Modified nucleotide compound, oligonucleotide thereof, and use thereof

Abstract

The present invention discloses a modified nucleotide compound having a structure as shown in a formula (I) or a pharmaceutically acceptable salt thereof or stereoisomer thereof, an oligonucleotide thereof, and a use thereof. By means of connecting the modified nucleotide compound provided by the present invention to a 5'-tail end of an oligonucleotide, the gene expression inhibition activity of the oligonucleotide inhibition can be improved, and stability of the oligonucleotide in vivo can be increased, thereby effectively increasing the druggability of the oligonucleotide in vivo.

Description

Modified nucleotide compound, oligonucleotide thereof and use thereof

[0001] The present invention claims priority to two Chinese patent applications filed with the China Patent Office on September 18, 2023, with application number 202311202774.8 and application name “Modified Nucleotide Compounds, Oligonucleotides Thereof and Applications Thereof”, and filed with the China Patent Office on February 7, 2024, with application number 202410174938.9 and application name “Modified Nucleotide Compounds, Oligonucleotides Thereof and Applications Thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine, and in particular relates to a modified nucleotide compound, its oligonucleotide and application thereof. Background Art

[0003] In 1998, Fire and Mello discovered that RNA interference (RNAi) is a very promising gene therapy tool. Small nucleic acid therapies such as small interfering RNA (siRNA), microRNA (miRNA), and antisense oligonucleotides (ASOs) are all potential gene silencing tools and have shown therapeutic promise for a wide range of diseases. Exogenously introduced double-stranded siRNAs consist of a sense strand and an antisense strand, which can be recognized by the endogenous RNAi machinery to exert gene silencing functions. siRNA binds to the Argonate 2 protein (AGO2) and other auxiliary protein residues to form an RNA-induced silencing complex (RISC). The activated RISC contains the antisense strand that specifically targets the target mRNA, and AGO2 subsequently cleaves the mRNA to achieve effective gene silencing (Matzke et al. Nature reviews. Genetics vol. 6, 1 (2005): 24-35.). RNAi-based therapy is the most effective and reliable tool for inhibiting target genes. Currently, many diseases including cancer, neurodegenerative diseases, autoimmune diseases and viral infections have been explored using siRNA therapy (Gangopadhyay S et al. RNA Biol. 2022; 19(1): 452-467.).

[0004] There are still several challenges in siRNA therapy, including its limited tolerance to nucleases in the body, off-target effects, difficulty in specific delivery to specific tissues or organs, and poor drug properties (Hammond SM et al., EMBO Mol Med. 2021; 13(4): e13243.). To address these challenges, chemical modifications have made significant contributions to the drug properties of siRNA and played an indispensable role in its clinical success. For example, almost all recently approved siRNA drugs have undergone extensive ribose 2'-position modifications, including 2'-O-methylation and 2'-fluoro modification, which have a strong impact on the physicochemical properties and overall efficacy of siRNA. In addition, the delivery of siRNA drugs is also an important guarantee for RNAi therapy. Currently, the most mature delivery system is N-oligosaccharide acetylgalactosamine (GalNAc). GalNAc can bind to the asialoglycoprotein receptor (ASGPR) specifically expressed by hepatocytes. After coupling with siRNA, it can achieve active delivery to liver cells (Debacker, Alexandre J et al. Molecular therapy vol. 28, 8 (2020): 1759-1771).

[0005] It is worth noting that when double-stranded siRNA is loaded into the RISC complex, the sense strand is removed, while the antisense strand remains stably bound to the RISC complex. The phosphate group (5'-P) at the 5' end of the antisense strand can be anchored in a binding pocket in the middle domain of the Ago2 protein, which is considered to be a key factor for the effective loading of siRNA into the RISC complex (Schirle NT et al., Science. 2012; 336(6084): 1037-1040.). However, the above-mentioned chemical modification, especially the GalNAc delivery system, causes the natural phosphate group 5'-P to be rapidly hydrolyzed by lysosomal acid phosphatase during transport in the endocytic pathway. Therefore, it is urgent to modify the phosphate group at the 5' end of the antisense strand to improve its metabolic stability (Nair JK et al., J Am Chem Soc. 2014; 136(49): 16958-16961.). It has been reported that 5'-(E)-vinyl phosphate (5'-VP), an analog of 5'-P, is a modification that can be used to stabilize single-stranded RNAi (ssRNAi) (Prakash TP et al., Nucleic Acids Res. 2015; 43(6): 2993-3011.). Subsequently, researchers tested four siRNA-GalNAc conjugates targeting apolipoprotein B (ApoB) and found that replacing 5'-P with the stable analog 5'-VP could increase the siRNA silencing effect by up to 20-fold in vitro; and in mice, 5'-VP could increase the in vivo activity of phosphate-dependent conjugates by up to 3-fold (Parmar R et al., Chembiochem. 2016; 17(11): 985-989.). It can be seen that the metabolically stable 5'-VP can enhance the stability of siRNA by promoting the loading of siRNA into the RISC complex, thereby enhancing the in vivo activity of siRNA-GalNAc conjugates, especially phosphate-dependent sequences. Currently, the most widely used 5'-VP modified nucleotides are mostly modified with 2'-O-methyl.

[0006] Summary of the Invention

[0007] Based on this, the present invention provides a new class of 5'-VP modified nucleotide compounds, which can enhance the activity of oligonucleotides in inhibiting gene expression.

[0008] In one aspect, the present invention provides a modified nucleotide compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,

[0009] in:

[0010] R 1 and R 2Each is independently selected from: H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkylmethyl;

[0011] R 3 Selected from: -OR 5 ;

[0012] R 4 Selected from: -N(R 6 )(R 7 );

[0013] R 5 Selected from: H, C1-C6 alkyl, C1-C6 alkyl substituted by cyano, C1-C6 alkyl substituted by halogen, C1-C6 alkyl substituted by hydroxy, C1-C6 alkyl substituted by C1-C6 alkoxy, C1-C6 alkyl substituted by amino, C1-C6 alkylamino substituted C1-C6 alkyl;

[0014] R 6 and R 7 Each is independently selected from: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkylmethyl;

[0015] X is selected from: -C(R 8 )(R 9 )-, -S-;

[0016] R 8 and R 9 Each is independently selected from: H, C1-C6 alkyl, halogen;

[0017] G is selected from: H, halogen, cyano, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkylmethyl, C1-C 10 Alkoxy, halogen-substituted C1-C 10 Alkyl, hydroxy substituted C1-C 10 Alkyl, C1-C6 alkoxy substituted C1-C 10 Alkyl, amino substituted C1-C 10 Alkyl, C1-C6 alkylamino substituted C1-C 10 Alkyl, cyano-substituted C1-C 10 alkyl;

[0018] Bx is a heterocyclic base.

[0019] In some embodiments, R 1 and R 2Each is independently selected from the group consisting of: H, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C6 cycloalkyl, and C3-C6 cycloalkylmethyl.

[0020] In some embodiments, R 1 and R 2 Each is independently selected from the group consisting of: H, methyl, ethyl, n-propyl, and isopropyl.

[0021] In some embodiments, R 5 Selected from: H, C1-C3 alkyl, C1-C3 alkyl substituted by cyano, C1-C3 alkyl substituted by halogen, C1-C3 alkyl substituted by hydroxy, C1-C3 alkyl substituted by C1-C3 alkoxy, C1-C3 alkyl substituted by amino, C1-C3 alkyl substituted by C1-C3 alkylamino.

[0022] In some embodiments, R 5 Selected from: H, methyl, ethyl, n-propyl, isopropyl, cyano-substituted methyl, cyano-substituted ethyl, cyano-substituted n-propyl.

[0023] In some embodiments, R 6 and R 7 Each is independently selected from the group consisting of: C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C6 cycloalkyl, and C3-C6 cycloalkylmethyl.

[0024] In some embodiments, R 6 and R 7 are independently selected from the group consisting of: methyl, ethyl, n-propyl, and isopropyl.

[0025] In some embodiments, X is selected from: -S-, -CH2-, -C(H)(F)-, -CF2-.

[0026] In some embodiments, G is selected from: H, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkylmethyl, C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, C1-C6 alkoxy-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, C1-C6 alkylamino-substituted C1-C6 alkyl, cyano-substituted C1-C6 alkyl.

[0027] In some embodiments, G is selected from the group consisting of: H, halogen, cyano, C1-C3 alkyl, C1-C3 alkyl substituted with halogen, C2-C3 alkenyl, C2-C3 alkynyl, C1-C3 alkoxy, and C1-C3 alkyl substituted with C1-C3 alkoxy.

[0028] In some embodiments, G is selected from the group consisting of: H, methyl, ethyl, propyl, isopropyl, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, vinyl, propenyl, methoxy, ethoxy, methoxymethyl, methoxyethyl, cyano, fluorine, and chlorine.

[0029] In some embodiments, X is -S-; G is selected from: C1-C3 alkyl.

[0030] In some embodiments, X is -CH2-; G is selected from the group consisting of C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C1-C3 alkoxy-substituted C1-C2 alkyl, fluorine-substituted methyl, cyano, fluorine, and chlorine.

[0031] In some embodiments, X and G together form -XG selected from the group consisting of methylthio, ethylthio, propylthio, methyl, ethyl, propyl, butyl, allyl, methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, methyl substituted with cyano, ethyl substituted with cyano, monofluoromethyl, difluoromethyl, monofluoroethyl, and difluoroethyl.

[0032] In some embodiments, Bx is selected from the group consisting of adenine, guanine, cytosine, uracil, thymine, methyladenine, 1-methylguanine, isoguanine, 7-methylguanine, methylcytosine, isocytosine, pseudouracil, 5-hydroxymethylcytosine, hypoxanthine, Q-substituted adenine, Q-substituted guanine, Q-substituted cytosine, Q-substituted uracil, and Q-substituted thymine; wherein Q is selected from the group consisting of H, benzoyl, and benzyloxymethyl.

[0033] In some embodiments, the modified nucleotide compound is selected from:

[0034] Wherein: Q is selected from: H, benzoyl, benzyloxymethyl.

[0035] In a second aspect, the present invention provides an oligonucleotide, wherein at least one 5'-terminal nucleotide monomer is linked to the oligonucleotide via a linker group by the modified nucleotide compound; the oligonucleotide has the following structural formula:

[0036] Where: R 1 、R 2 , Bx, X, and G are as described above;

[0037] A is the nucleotide chain in the oligonucleotide excluding the 5'-terminal nucleotide monomer;

[0038] T is an internucleoside linking group that connects the modified nucleotide compound to the oligonucleotide.

[0039] In some embodiments, T is selected from the group consisting of: a phosphodiester bond, and a phosphorothioate bond.

[0040] In some embodiments, the oligonucleotide is selected from one or more combinations of siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA, antimir, supermir, antagomir, ribozyme, triplex-forming oligonucleotide, decoy oligonucleotide, splicing switch oligonucleotide, immunostimulatory oligonucleotide, RNA activator, U1 adapter, and CRISPR Cas.

[0041] In some embodiments, the oligonucleotide is siRNA.

[0042] In some embodiments, the modified nucleotide compound is linked to the 5'-end of the antisense strand of the siRNA via a T linker.

[0043] In a third aspect, the present invention provides uses of the modified nucleotide compound and oligonucleotide, comprising:

[0044] The modified nucleotide compound is used in preparing oligonucleotides for inhibiting or activating gene expression.

[0045] In some embodiments, the oligonucleotide is selected from one or more combinations of siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA, antimir, supermir, antagomir, ribozyme, triplex-forming oligonucleotide, decoy oligonucleotide, splicing switch oligonucleotide, immunostimulatory oligonucleotide, RNA activator, U1 adapter, and CRISPR Cas.

[0046] In some embodiments, the oligonucleotide is siRNA.

[0047] In some embodiments, the modified nucleotide compound is linked to the 5'-end of the antisense strand of the siRNA via a covalent linker.

[0048] In some embodiments, the covalent linking group is a phosphodiester bond or a phosphorothioate bond.

[0049] The modified nucleotide compound or oligonucleotide of the present invention is used in the preparation of a drug for inhibiting or activating gene expression.

[0050] In a fourth aspect, the present invention provides a pharmaceutical preparation for inhibiting or activating gene expression, which is prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the oligonucleotide described in the present invention.

[0051] In a fifth aspect, the present invention provides a method for inhibiting or activating gene expression, comprising the steps of administering a safe and effective amount of the oligonucleotide of the present invention and / or the pharmaceutical preparation of the present invention.

[0052] The present invention obtains a new class of 5'-VP modified nucleotide compounds by subjecting 5'-VP modified nucleotide compounds to different 2'-modifications. The present invention discovered that the 2'-modification significantly affects the drugability of oligonucleotides. Linking the modified nucleotide compounds provided by the present invention to the 5'-end of oligonucleotides can enhance the activity of oligonucleotides in inhibiting gene expression and improve the stability of oligonucleotides in vivo, thereby effectively improving their drugability in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows the effects of different modified 11040 sequences on the expression of human PCSK9 in the serum of AAV8-hPCSK9 mice.

[0054] FIG2 shows the effects of different modified W4005 sequences on hCIDEB expression in the liver of AAV8-hCIDEB mice. DETAILED DESCRIPTION

[0055] The technical solutions of the present invention are further illustrated by specific examples below. Those skilled in the art should understand that the examples are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0056] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0057] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0058] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0059] In the compounds described herein, when any variable occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permissible so long as such combinations result in a stable compound. A line drawn from a substituent into a ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds may be attached only to any suitable carbon atom in an adjacent ring. It will be understood that one of ordinary skill in the art can select substituents and substitution patterns in the compounds of the present invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it will be understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stable.

[0060] As used herein, the term "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, the definition of "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched chain. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0061] The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0062] The term "alkoxy" refers to a group in which an alkyl group is directly connected to oxygen, that is, a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, etc.

[0063] The term "heterocyclic base" refers to the base portion that makes up a nucleoside, the most common of which are the two major categories of purines and pyrimidines, such as adenine, guanine, cytosine, uracil, and thymine, and derivatives thereof such as methyladenine, 1-methylguanine, isoguanine, 7-methylguanine, methylcytosine, isocytosine, pseudouracil, 5-hydroxymethylcytosine, and hypoxanthine, and substituents thereof such as substituted adenines, substituted guanines, substituted cytosines, substituted uracils, and substituted thymines.

[0064] The term "substituted" refers to the replacement of hydrogen radicals in a specified structure with the radical of a specified substituent.

[0065] As will be understood by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0066] The present invention includes free forms of compounds of formula (I), as well as pharmaceutically acceptable salts and stereoisomers thereof. Stereoisomers of the present invention are, depending on their structure, enantiomers, diastereomers, syn- / anti-isomers, cis- / trans-isomers, epimers, and (E)- / (Z)-isomers. Compounds of formula I may be used in the context of the present invention in the form of pure stereoisomers or in the form of any mixture of stereoisomers.

[0067] The term "free form" refers to an amine compound in a non-salt form. Pharmaceutically acceptable salts encompass not only exemplary salts of the specific compounds described herein, but also all typical pharmaceutically acceptable salts of the free forms of the compounds of formula (I) or formula (I). The free form of a specific salt of the compound can be isolated using techniques known in the art. For example, the free form can be regenerated by treating the salt with a suitable dilute aqueous base solution, such as a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous ammonia, and a dilute aqueous solution of sodium bicarbonate. The free form differs somewhat from its respective salt form in certain physical properties, such as solubility in polar solvents, but for the purposes of the invention, such acid and base salts are equivalent to their respective free forms in other pharmaceutical aspects.

[0068] Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic or acidic moiety by conventional chemical methods. Typically, salts of basic compounds are prepared by ion exchange chromatography or by reacting a free base with a stoichiometric amount or an excess of an inorganic or organic acid in the desired salt form in a suitable solvent or combination of solvents. Similarly, salts of acidic compounds are formed by reaction with a suitable inorganic or organic base.

[0069] Therefore, pharmaceutically acceptable salts of the compounds of this invention include conventional non-toxic salts of the compounds of this invention formed by reacting an alkaline compound of this invention with an inorganic or organic acid. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, and also include salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, and the like.

[0070] If the compound of the present invention is acidic, then suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, guaiac, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0071] Berg et al., "Pharmaceutical Salts," J. Pharm. Sci. '1977: 66: 1-19, describes in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.

[0072] Since deprotonated acidic moieties such as carboxyl groups in the compounds may be anionic under physiological conditions, and this charge may then be balanced by internal cationic protonated or alkylated basic moieties such as tetravalent nitrogen atoms, it should be noted that the compounds of the present invention are potential inner salts or zwitterions.

