Antiviral compounds and uses thereof

Abstract

The present disclosure relates to antiviral compounds and uses thereof. Specifically, the present disclosure relates to a β-D-N 4 The invention relates to a phosphate compound of hydroxycytidine, a pharmaceutical composition comprising such a compound, and the use of such a compound and a pharmaceutical composition comprising such a compound for inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

Description

Technical Field

[0001] The present invention relates to a β-DN having broad-spectrum antiviral activity. 4 The present invention relates to a phosphate ester compound of -hydroxycytidine, a pharmaceutical composition comprising such a compound, and the use of such a compound and a pharmaceutical composition comprising such a compound for inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof. Background Art

[0002] Viral infection is a major global public health problem. Viruses invade the body through a variety of pathways and multiply in susceptible host cells, causing host disease. 4 -Hydroxycytidine (NHC, EIDD-1931) is a derivative of cytosine nucleoside (i.e., cytidine) and has the following structural formula:

[0003]

[0004] This substance has been found to have a broad spectrum of antiviral activity (see, for example, Stuyver et al., Antimicrob Agents Chemother, 2003, 47(1):244-54; Purohit et al., J Med Chem, 2012, 55(22):9988-9997; Ivanov et al., Collection of Czechoslovak Chem Commun, 2006, 71(7):1099-1106 and Fox et al., JACS, 1959, 81:178-870). However, EIDD-1931 has strong hydrophilicity, which leads to its poor bioavailability and is not suitable as a lead compound for clinical drug development.

[0005] WO 2019 / 113462 discloses a N- 4 -Hydroxycytidine and its derivatives, among which the representative compound is the isobutyrate of EIDD-1931 EIDD2801, having the following structural formula:

[0006]

[0007] Compared with EIDD-1931, the oral bioavailability of EIDD2801 has been improved, but there is also the problem of being degraded by digestive enzymes (such as but not limited to hydrolases) and gastric acid in the gastrointestinal tract, which affects the absorption in the body. At the same time, EIDD2801 itself has no biological activity. EIDD2801 absorbed into the body can only play a role after being hydrolyzed to expose the active structure EIDD-1931, which makes the in vivo activity of EIDD2801 greatly limited by the rate and efficiency of hydrolysis.

[0008] Therefore, there is still a need in the art for additional compounds based on EIDD-1931 that are novel and have broad-spectrum antiviral activity. Summary of the invention

[0009] The present disclosure provides novel β-DN with broad-spectrum antiviral replication activity. 4 -A phosphate ester compound of hydroxycytidine (hereinafter referred to as the compound of the present disclosure).

[0010] Therefore, in a first aspect of the present disclosure, there is provided a compound of formula I or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof:

[0011]

[0012] in,

[0013] m is any integer from 6 to 20;

[0014] n is any integer from 1 to 5;

[0015] X is -O- or -NR0-;

[0016] R0 is H or C1-C6 alkyl;

[0017] Z is -O- or -SS-;

[0018] R is H, C1-C6 alkyl, C6-C 12 Aryl, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-;

[0019] R' is C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 12 aryl, 3 to 12 membered heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O or S, or 5 to 12 membered heteroaryl containing 1 or 2 heteroatoms independently selected from N, O or S;

[0020] The alkyl, cycloalkyl, aryl, heterocyclic or heteroaryl groups in the R or R' group are optionally substituted by 1, 2 or 3 groups independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0021] In some embodiments of this aspect, the compound of Formula I has the structure of Formula I-1:

[0022]

[0023] wherein the variables m, n, X and R are as defined above for compounds of formula I.

[0024] In other embodiments of this aspect, the compound of Formula I has the structure of Formula I-2:

[0025]

[0026] wherein the variables m, n, X and R are as defined above for compounds of formula I.

[0027] In some embodiments of this aspect, the pharmaceutically acceptable salt of the compound of any one of Formula I, I-1 or I-2 is an alkali metal salt (e.g., lithium, sodium, potassium, rubidium, cesium salt), an alkaline earth metal salt (e.g., magnesium, calcium, strontium, barium salt), an aluminum salt, an organic base salt (e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine, imidazole, morpholine, 2-hydroxyethylmorpholine, dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine, benzylamine, tetramethylammonium hydroxide salt) or an amino acid salt (e.g., lysine, arginine salt).

[0028] In a second aspect of the present disclosure, a pharmaceutical composition is provided, comprising a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, and one or more pharmaceutically acceptable carriers.

[0029] In some embodiments of this aspect, the pharmaceutical composition comprises two, three or more pharmaceutically acceptable carriers.

[0030] In some embodiments of this aspect, the pharmaceutical composition is prepared into a pharmaceutically acceptable dosage form (for example, by mixing a compound of any one of Formula I, I-1 or I-2 or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof with one or more pharmaceutically acceptable carriers and preparing it into a pharmaceutically acceptable dosage form according to conventional production methods in the pharmaceutical field).

[0031] In the third aspect of the present disclosure, provided is a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers, which is used for treating or preventing a disease.

[0032] In the fourth aspect of the present disclosure, provided is a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers, which is used for inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

[0033] In the fifth aspect of the present disclosure, provided is a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers for use in inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

[0034] In the sixth aspect of the present disclosure, provided is the use of a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers in the preparation of a medicament for inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

[0035] In the seventh aspect of the present disclosure, a method for preventing and / or treating viral infection in a patient in need thereof is provided, the method comprising administering to a subject in need thereof an effective amount of a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, and one or more pharmaceutically acceptable carriers.

[0036] In some embodiments of the fourth, fifth, sixth, and seventh aspects of the present disclosure, the virus is a coronavirus (e.g., a human coronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, including, for example, strains B.1.1.7, B.1.351, Delta variant B.1.617.2, Omicron variant B.1.1.529, or other SARS-CoV-2 variants), equine encephalitis virus (e.g., Eastern equine encephalitis virus, Western equine encephalitis virus, or Venezuelan equine encephalitis virus), Chikungunya virus, Ross River virus, Orthomyxoviridae virus (e.g., influenza virus, such as influenza A virus, influenza B virus, or influenza C virus), Paramyxoviridae virus (e.g., respiratory syncytial virus), Filovirus (e.g., Ebola virus, Marburg virus, or Kuwa virus), or Zika virus.

[0037] In an eighth aspect of the present disclosure, an intermediate compound for synthesizing a compound of any one of formulas I, I-1 or I-2 of the present disclosure is provided.

[0038] In some embodiments, the intermediate compound is a compound of Formula 2a:

[0039]

[0040] wherein m, n and Z are as defined above for compounds of formula I; R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl; A is chlorine or nitro; B is hydrogen, chlorine or nitro. Preferably, R1 is methyl, ethyl or phenyl, A is chlorine or nitro, and B is hydrogen.

[0041] In some embodiments, the intermediate compound is a compound of Formula 2a-1:

[0042]

[0043] wherein m, n, Z, R0 and R are as defined above for compounds of formula I; R1 is a straight or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R bR1 is independently H or C1-C6 alkyl. Preferably, R1 is methyl, ethyl or phenyl.

[0044] In some embodiments, the intermediate compound is a compound of Formula 4a:

[0045]

[0046] wherein m, n and Z are as defined above for compounds of formula I; R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl; A is chlorine or nitro; B is hydrogen, chlorine or nitro. Preferably, R1 is methyl, ethyl or phenyl, A is chlorine or nitro, and B is hydrogen.

[0047] In some embodiments, the intermediate compound is a compound of Formula 4a-1:

[0048]

[0049] wherein m, n, Z, R0 and R are as defined above for compounds of formula I; R1 is a straight or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)ORa 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl. Preferably, R1 is methyl, ethyl or phenyl.

[0050] In some embodiments, the intermediate compound is a compound of Formula 2-1a:

[0051]

[0052] wherein m, n and Z are as defined above for compounds of formula I; A is chloro or nitro; and B is hydrogen, chloro or nitro. Preferably, A is chloro or nitro and B is hydrogen.

[0053] In some embodiments, the intermediate compound is a compound of Formula 2-1a-1:

[0054]

[0055] wherein m, n, Z, R0 and R are as defined above for compounds of formula I.

[0056] In some embodiments, the intermediate compound is a compound of Formula 4-1a:

[0057]

[0058] wherein m and n are as defined above for compounds of formula I; A is chlorine or nitro; and B is hydrogen, chlorine or nitro. Preferably, A is chlorine or nitro and B is hydrogen.

[0059] In some embodiments, the intermediate compound is a compound of formula 4-1a-1:

[0060]

[0061] wherein m, n, Z, R0 and R are as defined above for compounds of formula I. DETAILED DESCRIPTION

[0062] definition

[0063] It should be understood that, unless otherwise specifically defined herein, the terms used herein should be given their generally recognized meanings in the art. Further, it should be understood that the terms used herein are intended only to describe specific embodiments and are not intended to limit the scope of the present invention.

[0064] Unless otherwise stated, in the event of a discrepancy between the structural formula and the chemical name of a compound described herein, the structural formula controls.

[0065] As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless otherwise stated.

[0066] Unless otherwise stated, the term "comprising (or its equivalents such as "including", "containing", etc.)" or "consisting essentially of includes the case of "consisting of".

[0067] Unless otherwise stated, the ranges described in the present disclosure include the endpoints of the range and each integer contained in the range. For example, "m is any integer from 6 to 20" means that m can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. For another example, "n is any integer from 1 to 5" means that n can be 1, 2, 3, 4 and 5. In addition, any sub-ranges consisting of these integers are intended to be included in the scope of the present disclosure.

[0068] As used herein, the term "compounds of the present disclosure" refers to any one or more compounds falling within the scope of any of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof.

[0069] The prefix "C x -C y " refers to the range of the number of carbon atoms contained in the group (the endpoints of the range and each integer contained in the range and any sub-ranges formed by these integers are intended to be included in the scope of the present disclosure). For example, "C1-C6 alkyl" means an alkyl group containing 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5 or 6 carbon atoms).

[0070] The term "alkyl" as used herein refers to a straight or branched saturated monovalent hydrocarbon radical having a specified number of carbon atoms. Alkyl groups typically contain 1-6 carbon atoms ("C1-C6 alkyl"), more preferably 1-5 carbon atoms ("C1-C5 alkyl"), 1-4 carbon atoms ("C1-C4 alkyl"), or 1-2 carbon atoms ("C1-C2 alkyl"). Examples of preferred C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl. Examples of preferred C1-C4 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0071] The term "alkenyl" as used herein refers to a straight or branched unsaturated monovalent hydrocarbon radical with a specified number of carbon atoms comprising one or more double bonds. Alkenyl generally contains 2-6 carbon atoms ("C2-C6 alkenyl"), more preferably 2-5 carbon atoms ("C2-C5 alkenyl"), 2-4 carbon atoms ("C2-C4 alkenyl") or 2 carbon atoms ("vinyl"). Preferred C2-C6 alkenyls include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadi-1-alkenyl, 1-pentene-3-yl, 2-pentene-1-yl, 3-pentene-1-yl, 3-pentene-2-yl, 1,3-pentadiene-1-yl, 1,4-pentadiene-3-yl, 1-hexene-3-yl and 1,4-hexadiene-1-yl.

[0072] The term "alkynyl" as used herein refers to a straight or branched unsaturated monovalent hydrocarbon radical having a specified number of carbon atoms and containing one or more triple bonds. Alkynyl groups typically contain 2-6 carbon atoms ("C2-C6 alkynyl"), more preferably 2-5 carbon atoms ("C2-C5 alkynyl"), 2-4 carbon atoms ("C2-C4 alkynyl"), or 2 carbon atoms ("ethynyl"). Preferred C2-C6 alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0073] As used herein, the term "alkoxy" refers to an alkyl group attached to a parent molecule via an oxygen atom (i.e., "-O-alkyl"), wherein "alkyl" is as defined above. Alkoxy groups typically contain 1 to 6 carbon atoms ("C1-C6 alkoxy"), more preferably 1 to 4 carbon atoms ("C1-C4 alkoxy"). For example, C1-C4 alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy, etc.

[0074] The term "halogen" used herein refers to fluorine, chlorine, bromine, iodine and the like, with fluorine and chlorine atoms being preferred.

[0075] The term "halo" as used herein refers to one or more hydrogen atoms in a substituent being replaced by one or more identical or different halogen atoms. "Halogen" is as defined above. For example, "halogenated C1-C6 alkyl" refers to a "C1-C6 alkyl" in which one or more hydrogen atoms are replaced by one or more identical or different halogen atoms, wherein "C1-C6 alkyl" is as defined above. For another example, "halogenated C1-C6 alkoxy" refers to a "C1-C6 alkoxy" in which one or more hydrogen atoms are replaced by one or more identical or different halogen atoms, wherein "C1-C6 alkoxy" is as defined above.

[0076] As used herein, the term "cyano" refers to a -CN group.

[0077] As used herein, the term "hydroxyl" refers to an -OH group.

[0078] As used herein, the term "nitro" refers to a -NO2 group.

[0079] As used herein, the term "aryl" refers to a monovalent hydrocarbon radical derived from a monocyclic or fused bicyclic or polycyclic ring system having well-known aromatic characteristics, wherein at least one ring contains a completely conjugated π-electron system. Fused aryl groups may include an aryl ring fused to a saturated or partially unsaturated carbocyclic or heterocyclic ring or to another aryl or heteroaryl ring, provided that the point of attachment to the parent molecule on such fused ring system is an atom of the aromatic portion of the ring system. Typically, aryl groups contain 6-12 carbon atoms ("C6-C 12 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and tetrahydronaphthyl.

[0080] The term "cycloalkyl" as used herein refers to a monovalent hydrocarbon group derived from a non-aromatic saturated carbocyclic ring system containing a specified number of carbon atoms, which can be a monocyclic, spirocyclic, bridged or fused bicyclic or polycyclic ring system connected to the parent molecule through the carbon atoms of the cycloalkyl ring. Typically, the cycloalkyl of the present disclosure contains 3-8 carbon atoms ("C3-C8 cycloalkyl"), preferably 3-7 carbon atoms ("C3-C7 cycloalkyl") or 3-6 carbon atoms ("C3-C6 cycloalkyl"). Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, etc.

[0081] The term "heteroatom" as used herein refers to a N, O or S atom.

[0082] The term "heterocyclyl" as used herein refers to a monovalent group derived from a non-aromatic ring structure containing the specified number of carbon atoms and further comprising at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms as ring members, wherein the heteroatom refers to N, O or S atoms and the S atom is optionally substituted with one or two oxo groups (i.e., S(O) q , wherein q is 0, 1 or 2). Such heterocyclic radicals may be partially unsaturated. Heterocyclic radicals include spirocyclic, bridged or fused rings formed with one or more other heterocyclic or carbocyclic rings, wherein such spirocyclic, bridged or fused rings themselves may be saturated, partially unsaturated or aromatic, provided that the point of attachment to the parent molecule is an atom of the heterocyclic moiety of such a ring system. Typically, "heterocyclic radicals" contain 3 to 12 ring atoms (i.e., 3 to 12 membered heterocyclic radicals), preferably contain 4 to 7 ring atoms (i.e., 4 to 7 membered heterocyclic radicals), and most preferably contain 5 or 6 ring atoms (i.e., 5 or 6 membered heterocyclic radicals), wherein the ring atoms include carbon and non-carbon heteroatoms. In certain embodiments, representative examples of heterocyclyl groups include azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, dihydrooxazolyl, dihydroisoxazolyl, dihydropyrrolyl, dihydroimidazolyl, dihydropyrazolyl, dihydrothiazolyl, piperidinyl, dihydropyridinyl, dihydropyrimidinyl, piperazinyl, dioxanyl, oxathianyl, azepanyl, diazepanyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, morpholinyl, and thiomorpholinyl, and the like.

[0083] The term "heteroaryl" as used herein refers to a monovalent group derived from an aromatic ring structure containing a specified number of carbon atoms and also including at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms as ring members, wherein the heteroatom refers to N, O or S atoms. Typically, heteroaryl contains 5-12 ring atoms ("5-12 membered heteroaryl"), preferably 5-10 ring atoms ("5-10 membered heteroaryl"), more preferably 5 or 6 ring atoms ("5 or 6 membered heteroaryl"), wherein the ring atoms include carbon and non-carbon heteroatoms. The heteroaryl is connected to the parent molecule through the ring atoms of the heteroaromatic ring, thereby maintaining aromaticity. The heteroaryl can also be fused to another aryl or heteroaryl ring, or to a saturated or partially unsaturated carbocyclic or heterocyclic ring, provided that the point of attachment to the base molecule on such a fused ring system is an atom of the heteroaromatic portion of the ring system. In certain embodiments, representative examples of heteroaryl include furanyl, imidazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thienyl, triazolyl, triazinyl, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzothiadiazolyl, benzothiazolyl, imidazopyridinyl, imidazopyrimidinyl, imidazopyridazinyl, cinnolinyl, furopyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, quinolinyl, thienopyridinyl, and the like.

[0084] The term "optionally" as used herein means that the situation described immediately after the term may occur, but may not occur. For example, "the alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl in the R or R' group is optionally replaced by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R bThe term "substituted by substituents" encompasses "the alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl in the R or R' group is replaced by 1, 2 or 3 groups independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxy, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b or "the alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl in the R or R' group is not substituted by 1, 2 or 3 groups independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b There are two cases of "substituent replacement".

[0085] The term "pharmaceutically acceptable" as used herein refers to those compounds or pharmaceutical compositions that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects, such as humans or other mammals, without causing excessive toxicity, irritation, allergic response or other problems, and with a reasonable benefit / risk ratio commensurate.

[0086] The term "pharmaceutically acceptable salt" as used herein refers to an acid or base addition salt formed by a compound of any one of Formula I, I-1 or I-2 and a pharmaceutically acceptable acid or base, which retains the biological effectiveness and properties of the parent compound, i.e., a compound of any one of Formula I, I-1 or I-2. The pharmaceutically acceptable salt includes, but is not limited to, alkali metal salts (e.g., lithium, sodium, potassium, rubidium, cesium salts), alkaline earth metal salts (e.g., magnesium, calcium, strontium, barium salts), aluminum salts, organic base salts (e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine, imidazole, morpholine, 2-hydroxyethylmorpholine, dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine, benzylamine, tetramethylamine hydroxide salts) or amino acid salts (e.g., lysine, arginine salts). The pharmaceutically acceptable salt of the compound of any one of Formula I, I-1 or I-2 of the present disclosure can be prepared as follows: an equal amount of the compound of any one of Formula I, I-1 or I-2 and a base providing the required counter ion are stirred in a solvent at 25-70° C. for 0.5-2 h, and then the solvent is evaporated under reduced pressure to obtain the pharmaceutically acceptable salt.

[0087] The term 'solvate' as used herein refers to a molecular complex comprising a compound of any of Formula I, I-1 or I-2 and one or more pharmaceutically acceptable solvent molecules (eg, ethanol). When the solvent is water, the term "hydrate" is employed.

[0088] The term "stereoisomer" as used herein refers to isomers produced by different spatial arrangements of atoms in a molecule. When a compound has an asymmetric carbon atom, enantiomers are produced; when a compound has a carbon-carbon double bond or a cyclic structure, cis-trans isomers are produced. The scope of the present disclosure includes enantiomers, diastereomers, racemates, cis-trans isomers, geometric isomers, epimers and mixtures thereof of all compounds of any one of Formulas I, I-1 or I-2.

[0089] The term "pharmaceutical composition" as used herein refers to a substance or material with a specific medical use that is composed of a pharmaceutically active ingredient (in the context of the present disclosure, a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate and stereoisomer thereof) and a pharmaceutically acceptable carrier in a certain proportion. The pharmaceutical composition can be prepared into a pharmaceutically acceptable dosage form, such as tablets, powders (including sterile powders for injection), capsules, granules, solutions, syrups, suppositories, injections, patches, etc., by any conventional technique in the art. The pharmaceutical compositions of the present disclosure can be administered to a subject (e.g., a human or non-human mammal) by any of a variety of routes of administration, including, for example, oral administration (e.g., in the form of tablets, capsules, powders, granules); absorption through mucosa (e.g., sublingual, nasal, anal, rectal, or vaginal) (e.g., in the form of suppositories, creams, or foams); parenteral administration (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous, or intrathecal injection); transdermal administration (e.g., as a patch applied to the skin); and topical administration (e.g., as a cream, ointment, spray applied to the skin, or as eye drops). The pharmaceutical compositions can also be formulated for administration by inhalation. In general, the pharmaceutical composition usually contains 0.1% to 99.5% by weight of the active ingredient (i.e., a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof), preferably 0.5% to 99.5% by weight, for example 1%, 1.5%, 2%, 5%, 10%, 15%, 20%, 25%, 30% or 50% by weight of the active ingredient.

[0090] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier (or excipient) that does not cause significant irritation to an organism and does not eliminate the biological activity and properties of the administered compound. Any commonly used pharmaceutically acceptable carrier can be used, and its selection depends on factors such as the specific mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form and is within the ordinary skills of those skilled in the art. Some examples of materials that can be used as pharmaceutically acceptable carriers include: starches, such as corn starch and potato starch; sugars, such as lactose, glucose and sucrose; cellulose and its derivatives, such as ethyl cellulose, sodium carboxymethyl cellulose and cellulose acetate; gelatin, gum arabic, guar gum, tragacanth gum; magnesium stearate, zinc stearate, talc; water, saline; oils, such as peanut oil, cottonseed oil, olive oil, sesame oil, corn oil and soybean oil; alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; buffers, such as sodium chloride, phosphate buffer solution, etc.

[0091] As used herein, the term "prevention" refers to preventing the appearance of a disease in a subject at risk of the disease or the recurrence of a disease that has disappeared.

[0092] The term "treating" as used herein means controlling, alleviating or relieving the pathological progression of a disease and prolonging the survival of a subject suffering from the disease.

[0093] The term "subject" as used herein refers to an animal individual to whom the compounds or pharmaceutical compositions of the present disclosure are intended to be administered, including but not limited to humans and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); other mammals, such as horses, cattle, pigs, sheep, goats, cats, dogs; and birds, such as chickens, ducks, geese, quails, turkeys. The preferred subject is a human.

