CN113164506A

CN113164506A - Dinucleotide compounds and prodrugs thereof

Info

Publication number: CN113164506A
Application number: CN201980079681.6A
Authority: CN
Inventors: 张寅生; 刘保民; 朱炎; 盖阔; 黄雨; 尧超; 童顺禹; 张洋; 王晓金
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority date: 2018-12-06
Filing date: 2019-12-06
Publication date: 2021-07-23
Anticipated expiration: 2039-12-06
Also published as: CN113164506B; WO2020114495A1

Abstract

Dinucleotide compounds and prodrugs thereof are disclosed. In particular to a dinucleotide compound shown in a general formula (I), a tautomer, a stereoisomer or a pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition thereof and a medical application thereof in treating hepatitis B virus infection, especially liver diseases caused by the hepatitis B virus infection.

Description

Dinucleotide compounds and prodrugs thereof

Cross Reference to Related Applications

This application claims priority and benefit to chinese patent application No. 201811487263.4, filed on 2018, 12/06/h, to the chinese intellectual property office, the disclosure of which is hereby incorporated by reference in its entirety.

Technical Field

The present application relates to dinucleotide compounds and prodrugs thereof, methods for preparing the same, pharmaceutical compositions containing the same, and uses thereof in treating Hepatitis B Virus (HBV) infection and HBV-related liver diseases.

Background

Chronic viral hepatitis b is currently incurable and controllable, and two anti-HBV drugs that have been clinically approved include interferon-alpha and nucleoside analogs, but there are usually problems of rapid emergence of drug resistance and toxicity due to dose limitations associated with treatment. Many current drugs also suffer from poor absorption, distribution, metabolism and/or excretion (ADME) properties, which prevent their wider use. Poor ADME properties are also a large reason for the failure of candidate drugs in clinical trials.

The existing oligonucleotide drugs such as SB9000 and precursor drug SB9200 thereof have safety and antiviral effects in chronic hepatitis B patients without liver cirrhosis, but the development of compounds with better anti-HBV activity, safety, prodrug release active compound speed and bioavailability, higher stability and certain liver targeting property is still needed.

Summary of The Invention

The present application relates to compounds of general formula I, tautomers, stereoisomers, or pharmaceutically acceptable salts thereof,

wherein R is¹Is selected from

R is selected from C_1-12Alkyl radical, C_1-12Alkoxy radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl, said R group being optionally substituted with one or more groups selected from: halogen, -OH, C_1-6Alkyl, - (OCH)₂CH ₂) _m-OC _1-6Alkyl, -NO₂、-CN、-NH ₂、-NH(C _1-6Alkyl), -N (C)_1-6Alkyl radical)₂、-C(O)C _1-6Alkyl, -C (O) OC_1-6Alkyl, -CONHC_1-6Alkyl, -SH, -SC_1-6Alkyl radical, C_3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl, wherein m is selected from 0, 1,2,3,4 or 5;

R ²selected from H, -OH, halogen, C_1-6Alkyl radical, C _1-6Alkoxy radical, C_3-10Cycloalkyl, 3-10 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl;

R ³is selected from

R ⁴Selected from H, C_1-6Alkyl radical, C_3-6Cycloalkyl or 3-6 membered heterocycloalkyl.

In another aspect, the present application relates to a pharmaceutical composition comprising a compound of general formula I of the present application, a tautomer, a 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 comprising administering to the mammal in need thereof a therapeutically effective amount of a compound of formula I, a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.

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

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

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

Detailed Description

wherein R is¹Is selected from

R ²selected from H, -OH, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_3-10Cycloalkyl, 3-10 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl;

R ³is selected from

The application relates to a compound of general formula I or a pharmaceutically acceptable salt thereof,

wherein R is¹Is selected from

R is selected from C_1-12Alkyl radical, C_1-12Alkoxy radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl, said R being optionally substituted with: halogen, -OH, C_1-6Alkyl, - (OCH)₂CH ₂) _m-OC _1-6Alkyl, -NO₂、-CN、-NH ₂、-NH(C _1-6Alkyl), -N (C)_1-6Alkyl radical)₂、-C(O)C _1-6Alkyl, -C (O) OC_1-6Alkyl, -CONHC_1-6Alkyl, -SH, -SC_1-6Alkyl radical, C_3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl, wherein m is selected from 0, 1,2,3,4 or 5;

R ³is selected from

In some embodiments of the present invention, the substrate is,

and

combine to form

Wherein

Is a monovalent cation. In some embodiments of the present invention, the substrate is,

may be an alkali metal cation or an ammonium cation, e.g. Na⁺、K ⁺、Li ⁺Or NH₄ ⁺. In some embodiments of the present invention, the substrate is,

can be Na⁺Or NH₄ ⁺(ii) a In some embodiments of the present invention, the substrate is,

may be NH₄ ⁺. In some embodiments, the pharmaceutically acceptable salt of the compound of formula I is selected from sodium or ammonium salts. In some embodiments, the pharmaceutically acceptable salt of the compound of formula I is selected from ammonium salts.

In some embodiments, R¹Is selected from

In some embodiments, m is selected from 0, 1,2, or 3; in some embodiments, m is selected from 0, 1 or 2; in some embodiments, m is 0.

In some embodiments, R is selected from C_1-6Alkyl radical, C_1-6Alkoxy radical, C_2-6Alkenyl or C_2-6Alkynyl, said R being optionally substituted with one or more groups selected from: - (OCH)₂CH ₂) _m-OCH ₃、C _3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl; in some embodiments, R is selected from C_1-6Alkyl or C_1-6Alkoxy, said R being optionally substituted by one or more C_3-6Cycloalkyl substitution; in some embodiments, R is selected from (CH)₃) ₂CHO-、(CH ₃CH ₂) ₂CHO-、(CH ₃) ₂CHCH ₂O-、(CH ₃CH ₂) ₂CHCH ₂O-、(CH ₃) ₃CCH ₂O-, cyclopropyl-CH₂O-、(CH ₃) ₂CH-or (CH)₃) ₃C-。

In some embodiments, R²Selected from H, -OH or halogen; in some embodiments, R²Selected from H or halogen; in some embodiments, R²Selected from H or fluorine. In some embodiments, R²Is selected from H.

In some embodiments, R³Is selected from

In some embodiments, R⁴Is selected from C_1-6An alkyl group; in some embodiments of the present invention, the substrate is,R ⁴is selected from C_1-3An alkyl group; in some embodiments, R⁴Is methyl.

In some embodiments, the pharmaceutically acceptable salt of the compound of formula I is selected from the sodium salt of the compound of formula I or the ammonium salt of the compound of formula I.

In some embodiments, the compound of general formula I, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof, of the present application is selected from the following compounds, tautomers, stereoisomers, or pharmaceutically acceptable salts thereof:

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

in another aspect, the present application relates to a pharmaceutical composition comprising a compound of general formula I of the present application, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical compositions of the present application further comprise a pharmaceutically acceptable excipient.

In another aspect, the present application relates to a method of treating or inhibiting HBV infection in a mammal, comprising administering to a mammal, preferably a human, in need thereof a therapeutically effective amount of a compound of formula I, a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above.

In another aspect, the present application relates to the use of a compound of formula I, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a medicament for the prevention or treatment of HBV infection in a mammal.

