WO2020103929A1 - Oligonucleotide and prodrug thereof - Google Patents

Abstract

Disclosed is an oligonucleotide and a prodrug thereof, and in particular, disclosed are: a compound shown by formula I, a tautomer thereof, a stereoisomer or a pharmaceutically acceptable salt thereof, a preparation method, a pharmaceutical composition and a medical use, comprising a use for treating a hepatitis B viral infection, especially hepatic diseases caused by a hepatitis B viral infection.

Description

Oligonucleotides and prodrugs

Cross-reference of related applications

This application requires the priority and rights of the Chinese Patent Application No. 201811408257.5 filed with the State Intellectual Property Office of China on November 23, 2018. The disclosure of the application is incorporated herein by reference in its entirety.

Technical field

The present application relates to oligonucleotides and prodrugs, methods for their preparation, pharmaceutical compositions containing the compounds, and their use in the treatment of hepatitis B virus (HBV) infection and liver diseases related to HBV.

Background technique

Chronic hepatitis B is currently incurable and can only be controlled. Two anti-HBV drugs that have been clinically approved include interferon alpha and nucleoside analogs, but there is usually a rapid emergence of drug resistance and dose-related toxicity of treatment problem. In addition, many current drugs suffer from poor absorption, distribution, metabolism, and / or excretion (ADME) properties, preventing their wider application. Poor ADME properties are also a major cause of failure of drug candidates in clinical trials.

Existing oligonucleotide drugs such as SB9000 and its prodrug SB9200 have both safety and antiviral effects in untreated chronic hepatitis B patients without cirrhosis, but there is still a need to develop better anti-HBV activity , Safety, prodrug release activity rate and bioavailability, higher stability and certain liver targeting compounds.

Summary of the invention

In one aspect, the present application relates to compounds of formula I, tautomers, stereoisomers or pharmaceutically acceptable salts thereof,

R ^{1 is} selected from

or

Wherein R is selected from the following groups: C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, C _6-10 Aryl or 5-10 membered heteroaryl, the R group is optionally substituted with one or more of the following groups: halogen, -OH, C _1-6 alkyl, C _1-6 alkoxy,- NO ₂ , -CN, -NH ₂ , -NH (C _1-6 alkyl), -N (C _1-6 alkyl) ₂ , -C (O) C _1-6 alkyl, -C (O) OC _1-6 alkyl, -CONHC _1-6 alkyl, -SH or -SC _1-6 alkyl;

R ² and R ³ are independently selected from H, -OH, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _6-10 aryl or 5-10 membered heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _{6 -10} aryl or 5-10 membered heteroaryl is optionally substituted with one or more substituents selected from halogen, -OH, -NO ₂ , -NH ₂ or C _1-6 alkoxy;

R ⁴ and R ⁵ are independently selected from H, -OH, -NH ₂ or -SH;

n is selected from 0, 1, 2, 3 or 4;

X is selected from -C (R ^a R ^b )-or -C (= CH ₂ )-;

Wherein R ^a and R ^b are independently selected from H, halogen, C _1-6 alkoxy or C _1-6 alkyl, the C _1-6 alkoxy or C _1-6 alkyl is optionally It is substituted by one or more substituents selected from C _1-6 alkoxy, halogen, -OH, -NH ₂ or -NO ₂ .

In another aspect, the present application relates to a pharmaceutical composition comprising the compound of formula I of the present application, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof.

In another aspect, the present application relates to a method of treating or inhibiting HBV infection in a mammal, including administering to the mammal in need of such treatment a therapeutically effective amount of a compound of formula I, a tautomer, a stereoisomer, or a pharmaceutically acceptable Accepted salt or the above pharmaceutical composition.

In another aspect, the present application relates to the use of compounds of formula I, their tautomers, stereoisomers or pharmaceutically acceptable salts thereof or the above-mentioned pharmaceutical compositions in the preparation of a medicament for preventing or treating HBV infection in mammals.

In another aspect, the present application relates to the use of compounds of formula I, their tautomers, stereoisomers or pharmaceutically acceptable salts thereof or the above-mentioned pharmaceutical compositions in the prevention or treatment of mammalian HBV infection.

In another aspect, the present application relates to a compound of Formula I, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described above for use in preventing or treating HBV infection in a mammal.

Detailed description of the invention

This application relates to compounds of formula I, their tautomers, stereoisomers or pharmaceutically acceptable salts thereof,

R ^{1 is} selected from

or

Wherein R is selected from the following groups: C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, C _6-10 Aryl or 5-10 membered heteroaryl, the R group is optionally substituted with one or more groups selected from halogen, -OH, C _1-6 alkyl, C _1-6 alkoxy Group, -NO ₂ , -CN, -NH ₂ , -NH (C _1-6 alkyl), -N (C _1-6 alkyl) ₂ , -C (O) C _1-6 alkyl, -C (O) OC _1-6 alkyl, -CONHC _1-6 alkyl, -SH or -SC _1-6 alkyl;

R ² and R ³ are independently selected from H, -OH, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _6-10 aryl or 5-10 membered heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _{6 -10} aryl or 5-10 membered heteroaryl is optionally substituted with one or more substituents selected from halogen, -OH, -NO ₂ , -NH ₂ or C _1-6 alkoxy;

R ⁴ and R ⁵ are independently selected from H, -OH, -NH ₂ or -SH;

n is selected from 0, 1, 2, 3 or 4;

X is selected from -C (R ^a R ^b )-or -C (= CH ₂ )-;

Wherein R ^a and R ^b are independently selected from H, halogen, C _1-6 alkoxy or C _1-6 alkyl, the C _1-6 alkoxy or C _1-6 alkyl is optionally It is substituted by one or more substituents selected from C _1-6 alkoxy, halogen, -OH, -NH ₂ or -NO ₂ .

In some embodiments,

versus

Combine to form

among them

It is a monovalent cation. In some embodiments,

It is Na ⁺ or NH ₄ ⁺ .

In some embodiments, R is selected from the following groups: C _1-6 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl , Phenyl, or 5-6 membered heteroaryl; in some embodiments, R is selected from C _1-4 alkyl; in some embodiments, R is isopropyl, methyl, or isobutyl.

In some embodiments, R ² and R ³ are each independently selected from H, —OH, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 member Heterocycloalkyl, C _6-10 aryl or 5-10 membered heteroaryl, the C _1-6 alkyl or C _1-6 alkoxy group is optionally selected from one or more of halogen, -OH , -NO ₂ , -NH ₂ or C _1-6 alkoxy substituents; in some embodiments, R ² and R ³ are each independently selected from H, halogen, C _1-6 alkyl or optional C _1-6 alkoxy substituted by C _1-6 alkoxy; in some embodiments, R ² and R ³ are each independently selected from H, F, CH _3- , CH ₃ O- or CH ₃ OCH ₂ CH ₂ O-; in some embodiments, R ² is H or F, and R ³ is CH _3- , CH ₃ O-, or CH ₃ OCH ₂ CH ₂ O-.

In some embodiments, R ⁴ and R ⁵ are each independently selected from H or -OH; in some embodiments, R ⁴ is H and R ⁵ is -OH.

In some embodiments, n is selected from 0, 1, or 2. In some embodiments, n is 1.

In some embodiments, R ^a and R ^b are each independently selected from H, halogen, C _1-3 alkoxy, or C _1-3 alkyl; in some embodiments, R ^a and R ^b are each independently H or halogen.

In some embodiments, X is -C (= CH ₂ )-.

In some embodiments, the pharmaceutically acceptable salt is selected from sodium or ammonium salts.

In some embodiments, the compound of formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof is selected from the compound of formula II, its tautomer, stereoisomer or Its pharmaceutically acceptable salts:

Wherein R ¹ , R ² or R ³ are as defined in the compound of formula I.

