CN113164506B - Dinucleotide compounds and prodrugs thereof - Google Patents

Dinucleotide compounds and prodrugs thereof Download PDF

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Publication number
CN113164506B
CN113164506B CN201980079681.6A CN201980079681A CN113164506B CN 113164506 B CN113164506 B CN 113164506B CN 201980079681 A CN201980079681 A CN 201980079681A CN 113164506 B CN113164506 B CN 113164506B
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compound
methyl
tetrahydrofuran
methoxyethoxy
purin
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CN113164506A (en
Inventor
张寅生
刘保民
朱炎
盖阔
黄雨
尧超
童顺禹
张洋
王晓金
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Abstract

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

Description

Dinucleotide compounds and prodrugs thereof
Cross Reference to Related Applications
The present application claims priority and equity to chinese patent application No. 201811487263.4 filed on date 12 and 06 in 2018 to the chinese national intellectual property agency, the disclosure of which is incorporated herein by reference in its entirety.
Technical Field
The present application relates to dinucleotide compounds and prodrugs thereof, to processes for their preparation, to pharmaceutical compositions containing the compounds, and to their use in the treatment of Hepatitis B Virus (HBV) infection and HBV-related liver diseases.
Background
Chronic viral hepatitis b is currently incurable and can only be controlled, and two anti-HBV drugs that have been accepted in the clinic include interferon-alpha and nucleoside analogues, but there are often rapid onset of resistance and dose-limiting toxicity issues associated with treatment. In addition, many current drugs suffer from poor absorption, distribution, metabolism and/or excretion (ADME) properties, preventing their wider use. Poor ADME properties are also a major cause of drug candidates failing in clinical trials.
The existing oligonucleotide drugs such as SB9000 and the precursor SB9200 thereof have both safety and antiviral effects in chronic hepatitis B patients without treatment and liver cirrhosis, but there is still a need to develop compounds with better anti-HBV activity, safety, prodrug release active compound speed and bioavailability, higher stability and certain liver targeting.
Summary of The Invention
The present application relates to compounds of the general formula I, their tautomers, stereoisomers or pharmaceutically acceptable salts thereof,
wherein R is 1 Selected from the group consisting of
R is selected from C 1-12 Alkyl, C 1-12 Alkoxy, 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, said R group optionally substituted with one or more groups selected from: halogen, -OH, C 1-6 Alkyl, - (OCH) 2 CH 2 ) m -OC 1-6 Alkyl, -NO 2 、-CN、-NH 2 、-NH(C 1-6 Alkyl), -N (C) 1-6 Alkyl group 2 、-C(O)C 1-6 Alkyl, -C (O) OC 1-6 Alkyl, -CONHC 1-6 Alkyl, -SH, -SC 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl or 5-10 membered heteroaryl, wherein m is selected from 0, 1, 2, 3, 4 or 5;
R 2 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;
R 3 selected from the group consisting of
R 4 Selected from H, C 1-6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocycloalkyl.
In another aspect, the present application relates to a pharmaceutical composition comprising a compound of formula I of the present application, a 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 comprising administering to a 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, stereoisomer or pharmaceutically acceptable salt thereof or a pharmaceutical composition as described above, for the manufacture 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, stereoisomer or 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 use in the prevention or treatment of HBV infection in a mammal.
Detailed Description
The present application relates to compounds of the general formula I, their tautomers, stereoisomers or pharmaceutically acceptable salts thereof,
wherein R is 1 Selected from the group consisting of
R is selected from C 1-12 Alkyl, C 1-12 Alkoxy, 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, said R group optionally substituted with one or more groups selected from: halogen, -OH, C 1-6 Alkyl, - (OCH) 2 CH 2 ) m -OC 1-6 Alkyl, -NO 2 、-CN、-NH 2 、-NH(C 1-6 Alkyl), -N (C) 1-6 Alkyl group 2 、-C(O)C 1-6 Alkyl, -C (O) OC 1-6 Alkyl, -CONHC 1-6 Alkyl, -SH, -SC 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl or 5-10 membered heteroaryl, wherein m is selected from 0, 1, 2, 3, 4 or 5;
R 2 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;
R 3 selected from the group consisting of
R 4 Selected from H, C 1 - 6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocycloalkyl.