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

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

In some embodiments of the present application, the treatment of HBV infection refers to controlling, reducing or eliminating HBV to ameliorate or cure liver disease in an infected mammal or patient.

Definition of

The following terms used in the present application have the following meanings, unless otherwise specified. A particular term should not be considered as ambiguous or unclear without special definition, but rather construed according to ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The term "substituted" means that any one or more hydrogen atoms on a particular atom or group is replaced with a substituent, so long as the valency of the particular atom or group is normal and the substituted compound is stable.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occurAnd generating a description that includes both the occurrence of the event or circumstance and the non-occurrence of the event or circumstance. For example, ethyl is "optionally" substituted with halo, meaning that ethyl may be unsubstituted (CH)₂CH ₃) Monosubstituted (e.g. CH)₂CH ₂F) Polysubstituted (e.g. CHFCH)₂F、CH ₂CHF ₂Etc.) or completely substituted (CF)₂CF ₃). It will be appreciated by those skilled in the art that any group containing one or more substituents will not incorporate any substitution or substitution pattern which is sterically impossible and/or cannot be synthesized.

Herein C_m-nMeaning that the moiety has an integer number of carbon atoms in the given range. E.g. "C_1-6By "is meant that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 2R, then there are separate options for each R.

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

The term "alkyl" refers to a group of formula C_nH _2n+1A hydrocarbon group of (1). The alkyl group may be linear or branched. For example, the term "C₁- ₁₂Alkyl "refers to an alkyl group containing 1 to 12 carbon atoms; the term "C₁- ₆Alkyl "means an alkyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkanoyl, alkylsulfonyl and alkylthio groups have the same definitions as above.

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

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

The term "alkynyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon group having at least one triple bond consisting of carbon atoms and hydrogen atoms. 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.ident.CH), 1, 3-butadiynyl (-C.ident.C-C.ident.CH), and the like.

The term "cycloalkyl" refers to a carbon ring that is fully saturated and may exist in the form of a single ring, fused ring, bridged ring, or spiro ring. Unless otherwise indicated, the carbocycle is typically a 3 to 10 membered ring or a 3-6 membered ring. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, and the like.

The term "heterocycloalkyl" refers to a cyclic group that is fully saturated and may exist as a single ring, fused ring, bridged ring, or spiro ring, which may have 1 to 4 heteroatoms selected from nitrogen, oxygen, and/or sulfur. Unless otherwise indicated, the heterocycloalkyl group is typically a 3 to 6 membered, 3 to 7 membered 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 heterocycloalkyl include, but are not limited to, oxiranyl, thietanyl, cycloazenyl, non-limiting examples of 4-membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thiabutinyl, examples of 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, examples of tetrahydropyrazolyl, 6-membered heterocycloalkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, 4-thialkyl, 1, 4-dioxanyl, thiomorpholinyl, 1, 3-dithianyl, 1, 4-dithianyl, and examples of 7-membered heterocycloalkyl include, but are not limited to, azepanyl, oxepanyl, thiepanyl. Monocyclic heterocycloalkyl groups having 5 or 6 ring atoms are preferred.

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

The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing as ring atoms at least one (e.g., 1,2,3,4, 5, or 1 to 3) heteroatom selected from N, O and S, the remaining ring atoms being C, and having at least one aromatic ring. The heteroaryl group can be a 5 to 10 membered aromatic ring containing 1 to 3 or 1 to 4 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, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

The term "preventing" means preventing the occurrence of a disease, disorder, or condition in a mammal, particularly when such mammal is susceptible to the disease, disorder, or condition, but has not yet been diagnosed as having the disease, disorder, or condition, or when such mammal is susceptible to being at risk of having the disease, disorder, or condition.

The term "treating" means administering a compound or formulation described herein to ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) inhibiting the disease or disease state, i.e., arresting its development;

(ii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "therapeutically effective amount" means an amount of a compound of the present application that (i) treats or prevents a particular disease, disease state, or condition, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, disease state, or condition, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, disease state, or condition described herein. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease, disease state or condition 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 with their own knowledge and this disclosure.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As the pharmaceutically acceptable salt, for example, a metal salt (for example, an alkali metal salt, an alkaline earth metal salt, an aluminum salt, an iron salt, or the like), an ammonium salt, a salt with an organic base (for example, organic amine), a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, or the like can be mentioned.

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

The term "pharmaceutically acceptable adjuvants" refers to those adjuvants which do not have a significant irritating effect on the organism and do not impair the biological activity and properties of the active compound. Suitable adjuvants 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 variations thereof such as "comprises" or "comprising," are to be understood in an open, non-exclusive sense, i.e., "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 interconvert via a low energy barrier. For example, proton tautomers (also referred to as proton transfer tautomers) include interconversion via proton migration, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is an imidazole moiety, wherein the proton can migrate between two ring nitrogens. Valence tautomers include interconversion by recombination of some of the bonding electrons.

The compounds of the present application may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are included within the scope of the present application. The compounds of the present application containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents.

The compounds of the present application may exist in specific geometric isomeric or stereoisomeric forms. The present application contemplates all such compounds, including tautomers, cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, as well as racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present application. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present application.

The pharmaceutical compositions of the present application can be prepared by combining the compounds 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, aerosols, and the like.

Typical routes of administration of a compound of the present application or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The pharmaceutical compositions of the present application can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

In some embodiments, the pharmaceutical composition is in a dosage form for oral administration. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These adjuvants enable the compounds of the present application to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient.

Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid adjuvants, optionally the mixture obtained is milled, if desired with further suitable adjuvants, and the mixture is then processed to granules, to give tablets or dragee cores. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

The pharmaceutical compositions may also be adapted for parenteral administration, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

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

The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The chemical reactions of the embodiments herein are carried out in a suitable solvent that is compatible with the chemical changes of the present application and the reagents and materials required therefor. In order to obtain the compounds of the present application, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

An important consideration in the art of synthetic route planning is the selection of suitable protecting Groups for reactive functional Groups (such as hydroxyl Groups in the present application), for example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed.) Hoboken, New Jersey: John Wiley & Sons, Inc. all references cited herein are incorporated herein in their entirety.

In some embodiments, the compounds of formula I herein may be prepared by one skilled in the art of organic synthesis by the following route:

route 1:

route 2:

wherein R is¹、R、R ²、R ⁴The definition is the same as that of the compound in the general formula I.

The following abbreviations are used in this application:

me represents a methyl group; TBDPS represents tert-butyldiphenylsilyl; DMSO represents dimethyl sulfoxide.

For clarity, the invention is further illustrated by examples, which do not limit the scope of the application. All reagents used herein were commercially available and used without further purification.

Detailed description of the preferred embodiments

Example 1: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R)) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (R) -ammonium thiophosphate

Step 1: (2R,3R,3aS,9aR) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3-hydroxy-2, 3,3a,9 a-tetrahydro-6H-furo [2',3':4,5] oxazolo [3,2-a ] pyrimidin-6-one

To a reaction flask were added (2R,3R,3aS,9aR) -3-hydroxy-2-hydroxymethyl-2, 3,3a,9 a-tetrahydro-6H-furo [2',3':4,5] oxazolo [3,2-a ] pyrimidin-6-one (30g), 4,4' - (chloro (phenyl) methylene) bis (methoxybenzene) (49.4g), N, N-dimethylpyridin-4-amine (0.12g), N, N-dimethylformamide (120ml) and pyridine (360ml) in this order, and the mixture was stirred at room temperature for 22 hours. The reaction was concentrated, the residue was taken up in dichloromethane and washed with 5% sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give a crude product, and purified by slurrying with methyl tert-butyl ether to give the title compound (45.74 g).