[0094] The term "effective amount" used herein refers to the amount of any one or more beneficial or desired symptoms of the intermediate pathological phenotype that is sufficient to affect the disease, its complications, or in the development process of the disease. The "effective amount" of any one of the compounds of Formula I, I-1 or I-2 or its pharmaceutically acceptable salt, hydrate, solvate or stereoisomer used will depend on the species of the object being treated, the severity of the disease, the frequency of application, the metabolic characteristics of the drug and other factors, and can be judged by the prescribing physician according to conventional practice. In general, the effective amount is usually in the range of about 0.001 to about 100 mg / kg body weight / day, preferably about 0.01 to about 50 mg / kg body weight / day (in single dose or divided dose). In some cases, the dosage level below the lower limit of the above range may be more than enough, and in other cases, it may be necessary to use a larger dosage without causing any harmful side effects, wherein such a larger dosage is usually divided into several smaller doses for use in one day. It should be noted that all numerical ranges mentioned in the present disclosure are intended to include both endpoints of the range, all integers within the range, and sub-ranges formed by these integers.

[0095] Detailed description of the technical solution disclosed herein

[0096] The following specific embodiments are provided to enable those skilled in the art to more clearly understand the content of the present disclosure. It should be noted that these embodiments are recorded only for the purpose of illustration and are not intended to limit the scope of protection of the present application.

[0097] The present disclosure generally relates to a β-DN having broad-spectrum antiviral activity. 4 The present invention relates to a phosphate ester compound of -hydroxycytidine, a pharmaceutical composition comprising such a compound, and the use of such a compound and a pharmaceutical composition comprising such a compound for inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

[0098] In a first aspect of the present disclosure, there is provided a compound of Formula I or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof:

[0099]

[0100] wherein the variables m, n, X, Z and R are as defined above for compounds of formula I.

[0101] In some embodiments of this aspect, the compound of Formula I has the structure of Formula I-1:

[0102]

[0103] wherein the variables m, n, X and R are as defined above for compounds of formula I.

[0104] In other embodiments of this aspect, the compound of Formula I has the structure of Formula I-2:

[0105]

[0106] wherein the variables m, n, X and R are as defined above for compounds of formula I.

[0107] In some embodiments, m is any integer from 6-20.

[0108] In some embodiments, m is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0109] In some embodiments, n is any integer from 1-5.

[0110] In some embodiments, n is 1, 2, 3, 4, or 5.

[0111] In some embodiments, X is -O- or -NR0-.

[0112] In some embodiments, X is -O-.

[0113] In some embodiments, X is -NR0-.

[0114] In some embodiments, R0 is H or C1-C6 alkyl.

[0115] In some embodiments, R0 is H.

[0116] In some embodiments, R0 is C1-C6 alkyl.

[0117] In some embodiments, R0 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl.

[0118] In some embodiments, R0 is methyl or ethyl.

[0119] In some embodiments, R0 is methyl.

[0120] In some embodiments, Z is -O- or -SS-.

[0121] In some embodiments, Z is -O-.

[0122] In some embodiments, Z is -SS-.

[0123] In some embodiments, R is H, C1-C6 alkyl, C6-C 12 Aryl, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-, wherein the alkyl or aryl in the R group is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0124] In some embodiments, R is H.

[0125] In some embodiments, R is C1-C6 alkyl, wherein the alkyl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0126] In some embodiments, R is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl.

[0127] In some embodiments, R is methyl or ethyl.

[0128] In some embodiments, R is methyl.

[0129] In some embodiments, R is C6-C 12 Aryl, wherein the aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0130] In some embodiments, R is phenyl, wherein the phenyl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)Ra 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0131] In some embodiments, R is phenyl, wherein the phenyl is optionally substituted with 1, 2 or 3 substituents independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxyl or nitro.

[0132] In some embodiments, R is phenyl, wherein the phenyl is optionally substituted with 1, 2 or 3 substituents independently selected from halogen, C1-C6 alkyl, haloC1-C6 alkyl, C1-C6 alkoxy, haloC1-C6 alkoxy, cyano, hydroxyl or nitro.

[0133] In some embodiments, R is phenyl, wherein the phenyl is optionally substituted with 1 substituent independently selected from halogen, halo-substituted C1-C6 alkyl, halo-substituted C1-C6 alkoxy, cyano, or nitro.

[0134] In some embodiments, R is phenyl optionally substituted with 1 substituent independently selected from halogen or nitro.

[0135] In some embodiments, R is phenyl optionally substituted with 1 substituent independently selected from chloro or nitro.

[0136] In some embodiments, R is R'-OC(O)-O-CH2-.

[0137] In some embodiments, R is R'-C(O)-O-CH2-.

[0138] In some embodiments, R' is C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 12 The alkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl groups in the R' group are optionally substituted by 1, 2 or 3 groups independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)ORa 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0139] In some embodiments, R' is C1-C6 alkyl, wherein the alkyl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxy, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0140] In some embodiments, R' is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl.

[0141] In some embodiments, R' is methyl, ethyl, isopropyl, or tert-butyl.

[0142] In some embodiments, R' is methyl, isopropyl, or tert-butyl.

[0143] In some embodiments, R' is a C3-C8 cycloalkyl group, wherein the cycloalkyl group is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxy, nitro, -NR a R b 、-C(=O)R a 、-C(=O)ORa 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0144] In some embodiments, R' is cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl.

[0145] In some embodiments, R' is cyclopentyl or cyclohexyl.

[0146] In some embodiments, R' is C6-C 12 Aryl, wherein the aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0147] In some embodiments, R' is phenyl, wherein the phenyl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a Rb 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0148] In some embodiments, R' is phenyl, wherein the phenyl is optionally substituted with 1, 2 or 3 substituents independently selected from halogen, C1-C6 alkyl, haloC1-C6 alkyl, C1-C6 alkoxy, haloC1-C6 alkoxy, cyano, hydroxyl or nitro.

[0149] In some embodiments, R' is phenyl, wherein the phenyl is optionally substituted with 1 substituent independently selected from halogen, halo-substituted C1-C6 alkyl, halo-substituted C1-C6 alkoxy, cyano, or nitro.

[0150] In some embodiments, R' is phenyl.

[0151] In some embodiments, R' is a 3- to 12-membered heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O, or S, wherein the heterocyclyl is optionally substituted by 1, 2, or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxy, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0152] In some embodiments, R' is a 5- or 6-membered heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O or S, wherein the heterocyclyl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxy, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0153] In some embodiments, R' is pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dihydropyrrolyl, dihydrofuranyl, dihydrothiophenyl, dihydropyrazolyl, dihydroimidazolyl, dihydrooxazolyl, isodihydrooxazolyl, dihydrothiazolyl, isodihydrothiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, dihydropyrazinyl, dihydropyrimidinyl, or dihydropyridazinyl, wherein the groups are optionally substituted by 1, 2, or 3 groups independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0154] In some embodiments, R' is pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dihydropyrrolyl, dihydrofuranyl, dihydrothiophenyl, dihydropyrazolyl, dihydroimidazolyl, dihydrooxazolyl, isodihydrooxazolyl, dihydrothiazolyl, isodihydrothiazolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, dihydropyrazinyl, dihydropyrimidinyl, or dihydropyridazinyl, wherein the groups are optionally substituted with 1, 2, or 3 substituents independently selected from halogen, C1-C6 alkyl, haloC1-C6 alkyl, C1-C6 alkoxy, haloC1-C6 alkoxy, cyano, hydroxy, nitro, -NH2, or -C(=O)NH2.

[0155] In some embodiments, R' is a 5- to 12-membered heteroaryl containing 1 or 2 heteroatoms independently selected from N, O, or S, wherein the heteroaryl is optionally substituted by 1, 2, or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxy, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0156] In some embodiments, R' is pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, triazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzoxadiazolyl, benzothiadiazolyl, benzothiazolyl, imidazopyridinyl, imidazopyrimidinyl, imidazole pyridazinyl, cinnolinyl, furopyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, quinolinyl, thienopyridinyl, wherein the group is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxy, nitro, -NRa R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

[0157] In some embodiments, R' is pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, triazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzoxadiazolyl, benzothiadiazolyl, benzothiazolyl, imidazopyridinyl, imidazopyrimid ... Pyridazinyl, cinnolinyl, furopyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, quinolinyl, thienopyridinyl, wherein the group is optionally substituted with 1, 2 or 3 substituents independently selected from halogen, C1-C6 alkyl, haloC1-C6 alkyl, C1-C6 alkoxy, haloC1-C6 alkoxy, cyano, hydroxyl, nitro, -NH2 or -C(=O)NH2.

[0158] In some embodiments, each R a and R b are independently H or C1-C6 alkyl.

[0159] In some embodiments, each R a and R b Independently H.

[0160] In some embodiments, each R a and R b are independently C1-C6 alkyl.

[0161] In some embodiments, each R a and R b is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

[0162] In some embodiments, each R a and R b is independently methyl or ethyl.

[0163] In some embodiments, each R a and R b is independently methyl.

[0164] In some embodiments:

[0165] m is any integer from 6 to 20;

[0166] n is any integer from 1 to 5;

[0167] X is -O- or -NR0-;

[0168] R0 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl;

[0169] R is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl optionally substituted with 1 substituent independently selected from halogen or nitro, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-;

[0170] R' is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or phenyl.

[0171] In some embodiments:

[0172] m is any integer from 6 to 20;

[0173] n is any integer from 1 to 5;

[0174] X is -O- or -NR0-;

[0175] R0 is H, methyl or ethyl;

[0176] R is H, methyl, ethyl, phenyl optionally substituted with 1 substituent independently selected from chloro or nitro, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-;

[0177] R' is methyl, ethyl, isopropyl, tert-butyl, cyclopentyl, cyclohexyl or phenyl.

[0178] In some embodiments:

[0179] m is any integer from 6 to 20;

[0180] n is any integer from 1 to 5;

[0181] X is -O- or -NR0-;

[0182] R0 is H or methyl;

[0183] R is H, methyl, phenyl optionally substituted with 1 substituent independently selected from chloro or nitro, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-;

[0184] R' is methyl, isopropyl, tert-butyl, cyclopentyl, cyclohexyl or phenyl.

[0185] In some embodiments of this aspect, the present disclosure provides a compound selected from Table 1 or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof:

[0186] Table 1

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] In some embodiments of this aspect, the pharmaceutically acceptable salt of the compound of any one of Formula I, I-1 or I-2 is an alkali metal salt (e.g., lithium, sodium, potassium, rubidium, cesium salt), an alkaline earth metal salt (e.g., magnesium, calcium, strontium, barium salt), an aluminum salt, an organic base salt (e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine, imidazole, morpholine, 2-hydroxyethylmorpholine, dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine, benzylamine, tetramethylammonium hydroxide salt) or an amino acid salt (e.g., lysine, arginine salt).

[0216] In a second aspect of the present disclosure, a pharmaceutical composition is provided, comprising a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, and one or more pharmaceutically acceptable carriers.

[0217] In some embodiments of this aspect, the pharmaceutical composition contains 0.1% by weight-99.5% by weight of active ingredient (i.e., a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof), preferably containing 0.5% by weight-99.5% by weight, more preferably 1% by weight-50% by weight, for example 1%, 1.5%, 2%, 5%, 10%, 15%, 20%, 25%, 30% by weight or 50% by weight of active ingredient. The remainder of the pharmaceutical composition is a pharmaceutically acceptable carrier. In some embodiments of this aspect, the pharmaceutical composition comprises two, three or more pharmaceutically acceptable carriers. Examples of the pharmaceutically acceptable carrier include, but are not limited to, conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, lubricants, colorants, fragrances, absorption promoters, surfactants, adsorption carriers, etc. in the pharmaceutical field.

[0218] In some embodiments of this aspect, the pharmaceutical composition is prepared into a pharmaceutically acceptable dosage form. For example, in some embodiments, a compound of any one of Formula I, I-1 or I-2 or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof is mixed with one or more pharmaceutically acceptable carriers and prepared into a pharmaceutically acceptable dosage form according to conventional production methods in the pharmaceutical field. Examples of the pharmaceutically acceptable dosage form include, but are not limited to, conventional solid dosage forms in the pharmaceutical field (such as, but not limited to, tablets, capsules, powders, granules, cachets and suppositories, etc.), liquid dosage forms (such as, but not limited to, solutions, syrups, suspensions, emulsions, elixirs, etc.). In addition, the pharmaceutical composition of the present disclosure can be prepared into a sustained-release dosage form to provide a controlled-rate release of the active ingredient, thereby optimizing the antiviral activity. Dosage forms suitable for sustained release include, but are not limited to, multilayer tablets containing layers with different disintegration rates, or capsules containing a porous polymer matrix that has been impregnated or encapsulated.

[0219] In some embodiments of this aspect, the content of the active ingredient in the pharmaceutical composition can vary in the range of about 1 mg to about 2000 mg. In some embodiments, the content can be 1 mg to 1000 mg, 2 mg to 500 mg, 2.5 mg to 100 mg, or 5 mg to 75 mg.

[0220] In the third aspect of the present disclosure, provided is a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers, which is used for treating or preventing a disease.

[0221] In the fourth aspect of the present disclosure, provided is a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers, which is used for inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

[0222] In the fifth aspect of the present disclosure, provided is a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers for use in inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

[0223] In the sixth aspect of the present disclosure, provided is the use of a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof and one or more pharmaceutically acceptable carriers in the preparation of a medicament for inhibiting viruses or for preventing and / or treating viral infections in subjects in need thereof.

[0224] In the seventh aspect of the present disclosure, a method for preventing and / or treating viral infection in a patient in need thereof is provided, the method comprising administering to a subject in need thereof an effective amount of a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, or a pharmaceutical composition comprising a compound of any one of Formula I, I-1 or I-2, or a pharmaceutically acceptable salt, hydrate, solvate or stereoisomer thereof, and one or more pharmaceutically acceptable carriers.

[0225] In some embodiments, the virus is a coronavirus (e.g., a human coronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, including, for example, strains B.1.1.7, B.1.351, Delta variant B.1.617.2, Omicron variant B.1.1.529, or other SARS-CoV-2 variants), equine encephalitis virus (e.g., Eastern equine encephalitis virus, Western equine encephalitis virus, or Venezuelan equine encephalitis virus), Chikungunya virus, Ross River virus, Orthomyxoviridae virus (e.g., influenza virus, such as influenza A virus, influenza B virus, or influenza C virus), Paramyxoviridae virus (e.g., respiratory syncytial virus), Filovirus (e.g., Ebola virus, Marburg virus, or Kuwa virus), or Zika virus.

[0226] In some embodiments, the virus is an influenza virus.

[0227] In some embodiments, the virus is an influenza A virus.

[0228] In some embodiments, the virus is influenza A(H1N1) virus.

[0229] In some embodiments, the virus is strain SC09.

[0230] In some embodiments, the virus is influenza A(H3N2) virus.

[0231] In some embodiments, the virus is strain FJ09.

[0232] In some embodiments, the virus is an influenza B virus.

[0233] In some embodiments, the virus is strain SC11.

[0234] In some embodiments, the virus is Zika virus.

[0235] In some embodiments, the virus is strain SZ01.

[0236] In some embodiments, the virus is a human coronavirus.

[0237] In some embodiments, the virus is HCoV-229E strain or HCoV-OC43 strain.

[0238] In some embodiments, the virus is severe acute respiratory syndrome coronavirus 2.

[0239] In some embodiments, the virus is strain B.1.1.7, strain B.1.351, or strain B.1.617.2.

[0240] The compounds or pharmaceutical compositions of the present disclosure may be administered to a subject (e.g., a human or non-human mammal) by any of a variety of routes of administration, including, for example, oral administration (e.g., in the form of tablets, capsules, powders, granules); absorption through mucosa (e.g., sublingual, nasal, anal, rectal, or vaginal) (e.g., in the form of suppositories, creams, or foams); parenteral administration (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous, or intrathecal injection); transdermal administration (e.g., as a patch applied to the skin); and topical administration (e.g., as a cream, ointment, spray applied to the skin, or as eye drops). The compounds or pharmaceutical compositions of the present disclosure may also be formulated for administration by inhalation.

[0241] If desired, the total daily dose can be split and administered in divided doses throughout the day. In some embodiments, the daily dose is administered in one dose. In other embodiments, the total daily dose is administered in two, three or more divided doses over a 24-hour period.

[0242] The attending physician may adjust the dosage and frequency of administration of the compounds or pharmaceutical compositions of the present invention, taking into account factors such as the patient's age, general health, weight, and severity of the symptoms to be treated. In general, the total daily dose of the compounds or pharmaceutical compositions of the present invention is usually about 0.1 to about 2000 mg of active ingredient / day, such as about 1 to about 1500 mg / day, about 10 to about 1250 mg / day, about 50 to about 1000 mg / day, administered in a single dose or 2, 3 or 4 divided doses.

[0243] The main beneficial effects of the compounds disclosed herein include the following aspects:

[0244] (1) The compounds disclosed herein have broad-spectrum antiviral activity and can effectively inhibit viruses such as the following: coronavirus (e.g., human coronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, including, for example, B.1.1.7 strain, B.1.351 strain, Delta variant B.1.617.2 strain, Omicron variant B.1.1.529 strain or other SARS-CoV-2 variants), equine encephalitis virus (e.g., Eastern equine encephalitis virus, Western equine encephalitis virus or Venezuelan equine encephalitis virus), Chikungunya virus, Ross River virus, Orthomyxoviridae virus (e.g., influenza virus, such as influenza A virus, influenza B virus or influenza C virus), Paramyxoviridae virus (e.g., respiratory syncytial virus), Filovirus (e.g., Ebola virus, Marburg virus or Kuwa virus) or Zika virus;

[0245] (2) The compounds disclosed herein have improved oral administration stability and enhanced antiviral activity: Before entering cells, EIDD-2801 is partially converted to EIDD-1931 under the action of the acid, alkali and digestive enzymes in the gastrointestinal tract, thereby affecting in vivo absorption and weakening antiviral activity. In contrast, the compounds disclosed herein will not be degraded by the acid, alkali and digestive enzymes in the gastrointestinal tract, can be absorbed in vivo as the original compound, and have enhanced cell permeability. Under the action of phospholipases in cells, they are converted to EIDD-1931 to exert antiviral effects, thereby improving antiviral activity;

[0246] (3) The compounds disclosed herein have high safety: The compounds disclosed herein are lipid drugs with improved pharmacokinetic properties (especially in vivo distribution), which can reduce the toxicity of nucleoside drugs to the kidneys and improve the targeting to the lungs and liver, thereby significantly reducing lung damage caused by viral infection, and thus have the characteristics of greater drug safety.

[0247] General Methods for Preparing Disclosed Compounds

[0248] According to the synthesis and purification methods known to those skilled in the art of organic synthesis, compounds of formula I can be prepared from commercially available or easily prepared starting materials. Methods that can be used to prepare compounds of formula I are described in the following schemes and embodiments. However, these specific methods do not constitute any limitation to the present invention. Those skilled in the art of organic synthesis will understand alternative synthetic routes. As references for synthetic methods, Steven D. Dong, Chin-Chung Lin, Mel Schroeder, Synthesis and evaluation of a new phosphorylated ribavirin prodrug, Antiviral Research 99 (2013) 18–26 and WO 2019 / 113462 can be mentioned.

[0249] Solution 1

[0250] Scheme 1 below shows a method for preparing compounds of formula I. This method is particularly suitable for preparing compounds of formula I wherein X is -O-.

[0251]

[0252] In the above schemes, the variables m, n, Z, X, R and R' are as defined above for compounds of Formula I. R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl; A is chlorine or nitro, and B is hydrogen, chlorine or nitro. Preferably, R1 is methyl, ethyl or phenyl, A is chlorine or nitro, and B is hydrogen.

[0253] Step 1: Preparation of the compound of intermediate formula 2a

[0254] The compound of formula 1d (which can be selected from 2-chlorophenyl dichlorophosphonyl, 2-nitrophenyl dichlorophosphonyl, 4-chlorophenyl dichlorophosphonyl, 4-nitrophenyl dichlorophosphonyl, 2,4-dichlorophenyl dichlorophosphonyl, 2,4-dinitrophenyl dichlorophosphonyl) and the compound of formula 1c (both of which can be directly commercially obtained) are reacted in an anhydrous solvent, and then the compound of formula 1a (prepared according to the method of Chemistry-A European Journal (2016), 22 (21), 7215-7224) and the compound of formula 1b (prepared according to the literature Kyle E. Giesler, et al., Liotta Reduction Sensitive Lipid Conjugates of Tenofovir: Synthesis, Stability, and Antiviral Activity; J. Med. Chem. 2016, 59, 7097-7110; US2012 / 0058976A1 or Susumu Tsushima, Chew. Pharm. Bu11. 30 (9) 3260-3270, 1982). The above reaction can obtain the compound of formula 2a. The anhydrous solvent required in the above reaction process includes but is not limited to anhydrous tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably tetrahydrofuran and 2-methyltetrahydrofuran. The reaction temperature is 0-50°C, preferably 0-35°C; the reaction time is 5-48 hours, preferably 10-20 hours.

[0255] Step 2: Preparation of the compound of intermediate formula 4a

[0256] Method 1

[0257] The compound of formula 2a is reacted with the compound of formula 1c in the presence of phosphorus oxychloride to obtain a compound of formula 3a. The compound of formula 3a is then reacted with hydroxylamine hydrochloride under alkaline conditions to obtain a compound of formula 4a. The solvent used in the reaction is acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably acetonitrile and dioxane. The reaction temperature is 0-35°C. The base required for the reaction can be N,N-diethylethylamine, triethylamine, pyridine (all of which can be directly commercially obtained) (refer to Steven D. Dong, Chin-Chung Lin, Mel Schroeder, Synthesis and evaluation of a new phosphorylated ribavirin prodrug, Antiviral Research 99 (2013) 18–26).

[0258] Method 2

[0259] In an alkaline environment and under the catalysis of 4-dimethylaminopyridine, the compound of formula 2a is reacted with the compound of formula 3b (2,4,5-triisopropylbenzenesulfonyl chloride, TBSCl) to obtain a compound of formula 3c. The compound of formula 3c is reacted with hydroxylamine hydrochloride under alkaline conditions to obtain a compound of formula 4a. The solvent used in the reaction is dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably dichloromethane, chloroform, 1,2-dichloroethane. The reaction temperature is 0-35°C. The base required for the reaction is imidazole, N,N-diethylethylamine, triethylamine, pyridine (all of which can be directly commercially obtained), preferably imidazole (reference WO 2019 / 113462).

[0260] Step 3: Preparation of the compound of intermediate formula 5a

[0261] The compound of formula 4a is treated with a compound of formula 4b (1,1,3,3-tetramethylguanidine) and a compound of formula 4c (2-pyridinecarboxaldehyde oxime) to remove the substituted phenyl moiety to obtain a compound of formula 5a. The solvent used in the reaction is tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone. The reaction temperature is 10-40°C (reference Steven D. Dong, Chin-Chung Lin, Mel Schroeder, Synthesis and evaluation of a new phosphorylated ribavirin prodrug, Antiviral Research 99 (2013) 18–26).