The present application relates to compounds of the general formula I or pharmaceutically acceptable salts thereof,
wherein R is 1 Selected from the group consisting of
R is selected from C 1-12 Alkyl, C 1-12 Alkoxy, 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, said R optionally substituted with: halogen, -OH, C 1-6 Alkyl, - (OCH) 2 CH 2 ) m -OC 1-6 Alkyl, -NO 2 、-CN、-NH 2 、-NH(C 1-6 Alkyl), -N (C) 1-6 Alkyl group 2 、-C(O)C 1-6 Alkyl, -C (O) OC 1-6 Alkyl, -CONHC 1-6 Alkyl, -SH, -SC 1-6 Alkyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl or 5-10 membered heteroaryl, wherein m is selected from 0, 1, 2, 3, 4 or 5;
R 2 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;
R 3 selected from the group consisting of
R 4 Selected from H, C 1 - 6 Alkyl, C 3-6 Cycloalkyl or 3-6 membered heterocycloalkyl.
In some embodiments of the present invention, in some embodiments,and->Combine to form->Wherein->Is a monovalent cation. In some embodiments, the->Can be an alkali metal cation or an ammonium cation, e.g., na + 、K + 、Li + Or NH 4 + . In some embodiments, the->Can be Na + Or NH 4 + The method comprises the steps of carrying out a first treatment on the surface of the In some embodiments, the->Can be NH 4 + . 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, R 1 Selected from the group consisting of
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 embodimentsIn the scheme, R is selected from C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl, said R optionally substituted with one or more groups selected from: - (OCH) 2 CH 2 ) m -OCH 3 、C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, C 6-10 Aryl or 5-10 membered heteroaryl; in some embodiments, R is selected from C 1-6 Alkyl or C 1-6 Alkoxy, said R optionally being substituted with one or more C 3-6 Cycloalkyl substitution; in some embodiments, R is selected from (CH 3 ) 2 CHO-、(CH 3 CH 2 ) 2 CHO-、(CH 3 ) 2 CHCH 2 O-、(CH 3 CH 2 ) 2 CHCH 2 O-、(CH 3 ) 3 CCH 2 O-, cyclopropyl-CH 2 O-、(CH 3 ) 2 CH-or (CH) 3 ) 3 C-。
In some embodiments, R 2 Selected from H, -OH or halogen; in some embodiments, R 2 Selected from H or halogen; in some embodiments, R 2 Selected from H or fluorine. In some embodiments, R 2 Selected from H.
In some embodiments, R 3 Selected from the group consisting of
In some embodiments, R 4 Selected from C 1-6 An alkyl group; in some embodiments, R 4 Selected from C 1-3 An alkyl group; in some embodiments, R 4 Is 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 compounds of formula I of the present application, their tautomers, stereoisomers, or pharmaceutically acceptable salts thereof, are selected from the following compounds, their tautomers, stereoisomers, or pharmaceutically acceptable salts thereof:
In some embodiments, the compounds of formula I of the present application, their tautomers, stereoisomers, or pharmaceutically acceptable salts thereof, are selected from the group consisting of:
in another aspect, the present application relates to a pharmaceutical composition comprising a compound of formula I of the present application, a tautomer, stereoisomer or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical compositions of the present application further comprise a pharmaceutically acceptable adjuvant.
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, stereoisomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, for the manufacture 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, stereoisomer or 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, stereoisomer or 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 application, the treatment of HBV infection refers to controlling, reducing or eliminating HBV to alleviate or cure liver disease in an infected mammal or patient.
Definition of the definition
The following terms used in the present application have the following meanings unless otherwise indicated. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.
The term "substituted" means that any one or more hydrogen atoms on a particular atom or group is substituted with a substituent, provided that the valence 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 occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl "optionally" substituted with halogen means that ethyl may be unsubstituted (CH 2 CH 3 ) Monosubstituted (e.g. CH 2 CH 2 F) Polysubstituted (e.g. CHFCH 2 F、CH 2 CHF 2 Etc.) or fully substituted (CF) 2 CF 3 ). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, no substitution or pattern of substitution is introduced that is sterically impossible and/or synthetic.
C herein m-n Meaning that the moiety has an integer number of carbon atoms in the given range. For example "C 1-6 "means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atomsCarbon atoms, 5 carbon atoms, or 6 carbon atoms.
When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if one group is substituted with 2R's, then each R has an independent option.
The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.