¹H NMR(500MHz,DMSO-d ₆):δ7.94(d,J＝7.5Hz,1H),7.31-7.26(m,4H),7.21-7.14(m,5H),6.86-6.83(m,4H),6.34(d,J＝6.0Hz,1H),5.95(d,J＝4.5Hz,1H),5.89(d,J＝7.0Hz,1H),5.22(d,J＝5.5Hz,1H),4.34-4.31(m,1H),4.25-4.20(m,1H),3.74(s,6H),2.98-2.95(m,1H),2.86-2.82(m,1H).

Step 2: 1- ((2R,3R,4R,5R) -5- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4-hydroxy-3- (2-methoxyethoxy) tetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione

A magnesium rod (7.28g) and 2-methoxyethyl-1-ol (236ml) were successively charged into a reaction flask, and reacted at 110 ℃ for 10 minutes. The reaction was cooled to room temperature and (2R,3aS, 9aR) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3-hydroxy-2, 3,3a,9 a-tetrahydro-6H-furo [2',3':4,5] oxazolo [3,2-a ] pyrimidin-6-one (35g) was added, heated to 80 ℃ and reacted for 9H. 600ml of water and 600ml of dichloromethane were added to the reaction solution under ice bath, the pH of the reaction solution was adjusted to about 7 with glacial acetic acid, and the organic phase was washed with water and saturated brine, respectively, dried over anhydrous sodium sulfate, filtered, concentrated to give a crude product, and purified by column chromatography (dichloromethane: methanol ═ 50:1) to give the title compound (32 g).

¹H NMR(500MHz,DMSO-d ₆):δ11.37(s,1H),7.73(d,J＝8.0Hz,1H),7.39(d,J＝8.0Hz,2H),7.33(t,J＝7.5Hz,2H),7.26-7.27(m,5H),6.91(d,J＝8.5Hz,4H),5.81-5.82(m,1H),5.31(d,J＝8.0Hz,1H),5.10(d,J＝6.5Hz,1H),4.20(q,J＝6.0Hz,1H),3.97-4.02(m,2H),3.75(s,6H),3.73-3.74(m,2H),3.49(t,J＝5.0Hz,2H),3.29-3.32(m,1H),3.25(s,4H).

And step 3: (2R,3R,4R,5R) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

1- ((2R,3R,4R,5R) -5- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4-hydroxy-3- (2-methoxyethoxy) tetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (32g) was dissolved in dichloromethane (150ml), and a solution of 3- ((di (diisopropylamino) phosphono) oxy) propionitrile (20.15g) and pyridin-1-ium 2,2, 2-trifluoroacetate (12.26g) in dichloromethane (50ml) was slowly added dropwise and the reaction solution was stirred at room temperature for 2H. Concentrating the reaction solution to obtain crude product, and performing column chromatography (using stone as column)Oily ether ethyl acetate 1:1) to give the title compound (15.11 g). MS (ESI) M/z 827.5[ M + Na ]] ⁺。

And 4, step 4: ((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl ((2R,3R,4R,5R) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (2-cyanoethyl) phosphite

(2R,3R,4R,5R) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (13g) was dissolved in anhydrous acetonitrile (240ml), and a solution of N- (9- ((2R,4S,5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (8.73g) in N, N-dimethylformamide (60ml) and a solution of 1H-tetrazole (2.615g) in acetonitrile (83ml) were successively added dropwise, the reaction was stirred at room temperature for 3 h. The reaction solution was concentrated to give a crude product, which was purified by column chromatography (dichlorosilane: methanol: 20:1) to give the title compound (3.86 g). MS (ESI) M/z 1081.3[ M + Na ]] ⁺。

And 5: o- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O- (2-cyanoethyl) thiophosphate

((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl ((2R,3R,4R,5R) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (2-cyanoethyl) phosphite (3.58g) was dissolved in acetonitrile (40ml), a solution of 3H-benzo [ c ] [1,2] dithiol-3-one 1, 1-dioxide (0.691g) in acetonitrile (10ml) was added dropwise, the reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated to give a crude product, which was purified by column chromatography (dichlorosilane: methanol: 20:1) to give the title compound (1.37 g).

¹H NMR(500MHz,DMSO-d ₆):δ11.42(s,1H),11.16(s,1H),8.73(d,J＝9.0Hz,1H),8.60(d,J＝8.5Hz,1H),8.05(d,J＝7.0Hz,2H),7.69-7.65(m,2H),7.57-7.54(m,2H),7.39-7.22(m,9H),6.89(t,J＝7.5Hz,4H),6.51(d,J＝5.5Hz,1H),5.84(s,1H),5.38(t,J＝9.5Hz,1H),5.10(s,1H),4.54-4.07(m,9H),3.74(s,3H),3.72(s,3H),3.64(s,1H),3.44-3.12(m,8H),2.92-2.84(m,3H),2.42(s,1H).

Step 6: o- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) phosphorothioate

O- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O- (2-cyanoethyl) thiophosphate (1.37g) was dissolved in methylene chloride (13ml), 2-dichloroacetic acid (0.248g) was added dropwise, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was adjusted to pH 7 with triethylamine, concentrated to give a crude product, which was purified by column chromatography (dichlorosilane: methanol 10:1) to give the title compound (0.51 g).

¹H NMR(500MHz,DMSO-d ₆):δ11.40(s,1H),11.16(s,1H),8.76(s,1H),8.63(s,1H),8.05(d,J＝8.0Hz,2H),7.87(d,J＝8.0Hz,1H),7.65(t,J＝7.5Hz,1H),7.56(t,J＝7.5Hz,2H),6.53(t,J＝6.5Hz,1H),5.91(d,J＝6.0Hz,1H),5.72(d,J＝8.0Hz,1H),5.59(d,J＝4.5Hz,1H),5.33(t,J＝5.0Hz,1H),5.04-5.01(m,1H),4.57-4.54(m,1H),4.38-4.11(m,7H),3.72-3.58(m,4H),3.43-3.39(m,2H),3.18(s,3H),2.96-2.91(m,3H),2.47-2.42(m,1H).

And 7: o- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (R) -phosphorothioate

O- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) thiophosphate (410mg) was resolved by means of a YMC high-pressure preparative chromatograph (column: YMC Triart C18 high-pressure dynamic axial compression column, 10 μm, 50X 235 mm; mobile phase: water and acetonitrile (25% acetonitrile isocratic elution for 50 minutes); detector: UV 260nm), the eluent was concentrated to give the title compound (146 mg).