[0262] Step 4: Preparation of the compound of intermediate formula 6a

[0263] The compound of formula 4a is reacted in a solvent to remove the hydroxyl protecting group to obtain the compound of formula 6a. The solvent used in the reaction can be ammonia methanol solution, ammonia ethanol solution or ammonia isopropanol solution. The reaction temperature is 20-80°C.

[0264] Step: 5 Preparation of the compound of formula I

[0265] Method 1

[0266] The compound of formula 5a is reacted in a solvent to remove the hydroxyl protecting group to obtain a compound of formula I. The solvent used in the reaction can be ammonia methanol solution, ammonia ethanol solution or ammonia isopropanol solution. The reaction temperature is 20-50°C.

[0267] Method 2

[0268] The compound of formula 6a is treated with a compound of formula 4b (1,1,3,3-tetramethylguanidine) and a compound of formula 4c (2-pyridinecarboxaldehyde oxime) to remove the substituted phenyl moiety to obtain a compound of formula I. The solvent used in the reaction is tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone. The reaction temperature is 10-40°C (reference Steven D. Dong, Chin-Chung Lin, Mel Schroeder, Synthesis and evaluation of a new phosphorylated ribavirinprodrug, Antiviral Research 99 (2013) 18-26).

[0269] Solution 2

[0270] Scheme 2 below shows another method for preparing compounds of formula I, wherein the compound of formula 1d is replaced by a compound of formula 1-1d compared to Scheme 1. This method is particularly suitable for preparing compounds of formula I wherein X is -NR0-.

[0271]

[0272] In the above schemes, the variables m, n, Z, R and R0 are as defined above for compounds of Formula I. R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl. Preferably, R1 is methyl, ethyl or phenyl.

[0273] Step 1: Preparation of the compound of intermediate formula 2a-1

[0274] The compound of formula 1-1d and the compound of formula 1c (both of which can be directly commercially obtained) are reacted in an anhydrous solvent, and then the compound of formula 1a and the compound of formula 1b are added for reaction. The above reaction can obtain a compound of formula 2a-1. The anhydrous solvent used in the reaction includes but is not limited to anhydrous tetrahydrofuran, 2-methyltetrahydrofuran, dichloromethane, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably tetrahydrofuran and 2-methyltetrahydrofuran. The reaction temperature is 0-50°C, preferably 0-35°C; the reaction time is 5-48 hours, preferably 10-20 hours.

[0275] Step 2: Preparation of the compound of intermediate formula 4a-1

[0276] Method 1

[0277] The compound of formula 2a-1 is reacted with the compound of formula 1c in the presence of phosphorus oxychloride to obtain the compound of formula 3a-1. The compound of formula 3a-1 is then reacted with hydroxylamine hydrochloride under alkaline conditions to obtain the compound of formula 4a-1. The solvent used in the reaction is acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably acetonitrile and dioxane. The reaction temperature is 0-35°C. The base required for the reaction can be N,N-diethylethylamine, triethylamine or pyridine (all of which can be directly commercially obtained).

[0278] Method 2

[0279] In an alkaline environment and under the catalysis of 4-dimethylaminopyridine, the compound of formula 2a-1 is reacted with the compound of formula 3b (2,4,5-triisopropylbenzenesulfonyl chloride, TBSCl) to obtain a compound of formula 3c-1. The compound of formula 3c-1 is reacted with hydroxylamine hydrochloride under alkaline conditions to obtain a compound of formula 4a-1. The solvent used in the reaction is dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably dichloromethane, chloroform, 1,2-dichloroethane. The reaction temperature is 0-35°C. The base required for the reaction can be imidazole, N,N-diethylethylamine, triethylamine, pyridine (all of which can be directly commercially obtained), preferably imidazole (refer to WO 2019 / 113462).

[0280] Step 3: Preparation of compounds of formula I

[0281] The compound of formula 4a-1 is reacted in a solvent to remove the hydroxyl protecting group to obtain a compound of formula I. The solvent used in the reaction can be ammonia methanol solution, ammonia ethanol solution or ammonia isopropanol solution. The reaction temperature is 20-50°C.

[0282] Solution 3

[0283] Scheme 3 below shows a method for preparing compounds of formula 1-2.

[0284]

[0285] In the above schemes, the variables m, n and R' are as defined above for compounds of formula I. A is chloro or nitro, and B is hydrogen, chloro or nitro. Preferably, A is chloro or nitro, and B is hydrogen.

[0286] Step 1: Preparation of the compound of intermediate formula 2-1a

[0287] The compound of formula 1d (which can be selected from 2-chlorophenyl dichlorophosphonyl, 2-nitrophenyl dichlorophosphonyl, 4-chlorophenyl dichlorophosphonyl, 4-nitrophenyl dichlorophosphonyl, 2,4-dichlorophenyl dichlorophosphonyl, 2,4-dinitrophenyl dichlorophosphonyl) and the compound of formula 1c (both of which can be directly commercially available) are reacted, and then the compound of formula 1-1a (prepared according to the method disclosed in WO2019 / 113462 or Angewandte Chemie, International Edition, 53 (19), 4862-4866; 2014) and the compound of formula 1-1b (prepared according to the method disclosed in US2012 / 0058976A1 or Susumu Tsushima, Chew.Pharm.I3u11.30 (9) 3260-3270, 1982) are added. Specifically, it can be prepared as follows: using raw material CH3 (CH2) m CH2OH reacts with methanesulfonyl chloride or p-toluenesulfonyl chloride in the presence of triethylamine to form substituted sulfonyl esters, which are then reacted with diol OH(CH2) n OH (m, n are as defined above for the compound of formula I) reacts under the action of sodium hydride). The above reaction can give the compound of formula 2-1a (see Susumu Tsushima, Chew. Pharm. Bu11. 30 (9) 3260-3270, 1982).

[0288] Step 2: Preparation of the compound of intermediate formula 4-1-a

[0289] Method 1

[0290] The compound of formula 2-1a is reacted with the compound of formula 1c in the presence of phosphorus oxychloride to obtain a compound of formula 3-1a. Then the compound of formula 3-1a is reacted with hydroxylamine hydrochloride under alkaline conditions to obtain a compound of formula 4-1-a. The solvent used in the reaction is acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably acetonitrile and dioxane. The reaction temperature is 0-35°C. The base required for the reaction can be N,N-diethylethylamine, triethylamine, pyridine (all of which can be directly commercially obtained) (refer to Steven D. Dong, Chin-Chung Lin, Mel Schroeder, Synthesis and evaluation of a new phosphorylated ribavirin prodrug, Antiviral Research 99 (2013) 18–26).

[0291] Method 2

[0292] In an alkaline environment and under the catalysis of 4-dimethylaminopyridine, the compound of formula 2-1a is reacted with the compound of formula 3b (2,4,5-triisopropylbenzenesulfonyl chloride, TBSCl) to obtain a compound of formula 3-1c. The compound of formula 3-1c is reacted with hydroxylamine hydrochloride under alkaline conditions to obtain a compound of formula 4-1-a. The solvent used in the reaction is dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably dichloromethane, chloroform, 1,2-dichloroethane. The reaction temperature is 0-35°C. The base required for the reaction is imidazole, N,N-diethylethylamine, triethylamine, pyridine (all of which can be directly commercially obtained), preferably imidazole.

[0293] Step 3: Preparation of the compound of intermediate formula 5-1a

[0294] The compound of formula 4-1-a is treated with the compound of formula 4b (1,1,3,3-tetramethylguanidine) and the compound of formula 4c (2-pyridinecarboxaldehyde oxime) to remove the substituted phenyl moiety to obtain the compound of formula 5-1a. The solvent used in the reaction is tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone. The reaction temperature is 10-40°C.

[0295] Step 4: Preparation of the compound of intermediate formula 6-1a

[0296] The compound of formula 4-1-a is reacted in a formic acid aqueous solution (concentration of 80%-90%, w / w) or a trifluoroacetic acid aqueous solution (concentration of 80%-90%, w / w) to remove the isopropyl methylene protecting group to obtain a compound of formula 6-1a. The reaction temperature is 20-80°C.

[0297] Step 5: Preparation of the compound of formula I-2

[0298] Method 1

[0299] The compound of formula 5-1a is reacted in a formic acid aqueous solution (concentration of 80%-90%, w / w) or a trifluoroacetic acid aqueous solution (concentration of 80%-90%, w / w) to remove the isopropyl methylene protecting group to obtain a compound of formula I-2. The reaction temperature is 20-80°C.

[0300] Method 2

[0301] The compound of formula 6-1a is treated with a compound of formula 4b (1,1,3,3-tetramethylguanidine) and a compound of formula 4c (2-pyridinecarboxaldehyde oxime) to remove the substituted phenyl moiety to obtain a compound of formula I-2. The solvent used in the reaction is tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone. The reaction temperature is 10-40°C (reference WO 2019 / 113462).

[0302] Solution 4

[0303] Scheme 4 below shows another method for preparing compounds of formula I-2, wherein the compound of formula 1-1d is used instead of the compound of formula 1d compared to Scheme 3. This method is particularly suitable for preparing compounds of formula I-2 wherein X is -NR0-.

[0304]

[0305] In the above schemes, the variables m, n, R and R0 are as defined above for compounds of Formula I.

[0306] Step 1: Preparation of the compound of intermediate formula 2-1a-1

[0307] The compound of formula 1-1d and the compound of formula 1c (both of which can be directly commercially obtained) are reacted, and then the compound of formula 1-1a (prepared according to the method disclosed in WO 2019 / 113462 or Angewandte Chemie, International Edition, 53 (19), 4862-4866; 2014) and the compound of formula 1-1b (prepared according to the method disclosed in US2012 / 0058976A1 or Susumu Tsushima, Chew. Pharm. I3u11.30 (9) 3260-3270, 1982) are added. Specifically, it can be prepared as follows: using the raw material CH3 (CH2) m CH2OH reacts with methanesulfonyl chloride or p-toluenesulfonyl chloride in the presence of triethylamine to form substituted sulfonyl esters, which are then reacted with diol OH(CH2) n OH (m, n are as defined above for the compound of formula I) reacts under the action of sodium hydride). The above reaction can obtain the compound of formula 2-1a-1 (refer to the document Susumu Tsushima, Chew. Pharm. Bu11. 30 (9) 3260-3270, 1982).

[0308] Step 2: Preparation of the compound of intermediate formula 4-1a-1

[0309] Method 1

[0310] The compound of formula 2-1a-1 is reacted with the compound of formula 1c in the presence of phosphorus oxychloride to obtain the compound of formula 3-1a-1. Then the compound of formula 3-1a-1 is reacted with hydroxylamine hydrochloride under alkaline conditions to obtain the compound of formula 4-1a-1. The solvent used in the reaction is acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably acetonitrile and dioxane. The reaction temperature is 0-35°C. The base required for the reaction can be N,N-diethylethylamine, triethylamine, pyridine (all of which can be directly commercially obtained) (refer to Steven D. Dong, Chin-Chung Lin, Mel Schroeder, Synthesis and evaluation of a new phosphorylated ribavirin prodrug, Antiviral Research 99 (2013) 18–26).

[0311] Method 2

[0312] In an alkaline environment and under the catalysis of 4-dimethylaminopyridine, the compound of formula 2-1a-1 is reacted with the compound of formula 3b (2,4,5-triisopropylbenzenesulfonyl chloride, TBSCl) to obtain a compound of formula 3-1c-1. The compound of formula 3-1c-1 is reacted with hydroxylamine hydrochloride under alkaline conditions to obtain a compound of formula 4-1a-1. The solvent used in the reaction is dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, N-methylpyrrolidone, toluene, 1,4-dioxane or a mixed solvent thereof, preferably dichloromethane, chloroform, 1,2-dichloroethane. The reaction temperature is 0-35°C. The base required for the reaction is imidazole, N,N-diethylethylamine, triethylamine, pyridine (all of which can be directly commercially obtained), preferably imidazole.

[0313] Step 3: Preparation of the compound of formula I-2

[0314] The compound of formula 4-1a-1 is reacted in an aqueous formic acid solution (concentration of 80%-90%, w / w) or an aqueous trifluoroacetic acid solution (concentration of 80%-90%, w / w) by removing the isopropyl methylene protecting group to obtain a compound of formula I-2. The reaction temperature is 20-80°C (refer to WO 2019 / 113462).

[0315] To this end, in an eighth aspect of the present disclosure, an intermediate compound for synthesizing a compound of any one of formulas I, I-1 or I-2 of the present disclosure is provided.

[0316] In some embodiments, the intermediate compound is a compound of Formula 2a:

[0317]

[0318] wherein m, n and Z are as defined above for compounds of formula I; R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl; A is chlorine or nitro; B is hydrogen, chlorine or nitro. Preferably, R1 is methyl, ethyl or phenyl, A is chlorine or nitro, and B is hydrogen.

[0319] In some embodiments, the intermediate compound is a compound of Formula 2a-1:

[0320]

[0321] wherein m, n, Z, R0 and R are as defined above for compounds of formula I; R1 is a straight or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl. Preferably, R1 is methyl, ethyl or phenyl.

[0322] In some embodiments, the intermediate compound is a compound of Formula 4a:

[0323]

[0324] wherein m, n and Z are as defined above for compounds of formula I; R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl; A is chlorine or nitro; B is hydrogen, chlorine or nitro. Preferably, R1 is methyl, ethyl or phenyl, A is chlorine or nitro, and B is hydrogen.

[0325] In some embodiments, the intermediate compound is a compound of Formula 4a-1:

[0326]

[0327] wherein m, n, Z, R0 and R are as defined above for compounds of formula I; R1 is a straight or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b R1 is independently H or C1-C6 alkyl. Preferably, R1 is methyl, ethyl or phenyl.

[0328] In some embodiments, the intermediate compound is a compound of Formula 2-1a:

[0329]

[0330] wherein m, n and Z are as defined above for compounds of formula I; A is chloro or nitro; and B is hydrogen, chloro or nitro. Preferably, A is chloro or nitro and B is hydrogen.

[0331] In some embodiments, the intermediate compound is a compound of Formula 2-1a-1:

[0332]

[0333] wherein m, n, Z, R0 and R are as defined above for compounds of formula I.

[0334] In some embodiments, the intermediate compound is a compound of Formula 4-1a:

[0335]

[0336] wherein m and n are as defined above for compounds of formula I; A is chlorine or nitro; and B is hydrogen, chlorine or nitro. Preferably, A is chlorine or nitro and B is hydrogen.

[0337] In some embodiments, the intermediate compound is a compound of formula 4-1a-1:

[0338]

[0339] wherein m, n, Z, R0 and R are as defined above for compounds of formula I.

[0340] Example

[0341] The present invention is further described in detail below using examples. Obviously, the described examples are only a part of the present invention, not all. These examples are only used to illustrate the present invention and should not be understood as limiting the scope of protection of the present invention. Based on the examples of the present invention, all other technical solutions obtained by those skilled in the art without creative work belong to the scope of protection of the present invention. The structures of the compounds in the examples are all 1 Determined by HNMR or MS.

[0342] In addition, it should be noted that those skilled in the art can easily prepare the specific compounds listed in Table 1 whose synthesis methods are not specifically illustrated in the Examples section by selecting appropriate starting materials and adopting appropriate steps, based on the general synthesis scheme shown in the "General Methods for Preparing the Disclosed Compounds" section and the exemplary synthesis methods for preparing the disclosed compounds shown in the following Examples.

[0343] Example 1

[0344] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2- Methyl (2-(hexadecyldithio)ethyl) phosphate (NTV-29)

[0345]

[0346] Step 1:

[0347] 2-Chlorophenyl ((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrakis Hydrofuran-2-yl)methyl (2-(hexadecyldithio))ethyl phosphate (A2)

[0348]

[0349] (1) (3R,4R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diacetate (2',3'-di-O-acetyluidine) (A1)

[0350]

[0351] Place 24.4g (100.0mmol) of dry uridine, 8.2g (120mmol) of imidazole, and 0.6g (4.9mmol) of 1,4-dimethylaminopyridine in 300ml of dry dichloromethane / pyridine (1:1, v / v) solution, stir and cool the reaction solution to about 0°C. Add 26g (120mmol) of tert-butyldimethylsilyl chloride in dichloromethane (50ml) to the above solution, and react at this temperature under nitrogen protection for about 6h. After the reaction is completed by TLC monitoring, add 1ml of methanol, stir for 0.5 hours, and add 22.4g (252mmol) of acetic anhydride. Raise the temperature of the reaction system to room temperature and react for 6 hours. After the reaction is completed, wash the reaction solution with water (3x100ml) and saturated brine (100ml) in turn. Dry the organic phase with anhydrous sodium sulfate and filter. Evaporate the filtrate to dryness under reduced pressure, and add n-hexane (300ml) to the residue for recrystallization. The solid was filtered out and dried to obtain 24.0 g of a nearly white solid product, 2′,3′-diacetyl 5′-O-tert-butyldimethylsilyl uridine, with a yield of 73.0%. 24.0 g (54.2 mmol) of 2′,3′-O-diacetyl 5′-O-tert-butyldimethylsilyl uridine was dissolved in 200 mL of dry THF, 3.2 ml (54.2 mmol) of acetic acid was added, and 109 ml (109 mmol) of a 1M tetrabutylammonium fluoride THF solution was slowly dripped into the above solution under stirring. After the addition was completed, stirring was continued for 1.5-2.5 h. After the reaction was basically completed by TLC monitoring, the solvent was evaporated to dryness under reduced pressure, the obtained solid was redissolved in 500 mL of ethyl acetate, washed three times with an equal volume of water, and washed once with saturated brine. The ethyl acetate layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was evaporated to dryness under reduced pressure. The obtained solid was recrystallized using isopropyl ether to obtain 16 g of a nearly white solid product 2′,3′-di-O-acetylureaside (A1) with a total yield of 65%.

[0352] (2) 2-Hexadecyl dithioethanol (1b-1)

[0353] C 16 H 33 SSCH2CH2OH

[0354] 1b-1

[0355] To a solution of hexadecyl-1-thiol (8.74 ml, 28.4 mmol) and mercaptoethanol (2 ml, 28.4 mmol) in MeOH / DCM (50:50, 200 mL) was added pyridine (4.94 ml, 56.8 mmol), followed by addition of iodine (7.21 g, 28.4 mmol) in portions until the solution remained brown and did not fade. The reaction solution was stirred at room temperature for 2 h, and the resulting solution was filtered. The filtrate was concentrated under reduced pressure, and the resulting solid was added to water and extracted with DCM. The organic layer was concentrated under reduced pressure and then purified by silica gel chromatography (using a hexanes / EtOAc (0-8%) gradient) to give compound 2-hexadecyl dithioethanol (1-1b) (3.98 g, 11.89 mmol, 41.9%) as a white solid powder. Melting point 50-51 ° C; MS (ESI) m / e [M+Na] +: 357.225.

[0356] (3) 2-Chlorophenyl (((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(hexadecyldithio)ethyl) phosphate (A2)

[0357] A solution of 1,2,4-triazole (1c) (13.8 g, 212 mmol) and triethylamine (10.1 g, 200 mmol) in dry tetrahydrofuran (200 mL) was added to a solution of 2-chlorophenyl dichlorophosphate (1d-1) (24.5 g, 100 mmol) in dry tetrahydrofuran (150 mL) pre-cooled to 0°C. The resulting reaction solution was reacted at room temperature for 1 hour. The generated solid was filtered off, and a solution of (3R,4R)-2-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diacetate (2',3'-di-O-acetyluidine) (A1) (32.8 g, 100 mmol) and 1-methylimidazole (8.4 ml, 100 mmol) in tetrahydrofuran (150 mL) was added to the resulting filtrate. After the addition, stir and react at room temperature for 1 hour. Add 2-hexadecyl dithioethanol (1b-1) (33.4 g, 100 mmol) to the reaction solution. Stir the resulting reaction solution at room temperature overnight. After the reaction is completed, concentrate under reduced pressure and dissolve the residue in ethyl acetate (400 mL). Wash the resulting solution with water (200 ml x 2), saturated sodium bicarbonate aqueous solution (200 ml x 2), water (200 ml x 2) and saturated brine (200 ml x 2) in sequence. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2-chlorophenyl (((3R, 4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(hexadecanedithio)ethyl) phosphate (A2) (53.0 g, 70%) as a colorless oil, which was directly used in the next step without further purification. The sample for structural identification can be purified by a chromatographic column (CH2Cl2 + 0 to 10% MeOH).

[0358] 1HNMR (600MHz, chloroform-d4 / methanol-d4): δ7.490-7.486 (m, 3H); 7.390-7.268 (dd, J=6Hz, J=54Hz, 1H); 7.199-7.161 (dd, J=7.8Hz, 1H); 6.182-6.046 (dd, J=6Hz, 7.2Hz, 1H); 5.778-5.702 (dd, J=7.2Hz, 1H); 5.455-4.339 (m, 5H); 3.079-2.959(m,J=6Hz,J=7.2Hz,2H); 2.778-2.689(m,J=4.2Hz,J=3Hz,2H); 2.156-2.142(d,J=2.4Hz,3H) ; 2.113-2.108 (d, J = 3Hz, 3H); 1.409-1.355 (m, J = 7.2Hz, 2H); 1.318 (s, 28H); 0.912-0.888 (t, J = 7.2Hz, 3H).

[0359] 13 CNMR (600MHz, chloroform-d4 / methanol-d4) δ169.665-169.413 (dd, J=17.4, J=59Hz); 162.387; 150.228 (d, J=10Hz)(d, J=10Hz); 146.210; 139.355-139.280 (d, J=45Hz); 130.859-130.839

[0360] (d,J=12Hz);128.199;126.500;125.422;121.509-121.494(d,J=9Hz);103.601;86.370-86.200( d, J=102Hz); 80.813-80.762 (d, J=30.6Hz); 72.496-72.457 (d, J=23.4Hz); 70.192-70.171 (d, J=1 2.6Hz); 67.382; 67.126; 67.086; 67.016; 39.255; 39.146; 37.926-37.879 (d, J = 28Hz); 31.929; 29 .699; 29.660; 29.607; 29.520; 29.363; 29.241; 29.144; 28.542; 22.690; 20.539; 20.368; 14.121.

[0361] 31 P:δ-6.845,-7.188.

[0362] MS (ESI) m / e [M+H]+ :835.3.

