Modified nucleoside compound, oligonucleotide prepared from modified nucleoside compound and application of modified nucleoside compound

Abstract

The invention provides a nucleoside phosphoramidite compound, which is a compound of a 2 '-phosphoramidite group and 3'-modified 5 '-(E)-vinyl phosphate. The nucleoside phosphoramidite compounds of the invention can be used for incorporation into a terminal position of an oligonucleotide such that the modified oligonucleotide hybridizes to a portion of the target mRNA, resulting in loss or down-regulation of normal function of the target mRNA.

Description

Technical Field

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

[0002] Oligonucleotides are polymers of nucleotides. As nucleic acid inhibitor molecules, oligonucleotides can regulate intracellular mRNA levels and have shown early promise in the treatment of genetic diseases, metabolic diseases, cancers, and viral infectious diseases. Nucleic acid inhibitor molecules can regulate mRNA expression via a set of different mechanisms, including RNA interference (RNAi).

[0003] RNAi is a conserved pathway found in most eukaryotes, in which double-stranded RNA molecules (dsRNA) inhibit the expression of target genes having a complementary sequence to the dsRNA. In a typical RNAi pathway, longer dsRNA is cleaved by an endonuclease (Dicer) into shorter RNA duplexes called small interfering RNAs ("siRNAs"). It has been demonstrated that siRNAs associate with endonucleases, trans-activation response RNA-binding proteins (TRBP), and Argonaute 2 ("Ago2") to form a complex, sometimes referred to as the RNA-induced silencing complex ("RISC"). Ago2 is an endonuclease that uses the antisense strand (also called the guide strand) of the siRNA to direct the sequence-specific cleavage of the target mRNA to cleave the target mRNA.

[0004] Over the years, a variety of double-stranded RNAi inhibitor molecule structures have been developed. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules that mimic natural siRNAs, where each strand has 19-25 nucleotides and includes at least one 3' overhang of 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed that are processed in vivo by endonucleases into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work has developed extended double-stranded nucleic acid inhibitor molecules, where at least one end of at least one strand extends beyond the double-stranded targeting region of the molecule, and one of the strands 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 WO2016 / 100401). Those structures include single-stranded extension structures (on one or both sides of the molecule) and double-stranded extension structures.

[0005] Single-stranded nucleic acid inhibitor molecules are also known in the art. For example, recent results have demonstrated the activity of ssRNAi inhibitor molecules (see, e.g., Matsui et al., 2016, 24(5):946-55). Also, antisense oligonucleotide molecules have been used for decades to reduce the expression of specific target genes. Pelechano and Steinmetz, Nature Review Genetics, 2013, 14:880-93, have developed many variations on these structural common themes for a range of targets. Other single-stranded nucleic acid inhibitor molecules include, for example, miRNAs, ribozymes, antagomirs, and aptamers, all of which are known in the art.

[0006] In some cases, chemical modifications have been introduced into nucleic acid inhibitor molecules to confer properties that may be desirable under specific conditions, such as those experienced after in vivo administration. These modifications include modifications designed to, for example, stabilize against nucleases, or other enzymes that degrade / affect the structure or activity of the oligonucleotide, increase cellular uptake of the oligonucleotide, or improve the pharmacokinetic properties of the oligonucleotide.

[0007] For example, synthetic oligonucleotides generally terminate with a 5'-hydroxyl or 3'-hydroxyl. It is possible to replace the terminal hydroxyl with a phosphate group, which can be used, for example, to attach a linker, adaptor, or label or to directly ligate the oligonucleotide to another nucleic acid. Additionally, it has been reported that a 5'-terminal phosphate group enhances the interaction between certain nucleic acid inhibitor molecules (see patents or articles: Cell 2012, 150, 883-894; Nucleic Acids Res. 2015, 43, 2993-3011; CN 109526222; US2023 / 0000895; WO2023003805; US11560563; WO2023003995; US20230020192; US20230024926; US11566248; WO2022 / 204429; WO2023039005; CN 113412268; CN113412268; CN109526222; WO2023003805, etc.) and Ago2. However, oligonucleotides with phosphate groups are generally prone to degradation via phosphatases or other enzymes, which may limit their bioavailability in vivo.

[0008] Accordingly, it is desirable to develop modifications to the 5'-terminal nucleotide of oligonucleotides, such as nucleic acid inhibitor molecules, that provide a functional role for the phosphate group, but that render the oligonucleotide more stable to the ambient conditions to which it is exposed when administered to a subject. These phosphate-modified nucleotides will be more resistant to phosphatases and other enzymes, while minimizing any negative impact on the function of the oligonucleotide (e.g., when used in an RNAi inhibitor molecule, minimizing any negative impact on knocking down the target gene). SUMMARY OF THE INVENTION

[0009] In view of the deficiencies of the prior art, the present invention aims to develop a nucleoside compound that can effectively enhance the activity of an oligonucleotide molecule (including but not limited to siRNA, antisense nucleic acid, saRNA (small activating nucleic acid), miRNA (microRNA), nucleic acid aptamer, etc.) when applied at one terminal position (preferably the 5'-position) of the oligonucleotide molecule, improve the corresponding target gene silencing efficiency, and simultaneously improve the resistance of the corresponding oligonucleotide to enzymatic cleavage by phosphatases, exonucleases, endonucleases, etc.

[0010] The present invention provides an oligonucleotide, which is a single-stranded or double-stranded nucleotide molecule, and each nucleotide in the single-stranded or double-stranded nucleotide molecule is a modified or unmodified nucleotide, wherein the double-stranded nucleotide molecule has 16 - 30 nucleotides; the antisense strand of the double-stranded nucleotide molecule is at least 14 nucleotides reverse complementary to a sequence in the target mRNA; in the direction from the 5'-terminal to the 3'-terminal, the 5'-terminal of the antisense strand of the double-stranded nucleotide molecule is replaced by a nucleotide of 2'-phosphoramidite group, 3'-modification, 5'-(E)-vinyl phosphate.

[0011] The double-stranded RNAi inhibitor molecule comprises an antisense strand and a sense strand. The antisense strand or a region thereof is partially, substantially or completely complementary to the corresponding region of the target nucleic acid. In addition, the antisense strand or a region thereof of the double-stranded RNAi inhibitor molecule is partially, substantially or completely complementary to the sense strand or a region thereof of the double-stranded RNAi inhibitor molecule. In certain embodiments, the antisense strand may also contain non-complementary nucleotides to the target nucleic acid. The non-complementary nucleotides may be on either side of the complementary sequence or may be on both sides of the complementary sequence. In certain embodiments, when the antisense strand or a region thereof is partially or substantially complementary to the sense strand or a region thereof, the non-complementary nucleotides may be located between one or more complementary regions, such as one or more mismatches. The antisense strand of the double-stranded RNAi inhibitor molecule is also referred to as the guide strand.

[0012] One object of the present invention is to provide a nucleoside compound of formula 1-a (nucleoside phosphoramidite compound).

[0013]

[0014] Wherein:

[0015] R 1 、R 2 、R 3 are each independently selected from C3-C10 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, or aminoacyl or substituted aminoacyl (preferably alkyl-substituted aminoacyl, for example, methylcarbamoyl, ethylcarbamoyl, etc.);

[0016] Nu is a base;

[0017] Z is -O-, -S- or -CR 4 R 5 , wherein R 4 and R 5 are each independently hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic group, or substituted or unsubstituted cycloalkyl;

[0018] X1 and X2 are each independently selected from -(CH2) n O-, or -(CH2) n S-, wherein n is any integer from 0 to 10 (for example, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0019] Y is O or S.

[0020] For the nucleoside compound represented by Formula 1-a according to the present invention, wherein R 1 、R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; preferably, R 1 、R 2 are each independently selected from C1-C6 alkyl, or substituted C1-C6 alkyl; more preferably, R 1 、R 2 are each independently selected from C1-C3 alkyl, or substituted C1-C3 alkyl; most preferably, R 1 、R 2 are each independently selected from methyl or ethyl.

[0021] For the nucleoside compound represented by Formula 1-a according to the present invention, wherein R 3is a C1-C6 alkyl group, a C1-C6 alkoxy group, or an aminoacyl group or a substituted aminoacyl group; preferably, R 3 is a C1-C3 alkyl group, a C1-C3 alkoxy group, or an alkyl-substituted aminoacyl group; more preferably, R 3 is methyl, ethyl, n-propyl, isopropyl, methoxy, methylcarbamoyl, or ethylcarbamoyl; most preferably, R 3 is methyl, methoxy, or methylcarbamoyl.

[0022] For the nucleoside compound represented by Formula 1-a according to the present invention, wherein X1 is -(CH2) n O-, or -(CH2) n S-, and n is any integer from 0 to 5; preferably, X1 is -(CH2) n O-, or -(CH2) n S-, and n is any integer from 0 to 3, more preferably, X1 is -(CH2) n O-, or -(CH2) n S-, and n is any integer from 0 to 2; most preferably, X1 is -O-, -CH2O-, -(CH2)2O-, or -S-.

[0023] For the nucleoside compound represented by Formula 1-a according to the present invention, wherein X2 is -(CH2) n O-, or -(CH2) n S-, and n is any integer from 0 to 5; preferably, X2 is -(CH2) n O-, or -(CH2) n S-, and n is any integer from 0 to 3, more preferably, X2 is -(CH2) n O-, or -(CH2) n S-, and n is any integer from 0 to 2; most preferably, X2 is -O- or -S-.

[0024] For the nucleoside compound represented by Formula 1-a according to the present invention, wherein Z is -O- or -S-.

[0025] For the nucleoside compound represented by Formula 1-a according to the present invention, wherein the base is selected from any one of the following bases:

[0026]

[0027] For the nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 2-a,

[0028]

[0029] Among them,

[0030] R 1 and R 2 each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0031] Nu is as defined above.

[0032] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 3-a,

[0033]

[0034] Among them,

[0035] R 1 and R 2 each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0036] Nu is as defined above.

[0037] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 4-a,

[0038]

[0039] Among them,

[0040] R 1 and R 2 each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl:

[0041] Nu is as defined above.

[0042] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 5-a,

[0043]

[0044] Wherein,

[0045] R 1 and R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0046] Nu is as defined above.

[0047] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 6-a,

[0048]

[0049] Wherein,

[0050] R 1 and R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0051] Nu is as defined above.

[0052] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 7-a,

[0053]

[0054] Wherein,

[0055] R 1 and R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0056] Nu is as defined above.

[0057] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 8-a,

[0058]

[0059] Wherein,

[0060] R 1 and R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0061] Nu is as defined above.

[0062] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 9-a,

[0063]

[0064] Wherein,

[0065] R 1 and R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0066] Nu is as defined above.

[0067] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 10-a,

[0068]

[0069] Wherein,

[0070] R 1 and R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0071] Nu is as defined above.

[0072] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds represented by Formula 11-a,

[0073]

[0074] Wherein,

[0075] R 1 、R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0076] Nu is as defined above.

[0077] The nucleoside compound represented by Formula 1-a according to the present invention, wherein the nucleoside compound represented by Formula 1-a of the present invention is selected from the nucleoside compounds shown below:

[0078]

[0079]

[0080] Another object of the present invention is to provide an oligonucleotide, wherein one terminal position (preferably the 5'-position) of the oligonucleotide is modified with the nucleoside compound represented by Formula 1-a.

[0081] The oligonucleotide according to the present invention, wherein the nucleoside compound has the following structure in the oligonucleotide

[0082]

[0083] Wherein R 1 、R 2 、R 3 、Z, X1, X2, Y and Nu are as defined above.

[0084] Another object of the present invention is to provide the use of the nucleoside compound represented by Formula 1-a for the preparation of an oligonucleotide.

[0085] The use according to the present invention, wherein the nucleoside compound has the following structure in the oligonucleotide

[0086]

[0087] Wherein R 1 、R 2 、R 3 、Z, X1, X2, Y and Nu are as defined above.

[0088] In the present invention, the oligonucleotide can be siRNA, antisense oligonucleotide, saRNA, miRNA, aptamer.

[0089] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0090] As used herein, the terms "carbon," "hydrogen," "oxygen," "sulfur," "nitrogen," or "F," "Cl," "Br," or "I" in connection with the groups and compounds of the present application include their isotopic forms, and the carbon, hydrogen, oxygen, sulfur, or nitrogen in the groups and compounds of the present application are optionally further substituted by one or more of their corresponding isotopes, where the isotopes of carbon include 12 C, 13 C, and 14 C; the isotopes of hydrogen include protium (H), deuterium (D, also known as heavy hydrogen), and tritium (T, also known as superheavy hydrogen); the isotopes of oxygen include 16 O, 17 O, and 18 O; the isotopes of sulfur include 32 S, 33 S, 34 S, and 36 S; the isotopes of nitrogen include 14 N and 15 N; the isotopes of fluorine include 17 F and 19 F; the isotopes of chlorine include 35 Cl and 37 Cl; the isotopes of bromine include 79 Br and 81 Br.

[0091] "Alkyl" means a straight-chain or branched-chain saturated aliphatic hydrocarbon group having 1 to 30 carbon atoms, preferably an alkyl group having 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 having 1 to 6 carbon atoms or an alkyl group having 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, straight-chain or branched-chain C 10-18 alkyl. The alkyl group may optionally be further substituted by one or more substituents.

[0092] "Alkoxy" means a group formed by linking an alkyl group to 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-pentyloxy, n-hexyloxy, cyclopropoxy, cyclobutoxy, straight-chain or branched-chain C 10-18 alkoxy. The alkoxy group may optionally be further substituted by one or more substituents.

[0093] "Cycloalkyl" refers to a saturated cycloalkyl group, the ring of which can be a monocyclic ring with 3 to 10 members (such as 3, 4, 5, 6, 7, 8, 9, 10 members), a bicyclic ring with 4 to 12 members (such as 4, 5, 6, 7, 8, 9, 10, 11, 12 members) or a polycyclic system with 10 to 20 members (such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 members). The ring carbon atoms preferably have 3 to 10 carbon atoms, and 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 by one or more substituents.

[0094] "Alkenyl" refers to an unsaturated hydrocarbon group containing at least one carbon-carbon double bond in its structure, which can be substituted or unsubstituted. The alkenyl moiety can be a straight-chain alkenyl or a branched-chain alkenyl. For example, -(C2-C6) alkenyl or -(C2-C4) alkenyl. -(C2-C6) alkenyl refers to an alkenyl having 2 to 6 carbon atoms, such as an alkenyl having 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms. Non-limiting examples of alkenyl include vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, n-hexenyl, etc. The alkenyl group may optionally be further substituted by one or more substituents.

[0095] "Alkynyl" refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond in its structure, which can be substituted or unsubstituted. The alkynyl moiety can be a straight-chain alkynyl or a branched-chain alkynyl. For example, -(C2-C6) alkynyl or -(C2-C4) alkynyl. -(C2-C6) alkynyl refers to an alkynyl having 2 to 6 carbon atoms, such as an alkynyl having 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms. Non-limiting examples of alkynyl include ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, n-hexynyl, etc. The alkynyl group may optionally be further substituted by one or more substituents.

[0096] Halogen includes F, Cl, Br and I.

[0097] Acyl refers to a structure formed by a functional group containing a carbonyl group connected to a substituent group, written as -COR'. In the present invention, R' represents an alkyl group, an alkenyl group, or an alkynyl group. The definitions of the alkyl group, alkenyl group, and alkynyl group are the same as the definitions of "alkyl group", "alkenyl group", and "alkynyl group" described above. Non-limiting examples of acyl include formyl, acetyl, propionyl, butyryl.

[0098] "Aryl" refers to an aromatic ring group having a conjugated planar ring system, which can be a monocyclic ring of 5 to 8 members (such as 5, 6, 7, 8 members), a bicyclic ring of 5 to 12 members (such as 5, 6, 7, 8, 9, 10, 11, 12 members) or a tricyclic ring system of 10 to 15 members (such as 10, 11, 12, 13, 14, 15 members), and which can be a bridged ring or a spiro ring. Non-limiting examples of aryl include phenyl and naphthyl. The aryl can optionally be further substituted by one or more substituents.

[0099] "Heteroaryl" refers to an aromatic ring group having a conjugated planar ring system and containing heteroatoms, which can be a monocyclic ring of 3 to 8 members (such as 3, 4, 5, 6, 7, 8 members), a bicyclic ring of 5 to 12 members (such as 5, 6, 7, 8, 9, 10, 11, 12 members) or a tricyclic ring system of 10 to 15 members (such as 10, 11, 12, 13, 14, 15 members), and contains 1 to 6 (such as 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, piperidinyl benzimidazolyl, benzopyridyl, pyrrolopyridyl. The heteroaryl can optionally be further substituted by one or more substituents.

