CN106543253B

CN106543253B - Anti-viral nucleoside phosphoramidate and its pharmaceutical composition and purposes

Info

Publication number: CN106543253B
Application number: CN201610976842.XA
Authority: CN
Inventors: 杨学聪; 赵蕾; 刘江; 黄新全
Original assignee: Individual
Current assignee: Suzhou Half Fish Health Technology Service Co ltd
Priority date: 2015-11-24
Filing date: 2016-11-07
Publication date: 2019-04-02
Anticipated expiration: 2035-11-24
Also published as: CN106543253A

Abstract

The present invention provides a kind of anti-viral nucleoside phosphoramidate and its pharmaceutical composition and purposes, ucleosides phosphoramidate compounds are nucleosides with phosphorus oxygen key connection phosphate, structure nucleoside phosphoramidate compound, its stereoisomer as described in general formula a, a1, a2, b, b1 and b2, its pharmaceutically acceptable salt, hydrate, solvate or crystallization.Novel nucleoside phosphoramidate provided by the invention is significantly superior to the Suo Feibuwei of clinical application in terms of anti-hepatitis activity, and in saccharide ring, chlorine atom replaces fluorine atom, and cytotoxicity is significantly reduced in surveyed cell line.Pass through the transformation and optimization to base, saccharide ring and prodrug moiety system, the anti-hepatitis activity of partially synthetic compound is higher than Suo Feibuwei 2-10 times, meanwhile in the optimization of metabolism key position, synthesis compound shows the stronger metabolic stability of peso Fei Buwei and chemical stability in blood plasma.

Description

Antiviral nucleoside phosphoramidates and pharmaceutical compositions and uses thereof

Technical Field

The invention relates to nucleoside phosphoramidate derivatives, and a pharmaceutical composition and application thereof.

Background

Hepatitis C Virus (HCV) is a new 3-4 million patients each year, and the world health organization estimates that more than 2 hundred million infected people worldwide and more than 1000 million patients in China, HCV belongs to the genus hepacivirus of the family Flaviviridae. Chronic hepatitis c virus infection is mild to inflammation, severe to cirrhosis and liver cancer. And various complications can occur when the hepatitis C cirrhosis is in the decompensation stage, such as ascites abdominal infection, upper gastrointestinal hemorrhage, hepatic encephalopathy, hepatorenal syndrome, hepatic failure and the like. The first treatment for HCV infection was interferon and ribavirin combination therapy, to which only 50% of the patients responded, with interferon having significant side effects such as flu-like symptoms, weight loss and fatigue weakness, and interferon and ribavirin combination therapy producing considerable side effects including hemolysis, anemia and fatigue.

In recent years the FDA has approved a number of HCV drugs including protease inhibitors, nucleoside and non-nucleoside polymerase inhibitors, and NS5A inhibitors. There are three FDA-approved protease inhibitor drugs: VX-950 (Telaprevir), SCH-503034 (Boceprevir) and TMC435(Simeprevir), protease inhibitors have the disadvantages of susceptibility to mutation, high toxicity, poor bioavailability and efficacy for individual gene types. Telaprevir has exited the market as the first generation of protease inhibitors. Highly active and broad-spectrum second-and third-generation protease inhibitors are mainly used as one of the components of combination drugs with other hepatitis C drugs.

NS5A inhibitors are a highly active class of anti-HCV agents. The most representative examples are BMS, Daclatasive, Gilead Ledipasvir and AbbVie Ombitasvir, which are mainly used as one of the drug components for the combined treatment of HCV because the drugs are easy to generate drug resistance when being taken alone.

The polymerase inhibitors of hepatitis C are generally divided into two types of nucleoside inhibitors and non-nucleoside inhibitors, only one nucleoside hepatitis C drug is clinically approved by FDA to be on the market, other nucleoside anti-hepatitis C virus drugs which are still in clinical test stage are MK-3682 of Davidon (IDX21437), Achillion pharmaceutical company ACH-3422 and AL-335 of Alios, the hepatitis C virus has the characteristics of multiple genotypes and rapid mutation, single drug treatment of hepatitis C has various defects such as quick drug resistance, low cure rate and long course of treatment for partial genotypes, the combined treatment scheme of combined drugs is mainly adopted at present to overcome the defects, Sovaldi approved by FDA is taken as a key component of combined drug, the combined drug combination of Sovaldi approved by FDA is taken as a combined drug target, the combined drug of Sovalvirbuvir, ribavirin and polyethylene glycol- α -interferon is a combined drug for non-ribavirin, the non-protein inhibitors of Nonpavirus protease, the non-ribavirin, the non-protein inhibitors of NS-2, the non-ribavirin-protein, the non-ribavirin-protein, the non-ribavirin protein, the non-ribavirin protein, the non-protein, the protein.

Deuterium is a naturally occurring hydrogen isotope in nature, and common drugs all contain trace amounts of deuterium isotopes. Deuterium is nontoxic and nonradioactive, is safe to human bodies, C-D bonds are more stable (6-9 times) than C-H bonds, the half-life period of the drug can be prolonged after hydrogen is replaced by deuterium, and the pharmacological activity is not influenced (the shape difference of H and D is small, J Med chem.2011,54, 2529-2591), in addition, deuterium drugs often show better bioavailability and less toxicity, and the metabolized active nucleoside triphosphate is more stable, so the curative effect of deuterium nucleoside phosphoramidate in clinical application is better than that of corresponding nucleoside drugs. For example, the nucleoside anti-hepatitis c virus drug ACH-3422, approved by the FDA in 2013 for clinical trials, is a deuterated compound and has higher bioavailability and longer half-life than the non-deuterated original compound (WO 201416978, WO 2014169280).

Based on the current state of the art, we have designed and prepared a novel deuterated nucleoside phosphoramidate represented by compound VI-1 b2. The structural characteristics, the preparation method, the antiviral activity experimental results and the application of the deuterated nucleoside phosphoramidate as a backbone component of the anti-hepatitis C virus combined drug and NS5A inhibitor and/or protease inhibitor in the aspect of antivirus are described in detail below.

Disclosure of Invention

The invention provides a deuterated nucleoside phosphoramidate compound for treating and/or preventing HCV infection, which has the structural characteristics shown in general formulas a, a1, a2, b1 and b2, can be metabolized into nucleotide triphosphates (with the structures shown in general formulas a and b) capable of inhibiting HCV virus replication through different metabolic pathways in vitro or in vivo as nucleoside prodrugs, and the deuterated nucleoside phosphoramidate can be used alone or combined with two or more compounds such as other hepatitis C virus-resistant protease inhibitors, polymerase inhibitors, helicase inhibitors, NS5A inhibitors, α -interferon, pegylated interferon and ribavirin.

The technical scheme of the invention is as follows:

a nucleoside phosphoramidate compound is characterized in that: nucleoside phosphoramidate compound, its stereoisomer, its pharmaceutically acceptable salt, hydrate, solvate or crystal, wherein nucleoside is connected with phosphate by phosphorus-oxygen bond, and structure is described by general formula a, a1, a2, b1 and b 2;

wherein,

R₁is C_1‐6Alkyl radical, C_3‐6Cycloalkyl, benzyl, deuterated C_1‐6Alkyl, deuterated C_3‐6Cycloalkyl or deuterated benzyl;

R₂is optionally substituted phenyl, biphenyl, heteroaryl or naphthyl; or, R₂Optionally substituted phenyl, biphenyl, heteroaryl or naphthyl which are deuterated, wherein the substituents can be halogen, azide, cyano, alkynyl, C_1‐4Alkyl radical, C_3‐4Cycloalkyl or C_1‐4An alkoxy group;

R₃selected from various natural or non-natural amino acid acyl groups, carboxylic acid acyl groups, polypeptide acyl groups or hydrogen;

R₄、R₅、R₆、R₇、R₈、R₉and R₁₀Independently selected from hydrogen or deuterium atoms;

R₁₁selected from methyl or deuterated methyl (CD)₃、CHD₂Or CH₂D)。

The above nucleoside phosphoramidate compounds are characterized in that: r₁Is isopropyl or deuterated isopropyl; r₂Selected from phenyl, biphenyl, 4-cyclopropylphenyl or deuterated phenyl, biphenyl, 4-cyclopropylphenyl; r₃Selected from the group consisting of hydrogen, acetyl, propionyl, butyryl, isobutyryl, alanyl, glycyl, prolyl, lysyl, phenylalanyl, leucyl, isoleucyl, valyl, tryptophanyl, tyrosyl, aspartyl, asparaginyl, glutamyl, glutaminyl, methionyl, threonyl, histaminyl, seryl, cysteinyl, arginyl, and β -alanyl.

The nucleoside phosphoramidate compounds described herein are characterized by the R at the C1' position in the nucleoside derivatives described by the general formulae a, a1 and a2 and b, b1 and b2₈As deuterium atoms, the compounds are preferably selected from the following formulae:

the nucleoside phosphoramidate compound is also characterized in that the C2' methyl R of the nucleoside derivative described by the general formulas a, a1, a2, b1 and b2₁₁Wherein the hydrogen atom is replaced by deuterium, preferably selected from the following formulae:

the nucleoside phosphoramidate compound may be R at C3' position of a nucleoside derivative represented by the general formulae a, a1, a2 and b, b1 or b2₆For deuterium, the compounds are preferably selected from the following formulae:

the nucleoside phosphoramidate compound may be R at C4' position of a nucleoside derivative represented by general formula a, a1, a2, b1 or b2₆As deuterium atoms, the compounds are preferably selected from the following formulae:

the nucleoside phosphoramidate compounds described in this patent application are also characterized by the R at the C5' position of nucleoside derivatives described by the general formulae a, a1, a2 and b, b1, b2₄And R₅As deuterium atoms, the compounds are preferably selected from the following formulae:

the nucleoside phosphoramidate compound may be R at positions C5 and C6 of the nucleoside derivative represented by general formulae a, a1, a2, b1 and b2₉And R₁₀Each independently a deuterium atom, the compound being preferably selected from the following structural formulae:

nucleoside phosphoramidate compounds described in the present patent application, which are also characterized in that the hydrogen atoms of the furanose ring of the nucleoside derivatives described by the general formulae a, a1, a2 and b, b1 and b2, which may simultaneously be in two positions, are replaced by deuterium, are preferably selected from the following formulae:

the nucleoside phosphoramidate compound may be a substituent R of a prodrug group of a nucleoside derivative phosphoramidate described by general formulas a, a1, a2, b1 and b2₁Is deuterated isopropyl or R₂For deuterated phenyl, the compounds are preferably selected from the following formulae:

the nucleoside phosphoramidate compounds described in this patent are also characterized by the substituent R at the hydroxyl group at the C3' position of the nucleoside derivatives described by the general formulas a, a1, a2 and b, b1, b2₃Is carboxylic acid acyl, natural amino acid acyl, β -propylAminoacyl, or hydrogen (i.e., unsubstituted hydroxyl at the 3' position), the compound is preferably selected from the following structural formulae:

the nucleoside phosphoramidate compound is mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation and a nano preparation so as to be suitable for oral or parenteral administration; methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes.

The pharmaceutical composition of the nucleoside phosphoramidate compound includes a nucleoside phosphoramidate and another therapeutic agent. The additional therapeutic agent is independently selected from the following: ribavirin, interferon, hepatitis c NS3 protease inhibitors, HCV reverse transcriptase NS5B non-nucleoside inhibitors, HCV reverse transcriptase NS5B nucleoside inhibitors, NS5A inhibitors, and potentiators of NS5A inhibitors, entry inhibitors, cyclosporin immunosuppressants, NS4A antagonists, NS4B inhibitors, cyclophilin inhibitors.

The present invention provides pharmaceutical compositions wherein NS5A inhibitors may include, but are not limited to, the following compounds:

the present invention provides pharmaceutical compositions wherein the protease inhibitor may include, but is not limited to, the following compounds:

the deuterated nucleoside phosphoramidate compound, the stereoisomer, the salt, the hydrate, the solvate or the crystal thereof can be mixed with a pharmaceutically acceptable carrier, a diluent or an excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The formulations may be administered by any route, for example by infusion or bolus injection, by a route of absorption through epithelial or cutaneous mucosa (e.g. oral mucosa or rectum, etc.). Administration may be systemic or local. Examples of the formulation for oral administration include solid or liquid dosage forms, specifically, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions and the like. The formulations may be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the art of pharmaceutical formulation.

The nucleoside phosphoramidate compounds provided by the present invention are useful alone, or in combination with other HCV therapeutic agents such as NS5A inhibitors or protease inhibitors, in the treatment of patients with Flaviviridae virus infections. Comprising administering to said patient a nucleoside phosphoramidate compound described by general formula a, a1, a2 and b, b1, b2, or a pharmaceutical composition comprising a compound of general formula a, a1, a2 and b, b1, b2, a stereoisomer, salt, hydrate, solvate or crystal thereof, effective to treat hepatitis c. In one embodiment, the present invention provides a method for the treatment and/or prevention of an infection by an RNA virus, such as a virus of the flaviviridae family, comprising administering to a patient in need of such treatment a compound of the present invention, a stereoisomer, salt, hydrate, solvate or crystal thereof, or a pharmaceutical composition thereof. In another embodiment, the present invention provides a method of inhibiting infection by an RNA virus, such as a virus of the flaviviridae family, comprising contacting said virus with a therapeutically effective amount of a compound of the present invention, a stereoisomer, salt, hydrate, solvate or crystal thereof, or a pharmaceutical composition thereof.