The term "alkyl" refers to a compound of the formula C n H 2n+1 Is a hydrocarbon group of (a). The alkyl group may be linear or branched. For example, the term "C 1-12 Alkyl "refers to an alkyl group containing 1 to 12 carbon atoms; the term "C 1-6 Alkyl "refers to an alkyl group containing 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, etc.). Similarly, the alkyl portion (i.e., alkyl) of alkoxy, alkylamino, dialkylamino, alkanoyl, alkylsulfonyl and alkylthio has the same definition as above.
The term "alkoxy" refers to an-O-alkyl group.
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 consisting of carbon and hydrogen atoms having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C.ident.CH), 1-propynyl (-C.ident.C-CH) 3 ) 2-propynyl (-CH) 2 -C.ident.CH), 1, 3-butadienyl (-C.ident.C-C.ident.CH), and the like.
The term "cycloalkyl" refers to a carbocycle that is fully saturated and may exist as 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 ring, a 3 to 7 membered ring, or a 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 groups include, but are not limited to, ethylene oxide, ethylene nitride, non-limiting examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, thietanyl, examples of 5-membered heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, tetrahydropyrazolyl, examples of 6-membered heterocycloalkyl groups include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, 4-thiaalkyl, 1, 4-dioxanyl, thiomorpholinyl, 1, 3-dithianyl, 1, 4-dithianyl, examples of 7-membered heterocycloalkyl groups include, but are not limited to, azepanyl, oxepinyl, thiepanyl. Preferred are monocyclic heterocycloalkyl groups having 5 or 6 ring atoms.
The term "aryl" refers to an all-carbon monocyclic or fused-polycyclic aromatic ring radical having a conjugated pi-electron system. For example, an 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, anthryl, and 1,2,3, 4-tetrahydronaphthalene, and the like.
The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one (e.g., 1,2,3,4, 5, or 1 to 3) heteroatom selected from N, O and S as a ring atom, the remaining ring atoms being C and having at least one aromatic ring. Heteroaryl groups may be 5 to 10 membered aromatic rings 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 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 "treatment" means administration of a compound or formulation of the 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, i.e., inhibiting its progression;
(ii) The disease or condition is alleviated, even if the disease or condition subsides.
The term "therapeutically effective amount" means an amount of a compound of the application that (i) treats or prevents a particular disease, disease state, or condition, (ii) reduces, ameliorates, or eliminates one or more symptoms of the particular disease, disease state, or condition, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, disease state, or condition described herein. The amount of a compound of the present application that constitutes a "therapeutically effective amount" will vary depending on the compound, the 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 one of ordinary skill in the art based on his own knowledge and the present 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.
Examples of pharmaceutically acceptable salts include metal salts (for example, alkali metal salts, alkaline earth metal salts, aluminum salts, iron salts, etc.), ammonium salts, salts with organic bases (for example, organic amines), 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 comprising one or more compounds of the application, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the application to an organism.
The term "pharmaceutically acceptable excipients" refers to those excipients which have no significant irritating effect on the organism and which do not impair the biological activity and properties of the active compound. Suitable excipients are well known to the person 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 "include" and variations thereof such as "comprises" or "comprising" are to be interpreted in an open, non-exclusive sense, i.e. "including but not limited to.
The compounds and intermediates of the application may also exist in different tautomeric forms and all such forms are included within the scope of the 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 known as proton transfer tautomers) include tautomers via proton transfer, such as keto-enol and imine-enamine isomerisation. A specific example of a proton tautomer is an imidazole moiety, where a proton can migrate between two ring nitrogens. Valence tautomers include tautomers by recombination of some bond-forming electrons.
The compounds of the application may be asymmetric, e.g., have one or more stereoisomers. Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are included within the scope of the application. The compounds of the application containing asymmetric carbon atoms can be isolated in optically pure or racemic form. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.
The compounds of the application may exist in particular geometric 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, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the 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 may be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, for example, in 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 the compounds of the application or pharmaceutically acceptable salts thereof or pharmaceutical compositions 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 may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, freeze-drying, and the like.
In some embodiments, the pharmaceutical composition is in the form of an oral dosage form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present application to be formulated into tablets, pills, troches, dragees, capsules, liquids, gels, slurries, suspensions and the like for oral administration to a patient.
The solid oral compositions may be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: the active compound is mixed with solid auxiliary materials, the resulting mixture is optionally milled, if desired with other suitable auxiliary materials, and the mixture is then processed to granules, giving a tablet or dragee core. 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 suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.