¹H NMR(500MHz,DMSO-d ₆):δ11.39(s,1H),11.17(s,1H),8.76(s,1H),8.63(s,1H),8.05(d,J＝8.0Hz,2H),7.87(d,J＝8.0Hz,1H),7.65(t,J＝7.5Hz,1H),7.56(t,J＝7.5Hz,2H),6.54(t,J＝6.5Hz,1H),5.92(d,J＝6.0Hz,1H),5.71(d,J＝8.0Hz,1H),5.57(d,J＝4.0Hz,1H),5.32(t,J＝5.0Hz,1H),5.03-4.99(m,1H),4.57-4.55(m,1H),4.42-4.13(m,7H),3.67-3.53(m,4H),3.33-3.32(m,2H),3.11(s,3H),2.96-2.91(m,3H),2.46-2.42(m,1H).

And 8: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R)) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (R) -ammonium thiophosphate

O- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (R) -phosphorothioate (13mg) was dissolved in aqueous ammonia (1ml), and the reaction mixture was stirred at room temperature for 17H. The reaction solution was adjusted to pH 8 with 50% aqueous acetic acid in ice bath, and extracted with ethyl acetate. The aqueous phase was separated and purified by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to give the title compound (70 mg).

¹H NMR(500MHz,DMSO-d ₆):δ11.34(s,1H),8.49(s,1H),8.15(s,1H),7.90(d,J＝8.5Hz,1H),7.23(s,2H),7.16(brs,4H),6.39-6.36(m,1H),5.86(d,J＝6.0Hz,1H),5.68(d,J＝8.0Hz,1H),5.37(s,1H),5.19(s,1H),4.76-4.72(m,1H),4.48(s,1H),4.18-4.16(m,1H),4.04-3.88(m,4H),3.77-3.73(m,1H),3.65-3.59(m,2H),3.54-3.50(m,1H),3.38-3.34(m,2H),3.14(s,3H),2.77-2.72(m,1H),2.30-2.25(m,1H).HRMS(ESI)m/z:632.1540[M+H] ⁺.

Example 2: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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) -ammonium thiophosphate

Step 1: o- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -phosphorothioate

Reference was made to the operation of step 7 in example 1 to give the title compound (158 mg).

Step 2: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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) -ammonium thiophosphate

Starting from O- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -thiophosphate, the title compound was obtained by following the procedure of step 8 in example 1.

¹H NMR(500MHz,DMSO-d ₆):δ11.34(s,1H),8.46(s,1H),8.15(s,1H),7.90(d,J＝8.0Hz,1H),7.24(s,2H),7.13(brs,4H),6.39-6.36(m,1H),5.85(d,J＝6.0Hz,1H),5.67(d,J＝6.0Hz,1H),5.36(s,1H),5.22(s,1H),4.76-4.72(m,1H),4.47(s,1H),4.15-4.14(m,1H),4.04-3.87(m,4H),3.84-3.80(m,1H),3.64-3.61(m,2H),3.58-3.54(m,1H),3.40(t,J＝5.0Hz,2H),3.18(s,3H),2.75-2.70(m,1H),2.29-2.25(m,1H).HRMS(ESI)m/z:632.1539[M+H] ⁺.

Example 3: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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

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

Triethylamine bis (perfluorophenyl) dithiophosphate (126g), (1R,4R,6S) -1-methyl-4- (prop-1-en-2-yl) -7-oxabicyclo [4.1.0] heptane (48.3g) and dibutyl hydrogen phosphate (48.9g) were successively added to a reaction flask, followed by slowly dropping 2, 2-dichloroacetic acid (60.0g) into the above solution and reacting at room temperature for 1 h. After the reaction mixture was concentrated to 500ml, 1000ml of n-hexane was added to the reaction mixture to dilute the mixture, and then the mixture was washed with a 10% potassium dihydrogen phosphate solution and a saturated saline solution. The organic phase was concentrated to 200ml, 200ml of methanol was added, and after further concentration to 50ml, 200ml of methanol was added, and further concentration to 50ml was carried out, and a solid was precipitated. The temperature is reduced to 0 ℃ in an ice bath and then filtered to obtain 93.48g of crude product. The crude product (93.48g) was dissolved in 225ml of dichloromethane, followed by addition of 374ml of n-heptane, and the crude product was completely dissolved. The solution was concentrated to 200ml and 374ml of n-heptane was added, and this operation was repeated 3 times. The solution was concentrated to 200ml and a large amount of solid precipitated which was stirred at room temperature for 2 h. Filtration and drying of the filter cake gave the title compound (62.58 g).

¹H NMR(500MHz,CDCl ₃):δ5.04(s,1H),4.87(s,1H),4.31-4.27(m,1H),2.64-2.62(m,1H),2.37-2.35(m,1H),2.11-1.85(m,5H),1.83(s,3H),1.68(s,3H).

Step 2: 1- ((2R,3R,4R,5R) -5- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R,3aS,6R,7aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiolan-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) -3 a-methyl-2- ((perfluorophenyl) thio) -6- (prop-1-en-2-yl) hexahydrobenzo [ d ] [1,3,2] oxathiolane 2-sulfide (37.6g) were dissolved in acetonitrile (645ml) and cooled in an ice bath. 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] azepine (12.81g) was slowly added dropwise to the reaction solution. The reaction was continued for 2h under ice-bath conditions. The reaction solution was washed with purified water and 10% potassium dihydrogen phosphate solution in this order. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate 1:1) to give the title compound (49.23 g).

¹H NMR(500MHz,CDCl ₃):δ8.92(s,1H),7.80(d,J＝8.2Hz,1H),7.70-7.63(m,4H),7.48-7.38(m,6H),6.12(d,J＝5.5Hz,1H),5.45-5.41(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＝11.9,2.0Hz,1H),3.90-3.79(m,3H),3.54(t,J＝4.5Hz,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).

And step 3: o- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3- ((tert-butyldiphenylsilyl) oxy) tetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- (((tert-butyldiphenylsilyl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O-hydro (S) -phosphorothioate

1- ((2R,3R,4R,5R) -5- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R,3aS,6R,7aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiolan-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2, 4- (1H,3H) -dione (25g), N- (9- ((2R,4S,5R) -4- ((tert-butyldiphenylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purine-6 -yl) benzamide (37.7g) was dissolved in acetonitrile (235ml), and 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] azepine (14.51g) was added at room temperature, and the reaction solution was stirred for 1 h. The reaction solution was diluted with ethyl acetate, washed successively with a saturated aqueous sodium bicarbonate solution and a saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by column chromatography (dichloromethane: methanol ═ 10:1) to give the title compound (33.01 g).

¹H NMR(500MHz,DMSO-d ₆):δ11.43(s,1H),11.17(s,1H),8.93(s,1H),8.73(s,1H),8.05(d,J＝7.5Hz,2H),7.67-7.53(m,13H),7.47-7.35(m,13H),6.64-6.61(m,1H),5.89(d,J＝6.0Hz,1H),5.24(d,J＝8.0Hz,1H),5.01-4.97(m,1H),4.72-4.57(m,1H),4.15(d,J＝4.0Hz,1H),4.08-4.02(m,2H),3.92-3.61(m,6H),3.55-3.51(m,1H),3.13(s,3H),2.68-2.64(m,1H),2.30-2.26(m,1H),1.06(s,9H),0.99(s,9H).