In some embodiments, the pharmaceutically acceptable salts of the compounds of Formula I of the present application are selected from sodium salts or ammonium salts.

In some embodiments, the compound of formula I of the present application, its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof is selected from:

In some embodiments, the compound of formula I of the present application, its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof is selected from:

In another aspect, the present application relates to a pharmaceutical composition comprising the compound of formula I of the present application, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition of the present application further comprises pharmaceutically acceptable excipients.

In another aspect, the present application relates to a method of treating or inhibiting HBV infection in mammals, including administering to a mammal in need of such treatment, preferably a human, a therapeutically effective amount of a compound of formula I, its tautomer, stereoisomer or A pharmaceutically acceptable salt, or the above pharmaceutical composition.

On the other hand, the present application relates to the use of the compound of formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing or treating HBV infection in mammals .

On the other hand, the present application relates to the use of the compound of formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition for preventing or treating HBV infection in mammals.

In another aspect, the present application relates to a compound of formula I for preventing or treating HBV infection in a mammal, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.

In some embodiments of the present application, the treatment of HBV infection refers to the control, reduction or elimination of HBV to alleviate or cure liver disease in infected mammals or patients.

definition

Unless otherwise stated, the following terms used in this application have the following meanings. A specific term should not be considered uncertain or unclear without a specific definition, but should be understood in accordance with the ordinary meaning in the art. When a trade name appears in this article, it is intended to refer to its corresponding trade product or its active ingredient.

The term "substituted" means that any one or more hydrogen atoms on a specific group are substituted with a substituent, as long as the valence state of the specific group is normal and the compound after substitution is stable.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes the occurrence or non-occurrence of the event or circumstance. For example, ethyl is "optionally" substituted with halogen, meaning that ethyl can be unsubstituted (CH ₂ CH ₃ ), mono-substituted (such as CH ₂ CH ₂ F), poly-substituted (such as CHFCH ₂ F, CH ₂ CHF ₂ etc.) or completely substituted (CF ₂ CF ₃ ). Those skilled in the art will understand that for any group that contains one or more substituents, it will not introduce any substitution or substitution pattern that is not possible in space and / or cannot be synthesized.

In this paper, C _mn is an integer number of carbon atoms in the given range. For example, "C _1-6 " means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent. So, for example, if a group is replaced by 2 Rs, then each R has an independent option.

The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "alkyl" refers to _a hydrocarbon group of the formula C _n H _{2n +.} The alkyl group may be linear or branched. For example, the term "C ₁ - ₆ alkyl" refers to (e.g., methyl, ethyl, n-propyl, isopropyl, alkyl containing 1 to 6 carbon atoms, n-butyl, isobutyl, sec-butyl, Tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.). Similarly, the alkyl portion (ie, alkyl) of alkoxy, alkylamino, dialkylamino, alkylsulfonyl, and alkylthio groups has the same definition as above.

The term "alkoxy" refers to -O-alkyl.

The term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like.

The term "alkynyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH ₃ ), 2-propynyl (-CH ₂ -C≡CH), 1,3-butadiynyl (-C≡CC≡CH) etc.

The term "cycloalkyl" refers to a carbocycle that is fully saturated and can exist in the form of a monocyclic, bridged or spiro ring. Unless otherwise indicated, the carbocyclic ring is usually a 3 to 10 membered ring or a 3 to 6 membered ring. Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamant Alkyl and so on.

The term "heterocycloalkyl" refers to a cyclic group that is fully saturated and can exist in the form of a monocyclic, bridged ring, or spiro ring, containing 1 to 4 heteroatoms selected from N, O, and S. Unless otherwise indicated, the heterocyclic ring is usually a 3 to 7 membered ring or 3 to 10 membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and / or nitrogen . Examples of 3-membered heterocyclic alkyl groups include, but are not limited to, ethylene oxide, epithioethyl, and cyclic azetyl groups, and non-limiting examples of 4-membered heterocyclic alkyl groups include, but are not limited to, azetidinyl, oxin Examples of cyclic groups, thibutane, 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidine Examples of groups, imidazolidinyl, tetrahydropyrazolyl, and 6-membered heterocycloalkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, Examples of 4-thioxanyl, 1,4-dioxane, thiomorpholinyl, 1,3-dithioalkyl, 1,4-dithioalkyl, and 7-membered heterocycloalkyl include However, it is not limited to azacycloheptyl, oxepanyl, and thioheptanyl. It is preferably a monocyclic heterocycloalkyl group having 5 or 6 ring atoms.

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. For example, the aryl group may have 6-20 carbon atoms, 6-14 carbon atoms, 6-12 carbon atoms, or 6-10 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, 1,2,3,4-tetrahydronaphthalene, and the like.

The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system, which contains at least one (eg 1, 2, or 1 to 3) heteroatoms selected from N, O, and S as ring atoms, and the remaining rings The atom is C and has at least one aromatic ring. The heteroaryl group may be a 5- to 10-membered ring containing 1 to 3 heteroatoms independently selected from N, O, and S. Preferred heteroaryl groups have a single 5- to 8-membered ring, or multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl , Tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.

The term "preventing" means preventing the occurrence of a disease or disease state in a mammal, especially when such mammals are susceptible to the disease state, but have not been diagnosed with the disease state.

The term "treatment" means administration of a compound or formulation described in this application to ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) Inhibiting a disease or disease state, that is, curbing its development;

(i) Relieve the disease or disease state even if the disease or disease state subsides.

The term "therapeutically effective amount" means (i) treatment or prevention of a specific disease, condition or disorder, (ii) reduction, improvement or elimination of one or more symptoms of a specific disease, condition or disorder, or (iii) prevention or delay The amount of a compound of the present application where one or more symptoms of a specific disease, condition or disorder described herein are onset. The amount of the compound of the present application constituting the "therapeutically effective amount" depends on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art It is determined by its own knowledge and this disclosure.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms, which are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues without Multiple toxicity, irritation, allergic reactions or other problems or complications are commensurate with a reasonable benefit / risk ratio.

Examples of pharmaceutically acceptable salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or salts thereof and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to an organism.

The term "pharmaceutically acceptable excipients" refers to those excipients that have no significant stimulating effect on the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The words "comprise" or "comprise" and their English variants such as comprises or comprising should be understood in an open, non-exclusive sense, ie "including but not limited to".

The compounds and intermediates of the present application may also exist in different tautomeric forms, and all such forms are included within the scope of the present application. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can be interconverted via a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) include tautomerization via proton migration, such as keto-enol and imine-enamine isomerization. A specific example of proton tautomers is an imidazole moiety, where protons can migrate between two ring nitrogens. Valence tautomers include interconversion through the recombination of some bonding electrons.

The compounds of the present application may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of the present application can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from the racemic mixture or synthesized by using chiral raw materials or chiral reagents.

The compounds of the present application may exist in specific geometric isomers or stereoisomeric forms. This application contemplates all such compounds, including tautomers, cis and trans isomers, (-)-and (+)-enantiomers, (R)-and (S) -enantiomers , Diastereomers, (D) -isomers, (L) -isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, All of these are within the scope of this application. Additional asymmetric carbon atoms may be present in the substituents such as alkyl. All these isomers and their mixtures are included within the scope of this application.

The pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous formulations, such as tablets, pills, capsules, powders , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

Typical routes for administering the compounds of the present application or pharmaceutically acceptable salts or pharmaceutical compositions thereof include, but are not limited to oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal Intramuscular, subcutaneous and intravenous administration.

The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method, and the like.

In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compound of the present application to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc. for oral administration to patients.

The solid oral composition can be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by mixing the active compound with solid auxiliary materials, optionally grinding the resulting mixture, adding other suitable auxiliary materials if necessary, and then processing the mixture into granules to obtain tablets Or the core of sugar coating. Suitable excipients include but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

The pharmaceutical composition may also be suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form.

