1 M in toluene, 26 mL, 25.95 mmol, 3.0 eq.) was slowly added dropwise to the reaction system and the reaction was continued for 5 hours until compound 7 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 8. The crude product was purified by column chromatography (PE / EA = 2 / 3) to obtain compound 8 (2.0 g, 4.4 mmol, 50.9% yield). ESI-MS: m / z 455.3 [M+H] +

[0235] Synthesis of compound 9:

[0236] Compound 8 (2.0 g, 4.40 mmol, 1.0 eq.) was added to a 250 ml round-bottom flask, and ultra-dry DCE (30 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (7.45 g, 22.00 mmol, 5.0 eq.), AgNO3 (747 mg, 4.40 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.33 g, 44 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was placed in an oil bath and heated to 80°C and allowed to react overnight. TLC and LCMS showed that compound 8 had reacted completely. The reaction was returned to room temperature and methanol was added to the reaction to quench the reaction. Ethyl acetate was added to the reaction to dilute it and filtered through celite to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 9. The crude product was purified by column chromatography (PE / EA=100 / 35) to give compound 9 (3.2 g, 4.23 mmol, 96% yield). ESI-MS: m / z 757.4 [M+H] +

[0237] Synthesis of compound 10:

[0238] Compound 9 (3.2 g, 4.23 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. Aqueous ammonia (50 mL) was then added to the reaction at room temperature. The reaction was stirred at room temperature for 3 hours until compound 9 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to afford crude product 10. This crude product was purified by column chromatography (PE / EA = 1 / 1) to afford compound 10 (2.9 g, 4.06 mmol, 96% yield).1 H NMR (400MHz, DMSO-d6) δ11.28 (s, 1H), 7.90 (d, J=8.1Hz, 1H), 7.46-7.39 (m, 2H ), 7.33-7.22 (m, 7H), 6.90-6.78 (m, 4H), 5.45 (d, J=3.6Hz, 1H), 4.14 (t, J=4.7 Hz, 1H), 3.74 (s, 6H), 3.68 (dd, J=9.2, 4.5Hz, 1H), 3.62-3.49 (m, 2H), 3.49-3. 39(m, 3H), 1.58(m, 2H), 1.39-1.20(m, 20H), 0.85(t, J=6.7Hz, 3H).ESI-MS: m / z 715.4[M+H] +

[0239] Synthesis of compound 11:

[0240] Dried compound 10 (2.9 g, 4.06 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred until completely dissolved. DIPEA (1.05 g, 8.12 mmol, 2.0 eq.) and DMAP (99 mg, 0.81 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.44 g, 6.09 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 10 was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 11. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 11 (3.16 g, 3.46 mmol, 85.2% yield). 1H NMR (400MHz, DMSO-d6) δ11.35 (dd, J=14.6, 2.2Hz, 1H), 7.93 (d, J=8.1Hz, 1H), 7.47-7.36 (m, 2H), 7. 33-7.23 (m, 7H), 6.89-6.83 (m, 4H), 5.71-5.51 (m, 2H), 4.34 (dt, J=8.8, 4.7Hz, 1H), 4.11-3.90 (m, 1H ), 3.84-3.76 (m, 1H), 3.75 (s, 6H), 3.69-3.36 (m, 6H), 2.71 (t, J=6.0Hz, 1H), 2.60-2.39 (m, 1H), 1.6 2-1.52 (m, 2H), 1.42-1.20 (m, 20H), 1.10 (t, J=6.6Hz, 6H), 1.02 (d, J=6.7Hz, 6H), 0.89-0.81 (m, 3H). 31 P NMR (162MHz, DMSO-d6) δ151.30, 148.65., ESI-MS: m / z 915.6[M+H] +

[0241] Example 2: Synthesis of Compound 16

[0242] Synthesis of compound 12:

[0243] Compound 10 (2.6 g, 3.64 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DMF (30 mL) was added and stirred to dissolve. Imidazole (991 mg, 14.56 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (1.10 g, 7.28 mmol, 2.0 eq.) was then slowly added portionwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS analysis indicated that compound 10 had reacted completely, and water was added to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 1) to obtain compound 12 (2.75 g, 3.32 mmol, 91.2% yield). ESI-MS: m / z 829.5[M+H] +

[0244] Synthesis of compound 13:

[0245] Compound 12 (2.75 g, 3.32 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry ACN (30 mL) was added and stirred until completely dissolved. TEA (672 mg, 6.64 mmol, 2.0 eq.) and DMAP (811 mg, 6.64 mmol, 2.0 eq.) were added to the reaction system and stirred uniformly. The reaction mixture was cooled to 0-5°C, and TPSCl (2.01 g, 6.64 mmol, 2.0 eq.) was slowly added portionwise. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 12. Aqueous ammonia (20 mL) was added to the reaction at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude product 13 (8.3 g, calculated based on 100% yield). ESI-MS: m / z 828.6 [M+H] +

[0246] Synthesis of compound 14:

[0247] Crude compound 13 (8.3 g, 1.0 eq.) was added to a 100 mL round-bottom flask, and pyridine (50 mL) was added and stirred until the crude compound 13 was completely dissolved. The reaction system was cooled to 0°C, and BzCl (933 mg, 6.64 mmol, 2.0 eq.) was slowly added dropwise to the reaction system and stirred at 0°C for 1 hour until the reaction of compound 13 was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 14 (2.35 g, 2.52 mmol, 76% total yield over two steps). ESI-MS: m / z 931.5 [M+H] +

[0248] Synthesis of compound 15:

[0249] Compound 14 (2.35 g, 2.52 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (30 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized with triethylamine (17 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS analysis showed that compound 14 had reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 15 (1.57 g, 1.92 mmol, 76.2% yield). ESI-MS: m / z 818.5 [M+H] +

[0250] Synthesis of compound 16:

[0251] Dried compound 15 (1.57 g, 1.92 mmol) was added to a 100 mL round-bottom flask, and ultra-dry DCM (20 mL) was added and stirred to dissolve. DIPEA (496 mg, 3.84 mmol, 2.0 eq.) and DMAP (47 mg, 0.38 mmol, 0.2 eq.) were added to the reaction, and the reaction was replaced with nitrogen. The reaction temperature was cooled to about 0°C, and CEP-Cl (682 mg, 2.93 mmol, 1.5 eq.) was slowly added dropwise. The reaction was allowed to react at this temperature under nitrogen for 1 hour until compound 15 was completely reacted. After the reaction was completed, saturated sodium bicarbonate aqueous solution was added to the reaction to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 16 (1.7 g, 1.67 mmol, 87% yield). 1H NMR (400MHz, DMSO-d6) δ11.27 (d, J=17.5Hz, 1H), 8.46 (dd, J=30.3, 7.5Hz, 1H), 8.01 (d, J=7.6Hz, 2H ), 7.62 (t, J=7.3Hz, 1H), 7.55-7.44 (m, 2H), 7.39-7.13 (m, 9H), 6.87-6.82 (m, 4H), 5.71 (d, J=25.0H z, 1H), 4.23-3.90 (m, 3H), 3.78-3.38 (m, 13H), 2.74 (dd, J=10.7, 5.6Hz, 1H), 2.61-2.32 (m, 1H), 1.6 1-1.50 (m, 2H), 1.38-1.19 (m, 20H), 1.12-1.04 (m, 9H), 0.96 (d, J=6.7Hz, 3H), 0.83 (t, J=6.6Hz, 3H), 31 P NMR (162MHz, DMSO-d6) δ149.87, 149.34, ESI-MS: m / z 1018.6[M+H] +

[0252] Example 3: Synthesis of Compound 21

[0253] Synthesis of compound 17:

[0254] Compound 6 (27 g, 54.81 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (250 mL) was added and stirred to dissolve. BSA (33.45 g, 164.43 mmol, 3.0 eq.) and A(Bz) (26.22 g, 109.62 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (12.18 g, 54.81 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 6. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 17. The crude product was purified by column chromatography (PE / EA = 1 / 1) to afford compound 17 (25 g, 37.23 mmol, 67.9% yield). 1H NMR (400MHz, DMSO-d6) δ11.24 (s, 1H), 8.76 (s, 1H), 8.42 (s, 1H), 8.06 (d, J=7.4Hz, 2H), 7.65 (t , J=7.4Hz, 1H), 7.55 (t, J=7.6Hz, 2H), 7.38-7.27 (m, 5H), 6.31 (d, J=1.7Hz, 1H), 4.78 (d, J=11. 8Hz, 1H), 4.60 (d, J=11.8Hz, 1H), 4.44 (dd, J=10.3, 4.6Hz, 1H), 3.83-3.67 (m, 2H), 3.46-3.39 ( m, 2H), 2.13 (s, 3H), 1.53-1.43 (m, 2H), 1.32-1.16 (m, 20H), 0.84 (t, J=6.7Hz, 3H). ESI-MS: m / z 672.4[M+H] +

[0255] Synthesis of compound 18:

[0256] Compound 17 (3.0 g, 4.47 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry DCM (30 mL) was added to the reaction flask. The reaction system was cooled to -10°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (13.4 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 17 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 18. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 18 (1.0 g, 1.72 mmol, 38.5% yield).

[0257] Synthesis of compound 19:

[0258] Compound 18 (1.0 g, 1.72 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (15 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (2.9 g, 8.6 mmol, 5.0 eq.), AgNO3 (0.29 g, 1.72 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (2.08 g, 17.2 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was placed in an oil bath and heated to 80°C and allowed to react overnight. TLC and LCMS showed that compound 18 had reacted completely. The reaction was returned to room temperature and methanol was added to the reaction to quench the reaction. Ethyl acetate was added to the reaction to dilute it and filtered through celite to obtain a filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain crude compound 19. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 19 (1.2 g, 1.36 mmol, 72% yield). ESI-MS: m / z 884.5 [M+H] +

[0259] Synthesis of compound 20:

[0260] Compound 19 (1.2 g, 1.36 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (10 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (0.76 mL, 4.08 mmol, 3.0 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 19 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 20. The crude product was purified by column chromatography to yield compound 20 (0.82 g, 0.97 mmol, 73% yield). ESI-MS: m / z 842.4 [M+H] +

[0261] Synthesis of compound 21:

[0262] Dried compound 20 (0.82 g, 0.97 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (10 mL) was added and stirred until completely dissolved. DIPEA (0.25 g, 1.94 mmol, 2.0 eq.) and DMAP (23.8 mg, 0.194 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (0.35 g, 1.46 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 20 was fully reacted. The reaction mixture was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 21. The crude product was purified by column chromatography to give compound 21 (0.74 g, 0.71 mmol, 73.2% yield). 1 H NMR (400MHz, DMSO-d6) δ11.21 (d, J=4.0Hz, 1H), 8.71 (d, J=5.9Hz, 1H), 8.60 (d, J=29.9Hz, 1H), 8.04 ( d, J=7.6Hz, 2H), 7.65 (t, J=7.3Hz, 1H), 7.57 (t, J=7.6Hz, 2H), 7.35 (t, J=8.6Hz, 2H), 7.21-7.12 (m, 7 H), 6.83-6.71 (m, 4H), 6.02 (dd, J=36.3, 3.4Hz, 1H), 4.78-4.31 (m, 2H), 3.81-3.36 (m, 15H), 2.67-2. 38(m, 2H), 1.62-1.48(m, 2H), 1.42-1.21(m, 18H), 1.12-0.99(m, 9H), 0.93-0.78(m, 6H).ESI-MS: m / z 1042.6 [M+H] +

[0263] Example 4: Synthesis of Compound 26

[0264] Synthesis of compound 22:

[0265] Compound 6 (20 g, 40.60 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (200 mL) was added and stirred to dissolve. BSA (24.78 g, 121.8 mmol, 3.0 eq.) and G(iBu) (17.96 g, 81.2 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (9.02 g, 40.6 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight at this temperature. TLC and LCMS analysis confirmed the complete reaction of compound 6. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 22. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 22 (18 g, 27.53 mmol, 67.8% yield). ESI-MS: m / z 654.5 [M+H] +

[0266] Synthesis of compound 23:

[0267] Compound 22 (5.0 g, 7.64 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry DCM (50 mL) was added to the reaction flask. The reaction system was cooled to -10°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (22.92 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 22 was fully reacted. The reaction was quenched with triethylamine and methanol at -10°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 23. The crude product was purified by column chromatography (PE / EA = 1 / 2) to yield compound 23 (2.6 g, 4.61 mmol, 60.3% yield). ESI-MS: m / z 564.3 [M+H] +

[0268] Synthesis of compound 24:

[0269] Compound 23 (2.6 g, 4.61 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (35 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (7.8 g, 23.05 mmol, 5.0 eq.), AgNO3 (0.8 g, 4.61 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.59 g, 46.1 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 23. The reaction was returned to room temperature and methanol was added to the reaction to quench the reaction. Ethyl acetate was added to the reaction and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product compound 24. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 24 (3.18 g, 3.67 mmol, 79.6% yield). ESI-MS: m / z 866.6 [M+H] +

[0270] Synthesis of compound 25:

[0271] Compound 24 (3.18 g, 3.67 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (30 mL) was added to the reaction and stirred until completely dissolved. 5.4 mol / L NaOMe solution (2.04 mL, 11.01 mmol, 3.0 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 24 was completely reacted. After the reaction was complete, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 25. The crude product was purified by column chromatography to obtain compound 25 (2.48 g, 3.01 mmol, 82.0% yield). ESI-MS: m / z 824.5 [M+H]+

[0272] Synthesis of compound 26:

[0273] Dried compound 25 (2.0 g, 2.43 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred until completely dissolved. DIPEA (0.63 g, 4.86 mmol, 2.0 eq.) and DMAP (59.4 mg, 0.486 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (0.86 g, 3.65 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 25 was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 26. The crude product was purified by column chromatography to give compound 26 (2.08 g, 2.06 mmol, 83.5% yield). 1 H NMR (400MHz, DMSO-d6) δ12.08 (s, 1H), 11.48 (s, 1H), 8.18 (d, J=29.6Hz, 1H), 7.52-7.11 (m, 9H), 6.93- 6.84 (m, 4H), 5.72 (dd, J=38.6, 4.8Hz, 1H), 5.21-5.04 (m, 1H), 4.53 (t, J=5.5Hz, 1H), 3.81-3.69 (m, 8H ), 3.61-3.34(m, 3H), 3.30-3.23(m, 2H), 3.11(d, J=8.4Hz, 1H), 2.79-2.64(m, 1H), 2.59-2.48(m, 2H), 2.46-2.35 (m, 1H), 1.51 (d, J=6.6Hz, 2H), 1.38-1.18 (m, 18H), 1.13-0.98 (m, 13H), 0.88-0.74 (m, 8H). 31 P NMR (162MHz, DMSO-d6) δ151.94, 148.86, ESI-MS: m / z 1024.6[M+H] +

[0274] Example 5: Synthesis of Compound 33

[0275] Synthesis of compound 27:

[0276] Compound 4 (16.6 g, 59.22 mmol, 1.0 eq.) was dissolved in DMF (170 mL) and stirred. The reaction mixture was cooled to 0°C and stirred for 30 minutes. NaH (2.84 g, 71.06 mmol, 1.2 eq.) was then added and stirred for 30 minutes. CHI (12.61 g, 88.83 mmol, 1.5 eq.) was slowly added dropwise to the reaction mixture. After addition, the reaction mixture was allowed to cool to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 4. The reaction mixture was slowly poured into ice water to quench the mixture. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 100 / 9) to afford compound 27 (15.0 g, 50.96 mmol, 86.1% yield). ESI-MS: m / z 295.2[M+H] +

[0277] Synthesis of compound 28:

[0278] Compound 27 (15.0 g, 50.96 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask. AcOH (136.5 mL) and Ac2O (26.01 g, 254.95 mmol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction mixture was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (1.37 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5°C. After the addition, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 27 was completely reacted. The reaction system was cooled to approximately -5°C and neutralized with aqueous ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 28. The crude product was purified by column chromatography (PE / EA=10 / 2) to give compound 28 (15.0 g, 44.33 mmol, 87.0% yield). ESI-MS: m / z 361.1 [M+Na] +

[0279] Synthesis of compound 29:

[0280] Compound 28 (15.0 g, 44.33 mmol, 1.00 eq.) and uracil (9.94 g, 88.66 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (160 mL) was added and stirred to dissolve. BSA (27.05 g, 132.99 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (9.85 g, 44.36 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 28. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 29. The crude product was purified by column chromatography (PE / EA = 5 / 2) to obtain compound 29 (15.0 g, 38.42 mmol, 86.7% yield). ESI-MS: m / z 391.1 [M+H] +

[0281] Synthesis of compound 30:

[0282] Compound 29 (15.0 g, 38.42 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Ultra-dry DCM (200 mL) was added to the reaction flask. The reaction system was cooled to -15°C and stirred at this temperature for 30 minutes. BCl3 (1 M in toluene, 115 mL, 115.26 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 29 was fully reacted. The reaction was quenched with triethylamine and methanol at -15°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 30. The crude product was purified by column chromatography (PE / EA = 2 / 3) to obtain compound 30 (5.0 g, 16.65 mmol, 43.3% yield). ESI-MS: m / z 301.2[M+H] +

[0283] Synthesis of compound 31:

[0284] Compound 30 (5.0 g, 16.65 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (60 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (11.28 g, 33.30 mmol, 2.0 eq.), AgNO3 (2.83 g, 16.65 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (20.18 g, 166.5 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 30. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product compound 31. The crude product was purified by column chromatography (PE / EA=100 / 35) to give compound 31 (8.0 g, 13.28 mmol, 79.7% yield). ESI-MS: m / z 603.2 [M+H] +

[0285] Synthesis of compound 32:

[0286] Compound 31 (8.0 g, 13.28 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. 7 M methanolic ammonia (50 mL) was then added to the reaction at room temperature. The reaction was stirred at room temperature for 3 hours until the reaction of compound 31 was complete. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to afford crude product 32. The crude product was purified by column chromatography (PE / EA = 1 / 1) to afford compound 32 (6.0 g, 10.71 mmol, 80.6% yield). ESI-MS: m / z 561.2 [M+H] +

[0287] Synthesis of compound 33:

[0288] Dry compound 32 (2.0 g, 3.57 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (20 mL) was added and stirred until completely dissolved. DIPEA (0.93 g, 7.14 mmol, 2.0 eq.) and DMAP (87 mg, 0.714 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and maintained under a nitrogen atmosphere. CEP-Cl (1.27 g, 5.36 mmol, 1.5 eq.) was added dropwise at room temperature and allowed to react for 30-60 minutes until compound 32 was fully reacted. The reaction mixture was quenched with saturated aqueous sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure to yield crude product 33. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 33 (2.1 g, 2.76 mmol, 77.3% yield). 1 H NMR (400MHz, DMSO-d6) δ11.34 (s, 1H), 7.86 (dd, J=18.9, 8.2Hz, 1H), 7.42-7.19 (m, 9H), 6.92-6.84 (m, 4H), 5.65-5.50 (m, 2H), 4.25-4.14 ( m, 1H), 4.00-3.46 (m, 14H), 3.35-3.30 (d, J=20Hz, 3H), 2.73-2.68 (m, 1H), 2.65-2.41 (m, 1H), 1.09 (t, J=6.1Hz, 6H), 1.01 (t, J=6.8Hz, 6H), 31 P NMR (162MHz, DMSO-d6) δ151.13, 148.52.ESI-MS: m / z 761.3[M+H] +

[0289] Example 6: Synthesis of Compound 38

[0290] Synthesis of compound 34:

[0291] Compound 32 (4.0 g, 7.14 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DMF (50 mL) was added and stirred to dissolve. Imidazole (1.94 g, 28.56 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (2.15 g, 14.28 mmol, 2.0 eq.) was then slowly added portionwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS analysis indicated that compound 32 had reacted completely, and the reaction was quenched by adding water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 1) to afford compound 34 (3.88 g, 5.75 mmol, 80.5% yield). ESI-MS: m / z 675.3[M+H] +

[0292] Synthesis of compound 35:

[0293] Compound 34 (3.88 g, 5.76 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry ACN (30 mL) was added and stirred until completely dissolved. TEA (1.16 g, 11.52 mmol, 2.0 eq.) and DMAP (1.40 g, 11.50 mmol, 2.0 eq.) were added to the reaction system and stirred uniformly. The reaction mixture was cooled to 0-5°C, and TPSCl (3.48 g, 11.50 mmol, 2.0 eq.) was slowly added in portions. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 34. Aqueous ammonia (40 mL) was added to the reaction at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude product 35 (5.1 g, calculated based on 100% yield). ESI-MS: m / z 674.3 [M+H] +

[0294] Synthesis of compound 36:

[0295] Crude compound 35 (5.1 g, 1.0 eq.) was added to a 100 mL round-bottom flask, and pyridine (50 mL) was added and stirred until the crude compound 35 was completely dissolved. The reaction system was cooled to 0°C, and BzCl (1.62 g, 11.50 mmol, 2.0 eq.) was slowly added dropwise to the reaction system and stirred at 0°C for 1 hour until the reaction of compound 35 was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 36 (3.7 g, 4.76 mmol, 82.8% total yield over two steps). ESI-MS: m / z 778.4 [M+H] +

[0296] Synthesis of compound 37:

[0297] Compound 36 (3.7 g, 4.76 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (40 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized with triethylamine (17 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS analysis showed that compound 36 had reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 37 (2.6 g, 3.92 mmol, 82.4% yield). ESI-MS: m / z 664.3 [M+H] +

[0298] Synthesis of compound 38:

[0299] Dried compound 37 (2.6 g, 3.92 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred to dissolve. DIPEA (1.0 g, 7.84 mmol, 2.0 eq.) and DMAP (96 mg, 0.784 mmol, 0.2 eq.) were added to the reaction, and the reaction was purged with nitrogen. The reaction temperature was cooled to approximately 0°C, and CEP-Cl (1.39 g, 5.88 mmol, 1.5 eq.) was slowly added dropwise. The reaction was allowed to react at this temperature under nitrogen for 1 hour until compound 37 was fully reacted. After completion of the reaction, saturated aqueous sodium bicarbonate solution was added to the reaction mixture to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 38 (2.3 g, 2.66 mmol, 67.9% yield).