In all methods of administration of the compounds of formula I described herein, the dosages administered daily are from 0.01mg/kg body weight to 200mg/kg body weight, either as individual doses or as 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 set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present application.
The chemical reactions of the embodiments of the present application are accomplished 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 on the basis of the embodiments already present.
An important consideration in the art of synthetic route planning is the selection of suitable protecting groups for reactive functionalities (e.g. hydroxyl groups in the present application), for example, reference may be made to Greene's Protective Groups in Organic Synthesis (4 th Ed). Hoboken, new Jersey: john Wiley & Sons, inc.
In some embodiments, the compounds of formula I of the present application may be prepared by one skilled in the art of organic synthesis by the following route:
route 1:
route 2:
wherein R is 1 、R、R 2 、R 4 The definition is the same as the compound of the general formula I.
The application adopts the following abbreviations:
me represents methyl; TBDPS represents tert-butyldiphenylsilyl; DMSO stands for dimethyl sulfoxide.
The application is further illustrated by examples, which are not intended to limit the scope of the application, for clarity. All reagents used in the present application are commercially available and can be 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: (2 r,3as,9 ar) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3-hydroxy-2, 3a,9 a-tetrahydro-6H-furo [2',3':4,5] oxazolo [3,2-a ] pyrimidin-6-one
To the reaction flask was added sequentially (2 r,3as,9 ar) -3-hydroxy-2-hydroxymethyl-2, 3a,9 a-tetrahydro-6H-furo [2',3':4,5] oxazolo [3,2-a ] pyrimidin-6-one (30 g), 4' - (chloro (phenyl) methylene) bis (methoxybenzene) (49.4 g), N, N-dimethylpyridin-4-amine (0.12 g), N, N-dimethylformamide (120 ml) and pyridine (360 ml), and stirred at room temperature for 22 hours. The reaction was concentrated, and the residue was dissolved in methylene chloride and washed with 5% sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give a crude product, which was purified by beating with methyl tert-butyl ether to give the title compound (45.74 g).
1 H 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
To the flask was added magnesium strip (7.28 g) and 2-methoxyethyl-1-ol (236 ml) in this order, and the mixture was reacted at 110℃for 10 minutes. The reaction solution was cooled to room temperature, and (2 r,3as,9 ar) -2- ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3-hydroxy-2, 3a,9 a-tetrahydro-6H-furo [2',3':4,5] oxazolo [3,2-a ] pyrimidin-6-one (35 g), heated to 80 ℃ and reacted for 9h. 600ml of water and 600ml of methylene chloride were added to the reaction solution in an ice bath, the pH of the reaction solution was adjusted to about 7 with glacial acetic acid, the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was purified by column chromatography (methylene chloride: methanol=50:1) to give the title compound (32 g).
1 H 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).
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) tetrahydrofuranl-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 (32 g) was dissolved in dichloromethane (150 ml), and a solution of 3- ((di (diisopropylamino) phosphono) oxy) propionitrile (20.15 g) and pyridine-1-ium 2, 2-trifluoroacetate (12.26 g) in dichloromethane (50 ml) was slowly added dropwise in this order, and the reaction solution was stirred at room temperature for 2h. The reaction solution was concentrated to obtain a crude product, which was purified by column chromatography (petroleum ether: ethyl acetate=1:1) to obtain the title compound (15.11 g). MS (ESI) m/z 827.5[ M+Na ] + 。
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) tetrahydrofuranl-3-yl (2-cyanoethyl) diisopropylphosphoramidite (13 g)) N- (9- ((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (8.73 g) in N, N-dimethylformamide (60 ml) and 1H-tetrazole (2.615 g) in acetonitrile (83 ml) were slowly added dropwise in this order, and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated to obtain a crude product, which was purified by column chromatography (dichloroganane: methanol=20:1) to obtain the title compound (3.86 g). MS (ESI) m/z 1081.3[ M+Na] + 。
Step 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 (3.58 g) was dissolved in acetonitrile (40 ml), a solution of 3H-benzo [ c ] [1,2] dithiol-3-one 1, 1-dioxide (0.691 g) in acetonitrile (10 ml) was added dropwise, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated to obtain a crude product, which was purified by column chromatography (dichloroganane: methanol=20:1) to obtain the title compound (1.37 g).