By "flaviviridae virus" is meant any virus of the flaviviridae family, including those that infect humans and non-human animals, such as flavivirus, pestivirus and hepatitis c virus, dengue virus. The compounds and compositions of the present invention are particularly useful in the therapeutic or prophylactic treatment of HCV and dengue virus.

The nucleoside phosphoramidate compound, the stereoisomer, the salt, the hydrate, the solvate or the crystal thereof provided by the invention can be used for preventing or treating viral infection, especially flavivirus infection diseases, and can be used for preparing medicaments for preventing and/or treating viral infection diseases, especially for preparing medicaments for preventing and/or treating HCV virus infection, such as HCV viral hepatitis diseases. Examples of such diseases are acute hepatitis c, chronic hepatitis c and mixed infections of hepatitis c and hepatitis b or hepatitis d.

Definition of terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "stereoisomer" refers to an isomer resulting from the different arrangement of atoms in a molecule. Including cis-trans isomers, enantiomers, and conformers. All stereoisomers are within the scope of the present invention. Individual stereoisomers of the compounds of the invention may be substantially free of other isomers or may be mixed, for example, as racemates or with all other stereoisomers.

The term "salt" refers to a pharmaceutically acceptable salt of a compound of the invention with an acid, which may be selected from: phosphoric acid, sulfuric acid, hydrochloric acid, hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid, succinic acid, fumaric acid, acetic acid, lactic acid, nitric acid, sulfonic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid, or the like.

The term "solvate" refers to a form of a compound of the present invention that forms a solid or liquid complex by coordination with a solvent molecule. Hydrates are a special form of solvates in which coordination occurs with water. Within the scope of the present invention, the solvate is preferably a hydrate.

The term "crystalline" refers to the various solid forms formed by the compounds of the present invention, including crystalline forms, amorphous forms. The term "alkyl" refers to a straight, branched or cyclic saturated hydrocarbon group, preferably a hydrocarbon group of 6 carbon atoms or less. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl, neopentyl, cyclohexyl, n-hexyl, isohexyl, 2, -methylbutyl and 2, 3-dimethylbutyl. The term "C1-6 alkyl" refers to a straight, branched, or cyclic saturated hydrocarbon group containing 1 to 6 carbon atoms. The term "C1-4 alkyl" refers to a straight, branched, or cyclic saturated hydrocarbon group containing 1 to 4 carbon atoms.

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

The term "haloalkyl" refers to an alkyl group substituted with at least one halogen atom.

The term "heterocyclyl" refers to a cyclic group containing at least one heteroatom, wherein the heteroatom is N, O or S, including mono-and fused heterocyclyl groups. Wherein the mono-heterocyclic group includes, but is not limited to, furan, thiophene, pyrrole, thiazole, imidazole, 1,2, 3-triazole, 1,2, 4-triazole, 1,2, 3-thiadiazole, oxazole, 1,2, 4-oxadiazole, 1,3, 4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, tetrahydrofuran, tetrahydropyrrole, piperidine, piperazine, morpholine, isoxazoline, etc. Fused heterocyclic groups include, but are not limited to, quinoline, isoquinoline, indole, benzofuran, benzothiophene, purine, acridine, carbazole, fluorene, chromene, fluorenone, quinoxaline, 3, 4-dihydronaphthalenone, dibenzofuran, hydrogenated dibenzofuran, benzoxazolyl, and the like.

The term "heteroaryl" refers to an aryl group containing at least one heteroatom, wherein the heteroatom is N, O or S. Deuterium is an isotope of hydrogen, has 2 times the atomic mass of the latter, and is more strongly bonded to carbon. Deuterated "and" deuterium "indicate that hydrogen is replaced with deuterium at the indicated position. One "deuterated substituent" is a substituent wherein at least one hydrogen is substituted for deuterium enriched by the percentage indicated.

Detailed Description

The present invention will be further illustrated in detail with reference to the following examples, but the present invention is not limited to these examples. The reagents and starting materials used in the present invention are commercially available.

Example 1.

Isopropyl [ (S) - (pentafluorophenoxy) (phenoxy) phosphoryl ] -L-alanine ester (3) and isopropyl [ (R) - (pentafluorophenoxy) (phenoxy) phosphoryl ] -D-alanine ester (6)

Phenyl dichlorophosphate (5.7 g, 25.7 mmol) and L-alanine isopropyl ester hydrochloride (4.32 g, 25.7 mmol) were added to a reaction flask, cooled to-70 deg.C, a solution of triethylamine (7.2 ml, 51.7 mmol) in dichloromethane (22 ml) was added dropwise over 1.5h, after which time the temperature was raised to room temperature, the reaction mixture was cooled to 0 ℃ after stirring overnight, a solution of pentafluorophenol (4.73 g, 25.7 mmol) and triethylamine (3.6 mL, 25.7 mmol) in dichloromethane (30mL) was added dropwise over 40 minutes, after stirring at 0 ℃ for 1 hour, warming to room temperature and stirring overnight, the triethylamine hydrochloride solid was removed by filtration, the solid cake was washed with dichloromethane (3X10mL), the filtrate was concentrated under reduced pressure, tert-butyl methyl ether (80mL) was added to the residue, and the resulting white solid was filtered and washed with tert-butyl methyl ether (3X10 mL). All filtrates are combined and concentrated, the obtained filter cake is added with 20% ethyl acetate-hexane mixed solvent (60mL), the solid obtained after filtration is washed by 5% sodium bicarbonate solution until the filtrate is neutral, then the filtrate is washed by water, the obtained solid is dried for 30 hours in vacuum at 55 ℃,15 mL ethyl acetate-pentane (1:5) mixed solvent is added into the dried solid, the mixture is stirred for 1 hour, the solid obtained after filtration is washed by ethyl acetate-pentane (1:5, 2X5mL), the solid is combined with the solid obtained at the first time, and the solid compound (3) is obtained after drying, and the purity is more than 99%.

The compound (6) was obtained by the same procedure as above using D-alanine isopropyl ester hydrochloride as a starting material. The nuclear magnetic data for compounds (3) and (6) are the same:

¹H NMR(400MHz,CDCl₃)δ(ppm):7.37(d,J＝7.6Hz,2H),7.34–7.19(m,3H),5.18–4.98(m,1H),4.29–4.10(m,1H),4.07–3.97(m,1H),1.48(d,J＝7.0Hz,3H),1.27(d,J＝5.9Hz,3H),1.26(d,J＝5.9Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐1.81.

example 2

N- (tert-butoxycarbonyl) -D-alanine (8a)

4.5g (50mmol) of D-alanine, 3.8g (55mmol), potassium hydroxide together with 13.0g (55mmol), di-tert-butyl carbonate were suspended in a mixed solvent of water (200mL) and THF (20mL) and stirred at room temperature overnight to give 9.6g of N-Boc-D-alanine as a white solid which was used directly in the next esterification reaction without purification.

N- (tert-butoxycarbonyl) -D-alanine-deuterated isopropyl ester-D₆(9a)

The aforementioned N-Boc-D-alanine 8a (2.18g,11.5mmol) was dissolved in dry dichloromethane 40mL, followed by the addition of hexadeuterated isopropanol-D₆(prepared by reduction of deuterated acetone with reference to CN 102010384, 15 mmol). The reaction was cooled to 5 ℃ and EDC (3.31g,17.2mmol) and DMAP (140mg,1.15mmol) were added, followed by stirring at room temperature overnight. The reaction was diluted with 300mL of ethyl acetate, the organic phase was washed with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography to give product 9a as a white solid:¹HNMR(CDCl₃,400MHz)δ5.11(s,br,1H),4.99(s,1H),4.22(s,br,1H),1.43(s,9H),1.32‐1.37(m,3H)；¹³CNMR(CDCl₃,400MHz)δ172.87,155.09,79.60,68.53,49.33,28.29,18.66.

d-alanine-deuterated isopropyl ester-D₆Hydrochloride (10a)

Compound 9a (230mg,1mmol) was dissolved in 20mL tetrahydrofuran, HCl in dioxane (4M, 1mL) was added at 0 deg.C and stirred at room temperature for two hours, and the solvent was evaporated under reduced pressure to give product 10a as an off-white solid which was used without further purification in the next step:¹HNMR(MeOD‐d₄,400MHz)δ5.01(s,1H),4.04(q,J＝7.2Hz,1H),1.53(d,J＝7.2Hz,3H).

((R) - (pentafluorophenoxy) (phenoxy) phosphoryl) -D-alanine-deuterated isopropyl ester-D₆(11a) And (11 a') Triethylamine (1.12mL,8mmol) was slowly added dropwise to a solution of Compound 10a (1.18g,3.8mmol) in dichloromethane (10mL) at-78 deg.C, followed by addition of phenoxyphosphoryl dichloride (0.58mL,3.8mmol) in dichloromethane (5mL) over one hour, and the reaction mixture was allowed to react at-78 deg.C for 30 minutes, then slowly warmed to 0 deg.C and stirred for 2-3 hours. Pentafluorophenol (700mg,3.8mmol) and triethylamine (0.5mL,3.8mmol) in dichloromethane (5mL) were slowly dropped into the above reaction solution through a dropping funnel at 0 ℃ over 30 minutes, and the reaction solution was further stirred at 0 ℃ for one day. The reaction solution was concentrated, the residue was dissolved in THF, the insoluble matter was filtered off, the filtrate was again concentrated, the residual pink solid was recrystallized from methyl t-butyl ether to obtain a white crystalline product 11a, and the residue in the mother liquor was separated by column chromatography to obtain the product 11 a'.

11a:¹H NMR(400MHz,CDCl₃)δ(ppm):7.38–7.35(m,2H),7.33–7.21(m,3H),4.87(s,1H),3.92(brs,1H),3.68–3.60(m,1H),1.41(d,J＝7.0Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐2.05.

11a’:¹H NMR(400MHz,CDCl₃)δ(ppm):7.40–7.36(m,2H),7.32–7.20(m,3H),4.91(m,1H),3.86(brs,1H),3.66–3.61(m,1H),1.43(d,J＝7.2Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐1.65.

Example 3

Respectively reacting deuterated alanine esters 9b, 9c and pentafluorophenol with phenoxyphosphoryl dichloride by the same method as 11a to obtain deuterated (pentafluorophenoxy) (phenoxy) phosphoryl) -isopropyl alanine 11b and 11c by a one-step method:

compound 11b and 11c have the same nmr spectra:

¹H NMR(400MHz,CDCl₃)δ(ppm):7.40–7.35(m,2H),7.33–7.20(m,3H),3.95(brs,1H),3.65–3.60(m,1H),1.43(s,6H),1.41(d,J＝7.0Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐1.99.

compound 11b 'has the same nmr spectrum as 11 c':

¹H NMR(400MHz,CDCl₃)δ(ppm):7.42–7.34(m,2H),7.32–7.19(m,3H),3.89(brs,1H),3.66–3.61(m,1H),1.45(s,6H),1.42(d,J＝7.0Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐1.63.

example 4

Respectively reacting alanine esters 9d, 9e and pentafluorophenol with deuterated phenoxyphosphoryl dichloride by the same method as 11a and 11a ', and preparing deuterated (pentafluorophenoxy) (phenoxy) phosphoryl) -isopropyl alanine 11d, 11d ' and 11e ' by a one-step method:

compound 11d has the same nmr spectrum as 11 e:

¹H NMR(400MHz,CDCl₃)δ(ppm):5.22–4.96(m,1H),4.25–4.09(m,1H),3.97–3.91(m,1H),1.47(d,J＝7.0Hz,3H),1.25(d,J＝6.0Hz,3H),1.24(d,J＝6.0Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐2.05.

compound 11d 'has the same nmr spectrum as 11 e':

¹HNMR(CDCl₃,400MHz)δ5.00‐5.07(m,1H),4.05‐4.19(m,1H),3.99(t,J＝9.4Hz,1H),1.48(d,J＝7.0Hz,3H)，1.22‐1.27(m,6H)；³¹PNMR(CDCl₃,400MHz)δ‐1.53。

example 5

Respectively reacting alanine esters 9f, 9g and pentafluorophenol with deuterated phenoxyphosphoryl dichloride by the same method as 11a to obtain deuterated (pentafluorophenoxy) (phenoxy) phosphoryl) -isopropyl alanine 11f, 11g, 11h and 11 h' by a one-step method:

compound 11f has the same nmr spectrum as 11 g:

¹HNMR(CDCl₃,400MHz)δ7.35(d,2H),7.22‐7.28(m,2H),5.00‐5.07(m,1H),4.02‐4.19(m,1H),3.97(t,1H),1.45(d,3H)，1.22‐1.27(m,6H)；³¹PNMR(CDCl₃,400MHz)δ‐1.53。

11h:¹H NMR(400MHz,CDCl₃)δ(ppm):7.40–7.36(m,2H),7.33–7.21(m,3H),3.98(brs,1H),3.65–3.59(m,1H),1.37(d,J＝7.0Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐1.97.