[0300] 1 H NMR (400MHz, DMSO-d6) δ11.18 (d, J=24.4Hz, 1H), 8.49 (dd, J=30.5, 7.3Hz, 1H), 7. 99(d, J=7.7Hz, 2H), 7.65-7.08(m, 14H), 6.85-6.75(m, 4H), 5.71-5.62(m, 1H), 4.2 2-3.82(m, 3H), 3.82-3.69(m, 8H), 3.61-3.39(m, 5H), 3.35-3.30(d, J=20Hz, 3H), 2 .75-2.70(m, 1H), 2.53-2.31(m, 1H), 1.08(t, J=6.1Hz, 6H), 1.00(t, J=6.8Hz, 6H), 31 P NMR (162MHz, DMSO-d6) δ149.52, 149.10, ESI-MS: m / z 864.4[M+H] +

[0301] Example 7: Synthesis of Compound 43

[0302] Synthesis of compound 39:

[0303] Compound 28 (15.0 g, 0.04 mol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (150 mL) was added and stirred to dissolve. BSA (24.4 g, 0.12 mol, 3.0 eq.) and A(Bz) (19.14 g, 0.08 mol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (8.9 g, 0.04 mol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 28. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 39. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 39 (20.0 g, 38.64 mmol, 87.2% yield). ESI-MS: m / z 518.2 [M+H] +

[0304] Synthesis of compound 40:

[0305] Compound 39 (20.0 g, 38.64 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Extra-dry DCM (200 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L boron trichloride solution in dichloromethane (116 mL, 115.92 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 39 was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 40. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 40 (9.7 g, 22.69 mmol, 58.7% yield). ESI-MS: m / z 428.2[M+H] +

[0306] Synthesis of compound 41:

[0307] Compound 40 (9.7 g, 22.69 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (15.4 g, 45.38 mmol, 2.0 eq.), AgNO3 (3.85 g, 22.69 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (27.50 g, 226.9 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 40. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product, compound 41. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 41 (14.5 g, 19.87 mmol, 87.6% yield). ESI-MS: m / z 730.3 [M+H] +

[0308] Synthesis of compound 42:

[0309] Compound 41 (14.5 g, 19.87 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (150 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (5.5 mL, 29.82 mmol, 1.5 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 41 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 42. The crude product was purified by column chromatography to yield compound 42 (12.0 g, 17.45 mmol, 87.8% yield). ESI-MS: m / z 688.3 [M+H] +

[0310] Synthesis of compound 43:

[0311] Dried compound 42 (3.0 g, 4.36 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred until completely dissolved. DIPEA (1.13 g, 8.72 mmol, 2.0 eq.) and DMAP (106.5 mg, 0.872 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.55 g, 6.56 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 42 was fully reacted. The reaction mixture was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 43. The crude product was purified by column chromatography to give compound 43 (3.4 g, 3.83 mmol, 87.8% yield). 1 H NMR (400MHz, DMSO-d6) δ11.21 (s, 1H), 8.75-8.54 (m, 2H), 8.05 (d, J=7.3Hz, 2 H), 7.68-7.50(m, 3H), 7.39-7.31(m, 2H), 7.29-7.15(m, 7H), 6.86-6.72(m, 4 H), 6.05 (dd, J=34.0, 3.2Hz, 1H), 4.74-4.30 (m, 2H), 3.87-3.78 (m, 1H), 3.79 -3.46(m, 12H), 3.30(d, J=24.0, 3H), 2.70-2.57(m, 2H), 1.11-0.87(m, 12H), 31 P NMR (162MHz, DMSO-d6) δ150.68, 149.45, ESI-MS: m / z 888.4[M+H] +

[0312] Example 8: Synthesis of Compound 48

[0313] Synthesis of compound 44:

[0314] Compound 28 (15.0 g, 0.04 mol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (150 mL) was added and stirred to dissolve. BSA (24.4 g, 0.12 mol, 3.0 eq.) and G(iBu) (17.70 g, 0.08 mol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (8.9 g, 0.04 mol, 1.0 eq.) was slowly added dropwise to the reaction. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight at this temperature. TLC and LCMS analysis confirmed the complete reaction of compound 28. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 44. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 44 (10.62 g, 21.26 mmol, 48.0% yield). ESI-MS: m / z 500.2 [M+H] +

[0315] Synthesis of compound 45:

[0316] Compound 44 (10.62 g, 21.26 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Extra-dry DCM (100 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (31.89 mL, 31.92 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 44 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 45. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 45 (2.03 g, 4.96 mmol, 23.3% yield). ESI-MS: m / z 410.2[M+H] +

[0317] Synthesis of compound 46:

[0318] Compound 45 (2.0 g, 4.89 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (25 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (3.32 g, 9.78 mmol, 2.0 eq.), AgNO3 (0.83 g, 4.89 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (5.93 g, 48.9 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 45. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product compound 46. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 46 (2.96 g, 4.16 mmol, 85.1% yield). ESI-MS: m / z 712.4 [M+H] +

[0319] Synthesis of compound 47:

[0320] Compound 46 (2.96 g, 4.16 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (30 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (2.31 mL, 12.48 mmol, 3.0 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 46 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 47. The crude product was purified by column chromatography to yield compound 47 (2.54 g, 3.79 mmol, 91.1% yield). ESI-MS: m / z 670.4 [M+H] +

[0321] Synthesis of compound 48:

[0322] Dried compound 47 (2.54 g, 3.79 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred until completely dissolved. DIPEA (0.98 g, 7.58 mmol, 2.0 eq.) and DMAP (93 mg, 0.758 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.35 g, 5.69 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 47 was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 48. The crude product was purified by column chromatography to give compound 48 (2.57 g, 2.96 mmol, 77.8% yield). 1 H NMR (400MHz, DMSO-d6) δ12.708 (s, 1H), 11.59 (s, 1H), 8.21 (d, J=29.6Hz, 1H), 7.55-7.18 ( m, 9H), 6.88-6.79 (m, 4H), 5.72 (dd, J=38.6, 4.8Hz, 1H), 5.25-5.12 (m, 1H), 4.50 (t, J=5.5H z, 1H), 3.80-3.68 (m, 8H), 3.62-3.35 (m, 3H), 3.30 (d, J=24.0, 3H), 3.25-3.20 (m, 1H), 3.1 2(d, J=8.4Hz, 1H), 2.78-2.65(m, 1H), 2.57(m, 1H), 2.44-2.35(m, 1H), 1.11-0.87(m, 18H), 31 P NMR (162MHz, DMSO-d6) δ151.65, 148.95, ESI-MS: m / z 870.5[M+H] +

[0323] Example 9: Synthesis of Compound 55

[0324] Synthesis of compound 49:

[0325] Compound 4 (36.0 g, 128.42 mmol, 1.0 eq.) was dissolved in DMF (400 mL) and stirred thoroughly. The reaction mixture was cooled to 0°C and stirred for 30 minutes. NaH (7.7 g, 192.63 mmol, 1.5 eq.) was added and stirred for 30 minutes. Tetradecane bromide (42.7 g, 154.10 mmol, 1.2 eq.) was slowly added dropwise to the reaction system. After addition, the reaction mixture was allowed to cool to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 4. The reaction mixture was slowly poured into ice water to quench the mixture. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 100 / 9) to afford compound 49 (55.8 g, 117.10 mmol, 91% yield). ESI-MS: m / z 477.5[M+H] +

[0326] Synthesis of compound 50:

[0327] Compound 49 (55.8 g, 117.10 mmol, 1.0 eq.) was added to a 1000 mL round-bottom three-necked flask. AcOH (500 mL) and ACO (60.0 g, 0.585 mol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction mixture was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (10 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0 and 5°C. After addition, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 49 was completely reacted. The reaction system was cooled to approximately -5°C and neutralized with aqueous ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 50. The crude product was purified by column chromatography (PE / EA=10 / 2) to give compound 50 (31.0 g, 59.58 mmol, 50.8% yield). ESI-MS: m / z 521.3 [M+H] +

[0328] Synthesis of compound 51:

[0329] Compound 50 (13.0 g, 0.025 mol, 1.00 eq.) and uracil (5.6 g, 0.05 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (130 mL) was added and stirred to dissolve. BSA (15.3 g, 0.075 mol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (5.56 g, 0.025 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 50. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 51. The crude product was purified by column chromatography (PE / EA = 5 / 2) to obtain compound 51 (11.0 g, 19.21 mmol, 76.9% yield). ESI-MS: m / z 573.5 [M+H] +

[0330] Synthesis of compound 52:

[0331] Compound 51 (11.0 g, 19.21 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Ultra-dry DCM (120 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 77 mL, 76.84 mmol, 4.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 51 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 52. The crude product was purified by column chromatography (PE / EA = 2 / 3) to obtain compound 52 (7.7 g, 15.96 mmol, 83.0% yield). ESI-MS: m / z 483.3 [M+H] +

[0332] Synthesis of compound 53:

[0333] Compound 52 (7.7 g, 15.96 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (80 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (27.04 g, 79.8 mmol, 5.0 eq.), AgNO3 (2.72 g, 15.96 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (19.4 g, 159.6 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 52. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product, compound 53. The crude product was purified by column chromatography (PE / EA=100 / 35) to give compound 53 (11.37 g, 14.48 mmol, 90.7% yield). ESI-MS: m / z 785.4 [M+H] +

[0334] Synthesis of compound 54:

[0335] Compound 53 (11.37 g, 14.48 mmol, 1.0 eq.) was added to a 250 ml round-bottom flask. Aqueous ammonia (50 mL) was then added to the reaction at room temperature. The reaction was stirred at room temperature for 3 hours until the reaction of compound 53 was complete. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 54. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 54 (10.0 g, 13.46 mmol, 93.0% yield). ESI-MS: m / z 743.4 [M+H] +

[0336] Synthesis of compound 55:

[0337] Dried compound 54 (5.0 g, 6.73 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (50 mL) was added and stirred until completely dissolved. DIPEA (1.74 g, 13.46 mmol, 2.0 eq.) and DMAP (164.4 mg, 1.346 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (2.40 g, 10.1 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 54 was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 55. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 55 (4.54 g, 4.82 mmol, 71.6% yield). 1 H NMR (400MHz, DMSO-d6) δ11.35 (d, J=14.5Hz, 1H), 7.93 (d, J=8.1Hz, 1H), 7.41 (dd, J=16.0, 7. 5Hz, 2H), 7.34-7.20 (m, 7H), 6.86 (t, J=8.5Hz, 4H), 5.73-5.49 (m, 2H), 4.41-4.30 (m, 1H), 4.1 0-3.90(m, 1H), 3.83-3.33(m, 15H), 2.71(t, J=6.0Hz, 1H), 2.60-2.39(m, 1H), 1.60-1.52(m, 2H), 1.42-1.21 (m, 22H), 1.10 (t, J=6.5Hz, 6H), 1.02 (d, J=6.7Hz, 6H), 0.84 (t, J=6.5Hz, 3H), 31 P NMR (162MHz, DMSO-d6) δ151.28, 148.64, ESI-MS: m / z 943.5[M+H] +

[0338] Example 10: Synthesis of Compound 60

[0339] Synthesis of compound 56:

[0340] Compound 54 (6.0 g, 8.08 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DMF (60 mL) was added and stirred to dissolve. Imidazole (1.65 g, 24.24 mmol, 3.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (1.83 g, 12.12 mmol, 1.5 eq.) was then slowly added portionwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS analysis indicated that compound 54 had reacted completely, and the reaction was quenched by adding water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 1) to afford compound 56 (5.6 g, 6.54 mmol, 81.0% yield). ESI-MS: m / z 857.5[M+H] +

[0341] Synthesis of compound 57:

[0342] Compound 56 (5.6 g, 6.54 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry ACN (60 mL) was added and stirred until completely dissolved. Triethylamine (1.32 g, 13.08 mmol, 2.0 eq.) and DMAP (1.60 g, 13.08 mmol, 2.0 eq.) were added to the reaction system and stirred uniformly. The reaction mixture was cooled to 0-5°C, and TPSCl (3.96 g, 13.08 mmol, 2.0 eq.) was slowly added in portions. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 56. Aqueous ammonia solution (40 mL) was added to the reaction at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude product 57 (9.0 g, calculated based on 100% yield). ESI-MS: m / z 856.5 [M+H] +

[0343] Synthesis of compound 58:

[0344] Crude compound 57 (9.0 g, 1.0 eq.) was added to a 100 mL round-bottom flask, and pyridine (50 mL) was added and stirred until the crude compound 57 was completely dissolved. The reaction system was cooled to 0°C, and BzCl (1.38 g, 9.81 mmol, 1.5 eq.) was slowly added dropwise to the reaction system and stirred at 0°C for 1 hour until the reaction of compound 57 was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 58 (5.5 g, 5.73 mmol, 87.6% total yield over two steps). ESI-MS: m / z 960.6 [M+H] +

[0345] Synthesis of compound 59:

[0346] Compound 58 (5.5 g, 5.73 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and THF (60 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (6.0 mL) was neutralized with triethylamine (20 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS analysis showed that compound 58 had reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 59 (4.6 g, 5.44 mmol, 94.9% yield). ESI-MS: m / z 846.5 [M+H] +

[0347] Synthesis of compound 60:

[0348] Dried compound 59 (1.0 g, 1.18 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (15 mL) was added with stirring to dissolve. DIPEA (305.1 mg, 2.36 mmol, 2.0 eq.) and DMAP (28.8 mg, 0.236 mmol, 0.2 eq.) were added to the reaction mixture, and the reaction was purged with nitrogen. The reaction mixture was cooled to approximately 0°C, and CEP-Cl (418.9 mg, 1.77 mmol, 1.5 eq.) was slowly added dropwise. The reaction mixture was allowed to react at this temperature under nitrogen for 1 hour until compound 59 was fully reacted. After completion of the reaction, saturated aqueous sodium bicarbonate solution was added to the reaction mixture to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 60 (1.1 g, 1.05 mmol, 89.0% yield). 1 H NMR (400MHz, DMSO-d6) δ11.25 (d, J=24.4Hz, 1H), 8.46 (dd, J=30.5, 7.3Hz, 1H), 8.01 (d, J= 7.7Hz, 2H), 7.66-7.10(m, 14H), 6.89-6.79(m, 4H), 5.75-5.66(m, 1H), 4.27-3.87(m, 3H), 3.81-3.67(m, 8H), 3.66-3.35(m, 7H), 2.77-2.70(m, 1H), 2.57-2.32(m, 1H), 1.62-1.49(m , 2H), 1.36-1.19 (m, 22H), 1.13-1.02 (m, 9H), 0.96 (d, J=6.7Hz, 3H), 0.83 (t, J=6.8Hz, 3H), 31 P NMR (162MHz, DMSO-d6) δ149.86, 149.33, ESI-MS: m / z 1046.6[M+H] +

[0349] Example 11: Synthesis of Compound 65

[0350] Synthesis of compound 61:

[0351] Compound 50 (8.0 g, 15.37 mmol, 1.00 eq.) was added to a 250 mL three-necked round-bottom flask. Ultra-dry ACN (80 mL) was added and stirred to dissolve. BSA (9.38 g, 46.11 mmol, 3.0 eq.) and A(Bz) (7.35 g, 30.74 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (3.4 g, 15.37 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 50. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 61. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 61 (7.8 g, 11.15 mmol, 72.6% yield). ESI-MS: m / z 700.4 [M+H] +

[0352] Synthesis of compound 62:

[0353] Compound 61 (7.8 g, 11.15 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry DCM (80 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L boron trichloride solution in dichloromethane (40 mL, 39.03 mmol, 3.5 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 61 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 62. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 62 (5.5 g, 9.03 mmol, 80.9% yield). ESI-MS: m / z 610.4 [M+H] +

[0354] Synthesis of compound 63:

[0355] Compound 62 (5.5 g, 9.03 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (60 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (7.65 g, 22.58 mmol, 2.5 eq.), AgNO3 (1.53 g, 9.03 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (10.94 g, 90.3 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 62. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product, compound 63. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 63 (6.6 g, 7.24 mmol, 80.2% yield). ESI-MS: m / z 912.5 [M+H] +

[0356] Synthesis of compound 64:

[0357] Compound 63 (6.6 g, 7.24 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. THF (70 mL) was added and the reaction was stirred until completely dissolved. A 5.4 mol / L NaOMe solution (2.01 mL, 10.86 mmol, 1.5 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 63 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 64. Column chromatography of the crude product afforded compound 64 (5.8 g, 6.67 mmol, 92.1% yield). ESI-MS: m / z 870.5 [M+H] +

[0358] Synthesis of compound 65:

[0359] Dried compound 64 (1.0 g, 1.15 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (10 mL) was added and stirred until completely dissolved. DIPEA (297.3 g, 2.3 mmol, 2.0 eq.) and DMAP (28.1 mg, 0.23 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (408.3 mg, 1.73 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 64 was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 65. The crude product was purified by column chromatography to give compound 65 (1.0 g, 0.93 mmol, 80.9% yield). 1 H NMR (400MHz, DMSO-d6) δ11.20 (d, J=4.0Hz, 1H), 8.70 (d, J=5.9Hz, 1H), 8.61 (d, J=29.9Hz, 1H), 8. 05 (d, J=7.6Hz, 2H), 7.64 (t, J=7.3Hz, 1H), 7.54 (t, J=7.6Hz, 2H), 7.37 (t, J=8.6Hz, 2H), 7.26-7.1 8(m, 7H), 6.84-6.72(m, 4H), 6.05(dd, J=36.3, 3.4Hz, 1H), 4.79-4.32(m, 2H), 3.80-3.35(m, 15H) , 2.69-2.39(m, 2H), 1.62-1.47(m, 2H), 1.40-1.20(m, 22H), 1.11-0.97(m, 9H), 0.92-0.79(m, 6H), 31 P NMR (162MHz, DMSO-d6) δ151.29, 149.24, ESI-MS: m / z 1070.6[M+H] +

[0360] Example 12: Synthesis of Compound 70

[0361] Synthesis of compound 66:

[0362] Compound 50 (10.0 g, 19.22 mmol, 1.00 eq.) was added to a 250 mL three-necked round-bottom flask. Ultra-dry ACN (80 mL) was added and stirred to dissolve. BSA (11.73 g, 57.66 mmol, 3.0 eq.) and G(iBu) (8.5 g, 38.44 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 50 minutes. TMSOTf (4.27 g, 19.22 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 50. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 66. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 66 (7.8 g, 11.45 mmol, 59.6% yield). ESI-MS: m / z 682.4 [M+H] +

[0363] Synthesis of compound 67:

[0364] Compound 66 (7.8 g, 11.45 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. DCM (80 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (40.1 mL, 40.1 mmol, 3.5 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 66 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 67. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 67 (3.8 g, 6.43 mmol, 56.2% yield). ESI-MS: m / z 592.4 [M+H] +

[0365] Synthesis of compound 68:

[0366] Compound 67 (3.8 g, 6.43 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (5.45 g, 16.1 mmol, 2.5 eq.), AgNO3 (1.1 g, 6.43 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (7.8 g, 64.3 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 67. The reaction was returned to room temperature and methanol was added to the reaction to quench the reaction. Ethyl acetate was added to the reaction and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product compound 68. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 68 (1.5 g, 1.68 mmol, 26.1% yield). ESI-MS: m / z 894.5 [M+H] +

[0367] Synthesis of compound 69:

[0368] Compound 68 (1.5 g, 1.68 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (30 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (0.46 mL, 2.52 mmol, 1.5 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 68 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain the crude product. The crude product was purified by column chromatography to obtain compound 69 (0.7 g, 0.82 mmol, 48.8% yield). ESI-MS: m / z 852.5 [M+H] +

[0369] Synthesis of compound 70:

[0370] Dried compound 69 (0.7 g, 0.82 mmol, 1.0 eq.) was added to a 25 mL round-bottom flask, and ultra-dry DCM (10 mL) was added and stirred until completely dissolved. DIPEA (212.0 g, 1.64 mmol, 2.0 eq.) and DMAP (20.04 mg, 0.164 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (291.1 mg, 1.23 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 69 was completely reacted. The reaction mixture was quenched with a saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 70. The crude product was purified by column chromatography to give compound 70 (0.56 g, 0.53 mmol, 64.6% yield). 1 H NMR (400MHz, DMSO-d6) δ12.09 (s, 1H), 11.53 (s, 1H), 8.19 (d, J=29.6Hz, 1H), 7.53-7.12 (m, 9H), 6.9 2-6.82 (m, 4H), 5.73 (dd, J=38.6, 4.8Hz, 1H), 5.22-5.05 (m, 1H), 4.54 (t, J=5.5Hz, 1H), 3.80-3.68 (m , 8H), 3.62-3.35 (m, 3H), 3.31-3.25 (m, 2H), 3.12 (d, J=8.4Hz, 1H), 2.78-2.68 (m, 1H), 2.57 (m, 2H), 2 .47-2.38(m, 1H), 1.50(d, J=6.6Hz, 2H), 1.37-1.19(m, 22H), 1.15-0.99(m, 13H), 0.89-0.75(m, 8H), 31 P NMR (162MHz, DMSO-d6) δ151.78, 148.87, ESI-MS: m / z 1052.6[M+H] +

[0371] Example 13: Synthesis of Compound 77

[0372] Synthesis of compound 71:

[0373] Compound 4 (35 g, 124.85 mmol, 1.0 eq.) was dissolved in DMF (350 mL) and stirred. The reaction mixture was cooled to 0°C and stirred for 30 minutes. NaH (7.5 g, 187.28 mmol, 1.5 eq.) was then added dropwise. Hexadecane bromide (45.75 g, 149.82 mmol, 1.2 eq.) was slowly added to the reaction system. After addition, the reaction mixture was allowed to cool to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 4. The reaction mixture was slowly poured into ice water to quench the mixture. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 100 / 9) to afford compound 71 (57.5 g, 114.0 mmol, 91.3% yield). ESI-MS: m / z 505.4 [M+H] +

[0374] Synthesis of compound 72:

[0375] Compound 71 (57.5 g, 114.0 mmol, 1.0 eq.) was added to a 1000 mL round-bottom three-necked flask, and AcOH (535 mL) and Ac2O (58.19 g, 569.98 mmol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction mixture was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (5.35 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0-5°C. After the addition, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 71 was completely reacted. The reaction system was cooled to approximately -5°C and neutralized with aqueous ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 72. The crude product was purified by column chromatography (PE / EA=10 / 2) to give compound 72 (47.5 g, 86.6 mmol, 76% yield). ESI-MS: m / z 549.4 [M+H] +

[0376] Synthesis of compound 73:

[0377] Compound 72 (20.3 g, 37.02 mmol, 1.00 eq.) and uracil (8.3 g, 74.04 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (200 mL) was added and stirred to dissolve. BSA (22.59 g, 111.06 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (8.23 g, 37.02 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 72. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 73. The crude product was purified by column chromatography (PE / EA = 5 / 2) to obtain compound 73 (20.0 g, 33.3 mmol, 90% yield). ESI-MS: m / z 601.4 [M+H] +

[0378] Synthesis of compound 74:

[0379] Compound 73 (10.0 g, 16.66 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Ultra-dry DCM (100 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 66.7 mL, 66.64 mmol, 4.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 73 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 74. The crude product was purified by column chromatography (PE / EA = 2 / 3) to obtain compound 74 (5.66 g, 11.09 mmol, 66.6% yield). ESI-MS: m / z 511.3 [M+H] +

[0380] Synthesis of compound 75:

[0381] Compound 74 (5.6 g, 11.09 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (70 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (7.52 g, 22.18 mmol, 2.0 eq.), AgNO3 (1.87 g, 11.09 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (13.4 g, 110.9 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 74. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product compound 75. The crude product was purified by column chromatography (PE / EA=100 / 35) to give compound 75 (8.9 g, 10.95 mmol, 98.8% yield). ESI-MS: m / z 813.5 [M+H] +

[0382] Synthesis of compound 76:

[0383] Compound 75 (8.9 g, 10.95 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Aqueous ammonia solution (70 mL) was then added to the reaction at room temperature. The reaction was stirred at room temperature for 3 hours until the reaction of compound 75 was complete. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 76. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 76 (7.3 g, 9.47 mmol, 86.5% yield). ESI-MS: m / z 771.5 [M+H] +

[0384] Synthesis of compound 77:

[0385] Dried compound 76 (3.3 g, 4.28 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (35 mL) was added and stirred until completely dissolved. DIPEA (1.11 g, 8.56 mmol, 2.0 eq.) and DMAP (105 mg, 0.856 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.52 g, 6.42 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 76 was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 77. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 77 (3.9 g, 4.02 mmol, 93.9% yield). 1 H NMR (400MHz, DMSO-d6) δ11.28 (d, J=14.5Hz, 1H), 7.90 (d, J=8.1Hz, 1H), 7.35 (dd, J=16.0, 7.5Hz, 2H), 7.30-7.16(m, 7H), 6.88(t, J=8.5Hz, 4H), 5.77-5.55(m, 2H), 4.42-4.32(m, 1H), 4.11-3.95( m, 1H), 3.84-3.68 (m, 8H), 3.62-3.35 (m, 7H), 2.68 (t, J=6.0Hz, 1H), 2.55-2.38 (m, 1H), 1.65-1.5 5 (m, 2H), 1.43-1.23 (m, 26H), 1.09 (t, J = 6.5Hz, 6H), 1.01 (d, J = 6.7Hz, 6H), 0.83 (t, J = 6.5Hz, 3H), 31 P NMR (162MHz, DMSO-d6) δ150.13, 148.46, ESI-MS: m / z 971.6[M+H] +

[0386] Example 14: Synthesis of Compound 82

[0387] Synthesis of compound 78:

[0388] Compound 76 (4.0 g, 5.19 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DMF (40 mL) was added and stirred to dissolve. Imidazole (1.06 g, 15.57 mmol, 3.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (1.17 g, 7.79 mmol, 1.5 eq.) was then slowly added portionwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS analysis indicated that compound 76 had reacted completely, and water was added to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 1) to afford compound 78 (2.9 g, 3.28 mmol, 63% yield). ESI-MS: m / z 885.5[M+H] +

[0389] Synthesis of compound 79:

[0390] Compound 78 (2.9 g, 3.28 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry ACN (30 mL) was added and stirred until completely dissolved. TEA (663 mg, 6.56 mmol, 2.0 eq.) and DMAP (801 mg, 6.56 mmol, 2.0 eq.) were added to the reaction system and stirred uniformly. The reaction mixture was cooled to 0-5°C, and TPSCl (1.98 g, 6.56 mmol, 2.0 eq.) was slowly added in portions. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 78. Aqueous ammonia (20 mL) was added to the reaction at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude product 79 (4.3 g, calculated based on 100% yield). ESI-MS: m / z 884.6 [M+H] +

[0391] Synthesis of compound 80:

[0392] Crude compound 79 (4.3 g, 1.0 eq.) was added to a 100 mL round-bottom flask, and pyridine (50 mL) was added and stirred until the crude compound 79 was completely dissolved. The reaction system was cooled to 0°C, and BzCl (922 mg, 6.56 mmol, 2.0 eq.) was slowly added dropwise to the reaction system and stirred at 0°C for 1 hour until the reaction of compound 79 was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 80 (2.0 g, 2.03 mmol, 61.8% total yield over two steps). ESI-MS: m / z 988.6 [M+H] +

[0393] Synthesis of compound 81:

[0394] Compound 80 (2.0 g, 2.03 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (20 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized with triethylamine (17 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS analysis showed that compound 80 had reacted completely. Water was added to the reaction, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 81 (1.37 g, 1.57 mmol, 77.3% yield). ESI-MS: m / z 874.5 [M+H] +

[0395] Synthesis of compound 82:

[0396] Dried compound 81 (1.37 g, 1.57 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (15 mL) was added and stirred to dissolve. DIPEA (405 mg, 3.14 mmol, 2.0 eq.) and DMAP (38.4 mg, 0.314 mmol, 0.2 eq.) were added to the reaction, and the reaction was purged with nitrogen. The reaction temperature was cooled to approximately 0°C, and CEP-Cl (557.4 mg, 2.35 mmol, 1.5 eq.) was slowly added dropwise. The reaction was allowed to proceed at this temperature under nitrogen for 1 hour until compound 81 was fully reacted. After completion of the reaction, saturated aqueous sodium bicarbonate solution was added to the reaction mixture to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 82 (1.0 g, 0.93 mmol, 59.2% yield). 1 H NMR (400MHz, DMSO-d6) δ11.24 (d, J=24.2Hz, 1H), 8.55-8.39 (m, 1H), 8.01 (d, J=7.7H z, 2H), 7.66-7.10 (m, 14H), 6.92-6.73 (m, 4H), 5.70 (d, J=26.4Hz, 1H), 4.25-3.88 (m , 3H), 3.81-3.35(m, 15H), 2.73(dd, J=10.4, 5.6Hz, 1H), 2.61-2.31(m, 1H), 1.55(dt , J=14.1, 7.1Hz, 2H), 1.37-1.18 (m, 26H), 1.13-0.94 (m, 12H), 0.84 (t, J=6.5Hz, 3H), 31 P NMR (162MHz, DMSO-d6) δ149.86, 149.32, ESI-MS: m / z 1074.6[M+H] +

[0397] Example 15: Synthesis of Compound 87

[0398] Synthesis of compound 83:

[0399] Compound 72 (17 g, 31.0 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (200 mL) was added and stirred to dissolve. BSA (18.91 g, 93 mmol, 3.0 eq.) and A(Bz) (14.83 g, 62.0 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (6.89 g, 31.0 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 72. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 83. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 83 (18.0 g, 24.74 mmol, 79.8% yield). ESI-MS: m / z 728.4 [M+H] +

[0400] Synthesis of compound 84:

[0401] Compound 83 (18.0 g, 24.74 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Extra-dry DCM (300 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L boron trichloride solution in dichloromethane (74.2 mL, 74.22 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 83 was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 84. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 84 (10.0 g, 15.68 mmol, 63.4% yield). ESI-MS: m / z 638.4[M+H] +

[0402] Synthesis of compound 85:

[0403] Compound 84 (10.0 g, 15.68 mmol, 1.0 eq.) was added to a 500 mL round-bottom flask, and ultra-dry DCE (150 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (10.63 g, 31.36 mmol, 2.0 eq.), AgNO3 (2.66 g, 15.68 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (19 g, 156.8 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 84. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product, compound 85. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 85 (13.0 g, 13.84 mmol, 88.3% yield). ESI-MS: m / z 940.5 [M+H] +

[0404] Synthesis of compound 86:

[0405] Compound 85 (13.0 g, 13.84 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. THF (130 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (3.84 mL, 20.76 mmol, 1.5 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 85 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain crude product 86. The crude product was purified by column chromatography to yield compound 86 (10.5 g, 11.7 mmol, 84.5% yield). ESI-MS: m / z 898.5 [M+H] +

[0406] Synthesis of compound 87:

[0407] Dried compound 86 (1.0 g, 1.11 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (10 mL) was added and stirred until completely dissolved. DIPEA (286.9 mg, 2.22 mmol, 2.0 eq.) and DMAP (26.9 mg, 0.22 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (395.3 g, 1.67 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 86 was completely reacted. The reaction mixture was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 87. The crude product was purified by column chromatography to give compound 87 (1.0 g, 0.91 mmol, 82.1% yield). 1 H NMR (400MHz, DMSO-d6) δ11.24-11.16 (m, 1H), 8.70 (d, J=6.5Hz, 1H), 8.61 (d, J=30.2Hz, 1H), 8.05 (d, J=7.6 Hz, 2H), 7.64 (t, J=7.3Hz, 1H), 7.54 (t, J=7.5Hz, 2H), 7.41-7.34 (m, 2H), 7.27-7.17 (m, 7H), 6.84-6.73 (m, 4 H), 6.05 (dd, J=35.6, 3.3Hz, 1H), 4.78-4.30 (m, 2H), 3.81-3.76 (m, 1H), 3.74-3.37 (m, 14H), 2.66 (t, J=6.1H z, 1H), 2.62-2.39 (m, 1H), 1.63-1.48 (m, 2H), 1.36-1.18 (m, 26H), 1.13-0.96 (m, 9H), 0.92-0.79 (m, 6H), 31P NMR (162MHz, DMSO-d6) δ151.26, 149.26, ESI-MS: m / z 1098.6[M+H] +

[0408] Example 16: Synthesis of Compound 92

[0409] Synthesis of compound 88:

[0410] Compound 72 (12.0 g, 21.88 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (130 mL) was added and stirred to dissolve. BSA (13.35 g, 65.65 mmol, 3.0 eq.) and G(iBu) (9.67 g, 43.76 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 50 minutes. TMSOTf (4.86 g, 21.88 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 72. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 88. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 88 (11.2 g, 15.79 mmol, 72.2% yield). ESI-MS: m / z 710.4 [M+H] +

[0411] Synthesis of compound 89:

[0412] Compound 88 (11.2 g, 15.79 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry DCM (100 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (47.4 mL, 47.37 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 88 was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 89. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 89 (6.3 g, 10.17 mmol, 64.4% yield). ESI-MS: m / z 620.5[M+H] +

[0413] Synthesis of compound 90:

[0414] Compound 89 (6.3 g, 10.17 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (70 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (6.89 g, 20.34 mmol, 2.0 eq.), AgNO3 (1.73 g, 10.17 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (12.3 g, 101.7 mmol, 10.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 89. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product, compound 90. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 90 (7.8 g, 8.46 mmol, 83.2% yield). ESI-MS: m / z 922.5 [M+H] +

[0415] Synthesis of compound 91:

[0416] Compound 90 (7.8 g, 8.46 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. THF (80 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (2.4 mL, 12.69 mmol, 1.5 eq.) was added to the reaction and stirred at room temperature for 3 hours until compound 90 was completely reacted. After completion of the reaction, the reaction system was concentrated under reduced pressure at 40°C to obtain the crude product. The crude product was purified by column chromatography to obtain compound 91 (6.43 g, 7.31 mmol, 86.4% yield). ESI-MS: m / z 880.5 [M+H] +

[0417] Synthesis of compound 92:

[0418] Dried compound 91 (3.0 g, 3.41 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred until completely dissolved. DIPEA (881 mg, 6.82 mmol, 2.0 eq.) and DMAP (83.3 mg, 0.682 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.21 g, 5.12 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 91 was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield crude product 92. The crude product was purified by column chromatography to give compound 92 (3.1 g, 2.87 mmol, 84.2% yield). 1 H NMR (400MHz, DMSO-d6) δ12.05 (s, 1H), 11.45 (s, 1H), 8.23 ​​(d, J=29.6Hz, 1H), 7.58-7.15 (m, 9H), 6.95 -6.89 (m, 4H), 5.66 (dd, J=38.6, 4.8Hz, 1H), 5.25-5.09 (m, 1H), 4.55 (t, J=5.5Hz, 1H), 3.81-3.65 (m, 8 H), 3.63-3.38 (m, 3H), 3.31-3.21 (m, 2H), 3.10 (d, J=8.4Hz, 1H), 2.74-2.65 (m, 1H), 2.58 (m, 2H), 2.4 8-2.31 (m, 1H), 1.51 (d, J=6.6Hz, 2H), 1.38-1.18 (m, 26H), 1.13-0.98 (m, 13H), 0.88-0.73 (m, 8H), 31P NMR (162MHz, DMSO-d6) δ151.58, 148.93, ESI-MS: m / z 1080.6[M+H] +

[0419] The following Example 17 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 3-a of the present invention, wherein the nucleoside compound represented by Formula 3-a is a 2'-phosphoramidite group, 3'-CH2ODMTr, 4'-modified TNA structure compound.

[0420] Example 17: Synthesis of Compound 104

[0421] The preparation route of compound 104A is as follows:

[0422] Nucleoside compound 104 is a 2′-phosphoramidite group, 3′-ODMTr, Nu=ABz, R=C 16 H 33 TNA structure compounds.

[0423] In the above preparation route, when compound 98 is used to prepare compound 99, R-Br is selected as C 16 H 33 -Br, when compound 100 was used to prepare compound 101, Nu was selected as adenine A, and the synthesis method of compound 87 in Example 15 was referred to. The structure and NMR data of the obtained nucleoside compound 104 are shown below:

[0424] 1 H NMR (400MHz, DMSO-d6) δ11.24-11.16 (m, 1H), 8.70 (d, J = 6.5Hz, 1H), 8.61 (d, J = 30.2Hz, 1H), 8.05 (d, J = 7.6Hz, 2 H), 7.64 (t, J=7.3Hz, 1H), 7.54 (t, J=7.5Hz, 2H), 7.41-7.34 (m, 2H), 7.27-7.17 (m, 7H), 6.84-6.73 (m, 4H), 6.05 (dd, J=35.6, 3.3Hz, 1H), 4.78-4.30(m, 2H), 4.13-3.88(m, 2H), 3.81-3.76(m, 1H), 3.74-3.37(m, 14H), 2.66(t, J=6.1Hz, 1H), 2.62-2.39(m, 1H), 1.63-1.48(m, 2H), 1.36-1.18(m, 26H), 1.13-0.96(m, 9H), 0.92-0.79(m, 6H), 31 P NMR (162MHz, DMSO-d6) δ150.26, 148.26, ESI-MS: m / z 1112.6[M+H] +

[0425] The following Example 18 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 4-a of the present invention, wherein the nucleoside compound represented by Formula 4-a is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0426] Example 18: Synthesis of Compound 116

[0427] The preparation route of compound 116A is as follows:

[0428] Nucleoside compound 116 is a 2′-phosphoramidite group, 3′-ODMTr, Nu=ABz, R=C 16 H 33 TNA structure compounds.

[0429] In the above preparation route, when compound 109 is used to prepare compound 110, R-Br is selected as C 16 H 33 -Br, when compound 111 was used to prepare compound 112, Nu was selected as adenine A, and the synthesis method of compound 87 in Example 15 was referred to. The structure and NMR data of the obtained nucleoside compound 116 are shown below:

[0430] 1 H NMR (400MHz, DMSO-d6) δ11.22 (d, J=4.0Hz, 1H), 8.72 (d, J=5.9Hz, 1H), 8.61 (d, J=29.9Hz, 1H), 8.0 3(d, J=7.6Hz, 2H), 7.64 (t, J=7.3Hz, 1H), 7.57 (t, J=7.6Hz, 2H), 7.35 (t, J=8.6Hz, 2H), 7.21-7.12 (m, 7H), 6.82-6.72 (m, 4H), 6.01 (dd, J=36.3, 3.4Hz, 1H), 4.79-4.32 (m, 2H), 3.83-3.37 (m, 17H), 2 .68-2.37(m, 2H), 1.63-1.47(m, 2H), 1.41-1.21(m, 22H), 1.13-0.99(m, 9H), 0.92-0.77(m, 6H).31P NMR (162MHz, DMSO-d6) δ151.83, 149.06.ESI-MS: m / z 1028.5[M+H] +

[0431] The following Example 19 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 5-a of the present invention, wherein the nucleoside compound represented by Formula 5-a is a 2'-phosphoramidite group, 3'-CH2ODMTr, 4'-modified TNA structure compound.

[0432] Example 19:

[0433] The following Example 20 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 6-a of the present invention, wherein the nucleoside compound represented by Formula 6-a is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0434] Example 20: Synthesis of Compound 135

[0435] The preparation route of compound 135A is as follows:

[0436] Nucleoside compound 135 is a 2′-phosphoramidite group, 3′-ODMTr, Nu=ABz, R=C 14 H 29 TNA structure compounds.

[0437] When compound 128 was used to prepare compound 129 through the above preparation route of Example 20, Nu was selected as ABz, wherein A was adenine, and when compound 132 was used to prepare compound 133, R-COOH was selected as C 14 H 29 -COOH, and the structure and NMR data of the nucleoside compound 135 prepared by the synthesis of compound 87 in reference example 15 are shown below:

[0438] 1 H NMR (400MHz, DMSO-d6) δ11.21 (d, J=4.0Hz, 1H), 8.71 (d, J=5.9Hz, 1H), 8.62 (d, J=29.9Hz, 1H), 8 .05-7.99 (m, 3H), 7.64 (t, J=7.3Hz, 1H), 7.57 (t, J=7.6Hz, 2H), 7.35 (t, J=8.6Hz, 2H), 7.21-7.12 (m, 7H), 6.82-6.72 (m, 4H), 6.01 (dd, J=36.3, 3.4Hz, 1H), 4.79-4.32 (m, 2H), 3.83-3.37 (m, 15H), 2.68-2.37(m, 2H), 1.63-1.47(m, 2H), 1.41-1.21(m, 20H), 1.13-0.99(m, 9H), 0.92-0.77(m, 6H). 31 P NMR (162MHz, DMSO-d6) δ151.92, 148.91.ESI-MS: m / z 1055.6[M+H] +

[0439] Example 21: Synthesis of Compound 143

[0440] The preparation route of compound 143A is as follows:

[0441] The following Example 22 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 8-a of the present invention, wherein the nucleoside compound represented by Formula 8-a is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0442] Example 22: Synthesis of Compound 154

[0443] The preparation route of compound 154A is as follows:

[0444] Nucleoside compound 154 is a 2′-phosphoramidite group, 3′-ODMTr, Nu=ABz, R=C 16 H 33 TNA structure compounds.

[0445] When compound 151 was used to prepare compound 152 by the above preparation route of Example 22, R was selected as C 16 H 33 When compound 146 was used to prepare compound 147, Nu was selected as ABz, where A was adenine. The structure and NMR data of the obtained nucleoside compound 154 are shown below:

[0446] 1 H NMR (400MHz, DMSO-d6) δ11.21 (d, J=4.0Hz, 1H), 8.71 (d, J=5.9Hz, 1H), 8.60 (d, J=29.9Hz, 1H), 8 .04 (d, J=7.6Hz, 2H), 7.65 (t, J=7.3Hz, 1H), 7.61-7.51 (m, 3H), 7.35 (t, J=8.6Hz, 2H), 7.21-7.12 (m, 7H), 6.83-6.71 (m, 4H), 6.02 (dd, J=36.3, 3.4Hz, 1H), 4.78-4.31 (m, 4H), 3.81-3.36 (m, 17H), 2.67-2.38(m, 2H), 1.62-1.48(m, 2H), 1.42-1.21(m, 22H), 1.12-0.99(m, 9H), 0.93-0.78(m, 6H). 31 P NMR (162MHz, DMSO-d6) δ151.63, 149.23.ESI-MS: m / z 1123.6[M+H] +

[0447] The following Example 23 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 9-a of the present invention, wherein the nucleoside compound represented by Formula 9-a is a 2'-phosphoramidite group, 3'-CH2ODMTr, and 4'-modified TNA structure compound.

[0448] Example 23:

[0449] The following Example 24 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 10-a of the present invention, wherein the nucleoside compound represented by Formula 10-a is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0450] Example 24:

[0451] The preparation route of compound 175A is as follows:

[0452] Nucleoside compound 175 is a 2′-phosphoramidite group, 3′-ODMTr, Nu=ABz, R=C 16 H 33 TNA structure compounds.