[0363] Step 2:

[0364] 2-Chlorophenyl ((3R,4R)-5-(2-oxo-4-(1H-1,2,4-triazol-1-yl)pyrimidin-1(2H)yl)-3,4-dichlorophenyl Acetoxytetrahydrofuran-2-yl)-methyl (2-(hexadecyldithio)ethyl) phosphate (A3)

[0365]

[0366] 2-Chlorophenyl ((3R, 4R) -5- (2,4-dioxo-3,4-dihydropyrimidin-1 (2H) yl) -3,4-diacetoxytetrahydrofuran-2-yl) methyl (2- (hexadecyldithio) ethyl) phosphate (A2) (83.5 g, 100.0 mmol) was dissolved in acetonitrile (400.0 mL), and 1,2,4-triazole (48.0 g, 700.0 mmol) and triethylamine (81 g, 800.0 mmol) were added, and the solid was dissolved by stirring. The reaction solution was cooled to 0°C, and phosphorus oxychloride (14.5 ml, 150.0 mmol) was slowly added dropwise under a nitrogen atmosphere. After the addition, the reaction solution was heated to room temperature, and stirring was continued for 2 hours to complete the reaction, and the reaction was monitored by TLC (100% EtOAc). After the reaction is complete, water (500 mL) and ethyl acetate (400 mL) are added to the reaction solution, and the mixture is stirred at room temperature for 15 minutes to quench the reaction. The separated organic layer is washed with water (2x100 mL), 0.5N HCl (200 mL) and saturated brine (2x100 mL) in sequence, dried over anhydrous magnesium sulfate and filtered, and the filtrate is concentrated to obtain the transition intermediate 2-chlorophenyl (((3R, 4R)-5-(2-oxo-4-(1H-1,2,4-triazol-1-yl) pyrimidin-1(2H) yl)-3,4-diacetoxytetrahydrofuran-2-yl) methyl (2-(hexadecanedithio) ethyl) phosphate (A3) (80.6 g, 100%) (which is used directly in the next step without purification). The sample used for structural identification can be purified by a chromatographic column (CH2Cl2 + 0 to 8% MeOH).

[0367] 1HNMR (600 MHz, chloroform-d4 / methanol-d4): δ9.293-9.275 (m, J=4.2 Hz, J=6.6 Hz, 1H); 8.317 (s, 1H); 8.152-8.131 (d, J=3.6 Hz, 1H); 7.648-7.041 (m, 5H); 6.315-6.191 (dd, J=4.2 Hz, 12 Hz, 1H); 5.486-5.31 0(m,2H);4.663-4.324(m,3H);3.00-2.947(m,J=6Hz,2H);2.737-2.665(m,J=7.2Hz,2H);2.13 7 (s, 3H); 2.113 (s, 3H); 1.358-1.334 (m, J = 7.4Hz, 2H); 1.260 (s, 30H); 0.881 (t, J = 7.2Hz, 3H).

[0368] 13 CNMR (600 MHz, chloroform-d4 / methanol-d4): δ 169.684 (dd, J = 10 Hz); 159.554; 154.541; 153.768; 146.675-146.595

[0369] (d, J=48Hz); 145.638; 143.320; 130.807-130.791 (d, J=9Hz); 128.202-128.158 (d, J=26.4Hz); 126.630-126.583 (d, J=28. 2); 125.290; 121.365-121.330 (d, J = 21Hz); 95.740; 93.291; 89.038-88.956 (d, J = 49.2Hz); 80.849-80.754 (t, J = 26.4Hz); 73.752; 69.433; 67.149; 66.776; 45.961; 42.305; 40.261; 39.231; 38.919-38.898 (d, J = 7Hz); 37.77737.696 (t, J = 25.8Hz) ;31.791;29.559;29.523;29.465;29.380;29.227;29.095;28.999;28.365;22.552;20.263-20.177(d,J=51.6Hz);13.920.

[0370] 31 P:δ-7.005,-7.314.

[0371] MS (ESI) m / e: 885.30 [M] + .

[0372] Step 3:

[0373] 2-Chlorophenyl (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrakis Hydrofuran-2-yl)methyl (2-(hexadecyldithio)ethyl) phosphate (A4)

[0374]

[0375] Add isopropanol (300mL) and triethylamine (7.5g, 75mmol) to the product (A3) (41.8g, 50mmol) obtained above, and stir at room temperature to dissolve the solid. Add hydroxylamine hydrochloride (5.2g, 75mmol), stir the reaction for 2.5 hours to obtain a light yellow reaction solution. Monitor the completion of the reaction by TLC (EtOAc). Concentrate under reduced pressure at a temperature of less than 45°C to remove the solvent. Dissolve the residue in ethyl acetate (300ml), wash the organic phase with water (150mlx2) and saturated brine (150mlx2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The obtained concentrate was recrystallized from petroleum ether to obtain 2-chlorophenyl((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl(2-(hexadecyldithio)ethyl)phosphate (A4) (38.3 g, 90%) as a white solid.

[0376] 1 HNMR (600 MHz, chloroform-d4 / methanol-d4): δ7.477-7.443 (m, J = 4.8 Hz, J = 7.8 Hz, 3H); 7.332-7.306 (t, J = 7.8 Hz, 1H); 7.272-7.160 (m, J = 8.4 Hz, 1H); 6.407-6.320, 5.8445.768 (m, J = 7.8 Hz, 1H); 6.181-6.059 (m, J = 7.8 Hz ,1H);5.454-5.205(m,3H);4.517-4.305(m,2H);2.991-2.957(m,J=6.6Hz,2H),2.718-2.690(m,J=7. 8Hz, 2H); 2.136 (s, 3H); 2.109 (s, 3H); 1.372 (m, J = 7.4Hz, 2H); 1.272 (s, 26H); 0.898 (t, J = 7.2Hz, 3H).

[0377] 13CNMR (600 MHz, chloroform-d4 / methanol-d4): δ 169.684 (dd, J = 10 Hz); 158.406; 155.294; 148.382; 147.301; 144.327; 130.931; 128.419; 126.692; 121.546; 91.434; 80.782; 72.370; 70.926; 70.137; 7 0.098; 69.369; 69.085; 68.969; 67.131; 54.971; 39.259; 37.875; 32.430; 31.791; 31.361; 29.707; 29.665; 29.623; 29.540; 29.336; 29.260; 29.157; 28.532; 22.696; 20.401; 14.124.

[0378] 31 P:δ-6.954,-7.206,-7.326.

[0379] MS (ESI) m / e: 850.30 [M+1] + .

[0380] Step 4:

[0381] (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2- methyl (2-(hexadecyldithio)ethyl) phosphate (A5)

[0382]

[0383] A solution of 1,1,3,3-tetramethylguanidine (4b) (12.9 g, 112.0 mmol) and 2-pyridinecarbaldehyde oxime (4c) (13.70 g, 112.0 mmol) in THF (120 ml) was added to a solution of 2-chlorophenyl (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(hexadecyldithio)ethyl) phosphate (A4) (17.0 g, 20 mmol) in THF (360 mL). The resulting reaction solution was stirred at room temperature overnight. After the reaction was completed as detected by TLC (dichloromethane:methanol=20:1, v / v), ethyl acetate (480 ml) was added to the reaction solution. l) and cold hydrochloric acid solution (pH = 1-2, 200 ml). Separate the organic layer, extract the aqueous layer with 100 ml of ethyl acetate / tetrahydrofuran (1:1) solution 3 times. Combine the organic layers and extract with 2N sodium hydroxide solution (200 ml×2). Combine the aqueous phases, acidify with hydrochloric acid to a pH of 1-2, and then extract with ethyl acetate (200 ml×2). Combine the extracts, dry over anhydrous sodium sulfate, filter and concentrate. The concentrate is recrystallized from petroleum ether to obtain an off-white solid (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(hexadecanedithio)ethyl) phosphate (A5) (10.35 g, 70%).

[0384] 1 HNMR (600 MHz, chloroform-d4 / methanol-d4): δ7.728-7.713 (m, J=5.4 Hz, J=3.6 Hz, 0.35 H); 7.565-7.550 (m, J=5.4 Hz, J=3.6 Hz, 0.35 H); 7.274-7.260, 7.035-7.021, 6.834-6.819 (td. J=8.4 Hz, 0.30 H); 5.988-5.328 (m, J=7.2 Hz, 0.3 H J=3 Hz, 2 H); 4.394-4.371 (dd, J =8.4Hz, J=7.2Hz, 2H); 4.215-4.109 (m, J=6.6Hz, J=7.2, 3H); 2.713-2.678 (m, J=6.6Hz,, 2H); 2.532-2.505 (m, J=8.4Hz, 2H) ; 2.316-2.265 (m, J = 7.8Hz, 2H); 2.171 (d, 3H); 2.037 (d, 3H); 1.416 (m, J = 7.4Hz, 2H); 1.272 (s, 26H); 0.883 (t, J = 7.2Hz, 3H).

[0385] 13 CNMR (600MHz, chloroform-d4 / methanol-d4): δ163.850; 149.382; 132.649; 127.665; 104.878; 72.861; 71.790; 70.217; 68.812; 66.924; 65.615; 62.537; 60.846; 56.043; 39 .099;36.165;32.934;32.067;31.793;29.707;30.655;29.553;29.517;29.221;29.093;29.004;28.896;28.405;27.735;23.385;22.542;20.542;13.889.

[0386] 31 P: δ-6.954,-1.106,-1.413,-1.627.

[0387] MS (-ESI) m / e: 738.30 [MH] + .

[0388] Step 5:

[0389] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran- 2-(hexadecyldithio)ethyl) methyl phosphate (NTV-29)

[0390]

[0391] ((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl(2-(hexadecyldithio)ethyl)phosphate (A5) (7.4 g, 10 mmol) was dissolved in methanol (50 mL). The reaction solution was stirred at room temperature to dissolve the material. Ammonia methanol solution (7 mol / L, 5 ml) was added and reacted at room temperature for 5 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure. 30 ml of aqueous hydrochloric acid solution (pH 1-2) was added to the obtained residue. The obtained solution was extracted with ethyl acetate (3 x 50 ml). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and recrystallized from methanol to obtain (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2-yl)methyl(2-(hexadecyldithio)ethyl)phosphate (NTV-29) (6.04 g, 90%)

[0392] 1HNMR (600 MHz, chloroform-d4 / methanol-d4): δ8.304-7.264 (qd, J=7.8 Hz, H); δ6.180-5.748 (dd, J=6.0 Hz, 7.8 Hz, 2H); 4.328-4.125 (m, 5H); 3.175-2.937 (dtJ=7.2 Hz, J=6.0 Hz, 2H); 2.841-2.679 (m, J=7.2 Hz 2H); 1.772-1.617 (m, J=7.2 Hz, 2H); 1.476-1.354 (m, J=7.2 Hz, J=6.0 Hz, 2H); 1.276 (s, 26H); 0.890 (t, J=7.2 Hz, 3H).

[0393] 13 CNMR (600 MHz, chloroform-d4 / methanol-d4): δ 163.649; 148.928; 134.091; 128.979; 122.135; 105.786; 103.191; 89.422; 74.500; 73.341; 69.152; 66.924; 56.036; 38.716; 38.527; 38.092; 31.552; 29.281; 29.253; 29.208; 29.141; 28.964; 28.865; 28.732; 28.088; 22.259; 13.338.

[0394] 31 P:δ-3.002,-2.964,-2.864.

[0395] MS (ESI) m / e: 656.2 [M+H] + .

[0396] Example 2

[0397] (((2R,3S,4R,5R)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2- Methyl (2-(octadecyldithio)ethyl) phosphate (NTV-37)

[0398]

[0399] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2-yl)methyl(2-(octadecandithioyl)ethyl)phosphate (NTV-37) was prepared in a similar manner to Example 1.

[0400] Step 1:

[0401] 2-Chlorophenyl(((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxy Tetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A2)

[0402]

[0403] Referring to the method for preparing (A2) described in step 1 of Example 1, 2-(octadecyldithio)ethanol (1b-2) was used instead of 2-hexadecyldithioethanol (1b-1) to react to obtain 2-chlorophenyl (((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A2).

[0404] Specifically, feed according to the following table:

[0405]

[0406] A solution of 1,2,4-triazole (1c) and triethylamine in dry tetrahydrofuran was added to a solution of 2-chlorophenyl dichlorophosphate (1d-1) in dry tetrahydrofuran previously cooled to 0°C. The resulting reaction solution was reacted at room temperature for 1 hour. The generated solid was filtered off. A solution of 2',3'-di-O-acetyluidine (A1) and 1-methylimidazole in tetrahydrofuran was added to the resulting filtrate. After the addition, the reaction was stirred at room temperature for 1 hour. Then, 2-octadecyl dithioethanol (1b-2) was added to the reaction solution, and the resulting reaction solution was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (200 mL). The resulting solution was washed with water (100 ml x 2), saturated sodium bicarbonate aqueous solution (100 ml x 2), water (100 ml x 2) and saturated brine (100 ml x 2) in sequence. The organic phase was separated, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 2-chlorophenyl (((3R, 4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A2) (29.2 g, yield 71%) as a colorless oil, which was directly used in the next step without further purification. The sample for structural identification can be purified by a chromatographic column (CH2Cl2 + 0 to 10% MeOH).

[0407] 11H NMR (600 MHz, chloroform-d4 / methanol-d4): δ 7.483 - 7.414 (m, J = 3 Hz, J = 8.4 Hz, 2H); 7.308 - 7.266 (dd, J = 1.8 Hz, J = 7.8 Hz, 1H); 7.199 - 7.161 (dd, J = 7.2 Hz, 1H); 6.099 - 6.064 (ddd, J = 6 Hz, 3.6 Hz, 1.8 Hz, 1H); 5.747 - 5.677 (ddd, J = 7.8 Hz, J = 1.8 Hz, J = 2.4 Hz, 1H); 5.439 - 5.339 (m, J = 3.6 Hz, 6.6 Hz, 4.2H, 1H); 5.323 - 5.269 (m, J = 3.6 Hz, 2.4 Hz, 7.2H, 1H); 4.542 - 4.460 (m, 4H); 2.992 - 2.947 (t, J = 6 Hz, 2H); 2.707 - 2.674 (m, J = 4.2 Hz, J = 2.4 J = 7.6 Hz, 2H); 2.135 - 2.129 (t, J = 1.8 Hz, 3H); 2.104 - 2.096 (t, J = 2.4 Hz, 3H); 1.683 - 1.630 (m, J = 7.2 Hz, J = 5.4 Hz, J = 2.4 Hz, 2H); 1.261 (s, 30H); 0.893 - 0.866 (t, J = 7.2 Hz, 3H).

[0408] 13CNMR (600 MHz, chloroform-d4 / methanol-d4): δ 169.766-169.743 (d, J = 13.8, Hz); 169.580-169.564 (d, J = 9.6 Hz); 150.402; 145.988-145.948 (d, J = 24 Hz); 139.674-139.585 (d, J = 53 .4Hz);130.707-130.690(d,J=10.2Hz);128.081;126.482;125.265-125.218(d,J =26.4Hz); 121.282; 103.233; 86.891-86.728 (d, J = 97.8Hz); 80.418-80.312 (t, J = 31.8Hz); 72.461; 69.848-69.832 (d, J=8.4.Hz); 67.241-67.203 (d, J=22.8Hz); 67 .066-67.025(d,J=24.6Hz); 67.033-66.962(dd,J=24.6Hz, J=7.2Hz); 39.055; 38. 888;37.69537.648(d,J=22.2Hz);31.752;29.516;29.476;29.424;29.341;29.18 0;29.056;28.953;28.326;22.502;20.211;20.075-20.067(d,J=8.4Hz);13.858.

[0409] 31 Pδ:-3.188,-3.445.

[0410] MS (ESI) m / e: 863.3 [M] + .

[0411] Step 2:

[0412] 2-Chlorophenyl(((3R,4R)-5-(2-oxo-4-(1H-1,2,4-triazol-1-yl)pyrimidin-1(2H)yl)-3,4- Diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A3)

[0413]

[0414] Referring to the method for preparing (A3) described in step 2 of Example 1, 2-chlorophenyl (((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio))ethyl) phosphate (1A2) was used instead of 2-chlorophenyl (((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio))ethyl) phosphate. 4-diacetoxytetrahydrofuran-2-yl)methyl(2-(hexadecanedithio)ethyl)phosphate (A2) was reacted to obtain 12.8 g of 2-chlorophenyl(((3R,4R)-5-(2-oxo-4-(1H-1,2,4-triazol-1-yl)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl(2-(octadecanedithio)ethyl)phosphate (1A3) as a colorless oil with a yield of 81%.

[0415] Specifically, feed according to the following table:

[0416]

[0417]

[0418] 1 HNMR (600MHz, chloroform-d4 / methanol-d4): δ9.297-9.285 (t, J=3.5Hz, 0.5H); 8.404 (m, 0.5H); 8.325-8.310 (dd, J=7.2Hz, J=1.8Hz, 0.5H); 7.465-7.439 (m, J=6.6Hz, J=2.4Hz, 1H); 7.444-7.379 (m, J=7.2Hz, J=1.8Hz, 1H); 7.321-7.273 (m, J=8.4Hz, J=2.4Hz, J=1.8Hz, 1H); 7.245-7.169 (m, J=3Hz, 1H); 7.141- 7.068 (dd, J=7.2Hz, J=36.6Hz, 1H); 6.296-5.987 (dd, J=7.8Hz, J=32.4Hz , J=11.4Hz, 1H),5.464-5.337(m,2H); 4.664-4.309(m,4H); 3.002-2.948( m, J=6.6Hz, 2H); 2.723-2.667 (m, J=7.2Hz, 2H); 2.145 (m, J=7.2Hz, 3H); 2 .139(s,3H);1.366-1.362(m,2H);1.272(s,28H);0.882(t,J=7.2Hz,3H).

[0419] 13CNMR (600MHz, chloroform-d4 / methanol-d4): δ169.898 (d, J=8.4Hz); 169.798; 159.751 (d, J=11.4Hz); 154.762; 153.923; 148.895 (d, J=46.2Hz); 145.459; 143.477; 130.981; 128.354 (d, J=22.4Hz); 126.828 (d, J=28.8Hz); 121.506 (t, J=29.4Hz); 95.944; 89.268 (d, J = 49.2Hz); 80.981 (t, J = 29.4Hz); 69.607; 67.302 (d, J = 57Hz); 66.961; 46.244; 39.096; 37.913 ;31.981;29.745;29.705;29.650;29.564;29.409;29.280;29.181;28.547;22.731;20.476(d,J=58.2Hz);14.080.

[0420] 31 P:δ-6.954,-3.039,-3.342.

[0421] MS (ESI) m / e: 914.50 [M] + .

[0422] Step 3:

[0423] 2-Chlorophenyl (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrakis Hydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A4)

[0424]

[0425] The target product was synthesized by referring to the method described in step 3 of Example 1. Specifically, the materials were added according to the following table:

[0426]

[0427] Isopropanol and triethylamine were added to the product (1A3) obtained above, and the mixture was stirred at room temperature to dissolve the solid. Hydroxylamine hydrochloride was added to the reaction solution, and a light yellow reaction solution was obtained after stirring for 1 hour. The reaction was monitored by TLC (EtOAc). The solvent was removed by concentrating under reduced pressure at a temperature less than 45°C. The residue was dissolved in ethyl acetate, and the organic layer was washed with water and saturated brine in sequence. The obtained organic solution was dried over anhydrous sodium sulfate, filtered and concentrated. The concentrate was recrystallized from petroleum ether to obtain a white solid 2-chlorophenyl (((3R, 4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A4) in a yield of 90%.

[0428] 1 HNMR (600MHz, methanol-d4): δ7.511-7.491 (dd, J=7.2Hz, 1H); 7.480-7.466 (d, J=7.2Hz, 1H); 7.340-7.327 ((dd, J=7.2Hz, 1H); 7.234-7.224((d,J=7.2Hz,1H),6.853-6.839(d,J=7.4Hz,1H); 5.968-5.940(d,J=6.0Hz,1H); 5.704-5.53(dd,J=16.8;Hz J=7.2Hz,1H);5.453-5.417(m,J=7.2Hz,1H);5.352-5.33(m,J=7.2Hz,1H);4.531- 4.471 (m, 3H); 4.315 (m, 2H); 3.017-3.000 (m, J = 4.2Hz, 3.6Hz, 2H); 2.727-2.698 (d t, J=7.2Hz, J=4.2Hz, 2H); 2.109-2.107 (d, J=1.2Hz, 3H); 2.071-2.068 (d, J=1.8Hz ,3H);1.672-1.648(t,J=7.2Hz,2H);1.434-1.243(m,30H);0.898(t,J=7.2Hz,3H).

[0429] 13CNMR (600MHz, methanol-d4): δ169.972 (d, J = 15 Hz), 169.818 (d, J = 12.6 Hz); 149.795; 146.256; 144.315; 130.591; 130.363 (d, J = 38. 4Hz); 128.160; 126.558; 125.162 (d, J = 27Hz); 121.435 (d, J = 24.6Hz); 102.213; 98.915; 88.690; 87.192 (d, J = 69.6Hz); 79.92 8(dd, J=16.2Hz, J=30Hz); 71.840; 69.886 (d, J=9.6hz); 69.718 (m, J=24.6Hz, J=16.8Hz, J=12Hz); 38.503; 37.641 (d, J=33Hz) ;31.729;29.415;29.426;29.363;19.298;29.141;28.983;28.891;28.807;28.135;22.395;19.132, (d,J=38.4Hz);13.119.

[0430] 31 P:δ-7.277,-7.413.

[0431] MS (ESI) m / e: 878.3237 [M+H] + .

[0432] Step 4:

[0433] (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2- methyl (2-(octadecyldithio)ethyl) phosphate (1A5)

[0434]

[0435] Referring to the method for preparing (A5) described in step 4 of Example 1, 2-chlorophenyl (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A4) was used instead of 2-chlorophenyl (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(hexadecyldithio)ethyl) phosphate (A4) to prepare compound (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A5):

[0436] Specifically, feed according to the following table:

[0437]

[0438] A solution of 1,1,3,3-tetramethylguanidine (4b) and 2-pyridinecarbaldehyde oxime (4c) in THF was added to a solution of 2-chlorophenyl (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (1A4) in THF. The resulting reaction solution was stirred at room temperature overnight. The completion of the reaction was detected by TLC (dichloromethane:methanol=20:1, v / v). After the reaction was completed, ethyl acetate and cold hydrochloric acid aqueous solution (pH=1-2) were added to the reaction solution. Separation The organic layer was extracted with ethyl acetate / tetrahydrofuran (1:1, v / v) solution for 3 times. The organic layers were combined and extracted with 2N sodium hydroxide solution. The aqueous phases were combined, acidified with hydrochloric acid to a pH of 1-2, and then extracted with ethyl acetate. The extracts were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The concentrate was recrystallized from petroleum ether to obtain an off-white solid (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidine-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecanedithio)ethyl) phosphate (1A5) 4.2 g (yield 65%).