11h’:¹H NMR(400MHz,CDCl₃)δ(ppm):7.42–7.34(m,2H),7.32–7.19(m,3H),3.89(brs,1H),3.66–3.60(m,1H),1.35(d,J＝7.0Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐1.60.

example 6

(4S,5R) -4- ((tert-butyldimethylsilyl) oxy) -5- (((tert-butyldimethylsilyl) oxy) methyl) -3-methyldihydrofuran-2 (3H) -one (13)

A solution of compound 12(18g, 50mmol) and THF (250mL) was cooled to-70 ℃ using a dry ice-acetone bath, lithium diisopropylamine reagent (2M in tetrahydrofuran, 65mL, 65mmol) was slowly added dropwise, stirring at this temperature continued for 20 minutes after addition was complete, and methyl iodide (9.23 g,65 mmol) was added dropwise. The mixture was stirred at-70 ℃ for half an hour, gradually warmed to-30 ℃ until the reaction was complete. After cooling to-70 ℃ and pouring into 150mL of saturated ammonium chloride solution, gradually warming to room temperature, the solvent was evaporated under reduced pressure, and the residue was diluted with n-hexane (300mL) and washed three times with brine (80 mL). The organic layer was separated, dried over sodium sulfate, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (0-10% ethyl acetate in hexane) to give product 13(9.54 g) as a white solid in 51% yield.¹H NMR(400MHz,CDCl₃)δppm:4.20(dt,J＝18.4,9.1Hz,1H),4.13–4.06(m,1H),3.97–3.90(m,1H),3.76(dd,J＝12.1,2.6Hz,1H),2.59(p,J＝7.3Hz,1H),1.27(d,J＝7.3Hz,3H),0.90(s,9H),0.89(s,9H),0.11(s,3H),0.10(s,3H),0.08(s,3H),0.07(s,3H).¹³C NMR(101MHz,CDCl₃)δppm:176.83,84.44,74.10,60.38,44.25,25.79,25.60,18.26,17.79,13.03,‐4.34,‐4.74,‐5.34,‐5.52.

(3R,4R,5R) -4- ((tert-butyldimethylsilyl) oxy-5- (((tert-butyldimethylsilyl) oxy) methyl) -3-chloro-3-methyldihydrofuran-2 (3H) -one (14)

Compound 13(9 g, 24.1 mmol) was dissolved in 120mL of dichloromethane and, after cooling to 0 deg.C, triethylamine (19.7 mL, 144.6mmol) and trimethylsilyl trifluoromethanesulfonate (13.1 mL, 72.3mmol) were added in that order. After the reaction mixture was stirred at 0 ℃ for 30 minutes, NCS (4.84 g, 36.2mmol) was added, stirred for 15 minutes, poured into 110 ml of a sodium hydrogen carbonate (5%) solution, washed twice with dichloromethane, the organic phase was separated off, dried over sodium sulfate and the solvent was evaporated off under reduced pressure. The residue was purified by column chromatography (0-10% ethyl acetate in hexane) to give compound 14a (2.66 g, yield 27%) which was isolated with little polarity; the compound was subsequently isolated as 14b (3.47 g, 35% yield).

Compound 14 a:

¹H NMR(400MHz,CDCl₃)δppm:4.32(d,J＝7.9Hz,1H),4.23(dt,J＝7.9,1.8Hz,1H),4.04(dd,J＝12.6,1.6Hz,1H),3.80(dd,J＝12.6,2.1Hz,1H),1.71(s,3H),0.94(s,9H),0.88(s,9H),0.14and 0.15(2s,6H),0.09and 0.07(2s,6H).

¹³C NMR(101MHz,CDCl₃)δppm:171.85,72.86,65.95,58.64,25.74,25.55,23.56,18.19,17.97,‐4.46,‐4.69,‐5.37,‐5.51.

compound 14 b:

¹H NMR(400MHz,CDCl₃)δppm:4.72(d,J＝7.0Hz,1H),4.07(dt,J＝6.9,2.8Hz,1H),3.99(dd,J＝12.2,2.7Hz,1H),3.82(d,J＝2.9Hz,1H),3.79(d,J＝2.9Hz,1H),1.67(s,3H),0.93(s,9H),0.90(s,9H),0.20(s,3H),0.15(s,3H),0.09(s,6H).

¹³C NMR(101MHz,CDCl₃)δppm:172.90,82.96,66.87,59.84,25.75,25.62,21.04,18.22,17.95,‐4.21,‐5.12,‐5.38,‐5.48.

(3R,4R,5R) -4- ((tert-butyldimethylsilyl) oxy-5- (((tert-butyldimethylsilyl) oxy) methyl) -3-chloro-3-methyldihydrofuran-2-ol (15)

A mixture of 14a (2.6 g, 6.36 mmol) and THF (35 mL) was cooled to 0 deg.C, DIBAL-H (13.0 mL, 13.0 mmol) was added, followed by reaction at 0 deg.C for 40 minutes, slowly warmed to room temperature and stirred for an additional hour. After cooling to 0 ℃, the reaction was quenched by the addition of 1N hydrochloric acid solution (10 mL). Adding ethyl acetate (80mL) and water (10mL), washing the organic layer with 1N hydrochloric acid solution (10mL), saturated sodium bicarbonate solution (20mL) and saturated brine (30mL), drying over anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a crude product, separating by column chromatography, eluting with hexane/ethyl acetate (20/1-8/1) to obtain 2.30 g of compound 15 in total, and obtaining the final productThe rate was 88%.¹H NMR(400MHz,CDCl₃)δ(ppm):5.13(d,J＝7.6Hz,1H),4.33(d,J＝7.2Hz,1H),4.06(d,J＝7.2Hz,1H),3.83(dd,J＝11Hz,J＝1.8Hz,1H),3.65(d,J＝11Hz,1H),3.52(d,J＝11.4Hz,1H),1.62(s,3H,‐CH₃),0.94(s,18H),0.09and 0.14(2s,12H).

(3R,4R,5R) -4- ((tert-butyldimethylsilyl) oxy-5- (((tert-butyldimethylsilyl) oxy) methyl) -3-3-chloro-3-methyltetrahydrofuran-2-yl-benzoate (16)

Compound 15(2.3 g, 5.6 mmol) was dissolved in 25mL of dichloromethane and cooled to 0 deg.C, triethylamine (1.14mL,8.4mmol) was added, benzoyl chloride (0.94 g, 6.7 mmol) was slowly added dropwise, allowed to warm to room temperature slowly and stirring was continued overnight. The reaction was quenched by the addition of 60mL of dichloromethane and 5% sodium bicarbonate (10 mL). The organic layer was washed with saturated sodium bicarbonate solution (10mL) and saturated brine (20mL), respectively, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give a crude product which was isolated by column chromatography and eluted with hexane/ethyl acetate (50/1 to 10/1) to give compound 16 in a total of 2.39g with a yield of 83%.¹H NMR(400MHz,CDCl₃)δ(ppm):8.17(d,J＝8Hz,2H)，7.60(t,J＝15.4Hz,1H),7.46(t,J＝15.4Hz,2H),6.30(s,1H),4.21‐4.23(m,1H),4.13(d,J＝5.2Hz,1H),3.91(dd,J＝12Hz and 2.4Hz,1H),3.81(dd,J＝12Hz and 2.4Hz,1H),0.99(s,9H,‐CH₃),0.94(s,9H,‐CH₃),0.94(s,18H),0.11(s,3H),0.12(s,3H),0.15(s,3H),0.2(s,3H)；¹³C NMR(100MHz,CDCl₃)δ(ppm):165.21,133.33,130.17,129.75,128.33,100.21,86.82,75.06,72.96,61.38,26.78,25.83,25.68,18.40,18.14,‐4.40,‐4.60,‐5.28,‐5.52.

6.5.1- ((2R,3R,4R,5R) -3-chloro-4-hydroxy-5- (hydroxymethyl) -3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (18a)

To a 100ml reaction flask were added uracil (672 mg, 6.0 mmol), acetonitrile (30ml) and N, O-bis-methylacetamide (2.92 ml,12.0mmol) under nitrogen. Heating to 60 deg.C, stirring for 10min until the turbid liquid is clearThe reaction was cooled to room temperature and compound 16(1.55 g,3.0 mmol) was added followed by trimethylsilyl trifluoromethanesulfonate (2.16mL,12.0 mmol). The reaction was stirred at room temperature for half an hour and then heated to reflux for 16 hours, after cooling quenched with saturated sodium bicarbonate, the organic phase separated, washed with water, dried, concentrated, the residue 17a dissolved in tetrahydrofuran, tetrabutylammonium fluoride (1.0M solution in tetrahydrofuran, 12ml, 12 mmol) added and stirred at room temperature for 1.5 hours. After concentration, the residue was purified by column chromatography (dichloromethane/methanol 30:1 to 20:1) to give the product 18a as a white solid (240 mg, 29% overall yield).¹HNMR(400MHz,DMSO‐D6)δ(ppm):11.49(s,1H),8.18(d,J＝8.0Hz,1H),6.22(s,1H),5.97(d,J＝5.6Hz,1H),5.65(d,J＝8.0Hz,1H),5.43(s,1H),3.84–3.92(m,3H),3.67(dd,J＝11.8,3.1Hz,1H),1.44(s,3H)；LCMS‐ESI⁺(M/z):277.2(M + 1). By the same method, compound 16 is coupled with deuterated uracil to obtain C-5 and/or C-6 deuterated nucleoside compounds 18 b-18 d on the following bases.

18b:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.47(s,1H),8.17(s,1H),6.22(s,1H),5.97(d,J＝5.6Hz,1H),5.43(s,1H),3.84–3.92(m,3H),3.67(dd,J＝11.8,3.1Hz,1H),1.44(s,3H).

18c:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.49(s,1H),6.22(s,1H),5.97(d,J＝5.6Hz,1H),5.64(s,1H),5.42(s,1H),3.84–3.94(m,3H),3.67(m,1H),1.44(s,3H).

18d:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.49(s,1H),6.22(s,1H),5.97(d,J＝5.6Hz,1H),5.43(s,1H),3.86–3.92(m,3H),3.64‐3.68(m,1H),1.44(s,3H).

Example 7

7.1.1- ((2R,3R,4R,5R) -4- ((tert-butyldimethylsilyl) oxy) -3-chloro-5- (hydroxymethyl) -3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (20)

To nucleoside compound 19(5.3g,19.21mmol) were added pyridine (30mL) and dichloromethane (30mL), and the solution was cooled to 0 ℃. To the solution was added 4, 4' -dimethoxytrityl chloride (7.16g, 21.14mmol), and the mixture was stirred at 0 ℃ overnight. Methanol (5mL) was added to quench the reaction, the reaction was concentrated to dryness under reduced pressure, and ethyl acetate (300mL) and water (30mL) were added to the residue. The organic layer was washed with brine (40mL) and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (1 OOmL). Imidazole (3.92g, 57.5mmol) and tert-butyldimethylsilyl chloride (4.34g, 28.8mmol) were added to the solution. After the reaction mixture was stirred at room temperature overnight, methanol (5mL) was added, the mixture was stirred for ten minutes, the solvent was removed under reduced pressure, and ethyl acetate (300mL) and water (50mL) were added to the residue. The organic layer was separated, dried over sodium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (0-40% ethyl acetate in hexane) to give 5 '-oxo-dimethoxytrityl-3' -oxo-tert-butyldimethylsilyl intermediate product. A solution of 5% trichloroacetic acid in dichloromethane (80mL) was added and the mixture stirred at room temperature for 2 hours, then water (10mL) was added and stirring continued at room temperature for 1 hour, methanol (5mL) was added slowly and the solution stirred at room temperature for l hours. The pH of the solution was adjusted to 7 with ammonia. The organic layer was separated, dried over sodium sulfate, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (0-5% methanol in dichloromethane) to give product 20(4.11 g) as a white solid in 55% yield over three steps.¹HNMR(400MHz,Methanol‐d₄)δ8.33(d,J＝8.2Hz,1H),6.35(s,1H),5.70(d,J＝8.1Hz,1H),4.20(d,J＝8.8Hz,1H),4.02–3.99(m,2H),3.75(dd,J＝12.5,1.8Hz,1H),1.51(s,3H),0.95(s,9H),0.18(s,3H),0.15(s,3H)；LCMS‐ESI⁺(m/z):391.4(M+H)⁺.