1 H 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) thiophosphate
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 (1.37 g) was dissolved in methylene chloride (13 ml), and 2, 2-dichloroacetic acid (0.248 g) was added dropwise thereto, and the reaction mixture was stirred at room temperature for 1 hour. The reaction solution was adjusted to pH 7 with triethylamine, concentrated to give a crude product, and purified by column chromatography (dichlorosilane: methanol=10:1) to give the title compound (0.51 g).
1 H 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).
Step 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) -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) phosphorothioate (410 mg) was resolved by YMC high pressure preparative chromatography (column: YMC Triart C18 high pressure dynamic axial compression column, 10 μm, 50X 235mm; mobile phase: water and acetonitrile (25% acetonitrile isocratic elution 50 min; detector: UV 260 nm), the eluent was concentrated to give the title compound (146 mg).
1 H 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).
Step 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) -thiophosphate (13 mg) was dissolved in aqueous ammonia (1 ml), and the reaction solution was stirred at room temperature for 17 hours. 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 C18 g reverse phase chromatography (water: acetonitrile=3:1) to give the title compound (70 mg).
1 H 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) -thiophosphate
The procedure of step 7 in example 1 was followed to obtain the title compound (158 mg).
1 H 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).
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
The title compound was obtained by the procedure of step 8 in example 1 starting from O- (((2 r,3S,5 r) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2 r,3r,4r,5 r) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2-hydroxymethyl-4- (2-methoxyethoxy) tetrahydrofuran-3-yl) (S) -thiophosphate.
1 H 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,7 aS) -3 a-methyl-2- ((perfluorophenyl) thio) -6- (prop-1-en-2-yl) hexahydrobenzo [ d ] [1,3,2] oxathiaphospholane 2-sulfide
Triethylamine bis (perfluorophenyl) dithiophosphate (126 g), (1R, 4R, 6S) -1-methyl-4- (prop-1-en-2-yl) -7-oxabicyclo [4.1.0] heptane (48.3 g) and dibutyl hydrogen phosphate (48.9 g) were successively added to chloroform (1200 ml) in a reaction flask, and then 2, 2-dichloroacetic acid (60.0 g) was slowly added dropwise to the above solution to react at room temperature for 1h. After the reaction solution was concentrated to 500ml, 1000ml of n-hexane was added to the reaction solution to dilute the solution, and the solution was washed with 10% potassium dihydrogen phosphate solution and saturated brine, respectively. 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, whereby a solid was precipitated. Cooling to 0 ℃ in an ice bath, and filtering to obtain 93.48g of crude product. The crude product (93.48 g) was dissolved in 225ml of dichloromethane, then 374ml of n-heptane was added, the crude product being completely dissolved. The solution was concentrated to 200ml and additional 374ml of n-heptane was added and the procedure repeated 3 times. After the solution was concentrated to 200ml, a large amount of solids precipitated and stirred at room temperature for 2 hours. Filtration and drying of the filter cake gave the title compound (62.58 g).
1 H 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-butyldiphenylsilanyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R, 3aS,6R,7 aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiaphospholan-2-yl) oxy) tetrahydrofuran-2-yl) pyrimidine-2, 4- (1H, 3H) -dione
1- ((2R, 3R,4R, 5R) -5- (((tert-butyldiphenylsilanyl) oxy) methyl) -4-hydroxy-3- (2-methoxyethoxy) tetrahydrofuran-2-yl) pyrimidine-2, 4 (1H, 3H) -dione (35 g) and (2S, 3aS,6R,7 aS) -3 a-methyl-2- ((perfluorophenyl) thio) -6- (prop-1-en-2-yl) hexahydrobenzo [ d ] [1,3,2] oxathiaphospholane 2-sulphide (37.6 g) were dissolved in acetonitrile (645 ml) and cooled in an ice bath. 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] azepine (12.81 g) was slowly added dropwise to the reaction mixture. The reaction was continued for 2h in ice bath. 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, and 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).
1 H 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).
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-hydrogen (S) -thiophosphate
1- ((2R, 3R,4R, 5R) -5- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R, 3aS,6R,7 aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiaphospholan-2-yl) oxy) tetrahydrofuran-2, 4- (1H, 3H) -dione (25 g), N- (9- ((2R, 4S, 5R) -4- ((tert-butyldiphenylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (37.7 g) was dissolved in acetonitrile (235 ml), and 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] aza (14.51 g) was added at room temperature and the reaction solution was stirred for 1H. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium hydrogencarbonate and 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 (33.01 g).