[0453] When compound 169 was prepared from compound 168 by the above preparation route of Example 24, R-Br was selected as C 16 H 33 -Br, when compound 170 is used to prepare compound 171, Nu is selected as ABz, wherein A is adenine, and the structure and NMR data of the nucleoside compound 175 prepared by the synthesis method of compound 87 in Example 15 are shown below:

[0454] 1 H NMR (400MHz, DMSO-d6) δ11.22 (d, J=17.5Hz, 1H), 8.47 (dd, J=30.3, 7.5Hz, 1H), 8.02 (d, J=7.6Hz, 2H ), 7.63 (t, J=7.3Hz, 1H), 7.56-7.43 (m, 2H), 7.38-7.12 (m, 9H), 6.88-6.81 (m, 4H), 5.72 (d, J=25.0H z, 1H), 4.24-3.91 (m, 3H), 3.77-3.39 (m, 17H), 2.75 (dd, J=10.7, 5.6Hz, 1H), 2.62-2.31 (m, 1H), 1.6 2-1.51 (m, 2H), 1.37-1.18 (m, 24H), 1.11-1.03 (m, 9H), 0.96 (d, J=6.7Hz, 3H), 0.82 (t, J=6.6Hz, 3H), 31 P NMR (162MHz, DMSO-d6) δ149.78, 149.21, ESI-MS: m / z 1058.6[M+H] +

[0455] The following Example 25 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 11-a of the present invention, wherein the nucleoside compound represented by Formula 11-a is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0456] Example 25:

[0457] The following Example 26 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 12-a of the present invention, wherein the nucleoside compound represented by Formula 12-a is a 1'-H, 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0458] Example 26:

[0459] The preparation route of compound 205A is as follows:

[0460] Nucleoside compound 205 is a 2′-phosphoramidite group, 3′-ODMTr, R=C 16 H 33 TNA structure compounds.

[0461] When compound 200 was prepared from compound 199 by the above preparation route of Example 26, R-Br was selected as C 16 H 33 -Br, the structure and NMR data of the prepared nucleoside compound 205 are shown below:

[0462] 1 H NMR (400MHz, DMSO-d6) δ7.35-7.21 (m, 9=H), 6.92-6.81 (m, 4H), 4.24-3.91 (m, 4H), 3.77-3.39 (m, 17H), 2.75 (dd, J=10.7, 5.6Hz, 1H), 2.62-2.31(m, 1H), 1.62-1.51(m, 2H), 1.37-1.18(m, 24H), 1.11-1.03(m, 9H), 0.96(d, J=6.7Hz, 3H), 0.82(t, J=6.6Hz, 3H), 31 P NMR (162MHz, DMSO-d6) δ150.13, 149.31, ESI-MS: m / z 805.5[M+H] +

[0463] The following Example 27 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 13-a of the present invention, wherein the nucleoside compound represented by Formula 13-a is a 2'-S-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0464] Example 27:

[0465] The following Example 28 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 14-a of the present invention, wherein the nucleoside compound represented by Formula 14-a is a 2'-phosphoramidite group, 3'-SDMTr, 4'-modified TNA structure compound.

[0466] Example 28:

[0467] The following Example 29 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 15-a of the present invention, wherein the nucleoside compound represented by Formula 15-a is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0468] Example 29:

[0469] The following Example 30 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 16-a of the present invention, wherein the nucleoside compound represented by Formula 16-a is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0470] Example 30:

[0471] The structures of some compounds prepared by the above preparation method are shown in Table A below (Formula 1-a nucleoside compound).

[0472] Here, Nu represents a base or is absent.

[0473] The following Examples 31-34 are preparation examples of the nucleoside compound represented by Formula 1-b of the present invention, wherein the nucleoside compound represented by Formula 1-b is a 2′-phosphoramidite group, 3′-ODMTr, and 4′-modified TNA structure compound.

[0474] Example 31: Synthesis of Compound 11'

[0475] Synthesis of compound 2':

[0476] Dried compound 1' (20.0 g, 133.22 mmol, 1.0 eq.) was dissolved in DMF (200 mL). Imidazole (22.67 g, 333.06 mmol, 2.5 eq.) was added to the reaction system, and the reaction mixture was cooled to 0°C and stirred for 30 minutes. Tert-butyldiphenylsilyl chloride (40.28 g, 146.54 mmol, 1.1 eq.) was then slowly added dropwise to the reaction system. After the addition, the ice bath was removed and the reaction temperature was raised to 60°C for overnight reaction. TLC analysis of the reaction indicated that compound 1' had reacted completely. Water was added to the reaction system, and the mixture was extracted twice with ethyl acetate. The organic phases were combined and washed with water and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 35.38 g of the crude intermediate. This crude product was dissolved in acetone (300 mL) and p-toluenesulfonic acid (1.15 g, 6.66 mmol, 0.05 eq.) was added, followed by 2,2-dimethoxypropane (69.37 g, 666.1 mmol, 5.0 eq.). After addition, the reaction was stirred at room temperature. TLC confirmed the complete reaction of the intermediate. Solid sodium bicarbonate was added to quench the p-toluenesulfonic acid, the solid was removed by filtration, and the mixture was concentrated to yield 38.24 g of the crude product. Column chromatography (PE / EA = 20 / 3) afforded compound 2' (19.27 g, 44.96 mmol, 33.75% yield). ESI-MS: m / z 451.2 [M+Na] +

[0477] Synthesis of compound 3':

[0478] Compound 2' (19.27 g, 44.96 mmol, 1.0 eq.) was dissolved in DMF (200 mL) and stirred until completely dissolved. The reaction system was cooled to approximately 0°C. 60% NaH (2.70 g, 67.44 mmol, 1.5 eq.) was slowly added portionwise to the reaction. After addition, the reaction system was stirred at 0°C for 30 minutes. BnBr (11.53 g, 67.44 mmol, 1.5 eq.) was slowly added dropwise to the reaction system, maintaining the reaction temperature at approximately 0°C. After addition, the reaction system was returned to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 2'. The reaction system was slowly poured into a saturated aqueous ammonium chloride solution at approximately 0°C. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield crude compound 3' (38.2 g), which was used directly in the next step. ESI-MS: m / z 519.3[M+H] +

[0479] Synthesis of compound 4':

[0480] Crude compound 3' (68.2 g) was dissolved in THF (200 mL) and stirred until completely dissolved. TBAF (1 M in THF, 50 mL, 50 mmol, 1.11 eq.) was added to the reaction mixture, and stirred at room temperature overnight until reaction of compound 3' was complete. The reaction solution was concentrated under reduced pressure to remove the solvent, and the resulting crude product was purified by column chromatography (PE / EA = 10 / 3) to afford compound 4' (10.77 g, 38.41 mmol, 85.43% yield over two steps). 1 H NMR (400MHz, DMSO-d6) δ7.42-7.26 (m, 5H), 5.85 (d, J=3.6Hz, 1H), 4.73 (t, J=5.2Hz, 1H), 4.69 (m, 2H), 4.5 2-4.41 (d, J=12Hz, 1H), 4.03 (m, 1H), 3.81 (d, J=3.2Hz, 1H), 3.70-3.51 (m, 2H), 1.32 (s, 3H), 1.25 (s, 3H).

[0481] Synthesis of compound 5':

[0482] Compound 4' (10.77 g, 38.41 mmol, 1.0 eq.) was dissolved in DMF (200 mL) and stirred. The reaction mixture was cooled to 0°C and stirred for 30 minutes. 60% NaH (1.84 g, 46.09 mmol, 1.2 eq.) was slowly added portionwise to the reaction mixture. Bromododecane (11.49 g, 46.09 mmol, 1.2 eq.) was then slowly added dropwise to the reaction system. After addition, the reaction mixture was allowed to cool to room temperature and allowed to react overnight. TLC confirmed the complete reaction of compound 4. The reaction mixture was slowly poured into ice water to quench the mixture. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 100 / 9) to afford compound 5' (15.74 g, 35.08 mmol, 91.33% yield). 1H NMR (400MHz, DMSO-d6) δ7.40-7.21(m, 5H), 5.78(d, J=3.8Hz, 1H), 4.74-4.61(m, 2H), 4.44 (d, J=12.0Hz, 1H), 4.22 (td, J=5.9, 3.2Hz, 1H), 3.89 (d, J=3.2Hz, 1H), 3.63 (dd, J=10.1, 5.4Hz, 1H), 3.52 (dd, J=10.1, 6.4Hz, 1H), 3.47-3.28 (m, 2H), 1 .48(m, 2H), 1.38(s, 3H), 1.21(d, J=11.6Hz, 21H), 0.88-0.80(m, 3H).ESI-MS: m / z 471.3[M+Na] +

[0483] Synthesis of compound 6':

[0484] Compound 5' (15.74 g, 35.08 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask. AcOH (180 mL) and Ac2O (17.91 g, 175.4 mmol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction mixture was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (2 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0-5°C. After the addition, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 5' was completely reacted. The reaction system was cooled to approximately -5°C and neutralized with aqueous ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=5 / 1) to give compound 6' (10.93 g, 22.19 mmol, 63.26% yield). 1 H NMR (400MHz, DMSO-d6) δ7.32-7.22 (m, 5H), 6.22 (d, J=8.4, 1H), 5.29-5.12 (m, 1H), 4.71-4.52 (m, 2H), 4.35-4.11 (m, 2H), 3.68-3 .41 (m, 2H), 3.41-3.34 (m, 2H), 2.05 (s, 3H), 2.01 (s, 3H), 1.50-1.41 (m, 2H), 1.32-1.18 (m, 18H), 1.22 (t, J=6.8, 3H). ESI-MS: m / z 493.3[M+H] +

[0485] Synthesis of compound 7':

[0486] Compound 6' (5.0 g, 10.15 mmol, 1.00 eq.) and U (2.28 g, 20.30 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (60 mL) was added and stirred to dissolve. BSA (6.19 g, 30.45 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (2.26 g, 10.15 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 6'. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 5 / 2) to obtain compound 7' (4.44 g, 8.15 mmol, 80.30% yield). 1 H NMR (400MHz, DMSO-d6) δ11.31 (s, 1H), 7.63 (d, J = 8.1Hz, 1H), 7.31-7.22 (m, 5H), 5.93 (d, J = 2.1Hz, 1H), 5 .60 (dd, J=8.1, 2.2Hz, 1H), 5.21 (t, J=1.9Hz, 1H), 4.69 (d, J=12.0Hz, 1H), 4.51 (d, J=12.0Hz, 1H), 4.31- 4.21 (m, 1H), 4.11 (dd, J=4.1, 1.6Hz, 1H), 3.72 (dd, J=10.6, 4.5Hz, 1H), 3.65 (dd, J=10.6, 6.1Hz, 1H), 3. 48-3.37 (m, 2H), 2.05 (s, 3H), 1.51-1.41 (m, 2H), 1.27-1.21 (m, 18H), 1.18 (t, J=7.1Hz, 3H). ESI-MS: m / z 545.3[M+H] +

[0487] Synthesis of compound 8':

[0488] Compound 7' (4.44 g, 8.15 mmol) was added to a 250 mL three-necked flask. Ultra-dry DCM (50 mL) was added to the reaction flask. The reaction system was cooled to -30°C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 25 mL, 24.45 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued until compound 7' was completely reacted. The reaction was quenched with triethylamine and methanol at -30°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with DCM, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 3) to obtain compound 8' (2.31 g, 5.08 mmol, 62.33% yield). ESI-MS: m / z 455.3 [M+H] +

[0489] Synthesis of compound 9':

[0490] Compound 8' (2.31 g, 5.08 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DCE (30 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (3.44 g, 10.16 mmol, 2.0 eq.), silver nitrate (863 mg, 5.08 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (3.08 g, 25.4 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 8'. The reaction was returned to room temperature and quenched by the addition of methanol. Ethyl acetate was added to the reaction mixture and the filtrate was filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=20 / 7) to give compound 9' (3.31 g, 4.37 mmol, 86.02% yield). ESI-MS: m / z 757.4 [M+H] +

[0491] Synthesis of compound 10':

[0492] Compound 9' (3.31 g, 4.37 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask and dissolved in 30 mL of THF. 0.97 mL of NaOMe solution (5.4 M in Methanol) (5.24 mmol, 1.2 eq.) was then added to the reaction mixture at room temperature. The reaction mixture was stirred at room temperature for 15 minutes until the reaction of compound 9' was complete. After completion of the reaction, the reaction mixture was poured into water and extracted with ethyl acetate. The organic phases were combined and washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure to yield a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to yield compound 10' (3.01 g, 4.21 mmol, 96.34% yield). 1 H NMR (400MHz, DMSO-d6) δ11.27 (s, 1H), 7.91 (d, J=8.1Hz, 1H), 7.47-7.38 (m, 2H ), 7.32-7.21 (m, 7H), 6.91-6.75 (m, 4H), 5.46 (d, J=3.6Hz, 1H), 4.16 (t, J=4.7 Hz, 1H), 3.76 (s, 6H), 3.66 (dd, J=9.2, 4.5Hz, 1H), 3.66-3.46 (m, 2H), 3.46-3. 38(m, 3H), 1.56(m, 2H), 1.36-1.21(m, 20H), 0.85(t, J=6.7Hz, 3H).ESI-MS: m / z 715.4[M+H] +

[0493] Synthesis of compound 11':

[0494] Dried compound 10' (3.01 g, 4.21 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred until completely dissolved. DIPEA (1.09 g, 8.42 mmol, 2.0 eq.) and DMAP (103 mg, 0.84 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.50 g, 6.32 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 10' was completely reacted. The reaction system was quenched by the addition of a saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 11' (3.22 g, 3.52 mmol, 83.61% yield). 1H NMR (400MHz, DMSO-d6) δ11.32 (dd, J=14.6, 2.2Hz, 1H), 7.92 (d, J=8.1Hz, 1H), 7.46-7.37 (m, 2H), 7. 35-7.25 (m, 7H), 6.88-6.84 (m, 4H), 5.73-5.52 (m, 2H), 4.33 (dt, J=8.8, 4.7Hz, 1H), 4.12-3.92 (m, 1H ), 3.84-3.75 (m, 1H), 3.76 (s, 6H), 3.66-3.39 (m, 6H), 2.72 (t, J=6.0Hz, 1H), 2.61-2.37 (m, 1H), 1.6 1-1.53 ​​(m, 2H), 1.41-1.21 (m, 20H), 1.11 (t, J=6.6Hz, 6H), 1.03 (d, J=6.7Hz, 6H), 0.88-0.80 (m, 3H). 31 P NMR (162MHz, DMSO-d6) δ151.12, 148.78., ESI-MS: m / z 913.5[M+H] +

[0495] Example 32: Synthesis of Compound 16'

[0496] Synthesis of compound 12':

[0497] Compound 10' (5.0 g, 6.99 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DMF (50 mL) was added and stirred to dissolve. Imidazole (1.90 g, 27.96 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (952 mg, 13.98 mmol, 2.0 eq.) was then slowly added portionwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS analysis indicated that compound 10' had reacted completely, and the reaction was quenched by adding water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 1) to obtain compound 12' (4.84 g, 5.84 mmol, 83.58% yield). ESI-MS: m / z 829.5[M+H] +

[0498] Synthesis of compound 13':

[0499] Compound 12' (4.84 g, 5.84 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry ACN (50 mL) was added and stirred until completely dissolved. TEA (1.18 g, 11.68 mmol, 2.0 eq.) and DMAP (1.43 g, 11.68 mmol, 2.0 eq.) were added to the reaction system and stirred uniformly. The reaction mixture was cooled to 0-5°C, and TPSCl (3.54 g, 11.68 mmol, 2.0 eq.) was slowly added in portions. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 12'. Aqueous ammonia (40 mL) was added to the reaction at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 13' (10.31 g, calculated based on 100% yield). ESI-MS: m / z 826.5 [MH] -

[0500] Synthesis of compound 14':

[0501] Crude compound 13' (10.31 g, 1.0 eq.) was added to a 100 mL round-bottom flask, and pyridine (100 mL) was added and stirred until the crude compound 13' was completely dissolved. The reaction system was cooled to 0°C, and BzCl (1.64 g, 11.68 mmol, 2.0 eq.) was slowly added dropwise to the reaction system. The mixture was stirred at 0°C for 1 hour until the reaction of compound 13' was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 14' (3.90 g, 4.18 mmol, total yield for two steps 71.58%). ESI-MS: m / z 930.5 [MH] -

[0502] Synthesis of compound 15':

[0503] Compound 14' (3.90 g, 4.18 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (30 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized with triethylamine (17 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS analysis showed that compound 14' had reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 15' (2.51 g, 3.07 mmol, 76.2% yield). ESI-MS: m / z 816.4 [MH] -

[0504] Synthesis of compound 16':

[0505] Dried compound 15' (2.51 g, 3.07 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. Ultra-dry DCM (30 mL) was added and stirred to dissolve. DIPEA (794 mg, 6.14 mmol, 2.0 eq.) and DMAP (75 mg, 0.61 mmol, 0.2 eq.) were added to the reaction mixture, and the atmosphere was purged with nitrogen. The reaction mixture was cooled to approximately 0°C, and CEP-Cl (1.09 g, 4.61 mmol, 1.5 eq.) was slowly added dropwise. The reaction mixture was allowed to react at this temperature under nitrogen for 1 hour until compound 15' was completely reacted. After completion of the reaction, saturated aqueous sodium bicarbonate solution was added to the reaction mixture to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 16' (2.68 g, 2.63 mmol, 85.67% yield). 1H NMR (400MHz, DMSO-d6) δ11.29 (d, J=17.5Hz, 1H), 8.41 (dd, J=30.3, 7.5Hz, 1H), 8.00 (d, J=7.6Hz, 2H ), 7.61 (t, J=7.3Hz, 1H), 7.57-7.41 (m, 2H), 7.36-7.11 (m, 9H), 6.88-6.81 (m, 4H), 5.72 (d, J=25.0Hz , 1H), 4.22-3.91 (m, 3H), 3.77-3.39 (m, 13H), 2.75 (dd, J=10.7, 5.6Hz, 1H), 2.62-2.31 (m, 1H), 1.62 -1.51 (m, 2H), 1.35-1.18 (m, 20H), 1.12-1.04 (m, 9H), 0.96 (d, J = 6.7Hz, 3H), 0.83 (t, J = 6.6Hz, 3H).3 1 P NMR (162MHz, DMSO-d6) δ149.74, 149.21.ESI-MS: m / z 1016.5[MH] -

[0506] Example 33: Synthesis of Compound 21'

[0507] Synthesis of compound 17':

[0508] Compound 6' (20.00 g, 40.60 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (250 mL) was added and stirred to dissolve. BSA (24.78 g, 121.80 mmol, 3.0 eq.) and A(Bz) (19.43 g, 81.20 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (9.02 g, 40.60 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS analysis confirmed the complete reaction of compound 6'. The reaction mixture was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to afford compound 17' (19.03 g, 28.32 mmol, 69.75% yield). 1H NMR (400MHz, DMSO-d6) δ11.25 (s, 1H), 8.75 (s, 1H), 8.45 (s, 1H), 8.05 (d, J=7.4Hz, 2H), 7.62 (t , J=7.4Hz, 1H), 7.52 (t, J=7.6Hz, 2H), 7.37-7.28 (m, 5H), 6.32 (d, J=1.7Hz, 1H), 4.79 (d, J=11. 8Hz, 1H), 4.61 (d, J=11.8Hz, 1H), 4.43 (dd, J=10.3, 4.6Hz, 1H), 3.85-3.68 (m, 2H), 3.48-3.41 ( m, 2H), 2.12 (s, 3H), 1.54-1.41 (m, 2H), 1.34-1.11 (m, 20H), 0.85 (t, J=6.7Hz, 3H). ESI-MS: m / z 672.4[M+H] +

[0509] Synthesis of compound 18':

[0510] Compound 17' (19.03 g, 28.32 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Extra-dry DCM (200 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L boron trichloride solution in dichloromethane (85 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 17' was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 18' (11.23 g, 19.31 mmol, 68.19% yield).