[0073] The pharmaceutical formulations or methods for inhibiting or activating gene expression provided herein comprise (administered to a patient or subject) a safe and effective amount of an active ingredient (i.e., the oligonucleotides described herein) and a pharmaceutically acceptable excipient. When administering the drug, a safe and effective amount of the active ingredient is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage at the time of administration is a pharmaceutically effective dosage. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0074] Here, "safe and effective amount" means an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. "Pharmaceutically acceptable excipients" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the various components of the composition to blend with the active ingredient (oligonucleotide) of the present invention, as well as with each other, without significantly reducing the efficacy of the active ingredient.

[0075] Pharmaceutically acceptable excipients include, but are not limited to, one or more of the following materials: at least one of solvents, excipients, fillers, extenders, binders, humectants, disintegrants, dissolution buffers, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents, and fragrances.

[0076] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0077] The targeted delivery ligand or other type of delivery carrier of the present invention is preferably an N-acetylgalactosamine derivative (GalNAc carrier). Other types of delivery carriers such as liposomes that are specifically delivered to liver cells can be used in the present invention as long as they can achieve the delivery of siRNA.

[0078] At present, the GalNAc carrier has been studied in depth. GalNAc-nucleic acid is a monoconjugate formed by a carbohydrate compound and a nucleic acid. N-acetylated galactosamine is covalently conjugated to the 3′ and / or 5′ end of the sense chain of siRNA of different sequences in a trivalent manner to form a GalNAc-siRNA drug, thereby achieving specific delivery to hepatocytes, and allowing the drug to enter the cell and exert its function through endocytosis.

[0079] In the following examples, the exemplary structures of the GalNAc ligand and the 3' end of the siRNA sense strand are as follows:

[0080] above represents double-stranded RNA, such as the siRNA of the present invention.

[0081] In the nucleic acid sequence table of the present invention, the abbreviations and structures of the nucleotide monomers used are as follows:

[0082] The following are specific examples.

[0083] Example 1: Preparation of (2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 1)

[0084] Step 1a: Preparation of (2R,3R,9aR)-3-hydroxy-2-(hydroxymethyl)-2,3,3a,9a-tetrahydro-6H-furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one (Compound 1-2): A mixture of 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-1) (30 g, 122.9 mmol), diphenyl carbonate (31.6 g, 147.5 mmol) and sodium bicarbonate (825 mg, 9.8 mmol) in N,N-dimethylformamide (150 mL) was stirred at 100° C. for 4 h. After cooling to room temperature, the mixture was added dropwise to MTBE (1.5 L) and stirred for 15 minutes. The solid was filtered, dispersed in methanol (150 mL) and stirred for 15 minutes. The solid was filtered and dried under vacuum to give an off-white solid product (2R,3R,9aR)-3-hydroxy-2-(hydroxymethyl)-2,3,3a,9a-tetrahydro-6H-furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one (23.0 g, 83.0% yield). LCMS (ESI): m / z = 227 [M+1] + .

[0085] Step 1b: Preparation of 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-3): (2R,3R,9aR)-3-hydroxy-2-(hydroxymethyl)-2,3,3a,9a-tetrahydro-6H-furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one (Compound 1-2) (10 g, 44.2 mmol) was dissolved in N,N-dimethylformamide (100 ml), sodium thiomethoxide (14.8 g, 212.4 mmol) was added, and the mixture was stirred at room temperature for 1 h. 6 M aqueous hydrochloric acid was added to adjust the pH to 6-7. The mixture was filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1) to obtain 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (12 g, 99.0% yield) as a pale yellow solid. LCMS (ESI): m / z = 275 [M+1] + .

[0086] Step 1c: Preparation of 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-4): Under nitrogen protection, 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-3) (12 g, 43.8 mmol) was dissolved in anhydrous pyridine (120 ml), 4,4'-bismethoxytrityl chloride (17.8 g, 52.6 mmol) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated, and the residue was dissolved in dichloromethane, washed with water, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / ethyl acetate = 1:1:1) to obtain 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (13.4 g, 53.1% yield) as a yellow foamy solid. LCMS (ESI): m / z = 575 [M-1]. - . 1H NMR (500MHz, DMSO-d6) δ11.35(s,1H),7.55(d,J=8.1Hz,1H),7.42(d,J=7.7 Hz,2H),7.28(tt,J=20.0,7.4Hz,7H),6.96-6.87(m,4H),5.97(d,J=5.2Hz,1 H),5.71(d,J=4.2Hz,1H),5.37(d,J=8.1Hz,1H),4.54(td,J=6.3,3.2Hz,1H ), 4.19 (q, J = 4.8Hz, 1H), 3.74 (s, 6H), 3.40-3.35 (m, 1H), 3.30-3.24 (m, 2H).

[0087] Step 1d: Preparation of 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-5): 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl) -4-Hydroxy-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-4) (13.4 g, 23.3 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (7.3 ml, 48.8 mmol) were dissolved in anhydrous dichloromethane (270 ml), tert-butyldimethylsilyl chloride (7.0 g, 46.5 mmol) was added, and the mixture was stirred at room temperature overnight. The mixture was washed with water, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10:1) to afford 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (10.2 g, 63.3% yield) as a white foamy solid. LCMS (ESI): m / z = 689 [M-1]. - . 1H NMR (500MHz, CDCl3) δ8.62(d,J=11.6Hz,1H),7.50(d,J=8.2Hz,1H),7.41-7.31(m,2H),7.23(t,J=8.8Hz,3H),7.19- 7.15(m,2H),7.11(dd,J=13.0,5.8Hz,1H),7.04(d,J=8.7Hz,1H),6.77-6.66(m,4H),5.63(t,J=2.9Hz,1H),5.28(dd, J=8.1,2.2Hz,1H),4.57-4.39(m,1H),4.28-4.19(m,1H),3.67(s,6H),3.44(dd,J=10.7,5.7Hz,1H),3.28(dd,J=10. 6,3.1Hz,1H),3.00(ddd,J=78.1,6.6,4.5Hz,1H),1.92(d,J=12.7Hz,3H),0.77(d,J=9.6Hz,9H),0.06--0.10(m,6H).

[0088] Step 1e: Preparation of 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-6): 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 1-5) (9.18 g, 13.3 mmol) was dissolved in dichloromethane (92 ml), dichloroacetic acid (4.4 ml, 53.2 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was adjusted to pH 7-8 with saturated aqueous sodium bicarbonate solution, and the layers were separated. The organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 30:1) to afford 1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (4.47 g, 86.6% yield) as a yellow solid. LCMS (ESI): m / z = [M+1] + . 1H NMR (500MHz, DMSO-d6) δ11.26(s,1H),7.85(d,J=8.2Hz,1H),5.61(dd,J=8.4,3.3Hz,2H),5.01(t,J=4.4Hz,1H),4 .27-4.16(m,2H),3.59(tdd,J=11.9,10.1,8.1,4.4Hz,2H),2.05(s,3H),0.75(s,9H),-0.00(s,3H),-0.12(s,3H).

[0089] Step 1f: Preparation of dimethyl ((E)-2-((2R, 3R, 4R, 5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 1-7): 1-((2R, 3R, 4R, 5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-(methylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione (Compound 1-6) (4.4 g, 11.3 mmol) was dissolved in anhydrous dichloromethane (120 ml), and Desmartine reagent (7.21 g, 17.0 mmol) was added at 0-5°C, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with saturated aqueous sodium thiosulfate and adjusted to pH 7-8 by adding saturated aqueous sodium bicarbonate. After separation, the organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to yield crude product 1-6A. Tetramethylmethylene diphosphate (6.54 g, 22.7 mmol) was dissolved in anhydrous tetrahydrofuran (100 ml), and potassium tert-butoxide (2.54 g, 22.7 mmol) was added at 0-5°C. The mixture was stirred at room temperature for 15 minutes. A solution of 1-6A in anhydrous tetrahydrofuran (20 ml) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was added to saturated sodium chloride solution, extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / acetone = 5:1) to obtain dimethyl ((E)-2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (2.6 g, 46.7% yield) as an off-white foamy solid. LCMS (ESI): m / z = 493 [M+1]. + .

[0090] Step 1g: Preparation of dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 1-8): To dimethyl ((E)-2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate To a mixture of 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 1-7) (1.6 g, 3.25 mmol) and N,N-diisopropylethylamine (545 mg, 4.23 mmol) in anhydrous dichloromethane (32 ml) was added benzoyl chloride (453 μl, 3.90 mmol), and the mixture was stirred at room temperature overnight. The reaction solution was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / methanol = 50:50:1 to dichloromethane / methanol = 50:1) to obtain dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (1.91 g, 98.9% yield) as an off-white foamy solid. LCMS (ESI): m / z = 597 [M+H]. + .

[0091] Step 1h: Preparation of dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 1-9): To dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate To a mixture of 4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 1-8) (1.9 g, 3.19 mmol) and triethylamine (1.2 ml, 4.97 mmol) in tetrahydrofuran (40 ml) was added triethylamine trihydrofluoride (2.67 ml, 15.9 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / methanol = 50:50:1 to dichloromethane / methanol = 50:1) to afford dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (1.2 g, 76.1% yield) as an off-white foamy solid. LCMS (ESI): m / z = [M+1] + . 1 H NMR (500MHz, CDCl3) δ8.01-7.90(m,2H),7.76(d,J=8.2Hz,1H),7.67(t,J=7.4Hz ,1H),7.52(t,J=7.8Hz,2H),6.83(ddd,J=22.1,17.2,4.7Hz,1H),6.10-5.99(m,1 H),5.87(d,J=8.3Hz,1H),5.72(d,J=2.1Hz,1H),5.14(dd,J=5.2,2.9Hz,1H),4. 38(d,J=2.1Hz,1H),3.93-3.56(m,7H),3.32(dd,J=5.4,2.1Hz,1H),2.16(s,3H).

[0092] Step 1i: Preparation of (2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 1): To dimethyl((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite To a mixture of 3-(4-(2-(methylthio)tetrahydrofuran-2-yl)-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 1-9) (1.25 g, 2.59 mmol) and N,N-diisopropylethylamine (1.34 g, 10.4 mmol) in anhydrous tetrahydrofuran (20 ml) was added cyanoethyl-N,N-diisopropylchlorophosphoramidite (600 μl, 2.70 mmol), and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated in vacuo at 30°C, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / acetone = 3:1, supplemented with 2% triethylamine; petroleum ether / dichloromethane / acetone = 3:1:1, supplemented with 2% triethylamine) to afford (2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (1.60 g, 90% yield) as a white foamy solid. LCMS (ESI): m / z = 600 [M-83+1]. + and m / z = 547 [M-135+1] + . 1 H NMR (500MHz, CDCl3) δ7.94(dd,J=11.0,7.9Hz,2H),7.84(d,J=8.3Hz,1H),7.66(q,J=7.1Hz,1H),7.51(q,J=7.4Hz,2 H),6.87(dddd,J=21.9,17.1,9.3,4.2Hz,1H),6.15-5.99(m,2H),5.88(t,J=8.8Hz,1H),5.11(dd,J=17.9,4.4Hz,1H) ,4.54(dd,J=16.4,9.6Hz,1H),3.94-3.81(m,1H),3.79(d,J=2.0Hz,6H),3.75-3.69(m,1H),3.60(ddt,J=23.5,10.4 ,6.7Hz,2H),3.41(dd,J=56.0,5.1Hz,1H),2.55(dt,J=27.8,5.7Hz,2H),2.21(d,J=14.2Hz,3H),1.23-1.11(m,12H).

[0093] Example 2: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-propyltetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 2)

[0094] Step 2a: Preparation of O-((6aR,8R,9R,9aR)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)O-phenylthiocarbonyl ester (Compound 2-2): Under nitrogen protection, at 10°C, add 1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetrahydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)O-phenylthiocarbonyl ester to the reaction mixture. To a mixture of isopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-1) (50.0 g, 102.74 mmol, 1.0 eq) and 4-dimethylaminopyridine (25.1 g, 205.48 mmol, 2.0 eq) in dry acetonitrile (600 ml) was added dropwise phenylchlorothioformate (23.94 g, 138.70 mmol, 1.35 eq). The mixture was stirred at room temperature overnight. The mixture was filtered through celite. The filter cake was washed three times with ethyl acetate, and the filtrate was then washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1 to 5 / 1) to give O-((6aR,8R,9R,9aR)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)O-phenylthiocarbonyl ester (59.67 g, yield: 93.2%) as a white solid. LCMS (ESI): m / z = 623 [M+1]. + .

[0095] Step 2b: Preparation of 1-((6aR,8R,9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-3): Under nitrogen protection, at room temperature, add O-((6aR,8R,9R,9aR)-8-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4, To a mixture of 4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl) O-phenylthiocarbonyl ester (Compound 2-2) (30.0 g, 48.17 mmol, 1.0 eq) and allyltributylstannane (79.8 g, 240.85 mmol, 5.0 eq) in dry toluene (450 ml) was added azobisisobutyronitrile mixture (3.95 g, 24.09 mmol, 0.5 eq in 50 ml of toluene). The mixture was stirred at 80°C for 5.0 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1 to 1 / 1) to give 1-((6aR,8R,9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (17.4 g, yield: 70.4%) as a white solid. LCMS (ESI): m / z = 511 [M+1] +

[0096] Step 2c: Preparation of 1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-propyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-4): Add 1-((6aR,8R,9R,9aS)-9-allyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine to a 1-(1H,3H)-dione mixture at room temperature. Platinum dioxide (357 mg, 1.57 mmol, 0.2 eq) was added to a solution of 2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-3) (4.0 g, 7.84 mmol, 1.0 eq) in methanol (50 ml). The mixture was stirred under hydrogen at room temperature overnight. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give 1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-propyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (4.1 g, crude) as a white solid. LCMS (ESI): m / z = 513 [M+1]. +

[0097] Step 2d: Preparation of 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-propyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-5): 1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9- To a mixture of propyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-4) (4.1 g, 8.0 mmol, 1.0 eq) and diisopropylethylamine (2.27 g, 17.6 mmol, 2.2 eq) in dichloromethane (50 ml) was added benzoyl chloride (1.69 g, 12.0 mmol, 1.5 eq). The mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to give 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-propyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (5.3 g, crude) as a yellow oil. LCMS (ESI): m / z = 617 [M+1] + .

[0098] Step 2e: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-6): To a solution of 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-propyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-5) (5.3 g, 8.0 mmol, 1.0 eq) in tetrahydrofuran (60 mL) was added tetrabutylammonium fluoride (2.27 g, 7.2 mmol, 0.9 eq) at room temperature. The mixture was stirred at room temperature for two hours. The mixture was diluted with ethyl acetate, washed with water and saturated brine, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 3 to 1 / 15) to obtain 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (2.5 g, yield: 83.6%) as a white solid. LCMS (ESI): m / z = 375 [M+1] + .

[0099] Step 2f: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-7): To a solution of 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-6) (2.5 g, 6.69 mmol, 1.0 eq) in pyridine (30 mL) was added 4,4'-dimethoxytriphenylmethane chloride (2.73 g, 8.03 mmol, 1.2 eq). The mixture was stirred at room temperature for three hours. The pyridine was removed by concentration under reduced pressure, and the residue was dissolved in ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to obtain 3-benzoyl-1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (4.25 g, yield: 93.8%) as a yellow solid. LCMS (ESI): m / z = 677 [M+1] + .

[0100] Step 2g: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-8): Under nitrogen protection, 3-benzoyl-1-((2R,3R,4S,5R)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione was added. To a mixture of (4-hydroxy-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-7) (3.96 g, 5.86 mmol, 1.0 equiv) and imidazole (2.0 g, 29.3 mmol, 5.0 equiv) in dry dimethylformamide (13 ml) was added tert-butyldimethylsilyl chloride (2.21 g, 14.65 mmol, 2.5 equiv). The mixture was stirred at 36°C overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1 to 4 / 1, with 2% triethylamine added) to afford 3-benzoyl-1-((2R,3R,4S,5R)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3.31 g, yield: 68.8%) as a yellow solid. LCMS (ESI): m / z = 791 [M+1]. + .

[0101] Step 2h: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-9): To a solution of 3-benzoyl-1-((2R,3R,4S,5R)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-8) (3.31 g, 4.18 mmol, 1.0 eq) in dichloromethane (50 mL) was added dichloroacetic acid (5 mL, 63.7 mmol, 15.0 eq). The mixture was stirred at room temperature for two hours. Saturated aqueous sodium carbonate solution was added for neutralization, and the layers were separated. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1 to 1 / 5) to obtain 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.59 g, yield: 77.9%) as a white solid. LCMS (ESI): m / z = 489 [M+1]. + .