1 H 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).
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 the reaction flask were successively 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-hydrogen (S) -thiophosphate (25 g), tetrabutylammonium fluoride trihydrate (19.52 g) and tetrahydrofuran (17 ml), and 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 into the next step without treatment.
Step 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
To the reaction flask were successively added 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 (36 g) and aqueous ammonia (260 ml), and the mixture was stirred at room temperature for reaction for 16 hours. Under ice bath, 50% aqueous acetic acid solution was added dropwise to the reaction solution, and the pH was adjusted to about 8. The reaction solution was extracted twice with ethyl acetate, and an aqueous phase was separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1) and concentrated to give the title compound (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 the reaction flask was added in order 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 (0.30 g), sodium ion exchange resin (6.0 g) and deionized water (5 ml), and stirred at room temperature for 1H. The aqueous phase was filtered and purified by Biotage C18 g reverse phase chromatography (water: acetonitrile=3:1) and concentrated to give the title compound (0.16 g).
1 H 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) -thiophosphate sodium
Step 1: o- (((2R, 3R,4R, 5R) -5- (6-benzoylamino-9H-purin-9-yl) -3- ((tert-butyldiphenylsilanyl) 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-hydrogen (S) -thiophosphate
1- ((2R, 3R,4R, 5R) -5- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R, 3aS,6R,7 aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiolan-2-yl) oxy) tetrahydrofuran-2, 4- (1H, 3H) -dione (1.50 g) and N- (9- ((2R, 3R,4R, 5R) -4- ((tert-butyldiphenylsilyl) oxy) -3-fluoro-5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purin-6-yl) benzamide (2.33 g) were dissolved in acetonitrile (30 ml), 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] azepin (0.87 g) were added at room temperature, and stirred and reacted for 1H. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium hydrogencarbonate and 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).
1 H 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
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To the reaction flask were successively 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-hydrogen (S) -thiophosphate (1.80 g), tetrabutylammonium fluoride trihydrate (1.15 g) and tetrahydrofuran (15 ml), and the reaction solution 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 taken directly into the next step without purification.
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 the reaction flask were successively 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 (2.10 g) and aqueous ammonia (15 ml), and the mixture was stirred at room temperature and reacted overnight. The reaction solution was adjusted to pH 8 with 50% aqueous acetic acid in ice bath. The reaction mixture was washed with ethyl acetate, and an aqueous phase was separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1) and concentrated to give the title compound (0.90 g). MS (ESI) m/z:648.7[ M-H ] ] - .
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) -thiophosphate sodium
To the reaction flask was added in order 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 (0.30 g), sodium ion resin (6.0 g) and deionized water (5 ml) and stirred at room temperature for 1H. The aqueous phase was filtered and purified by Biotage C18 g reverse phase chromatography (water: acetonitrile=3:1) and concentrated to give the title compound (0.15 g).
1 H 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) -thiophosphate sodium
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-hydrogen (S) -thiophosphate
1- ((2R, 3R,4R, 5R) -5- (((tert-butyldiphenylsilyl) oxy) methyl) -3- (2-methoxyethoxy) -4- (((2R, 3aS,6R,7 aS) -3 a-methyl-6- (prop-1-en-2-yl) -2-sulfonylhexahydrobenzo [ d ] [1,3,2] oxathiolan-2-yl) oxy) tetrahydrofuran-2, 4- (1H, 3H) -dione (1.50 g) 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 (2.20 g) were dissolved in acetonitrile (20 ml), 2,3,4,6,7,8,9, 10-octahydropyrimido [1,2-a ] azepin (0.87 g) was added at room temperature and stirred for 1H. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium hydrogencarbonate and 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 (2.10 g).
1 H 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) tetrahydrofurane-3-yl) O- (((2R, 3S, 5R) -3-hydroxy-5- (2-isobutyrylamino-6-oxo-1, 6-dihydro-9H-purin-9-yl) tetrahydrofurane-2-yl) methyl) (S) -tetrabutylammonium thiophosphate
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To the reaction flask was added in order 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-hydrogen (S) -thiophosphate (1.80 g), tetrabutylammonium fluoride trihydrate (1.43 g) and tetrahydrofuran (20 ml), and the reaction mixture was stirred overnight at room temperature. The reaction solution was diluted with water, washed with diethyl ether and the aqueous phase was concentrated to give 2.20g of the title compound, which was taken directly into the next step without purification.