[0511] Synthesis of compound 19':

[0512] Compound 18' (11.23 g, 19.31 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Ultra-dry DCE (100 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (13.09 g, 38.62 mmol, 2.0 eq.), silver nitrate (3.28 g, 19.31 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (11.70 g, 96.55 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 18'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 19' (13.85 g, 15.67 mmol, 81.15% yield). ESI-MS: m / z 882.4 [MH] -

[0513] Synthesis of compound 20':

[0514] Compound 19' (13.85 g, 15.67 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. THF (150 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (3.48 mL, 18.80 mmol, 1.2 eq.) was added to the reaction and stirred at room temperature until compound 19' was completely dissolved. After completion of the reaction, the reaction mixture was extracted twice with water and ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain compound 20' (12.13 g, 14.41 mmol, 91.96% yield). ESI-MS: m / z 840.4 [MH] -

[0515] Synthesis of compound 21':

[0516] Dried compound 20' (2.0 g, 2.38 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (20 mL) was added and stirred until completely dissolved. DIPEA (615 mg, 4.76 mmol, 2.0 eq.) and DMAP (59 mg, 0.48 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (845 mg, 3.57 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 20' was completely reacted. The reaction system was quenched with a saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography to obtain compound 21' (1.92 g, 1.84 mmol, 77.31% yield). 1 H NMR (400MHz, DMSO-d6) δ11.25 (d, J=4.0Hz, 1H), 8.72 (d, J=5.9Hz, 1H), 8.61 (d, J=29.9Hz, 1H), 8. 14 (d, J=7.6Hz, 2H), 7.61 (t, J=7.3Hz, 1H), 7.52 (t, J=7.6Hz, 2H), 7.33 (t, J=8.6Hz, 2H), 7.22-7.1 4(m, 7H), 6.81-6.61(m, 4H), 6.03(dd, J=36.3, 3.4Hz, 1H), 4.74-4.32(m, 2H), 3.82-3.39(m, 15H) , 2.67-2.35(m, 2H), 1.61-1.49(m, 2H), 1.41-1.20(m, 18H), 1.13-0.98(m, 9H), 0.92-0.77(m, 6H). 31 P NMR (162MHz, DMSO-d6) δ149.64, 148.91.ESI-MS: m / z 1040.5[MH] -

[0517] Example 34: Synthesis of Compound 26'

[0518] Synthesis of compound 22':

[0519] Compound 6' (20 g, 40.60 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (200 mL) was added and stirred to dissolve. BSA (24.78 g, 121.8 mmol, 3.0 eq.) and G(iBu) (17.96 g, 81.2 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (9.02 g, 40.6 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight at this temperature. TLC and LCMS analysis confirmed the complete reaction of compound 6. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 22' (19.22 g, 29.40 mmol, 72.41% yield). ESI-MS: m / z 654.4 [M+H] +

[0520] Synthesis of compound 23':

[0521] Compound 22' (10.00 g, 15.29 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry DCM (100 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (45.87 mL, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 22' was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 23' (6.12 g, 10.86 mmol, 71.03% yield). ESI-MS: m / z 564.3[M+H] +

[0522] Synthesis of compound 24':

[0523] Compound 23' (6.12 g, 10.86 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Ultra-dry DCE (70 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (7.36 g, 21.72 mmol, 2.0 eq.), AgNO3 (1.84 g, 10.86 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (6.58 g, 54.30 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred evenly. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 23'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 24' (8.18 g, 9.46 mmol, 87.11% yield). ESI-MS: m / z 864.5 [MH] -

[0524] Synthesis of compound 25':

[0525] Compound 24' (3.18 g, 3.67 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (30 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (0.81 mL, 4.40 mmol, 1.2 eq.) was added to the reaction and stirred at room temperature until compound 24' was completely dissolved. After completion of the reaction, the reaction mixture was extracted twice with water and ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain compound 25' (2.68 g, 3.26 mmol, 88.83% yield). ESI-MS: m / z 822.4 [MH] -

[0526] Synthesis of compound 26':

[0527] Dried compound 25' (2.68 g, 3.26 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (30 mL) was added and stirred until completely dissolved. DIPEA (843 mg, 6.52 mmol, 2.0 eq.) and DMAP (79 mg, 0.65 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.16 g, 4.89 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 25' was completely reacted. The reaction system was quenched with a saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain compound 26' (2.98 g, 2.91 mmol, 89.26% yield). 1 H NMR (400MHz, DMSO-d6) δ12.09 (s, 1H), 11.49 (s, 1H), 8.15 (d, J=29.6Hz, 1H), 7.53-7.12 (m, 9H), 6.92- 6.87 (m, 4H), 5.73 (dd, J=38.6, 4.8Hz, 1H), 5.22-5.01 (m, 1H), 4.52 (t, J=5.5Hz, 1H), 3.82-3.67 (m, 8H ), 3.66-3.39(m, 3H), 3.26-3.21(m, 2H), 3.10(d, J=8.4Hz, 1H), 2.79-2.66(m, 1H), 2.57-2.47(m, 2H), 2.47-2.33 (m, 1H), 1.50 (d, J=6.6Hz, 2H), 1.36-1.16 (m, 18H), 1.14-0.99 (m, 13H), 0.89-0.73 (m, 8H). 31 P NMR (162MHz, DMSO-d6) δ151.33, 148.56, ESI-MS: m / z 1022.6[MH] -

[0528] Example 35: Synthesis of Compound 33'

[0529] Synthesis of compound 27':

[0530] Compound 4' (30.00 g, 103.02 mmol, 1.0 eq.) was dissolved in DMF (300 mL) and stirred. The reaction mixture was cooled to 0°C and stirred for 30 minutes. 60% NaH (4.94 g, 123.62 mmol, 1.2 eq.) was then added to the reaction mixture and stirred for 30 minutes. Compound CH3I (21.93 g, 154.53 mmol, 1.5 eq.) was slowly added dropwise to the reaction mixture. After addition, the reaction mixture was allowed to cool to room temperature and allowed to react overnight. TLC analysis confirmed the complete reaction of compound 4'. The reaction mixture was slowly poured into ice water to quench the mixture. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA=100 / 9) to give compound 27' (28.32 g, 96.21 mmol, 93.39% yield). ESI-MS: m / z 295.2 [M+H] +

[0531] Synthesis of compound 28':

[0532] Compound 27' (28.32 g, 96.21 mmol, 1.0 eq.) was added to a 500 mL round-bottom three-necked flask. AcOH (300 mL) and Ac2O (49.11 g, 481.05 mmol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction mixture was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (3 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0-5°C. After addition, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 27' was completely reacted. The reaction system was cooled to approximately -5°C and neutralized with aqueous ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=10 / 2) to give compound 28' (21.82 g, 64.49 mmol, 67.03% yield). ESI-MS: m / z 339.1 [M+H] +

[0533] Synthesis of compound 29':

[0534] Compound 28' (5.0 g, 14.78 mmol, 1.00 eq.) and uracil (3.31 g, 29.56 mmol, 2.0 eq.) were added to a 250 mL three-necked round-bottom flask. Ultra-dry ACN (80 mL) was added and stirred to dissolve. BSA (9.02 g, 44.34 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (3.29 g, 14.78 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis confirmed the complete reaction of compound 28'. The reaction was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate solution was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 5 / 2) to obtain compound 29' (5.03 g, 12.88 mmol, 87.14% yield). ESI-MS: m / z 391.2 [M+H] +

[0535] Synthesis of compound 30':

[0536] Compound 29' (5.03 g, 12.88 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Ultra-dry DCM (50 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. Boron trichloride (1M intoluene, 38.64 mL, 38.64 mmol, 3.0 eq.) was slowly added dropwise to the reaction system, and the reaction was continued for 5 hours until compound 29' was completely reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 3) to obtain compound 30' (2.44 g, 8.13 mmol, 63.13% yield). ESI-MS: m / z 301.1[M+H] +

[0537] Synthesis of compound 31':

[0538] Compound 30' (2.44 g, 8.13 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. Ultra-dry DCE (50 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (5.51 g, 16.26 mmol, 2.0 eq.), AgNO3 (1.38 g, 8.13 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (4.93 g, 40.65 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred uniformly. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 30'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=20 / 7) to give compound 31' (4.33 g, 7.20 mmol, 88.56% yield). ESI-MS: m / z 601.2 [MH] -

[0539] Synthesis of compound 32':

[0540] Compound 31' (4.33 g, 7.20 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. THF (50 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (1.60 mL, 8.64 mmol, 1.2 eq.) was added to the reaction and stirred at room temperature until compound 31' was completely reacted. After completion of the reaction, the reaction mixture was extracted twice with water and ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain compound 32' (3.91 g, 6.99 mmol, 97.08% yield). ESI-MS: m / z 559.2 [MH] -

[0541] Synthesis of compound 33':

[0542] Dried compound 32' (3.91 g, 6.99 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCM (40 mL) was added and stirred until completely dissolved. DIPEA (1.81 g, 13.98 mmol, 2.0 eq.) and DMAP (171 mg, 1.40 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.27 g, 5.36 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 32' was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 33' (4.02 g, 5.29 mmol, 75.68% yield). 1 H NMR (400MHz, DMSO-d6) δ11.33 (s, 1H), 7.88 (dd, J=18.9, 8.2Hz, 1H), 7.43-7.18 (m, 9H), 6.93-6.85 (m, 4H), 5.66-5.46 (m, 2H), 4.26-4.13 ( m, 1H), 4.01-3.43 (m, 14H), 3.35-3.30 (d, J=20Hz, 3H), 2.72-2.69 (m, 1H), 2.63-2.42 (m, 1H), 1.08 (t, J=6.1Hz, 6H), 1.00 (t, J=6.8Hz, 6H), 31 P NMR (162MHz, DMSO-d6) δ151.23, 149.32.ESI-MS: m / z 759.3[MH] -

[0543] Example 36: Synthesis of Compound 38'

[0544] Synthesis of compound 34':

[0545] Compound 32' (4.0 g, 7.14 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry DMF (50 mL) was added and stirred to dissolve. Imidazole (1.94 g, 28.56 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (2.15 g, 14.28 mmol, 2.0 eq.) was then slowly added portionwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS analysis indicated that compound 32' had reacted completely, and the reaction was quenched by adding water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 2 / 1) to afford compound 34' (3.93 g, 5.82 mmol, 81.51% yield). ESI-MS: m / z 673.3[MH] -

[0546] Synthesis of compound 35':

[0547] Compound 34' (3.93 g, 5.82 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask, and ultra-dry ACN (40 mL) was added and stirred until completely dissolved. TEA (1.18 g, 11.64 mmol, 2.0 eq.) and DMAP (1.42 g, 11.64 mmol, 2.0 eq.) were added to the reaction system and stirred uniformly. The reaction mixture was cooled to 0-5°C, and TPSCl (3.53 g, 11.64 mmol, 2.0 eq.) was slowly added in portions. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 34'. Aqueous ammonia (50 mL) was added to the reaction at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 35' (6.21 g, calculated based on 100% yield). ESI-MS: m / z 672.3 [MH] -

[0548] Synthesis of compound 36':

[0549] Crude compound 35' (6.21 g, 1.0 eq.) was added to a 250 mL round-bottom flask, and pyridine (60 mL) was added. The mixture was stirred until the crude compound 35' was completely dissolved. The reaction system was cooled to 0°C, and BzCl (1.64 g, 11.64 mmol, 2.0 eq.) was slowly added dropwise to the reaction system. The mixture was stirred at 0°C for 1 hour until the reaction of compound 35' was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude compound. The crude product was purified by column chromatography (PE / EA = 1 / 1) to afford compound 36' (3.82 g, 4.91 mmol, 84.36% total yield over two steps). ESI-MS: m / z 776.3 [MH] -

[0550] Synthesis of compound 37':

[0551] Compound 36' (3.82 g, 4.91 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (40 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized with triethylamine (20 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS analysis showed that compound 36' had reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 37' (2.72 g, 4.10 mmol, 83.50% yield). ESI-MS: m / z 662.3 [MH] -

[0552] Synthesis of compound 38':

[0553] Dried compound 37' (2.72 g, 4.10 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. Ultra-dry DCM (30 mL) was added and stirred to dissolve. DIPEA (1.06 g, 8.2 mmol, 2.0 eq.) and DMAP (100 mg, 0.82 mmol, 0.2 eq.) were added to the reaction mixture, and the reaction was purged with nitrogen. The reaction mixture was cooled to approximately 0°C, and CEP-Cl (1.46 g, 6.15 mmol, 1.5 eq.) was slowly added dropwise. The reaction mixture was allowed to react at this temperature under nitrogen for 1 hour until compound 37' was completely reacted. After completion of the reaction, saturated aqueous sodium bicarbonate solution was added to the reaction mixture to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 38' (2.3 g, 2.66 mmol, 67.9% yield). 1 H NMR (400MHz, DMSO-d6) δ11.16 (d, J=24.4Hz, 1H), 8.44 (dd, J=30.5, 7.3Hz, 1H), 7. 98(d, J=7.7Hz, 2H), 7.66-7.09(m, 14H), 6.86-6.74(m, 4H), 5.72-5.63(m, 1H), 4.2 4-3.83(m, 3H), 3.82-3.68(m, 8H), 3.62-3.39(m, 5H), 3.35-3.30(d, J=20Hz, 3H), 2 .76-2.68(m, 1H), 2.52-2.31(m, 1H), 1.07(t, J=6.1Hz, 6H), 1.01(t, J=6.8Hz, 6H), 31 P NMR (162MHz, DMSO-d6) δ149.91, 149.03, ESI-MS: m / z 864.4[MH] -

[0554] Example 37: Synthesis of Compound 43'

[0555] Synthesis of compound 39':

[0556] Compound 28' (10.00 g, 29.55 mol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (150 mL) was added and stirred to dissolve. BSA (18.03 g, 88.65 mmol, 3.0 eq.) and A(Bz) (14.14 g, 59.10 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (6.57 g, 29.55 mol, 1.0 eq.) was then slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS analysis confirmed the complete reaction of compound 28'. The reaction mixture was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 39' (10.11 g, 19.53 mmol, 66.09% yield). ESI-MS: m / z 518.2 [M+H] +

[0557] Synthesis of compound 40':

[0558] Compound 39' (10.11 g, 19.53 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Extra-dry DCM (200 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (58.59 mL, 58.59 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. The reaction was continued for 5 hours until compound 39' was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 40' (5.21 g, 12.19 mmol, 62.42% yield). ESI-MS: m / z 428.2[M+H] +

[0559] Synthesis of compound 41':

[0560] Compound 40' (5.21 g, 12.19 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Ultra-dry DCE (80 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (8.26 g, 24.38 mmol, 2.0 eq.), AgNO3 (2.07 g, 12.19 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (7.39 g, 60.95 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred until uniform. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 40'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 41' (7.75 g, 10.63 mmol, 87.20% yield). ESI-MS: m / z 728.3 [MH] -

[0561] Synthesis of compound 42':

[0562] Compound 41' (7.75 g, 10.63 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. THF (80 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (2.36 mL, 12.76 mmol, 1.2 eq.) was added to the reaction and stirred at room temperature until compound 41' was completely dissolved. After completion of the reaction, the reaction mixture was extracted twice with water and ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain compound 42' (6.81 g, 9.92 mmol, 93.32% yield). ESI-MS: m / z 686.3 [MH] -

[0563] Synthesis of compound 43':

[0564] Dried compound 42' (2.0 g, 2.91 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (20 mL) was added and stirred until completely dissolved. DIPEA (752 mg, 5.82 mmol, 2.0 eq.) and DMAP (71 mg, 0.58 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.55 g, 6.56 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 42' was completely reacted. The reaction mixture was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography to obtain compound 43' (2.24 g, 2.53 mmol, 86.94% yield). 1 H NMR (400MHz, DMSO-d6) δ11.25 (s, 1H), 8.73-8.52 (m, 2H), 8.01 (d, J=7.3Hz, 2 H), 7.69-7.51(m, 3H), 7.38-7.32(m, 2H), 7.28-7.11(m, 7H), 6.87-6.72(m, 4 H), 6.03 (dd, J=34.0, 3.2Hz, 1H), 4.73-4.31 (m, 2H), 3.85-3.79 (m, 1H), 3.78 -3.45(m, 12H), 3.31(d, J=24.0, 3H), 2.71-2.56(m, 2H), 1.12-0.88(m, 12H), 31 P NMR (162MHz, DMSO-d6) δ150.71, 149.12, ESI-MS: m / z 886.4[MH] -

[0565] Example 38: Synthesis of Compound 48'

[0566] Synthesis of compound 44':

[0567] Compound 28' (10.00 g, 29.55 mmol, 1.00 eq.) was added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (150 mL) was added and stirred to dissolve. BSA (18.03 g, 88.65 mmol, 3.0 eq.) and G(iBu) (13.07 g, 59.10 mmol, 2.0 eq.) were then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (6.57 g, 29.55 mmol, 1.0 eq.) was slowly added dropwise to the reaction. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS analysis confirmed the complete reaction of compound 28'. The reaction mixture was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 44' (9.62 g, 19.26 mmol, 65.18% yield). ESI-MS: m / z 500.2 [M+H] +

[0568] Synthesis of compound 45':

[0569] Compound 44' (9.62 g, 19.26 mmol, 1.0 eq.) was added to a 250 mL three-necked flask. Extra-dry DCM (100 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. A 1.0 mol / L BCl3 solution in dichloromethane (57.78 mL, 57.78 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. The reaction was continued for 5 hours until compound 44' was fully reacted. The reaction was quenched with triethylamine and methanol at -20°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 45' (5.12 g, 12.51 mmol, 64.95% yield). ESI-MS: m / z 410.2[M+H] +

[0570] Synthesis of compound 46':

[0571] Compound 45' (5.12 g, 12.51 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Ultra-dry DCE (80 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (8.48 g, 25.02 mmol, 2.0 eq.), AgNO3 (2.13 g, 12.51 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (7.58 g, 62.55 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred until uniform. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 45'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=2 / 3) to give compound 46' (7.37 g, 10.35 mmol, 82.73% yield). ESI-MS: m / z 710.3 [MH] -

[0572] Synthesis of compound 47':

[0573] Compound 46' (7.37 g, 10.35 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. THF (80 mL) was added to the reaction and stirred until completely dissolved. A 5.4 mol / L NaOMe solution (2.30 mL, 12.42 mmol, 1.2 eq.) was added to the reaction and stirred at room temperature until compound 46' was completely dissolved. After completion of the reaction, the reaction mixture was extracted twice with water and ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain compound 47' (6.58 g, 9.82 mmol, 91.48% yield). ESI-MS: m / z 668.3 [MH] -

[0574] Synthesis of compound 48':

[0575] Dried compound 47' (2.00 g, 2.99 mmol, 1.0 eq.) was added to a 50 mL round-bottom flask, and ultra-dry DCM (20 mL) was added and stirred until completely dissolved. DIPEA (773 mg, 5.98 mmol, 2.0 eq.) and DMAP (73 mg, 0.60 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.06 g, 4.49 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 47' was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography to obtain compound 48' (2.27 g, 2.61 mmol, 87.29% yield). 1 H NMR (400MHz, DMSO-d6) δ12.08 (s, 1H), 11.52 (s, 1H), 8.22 (d, J=29.6Hz, 1H), 7.56-7.14 (m, 9H), 6.89-6.78 (m, 4H), 5.73 (dd, J=38.6, 4.8Hz, 1H), 5.23-5.11 (m, 1H), 4.51 (t, J=5.5Hz, 1 H), 3.81-3.66 (m, 8H), 3.61-3.38 (m, 3H), 3.31 (d, J=24.0, 3H), 3.23-3.19 (m, 1H), 3.11 (d, J=8.4Hz, 1H), 2.79-2.63(m, 1H), 2.59-2.49(m, 1H), 2.43-2.33(m, 1H), 1.12-0.88(m, 18H), 31 P NMR (162MHz, DMSO-d6) δ151.35, 149.21, ESI-MS: m / z 868.4[MH] -

[0576] Example 39: Synthesis of Compound 56'

[0577] Synthesis of compound 49':

[0578] Compound 4' (45.00 g, 160.53 mmol, 1.00 eq.) was added to a 500 mL round-bottom flask. Toluene (400 mL) was added and stirred to dissolve. Imidazole (38.25 g, 561.86 mmol, 3.5 eq.) and PPh3 (50.53 g, 192.64 mmol, 1.2 eq.) were then added. 12 (48.9 g, 192.64 mmol, 1.2 eq.) was then slowly added. After completion of addition, the reaction system was heated to 70°C in an oil bath and stirred for 3 hours. TLC and LCMS analysis confirmed the complete reaction of compound 4'. The reaction was removed from the oil bath and cooled to room temperature. Aqueous sodium sulfite solution was added to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 4) to give compound 49' (54.40 g, 139.41 mmol, 86.8% yield). ESI-MS: m / z 391.2 [M+H] +

[0579] Synthesis of compound 50':

[0580] Compound 49' (54.40 g, 139.42 mmol, 1.0 eq.) was dissolved in MeOH (500 mL). KCO (19.34 g, 139.42 mmol, 1.0 eq.) and Pd / C (10% W, 5.4 g) were then added to the reaction mixture. Hydrogen was introduced and the reaction was stirred at room temperature until the reaction of compound 49' was complete. The mixture was filtered through Celite, concentrated, and extracted twice with ethyl acetate. The salt was removed by filtration and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 4 / 1) to afford compound 50' (33.20 g, 125.61 mmol, 90.1% yield). 1 H NMR (400MHz, DMSO-d6) δ7.51-7.20 (m, 5H), 5.81 (d, J=4.0Hz, 1H), 4.72-4.64 (m, 2H), 4.49 (d, J=12.0Hz, 1 H), 4.25-4.16 (m, 1H), 3.71 (d, J=3.1Hz, 1H), 1.38 (s, 3H), 1.25 (s, 3H), 1.20 (d, J=6.4Hz, 3H). ESI-MS: m / z 265.3[M+H] +

[0581] Synthesis of compound 51':

[0582] Compound 50' (33.20 g, 125.61 mmol, 1.0 eq) was added to a 500 mL round-bottom three-necked flask. AcOH (300 mL) and Ac2O (64.12 g, 628.05 mmol, 5.0 eq.) were added to the reaction system and stirred until completely dissolved. The reaction mixture was cooled to 0°C and stirred at this temperature for 30 minutes. Concentrated sulfuric acid (10 mL) was slowly added dropwise to the reaction system, maintaining the temperature between 0-5°C. After addition, the reaction system was allowed to return to room temperature and stirred for 8 hours until compound 50' was completely reacted. The reaction system was cooled to approximately -5°C and neutralized with aqueous ammonia to a pH of approximately 7. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=4 / 1) to give compound 51' (21.20 g, 68.76 mmol, 54.7% yield). ESI-MS: m / z 309.3 [M+H] +

[0583] Synthesis of compound 52':

[0584] Compound 51' (10.00 g, 32.43 mmol, 1.00 eq.) and uracil (7.27 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (100 mL) was added and stirred to dissolve. BSA (19.79 g, 97.29 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction process was carried out under nitrogen protection. After the reaction was completed, the reaction system was placed in a 0°C ice-water bath and stirred for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was placed in an oil bath and slowly heated to 80°C, where it remained overnight. TLC and LCMS analysis showed that compound 51' had reacted completely. The reaction mixture was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 3) to afford compound 52' (9.61 g, 26.64 mmol, 82.1% yield). 1H NMR (400MHz, DMSO-d6) δ11.36 (s, 1H), 7.46-7.21 (m, 6H), 6.32 (d, J = 8.0Hz, 1H), 5.90 (t, J = 9.0Hz, 1H), 5.18 (s, 1H), 4.77 (d, J = 11.5Hz, 1H), 4.58 (t, J=11.0Hz, 1H), 4.31-4.18 (m, 1H), 3.94 (d, J=3.4Hz, 1H), 2.11 (s, 3H), 1.33 (d, J=6.3Hz, 3H). ESI-MS: m / z 361.34[M+H] +