[0102] Step 2i: Preparation of (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-propyltetrahydrofuran-2-carbaldehyde (Compound 2-10): Under a nitrogen atmosphere, sulfur trioxide pyridine complex (3.29 g, 21.0 mmol, 8.0 eq) was added to dry dimethyl sulfoxide (12 ml, 169.0 mmol, 64.0 eq) and the mixture was stirred at room temperature for one hour. Under a nitrogen atmosphere, this mixture was added to a dichloromethane mixture of 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-propyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-9) (1.26 g, 2.58 mmol, 1.0 eq) and diisopropylethylamine (3.8 ml, 21.9 mmol, 8.5 eq) at 10°C. The mixture was stirred at 10°C for one hour. Ethyl acetate was added, and the mixture was washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to give (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-propyltetrahydrofuran-2-carbaldehyde (1.33 g, crude) as a yellow oil. LCMS (ESI): m / z = 487 [M+1] + .

[0103] Step 2j: Preparation of (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-propyltetrahydrofuran-2-yl)vinyl)phosphonic acid dimethyl ester (Compound 2-11): Under nitrogen protection, potassium tert-butoxide (578 mg, 5.16 mmol, 2.0 equiv) was added to a solution of tetramethylmethylene diphosphate (1.20 g, 5.16 mmol, 2.0 equiv) in dry tetrahydrofuran (20 ml) at room temperature, and the mixture was stirred for one hour. (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-propyltetrahydrofuran-2-carbaldehyde (Compound 2-10) (1.33 g, 2.58 mmol, 1.0 eq) was dissolved in dry tetrahydrofuran (5 mL) and added to the above mixture. The mixture was stirred at room temperature for one hour. The reaction mixture was quenched with aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 20 / 1) to give dimethyl (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-propyltetrahydrofuran-2-yl)vinyl)phosphonate (1.08 g, yield: 70.5%) as a white solid. LCMS (ESI): m / z = 593 [M+1] + .

[0104] Step 2k: Preparation of dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-propyltetrahydrofuran-2-yl)vinyl)phosphonate (Compound 2-12): To (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-propyltetrahydrofuran-2-yl)vinyl)phosphonate To a solution of dimethyl (1-(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-propyltetrahydrofuran-2-yl)vinyl)phosphonate (Compound 2-11) (1.24 g, 2.10 mmol, 1.0 eq) in tetrahydrofuran (12.5 ml) were added triethylamine (850 mg, 8.42 mmol, 4.0 eq) and triethylamine trihydrofluoride (2.38 g, 14.7 mmol, 7.0 eq). The mixture was stirred at room temperature overnight. The reaction mixture was neutralized with saturated aqueous sodium carbonate solution, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 15 / 1) to obtain dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-propyltetrahydrofuran-2-yl)vinyl)phosphonate (772 mg, yield: 80.6%) as a white solid. LCMS (ESI): m / z = 479 [M+1]. + .

[0105] Step 21: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-propyltetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 2): Under nitrogen protection, dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-propyltetrahydrofuran-2-yl)vinyl)phosphonate (Compound 2-12) (494 mg, 1.03 mmol, 1.0 equiv) was added. To a mixture of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (778 mg, 2.58 mmol, 2.5 eq) in dichloromethane (10 mL) was added 5-ethylthio-1H-tetrazole (202 mg, 1.55 mmol, 1.5 eq). The mixture was stirred at room temperature for two hours. The mixture was diluted with ethyl acetate and washed with aqueous sodium bicarbonate and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / acetone = 3 / 1 / 1, with 2% triethylamine added) to give (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-propyltetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite as a white solid (532 mg, yield: 75.4%). LCMS (ESI): m / z = 596 [M-100+18]. + . 1 H NMR (500MHz, CDCl3) δ7.93 (d, J = 7.6Hz, 2H), 7.65 (t, J = 7.4Hz, 1H), 7.50 (t, J = 7.8Hz, 2H), 7.38 (dd, J = 10.8, 8. 4Hz,1H),7.00-6.81(m,1H),6.05(dt,J=34.6,12.7Hz,2H),5.92(dd,J=8.2,1.6Hz,1H),4.75(d,J=24.6Hz,1H) ,4.43-4.33(m,1H),3.87(td,J=13.5,6.4Hz,1H),3.81-3.54(m,9H),2.71-2.57(m,2H),2.11(dd,J=25.5,20.8 Hz,1H),1.68(dd,J=13.4,7.7Hz,2H),1.38-1.31(m,2H),1.18(t,J=7.0Hz,12H),0.92(dd,J=13.0,6.9Hz,3H).

[0106] Example 3: Preparation of (2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 3)

[0107] Step 3a: Preparation of 1-((6aR,8R,9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-1): At room temperature, add 1-((6aR,8R,9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)3-benzoylpyrimidine-2,4(1H,3H)-dione to the mixture. Benzoyl chloride (3.02 g, 21.47 mmol, 1.5 eq) was added to a mixture of 6H-(1H,3H)-tetrahydro-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 2-3) (7.3 g, 14.31 mmol, 1.0 eq) and diisopropylethylamine (4.06 g, 31.48 mmol, 2.2 eq) in dichloromethane (73 ml). The mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to give 1-((6aR,8R,9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (9.94 g, crude) as a yellow oil. LCMS (ESI): m / z = 615 [M+1] + .

[0108] Step 3b: Preparation of 1-((2R,3R,4S,5R)-3-allyl-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-2): To a solution of 1-((6aR,8R,9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-1) (9.94 g, 14.31 mmol, 1.0 eq) in tetrahydrofuran (90 ml) was added tetrabutylammonium fluoride (3.61 g, 11.45 mmol, 0.8 eq) at room temperature. The mixture was stirred at room temperature for two hours. The mixture was diluted with ethyl acetate, washed with water and saturated brine, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 3 to 1 / 15) to obtain a white solid product, 1-((2R,3R,4S,5R)-3-allyl-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (4.42 g, yield: 83.0%). LCMS (ESI): m / z = 373 [M+1] + .

[0109] Step 3c: Preparation of 1-((2R,3R,4S,5R)-3-allyl-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-3): To a solution of 1-((2R,3R,4S,5R)-3-allyl-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-1) (4.42 g, 11.89 mmol, 1.0 eq) in pyridine (50 mL) was added 4,4'-dimethoxytriphenylmethane chloride (4.84 g, 14.27 mmol, 1.2 eq) at room temperature. The mixture was stirred at room temperature for three hours. The pyridine was removed by concentration under reduced pressure, and the residue was dissolved in ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to obtain a yellow solid product, 1-((2R,3R,4S,5R)-3-allyl-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (7.4 g, yield: 92.2%). LCMS (ESI): m / z = 675 [M+1] + .

[0110] Step 3d: Preparation of 1-((2R,3R,4S,5R)-3-allyl-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-4): Under nitrogen protection, 1-((2R,3R,4S,5R)-3-allyl-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione was added. To a mixture of (1-(phenyl)-(1-(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-3) (7.4 g, 10.98 mmol, 1.0 equiv) and imidazole (3.74 g, 54.9 mmol, 5.0 equiv) in dry dimethylformamide (24 ml) was added tert-butyldimethylsilyl chloride (4.14 g, 27.45 mmol, 2.5 equiv). The mixture was stirred at 36°C overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1 to 4 / 1, with 2% triethylamine added) to afford 1-((2R,3R,4S,5R)-3-allyl-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (6.70 g, yield: 77.3%) as a yellow solid. LCMS (ESI): m / z = 789 [M+1]. + .

[0111] Step 3e: Preparation of 1-((2R,3R,4S,5R)-3-allyl-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-5): To a solution of 1-((2R,3R,4S,5R)-3-allyl-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-4) (6.7 g, 8.50 mmol, 1.0 equiv) in dichloromethane (100 mL) was added dichloroacetic acid (10 mL, 127.5 mmol, 15.0 equiv). The mixture was stirred at room temperature for two hours. Saturated aqueous sodium carbonate was added for neutralization, the layers were separated, the organic phase was dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1 to 1 / 5) to obtain 1-((2R,3R,4S,5R)-3-allyl-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (3.29 g, yield: 79.4%) as a white solid. LCMS (ESI): m / z = 487 [M+1] + .

[0112] Step 3f: Preparation of (2S,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-carbaldehyde (Compound 3-6): Under a nitrogen atmosphere, sulfur trioxide pyridine complex (5.90 g, 37.1 mmol, 5.5 eq) was added to dry dimethyl sulfoxide (20 ml, 282 mmol, 42.0 eq) and the mixture was stirred at room temperature for one hour. Under a nitrogen atmosphere, this mixture was added to a dichloromethane mixture of 1-((2R,3R,4S,5R)-3-allyl-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-5) (3.29 g, 6.67 mmol, 1.0 eq) and diisopropylethylamine (10 ml, 57.46 mmol, 8.5 eq) at 10°C. The mixture was stirred at 10°C for one hour. Ethyl acetate was added to the mixture, and the mixture was washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to give (2S,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-carbaldehyde (3.56 g, crude) as a yellow oil. LCMS (ESI): m / z = 485 [M+1] + .

[0113] Step 3g: Preparation of (E)-2-((2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)vinyl)phosphonic acid dimethyl ester (Compound 3-7): Under nitrogen protection, potassium tert-butoxide (1.50 g, 13.52 mmol, 2.0 equiv) was added to a solution of tetramethylmethylene diphosphate (3.14 g, 13.52 mmol, 2.0 equiv) in dry tetrahydrofuran (50 ml) at room temperature, and the mixture was stirred for one hour. (2S,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-carbaldehyde (Compound 3-6) (3.56 g, 6.76 mmol, 1.0 eq) was dissolved in dry tetrahydrofuran (10 ml) and added to the above mixture. The mixture was stirred at room temperature for one hour. The reaction mixture was quenched with aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 20 / 1) to give dimethyl (E)-2-((2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)vinyl)phosphonate (3.06 g, yield: 77.6%) as a white solid. LCMS (ESI): m / z = 591 [M+1] + .

[0114] Step 3h: Preparation of dimethyl ((E)-2-((2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 3-8): To (E)-2-((2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4 To a solution of dimethyl (1-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 3-7) (1.87 g, 3.17 mmol, 1.0 eq) in tetrahydrofuran (13 ml) were added triethylamine (1.92 g, 8.42 mmol, 6.0 eq) and triethylamine trihydrofluoride (3.83 g, 23.78 mmol, 7.5 eq). The mixture was stirred at room temperature overnight. Saturated aqueous sodium carbonate solution was added for neutralization and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 15 / 1) to obtain dimethyl ((E)-2-((2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (1.36 g, yield: 83.4%) as a white solid. LCMS (ESI): m / z = 477 [M+1]. + .

[0115] Step 3i: Preparation of (2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 3): Under nitrogen protection, add ((E)-2-((2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite To a mixture of dimethyl (2-(2-hydroxy-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 3-8) (691 mg, 1.45 mmol, 1.0 equiv) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.09 g, 3.63 mmol, 2.5 equiv) in dichloromethane (14 ml) was added 5-ethylthio-1H-tetrazole (283 mg, 2.18 mmol, 1.5 equiv). The mixture was stirred at room temperature for two hours. The mixture was diluted with ethyl acetate and washed with aqueous sodium bicarbonate and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / acetone = 3 / 1 / 1, with 2% triethylamine added) to give (2R,3S,4R,5R)-4-allyl-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite as a white solid (632 mg, yield: 64.4%). LCMS (ESI): m / z = 594 [M-100+18]. + . 1 H NMR (500MHz, CDCl3) δ7.86(d,J=8.0Hz,2H),7.58(t,J=7.4Hz,1H),7.43(t,J=7.8Hz,2H),7.29(dd,J=13 .9,8.3Hz,1H),6.95-6.72(m,1H),5.99(dt,J=34.0,12.5Hz,2H),5.84(dd,J=8.1,5.1Hz,1H),5.74-5.5 7(m,1H),5.10-4.98(m,2H),4.66(d,J=40.9Hz,1H),4.40-4.28(m,1H),3.86-3.76(m,1H),3.76-3.46(m ,9H),2.67-2.52(m,2H),2.50-2.31(m,1H),2.18(ddt,J=21.4,14.8,8.1Hz,2H),1.12(t,J=7.0Hz,12H).

[0116] Example 4: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methoxymethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 4)

[0117] Step 4a: Preparation of 3-benzoyl-1-((6aR, 8R, 9R, 9aS)-9-((E)-3-(3,5-dioxo-4-phenyl-1,2,4-triazolidin-1-yl)prop-1-en-1-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-1): Under nitrogen protection, 1-((6aR, 8R, 9R, 9aS)-9-((E)-3-(3,5-dioxo-4-phenyl-1,2,4-triazolidin-1-yl)prop-1-en-1-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione was reacted with 1-((6aR, 8 ... ,9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-1) (14.0 g, 22.768 mmol, 1.0 eq) in dichloromethane (200 ml) was added 4-phenyl-1,2,4-triazolidinyl-3,5-dione (5.98 g, 34.152 mmol, 1.5 eq). The mixture was stirred at room temperature overnight in the dark. The mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 8 / 1 to 5 / 1) to give 3-benzoyl-1-((6aR,8R,9R,9aS)-9-((E)-3-(3,5-dioxo-4-phenyl-1,2,4-triazolidin-1-yl)prop-1-en-1-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (15.57 g, yield: 86.6%) as a yellow solid product. LCMS (ESI): m / z = 790 [M+1] + .

[0118] Step 4b: Preparation of 3-benzoyl-1-((6aR, 8R, 9R, 9aS)-9-(hydroxymethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-3): A mixture of 3-benzoyl-1-((6aR, 8R, 9R, 9aS)-9-((E)-3-(3,5-dioxo-4-phenyl-1,2,4-triazolidinyl-1- A solution of (6aR,8R benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (compound 4-1) (19.70 g, 24.937 mmol, 1.0 eq) in dichloromethane (250 ml) was stirred at -70°C for 20 minutes under an ozone atmosphere to obtain the intermediate (6aR,8R benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-carbonyl (compound 4-2). The reaction solution was diluted with methanol (250 ml) and sodium borohydride (3.79 g, 99.748 mmol, 4.0 eq) was added portionwise at -70°C. The mixture was stirred at room temperature for 10 minutes. The reaction was quenched with water and extracted with dichloromethane. The organic phase was washed with water and saturated brine and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 6 / 1 to 2 / 1) to give a white solid product, 3-benzoyl-1-((6aR, 8R, 9R, 9aS)-9-(hydroxymethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (9.02 g, yield: 59.8%). LCMS (ESI): m / z=605[M+1] + .

[0119] Step 4c: Preparation of 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-(methoxymethyl)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-4): In a 250 mL flask, 36 mL of 40% potassium hydroxide solution and 120 mL of dichloromethane were added. The mixture was cooled to 0°C and 12 g of crushed nitrosomethylurea was added portionwise while maintaining the temperature. After the addition was complete, the organic layer was quickly separated. The aqueous layer was extracted once with dichloromethane. The combined organic layers were dried over potassium hydroxide at 0°C for 2 hours to obtain a diazomethane solution containing approximately 3.2 g (75.89 mmol, 9.0 eq). The prepared diazomethane solution (67.46 mmol, 8.0 eq) was added portionwise to a solution of 3-benzoyl-1-((6aR, 8R, 9R, 9aS)-9-(hydroxymethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-3) (5.10 g, 8.432 mmol, 1.0 eq) and boron trifluoride-etherate (0.3 ml, 0.843 mmol, 0.1 eq) in dry dichloromethane (30 ml) at -5°C. The mixture was stirred at 0°C for 0.5 h. The reaction solution was washed sequentially with 10% sodium bicarbonate solution and saturated brine. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1 to 4 / 1) to give 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-(methoxymethyl)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (3.80 g, yield: 72.8%) as a white solid. LCMS (ESI): m / z = 619 [M+1] + .

[0120] Step 4d: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-bis(Compound 4-5): 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-(methoxymethyl)tetrahydro-6H-furan[3 To a solution of [1,3,5,2,4]trioxadisiloxane-8-yl]pyrimidine-2,4(1H,3H)-dione (Compound 4-4) (3.80 g, 6.139 mmol, 1.0 eq) in tetrahydrofuran (40 ml) were added triethylamine (2.48 g, 24.56 mmol, 4.0 eq) and triethylamine trihydrofluoride (6.92 g, 42.973 mmol, 7.0 eq). The mixture was stirred at room temperature overnight. Saturated aqueous sodium carbonate solution was added for neutralization, and ethyl acetate was added for extraction. The organic phase was washed with water and saturated brine, dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 80 / 1 to 10 / 1) to give 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.62 g, yield: 97%) as a white solid product. LCMS (ESI): m / z = 377 [M+1] + .