(2S,3R,4R,5R) -3- ((tert-butyldimethylsilyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-carboxylic acid (21)

To compound 20(694mg,1.78mmol), bisacetoxyiodobenzene (1.38 g, 4.3mmol) and 2,2, 6, 6-tetramethyl-1-piperidinyloxy (69mg,0.43mmol) were added acetonitrile (4mL) and water (4mL), the mixture stirred at room temperature for 2h until the reaction was complete, the solution was cooled to 0 deg.C, 0.5N potassium hydroxide solution (30mL) was added, back-extracted with dichloromethane (30mL X3), the aqueous solution was neutralized to pH 2 with 2N HCl, filtered, washed with water and dried to give product 21(590mg) as a white solid in 85% yield. LCMS-ESI⁺(m/z):405.3(M+H)⁺。

7.3.1- ((2R,3R,4R,5R) -4- ((tert-butyldimethylsilyl) oxy) -3-chloro-5- (hydroxymethyl-d₂) -3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4- (1H,3H) -dione (22)

A mixture of compound 21(530mg, 1.31mmol) and THF (50mL) was cooled to 0 deg.C and methyl chloroformate (0.44mL) was slowly added dropwise followed by triethylamine (0.3mL, 2.2mmol) and the mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 ℃ and sodium deuteroborohydride (0.35g) was added in one portion, followed by slowly dropping heavy water at 0 ℃ until the sodium deuteroborohydride was dissolved. The reaction mixture was reacted at 0 ℃ until completion, the solvent was evaporated under reduced pressure, ethyl acetate (300mL) and water (50mL) were added to the residue, the organic layer was washed with a 1N hydrochloric acid solution (10mL), a saturated sodium bicarbonate solution (20mL) and a saturated saline (30mL), respectively, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (0-50% ethyl acetate in hexane) to give 22(0.35g) as a white solid product in a yield of 68%.¹H NMR(400MHz,Methanol‐d₄)δ8.33(d,J＝8.1Hz,1H),6.35(s,1H),4.19(d,J＝8.8Hz,1H),4.00(d,J＝8.8Hz,1H),1.51(s,3H),0.95(s,9H),0.18(s,3H),0.15(s,3H)；LCMS‐ESI⁺(m/z):393.4(M+H)⁺.

7.4. Isopropyl ((S) - ((((2R, 3R,4R,5R) -3- ((tert-butyldimethylsilyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -L-alanine ester (23)

To a 10mL reaction tube were added nucleoside 22(78.6mg,0.20mmol) and 1.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (0.6mL of a 1M solution in THF, 0.6mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 3(145mg, 0.32mmol) in 1mL THF at 0 ℃. The resulting clear reaction solution was warmed to 35 ℃ and stirred for 1 day. Adding saturated NH₄Cl (3mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (60 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with water (10mL), saturated NaHCO₃(2X1OmL), brine (1OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-5% methanol in dichloromethane) to give the product 23(81mg) as a white solid in a yield of 61%).¹H NMR(400MHz,CDCl₃)δ7.70(d,J＝8.1Hz,1H),7.36‐7.32(m,2H),7.24‐7.17(m,3H),6.42(s,1H),5.67(d,J＝8.1Hz,1H),5.03–4.99(m,1H),4.19‐4.16(m,1H),4.02‐3.96(m,3H),1.51(s,3H),1.37(d,J＝6.0Hz,3H),1.24(d,J＝6.2Hz,6H),0.94(s,9H),0.16(s,3H),0.15(s,3H)；³¹P NMR(162MHz,CDCl₃)δ2.13；LCMS‐ESI⁺(m/z):662.5(M+H)⁺.

7.5. Isopropyl ((S) - ((((2R, 3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -L-alanine ester (VI-1 a1)

A pre-cooled 90% aqueous trifluoroacetic acid solution (4mL) was added to compound 23(81mg, 0.122mmol) at 0 deg.CThe mixture was stirred at room temperature overnight to completion. The solvent was evaporated under reduced pressure, the residue was cooled to 0 ℃, neutralized with sodium hydrogencarbonate, and the mixture was extracted with ethyl acetate and water. The combined organic layers were dried over sodium sulfate and the resulting residue was concentrated and purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give the product VI-1 a1(39.5mg) as a white solid in 59% yield.¹H NMR(400MHz,CDCl₃)δ7.61(d,J＝8.2Hz,1H),7.39‐7.35(m,2H),7.24‐7.21(m,3H),6.43(s,1H),5.69(d,J＝8.2Hz,1H),5.05–5.01(m,1H),4.20‐4.17(m,1H),4.00‐3.92(m,3H),1.59(s,3H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.22；LCMS‐ESI⁺(m/z):548.5(M+H)⁺.

7.6. Isopropyl ((R) - ((((2R, 3R,4R,5R) -3- ((tert-butyldimethylsilyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -D-alanine ester (24)

To a 10mL reaction tube were added nucleoside 22(30mg,0.078mmol) and 0.5mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (0.25mL of a 1M solution in THF, 0.25mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 6(60mg, 0.132mmol) in 1mL THF at 0 ℃. The resulting clear reaction solution was warmed to 35 ℃ and stirred for 1 day. Adding saturated NH₄Cl (3mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (60 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with water (10mL), saturated NaHCO₃(2X1OmL), brine (1OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-5% methanol in dichloromethane) to give product 24 as a white solid (33.5mg, yield 65%).¹HNMR(400MHz,Methanol‐d₄)δ7.79(d,J＝8.1Hz,1H),7.41‐7.37(m,2H),7.29‐7.19(m,3H),6.40(s,1H),5.75(d,J＝8.1Hz,1H),4.97–4.93(m,1H),4.19‐4.16(m,1H),4.09–4.66(m,1H),3.89‐3.85(m,1H),1.51(s,3H),1.21(d,J＝7.2Hz,3H),1.21(d,J＝6.5Hz,3H),1.19(d,J＝6.6Hz,3H),0.95(s,9H),0.16(s,3H),0.12(s,3H)；³¹P NMR(162MHz,CDCl₃)δ3.04；LCMS‐ESI⁺(m/z):662.4(M+H)⁺.

7.7. Isopropyl ((R) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -D-alanine ester (VI-1 b2)

A pre-cooled 90% aqueous trifluoroacetic acid solution (2mL) was added to compound 24(33.5mg, 0.05mmol) at 0 deg.C, and the mixture was stirred at room temperature overnight to completion. The solvent was evaporated under reduced pressure, the residue was cooled to 0 ℃, neutralized with sodium hydrogencarbonate, and the mixture was extracted with ethyl acetate and water. The combined organic layers were dried over sodium sulfate and the resulting residue was concentrated and purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give product VI-1 b2 as a white solid (16.1mg, 58% yield).¹H NMR(400MHz,CDCl₃)δ8.95(brs,1H),7.47(d,J＝8.2Hz,1H),7.38‐7.34(m,2H),7.26‐7.18(m,3H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.17‐4.14(m,1H),4.06‐4.01(m,1H),3.95‐3.86(m,2H),1.51(s,3H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS‐ESI⁺(m/z):548.5(M+H)⁺.

7.8.1- ((2R,3R,4R,5R) -3-chloro-4-hydroxy-5- (hydroxymethyl-d)₂) -3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (25)

A solution of compound 22(51mg, 0.13mmol) in THF (10mL) was cooled to 0 ℃, tetrabutylammonium fluoride (0.1mL) was added dropwise, the mixture was stirred at room temperature for 2h until the reaction was complete, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give product 25(29.7mg) as a white solid in 82% yield. LCMS-ESI⁺(m/z):279.1(M+H)⁺.

Example 8.

Isopropyl-d₆- ((R) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine ester (IX-2 b2)

To a 10mL reaction tube were added nucleoside 18a (30mg,0.11mmol) and 0.7mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (0.15mL of a 1M solution in THF, 0.15mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 11a (60mg, 0.132mmol) in 1mL THF at 0 ℃. The resulting clear reaction solution was warmed to 35 ℃ and stirred for 1 day. Adding saturated NH₄Cl (3mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (60 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with water (10mL), saturated NaHCO₃(2X1OmL), brine (1OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-5% methanol in dichloromethane) to give the product IX-2 b2(27.5mg, yield 45%) as a white solid.¹H NMR(400MHz,CDCl₃)δ8.96(brs,1H),7.48(d,J＝8.2Hz,1H),7.39‐7.35(m,2H),7.26‐7.18(m,3H),6.42(s,1H),5.57(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.17‐4.14(m,1H),4.08‐4.03(m,1H),3.94‐3.88(m,2H),1.52(s,3H),1.38(d,J＝6.9Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.90；LCMS‐ESI⁺(m/z):552.4(M+H)⁺.

Example 9

(2R,3R,4R) -5-acetoxy-2- ((benzoyloxy) methyl) -4-chloro-4-methyltetrahydrofuran-3-yl-5-d-benzoate (28)

A mixture of 26(3.89 g, 10.0 mmol) and THF (45 mL) was cooled to 0 deg.C and Li (t-BuO) was added₃-D (21.0 mmoles, from LiAlD)₄And t-butanol) followed by reaction at 0 ℃ for 20 minutes, slowly warmed to room temperature and stirred for three hours. After cooling to 0 ℃, the reaction was quenched by addition of saturated ammonium chloride solution (10 mL). Ethyl acetate (80mL) and water (10mL) were added to the reaction solution, and the organic layer was washed with ammonium chloride solution (10mL), saturated sodium bicarbonate solution (20mL) and saturated brine (30mL), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. To the resulting crude product 27, 35ml of pyridine and 5ml of acetic anhydride were added at zero degrees, and stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was subjected to column chromatography with hexane/ethyl acetate (50/1 to 10/1) to afford compound 28 in a total of 3.64 g, 84% yield.

((2R,3R,4R,5R) -3- (benzoyloxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl-5-d) benzoate (30)

HMDS (2.25 g, 14mmol) is added to a solution of N-benzoylcytosine (3 g, 14mmol), ammonium sulfate (40 mg, 0.3 mmol) in isopropyl acetate (32 ml) at reflux temperature over 30-60 minutes, the reaction is refluxed for 4-5 hours until the turbid solution is clear, the solution is concentrated under reduced pressure to about 10ml, and then addedAfter a solution of compound 28(3.03 g, 7mmol) in isopropyl acetate (100mL) was added and the mixture was concentrated again, the residue was dissolved in dichloroethane (40mL), tin tetrachloride (4.6g,17mmol) was added and the reaction mixture was reacted at 75-80 ℃ for 24 hours. Cooling the reaction solution, adding acetic acid water to quench, separating organic phase, washing, drying, concentrating, dissolving the crude product 29 in 5ml 80% acetic acid water solution, stirring the reaction solution at 110 deg.C for 24 hr, concentrating, evaporating to remove solvent, and purifying by column chromatography (dichloromethane: methanol 20:1) to obtain white solid 30(0.81 g, 24%), LCMS-ESI⁺(m/z):486.1(M+H)⁺。

9.3.1- ((2R,3R,4R,5R) -3-chloro-4-hydroxy-5- (hydroxymethyl) -3-methyltetrahydrofuran-2-yl-2-d) pyrimidine-2, 4(1H,3H) -dione (31)

Deuterated nucleoside compound 30(0.8 g, 1.65 mmol) was dissolved in 20ml of methanolic ammonia solution, stirred at room temperature overnight, concentrated to remove the solvent and purified by column chromatography (dichloromethane: methanol 20:1 to 10:1) to obtain 31(397 mg, yield 87%) as a white solid. LCMS-ESI⁺(m/z):278.2(M+H)⁺。

9.4. Isopropyl ((S) - ((((2R, 3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-5-d) methoxy) (phenoxy) phosphoryl) -L-alanine ester (I-1 a1)

By the preparation method of the compound IX-2 b2, the deuterated nucleoside compound 31 and the phosphoramidate compound 3 are coupled under the action of a tert-butyl magnesium chloride Grignard reagent to obtain a white solid product I-1 a 1. LCMS-ESI (M/z):547.3(M + H)⁺。¹H NMR(400MHz,CDCl₃)δ7.62(d,J＝8.2Hz,1H),7.40‐7.34(m,2H),7.24‐7.20(m,3H),5.70(d,J＝8.2Hz,1H),5.05–5.00(m,1H),4.20‐4.18(m,1H),4.00‐3.90(m,3H),1.60(s,3H),1.37(d,J＝6.1Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.23.

Likewise, we also prepared the following respective 1' -deuterated nucleoside compounds:

LCMS‐ESI⁺(m/z):547.3(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.60(d,J＝8.2Hz,1H),7.20‐7.40(m,5H),5.70(d,J＝8.2Hz,1H),4.20‐4.17(m,1H),4.00‐3.88(m,3H),1.60(s,3H),1.25‐1.36(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.23.

LCMS‐ESI⁺(m/z):553.3(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.62(d,J＝8.2Hz,1H),7.41‐7.35(m,2H),7.26‐7.20(m,3H),5.70(d,J＝8.2Hz,1H),5.03(s,1H),4.20‐4.18(m,1H),4.00‐3.90(m,3H),1.60(s,3H),1.37(d,J＝6.1Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.24.

LCMS‐ESI⁺(m/z):552.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.62(d,J＝8.2Hz,1H),5.70(d,J＝8.2Hz,1H),5.05–5.00(m,1H),4.20‐4.14(m,1H),4.00‐3.90(m,3H),1.60(s,3H),1.20‐1.37(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.23.

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.61(s,1H),7.40‐7.34(m,2H),7.24‐7.20(m,3H),5.05–5.00(m,1H),4.20‐4.18(m,1H),4.00‐3.90(m,3H),1.60(s,3H),1.37(d,J＝6.1Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.22.

LCMS‐ESI⁺(m/z):548.3(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.20‐7.42(m,5H),5.70(s,1H),5.05–5.00(m,1H),4.20‐4.18(m,1H),4.00‐3.90(m,3H),1.60(s,3H),1.20‐1.43(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.23.