[0439] 1 HNMR (600 MHz, chloroform-d4 / methanol-d4): δ7.846-7.723 (m, J=7.2 Hz, 1H); 6.165-5.413 (mJ=7.8 Hz, 2H); 4.710-4.386 (m, J=4.2 Hz, J=3 Hz, J=14.2 Hz, 5H); 4.189-4.176 (m, J=3 Hz, J= 4.8Hz,2H);3.916-3.893(m,J=8.4Hz,2H);3.811-3.773(m,J=4.8Hz,,2H),2.132(d , 3H); 2.096 (d, 3H); 1.408 (m, J = 7.4Hz, 2H); 1.261 (s, 30H); 0.882 (t, J = 7.2Hz, 3H).

[0440] 13CNMR (600 MHz, chloroform-d4 / methanol-d4): δ 170.158; 166.927; 157.177; 133.281; 128.768; 124.491; 120.504; 116.385; 96.866; 64.274; 63.160; 62.814 (J = 27.6 Hz); 62.689 (J=35.46Hz); 52.024; 33.991; 31.794; 29.556; 29.518; 29.477; 29.3 83; 29.350; 29.171; 29.041; 24.818; 22.545; 16.184 (m, J=13.8Hz); 13.996.

[0441] 31 P:δ, 25.554; 24.651.

[0442] Step 5:

[0443] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2- Methyl (2-(octadecyldithio)ethyl) phosphate (NTV-37)

[0444]

[0445] Referring to the method for preparing compound NTV-29 described in Step 5 of Example 1, (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl(2-(octadecyldithio))ethyl phosphate (1A5) was reacted with a methanol solution of ammonia to obtain the target product (NTV-37).

[0446] Specifically, (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl(2-(octadecyldithio))ethyl phosphate (1A5) was dissolved in methanol and stirred at room temperature to dissolve the material. A methanol solution of ammonia (7 mol / L) was added and reacted at room temperature for 5 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure. A hydrochloric acid solution was added to the residue. 30 ml of liquid (pH 1-2) was added to dissolve the residue. The aqueous solution was extracted with ethyl acetate three times, and the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and recrystallized with methanol to obtain ((2R, 3S, 4R, 5S)-3, 4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2-yl)methyl (2-(octadecanedithio)ethyl) phosphate (NTV-37) (yield 95%).

[0447] 1HNMR (600 MHz, chloroform-d4 / methanol-d4): δ8.061 (d, J = 8.4 Hz, 0.2H); δ7.572 (dd, J = 7.8 Hz, 3.0 Hz, 0.8H); 5.997 (d, J = 7.8 Hz, 0.2H); 5.764 (d, J = 8.4 Hz, 0.8H); 5.699 (d, J = 8.4 Hz, 0.8H); 5.573 (d, J = 8.4 Hz, 0.2 H); 4.048-3.95 (m, 5H); 3.166-3.154 (m, J = 2.4Hz, 2H); 2.724-2.754 (t, J = 7.2Hz, 2H); 2.493-2.7 17 (t, J = 7.2 Hz, 2H); 1.406-1.489 (t, J = 7.2 Hz, 2H); 1.068-1.196 (m, 30H); 0.688 (t, J = 7.2 Hz, 3H).

[0448] 13 CNMR (600 MHz, chloroform-d4 / methanol-d4):δ156.34;154.804;147.446;128.561;125.075;119.237;113.345;92.684;89.755;74.407;69.327;64.981;39.214;38.991;31.916;29.676;29.639;29.603;29.574;29.526;29.341;29.253;29.208;28.510;22.656;13.955.

[0449] 31 P: δ2.800, 2.667.

[0450] MS (ESI) m / e: 700.29 [M+H] + .

[0451] Example 3

[0452] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2- 2-(octadecyldithio)ethyl)(methylcarbonatemethyl)phosphate (NTV-59)

[0453]

[0454] The synthesis was carried out according to the following route:

[0455]

[0456] Step 1:

[0457] Methyl chloromethyl carbonate

[0458] Place chloromethyl chloroformate (1.5 g, 11.72 mmol) and methanol (375 mg, 11.72 mmol) in a reaction flask, add 15 ml of anhydrous ether to dissolve. Slowly add pyridine (0.93 g, 11.72 mmol) at 0°C. After addition, warm the reaction solution to room temperature and stir to react overnight. Add water to dilute, wash three times with 1% (w / v) citric acid aqueous solution, saturated sodium bicarbonate aqueous solution and saturated brine in sequence, separate the organic layer, dry over anhydrous sodium sulfate, filter and concentrate to obtain 1.32 g of product, yield: 90.8%.

[0459] Step 2:

[0460] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2- 2-(octadecyldithio)ethyl)(methylcarbonatemethyl)phosphate (NTV-59)

[0461] Compound NTV-37 (341.8 mg, 0.5 mmol), chloromethyl methyl carbonate (513.8 mg, 4.14 mmol), potassium carbonate (572.7 mg, 4.14 mmol), potassium iodide (68.1 mg, 0.41 mmol) and DMF (10 ml) were added to the reaction flask. The reaction solution was heated to 60 ° C and reacted overnight. The reaction was completed by TLC. The reaction solution was cooled to room temperature, diluted with excess water, and extracted with ethyl acetate (3x30 ml). The organic phases were combined, washed with saturated brine and concentrated, and purified by silica gel column chromatography to obtain 154 mg of product, yield: 40%.

[0462] 1 HNMR (600MHz, methanol-d4): δ7.961 (d, J=8.4Hz, 0.2H); δ7.472 (dd, J=7.8Hz, 3.0Hz, 0.8H); 6.097 (d, J=7.8Hz, 0.2H); 5.704 (d, J=8.4Hz, 0.8H); 5.629 (d, J=8.4Hz, 0.8H); 5.583 (d, J=8.4Hz, 0.2H); 4.028-3.85(m,8H);3.164-3.152(m,J=2.4Hz,4H);2.724-2.754(t,J=7.2Hz,2H);2.493-2.717 (t, J=7.2Hz, 2H); 1.406-1.489 (t, J=7.2Hz, 2H); 1.068-1.196 (m, 30H); 0.688 (t, J=7.2Hz, 3H).

[0463] MS (ESI) m / e: 771.32 [M] + .

[0464] Example 4

[0465] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2- 2-(octadecyldithio)ethyl)(N,N-dimethylamino)phosphate (NTV-84)

[0466]

[0467] Referring to the reaction route of Example 2, 1d-2 was used instead of 1d-1 to carry out the reaction to obtain compound NTV-84.

[0468] Step 1:

[0469] N,N-dimethylamino(((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetyl Oxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (A2-1)

[0470]

[0471] Feed according to the table below:

[0472]

[0473] A solution of 1,2,4-triazole (1c) and triethylamine in dry tetrahydrofuran is added to a solution of N,N-dimethylaminodichlorophosphate (1d-2) in dry tetrahydrofuran previously cooled to 0°C. The resulting reaction solution is reacted at room temperature for 1 hour. The generated solid is filtered off. A solution of 2',3'-di-O-acetyluidine (A1) and 1-methylimidazole in tetrahydrofuran is added to the resulting filtrate. After the addition, the reaction is stirred at room temperature for 1 hour. Then, 2-octadecyl dithioethanol (1b-2) is added to the reaction solution, and the resulting reaction solution is stirred at room temperature overnight. The reaction solution is concentrated under reduced pressure, and the residue is dissolved in ethyl acetate (200 mL). The resulting solution is washed with water (100 ml x 2), saturated sodium bicarbonate aqueous solution (100 ml x 2), water (100 ml x 2) and saturated brine (100 ml x 2) in sequence. The organic phase is separated, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give N,N-dimethylamino(((3R,4R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl(2-(octadecyldithio)ethyl)phosphate (A2-1) (39.24 g, yield 75%) as a colorless oil, which was directly used in the next reaction without further purification.

[0474] MS (ESI) m / e: 779.36 [M] + .

[0475] Step 2:

[0476] N,N-dimethylamino(((3R,4R)-5-(2-oxo-4-(1H-1,2,4-triazol-1-yl)pyrimidin-1(2H)yl)- 3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecyldithio)ethyl) phosphate (A3-1)

[0477]

[0478] The reaction was carried out according to the method for preparing (1A3) described in step 2 of Example 2 to obtain the intermediate (A3-1). Specifically, the materials were added according to the following table:

[0479]

[0480] The intermediate (A2-1) was dissolved in acetonitrile with stirring. After dissolution, 1,2,4-triazole and triethylamine were added, and the reaction solution was stirred to dissolve the solid. The reaction solution was cooled to 0°C, and phosphorus oxychloride was slowly added dropwise under a nitrogen atmosphere. After the addition, the temperature of the reaction solution was raised to room temperature, and stirring was continued for 2 hours to complete the reaction. The reaction was monitored by TLC (100% EtOAc). After the reaction was completed, water and ethyl acetate were added and stirred at room temperature for 15 minutes to quench the reaction. The organic layer was separated, washed with water, 0.5N HCl and saturated brine in turn, dried over anhydrous magnesium sulfate, filtered and concentrated to obtain a white solid (A3-1) (33 g, yield 80%), which did not need to be purified and was directly used in the next step.

[0481] MS (ESI) m / e: 830.38 [M] + .

[0482] Step 3:

[0483] N,N-Dimethylamino(((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxy 2-((2-(octadecyldithio)ethyl)-1,4-dihydrofuran-2-yl)methyl(2-(octadecyldithio)ethyl)phosphate (A4-1)

[0484]

[0485] The reaction was carried out according to the method for preparing (1A4) described in step 3 of Example 2 to obtain the intermediate (A4-1). Specifically, the materials were added according to the following table:

[0486]

[0487] Isopropanol and triethylamine were added to the product (A3-1) obtained above, and the mixture was stirred at room temperature to dissolve the solid. Hydroxylamine hydrochloride was added to the reaction solution, and a light yellow reaction solution was obtained after stirring for 1 hour. The reaction was monitored by TLC (EtOAc). The solvent was removed by concentrating under reduced pressure at a temperature of less than 45°C. The residue was dissolved in ethyl acetate, and the organic layer was washed with water and saturated brine in turn. The obtained organic solution was dried over anhydrous sodium sulfate, filtered and concentrated. The concentrate was recrystallized from petroleum ether to obtain 7.3 g of white solid N,N-dimethylamino (((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidine-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl (2-(octadecanedisulfide)ethyl) phosphate (A4-1), with a yield of 91%.

[0488] MS (ESI) m / e: 794.37 [M] + .

[0489] Step 4:

[0490] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)yltetrahydrofuran-2- 2-(octadecyldithio)ethyl)(N,N-dimethylamino)phosphate (NTV-84)

[0491] 4 g of N,N-dimethylamino(((3R,4R)-5-(2-oxo-4-(hydroxyamino)pyrimidin-1(2H)yl)-3,4-diacetoxytetrahydrofuran-2-yl)methyl(2-(octadecyldithio)ethyl)phosphate (A4-1) was dissolved in 30 ml of methanol. The reaction solution was stirred at room temperature to dissolve the material. 10 ml (7 mol / L) of ammonia methanol solution was added and reacted at room temperature for 5 h. After the reaction was completed, it was concentrated under reduced pressure and the residue was added to 30 ml of water. The resulting aqueous solution was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and recrystallized from methanol to obtain 2.85 g of compound (NTV-84) (yield 80%).

[0492] 1 HNMR (600 MHz, methanol-d4): δ 7.861-7.562 (m J=8.4Hz, 3.0Hz, 1H); 7.572-5.997 (m, J=7.8Hz, 1H) 5.764-5.719 (m, J=8.4H z,1H);4.048-3.95(m,5H);3.166-3.154(m,J=2.4Hz,2H);2.724-2.754(t, J=7.2Hz,2H);2.652(s,3H);2.602(s,3H);2.487-2.713(t,J=7.2Hz,2H);1 .402-1.477(t,J=7.2Hz,2H); 1.068-1.196(m,30H); 0.688(t,J=7.2Hz,3H).

[0493] MS (ESI) m / e: 710.35 [M] + .

[0494] Example 5

[0495] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-(hexadecyldithio)ethyl) phosphate sodium salt (II-1-1)

[0496]

[0497] To (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yltetrahydrofuran-2-yl)methyl (2-(hexadecyldithio)ethyl) phosphate (NTV-29) (6.56 g, 10 mmol) was added 50 ml of anhydrous ethanol. The reactant was heated to 60°C with stirring to dissolve it. At this temperature, 2NNaOH ethanol solution (5 ml) was added to the solution and stirred for 10 minutes. The temperature of the reaction solution was lowered to room temperature, left to stand at 4°C overnight, and filtered to obtain (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (2-(hexadecyldithio)ethyl) phosphate sodium salt alkoxy) (II-1-1) as a white solid (5.9 g, 92%).

[0498] Example 6

[0499] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methyl (3-(octadecyldithio)ethyl) phosphate ammonium salt (II-1-2)

[0500]

[0501] A flask was charged with (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl(2-(octadecyldithio)ethyl)phosphate (I-2) (6.83 g, 0.01 mol), 2-propanol (50 ml) and 28-30% (w / w) aqueous ammonia solution (1 ml). The reaction solution was stirred and refluxed at 62-80°C to achieve dissolution (over about 10 minutes). The solution was allowed to stand for more than 15 minutes without stirring under reflux. The solution was cooled at a temperature below 25°C for 8-24 hours, and then the mixture was cooled to 5±5°C and maintained for at least 1 hour. The product was filtered and washed with cooled 2-propanol (0±5°C, 30 ml). The white solid product was dried at 30-35°C for 25 hours to give (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl(3-(octadecyldithio)ethyl)phosphate ammonium salt (II-1-2) (6.0 g, 92.3%).

[0502] Example 7

[0503] ((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methyl (3-(hexadecyloxy)propyl) phosphate (NTV-118)

[0504]

[0505] Step 1:

[0506] 2-Chlorophenyl (((3aR,4R,6S,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dihydro- Methyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)(3-(hexadecyloxy)propyl)phosphate (1-1A2)

[0507]

[0508] (1) 1-((3aR,4S,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)pyrimidine-2,4(1H,3H)-dione (1-1a)

[0509]

[0510] 1-((3R,4S)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidine-2,4(1H,3H)-dione (uridine) (48.4 g, 200 mmol) and acetone (2000 mL) were added to a reaction flask. Sulfuric acid (98%, 2 mL) was added to the resulting slurry under stirring at room temperature. The reaction solution was stirred at room temperature for 48 hours. After a clear solution was obtained, the pH of the reaction solution was adjusted to 8 with triethylamine. The solution was concentrated under reduced pressure to obtain a light yellow oily residue. The residue was washed with ethyl acetate (400 mL). mL) was dissolved. The resulting solution was washed with water (200 ml x 2), saturated sodium bicarbonate aqueous solution (200 ml x 2), water (200 ml x 2) and saturated brine (200 ml x 2) in sequence. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain white 1-((3aR,4S,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxolan-4-yl)pyrimidine-2,4(1H,3H)-dione (1-1a) (33 g).

[0511] (2) 3-(Hexadecyloxy)propan-1-ol (1-1b-1)

[0512] HOCH2CH2CH2O-CH2(CH2) 14 CH3

[0513] 1-1b-1

[0514] Reaction route:

[0515]

[0516] I. 1-(Hexadecyl) p-Toluenesulfonate

[0517]

[0518] Under nitrogen protection, 1-(hexadecyl) alcohol (48.4 g), anhydrous dichloromethane (400 mL) and triethylamine (26.3 g) were added to the reactor. The reaction mixture was cooled to 0 ± 5 ° C. A solution of p-toluenesulfonyl chloride (41 g) in dichloromethane (200 ml) was added dropwise at this temperature. After addition, the reaction mixture was warmed to 20-30 ° C and stirred for 2 hours. The reaction solution was maintained at 20-30 ° C and diluted with water (150 mL). The organic layer was separated, washed with water (50 ml) and concentrated to dryness to obtain 1-(hexadecyl) p-toluenesulfonate (48.7 g, 96%) as a light yellow solid.

[0519] II. 3-(Hexadecyloxy)propan-1-ol (1-1b-1)

[0520] Under nitrogen, l,3-propanediol (40.7 g) and N-methylformamide (NMF) (300 ml) were added to the reactor. The reaction solution was cooled to 0±5°C and sodium hydride (10.7 g, 60%) was carefully added in batches. After the addition was completed, the reaction solution was stirred at room temperature for another 2 hours. A solution of hexadecyl p-toluenesulfonate (52.8 g) dissolved in DMF (100 ml) was slowly added to the reaction mixture at 20-35°C. The solution was stirred at 20-35°C for 24 hours. The reaction mixture was cooled to 0±5°C, slowly diluted with water (150 ml), and extracted with ethyl acetate (2x250 ml). The organic phase was washed with water (200 ml), dried over anhydrous Na2SO4 and concentrated to obtain a brown oil. The crude product was dissolved in methanol (200 ml) and placed at 0-30°C for 12 hours. The solid impurities generated were filtered and discarded, and the filtrate was concentrated. To the concentrate was added acetonitrile (400 ml), and the mixture was allowed to stand for 16 hours at 5-15° C. The solid was filtered and dried at 25-30° C. to give 3-(hexadecyloxy)propan-1-ol (1-1b-1) (31.1 g, 77%) as a white solid.

[0521] (3) 2-Chlorophenyl (((3aR,4R,6S,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2- dimethyltetrahydrofuro[3,4-d]1,3]dioxolan-4-yl)methyl)(3-(hexadecyloxy)propyl)phosphoric acid Ester (1-1A2)

[0522]

[0523] A solution of 1,2,4-triazole (1c) (13.8 g, 2002.12 mmol) and triethylamine (10.1 g, 200 mmol) in dry tetrahydrofuran (200 mL) was added to a solution of 2-chlorophenyl dichlorophosphate (1d-1) (24.5 g, 100 mmol) in dry tetrahydrofuran (150 mL). The resulting reaction solution was reacted at room temperature for 1 hour. The generated solid was filtered off, and a solution of 1-((3aR,4S,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)pyrimidine-2,4(1H,3H)-dione (1-1a) (28.4 g, 100 mmol) and 1-methylimidazole (8.4 ml, 100 mmol) in tetrahydrofuran (150 mL) was added to the obtained filtrate. After the addition, the reaction was stirred at room temperature for 1 hour. 3-(hexadecyloxy)propan-1-ol (1b-3) (30 g, 100 mmol) was added to the reaction solution. The obtained reaction solution was stirred at room temperature overnight, and after the reaction was completed, it was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (400 mL). The resulting solution was washed with water (200 ml x 2), saturated sodium bicarbonate aqueous solution (200 ml x 2), water (200 ml x 2) and saturated brine (200 ml x 2) in sequence. The organic layer was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 2-chlorophenyl (((3aR, 4R, 6S, 6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxolan-4-yl)methyl (3-(hexadecyloxy)propyl) phosphate (1-1A2) (53 g, 70%) as a colorless oil, which was directly used in the next step without further purification.

[0524] 11H NMR (600 MHz, / methanol-d4): δ 7.615 (dd, J = 8.4 Hz, J = 3.6 Hz, 1H); 7.510 (d, J = 7.8 Hz, 1H); 7.326 (d, J = 7.8 Hz, 1H); 7.228 (d, J = 7.8 Hz, 1H), 5.771 (ddd, J = 1.8, J = 4.8, J = 6 Hz, 1H); 5.659 (dd, J = 7.8 Hz, 1H); 5.087 (ddd, J = 25.8 Hz, J = 7.8 Hz, 1H); 4.418 - 4.479 (m, J = 7.8 Hz, J = 3.6 Hz, 2H); 4.389 - 4.342 (m, J = 6.0 Hz, J = 4.2 Hz, 2H); 3.652 (t, J = 6.6 Hz, 2H); 3.545 - 3.501 (m, 2H), 3.454 - 3.401 (m, 2H); 1.988 - 1.953 (m, 2H); 1.558 (s, 6H); 1.366 - 1.361 (m, 2x2H); 1.352 - 1.228 (m, 26H); 0.87 (t, J = 7.2 Hz, 3H).

[0525] 13 13C NMR (600 MHz, / methanol-d4): 164.693 (d, J = 13 Hz); 150.454; 146.399; 146.357; 143.445 (t, J = 30 Hz); 143.268; 130,485; 127.967; 126.294 (d, J = 30 Hz); 121.260 (t, J = 7.2 Hz); 114.031 (t, J = 6 Hz); 101.490; 94.577; 94.839; 85.482 (d, J = 30 Hz); 85.635 (d, J = 30 Hz); 84.271; 81; 008 (d, J = 24 Hz); 70.736; 70.695; 68.348 (t, J = 27 Hz); 67.404; 66.613 (d, J = 25 Hz); 65.815 (d, J = 22 Hz); 58.787; 32.320; 31.702; 30.163 (d, J = 27 Hz); 29.423; 29.410; 29.396; 29.369; 29.230; 26.033; 25.903; 24.124; 22.364; 13.103.

[0526] 31 31P NMR (600 MHz, CD3OD): δ -7.173; -7.185.

[0527] MS (ESI) m / e: 757.35803 [M+H] +.

[0528] Step 2:

[0529] 2-Chlorophenyl(3-(hexadecyloxy)propyl)((3aR,4R,6S,6aR)-6-(4-(1,2,4-triazolyl)-2-oxy 1-(2H)-pyrimidin-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methylphosphonic acid Ester (1-1A3)

[0530]

[0531] 2-Chlorophenyl (((3aR,4R,6S,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl (3-(hexadecyloxy)propyl) phosphate (1-1A2) (76 g, 100.0 mmol) was dissolved in acetonitrile (400.0 mL) under stirring. After dissolution, 1,2,4-triazole (1c) (48.0 g, 700.0 mmol) and triethylamine were added. Amine (81g, 800.0mmol) was added and the solid was dissolved under stirring. The reaction solution was cooled to 0°C and phosphorus oxychloride (14.5ml, 150.0mmol) was slowly added dropwise under a nitrogen atmosphere. After the addition, the reaction solution was allowed to warm to room temperature and stirred for 2 hours to allow the reaction to proceed completely. The reaction was monitored by TLC (100% EtOAc). After the reaction was completed, water (500mL) and ethyl acetate (400mL) were added and stirred at room temperature for 15 minutes to quench the reaction. The organic layer was separated and the organic phase was washed with water (2x100 mL), 0.5N HCl (200 mL) and saturated brine (2x100 mL), dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to obtain the transitional intermediate 2-chlorophenyl (3- (hexadecyloxy) propyl) ((3aR, 4R, 6S, 6aR) -6- (4- (1,2,4-triazolyl) -2-oxopyrimidin-1 (2H) yl) -2,2- dimethyltetrahydrofuran [3,4-d] [1,3] dioxolan-4-yl) methyl) phosphate (1-1A3) (80.6 g, 100%), which was used directly in the next step without purification.