[0121] Step 4e: 3-Benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-bis(Compound 4-6) Preparation of: To a mixture of 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-5) (1.7 g, 4.517 mmol, 1.0 eq) and 1,8-diazacyclo[5,4,0]undecene-7 (2.75 g, 18.068 mmol, 4.0 eq) in dichloromethane (16 ml) was added tert-butyldimethylsilyl chloride (1.88 g, 12.471 mmol, 3.0 eq) under nitrogen protection at 0°C. The mixture was stirred at room temperature for 7 hours. The mixture was diluted with dichloromethane and washed with water and saturated brine. The organic phase was concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1 to 10 / 1) to give 3-benzoyl-1-((2R,3R,4S,5R)-4-(((tert-butyldimethylsilyl)oxy)-5-((((tert-butyldimethylsilyl)oxy)methyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.90 g, yield: 74.5%) as a white solid product. LCMS (ESI): m / z = 605 [M+1] + .

[0122] Step 4f: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-7): 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxymethyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-6) (1.90 g, 3.069 mmol, 1.0 equiv) in tetrahydrofuran (20 To the 4-nitro-2-nitro-1H-pyrimidine-2,4-dione (1.3 g, yield: 86.6%) was added trifluoroacetic acid / water (5 ml / 5 ml) to the 4-nitro-2-nitro-1H-pyrimidine-2,4-dione (1.3 g, yield: 86.6%). LCMS (ESI): m / z=491[M+1] + .

[0123] Step 4g: Preparation of (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(methoxymethyl)tetrahydrofuran-2-carboxylic acid (Compound 4-8): Under nitrogen protection, sulfur trioxide pyridine (1.17 g, 7.338 mmol, 4.0 eq) was added to dry dimethyl sulfoxide (10 ml), and the mixture was stirred at room temperature for 15 minutes. The mixture was added to a solution of 3-benzoyl-1-((2R,3R,4S,5R)-4-(((tert-butyldimethyl)oxy)-5-(hydroxymethyl)-3-(methoxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-7) (900 mg, 1.834 mmol, 1.0 eq) and N,N-diisopropylethylamine (1.89 g, 14.672 mmol, 8.0 eq) in dry dichloromethane at 0°C. The mixture was stirred at 0°C for 1.0 hour. Ethyl acetate was added and washed with water and saturated brine. The organic phase was dried and concentrated under vacuum to give a light yellow solid product (2S, 3S, 4R, 5R) -5- (3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -3- (tert-butyldimethylsilyl) oxy) -4- (methoxymethyl) tetrahydrofuran-2-carbaldehyde (920 mg, crude). LCMS (ESI): m / z = 489 [M + 1] + .

[0124] Step 4h: Preparation of dimethyl (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(methoxymethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 4-9): To a solution of tetramethylmethylene diphosphate (936 mg, 4.035 mmol, 2.2 equiv) in dry tetrahydrofuran (20 ml) was added potassium tert-butoxide (411 mg, 3.668 mmol, 2.0 equiv) under nitrogen protection at room temperature, and the mixture was stirred at room temperature for 15 minutes. (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(methoxymethyl)tetrahydrofuran-2-carbaldehyde (Compound 4-8) (896 mg, 1.834 mmol, 1.0 eq) was added. The mixture was stirred at room temperature for 15 minutes. The reaction solution was quenched with aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 300 / 1 to 100 / 1) to give dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((tert-butyldimethylsilyl)oxy)-4-(methoxymethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (903 mg, yield: 82.84%) as a white solid product. LCMS (ESI): m / z = 595 [M+1] + .

[0125] Step 4i: Preparation of ((E)-2-((2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methoxymethyl)tetrahydrofuran-2-yl)vinyl)phosphonic acid dimethyl (Compound 4-10): Under nitrogen protection, to (E)-2-(((2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dihydropyrimidin-1( To a solution of dimethyl (2H)-((tert-butyldimethylsilyl)oxy)-4-(methoxymethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 4-9) (903 mg, 1.518 mmol, 1.0 eq) in dry tetrahydrofuran (10 ml) were added triethylamine (613 mg, 6.072 mmol, 4.0 eq) and triethylamine trihydrofluoride (1.71 g, 10.629 mmol, 7.0 eq). The mixture was stirred at room temperature overnight. The reaction solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried, and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 30 / 1) to give the product, dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methoxymethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (608 mg, 83% yield) as a white solid. LCMS (ESI): m / z = 481 [M+1]. + .

[0126] Step 4j: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methoxymethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 4): Under nitrogen protection, add ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methoxymethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite To a mixture of dimethyl (3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(methoxymethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 4-10) (608 mg, 1.265 mmol, 1.0 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.14 g, 3.796 mmol, 3.0 eq) in dichloromethane (15 ml) was added tetrazole (133 mg, 1.898 mmol, 1.5 eq). The mixture was stirred at room temperature for 2 hours, diluted with ethyl acetate, and then washed with aqueous sodium bicarbonate and saturated brine. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether / acetone = 2 / 5 / 1 to 2 / 2 / 1, supplemented with 2% Et3N) to give (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(methoxymethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite as a white solid (816 mg, 94% yield). LCMS (ESI): m / z = 599 [M-100+18] + . 1 H NMR (500MHz, CDCl3) δ7.87(d,J=7.4Hz,2H),7.58(t,J=7.4Hz,1H),7.43(t,J=7.7Hz, 2H),7.33(t,J=7.7Hz,1H),6.93-6.72(m,1H),6.11(t,J=7.4Hz,1H),6.03-5.88(m,1 H),5.82(t,J=8.1Hz,1H),4.63(d,J=44.5Hz,1H),4.42-4.32(m,1H),3.86-3.75(m,1 H),3.72-3.60(m,7H),3.60-3.51(m,3H),3.22(d,J=15.4Hz,3H),2.61-2.36(m,4H), 1.12(t,J=5.1Hz,12H).

[0127] Example 5: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(cyanomethyl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 5)

[0128] Step 5a: Preparation of 3-benzoyl-1-((6aR,8R,9R,9aS)-9-(2-hydroxyethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilafuran-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 5-1): At -70°C, 1-((6aR,8R,9aS)-9-(2-hydroxyethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilafuran-8-yl)pyrimidine-2,4(1H,3H)-dione was added. A solution of (9R,9aS)-9-allyl-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilafuran-8-yl)-3-benzoylpyrimidine-2,4(1H,3H)-dione (Compound 3-1) (20.0 g, 32.57 mmol, 1.0 eq) in methanol (280 mL) was bubbled with ozone for 25 minutes. Thin-layer chromatography indicated the absence of starting material. Dimethyl sulfide (40 mL) was added dropwise, and the mixture was warmed to 10°C. The reaction mixture was stirred for 30 minutes. The mixture was then cooled to -70°C. Sodium borohydride (2.35 g, 61.88 mmol, 1.9 eq) was added portionwise, and the mixture was allowed to warm to room temperature. The reaction mixture was stirred for 30 minutes. Ammonium chloride solution was added, and the mixture was concentrated under reduced pressure to remove methanol. The residue was extracted with dichloromethane and washed with water and saturated brine. The organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1 to 2 / 1) to obtain 3-benzoyl-1-((6aR,8R,9R,9aS)-9-(2-hydroxyethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilafuran-8-yl)pyrimidine-2,4(1H,3H)-dione (9.2 g, yield: 45.7%) as a white solid. LCMS (ESI): m / z = 619 [M+1] + .

[0129] Step 5b: 2-((6aR,8R,9R,9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4- Preparation of tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetaldehyde (Compound 5-2): A mixture of 3-benzoyl-1-((6aR,8R,9R,9aS)-9(2-hydroxyethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 5-1) (2.5 g, 4.04 mmol, 1.0 eq) and 2-iodoacylbenzoic acid (1.36 g, 4.85 mmol, 1.2 eq) in dry dimethyl sulfoxide (25 ml) was stirred at room temperature for 1.5 hours. The mixture was quenched with saturated aqueous sodium bisulfite solution, and the pH was adjusted to 7 with saturated aqueous sodium bicarbonate solution. The mixture was extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the product 2-((6aR,8R,9R,9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetaldehyde as a light yellow solid, which was used directly in the next step. (2.48 g, yield: 99.5%). LCMS (ESI): m / z = 617 [M+1] + .

[0130] Step 5c: Preparation of (Z)-2-((6aR, 8R, 9R, 9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetaldehyde oxime (Compound 5-3): 2-(6aR, 8R, 9R, 9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetaldehyde oxime was added at room temperature. A mixture of ethanol / water (25 mL / 25 mL) containing 4-(4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)-acetaldehyde (Compound 5-2) (2.48 g, 4.019 mmol, 1.0 eq) and hydroxylamine hydrochloride (1.12 g, 16.078 mmol, 4.0 eq) was stirred for 15 minutes. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain (Z)-2-((6aR,8R,9R,9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetaldehyde oxime (2.40 g, 94.5% yield) as a pale yellow solid, which was used directly in the next step. LCMS (ESI): m / z = 632 [M+1]. + .

[0131] Step 5d: Preparation of 2-(6aR, 8R, 9R, 9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetonitrile (Compound 5-4): Under nitrogen protection, (Z)-2-((6aR, 8R, 9R, 9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetonitrile A mixture of 2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetaldehyde oxime (Compound 5-3) (2.40 g, 3.798 mmol, 1.0 eq) and copper acetate (1.52 g, 7.596 mmol, 2.0 eq) in acetonitrile (30 ml) was stirred at 60 degrees Celsius for 5 hours. The mixture was cooled to room temperature and diluted with water, and extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to give 2-((6aR,8R,9R,9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetonitrile (2.0 g, yield: 85.8%) as a pale yellow solid. LCMS (ESI): m / z = 614 [M+1] + .

[0132] Step 5e: Preparation of 2-(2R,3R,4S,5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydro-pyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)acetonitrile (Compound 5-5): Under nitrogen protection and at zero degrees Celsius, 2-(6aR,8R,9R,9aS)-8-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-9-yl)acetonitrile (Compound 5-4) (1.76 g, 2.867 mmol, 1.0 equiv) was added to the mixture. To a solution of dry tetrahydrofuran (60 ml) was added triethylamine trihydrofluoride (4.62 g, 28.67 mmol, 10.0 equivalents) dropwise. The mixture was stirred at room temperature overnight. The reaction solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 200 / 1 to 20 / 1) to give a white solid product 2-((2R, 3R, 4S, 5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)acetonitrile (1.0 g, yield: 84.7%). LCMS (ESI): m / z = 372 [M+1] + .

[0133] Step 5f: Preparation of 2-(2R,3R,4S,5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydro-pyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-((tert-butyldimethylsilyl)oxymethyl)tetrahydrofuran-3-yl)acetonitrile (Compound 5-6): Under nitrogen protection and zero degrees Celsius, 2-((2R,3R,4S,5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydro-pyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-((tert-butyldimethylsilyl)oxymethyl)tetrahydrofuran-3-yl)acetonitrile was added. To a solution of (1.0 g, 2.693 mmol, 1.0 equiv) and imidazole (1.46 g, 21.544 mmol, 8.0 equiv) in dry dichloromethane (10 ml) was added tert-butyldimethylsilyl chloride (2.44 g, 16.157 mmol, 6.0 equiv). The mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with water and saturated brine. The organic phase was concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 30 / 1 to 5 / 1 + 2% dichloromethane) to give 2-((2R,3R,4S,5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxymethyl)tetrahydrofuran-3-yl)acetonitrile (1.12 g, yield: 69.4%) as a white solid product. LCMS (ESI): m / z = 600 [M+1] + .

[0134] Step 5g: Preparation of 2-((2R, 3R, 4S, 5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)acetonitrile (Compound 5-7): Under nitrogen protection at zero degrees Celsius, 2-((2R, 3R, 4S, 5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxymethyl)tetrahydrofuran-3-yl)acetonitrile (Compound 5-6) (1.12 g, 1.867 mmol, 1.0 equiv) in tetrahydrofuran ( To the mixture was added trifluoroacetic acid / water (4 ml / 4 ml) (16 ml) solution. The mixture was stirred at zero degrees Celsius for 4 hours. The reaction solution was quenched with saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate. The organic phase was washed with saturated brine and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to give a white solid product 2-((2R, 3R, 4S, 5R)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)acetonitrile (760 mg, yield: 84%). LCMS (ESI): m / z = 486 [M+1] + .

[0135] Step 5h: Preparation of 2-((2R,3R,4S,5S)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-formyltetrahydrofuran-3-yl)acetonitrile (Compound 5-8): Under nitrogen protection, sulfur trioxide pyridine (995 mg, 6.260 mmol, 6.0 eq) was added to dry dimethyl sulfoxide (10 ml), and the mixture was stirred at room temperature for 15 minutes. Under nitrogen protection at 0°C, the mixture was added to a mixture of 2-((2R,3R,4S,5R)-2-(3-benzoyl-2,4-dihydropyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)acetonitrile (Compound 5-7) (760 mg, 1.565 mmol, 1.0 eq) and N,N-diisopropylethylamine (1.62 g, 12.52 mmol, 8.0 eq) in dry dichloromethane (15 ml). The mixture was stirred at 0°C for 1.0 hour. Ethyl acetate was added, and the mixture was washed with water and saturated brine. The organic phase was dried and concentrated under vacuum to give 2-((2R,3R,4S,5S)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(((tert-butyldimethylsilyl)oxy)-5-formyltetrahydrofuran-3-yl)acetonitrile (716 mg, crude) as a pale yellow solid. LCMS (ESI): m / z = 484 [M+1] + .

[0136] Step 5i: Preparation of dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(cyanomethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 5-9): To a solution of tetramethylmethylene diphosphate (756 mg, 3.257 mmol, 2.2 eq) in dry tetrahydrofuran (20 mL) was added potassium tert-butoxide (332 mg, 2.96 mmol, 2.0 eq) under nitrogen at room temperature. The mixture was stirred at room temperature for 15 minutes. 2-((2R,3R,4S,5S)-2-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((tert-butyldimethylsilyl)oxy)-5-formyltetrahydrofuran-3-yl)acetonitrile (Compound 5-8) (716 mg, 1.480 mmol, 1.0 eq) was dissolved in dry tetrahydrofuran (5 ml) and added to the above mixture. The mixture was stirred at room temperature for 15 minutes. The reaction mixture was quenched with aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 300 / 1 to 100 / 1) to give the product, dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((tert-butyldimethylsilyl)oxy)-4-(cyanomethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (578 mg, yield: 66.3%) as a white solid. LCMS (ESI): m / z = 590 [M+1]. + .

[0137] Step 5j: Preparation of dimethyl ((E)-2-((2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(cyanomethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 5-10): Under nitrogen protection at zero degrees Celsius, to ((E)-2-((2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(cyanomethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate To a solution of dimethyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(cyanomethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 5-9) (578 mg, 0.980 mmol, 1.0 eq) in dry tetrahydrofuran (25 ml) was added dropwise triethylamine trihydrofluoride (1.57 g, 9.802 mmol, 10.0 eq). The mixture was stirred at room temperature overnight. The reaction solution was diluted with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried, and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 200 / 1 to 30 / 1) to give the product ((E)-dimethyl 2-(((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(cyanomethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (387 mg, yield: 83%) as a white solid. LCMS (ESI): m / z = 476 [M+1] + .

[0138] Step 5k: Preparation of (2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(cyanomethyl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphinyl (Compound 5): Under nitrogen protection, to ((E)-2-(((2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(cyanomethyl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphinyl To a solution of dimethyl)-4-(cyanomethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 5-10) (475 mg, 0.999 mmol, 1.0 eq) in dry dichloromethane (3 ml) was added 4,5-dicyanoimidazole (178 mg, 1.499 mmol, 1.5 eq). The mixture was stirred at room temperature for 15 minutes. 3-(bis(diisopropylamino)phosphino)oxy)propionitrile (903 mg, 2.997 mmol, 3.0 eq) was added to the above solution. The reactants were stirred at room temperature for 1.0 hour. The mixture was diluted with ethyl acetate and washed with aqueous sodium bicarbonate solution and saturated brine. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether / acetone = 2 / 6 / 1 to 2 / 0.5 / 1, with 2% triethylamine added) to give (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(cyanomethyl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite as a white solid (488 mg, 72.3% yield). LCMS (ESI): m / z = 593 [M-100+18] + . 1 H NMR (500MHz, CDCl3) δ7.91(d,J=8.0Hz,2H),7.59(t,J=7.4Hz,1H),7.43(t,J=7 .7Hz,2H),7.34(t,J=7.7Hz,1H),6.82(dddd,J=41.4,22.0,17.2,4.6Hz,1H),6. 06-5.93(m,2H),5.88(t,J=8.5Hz,1H),4.71(d,J=51.3Hz,1H),4.49-4.35(m,1H ),3.87-3.77(m,1H),3.72-3.51(m,8H),2.82-2.40(m,6H),1.18-1.05(m,12H).