[0100] "Heterocyclic group" 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 that of "heteroaryl" above; when it is a non-aromatic heterocycle, it can be a monocyclic ring of 3 to 10 members (such as 3, 4, 5, 6, 7, 8, 9, 10 members), a bicyclic ring of 4 to 12 members (such as 4, 5, 6, 7, 8, 9, 10, 11, 12 members) or a tricyclic ring system of 10 to 15 members (such as 10, 11, 12, 13, 14, 15 members), and contains 1 to 4 (such as 1, 2, 3, 4) heteroatoms selected from N, O or S, preferably a heterocyclic group of 3 to 8 members. Non-limiting examples of "heterocyclic group" or "heterocycle" include oxetanyl, azetidinyl, thietanyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxanyl, azepanyl, oxepanyl, thiepanyl, triazolyl, pyridyl, piperidyl, furyl, thienyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, 1,3-dithianyl, dihydrofuryl, dithiolanyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzimidazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuryl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolyl, pyrazolinyl, dithianyl, dithiolanyl, dihydrothienyl, pyrazolyl, imidazolinyl, imidazolyl, 1,2,3,4-tetrahydroisoquinolinyl. The said "heterocyclic group" or "heterocycle" may optionally be further substituted by one or more substituents.

[0101] When the "alkyl", "alkoxy", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl", "heterocyclic group" described above are substituted, they are optionally further substituted by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents selected from F, Cl, Br, I, hydroxy, mercapto, 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, C5-10 heteroaryl.

[0102] "Optionally" or "optionally" means that the subsequent described event or condition can but does not necessarily occur, and this description includes the situation where the event or condition occurs and the situation where it does not occur. For example, "heterocyclic group optionally substituted by alkyl" means that the alkyl may or may not be present, and this description includes the situation where the heterocyclic group is substituted by alkyl and the situation where the heterocyclic group is not substituted by alkyl.

[0103] "Base" refers to the heterocyclic base moiety in nucleosides and nucleotides. In natural nucleic acids, the bases are the purine moieties in nucleosides (such as adenine and guanine) and the pyrimidine moieties (such as uracil, thymine, and cytosine). In this article, the term "base" also encompasses modified bases that can be different from the naturally occurring nucleobases but are functional during nucleic acid hybridization. In such cases, "base" refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Descriptions of such variants can be found, for example, in Hirao et al. (2012) Accounts of Chemical Research, Volume 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1. In some cases, the base moiety can be modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or a substituted pyrimidine, such as bases selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolylcytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolyluracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine. The base moiety can be indicated by the letter code of each corresponding base (such as A, T, G, C, or U), where each letter can optionally include functionally equivalent modified bases. For example, in the structures exemplified in this disclosure, the base moiety can be selected from A, T, G, C, and U.

[0104] Beneficial effects

[0105] The present invention provides modified nucleoside compounds that can be used for incorporation into the 5'-end of oligonucleotides such that the modified oligonucleotides hybridize to a portion of the target RNA, resulting in the loss or downregulation of the normal function of the target RNA. Such oligonucleotides can also be included in double-stranded compositions. Specifically, the nucleoside compounds represented by Formula 1-a of the present invention can be applied in oligonucleotides (including but not limited to siRNA, antisense nucleic acids, saRNA, miRNA, nucleic acid aptamers, etc.), can effectively improve the activity of oligonucleotides, improve the corresponding target gene silencing efficiency, and at the same time improve the resistance of the corresponding oligonucleotides to enzymes such as phosphatases, exonucleases, and endonucleases. Through cell and mouse experiments, the in vitro and in vivo activities, pharmacokinetic properties, and bioavailability of oligonucleotide drugs prepared with the nucleoside compounds represented by Formula 1-a of the present invention are better improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1Aand Figure 1B The IC 50 value showing the in vitro inhibition rate of the compound of the present invention.

[0107] Figure 2 Showing the level of TTR protein in the serum of mice after administration of the compound of the present invention. Detailed Description of the Invention

[0108] Unless otherwise specified, the instruments used in the present invention are conventional instruments, and the reagents used are conventional reagents.

[0109] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0110] The NMR shift (δ) is given in units of 10 -6 (ppm). The NMR measurement was performed using a (Broker Avance III 400) nuclear magnetic resonance spectrometer, and the solvents for measurement were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS);

[0111] The MS measurement was performed using (Agilent 6120B (ESI) and Agilent 6120B (APCI));

[0112] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specifications of the silica gel plate used in thin layer chromatography (TLC) are 0.15 mm - 0.20 mm, and the specifications of the silica gel plate used for thin layer chromatography separation and purification of products are 0.4 mm - 0.5 mm;

[0113] Column chromatography generally uses Yantai Huanghai silica gel with 200 - 300 mesh as the carrier.

[0114] Oligo solid-phase synthesis was all completed on an LK-48E synthesizer (Lingkun).

[0115] Explanation of the abbreviated names of the materials used in the examples:

[0116]

[0117]

[0118] The content of the present invention will be described in detail below through examples. For those not specified in the examples, the experimental methods under conventional conditions were used. The examples given are for better illustration of the content of the present invention, but it should not be understood that the content of the present invention is limited to the examples given. Non-essential improvements and adjustments made by those skilled in the art to the implementation schemes based on the above invention content still fall within the protection scope of the present invention.

[0119] Examples

[0120] The following Examples 1-4 are preparation examples of the nucleoside compounds represented by Formula 2-a of the present invention, wherein the nucleoside compound represented by Formula 2-a is a compound of 5'-(E)-VP-3'-OMe-2'-phosphoramidite group

[0121] Example 1: Synthesis of Compound 13 (Uvp1)

[0122]

[0123] Synthesis of Compound 2:

[0124] Dissolve Compound 1 (95.0 g, 0.50 mol, 1.0 eq.) in ultradry DCM (950 mL) and stir evenly. Then add imidazole (85.1 g, 1.25 mol, 2.5 eq.) to the reaction and stir at room temperature for 30 minutes. Then slowly add TBSCl (90.4 g, 0.60 mol, 1.2 eq.) in batches, and react overnight. Cool the reaction to 0 °C, add saturated aqueous NaHCO3 solution to quench the reaction, then add DCM (500 mL) and water (1000 mL) for liquid separation to obtain the organic phase. The organic phase is washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent is evaporated by a rotary evaporator to obtain the crude product. The crude product is purified by flash column chromatography (PE∶EA = 4∶1) to obtain Compound 2 (136.9 g, 0.45 mol, 90% yield). ESI-MS: m / z 305.2 [M+H] +

[0125] Synthesis of Compound 3:

[0126] NaH (60% dispersion in mineral oil, 27.0 g, 0.675 mol, 1.5 eq.) was added to a three-necked flask and placed in an ice bath. Then, DMF (1.1 L) was added dropwise under the ice bath. After the addition was complete, the mixture was stirred at this temperature for 30 minutes. Subsequently, a DMF solution of compound 2 (136.9 g, 0.45 mol, 250 mL of DMF) was added dropwise. After the addition was complete, the reaction was carried out at 0 - 5 °C for 30 min. Then, CH3I (95.8 g, 0.675 mol, 1.5 eq.) was slowly added dropwise while controlling the internal temperature of the reaction not to exceed 5 °C. After the addition was complete, the reaction was restored to room temperature and reacted overnight. The reaction solution was slowly poured into a saturated NH4Cl aqueous solution to quench the reaction. After quenching, EA (3.5 L) and water (2.5 L) were added for extraction and liquid separation to obtain the organic phase. The organic phase was washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure using a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 10∶1) to obtain compound 3 (114.8 g, 0.361 mol, 80.2% yield). ESI-MS: m / z 319.2 [M+H] +

[0127] Synthesis of compound 4:

[0128] Compound 3 (114.8 g, 0.361 mol, 1.0 eq.) was dissolved in AcOH (1500 mL). Then, the temperature was lowered to 0 °C, and Ac2O (184.3 g, 1.805 mol, 5.0 eq.) and concentrated H2SO4 (40 mL) were slowly added dropwise. After the addition was complete, the reaction was slowly restored to room temperature and reacted overnight until the raw materials disappeared. Then, the reaction solution was slowly added dropwise to an ice-cold aqueous NaHCO3 solution to quench the reaction until the pH of the reaction solution was 7 - 8. After quenching, EA (2.0 L) and water (500 mL) were added for extraction. The aqueous phase was then extracted twice with EA (1.0 L). Then, the organic phases were combined. The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product. The crude product was purified by flash column chromatography (PE∶EA = 5∶1) to obtain compound 4 (73.2 g, 0.251 mol, 69.5% yield). ESI-MS: m / z 291.1 [M+H] +

[0129] Synthesis of compound 5:

[0130] Dissolve compound 4 (29.2 g, 100.65 mmol, 1.0 eq.), Uracil (22.56 g, 201.30 mmol, 2.0 eq.) in ACN (400 mL), then add BSA (30.7 g, 150.98 mmol, 1.5 eq.). Place the reaction at 50 °C for 1 h, then cool to 0 °C. Slowly add a solution of TMSOTf (22.37 g, 100.65 mmol, 1.0 eq.) dropwise to the reaction. After the addition is complete, reflux the reaction in an oil bath for 3 h. Quench the reaction by adding saturated aqueous NaHCO3. Then add EA (300 mL) and water (200 mL) for extraction and separation. Extract the aqueous phase twice with EA (350 mL). Then combine the organic phases. Wash the organic phase with saturated brine and dry over anhydrous sodium sulfate. Rotavap to remove the solvent to obtain the crude product. Purify the crude product by column chromatography (PE∶EA = 1∶1) to obtain compound 5 (27.8 g, 81.26 mmol, 80.7% yield). ESI-MS: m / z 343.1 [M+H] +

[0131] Synthesis of compound 6:

[0132] Dissolve compound 5 (27.2 g, 79.51 mmol, 1.0 eq.) in MeOH (100 mL), then cool to 0 °C. Add sodium methoxide (21.48 g, 397.55 mmol, 5.0 eq.). After addition, slowly warm to room temperature and react overnight. Cool the reaction to about 0 °C. Slowly add 1 mol / L dilute hydrochloric acid to neutralize the reaction to pH about 7. Extract the reaction twice with ethyl acetate. Combine the organic phases. Wash the organic phase with water, saturated brine, dry over anhydrous sodium sulfate and remove the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography (DCM∶MeOH = 20∶1) to obtain compound 6 (18.5 g, 71.68 mmol, 90.2% yield). ESI-MS: m / z 259.1 [M+H] +

[0133] Synthesis of compound 7:

[0134] The original compound 6 (18.2 g, 71.68 mmol, 1.0 eq.) was dissolved in ultradry DMF (200 mL), and then imidazole (29.30 g, 430.08 mmol, 6.0 eq.) and TBSCl (32.27 g, 214.08 mmol, 3.0 eq.) were added successively. The reaction was carried out overnight. The reaction was cooled to 0 °C, quenched with saturated aqueous NaHCO3 solution, and then extracted and separated with ethyl acetate (250 mL) and water (150 mL). The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined. The combined organic phase was washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure using a rotary evaporator to obtain the crude compound 7 (31.6 g). ESI-MS: m / z 487.3 [M+H] +

[0135] Synthesis of compound 8:

[0136] The crude compound 7 (31.6 g) was dissolved in THF (300 mL), and then cooled to -5 °C. A solution of TFA / H2O (v∶v = 1∶1, 100 mL) was slowly added dropwise. After the addition was complete, the reaction was continued at this temperature for 1 h until the raw materials disappeared. Then, ammonia water was slowly added dropwise for quenching until the reaction solution was neutral. After quenching, extraction was carried out with EA (200 mL) and water (100 mL). The aqueous phase was then extracted twice with EA (100 mL). Then, the organic phases were combined. The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product, and the crude product was purified by column chromatography (PE∶EA = 1∶2) to obtain compound 8 (18.5 g, 49.71 mmol, total yield of two steps 69.3%). ESI-MS: m / z 373.2 [M+H] +

[0137] Synthesis of compound 9:

[0138] Compound 8 (3.7 g, 9.94 mmol, 1.0 eq.) was dissolved in ACN (40 mL), and then IBX (5.57 g, 19.88 mmol, 2.0 eq.) was added. Then, the reaction was refluxed in an oil bath for 2 h until compound 8 reacted completely. The reaction solution was cooled to room temperature and then filtered. The filter cake was washed twice with acetonitrile, and the organic phases were combined. After the organic phase was evaporated to dryness, the crude compound 9 (3.5 g) was obtained and used directly in the next step without purification. ESI-MS: m / z 371.2 [M+H] +

[0139] Synthesis of compound 11:

[0140] Compound 10 (5.73 g, 19.88 mmol, 2.0 eq.) was dissolved in THF (56 mL), and then the temperature was lowered to 0 °C. NaH (60% dispersion in mineral oil, 1.59 g, 39.76 mmol, 2.0 eq.) was slowly added. After reacting for 30 min, a THF solution of compound 9 (3.5 g, 10 mL THF) was added dropwise, and then the reaction was slowly warmed to room temperature and continued for 2 h until the raw materials disappeared. The reaction was quenched by adding a saturated aqueous solution of NH4Cl. After quenching, extraction was carried out with EA (100 mL) and water (50 mL). The aqueous phase was further extracted twice with EA (50 mL). Then the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product, and the crude product was purified by column chromatography (PE∶EA = 2∶1) to obtain compound 11 (4.2 g, 8.33 mmol, total yield of two steps 83.8%). 1 H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 5.90 (d, J = 1.6 Hz, 1H), 5.60 (m, 1H), 4.76 (m, 1H), 4.29 (s, 1H), 4.06 (s, 1H), 4.00 (m, 4H), 3.30 (s, 3H), 2.66 (m, 2H), 1.21 (m, 6H), 0.85 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H), 31 P NMR (162 MHz, DMSO-d6) δ 26.73, ESI-MS: m / z 505.2 [M+H] +

[0141] Synthesis of compound 12:

[0142] Compound 11 (4.2 g, 8.33 mmol, 1.0 eq.) was dissolved in THF (42 mL), and then TBAF solution (1.0 mol / L in THF, 10 mL, 10.0 mmol, 1.2 eq.) was added dropwise. After reacting at room temperature for 1 h, the solvent was evaporated to obtain the crude product, and the crude product was purified by normal-phase column chromatography (PE∶EA = 1∶10) to obtain compound 12 (2.9 g, 7.43 mmol, yield 89.2%). 11H NMR (400 MHz, CD3OD) δ 7.58 (d, J = 8.1 Hz, 1H), 6.86 - 6.75 (m, 1H), 6.07 - 5.98 (m, 1H), 5.82 (s, 1H), 5.57 (d, J = 8.1 Hz, 1H), 4.84 (m, 1H), 4.20 (s, 1H), 4.01 (m, 4H), 3.72 (d, J = 3.4 Hz, 1H), 3.25 (s, 3H), 1.24 (t, J = 7.0 Hz, 6H), 31 31P NMR (161 MHz, CD3OD) 618.07, ESI-MS: m / z 391.1 [M+H] +

[0143] Synthesis of Compound 13:

[0144] Dissolve Compound 12 (2.9 g, 7.43 mmol) in DCM (30 mL), then add DCI (0.70 g, 5.94 mmol, 0.8 eq.), P-REAGENT (2.69 g, 8.92 mmol, 1.2 eq.). After reacting at room temperature for 1 h, evaporate the solvent to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 1∶1) to obtain Compound 13 (2.5 g, 4.32 mmol, 58.1% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.52 (m, 1H), 6.82 (m, 1H), 6.15 - 6.05 (m, 1H), 5.95 - 5.90 (m, 1H), 5.67 (m, 1H), 4.84 (m, 1H), 4.46 (dd, J = 10.6 Hz, 1H), 4.02 - 3.91 (m, 1H), 3.77 (m, 2H), 3.60 (m, 2H), 3.32 (s, 5H), 2.80 (m, 2H), 1.28 - 1.09 (m, 19H), 31 31P NMR (161 MHz, DMSO-d6) δ 149.84, 149.74, 16.81, 16.79, ESI-MS: m / z 590.2 [M+H] +

[0145] Example 2: Synthesis of Compound 21 (Avp3)

[0146]

[0147] Synthesis of Compound 14:

[0148] Dissolve compound 4 (14.6 g, 50.32 mmol) and ABz (18.06 g, 75.48 mmol, 1.5 eq.) in ACN (150 mL), then add BSA (20.47 g, 100.64 mmol, 2.0 eq.). Place the reaction in an oil bath at 50 °C and react for 1 h, then cool to 0 °C. Dropwise add a solution of TMSOTf (11.18 g, 50.32 mmol, 1.0 eq.) to the reaction. After the addition is complete, place the reaction in an oil bath and reflux for 3 h until compound 4 reacts completely. Quench the reaction by adding saturated aqueous NaHCO3, then add ethyl acetate (300 mL) and water (200 mL) for extraction and separation to obtain the organic phase. Extract the aqueous phase twice with ethyl acetate (150 mL), then combine the organic phases. Wash the combined organic phases with saturated brine and dry over anhydrous sodium sulfate. Rotavaporize the solvent to obtain the crude product. Purify the crude product by column chromatography (PE∶EA = 2∶1) to obtain compound 14 (14.1 g, 30.05 mmol, 59.7% yield). ESI-MS: m / z 470.2 [M+H] +

[0149] Synthesis of compound 15:

[0150] Dissolve compound 14 (14.1 g, 30.05 mmol, 1.0 eq.) in MeOH (100 mL), then cool to 0 °C. Add sodium methoxide (8.12 g, 150.25 mmol, 5.0 eq.). After the addition, slowly warm the reaction to room temperature and react overnight until compound 14 reacts completely. Cool the reaction to 0 - 5 °C and neutralize the reaction with 1 M dilute hydrochloric acid to a pH of about 7. Pour the mixture into water and extract twice with ethyl acetate. Combine the organic phases. Wash the organic phases with water, saturated brine, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography (DCM∶MeOH = 20∶1) to obtain compound 15 (10.5 g, 27.26 mmol, 90.7% yield). ESI-MS: m / z 386.1 [M+H] +

[0151] Synthesis of compound 16:

[0152] Compound 15 (10.5 g, 27.26 mmol, 1.0 eq.) was dissolved in ultradry DMF (100 mL). Then imidazole (11.14 g, 163.56 mmol, 6.0 eq.) and TBSCl (12.33 g, 81.78 mmol, 3.0 eq.) were added successively. After the reaction overnight until the raw materials were completely consumed, the reaction was cooled to 0 °C, and saturated aqueous NaHCO3 was added to quench the reaction. Then ethyl acetate (100 mL) and water (100 mL) were added, and the organic phase was obtained by liquid separation extraction. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined. The combined organic phase was washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated by a rotary evaporator to obtain crude compound 16 (16.6 g, the crude product was directly used in the next step without purification). ESI-MS: m / z 614.3 [M+H] +

[0153] Synthesis of compound 17:

[0154] The crude compound 16 (16.6 g) was dissolved in THF (160 mL). Then the temperature was cooled to -5 °C, and a solution of TFA / H2O (v∶v = 1∶1, 100 mL) was slowly added dropwise. After the addition was completed, the reaction continued at this temperature for 1 h until compound 16 reacted completely. Then ammonia water was slowly added dropwise to quench the reaction, and the internal temperature of the reaction was maintained below 5 °C until the pH of the reaction solution was 7 - 8. After quenching, ethyl acetate (200 mL) and water (100 mL) were added for extraction and liquid separation to obtain the organic phase. Then the aqueous phase was extracted twice with EA (100 mL), and the organic phases were combined. The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. 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 17 (9.7 g, 19.43 mmol, total yield of two steps 71.3%). ESI-MS: m / z 500.2 [M+H] +

[0155] Synthesis of compound 18:

[0156] Compound 17 (9.7 g, 19.43 mmol, 1.0 eq.) was dissolved in ACN (100 mL). Then IBX (10.88 g, 38.86 mmol, 2.0 eq.) was added, and the reaction was refluxed in an oil bath for 2 h until compound 17 reacted completely. The reaction solution was slowly restored to room temperature and then filtered. The filter cake was washed twice with ethyl acetate, and the filtrates were combined. After the filtrate was evaporated to dryness, compound 18 (9.5 g, 19.11 mmol, yield 98.4%) was obtained. ESI-MS: m / z 498.2 [M+H] +

[0157] Synthesis of compound 19:

[0158] Compound 10 (11.01 g, 38.22 mmol, 2.0 eq.) was dissolved in THF (100 mL), then the temperature was lowered to 0 °C, and NaH (60% dispersion in mineral oil, 1.15 g, 28.67 mmol, 1.5 eq.) was added. After reacting for 30 min, a THF solution of compound 18 (9.5 g, 19.11 mmol, 30 mL THF) was added dropwise, and then the temperature was slowly raised to room temperature and reacted for 2 h until the raw materials disappeared. The reaction was quenched by adding a saturated aqueous solution of NH4Cl. After quenching, EA (100 mL) and water (50 mL) were added for extraction, and the aqueous phase was extracted twice with ethyl acetate (50 mL). Then the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude product, and the crude product was purified by column chromatography (PE:EA = 2:1) to obtain compound 19 (6.1 g, 9.66 mmol, 50.5% yield). ESI-MS: m / z 632.3 [M+H] +

[0159] Synthesis of compound 20:

[0160] Compound 19 (6.1 g, 9.66 mmol, 1.0 eq.) was dissolved in THF (60 mL), and then TBAF (1.0 mol / L in THF, 15.0 mL, 14.49 mmol, 1.5 eq.) was added dropwise. After reacting at room temperature for 1 h, the solvent of the reaction was evaporated under reduced pressure at low temperature to obtain a crude product, and the crude product was purified by column chromatography (PE∶EA = 1∶10) to obtain compound 20 (4.3 g, 8.31 mmol, 86.0% yield). ESI-MS: m / z518.2 [M+H] +

[0161] Synthesis of compound 21:

[0162] Compound 20 (4.3 g, 8.31 mmol, 1.0 eq.) was dissolved in DCM (40 mL), and then DCI (785 mg, 6.65 mmol, 0.8 eq.) and P-REAGENT (3.0 g, 9.97 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent of the reaction was evaporated under reduced pressure at low temperature to obtain a crude product, and the crude product was purified by column chromatography (PE∶EA = 1∶1) to obtain compound 21 (4.1 g, 5.72 mmol, 68.8% yield). ESI-MS: m / z 718.3 [M+H] +

[0163] Example 3: Synthesis of compound 29 (Gvp4)

[0164]

[0165] Synthesis of Compound 22:

[0166] Dissolve Compound 4 (14.6 g, 50.33 mmol, 1.0 eq.) and GiBu (16.69 g, 75.50 mmol, 1.5 eq.) in ACN (200 mL), then add BSA (15.36 g, 75.50 mmol, 1.5 eq.). Place the reaction in an oil bath at 50 °C and react for 1 h. Then cool the reaction to 0 °C and dropwise add a solution of TMSOTf (11.19 g, 50.33 mmol, 1.0 eq.). After the addition is complete, place the reaction in an oil bath and reflux for 3 h until Compound 4 reacts completely. Then restore the reaction to room temperature and quench it with saturated aqueous NaHCO3. Add ethyl acetate (300 mL) and water (200 mL) for extraction and liquid separation to obtain the organic phase. Extract the aqueous phase twice with ethyl acetate (150 mL). Then combine the organic phases. Wash the combined organic phases with saturated brine and dry over anhydrous sodium sulfate. Rotavap to remove the solvent to obtain the crude product. The crude product is slurried with ethyl acetate to obtain Compound 22 (13.1 g, 29.04 mmol, 57.7% yield). ESI-MS: m / z 452.2 [M+H] +

[0167] Synthesis of Compound 23:

[0168] Dissolve Compound 22 (13.1 g, 29.04 mmol, 1.00 eq.) in MeOH (130 mL). Then cool the reaction to 0 °C and add sodium methoxide (7.84 g, 145.20 mmol, 5.0 eq.). React at room temperature overnight. Neutralize the reaction with 1 mol / L dilute hydrochloric acid to a pH of about 7. Extract the mixed system twice with ethyl acetate and combine the organic phases. Wash the organic phases with water, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography (DCM∶MeOH = 20∶1) to obtain Compound 23 (9.5 g, 25.87 mmol, 89.1% yield). ESI-MS: m / z 368.2 [M+H] +

[0169] Synthesis of Compound 24:

[0170] Compound 23 (9.5 g, 25.87 mmol, 1.0 eq.) was dissolved in ultradry DMF (100 mL), then imidazole (10.57 mmol, 155.22 mmol, 6.0 eq.) and TBSCl (11.7 g, 77.61 mmol, 3.0 eq.) were added successively. The reaction was carried out overnight, cooled to 0 °C, quenched with saturated aqueous NaHCO3, and then extracted with ethyl acetate (300 mL) and water (300 mL). The organic phase was washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the crude product, which was recrystallized simply to obtain Compound 24 (15.6 g, used directly in the next step without column chromatography purification). ESI-MS: m / z 596.3 [M+H] +

[0171] Synthesis of Compound 25:

[0172] Compound 24 (15.6 g) was dissolved in THF (150 mL), then cooled to -5 °C, and a solution of TFA / H2O (v∶v = 1∶1, 100 mL) was added dropwise slowly, controlling the internal temperature of the reaction not to exceed 5 °C. After the addition was completed, the reaction was continued at this temperature for 1 h until the raw materials disappeared. Then ammonia water was added dropwise slowly for quenching until the reaction solution was neutral. After quenching, it was extracted with ethyl acetate (200 mL) and water (100 mL) to obtain the organic phase. The aqueous phase was extracted twice with ethyl acetate (100 mL), and then the organic phases were combined. The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the crude product, and the crude product was purified by column chromatography (DCM∶MeOH = 20∶1) to obtain Compound 25 (8.7 g, 18.08 mmol, total yield of two steps 69.9%). ESI-MS: m / z 482.2 [M+H] +

[0173] Synthesis of Compound 26:

[0174] Compound 25 (8.7 g, 18.08 mmol, 1.0 eq.) was dissolved in ACN (90 mL), then IBX (10.13 g, 36.16 mmol, 2.0 eq.) was added, and then the reaction was refluxed in an oil bath for 2 h until the raw materials disappeared. The reaction solution was cooled to room temperature and then filtered. The filter cake was washed twice with ethyl acetate, and the filtrates were combined. After the filtrate was evaporated to dryness, Compound 26 (7.5 g, 15.65 mmol, yield 86.6%) was obtained. ESI-MS: m / z 480.2 [M+H] +

[0175] Synthesis of Compound 27:

[0176] Compound 10 (9.02 g, 31.30 mmol, 2.0 eq.) was dissolved in THF (100 mL), and then the temperature was lowered to 0 °C. NaH (60% dispersion in mineral oil, 1.25 g, 31.30 mmol, 2.0 eq.) was added. After reacting for 30 min, a solution of compound 26 (7.5 g, 15.65 mmol, 1.0 eq., 30 mL THF) in THF was added dropwise. Then, the reaction mixture was slowly warmed to room temperature and reacted for 2 h until the starting materials disappeared. The reaction was quenched by adding saturated NH4Cl solution. After quenching, EA (100 mL) and water (50 mL) were added for extraction. The aqueous phase was extracted twice with EA (50 mL). Then, the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product, which was purified by column chromatography (PE∶EA = 1∶1) to obtain compound 27 (4.4 g, 7.17 mmol, 45.8% yield). ESI-MS: m / z 614.3 [M+H] +

[0177] Synthesis of compound 28:

[0178] Compound 27 (4.4 g, 7.17 mmol, 1.0 eq.) was dissolved in THF (45 mL), and then TBAF (1.0 mol / L in THF, 10.8 mL, 10.76 mmol, 1.5 eq.) was added dropwise. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain the crude product, which was purified by column chromatography (EA) to obtain compound 28 (3.2 g, 6.41 mmol, 89.4% yield). ESI-MS: m / z 500.2 [M+H ] +

[0179] Synthesis of compound 29:

[0180] Compound 28 (3.2 g, 6.41 mmol, 1.0 eq.) was dissolved in DCM (30 mL), and then DCI (606 mg, 5.13 mmol, 0.8 eq.) and R-REAGENT (2.32 g, 7.69 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain the crude product, which was purified by column chromatography (PE∶EA = 1∶2) to obtain compound 29 (2.2 g, 3.15 mmol, 49.1% yield). ESI-MS: m / z 700.3 [M+H] +

[0181] Example 4: Synthesis of compound 36 (Cvp2)

[0182]

[0183] Synthesis of compound 30:

[0184] Compound 7 (21.2 g, 43.60 mmol, 1.0 eq.) was dissolved in ACN (200 mL), then triazole (18.07 g, 261.6 mmol, 6.0 eq.) and triethylamine (26.47 g, 261.6 mmol, 6.0 eq.) were added successively. Then the temperature was lowered to 0 °C, and POCl3 (13.37 g, 87.20 mmol, 2.0 eq.) was slowly added dropwise. The reaction was carried out at 0 °C for 2 h until the raw materials disappeared. Then ammonia water (200 mL) was added and the reaction was carried out overnight. Then EA (500 mL) and water (200 mL) were added for extraction. The organic phase was washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated by a rotary evaporator to obtain the crude product of compound 30 (20.6 g, which was directly used for the next step without purification). ESI-MS: m / z 486.3 [M+H] +

[0185] Synthesis of compound 31:

[0186] Compound 30 (20.6 g) was dissolved in pyridine (200 mL). The temperature was lowered to 0 °C, and benzoyl chloride (12.26 g, 87.20 mmol, 2.0 eq.) was added. The reaction was carried out overnight. Saturated aqueous NaHCO3 was added to quench the reaction. Then EA (500 mL) and water (250 mL) were added for extraction. The organic phase was washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated by a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 3∶1) to obtain compound 31 (17.5 g, 29.70 mmol, total yield of two steps 68.1%). ESI-MS: m / z 590.3 [M+H] +

[0187] Synthesis of compound 32:

[0188] Compound 31 (11.8 g, 20.02 mmol) was dissolved in THF (100 mL). Then the temperature was lowered to -5 °C, and a solution of TFA / H2O (v / v = 1∶1, 100 mL) was slowly added dropwise. After the addition was completed, the reaction was continued at this temperature for 1 h. Then ammonia water was slowly added dropwise to quench the reaction until the pH of the reaction solution was about 7. After quenching, EA (200 mL) and water (100 mL) were added for extraction. The aqueous phase was extracted twice with EA (100 mL). Then the organic phases were combined. The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 1∶2) to obtain compound 32 (6.7 g, 14.10 mmol, yield 70.4%). ESI-MS: m / z 476.2 [M+H] +

[0189] Synthesis of Compound 33:

[0190] Dissolve Compound 32 (6.7 g, 14.10 mmol, 1.0 eq.) in ACN (70 mL), then add IBX (7.9 g, 28.20 mmol, 2.0 eq.), and then reflux the reaction in an oil bath for 2 h. Cool the reaction solution to room temperature and then filter. Wash the filter cake twice with ethyl acetate and combine the filtrates. After rotary evaporation of the filtrate, Compound 33 (6.3 g, 13.31 mmol) is obtained and used directly in the next step without purification. ESI-MS: m / z 474.2 [M+H] +

[0191] Synthesis of Compound 34:

[0192] Dissolve Compound 10 (7.67 g, 26.62 mmol, 2.0 eq.) in THF (60 mL), then cool to 0 °C, add NaH (60% dispersion in mineral oil, 1.07 g, 26.62 mmol, 2.0 eq.). After reacting for 30 min, dropwise add a THF solution of Compound 33 (6.3 g, 13.31 mmol, in 120 mL THF), then slowly warm to room temperature and react for 2 h. Quench the reaction with saturated aqueous NH4Cl solution. After quenching, add EA (100 mL) and water (50 mL) for extraction, and then extract the aqueous phase twice with EA (50 mL). Then combine the organic phases, wash the organic phases with saturated brine and dry over anhydrous sodium sulfate. Rotary evaporate the solvent to obtain the crude product, and purify the crude product by column chromatography (PE∶EA = 2∶1) to obtain Compound 34 (3.0 g, 4.94 mmol, 37.0% yield). ESI-MS: m / z 608.3 [M+H] +

[0193] Synthesis of Compound 35:

[0194] Dissolve Compound 34 (3.0 g, 4.94 mmol, 1.0 eq.) in THF (30 mL), then dropwise add TBAF (1.0 mol / L in THF, 7.4 mL, 7.41 mmol, 1.5 eq.). After reacting at room temperature for 1 h, rotary evaporate the solvent at low temperature to obtain the crude product. Purify the crude product by column chromatography (PE∶EA = 1∶10) to obtain Compound 35 (2.2 g, 4.46 mmol, 90.3% yield). ESI-MS: m / z 494.2 [M+H] +

[0195] Synthesis of Compound 36:

[0196] Compound 35 (2.2 g, 4.46 mmol, 1.0 eq.) was dissolved in DCM (30 mL), then DCI (422 mg, 3.57 mmol, 0.8 eq.) and CEP (1.61 g, 5.35 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent was evaporated to dryness to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 1∶1) to obtain compound 36 (2.1 g, 3.03 mmol, 67.9% yield). ESI-MS: m / z 694.3 [M+H] +

[0197] The following Examples 5-8 are the preparation examples of the nucleoside compounds represented by Formula 6-a of the present invention, wherein the nucleoside compound represented by Formula 6-a is a compound of 5'-(E)-VP-3'-OMOE-2'-phosphoramidite group

[0198] Example 5: Synthesis of Compound 46 (Uvp17)

[0199]

[0200] Synthesis of Compound 37:

[0201] NaH (60% dispersion in mineral oil, 43.2 g, 1.08 mol, 1.5 eq.) was added to a three-necked flask, and then DMF (1.1 L) was added dropwise under an ice bath. Subsequently, a DMF solution of compound 2 (136.8 g, 0.720 mol, 250 mL of DMF) was added dropwise. After reacting for 30 min, 2-bromoethyl methyl ether (89.4 g, 0.648 mol, 0.9 eq.) was added dropwise, and the reaction was carried out overnight at room temperature. Then it was slowly poured into a saturated NH4Cl aqueous solution to quench the reaction. After quenching, EA (3.5 L) and water (2.5 L) were added for extraction. The organic phase was washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness with a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 10∶1) to obtain compound 37 (130.3 g, 0.360 mol, 50.0% yield). ESI-MS: m / z 363.2 [M+H] +

[0202] Synthesis of Compound 38:

[0203] Compound 37 (130.3 g, 0.36 mol, 1.0 eq.) was dissolved in AcOH (1300 mL), and then the temperature was lowered to 0 °C. Ac2O (147.0 g, 1.44 mol, 4.0 eq.) and concentrated H2SO4 (40.0 mL) were added dropwise. The reaction was carried out at room temperature overnight until the raw materials disappeared. Then the reaction solution was slowly added dropwise to saturated, ice-cold aqueous NaHCO3 solution for quenching until the reaction solution was neutral. After quenching, EA (2.0 L) and water (2000 mL) were added for extraction. The aqueous phase was extracted twice with EA (800 mL). Then the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product, and the crude product was purified by column chromatography (PE∶EA = 5∶1) to obtain compound 38 (94.6 g, 0.283 mol, 78.6% yield). ESI-MS: m / z 335.1 [M+H] +

[0204] Synthesis of compound 39:

[0205] Compound 38 (30.0 g, 89.79 mmol, 1.0 eq.) and Uracil (20.13 g, 179.58 mmol, 2.0 eq.) were dissolved in ACN (300 mL). Then BSA (27.40 g, 134.69 mmol, 1.5 eq.) was added, and the reaction was carried out at 50 °C for 1 h. Then the temperature was lowered to 0 °C. A solution of TMSOTf (19.96 g, 89.79 mmol, 1.0 eq.) was added dropwise. After the addition was completed, the reaction was refluxed (80 °C) in an oil bath for 3 h until the raw materials disappeared. The reaction was quenched by adding saturated aqueous NaHCO3 solution. Then ethyl acetate (300 mL) and water (200 mL) were added for extraction. The aqueous phase was extracted twice with ethyl acetate (150 mL). Then the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product, and the crude product was purified by normal-phase purification (PE∶EA = 1∶1) to obtain compound 39 (29.1 g, 75.36 mmol, 83.9% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 5.92 (d, J = 1.3 Hz, 1H), 5.64 (dd, J = 8.1, 1.4 Hz, 1H), 5.09 (s, 1H), 4.38 - 4.24 (m, 3H), 4.06 (d, J = 2.3 Hz, 1H), 3.77 - 3.60 (m, 2H), 3.47 - 3.36 (m, 2H), 3.22 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H). ESI-MS: m / z 387.1 [M+H] +

[0206] Synthesis of Compound 40:

[0207] Dissolve Compound 39 (29.1 g, 75.36 mmol, 1.0 eq.) in MeOH (200 mL), then cool the temperature to 0 °C, add sodium methoxide (20.35 g, 376.8 mmol, 5.0 eq.), react at room temperature overnight. After the reaction is completed, neutralize the reaction with 1 mol / L dilute hydrochloric acid to about pH 7. Extract the mixed system twice with ethyl acetate, and combine the organic phases. The organic phase is washed with water and saturated brine, dried over anhydrous sodium sulfate and the solvent is removed under reduced pressure to obtain the crude product. The crude product is purified by column chromatography (EA) to obtain Compound 40 (20.9 g, 69.18 mmol, 91.8% yield). ESI-MS: m / z 303.1 [M+H] +

[0208] Synthesis of Compound 41:

[0209] Dissolve Compound 40 (20.9 g, 69.18 mmol, 1.0 eq.) in ultradry DMF (200 mL), then successively add imidazole (28.26 g, 415.08 mmol, 6.0 eq.) and TBSCl (31.28 g, 207.54 mmol, 3.0 eq.). After the reaction overnight until the raw materials are completely consumed, cool the temperature to 0 °C, add saturated aqueous NaHCO3 solution to quench the reaction, then add ethyl acetate (250 mL) and water (250 mL) for extraction and separation to obtain the organic phase. The organic phase is washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent is evaporated to dryness with a rotary evaporator to obtain the crude product. The crude product is obtained by flash column chromatography to obtain Compound 41 (31.2 g, 58.84 mmol, 85.1% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 5.67 (d, J = 2.0 Hz, 1H), 5.58 (d, J = 8.1 Hz, 1H), 4.30 (s, 1H), 4.19 (q, J = 5.0 Hz, 1H), 3.96 - 3.78 (m, 3H), 3.64 - 3.55 (m, 2H), 3.42 - 3.33 (m, 2H), 3.20 (s, 3H), 0.87 (d, J = 9.6 Hz, 18H), 0.10 - 0.06 (m, 12H). ESI-MS: m / z 531.3 [M+H] +

[0210] Synthesis of Compound 42:

[0211] Dissolve compound 41 (10.6 g, 19.99 mmol, 1.0 eq.) in THF (100 mL), then cool the temperature to -5 °C, and slowly add dropwise the solution of TFA / H₂O (v / v = 1∶1, 100 mL). Control the internal temperature not to exceed 5 °C throughout the process. After the addition is completed, continue the reaction at this temperature for 1 h until compound 41 reacts completely. Then, at low temperature, slowly add dropwise ammonia water for quenching until the reaction solution is neutral. After quenching, add EA (200 mL) and water (100 mL) for extraction, and then extract the aqueous phase with ethyl acetate (100 mL) twice. Then combine the organic phases. The organic phases are washed with saturated brine and dried over anhydrous sodium sulfate. Rotate to dry the solvent to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 1∶2) to obtain compound 42 (6.2 g, 14.90 mmol, 74.5% yield). 1 H NMR (400 MHz, DMSO-d₆) δ 11.30 (s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 5.63 (s, 1H), 5.58 (d, J = 8.1 Hz, 1H), 4.82 (t, J = 5.6 Hz, 1H), 4.30 (s, 1H), 4.18 (dd, J = 9.3, 5.7 Hz, 1H), 3.81 - 3.52 (m, 5H), 3.42 - 3.34 (m, 2H), 3.20 (s, 3H), 0.87 (s, 9H), 0.11 (d, J = 3.3 Hz, 6H). ESI-MS: m / z 417.2 [M + H] +

[0212] Synthesis of compound 43:

[0213] Dissolve compound 42 (6.2 g, 14.90 mmol, 1.0 eq.) in ACN (60 mL), then add IBX (8.34 g, 29.8 mmol, 2.0 eq.), and then reflux the reaction in an oil bath for 2 h until compound 42 reacts completely. Cool the reaction to room temperature and then filter. Wash the filter cake with ethyl acetate twice and combine the filtrates. After rotating to dry the filtrates, obtain the crude product compound 43 (6.0 g, 14.49 mmol, 97.2% yield). The crude product is directly used in the next step without purification. ESI-MS: m / z 415.2 [M + H] +

[0214] Synthesis of compound 44:

[0215] Compound 10 (8.35 g, 28.98 mmol, 2.0 eq.) was dissolved in THF (80 mL), then the temperature was lowered to 0 °C, and NaH (60% dispersion in mineral oil, 1.16 g, 28.98 mmol, 2.0 eq.) was added. After reacting for 30 min, a THF solution of compound 43 (6.0 g, 14.49 mmol, 20 mL THF) was added dropwise, and then the temperature was slowly raised to room temperature and reacted for 2 h until the raw materials disappeared. The reaction was quenched by adding saturated NH4Cl aqueous solution. After quenching, ethyl acetate (100 mL) and water (50 mL) were added for extraction, and the aqueous phase was extracted twice with EA (50 mL). Then the organic phases were combined, and the combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude product, and the crude product was purified by column chromatography (PE∶EA = 2∶1) to obtain compound 44 (4.3 g, 7.84 mmol, 54.1% yield). ESI-MS: m / z 549.2 [M+H] +

[0216] Synthesis of compound 45:

[0217] Compound 44 (4.3 g, 7.84 mmol, 1.0 eq.) was dissolved in THF (43 mL), and then TBAF (1.0 mol / L in THF, 15.7 mL, 15.68 mmol, 2.0 eq.) was added dropwise. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain a crude product, and the crude product was purified by column chromatography (PE∶EA = 1∶10) to obtain compound 45 (3.1 g, 7.14 mmol, 91.1% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 6.93 - 6.77 (m, 1H), 6.20 - 6.05 (m, 1H), 6.00 (d, J = 4.2 Hz, 1H), 5.81 (d, J = 0.9 Hz, 1H), 5.65 - 5.54 (m, 1H), 4.83 (d, J = 3.3 Hz, 1H), 4.21 (d, J = 3.6 Hz, 1H), 4.02 (m, 4H), 3.89 (d, J = 3.6 Hz, 1H), 3.65 - 3.49 (m, 2H), 3.43 - 3.33 (m, 2H), 3.20 (s, 3H), 1.20 (t, J = 7.2 Hz, 6H). 31 31P NMR (162 MHz, DMSO-d6) δ 16.97. ESI-MS: m / z 435.2 [M+H] +

[0218] Synthesis of compound 46:

[0219] Compound 45 (3.1 g, 7.14 mmol, 1.0 eq.) was dissolved in DCM (30 mL), then DCI (674 mg, 5.71 mmol, 0.8 eq.) and P-REAGENT (2.58 g, 8.57 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 1∶1) to obtain compound 46 (3.1 g, 4.89 mmol, 68.5% yield). 1 HNMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.61 (dd, J = 8.1, 6.0 Hz, 1H), 6.96 - 6.80 (m, 1H), 6.18 (dd, J = 22.3, 17.3 Hz, 1H), 5.98 - 5.89 (m, 1H), 4.88 - 4.81 (m, 1H), 4.52 - 4.38 (m, 1H), 4.17 - 3.98 (m, 2H), 4.01 (m, 4H), 3.88 - 3.71 (m, 2H), 3.67 - 3.54 (m, 3H), 3.53 - 3.34 (m, 2H), 3.19 (d, J = 2.3 Hz, 3H), 2.89 (t, J = 5.8 Hz, 1H), 2.78 (dd, J = 12.6, 6.2 Hz, 2H), 1.24 - 1.10 (m, 18H). 31 P NMR (162 MHz, DMSO) δ 149.99, 149.74, 16.73, 13.86. ESI-MS: m / z 635.3 [M+H] +

[0220] Example 6: Synthesis of Compound 53 (Cvp18)

[0221]

[0222] Synthesis of Compound 47:

[0223] Dissolve compound 41 (20.6 g, 38.85 mmol, 1.0 eq.) in ACN (200 mL), then successively add triazole (16.1 g, 233.1 mmol, 6.0 eq.) and triethylamine (23.59 g, 233.1 mmol, 6.0 eq.). Then cool the temperature to 0 °C and slowly add dropwise POCl3 (11.91 g, 77.70 mmol, 2.0 eq.). React under an ice bath for 2 h until the raw materials disappear. Then add ammonia water (200 mL) and react overnight. Add ethyl acetate (500 mL) and water (200 mL) for extraction and liquid separation to obtain the organic phase. The organic phase is washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. Rotate and evaporate the solvent to obtain the crude product of compound 47 (21.6 g). The crude product is directly used in the next reaction without purification. ESI-MS: m / z 530.3 [M+H] +

[0224] Synthesis of compound 48:

[0225] Dissolve the crude product of compound 47 (21.6 g) in pyridine (200 mL), cool the temperature to 0 °C, add benzoyl chloride (10.92 g, 77.70 mmol, 2.0 eq.). After reacting overnight until the raw materials are completely consumed, add saturated aqueous NaHCO3 to quench the reaction. Then add ethyl acetate (500 mL) and water (250 mL) for extraction and liquid separation to obtain the organic phase. The organic phase is washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate. Rotate and evaporate the solvent to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 3∶1) to obtain compound 48 (14.1 g, 22.26 mmol, total yield of two steps 57.3%). ESI-MS: m / z 634.3 [M+H] +

[0226] Synthesis of compound 49:

[0227] Dissolve compound 48 (14.1 g, 22.26 mmol, 1.0 eq.) in THF (100 mL), then cool the temperature to -5 °C and slowly add dropwise the TFA / H2O (v / v = 1∶1, 100 mL) solution. Control the internal temperature of the reaction not to exceed 5 °C throughout the process. After the addition is completed, continue to react at this temperature for 1 h until the raw materials disappear. At about 0 °C, slowly add dropwise ammonia water to quench the reaction until the reaction solution is neutral. After quenching, add EA (200 mL) and water (100 mL) for extraction, and then extract the aqueous phase twice with EA (100 mL). Then combine the organic phases. The organic phase is further washed with saturated brine and dried over anhydrous sodium sulfate. Rotate and evaporate the solvent to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 1∶2) to obtain compound 49 (7.3 g, 14.06 mmol, yield 63.2%). ESI-MS: m / z 520.2 [M+H]+

[0228] Synthesis of Compound 50:

[0229] Dissolve Compound 49 (7.3 g, 14.06 mmol, 1.0 eq.) in ACN (70 mL), then add IBX (7.87 g, 28.12 mmol, 1.0 eq.), and then reflux the reaction in an oil bath for 2 h until Compound 49 reacts completely. The reaction solution is cooled to room temperature and then filtered. The filter cake is washed twice with ethyl acetate, and the filtrates are combined. After rotary evaporation of the filtrate, Compound 50 (7.1 g, 13.73 mmol, 97.7% yield) is obtained. The crude product is directly used in the next step without purification. ESI-MS: m / z 518.2 [M+H] +

[0230] Synthesis of Compound 51:

[0231] Dissolve Compound 10 (7.91 g, 27.46 mmol, 2.0 eq.) in THF (70 mL), then cool to 0 °C, add NaH (60% dispersion in mineral oil, 1.10 g, 27.46 mmol, 2.0 eq.). After reacting for 30 min, a THF solution of Compound 50 (7.1 g, 13.73 mmol, 20 mL THF) is added dropwise, and then the reaction is slowly warmed to room temperature and reacted for 2 h until Compound 50 reacts completely. The reaction is quenched by adding saturated aqueous NH4Cl solution. After quenching, EA (100 mL) and water (50 mL) are added for extraction, and the aqueous phase is extracted twice with EA (50 mL). Then the organic phases are combined, washed with saturated brine and dried over anhydrous sodium sulfate. After rotary evaporation of the solvent, a crude product is obtained. The crude product is purified by column chromatography (PE:EA = 2:1) to obtain Compound 51 (5.3 g, 8.14 mmol, 59.3% yield). ESI-MS: m / z 652.3 [M+H] +

[0232] Synthesis of Compound 52:

[0233] Dissolve Compound 51 (5.3 g, 8.14 mmol, 1.0 eq.) in THF (50 mL), then add dropwise TBAF (1.0 mol / L in THF, 12.2 mL, 12.21 mmol, 1.5 eq.). After reacting at room temperature for 1 h, the solvent is removed by rotary evaporation at low temperature to obtain a crude product. The crude product is purified by column chromatography (EA∶MeOH = 10∶1) to obtain Compound 52 (3.7 g, 6.89 mmol, 84.6% yield). ESI-MS: m / z 538.2 [M+H] +

[0234] Synthesis of Compound 53:

[0235] Dissolve Compound 52 (3.7 g, 6.89 mmol, 1.0 eq.) in DCM (37 mL), then add DCI (651 mg, 5.51 mmol, 0.8 eq.) and P-REAGENT (2.49 g, 8.27 mmol, 1.2 eq.). After reacting at room temperature for 1 h, rotary evaporate the solvent under reduced temperature to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 2∶1) to obtain Compound 53 (3.8 g, 5.15 mmol, 74.7% yield). ESI-MS: m / z 738.3 [M+H] +

[0236] Example 7: Synthesis of Compound 61 (Avp19)

[0237]

[0238] Synthesis of Compound 54:

[0239] Dissolve Compound 38 (16.7 g, 49.98 mmol, 1.0 eq.) and ABz (23.91 g, 99.96 mmol, 2.0 eq.) in ACN (200 mL), then add BSA (15.25 g, 74.97 mmol, 1.5 eq.). Place the reaction in an oil bath at 50 °C and react for 1 h, then cool to 0 °C. Dropwise add a solution of TMSOTf (11.10 g, 49.98 mmol, 1.0 eq.) to the reaction. After the addition is complete, reflux the reaction in an oil bath for 3 h until Compound 38 reacts completely. After the reaction is completed, restore the reaction to room temperature and quench it with saturated aqueous NaHCO3. Then add ethyl acetate (300 mL) and water (200 mL) for extraction and separation to obtain the organic phase. Extract the aqueous phase twice with EA (150 mL), then combine the organic phases. The organic phase is washed with saturated brine and dried over anhydrous sodium sulfate. Rotary evaporate the solvent to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 2∶1) to obtain Compound 54 (20.6 g, 40.14 mmol, 80.3% yield). ESI-MS: m / z 514.2 [M+H] +

[0240] Synthesis of Compound 55:

[0241] Compound 54 (20.6 g, 40.14 mmol, 1.0 eq.) was dissolved in MeOH (200 mL), and then the temperature was lowered to 0 °C. Sodium methoxide (10.84 g, 200.7 mmol, 5.0 eq.) was added, and the reaction was carried out at room temperature overnight. The reaction was cooled to about 0 °C, and a 1 mol / L dilute hydrochloric acid solution was slowly added dropwise to neutralize the reaction to pH about 7. Ethyl acetate was added to the reaction for extraction twice, and the organic phases were combined. The organic phase was washed with water, 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 (DCM∶MeOH = 20∶1) to obtain compound 55 (13.4 g, 31.22 mmol, 77.8% yield). ESI-MS: m / z 430.2 [M+H] +

[0242] Synthesis of compound 56:

[0243] Compound 55 (13.4 g, 31.22 mmol, 1.0 eq.) was dissolved in ultradry DMF (140 mL), and then imidazole (12.75 g, 187.32 mmol, 6.0 eq.) and TBSCl (14.12 g, 93.66 mmol, 3.0 eq.) were added in sequence. After the reaction overnight until the raw materials were completely consumed, the temperature was lowered to 0 °C, and a saturated NaHCO3 aqueous solution was added to quench the reaction. Then ethyl acetate (100 mL) and water (100 mL) were added for extraction and liquid separation to obtain the organic phase. The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the crude product, which was purified by flash column chromatography to obtain compound 56 (18.6 g, 28.30 mmol, 90.6% yield). ESI-MS: m / z 658.3 [M+H] +

[0244] Synthesis of compound 57:

[0245] Dissolve compound 56 (18.6 g, 28.30 mmol, 1.0 eq.) in THF (200 mL), then cool the temperature to -5 °C, and slowly add dropwise the solution of TFA / H2O (v∶v = 1∶1, 100 mL). Control the reaction temperature not to exceed 5 °C throughout the process. After the addition is completed, continue to react at this temperature for 1 h until compound 56 completely reacts. After the reaction is completed, slowly add dropwise ammonia water to quench the reaction at 0 °C until the reaction solution is neutral. After quenching, add ethyl acetate (200 mL) and water (100 mL) for extraction, and then extract the aqueous phase twice with ethyl acetate (100 mL). Then combine the organic phases. The organic phase is washed with saturated brine and dried over anhydrous sodium sulfate. Rotate to dry the solvent to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 1∶1) to obtain compound 57 (10.7 g, 19.70 mmol, 69.6% yield). ESI-MS: m / z 544.3 [M+H] +

[0246] Synthesis of compound 58:

[0247] Dissolve compound 57 (10.7 g, 19.70 mmol, 1.0 eq.) in ACN (100 mL), then add IBX (11.03 g, 39.40 mmol, 2.0 eq.), and then reflux the reaction in an oil bath for 2 h until compound 57 completely reacts. After the reaction is completed, cool the reaction to room temperature and then filter. Wash the filter cake twice with ethyl acetate, and combine the filtrates. After rotating the filtrate to dryness, compound 58 (10.3 g, 19.03 mmol, 96.6% yield) is obtained. ESI-MS: m / z 542.2 [M+H] +

[0248] Synthesis of compound 59:

[0249] Compound 10 (10.96 g, 38.06 mmol, 2.0 eq.) was dissolved in THF (100 mL), then the temperature was lowered to 0 °C, and NaH (60% dispersion in mineral oil, 1.52 g, 38.06 mmol, 2.0 eq.) was added. After reacting for 30 min, a solution of compound 58 (10.3 g, 19.03 mmol, 30 mL THF) in THF was added dropwise, and then the temperature was slowly raised to room temperature and reacted for 2 h until the raw materials disappeared. After the reaction was complete, the reaction was quenched with a saturated aqueous solution of NH4Cl. After quenching, ethyl acetate (100 mL) and water (50 mL) were added for extraction, and the aqueous phase was extracted twice with EA (100 mL). Then the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude product, and the crude product was purified by column chromatography (PE∶EA = 2∶1) to obtain compound 59 (7.4 g, 10.96 mmol, 57.6% yield). ESI-MS: m / z 676.3 [M+H] +

[0250] Synthesis of compound 60:

[0251] Compound 59 (7.4 g, 10.96 mmol, 1.0 eq.) was dissolved in THF (74 mL), and then TBAF (1.0 mol / L in THF, 21.9 mL, 21.92 mmol, 2.0 eq.) was added dropwise. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain a crude product, and the crude product was purified by column chromatography (EA) to obtain compound 60 (4.3 g, 7.66 mmol, 69.9% yield). ESI-MS: m / z 562.2 [M+H] +

[0252] Synthesis of compound 61:

[0253] The starting material 60 (4.3 g, 7.66 mmol) was dissolved in DCM (40 mL), and then DCI (724 mg, 6.13 mmol, 0.8 eq.) and P-REAGENT (2.77 g, 9.19 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain a crude product, and the crude product was purified by column chromatography (PE∶EA = 1∶1) to obtain compound 61 (4.2 g, 5.52 mmol, 72.1% yield). ESI-MS: m / z762.3 [M+H] +

[0254] Example 8: Synthesis of compound 69 (Gvp20)

[0255]

[0256] Synthesis of compound 62:

[0257] Dissolve compound 38 (16.7 g, 49.98 mmol) and GiBu (16.58 g, 74.97 mmol, 1.5 eq.) in ACN (200 mL), then add BSA (15.25 g, 74.97 mmol, 1.5 eq.). Place the reaction in an oil bath at 50 °C and react for 1 h, then cool to 0 °C. Slowly add a solution of TMSOTf (11.11 g, 49.98 mmol, 1.0 eq.) dropwise to the reaction. After the addition is complete, reflux the reaction in an oil bath for 3 h until compound 38 has completely reacted. After the reaction is complete, restore the reaction to room temperature and quench it with saturated aqueous NaHCO3. Then add ethyl acetate (300 mL) and water (200 mL) for extraction. Extract the aqueous phase twice with ethyl acetate (150 mL). Then combine the organic phases. Wash the organic phase with saturated brine and dry it over anhydrous sodium sulfate. Rotavap the solvent to obtain the crude product. The crude product is slurried with EA to obtain compound 62 (10.8 g, 21.81 mmol, 43.6% yield). ESI-MS: m / z 496.2 [M+H] +

[0258] Synthesis of compound 63:

[0259] Dissolve compound 62 (10.8 g, 22.18 mmol, 1.0 eq.) in MeOH (100 mL), then cool to 0 °C. Add sodium methoxide (6.0 g, 110.9 mmol, 5.0 eq.). React at room temperature overnight. Cool the reaction to 0 °C and add 1 mol / L dilute hydrochloric acid dropwise to the reaction to neutralize it until the pH is about 7. Extract the reaction twice with ethyl acetate and combine the organic phases. Wash the organic phase with water, saturated brine, dry it over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain the crude product. The crude product is purified by column chromatography (DCM∶MeOH = 20∶1) to obtain compound 63 (8.1 g, 19.70 mmol, 88.8% yield). ESI-MS: m / z 412.2 [M+H] +

[0260] Synthesis of compound 64:

[0261] Compound 63 (8.1 g, 19.70 mmol, 1.0 eq.) was dissolved in DMF (100 mL), and then imidazole (8.05 g, 118.2 mmol, 6.0 eq.) and TBSCl (8.91 g, 59.10 mmol, 1.0 eq.) were added successively. The reaction was carried out overnight until Compound 63 reacted completely. Then the temperature was lowered to 0 °C, and saturated aqueous NaHCO3 was added to the reaction to quench the reaction. Then ethyl acetate (300 mL) and water (300 mL) were added for extraction. The organic phase was washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated by a rotary evaporator to obtain crude Compound 64 (11.4 g), and the crude product was directly used in the next step without purification. ESI-MS: m / z 640.4 [M+H] +

[0262] Synthesis of Compound 65:

[0263] Compound 64 (11.4 g) was dissolved in THF (100 mL), and then the temperature was lowered to -5 °C. A solution of TFA / H2O (v∶v = 1∶1, 100 mL) was slowly added dropwise to the reaction while controlling the internal temperature of the reaction not to exceed 5 °C. After the addition was completed, the reaction was continued at this temperature for 1 h until Compound 64 reacted completely. Then ammonia water was slowly added dropwise to the reaction to quench it until the reaction solution was neutral. After quenching, ethyl acetate (200 mL) and water (100 mL) were added for extraction, and then the aqueous phase was extracted twice with ethyl acetate (100 mL). Then the organic phases were combined, and the combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude product, and the crude product was purified by column chromatography (DCM∶MeOH = 20∶1) to obtain Compound 65 (6.3 g, 11.99 minol, total yield of two steps 60.9%). ESI-MS: m / z 526.3 [M+H] +

[0264] Synthesis of Compound 66:

[0265] Compound 65 (6.3 g, 11.99 mmol, 1.0 eq.) was dissolved in ACN (60 mL), and then IBX (6.71 g, 23.98 mmol, 2.0 eq.) was added. Then the reaction was refluxed in an oil bath for 2 h until Compound 65 reacted completely. The reaction solution was cooled to room temperature and then filtered. The filter cake was washed twice with ethyl acetate, and the filtrates were combined. After the filtrate was evaporated to dryness, crude Compound 66 (6.1 g) was obtained, and the crude product was directly used in the next step without purification. ESI-MS: m / z 524.3 [M+H] +

[0266] Synthesis of Compound 67:

[0267] Compound 10 (6.91 g, 23.98 mmol, 2.0 eq.) was dissolved in THF (100 mL), then the temperature was lowered to 0 °C, and NaH (60% dispersion in mineral oil, 0.96 g, 23.98 mmol, 2.0 eq.) was added. After reacting for 30 min, a THF solution of Compound 66 (6.1 g, 20 mL THF) was added dropwise, and then the temperature was slowly raised to room temperature and reacted for 2 h until the raw materials disappeared. The reaction was quenched by adding a saturated NH4Cl aqueous solution. After quenching, extraction was carried out with ethyl acetate (100 mL) and water (50 mL), and then the aqueous phase was extracted twice with ethyl acetate (50 mL). Then the organic phases were combined, and the combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude product, and the crude product was purified by normal-phase purification (PE∶EA = 1∶1) to obtain Compound 67 (4.0 g, 6.09 mmol, total yield of two steps 50.8%). ESI-MS: m / z 658.3 [M+H] +

[0268] Synthesis of Compound 68:

[0269] Compound 67 (4.0 g, 6.09 mmol) was dissolved in THF (40 mL), then TBAF (1.0 mol / L in THF, 9.1 mL, 9.14 mmol, 1.5 eq.) was added dropwise. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain a crude product, and the crude product was purified by column chromatography (EA) to obtain Compound 68 (3.0 g, 5.52 mmol, yield 90.6%). ESI-MS: m / z 544.2 [M+H] +

[0270] Synthesis of Compound 69:

[0271] Compound 68 (3.0 g, 5.52 mmol, 1.0 eq.) was dissolved in DCM (30 mL), then DCI (522 mg, 4.42 mmol, 0.8 eq.) and P-REAGENT (2.0 g, 6.62 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain a crude product, and the crude product was purified by column chromatography (PE∶EA = 1∶2) to obtain Compound 69 (2.6 g, 3.50 mmol, yield 63.4%). ESI-MS: m / z 744.3 [M+H] +

[0272] The following Examples 9-12 are preparation examples of the nucleoside compounds shown by Formula 7-a of the present invention, wherein the nucleoside compounds shown by Formula 7-a are compounds with a 5'-(E)-VP-3'-ONMA-2'-phosphoramidite group.

[0273] Example 9: Synthesis of Compound 79 (Uvp21)

[0274]

[0275] Synthesis of Compound 70:

[0276] Add NaH (60% dispersion in mineral oil, 25.64 g, 0.641 mol, 1.5 eq.) to a three-necked flask, then dropwise add ultra-dry DMF (1.1 L) at 0 - 5 °C. Subsequently, dropwise add Compound 2 (130.0 g, 0.427 mol, 250 mL of DMF) to the reaction. After reacting for 30 min, dropwise add 2-bromo-N-methylacetamide (77.29 g, 0.512 mol, 1.2 eq.). After addition, restore the reaction to room temperature and react overnight until Compound 2 reacts completely. Cool the reaction to 0 °C and add saturated NH₄Cl aqueous solution to quench the reaction. After quenching, add ethyl acetate (3.5 L) and water (2.5 L) for extraction and liquid separation to obtain the organic phase. The aqueous phase is extracted twice with ethyl acetate, and the organic phases are combined. The combined organic phase is washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent is evaporated off using a rotary evaporator to obtain the crude product. The crude product is purified by flash column chromatography (PE∶EA = 10∶1) to obtain Compound 70 (118.7 g, 0.316 mmol, 74.0% yield). 1 ¹H NMR (400 MHz, DMSO-d₆) δ 7.50 (q, J = 4.0 Hz, 1H), 5.85 (d, J = 4.0 Hz, 1H), 4.70 (d, J = 4.0 Hz, 1H), 4.10 - 4.00 (m, 2H), 3.92 - 3.86 (m, 3H), 3.79 - 3.74 (m, 1H), 2.63 (d, J = 8.0 Hz, 3H), 1.39 (s, 3H), 1.25 (s, 3H), 0.86 (s, 9H), 0.05 (s, 6H). ESI-MS: m / z 376.2 [M + H] +

[0277] Synthesis of Compound 71:

[0278] Dissolve compound 70 (118.7 g, 0.316 mmol, 1.0 eq.) in AcOH (1100 mL), then cool the temperature to 0 °C, and add dropwise Ac2O (161.3 g, 1.58 mol, 5.00 eq.) and concentrated H2SO4 (40.0 mL). After the addition, slowly restore the reaction to room temperature and react overnight until the raw materials disappear. Then slowly add dropwise the reaction solution into an ice-cold aqueous solution of NaHCO3 to quench the reaction until the reaction solution is neutral. After quenching, add ethyl acetate (3.0 L) and extract and separate the organic phase. Then extract the aqueous phase twice with EA (1000 mL). Then combine the organic phases. The organic phase is further washed with saturated brine and dried over anhydrous sodium sulfate. Rotate to dry the solvent to obtain the crude product. The crude product is purified by column chromatography (PE:EA = 10:1) to obtain compound 71 (86.9 g, 0.250 mol, 79.1% yield). ESI-MS: m / z 348.1 [M+H] +

[0279] Synthesis of compound 72:

[0280] Dissolve compound 71 (25.2 g, 72.60 mmol, 1.0 eq.) and Uracil (12.21 g, 108.9 mmol, 1.5 eq.) in ACN (300 mL), then add BSA (22.15 g, 108.9 mmol, 1.5 eq.). Place the reaction in an oil bath at 50 °C and react for 1 h. Then cool the temperature to 0 °C, and add dropwise TMSOTf (16.14 g, 72.60 mmol, 1.0 eq.) to the reaction. After the addition is complete, place the reaction in an oil bath and reflux for 3 h until compound 71 reacts completely. After the reaction is completed, cool the reaction to room temperature and add a saturated aqueous solution of NaHCO3 to quench the reaction. Then add EA (300 mL) and water (200 mL) for extraction. Extract the aqueous phase twice with EA (350 mL). Then combine the organic phases. The organic phase is further washed with saturated brine and dried over anhydrous sodium sulfate. Rotate to dry the solvent to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 2∶1) to obtain compound 72 (21.1 g, 52.86 mmol, 72.8% yield). 11H NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 4.0 Hz, 1H), 5.93 (d, J = 4.0 Hz, 1H), 4.61 (d, J = 8.0 Hz, 1H), 5.20 (s, 1H), 4.50 - 4.40 (m, 2H), 4.33 - 4.29 (m, 1H), 4.14 - 4.08 (m, 2H), 3.98 - 3.94 (m, 1H), 2.62 (d, J = 4.0 Hz, 3H), 2.07 (d, J = 12.0 Hz, 6H). ESI-MS: m / z 400.1 [M+H] +

[0281] Synthesis of Compound 73:

[0282] Dissolve Compound 72 (21.1 g, 52.86 mmol, 1.0 eq.) in MeOH (100 mL), then cool the temperature to 0 °C, add sodium methoxide (14.28 g, 264.3 mmol, 5.0 eq.). After addition, slowly restore the reaction to room temperature and react overnight. After the reaction is completed, cool the reaction to 0 °C, then neutralize the reaction with 1 mol / L dilute hydrochloric acid to about pH 7. Extract the reaction twice with ethyl acetate, and combine the organic phases. The organic phase is washed with water, saturated brine, dried over anhydrous sodium sulfate and the solvent is removed under reduced pressure to obtain the crude product. The crude product is purified by column chromatography (EA) to obtain Compound 73 (15.0 g, 47.60 mmol, 90.0% yield). ESI-MS: m / z 316.1 [M+H] +

[0283] Synthesis of Compound 74:

[0284] Dissolve Compound 73 (15.0 g, 47.60 mmol, 1.0 eq.) in ultra-dry DMF (150 mL), then successively add imidazole (19.44 g, 285.6 mmol, 6.0 eq.), TBSCl (21.52 g, 142.8 mmol, 3.0 eq.). After reacting overnight until the raw materials are completely consumed, cool the reaction to 0 °C, add saturated aqueous NaHCO3 solution to quench the reaction, then add ethyl acetate (350 mL) and water (150 mL) for extraction and separation to obtain the organic phase. The aqueous phase is extracted twice with ethyl acetate, and the organic phases are combined. The organic phase is washed twice with water, washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent is evaporated to dryness with a rotary evaporator to obtain the crude product. The crude product is purified by flash column chromatography to obtain Compound 74 (24.1 g, 44.36 mmol, 93.2% yield). 11H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 4.0 Hz, 1H), 5.59 (s, 1H), 5.45 (d, J = 8.0 Hz, 1H), 4.37 (s, 1H), 4.12 - 4.09 (m, 1H), 3.99 - 3.84 (m, 4H), 3.70 - 3.69 (m, 1H), 2.54 (d, J = 4.0 Hz, 3H), 0.79 (d, J = 8.0 Hz, 18H), 0.03 - 0.00 (m, 12H). ESI-MS: m / z 544.3 [M+H] +

[0285] Synthesis of Compound 75:

[0286] Dissolve Compound 74 (10.9 g, 20.06 mmol, 1.0 eq.) in THF (100 mL), then cool the temperature to -5 °C, slowly add dropwise the solution of TFA / H2O (v∶v = 1∶1, 100 mL). After the addition is completed, continue to react at this temperature for 1 h until the raw materials disappear. Then slowly add dropwise ammonia water for quenching until the reaction solution is neutral. After quenching, add EA (200 mL) and water (100 mL) for extraction, and then extract the aqueous phase with EA (100 mL) twice. Then combine the organic phases, wash the organic phases with saturated brine and dry over anhydrous sodium sulfate, and rotary evaporate the solvent to obtain the crude product. The crude product is purified by column chromatography (PE∶EA = 1∶1) to obtain Compound 75 (6.5 g, 15.14 mmol, 75.5% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 7.57 - 7.53 (m, 2H), 5.54 (s, 1H), 5.44 (d, J = 8.0 Hz, 1H), 4.97 (t, J = 8.0 Hz, 1H), 4.31 (s, 1H), 4.11 - 4.07 (m, 1H), 3.94 - 3.81 (m, 2H), 3.73 - 3.64 (m, 3H), 2.50 (d, J = 8.0 Hz, 3H), 0.75 (s, 9H), 0.00 (d, J = 4.0 Hz, 6H). ESI-MS: m / z 430.2 [M+H] +

[0287] Synthesis of Compound 76:

[0288] Dissolve compound 75 (6.5 g, 15.14 mmol, 1.0 eq.) in ACN (60 mL), then add IBX (8.50 g, 30.28 mmol, 2.0 eq.). Then reflux the reaction in an oil bath for 2 h until the raw materials disappear. Cool the reaction solution to room temperature and then filter. Wash the filter cake twice with ethyl acetate and combine the filtrates. After rotary evaporation of the filtrate, crude compound 76 (6.1 g) is obtained. The crude product is directly used in the next reaction without purification. ESI-MS: m / z 428.2 [M+H] +

[0289] Synthesis of compound 77:

[0290] Dissolve compound 10 (8.72 g, 30.28 mmol, 2.0 eq.) in THF (82 mL), then cool to 0 °C. Slowly add a solution of NaH (60% dispersion in mineral oil, 1.21 g, 30.28 mmol, 2.0 eq.) in portions. After reacting for 30 min, dropwise add a THF solution of crude compound 76 (6.1 g, 15 mL THF). Then slowly warm to room temperature and react for 2 h until the raw materials disappear. Quench the reaction with an aqueous NH4Cl solution. After quenching, add EA (100 mL) and water (50 mL) for extraction. Then extract the aqueous phase twice with EA (100 mL). Then combine the organic phases. Wash the organic phase with saturated brine and dry over anhydrous sodium sulfate. Rotary evaporate the solvent to obtain a crude product. The crude product is purified by normal-phase chromatography (PE∶EA = 2∶1) to obtain compound 77 (8.1 g, 14.43 mmol, total yield of two steps 47.7%). ESI-MS: m / z 562.2 [M+H] +

[0291] Synthesis of compound 78:

[0292] Dissolve compound 77 (8.1 g, 14.43 mmol) in THF (81 mL), then dropwise add TBAF (1.0 mol / L in THF, 21.7 mL, 21.65 mmol, 2.0 eq.). After reacting at room temperature for 1 h, rotary evaporate the solvent at low temperature to obtain a crude product. The crude product is purified by column chromatography (EA) to obtain compound 78 (5.8 g, 12.97 mmol, yield 89.9%). 11H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 4.0 Hz, 1H), 7.07 - 6.96 (m, 1H), 6.23 - 6.13 (m, 1H), 6.01 (d, J = 4.0 Hz, 1H), 5.79 (s, 1H), 5.62 - 5.57 (m, 5H), 4.84 - 4.83 (m, 1H), 4.36 - 4.35 (m, 1H), 3.98 - 3.86 (m, 3H), 2.61 (d, J = 8.0 Hz, 3H), 1.15 (m, 6H), 31 31P NMR (162 MHz, DMSO-d6): δ 17.0, ESI-MS: m / z 448.1 [M+H] +

[0293] Synthesis of Compound 79:

[0294] Dissolve Compound 78 (2.0 g, 4.47 mmol) in DCM (20 mL), then add DCI (423 mg, 3.58 mmol, 0.8 eq.), P-REAGENT (1.62 g, 5.36 mmol, 1.2 eq.). After reacting at room temperature for 1 h, evaporate the solvent to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 1∶2) to obtain Compound 79 (2.1 g, 3.24 mmol, 72.5% yield). 1 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 7.72 - 7.67 (m, 1H), 7.53 - 7.50 (m, 1H), 7.10 - 6.96 (m, 1H), 6.27 - 6.17 (m, 1H), 5.91 (d, J = 20.0 Hz, 1H), 5.63 - 5.57 (m, 5H), 4.83 (s, 1H), 4.70 - 4.59 (m, 1H), 4.18 - 4.06 (m, 1H), 4.01 - 3.87 (m, 2H), 3.83 - 3.71 (m, 2H), 3.64 - 3.53 (m, 2H), 2.79 - 2.76 (m, 2H), 2.61 - 2.59 (m, 3H), 1.15 - 1.11 (m, 18H), 31 31P NMR (162 MHz, DMSO-d6): δ 150.5, 150.1, 16.8, ESI-MS: m / z 648.3 [M+H] +

[0295] Example 10: Synthesis of Compound 86 (Cvp22)

[0296]

[0297] Synthesis of Compound 80:

[0298] Dissolve Compound 74 (13.2 g, 24.30 mmol, 1.0 eq.) in ACN (150 mL), then successively add triazole (10.07 g, 145.8 mmol, 6.0 eq.) and triethylamine (14.75 g, 145.8 mmol, 6.0 eq.). Then cool the temperature to 0 °C and slowly add dropwise POCl3 (7.45 g, 48.60 mmol, 2.0 eq.). React for 2 h under an ice bath until the raw materials disappear. Then add ammonia water (200 mL) and react overnight. Then add ethyl acetate (500 mL) and water (200 mL) for extraction and liquid separation to obtain the organic phase. The organic phase is washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent is evaporated to dryness using a rotary evaporator to obtain crude Compound 80 (13.2 g). The crude product is directly used in the next reaction without purification. ESI-MS: m / z 543.3 [M+H] +

[0299] Synthesis of Compound 81:

[0300] Dissolve Compound 80 (13.2 g) in pyridine (200 mL), cool the temperature to 0 °C, add benzoyl chloride (6.83 g, 48.60 mmol, 2.0 eq.). After the reaction overnight until the raw materials are completely consumed, add saturated aqueous NaHCO3 to quench the reaction. Then add ethyl acetate (500 mL) and water (250 mL) for extraction and liquid separation to obtain the organic phase. The organic phase is washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent is evaporated to dryness using a rotary evaporator to obtain the crude product. The crude product is purified by column chromatography (PE:EA = 3:1) to obtain Compound 81 (11.0 g, 17.02 mmol, total yield of two steps 70.0%). ESI-MS: m / z 647.3 [M+H] +

[0301] Synthesis of Compound 82:

[0302] Compound 81 (11.0 g, 17.02 mmol) was dissolved in THF (100 mL), and then the temperature was lowered to -5 °C. A solution of TFA / H2O (v / v = 1:1, 100 mL) was slowly added dropwise. After the addition was completed, the reaction was continued under an ice bath for 1 h until the raw materials disappeared. Then, ammonia water was slowly added dropwise for quenching until the reaction solution was neutral. After quenching, EA (200 mL) and water (100 mL) were added for extraction, and the aqueous phase was extracted twice with EA (100 mL). Then, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography (PE:EA = 1:2) to obtain Compound 82 (6.3 g, 11.84 mmol, 69.6% yield). ESI-MS: m / z 533.2 [M+H] +

[0303] Synthesis of Compound 83:

[0304] Compound 82 (6.3 g, 11.84 mmol) was dissolved in ACN (70 mL), and then IBX (6.63 g, 23.68 mmol, 2.0 eq.) was added. Then, the mixture was refluxed in an oil bath for 2 h until the raw materials disappeared. The reaction solution was cooled to room temperature and then filtered. The filter cake was washed twice with ethyl acetate, and the filtrates were combined. The solvent of the filtrate was evaporated to obtain the crude product of Compound 83 (6.0 g). ESI-MS: m / z 531.2 [M+H] +

[0305] Synthesis of Compound 84:

[0306] Compound 10 (6.82 g, 23.68 mmol, 2.0 eq.) was dissolved in THF (70 mL), and then the temperature was lowered to 0 °C. NaH (60% dispersion in mineral oil, 0.95 g, 23.68 mmol, 2.0 eq.) was added. After reacting for 30 min, a THF solution of Compound 83 (6.0 g, 20 mL THF) was added dropwise. Then, the temperature was slowly raised to room temperature and the reaction was continued for 2 h until the raw materials disappeared. The reaction was quenched by adding a saturated NH4Cl aqueous solution. After quenching, EA (100 mL) and water (50 mL) were added for extraction, and the aqueous phase was extracted twice with EA (100 mL). Then, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography (PE:EA = 2:1) to obtain Compound 84 (4.3 g, 6.47 mmol, total two-step yield 65.6%). ESI-MS: m / z 665.3 [M+H] +

[0307] Synthesis of Compound 85:

[0308] Compound 84 (4.3 g, 6.47 mmol) was dissolved in THF (43 mL), and then TBAF (1.0 mol / L in THF, 9.7 mL, 9.71 mmol, 1.5 eq.) was added dropwise. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain the crude product. The crude product was purified by column chromatography (EA∶MeOH = 10∶1) to obtain compound 85 (3.2 g, 5.82 mmol, 90.0% yield). ESI-MS: m / z 551.2 [M+H] +

[0309] Synthesis of compound 86:

[0310] Compound 85 (3.2 g, 5.82 mmol, 1.0 eq.) was dissolved in DCM (32 mL), and then DCI (550 mg, 4.66 mmol, 0.8 eq.) and P-REAGENT (2.10 g, 6.98 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 1∶1) to obtain compound 86 (3.3 g, 4.40 mmol, 75.6% yield). ESI-MS: m / z 751.3 [M+H] +

[0311] Example 11: Synthesis of compound 94 (Avp23)

[0312]

[0313] Synthesis of compound 87:

[0314] Compound 71 (15.2 g, 43.79 mmol, 1.0 eq.) and ABz (15.72 g, 65.69 mmol, 1.5 eq.) were dissolved in ACN (200 mL), and then BSA (1.36 g, 65.69 mmol, 1.5 eq.) was added. The reaction was placed in an oil bath at 50 °C for 1 h, and then cooled to 0 °C. A solution of TMSOTf (9.73 g, 43.79 mmol, 1.0 eq.) was added dropwise to the reaction. After the addition was complete, the reaction was refluxed in an oil bath for 3 h until the raw materials disappeared. After the reaction was completed, the reaction was cooled to room temperature, and saturated aqueous NaHCO3 was added to quench the reaction. Then, ethyl acetate (300 mL) and water (200 mL) were added for extraction and liquid separation to obtain the organic phase. The aqueous phase was extracted twice with EA (150 mL), and then the organic phases were combined. The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product. The crude product was purified by column chromatography (PE∶EA = 2∶1) to obtain compound 87 (16.1 g, 30.60 mmol, 69.9% yield). ESI-MS: m / z 527.2 [M+H]+

[0315] Synthesis of Compound 88:

[0316] Dissolve Compound 87 (16.1 g, 30.60 mmol) in MeOH (100 mL), then cool the temperature to 0 °C, add sodium methoxide (8.27 g, 153.0 mmol, 5.0 eq.), and after addition, slowly restore the reaction to room temperature and react overnight until Compound 87 completely reacts. Cool the reaction to 0 °C, and dropwise add 1 mol / L dilute hydrochloric acid to neutralize the reaction until the pH is about 7. Extract the reaction twice with ethyl acetate, and combine the organic phases. The organic phase is washed with water, saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain the crude product. The crude product is purified by column chromatography (DCM∶MeOH = 20∶1) to obtain Compound 88 (10.8 g, 24.43 mmol, 79.8% yield). ESI-MS: m / z 443.2 [M+H] +

[0317] Synthesis of Compound 89:

[0318] Dissolve Compound 88 (10.8 g, 24.43 mmol, 1.0 eq.) in ultra-dry DMF (120 mL), then successively add imidazole (9.98 g, 146.58 mmol, 6.0 eq.) and TBSCl (11.05 g, 73.29 mmol, 3.0 eq.). After reacting overnight until the raw materials are completely consumed, cool the temperature to 0 °C, add saturated aqueous NaHCO3 to quench the reaction, then add ethyl acetate (200 mL) and water (100 mL) to extract and separate to obtain the organic phase. The aqueous phase is extracted twice with ethyl acetate, and the organic phases are combined. The organic phase is washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent is evaporated to dryness with a rotary evaporator to obtain the crude product of Compound 89 (16.4 g). The crude product is directly used in the next step without purification. ESI-MS: m / z 671.3 [M+H] +

[0319] Synthesis of Compound 90:

[0320] Dissolve compound 89 (16.4 g) in THF (150 mL), then cool the temperature to -5 °C, slowly add dropwise the solution of TFA / H2O (v∶v = 1∶1, 100 mL). After the addition is complete, continue to react at this temperature for 1 h until compound 89 completely reacts. Then slowly add dropwise ammonia water for quenching until the reaction solution is neutral. After quenching, add ethyl acetate (200 mL) and water (100 mL) for extraction, and then extract the aqueous phase with ethyl acetate (100 mL) twice. Then combine the organic phases, and the organic phases are washed with saturated brine and dried over anhydrous sodium sulfate. Rotate to dry the solvent to obtain the crude product, and the crude product is purified by column chromatography (PE∶EA = 1∶1) to obtain compound 90 (9.5 g, 17.08 mmol, the total yield of two steps is 69.9%). ESI-MS: m / z 557.3 [M+H] +

[0321] Synthesis of compound 91:

[0322] Dissolve compound 90 (9.5 g, 17.08 mmol, 1.0 eq.) in ACN (100 mL), then add IBX (9.57 g, 34.16 mmol, 2.0 eq.), and then reflux the reaction in an oil bath for 2 h until compound 90 completely reacts. Cool the reaction solution to room temperature and then filter. Wash the filter cake with acetonitrile twice and combine the filtrates. After rotating the filtrate to dryness, obtain compound 91 (9.2 g, crude compound, directly used in the next step without purification). ESI-MS: m / z 555.2 [M+H] +

[0323] Synthesis of compound 92:

[0324] Dissolve compound 10 (9.84 g, 34.16 mmol, 2.0 eq.) in THF (100 mL), then cool the temperature to 0 °C, add NaH (60% dispersion in mineral oil, 1.37 g, 34.16 mmol, 2.0 eq.). After reacting for 30 min, add dropwise the THF solution of compound 91 (9.2 g, 30 mL THF), and then slowly raise the temperature to room temperature and react for 2 h until the raw materials disappear. Add saturated NH4Cl aqueous solution to the reaction to quench the reaction. After quenching, add EA (200 mL) and water (100 mL) for extraction, and then extract the aqueous phase with ethyl acetate (100 mL) twice. Then combine the organic phases, and the organic phases are washed with saturated brine and dried over anhydrous sodium sulfate. Rotate to dry the solvent to obtain the crude product, and the crude product is purified by column chromatography (PE∶EA = 2∶1) to obtain compound 92 (5.9 g, 8.57 mmol, the total yield of two steps is 50.2% yield). ESI-MS: m / z 689.3 [M+H] +

[0325] Synthesis of Compound 93:

[0326] Dissolve Compound 92 (5.9 g, 8.57 mmol) in THF (60 mL), then add dropwise TBAF (1.0 mol / L in THF, 12.9 mL, 12.86 mmol, 1.5 eq.). After reacting at room temperature for 1 h, the solvent was removed by rotary evaporation under reduced temperature to obtain a crude product, which was purified by column chromatography (EA) to obtain Compound 93 (3.4 g, 5.92 mmol, 69.1% yield). ESI-MS: m / z 575.2 [M+H] +

[0327] Synthesis of Compound 94:

[0328] Dissolve Compound 93 (3.4 g, 5.92 mmol) in DCM (40 mL), then add DCI (560 mg, 4.74 mmol, 0.8 eq.) and P-REAGENT (2.14 g, 7.10 mmol, 1.2 eq.). After reacting at room temperature for 1 h, the solvent was removed by rotary evaporation under reduced temperature to obtain a crude product, which was purified by column chromatography (PE∶EA = 1∶1) to obtain Compound 94 (3.2 g, 4.13 mmol, 69.8% yield). ESI-MS: m / z 775.3 [M+H] +

[0329] Example 12: Synthesis of Compound 102 (Gvp24)

[0330]

[0331] Synthesis of Compound 95:

[0332] Dissolve compound 71 (17.4 g, 50.13 mmol, 1.0 eq.), GiBu (16.63 g, 75.20 mmol, 1.5 eq.) in ACN (200 mL), then add BSA (15.30 g, 75.20 mmol, 1.5 eq.). Place the reaction in an oil bath at 50 °C for 1 h, then cool it to 0 °C. Dropwise add a solution of TMSOTf (11.14 g, 50.13 mmol, 1.0 eq.) to the reaction. After the addition is complete, reflux the reaction in an oil bath for 3 h until compound 71 reacts completely. After the reaction is completed, cool the reaction to room temperature and quench the reaction by adding saturated aqueous NaHCO3. Then add ethyl acetate (300 mL) and water (200 mL) for extraction. Extract the aqueous phase twice with ethyl acetate (150 mL). Then combine the organic phases. Wash the organic phase with saturated brine and dry it over anhydrous sodium sulfate. Rotavaporize the solvent to obtain the crude product. The crude product is slurried with EA to obtain compound 95 (11.6 g, 22.83 mmol, 45.5% yield). ESI-MS: m / z 509.2 [M+H] +

[0333] Synthesis of compound 96:

[0334] Dissolve compound 95 (11.6 g, 22.83 mmol, 1.0 eq.) in MeOH (100 mL), then cool it to 0 °C. Add sodium methoxide (6.17 g, 114.15 mmol, 5.0 eq.). React at room temperature overnight until compound 95 reacts completely. Cool the reaction to about 0 °C and slowly add 1 mol / L dilute hydrochloric acid solution dropwise to the reaction to neutralize the reaction to pH about 7. Extract the reaction twice with ethyl acetate. Combine the organic phases. Wash the organic phase with water, saturated brine, dry it over anhydrous sodium sulfate and remove the solvent under reduced pressure to obtain the crude product. Purify the crude product by column chromatography (DCM∶MeOH = 20∶1) to obtain compound 96 (8.1 g, 19.10 mmol, 83.7% yield). ESI-MS: m / z 425.2 [M+H] +

[0335] Synthesis of compound 97:

[0336] Compound 96 (8.1 g, 19.10 mmol, 1.0 eq.) was dissolved in ultradry DMF (100 mL), and then imidazole (7.80 g, 114.6 mmol, 6.0 eq.) and TBSCl (8.64 g, 57.3 mmol, 3.0 eq.) were added successively. After reacting overnight until the raw materials were completely consumed, the temperature was lowered to 0 °C, and the reaction was quenched by adding saturated aqueous NaHCO3 solution. Then, ethyl acetate (300 mL) and water (300 mL) were added for extraction. The organic phase was washed twice with water, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure using a rotary evaporator to obtain the crude product of compound 97 (11.4 g). The crude product was directly used in the next reaction without purification. ESI-MS: m / z 653.3 [M+H] +

[0337] Synthesis of compound 98:

[0338] The crude product of compound 97 (11.4 g) was dissolved in THF (100 mL), and then the temperature was lowered to -5 °C. A solution of TFA / H2O (v∶v = 1∶1, 100 mL) was slowly added dropwise. After the addition was complete, the reaction was continued at this temperature for 1 h until compound 97 reacted completely. Then, ammonia water was slowly added dropwise for quenching until the reaction solution was neutral. After quenching, ethyl acetate (200 mL) and water (100 mL) were added for extraction, and the aqueous phase was extracted twice with EA (100 mL). Then, the organic phases were combined, and the combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain the crude product, and the crude product was purified by column chromatography (DCM∶MeOH = 20∶1) to obtain compound 98 (6.7 g, 12.45 mmol, total yield of two steps 65.2%). ESI-MS: m / z 539.3 [M+H] +

[0339] Synthesis of compound 99:

[0340] Compound 98 (6.7 g, 12.45 mmol) was dissolved in ACN (60 mL), and then IBX (6.97 g, 24.9 mmol, 2.0 eq.) was added. Then, the reaction was refluxed in an oil bath for 2 h until compound 98 reacted completely. The reaction solution was cooled to room temperature and then filtered. The filter cake was washed twice with acetonitrile, and the filtrates were combined. After the filtrate was evaporated to dryness, compound 99 (6.1 g, crude product, directly used in the next reaction without purification) was obtained. ESI-MS: m / z 537.2 [M+H] +

[0341] Synthesis of compound 100:

[0342] Compound 10 (7.17 g, 24.9 mmol, 2.0 eq.) was dissolved in THF (100 mL), and then the temperature was lowered to 0 °C. NaH (60% dispersion in mineral oil, 1.0 g, 24.9 mmol, 2.0 eq.) was added. After reacting for 30 min, a THF solution of crude compound 99 (6.1 g, 20 mL THF) was added dropwise. Then, the temperature was slowly raised to room temperature and the reaction was carried out for 2 h until the raw materials disappeared. The reaction was quenched by adding an NH4Cl solution. After quenching, extraction was carried out with EA (100 mL) and water (50 mL). The aqueous phase was then extracted twice with EA (50 mL). Then, the organic phases were combined. The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude product. The crude product was purified by column chromatography (PE∶EA = 1∶1) to obtain compound 100 (3.8 g, 5.67 mmol, total yield of two steps 45.5%). ESI-MS: m / z 671.3 [M+H] +

[0343] Synthesis of compound 101:

[0344] Compound 100 (3.8 g, 5.67 mmol) was dissolved in THF (40 mL), and then TBAF (1.0 mol / L in THF, 8.5 mL, 8.51 mmol) was added dropwise. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain a crude product. The crude product was purified by column chromatography (EA) to obtain compound 101 (2.5 g, 4.49 mmol, yield 79.2%). ESI-MS: m / z 557.2 [M+H] +

[0345] Synthesis of compound 102:

[0346] Compound 101 (2.5 g, 4.49 mmol) was dissolved in DCM (25 mL), and then DCI (424 mg, 3.59 mmol, 0.8 eq.) and P-REAGENT (1.62 g, 5.39 mmol, 1.2 eq.) were added. After reacting at room temperature for 1 h, the solvent was evaporated under reduced pressure at low temperature to obtain a crude product. The crude product was purified by column chromatography (PE∶EA = 1∶2) to obtain compound 102 (2.0 g, 2.64 mmol, yield 58.8%). ESI-MS: m / z 757.3 [M+H] +

[0347] The following Example 13 is a preparation example (synthesis route) of the nucleoside compound shown in Formula 3-a of the present invention, wherein the nucleoside compound shown in Formula 3-a is a compound with a 5'-(E)-VP-3'-CH2OMe-2'-phosphoramidite group.

[0348] Example 13: (Uvp5, Cvp6, Avp7, Gvp8)

[0349]

[0350] The nucleoside compound Uvp5 has a Nu = U base and a compound with the structure shown in 115.

[0351] Through the above preparation route, when preparing compound 108 from compound 107, the Nu selects the U base, and the structure and NMR data of the obtained nucleoside compound Uvp5 are as follows:[[]]

[0352]

[0353] 1 H NMR(400MHz, DMSO-d6)δ11.41(s, 1H), 7.50(m, 1H), 6.80(m, 1H), 6.13 - 6.05(m, 1H), 5.92 - 5.90(m, 1H), 5.65(m, 1H), 4.80(m, 1H), 4.45(m, 1H), 4.02 - 3.91(m, 3H), 3.80(m, 2H), 3.77(m, 2H), 3.60(m, 2H), 3.32(s, 5H), 2.80(m, 2H), 1.28 - 1.09(m, 18H); 31 P NMR(161MHz, DMSO-d6)δ149.88, 149.82, 16.74, 16.71; ESI-MS: m / z 605.7[M + H] + .

[0354] The nucleoside compound Avp7 has a Nu = A base and a compound with the structure shown in 115.

[0355] Through the above preparation route, when preparing compound 108 from compound 107, the Nu selects the Bz-protected A base, and the structure and NMR data of the obtained nucleoside compound Avp7 are as follows:[[]]

[0356]

[0357] 11H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.03 (s, 1H), 7.86 - 7.89 (m, 3H), 7.54 - 7.60 (m, 3H), 6.15 - 6.05 (m, 1H), 5.95 - 5.90 (m, 1H), 5.67 (m, 1H), 4.84 (m, 1H), 4.46 (dd, J = 10.6 Hz, 1H), 4.02 - 3.91 (m, 1H), 3.77 (m, 2H), 3.60 (m, 2H), 3.32 (s, 5H), 2.80 (m, 2H), 1.28 - 1.09 (m, 19H), 31 31P NMR (161 MHz, DMSO-d6) δ 149.84, 149.74, 16.81, 16.79, ESI-MS: m / z 732.7 [M+H] + 。

[0358] Example 14 below is a preparation example (synthetic route) of the nucleoside compound shown by Formula 8-a of the present invention. The nucleoside compound shown by Formula 8-a is a compound of 5'-(E)-VP-3'-OMe-4-S-2'-phosphoramidite group.

[0359] Example 14: (Uvp25, Cvp26, Avp27, Gvp28)

[0360]

[0361] The nucleoside compound Uvp25 has a Nu = U base and has the structure shown in 128.

[0362] Through the above preparation route, when preparing compound 121 from compound 120, the Nu selects the U base, and the structure and NMR data of the obtained nucleoside compound Uvp25 are as follows:

[0363]

[0364] 1 1H NMR (400 MHz, CDCl3) δ 8.1 (s, 1H), 7.9 (s, 1H), 6.05 (m, 1H), 5.55 (m, 1H), 4.32 (m, 1H), 3.80 (m, 2H), 3.55 (m, 3H), 3.32 (m, 4H), 2.80 (m, 4H), 2.6 (m, 2H), 2.4 (m, 2H), 1.26 - 1.01 (m, 18H); 31 31P NMR (161 MHz, CDCl3) δ 150.02, 149.94, 16.48, 16.46, ESI-MS: m / z 607.6 [M+H] +。

[0365] Example 15 below is a preparation example (synthetic route) of the nucleoside compound shown in Formula 4-a of the present invention, wherein the nucleoside compound shown in Formula 4-a is a compound with a 5'-(E)-VP-3'-OMe-2'-phosphoramidite group.

[0366] Example 15: (Uvp9)

[0367]

[0368] The nucleoside compound Uvp9 has Nu = U base and a compound with the structure shown in 139. The structure and NMR data are as follows:

[0369]

[0370] 1 H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 7.72 (m, 1H), 6.84 (m, 1H), 6.10 (m, 1H), 5.80 (m, 1H), 5.72 (m, 1H), 5.22 (m, 2H), 4.93 (m, 1H), 4.62 (m, 1H), 4.01 (m, 3H), 3.81 (m, 2H), 3.32 (m, 5H), 2.80 (m, 2H), 1.26 - 1.09 (m, 18H); 31 P NMR (161 MHz, DMSO-d6) δ 121.75, 121.51, 15.23, 15.11; ESI-MS: m / z 607.6 [M+H] +

[0371] Example 16 below is a preparation example (synthetic route) of the nucleoside compound shown in Formula 5-a of the present invention, wherein the nucleoside compound shown in Formula 5-a is a compound with a 5'-(E)-VP-3'-SMe-2'-phosphoramidite group.

[0372] Example 16: (Uvp13, Cvp14, Avp15, Gvp16)

[0373]

[0374] The nucleoside compound Uvp13 has Nu = U base and a compound with the structure shown in 151.

[0375] Through the above preparation route, the starting compound 140 is a compound with Nu = U base. The structure and NMR data of the prepared nucleoside compound Uvp13 are as follows:

[0376]

[0377] 1 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 7.60 (m, 1H), 6.74 (m, 1H), 6.13 - 6.02 (m, 1H), 5.95 - 5.90 (m, 1H), 5.67 (m, 1H), 4.84 (m, 1H), 4.52 (m, 1H), 4.03 - 3.91 (m, 5H), 3.77 (m, 2H), 3.32 (m, 2H), 3.22 (m, 3H), 2.80 (m, 2H), 1.28 - 1.09 (m, 18H); 31 31P NMR (161 MHz, DMSO-d6) δ 150.23, 150.01, 16.73, 16.71; ESI-MS: m / z 607.6 [M + H] +

[0378] Example 17: (Uvp29, Cvp30, Avp31, Gvp32)

[0379]

[0380] The nucleoside compound Uvp29 has a Nu = U base and is a compound with the structure shown in 164.

[0381] Through the above preparation route, when preparing compound 157 from compound 156, the Nu selects the U base, and the structure and NMR data of the obtained nucleoside compound Uvp29 are as follows:

[0382]

[0383] 1 1H NMR (400 MHz, CDCl3) δ 8.1 (s, 1H), 7.9 (s, 1H), 6.05 (m, 1H), 5.55 (m, 1H), 4.32 (m, 1H), 3.80 (m, 2H), 3.55 (m, 5H), 3.32 (m, 4H), 2.80 (m, 4H), 2.6 (m, 2H), 2.4 (m, 2H), 1.26 - 1.01 (m, 18H); 31 31P NMR (161 MHz, CDCl3) δ 150.24, 149.84, 16.51, 16.49, ESI-MS: m / z 621.5 [M + H] +

[0384] Example 18 below is a preparation example (synthetic route) of the nucleoside compound shown in Formula 10-a of the present invention, wherein the nucleoside compound shown in Formula 10-a is a compound with a 5'-(E)-VP-3'-OMe-2'-phosphoramidite group.