And 4, step 4: o- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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

To a reaction flask were added O- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3- ((tert-butyldiphenylsilyl) oxy) tetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- (((tert-butyldiphenylsilyl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O-hydro (S) -thiophosphate (25g), tetrabutylammonium fluoride trihydrate (19.52g) and tetrahydrofuran (17ml) in this order, the reaction solution was stirred at room temperature for 17 hours. The reaction solution was diluted with water and washed with diethyl ether. The aqueous phase was concentrated to give 36g of the title compound, which was taken directly to the next step without treatment.

And 5: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (36g) and aqueous ammonia (260ml) were added successively to the reaction flask, and the mixture was stirred at room temperature for 16 hours. 50% acetic acid aqueous solution was added dropwise to the reaction solution in ice bath to adjust the pH to about 8. The reaction solution was extracted twice with ethyl acetate, the aqueous phase was separated and purified by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1), and the title compound was obtained after concentration (20.27 g). MS (ESI) M/z 630.5[ M-H ]] ^-.

Step 6: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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

To a reaction flask were added O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (0.30g), a sodium ion exchange resin (6.0g), and deionized water (5ml) in this order, and stirred at room temperature for 1H. Filtration and purification of the aqueous phase by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) gave the title compound (0.16g) after concentration.

¹H NMR(500MHz,DMSO-d ₆):δ11.36(br,1H),8.46(s,1H),8.15(s,1H),7.82(d,J＝8.0Hz,1H),7.25(s,2H),6.37(t,J＝7.0Hz,1H),5.85(d,J＝5.5Hz,1H),5.60(d,J＝8.0Hz,1H),5.40(s,1H),5.26(s,1H),4.81-4.61(m,1H),4.47(d,J＝6.0Hz,1H),4.13(t,J＝3.0Hz,1H),4.01-3.82(m,5H),3.61(s,2H),3.59-3.54(m,1H),3.39(t,J＝5.0Hz,2H),3.18(s,3H),2.74-2.71(m,1H),2.29-2.26(m,1H).MS(ESI)m/z:630.5[M-H] ^-.

Example 4: o- (((2R,3R,4R,5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-3-hydroxytetrahydrofuran-2-yl) 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

Step 1: o- (((2R,3R,4R,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3- ((tert-butyldiphenylsilyl) oxy) -4-fluorotetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- (((tert-butyldiphenylsilyl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O-hydro (S) -thiophosphate

1- ((2R,3R,4R,5R) -5- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R,3aS,6R,7aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiolan-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2, 4- (1H,3H) -dione (1.50g) and N- (9- ((2R,3R,4R,5R) -4- ((tert-butyldiphenylsilyl) oxy) -3-fluoro-5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H) -9 -purin-6-yl) benzamide (2.33g) was dissolved in acetonitrile (30ml), and 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] azepine (0.87g) was added at room temperature, stirred and reacted for 1 h. The reaction solution was diluted with ethyl acetate, washed with a saturated aqueous sodium bicarbonate solution and a saturated brine in this order, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by column chromatography (dichloromethane: methanol ═ 10:1) to give the title compound (2.00 g).

¹H NMR(500MHz,DMSO-d ₆):δ11.42(d,J＝2.0Hz,1H),11.21(s,1H),8.90(s,1H),8.69(s,1H),8.05(d,J＝7.5Hz,2H),7.69-7.47(m,13H),7.47-7.34(m,12H),6.47(dd,J＝14.5,5.0Hz,1H),5.88(d,J＝6.0Hz,1H),5.40(m,1H),5.22(d,J＝8.0Hz,1H),4.97(dt,J＝10.0,4.0Hz,1H),4.74(dt,J＝8.0,4.0Hz,1H),4.20(s,1H),4.11-4.02(m,2H),3.96(t,J＝9.5Hz,1H),3.86-3.77(m, 3H),3.69-3.67(m,1H),3.55-3.53(m,1H),3.37(t,J＝5.0Hz,2H),3.14(s,3H),1.09(s,9H),1.00(s,9H).

Step 2: o- (((2R,3R,4R,5R) -5- (6-benzoylamino-9H-purin-9-yl) -4-fluoro-3-hydroxytetrahydrofuran-2-yl) 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

To a reaction flask were added O- (((2R,3R,4R,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3- ((tert-butyldiphenylsilyl) oxy) -4-fluorotetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- (((tert-butyldiphenylsilyl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O-hydro (S) -thiophosphate (1.80g), tetrabutylammonium fluoride trihydrate (1.15g) and tetrahydrofuran (15ml) in this order, the reaction was stirred at room temperature overnight. The reaction solution was diluted with water and washed with diethyl ether. The aqueous phase was concentrated to give 2.10g of the title compound, which was directly taken to the next step without purification.

And step 3: o- (((2R,3R,4R,5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-3-hydroxytetrahydrofuran-2-yl) 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

To a reaction flask were added O- (((2R,3R,4R,5R) -5- (6-benzoylamino-9H-purin-9-yl) -4-fluoro-3-hydroxytetrahydrofuran-2-yl) 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 (2.10g) and aqueous ammonia (15ml) in this order, and the mixture was stirred at room temperature and reacted overnight. The reaction was adjusted to pH 8 with 50% aqueous acetic acid in ice bath. The reaction mixture was washed with ethyl acetate, the aqueous phase was separated and purified by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) and concentrated to obtain the title compound (0.90 g). MS (ESI) M/z 648.7[ M-H ]] ^-.

And 4, step 4: o- (((2R,3R,4R,5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-3-hydroxytetrahydrofuran-2-yl) 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- (((2R,3R,4R,5R) -5- (6-amino-9H-purin-9-yl) -4-fluoro-3-hydroxytetrahydrofuran-2-yl) 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 (0.30g), sodium ion resin (6.0g) and deionized water (5ml) were added successively to the reaction flask and stirred at room temperature for 1H. Filtration and purification of the aqueous phase by reverse phase chromatography on a Biotage C18120 g column (water: acetonitrile 3:1) gave the title compound (0.15g) after concentration.

¹H NMR(500MHz,DMSO-d ₆):δ11.33(s,1H),8.48(s,1H),8.16(s,1H),7.89(d,J＝8.0Hz,1H),7.33(s,2H),6.24(dd,J＝16.5,3.0Hz,1H),5.85(d,J＝5.5Hz,2H),5.65(d,J＝8.0Hz,1H),5.50-5.40(m,1H),5.24(d,J＝5.5Hz,1H),4.74(dd,J＝10.0,5.0Hz,1H),4.53(d,J＝16.0Hz,1H),4.17-4.09(m,2H),4.09-3.96(m,3H),3.84-3.81(m,1H),3.62-3.54(m,3H),3.40-3.38(m,2H),3.18(s,3H).MS(ESI)m/z:648.7[M-H] ^-.

Example 5: o- (((2R,3S,5R) -5- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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

Step 1: o- (((2R,3S,5R) -3- ((tert-butyldiphenylsilyl) oxy) -5- (2-isobutyrylamino-6-oxo-1, 6-dihydro-9H-purin-9-yl) tetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- (((tert-butyldiphenylsilyl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O-hydro (S) -thiophosphate

1- ((2R,3R,4R,5R) -5- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R,3aS,6R,7aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiolan-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2, 4- (1H,3H) -dione (1.50g) and N- (9- ((2R,4S,5R) -4- ((tert-butyldiphenylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-2-yl) -6-oxo-6, 9-dihydro-1H-purin-2-yl) isobutyramide (2.20g) was dissolved in acetonitrile (20ml), and 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] azepine (0.87g) was added at room temperature, stirred and reacted for 1H. The reaction solution was diluted with ethyl acetate, washed successively with a saturated aqueous sodium bicarbonate solution and a saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by column chromatography (dichloromethane: methanol ═ 10:1) to give the title compound (2.10 g).