LCMS‐ESI⁺(m/z):549.3(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.40‐7.33(m,2H),7.26‐7.20(m,3H),5.05–5.00(m,1H),4.20‐4.18(m,1H),4.00‐3.90(m,3H),1.60(s,3H),1.19‐1.40(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.24.

9.5. isopropyl ((R) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-5-D) methoxy) (phenoxy) phosphoryl) -D-alanine ester (I-1 b1)

By adopting the preparation method of the compound IX-2 b2, the deuterated nucleoside compound 31 and the phosphoramidate compound 6 are coupled under the action of a tert-butyl magnesium chloride Grignard reagent to obtain a white solid product I-1 b2 which is LCMS-ESI⁺(m/z)547.2(M+H)⁺；¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.46(d,J＝8.2Hz,1H),7.39‐7.33(m,2H),7.25‐7.18(m,3H),5.55(d,J＝8.2Hz,1H),5.05–5.00(m,1H),4.42‐4.60(m,2H),4.18‐4.15(m,1H),3.65‐3.96(m,3H),1.57(s,3H),1.25‐1.37(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.93.

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.46(d,J＝8.2Hz,1H),7.19‐7.39(m,5H),5.55(d,J＝8.2Hz,1H),4.39‐4.53(m,2H),4.18‐4.15(m,1H),3.67‐3.96(m,4H),1.45(s,3H),1.20‐1.37(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.93.

LCMS‐ESI⁺(m/z):553.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.90(s,1H),7.48(d,J＝8.2Hz,1H),7.39‐7.30(m,2H),7.25‐7.18(m,3H),5.57(d,J＝8.2Hz,1H),5.05(s,1H),4.40‐4.55(m,2H),4.18‐4.15(m,1H),3.65‐3.99(m,4H),1.51(s,3H),1.37(d,J＝6.8Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.63.

LCMS‐ESI⁺(m/z):552.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.46(d,J＝8.2Hz,1H),5.55(d,J＝8.2Hz,1H),5.05–5.00(m,1H),4.36‐4.55(m,2H),4.18‐4.12(m,1H),3.65–4.01(m,4H),1.51(s,3H),1.25‐1.37(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.96.

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.80(s,1H),7.46(d,1H),7.34‐7.36(d,2H),7.22‐7.26(t,3H),5.57(d,1H),5.05(m,1H),4.39‐4.53(m,2H),4.16‐4.18(m,1H),3.69‐4.06(m,4H),1.51(s,3H),1.37(d,3H),1.26(d,3H x2)；³¹P NMR(162MHz,CDCl₃)δ3.62.

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.45(s,1H),7.40‐7.30(m,2H),7.25‐7.16(m,3H),5.05–5.00(m,1H),4.34‐4.58(m,2),4.18‐4.15(m,1H),3.65–4.06(m,4H),1.51(s,3H),1.25‐1.37(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.93.

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.39‐7.33(m,2H),7.25‐7.18(m,3H),5.56(s,1H),5.05–5.00(m,1H),4.36‐4.60(m,2H),4.18‐4.15(m,1H),3.65–4.06(m,4H),1.51(s,3H),1.24‐1.37(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.93.

LCMS‐ESI⁺(m/z):549.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.39‐7.33(m,2H),7.25‐7.18(m,3H),5.05–5.00(m,1H),4.36‐4.60(m,2H),4.18‐4.15(m,1H),3.65–4.01(m,4H),1.51(s,3H),1.26‐1.37(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.93.

example 10

Isopropyl ((R) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3)4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-5-D) methoxy) (phenoxy) phosphoryl) -D-alanine ester-D₁(VII‐1b2)

By adopting the preparation method of the compound IX-2 b2, the deuterated nucleoside compound 18b and the deuterated phosphoramidate compound 11c are respectively coupled under the action of a tert-butyl magnesium chloride Grignard reagent to obtain a white solid product VII-1 b2. LCMS-ESI⁺(m/z):548.2(M+H)⁺；¹H NMR(400MHz,CDCl₃)δ7.60(s,1H),7.39‐7.35(m,2H),7.24‐7.21(m,3H),6.43(s,1H),4.37‐4.52(m,2H),4.10–4.16(m,1H),3.66‐3.96(m，4H),1.59(s,3H),1.25‐1.36(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.32；

Similarly, we also prepared the following C-5 deuterated nucleoside compounds for each base:

LCMS‐ESI⁺(m/z):553.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.60(s,1H),7.39‐7.35(m,2H),7.24‐7.21(m,3H),6.43(s,1H),5.03(s,1H),4.37‐4.52(m,2H),4.13–4.17(m,1H),3.66‐3.96(m,4H),1.59(s,3H),1.36(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.32；

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.60(s,1H),7.39‐7.35(m,2H),7.24‐7.21(m,3H),6.43(s,1H),4.42‐4.50(m,2H),4.17–4.20(m,1H),3.69‐3.96(m，3H),1.59(s,3H),1.24‐1.36(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.47；

LCMS‐ESI⁺(m/z):553.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.60(s,1H),7.39‐7.35(m,2H),7.24‐7.21(m,3H),6.43(s,1H),5.03(s,1H),4.37‐4.59(m,2H),4.19–4.15(m,1H),3.66‐3.99(m,4H),1.59(s,3H),1.36(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.47。

LCMS‐ESI⁺(m/z):552.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ7.60(s,1H),6.43(s,1H),5.00‐5.05(m，1H),4.37‐4.52(m,2H),4.10–4.02(m,1H),3.66‐3.96(m,4H),1.59(s,3H),1.25‐1.36(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.34。

example 11

Isopropyl ((R) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-6-D) methoxy) (phenoxy) phosphoryl) -D-alaninate-D₁

By adopting the preparation method of the compound IX-2 b2, the deuterated nucleoside compound 18c and the deuterated phosphoramidate compound are respectively coupled under the action of a tert-butyl magnesium chloride Grignard reagent to obtain a white solid product VII-2 b2 and LCMS-ESI⁺(m/z)548.2(M+H)⁺；¹H NMR(400MHz,CDCl₃)δ8.06(brs,1H),7.39‐7.35(m,2H),7.24‐7.21(m,3H),6.43(s,1H),5.56(s,1H),4.37‐4.52(m,2H),4.10–4.02(m,1H),3.66‐3.96(m,4H),1.59(s,3H),1.24‐1.36(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.32；

Similarly, we also prepared the following C-6 deuterated nucleoside compounds for each base:

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.20‐7.39(m,5H),6.43(s,1H),5.68(s,1H),4.42‐4.50(m,2H),4.17–4.20(m,1H),3.69‐3.96(m，3H),1.59(s,3H),1.22‐1.36(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.48；

LCMS‐ESI⁺(m/z):553.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.04(brs,1H),7.20‐7.37(m,5H),6.44(s,1H),5.67(s,1H),5.03(s,1H),4.42‐4.50(m,2H),4.17–4.20(m,1H),3.69‐3.96(m，3H),1.57(s,3H),1.35(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.48；

LCMS‐ESI⁺(m/z):548.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ6.43(s,1H),5.56(s,1H),5.00‐5.04(m,1H),4.37‐4.52(m,2H),4.16–4.08(m,1H),3.66‐3.96(m,4H),1.59(s,3H),1.22‐1.36(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.32；

example 12

Isopropyl ((R) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidine-1 (2H)-yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-5, 6-d₂) Methoxy) (phenoxy) phosphoryl) -D-alanine ester-D₁(VII‐3b2)

By adopting the preparation method of the compound IX-2 b2, the deuterated nucleoside compound 18d and the deuterated phosphoramidate compound are respectively coupled under the action of a tert-butyl magnesium chloride Grignard reagent, and a white solid product VII-3 b2 can be obtained. LCMS-ESI⁺(m/z)549.2(M+H)⁺；¹H NMR(400MHz,CDCl₃)δ8.06(brs,1H),7.20‐7.39(m,5H),6.43(s,1H),5.00‐5.04(m,1H),4.37‐4.52(m,2H),4.16‐4.10(m,1H),3.66‐3.96(m，4H),1.59(s,3H),1.21‐1.39(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.33；

LCMS‐ESI⁺(m/z):549.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.06(brs,1H),7.20‐7.39(m,5H),6.43(s,1H),4.42‐4.50(m,2H),4.17–4.20(m,1H),3.69‐3.96(m,3H),1.59(s,3H),1.22‐1.37(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.48；

LCMS‐ESI⁺(m/z):554.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ8.06(brs,1H),7.20‐7.39(m,5H),6.43(s,1H),5.02(s,1H),4.37‐4.52(m,2H),4.16‐4.10(m,1H),3.65‐3.94(m,3H),1.59(s,3H),1.35(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.32；

LCMS‐ESI⁺(m/z):553.2(M+H)⁺。¹H NMR(400MHz,CDCl₃)δ6.43(s,1H),5.00‐5.04(m,1H),4.37‐4.52(m,2H),4.16‐4.12(m,1H),3.66‐3.96(m,3H),1.59(s,3H),1.21‐1.39(m,9H)；³¹P NMR(162MHz,CDCl₃)δ3.33；

example 13

Preparation of 1- ((6aR,8R) -9-hydroxy-2, 2,4, 4-tetraisopropyltetrahydro-6H-furan [3, 2-f ] [1, 3,5,2,4] trioxasin-8-yl) pyrimidine-2, 4(1H,3H) -dione (32a)

Uridine 31a (10.0g, 41mmol) and anhydrous pyridine (50mL) were co-evaporated to dryness and resuspended in anhydrous pyridine (60 mL). To this stirred fine suspension was added 1, 3-dichloro-1, 1,3, 3-tetraisopropyl disiloxane (13.5g, 48.3mmol) internally at ambient temperature. After stirring the fine suspension at ambient temperature for 14h, it was quenched by addition of methanol (3mL) and then concentrated under reduced pressure. The residue was partitioned between ethyl acetate (150mL) and water (200 mL). The organic layer was further washed with 5% hydrochloric acid (2 × 100mL), brine (50mL), dried over solid sodium sulfate, filtered, and concentrated under reduced pressure to the crude product. Column chromatography (20-50% ethyl acetate-hexanes). This gave 32a as a white foamy solid, 14.5g (75%).¹H NMR(400MHz,CDCl₃)δppm:1.10‐1.19[m,28H,4X‐CH(CH₃)₂],4.38‐4.69(m,6H,2’‐H,3’‐H,4’‐H,5’‐H；and 2’‐OH),5.95‐6.05(m,2H,5‐H and 1’‐H),8.05(d,1H,6‐H),10.25(s,1H,‐NH).LCMS‐ESI⁺(m/z):487.2(M+H)⁺

In the same manner, compounds 32b, 32c and 32d were obtained.

32b:¹H NMR(400MHz,CDCl₃)δppm:1.10‐1.20[m,28H,4X‐CH(CH₃)₂],4.40‐4.70(m,6H,2’‐H,3’‐H,4’‐H,5’‐H；and 2’‐OH),5.95‐6.04(m,1H,1’‐H),8.05(d,1H,6‐H),10.30(s,1H,‐NH)；LCMS‐ESI(m/z):488.2(M+H)⁺

32c:¹H NMR(400MHz,CDCl₃)δppm:1.08‐1.20[m,28H,4X‐CH(CH₃)₂],4.39‐4.71(m,6H,2’‐H,3’‐H,4’‐H,5’‐H；and 2’‐OH),5.95‐6.05(m,2H,5‐H and 1’‐H),10.26(s,1H,‐NH)；LCMS‐ESI(m/z):488.2(M+H)⁺

32d:¹H NMR(400MHz,CDCl₃)δppm:1.11‐1.21[m,28H,4X‐CH(CH₃)₂],4.38‐4.71(m,6H,2’‐H,3’‐H,4’‐H,5’‐H；and 2’‐OH),5.97‐6.06(m,2H,5‐H and 1’‐H),10.27(s,1H,‐NH)；LCMS‐ESI(m/z):489.2(M+H)⁺.

Preparation of 1- ((6aR,8R, 9aR) -2, 2,4, 4-tetraisopropyl-9-oxotetrahydro-6H-furan- [3, 2-f ] [1, 3,5,2,4] trioxasid-8-yl) pyrimidine-2, 4(1H,3H) -dione (33a)

To a dry three-neck round bottom flask were added anhydrous dichloromethane (60mL) and dimethyl sulfoxide (3.1g, 39.4 mmol). The solution was cooled to-78 ℃ in a dry ice/acetone bath under a nitrogen atmosphere. Trifluoroacetic anhydride (7.8g, 37mmol) was added via syringe over 10min to give a turbid mixture. Uridine derivative II-22 a (14.5g, 3) was added dropwise at-78 deg.C0mmol) in dichloromethane (60 mL). Is uniformly mixed inStirring was continued for 1.5h, then triethylamine (9.0mL) was added dropwise, and after stirring at low temperature for 1h, the cooling bath was removed and the reaction mixture was slowly heated to ambient temperature. The reaction was quenched by the addition of saturated aqueous ammonium chloride (18 mL). Water (20mL) was added and the organic layer was separated. The aqueous layer was extracted again with dichloromethane. The combined organic layers were washed with water, brine and dried over sodium sulfate. The crude oily product residue was purified by column chromatography (15-60% ethyl acetate/hexanes) to give 33a as a yellow orange solid, 13.2g (91% yield).¹H NMR(400MHz,CDCl₃)δppm:0.96‐1.15[m,28H,4X‐CH(CH₃)₂],4.02‐4.17(m,3H,4’‐H,5’‐H),4.95‐5.05(m,2H,3’‐H and1’‐H),5.70(d,1H,5‐H),7.17(d,1H,6‐H),9.1(s,1H,‐NH).