[0139] Example 6: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(fluoromethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 6)

[0140] Step 6a: Preparation of 3-benzoyl-1-((6aR, 8R, 9R, 9aS)-9-(fluoromethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furan[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 6-1): Under nitrogen protection and at zero degrees Celsius, 3-benzoyl-1-((6aR, 8R, 9R, To a solution of (9aS)-9-(hydroxymethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 4-3) (2 g, 3.3 mmol, 1.0 eq) in dry dichloromethane (80 mL) was added diethylaminosulfur trifluoride (1.6 g, 9.9 mmol, 3.0 eq) dropwise. The mixture was stirred at 0°C for 30 minutes. The mixture was diluted with dichloromethane and washed with water and saturated brine. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to give 3-benzoyl-1-((6aR,8R,9R,9aS)-9-(fluoromethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (1.09 g, yield: 54.5%) as a white solid. LCMS (ESI): m / z = 607 [M+1] + .

[0141] Step 6b: Preparation of 3-benzoyl-1-((2R, 3R, 4S, 5R)-3-(fluoromethyl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione (Compound 6-2): Under nitrogen protection, at 0°C, 3-benzoyl-1-((6aR, 8R, 9R, 9aS)-9-(fluoromethyl)-2,2,4,4-tetraisopropyltetrahydro-6- To a mixture of H-furo[3,2-f][1,3,5,2,4]trioxadisiloxane-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 6-1) (1.09 g, 1.8 mmol, 1.0 eq) and triethylamine (725 mg, 7.2 mmol, 4.0 eq) in dry tetrahydrofuran (10 ml) was added triethylamine hydrogen fluoride (2.0 g, 12.6 mmol, 7.0 eq). The mixture was stirred at room temperature overnight. A saturated solution of NaHCO3 was added to adjust the pH to 6-7, the mixture was extracted with EtOAc, and the mixture was washed with water and saturated brine. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 20 / 1) to give 3-benzoyl-1-((2R,3R,4S,5R)-3-(fluoromethyl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (585.9 mg, yield: 89.6%) as a white solid. LCMS (ESI): m / z = 365 [M+1] + .

[0142] Step 6c: Preparation of 3-benzoyl-1-((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)-5-((tert-butyldimethylsilyl)oxy)methyl)-3-(fluoromethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione (Compound 6-3): Under nitrogen protection, at room temperature, 3-benzoyl-1-((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)methyl)-3-(fluoromethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione was added. To a mixture of R)-3-(fluoromethyl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 6-2) (1.14 g, 3.1 mmol, 1.0 eq) and imidazole (1.3 g, 18.8 mmol, 6.0 eq) in dichloromethane (10 ml) was added tert-butyldimethylsilyl chloride (1.9 g, 12.5 mmol, 4.0 eq). The mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane and washed with water and saturated brine. The organic phase was dried and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to give 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-((tert-butyldimethylsilyl)oxy)methyl)-3-(fluoromethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.1 g, yield: 61%) as a white solid product. LCMS (ESI): m / z = 593 [M+1] + .

[0143] Step 6d: Preparation of 3-benzoyl-1-((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)-3-(fluoromethyl)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione (Compound 6-4): Under nitrogen protection, to a solution of 3-benzoyl-1-((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)-5-((tert-butyldimethylsilyl)oxy)methyl)-3-(fluoromethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione (Compound 6-3) (1.48 g, 2.49 mmol, 1.0 equiv) in tetrahydrofuran (50 ml) was added trifluoroacetic acid / water = 1 / 1 (5 ml / 5 ml) at 0°C. The mixture was stirred at 0°C for three hours. A saturated sodium carbonate solution was added to an ice bath to adjust the pH to 8, extracted with ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under vacuum, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1 to 2 / 1) to give a white solid product 3-benzoyl-1-((2R, 3R, 4S, 5R)-4-((tert-butyldimethylsilyl)oxy)-3-(fluoromethyl)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H, 3H)-dione (1.07 g, yield: 89.9%). LCMS (ESI): m / z = 479 [M+1] + .

[0144] Step 6e: Preparation of (2S, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(fluoromethyl)tetrahydrofuran-2-carbaldehyde (Compound 6-5): Under nitrogen protection, sulfur trioxide pyridine complex (1.4 g, 8.9 mmol, 4.0 eq) was added to dry dimethyl sulfoxide (10 ml), and the mixture was stirred at room temperature for thirty minutes. Under nitrogen protection at 0°C, this mixture was added to a dichloromethane mixture of 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-3-(fluoromethyl)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 6-4) (1.07 g, 2.3 mmol, 1.0 eq) and diisopropylethylamine (2.3 g, 17.9 mmol, 8.0 eq). The mixture was stirred at 0°C for one hour. Ethyl acetate was added, and the mixture was washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to give 2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(fluoromethyl)tetrahydrofuran-2-carbaldehyde (1.07 g, crude) as a yellow oily product. LCMS (ESI): m / z = 477 [M+1] + .

[0145] Step 6f: Preparation of (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(fluoromethyl)tetrahydrofuran-2-yl)vinyl)phosphonic acid dimethyl ester (Compound 6-6): To a solution of tetramethylmethylene diphosphate (1.14 g, 4.9 mmol, 2.2 equiv) in dry tetrahydrofuran (10 ml) was added potassium tert-butoxide (502 mg, 4.4 mmol, 2.0 equiv) under nitrogen protection at room temperature, and the mixture was stirred for thirty minutes. (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(fluoromethyl)tetrahydrofuran-2-carbaldehyde (1.07 g, 2.2 mmol, 1.0 equiv) was dissolved in dry tetrahydrofuran (10 ml) and added to the above mixture. The mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched with aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 20 / 1) to give dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(fluoromethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (687.5 mg, yield: 52.8%) as a white solid. LCMS (ESI): m / z = 583 [M+1] + .

[0146] Step 6g: Preparation of dimethyl (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(fluoromethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 6-7): To (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(fluoromethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate To a solution of dimethyl (1-(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(fluoromethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 6-6) (581 mg, 0.99 mmol, 1.0 eq) in tetrahydrofuran (5 ml) were added triethylamine (402.6 mg, 3.98 mmol, 4.0 eq) and triethylamine trihydrofluoride (1.1 g, 6.9 mmol, 7.0 eq). The mixture was stirred at room temperature overnight. The reaction mixture was neutralized with saturated aqueous sodium carbonate solution, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 15 / 1) to obtain dimethyl (E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(fluoromethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (370 mg, yield: 79.3%) as a white solid. LCMS (ESI): m / z = 469 [M+1]. + .

[0147] Step 6h: Preparation of (2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(fluoromethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 6): Under nitrogen protection, to (E)-2-((2R, 3S, 4R, 5R)-5-(3-benzoyl-2,4-dioxo- To a mixture of dimethyl (3,4-dihydropyrimidin-1(2H)-yl)-4-(fluoromethyl)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 6-7) (370 mg, 0.79 mmol, 1.0 equiv) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (714.9 mg, 2.4 mmol, 3.0 equiv) in dichloromethane (10 ml) was added tetrazole (83 mg, 1.2 mmol, 1.5 equiv). The mixture was stirred at room temperature for one hour. The mixture was diluted with ethyl acetate and washed with aqueous sodium bicarbonate and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / acetone = 5 / 2 / 1 to 2 / 2 / 1, with 2% triethylamine added) to give (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(fluoromethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite) as a white solid (470 mg, yield: 89%). LCMS (ESI): m / z = 586 [M-100+18]. + . 1 H NMR(500MHz, CDCl3)δ7.87(t,J=7.3Hz,2H),7.58(t,J=7.3Hz,1H),7.42(dd,J=16.3,8.6Hz,2H), 7.39-7.33(m,1H),6.84(dddd,J=27.0,22.0,17.2,4.5Hz,1H),6.12(dd,J=15.1,6.2Hz,1H),5.97 (dd,J=37.9,18.8Hz,1H),5.84(t,J=8.2Hz,1H),4.83-4.69(m,1H),4.68-4.61(m,1H),4.57-4.4 1(m,2H),3.81(tt,J=16.3,8.0Hz,1H),3.73-3.49(m,8H),2.76-2.48(m,4H),1.16-1.04(m,12H).

[0148] Example 7: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(2-methoxyethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 7)

[0149] Step 7a: Preparation of 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-(2-methoxyethyl)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadiazolidin-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-1): 1-Methyl-1-nitrosourea (8.24 g, 79.9 mmol, 12.35 eq) was added portionwise to a mixture of 24 mL of aqueous potassium hydroxide and 80 mL of dichloromethane at -5°C. The mixture was stirred for 30 minutes. The layers were separated, and the organic phase was dried over potassium hydroxide at below 0°C for 2 hours. The organic phase was added portionwise to a mixture of 3-benzoyl-1-((6aR,8R,9R,9aS)-9-(2-hydroxyethyl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilafuran-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 5-1) (4.0 g, 6.47 mmol, 1.0 eq) and boron trifluoride etherate (197.8 mg, 0.65 mmol, 0.1 eq) in 24 mL of dichloromethane at 0°C. The mixture was stirred at 0°C for 1 hour and quenched with saturated sodium bicarbonate solution. The organic phase was washed with water and saturated brine and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1 to 2 / 1) to give 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-(2-methoxyethyl)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadiazolidin-8-yl)pyrimidine-2,4(1H,3H)-dione (2.86 g, yield: 69.9%) as a white solid. LCMS (ESI): m / z = 633 [M+1]. + .

[0150] Step 7b: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-hydroxymethyl-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-2): Under nitrogen protection, 3-benzoyl-1-((6aR,8R,9R,9aS)-2,2,4,4-tetraisopropyl-9-(2-methoxyethyl)tetrahydrofuran-6H To a solution of 2-furo[3,2-f][1,3,5,2,4]trioxadiazolidin-8-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-1) (2.9 g, 4.59 mmol, 1.0 eq) in tetrahydrofuran (60 ml) were added triethylamine (2.78 g, 27.54 mmol, 6.0 eq) and triethylamine trihydrofluoride (5.54 g, 34.41 mmol, 7.5 eq). The mixture was stirred at room temperature overnight. Saturated aqueous sodium carbonate solution was added for neutralization, followed by extraction with ethyl acetate. The organic phase was washed with water and saturated brine, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 25 / 1) to give 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-hydroxymethyl-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.78 g, yield: 99.4%) as a white solid. LCMS (ESI): m / z = 391 [M+1] + .

[0151] Step 7c: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-5-(((4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-3): To a solution of 3-benzoyl-1-((2R,3R,4S,5R)-4-hydroxy-5-hydroxymethyl-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-2) (2.0 g, 5.12 mmol, 1.0 eq) in pyridine (20 mL) was added 4,4'-dimethoxytriphenylmethane chloride (2.08 g, 6.14 mmol, 1.2 eq) under nitrogen. The mixture was stirred at room temperature for three hours. The pyridine was removed by concentration under reduced pressure, and the residue was dissolved in ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1 to 1 / 1) to obtain 3-benzoyl-1-((2R,3R,4S,5R)-5-(((4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3.14 g, yield: 88.4%) as a yellow solid. LCMS (ESI): m / z = 693 [M+1] + .

[0152] Step 7d: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-4): Under nitrogen protection, 3-benzoyl-1-((2R,3R,4S,5R)-5-(((4-methoxyphenyl) To a mixture of (phenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-3) (1.85 g, 2.67 mmol, 1.0 equiv) and imidazole (909 mg, 13.36 mmol, 5.0 equiv) in dry dimethylformamide (5.6 ml) was added tert-butyldimethylsilyl chloride (1.0 g, 6.68 mmol, 2.5 equiv). The mixture was stirred at 36°C overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1 to 4 / 1, with 2% triethylamine added) to give 3-benzoyl-1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.33 g, yield: 61.8%) as a yellow solid. LCMS (ESI): m / z = 807 [M+1]. + .

[0153] Step 7e: Preparation of 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-5): To a solution of 3-benzoyl-1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-4) (1.34 g, 1.66 mmol, 1.0 eq) in dichloromethane (15 mL) was added 1.5 mL of dichloroacetic acid. The mixture was stirred at room temperature for two hours. Saturated aqueous sodium bicarbonate solution was added for neutralization, and the layers were separated. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1 to 1 / 5) to obtain 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (680 mg, yield: 81.1%) as a white solid. LCMS (ESI): m / z = 505 [M+1] + .

[0154] Step 7f: Preparation of (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(2-methoxyethyl)tetrahydrofuran-2-carbaldehyde (Compound 7-6): Under nitrogen protection, sulfur trioxide pyridine complex (2.61 g, 16.4 mmol, 8.0 eq) was added to dry dimethyl sulfoxide (8.6 ml, 121.0 mmol, 59.0 eq), and the mixture was stirred at room temperature for one hour. Under nitrogen, this mixture was added to a dichloromethane mixture of 3-benzoyl-1-((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-(2-methoxyethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 7-5) (1.03 g, 2.05 mmol, 1.0 eq) and diisopropylethylamine (2.38 g, 18.43 mmol, 9.0 eq) at 0°C. The mixture was stirred at 0°C for one hour. Ethyl acetate was added, and the mixture was washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure to give (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(2-methoxyethyl)tetrahydrofuran-2-carbaldehyde (1.09 g, crude) as a yellow oil. LCMS (ESI): m / z = 503 [M+1] + .

[0155] Step 7g: Preparation of dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(2-methoxyethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 7-7): To a solution of tetramethylmethylene diphosphate (1.0 g, 4.34 mmol, 2.0 equiv) in dry tetrahydrofuran (16 ml) was added potassium tert-butoxide (486 mg, 4.34 mmol, 2.0 equiv) under nitrogen protection and room temperature, and the mixture was stirred for one hour. (2S,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(2-methoxyethyl)tetrahydrofuran-2-carbaldehyde (Compound 7-6) (1.09 g, 2.17 mmol, 1.0 eq) was dissolved in dry tetrahydrofuran (5 ml) and added to the above mixture. The mixture was stirred at room temperature for one hour. The reaction mixture was quenched with aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 80 / 1 to 60 / 1) to obtain dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(2-methoxyethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (975 mg, yield: 73.8%) as a white solid. LCMS (ESI): m / z = 609 [M+1] + .

[0156] Step 7h: Preparation of dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(2-methoxyethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 7-8): Under nitrogen protection, add ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(2-methoxyethyl)tetrahydrofuran-2-yl)vinyl)phosphonate to the mixture. To a solution of dimethyl (2-((tert-butyldimethylsilyl)oxy)-4-(2-methoxyethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 7-7) (975 mg, 1.60 mmol, 1.0 eq) in tetrahydrofuran (40 ml) were added triethylamine (971 mg, 9.61 mmol, 6.0 eq) and triethylamine trihydrofluoride (1.94 g, 12.0 mmol, 7.5 eq). The mixture was stirred at room temperature overnight. The reaction mixture was neutralized with saturated aqueous sodium carbonate solution, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 15 / 1) to obtain dimethyl ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(2-methoxyethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (636 mg, yield: 80.3%) as a white solid. LCMS (ESI): m / z = 495 [M+1]. + .

[0157] Step 7i: Preparation of (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(2-methoxyethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 7): Under nitrogen, ((E)-2-((2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(2-methoxyethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite To a mixture of dimethyl (2-(2H)-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-(2-methoxyethyl)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 7-8) (554 mg, 1.12 mmol, 1.0 equiv) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.01 g, 3.36 mmol, 3.0 equiv) in dichloromethane (13 ml) was added 5-ethylthio-1H-tetrazole (219 mg, 1.68 mmol, 1.5 equiv). The mixture was stirred at room temperature for two hours. The mixture was diluted with ethyl acetate and washed with aqueous sodium bicarbonate and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / acetone = 3 / 1 / 1, with 2% triethylamine added) to give (2R,3S,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(2-methoxyethyl)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite as a white solid (659 mg, yield: 84.7%). LCMS (ESI): m / z = 612 [M-100+18]. + . 1H NMR (500MHz, CDCl3) δ7.88(d,J=8.1Hz,2H),7.58(t,J=7.4Hz,1H),7.43(t,J=7.7Hz,2H),7.33(dd,J=10.7,8.4Hz,1H),6.93-6.74(m,1 H),6.13-6.04(m,1H),5.96(dd,J=34.8,17.8Hz,1H),5.84(dd,J=8.2,3.5Hz,1H),4.67(d,J=33.2Hz,1H),4.39-4.26(m,1H),3.80(td, J=13.5,6.8Hz,1H),3.73-3.47(m,9H),3.41-3.26(m,2H),3.22(d,J=5.8Hz,3H),2.58(dt,J=12.4,6.6Hz,2H ),2.34(ddd,J=21.6,14.0,6.8Hz,1H),1.99-1.76(m,1H),1.64(dt,J=20.8,6.7Hz,1H),1.16-1.03(m,12H).