[0585] Synthesis of compound 53':

[0586] Dry compound 52' (9.61 g, 26.64 mmol, 1.0 eq) was added to a 500 mL three-necked flask. Ultra-dry DCM (100 mL) was added to the reaction flask. The reaction system was cooled to -20°C and stirred at this temperature for 30 minutes. BCl3 (1 M in toluene, 80 mL, 79.92 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After completion of the addition, the temperature was returned to -10°C and the reaction was continued until compound 52' was fully reacted. The reaction was quenched with triethylamine and methanol at -10°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 53' (5.01 g, 18.5 mol, 69.4% yield). 1 H NMR (400MHz, DMSO-d6) δ11.36 (s, 1H), 7.59 (s, 1H), 6.32 (d, J = 8.0Hz, 1H), 5.90 (t, J = 9.0Hz, 1H), 5.18 (s, 1H), 4 .49 (d, J=7.8Hz, 1H), 4.31-4.18 (m, 1H), 3.94 (d, J=3.4Hz, 1H), 2.11 (s, 3H), 1.33 (d, J=6.3Hz, 3H). ESI-MS: m / z 271.24[M+H]

[0587] Synthesis of compound 54':

[0588] Compound 53' (5.01 g, 18.5 mmol, 1.0 eq) was added to a 100 mL round-bottom flask, and ultra-dry DCE (100 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (12.54 g, 37.0 mmol, 2.0 eq.), AgNO3 (3.14 g, 18.5 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (11.21 g, 92.5 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred evenly at room temperature. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 53'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 54' (7.30 g, 12.75 mmol, 70% yield). 1 H NMR (400MHz, DMSO-d6) δ11.36 (s, 1H), 7.46 (d, J=7.5Hz, 3H), 7.39-7.22 (m, 7H), 6.92 (dd, J=8.9, 7.3Hz, 4H), 6.32 (d, J=8.0Hz, 1H), 5.9 0 (t, J=9.0Hz, 1H), 5.18 (s, 1H), 4.31-4.18 (m, 1H), 3.94 (d, J=3.4Hz, 1H), 3.81 (s, 6H), 2.11 (s, 3H), 1.33 (d, J=6.3Hz, 3H). ESI-MS: m / z 573.6[MH] -

[0589] Synthesis of compound 55':

[0590] Compound 54' (7.30 g, 12.75 mmol, 1.0 eq.) was dissolved in THF (100 mL). NaOMe (826.51 mg, 15.3 mmol, 1.2 eq.) was then added to the reaction mixture and stirred at room temperature until the reaction of compound 54' was complete. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 2) to obtain compound 55' (6.21 g, 11.68 mmol, 91.61% yield). 1H NMR (400MHz, DMSO-d6) δ11.36 (s, 1H), 7.46 (d, J=7.5Hz, 3H), 7.39-7.22 (m, 7H), 6.92 (dd, J=8.9, 7.3Hz, 4H), 6.32 (d, J=8.0Hz, 1H), 5.90 (t, J=9.0Hz, 1H), 5.30 (d, J=3.8Hz, 1H), 5.18 (s, 1H), 4.31-4.18 (m, 1H), 3.94 (d, J=3.4Hz, 1H), 3.81 (s, 6H), 1.33 (d, J=6.3Hz, 3H). ESI-MS: m / z 531.6[MH] -

[0591] Synthesis of compound 56':

[0592] Dried compound 55' (2.00 g, 3.77 mmol, 1.0 eq.) was added to a 100 ml round-bottom flask, and ultra-dry DCM (20 mL) was added and stirred until completely dissolved. DIPEA (974.5 mg, 7.54 mmol, 2.0 eq.) and DMAP (92.12 mg, 0.754 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.34 g, 5.66 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 55' was completely reacted. The reaction mixture was quenched with a saturated aqueous sodium bicarbonate solution and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 56' (2.31 g, 3.15 mmol, 83.55% yield). 1 H NMR (400MHz, DMSO-d6) δ11.36 (s, 1H), 7.46 (d, J=7.5Hz, 3H), 7.39-7.22 (m, 7H), 6.92 (dd, J=8.9, 7.3Hz, 4H), 6.32 (d, J=8.0Hz, 1H), 5.90 (t, J=9 .0Hz, 1H), 5.18 (s, 1H), 4.31-4.18 (m, 1H), 3.99-3.80 (m, 9H), 2.95-2.7 0 (m, 2H), 2.60-2.49 (m, 2H), 1.33 (d, J=6.3Hz, 3H), 1.12-0.92 (m, 12H). 31P NMR (162MHz, DMSO-d6) δ150.02(s), 149.73(s).ESI-MS: m / z 731.8[MH] -

[0593] Example 40: Synthesis of Compound 61'

[0594] Synthesis of compound 57':

[0595] Compound 55' (1.98 g, 3.74 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DMF (30 mL) was added and stirred to dissolve. Imidazole (1.02 g, 14.96 mmol, 4.0 eq.) was added at room temperature and stirred for 10 minutes. TBSCl (1.13 g, 7.48 mmol, 2.0 eq.) was then slowly added portionwise to the reaction system, and the reaction was allowed to proceed overnight at room temperature under nitrogen. TLC and LCMS analysis indicated that compound 55' had reacted completely, and water was added to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to yield compound 57' (2.15 g, 3.32 mmol, 88.77% yield). ESI-MS: m / z 645.8[M+H] -

[0596] Synthesis of compound 58':

[0597] Compound 57' (2.15 g, 3.32 mmol, 1.0 eq.) was added to a 100 ml round-bottom flask, and ultra-dry ACN (30 mL) was added and stirred until completely dissolved. TEA (672 mg, 6.64 mmol, 2.0 eq.) and DMAP (811 mg, 6.64 mmol, 2.0 eq.) were then added to the reaction system and stirred until uniformly dissolved. The reaction mixture was cooled to 0-5°C, and TPSCl (2.01 g, 6.64 mmol, 2.0 eq.) was slowly added portionwise. After the addition, the ice bath was removed and the mixture was allowed to return to room temperature. The reaction was stirred at room temperature overnight. TLC confirmed the complete reaction of compound 57'. Aqueous ammonia (20 mL) was added to the reaction mixture at room temperature and stirred for approximately 12 hours until the intermediate was completely reacted. Saturated brine was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude compound 58 (2.42 g, calculated based on 100% yield). ESI-MS: m / z 644.9 [M+H] +

[0598] Synthesis of compound 59':

[0599] Crude compound 58' (2.42 g) was added to a 100 mL round-bottom flask, pyridine (50 mL) was added, and the mixture was stirred until the crude compound 58' was completely dissolved. The reaction system was cooled to 0°C, and BzCl (933 mg, 6.64 mmol, 2.0 eq.) was slowly added dropwise to the reaction system. The mixture was stirred at 0°C for 1 hour until the reaction of compound 58' was complete. Nitrogen was used throughout the reaction. The reaction system was returned to room temperature and quenched by adding methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 1) to obtain compound 59' (1.81 g, 2.41 mmol, 72.59% yield over two steps). ESI-MS: m / z 752.93 [M+-H] -

[0600] Synthesis of compound 60':

[0601] Compound 59' (1.81 g, 2.41 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and THF (20 mL) was added and stirred until completely dissolved. Triethylamine trihydrofluoride (5.0 mL) was neutralized with triethylamine (17 mL) until alkaline and then added to the above reaction system. The reaction was placed in a 40°C oil bath and stirred overnight under nitrogen. TLC and LCMS analysis showed that compound 59' had reacted completely. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water, saturated sodium bicarbonate aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 3) to obtain compound 60' (1.16 g, 1.83 mmol, 75.93% yield). 1 HNMR (400MHz, DMSO-d6) δ11.36 (s, 1H), 7.46 (d, J=7.5Hz, 3H), 7.92-7.72 (m, 2H ), 7.69-7.46(m, 3H), 7.39-7.22(m, 7H), 6.92(dd, J=8.9, 7.3Hz, 4H), 6.32(d, J= 8.0Hz, 1H), 5.90 (t, J=9.0Hz, 1H), 5.30 (d, J=3.8Hz, 1H), 5.18 (s, 1H), 4.31-4.1 8 (m, 1H), 3.94 (d, J = 3.4Hz, 1H), 3.81 (s, 6H), 1.33 (d, J = 6.3Hz, 3H). ESI-MS: m / z 634.7[MH] -

[0602] Synthesis of compound 61':

[0603] Dry compound 60' (1.16 g, 1.83 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask. Ultra-dry DCM (20 mL) was added and stirred to dissolve. DIPEA (473 mg, 3.66 mmol, 2.0 eq.) and DMAP (45 mg, 0.37 mmol, 0.2 eq.) were added to the reaction mixture, and the atmosphere was purged with nitrogen. The reaction mixture was cooled to approximately 0°C, and CEP-Cl (651 mg, 2.75 mmol, 1.5 eq.) was slowly added dropwise. The reaction mixture was allowed to react at this temperature under nitrogen for 1 hour until compound 60' was completely reacted. After completion of the reaction, saturated aqueous sodium bicarbonate solution was added to the reaction mixture to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 1) to give compound 61' (1.22 g, 1.46 mmol, 79.78% yield). 1 H NMR (400MHz, DMSO-d6) δ11.36 (s, 1H), 7.92-7.72 (m, 2H), 7.69-7.46 (m, 3H), 7.41 (d, J=7.5Hz, 3H), 7.39-7.22 (m, 7H), 6.92 (dd, J=8.9, 7.3Hz, 4H), 6.32 (d, J=8.0H z, 1H), 5.90 (t, J=9.0Hz, 1H), 5.18 (s, 1H), 4.31-4.18 (m, 1H), 3.99-3.80 (m, 9H), 2.95-2.70 (m, 2H), 2.60-2.49 (m, 2H), 1.33 (d, J=6.3Hz, 3H), 1.12-0.92 (m, 12H). 31 P NMR (162MHz, DMSO-d6) δ149.92(s), 149.69(s).ESI-MS: m / z 834.9[MH] -

[0604] Example 41: Synthesis of Compound 66'

[0605] Synthesis of compound 62':

[0606] Compound 51' (10.00 g, 32.43 mmol, 1.00 eq.) and A(Bz) (15.52 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (150 mL) was added and stirred to dissolve. BSA (19.79 g, 97.29 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS analysis confirmed the complete reaction of compound 51'. The reaction mixture was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 5) to afford compound 62' (11.40 g, 23.38 mmol, 72.1% yield). 1 H NMR (400MHz, DMSO-d6) δ11.17 (s, 1H), 8.35 (s, 1H), 8.21 (s, 1H), 8.01-7. 87 (m, 2H), 7.69-7.47 (m, 3H), 7.46-7.21 (m, 5H), 5.90 (t, J=9.0Hz, 1H), 5. 18 (s, 1H), 4.77 (d, J = 11.5Hz, 1H), 4.58 (t, J = 11.0Hz, 1H), 4.31-4.18 (m, 1 H), 3.94 (d, J=3.4Hz, 1H), 2.11 (s, 3H), 1.33 (d, J=6.3Hz, 3H). ESI-MS: m / z 488.5[M+H] +

[0607] Synthesis of compound 63':

[0608] Compound 62' (11.40 g, 23.38 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Ultra-dry DCM (100 mL) was added to the reaction flask. The reaction system was cooled to -80°C and stirred at this temperature for 30 minutes. BCl3 (1 Mintoluene, 70 mL, 70.14 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After completion of the addition, the temperature was returned to -10°C and the reaction was continued until compound 62' was fully reacted. The reaction was quenched with triethylamine and methanol at -10°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 10) to obtain compound 63' (4.80 g, 12.08 mmol, 51.67% yield). ESI-MS: m / z 398.4 [M+H] +

[0609] Synthesis of compound 64':

[0610] Compound 63' (4.80 g, 12.08 mmol, 1.0 eq.) was added to a 250 mL round-bottom flask. Ultra-dry DCE (50 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (8.19 g, 24.16 mmol, 2.0 eq.), AgNO3 (2.05 g, 12.08 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (7.32 g, 60.40 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred uniformly. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 63'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 3) to give compound 64' (6.20 g, 8.86 mmol, 73.34% yield). ESI-MS: m / z 700.7 [MH] -

[0611] Synthesis of compound 65':

[0612] Compound 64' (6.20 g, 8.86 mmol, 1.0 eq.) was dissolved in THF (100 mL). Aqueous ammonia solution was then added to the reaction mixture and stirred at room temperature until the reaction of compound 64' was complete. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 5) to obtain compound 65' (5.21 g, 7.91 mmol, 90% yield). 1 H NMR (400MHz, DMSO-d6) δ11.17 (s, 1H), 8.35 (s, 1H), 8.21 (s, 1H), 8.01-7.87 (m, 2H), 7.69-7.47 (m, 3H), 7.33-7.22 (m, 9H), 6.94-6.82 (m, 4H), 5.90 (t, J =9.0Hz, 1H), 5.23(d, J=3.8Hz, 1H), 5.18(s, 1H), 4.31-4.18(m, 1H), 3.94(d , J=3.4Hz, 1H), 3.82 (s, 6H), 1.33 (d, J=6.3Hz, 3H).ESI-MS: m / z660.2[M+H] -

[0613] Synthesis of compound 66':

[0614] Dried compound 65' (5.21 g, 7.91 mmol, 1.0 eq.) was added to a 100 ml round-bottom flask, and ultra-dry DCM (50 mL) was added and stirred until completely dissolved. DIPEA (2.04 g, 15.82 mmol, 2.0 eq.) and DMAP (193 mg, 1.58 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (2.81 g, 11.87 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes until compound 65' was completely reacted. The reaction system was quenched by the addition of a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA=1 / 3) to give compound 66' (5.0 g, 5.83 mmol, 74% yield). 1H NMR (400MHz, DMSO-d6) δ11.17(s, 1H), 8.35(s, 1H), 8.21(s, 1H), 8.01-7.87(m , 2H), 7.69-7.47(m, 3H), 7.33-7.22(m, 9H), 6.94-6.82(m, 4H), 5.90(t, J=9.0H z, 1H), 5.18 (s, 1H), 4.31-4.18 (m, 1H), 3.99-3.85 (m, 3H), 3.82 (s, 6H), 2.96-2 .74 (m, 2H), 2.73-2.66 (m, 2H), 1.33 (d, J = 6.3Hz, 3H), 1.06 (d, J = 12.0Hz, 12H). 31 P NMR (162MHz, DMSO-d6) δ150.91(s), 149.82(s).ESI-MS: m / z 858.9[MH] -

[0615] Example 42: Synthesis of Compound 71'

[0616] Synthesis of compound 67':

[0617] Compound 51' (10.00 g, 32.43 mmol, 1.00 eq.) and G(iBu) (14.3 g, 64.86 mmol, 2.0 eq.) were added to a 500 mL three-necked round-bottom flask. Ultra-dry ACN (150 mL) was added and stirred to dissolve. BSA (19.8 g, 97.29 mmol, 3.0 eq.) was then added. The reaction system was heated to 80°C in an oil bath and stirred at this temperature for 1 hour. The entire reaction was carried out under nitrogen. After completion of the reaction, the reaction system was stirred in a 0°C ice-water bath for 30 minutes. TMSOTf (7.21 g, 32.43 mmol, 1.0 eq.) was slowly added dropwise to the reaction system. After the addition, the reaction system was slowly heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS analysis confirmed the complete reaction of compound 51'. The reaction mixture was removed from the oil bath and cooled to room temperature. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The system was extracted with ethyl acetate, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 6) to afford compound 67' (11.60 g, 24.71 mmol, 76.2% yield). 1H NMR (400MHz, DMSO-d6) δ12.09 (s, 1H), 11.53 (s, 1H), 7.91 (s, 1H), 7.46-7.2 1(m, 5H), 5.90(t, J=9.0Hz, 1H), 5.18(s, 1H), 4.77(d, J=11.5Hz, 1H), 4.58( t, J=11.0Hz, 1H), 4.31-4.18 (m, 1H), 3.94 (d, J=3.4Hz, 1H), 2.87-2.53 (m, 1 H), 2.11 (s, 3H), 1.33 (d, J = 6.3Hz, 3H), 1.10 (d, J = 12.9Hz, 6H). ESI-MS: m / z 470.5[M+H] +

[0618] Synthesis of compound 68':

[0619] Compound 67' (11.60 g, 24.71 mmol, 1.0 eq.) was added to a 500 mL three-necked flask. Ultra-dry DCM (150 mL) was added to the reaction flask. The reaction system was cooled to -78°C and stirred at this temperature for 30 minutes. Boron trichloride (1 M in toluene, 74 mL, 74.13 mmol, 3.0 eq.) was slowly added dropwise to the reaction system. After completion of the addition, the temperature was returned to -10°C and the reaction was continued until compound 67' was fully reacted. The reaction was quenched with triethylamine and methanol at -78°C. The reaction mixture was returned to room temperature and water was added. The mixture was extracted with dichloromethane, and the organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA = 1 / 20) to obtain compound 68' (5.61 g, 14.76 mmol, 60% yield). ESI-MS: m / z 380.37 [M+H] +

[0620] Synthesis of compound 69':

[0621] Compound 68' (5.61 g, 14.76 mmol, 1.0 eq.) was added to a 100 mL round-bottom flask, and ultra-dry DCE (50 mL) was added to the reaction flask and stirred until completely dissolved. DMTrCl (10.0 g, 29.52 mmol, 2.0 eq.), AgNO3 (2.51 g, 14.76 mmol, 1.0 eq.), and 2,4,6-trimethylpyridine (8.9 g, 73.8 mmol, 5.0 eq.) were added to the reaction at room temperature and stirred evenly. The reaction was heated to 80°C in an oil bath and allowed to react overnight. TLC and LCMS confirmed the complete reaction of compound 68'. The reaction was returned to room temperature and quenched by the addition of methanol. The reaction was diluted with ethyl acetate and filtered through celite to obtain the filtrate. The filter cake was washed twice with ethyl acetate. The combined filtrates were concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (PE / EA=1 / 10) to give compound 69' (7.20 g, 10.56 mmol, 71.5% yield). ESI-MS: m / z 682.7 [MH] -

[0622] Synthesis of compound 70':

[0623] Compound 69' (7.20 g, 10.56 mmol, 1.0 eq.) was dissolved in THF (100 mL). Aqueous ammonia was then added to the reaction mixture and stirred at room temperature until the reaction of compound 69' was complete. Water was added to the reaction system, and the reaction mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (PE / EA = 1 / 20) to obtain compound 70' (5.30 g, 8.29 mmol, 78.5% yield). 1 H NMR (400MHz, DMSO-d6) δ12.09 (s, 1H), 11.53 (s, 1H), 7.91 (s, 1H), 7.38-7.20 (m, 9H), 6.96-6.74 (m, 4H), 5.90 (t, J=9.0Hz, 1H), 5.30 (d, J=3.8Hz, 1H ), 5.18 (s, 1H), 4.31-4.18 (m, 1H), 3.94 (d, J = 3.4Hz, 1H), 3.80 (s, 6H), 2.8 7-2.53 (m, 1H), 1.33 (d, J=6.3Hz, 3H), 1.10 (d, J=12.9Hz, 6H).ESI-MS: m / z 640.7[MH] -

[0624] Synthesis of compound 71':

[0625] Dried compound 70' (2.00 g, 3.13 mmol, 1.0 eq.) was added to a 100 ml round-bottom flask, and ultra-dry DCM (20 mL) was added and stirred until completely dissolved. DIPEA (809 mg, 6.26 mmol, 2.0 eq.) and DMAP (77 mg, 0.63 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for 15 minutes. The reaction system was purged with nitrogen and carried out under a nitrogen atmosphere. CEP-Cl (1.1 g, 4.70 mmol, 1.5 eq.) was added dropwise to the reaction mixture at room temperature and allowed to react for 30-60 minutes at room temperature until compound 70' was completely reacted. The reaction system was quenched with a saturated aqueous solution of sodium bicarbonate and extracted twice with dichloromethane. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by column chromatography (PE / EA=1 / 3) to give compound 71' (2.01 g, 2.38 mmol, 76% yield). 1 H NMR (400MHz, DMSO-d6) δ12.09 (s, 1H), 11.53 (s, 1H), 7.91 (s, 1H), 7.38-7.2 0 (m, 9H), 6.96-6.74 (m, 4H), 5.90 (t, J = 9.0Hz, 1H), 5.30 (d, J = 3.8Hz, 1H), 5. 18(s, 1H), 4.31-4.18(m, 1H), 4.00-3.83(m, 3H), 3.80(s, 6H), 2.93-2.53(m, 5H), 1.33 (d, J=6.3Hz, 2H), 1.10 (d, J=12.9Hz, 6H), 1.06 (d, J=12.0Hz, 12H). 31 P NMR (162MHz, DMSO-d6) δ151.11(s), 149.91(s).ESI-MS: m / z 840.9[MH] -

[0626] The following Example 43 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 3-b of the present invention, wherein the nucleoside compound represented by Formula 3-b is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0627] Example 43:

[0628] The following Example 44 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 4-b of the present invention, wherein the nucleoside compound represented by Formula 4-b is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0629] Example 44:

[0630] The following Example 45 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 5-b of the present invention, wherein the nucleoside compound represented by Formula 5-b is a compound having a 2'-phosphoramidite group, a 3'-ODMTr, and a 4'-modified TNA structure.