In all methods of administration of the compounds of general formula I described herein, the daily dose is 0.01 to 200 mg / kg body weight, either in separate or divided doses.

The compounds of the present application can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalently, preferred embodiments include but are not limited to the examples of the present application.

The chemical reaction of the specific embodiment of the present application is completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present application and the reagents and materials required. In order to obtain the compound of the present application, it is sometimes necessary for a person skilled in the art to modify or select a synthesis step or a reaction scheme based on existing embodiments.

An important consideration in the planning of synthetic routes in the field is the selection of suitable protecting groups for reactive functional groups (such as the hydroxyl group in this application). For example, refer to Greene's Protective Groups Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this application are incorporated into this application as a whole.

In some embodiments, the compound of the general formula II of the present application can be prepared by a person skilled in the art of organic synthesis through the following route:

Route 1:

This application uses the following acronyms:

Bz stands for benzoyl; DMTr stands for 4,4'-dimethoxytriphenylmethyl; Me stands for methyl; TBDPS stands for tert-butyldiphenylsilyl; DMSO stands for dimethyl sulfoxide.

For the sake of clarity, the present invention is further illustrated with examples, but the examples do not limit the scope of the present application. All reagents used in this application are commercially available and can be used without further purification.

Specific implementation plan

Example 1: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl)- 2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) ammonium thiophosphate

Step 1: N- (9-((1S, 3R, 4S) -4-hydroxy-3-hydroxymethyl-2-methylenecyclopentyl) -6-oxo-6,9-dihydro-1H -Purin-2-yl) benzamide

2-amino-9-((1S, 3R, 4S) -4-hydroxy-3-hydroxymethyl-2-methylenecyclopentyl) -1,9-dihydro-6H-purin-6-one (5.0g) was mixed with pyridine (80ml). The temperature was lowered in an ice bath, trimethylchlorosilane (14.69g) was added dropwise thereto, and the reaction was continued at room temperature for 3h after the drop. The temperature was lowered in an ice bath, and benzoyl chloride (4.31 g) was slowly added dropwise. After the addition, the reaction was allowed to proceed overnight. The reaction solution was cooled to 0 ° C in an ice bath, and water (50 ml) was slowly added, and stirred at 0 ° C for 30 min. The ice bath was removed, and ammonia water (85 g) was added to the reaction solution, and stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure, and the resulting concentrate was added with water (50 ml) to beat. After filtration, the filter cake was washed successively with water and ether, and dried to obtain the title compound (5.8 g). MS (ESI) m / z: 380.3 [MH] ^- .

Step 2: (2R, 3R, 4R, 5R) -2-((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2,4-dioxo- 3,4-dihydropyrimidine-1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

Add 1-((2R, 3R, 4R, 5R) -5-((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4-hydroxy-3- to the reaction flask Methoxytetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -dione (1.0 g), after replacing with nitrogen, dichloromethane (20 ml) was added. A methylene chloride solution (10ml) of ((bis (diisopropylamino) phosphino) oxy) propionitrile (0.64g) and trifluoroacetic acid pyridinium salt (0.41g) was added dropwise to the reaction solution and reacted at room temperature 3h. The reaction solution was concentrated, and the crude product was purified by column chromatography (petroleum ether: ethyl acetate = 2: 1) to obtain the title compound (1.2 g). MS (ESI) m / z: 783.4 [M + Na] ⁺ .

Step 3: ((1R, 3S, 5S) -3- (2-Benzamido-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Methylcyclopentyl) methyl ((2R, 3R, 4R, 5R) -2-((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2, 4-dioxo-3,4-dihydropyrimidine-1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl) (2-cyanoethyl) phosphite

N- (9-((1S, 3R, 4S) -4-hydroxy-3-hydroxymethyl-2-methylenecyclopentyl) -6-oxo-6,9-dihydro-1H-purine -2-yl) benzamide (1.263g) was dissolved in a mixed solvent of anhydrous acetonitrile (10ml) and anhydrous N, N-dimethylformamide (5ml), and anhydrous acetonitrile (10ml × 3) was added in total Boil 3 times. Under the protection of nitrogen, (2R, 3R, 4R, 5R) -2-((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2,4-di Oxy-3,4-dihydropyrimidin-1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (2.4g) Aqueous acetonitrile (10ml) solution and 0.45M1H-tetrazole (0.4g) in acetonitrile solution (12.69ml) were added dropwise to the reaction solution and reacted at room temperature for 2h. The reaction solution was concentrated, and the crude product was purified by column chromatography (dichloromethane: methanol = 95: 5) to obtain the title compound (1.41 g).

Step 4: O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy- 2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl) O- (2-cyanoethyl) sulfur Phosphoric acid ester

In the reaction flask, place ((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2 -Methylenecyclopentyl) methyl ((2R, 3R, 4R, 5R) -2-((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- ( 2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl) (2-cyanoethyl) phosphite (0.90g) Dissolved in anhydrous acetonitrile (5ml). Under the protection of nitrogen, a solution of 3H-benzo [c] [1,2] dithiol-3-one 1,1-dioxide (0.177g) in anhydrous acetonitrile (3ml) was dropped into the reaction solution at room temperature Reaction 1h. The reaction solution was concentrated, and the crude product was purified by column chromatography (dichloromethane: methanol = 90: 10) to obtain the title compound (0.7 g).

Step 5: O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy- 2-methylenecyclopentyl) methyl) O- (2-cyanoethyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-di Hydropyrimidine-1 (2H) -yl) -2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) phosphorothioate

O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5 -(2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl) O- (2-cyanoethyl) thiophosphoric acid The ester (0.7) was dissolved in dichloromethane (8 ml), a solution of dichloroacetic acid (0.129 g) in dichloromethane (1 ml) was added dropwise to the reaction, and reacted at room temperature for 4 h. The temperature was lowered in an ice bath, and triethylamine was added dropwise to the reaction to adjust the pH to neutral. The reaction solution was concentrated, and the crude product was purified by column chromatography (dichloromethane: methanol = 90:10) to obtain the title compound (0.25 g). MS (ESI) m / z: 771.5 [M + H] ⁺ .

Step 6: O-(((1R, 3S, 5S) -3- (2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Methylcyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2 -Hydroxymethyl-4-methoxytetrahydrofuran-3-yl) ammonium thiophosphate

Add O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5 to the reaction bottle in sequence -Hydroxy-2-methylenecyclopentyl) methyl) O- (2-cyanoethyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3, 4-dihydropyrimidine-1 (2H) -yl) -2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) phosphorothioate (210mg) and ammonia (28%) (1ml) at room temperature React for 5 days. A 50% acetic acid solution was added dropwise to the reaction solution under an ice bath to adjust the pH to about 8. The reaction solution was extracted twice with ethyl acetate, and the aqueous phase was separated. The aqueous phase was separated and purified using a Biotage C18 30g reverse-phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (120 mg). ¹ H NMR (500 MHz, DMSO-d ₆ ): δ 11.35 (s, 1H), 10.52 (s, 1H), 7.92 (dd, J = 5.0 Hz, 8.0 Hz, 1H), 7.71 (d, J = 3.0 Hz, 1H), 7.18 (brs, 4H), 6.43 (s, 2H), 5.88 (d, J = 6.0Hz, 1H), 5.69 (d, J = 8.5Hz, 1H), 5.37 (m, 1H), 5.26 (d, J = 5.0 Hz, 1H), 5.13 (s, 1H), 4.93 (s, 1H), 4.77 (m, 1H), 4.57 (d, J = 7.0 Hz, 1H), 4.29 (s, 1H ), 4.22 (d, J = 2.5 Hz, 1H), 4.07 (d, J = 9.5 Hz, 1H), 3.90 (t, J = 6.0 Hz, 2H), 3.65 (s, 2H), 3.38 (s, 3H ), 2.69 (s, 1H), 2.37 (m, 1H), 2.04 (m, 1H). MS (ESI) m / z: 614.5 [M + H] ⁺ .