[0158] Example 8: Preparation of (2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(ethylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 8)

[0159] Step 8a: Preparation of 1-((2R,4R,5R)-3-(ethylthio)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-1): To a solution of ethanethiol (2.1 g, 331.6 mmol, 1.0 eq) in dimethylformamide (150 mL) was added sodium hydride (13.3 g, 331.6 mmol, 5.0 eq) under nitrogen at 0°C. The mixture was stirred at room temperature for 1 hour. A solution of (2R,3R,3aR,9aR)-3-hydroxy-2-(hydroxymethyl)-2,3,3a,4a,5,9a-hexahydro-6H-furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one (Compound 1-2) (15 g, 66.3 mmol, 1.0 eq) in dimethylformamide (10 mL) was added. The mixture was stirred at room temperature for 1 hour. The pH was adjusted to 6 with 6M hydrochloric acid solution, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1 to 8 / 1) to obtain 1-((2R,4R,5R)-3-(ethylthio)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (21 g, crude) as a yellow solid. LCMS (ESI): m / z=289[M+1] + .

[0160] Step 8b: Preparation of 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(ethylthio)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-2): To a solution of 1-((2R,4R,5R)-3-(ethylthio)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-1) (21 g, 66.3 mmol, 1.0 eq) in pyridine (200 mL) was added 4,4'-dimethoxytriphenylmethane chloride (26.92 g, 79.6 mmol, 1.2 eq) at room temperature. The mixture was stirred at room temperature overnight. Pyridine was removed by concentration under reduced pressure, and the residue was dissolved in ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / ethyl acetate = 1 / 1 / 1 to 1 / 1 / 3) to give 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(ethylthio)-4-hydroxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3.47 g, yield: 8.87%) as a yellow solid. LCMS (ESI): m / z = 591 [M+1] + .

[0161] Step 8c: Preparation of 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-3): Under nitrogen protection, 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione was added. To a mixture of 1,8-(1,4-dimethyl-1,4-dione)-2,4-(1H,3H)-dione (Compound 8-2) (3.47 g, 5.88 mmol, 1.0 equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene (1.89 g, 12.35 mmol, 2.1 equiv) in dry dichloromethane (35 ml) was added tert-butyldimethylsilyl chloride (1.78 g, 11.76 mmol, 2.0 equiv). The mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 8 / 1 to 4 / 1, with 2% triethylamine added) to afford 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3.49 g, yield: 84.2%) as a white solid. LCMS (ESI): m / z = 705 [M+1]. + .

[0162] Step 8d: Preparation of 3-benzoyl-1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-bis(Compound 8-4): At room temperature, add 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-bis(Compound 8-4) to the reaction mixture. Benzoyl chloride (1.05 g, 7.44 mmol, 1.5 eq) was added to a mixture of 4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-3) (3.49 g, 4.96 mmol, 1.0 eq) and diisopropylethylamine (1.41 g, 10.91 mmol, 2.2 eq) in dichloromethane (35 ml). The mixture was stirred at room temperature overnight. The mixture was diluted with ethyl acetate and washed with water and saturated brine. The organic phase was dried and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1 to 6 / 1, with 2% triethylamine added) to afford 3-benzoyl-1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (3.97 g, yield: 99.2%) as a white solid. LCMS (ESI): m / z = 809 [M+1]. + .

[0163] Step 8e: Preparation of 3-benzoyl-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-5): To a solution of 3-benzoyl-1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-4) (3.97 g, 4.91 mmol, 1.0 eq) in dichloromethane (40 mL) was added 4.0 mL of dichloroacetic acid. The mixture was stirred at room temperature for two hours. Saturated aqueous sodium bicarbonate solution was added for neutralization, and the layers were separated. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to give 3-benzoyl-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.9 g, yield: 76.3%) as a white solid. LCMS (ESI): m / z = 507 [M+1]. + .

[0164] Step 8f: Preparation of (2S,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(ethylthio)tetrahydrofuran-2-carbaldehyde (Compound 8-6): Dess-Martin periodinane (2.15 g, 5.07 mmol) was added to the mixture under nitrogen at 0°C. , 1.5 equiv) was added to a solution of 3-benzoyl-1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-3-(ethylthio)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (Compound 8-5) (1.71 g, 3.38 mmol, 1.0 equiv) in tetrahydrofuran (40 ml). The mixture was stirred at 0°C for 1.5 hours. Saturated sodium thiosulfate solution and dichloromethane were added, and the organic phase was washed with water and saturated sodium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give (2S,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(ethylthio)tetrahydrofuran-2-carbaldehyde (1.9 g, crude) as a yellow oil. LCMS (ESI): m / z = 505 [M+1] + .

[0165] Step 8g: Preparation of dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(ethylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 8-7): To a solution of tetramethylmethylene diphosphate (1.57 g, 6.67 mmol, 2.0 equiv) in dry tetrahydrofuran (40 ml) was added potassium tert-butoxide (757 mg, 6.67 mmol, 2.0 equiv) under nitrogen protection at room temperature, and the mixture was stirred at room temperature for 1 hour. A solution of (2S,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(ethylthio)tetrahydrofuran-2-carbaldehyde (Compound 8-6) (1.9 g, 3.38 mmol, 1.0 eq) in dry tetrahydrofuran (5 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with aqueous ammonium chloride and extracted with ethyl acetate. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 80 / 1 to 50 / 1) to give dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(ethylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (1.77 g, yield: 85.5%) as a yellow solid. LCMS (ESI): m / z = 611 [M+1] + .

[0166] Step 8h: Preparation of dimethyl ((E)-2-((2R, 3R, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(ethylthio)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 8-8): Under nitrogen protection, to ((E)-2-((2R, 3R, 4R, 5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(ethylthio)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate To a solution of dimethyl pyrimidin-1(2H)-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(ethylthio)tetrahydrofuran-2-yl)vinyl)phosphonate (Compound 8-7) (1.77 g, 2.93 mmol, 1.0 eq) in tetrahydrofuran (30 ml) were added triethylamine (1.78 g, 17.58 mmol, 6.0 eq) and triethylamine trihydrofluoride (3.54 g, 21.98 mmol, 7.5 eq). The mixture was stirred at room temperature overnight. The mixture was neutralized with saturated aqueous sodium carbonate solution, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1 to 15 / 1) to obtain dimethyl ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-(ethylthio)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (1.05 g, yield: 72.4%). LCMS (ESI): m / z = 497 [M+1]. + .

[0167] Step 8i: Preparation of (2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(ethylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite (Compound 8): Under nitrogen protection, to ((E)-2-((2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(ethylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite To a mixture of dimethyl (dihydropyrimidin-1(2H)-yl)-4-(ethylthio)-3-hydroxytetrahydrofuran-2-yl)vinyl)phosphonate (Compound 8-8) (886 mg, 1.79 mmol, 1.0 equiv) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.61 g, 5.35 mmol, 3.0 equiv) in dichloromethane (15 ml) was added 5-ethylthio-1H-tetrazole (154 mg, 1.19 mmol, 1.5 equiv). The mixture was stirred at room temperature for two hours. The mixture was diluted with ethyl acetate and washed with aqueous sodium bicarbonate and saturated brine. The organic phase was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane / acetone = 3 / 1 / 1, with 2% triethylamine added) to give (2R,3R,4R,5R)-5-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((E)-2-(dimethoxyphosphoryl)vinyl)-4-(ethylthio)tetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite as a white solid (1.2 g, yield: 96.0%). LCMS (ESI): m / z = 561 [M-100+18] + . 1 H NMR (500MHz, CDCl3) δ7.87 (dd, J=9.8, 8.6Hz, 2H), 7.76 (dd, J=8.3, 2.1Hz, 1H), 7.59 (dd, J=13. 6,7.3Hz,1H),7.43(q,J=7.4Hz,2H),6.87-6.73(m,1H),6.05-5.89(m,2H),5.81(dd,J=9.8,8. 5Hz,1H),5.05(dd,J=22.7,3.5Hz,1H),4.47(t,J=10.5Hz,1H),3.82-3.61(m,8H),3.51(dddd, J=65.9,55.9,32.2,5.9Hz,3H),2.62-2.43(m,4H),1.20(d,J=8.3Hz,3H),1.12-1.03(m,12H).

[0168] Example 9: Preparation of (R)-2-((R)-1-benzoyl-5-methyl-2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphoramidite (Compound 9)

[0169] Step 9a: Preparation of 3-benzoyl-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-2): 5-methylpyrimidine-2,4(1H,3H)-dione (10 g, 79.4 mmol) was dissolved in anhydrous pyridine and anhydrous toluene, and benzoyl chloride was added. The mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was dissolved in 320 ml of 1,4-dioxane, and aqueous potassium carbonate solution (180 ml) was added. The mixture was stirred at room temperature overnight. Extracted with dichloromethane, the organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was slurried with dichloromethane, the solid was filtered, and vacuum dried to give the product 3-benzoyl-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-2). LCMS (ESI): m / z=231[M+1] + .

[0170] 1 H NMR (500MHz, DMSO-d6), δ11.33(s,1H),7.95(d,J=9.5Hz,2H),7.77(t,J=7.5Hz,1H),7.64-7.58(m,2H),7.52(s,1H),1.82(s,3H).

[0171] Step 9b: Preparation of (R)-tert-butyl((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)diphenylsilane (Compound 9-4): Under nitrogen, tert-butyldiphenylchlorosilane was added to a mixture of (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (5.0 g, 38 mmol), 4-dimethylaminopyridine (370 mg, 3.0 mmol), and triethylamine in dichloromethane. The mixture was stirred at room temperature overnight. The mixture was washed sequentially with sodium bicarbonate solution and water, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography to provide (R)-tert-butyl((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)diphenylsilane (Compound 9-4).

[0172] Step 9c: (R)-3-((tert-Butyldiphenylsilyl)oxy)propane-1,2-diol (Compound 9-5) To a solution of (R)-tert-butyl((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)diphenylsilane (Compound 9-4) (12 g, 32 mmol) in dichloromethane was added a methanolic solution of concentrated hydrochloric acid, and the mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to provide (R)-3-((tert-Butyldiphenylsilyl)oxy)propane-1,2-diol (Compound 9-5).

[0173] 1 H NMR (500MHz, DMSO-d6), δ7.70-7.62(m,4H),7.50-7.39(m,6H),4.61(d,J=4.5Hz,1H),4.43(t,J=5.6 Hz,1H),3.66-3.54(m,3H),3.49(dt,J=10.2,4.8Hz,1H),3.39(dt,J=10.6,4.9Hz,1H),1.00(s,9H).

[0174] Step 9d: Preparation of (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol (Compound 9-6): To a mixture of (R)-3-((tert-butyldiphenylsilyl)oxy)propane-1,2-diol (Compound 9-5) (3.0 g, 9.1 mmol) and 4-dimethylaminopyridine (77 mg, 0.63 mmol) in anhydrous pyridine (40 ml) was added 4,4 , -bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol (Compound 9-6) was obtained by adding 1-bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol to the mixture. The mixture was stirred at room temperature for 3 hours, and then 10 ml of methanol was added and stirred for 0.5 hours. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol (Compound 9-6).

[0175] 1 H NMR(500MHz,DMSO-d6),δ7.65-7.49(m,4H),7.49-7.34(m,8H),7.31-7.24(m,6H),7.21(dd,J=5.8,3.8Hz,1H),6.88-6.81(m,4H) ,3.81(d,J=5.4Hz,1H),3.72(s,7H),3.63(d,J=5.6Hz,2H),3.13(dd,J=9.1,4.8Hz,1H),3.00(dd,J=9.1,5.7Hz,1H),0.88(s,9H).

[0176] Step 9e: Preparation of (R)-3-benzoyl-1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-7): Under nitrogen protection, to a mixture of 3-benzoyl-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-2) (800 mg, 3.47 mmol), (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol (Compound 9-6) (1.98 g, 3.13 mmol) and triphenylphosphine (911 mg, 3.47 mmol) in anhydrous tetrahydrofuran was added diisopropyl azodicarboxylate, and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give (R)-3-benzoyl-1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)prop-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-7).

[0177] 1 H NMR (500MHz, Chloroform-d), δ7.94-7.83 (m, 3H), 7.62 (dt, J = 8.0, 1.5Hz, 6H), 7.53 (s,2H),7.49-7.39(m,11H),7.38-7.32(m,3H),7.29(dd,J=8.5,2.7Hz,4H),7.18(d,J=2.1Hz,5H),6.85-6.82(m,5H),4.98(h ,J=5.4,4.6Hz,2H),3.89(d,J=4.8Hz,1H),3.80(s,7H),3.78-3.71(m,1H),3.55-3.44(m,1H),1.97-1.93(m,3H),1.09(s,9H).

[0178] Step 9f: Preparation of (S)-3-benzoyl-1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-8): To a solution of (R)-3-benzoyl-1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-7) (1.4 g, 1.63 mmol) in tetrahydrofuran (14 mL) was added tetrabutylammonium fluoride. The mixture was stirred at room temperature for 0.5 h. Water was added and the mixture was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give (S)-3-benzoyl-1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-8) as a white foamy solid product. LCMS (ESI): m / z=605[M-1]-

[0179] 1 H NMR(500MHz,Chloroform-d),δ7.93-7.87(m,2H),7.62(t,J=7.5Hz,1H),7.43(t,J=7.7Hz,2H),7.37(d,J=7.3Hz,2H),7.34(s,1H),7.32-7.27( m,4H),7.26(s,4H),6.84(d,J=8.4Hz,4H),4.73-4.65(m,1H),4.04-3.8 7(m,2H),3.80(s,6H),3.53(ddd,J=36.4,10.4,5.6Hz,2H),1.92(s,3H).

[0180] Step 9g: Preparation of (R)-2-((R)-1-benzoyl-5-methyl-2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphoramidite (Compound 9): Under nitrogen protection, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added to a mixture of (S)-3-benzoyl-1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (Compound 9-8) (0.55 g, 0.91 mmol) and N,N-diisopropylethylamine (585 mg, 4.54 mmol) in tetrahydrofuran, and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give (R)-2-((R)-1-benzoyl-5-methyl-2,6-dioxo-1,2,3,6-tetrahydropyridin-3-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphoramidite (Compound 9) as a white foamy solid. LCMS (ESI): m / z = 669 [M-137] and m / z = 722 [M-84]. - . 1 H NMR(500MHz,Chloroform-d),δ7.94-7.86(m,2H),7.62(t,J=7.4Hz,1H),7.48-7.41(m,2H),7.38(d d,J=7.5,3.5Hz,2H),7.34-7.27(m,4H),7.23(d,J=7.1Hz,1H),7.16(d,J=5.7Hz,1H),6.83(dt,J=8 .8,2.0Hz,4H),5.00-4.87(m,1H),3.98-3.82(m,2H),3.79(s,6H),3.77-3.65(m,2H),3.58-3.44(m ,4H),2.54(dt,J=10.0,6.2Hz,2H),1.89(d,J=5.1Hz,3H),1.20-1.13(m,6H),1.09(d,J=6.8Hz,6H).

[0181] Example 10: Preparation of (R)-2-(6-benzamido-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphoramidite (Compound 10)

[0182] To a mixture of N-(9H-purin-6-yl)benzamide (Compound 10-1) (2.08 g, 1.0 equiv), (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol (Compound 9-6) (5.5 g, 1.0 equiv) and triphenylphosphine (4.6 g, 2.0 equiv) in dioxane was added diethyl azodimethylformate, and the mixture was sonicated for half an hour. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (R)-N-(9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-9H-purin-6-yl)benzamide (Compound 10-2). LCMS (ESI): m / z = 855 [M+1] + .

[0183] To a solution of (R)-N-(9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-9H-purin-6-yl)benzamide (Compound 10-2) (2.6 g, 1.0 eq) in dichloromethane (210 mL) was added tetrabutylammonium fluoride (12 mL, 4.0 eq, 1 mol / L tetrahydrofuran solution), and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, dissolved in dichloromethane, washed with saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by silica gel column chromatography to afford (S)-N-(9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-9H-purin-6-yl)benzamide (Compound 10-3) as an off-white foamy solid. LCMS (ESI): m / z=616[M+1] + . 1 H NMR (500MHz, Chloroform-d) δ, 9.12 (s, 1H), 8.68 (s, 1H), 8.13 (s, 1H), 8.07-8.00 (m, 2H), 7.62 (t, J = 7.4Hz, 1H), 7.53 (t, J=7.6Hz,2H),7.23-7.07(m,9H),6.72(dd,J=11.5,8.6Hz,4H),4.72-4.57(m,2H),4.22-4.07(m,2H),3.81-3.65(m,8H).