[0631] Example 45:

[0632] The following Example 46 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 6-b of the present invention, wherein the nucleoside compound represented by Formula 6-b is a compound having a 2'-phosphoramidite group, a 3'-ODMTr, and a 4'-modified TNA structure.

[0633] Example 46:

[0634] The following Example 47 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 7-b of the present invention, wherein the nucleoside compound represented by Formula 7-b is a compound having a 2'-phosphoramidite group, a 3'-ODMTr, and a 4'-modified TNA structure.

[0635] Example 47:

[0636] The following Example 48 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 8-b of the present invention, wherein the nucleoside compound represented by Formula 8-b is a 1'-H, 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0637] Example 48:

[0638] The following Example 49 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 9-b of the present invention, wherein the nucleoside compound represented by Formula 9-b is a 2'-S-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0639] Example 49:

[0640] The following Example 50 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 10-b of the present invention, wherein the nucleoside compound represented by Formula 10-b is a 2'-phosphoramidite group, 3'-SDMTr, 4'-modified TNA structure compound.

[0641] Example 50:

[0642] The following Example 51 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 11-b of the present invention, wherein the nucleoside compound represented by Formula 11-b is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0643] Example 51:

[0644] The following Example 52 is a preparation example (synthetic route) of the nucleoside compound represented by Formula 12-b of the present invention, wherein the nucleoside compound represented by Formula 12-b is a 2'-phosphoramidite group, 3'-ODMTr, 4'-modified TNA structure compound.

[0645] Example 52:

[0646] Example 53: Synthesis of Compound 188'

[0647] Nucleoside compound 188' is a 2'-phosphoramidite group, 3'-ODMTr, Nu = A, R = C 14 H 29 The preparation route refers to Examples 31 and 33, except that the raw material C of Example 31 is used to synthesize compound 5' from compound 4'. 12 H 25 Br changed to C 14 H 29 Br, the remaining steps are similar to those in Examples 31 and 33, and the structure and NMR data of the obtained nucleoside compound 188' are shown below:

[0648] 1 H NMR (400MHz, DMSO-d6) δ11.26 (d, J=4.0Hz, 1H), 8.74 (d, J=5.9Hz, 1H), 8.56 (d, J=29.9Hz, 1H), 8. 24 (d, J=7.6Hz, 2H), 7.65 (t, J=7.3Hz, 1H), 7.53 (t, J=7.6Hz, 2H), 7.36 (t, J=8.6Hz, 2H), 7.24-7.1 2(m, 7H), 6.80-6.61(m, 4H), 6.03(dd, J=36.3, 3.4Hz, 1H), 4.74-4.32(m, 2H), 3.82-3.39(m, 15H) , 2.67-2.35(m, 2H), 1.61-1.49(m, 2H), 1.41-1.20(m, 22H), 1.03-0.96(m, 9H), 0.90-0.76(m, 6H). 31P NMR (162MHz, DMSO-d6) δ149.66, 148.93.ESI-MS: m / z 1070.5[M+H] +

[0649] Example 54: Synthesis of Compound 189'

[0650] Nucleoside compound 189' is a 2'-phosphoramidite group, 3'-ODMTr, Nu = A, R = C 16 H 33 The preparation route refers to Examples 31 and 33, except that the raw material C of Example 31 is used to synthesize compound 5' from compound 4'. 12 H 25 Br changed to C 16 H 33 Br, the remaining steps are similar to those in Examples 31 and 33, and the structure and NMR data of the obtained nucleoside compound 189' are as follows:

[0651] 1 H NMR (400MHz, DMSO-d6) δ11.19 (d, J=4.0Hz, 1H), 8.71 (d, J=5.9Hz, 1H), 8.49 (d, J=29.9Hz, 1H), 8. 27 (d, J=7.6Hz, 2H), 7.35 (t, J=7.3Hz, 1H), 7.55 (t, J=7.6Hz, 2H), 7.37 (t, J=8.6Hz, 2H), 7.25-7.1 7 (m, 7H), 6.81-6.60 (m, 4H), 6.05 (dd, J=36.3, 3.4Hz, 1H), 4.75-4.33 (m, 2H), 3.89-3.37 (m, 15H) , 2.69-2.32(m, 2H), 1.62-1.45(m, 2H), 1.43-1.22(m, 26H), 1.13-0.79(m, 9H), 0.91-0.77(m, 6H). 31 P NMR (162MHz, DMSO-d6) δ149.29, 148.76.ESI-MS: m / z 1098.4[M+H] +

[0652] The structures of some compounds prepared by the above preparation method are shown in Table B below (nucleoside compounds of Formula 1-b).

[0653] Here, Nu represents a base or is absent.

[0654] Experimental part

[0655] The following experiments 1 to 7 are experiments on the nucleoside compound of formula 1-a, and the following experiments 8 to 13 are experiments on the nucleoside compound of formula 1-b.

[0656] Experiment 1: In vitro evaluation of siRNA sequence synthesis

[0657] The synthesis of siRNA is no different from the conventional phosphoramidite solid-phase synthesis method. When synthesizing modified nucleotides at various positions of the SS chain and AS chain, the above-mentioned synthesized phosphoramidite monomers are used to replace the original nucleotides of the parent sequence.

[0658] The synthesis process is briefly described as follows: On an LK-48E synthesizer (Lingkun), starting with the Universal CPG vector, nucleoside phosphoramidite monomers were linked one by one according to the synthesis procedure. With the exception of the syntheses of LT3-A, U, G, and C (corresponding to compounds 21, 11, 26, and 16, respectively) described above, the remaining nucleoside monomer raw materials, including 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activating agent (0.6 M in acetonitrile), a 0.22 M PADS solution in a 1:1 volume ratio with collidine (Suzhou Kelema) was used as the sulfurizing agent, and an iodine pyridine / water solution (Kelema) was used as the oxidizing agent.

[0659] After solid-phase synthesis, the oligoribonucleotide was cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The supernatant was then centrifuged and transferred to another centrifuge tube. After concentration and evaporation to dryness, the product was purified by C18 reverse-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected and lyophilized, identified as the target product by LCMS, and quantified by UV (260 nm).

[0660] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equal molar ratio, and the resulting double-stranded siRNA was dissolved in 1xPBS and adjusted to the required concentration for the experiment.

[0661] The modification schemes used in Table 1 are as follows:

[0662] "m" means the nucleotide adjacent to the left of the letter m is modified with 2'-OMe, "f" means the nucleotide adjacent to the left of the letter f is modified with 2'-F, "s" means the two nucleotides adjacent to the left and right of the letter s are modified with PS backbone, and "LT3" means 4'-(R)-CH2OC 12 H 25-TNA modification.

[0663] Among them, CM0412-0000 had no nucleoside compound modification in the positive chain compared with other groups and served as the control group.

[0664] Table 1: siRNA sequences used in in vitro screening

[0665] Experiment 2: In vitro silencing efficacy of TTR target mRNA and siRNA duplex containing LT3--A, U, G, C

[0666] Activity screening steps:

[0667] Cell culture and transfection:

[0668] Hepa1-6 cells (ATCC, product catalog number CRL1830) were cultured in DMEM (Invitrogen, product catalog number 11965092) containing 10% FBS (Ausgenex, product catalog number FBS500-S) and 1% penicillin-streptomycin at 37°C and 5% CO2 until close to confluence. The cells were then digested and resuspended in cell seeding medium (DMEM + 10% FBS), and 5000 cells / well / 90 μL were seeded into 96-well cell culture plates (Corning, product catalog number 3904) and cultured overnight at 37°C and 5% CO2. Transfection conditions were as follows: 5 μL of siRNA per well was mixed with 4.6 μL of Opti-MEM (Gibco, catalog number 1985-070) and 0.4 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, catalog number 13778-150) and incubated at room temperature for 15 minutes. This mixture was then added to 10 μL per well of a 96-well plate prepared the day before. Cultures were incubated at 37°C in 5% CO₂ for 48 hours. Final concentrations of siRNAs used in the experiments were 10 nM, 1 nM, and 0.1 nM.

[0669] RNA extraction and reverse transcription into cDNA:

[0670] Lyse cells and extract RNA according to the instructions for Cells-to-Ct bulk lysis reagents (Invitrogen, catalog number 4391851C). 1) Aspirate the old medium from each well and rinse twice with 100 μL of PBS. 2) Carefully aspirate the PBS. Add 50 μL of lysis buffer to each well. 3) Shake at 500 rpm at room temperature for 5 minutes. 4) Add 5 μL of stop solution to each well and shake at 500 rpm at room temperature for 2 minutes. If proceeding immediately with cDNA synthesis, place the plate on ice.

[0671] Reverse transcription was performed according to the instructions for Cells-to-CT Bulk Fast Advanced RT Reagent (Invitrogen, catalog number A39110): a mixture (per well: 25 μL 2×Fast Advanced RT Buffer, 2.5 μL 20×RT Fast Advanced Enzyme Mix, and 22.5 μL total RNA) was prepared. cDNA was synthesized using a Biometra TAdvanced 96SG (analytikjena) using the following steps: 37°C for 30 min, 95°C for 5 min, and 4°C hold. cDNA was stored at -20°C or immediately analyzed by real-time PCR.

[0672] Real-time PCR:

[0673] 4.33 μL of cDNA, 0.17 μL of 60X GAPDH TaqMan probe (Invitrogen, catalog number 4448491), 0.5 μL of 20X TTR TaqMan probe (Invitrogen, catalog number 4351370) and 5 μL of FAST PCR Master Mix (Applied Biosystems, catalog number 4444965) was added to a 96-well plate (Axygen, catalog number PCR-96-FLT-C) for real-time PCR detection. 7 (Applied Biosystems), the system program was: 50°C for 2 min, 95°C for 20 s, and 40 cycles of 90°C for 1 s, 60°C for 20 s. Unless otherwise stated, each siRNA was transfected in triplicate.

[0674] Data analysis: Real-time data were analyzed using the ΔΔCt method and normalized with the mock group data. The specific calculation method is: ΔCt = Ct (target gene) - Ct (GAPDH), ΔΔCt = ΔCt (test sample) - ΔCt (Mock), Relative mRNA expression to Mock = 2 -ΔΔCt , %Inhibition vs. Mock = {1-Expression fold (test sample) / Expression fold (Mock)} * 100. The final results are shown in Table 2.

[0675] Table 2: q-PCR in vitro activity screening data

[0676] Results: When the modifications (LT3-A, U, G, C) were added to the siRNA sequence, they were able to enhance the activity of the siRNA sequence. Even at a low concentration (0.1 nM), some sequences still had a silencing efficiency greater than 60%.

[0677] Experiment 3: Synthesis of single-stranded oligonucleotides for 3'-exonuclease stability evaluation

[0678] The synthesis of single-stranded oligonucleotides is the same as the conventional phosphoramidite solid phase synthesis method. When synthesizing single-stranded oligonucleotides with LT3 modification at the 3'-terminus, the above-synthesized phosphoramidite monomer (LT3) is used to replace the original nucleotide of the parent sequence.

[0679] The synthesis process is briefly described as follows: On an LK-48E synthesizer (Lingkun), starting with the Universal CPG (Dynaxin) vector, nucleoside phosphoramidite monomers were linked one by one according to the synthesis procedure. With the exception of the syntheses of LT3-A, U, G, and C (corresponding to compounds 21, 11, 26, and 16, respectively) described above, the remaining nucleoside monomer raw materials, including 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M in acetonitrile), a 0.22 M PADS solution in a 1:1 volume ratio with collidine (Suzhou Kelema) was used as the sulfurization reagent, and an iodine pyridine / water solution (Kelema) was used as the oxidant.

[0680] After solid-phase synthesis, the oligoribonucleotide was cleaved from the solid support and soaked in a 3:1 28% ammonia and ethanol solution at 50°C for 16 hours. The supernatant was then centrifuged and transferred to another centrifuge tube. After concentration and evaporation, it was purified by C18 reverse-phase chromatography with a mobile phase of 0.1M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected and lyophilized, identified as the target product by LCMS, and quantified by UV (260nm). The resulting single-stranded oligonucleotide was dissolved in 1xPBS and adjusted to the required concentration for the experiment.

[0681] The modification schemes used in Table 3 are as follows:

[0682] "m" means the nucleotide adjacent to the left of the letter m is modified with 2'-OMe, "f" means the nucleotide adjacent to the left of the letter f is modified with 2'-F, "s" means the two nucleotides adjacent to the left and right of the letter s are modified with PS backbone, and "LT3" means 4'-(R)-CH2OC 12 H 25 -TNA modification.

[0683] Among them, CM0412-1000 was not modified with nucleoside compounds compared with other groups and served as the control group.

[0684] Table 3: Oligonucleotide sequences used to investigate 3'-exonuclease stability

[0685] Experiment 4: 3'-Exonuclease Stability Test of Single-Stranded Sequences

[0686] Stability evaluation steps:

[0687] After the samples (oligonucleotide molecules CM0412-1000, CM0412-1001, and CM0412-1002) were dissolved in PBS (BOSTER, Lot. 17F24C21), the compound concentrations were determined using a microplate reader, and the working solution was diluted to 300 ng / μL using PBS.

[0688] The test samples (CM0412-1000, CM0412-1001, and CM0412-1002, see Table 3) were co-incubated with the enzyme system: 43 μL of the enzyme system (Sigma, Lot. P4506) and 7 μL of the sample to be tested were added to each tube (N=2 (two replicates per sample), vortexed, and incubated in a 37°C incubator. 50 μL of each tube from the first step was placed in another EP tube, and 5 μL of 0.5 M EDTA solution was added to each tube. Mix well, and immediately place in liquid nitrogen to rapidly cool the reaction to serve as a 0-hour self-control. The remaining samples were placed in a 37°C incubator. Samples were removed at various time points, and 5 μL of 0.5 M EDTA was added to each tube. Mix well, and immediately place in liquid nitrogen to rapidly cool the reaction to terminate the reaction.

[0689] Biological sample pretreatment: Prepare RNA extraction reagent: 25 mL of saturated phenol aqueous solution, 24 mL of chloroform, and 1 mL of isoamyl alcohol, and mix thoroughly. After thawing the frozen samples at room temperature, add 10 μL of internal standard compound (quality control compound, i.e., siRNA sequence: antisense strand: CmsUfsCmGmGfUmAfCmAmUmGmCfAmAfUmCmsCmsCm; sense strand: GmsGmGmAmUmUfGmCfAfUfGmUmAmAmCmCmGmsAmsGm) to each tube. Vortex mix and centrifuge for 10 minutes. The supernatant is then collected for high-resolution liquid chromatography-mass spectrometry analysis. The remaining sample volume at each time point is shown in Table 4 and Figure 1.

[0690] Table 4: Remaining amount of test samples at various time points

[0691] Results: When the modification (LT3) was added to the 3'-end of the single-stranded oligonucleotide, it could significantly improve the stability of the oligonucleotide single-stranded oligonucleotide against 3'-exonuclease.

[0692] The remaining LT0-LT2, LT4-LT106 nucleoside compounds have the same or similar sugar backbone structures and the same 2,3-phosphate linkage mode in the sequence, and their resistance to nucleases is also improved.

[0693] Experiment 5: Synthesis of siRNA sequences for in vivo evaluation in wild-type mice

[0694] The synthesis of siRNA is no different from the conventional phosphoramidite solid-phase synthesis method. When synthesizing modified nucleotides at various positions of the SS chain and AS chain, the above-mentioned synthesized phosphoramidite monomers are used to replace the original nucleotides of the parent sequence.

[0695] The synthesis process is briefly described as follows: On an LK-48E synthesizer (Lingkun), starting from the Universal CPG (Dynaxin) vector, nucleoside phosphoramidite monomers were linked one by one according to the synthesis procedure. Except for the above-described LT3-A, U, G, C (corresponding to compounds 21, 11, 26, and 16, respectively), LT0-A, U, G, C (corresponding to compounds 43, 33, 48, and 38, respectively), LT5-A, U, G, C (corresponding to compounds 65, 55, 70, and 60, respectively), LT7-A, U, G, C (corresponding to compounds 87, 77, 82, and 82, respectively), LT16-A (corresponding to compound 116), LT25-A (corresponding to compound 104), LT41-A (corresponding to compound 135), LT88-A (corresponding to compound 154), LT93-A (corresponding to compound 175), and LT97-A (corresponding to compound 205), the remaining nucleoside monomer raw materials, such as 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei, and L96 was purchased from WuXi AppTec. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M acetonitrile solution), 0.22 M PADS dissolved in a 1:1 volume ratio and collidine (Suzhou Kelema) solution was used as the sulfurization reagent, and iodine pyridine / water solution (Kelema) was used as the oxidant.

[0696] After solid-phase synthesis, the oligoribonucleotide was cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The supernatant was then centrifuged and transferred to another centrifuge tube. After concentration and evaporation to dryness, the product was purified by C18 reverse-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected and lyophilized, identified as the target product by LCMS, and quantified by UV (260 nm).

[0697] The obtained single-stranded oligonucleotide and sodium acetate were ultrafiltered through a 3KD ultrafiltration tube to exchange salt, and annealed according to the equimolar ratio and complementary pairing. Finally, the obtained double-stranded siRNA was dissolved in water and adjusted to the required concentration for use in the experiment.

[0698] The modification scheme used in Table 5 is as follows:

[0699] "m" means the nucleotide adjacent to the left of the letter m is modified with 2'-OMe, "f" means the nucleotide adjacent to the left of the letter f is modified with 2'-F, "s" means the two nucleotides adjacent to the left and right of the letter s are modified with PS backbone, and "LT3" means 4'-(R)-CH2OC 12 H 25 -TNA modification, "LT0" is 4'-(R)-CH2OMe-TNA modification, "LT5" is 4'-(R)-CH2OC 14 H 29 -TNA modification, "LT7" is 4'-(R)-CH2OC 16 H 33 -TNA modification, "LT1, LT2, LT4, LT6" and "LT8 to LT106" modifications are detailed in the corresponding compound structure list in the specific implementation method.

[0700] Among them, CM0412-2000 was used as the control group because its positive chain was not modified with nucleoside compounds compared with other groups.

[0701] Table 5: siRNA sequences used for in vivo evaluation in wild-type mice

[0702] Experiment 6: In vivo evaluation of the activity of TTR target mRNA and siRNA duplexes containing LT-4'-TNA modified nucleosides

[0703] Evaluation of siRNA sequence activity in wild-type mice:

[0704] The in vivo activity of the above compounds (CM0412-2000-2032, see Table 5) was evaluated using wild-type C57BL / 6 mice (Beijing SPIEF Biotechnology Co., Ltd.).

[0705] Six to eight-week-old C57BL / 6 mice were injected subcutaneously with a single 1 mg / kg dose of a compound (compounds listed in Table 5), and orbital blood was collected using an EP tube before dosing and on days 7, 14, 21, 28, 35, 42, and 56 after dosing. After the blood samples were allowed to stand at room temperature for two hours, they were centrifuged at 5500 rpm for 10 minutes at 4°C, and serum was separated and collected for the detection of TTR levels in the animal serum.

[0706] Serum TTR levels were analyzed using a TTR enzyme-linked immunosorbent assay (ELISA). The ELISA was performed using the Abcam Mouse Prealbumin ELISA Kit (ab282297), and all sample testing was performed according to the kit instructions. The absorbance at 450 nm was read on a Tecan SPARK microplate reader, and the data from the standard (from the ELISA kit) were fitted to a parametric standard curve to determine serum TTR protein levels (in μg / mL). The protein content of each animal was compared with its corresponding pre-dose serum protein content to determine the percentage of TTR remaining relative to pre-dose levels.

[0707] The experimental results are shown in Figure 2.

[0708] Results: This type of modified nucleoside compound can enhance the silencing activity of siRNA sequences in vivo and has a certain long-term effect.

[0709] Experiment 7: Evaluation of liver exposure of wild-type mice with TTR target mRNA and siRNA duplexes containing LT-4'-TNA modified nucleosides

[0710] Liver exposure evaluation in wild-type mice:

[0711] Wild-type C57BL / 6 mice (Beijing Biotechnology Co., Ltd.) were used to compare the exposure levels of the above compounds (Table 5) in the liver.

[0712] 6-8 week old C57BL / 6 mice were subcutaneously injected with a single dose of 3 mg / kg of the compound (CM0412-2001). The mice were killed 1h, 4h, 8h, 24h, Day 3, Day 7, and Day 14 after administration, and the liver tissues were collected after autopsy.

[0713] After dissection, the liver was harvested, and the median lobe was used for experiments. The harvested liver tissue was homogenized in a 24-bead tissue homogenizer (Bertin Technologies (Montigny-le-Bretonneux, France)) using a homogenization buffer (100 mM Tris and 1 mM MgCl2, pH 6.0) at a ratio of 200 mg of liver tissue to 1 mL of homogenization buffer.

[0714] Before SPE sample preparation, all samples were spiked with an internal standard at a concentration of 2.5 mg / mL (1 mL of a 0.5 mg / mL internal standard solution was added to 200 mL of liver homogenate per well). Samples were processed by solid-phase extraction (96-well Clarity OTX SPE plates (100 mg / well), purchased from Phenomenex (Torrance, CA, USA)) and analyzed by liquid chromatography-mass spectrometry (LC-MS) using Waters ACQUITY Premier, ACQUITY Premier Oligonucleotide BEH C18, and Waters ACQUITY Premier+Xevo TQ-Absolute LC-MS.

[0715] The liver exposure was calculated based on the measured drug concentrations at each time point, and the exposure was compared between the compounds.

[0716] The operation of solid phase extraction is specifically referred to the instructions. The buffer involved is prepared at room temperature. The information is as follows:

[0717] Equilibration buffer: 50 mM NaH2PO4, 2 mM NaN3, 10 mM K2EDTA in water (pH = 5.5)

[0718] Wash buffer 1: 50 mM NaH2PO4 in 50:50 (v:v) water:acetonitrile (pH = 5.5)

[0719] Wash buffer 2: 50 mM NH4HCO3 in 20:80 (v:v) water:acetonitrile (pH=5.5)

[0720] Elution buffer: 100 mM NH4HCO3, 10 mM TCEP in 50:40:10 (v:v:v) water: acetonitrile: tetrahydofuran (pH = 9)

[0721] All buffers were pH adjusted using ammonium hydroxide or glacial acetic acid.