In the same manner, compounds 33b, 33c and 33d were obtained.

33b：¹H NMR(400MHz,CDCl₃)δppm:0.99‐1.17[m,28H,4X‐CH(CH₃)₂],4.03‐4.16(m,3H,4’‐H,5’‐H),4.96‐5.05(m,2H,3’‐H and 1’‐H),7.15(d,1H,6‐H),9.2(s,1H,‐NH).

33c：¹H NMR(400MHz,CDCl₃)δppm:0.97‐1.17[m,28H,4X‐CH(CH₃)₂],4.01‐4.17(m,3H,4’‐H,5’‐H),4.94‐5.07(m,2H,3’‐H and 1’‐H),5.65(d,1H,5‐H),9.15(s,1H,‐NH).

33d：¹H NMR(400MHz,CDCl₃)δppm:0.95‐1.16[m,28H,4X‐CH(CH₃)₂],4.04‐4.20(m,3H,4’‐H,5’‐H),4.91‐5.04(m,2H,3’‐H and 1’‐H),9.25(s,1H,‐NH).

Preparation of 1- ((6aR,8R, 9aR) -2, 2,4, 4-tetraisopropyl-9-methylenetetrahydro-6H-furan- [3, 2-f ] [1, 3,5,2,4] trioxasin-8-yl) pyrimidine-2, 4(1H,3H) -dione (34a)

To 12ml of dimethyl sulfoxide was added sodium hydride (0.4g, 60%, 10mmol), heated to 65 ℃, stirred until the sodium hydride was completely dissolved, cooled to room temperature, and triphenylphosphine methyl bromide (4.0g,11mmol) was added with stirring. After 45 min, compound 33a (1.4g,2.8mmol) was added and the mixture was heated to 50 ℃ and stirred for 1h, followed by removal of the solvent under reduced pressure. To the residue was added 80ml of water, the pH was adjusted to neutral with acetic acid, extracted three times with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure, and the residue was purified by column chromatography (10-40% ethyl acetate/hexane) to obtain compound 34a,0.91g (yield 67%).¹H NMR(400MHz,CDCl₃)δppm:0.81‐1.11[m,28H,4X‐CH(CH₃)₂],3.60(m,1H)4.02(m,2H,5’‐H),4.75(d,1H),5.38(m,2H),5.62(d,1H,5‐H),7.30(d,1H,6‐H),9.70(s,1H,‐NH).

In the same manner, compounds 34b, 34c and 34d were obtained.

34b:¹H NMR(400MHz,CDCl₃)δppm:0.83‐1.12[m,28H,4X‐CH(CH₃)₂],3.62(m,1H)4.03(m,2H,5’‐H),4.75(d,1H),5.40(m,2H),7.32(d,1H,6‐H),9.66(s,1H,‐NH).

34c:¹H NMR(400MHz,CDCl₃)δppm:0.83‐1.13[m,28H,4X‐CH(CH₃)₂],3.60(m,1H)4.04(m,2H,5’‐H),4.75(d,1H),5.39(m,2H),5.60(d,1H,5‐H),9.72(s,1H,‐NH).

34d:¹H NMR(400MHz,CDCl₃)δppm:0.82‐1.12[m,28H,4X‐CH(CH₃)₂],3.61(m,1H)4.01(m,2H,5’‐H),4.76(d,1H),5.41(m,2H),9.73(s,1H,‐NH).

13.41- ((2R,5R) -4- ((tert-butyldimethylsilyl) oxy) -5- (((tert-butyldimethylsilyl) oxy) methyl) -3-methylenetetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (36a)

Compound 34a (4.82g,10mmol) was dissolved in 50mL of tetrahydrofuran, and 25mL of tetrabutylammonium fluoride solution (1.0M,25mmol) was slowly added dropwise to the solution to effect reactionThe mixture was stirred at room temperature for 30 minutes, the solvent was distilled off under reduced pressure, and then the mixture was placed on a funnel containing silica gel and rinsed with 100ml (dichloromethane-methanol, 20: 1). After concentration to dryness, 40ml DMF was added and cooled to zero, 3.4g (50mmol) imidazole and 3.75 g (25 mmol) tert-butyldimethylsilylchloride were added, stirred overnight, 100ml water was added, extracted three times with ethyl acetate, the combined organic phases were washed twice with water, dried over anhydrous sodium sulfate, filtered, the solvent was removed, and dried under vacuum to give crude 36a, 3.41 g (71%) which was used directly in the next reaction. LCMS-ESI⁺(m/z):469.2(M+H)⁺.

In the same manner, compounds 36b, 36c and 36d were obtained.

36b:LCMS‐ESI⁺(m/z):470.2(M+H)⁺.

36c:LCMS‐ESI⁺(m/z):470.2(M+H)⁺.

36d:LCMS‐ESI⁺(m/z):471.2(M+H)⁺.

13.51- ((2R,3R,5R) -4- ((tert-butyldimethylsilyl) oxy) -5- (((tert-butyldimethylsilyl) oxy) methyl) -3-chloro-3-methyl-d) -tetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (39a)

Compound 36a (2.34 g, 5mmol), 37(107 mg, 0.18mmol) and 2.5 g (13.1 mmol) of p-toluenesulfonyl chloride were dissolved in 15ml of dioxane, stirred for 10 minutes, and then trideuteridophenylsilane (0.65 g, 6mmol, prepared according to the methods of Tetrahedron,70(19), 3185-3190; 2014) dissolved in 5ml of ethanol was added dropwise. After the reaction mixture was stirred for 5 hours, 100ml of ethyl acetate was added, washed twice with 30ml of brine, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated, the residue was dissolved in tetrahydrofuran, tetrabutylammonium fluoride (1.0M tetrahydrofuran solution, 12ml, 12 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After concentration, the residue was purified by column chromatography (dichloromethane/methanol 30:1 to 20:1) to give the product 39a as a white solid (222 mg, 16% overall yield).¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.51(s,1H),8.19(d,J＝8.0Hz,1H),6.22(s,1H),5.98(d,J＝5.6Hz,1H),5.67(d,J＝8.0Hz,1H),5.45(s,1H),3.85–3.93(m,3H),3.66(dd,J＝11.8,3.1Hz,1H),1.42(s,2H)；LCMS‐ESI⁺(m/z):278.2(M+1)。

In the same manner, compounds 39b, 39c and 39d were obtained.

39b:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.48(s,1H),8.20(d,J＝8.0Hz,1H),6.24(s,1H),5.96(d,J＝5.6Hz,1H),5.46(s,1H),3.81–3.93(m,3H),3.67(dd,J＝11.8,3.1Hz,1H),1.43(s,2H)；LCMS‐ESI⁺(m/z):279.2(M+H)⁺.

39c:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.52(s,1H),6.21(s,1H),5.99(d,J＝5.6Hz,1H),5.65(d,J＝8.0Hz,1H),5.44(s,1H),3.86–3.92(m,3H),3.64(dd,J＝11.8,3.1Hz,1H),1.43(s,2H).LCMS‐ESI⁺(m/z):279.2(M+H)⁺.

39d:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.48(s,1H),6.24(s,1H),6.0(d,J＝5.6Hz,1H),5.44(s,1H),3.85–3.94(m,3H),3.64(dd,J＝11.8,3.2Hz,1H),1.41(s,2H)；LCMS‐ESI⁺(m/z):280.2(M+H)⁺.

Example 14

14.1 preparation of l- ((2R, 3S, 4R,5R) -3, 4-dihydroxy-5- (hydroxymethyl) -3-¹³C-Perdeuteromethyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (40a)

Metallic magnesium (0.8g, 33 mmol) was washed with 5% aqueous hydrochloric acid and dried, and then placed in a three-necked round bottom flask equipped with a condenser. The flask was filled with argon and dry ether (20mL) was added. To the slow addition of deuterium-depleted methyl iodide (3.9g, 27 mmol), which was addedAn exothermic reaction occurs. After the reaction mixture was cooled, the supernatant was transferred to a solution of dry compound 33a (2.43g, 5mmol) in dry tetrahydrofuran (40ml) at-50 ℃. The temperature was raised to-40 ℃ and the mixture was stirred at-40 to-25 ℃ for 6 hours. After completion of the reaction, the mixture was diluted with 200mL of ethyl acetate at-50 ℃ and then brine (50mL) was slowly added. The organic layer was separated, then washed with a saturated aqueous solution of ammonium chloride, and dried over sodium sulfate. After filtration and concentration under reduced pressure, the residue was dissolved in 50ml of methanol. Ammonium fluoride (2.4g) and tetrabutylammonium fluoride (80mg) were added. The resulting mixture was stirred at 90 ℃ for 12h, concentrated under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (methanol: dichloromethane ═ 1:30 to 1:10) to give compound 40a (503 mg, 39%) as a white solid.¹H NMR(400MHz,DMSO‐d₆)δ(ppm):11.21(s，lH，NH)，7.67(d，lH，J＝8.4Hz，H‐6)，5.74(d，lH，J＝2.4Hz，H‐l’)，5.6(d，1H，J＝8Hz，H‐5)，5.5(d，1H，J＝5.0Hz，3’‐OH)，5.25(d，1H，J＝2.4Hz,2’‐OH),5.15(t,1H,J＝5.6Hz,5'‐OH),3.7‐3.6(m,4H).LCMS‐ESI⁺(m/z):262.2(M+H)⁺.

In the same manner, compounds 40b, 40c and 40d were obtained.

40b:¹H NMR(400MHz,DMSO‐d₆)δ(ppm):11.23(s，lH，NH)，7.70(d，lH，J＝8.4Hz，H‐6)，5.71(d，lH，J＝2.4Hz，H‐l’)，5.51(d，1H，J＝5.0Hz，3’‐OH)，5.25(d，1H，J＝2.4Hz,2’‐OH),5.16(t,1H,J＝5.6Hz,5'‐OH),3.68‐3.61(m,4H).LCMS‐ESI⁺(m/z):263.2(M+H)⁺.

40c:¹H NMR(400MHz,DMSO‐d₆)δ(ppm):11.22(s，lH，NH)，5.68(d，lH，J＝2.4Hz，H‐l’)，5.62(d，1H，J＝8Hz，H‐5)，5.51(d，1H，J＝5.0Hz，3’‐OH)，5.30(d，1H，J＝2.4Hz,2’‐OH),5.14(t,1H,J＝5.6Hz,5'‐OH),3.71‐3.63(m,4H).LCMS‐ESI⁺(m/z):263.2(M+H)⁺.

40d:¹H NMR(400MHz,DMSO‐d₆)δ(ppm):11.19(s，lH，NH),5.76(d，lH，J＝2.4Hz，H‐l’)，5.52(d，1H，J＝5.0Hz，3’‐OH)，5.30(d，1H，J＝2.4Hz,2’‐OH),5.21(t,1H,J＝5.6Hz,5'‐OH),3.71‐3.62(m,4H).LCMS‐ESI⁺(m/z):264.2(M+H)⁺.

14.2 preparation of ((2R,3R, 4S,5R) -3-acetoxy-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1- (2H) -yl) -4-hydroxy-4-per-deuteromethyltetrahydrofuran-2-yl) methyl acetate (41a)

To a solution of compound 40a (1.29 g, 5mmol) in anhydrous pyridine (25mL) was added acetic anhydride (2mL) at ambient temperature. The resulting mixture was stirred at ambient temperature overnight, diluted with ethyl acetate, washed with water and dried over anhydrous sodium sulfate. After filtration and concentration, the residue was purified by column chromatography (methanol: dichloromethane ═ 1:40 to 1:10) to give compound 41a (188 mg, 55%) as a white solid. LCMS-ESI⁺(m/z):346.2(M+H)⁺.

In the same manner, compounds 41b, 41c and 41d were obtained.

41b:LCMS‐ESI⁺(m/z):347.2(M+H)⁺.

41c:LCMS‐ESI⁺(m/z):347.2(M+H)⁺.

41d:LCMS‐ESI⁺(m/z):348.2(M+H)⁺.

14.3 preparation of ((2R,3R,4R,5R) -3-acetoxy-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1- (2H) -yl) -3-chloro-4-hydroxy-5- (hydroxymethyl) 3-deuteromethyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (43a)

To compound 41a (172 mg,0.5mmol) was dissolved in 5ml of anhydrous tetrahydrofuran and cooled to zero degrees, anhydrous hydrogen chloride gas was slowly bubbled through for about one hour. After evaporation of the solvent, 3ml of methanol and 1ml of n-butylamine were added. The resulting mixture was stirred at room temperature overnight, concentrated under reduced pressure, and the residue was purified by column chromatography (methanol: dichloromethane ═ 1:20 to 1)10) to give compound 43a (60mg, 43%) as a white solid.¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.49(s,1H),8.18(d,J＝8.0Hz,1H),6.22(s,1H),5.97(d,J＝5.6Hz,1H),5.65(d,J＝8.0Hz,1H),5.43(s,1H),3.84–3.92(m,3H),3.67(dd,J＝11.8,3.1Hz,1H).LCMS‐ESI⁺(m/z):280.2(M+1)

In the same manner, compounds 43b, 43c and 43d were obtained.