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 (2.20 g) was dissolved in aqueous ammonia (15 ml), stirred at 40℃and reacted for 36H under ice bath, the reaction solution was adjusted to pH 8 with 50% aqueous acetic acid, the reaction solution was washed with ethyl acetate, the aqueous phase was separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1) to give the title compound (0.17 g).
MS(ESI)m/z：646.6[M-H] - .
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) -thiophosphate sodium
To the reaction flask was added in order 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 (0.30 g), sodium ion exchange resin (6.0 g) and deionized water (5 ml) and stirred at room temperature for 1H. The aqueous phase was filtered and purified by Biotage C18 g reverse phase chromatography (water: acetonitrile=3:1) and concentrated to give the title compound (0.16 g).
1 H 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) methyl isopropyl 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- (((2 r,3s,5 r) -5- (6-benzoylamino-9H-purin-9-yl) -3-hydroxytetrahydrofuran-2-yl) methyl) O- (2-cyanoethyl) O- ((2 r,3r,4r,5 r) -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 example 1 was referenced to give 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) methyl isopropyl 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 (1.56 g) was dissolved in a mixed solution of water (150 ml) and isopropyl alcohol (39 ml), iodomethyl isopropyl carbonate (1.761 g) was added dropwise, reacted at room temperature for 24 hours, concentrated to remove isopropyl alcohol, and the residue was extracted three times with n-hexane. The aqueous phase was separated and purified by Biotage C18 g reverse phase chromatography (water: acetonitrile=3:1) to give the title compound (1.39 g). MS (ESI) m/z 748.2[ M+H ] ] + .
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) methyl isopropyl 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) methyl isopropyl carbonate (1.39 g) was resolved by YMC high pressure preparative chromatography (column: CHIRAL ART Amylose-SA,5 μm, 30X 250mm; mobile phase: absolute ethanol and n-hexane (50% n-hexane isocratic elution for 45 min); a detector: UV 254 nm) to give the title compound (0.527 g).
1 H NMR(500MHz，CD 3 OD)：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) methyl isopropyl carbonate
With reference to the procedure of step 3 of example 6, the title compound (0.456 g) was additionally obtained.
1 H NMR(500MHz，CD 3 OD)：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 (1.0 g) was dissolved in a mixed solution of water (120 ml) and isopropanol (30 ml), iodomethylpent-3-ylcarbonate (0.935 g) was added dropwise, reacted at room temperature for 24 hours, and concentrated to remove isopropanol. After the aqueous phase was extracted three times with n-hexane, it was separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1), and concentrated to give the title compound (0.400 g).
1 H NMR(500MHz，CD 3 OD)：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 (1.0 g) was dissolved in a mixed solution of water (100 ml) and isopropyl alcohol (26 ml), and iodomethyl isobutyl carbonate (0.887 g) was added dropwise thereto for reaction at room temperature for 24 hours. After isopropanol was removed by concentration, the aqueous phase was extracted three times with n-hexane and separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1), the title compound (0.419 g) was obtained after concentration.
1 H NMR(500MHz，CD 3 OD)：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 (1.0 g) was dissolved in a mixed solution of water (120 ml) and isopropanol (30 ml), and 2-ethylbutyl (iodomethyl) carbonate (0.983 g) was added dropwise at room temperature for 24 hours. After isopropanol was removed by concentration, the aqueous phase was extracted three times with n-hexane and separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1), the title compound (0.440 g) was obtained after concentration.
1 H NMR(500MHz，CD 3 OD)：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) methyl neopentyl 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 (1.0 g) was dissolved in a mixed solution of water (120 ml) and isopropanol (30 ml), and iodomethyl neopentyl carbonate (0.935 g) was added dropwise thereto for reaction at room temperature for 24 hours. After isopropanol was removed by concentration, the aqueous phase was extracted three times with n-hexane and separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1), and concentrated to give the title compound (0.250 g).
1 H NMR(500MHz，CD 3 OD)：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 (1.0 g) was dissolved in a mixed solution of water (100 ml) and isopropyl alcohol (26 ml), and cyclopropylmethyl (iodomethyl) carbonate (0.880 g) was added dropwise thereto for reaction at room temperature for 24 hours. After isopropanol was removed by concentration, the aqueous phase was extracted three times with n-hexane and separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1), the title compound (0.400 g) was obtained after concentration.