¹H NMR(500MHz,DMSO-d ₆):δ12.12(s,1H),12.04(s,1H),11.41(s,1H),8.29(s,1H),7.66(d,J＝7.0Hz,2H),7.63-7.57(m,7H),7.48-7.38(m,12H),6.35-6.32(m,1H),5.84(d,J＝6.0Hz,1H),5.25(d,J＝8.0Hz,1H),4.96-4.92(m,1H),4.71(d,J＝4.0Hz,1H),4.09-4.01(m,3H),3.86-3.70(m,5H),3.53-3.49(m,1H),3.37-3.36(m,2H),3.13(s,3H),2.95-2.90(m,1H),2.85-2.80(m,1H),2.14-2.10(m,1H),1.11(d,J＝7.5Hz,3H),1.07(d,J＝7.5Hz,3H),1.05(s,9H),1.01(s,9H).

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

To a reaction flask were added O- (((2R,3S,5R) -3- ((tert-butyldiphenylsilyl) oxy) -5- (2-isobutyrylamino-6-oxo-1, 6-dihydro-9H-purin-9-yl) tetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R) -2- (((tert-butyldiphenylsilyl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O-hydro (S) -thiophosphate (1.80g) in this order, Tetrabutylammonium fluoride trihydrate (1.43g) and tetrahydrofuran (20ml), and the reaction solution was stirred overnight at room temperature. The reaction was diluted with water, washed with diethyl ether and the aqueous phase was concentrated to give the title compound 2.20g which was directly used in the next step without purification.

And step 3: o- (((2R,3S,5R) -5- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H-yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) O- (((2R,3S,5R) -3-hydroxy-5- (2-isobutyrylamino-6-oxo-1, 6-dihydro-9H-purin-9-yl) tetrahydrofuran-2-yl) methyl) (S) -tetrabutylammonium thiophosphate (2.20g) is dissolved in aqueous ammonia (15ml), stirred at 40 ℃ and reacted for 36H under ice-bath, the reaction solution is adjusted to pH 8 with 50% aqueous acetic acid solution, the reaction solution was washed with ethyl acetate. The aqueous phase was separated and purified by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to give the title compound (0.17 g).

MS(ESI)m/z:646.6[M-H] ^-.

And 4, step 4: o- (((2R,3S,5R) -5- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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- (((2R,3S,5R) -5- (2-amino-6-oxo-1, 6-dihydro-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (0.30g), sodium ion exchange resin (6.0g) and deionized water (5ml) were added to the reaction flask in this order, and stirred at room temperature for 1H. Filtration and purification of the aqueous phase by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) gave the title compound (0.16g) after concentration.

¹H NMR(500MHz,DMSO-d ₆):δ11.11(brs,2H),7.97(s,1H),7.89(d,J＝8.0Hz,1H),6.55(s,2H),6.16-6.13(m,1H),5.85(d,J＝5.5Hz,1H),5.67(d,J＝8.5Hz,1H),5.31(s,1H),5.24(s,1H),4.73-4.71(m,1H),4.42(s,1H),4.15(s,1H),4.02(t,J＝5.0Hz,1H),3.96-3.92(m,3H),3.83-3.81(m,1H),3.62(s,2H),3.56-3.54(m,1H),3.39-3.38(m,2H),3.18(s,3H),2.63-2.58(m,1H),2.19-2.15(m,1H).MS(ESI)m/z:646.6[M-H] ^-.

Example 6: (((R) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisopropyl carbonate

Step 1: o- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R)) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) ammonium thiophosphate

Starting from O- (((2R,3S,5R) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) thiophosphate, the procedure of step 8 in reference example 1 gave the title compound. HRMS (ESI) M/z 632.1539[ M + H ]] ⁺.

Step 2: ((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisopropyl carbonate

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- ((2R,3R,4R,5R)) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) ammonium thiophosphate (1.56g) was dissolved in a mixed solution of water (150ml) and isopropanol (39ml), iodomethylisopropyl carbonate (1.761g) was added dropwise, and the mixture was subjected to reverse osmosis at room temperatureAfter 24h, it was concentrated to remove isopropanol and the residue was extracted three times with n-hexane. The aqueous phase was separated and purified by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to give the title compound (1.39 g). MS (ESI) M/z 748.2[ M + H ]] ⁺.

And step 3: (((R) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisopropyl carbonate

((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisopropyl carbonate (1.39g) was resolved by YMC high pressure preparative chromatography (column: CHIRAL ART Amylose-SA, 5 μm, 30X 250 mm; mobile phase: absolute ethanol and n-hexane (50% n-hexane isocratically eluted for 45 minutes); a detector: UV 254nm) to give the title compound (0.527 g).

¹H NMR(500MHz,CD ₃OD):δ8.32(s,1H),8.24(s,1H),8.01(d,J＝8.0Hz,1H),6.49(t,J＝6.5Hz,1H),6.03(d,J＝6.0Hz,1H),5.74(d,J＝8.0Hz,1H),5.53-5.46(m,2H),5.10-5.05(m,1H),4.90-4.87(m,1H),4.74-4.70(m,1H),4.52-4.46(m,1H),4.44-4.40(m,1H),4.38-4.35(m,1H),4.31-4.24(m,2H),3.82-3.76(m,3H),3.75-3.71(m,1H),3.53-3.48(m,2H),3.29(s,3H),2.99-2.91(m,1H),2.56-2.50(m,1H),1.27-1.24(m,6H).HRMS(ESI)m/z:748.2002[M+H] ⁺.

Example 7: (((S) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylisopropyl carbonate

The title compound (0.456g) was additionally obtained by following the procedure of example 6, step 3.

¹H NMR(500MHz,CD ₃OD):δ8.35(s,1H),8.24(s,1H),8.01(d,J＝8Hz,1H),6.50(t,J＝6.5Hz,1H),6.06(d,J＝6Hz,1H),5.74(d,J＝8Hz,1H),5.50-5.42(m,2H),5.13-5.08(m,1H),4.90-4.88(m,1H),4.74-4.70(m,1H),4.54-4.46(m,2H),4.36(t,J＝5.5Hz,1H),4.31-4.28(m,1H),4.26-4.23(m,1H),3.86-3.77(m,3H),3.73-3.68(m,1H),3.50-3.44(m,2H),3.25(s,3H),2.96-2.89(m,1H),2.56-2.50(m,1H),1.28(s,3H),1.27(d,J＝6.5Hz,6H).

HRMS(ESI)m/z:748.2034[M+H] ⁺.

Example 8: ((S) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylpent-3-ylcarbonate

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (1.0g) was dissolved in a mixed solution of water (120ml) and isopropanol (30ml), iodomethylpent-3-yl carbonate (0.935g) was added dropwise, reacted at room temperature for 24H, and concentrated to remove the isopropanol. The aqueous phase was extracted three times with n-hexane, and then separated and purified by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1), and concentrated to obtain the title compound (0.400 g).