[0532] Step 3:

[0533] 2-Chlorophenyl(3-(hexadecyloxy)propyl)(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidine 2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1A4)

[0534]

[0535] The product (1-1A3) (40.6 g, 50 mmol) obtained above was added to a mixture of isopropanol (300 mL) and triethylamine (7.5 g, 75 mmol) and stirred at room temperature to dissolve the solid. Then, hydroxylamine hydrochloride (5.2 g, 75 mmol) was added and stirred for 1-2 hours to obtain a light yellow reaction solution, and the reaction was monitored by TLC (EtOAc). The solvent was removed by concentrating under reduced pressure at a temperature less than 45°C. The residue was dissolved in ethyl acetate (300 ml), and the organic phase was washed with water (150 ml x 2), saturated brine (150 ml x 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The concentrate was recrystallized from petroleum ether to give 2-chlorophenyl(3-(hexadecyloxy)propyl)(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1A4) (34.7 g, 90%) as a white solid.

[0536] 1 HNMR (600MHz, / methanol-d4): δ7.517-7.504(d,J=7.8Hz,1H); 7.459-7.445(d,J=7.8Hz,1H); 7.347-7.321(t,J=7.8Hz,1H); 7.245-7.232(d,J=7.8H z, 1H); 6.851-6.826 (t, J = 7.8Hz, 1H); 5.700-5.691 (t, J = 3Hz, 1H); 5.576-5.537 (dd, J = 7.8Hz, 1H); 5.014-4.944 (ddd, J = 33Hz, J = 1.8Hz, 1H), 4 .443-4.415 (dd, J=6Hz, J=4.2Hz, 2H); 4.382-4.349 (dd, J=6.6Hz, 2H); 4.298-4.249 (m, J=3.6Hz, J=2.5Hz, 1H); 3.582-3.509 (t, J=6.6Hz, 2H); 3.428-3.407(t,J=6.6Hz,2H); 1.996-1.965(m,J=6.6,2H); 1.551(s,6H ); 1.398-1.327 (m, 2x2H); 1.342-1.300 (m, 26H); 0.87 (t, J = 7.2Hz, 3H).

[0537] 13CNMR (600MHz, / methanol-d4): δ 149.678; 148.760; 146.369; 144.751; 132.764; 130.475; 127.985; 126.793; 121.285; 114.067; 98.126; 93.290; 84.605; 83.846; 83.792; 80.816; 80 .766; 79.646; 70.730; 68.402; 66.557; 65.812; 65.775; 31.685; 30.150; 30.104; 29.393; 29.381; 29.346; 29.202; 29.086; 26.153; 26.084; 25.886; 24.130; 22.345; 13.057.

[0538] 31 P NMR (600MHz, CD3OD): δ-7.135,-7.196.

[0539] MS (ESI) m / e: 772.36597 [M+H] + .

[0540] Step 4:

[0541] 3-(Hexadecyloxy)propyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidine-1(2H)- 2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxolan-4-yl)methyl)

[0542] Phosphate ester (1-1A5)

[0543]

[0544] A solution of 1,1,3,3-tetramethylguanidine (4b) (12.9 g, 112.0 mmol) and 2-pyridinecarbaldehyde oxime (4c) (13.70 g, 112.0 mmol) in THF (120 ml) was added to a solution of 2-chlorophenyl(3-(hexadecyloxy)propyl)(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1A4) (15.4 g, 20 mmol) in THF (360 mL), and the resulting reaction solution was stirred at room temperature overnight. After the reaction was completed, ethyl acetate (480 ml) and cold hydrochloric acid aqueous solution (pH = 1-2, 200 ml) were added to the reaction solution by monitoring by TLC (dichloromethane: methanol = 20:1, v / v). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate / tetrahydrofuran (1:1, v / v) solution (100 ml × 3). The organic layers were combined and extracted with 2N sodium hydroxide solution (200 ml × 2). The aqueous phases were combined, acidified with hydrochloric acid to a pH of 1-2, and extracted with ethyl acetate (200 ml × 2). The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The concentrate was recrystallized from petroleum ether to give an off-white solid 3-(hexadecyloxy)propyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1A5) (9.2 g, 70%).

[0545] 1 H NMR (600MHz, CD3OD): δ7.765-7.752(d,J=7.8Hz,0.2H); δ7.608-7.594(d,J=8.4Hz,0.8H); 5.903-5.868(dd ,J=2.4,Hz,J=18Hz,1H);5.854-5.726(dd,J=8.4Hz,J=68Hz,1H);4.421-4.401(m,1H);4.179-4.145(m,2H) ;4.127-4.067(m,2H);4.044-4.011(dd,J=6.0Hz,2H);3.535-3.532(t,J=6.6Hz,2H);3.445-3.427(t,J=6. 6Hz, 2H); 1.934-1.886 (m, J = 6.6Hz, 2H); 1.570 (s, 6H); 1.375 (s, 2H); 1.308 (m, 26H); 0.913 (t, J = 7.2Hz, 3H).

[0546] 13 C NMR (600MHz, CD3OD): δ156.255; 152.349; 142.464; 138.394; 113.857; 11 3.638;109.757;101.583;93.084;85.493;84.449;84.230;80.958;70.7 42;66.577;66.436;65.674;63.339;63.297;31.689;30.542;30.493,29 .402;29.373;29.252;29.091;26.122;25.903;24.128;22.350;13.066;

[0547] 31 P NMR (600MHz, CD3OD): δ-0.548,-0.547.

[0548] MS (ESI) m / e: 662.37604 [M+H] + ; 684.35162[M+Na] + .

[0549] Step 5:

[0550] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-yl)methyl (3-(hexadecyloxy)propyl) phosphate (NTV-118)

[0551] 3-(Hexadecyloxy)propyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1A5) (6.6 g, 10 mmol) was dissolved in TFA:H2O (9:1, 80 mL), and the reaction solution was stirred at room temperature for 45 minutes. The reaction solution was concentrated under reduced pressure, and the concentrate was concentrated under reduced pressure twice with toluene (100 ml). Methanol (100 ml) was added to the residue, and the mixture was concentrated under reduced pressure three times and then recrystallized from ethanol to obtain (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (3-(hexadecyloxy)propyl) phosphate (NTV-118) (5.6 g, 90%).

[0552] 1HNMR (600MHz, CD3OD): δ8.013 (d, J=8.4Hz, 0.2H); δ7.229 (d, J=8.4Hz, 0.8H); 5.993 (d, J=4.8, Hz, 0.2H); 5.96 1(d,J=4.8Hz,0.8H);5.813(d,J=8.4Hz,0.2H);5.701(d,J=8.4Hz,0.8H);4.226(m,1H);4.068(m,1H);4.028(m 1H); 3.966 (dd, J = 6.0Hz, 2H); 3.543 (t, J = 6.0Hz, 2H); 3.434 (t, J = 6.6Hz, 2H); 1.912 (m ,J=6.6Hz,2H);1.579(m,J=6.6Hz,2H);1.357-1.900(m,26H);0.913(t,J=7.2Hz,3H).

[0553] 13 C NMR (600MHz, CD3OD): δ150.505; 144.958; 141.094; 130.706; 101.728; 98.252; 87.382; 83.467 (d, J=59Hz); 74.484; 73.005; 70.2 52;70.633;70.390;66.971;64.782;62.459;62.422;30.767;30.717;29.737;29.263;29.090;25.893;22.349;22.394;13.068.

[0554] 31 P NMR (600MHz, CD3OD): δ0.328, 0.293.

[0555] MS (ESI) m / e: 622.34363 [M+H] + .

[0556] Example 8

[0557] ((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-(octadecyloxy)ethyl) methyl phosphate (NTV-125)

[0558]

[0559] A synthetic method similar to that of Example 7 was used to prepare (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (2-(octadecyloxy)ethyl) phosphate (NTV-125).

[0560] Step 1:

[0561] 2-Chlorophenyl (((3aR,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dimethyltetrakis Hydrogen furano[3,4-d][1,3]dioxolan-4-yl)methyl)(2-(octadecyloxy)ethyl)phosphate (1-1B2)

[0562]

[0563] (1) 3-(Octadecyloxy)ethan-1-ol (1-1b-2)

[0564] H2CH2COH-O-CH2(CH2) 16 CH3

[0565] 1-1b-2

[0566] I. Octadecyl p-toluenesulfonate

[0567] Under nitrogen protection, 1-octadecyl alcohol (27.4 g), anhydrous dichloromethane (300 mL) and triethylamine (30 g) were added to the reactor. The reaction mixture was cooled to 0 ± 5 ° C. A solution of p-toluenesulfonyl chloride (22 g) in dichloromethane (200 ml) was added dropwise at this temperature. After addition, the reaction mixture was warmed to 20-30 ° C and stirred for 2 hours. The reaction solution was maintained at 20-30 ° C and diluted with water (150 mL). The organic layer was separated, washed with water (50 ml) and concentrated to dryness to obtain octadecyl p-toluenesulfonate (34.6 g, 90%) as a light yellow solid.

[0568] II. 2-(Octadecyloxy)ethanol (1-1b-2)

[0569] Under nitrogen, l, 2-ethylene glycol (12.2 g) and NMF (300 ml) were added to the reactor. The reaction solution was cooled to 0 ± 5 ° C, and sodium hydride (10.7 g, 60%) was carefully added in batches. After the addition was completed, the reaction solution was stirred at room temperature for another 2 hours. A solution of octadecyl p-toluenesulfonate (42.68 g) dissolved in DMF (100 ml) was slowly added to the reaction mixture at 20-55 ° C. The solution was stirred at 20-35 ° C for 24 hours. The reaction mixture was cooled to 0 ± 5 ° C, slowly diluted with water (150 ml), and extracted with ethyl acetate (2x250 ml). The organic phase was washed with water (200 ml), dried over anhydrous Na2SO4 and concentrated to obtain a brown oil. The crude product was dissolved in methanol (200 ml) and placed at 0-30 ° C for 12 hours. The solid impurities generated were filtered and discarded, and the filtrate was concentrated. To the concentrate was added acetonitrile (400 ml), and the mixture was allowed to stand at 5-15° C. for 16 hours. The solid was filtered and dried at 25-30° C. to give 2-(octadecyloxy)ethanol (1-1b-2) (25.3 g, 81.6%) as a white solid.

[0570] (2) 2-Chlorophenyl (((3aR,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)(2-(octadecyloxy)ethyl) phosphate (1-1B2)

[0571] Referring to the method for preparing 2-chlorophenyl(((3aR,4R,6S,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)(3-(hexadecyloxy)propyl)phosphate alkoxy)(1-1A2) described in step 1 of Example 7, 2-(octadecyloxy) Ethanol (1-1b-2) is used instead of 3-(hexadecyloxy)propan-1-ol (1-1b-1) to react to give 2-chlorophenyl (((3aR,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl (2-(octadecyloxy)ethyl) phosphate (1-1B2).

[0572] Specifically, feed according to the following table:

[0573]

[0574] A solution of 1,2,4-triazole (1c) and triethylamine in dry tetrahydrofuran is added to a solution of 2-chlorophenyl dichlorophosphate (1d-1) in dry tetrahydrofuran. The resulting reaction solution is reacted at room temperature for 1 hour. The generated solid is filtered off, and a solution of 1-[(3aR,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofurano[3,4-d][l,3]dioxolan-4-yl]pyrimidine-2,4-dione (1-1a) and 1-methylimidazole in tetrahydrofuran is added to the resulting filtrate. After the addition is completed, the reaction is stirred at room temperature for 1 hour. 2-(octadecyloxy)ethanol (1-1b-2) is added to the reaction solution. The resulting reaction solution is stirred at room temperature overnight, and after the reaction is completed, the solution is concentrated under reduced pressure, and the residue is dissolved in ethyl acetate. The resulting solution is washed with water, saturated sodium bicarbonate aqueous solution, water, and saturated brine in sequence. The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-chlorophenyl (((3aR, 4R, 6S, 6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H) yl)-2,2-dimethyltetrahydrofuran [3,4-d][1,3] dioxolan-4-yl) methyl (2-(octadecyloxy) ethyl) phosphate (1-1B2) (17.5 g, 65%) as a colorless oil, which can be directly used in the next step without further purification. The sample for structural identification can be purified by a chromatographic column (CH2Cl2 + 0 to 8% MeOH).

[0575] 1 HNMR (600MHz, CD3OD): δ7.589-7.566 (dd, J=7.8Hz, 1H); 7.473-7.438 (m, J=7.8H z, J=1.8Hz, 2H); 7.301-7.275 (ddd, J=7.8Hz, J=1.8Hz, 1H); 5.767-5.759 (t, J=2 .4Hz,1H);5.654-5.614(dd,J=7.8,Hz,1H);4.480-4.412(m,,5H);3.672-3.642 (m, J=3Hz, 2H); 3.494-3.447 (t, J=6.6Hz, 2H); 1.569-1.546 (m, J=6.6Hz, J=7.2Hz 2H); 1.533 (s, 3H); 1.337 (m, 2H); 1.337 (s, 3H); 1.278 (m, 30H); 0.892 (t, J = 7.2Hz, 3H).

[0576] 1313C NMR (600 MHz, CD3OD): δ 164.625 (d, J = 12 Hz); 150.467; 146.422 (d, J = 25.8 Hz); 143.214; 143.004; 130.470; 127.956; 126.337 (d, J = 26.4 Hz); 125.252 (m, J = 19.2 Hz, J = 9 Hz); 121.348 (m, J = 9 Hz); 114.053; 101.594; 94.530; 94.264; 85.628 (d, J = 30.6 Hz); 85.453 (d, J = 31.8 Hz); 80.986 (d, J = 33 Hz); 84.282 (m, J = 25.8 Hz); 71.031; 69.004 (m, J = 27 Hz); 68.499 (t, J = 25.2 Hz); 68.379 (t, J = 25.2 Hz); 31.736; 29.468; 29.437; 29.423; 29.402; 29.278; 29.148; 26.146; 25.874; 24.232; 22.400; 13.182.

[0577] 31 P: δ -7.1610; -7.1962.

[0578] MS (ESI) m / e: 771.40 [M+H] + 。

[0579] Step 2:

[0580] 2-Chlorophenyl(((3aR,4R,6S,6aR)-6-(4-(1H-1,2,4-triazol-1-yl)-2-oxopyrimidine-1(2H)- 2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxolan-4-yl)methyl(2-(octadecyloxy)ethyl Phosphate (1-1B3)

[0581]

[0582] Referring to the method for preparing 2-chlorophenyl(3-(hexadecyloxy)propyl)((3aR,4R,6S,6aR)-6-(4-(1,2,4-triazolyl)-2-oxopyrimidin-1(2H)yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1A3) as described in step 2 of Example 7, 2-chlorophenyl(((3aR,4R,6S,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl(2-(octadecyloxy)ethyl)phosphate alkoxy) (1-1B 2) Replacing 2-chlorophenyl (((3aR,4R,6S,6aR)-6-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl (3-(hexadecyloxy)propyl)phosphate alkoxy) (1-1A2) to obtain 2-chlorophenyl (((3aR,4R,6S,6aR)-6-(4-(1H-1,2,4-triazol-1-yl)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl (2-(octadecyloxy)ethyl)phosphate (1-1B3).

[0583] Specifically, feed according to the following table:

[0584]

[0585] 1HNMR(600 MHz,CD3OD):δ9.297-9.285(t,J=3.5Hz,0.5H);8.404(m,0.5H);8.325-8.310(dd,J=7.2Hz,J=1.8Hz,0.5H);7.465-7.439(m,J=6.6Hz,J=2.4Hz,1H);7.444-7.379(m,J=7.2Hz,J=1.8Hz,1H);7.321-7.273(m,J=8.4Hz,J=2.4Hz,J=1.8.Hz1H);7.245-7.169(m,J=3Hz,1H);7.141-7.068(dd,J=7.2Hz,J=36.6Hz,1H);6.296-5.987(dd,J=7.8Hz,J=32.4Hz,J=11.4Hz1H);5.464-5.337(m,2H);4.664-4.309(m,4H);3.002-2.948(m,J=6.6Hz,2H);2.723-2.667(m,J=7.2Hz,2H);2.145(m,J=7.2Hz,3H);2.139(s,3H);1.366-1.362(m,2H);1.272(s,28H);0.882(t,J=7.2 Hz,3H)。

[0586] 13 C NMR(600 MHz,CD3OD):δ159.838;154.938;153.533(d,J=19.8Hz);149.705;146.299(d,J=25.2Hz);143.377(d,J=15Hz);130.482(d,J=37.8Hz);127.976(d,J=27.6Hz);126.376;126.234;125.107(tJ=30Hz);121.247(d,J=9Hz);113.842;97.068;96.734;94.845(d,J=19.8Hz);86.964(d,J=31.2Hz);86.804(d,J=31.2Hz);85.271;85.159;81.106;81.006;71.079;68.981(m,J=26.4Hz);68.600;68.500(m,J=24Hz,);31.720;29.440;29.427;29.384;29.370;29.246;29.128;26.155(d,J=10.4Hz);25.845;24.247(d,J=18Hz);22.393;13.223。

[0587] 31 P:δ-7.195;-7.291.

[0588] MS (ESI) m / e: 822.40 [M] + .

[0589] Step 3:

[0590] 2-Chlorophenyl[2-(octadecyloxy)ethyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidine-1 (2H)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)

[0591] Methyl) phosphate (1-1B4)

[0592]

[0593] Feed according to the table below:

[0594]

[0595] The product (1-1B3) (8.2 g, 10 mmol) obtained above was added to isopropanol (100 mL) and stirred at room temperature to dissolve the solid. Then, hydroxylamine hydrochloride (1.1 g, 15 mmol) and sodium hydroxide (0.6 g, 15 mmol) were added, and a light yellow reaction solution was obtained after stirring for 1 hour, and the reaction was monitored by TLC (EtOAc). The solvent was removed by concentrating under reduced pressure at a temperature less than 45°C. The residue was dissolved in ethyl acetate (100 ml), and the organic phase was washed with water (50 ml x 2), saturated brine (50 ml x 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The concentrate was recrystallized from petroleum ether to give 2-chlorophenyl(2-(octadecyloxy)ethyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1B4) (7.3 g, 93%) as a white solid.

[0596] 1HNMR(600MHz,CD3OD):δ7.586-7.573(m,J=5.4Hz,J=6Hz0.5H);8.404(m,0.5H);8.325-8.310(dd,J=7.2Hz,J=1.8Hz,0.5H);7.465-7.439(m,J=6.6Hz,J=2.4Hz,1H);7.444-7.379(m,J=7.2Hz,J=1.8Hz,1H);7.321-7.273(m,J=8.4Hz,J=2.4Hz,J=1.8.Hz,1H);7.245-7.169(mJ=3Hz,1H);7.141-7.068(dd,J=7.2Hz,J=36.6Hz,1H);6.296-5.987(dd,J=7.8Hz,J=32.4Hz,J=11.4Hz,1H);5.464-5.337(m,2H);4.664-4.309(m,4H);3.002-2.948(m,J=6.6Hz,2H);2.723-2.667(m,J=7.2Hz,2H);2.145(m,J=7.2Hz,3H);2.139(s,3H);1.366-1.362(m,2H);1.272(s,28H);0.882(t,J=7.2Hz,3H)。

[0597] 1313C NMR (600 MHz, CD3OD): δ 150.477; 149.688; 146.437 (m, J = 7.8 Hz, J = 38 Hz); 144.757; 143.254; 143.042; 132.875; 132.686; 130.466; 127.970 (d, J = 7.2 Hz); 126.322 (d, J = 32.4 Hz); 125.264 (t, J = 28.2 Hz); 121.397 (dd, J = 9 Hz, J = 16.8 Hz); 114.046 (d, J = 9 Hz); 101.604; 101.624; 98.240 (d, J = 23.4 Hz); 94.589; 94.318; 93.424; 93.132; 84.665 (d, J = 30 Hz); 84.496 (d, J = 31.2 Hz); 84.219; 83.817 (d, J = 27.6 Hz); 80.997 (d, J = 32.4 Hz); 80.833 (d, J = 35.4 Hz); 71.049; 69.011 (d, J = 27 Hz); 68.452 (m, J = 7.8 Hz, J = 17.4 Hz, J = 9 Hz); 31.750; 29.452; 29.445; 29.418; 29.229; 29.165; 26.242 (J = 33 Hz); 25.889; 24.319 (d, J = 33.6 Hz); 22.451; 13.218.

[0598] 31 P: δ -7.181; -7.193.

[0599] MS (ESI) m / e: 785.3801 [M] + 。

[0600] Step 4:

[0601] 2-(Octadecyloxy)ethyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidine-1(2H)- 2,2-dimethyltetrahydrofurano[3,4-d][1,3]dioxolan-4-yl)methyl)

[0602] Phosphate ester (1-1B5)

[0603]

[0604] Referring to the method described in step 4 of Example 7, 1-1B5 was prepared using 2-chlorophenyl(2-(octadecyloxy)ethyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate)(1-1B4) instead of 2-chlorophenyl(3-(hexadecyloxy)propyl)(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate)(1-1A4) (white solid product, 3.1 g, 60% yield).

[0605] Specifically, feed according to the following table:

[0606]

[0607] 1 HNMR (600MHz, CD3OD): δ7.764-7.750 (d, J=8.4Hz, 0.2H); 7.612-7.599 (d, J=8.4Hz, 0.8H); 5.91 8-5.910 (d, J=4.8, Hz, 0.2H); 5.901-5.896 (d, J=3Hz, 0.2H); 5.872-5.867 (d, J=8.4Hz, 0.8H); 5. 854-5.840(d,J=8.4Hz,0.8H);4.168-4.008(m,,5H);3.615-3.600(t,J=4.2Hz,2H);3.482-3.4 60(t,J=6.6Hz,2H); 1.548(s,3H); 1.353(s,3H); 1.2845(m,30H); 0.908-0.884(t,J=7.2Hz,3H).