Example 2: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5-hydroxy-2-methylene ring Amyl) methyl) O-((2R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -yl) -2-hydroxymethyl- 4-methoxytetrahydrofuran-3-yl) (S) -sodium thiophosphate

Step 1: (2S, 3aS, 6R, 7aS) -3a-methyl-2-((perfluorophenyl) thio) -6- (prop-1-en-2-yl) hexahydrobenzo [d ] [1,3,2] oxathiophosphorane 2-sulfide

In the reaction flask, sequentially put triethylamine bis (perfluorophenyl) phosphorodithioate (126g), (1R, 4R, 6S) -1-methyl-4- (prop-1-en-2-yl ) -7-oxabicyclo [4.1.0] heptane (48.3g), dibutyl hydrogen phosphate (48.9g) was added to chloroform (1200ml), then 2,2-dichloroacetic acid (60.0g) Slowly added dropwise to the above solution, the reaction at room temperature for 1h. After the reaction solution was concentrated to 500 ml, 1000 ml of n-hexane was added to the reaction solution for dilution, and then washed with 10% potassium dihydrogen phosphate solution and saturated saline, respectively. The organic phase was concentrated to 200 ml, 200 ml of methanol was added, and after further concentration to 50 ml, 200 ml of methanol was added, and then concentrated to 50 ml, and a solid precipitated. After cooling to 0 ° C in an ice bath, it was filtered. The crude product was 93.48g. The crude product (93.48g) was dissolved in 225ml of dichloromethane, and then 374ml of n-heptane was added, and the crude product was completely dissolved. The solution was concentrated to 200 ml, and then 374 ml of n-heptane was added, and this operation was repeated 3 times. After the solution was concentrated to 200 ml, a large amount of solid precipitated, and the mixture was stirred at room temperature for 2 h. Filter and dry the filter cake to give the title compound (62.58g). ¹ H NMR (500MHz, CDCl ₃ ): δ5.04 (s, 1H), 4.87 (s, 1H), 4.30-4.280 (m, 1H), 2.63 (s, 1H), 2.37-2.34 (m, 1H) , 2.07-1.85 (m, 4H), 1.81 (s, 3H), 1.79-1.73 (m, 1H), 1.68 (s, 3H)

Step 2: 1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3-methoxy-4-(((2R, 3aS, 6R, 7aS) -3a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [d] [1,3,2] oxathiophosphorus heterocycle Pentane-2-oxy) oxy) tetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H))-dione

1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -4-hydroxy-3-methoxytetrahydrofuran-2-yl) Pyrimidine-2,4 (1H, 3H) -dione (6.44g) and (2S, 3aS, 6R, 7aS) -3a-methyl-2-((perfluorophenyl) thio) -6- (propylene -1-en-2-yl) hexahydrobenzo [d] [1,3,2] oxathiophosphorane 2-sulfide (7.5g) was dissolved in acetonitrile (156ml), and cooled to 0 ℃. 2,3,4,6,7,8,9,10-octahydropyrimido [1,2-a] aza (2.57g) was added, and the reaction was stirred at 0 ° C for 5h. The reaction solution was diluted with ethyl acetate and washed successively with purified water and 10% potassium dihydrogen phosphate solution. The organic phase was dried over anhydrous sodium sulfate, and after filtration, the organic phase was concentrated to obtain a crude product. Purification by column chromatography (petroleum ether: ethyl acetate = 1: 1) gave the title compound (4.53 g). ¹ H-NMR (500 MHz, CDCl ₃ ): δ 8.72 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.69-7.64 (m, 4H), 7.48-7.40 (m, 6H), 6.11 (d, J = 5.0Hz, 1H), 5.42-5.37 (m, 2H), 5.05 (s, 1H), 4.91 (s, 1H), 4.59-4.55 (m, 1H), 4.27 (d, J = 4.5Hz, 1H), 4.04-4.01 (m, 1H), 3.97 (t, J = 5.0Hz, 1H), 3.89-3.86 (m, 1H), 3.52 (s, 3H), 2.59 (s, 1H), 2.28 (d, J = 14.0Hz, 1H), 2.18-2.12 (m, 1H), 1.98-1.90 (m, 3H), 1.78 (m, 4H), 1.71 (s, 3H), 1.11 (s, 9H) .

Step 3: O-(((1R, 3S, 5S) -3- (2-benzoyl-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-((tert Butyldiphenylsilyl) oxy) -2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-(((tert-butyldiphenylsilane Yl) oxy) methyl) -5- (2,4-dioxo-3,4-dihydropyrimidine-1) (2H) -yl) -4-methoxytetrahydrofuran-3-yl) O- Hydrogen (S)-phosphorothioate

To the reaction flask, add 1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3-methoxy-4- ( ((2R, 3aS, 6R, 7aS) -3a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [d] [1,3,2] oxysulfur Heterophosphorane-2-oxy) oxy) tetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H))-dione (4.5g), N- (9-((1S, 3R , 4S) -4-((tert-butyldiphenylsilyl) oxy) -3- (hydroxymethyl) -2-methylenecyclopentyl) -6-oxo-6,9-dihydro -1H-purinepyridin-2-yl) benzamide (7.51g), then add anhydrous acetonitrile, azeotropically remove water, repeat twice. Acetonitrile (52ml) was added, and 2,3,4,6,7,8,9,10-octahydropyrimido [1,2-a] aza (2.77g) was slowly added dropwise at room temperature. After reaction for 3h, ethyl acetate was added for dilution. The organic phase was washed with saturated sodium bicarbonate solution and saturated brine in this order, and the organic layer was dried over anhydrous sodium sulfate. The crude product was purified by column chromatography (dichloromethane: methanol = 10: 1) to obtain the title compound (5.24 g). ¹ H NMR (500 MHz, DMSO-d ₆ ): δ 12.53 (s, 1H), 12.40 (s, 1H), 11.42 (d, J = 2.0 Hz, 1H), 8.23-8.14 (m, 2H), 7.68 -7.53 (m, 10H), 7.51-7.34 (m, 15H), 5.88 (d, J = 6.0 Hz, 1H), 5.59 (t, J = 10.0 Hz, 1H), 5.25 (d, J = 8.0 Hz, 1H), 5.12 (s, 1H), 4.96 (m, 1H), 4.71 (s, 1H), 4.67 (s, 1H), 4.20 (d, J = 9.0Hz, 1H), 4.12 (d, J = 3.5 Hz, 1H), 3.87-3.77 (m, 1H), 3.76 (s, 2H), 3.57-3.52 (m, 1H), 3.47 (t, J = 6.0Hz, 1H), 3.26 (s, 3H), 2.05 -1.96 (m, 1H), 1.91 (m, 1H), 1.03 (s, 9H), 1.01 (s, 9H).

Step 4: O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy- 2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-hydroxymethyl-5- (2,4-dioxo-3,4-dihydropyrimidine- 1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate

To the reaction flask, sequentially add O-(((1R, 3S, 5S) -3- (2-benzoyl-6-oxo-1,6-dihydro-9H-purin-9-yl) -5 -((Tert-butyldiphenylsilyl) oxy) -2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-(((tert-butyl di Phenylsilyl) oxy) methyl) -5- (2,4-dioxo-3,4-dihydropyrimidine-1 (2H))-(4-methoxytetrahydrofuran-3-yl) O-hydrogen (S) -thiophosphoric acid ester (5.2g), tetrabutylammonium fluoride trihydrate (3.43g) and tetrahydrofuran (35ml), the reaction was stirred at room temperature for 40h, water (100ml) was added to the reaction solution, It was then extracted twice with ether (50 ml). The aqueous phase was concentrated to obtain the title compound 7.09 g, which was directly used in the next step without treatment.