¹H NMR(500MHz,CD ₃OD):δ8.35(s,1H),8.24(s,1H),8.00(d,J＝8.0Hz,1H),6.50(t,J＝6.5Hz,1H),6.06(d,J＝6.5Hz,1H),5.74(d,J＝8.0Hz,1H),5.55-5.43(m,2H),5.17-5.09(m,1H),4.74-4.69(m,1H),4.63-4.57(m,1H),4.55-4.46(m,2H),4.39-4.35(m,1H),4.32-4.29(m,1H),4.27-4.22(m,1H),3.87-3.74(m,3H),3.73-3.66(m,1H),3.51-3.41(m,2H),3.25(s,3H),2.95-2.87(m,1H),2.58-2.50(m,1H),1.69-1.53(m,4H),0.89(t,J＝7.0Hz,6H).MS(ESI)m/z:776.5[M+H] ⁺.

Example 9: (((S) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methyl isobutyl carbonate

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (1.0g) was dissolved in a mixed solution of water (100ml) and isopropanol (26ml), iodomethyl isobutyl carbonate (0.887g) was added dropwise and reacted at room temperature for 24 hours. The mixture was concentrated to remove isopropanol, and the aqueous phase was extracted three times with n-hexane and then subjected to separation and purification by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to obtain the title compound (0.419g) after concentration.

¹H NMR(500MHz,CD ₃OD):δ8.35(s,1H),8.24(s,1H),8.00(d,J＝8.0Hz,1H),6.50(t,J＝6.5Hz,1H),6.06(d,J＝6.0Hz,1H),5.74(d,J＝8.0Hz,1H),5.50-5.46(m,2H),5.13-5.10(m,1H),4.74-4.70(m,1H),4.59-4.45(m,2H),4.36(t,J＝5.5Hz,1H),4.31-4.29(m,1H),4.25-4.24(m,1H),3.94(d,J＝6.5Hz,2H),3.85-3.68(m,4H),3.52-3.45(m,2H),3.25(s,3H),2.95-2.89(m,1H),2.56-2.51(m,1H),1.94-1.88(m,1H),0.91(d,J＝6.5Hz,6H).MS(ESI)m/z:760.6[M+H] ⁺.

Example 10: (((S) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methyl (2-ethylbutyl) carbonate

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (1.0g) was dissolved in a mixed solution of water (120ml) and isopropanol (30ml), and 2-ethylbutyl (iodomethyl) carbonate (0.983g) was added dropwise and reacted at room temperature for 24 hours. The mixture was concentrated to remove isopropanol, and the aqueous phase was extracted three times with n-hexane and then subjected to separation and purification by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to obtain the title compound (0.440g) after concentration.

¹H NMR(500MHz,CD ₃OD):δ8.35(s,1H),8.24(s,1H),8.00(d,J＝8.0Hz,1H),6.50(t,J＝ 6.5Hz,1H),6.06(d,J＝6.5Hz,1H),5.74(d,J＝8.5Hz,1H),5.54-5.45(m,2H),5.14-5.08(m,1H),4.74-4.69(m,1H),4.56-4.45(m,2H),4.39-4.34(m,1H),4.32-4.29(m,1H),4.26-4.22(m,1H),4.12-4.07(m,2H),3.86-3.81(m,1H),3.80-3.75(m,2H),3.74-3.68(m,1H),3.51-3.42(m,2H),3.25(s,3H),2.95-2.87(m,1H),2.57-2.50(m,1H),1.53-1.44(m,1H),1.37-1.26(m,4H),0.88(t,J＝7.5Hz,6H).MS(ESI)m/z:790.6[M+H] ⁺.

Example 11: (((S) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methylneopentyl carbonate

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (1.0g) was dissolved in a mixed solution of water (120ml) and isopropanol (30ml), iodomethyl neopentyl carbonate (0.935g) was added dropwise, and reacted at room temperature for 24 hours. The mixture was concentrated to remove isopropanol, and the aqueous phase was extracted three times with n-hexane and then subjected to separation and purification by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to obtain the title compound (0.250g) after concentration.

¹H NMR(500MHz,CD ₃OD):δ8.35(s,1H),8.25(s,1H),8.00(d,J＝8.0Hz,1H),6.50(t,J＝6.0Hz,1H),6.06(d,J＝6.0Hz,1H),5.74(d,J＝8.0Hz,1H),5.54-5.44(m,2H),5.16-5.08(m,1H),4.75-4.69(m,1H),4.56-4.45(m,2H),4.40-4.35(m,1H),4.33-4.29(m,1H),4.26-4.22(m,1H),3.90-3.86(m,2H),3.84-3.76(m,3H),3.74-3.69(m,1H),3.52-3.42(m,2H),3.26(s,3H),2.97-2.88(m,1H),2.59-2.49(m,1H),0.92(s,9H).HRMS(ESI)m/z:776.2350[M+H] ⁺.

Example 12: ((S) - ((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methyl (cyclopropylmethyl) carbonate

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (1.0g) was dissolved in a mixed solution of water (100ml) and isopropanol (26ml), cyclopropylmethyl (iodomethyl) carbonate (0.880g) was added dropwise and reacted at room temperature for 24 hours. The mixture was concentrated to remove isopropanol, and the aqueous phase was extracted three times with n-hexane and then subjected to separation and purification by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to obtain the title compound (0.400g) after concentration.

¹H NMR(500MHz,CD ₃OD)δ8.35(s,1H),8.24(s,1H),8.01(d,J＝8.0Hz,1H),6.50(t,J＝6.5Hz,1H),6.06(d,J＝6.0Hz,1H),5.75(d,J＝8.0Hz,1H),5.49-5.45(m,2H),5.16-5.06(m,1H),4.74-4.71(m,1H),4.54-4.46(m,2H),4.36(t,J＝5.5Hz,1H),4.32-4.29(m,1H),4.26-4.23(m,1H),4.03-3.96(m,2H),3.86-3.67(m,4H),3.50-3.43(m,2H),3.25(s,3H),2.93-2.91(m,1H),2.56-2.52(m,1H),1.18-1.10(m,1H),0.58-0.51(m,2H),0.31-0.29(m,2H).

MS(ESI)m/z:760.6[M+H] ⁺.

Example 13: (((S) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) methyl isobutyrate

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (1.0g) was dissolved in a mixed solution of water (120ml) and isopropanol (30ml), iodomethyl isobutyrate (0.784g) was added dropwise, and reacted at room temperature for 24 hours. The mixture was concentrated to remove isopropanol, and the aqueous phase was extracted three times with n-hexane and then subjected to separation and purification by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to obtain the title compound (0.330g) after concentration.

¹H NMR(500MHz,CD ₃OD):δ8.34(s,1H),8.24(s,1H),8.01(d,J＝8.0Hz,1H),6.50(t,J＝6.5Hz,1H),6.06(d,J＝6.0Hz,1H),5.74(d,J＝8.0Hz,1H),5.42-5.38(m,2H),5.10(s,1H),4.72(s,1H),4.49-4.47(m,2H),4.37(s,1H),4.28(s,1H),4.24(s,1H),3.82-3.80(m,2H),3.76-3.69(m,2H),3.51-3.44(m,2H),3.26(s,3H),2.97-2.92(m,1H),2.59-2.51(m,2H),1.14-1.12(m,6H).MS(ESI)m/z:732.5[M+H] ⁺.