[0608] 13 C NMR (600MHz, CD3OD): δ150.898; 148.265; 142.295; 138.749; 113.084; 113.527; 101.624; 93.106; 92.892; 92.762; 85.524; 84.525; 81.0 36;71.035;69.792;65.913;65.688;65.384;31.629;29.476;29.387;29.363;29.249;29.075;26.132;25.867;24.135;22.337;13.049.

[0609] 31 P NMR (600MHz, CD3OD): δ-0.345,-0.545.

[0610] MS (ESI) m / e: 676.3802 [M+H] + .

[0611] Step 5:

[0612] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-(octadecyloxy) alkoxy) ethyl) phosphate (NTV-125)

[0613]

[0614] Referring to the method described in Step 5 of Example 7, 2-(octadecyloxy)ethyl (((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxolan-4-yl)methyl)phosphate (1-1B5) was reacted with trifluoroacetic acid to obtain the target product (NTV-125).

[0615] Specifically, feed according to the following table:

[0616]

[0617] 2-(Octadecyloxy)ethyl(((3aR,4R,6S,6aR)-6-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)phosphate (1-1B5) was dissolved in TFA:H2O (9:1, 80 mL), and the reaction solution was stirred at room temperature for 45 minutes. The reaction solution was concentrated under reduced pressure, and the concentrate was concentrated under reduced pressure twice with toluene. Methanol (100 ml) was added to the residue, and the mixture was concentrated under reduced pressure three times and recrystallized from ethanol to give an off-white solid (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl (3-(octadecyloxy)ethyl) phosphate (NTV-125) (3.5 g, 92%).

[0618] 11H NMR (600 MHz, CD3OD): δ 7.834 (s, 0.2H); 7.683 - 7.670 (d, J = 7.8 Hz, 0.8H); 5.960 - 5.952 (d, J = 4.8 Hz, 0.2H); 5.928 - 5.921 (d, J = 4.2 Hz, 0.8H); 5.877 - 5.864 (d, J = 7.8 Hz, 1H); 4.219 - 4.026 (m, 5H); 3.624 - 3.615 (t, J = 5.4 Hz, 2H); 3.490 - 3.467 (t, J = 7.2 Hz, 2H); 1.583 - 1.537 (m, J = 7.2 Hz, 2H); 1.368 - 1.229 (m, 30H); 0.906 - 0.883 (t, J = 7.2 Hz, 3H).

[0619] 13 13C NMR (600 MHz, CD3OD): δ 149.601; 146.098; 141.054; 136.531; 125.935; 118.695; 114.004; 109.279; 105.619; 94.171; 88.764; 78.038; 77.821; 77.604, 73.894; 71.116; 69.893; 65.035; 31.701; 29.442; 29.281; 29.102; 25.859, 22.372; 13.182.

[0620] 31 31P NMR (600 MHz, CD3OD): δ 0.757, 0.231.

[0621] MS (ESI) m / e: 636.35 [M + H] + .

[0622] Example 9

[0623] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-(2-yl)methyl)(3-(hexadecyloxy)alkoxy)propyl)phosphate sodium salt (II-2-1)

[0624]

[0625] To (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl(3-(hexadecyloxy)alkoxy)propyl)phosphate (NTV-118) (6.21 g, 10 mmol) was added 50 ml of anhydrous ethanol. The reactant was heated to 60°C under stirring to dissolve it. At this temperature, 2N NaOH ethanol solution (5 ml) was added to the solution and stirred for 10 minutes. The reaction liquid temperature was lowered to room temperature, placed at 4°C overnight, and filtered to obtain (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl(3-(hexadecyloxy)alkoxy)propyl)phosphate sodium salt (II-2-1) as a white solid (5.9 g, 92%).

[0626] Example 10

[0627] (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran- 2-(octadecyloxy) alkoxy) ethyl) phosphate monoammonium salt (II-2-2)

[0628]

[0629] A flask was charged with (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl(2-(octadecyloxy)alkoxy)ethyl)phosphate (NTV-125) (6.3 g, 0.01 mol), 2-propanol (50 ml) and 28-30% (w / w) aqueous ammonia solution (1 ml). The reaction solution was stirred and refluxed at 62-80°C to achieve dissolution (over about 10 minutes). The solution was allowed to stand for more than 15 minutes without stirring under reflux. The solution was cooled at a temperature below 25°C for 8-24 hours, and then the mixture was cooled to 5±5°C and maintained for at least 1 hour. The product was filtered and washed with cooled 2-propanol (5±5°C, 30 ml). The white solid product was dried at 30-35°C for 25 hours. The product (((2R,3S,4R,5S)-3,4-dihydroxy-5-(4-hydroxyamino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl(2-(octadecyloxy)alkoxy)ethyl)phosphate monoammonium salt (II-2-2) (6.0 g, 92.3%) was obtained.

[0630] Embodiment 11

[0631] Tablet dosage form

[0632] The compound of Example 1 (which may be replaced by any other compound of the present disclosure or other specific compound disclosed in Table 1) is used as an active ingredient to prepare exemplary tablet dosage forms of different specifications of the present disclosure (which are immediate-release tablets). The components of the exemplary tablet dosage forms of different specifications and their functions and amounts are shown in Tables 2-4 below, and they can be prepared by a method comprising the following steps: (1) mixing predetermined amounts of the components to prepare a blend; and (2) compressing the blend by a conventional compression method to obtain the tablet dosage form.

[0633] Table 2 Composition of tablets containing 20 mg of the compound of Example 1 (per 1000 tablets)

[0634]

[0635] Table 3 Composition of tablets containing 50 mg of the compound of Example 1 (per 1000 tablets)

[0636]

[0637]

[0638] Table 4 Composition of tablets containing 100 mg of the compound of Example 1 (per 1000 tablets)

[0639]

[0640] Example 12 Antiviral Cell Protection Experiment

[0641] In order to study the antiviral efficacy of the compounds disclosed in the present invention in vitro, the cytopathic effect (CPE) assay was used to determine the maximum non-toxic concentration (TC) of the exemplary compounds on cells. 50 ), the half inhibitory concentration (IC 50 ) and selectivity index (SI), with ribavirin (RBV) as the positive control drug and β-DN 4 -Hydroxycytidine (NHC, EIDD-1931) was used as a reference drug.

[0642] The cells used in the experiment are mosquito-derived cells (C6 / 36), African green monkey kidney cells (VeroE6), canine kidney cells (MDCK), human liver cancer cells (Huh7 cells) and human lung cancer cells (H460 cells). The basic conditions of the cell culture are shown in Table 5 below:

[0643] Table 5 Basic information of cell culture

[0644]

[0645] The viruses used in the experiment were influenza A virus (H1N1 influenza SC09 strain and H3N2 influenza FJ09 strain), influenza B virus (SC11 strain), Zika virus (SZ01 strain), human coronavirus (HCoV-229E strain, HCoV-OC43 strain; and B.1.1.7 strain, B.1.351 strain and B.1.617.2 strain belonging to the new coronavirus (severe acute respiratory syndrome coronavirus 2, 2019-nCoV)).

[0646] Experiment 1: In vitro anti-influenza virus experiment

[0647] This experimental example measured the inhibitory activity of the exemplary compounds of the present disclosure against influenza A virus (H1N1 influenza SC09 strain and H3N2 influenza FJ09 strain) and influenza B virus (SC11 strain).

[0648] 1. Cell recovery and culture

[0649] Prepare DMEM complete cell culture medium (containing 10% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin) and MDCK cell maintenance medium (containing 2% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin) for later use. Take out the small tube of frozen cells from liquid nitrogen, quickly place it in a 37°C water bath and shake it quickly until it is completely dissolved (try to be within 1 minute). Take 1ml of the cell suspension and place it in a centrifuge tube, add 5ml of complete cell culture medium and centrifuge (2000rpm, 5min, at room temperature). Discard the supernatant, add 2ml of complete culture medium, blow it evenly with a pipette, and transfer it to a cell culture flask (25cm 2 ), make up the complete culture medium to 3ml, place in a cell culture incubator at 37℃ and 5% CO2, and replace with new complete culture medium after 24h. After the cells grow into a monolayer, discard the original culture medium, wash with PBS 2-3 times, and blow the cells vigorously to make them fall off. Repeatedly blow and aspirate until the cells are fully separated from the wall and evenly dispersed, take an appropriate amount of cell suspension and place it in a new cell culture bottle, and make up the complete culture medium. After passaging at a certain ratio (usually 1:2 or 1:3), place in a cell culture incubator at 37℃ and 5% CO2 for culture.

[0650] 2. Influenza virus passage

[0651] Take out the freezing tube of influenza virus strain SC09 from the -80℃ freezer and thaw it on ice for later use. Take out the culture bottle containing MDCK cells grown into a monolayer, pour out the culture fluid, and wash with PBS 1-2 times. Transfer the virus liquid to the culture bottle, flatten the bottom wall, add an appropriate amount of cell maintenance fluid, and place it in a cell culture incubator with corresponding conditions for culture. Take it out every day and observe it under a microscope until all cells are diseased (lasting 36 hours). Collect the virus liquid by blowing, centrifuge (4000rpm, 10min, 4℃), take the supernatant and filter it (the pore size of the filter membrane is 0.2μm), and divide it into 200μl / tube in freezing tubes for later use. The subculture operation of FJ09 strain and SC11 strain is the same as above.

[0652] 3. Influenza virus titer determination

[0653] MDCK cells were cultured at 3×10 5 The cells were inoculated into a 96-well plate at a concentration of 1 cell / ml. Each well was inoculated with 100 μl of cell suspension and cultured in a cell culture incubator at 37°C and 5% CO2 for 1-2 days to grow into a monolayer. The small tube containing the virus solution was taken out from the -80°C refrigerator, thawed in running tap water, and diluted 10 times with cell maintenance solution to obtain the first virus titer. The 96-well plate with monolayer cells was taken out, the supernatant was poured out, and 180 μl of cell maintenance solution was added to each well except the three sub-wells of the first titer. 20 μl of virus solution was drawn from the previous titer to the next concentration, and mixed evenly by pipetting. No virus solution was added to the last three sub-wells, which served as the cell control group. The 96-well plate was cultured in a cell culture incubator and observed under a microscope every day until no new cytopathic effect (CPE) wells appeared. As long as lesions appeared in the wells, they were recorded as "+". The wells with CPE at each dilution were counted, and the TCID of the virus was calculated by the Karber method. 50 value.

[0654] 4. Maximum non-toxic concentration of the test compound to cells (TC0)

[0655] (1) Preparation of drug solution: Take appropriate amount of compound (positive control and test substance), prepare into 25 mg / ml stock solution with DMSO, filter sterilize (filter pore size 0.2 μm), and store at 4°C.

[0656] (2) Preparation of neutral red dye solution: Dilute 1% neutral red solution 100 times using cell maintenance medium.

[0657] (3) Preparation of acetic acid ethanol solution: measure 5 ml of water, 5 ml of anhydrous ethanol and 100 μl of glacial acetic acid respectively, mix well.

[0658] (4) Method for determining the TC0 value of the compound: Six concentration gradients were set using the ten-fold dilution method, with three secondary wells for each concentration. The 96-well plate with monolayer cells was removed and the culture medium in the wells was discarded. 180 μl of cell maintenance medium was added to each well except the three secondary wells with the maximum concentration. The compound mother solution was diluted 500 times with the cell maintenance medium to obtain the maximum experimental concentration. For the three secondary wells with the maximum concentration, 200 μl of 50 μg / ml compound solution was added to each well. 20 μl of liquid was drawn from the well with the maximum concentration to the well with the second concentration and mixed evenly; then 20 μl of liquid was drawn from the well with the second concentration to the well with the third concentration, and so on, to complete the ten-fold decreasing dilution of the concentration, and a cell control well was set. The 96-well plate was placed in a cell culture incubator at 37°C and 5% CO2 for 72 h. The OD value of the cells in each well was determined by the neutral red staining method. Take out the 96-well plate that has reached the end point of the experiment, discard the original liquid in the well, add neutral red dye solution (100μl / well), and incubate in a cell culture incubator at 37°C and 5% CO2. After 1 hour, take out the 96-well plate, discard the neutral red dye solution, and wash the neutral red in the well with PBS solution 2-3 times, discard PBS, and add acetic acid ethanol solution for color development (100μl / well). Use an enzyme marker to measure the OD value at a wavelength of 544nm. Reference for judgment results: The OD value of the experimental group ≥90% of the OD value of the control group is judged to be the maximum non-toxic concentration TC0 value.

[0659] 5. The half cytotoxic concentration (CC) of the test compound 50 )

[0660] (1) Preparation of drug solution: Take appropriate amount of compound (positive control and test substance), prepare into 25 mg / ml stock solution with DMSO, filter sterilize (filter pore size 0.2 μm), and store at 4°C.

[0661] (2) Preparation of neutral red dye solution: Dilute 1% neutral red solution 100 times using cell maintenance medium.

[0662] (3) Preparation of acetic acid ethanol solution: measure 5 ml of water, 5 ml of anhydrous ethanol and 100 μl of glacial acetic acid respectively, mix well.

[0663] (4) CC of the compound 50Method for determining the value: Use the double dilution method to set 5 concentration gradients, and set 3 secondary wells for each concentration. Take out the 96-well plate with monolayer cells and discard the culture medium in the well. Add 100μl of cell maintenance solution to each well except the 3 secondary wells with the maximum concentration. Dilute the compound mother solution 500 times with the cell maintenance solution to be the maximum experimental concentration. For the 3 secondary wells with the maximum concentration, add 200μl of 50μg / ml compound solution to each well. Pipette 100μl of liquid from the well with the maximum concentration to the well with the second concentration and mix evenly; then pipette 100μl of liquid from the well with the second concentration to the well with the third concentration, and so on, to complete the double dilution of the concentration, and set up a cell control well. Place the 96-well plate in a cell culture incubator at 37℃ and 5% CO2, and observe it under a microscope every day until no new cell lesions appear, which is the end point of the experiment. Determine the OD value of the cells in each well by neutral red staining. Take out the 96-well plate that has reached the end point of the experiment, discard the original liquid in the well, add neutral red dye solution (100μl / well), and incubate in a cell culture incubator at 37°C and 5% CO2. After 1 hour, take out the 96-well plate, discard the neutral red dye solution, and wash the neutral red in the well with PBS solution 2-3 times, discard PBS, and add acetic acid ethanol solution for color development (100μl / well). Use an enzyme marker to measure the OD value at a wavelength of 544nm. After measuring the OD value, calculate the cell survival rate, the formula is as follows:

[0664] Cell survival rate (%) = (OD 544 Value / average OD of cell control wells 544 value)×100%

[0665] GraphPad Prism 5.0 software was used to calculate the half cytotoxic concentration (CC) of the compounds. 50 value).

[0666] 6. The half inhibitory concentration (IC 50 )

[0667] Prepare a 96-well plate with cells grown in a monolayer. Discard the original complete culture medium in the plate and dilute it to 10-100 TCID 50The virulent virus solution infects the cells in the virus control well, the positive drug control well and the experimental well (100 μl / well). Then, add the cell maintenance solution (100 μl / well) to the virus control well. Add the test compound sample solution with the selected TC0 concentration as the final concentration to the sample experimental group and dilute it by two times, dilute it downward by four concentration gradients, that is, 5 concentration gradients of the experimental group are tested. Add cell maintenance solution (200 μl / well) to the cell control well, and set up 5 blank wells as blank control groups. The virus control well and the normal cell control well are each set up with 5 sub-wells, and the experimental group is set up with 3 sub-wells for each concentration. The 96-well plate is placed in a 37°C, 5% CO2 incubator for culture. Observe and record the cell pathological changes under an inverted microscope every day. The experimental endpoint is when 4 "+" cell pathological changes appear in the virus control well. After staining with neutral red and developing with acetic acid ethanol solution, the OD value of the cells in each well is measured at a wavelength of 544nm, and the inhibition rate is calculated according to the following formula:

[0668] Inhibition rate (%) = (OD 544 Value - Average OD of virus control wells 544 Value / (average OD of cell control wells 544 Value - Average OD of virus control wells 544 value)×100%

[0669] GraphPad Prism 5.0 software was used to calculate the half inhibitory concentration (IC 50 value).

[0670] 7. Summary of experimental results

[0671] The results of the above experiments are summarized in Table 6 below:

[0672] Table 6 Summary of in vitro anti-influenza virus experimental results

[0673]

[0674] From the results in the above table, it can be seen that under the conditions of this experiment, the tested compounds have inhibitory effects on the cytopathic effects caused by influenza virus SC09, FJ09 and SC11 strains. Especially when compared with the positive control drug EIDD-1931, except for the compound NTV-118, which has equivalent activity to EIDD-1931, the other tested compounds all showed stronger inhibitory effects on the cytopathic effects caused by viral infection.

[0675] Experiment 2: In vitro anti-Zika virus experiment

[0676] This experimental example determined the inhibitory activity of the exemplary compounds of the present disclosure against Zika virus (SZ01 strain).

[0677] 1. Cell recovery and culture

[0678] In this experiment, C6 / 36 cells were used to culture Zika virus, and Vero cells were used for efficacy testing.

[0679] (1) Recovery and culture of C6 / 36 cells

[0680] Prepare RPMI-1640 complete cell culture medium (containing 10% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin) and RPMI-1640 cell maintenance medium (containing 2% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin) for later use. Take out the vial of frozen cells from liquid nitrogen, quickly place it in a 37°C water bath and shake it quickly until it is completely dissolved (try to be within 1 minute). Take 1ml of the cell suspension in a centrifuge tube, add 5ml of complete cell culture medium and centrifuge (2000rpm, 5min, at room temperature). Discard the supernatant, add 2ml of complete culture medium, blow it evenly with a pipette, and transfer it to a cell culture flask (25cm 2 ), add complete culture medium to 3ml, place in a cell culture incubator at 28℃, 5% CO2, and replace with new complete culture medium after 24h. After the cells grow into a monolayer, discard the original culture medium, wash with PBS 2-3 times, and blow the cells vigorously to make them fall off. Repeatedly blow and aspirate until the cells are fully separated from the wall and evenly dispersed, draw an appropriate amount of cell suspension into a new cell culture bottle, add complete culture medium, pass it in a certain ratio (usually 1:2 or 1:3), and place it in a cell culture incubator at 28℃, 5% CO2 for culture. The above operations must be performed under sterile conditions.

[0681] (2) Recovery and culture of Vero cells

[0682] Prepare DMEM complete cell culture medium (containing 10% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin) and DMEM cell maintenance medium (containing 2% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin) for use. The specific operation method is the same as the recovery and culture of C6 / 36 cells, the only difference is the setting of cell culture conditions. Vero cells are cultured in a cell culture incubator at 37°C and 5% CO2.

[0683] 2. Zika virus passage

[0684] Take out the cryopreserved tube of Zika virus SZ01 strain from the -80℃ refrigerator and thaw it on ice for later use. Take out the culture bottle containing Vero cells grown into a monolayer, pour out the culture fluid, and wash with PBS 1-2 times. Transfer the virus liquid to the culture bottle, flatten the bottom wall, add an appropriate amount of cell maintenance fluid, and place it in a cell culture incubator with corresponding conditions for culture. Take it out every day and observe it under a microscope until all the cells are diseased (lasting 7 days). Repeat the freezing and thawing of the virus liquid 3 times (-80℃ for 30min; 37℃ thaw; -80℃ for 30min), then centrifuge it in a liquid state (4000rpm, 10min, 4℃), take the supernatant and filter it (the pore size of the filter membrane is 0.2μm), and divide it into 200μl / tube in cryopreserved tubes for later use.

[0685] 3. Zika virus titer determination

[0686] Vero cells were cultured at 3 × 10 5 The cells were inoculated into a 96-well plate at a concentration of 1 cell / ml. Each well was inoculated with 100 μl of cell suspension and cultured in a cell culture incubator at 37°C and 5% CO2 for 1-2 days to grow into a monolayer. The small tube containing the virus solution was taken out from the -80°C refrigerator, thawed, and diluted 10 times with cell maintenance solution to obtain the first virus titer. The 96-well plate with monolayer cells was taken out, the supernatant was poured out, and 180 μl of cell maintenance solution was added to each well except the three sub-wells of the first titer. 20 μl of virus solution was drawn from the previous titer to the next concentration, and mixed evenly by pipetting. No virus solution was added to the last three sub-wells, which served as the cell control group. The 96-well plate was cultured in a cell culture incubator and observed under a microscope every day until no new cytopathic effect (CPE) wells appeared. As long as lesions appeared in the wells, they were recorded as "+". The wells with CPE at each dilution were counted, and the TCID of the virus was calculated by the Karber method. 50 value.

[0687] Based on the observed CPE results, the virus TCID was calculated using the following formula 50 value:

[0688] LogTCID 50 =Ld(s-0.5)

[0689] Where L is the log value of the lowest titer, d is the titer dilution to the power of 10, and s is the sum of the proportions of cytopathic wells with different titers.

[0690] The determination method of cytopathic effect is shown in Table 7 below:

[0691] Table 7 Cytopathicity determination method (CPE method)

[0692] Cytopathic ratio Recording Method <25％ + 25％-50％ ++ 50％-75％ +++ >75％ ++++

[0693] Using the CPE method, the pathological changes of the cells were observed and recorded, and the experimental results were recorded and summarized in the following Table 8:

[0694] Table 8 TCID of Zika virus SZ01 strain 50 value

[0695]

[0696] Calculate the TCID of Zika virus from the data in Table 8 50 value:

[0697] LogTCID 50 =-1-1(5.33-0.5)=-5.83,

[0698] TCID 50 =l0 -5.83 (Calculated based on 180 μl of virus liquid).

[0699] 4. Maximum non-toxic concentration of the test compound to cells (TC0)

[0700] (1) Preparation of drug solution: Take appropriate amount of compound (positive control and test substance), prepare into 25 mg / ml stock solution with DMSO, filter sterilize (filter pore size 0.2 μm), and store at 4°C.

[0701] (2) Preparation of neutral red dye solution: Dilute 1% neutral red solution 100 times using cell maintenance medium.

[0702] (3) Preparation of acetic acid ethanol solution: measure 5 ml of water, 5 ml of anhydrous ethanol and 100 μl of glacial acetic acid respectively, mix well.