Step 5: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylcyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2 -Hydroxymethyl-4-methoxytetrahydrofuran-3-yl) (S) -Tetrabutylammonium thiophosphate

Add O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5 to the reaction bottle in sequence -Hydroxy-2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-hydroxymethyl-5- (2,4-dioxo-3,4-di Hydropyrimidine-1 (2H) -yl) -4-methoxytetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (7g) and ammonia water (50ml), the mixture was heated to 50 ° C and stirred for 68h . A 50% acetic acid aqueous solution was added dropwise to the reaction solution under an ice bath to adjust the pH to about 8. The reaction solution was extracted twice with ethyl acetate, and the aqueous phase was separated. The aqueous phase was separated and purified using a Biotage C18 120g reverse-phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (3.39g). MS (ESI) m / z: 614.5 [M + H] ⁺ .

Step 6: O-(((1R, 3S, 5S) -3- (2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Methylcyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2 -Hydroxymethyl-4-methoxytetrahydrofuran-3-yl) (S) -Sodium thiophosphate

To the reaction flask, sequentially add O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxyl -2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidine-1 (2H)- Yl) -2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (0.2g), sodium ion exchange resin (4g) and deionized water (4ml) , Stir at room temperature for 1h. Filtered, Biotage C18 120g aqueous phase was purified reverse-phase column separation (water: acetonitrile = 3: 1) to give the title compound ^{(80mg) 1 H NMR (500MHz} , DMSO-d 6): δ10.69 (br, 1H) , 7.91 (dd, J = 8.5, 2.5 Hz, 1H), 7.72-7.09 (m, 1H), 6.45 (s, 2H), 5.86 (d, J = 5.7 Hz, 1H), 5.71-5.60 (m, 1H) ), 5.36-5.27 (m, 2H), 5.11 (s, 1H), 4.94 (d, J = 3.0 Hz, 1H), 4.76 (d, J = 11.0 Hz, 1H), 4.55 (s, 1H), 4.28 (s, 1H), 4.21 (s, 1H), 4.01 (q, J = 9.0Hz, 1H), 3.95-3.79 (m, 2H), 3.65 (s, 2H), 3.38 (s, 3H), 2.70 ( s, 1H), 2.34 (s, 1H), 2.06-1.98 (m, 2H). MS (ESI) m / z: 614.5 [M + H] ⁺ .

Example 3: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl)- 2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -sodium phosphorothioate

Step 1: 1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy)- 4-(((2R, 3aS, 6R, 7aS) -3a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [d] [1,3,2 ] Oxathiophosphoran-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2,4- (1H, 3H) -dione

1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -4-hydroxy-3- (2-methoxyethoxy ) Tetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -dione (35g) and (2S, 3aS, 6R, 7aS) -3a-methyl-2-((perfluorophenyl) thio ) -6- (prop-1-en-2-yl) hexahydrobenzo [d] [1,3,2] oxaphosphorane 2-sulfide (37.6g) dissolved in acetonitrile (645ml ), Cooling in an ice bath. Slowly add 2,3,4,6,7,8,9,10-octahydropyrimido [1,2-a] azazepine (12.81g) to the above reaction solution slowly. The reaction was continued for 2h under ice bath conditions. The reaction solution was washed successively with purified water and 10% potassium dihydrogen phosphate solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1: 1) to obtain the title compound (49.23g). ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.92 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.70-7.63 (m, 4H), 7.48-7.38 (m, 6H), 6.12 ( d, J = 5.5Hz, 1H), 5.41-5.45 (m, 1H), 5.38 (dd, J = 8.3, 1.9Hz, 1H), 5.05 (s, 1H), 4.92 (s, 1H), 4.55-4.59 (m, 1H), 4.32-4.22 (m, 2H), 3.98 (dd, J = 11.9, 2.0 Hz, 1H), 3.90-3.79 (m, 3H), 3.54 (t, J = 4.5 Hz, 2H), 3.30 (s, 3H), 2.59 (s, 1H), 2.33-2.24 (m, 1H), 2.14 (td, J = 13.4, 4.2Hz, 1H), 2.00-1.88 (m, 3H), 1.79 (s, 3H), 1.71 (d, J = 4.9Hz, 3H), 1.11 (s, 9H), 0.91-0.82 (m, 1H).

Step 2: O-(((1R, 3S, 5S) -3- (2-benzoyl-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-((tert Butyldiphenylsilyl) oxy) -2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-(((tert-butyldiphenylsilane Yl) oxy) methyl) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran- 3-yl) O-hydrogen (S) -phosphorothioate

1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- ( ((2R, 3aS, 6R, 7aS) -3a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [d] [1,3,2] oxysulfur Heterophosphorane-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -dione (1.5g) and N- (9-((1S, 3R, 4S ) -4-((tert-butyldiphenylsilyl) oxy) -3-hydroxymethyl-2-methylenecyclopentyl) -6-oxo-6,9-dihydro-1H-purine Pyridin-2-yl) benzamide (2.36g) was dissolved in acetonitrile (20ml), and 2,3,4,6,7,8,9,10-octahydropyrimido [1,2-a] was added at room temperature Azalea (0.87g), stirred for 2h. The reaction solution was diluted with ethyl acetate, washed sequentially with saturated sodium bicarbonate solution and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was subjected to column chromatography (dichloromethane: methanol = 10: 1) Purification afforded the title compound (1.98g). ¹ H-NMR (500 MHz, DMSO-d ₆ ): δ 12.57 (s, 1H), 12.41 (s, 1H), 11.41 (s, 1H), 8.21-8.18 (m, 3H), 7.66-7.61 (m , 8H), 7.57-7.54 (m, 2H), 7.47 (t, J = 7.5Hz, 3H), 7.43-7.38 (m, 11H), 5.86 (d, J = 6.0Hz, 1H), 5.59 (t, J = 9.5 Hz, 1H), 5.22 (d, J = 8.0 Hz, 1H), 5.12 (s, 1H), 4.91-4.89 (m, 1H), 4.71 (s, 1H), 4.67 (s, 1H), 4.23 (s, 1H), 4.11 (s, 1H), 3.98 (t, J = 5.5Hz, 1H), 3.75-3.69 (m, 4H), 3.46-3.45 (m, 1H), 3.27-3.26 (m, 2H), 3.10 (s, 3H), 2.90 (s, 1H), 2.79 (s, 1H), 2.04-2.00 (m, 1H), 1.04 (s, 9H), 1.00 (s, 9H).

Step 3: O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy- 2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl ) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate

Add O-(((1R, 3S, 5S) -3- (2-benzoyl-6-oxo-1,6-dihydro-9H-purin-9-yl) -5- ((Tert-butyldiphenylsilyl) oxy) -2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-(((tert-butyldiphenyl Silyl) oxy) methyl) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy ) Tetrahydrofuran-3-yl) O-hydrogen (S) -thiophosphoric acid ester (1.80g), tetrabutylammonium fluoride trihydrate (1.38g) and tetrahydrofuran (20ml), reacted at room temperature overnight. The reaction solution was diluted with water, washed with ether, and the aqueous phase was concentrated to obtain 2.40 g of the title compound, which was directly used in the next step without purification.

Step 4: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylcyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2 -Hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate

O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl)- 2-Hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (2.40g) was dissolved in ammonia water (15ml) and reacted at room temperature for 24h. Under an ice bath, the reaction solution was adjusted to pH 8 with 50% acetic acid aqueous solution, and washed with ethyl acetate. The aqueous phase was separated, and the aqueous phase was separated and purified using a Biotage C18 120g reverse-phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.91g). MS (ESI) m / z: 656.7 [MH] ^- .