Example 14: (((S) - (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methoxy) ((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) oxy) phosphoryl) thio) pivalic acid methyl ester

O- (((2R,3S,5R) -5- (6-amino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) 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 (1.0g) was dissolved in a mixed solution of water (120ml) and isopropanol (30ml), iodomethyl pivalate (0.832g) was added dropwise, and the reaction was carried out at room temperature for 24 hours. The mixture was concentrated to remove isopropanol, and the aqueous phase was extracted three times with n-hexane and then subjected to separation and purification by Biotage C18120 g reverse phase chromatography (water: acetonitrile 3:1) to obtain the title compound (0.550g) after concentration.

¹H NMR(500MHz,CD ₃OD):δ8.34(s,1H),8.24(s,1H),8.00(d,J＝8.0Hz,1H),6.49(t,J＝6.5Hz,1H),6.06(d,J＝6.0Hz,1H),5.74(d,J＝8.5Hz,1H),5.43(s,1H),5.39(s,1H),5.11-5.09(m,1H),4.72-4.71(m,1H),4.50-4.47(m,2H),4.36(t,J＝5.5Hz,1H),4.28-4.27(m,1H),4.24-4.23(m,1H),3.84-3.80(m,2H),3.76-3.69(m,2H),3.51-3.44(m,2H),3.26(s,3H),2.98-2.93(m,1H),2.56-2.51(m,1H),1.18(s,9H).MS(ESI)m/z:746.6[M+H] ⁺.

Screening for in vitro anti-hepatitis B virus activity

1. Experimental Material

1.1 cells

HepG2.2.15 cells

1.2 Compounds

Test compounds: the resulting compound was prepared in 20mM stock solution in dimethyl sulfoxide (DMSO).

1.3 reagents

Reagents used in this experiment included QIAamp 96 DNA Blood Kit (12) (Cat Qiagen-51162), FastStart Universal Probe Master (Cat Roche-04914058001), CellTiter-Blue detection reagent (Cat Promega-G808B).

2. Experimental methods

2.1 dilution of the compound: the test compound was always at a concentration of 100. mu.M, 3-fold dilution, 8 concentrations.

3.2. In vitro anti-HBV activity assay and cytotoxicity assay: the first day, HepG2.2.15 cells (4X 10) were seeded⁴Cells/well) to 96-well plates at 37 ℃, 5% CO₂The culture was carried out overnight. The following day, fresh medium containing different concentrations of the compounds was added to the culture wells. On the third to ten days, old culture medium in the culture wells was aspirated every day, and fresh culture medium containing compounds at various concentrations was added. The eleventh day, the supernatant in the culture well was collected for extracting HBV DNA in the supernatant. The qPCR experiment detects the HBV DNA content in the supernatant of HepG2.2.15. After collecting cell supernatant, adding CellTiter-Blue reagent, and detecting the fluorescence value of each hole by an enzyme-labeling instrument after incubation.

Percent inhibition:

inhibition [% ], or ] -% ] 0..

Percent cell viability:

cell viability = (fluorescence of sample wells-fluorescence of media control)/(fluorescence of DMSO control-fluorescence of media control) × 100%.

The results of the experiment are shown in table 1.

Table 1.

EXAMPLES Compounds	EC ₅₀(nM)	CC ₅₀(μM)
Example 1	5239	>100
Example 2	6355	>100

Claims

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

wherein R is¹Is selected from
And R is selected from C_1-12Alkyl radical, C_1-12Alkoxy radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl, said R being optionally substituted with one or more groups selected from: halogen, -OH, C_1-6Alkyl, - (OCH)₂CH ₂) _m-OC _1-6Alkyl, -NO₂、-CN、-NH ₂、-NH(C _1-6Alkyl), -N (C)_1-6Alkyl radical)₂、-C(O)C _1-6Alkyl, -C (O) OC_1-6Alkyl, -CONHC_1-6Alkyl, -SH, -SC_1-6Alkyl radical, C_3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl, wherein m is selected from 0, 1,2,3,4 or 5;

R ²selected from H, -OH, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_3-10Cycloalkyl, 3-10 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl;

R ³is selected from
And

R ⁴selected from H, C_1-6Alkyl radical, C_3-6Cycloalkyl or 3-6 membered heterocycloalkyl.
The compound of claim 1, wherein
And
combine to form
Wherein
Is a monovalent cation; or
Is an alkali metal cation or an ammonium cation; or
Is Na⁺、K ⁺、Li ⁺Or NH4⁺(ii) a Or
Is Na⁺Or NH4⁺(ii) a Or
Is NH4⁺。
The compound of any one of claims 1-2, wherein m is selected from 0, 1,2, or 3; or m is selected from 0, 1 or 2; or m is 0.
The compound of any one of claims 1-3, wherein R is selected from C_1-6Alkyl radical, C_1-6Alkoxy radical, C_2-6Alkenyl or C_2-6Alkynyl, said R being optionally substituted with one or more groups selected from- (OCH)₂CH ₂) _m-OCH ₃、C _3-6Cycloalkyl, 3-6 membered heterocycloalkyl, C_6-10Aryl or 5-10 membered heteroaryl; or R is selected from C_1-6Alkyl or C_1-6Alkoxy, and said R is optionally substituted with one or more C_3-6Cycloalkyl substitution; or R is selected from (CH)₃) ₂CHO-、(CH ₃CH ₂) ₂CHO-、(CH ₃) ₂CHCH ₂O-、(CH ₃CH ₂) ₂CHCH ₂O-、(CH ₃) ₃CCH ₂O-, cyclopropyl-CH₂O-、(CH ₃) ₂CH-or (CH)₃) ₃C-。
The compound of any one of claims 1-4, wherein R²Selected from H, -OH or halogen; or R²Selected from H or halogen; or R²Selected from H or fluorine; or R²Is H.
The compound of any one of claims 1-5, wherein R⁴Is selected from C_1-6An alkyl group; or R⁴Is selected from C_1-3An alkyl group; or R⁴Is methyl.
The compound of any one of claims 1-6, selected from the following compounds, tautomers, stereoisomers, or pharmaceutically acceptable salts thereof:
the compound of any one of claims 1-6, selected from the group consisting of:
a pharmaceutical composition comprising a compound of any one of claims 1-8, a tautomer, stereoisomer, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
A method of treating or inhibiting HBV infection in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 8, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 9.
Use of a compound of any one of claims 1-8, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 9, in the manufacture of a medicament for the prevention or treatment of HBV infection in a mammal; optionally, the preventing or treating of HBV infection in a mammal refers to controlling, reducing or eliminating HBV to prevent, ameliorate or cure liver disease in an infected mammal.
A compound of any one of claims 1-8, a tautomer, a stereoisomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 9 for use in the prevention or treatment of HBV infection in a mammal; optionally, the preventing or treating HBV infection in a mammal refers to controlling, reducing or eliminating HBV to prevent, ameliorate or cure liver disease in an infected mammal.