1 H NMR(500MHz，CD 3 OD)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) isobutyric 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 (1.0 g) was dissolved in a mixed solution of water (120 ml) and isopropyl alcohol (30 ml), and iodomethyl isobutyrate (0.784 g) was added dropwise thereto for reaction at room temperature for 24 hours. After isopropanol was removed by concentration, the aqueous phase was extracted three times with n-hexane and separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1), and concentrated to give the title compound (0.330 g).
1 H NMR(500MHz，CD 3 OD)：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 (1.0 g) was dissolved in a mixed solution of water (120 ml) and isopropyl alcohol (30 ml), and iodomethyl pivalate (0.832 g) was added dropwise at room temperature for reaction for 24 hours. After isopropanol was removed by concentration, the aqueous phase was extracted three times with n-hexane and separated and purified by a Biotage C18 g reverse phase chromatography column (water: acetonitrile=3:1), the title compound (0.550 g) was obtained after concentration.
1 H NMR(500MHz，CD 3 OD)：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] + .
In vitro anti-hepatitis B virus activity screening
1. Experimental materials
1.1 cells
HepG2.2.15 cells
1.2 Compounds
Test compounds: the compound prepared in the examples was formulated as a 20mM stock solution using dimethyl sulfoxide (DMSO).
1.3 reagents
The reagents used in this experiment included QIAamp 96 DNA Blood Kit (12) (Qiagen-51162), fastStart Universal Probe Master (Roche-04914058001), cellTiter-Blue detection reagent (Promega-G808B).
2. Experimental method
2.1 dilution of compound: the initial final concentration of test compound was 100. Mu.M, 3-fold dilution, 8 concentrations.
3.2. In vitro anti-HBV activity assay and cytotoxicity assay: on the first day, hepG2.2.15 cells (4X 10) 4 Cell/well) to 96-well plates at 37 ℃,5% co 2 Culturing overnight. The next day, fresh culture broth containing different concentrations of the compounds was added to the culture wells. For the third to ten days, the old culture medium in the culture wells was aspirated daily, and fresh culture medium containing different concentrations of the compound was added. On the tenth day, the supernatant from the culture well was collected for extraction of HBV DNA from the supernatant. qPCR assay to detect HepG2.2.15 supernatantHBV DNA content in (B) cells. After collecting cell supernatant, cellTiter-Blue reagent is added, and after incubation, the fluorescence value of each well is detected by an enzyme-labeled instrument.
Percentage of inhibition:
inhibition% = (HBV DNA content in 1-sample/HBV DNA content in DMSO control) ×100%.
Percentage of cell viability:
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 5239 ＞100
Example 2 6355 ＞100

Claims (17)

1. A compound of formula I or a pharmaceutically acceptable salt thereof,
wherein R is 1 Selected from the group consisting ofR 2 Selected from H, -OH or halogen;
R 3 selected from the group consisting ofAnd
R 4 selected from C 1-3 An alkyl group.
2. The compound of claim 1, whereinAnd->Combine to form->Wherein->Is a monovalent cation.
3. The compound of claim 2, whereinAnd->Combine to form->Wherein->Is an alkali metal cation or an ammonium cation.
4. The compound of claim 2, whereinAnd->Combine to form->Wherein->Is Na (Na) + 、K + 、Li + Or NH4 + 。
5. The compound of claim 2, whereinAnd->Combine to form->Wherein->Is Na (Na) + Or NH4 + 。
6. The compound of claim 2, whereinAnd->Combine to form->Wherein->Is NH4 + 。
7. The compound of claim 1, wherein R 2 Selected from H or halogen.
8. The compound of claim 7, wherein R 2 Selected from H or fluorine.
9. The compound of claim 7, wherein R 2 Selected from H.
10. The compound of claim 1, wherein R 4 Is methyl.
11. The compound of claim 1, selected from the following compounds or pharmaceutically acceptable salts thereof:
12. the compound of claim 1, selected from the group consisting of:
13. a pharmaceutical composition comprising a compound of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
14. Use of a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, in the manufacture of a medicament for the prevention or treatment of HBV infection in a mammal.
15. The use of claim 14, wherein the prevention or treatment of HBV infection in a mammal is controlling, reducing or eliminating HBV to prevent, ameliorate or cure liver disease in the infected mammal.
16. A compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for use in the prevention or treatment of HBV infection in a mammal.
17. A compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for use in the prevention or treatment of HBV infection in a mammal, wherein the prevention or treatment of HBV infection in a mammal is the control, reduction or elimination of HBV to prevent, ameliorate or cure liver disease in the infected mammal.
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