43b:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.51(s,1H),8.21(d,J＝8.0Hz,1H),6.23(s,1H),5.98(d,J＝5.6Hz,1H),5.42(s,1H),3.84–3.93(m,3H),3.68(dd,J＝11.8,3.1Hz,1H)；LCMS‐ESI⁺(m/z):281.2(M+H)⁺.

43c:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.49(s,1H),6.22(s,1H),5.97(d,J＝5.6Hz,1H),5.65(d,J＝8.0Hz,1H),5.43(s,1H),3.84–3.92(m,3H),3.67(dd,J＝11.8,3.1Hz,1H)；LCMS‐ESI⁺(m/z):281.2(M+H)⁺.

43d:¹H NMR(400MHz,DMSO‐D6)δ(ppm):11.48(s,1H),6.23(s,1H),6.0(d,J＝5.6Hz,1H),5.45(s,1H),3.84–3.94(m,3H),3.65(dd,J＝11.6,3.2Hz,1H)；LCMS‐ESI⁺(m/z):282.2(M+H)⁺.

By using the preparation method of the compound IX-2 b2, the deuterated nucleoside compounds 39a and 43a are respectively coupled with phosphoramidate compounds 3, 6 or 11 a-g under the action of a tert-butyl magnesium chloride Grignard reagent, and the corresponding 13 deuterated nucleoside phosphoramidates can be obtained.

1H NMR(400MHz,CDCl3)δ7.61(d,J＝8.2Hz,1H),7.39-7.35(m,2H),7.24-7.21(m,3H),6.43(s,1H),5.69(d,J＝8.2Hz,1H),5.05–5.01(m,1H),4.55-4.32(m,2H),4.20-4.17(m,1H),4.00-3.92(m,3H),1.59(s,2H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；31PNMR(162MHz,CDCl3)δ3.25；LCMS-ESI+(m/z):547.2(M+H)+.

¹H NMR(400MHz,CDCl₃)δ8.99(brs,1H),7.47(d,J＝8.2Hz,1H),7.38-7.34(m,2H),7.26-7.18(m,3H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.51(s,2H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS-ESI⁺(m/z):547.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.91(brs,1H),7.43(d,J＝8.2Hz,1H),7.35-7.32(m,2H),7.24-7.15(m,3H),6.36(s,1H),5.54(d,J＝8.2Hz,1H),5.04–4.95(m,1H),4.50-4.39(m,2H),4.14-4.10(m,1H),4.04-4.00(m,1H),3.93-3.83(m,2H),1.50(s,2H),1.35(d,J＝6.9Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.92；LCMS-ESI⁺(m/z):553.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.95(brs,1H),7.51(d,J＝8.2Hz,1H),7.41-7.38(m,2H),7.26-7.18(m,3H),6.40(s,1H),5.59(d,J＝8.2Hz,1H),4.57-4.41(m,2H),4.21-4.17(m,1H),4.1-4.11(m,1H),3.99-3.86(m,2H),1.55(s,2H),1.39(d,J＝6.9Hz,3H),1.26(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS-ESI⁺(m/z):548.5(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.02(brs,1H),7.47(d,J＝8.2Hz,1H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.51(s,2H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS-ESI⁺(m/z):552.5(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.0(brs,1H),7.47(d,J＝8.2Hz,1H),7.38-7.34(m,2H),7.26-7.18(m,2H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.51(s,2H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS-ESI⁺(m/z):548.5(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ7.61(d,J＝8.2Hz,1H),7.39-7.35(m,2H),7.24-7.21(m,3H),6.43(s,1H),5.69(d,J＝8.2Hz,1H),5.05–5.01(m,1H),4.55-4.32(m,2H),4.20-4.17(m,1H),4.00-3.92(m,3H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.32；LCMS-ESI⁺(m/z):549.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.88(brs,1H),7.44(d,J＝8.2Hz,1H),7.38-7.34(m,2H),7.26-7.18(m,3H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS-ESI⁺(m/z):549.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.99(brs,1H),7.47(d,J＝8.2Hz,1H),7.38-7.34(m,2H),7.26-7.18(m,3H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.37(d,J＝6.9Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.92；LCMS-ESI⁺(m/z):555.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ7.61(d,J＝8.2Hz,1H),7.39-7.35(m,2H),7.24-7.21(m,3H),6.43(s,1H),5.69(d,J＝8.2Hz,1H),4.55-4.32(m,2H),4.20-4.17(m,1H),4.00-3.92(m,3H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.22；LCMS-ESI⁺(m/z):550.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.99(brs,1H),7.47(d,J＝8.2Hz,1H),7.38-7.34(m,2H),7.26-7.18(m,3H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS-ESI⁺(m/z):550.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.01(brs,1H),7.47(d,J＝8.2Hz,1H),6.43(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.91；LCMS-ESI⁺(m/z):554.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.10(brs,1H),7.54(d,J＝8.2Hz,1H),7.38-7.34(m,2H),7.26-7.18(m,2H),6.45(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53-4.39(m,2H),4.17-4.14(m,1H),4.06-4.01(m,1H),3.95-3.86(m,2H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.88；LCMS-ESI⁺(m/z):550.3(M+H)⁺.

Example 15

15.1.1- ((2R,3R,4R,5R) -5- (((tert-butyldimethylsilyl) oxy) methyl) -3-chloro-4-hydroxy-3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (44)

2 ' -dehydroxy-2 ' -chloro-2 ' -C-methyluridine 18a (2g,7.22mmol) and pyridine (20mL) were charged into a reaction flask, tert-butyldimethylsilyl chloride (2.39g,15.88mmol) was added in portions at 0 ℃ to the flask, the mixture was warmed to room temperature, and the reaction mixture was stirred overnight. Methanol (1.7mL) was added thereto, the mixture was stirred for 10 minutes, the solvent was evaporated under reduced pressure, ethyl acetate (40mL) was added, the mixture was washed with saturated sodium hydrogencarbonate, water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography to give compound 44(2.3g, 82%).¹H NMR(400Hz,CD₃OD):δ8.30(d,J＝8.1Hz，1H)，6.32(s,1H),5.69(d,J＝8.1Hz,1H),4.18(d,J＝8.7Hz，1H),4.05‐3.98(m,2H),3.72(dd,J＝12.5Hz，1.8Hz，1H),1.52(s,3H),0.95(s,9H),0.18‐0.15(2s,6H)；LCMS‐ESI(m/z):391.1(M+H)⁺。

15.2.1- ((2R, 3S, 5R) -5- (((tert-butyldimethylsilyl) oxy) methyl) -3-chloro-3-methyl-4-oxotetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (45)

Compound 44(2g,5.11mmol) and dichloromethane (20mL) were added to the reaction flask followed by desssmatin Periodinane (DMP) (3.25g,7.67mmol), the reaction stirred overnight, concentrated, washed with ethyl acetate, washed with a saturated solution of sodium thiosulfate and sodium bicarbonate, the organic phase dried, concentrated, filtered briefly over a silica gel column to collect compound 45(1.5g, 76%).¹H NMR(400Hz,CD₃OD):δ8.30(d,J＝8.2Hz，1H)，6.34(s,1H),5.72(d,J＝8.2Hz，1H),4.02(m,2H),3.75(dd,J＝12.5，1.9Hz，1H),1.52(s,3H),0.95(s,9H),0.16(s,3H)，0.08(s,3H)；LCMS‐ESI(m/z):389.1(M+H)⁺。

15.3.1- ((2R,3R,5R) -5- (((tert-butyldimethylsilyl) oxy) methyl) -3-chloro-4-hydroxy-3-methyltetrahydrofuran-2-yl-4-d) pyrimidine-2, 4(1H,3H) -dione (46)

Sodium deuteroborohydride (0.45g,10.8mmol) was added to an ice-cooled solution of compound 45(1.4g,3.60mmol) and ethanol (10mL), stirred at 0 ℃ for 2 hours, quenched by addition of ammonium chloride, ethanol evaporated under reduced pressure, the residue diluted with ethyl acetate (30mL), saturated sodium bicarbonate and saturated brine washed, the organic phase dried over anhydrous sodium sulfate and concentrated to afford compound 46 as two 1:1 isomer 1.2g, was directly subjected to the next reaction without purification. LCMS-ESI (M/z):392.1(M + H)⁺。

15.4.1- ((2R,3R, 4S,5R) -3-chloro-4-hydroxy-5- (hydroxymethyl) -3-methyltetrahydrofuran-2-yl-4-d) pyrimidine-2, 4(1H,3H) -dione (47)

Compound 46(1.2g,3.06mmol) and THF (5mL) were charged to a reaction flask, triethylamine trihydrofluoride (1.55mL,9.53mmol) was added at room temperature, the reaction was stirred at room temperature overnight, concentrated, and the residue was purified by column chromatography (0-6% methanol in dichloromethane) to give compound 47 (0).39g,46％)。¹H NMR(400Hz,DMSO‐d6):δ11.48(s,1H),8.18(d,J＝8.0Hz，1H),6.23(s,1H),5.68(d,J＝8.0Hz，1H),5.97(d,J＝5.6Hz,1H),5.42(s,1H),4.02‐3.84(m,2H),3.69(dd,J＝11.8,3.1Hz,1H),1.44(s,3H)；LCMS‐ESI(m/z):278.2(M+H)⁺。

((S) - (((2R, 3S, 4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-3-d) methoxy) (phenoxy) phosphoryl) -L-alanine isopropyl ester (IV-1 a1)

Compound 47(200mg, 0.72mmol) and anhydrous THF (5.0mL) were added to the reaction flask and cooled to 0 ℃ in an ice-water bath. Magnesium tert-butylchloride Grignard reagent (2.16mL of 1M THF, 2.16mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 3(1.05g, 2.0mmol) in THF (5mL) at 0 ℃. The reaction solution was warmed up, stirred for 24 hours, and saturated NH was added₄Cl (20mL), stirred for 5 min. The mixture was extracted with ethyl acetate (20mL x2), the organic phases separated, the organic phases combined, washed with water (30mL), saturated NaHCO₃(3OmLx2), brine (3OmL), dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography (0-10% methanol in dichloromethane) of the residue to give the product IV-1 a1 as a white solid, 220.5mg, 56%.¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.61(d,J＝8.2Hz,1H),7.39‐7.22(m,5H),6.41(s,1H),5.68(d,J＝8.2Hz,1H),5.06‐5.01(m,1H),4.61‐4.44(m,2H),3.91‐4.01(m,2H),3.76‐3.68(m,1H),1.59(s,3H),1.36(d,J＝7.04Hz,3H)，1.26‐1.24(m，6H)；LCMS‐ESI(m/z):547.1(M+H)⁺。

((R) - (((2R, 3S, 4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-3-D) methoxy) (phenoxy) phosphoryl) -D-isopropyl alaninate (IV-1 b2) Compound 47(200mg, 0.72mmol) and anhydrous THF (5.0mL) were added to the reaction flask and cooled to 0 ℃ in an ice-water bath. Magnesium tert-butylchloride Grignard reagent (2.16mL of 1M THF, 2.16mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30minSubsequently, a solution of phosphorus reagent 6(1.05g, 2.0mmol) in THF (5mL) was added dropwise at 0 ℃. The reaction solution was warmed up, stirred for 24 hours, and saturated NH was added₄Cl (20mL), stirred for 5 min. The mixture was extracted with ethyl acetate (20mL x2), the organic phases separated, the organic phases combined, washed with water (30mL), saturated NaHCO₃(3OmLx2), brine (30mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (0-10% methanol in dichloromethane) of the residue to give the product IV-1 b2 as a white solid, 232.3mg, 59%.¹H NMR(400MHz,CDCl₃)δ8.06(brs,1H),7.43(d,J＝8.2Hz,1H),7.38‐7.26(m,5H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53‐4.39(m,2H),3.96‐3.82(m,2H),3.74‐3.66(m,2H),1.51(s,3H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；LCMS‐ESI(m/z):547.2(M+H)⁺.