[0385] Example 18: (Uvp33, Cvp34, Avp35, Gvp36)

[0386]

[0387] The nucleoside compound Uvp33 is a compound with Nu = U base and has the structure shown in 173.

[0388] Through the above preparation route, when preparing compound 165 from compound 4, Nu is selected as the U base, and the structure and NMR data of the obtained nucleoside compound Uvp33 are as follows:

[0389]

[0390] 1 H NMR (400 MHz, DMSO-d6) δ 11.41 (s, 1H), 7.52 (m, 1H), 6.82 (m, 1H), 6.15 - 6.05 (m, 1H), 5.95 - 5.90 (m, 1H), 5.67 (m, 1H), 4.84 (m, 1H), 4.46 (m, 1H), 4.03 - 3.91 (m, 5H), 3.77 (m, 2H), 3.60 (m, 2H), 3.32 (s, 5H), 2.80 (m, 2H), 1.28 - 1.09 (m, 18H); 31 P NMR (161 MHz, DMSO-d6) δ 149.87, 149.80, 67.43, 67.33, ESI-MS: m / z 607.2 [M + H] +

[0391] Example 19 below is a preparation example (synthetic route) of the nucleoside compound shown in Formula 11-a of the present invention, wherein the nucleoside compound shown in Formula 11-a is a compound with a 5'-(E)-VP-3'-OMe-2'-phosphoramidite group.

[0392] Example 19: (Uvp37)

[0393]

[0394] Example 20 below is a preparation example (synthetic route) of the nucleoside compound shown in Formula 11-a of the present invention, wherein the nucleoside compound shown in Formula 11-a is a compound with a 5'-(E)-VP-3'-OMe-2'-phosphoramidite group.

[0395] Example 20: (Uvp41)

[0396]

[0397] The nucleoside compound Uvp41 is a compound with the structure shown in 204 where R = Me.

[0398] Through the above preparation route, when preparing compound 195 from compound 194, MeI was selected as the R reagent, and the structure and NMR data of the obtained nucleoside compound Uvp41 are as follows:

[0399]

[0400] 1 H NMR(400MHz, DMSO-d6)δ11.40(s, 1H), 7.51(m, 1H), 6.80(m, 1H), 6.12 - 6.03(m, 1H), 5.93 - 5.90(m, 1H), 5.66(m, 1H), 4.82(m, 1H), 4.40(dd, J = 10.6Hz, 1H), 4.02 - 3.91(m, 1H), 3.82(m, 2H), 3.32(s, 5H), 2.80(m, 2H), 1.24 - 1.09(m, 18H); 31 P NMR(161MHz, DMSO-d6)δ149.82, 149.71, 16.80, 16.78; ESI-MS: m / z 563.5[M + H] +

[0401] Experimental section

[0402] Experiment 1: Synthesis of siRNA sequences used for in vitro evaluation

[0403] The synthesis of siRNA is no different from the usual phosphoramidite solid-phase synthesis method. When modifying nucleotides at the 5'-terminal position of the AS strand, the above-synthesized Uvp, Cvp, Avp, and Gvp phosphoramidite monomers are used to replace the original nucleotides in the parent sequence.

[0404] A brief description of the synthesis process is as follows: Starting from the Universal CPG support on the LK-48E synthesizer (Lingkun), nucleoside phosphoramidite monomers are linked one by one according to the synthesis program. Except for the above-mentioned synthesized Uvp, Cvp, Avp, and Gvp phosphoramidite monomers, the remaining nucleoside monomer raw materials such as 2'-F RNA and 2'-O-methyl RNA nucleoside phosphoramidite monomers are all purchased from Shanghai Zhaowei, and L96 is purchased from WuXi AppTec. 5'-Ethylthio-1H-tetrazole (ETT) is used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio and collidine (Suzhou Kelema) solution is used as the sulfurization reagent, and iodine pyridine / aqueous solution (Kelema) is used as the oxidant.

[0405] After solid-phase synthesis is completed, the oligoribonucleotide is cleaved from the solid support, soaked in a 3:1 solution of 28% ammonia water and ethanol at 50°C for 16 hours, then centrifuged, and the supernatant is transferred to another centrifuge tube and concentrated to dryness by evaporation. Then, the above-concentrated sample is dissolved in acetonitrile / pyridine (V:V = 1:1), 0.5mol / L TMSBr acetonitrile solution is added to remove the ethyl protecting group in the compound, the system is quenched with triethylamine, and purified by C18 reverse-phase chromatography with the mobile phase being 0.1mol / L TEAA and acetonitrile. After the target oligonucleotide is collected, freeze-dried, and identified as the target product by LCMS, it is quantified by UV (260nm).

[0406] The obtained single-stranded oligonucleotide is annealed according to the complementary pairing at an equimolar ratio, and finally the obtained double-stranded siRNA is dissolved in 1xPBS and adjusted to the required concentration for experiments.

[0407] The modification schemes used in Table 1 are as follows:

[0408] "m" indicates that the nucleotide adjacent to the left of the letter m is a 2'-OMe modification, "f" indicates that the nucleotide adjacent to the left of the letter f is a 2'-F modification, "s" indicates that the linkage between the two nucleotides adjacent to the left and right of the letter s or between the phosphoramidite monomers described in the present invention is a phosphorothioate linkage, and "Uvp1-41, Avp3, Avp7" are nucleoside compounds modified with 5'-(E)-VP-2'-phosphoramidite, as described above.

[0409] Table 1: siRNA molecular sequences used for in vitro screening

[0410]

[0411]

[0412] Experiment 2: In vitro silencing efficacy of TTR target mRNA and modified siRNA duplexes

[0413] PMH transfection:

[0414] Thaw and resuscitate PMH cells (Milestone, cat# CMH - 100CBP - PQ) with mouse primary hepatocyte thawing medium (Milestone, cat# HEP024). Add mouse primary hepatocyte medium (Milestone, cat# CMHEP054), and seed 40,000 cells per well in a 96 - well plate, 90 μL per well. Culture at 37 °C in 5% CO₂ for 6 h. After 6 h, change to hepatocyte maintenance medium (Milestone, cat# CMHEP064) and continue to culture overnight. The transfection conditions are as follows: Mix 4.9 μl of Opti - MEM (Gibco, cat# 31985088) and 0.1 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA, cat# 13778 - 150) with 5 μL of siRNAs per well and incubate at room temperature for 15 minutes. Then add this mixture to 10 μL per well in the 96 - well plate seeded the previous day. Incubate at 37 °C in 5% CO₂ for 48 h. The final concentrations in the siRNAs experiment are shown in Table 2 below.

[0415] RNA extraction and reverse transcription into cDNA:

[0416] Extract RNA by cell lysis according to the Cells - to - Ct bulk lysis reagents (Invitrogen#4391851C) instruction manual. 1) Aspirate the old medium in each well, and add 100 μL of PBS to wash twice. 2) Carefully aspirate the PBS. Add 50 μL of lysis buffer to each well. 3) Vortex at 500 rpm for 5 minutes at room temperature. 4) Add 5 μL of stop solution to each well and vortex at 500 rpm for 2 minutes at room temperature. If cDNA synthesis is to be carried out immediately, place the plate on ice.

[0417] Reverse transcription was performed according to the instructions of Cells-to-CT Bulk Fast Advanced RT Reagent (Invitrogen #A39110): Prepare the mixture (per well: 25 μL of 2× Fast Advanced RT Buffer, 2.5 μL of 20× RT Fast Advanced Enzyme Mix, and 22.5 μL of total RNA). Use Biometra TAdvanced 96SG (analytikjena) to synthesize cDNA through the following steps: 30 min at 37 °C, 5 min at 95 °C, and hold at 4 °C. Store the cDNA at -20 °C or perform real-time PCR analysis immediately.

[0418] Real-time PCR:

[0419] Mix 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, cat# 4351370), and FAST PCR Master Mix (Applied Biosystems, cat# 4444965) and add them to a 96-well plate (Axygen, cat#PCR-96-FLT-C) for real-time PCR detection. The instrument used is 7 (Applied Biosystems), and the system program is: 2 min at 50 °C, 20 s at 95 °C, and 40 cycles of 1 s at 90 °C and 20 s at 60 °C. Unless otherwise stated, each siRNA is transfected in triplicate.

[0420] Data analysis: Analyze the real-time data using the ΔΔCt method and normalize these data with the data of the mock (PBS control group). 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 v.s. Mock = {1 - Expression fold (test sample) / Expression fold (Mock)} * 100. The final results are shown in Table 2.

[0421] Table 2: q-PCR in vitro activity screening data

[0422]

[0423]

[0424] Among them, compounds CM05-0000 and CM05-0001 were tested at multiple concentrations (0.000128, 0.00064, 0.0032, 0.016, 0.08, 0.4, 2.0, 10.0 nM), and the inhibition rate results at each concentration were fitted by Graphpad Prism software to obtain the IC 50 value, as Figure 1A and Figure 1B shown.

[0425] This type of modified nucleoside compound can significantly improve the activity of siRNA sequences.

[0426] Experiment 3: Synthesis of siRNA sequences for in vivo evaluation in wild mice

[0427] The synthesis of siRNA is no different from the usual phosphoramidite solid-phase synthesis method. When modifying nucleotides at the 5'-end position of the AS strand, the above-synthesized Uvp, Cvp, Avp, and Gvp phosphoramidite monomers are used to replace the original nucleotides in the parent sequence.

[0428] The synthesis process is briefly described as follows: Starting with a Universal CPG support on an LK-48E synthesizer (Lingkun), nucleoside phosphoramidite monomers are linked one by one according to the synthesis program. Except for the above-synthesized Uvp, Cvp, Avp, and Gvp phosphoramidite monomers, the remaining nucleoside monomer raw materials such as 2'-F RNA and 2'-O-methyl RNA nucleoside phosphoramidite monomers are purchased from Shanghai Zhaowei, and L96 is purchased from WuXi AppTec. 5'-Ethylthio-1H-tetrazole (ETT) is used as the activator (0.6M acetonitrile solution), 0.22M PADS dissolved in a 1:1 volume ratio with trimethylpyridine (Suzhou Keloma) solution is used as the sulfurization reagent, and an iodine pyridine / water solution (Keloma) is used as the oxidant.

[0429] After solid-phase synthesis, the oligoribonucleotide is cleaved from the solid support, soaked in a 3:1 solution of 28% ammonia water and ethanol at 50°C for 16 hours, then centrifuged, and the supernatant is transferred to another centrifuge tube and concentrated to dryness by evaporation. Then, the above-concentrated sample is dissolved in acetonitrile / pyridine (v:v = 1:1), 0.5 mol / L TMSBr acetonitrile solution is added to remove the ethyl protecting group in the compound, the system is quenched with triethylamine, and purified by C18 reversed-phase chromatography with a mobile phase of 0.1 mol / L TEAA and acetonitrile. The target oligonucleotide is collected, freeze-dried, identified as the target product by LCMS, and quantified by UV (260 nm).

[0430] The obtained single-stranded oligonucleotides are annealed according to the complementary pairing at an equimolar ratio. Finally, the obtained double-stranded siRNA is dissolved in 1xPBS and adjusted to the concentration required for the experiment for standby.

[0431] The modification schemes used in Table 3 are as follows:

[0432] "m" indicates that the nucleotide adjacent to the left of the letter m is a 2'-OMe modification, "f" indicates that the nucleotide adjacent to the left of the letter f is a 2'-F modification, "s" indicates that the linkage between the two nucleotides adjacent to the left and right of the letter s or between the phosphoramidite monomers described in the present invention is a phosphorothioate linkage, and "Uvp1, Uvp5, Uvp17, Uvp21, Uvp25, Uvp33" are nucleoside compounds modified with 5'-(E)-VP-2'-phosphoramidite, as described above, and Vp0 is a U base 5'-(E)-VP-2'-phosphoramidite with a conventional structure.

[0433] Table 3: siRNA molecular sequences used for evaluation in wild mice

[0434]

[0435]

[0436] Experiment 4: In vivo activity evaluation of TTR target mRNA and siRNA duplexes containing Uvp-modified nucleosides

[0437] Activity evaluation of siRNA sequences in wild-type mice:

[0438] The activities of the above compounds (CM05-0000 to CM05-0006, CM05-0011, CM05-1000, see Table 3) were evaluated in vivo using wild-type C57BL / 6 mice (Spebefu (Beijing) Biotechnology Co., Ltd.).

[0439] C57BL / 6 mice at 6 to 8 weeks of age were subcutaneously injected once with a dose of 0.5 mg / kg or 1 mg / kg of the compound (the compounds in Table 3), and orbital blood was collected using EP tubes before dosing and on days 7, 14, 21, 28, 35, 42, 56, 63, and 70 after dosing. After the blood samples were allowed to stand at room temperature for two hours, they were centrifuged at 4°C and 5500 rpm for 10 min to separate and collect the serum for detecting the TTR level in the animal serum.

[0440] The detection of TTR levels in serum was analyzed by TTR enzyme-linked immunosorbent assay (ELISA). ELISA was performed using the Abcam Mouse Prealbumin ELISA Kit (ab282297), and all sample tests were performed according to the instructions of the test kit. The absorbance at 450 nm was read on a Tecan SPARK microplate reader, and the data of the standard (from the above ELISA kit) was fitted into a parameter standard curve to determine the serum TTR protein level (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. The test results are shown in Figure 2 .

[0441] result: Figure 2 This indicates that this type of modified nucleoside compound can enhance the silencing activity of siRNA sequences in vivo.

Claims

1. A nucleoside compound represented by formula 1-a in: R 1 , R 2 , R 3 Each is independently selected from C3-C10 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, or aminoacyl or substituted aminoacyl; Nu is a base; Z is -O-, -S- or -CR 4 R 5 , where R 4 and R 5 are each independently hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted cycloalkyl; X1 and X2 are each independently selected from -(CH2) n O-, or -(CH2) n S-, where n is any integer from 0 to 10; Y is O or S.

2. The nucleoside compound of formula 1-a according to claim 1, wherein R 1 , R 2 are each independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; preferably, R 1 , R 2 are each independently selected from C1-C6 alkyl, or substituted C1-C6 alkyl; more preferably, R 1 , R 2 Each is independently selected from C1-C3 alkyl, or substituted C1-C3 alkyl.

3. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein R 3 is C1-C6 alkyl, C1-C6 alkoxy, or aminoacyl or substituted aminoacyl; preferably, R 3 is C1-C3 alkyl, C1-C3 alkoxy, or alkyl-substituted aminoacyl; more preferably, R 3 It is methyl, ethyl, n-propyl, isopropyl, methoxy, methylcarbamoyl, or ethylcarbamoyl.

4. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein X1 is -(CH2) n O-, or -(CH2) n S-, n is any integer from 0 to 5; preferably, X1 is -(CH2) n O-, or -(CH2) n S-, n is any integer from 0 to 3.

5. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein X2 is -(CH2) n O-, or -(CH2) n S-, n is any integer from 0 to 5; preferably, X2 is -(CH2) n O-, or -(CH2) n S-, n is any integer from 0 to 3.

6. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein Z is -O- or -S-.

7. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein The base is selected from any of the following bases:

8. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 2-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

9. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 3-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

10. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 4-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

11. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 5-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl: Nu is as defined in claim 1.

12. The nucleoside compound of formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 6-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

13. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 7-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

14. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 8-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

15. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 9-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

16. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 10-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

17. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the nucleoside compound represented by formula 11-a, in, R 1 , R 2 Each is independently selected from C3-C10 cycloalkyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, or C2-C6 alkynyl or substituted C2-C6 alkynyl; Nu is as defined in claim 1.

18. The nucleoside compound represented by formula 1-a according to claim 1 or 2, wherein The nucleoside compound represented by formula 1-a is selected from the following nucleoside compounds:

19. An oligonucleotide, wherein one terminal position of the oligonucleotide is substituted by a nucleoside compound represented by formula 1-a as claimed in any one of claims 1 to 18.

20. The oligonucleotide according to claim 19, wherein the nucleoside compound has the following structure in the oligonucleotide Where R 1 , R 2 , R 3 , Z, X1, X2, Y and Nu are as defined in claim 1.

21. Use of the nucleoside compound represented by formula 1-a according to any one of claims 1 to 18 for preparing oligonucleotides.

22. The use according to claim 21, wherein the nucleoside compound has the following structure in an oligonucleotide Where R 1 , R 2 , R 3 , Z, X1, X2, Y and Nu are as defined in claim 1.

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