Step 5: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylcyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2 -Hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -sodium thiophosphate

O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Cyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2-hydroxyl Methyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (0.35g) was dissolved in deionized water (7ml), to which was added the sodium form Ion exchange resin (7g) was stirred at room temperature for 1h and then filtered. The aqueous phase was separated and purified using a Biotage C18 120g reverse phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.17g). ¹ H-NMR (500 MHz, DMSO-d ₆ ): δ 10.87 (brs, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 6.46 (s, 2H), 5.85 ( d, J = 5.5 Hz, 1H), 5.67 (d, J = 8.5 Hz, 1H), 5.38-5.33 (m, 1H), 5.27 (t, J = 5.0 Hz, 1H), 5.11 (s, 1H), 4.94 (s, 1H), 4.74-4.72 (m, 1H), 4.55 (s, 1H), 4.27 (s, 1H), 4.20 (s, 1H), 4.05-3.98 (m, 2H), 3.94-3.85 ( m, 2H), 3.65 (s, 2H), 3.61-3.57 (m, 1H), 3.43-3.42 (m, 2H), 3.20 (s, 3H), 2.70 (s, 1H), 2.39-2.34 (m, 1H), 2.06-2.02 (m, 1H) .MS (ESI) m / z: 656.7 [MH] ^- .

Example 4: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) 4-fluoro-2-hydroxymethyl-4-methyltetrahydrofuran-3-yl) (S) -sodium phosphorothioate

Step 1: 1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3-fluoro-4-hydroxy-3-methyltetrahydrofuran -2-yl) pyrimidine-2,4 (1H, 3H) -dione

1-((2R, 3R, 4R, 5R) -3-fluoro-4-hydroxy-5-hydroxymethyl-3-methyltetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -di Ketone (5g) and 4-dimethylaminopyridine (0.469g) were dissolved in pyridine (20ml), and tert-butyldiphenylchlorosilane (6.47g) was slowly added dropwise thereto. After stirring at room temperature for 12 hours, the reaction solution was diluted with ethyl acetate and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography (dichloromethane: methanol = 20: 1) to obtain the title compound (9.22g). ¹ H-NMR (500 MHz, DMSO-d ₆ ): δ 11.54 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 7.0 Hz, 2H), 7.63 (d, J = 6.5 Hz, 2H), 7.51-7.41 (m, 6H), 6.08 (d, J = 19.0 Hz, 1H), 5.85 (s, 1H), 5.11 (brs, 1H), 4.11-4.05 (m, 1H) ), 4.03-3.96 (m, 2H), 3.94-3.89 (m, 1H), 1.31 (d, J = 22.5 Hz, 3H), 1.01 (s, 9H).

Step 2: 1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3-fluoro-3-methyl-4-(( (2R, 3aS, 6R, 7aS) -3a-methyl-6- (prop-1-en-2-yl) -2-thiohexahydrobenzo [d] [1,3,2] oxathia Phosphalolan-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -dione

1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3-fluoro-4-hydroxy-3-methyltetrahydrofuran-2- Group) pyrimidine-2,4 (1H, 3H) -dione (0.9g) and (2S, 3aS, 6R, 7aS) -3a-methyl-2-((perfluorophenyl) thio) -6- (Prop-1-en-2-yl) hexahydrobenzo [d] [1,3,2] oxathiophosphorane 2-sulfide (1.048g) dissolved in acetonitrile (16ml), 0 ℃ Next, 2,3,4,6,7,8,9,10-octahydropyrimido [1,2-a] azazepine (0.357g) was added, and the reaction was stirred for 30 minutes. The reaction solution was diluted with ethyl acetate and washed with 10% potassium dihydrogen phosphate solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate = 1: 1) to obtain the title compound (1.26 g). ¹ H-NMR (500 MHz, CDCl ₃ ): δ 9.59 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 7.0 Hz, 2H), 7.66 (d, J = 7.0Hz, 2H), 7.47-7.37 (m, 6H), 6.32 (d, J = 18.5Hz, 1H), 5.39-5.27 (m, 1H), 5.08 (d, J = 8.0Hz, 1H), 5.01 ( s, 1H), 4.87 (s, 1H), 4.59-4.53 (m, 1H), 4.23-4.21 (m, 1H), 4.16-4.12 (m, 1H), 3.84-3.82 (m, 1H), 2.61 ( brs, 1H), 2.36-2.29 (m, 1H), 2.16-2.08 (m, 1H), 2.02-1.95 (m, 2H), 1.94-1.88 (m, 2H), 1.77 (s, 3H), 1.73 ( s, 3H), 1.52 (d, J = 22.0Hz, 3H), 1.11 (s, 9H).

Step 3: O-(((1R, 3S, 5S) -3- (2-benzoyl-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-((tert Butyldiphenylsilyl) oxy) -2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-(((tert-butyldiphenylsilane Yl) oxy) methyl) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-fluoro-4-methyltetrahydrofuran-3-yl) O-hydrogen (S) -phosphorothioate

1-((2R, 3R, 4R, 5R) -5-(((tert-butyldiphenylsilyl) oxy) methyl) -3-fluoro-3-methyl-4-(((2R, 3aS, 6R, 7aS) -3a-methyl-6- (prop-1-en-2-yl) -2-thiohexahydrobenzo [d] [1,3,2] oxathia heterocyclic Pentane-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2,4 (1H, 3H) -dione (1.2g) and N- (9-((1S, 3R, 4S) -4- ((Tert-butyldiphenylsilyl) oxy) -3- (hydroxymethyl))-2-methylenecyclopentyl) -6-oxo-6,9-dihydro-1H-purine -2-yl) benzamide (1.997g) dissolved in acetonitrile (15ml), add 2,3,4,6,7,8,9,10-octahydropyrimido [1,2-a] nitrogen at room temperature Zhazhuo (0.736g), stirring and reacting for 30 minutes. The reaction solution was diluted with ethyl acetate and washed with saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography (dichloromethane: methanol = 10: 1) to obtain the title compound (1.14g). ¹ H-NMR (500 MHz, DMSO-d ₆ ): δ 12.56 (s, 1H), 12.43 (s, 1H), 11.49 (s, 1H), 8.17 (s, 1H), 8.14 (d, J = 7.5 Hz, 2H), 7.74-7.71 (m, 3H), 7.69-7.61 (m, 6H), 7.60-7.55 (m, 1H), 7.50-7.45 (m, 2H), 7.44-7.34 (m, 12H), 6.02 (d, J = 18.5 Hz, 1H), 5.64-5.56 (m, 1H), 5.06 (s, 1H), 4.98-4.83 (m, 1H), 4.80-4.68 (m, 2H), 4.63 (s, 1H), 4.14-4.05 (m, 1H), 4.01-3.87 (m, 4H), 3.01-2.89 (m, 1H), 2.78 (s, 1H), 2.08-1.99 (m, 1H), 1.19-1.14 ( m, 3H), 1.06 (s, 9H), 1.00 (s, 9H).

Step 4: O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy- 2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl ) -4-fluoro-2-hydroxymethyl-4-methyltetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate

To the reaction flask, sequentially add O-(((1R, 3S, 5S) -3- (2-benzoyl-6-oxo-1,6-dihydro-9H-purin-9-yl) -5 -((Tert-butyldiphenylsilyl) oxy) -2-methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -2-(((tert-butyl di Phenylsilyl) oxy) methyl) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-fluoro-4-methyltetrahydrofuran- 3-yl) O-hydrogen (S) -thiophosphoric acid ester (1g), tetrabutylammonium fluoride trihydrate (0.791g) and tetrahydrofuran (9ml), stirred at room temperature overnight to react. The reaction solution was diluted with water, washed with ether, and the aqueous phase was concentrated to obtain 3 g of the title compound, which was directly used in the next step without purification.