[0703] (4) Method for determining the TC0 value of the compound: Six concentration gradients were set using the ten-fold dilution method, with three secondary wells for each concentration. The 96-well plate with monolayer cells was removed and the culture medium in the wells was discarded. 180 μl of cell maintenance medium was added to each well except the three secondary wells with the maximum concentration. The compound mother solution was diluted 500 times with the cell maintenance medium to obtain the maximum experimental concentration. For the three secondary wells with the maximum concentration, 200 μl of 50 μg / ml compound solution was added to each well. 20 μl of liquid was drawn from the well with the maximum concentration to the well with the second concentration and mixed evenly; then 20 μl of liquid was drawn from the well with the second concentration to the well with the third concentration, and so on, to complete the ten-fold decreasing dilution of the concentration, and a cell control well was set. The 96-well plate was placed in a cell culture incubator at 37°C and 5% CO2 for 72 h. The OD value of the cells in each well was determined by the neutral red staining method. Take out the 96-well plate that has reached the end point of the experiment, discard the original liquid in the well, add neutral red dye (100μl / well), and incubate in a cell culture incubator at 37°C and 5% CO2. After 1 hour, take out the 96-well plate, discard the neutral red dye, and wash the neutral red in the well with PBS solution 2-3 times, discard PBS, and add acetic acid ethanol solution for color development (100μl / well). Use an enzyme marker to measure the OD value at a wavelength of 544nm. Judgment result reference: The OD value of the experimental group ≥ 90% of the OD value of the control group is judged to be the maximum non-toxic concentration TC0 value. In addition, some compounds will be found to be inhibited in the cell state or cytopathic phenomenon in the range of 100μg / ml to 1ng / ml, and the maximum non-toxic concentration range needs to be determined again.

[0704] 5. The half cytotoxic concentration (CC) of the test compound 50 )

[0705] (1) Preparation of drug solution: Take appropriate amount of compound (positive control and test substance), prepare into 25 mg / ml stock solution with DMSO, filter sterilize (filter pore size 0.2 μm), and store at 4°C.

[0706] (2) Preparation of neutral red dye solution: Dilute 1% neutral red solution 100 times using cell maintenance medium.

[0707] (3) Preparation of acetic acid ethanol solution: measure 5 ml of water, 5 ml of anhydrous ethanol and 100 μl of glacial acetic acid respectively, mix well.

[0708] (4) CC of the compound 50Method for determining the value: Use the double dilution method to set 5 concentration gradients, and set 3 secondary wells for each concentration. Take out the 96-well plate with monolayer cells and discard the culture medium in the well. Add 100μl of cell maintenance solution to each well except the 3 secondary wells with the maximum concentration. Dilute the mother solution 500 times with the cell maintenance solution to obtain the maximum experimental concentration. For the 3 secondary wells with the maximum concentration, add 200μl of 50μg / ml compound solution to each well. Pipette 100μl of liquid from the well with the maximum concentration to the well with the second concentration and mix well; then pipette 100μl of liquid from the well with the second concentration to the well with the third concentration, and so on, to complete the double dilution of the concentration, and set up a cell control well. Place the 96-well plate in a cell culture incubator at 37℃ and 5% CO2, and observe it under a microscope every day until no new cell lesions appear, which is the end point of the experiment. Determine the OD value of the cells in each well by neutral red staining. Take out the 96-well plate that has reached the end point of the experiment, discard the original liquid in the well, add neutral red dye solution (100μl / well), and incubate in a cell culture incubator at 37°C and 5% CO2. After 1 hour, take out the 96-well plate, discard the neutral red dye solution, and wash the neutral red in the well with PBS solution 2-3 times, discard PBS, and add acetic acid ethanol solution for color development (100μl / well). Use an enzyme marker to measure the OD value at a wavelength of 544nm. After measuring the OD value, calculate the cell survival rate, the formula is as follows:

[0709] Cell survival rate (%) = (OD 544 Value / average OD of cell control wells 544 value)×100%

[0710] GraphPad Prism 5.0 software was used to calculate the half cytotoxic concentration (CC 50 value).

[0711] (5) Compound CC 50 The results of the value determination:

[0712] The results of the above experiments are summarized in Table 9 below:

[0713] Table 9 CC of test compounds 50 value

[0714] Compound <![CDATA[CC 50 (μM)±SD]]> Ribavirin 1358±13.92 EIDD-1931 11.23±0.22 NTV-16 43.23±2.23 NTV-29 13.20±0.94 NTV-37 10.51±1.12 NTV-118 18.40±2.04

[0715] The cytotoxicity results of the tested compounds recorded in the above table show that the cytotoxicity of compounds NTV-16, NTV-29, NTV-37 and NTV-118 is less than or equivalent to that of the positive control drug EIDD-1931.

[0716] 6. The half inhibitory concentration (IC50 )

[0717] TCID of Zika virus 50 =l0 -5.83 Dilute the virus stock solution to 10-100 TCID with cell maintenance medium 50 . Use 10% of the original solution -4 Concentration for infection reaction.

[0718] Prepare a 96-well plate with cells grown in a monolayer. Discard the original complete culture medium in the plate, and infect the cells in the virus control well, positive drug control well, and experimental well with the diluted virus solution (100 μl / well). Then, add cell maintenance solution (100 μl / well) to the virus control well. Add the test compound sample solution with the selected TC0 concentration as the final concentration to the sample experimental group and dilute it by 1 / 2 times, and dilute it downward to four concentration gradients (100 μl / well), that is, 5 concentration gradients of the experimental group are tested. Add cell maintenance solution (200 μl / well) to the cell control well, and set up 5 blank wells as blank control groups. Set up 5 sub-wells for the virus control well and the normal cell control well, and set up 5 sub-wells for each concentration in the experimental group. Place the 96-well plate in a 37°C, 5% CO2 incubator for culture. Observe and record the cytopathic condition under an inverted microscope every day. The experiment ends when 4 "+" cytopathic conditions appear in the virus control well. After staining with neutral red and developing with acetic acid ethanol solution, the OD value of cells in each well was measured at a wavelength of 544 nm, and the inhibition rate was calculated according to the following formula:

[0719] Inhibition rate (%) = (OD 544 Value - Average OD of virus control wells 544 value) / (average OD of cell control wells 544 Value - Average OD of virus control wells 544 value)×100%

[0720] GraphPad Prism 5.0 software was used to calculate the half inhibitory concentration (IC 50 value).

[0721] 7. Summary of experimental results

[0722] The results of the above experiments are summarized in Table 10 below:

[0723] Table 10 Summary of in vitro anti-Zika virus (SZ01) strain experimental results

[0724] Compound <![CDATA[IC 50 (μM)±SD]]> <![CDATA[CC 50 (μM)±SD]]> SI Ribavirin >400μM 1358±13.92 - EIDD-1931 1.36±0.42 11.23±0.22 8.25 NTV-29 0.65±0.12 13.20±0.94 20.3 NTV-37 1.05±0.38 10.51±1.12 10 NTV-118 2.25±0.62 18.40±2.04 8.2

[0725] It can be seen from the results in the above table that, under the conditions of this experiment, the in vitro anti-Zika virus activity of the tested compounds is better than or equivalent to that of the positive control drug EIDD-1931, while ribavirin did not show an anti-Zika virus effect.

[0726] Experiment 3: In vitro anti-coronavirus activity assay

[0727] This experimental example measured the inhibitory activity of the exemplary compounds of the present disclosure against human coronavirus (HCoV-229E strain and HCoV-OC43 strain).

[0728] 1. Experimental Preparation

[0729] (1) Cells: Human liver cancer cell Huh7 cells and human lung cancer cell H460 cells were subcultured and preserved in this experiment, and cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibiotics (penicillin and streptomycin) at 37°C in a 5% CO2 incubator. The cells were subcultured every 2-3 days.

[0730] (2) Virus strains: HCoV-229E strain was passaged in Huh7 cells and stored in a -80°C refrigerator. HCoV-OC43 strain was passaged in H460 cells and also stored in a -80°C refrigerator.

[0731] (3) Reagents: DMEM liquid culture medium, fetal bovine serum, penicillin and streptomycin solution, PBS (PH=7.4) and 0.25% trypsin-EDTA are all commercially available.

[0732] (4) Cell culture: Taking H460 cells as an example, add 3 ml of 0.25% trypsin-EDTA to a culture flask full of H460 cells and digest at 7°C for 1-2 minutes. Discard the digestion solution, add culture solution and pipette, subculture at a ratio of 1:3, and subculture every 2-3 days. When seeding, prepare 150,000 cells / ml culture solution, inoculate in a 96-well cell culture plate, 0.1 ml per well, and culture overnight at 37°C, 5% CO2 in a cell culture incubator. Experiments were performed after the cells grew into a monolayer. Huh7 cells were also cultured according to the above method.

[0733] 2. The half cytotoxic concentration (CC) of the test compound 50 )

[0734] (1) Preparation of drug solution: Take appropriate amount of compound (positive control and test substance), prepare into 25 mg / ml stock solution with DMSO, filter sterilize (filter pore size 0.2 μm), and store at 4°C.

[0735] (2) Preparation of neutral red dye solution: Dilute 1% neutral red solution 100 times using cell maintenance medium.

[0736] (3) Preparation of acetic acid ethanol solution: measure 5 ml of water, 5 ml of anhydrous ethanol and 100 μl of glacial acetic acid respectively, mix well.

[0737] (4) CC of the compound 50 Method for determining the value: Use the double dilution method to set 5 concentration gradients, and set 3 secondary wells for each concentration. Take out the 96-well plate with monolayer cells and discard the culture medium in the well. Add 100μl of cell maintenance solution to each well except the 3 secondary wells with the maximum concentration. Dilute the compound mother solution 500 times with the cell maintenance solution to be the maximum experimental concentration. For the 3 secondary wells with the maximum concentration, add 200μl of 50μg / ml compound solution to each well. Pipette 100μl of liquid from the well with the maximum concentration to the well with the second concentration and mix evenly; then pipette 100μl of liquid from the well with the second concentration to the well with the third concentration, and so on, to complete the double dilution of the concentration, and set up a cell control well. Place the 96-well plate in a cell culture incubator at 37℃ and 5% CO2, and observe it under a microscope every day until no new cell lesions appear, which is the end point of the experiment. Determine the OD value of the cells in each well by neutral red staining. Take out the 96-well plate that has reached the end point of the experiment, discard the original liquid in the well, add neutral red dye solution (100μl / well), and incubate in a cell culture incubator at 37°C and 5% CO2. After 1 hour, take out the 96-well plate, discard the neutral red dye solution, and wash the neutral red in the well with PBS solution 2-3 times, discard PBS, and add acetic acid ethanol solution for color development (100μl / well). Use an enzyme marker to measure the OD value at a wavelength of 544nm. After measuring the OD value, calculate the cell survival rate, the formula is as follows:

[0738] Cell survival rate (%) = (OD 544 Value / average OD of cell control wells 544 value)×100%

[0739] GraphPad Prism 5.0 software was used to calculate the median cytotoxic concentration (CC 50 value).

[0740] 3. The half inhibitory concentration (IC 50 )

[0741] The experiments were conducted in Huh7 cells and H460 cells. Huh7 cells and H460 cells were cultured at 1.5x10 4 The cells were seeded in a 96-well plate at a concentration of 100 cells / well. 50The virus solution of HCoV-229E strain and HCoV-OC43 strain was used to infect Huh7 cells and H460 cells in 96-well plates. The test compound was diluted with maintenance solution and administered at the same time of infection for determination: the test compound was tested with samples of 8 doses diluted three times, and 2 parallel wells were set for each dose. When the lesions of the virus control group reached 4+ (according to the cell death ratio, they were marked as 4+ (cell death ratio 75%-100%), 3+ (cell death ratio 50%-75%), 2+ (cell death ratio 25%-50%), 1+ (cell death ratio 0-25%), 0+ (all cells survived)), the results were observed, the data were recorded, and the half-maximal inhibitory concentration of the test compound on the virus was calculated according to the following formula using the Reed-Muench method, and the selection index SI value (SI=CC 50 / IC 50 ):

[0742]

[0743] Where: A = drug concentration at which cumulative inhibition rate is <50%, B = inhibition rate at which cumulative inhibition rate is >50%, C = inhibition rate at which cumulative inhibition rate is <50%, and D = log dilution factor.

[0744] 4. Summary of experimental results

[0745] The results of the above experiments are summarized in Table 11 below:

[0746] Table 11 Summary of in vitro anti-coronavirus experimental results

[0747]

[0748] It can be seen from the results in the above table that under the conditions of this experiment, the in vitro anti-coronavirus activity of the tested compounds is better than or equivalent to that of the positive control drug EIDD-1931.

[0749] Experiment 4: Determination of in vitro anti-novel coronavirus (2019-nCoV-2) activity

[0750] This experimental example determined the inhibitory activity of the exemplary compounds disclosed herein against the novel coronavirus (2019-nCoV-2) B.1.1.7 strain, B.1.351 strain, and B.1.617.2 strain.

[0751] 1. Virus culture and virus titer determination

[0752] African green monkey kidney cells (VeroE6) were cultured in a 37°C, 5% CO2 incubator with DMEM containing 10% fetal bovine serum (FBS), glutamine and penicillin-streptomycin. After the cells grew to a fully confluent monolayer, the culture medium was removed, washed 2-3 times with PBS, and then the virus solution was added and incubated at 37°C for 1 hour. After the culture medium was removed, DMEM containing 2% FBS was added and cultured until cytopathic effects were observed and >75% of the cell monolayer was destroyed. The virus culture was harvested and stored at -80°C for later use.

[0753] VeroE6 cells (density of 2-3×10 4 After the cells have grown into a monolayer, they are washed 2-3 times with PBS and 100 μl of different dilutions (10 -1 ~10 -10 ) of the virus solution, set up 6 to 8 replicate wells for each dilution, and culture in a 37°C, 5% CO2 incubator. Observe and record the cytopathic effect every day until no new cytopathic wells appear. Calculate the TCID of the virus solution using the Reed-Muench or Karber method 50 .

[0754]

[0755] 2. The half cytotoxic concentration (CC) of the test compound 50 )

[0756] Vero E6 cells were seeded in 96-well plates as described above. After culturing to confluent monolayers, the cells were washed with PBS and then 200 μl of 2% FBSDMEM solution of different concentrations of compounds were added. After 5-6 days of culture, 3 μl of 1.5% neutral red solution was added and incubated for another 24 hours before fixation with 10% formaldehyde saline solution. The cells were washed 2-3 times with PBS to remove residual neutral red, and 200 μl of acidified ethanol (composed of 50% ethanol, 49% water and 1% glacial acetic acid) was added. The cells were shaken for 10 minutes or until the neutral red was completely dissolved, and the OD was measured using a microplate reader. 540 The dose-effect curve was fitted by four-parameter logistic regression, and the half cytotoxic concentration (CC 50 ).

[0757] The CC of a drug is calculated using the following formula: 50 :

[0758]

[0759] Finally, GraphpadPrism 7 software was used to perform S-fitting analysis on the data and calculate the half cytotoxic concentration (CC50 value).

[0760] 3. The half inhibitory concentration (IC 50 )

[0761] As described above, Vero E6 cells were seeded in a 96-well plate and cultured overnight. After the cells grew into a monolayer, 100 μl of a 2% FBSDMEM solution of the compound diluted 1 / 2 was added to 6 rows, with 4 replicate wells for each concentration. After incubation for 1 hour, 100 μl of virus solution (~100×TCID 50 ), and add 200 μl 2% FBSDMEM culture medium to the remaining row. After 5-6 days of culture, stain with neutral red and measure OD 540 The half inhibitory concentration (IC 50 ), and calculate the selection index SI value (SI = CC 50 / IC 50 ).

[0762] Inhibition rate (IC): (%) = (OD 540 Value - Average OD of virus control wells 540 value) / (average OD of cell control wells 540 Value - Average OD of virus control wells 540 value)×100%

[0763] GraphPad Prism 5.0 software was used to calculate the half inhibitory concentration (IC 50 value).

[0764] 4. Summary of experimental results

[0765] The results of the above experiments are summarized in Table 12 below:

[0766] Table 12 Summary of in vitro anti-novel coronavirus experimental results

[0767]

[0768] It can be seen from the results in the above table that under the conditions of this experiment, the in vitro anti-novel coronavirus activity of the tested compounds is better than that of the positive control drug EIDD-1931.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt or stereoisomer thereof: in, m is any integer from 6 to 20; n is any integer from 1 to 5; X is -O- or -NR0-; R0 is H or C1-C6 alkyl; Z is -SS-; R is H, C1-C6 alkyl, C6-C 12 Aryl, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-; R' is C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 12 aryl, 3 to 12 membered heterocyclyl containing 1 or 2 heteroatoms independently selected from N, O or S, or 5 to 12 membered heteroaryl containing 1 or 2 heteroatoms independently selected from N, O or S; The alkyl, cycloalkyl, aryl, heterocyclic or heteroaryl groups in the R or R' group are optionally substituted by 1, 2 or 3 groups independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

2. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X is -O-.

3. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X is -NR0-, and wherein R0 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

4. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R is H.

5. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R is a C1-C6 alkyl group, wherein the alkyl group is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

6. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R is C6-C 12 Aryl, wherein the aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

7. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R is R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-.

8. The compound according to claim 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R' is a C1-C6 alkyl group, wherein the alkyl group is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

9. The compound according to claim 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R' is a C3-C8 cycloalkyl group, wherein the cycloalkyl group is optionally substituted by 1, 2 or 3 groups independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

10. The compound according to claim 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R' is C6-C 12 Aryl, wherein the aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

11. The compound according to claim 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R' is a 3 to 12 membered heterocyclic group containing 1 or 2 heteroatoms independently selected from N, O or S, wherein the heterocyclic group is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halo C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

12. The compound according to claim 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R' is a 5- to 12-membered heteroaryl group containing 1 or 2 heteroatoms independently selected from N, O or S, wherein the heteroaryl group is optionally substituted by 1, 2 or 3 groups independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkoxy, cyano, hydroxy, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

13. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each R a and R b is independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

14. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: m is any integer from 6 to 20; n is any integer from 1 to 5; X is -O- or -NR0-; R0 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; R is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, phenyl optionally substituted with 1 substituent independently selected from halogen or nitro, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-; R' is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or phenyl.

15. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: m is any integer from 6 to 20; n is any integer from 1 to 5; X is -O- or -NR0-; R0 is H, methyl or ethyl; R is H, methyl, ethyl, phenyl optionally substituted with 1 substituent independently selected from chloro or nitro, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-; R' is methyl, ethyl, isopropyl, tert-butyl, cyclopentyl, cyclohexyl or phenyl.

16. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: m is any integer from 6 to 20; n is any integer from 1 to 5; X is -O- or -NR0-; R0 is H or methyl; R is H, methyl, phenyl optionally substituted with 1 substituent independently selected from chloro or nitro, R'-OC(O)-O-CH2- or R'-C(O)-O-CH2-; R' is methyl, isopropyl, tert-butyl, cyclopentyl, cyclohexyl or phenyl.

17. The compound according to claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is any one of the compounds described in the following table:

18. A compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is any one of the following compounds:

19. The compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: The pharmaceutically acceptable salt is an alkali metal salt, an alkaline earth metal salt, an aluminum salt, an organic base salt or an amino acid salt.

20. The compound according to claim 19, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the alkali metal salt is a lithium, sodium, potassium, rubidium or cesium salt.

21. The compound according to claim 19 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the alkaline earth metal salt is a magnesium, calcium, strontium or barium salt.

22. The compound according to claim 19, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the organic base salt is ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, diethylamine, diethylaminoethanol, ethylenediamine, imidazole, morpholine, 2-hydroxyethylmorpholine, dibenzylethylenediamine, trimethylamine, piperidine, pyrrolidine, benzylamine or tetramethylammonium hydroxide salt.

23. The compound according to claim 19 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the amino acid salt is lysine, arginine or an arginine salt.

24. A pharmaceutical composition comprising a compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt or stereoisomer thereof and one or more pharmaceutically acceptable carriers.

25. Use of a compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt or stereoisomer thereof or a pharmaceutical composition according to claim 24 in the preparation of a medicament for inhibiting a virus or for preventing and / or treating a viral infection in a subject in need thereof.

26. The use according to claim 25, wherein the virus is a coronavirus, equine encephalitis virus, Chikungunya virus, Ross River virus, Orthomyxoviridae virus, Paramyxoviridae virus, Filovirus or Zika virus.

27. The method of claim 26, wherein the virus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, influenza virus, respiratory syncytial virus, Ebola virus, Marburg virus or Kuwaiti virus.

28. The use according to claim 26, wherein the virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

29. The use according to claim 26, wherein the virus is influenza A virus, influenza B virus or influenza C virus.

30. The use according to claim 26, wherein the virus is influenza A (H1N1) virus.

31. The use according to claim 26, wherein the virus is strain SC09.

32. The use according to claim 26, wherein the virus is influenza A(H3N2) virus.

33. The use according to claim 26, wherein the virus is strain FJ09.

34. The use according to claim 26, wherein the virus is the SC11 strain.

35. The use according to claim 26, wherein the virus is strain SZ01.

36. The use according to claim 26, wherein the virus is HCoV-229E strain or HCoV-OC43 strain.

37. The use according to claim 26, wherein the virus is strain B.1.1.7, strain B.1.351, strain B.1.617.2 or strain B.1.1.

529.

38. Compounds of formula 2a: wherein m, n and Z are as defined in claim 1; R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl; A is chlorine or nitro; B is hydrogen, chlorine or nitro.

39. Compounds of formula 2a-1: wherein m, n, Z, R0 and R are as defined in claim 1; R1 is a straight or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

40. Compounds of formula 4a: wherein m, n and Z are as defined in claim 1; R1 is a linear or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl; A is chlorine or nitro; B is hydrogen, chlorine or nitro.

41. Compounds of formula 4a-1: wherein m, n, Z, R0 and R are as defined in claim 1; R1 is a straight or branched C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C6-C 12 Aryl, the alkyl, alkenyl, alkynyl or aryl is optionally substituted by 1, 2 or 3 independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, cyano, hydroxyl, nitro, -NR a R b 、-C(=O)R a 、-C(=O)OR a 、-OC(=O)R a 、-C(=O)NR a R b 、-SR a 、-S(=O)R a 、-S(=O)2R a and -S(=O)2NR a R b Substituents, where each R a and R b are independently H or C1-C6 alkyl.

42. Compound of formula 2-1a: wherein m, n and Z are as defined in claim 1; A is chlorine or nitro; and B is hydrogen, chlorine or nitro.

43. Compound of formula 2-1a-1: wherein m, n, Z, R0 and R are as defined in claim 1.

44. Compound of formula 4-1a-1: wherein m, n, Z, R0 and R are as defined in claim 1.

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