[0184] Under nitrogen, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite was added to a mixture of (S)-N-(9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-9H-purin-6-yl)benzamide (Compound 10-3) (1.3 g, 1.0 equiv) and N,N-diisopropylethylamine (1.36 g, 5.0 equiv) in dichloromethane (13 ml). The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give (R)-2-(6-benzamido-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)propyl(2-cyanoethyl)diisopropylphosphoramidite (Compound 10) as an off-white foamy solid. LCMS (ESI): m / z = 733 [M-83]. - .

[0185] 1 H NMR (500MHz, Chloroform-d) δ, 9.06 (s, 1H), 8.75 (d, J = 4.0Hz, 1H), 8.09 (dt, J = 43.0, 5.5Hz, 3H), 7.76-7.49 (m, 3H ),7.26(s,3H),7.23-7.13(m,6H),6.76(dd,J=8.3,4.2Hz,4H),5.17-4.96(m,1H),4.25(ddt,J=23.2,12.2,6.1Hz, 1H),4.08(ddd,J=38.7,10.9,5.6Hz,1H),3.77(d,J=4.6Hz,6H),3.74-3.70(m,1H),3.65-3.56(m,2H),3.46(dq,J =11.7,6.0Hz,2H),2.61(d,J=17.1Hz,1H),2.50(dq,J=17.4,5.9Hz,2H),1.26(d,J=4.5Hz,5H),1.16-1.07(m,7H).

[0186] Example 11: Preparation of (S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(4-isobutyramido-2-oxopyrimidin-1(2H)-yl)propyl(2-cyanoethyl)diisopropylphosphoramidite (Compound 11)

[0187] To a solution of cytosine (Compound 11-1) (2.0 g, 1.0 eq) in pyridine (100 mL) was added isobutyric anhydride (6.0 mL, 2.0 eq). The mixture was heated to 115°C and stirred for 2 hours. After cooling to room temperature, it was concentrated under reduced pressure. Methanol was added and stirred at room temperature for 15 minutes. The solid was filtered and dried under vacuum to give N-(2-oxo-1,2-dihydropyrimidin-4-yl)isobutyramide (Compound 11-2) as an off-white solid. LCMS (ESI): m / z = 182 [M+1] + .

[0188] 1 H NMR (500MHz, DMSO-d6) δ, 11.45 (s, 1H), 10.69 (s, 1H), 7.79 (d, J = 7.0Hz, 1H), 7.13 (d, J = 7.1Hz, 1H), 2.71 (hept, J = 6.8Hz, 1H), 1.06 (d, J = 6.9Hz, 6H).

[0189] Under nitrogen protection and in an ice bath, diisopropyl azodicarboxylate was added to a mixture of N-(2-oxo-1,2-dihydropyrimidin-4-yl)isobutyramide (compound 11-2) (2.0 g, 1.0 equiv), (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol (compound 9-6) (6.9 g, 1.0 equiv) and triphenylphosphine (3.74 g, 1.3 equiv) in dioxane, and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain (S)-N-(1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)isobutyramide (Compound 11-3) as an off-white foamy solid. LCMS (ESI): m / z = 794 [M-83] - .

[0190] 1H NMR(500MHz,Chloroform-d)δ8.34(d,J=5.5Hz,1H),7.81-7.71(m,2H),7.65-7.57( m,4H),7.45-7.36(m,4H),7.31-7.29(m,5H),7.25-7.14(m,5H),6.86-6.81(m,2H), 6.80-6.72(m,4H),5.43(t,J=5.2Hz,1H),3.99(qd,J=10.6,5.4Hz,2H),3.76(d,J=2.4H z,6H),3.53-3.41(m,2H),2.55(dq,J=13.9,6.9Hz,1H),1.27-1.24(m,6H),0.94(s,9H).

[0191] Tetrabutylammonium fluoride was added to a solution of (S)-N-(1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)isobutyramide (Compound 11-3) (3.6 g, 1.0 equivalent) in tetrahydrofuran (215 ml). The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was dissolved in dichloromethane, washed with saturated aqueous NaHCO₃, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain (R)-N-(1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)isobutyramide (Compound 11-4) as an off-white foamy solid. LCMS (ESI): m / z=556[M-1] - .

[0192] 1 H NMR(500MHz,Chloroform-d)δ8.42-8.31(m,1H),8.14(s,1H),7.92-7.80(m,1H),7.47-7.38(m,1H),7.32-7.25(m,6H),7.17(d,J=8.8Hz,3H),6.91-6. 71(m,4H),5.31-5.13(m,1H),4.01-3.87(m,3H),3.79(d,J=11.4Hz,6H),3. 41(ddd,J=30.1,9.8,5.3Hz,1H),2.59-2.54(m,1H),1.25(d,J=7.0Hz,6H).

[0193] Under nitrogen, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added to a mixture of (R)-N-(1-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)isobutyramide (Compound 11-4) (1.6 g, 1.0 equiv) and N,N-diisopropylethylamine (1.82 g, 5.0 equiv) in tetrahydrofuran. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain (S)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(4-isobutyramido-2-oxopyrimidin-1(2H)-yl)propyl(2-cyanoethyl)diisopropylphosphoramidite (Compound 11) as a white foamy solid. LCMS (ESI): m / z=620 [M-137] - .

[0194] 1 H NMR(500MHz,Chloroform-d)δ8.36(d,J=5.8Hz,1H),7.95(d,J=5.6Hz,1H),7.78(d,J=5.8Hz,1H),7.40(dd,J=8.1,3.9Hz, 2H),7.29(dt,J=9.1,2.9Hz,4H),7.22(d,J=5.1Hz,3H),7.17(dt,J=8.1,4.2Hz,1H),6.78(dt,J=9.0,4.6Hz,4H),5.49(h,J =5.5Hz,1H),4.08-3.97(m,1H),3.88(dq,J=13.0,6.9Hz,1H),3.76(d,J=6.7Hz,6H),3.71(p,J=6.3Hz,2H),3.52(dq,J=10 .7, 6.8Hz, 2H), 3.40 (dq, J=11.3, 6.2, 5.4Hz, 2H), 2.54 (tq, J=13.1, 6.8Hz, 3H), 1.25 (d, J=6.9Hz, 6H), 1.16-1.07 (m, 12H).

[0195] Example 12: Preparation of 9-(2S)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)propan-2-yl)-2-isobutyrylamino-9H-purin-6-yldiphenylcarbamate (Compound 12)

[0196] To a solution of N-(6-oxo-6,9-dihydro-3H-purin-2-yl)isobutyramide (Compound 12-1) (2.0 g, 1.0 eq) in N,N-dimethylformamide (10 ml) was added acetic anhydride (2.22 ml, 2.6 eq). The mixture was heated to 100°C and stirred for 2 hours. After cooling to room temperature, the solid was filtered, washed with a small amount of ethanol, and dried in vacuo to afford N-(9-acetyl-6-oxo-6,9-dihydro-3H-purin-2-yl)isobutyramide (Compound 12-2) as an off-white solid.

[0197] 1 H NMR (500MHz, DMSO-d6) δ, 12.26 (s, 1H), 11.66 (s, 1H), 8.44 (s, 1H), 2.83 (s, 4H), 1.14 (d, J = 6.8Hz, 6H).

[0198] To a mixture of N-(9-acetyl-6-oxo-6,9-dihydro-3H-purin-2-yl)isobutyramide (Compound 12-2) (1.0 g, 1.0 equiv) and N,N-diisopropylethylamine (0.98 g, 2.0 equiv) in pyridine was added diphenylcarbamoyl chloride (1.2 equiv), and the mixture was stirred at room temperature overnight. Ethanol (10 ml) was added to the mixture, and after stirring at room temperature for 10 minutes, the solvent was removed by concentration under reduced pressure. Ethanol and water were added, and the mixture was heated to 100°C and stirred for 2 hours. After cooling to room temperature, the solid was filtered and dried under vacuum to obtain the off-white solid product, 2-isobutyramide-9H-purin-6-yldiphenylcarbamate (Compound 12-3). LCMS (ESI): m / z = 417 [M+1] + .

[0199] 1 H NMR (500MHz, DMSO-d6) δ13.51(s,1H),10.53(s,1H),8.40(s,1H),7.46(dt,J=15. 3,7.7Hz,8H),7.32(t,J=7.3Hz,2H),2.80(p,J=6.9Hz,1H),1.09(d,J=6.8Hz,6H).

[0200] Under nitrogen, diisopropyl azodicarboxylate was added to a mixture of 2-isobutyramide-9H-purin-6-yldiphenylcarbamate (Compound 12-3) (1.45 g, 1.1 equiv), (R)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-ol (Compound 9-6) (2.0 g, 1.0 equiv), and triphenylphosphine (1.66 g, 2.0 equiv) in dioxane. The mixture was heated to 65°C and stirred overnight. The mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give an off-white foamy solid product (S)-9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-2-isobutyramido-9H-purin-6-yldiphenylcarbamate (Compound 12-4).

[0201] 1 H NMR(500MHz,Chloroform-d)δ7.95(s,1H),7.76(s,1H),7.72-7.63(m,2H),7.55(td,J=7.3,1.5Hz,1H),7.51-7.40(m,9H),7.39-7.3 2(m,6H),7.22(d,J=2.8Hz,2H),7.20-7.14(m,3H),7.10(dd,J=10.3,8.7Hz,4H),6.76-6.66(m,4H),4.78(q,J=5.6Hz,1H),4.13-4.03 (m,2H),3.73(d,J=4.0Hz,6H),3.71-3.67(m,1H),3.56(dd,J=9.9,4.5Hz,1H),3.07(s,1H),1.19(dd,J=6.8,1.8Hz,6H),0.91(s,9H).

[0202] Tetrabutylammonium fluoride was added to a solution of (S)-9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldiphenylsilyl)oxy)propan-2-yl)-2-isobutyramido-9H-purin-6-yldiphenylcarbamate (Compound 12-4) (2.36 g, 1.0 eq) in tetrahydrofuran, and the mixture was stirred at room temperature for 20 minutes. Dichloromethane was added, the mixture was washed with a saturated aqueous solution of NaHCO₃, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give (R)-9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-2-isopyramido-9H-purin-6-yldiphenylcarbamate (Compound 12-5) as an off-white foamy solid. LCMS (ESI): m / z = 793 [M+1]. + .

[0203] 1 H NMR(500MHz,Chloroform-d)δ8.01(d,J=7.0Hz,2H),7.45(d,J=7.8Hz,4H),7.37(d,J= 7.7Hz,4H),7.26-7.19(m,4H),7.13(dt,J=23.9,8.2Hz,5H),7.05(d,J=8.5Hz,2H),6. 67(dd,J=16.7,8.5Hz,4H),5.78(s,1H),4.38(dd,J=9.0,4.4Hz,1H),4.17-4.10(m,2H ),3.82-3.72(m,2H),3.70(d,J=8.6Hz,6H),2.56(p,J=6.9Hz,1H),1.28-1.23(m,6H).

[0204] Under nitrogen, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite was added to a dichloromethane solution of (R)-9-(1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypropyl-2-yl)-2-isopyramido-9H-purin-6-yldiphenylcarbamate (Compound 12-5) (1.42 g, 1.0 equiv) and N,N-diisopropylethylamine (1.16 g, 5.0 equiv), and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 9-(2S)-1-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)propan-2-yl)-2-isobutyrylamino-9H-purin-6-yldiphenylcarbamate (Compound 12) as an off-white foamy solid. LCMS (ESI): m / z=661[M-331] - .

[0205] 1H NMR(500MHz,Chloroform-d)δ8.06(d,J=8.6Hz,1H),7.87(d,J=7.5Hz,1H),7.44(d,J=7.9Hz,4H),7.35(t,J=7.7Hz,4H),7.27( s,2H),7.25-7.19(m,4H),7.13(td,J=8.9,6.3Hz,5H),6.73(ddd,J=8.4,5.4,2.4Hz,4H),4.91(t,J=5.9Hz,1H),4.26-4.15(m,1 H),4.05-3.85(m,1H),3.74(d,J=2.6Hz,6H),3.61(s,2H),3.60-3.51(m,2H),3.49-3.38(m,2H),3.07(s,1H),2.51(t,J=6.3Hz ,1H),2.45(td,J=6.2,3.4Hz,1H),1.26-1.23(m,6H),1.10(dd,J=6.8,4.6Hz,6H),1.03(d,J=6.8Hz,3H),0.98(d,J=6.8Hz,3H).

[0206] Example 13 Synthesis of siRNA

[0207] Oligonucleotides were synthesized using a solid-phase oligonucleotide synthesis protocol on a 12-channel nucleic acid synthesizer from Beijing Qingke Biotechnology Co., Ltd. 0.2-1 μmol of oligonucleotides were synthesized. siRNA sequences containing a GalNAc ligand were synthesized on a prepacked CPG column coupled to GalNAc. Aminolysis reagent was added to the synthesized oligonucleotide and incubated at 45-80°C to separate the oligonucleotide from the solid support and free the oligonucleotide. The crude oligonucleotide was then precipitated with ethanol, and the supernatant discarded. This process was repeated twice to obtain the crude oligonucleotide, which was then resuspended in DEPC-treated water. The crude oligonucleotide was purified by ion-pairing HPLC, and the collected product was dried to a powder in a vacuum centrifuge. The purified product was dissolved in DEPC-treated water and analyzed by time-of-flight LC-MS. The oligonucleotide concentration was determined, and the required volumes of equimolar amounts of the sense and antisense strands were calculated. The equimolar amounts of the sense and antisense strands were mixed and annealed to form a duplex by heating at 95°C for 5 minutes and then cooling to room temperature.

[0208] The sequences of modified siRNA duplexes synthesized by this method are shown in Tables 1 to 4. All duplexes contain a GalNAc ligand for liver-targeted delivery.

[0209] Table 1 shows the modified sequences of siRNA targeting TMPRSS6, and the corresponding unmodified sequences are as follows:

[0210] Table 2 shows the modified sequences of siRNA targeting CIDEB, and the corresponding unmodified sequences are as follows:

[0211] Table 3 shows the modified sequences of siRNA targeting ANGPTL3, and the corresponding unmodified sequences are as follows:

[0212] Table 4 shows the modified sequences of siRNA targeting PCSK9, and the corresponding unmodified sequences are as follows:

[0213] Table 1 Modified sequences of siRNA targeting TMPRSS6

[0214] Table 2 Modified sequences of siRNA targeting CIDEB

[0215] Table 3 Modified sequences of siRNA targeting ANGPTL3

[0216] Table 4 Modified sequences of siRNA targeting PCSK9

[0217] Example 14 Testing the in vitro activity of siRNA in Hep3B cells using liposome transfection

[0218] Cell culture and 96-well plate transfection: In vitro experiments were performed in Hep3B cells using MEM (Basal Media, REF. L130KJ) + 10% FBS + 1X penicillin-streptomycin (Gibco, Cat. 15070-063) + 1X non-essential amino acids (cell cook, Cat. CM1008L). Cells were trypsinized when they reached 80% confluence, and cell density was determined using a Scepter automated cell counter (Millipore, #PHCC00000). Simultaneously, 2 μL of siRNA was mixed with 68 μL of Opti-MEM and 5 μL of INTERFERin (Polyplus transfection, REF. 409-10) in a 96-well plate and incubated at room temperature for 10 minutes. Then, 125 μL of Hep3B complete cell culture medium (containing 7,000 cells) was added to each well, and the 96-well plate was incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0219] 96-well plate RNA extraction and reverse transcription: Dynabeads mRNA DIRECT kit (Ambion) was used to extract mRNA from 96-well plate cells. The culture medium in the 96-well plate was aspirated, washed once with DPBS, and 100 μL cell lysis buffer was added to each well, followed by 20 μL Beads were shaken on an oscillator, and the 96-well plate was placed on a magnetic separation rack. The lysate in the wells was aspirated, and 100 μL of washing buffer A was added to each well. After blowing, the plate was placed on a magnetic separation rack, and the washing buffer A was aspirated. The beads were then blown up with washing buffer B and transferred to a new 96-well plate. The plate was placed on a magnetic separation rack, and the washing buffer B was aspirated. The beads were then blown up with buffer B and transferred to a 96-well PCR plate. At the same time, the reversal reagent was prepared, and the 96-well PCR plate was placed on a magnetic separation rack. The washing buffer B was aspirated, and 20 μL of reversal reagent was added to each well. The plate was sealed with a sealing film and incubated at 25°C for 10 minutes on a PCR instrument. After incubation for two hours at 37°C, the plate was heated at 85°C for 5 minutes, and then cooled to 4°C. The reverse transcription was completed.