Step 5: O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylcyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4 -Fluoro-2-hydroxymethyl-4-methyltetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate

O-(((1R, 3S, 5S) -3- (2-benzoylamino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylenecyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl)- 4-Fluoro-2-hydroxymethyl-4-methyltetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (3g) was dissolved in ammonia water (20ml) and stirred at room temperature for 17h. Under an ice bath, the reaction solution was adjusted to pH 8 with 50% acetic acid aqueous solution, and extracted with ethyl acetate. The aqueous phase was separated and purified using a Biotage C18 120g reverse-phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.5g). MS (ESI) m / z: 614.5 [MH] ^- .

Step 6: O-(((1R, 3S, 5S) -3- (2-Amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Methylcyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4 -Fluoro-2-hydroxymethyl-4-methyltetrahydrofuran-3-yl) (S) -sodium thiophosphate

O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Cyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -4-fluoro -2-hydroxymethyl-4-methyltetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (0.3g) was dissolved in deionized water (4ml), to which was added sodium ion exchange resin ( 6g), stirred at room temperature for 1h and filtered, and the aqueous phase was separated and purified using a Biotage C18 120g reverse phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.1g). ¹ H-NMR (500 MHz, D ₂ O): δ 7.84 (s, 1H), 7.82 (d, J = 8 Hz, 1H), 6.14 (d, J = 19.5 Hz, 1H), 5.85 (d, J = 8Hz, 1H), 5.39 (t, J = 8Hz, 1H), 5.32 (s, 1H), 4.87 (s, 1H), 4.68-4.62 (m, 1H), 4.49-4.44 (m, 1H), 4.19- 1.14 (m, 2H), 4.14-4.09 (m, 1H), 4.02-3.98 (m, 1H), 3.92-3.86 (m, 1H), 2.86 (s, 1H), 2.45-2.37 (m, 1H), 2.30-2.23 (m, 1H), 1.39 (d, J = 23.5 Hz, 3H). MS (ESI) m / z: 614.5 [MH] ^- .

Example 5: (((((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2- Methylenecyclopentyl) methoxy) (((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl)- 2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisopropyl carbonate

O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Cyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2-hydroxyl Methyl-4-methoxytetrahydrofuran-3-yl) ammonium thiophosphate (0.80g) was dissolved in a mixed solution of water (100ml) and isopropanol (26ml), and iodomethyl isopropyl carbonate ( 0.845g), react at room temperature for 24h. Isopropanol was removed by concentration, and the aqueous phase was extracted three times with n-hexane, and then separated and purified using a Biotage C18 120g reverse phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.536g). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.44 (s, 1H), 10.60 (s, 1H), 7.91 (t, J = 7.5 Hz, 1H), 7.69 (d, J = 14.0 Hz, 1H) , 6.38 (s, 2H), 5.94 (dd, J = 6.5, 2.0 Hz, 1H), 5.73 (d, J = 8.0 Hz, 1H), 5.53-5.51 (m, 2H), 5.40 (dt, J = 10.0 , 6.5Hz, 2H), 5.23-5.21 (m, 1H), 5.15 (dd, J = 6.0, 3.0Hz, 1H), 5.11-5.02 (m, 1H), 4.83-4.80 (m, 1H), 4.66 ( d, J = 2.5Hz, 1H), 4.30-4.21 (m, 3H), 4.15-4.13 (m, 2H), 3.77-3.58 (m, 2H), 3.38 (s, 3H), 2.85-2.84 (m, 1H), 2.34-2.32 (m, 1H), 2.12-2.13 (m, 1H), 1.22 (dd, J = 6.0, 2.5Hz, 6H). MS (ESI) m / z: 730.5 [M + H] ⁺ .

Example 6: ((((S)-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5- Hydroxy-2-methylenecyclopentyl) methoxy) (((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -Yl) -2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisopropyl carbonate

O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Cyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2-hydroxyl Methyl-4-methoxytetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (1g) was dissolved in a mixed solution of water (100ml) and isopropanol (25ml), and iodomethyl was added dropwise Isopropyl carbonate (0.856g), react at room temperature for 24h. The isopropyl alcohol was removed by concentration, and the aqueous phase was extracted three times with n-hexane, and then separated and purified using a Biotage C18 120g reverse phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.4g). ¹ H-NMR (500 MHz, CD ₃ OD): δ 8.05 (d, J = 8 Hz, 1H), 7.79 (s, 1H), 6.07 (d, J = 6 Hz, 1H), 5.76 (d, J = 8 Hz , 1H), 5.62-5.49 (m, 3H), 5.36 (s, 1H), 5.21-5.16 (m, 1H), 4.94-4.91 (m, 1H), 4.59-4.52 (m, 1H), 4.51-4.44 (m, 2H), 4.37-4.35 (m, 1H), 4.23-4.18 (m, 1H), 3.87-3.79 (m, 2H), 3.52 (s, 3H), 3.02-2.97 (m, 1H), 2.61 -2.54 (m, 1H), 2.32-2.24 (m, 1H), 1.88-1.78 (m, 1H), 1.34-1.27 (m, 6H) .MS (ESI) m / z: 730.5 [M + H] ⁺ .

Example 7: ((((S)-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5- Hydroxy-2-methylenecyclopentyl) methoxy) (((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -Yl) -2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisobutyl carbonate

O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Cyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2-hydroxyl Methyl-4-methoxytetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (0.85g) was dissolved in a mixed solution of water (120ml) and isopropanol (30ml), and iodine was added dropwise Methyl isobutyl carbonate (0.770g), react at room temperature for 24h. Isopropanol was removed by concentration, and the aqueous phase was extracted three times with n-hexane, and then separated and purified using a Biotage C18 120g reverse-phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.15 g). ¹ H-NMR (500 MHz, CD ₃ OD): δ 8.05 (d, J = 8.0 Hz, 1H), 7.80 (s, 1H), 6.07 (d, J = 6.0 Hz, 1H), 5.70 (d, J = 8.0Hz, 1H), 5.63-5.55 (m, 3H), 5.36 (s, 1H), 5.20-5.19 (m, 1H), 4.89 (s, 1H), 4.57-4.49 (m, 3H), 4.35 ( s, 1H), 4.20 (t, J = 5.0Hz, 1H), 3.99 (d, J = 6.5Hz, 2H), 3.86-3.81 (m, 2H), 3.52 (s, 3H), 3.00 (s, 1H ), 2.60-2.55 (m, 1H), 2.30-2.26 (m, 1H), 1.97-1.92 (m, 1H), 0.95 (s, 3H), 0.93 (s, 3H). MS (ESI) m / z : 744.6 [M + H] ⁺ .

Example 8: (((S)-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5- Hydroxy-2-methylenecyclopentyl) methoxy) (((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidine-1 (2H) -Yl) -2-hydroxymethyl-4-methoxytetrahydrofuran-3-yl) oxy) phosphoryl) thio) methyl methyl carbonate

O-(((1R, 3S, 5S) -3- (2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) -5-hydroxy-2-methylene Cyclopentyl) methyl) O-((2R, 3R, 4R, 5R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -2-hydroxyl Methyl-4-methoxytetrahydrofuran-3-yl) (S) -tetrabutylammonium thiophosphate (1.0g) is dissolved in a mixed solution of water (112ml) and isopropanol (30ml), and iodine is added dropwise Methyl methyl carbonate (0.758g), react at room temperature for 16h. Isopropanol was removed by concentration, and the aqueous phase was extracted three times with n-hexane and then separated and purified using a Biotage C18 120g reverse-phase chromatography column (water: acetonitrile = 3: 1) to obtain the title compound (0.21g). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.44 (s, 1H), 10.61 (s, 1H), 7.91 (s, 1H), 7.68 (s, 1H), 6.41 (s, 2H), 5.94 ( s, 1H), 5.73 (s, 1H), 5.52 (d, J = 19.5 Hz, 2H), 5.40 (s, 2H), 5.23 (s, 1H), 5.11 (d, J = 27.0 Hz, 2H), 4.66 (s, 1H), 4.26 (t, J = 21.5Hz, 3H), 4.14 (s, 1H), 3.77 (s, 2H), 3.65 (s, 2H), 3.39 (s, 3H), 2.86 (s , 1H), 2.51 (s, 2H), 2.31 (s, 1H), 2.11 (s, 1H).