[0722] The experimental results are shown in Figure 3.

[0723] Results: This type of modified nucleoside compound can maintain or increase the exposure of siRNA sequences in mouse liver.

[0724] Experiment 8: In vitro evaluation of siRNA sequence synthesis

[0725] The synthesis of siRNA is no different from the conventional phosphoramidite solid-phase synthesis method. When synthesizing modified nucleotides at various positions of the SS chain and AS chain, the above-mentioned synthesized phosphoramidite monomers are used to replace the original nucleotides of the parent sequence.

[0726] The synthesis process is briefly described as follows: Nucleoside phosphoramidite monomers were linked one by one according to the synthesis procedure, starting with the Universal CPG vector on an LK-48E synthesizer (Lingkun). With the exception of the syntheses of DT3-A (corresponding to compound 21'), U (corresponding to compound 11'), G (corresponding to compound 26'), and C (corresponding to compound 16'), DT0-A (corresponding to compound 43'), U (corresponding to compound 33'), G (corresponding to compound 48'), and C (corresponding to compound 38'), and DT10-A (corresponding to compound 66'), U (corresponding to compound 56'), G (corresponding to compound 71'), and C (corresponding to compound 61') described above, all other nucleoside monomer raw materials, including 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M acetonitrile solution), 0.22 M PADS dissolved in a 1:1 volume ratio and collidine (Suzhou Kelema) solution was used as the sulfurization reagent, and iodine pyridine / water solution (Kelema) was used as the oxidant.

[0727] After solid-phase synthesis, the oligoribonucleotide was cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The supernatant was then centrifuged and transferred to another centrifuge tube. After concentration and evaporation to dryness, the product was purified by C18 reverse-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected and lyophilized, identified as the target product by LCMS, and quantified by UV (260 nm).

[0728] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equal molar ratio, and the resulting double-stranded siRNA was dissolved in 1xPBS and adjusted to the required concentration for the experiment.

[0729] The modification scheme used in Table 6 is as follows:

[0730] "m" means the nucleotide adjacent to the left of the letter m is modified with 2'-OMe, "f" means the nucleotide adjacent to the left of the letter f is modified with 2'-F, "s" means the two nucleotides adjacent to the left and right of the letter s are modified with PS backbone, and "DT3" means 4'-(S)-CH2OC 12 H 25 -TNA modification.

[0731] Among them, DM0412-0000 had no nucleoside compound modification in the positive chain compared with other groups and served as the control group.

[0732] Table 6: siRNA molecule sequences used in in vitro screening

[0733] Experiment 9: In vitro silencing efficacy of TTR target mRNA with siRNA duplex containing DT3-Nu

[0734] Activity screening steps:

[0735] Cell culture and transfection:

[0736] Hepa1-6 cells (ATCC, product catalog number CRL1830) were cultured in DMEM (Invitrogen, product catalog number 11965092) containing 10% FBS (Ausgenex, product catalog number FBS500-S) and 1% penicillin-streptomycin at 37°C and 5% CO2 until close to confluence. The cells were then digested and resuspended in cell seeding medium (DMEM + 10% FBS) and 5000 cells / well / 90 μL were seeded into 96-well cell culture plates (Corning, product catalog number 3904) and cultured overnight at 37°C and 5% CO2. Transfection conditions were as follows: 4.6 μL of Opti-MEM (Gibco, catalog number 31985-070) and 0.4 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, catalog number 13778-150) were mixed with 5 μL of siRNA per well and incubated at room temperature for 15 minutes. This mixture was then added to 10 μL per well of a 96-well plate prepared the day before. Cultures were incubated at 37°C in 5% CO₂ for 48 hours. Final concentrations of siRNAs used in the experiments were 10 nM, 1 nM, and 0.1 nM.

[0737] RNA extraction and reverse transcription into cDNA:

[0738] Lyse cells and extract RNA according to the instructions for Cells-to-Ct bulk lysis reagents (Invitrogen, catalog number 4391851C). 1) Aspirate the old medium from each well and rinse twice with 100 μL of PBS. 2) Carefully aspirate the PBS. Add 50 μL of lysis buffer to each well. 3) Shake at 500 rpm at room temperature for 5 minutes. 4) Add 5 μL of stop solution to each well and shake at 500 rpm at room temperature for 2 minutes. If proceeding immediately with cDNA synthesis, place the plate on ice.

[0739] Reverse transcription was performed according to the instructions for Cells-to-CT Bulk Fast Advanced RT Reagent (Invitrogen, catalog number A39110): a mixture (per well: 25 μL 2×Fast Advanced RT Buffer, 2.5 μL 20×RT Fast Advanced Enzyme Mix, and 22.5 μL total RNA) was prepared. cDNA was synthesized using a Biometra TAdvanced 96SG (analytikjena) using the following steps: 37°C for 30 min, 95°C for 5 min, and 4°C hold. cDNA was stored at -20°C or immediately analyzed by real-time PCR.

[0740] Real-time PCR:

[0741] 4.33 μL of cDNA, 0.17 μL of 60X GAPDH TaqMan probe (Invitrogen cat, cat#4448491), 0.5 μL of 20X TTR TaqMan probe (Invitrogen, product catalog number 4351370) and 5 μL FAST PCR Master Mix (Applied Biosystems, catalog number 4444965) was added to a 96-well plate (Axygen, catalog number PCR-96-FLT-C) for real-time PCR detection. 7 (Applied Biosystems), the system program was: 50°C for 2 min, 95°C for 20 s, and 40 cycles of 90°C for 1 s, 60°C for 20 s. Unless otherwise stated, each siRNA was transfected in triplicate.

[0742] Data analysis: Real-time data were analyzed using the ΔΔCt method and normalized with the mock group data. The specific calculation method is: ΔCt = Ct (target gene) - Ct (GAPDH), ΔΔCt = ΔCt (test sample) - ΔCt (Mock), Relative mRNA expression to Mock = 2 -ΔΔCt , %Inhibition vsMock = {1-Expression fold (test sample) / Expression fold (Mock)} * 100. The final results are shown in Table 7.

[0743] Table 7: q-PCR in vitro activity screening data

[0744] Results: When these modifications (DT0-A, U, G, C, DT3-A, U, G, C, and DT10-A, U, G, C) were added to siRNA sequences, they enhanced their activity. Even at a low concentration (0.1 nM), some sequences still exhibited greater than 60% silencing efficiency.

[0745] Experiment 10: Synthesis of single-stranded oligonucleotides for 3'-exonuclease stability evaluation

[0746] The synthesis of single-stranded oligonucleotides is the same as the conventional phosphoramidite solid phase synthesis method. When synthesizing single-stranded oligonucleotides with DT3 modification at the 3'-terminus, the above-synthesized phosphoramidite monomer (DT3) is used to replace the original nucleotide of the parent sequence.

[0747] The synthesis process is briefly described as follows: Nucleoside phosphoramidite monomers were linked one by one according to the synthesis protocol, starting with the Universal CPG (Dynaxin) vector on an LK-48E synthesizer (Lingkun). With the exception of the synthesis of DT3-U (corresponding to compound 11') described above, all other nucleoside monomer starting materials, including 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M in acetonitrile), a 0.22 M PADS solution in a 1:1 volume ratio with collidine (Suzhou Kelema) was used as the sulfurization reagent, and an iodine pyridine / water solution (Kelema) was used as the oxidant.

[0748] After solid-phase synthesis, the oligonucleotide was cleaved from the solid support and soaked in a 3:1 28% ammonia and ethanol solution at 50°C for 16 hours. The supernatant was then centrifuged and transferred to another centrifuge tube. After concentration and evaporation, it was purified by C18 reverse phase chromatography with a mobile phase of 0.1M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected and lyophilized, identified as the target product by LCMS, and quantified by UV (260nm). The resulting single-stranded oligonucleotide was dissolved in 1xPBS and adjusted to the required concentration for use in the experiment.

[0749] The modification scheme used in Table 8 is as follows:

[0750] "m" means the nucleotide adjacent to the left of the letter m is modified with 2'-OMe, "f" means the nucleotide adjacent to the left of the letter f is modified with 2'-F, "s" means the two nucleotides adjacent to the left and right of the letter s are modified with PS backbone, and "DT3" means 4'-(S)-CH2OC 12 H 25 -TNA modification.

[0751] Among them, DM0412-0000 was not modified with nucleoside compounds compared with other groups and served as the control group.

[0752] Table 8: Oligonucleotide sequences used to investigate 3'-exonuclease stability

[0753] Experiment 11: 3'-Exonuclease Stability Test of Single-Stranded Sequences

[0754] Stability evaluation steps:

[0755] After the samples (oligonucleotide molecules DM0412-1000, DM0412-1001, and DM0412-1002) were dissolved in PBS (BOSTER, Lot. 17F24C21), the compound concentrations were determined using a microplate reader, and the working solution was diluted to 300 ng / μL using PBS.

[0756] The test samples (DM0412-1000, DM0412-1001, DM0412-1002, see Table 8) were co-incubated with the enzyme system: 43 μL of the enzyme system (Sigma, Lot. P4506) and 7 μL of the test sample (two replicates for each sample) were added to the EP tube, vortexed to mix, and incubated in a 37°C incubator.

[0757] Termination of the reaction: At each time point of the stability study, 50 μL was taken out from the incubation tube and placed in another EP tube. 5 μL of EDTA (0.5 M) solution was added, mixed, and immediately placed in liquid nitrogen for rapid cooling to terminate the reaction.

[0758] Biological sample pretreatment: After thawing the frozen samples at room temperature, 10 μL of internal standard compound (quality control compound, i.e., siRNA sequence: antisense chain: CmsUfsCmGmGfUmAfCmAmUmGmCfAmAfUmCmsCmsCm; sense chain: GmsGmGmAmUmUfGmCfAfUfGmUmAmAmCmCmGmsAmsGm) was added to each tube. 150 μL of Tris and 150 μL of extraction solvent (phenol:chloroform:isoamyl alcohol = 25:24:1) were added. After vortex mixing, the tubes were centrifuged for 10 minutes. The supernatant was collected for high-resolution liquid chromatography-mass spectrometry analysis. The remaining amount of test sample at each time point is shown in Table 9.

[0759] Table 9: Remaining amount of test samples at various time points

[0760] Results: When the modification (DT3-U) was added to the 3'-end of the single-stranded oligonucleotide, it could significantly improve the stability of the oligonucleotide single-stranded oligonucleotide against 3'-exonuclease.

[0761] Experiment 12: Synthesis of siRNA sequences for in vivo evaluation in wild-type mice

[0762] The synthesis process is briefly described as follows: Nucleoside phosphoramidite monomers were linked one by one according to the synthesis procedure, starting with the Universal CPG (Dynaxin) vector on an LK-48E synthesizer (Lingkun). With the exception of the syntheses of DT3-A (corresponding to compound 21'), U (corresponding to compound 11'), G (corresponding to compound 26'), C (corresponding to compound 16'), DT0-A (corresponding to compound 43'), U (corresponding to compound 33'), DT5-A (corresponding to compound 188'), DT7-A (corresponding to compound 189'), and DT10-A (corresponding to compound 66') described above, all other nucleoside monomer raw materials, including 2'-F RNA and 2'-O-methyl RNA, were purchased from Shanghai Zhaowei, and L96 was purchased from WuXi AppTec. 5'-Ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M acetonitrile solution), 0.22 M PADS dissolved in a 1:1 volume ratio and collidine (Suzhou Kelema) solution was used as the sulfurization reagent, and iodine pyridine / water solution (Kelema) was used as the oxidant.

[0763] After solid-phase synthesis, the oligoribonucleotide was cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The supernatant was then centrifuged and transferred to another centrifuge tube. After concentration and evaporation to dryness, the product was purified by C18 reverse-phase chromatography using a mobile phase of 0.1 M TEAA and acetonitrile. DMTr was removed using a 3% trifluoroacetic acid solution. The target oligonucleotide was collected and lyophilized, identified as the target product by LCMS, and quantified by UV (260 nm).

[0764] The obtained single-stra...

Claims

1. A nucleoside compound represented by formula (1), or a stereoisomer thereof: in: R 1 , R 2 , R 3 , R 4 Each is independently selected from hydrogen, halogen, cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; R 5 is selected from hydrogen, halogen, cyclopropane, cyclobutane, C1-C30 alkyl, substituted C1-C30 alkyl, C1-C30 alkoxy, substituted C1-C30 alkoxy, C2-C30 alkenyl, substituted C2-C30 alkenyl, C2-C30 alkynyl or substituted C2-C30 alkynyl; Nu represents base or absence; Z is -O-, -S-, or -NR 10 or -CR 10 R 11 , where R 10 and R 11 are each independently selected from hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle or substituted or unsubstituted cycloalkyl; X1, X2 are each independently selected from -(CH2) n O- or -(CH2) n S-, where n is any integer from 0 to 10; X3 is selected from -(CH2) n O-, -(CH2) n -、-(CH2) n S-, -(CH2) n NH(CO)-、-(CH2) n (CO)NH- or -(CH2) n O(CH2) n -A-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclyl or a substituted or unsubstituted cycloalkyl, and n is any integer from 0 to 10.

2. The nucleoside compound according to claim 1, which is a compound selected from Formula 1-a or 1-b: in, R 1 , R 2 , R 3 , R 4 , R 5 , Nu, X1, X2 and X3 are as defined in claim 1.

3. The nucleoside compound of formula 1-a or 1-b according to claim 2, wherein: Z is -O-, -S- or -N(COR')-, wherein R' is a C1-C5 alkyl group; preferably, R' is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl; X1, X2 are each independently selected from -(CH2) n O- or -(CH2) n S-, wherein n is any integer from 0 to 5; preferably, n is selected from 0, 1, 2, 3, 4 or 5; X3 is selected from -(CH2) n -、-(CH2) n O-, -(CH2) n S-, -(CH2) n NH(CO)-、-(CH2) n (CO)NH- or -(CH2) n O(CH2) n -A-, wherein A is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted heterocyclyl or a substituted or unsubstituted cycloalkyl, and wherein n is any integer from 0 to 5; preferably, n is selected from 0, 1, 2, 3, 4 or 5.

4. The nucleoside compound according to any one of claims 1 to 3, wherein: Z is -O-, -S- or -N(COCH3)-; X1 is -O-, -S- or -CH2O-; X2 is -O- or -S-; X3 is -O-, -CH2-, -CH2O-, -CH2S-, -CH2NH(CO)-, -(CO)NH- or -CH2OCH2-heteroaryl-; preferably, X3 is -O-, -CH2O-, -CH2S-, -CH2NH(CO)-, -(CO)NH- or -CH2OCH2-heteroaryl-; R 1 , R 2 , R 3 is hydrogen; R 4 is hydrogen or methoxy; R 5 is hydrogen, methyl, halogen, or C10-C18 alkyl; preferably, R 5 is methyl or C10-C18 alkyl; more preferably, R 5 is methyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl or C18 alkyl; more preferably, R 5 is methyl, C10 straight chain alkyl, C11 straight chain alkyl, C12 straight chain alkyl, C13 straight chain alkyl, C14 straight chain alkyl, C15 straight chain alkyl, C16 straight chain alkyl, C17 straight chain alkyl or C18 straight chain alkyl.

5. The nucleoside compound according to any one of claims 1 to 4, wherein Nu is a base, and the base is selected from:

6. The nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 2-a or Formula 2-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

7. The nucleoside compound represented by formula 1-a according to any one of claims 2 to 5, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 3-a, formula 5-a, formula 8-a or formula 9-a, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

8. The nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 4-a or Formula 3-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

9. The nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 6-a or Formula 4-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

10. The nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 7-a or Formula 5-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

11. The nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 10-a or Formula 6-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

12. [Corrected 18.10.2024 according to Rule 91] A nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 11-a or Formula 7-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; R' is a C1-C5 alkyl group; preferably, R' is a methyl group, an ethyl group, a n-propyl group or an isopropyl group; Nu is as defined in claim 1.

13. [Corrected 18.10.2024 according to Rule 91] A nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 12-a or 8-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group or a C18 alkyl group.

14. [Corrected 18.10.2024 according to Rule 91] A nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 13-a or Formula 9-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

15. [Corrected 18.10.2024 according to Rule 91] A nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 14-a or Formula 10-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

16. [Corrected 18.10.2024 according to Rule 91] A nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 15-a or Formula 11-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

17. [Corrected 18.10.2024 according to Rule 91] A nucleoside compound represented by Formula 1-a or Formula 1-b according to any one of claims 2 to 5, wherein: The nucleoside compound represented by Formula 1-a or Formula 1-b is respectively a nucleoside compound represented by Formula 16-a or 12-b, R is a C1-C30 alkyl group, a substituted C1-C30 alkyl group, a C1-C30 alkoxy group, a substituted C1-C30 alkoxy group, a C2-C30 alkenyl group, a substituted C2-C30 alkenyl group, a C2-C30 alkynyl group, or a substituted C2-C30 alkynyl group; preferably, R is a methyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, or a C18 alkyl group; Nu is as defined in claim 1.

18. [Corrected 18.10.2024 according to Rule 91] The nucleoside compound represented by formula 1-a according to claim 2 or 3, wherein The nucleoside compound represented by formula 1-a is the nucleoside compound shown below: wherein Nu is as defined in claim 1.

19. [Corrected 18.10.2024 according to Rule 91] The nucleoside compound represented by formula 1-a according to claim 2 or 3, wherein The nucleoside compound represented by formula 1-a is the nucleoside compound shown below:

20. [Corrected 18.10.2024 according to Rule 91] The nucleoside compound represented by formula 1-b according to claim 2 or 3, wherein The nucleoside compound represented by formula 1-b is the nucleoside compound shown below: wherein Nu is as defined in claim 1.

21. [Corrected 18.10.2024 according to Rule 91] The nucleoside compound of formula 1-b according to claim 2 or 3, wherein The nucleoside compound represented by formula 1-b is the nucleoside compound shown below:

22. [Corrected as of 18.10.2024 in accordance with Rule 91] An oligonucleotide, wherein the oligonucleotide is prepared using at least one nucleoside compound represented by Formula 1-a or Formula 1-b as described in any one of claims 2 to 21 as an intermediate; preferably, the oligonucleotide is siRNA, antisense nucleic acid, saRNA, miRNA, or nucleic acid aptamer.

23. [Corrected 18.10.2024 according to Rule 91] The oligonucleotide according to claim 22, wherein the nucleoside compound has a structure of Formula 17-a or Formula 18-a in the oligonucleotide, and is Connect to the rest of the oligonucleotide, Where R 1 , R 2 , R 3 , R 4 , R 5 , Z, X1, X2, X3 and Nu are as defined in claim 1; Preferably, The oligonucleotide is siRNA; The nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA sense strand; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st nucleotide at the 5' end of the siRNA sense strand; Alternatively, the nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA antisense chain; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

24. [Corrected 18.10.2024 according to Rule 91] The oligonucleotide according to claim 22, wherein the nucleoside compound has a structure of Formula 13-b or Formula 14-b in the oligonucleotide, and is Connect to the rest of the oligonucleotide, Where R 1 , R 2 , R 3 , R 4 , R 5 , Z, X1, X2, X3 and Nu are as defined in claim 1; Preferably, The oligonucleotide is siRNA; The nucleoside compound has a structure such as Formula 13-b or Formula 14-b in the siRNA sense strand; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st nucleotide at the 5' end of the siRNA sense strand; Alternatively, the nucleoside compound has a structure of Formula 13-b or Formula 14-b in the siRNA antisense chain; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

25. [Corrected on 18.10.2024 according to Rule 91] Use of a nucleoside compound represented by Formula 1-a or Formula 1-b as claimed in any one of claims 2 to 21 for preparing an oligonucleotide.

26. [Corrected 18.10.2024 according to Rule 91] The use according to claim 25, wherein the nucleoside compound has the structure of Formula 17-a or Formula 18-a in the oligonucleotide, and is Connect to the rest of the oligonucleotide, Where R 1 , R 2 , R 3 , R 4 , R 5 , Z, X1, X2, X3 and Nu are as defined in claim 1; Preferably, The oligonucleotide is siRNA; The nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA sense strand; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st nucleotide at the 5' end of the siRNA sense strand; Alternatively, the nucleoside compound has a structure such as Formula 17-a or Formula 18-a in the siRNA antisense chain; more preferably, the structure of Formula 17-a or Formula 18-a is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 17-a or Formula 18-a is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

27. [Corrected 18.10.2024 according to Rule 91] The use according to claim 25, wherein the nucleoside compound has the structure of Formula 13-b or Formula 14-b in the oligonucleotide, and is Connect to the rest of the oligonucleotide, Where R 1 , R 2 , R 3 , R 4 , R 5 , Z, X1, X2, X3 and Nu are as defined in claim 1; Preferably, The oligonucleotide is siRNA; The nucleoside compound has a structure such as Formula 13-b or Formula 14-b in the siRNA sense strand; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th or 21st nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 12th, 13th, 14th or 15th nucleotide at the 5' end of the siRNA sense strand; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st nucleotide at the 5' end of the siRNA sense strand; Alternatively, the nucleoside compound has a structure of Formula 13-b or Formula 14-b in the siRNA antisense chain; more preferably, the structure of Formula 13-b or Formula 14-b is the 1st, 2nd, 3rd, 4th, 5th or 6th nucleotide at the 3' end of the siRNA antisense chain; further preferably, the structure of Formula 13-b or Formula 14-b is the 1st or 2nd nucleotide at the 3' end of the siRNA antisense chain.

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