[0220] Real-time fluorescence quantitative PCR: After reverse transcription, place the 96-well plate on a magnetic separation rack until the beads are adsorbed to the bottom. Aspirate the reverse transcription reagent, add the prepared QPCR system to the 96-well PCR plate, seal the plate with a sealing film, and perform PCR on a StepOnePlus real-time PCR system (applied biosystems).

[0221] Data were analyzed using the ΔΔCt method and normalized to cells transfected with 1 nM of a negative control sequence.

[0222] The sequence of the negative control AD-1955 is: CUUACGCUGAGUACUUCGAdTdT;

[0223] UCGAAGUACUCAGCGUAAGdTdT.

[0224] The primers and probes for each human gene are shown in Tables 5 and 6.

[0225] Table 5 Primers for human genes

[0226] Table 6 Probes for human genes

[0227] Table 7 Activity results of 0.1 nM siRNA delivered by liposomes in Hep3B cells

[0228] From the results in Table 7, it can be seen that in Hep3B cells, by introducing VPU-S into the 2090 sequence, the activity of the sequence can be significantly improved, and the improvement effect is better than that of VPU.

[0229] Example 15 Testing the in vitro activity of siRNA using liposome transfection or GalNAc delivery in wild-type mice, AAV8-hTMPRSS6, AAV8-hCIDEB, humanized ANGPTL3, or humanized PCSK9 mice

[0230] First, the hTMPRSS6 (human TMPRSS6) gene, hCIDEB (human CIDEB) gene, or Hangptl3 (human ANGPTL3) gene was integrated into a liver-targeted AAV8 expression vector. The virus (VectorBuilder, AAV8LP) was then used to infect mice. After 14 days, transgenic mice stably expressing hANGPTL3, hTMPRSS6, or hCIDEB were obtained. Humanized ANGPTL3 or humanized PCSK9 mice were purchased from Shanghai Model Organisms.

[0231] Mouse primary hepatocyte extraction: Mouse hepatocytes were extracted by collagenase digestion via inferior vena cava perfusion. Viable primary mouse hepatocytes were filtered through a 70 μm tissue cell strainer (BIOLOGIX, 15-1070) and resuspended in DMEM (Basal Media, REF. L110KJ) supplemented with 10% FBS and 1X penicillin-streptomycin (Gibco, Cat. 15070-063). Cell density was determined using a Scepter automated cell counter (Millipore, #PHCC00000). See Example 14 for cell plating and sample testing methods.

[0232] Table 8 Results of the GalNAc-delivery activity test of 1 nM siRNA in AAV8-hTMPRSS6 mouse primary hepatocytes

[0233] The experimental results in Table 8 show that in AAV8-hTMPRSS6 mouse primary hepatocytes, compared with sequences without VPU, the activity of the sequence can be significantly improved by introducing VPU-S, VPU-6 or VPU-7 into the 2090 sequence, and the improvement effect is better than that of VPU.

[0234] Table 9 Results of the GalNAc-delivery activity test of 0.1 nM siRNA in AAV8-hTMPRSS6 mouse primary hepatocytes

[0235] The experimental results in Table 9 show that in AAV8-hTMPRSS6 mouse primary hepatocytes, the activity of the sequence can be significantly improved by introducing VPU-S, VPU-6 or VPU-7 into the 2051 sequence, and the improvement effect is better than that of VPU.

[0236] Table 10 Results of the GalNAc-delivery activity experiment of 1 nM siRNA in AAV8-hCIDEB mouse primary hepatocytes

[0237] Table 11 Activity results of 0.1 nM siRNA delivered by liposomes in AAV8-hCIDEB mouse primary hepatocytes

[0238] The experimental results in Tables 10 and 11 show that in AAV8-hCIDEB mouse primary hepatocytes, the introduction of VPU-9, VPU-10, VPU-11 and VPU-S into the W4005 sequence can make the sequence activity better than the sequence introducing VPU.

[0239] Table 12 Results of the GalNAc-delivery activity assay for 1 nM siRNA in humanized ANGPTL3 mouse primary hepatocytes

[0240] The experimental results in Table 12 show that in humanized ANGPTL3 mouse primary hepatocytes, the introduction of VPU-S and VPU-12 into the 7061 sequence can improve the sequence activity compared to the sequence introducing VPU.

[0241] Table 13 Results of the GalNAc-delivery activity assay for 1 nM siRNA in AAV8-hANGPTL3 mouse primary hepatocytes

[0242] Table 14 Results of the GalNAc-mediated delivery of 1 nM siRNA in AAV8-hANGPTL3 primary mouse hepatocytes

[0243] The experimental results in Tables 13 and 14 show that in AAV8-hANGPTL3 primary mouse hepatocytes, the introduction of VPU-S, VPU-9, VPU-10, and VPU-11 into the 7061 sequence can improve the sequence activity compared to the sequence introducing VPU. Among them, VPU-10 has the best effect in improving the activity of 7061, while VPU-8 has no effect on the activity of the 7061 sequence.

[0244] Table 15 Results of the GalNAc-delivery activity experiment of 1 nM siRNA in AAV8-hANGPTL3 mouse primary hepatocytes

[0245] The experimental results in Table 15 show that in AAV8-hANGPTL3 mouse primary hepatocytes, the activity of the sequence can be improved by introducing VPU-10 into the 7079 sequence. However, relatively speaking, the improvement of the activity of the 7079 sequence by VPU is not as obvious as that by VPU-10.

[0246] Table 16 Results of the GalNAc-delivery activity assay for 1 nM siRNA in humanized PCSK9 mouse primary hepatocytes

[0247] The experimental results in Table 16 show that in humanized PCSK9 mouse primary hepatocytes, the improvement of sequence activity by introducing VPU-S and VPU-12 into the 11040 sequence is more obvious than that of VPU.

[0248] Example 16 Evaluation of the Effects of Different Modifications 11040 on Human PCSK9 Expression in Serum in AAV8-hPCSK9 Mice

[0249] 1. Adenovirus-integrated hPCSK9

[0250] By 1x10 11 Transgenic mice stably expressing hPCSK9 were generated by infecting mice with ultrapurified recombinant AAV8 viral particles [Vector Builder]. 100 μl of virus was added to 3.9 mL of DPBS, and 200 μl of the diluted virus solution was injected intravenously into each mouse.

[0251] 2. Experimental Grouping and Medication

[0252] Fourteen days after virus injection, mice were bled by tail removal, allowed to stand at room temperature for 30 minutes, and centrifuged at 1000×g for 10 minutes. The serum supernatant was aliquoted and stored frozen at -80°C. Serum samples were diluted 1000-fold, and hPCSK9 expression in mouse serum was assessed using a PCSK9 double-antibody ELISA kit (Proteintech, Cat. No. KE00278). Based on hPCSK9 expression, mice were divided into groups of four. siRNA was dissolved in PBS at a concentration of 0.2 mg / kg, and mice were subcutaneously injected with 1 mg / kg of the siRNA solution.

[0253] 3. ELISA test

[0254] After the siRNA injection, as shown in Figure 1 , a small amount of blood was collected by tail cutting and allowed to stand at room temperature for 30 min. The supernatant serum was collected and diluted 1000-fold before hPCSK9 expression was detected by ELISA.

[0255] The results, as shown in Figure 1, show that introducing VPU-S and VPU-12 significantly boosts the activity of the 11040 sequence compared to introducing VPU into the 11040 sequence. VPU-S significantly boosts the activity of the 11040 sequence compared to VPU-12, while VPU-8, while also improving the activity of the 11040 sequence, is less effective than VPU.

[0256] Example 17 Evaluation of the Effects of Differently Modified W4005 on hCIDEB Expression in the Liver in AAV8-hCIDEB Mice

[0257] 1. Adenoviral integration of hCIDEB

[0258] By 1.5×10 11 Purified recombinant AAV8 viral particles were injected into 6-8 week old female mice (C57BL / 6) to obtain transgenic mice stably expressing hCIDEB.

[0259] 2. Medication

[0260] Fourteen days after virus injection, mice were divided into groups of 4. siRNA was dissolved in saline and injected subcutaneously at a dose of 1 mg / kg.

[0261] 3. Liver sampling and testing

[0262] Fifty-seven days after siRNA injection, liver tissue was harvested and RNA was extracted using the TRI REAGENT (MRC, Catalog No. TR118). The extracted RNA was reverse-transcribed into cDNA using the PrimeScript RT-PCR Kit (Takara, Catalog No. RR047A). The prepared QPCR system was added to a 96-well PCR plate, sealed with a film, and QPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) to measure hCIDEB expression.

[0263] As shown in Figure 2, compared with the introduction of VPU into the W4005 sequence, the introduction of VPU-S and VPU-12 can more significantly enhance the activity of the W4005 sequence, while the activity of VPU-8 in vivo is comparable to that of VPU.

[0264] In summary, the present invention found that the introduction of VPUS, VPU-6, VPU-7, VPU-9, VPU-10, VPU-11 and VPU-12 at the 5' end of the antisense strand of siRNA has a better effect on improving siRNA activity than VPU, while the activity of VPU-8 is comparable to that of VPU both in vivo and in vitro.

[0265] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0266] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A modified nucleotide compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, in: R 1 and R 2 Each is independently selected from: H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkylmethyl; R 3 Selected from: -OR 5 ; R 4 Selected from: -N(R 6 )(R 7 ); R 5 Selected from: H, C1-C6 alkyl, C1-C6 alkyl substituted by cyano, C1-C6 alkyl substituted by halogen, C1-C6 alkyl substituted by hydroxy, C1-C6 alkyl substituted by C1-C6 alkoxy, C1-C6 alkyl substituted by amino, C1-C6 alkyl substituted by C1-C6 alkylamino; R 6 and R 7 Each is independently selected from: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkylmethyl; X is selected from: -C(R 8 )(R 9 )-, -S-; R 8 and R 9 Each is independently selected from: H, C1-C6 alkyl, halogen; G is selected from: H, halogen, cyano, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkylmethyl, C1-C 10 Alkoxy, halogen-substituted C1-C 10 Alkyl, hydroxy substituted C1-C 10 Alkyl, C1-C6 alkoxy substituted C1-C 10 Alkyl, amino substituted C1-C 10 Alkyl, C1-C6 alkylamino substituted C1-C 10 Alkyl, cyano substituted C1-C 10 alkyl; Bx is a heterocyclic base.

2. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 1 and R 2 Each of the following is independently selected from the group consisting of: H, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C6 cycloalkyl, and C3-C6 cycloalkylmethyl.

3. The modified nucleotide compound according to claim 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 1 and R 2 Each is independently selected from: H, methyl, ethyl, n-propyl, isopropyl.

4. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 5 Selected from: H, C1-C3 alkyl, C1-C3 alkyl substituted by cyano, C1-C3 alkyl substituted by halogen, C1-C3 alkyl substituted by hydroxy, C1-C3 alkyl substituted by C1-C3 alkoxy, C1-C3 alkyl substituted by amino, C1-C3 alkyl substituted by C1-C3 alkylamino.

5. The modified nucleotide compound according to claim 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 5 Selected from: H, methyl, ethyl, n-propyl, isopropyl, methyl substituted with cyano, ethyl substituted with cyano, n-propyl substituted with cyano.

6. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 6 and R 7 Each of the following is independently selected from the group consisting of: C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C3-C6 cycloalkyl, and C3-C6 cycloalkylmethyl.

7. The modified nucleotide compound according to claim 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: R 6 and R 7 Each of the following is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.

8. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: X is selected from: -S-, -CH2-, -C(H)(F)-, -CF2-.

9. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: G is selected from the group consisting of: H, halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C3-C6 cycloalkylmethyl, C1-C6 alkoxy, C1-C6 alkyl substituted with halogen, C1-C6 alkyl substituted with hydroxy, C1-C6 alkyl substituted with C1-C6 alkoxy, C1-C6 alkyl substituted with amino, C1-C6 alkyl substituted with C1-C6 alkylamino, and C1-C6 alkyl substituted with cyano.

10. The modified nucleotide compound according to claim 9, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: G is selected from the group consisting of: H, halogen, cyano, C1-C3 alkyl, C1-C3 alkyl substituted with halogen, C2-C3 alkenyl, C2-C3 alkynyl, C1-C3 alkoxy, and C1-C3 alkyl substituted with C1-C3 alkoxy.

11. The modified nucleotide compound according to claim 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: G is selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, vinyl, propenyl, methoxy, ethoxy, methoxymethyl, methoxyethyl, cyano, fluorine, and chlorine.

12. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: X is -S-; G is selected from: C1-C3 alkyl.

13. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: X is -CH2-; G is selected from the group consisting of: C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C1-C3 alkoxy-substituted C1-C2 alkyl, fluorine-substituted methyl, cyano, fluorine, and chlorine.

14. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: X and G together form -XG selected from the group consisting of methylthio, ethylthio, propylthio, methyl, ethyl, propyl, butyl, allyl, methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl, cyano-substituted methyl, cyano-substituted ethyl, monofluoromethyl, difluoromethyl, monofluoroethyl, and difluoroethyl.

15. The modified nucleotide compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: Bx is selected from the group consisting of adenine, guanine, cytosine, uracil, thymine, methyladenine, 1-methylguanine, isoguanine, 7-methylguanine, methylcytosine, isocytosine, pseudouracil, 5-hydroxymethylcytosine, hypoxanthine, Q-substituted adenine, Q-substituted guanine, Q-substituted cytosine, Q-substituted uracil, Q-substituted thymine; wherein Q is selected from the group consisting of H, benzoyl, and benzyloxymethyl.

16. The modified nucleotide compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The modified nucleotide compound is selected from: Wherein: Q is selected from: H, benzoyl, benzyloxymethyl.

17. An oligonucleotide, characterized in that At least one 5'-terminal nucleotide monomer is connected to the oligonucleotide by a modified nucleotide compound according to any one of claims 1 to 16 through a linking group; the structural formula of the oligonucleotide is as follows: Where: R 1 , R 2 , Bx, X, and G as described in any one of claims 1-16; A is the nucleotide chain in the oligonucleotide excluding the 5'-terminal nucleotide monomer; T is a linking group that connects the modified nucleotide compound to the internucleoside of the oligonucleotide.

18. The oligonucleotide according to claim 17, characterized in that T is selected from the group consisting of: phosphodiester bond, phosphorothioate bond.

19. The oligonucleotide according to claim 17 or 18, characterized in that The oligonucleotide is selected from: siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA, antimir, supermir, antagomir, ribozyme, triplex-forming oligonucleotide, decoy oligonucleotide, splicing switch oligonucleotide, immunostimulatory oligonucleotide, RNA activator, U1 adapter, or one or more combinations of CRISPR Cas.

20. The oligonucleotide according to claim 19, characterized in that The oligonucleotide is siRNA.

21. The oligonucleotide according to claim 20, characterized in that The modified nucleotide compound is linked to the 5'-end of the antisense strand of the siRNA via a T linker.

22. Use of the modified nucleotide compound according to any one of claims 1 to 16 in the preparation of an oligonucleotide for inhibiting or activating gene expression.

23. The use according to claim 22, characterized in that The oligonucleotide is selected from: siRNA, shRNA, antisense oligonucleotide, microRNA, anti-microRNA, antimir, supermir, antagomir, ribozyme, triplex-forming oligonucleotide, decoy oligonucleotide, splicing switch oligonucleotide, immunostimulatory oligonucleotide, RNA activator, U1 adapter, or one or more combinations of CRISPR Cas.

24. The use according to claim 23, characterized in that The oligonucleotide is siRNA.

25. The use according to claim 24, characterized in that The modified nucleotide compound is linked to the 5'-end of the antisense strand of the siRNA via a covalent linking group.

26. The use according to claim 25, characterized in that The covalent linking group is a phosphodiester bond or a phosphorothioate bond.

27. Use of the modified nucleotide compound according to any one of claims 1 to 16, or the oligonucleotide according to any one of claims 17 to 21, in the preparation of a drug for inhibiting or activating gene expression.

28. A pharmaceutical preparation for inhibiting or activating gene expression, characterized in that: The composition is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients comprise the oligonucleotide according to any one of claims 17 to 21.

29. A method for inhibiting or activating gene expression, characterized in that: The method comprises the following steps: administering a safe and effective amount of the oligonucleotide according to any one of claims 17 to 21 and / or the pharmaceutical preparation according to claim 28.

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