¹³ C NMR (126MHz, DMSO-d ₆ ) δ163.36,157.28,154.56,153.94,151.80,151.07,149.49,140.64,136.43,116.84,111.42,103.01,85.79,84.00,81.12,75.35,70.31,68.88,67.66,60.91 , 58.47,55.89,55.38,51.81,39.05.

LCMS, ([M + H] ⁺ ) m / z: 702.5.

Screening of anti-hepatitis B virus activity in vitro

1. Experimental materials

1.1 cells

HepG2.2.15 cells

1.2 Compound

Test compound: The compound prepared in the example was formulated into 20 mM mother liquor with dimethyl sulfoxide (DMSO).

1.3 Reagent

The reagents used in this experiment include QIAamp 96 DNA Kit (12) (Cat. No. Qiagen-51162), FastStart Universal Probe (Cat. No. Roche-04914058001), CellTiter-Blue detection reagent (Cat. No. Promega-G808B)

2. Experimental method

2.1 Compound dilution: The concentration of the test compound is always 100 μM, 3 times dilution, 8 concentrations.

3.2. In vitro anti-HBV activity experiment and cytotoxicity experiment: On the first day, HepG2.2.15 cells (4 × 10 ⁴ cells / well) were seeded into 96-well plates, and incubated at 37 ° C., 5% CO ₂ overnight. The next day, add fresh medium containing different concentrations of compounds to the culture wells. On the third to tenth days, the old culture medium in the culture wells is sucked up every day, and fresh culture medium containing compounds of different concentrations is added. On the eleventh day, the supernatant in the culture well was collected and used to extract HBV DNA in the supernatant. The qPCR experiment detected the HBV DNA content in the supernatant of HepG2.2.15. After collecting the cell supernatant, CellTiter-Blue reagent is added, and the microplate reader detects the fluorescence value of each well after incubation. Suppression percentage:

Inhibition rate% = (1-HBV DNA content in the sample / HBV DNA content in the DMSO control group) x 100%.

Cell viability percentage:

Cell viability% = (Fluorescence value of sample well-Fluorescence value of medium control) / (Fluorescence value of DMSO control group-Fluorescence value of medium control) × 100%.

The experimental results are shown in Table 1.

Table 1.

Example compounds	EC 50 (nM)	CC 50 (μM)
Example 1	441	> 100

Claims (15)

1. A compound of formula I, its tautomers, stereoisomers or pharmaceutically acceptable salts thereof,

   

   R ^{1 is} selected from

   

   Wherein R is selected from the following groups: C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, C _6-10 Aryl or 5-10 membered heteroaryl, the R group is optionally substituted with one or more of the following groups: halogen, -OH, C _1-6 alkyl, C _1-6 alkoxy,- NO ₂ , -CN, -NH ₂ , -NH (C _1-6 alkyl), -N (C _1-6 alkyl) ₂ , -C (O) C _1-6 alkyl, -C (O) OC _1-6 alkyl, -CONHC _1-6 alkyl, -SH or -SC _1-6 alkyl;

   R ² and R ³ are independently selected from H, -OH, halogen, C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _6-10 aryl or 5-10 membered heteroaryl, the C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _{6 -10} aryl or 5-10 membered heteroaryl is optionally substituted with one or more substituents selected from halogen, -OH, -NO ₂ , -NH ₂ or C _1-6 alkoxy;

   R ⁴ and R ⁵ are independently selected from H, -OH, -NH ₂ or -SH;

   n is selected from 0, 1, 2, 3 or 4;

   X is selected from -C (R ^a R ^b )-or -C (= CH ₂ )-;

   Wherein R ^a and R ^b are independently selected from H, halogen, C _1-6 alkoxy or C _1-6 alkyl, the C _1-6 alkoxy or C _1-6 alkyl is optionally It is substituted by one or more substituents selected from C _1-6 alkoxy, halogen, -OH, -NH ₂ or -NO ₂ .
2. The compound of formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein

   

   versus

   

   Combine to form

   

   among them

   

   It is a monovalent cation.
3. The compound of formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein R is selected from the group consisting of C _1-6 alkyl, C _2-4 ene Group, C _2-4 alkynyl, C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, phenyl or 5-6 membered heteroaryl; or R selected from C _1-4 alkyl; or R It is selected from isopropyl, methyl or isobutyl.
4. The compound of formula I according to any one of claims 1 to 3, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof, wherein R ² and R ³ are independently selected from H, -OH , Halogen, C _1-6 alkyl, C _1-6 alkoxy, C _3-10 cycloalkyl, 3-10 membered heterocycloalkyl, C _6-10 aryl or 5-10 membered heteroaryl, The C _1-6 alkyl or C _1-6 alkoxy is optionally substituted with one or more substituents of halogen, -OH, -NO ₂ , -NH ₂ or C _1-6 alkoxy; or R ² and R ³ are each independently selected from H, halogen, C _1-6 alkyl or C _1-6 alkoxy optionally substituted with C _1-6 alkoxy; or R ² and R ³ are each independently Selected from H, F, CH _3- , CH ₃ O- or CH ₃ OCH ₂ CH ₂ O-; or R ² is H or F, R ³ is CH _3- , CH ₃ O- or CH ₃ OCH ₂ CH ₂ O-.
5. The compound of formula I according to any one of claims 1 to 4, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof, wherein R ⁴ and R ⁵ are independently selected from H or -OH ; Or R ⁴ is H, R ⁵ is -OH.
6. The compound of formula I, a tautomer, a stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein n is selected from 0, 1, or 2; or n is 1.
7. The compound of formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein R ^a and R ^b are independently selected from H, halogen, C _1-3 alkoxy or C _1-3 alkyl; or R ^a and R ^b are each independently selected from H or halogen.
8. The compound of formula I, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein X is -C (= CH ₂ )-.
9. The compound of formula I according to any one of claims 1-8, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from sodium salt or ammonium salt.
10. The compound of formula I, its tautomer, stereoisomer or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein the compound of formula I, its tautomer and stereoisomer Isomers or pharmaceutically acceptable salts thereof are selected from compounds of formula II, tautomers, stereoisomers or pharmaceutically acceptable salts thereof:

    
11. The following compounds, their tautomers, stereoisomers or their pharmaceutically acceptable salts:

    
12. The following compounds, their tautomers, stereoisomers or their pharmaceutically acceptable salts:

    

    
13. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1-12, a tautomer, a stereoisomer or a pharmaceutically acceptable salt thereof.
14. The compound of formula I according to any one of claims 1-12, its tautomer, stereoisomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13 is prepared for prevention or treatment Use in medicine of mammalian HBV infection; optionally, the prevention or treatment of mammalian HBV infection refers to the control, reduction or elimination of HBV to prevent, alleviate or cure liver disease of infected mammals.
15. The compound of formula I, its tautomer, stereoisomer, or pharmaceutically acceptable salt thereof according to any one of claims 1-12 for use in the prevention or treatment of HBV infection in a mammal, or claim 13 Pharmaceutical composition; optionally, the prevention or treatment of mammalian HBV infection refers to the control, reduction or elimination of HBV to prevent, alleviate or cure the liver disease of the infected mammal.