In the same manner, the following compounds were synthesized:

¹H NMR(400MHz,CDCl₃)δ8.15(s,1H),7.60(d,J＝8.2Hz,1H),7.40‐7.21(m,5H),6.42(s,1H),5.68(d,J＝8.2Hz,1H),4.60‐4.43(m,2H),4.02‐3.91(m,2H),3.76‐3.69(m,1H),1.58(s,3H),1.36(d,J＝7.0Hz,3H)，1.25‐1.28(m，6H)；LCMS‐ESI(m/z):548.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.10(s,1H),7.62(d,J＝8.1Hz,1H),7.39‐7.22(m,5H),6.42(s,1H),5.69(d,J＝8.1Hz,1H),5.04(s,1H),4.60‐4.43(m,2H),4.01‐3.91(m,2H),3.76‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H)；LCMS‐ESI(m/z):553.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.17(s,1H),7.63(s,1H),7.39‐7.22(m,5H),6.42(s,1H),5.06‐5.01(m,1H),4.62‐4.44(m,2H),4.01‐3.91(m,2H),3.76‐3.68(m,1H),1.60(s,3H),1.36(d,J＝7.0Hz,3H)，1.26‐1.24(m，6H)；LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.16(s,1H),7.39‐7.22(m,5H),6.41(s,1H),5.70(s,1H),5.06‐5.01(m,1H),4.61‐4.44(m,2H),4.01‐3.91(m,2H),3.76‐3.68(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H)，1.26‐1.25(m，6H)；LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.20(s,1H),7.39‐7.22(m,5H),6.41(s,1H),5.06‐5.02(m,1H),4.61‐4.44(m,2H),4.01‐3.91(m,2H),3.76‐3.68(m,1H),1.60(s,3H),1.35(d,J＝7.05Hz,3H)，1.27‐1.24(m，6H)；LCMS‐ESI(m/z):549.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.62(d,J＝8.2Hz,1H),7.40‐7.23(m,4H),6.41(s,1H),5.69(d,J＝8.2Hz,1H),5.07‐5.03(m,1H),4.61‐4.44(m,2H),4.01‐3.91(m,2H),3.76‐3.68(m,1H),1.59(s,3H),1.35(d,J＝7.0Hz,3H)，1.26‐1.24(m，6H)；LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.60(d,J＝8.2Hz,1H),6.40(s,1H),5.68(d,J＝8.2Hz,1H),5.08‐5.02(m,1H),4.61‐4.44(m,2H),4.01‐3.92(m,2H),3.76‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H)，1.26‐1.25(m，6H)；LCMS‐ESI(m/z):552.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.61(d,J＝8.1H_z,1H),7.30(m,2H),6.40(s,1H),5.69(d,J＝8.1Hz,1H),5.07‐5.01(m,1H),4.61‐4.44(m,2H),4.01‐3.91(m,2H),3.76‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.03Hz,3H)，1.26‐1.24(m，6H)；LCMS‐ESI(m/z):550.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.61(d,J＝8.1H_z,1H),7.39‐7.22(m,5H),6.40(s,1H),5.68(d,J＝8.1Hz,1H),5.06‐5.01(m,1H),4.62‐4.46(m,2H),4.01‐3.92(m,1H),3.72‐3.69(m,1H),1.60(s,3H),1.37(d,J＝7.04Hz,3H)，1.26‐1.24(m，6H)；LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.07(brs,1H),7.44(d,J＝8.2Hz,1H),7.38‐7.26(m,5H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),4.53‐4.39(m,2H),3.96‐3.83(m,2H),3.75‐3.66(m,2H),1.51(s,3H),1.37(d,J＝7.0Hz,3H),1.25(s,6H)；LCMS‐ESI(m/z):548.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.06(brs,1H),7.44(d,J＝8.1Hz,1H),7.39‐7.25(m,5H),6.40(s,1H),5.56(d,J＝8.1Hz,1H),5.06(s,1H),4.53‐4.40(m,2H),3.96‐3.83(m,2H),3.74‐3.67(m,2H),1.51(s,3H),1.38(d,J＝6.9Hz,3H)；LCMS‐ESI(m/z):553.3(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.10(brs,1H),7.43(s,1H),7.38‐7.27(m,5H),6.39(s,1H),5.06–5.01(m,1H),4.53‐4.39(m,2H),3.96‐3.82(m,2H),3.74‐3.66(m,2H),1.51(s,3H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；LCMS‐ESI(m/z):548.1(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.10(brs,1H),7.40‐7.26(m,5H),6.40(s,1H),5.56(s,1H),5.06–5.00(m,1H),4.53‐4.39(m,2H),3.96‐3.82(m,2H),3.74‐3.66(m,2H),1.51(s,3H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；LCMS‐ESI(m/z):548.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.12(brs,1H),7.40‐7.26(m,5H),6.39(s,1H),5.06–5.00(m,1H),4.53‐4.39(m,2H),3.96‐3.82(m,2H),3.74‐3.66(m,2H),1.52(s,3H),1.36(d,J＝6.9Hz,3H),1.25(d,J＝6.28Hz,6H)；LCMS‐ESI(m/z):549.3(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.01(brs,1H),7.43(d,J＝8.2Hz,1H),7.40‐7.27(m,4H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53‐4.39(m,2H),3.96‐3.82(m,2H),3.74‐3.66(m,2H),1.50(s,3H),1.37(d,J＝6.9Hz,3H),1.26(d,J＝6.3Hz,6H)；LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.08(brs,1H),7.42(d,J＝8.2Hz,1H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.00–5.06(m,1H),4.53‐4.39(m,2H),3.96‐3.82(m,2H),3.74‐3.66(m,2H),1.52(s,3H),1.36(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；LCMS‐ESI(m/z):552.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.07(brs,1H),7.43(d,J＝8.18Hz,1H),7.34(m,2H),6.40(s,1H),5.57(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53‐4.39(m,2H),3.96‐3.81(m,2H),3.74‐3.67(m,2H),1.52(s,3H),1.36(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；LCMS‐ESI(m/z):550.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.03(brs,1H),7.43(d,J＝8.2Hz,1H),7.38‐7.26(m,5H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.00(m,1H),4.53‐4.39(m,2H),3.86(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.37(d,J＝6.9Hz,3H),1.25(d,J＝6.3Hz,6H)；LCMS‐ESI⁺(m/z):547.2(M+H)⁺.

example 16

(2S,3R,4R,5R) -3- ((tert-butyldimethylsilane) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-carbaldehyde (48)

Compound 18(2.8g,7.16mmol) and dichloromethane (30mL) were added to the reaction flask followed by desssmatin Periodinane (DMP) (4.55g,10.73mmol), the reaction stirred for 3 hours, concentrated, washed with ether, washed three times with a saturated solution of sodium thiosulfate and sodium bicarbonate, the organic phase dried, concentrated, filtered briefly through a silica gel column and compound 48(1.8g, 65%) was collected. LCMS-ESI (M/z):389.1(M + H)⁺。

(2S, 3S, 4R,5R) -3- ((tert-butyldimethylsilane) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-d-2-carbaldehyde (49)

Pyridine (61.5mL, 762.5mmol) and heavy water (16.42mL, 821.2mmol) were added sequentially to a reaction flask for compound 48(1.2g,3.08 mmol). The reaction solution was reacted at 50 ℃ for 28 hours, and the completion of the reaction was checked by a hydrogen spectrum. When the reaction was completed, pyridine and water were distilled off under reduced pressure, and residual pyridine was distilled off with deuterated acetone and deuterated chloroform to obtain crude compound 49 (1.24g), which was directly subjected to the next reaction. LCMS-ESI (M/z):390.1(M + H)⁺。

16.3.1- ((2R,3R, 4S,5R) -4- ((tert-butyldimethylsilyl) oxy) -3-chloro-5- (hydroxymethyl) -3-methyltetrahydrofuran-2-yl-5-d) pyrimidine-2, 4(1H,3H) -dione (50)

Sodium borohydride (0.29g,7.69mmol) was added to an ice-bath solution of compound 49(1.0g,2.56mmol) and deuterated methanol (25.6mL) and stirred at room temperature for 30 minutes, followed by the addition of sodium borohydride (96.8mg,2.56mmol) in small portions until the reaction was complete. The reaction was quenched by addition of ammonium chloride, ethanol was evaporated under reduced pressure, the residue was extracted with ethyl acetate (30mL), and the organic phase was washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound 50(0.8g, 80%).¹H NMR(400Hz,CD₃OD):δ8.33(d,J＝8.2Hz,1H),6.35(s,1H),5.69(d,J＝8.2Hz,1H),4.23(d,J＝8.8Hz,1H),4.02‐3.98(m,1H),3.75(dd,J＝12.5,1.8Hz,1H),1.51(s,3H),0.95(s,9H),0.17(s,3H),0.15(s,3H)。LCMS‐ESI(m/z):392.1(M+H)⁺。

((S) - ((((2R, 3R,4R,5R) -3- ((tert-butyldimethylsilyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl-2-d) methoxy) (phenoxy) phosphoryl) -L-alanine isopropyl ester (51)

Tert-butyl magnesium chloride Grignard reagent (1.53mL, 1.53mmol) was added slowly dropwise to an ice-bath solution of compound 50(200mg,0.51mmol) and anhydrous THF (3.0 mL). The reaction mixture was stirred at O ℃ for 30min, then a solution of phosphorus reagent 3(888mg, 1.68mmol) in THF (3mL) was added dropwise at 0 ℃. The reaction mixture was stirred at room temperature for 24 h, saturated ammonium chloride (15mL) was added, stirred for 5 min, the mixture was extracted with ethyl acetate (50mL x3), the organic phases were combined, the combined organic layers were washed with water (30mL), saturated sodium bicarbonate (3O mL x2), brine (3O mL), dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give 51(175mg, 52%) as a white solid.¹H NMR(400MHz,CDCl₃)δ7.71(d,J＝8.1Hz,1H),7.36‐7.17(m,5H),6.41(s,1H),5.65(d,J＝8.1Hz,1H),5.04‐4.99(m,1H),4.56‐4.43(m,2H),4.19‐4.17(m,1H),4.06‐3.97(m,1H),3.78‐3.69(m,1H),1.51(m,3H),1.37(d,J＝6.0Hz,3H),1.24(d,J＝6.2Hz,6H),0.94(s,9H),0.17(s,3H),0.15(s,3H)。LCMS‐ESI(m/z):661.2(M+H)⁺。

((S) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl-2-d) methoxy) (phenoxy) phosphoryl) -L-alanine isopropyl ester (V-1 a1)

A pre-cooled 90% aqueous trifluoroacetic acid solution (5mL) was added to compound 51(100mg, 0.15mmol) at 0 deg.C, and the mixture was stirred at room temperature overnight to completion. The reaction was evaporated under reduced pressure to remove the solvent, the residue was cooled to 0 ℃, neutralized with sodium bicarbonate, added with water and extracted with ethyl acetate. Combining the organic layers, drying over sodium sulfate, concentrating to give a residue, and purifying by silica gel column chromatography (0-10% methanol in water)Methyl chloride) to give the product V-1 a1(50mg, 61%) as a white solid.¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.60(d,J＝8.18Hz,1H),7.39‐7.21(m,5H),6.42(s,1H),5.69(d,J＝8.2Hz,1H),5.06–5.01(m,1H),4.58‐4.43(m,2H),4.20‐4.18(m,1H),4.00‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):547.1(M+H)⁺

((R) - (((2R,3R,4R,5R) -3- ((tert-butyldimethyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl-2-D) methoxy) (phenoxy) phosphoryl) -D-isopropyl propionate (52)

Magnesium tert-butylchloride Grignard reagent (2.29mL, 2.29mmol) was added slowly dropwise to a solution of compound 50(300mg,0.76mmol) and anhydrous THF (5mL) in an ice bath. The reaction mixture was stirred at 0 ℃ for 30min, then a solution of phosphorus reagent 6(1.33g, 2.52mmol) in THF (5mL) was added dropwise at 0 ℃. The reaction mixture was stirred at room temperature for 24 h, saturated ammonium chloride (20mL) was added, stirred for 5 min, the mixture was extracted with ethyl acetate (60mL x3), the organic phases were combined, the combined organic layers were washed with water (30mL), saturated sodium bicarbonate (35mL x2), brine (4O mL), dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give 52 as a white solid (273mg, 54%).¹H NMR(400MHz,CD₃OD)δ7.79(d,J＝8.1Hz,1H),7.41–7.19(m,5H),6.40(s,1H),5.75(d,J＝8.1Hz,1H),4.97–4.93(m,1H),4.53‐4.39(m,1H),4.19‐4.15(m,1H),4.09‐3.85(m,1H),3.74–3.66(m,1H),1.51(s,3H),1.21(d,J＝7.2Hz,3H),1.20(d,J＝6.5Hz,3H),1.19(d,J＝6.6Hz,3H),0.95(s,9H),0.16(s,3H),0.12(s,3H)；LCMS‐ESI(m/z):661.2(M+H)⁺。

((R) - (((2R,3R,4R,5R) -3- ((tert-butyldimethyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl-2-D) methoxy) (phenoxy) phosphoryl) -D-isopropyl alaninate (V-1 b2)

At 0 deg.CA pre-cooled 90% aqueous trifluoroacetic acid solution (5mL) was added to compound 52(100mg, 0.15mmol), and the mixture was stirred at room temperature overnight to completion. The reaction was evaporated under reduced pressure to remove the solvent, the residue was cooled to 0 ℃, neutralized with sodium bicarbonate, added with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to give a residue which was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product V-1 b2 as a white solid, 49mg, 59%.¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.43(d,J＝8.2Hz,1H),7.43‐7.19(m,5H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.01(m,1H),4.53‐4.39(m,2H),4.17‐4.14(m,1H),3.96‐3.82(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(d,J＝6.25Hz,6H)。LCMS‐ESI(m/z):547.1(M+H)⁺。

Using the same coupling procedure, nucleoside 50 was reacted with phosphorus reagents 3, 6, 11 a-g to give the following product:

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.61(d,J＝8.18Hz,1H),7.39‐7.21(m,5H),6.42(s,1H),5.69(d,J＝8.2Hz,1H),4.58‐4.43(m,2H),4.19‐4.18(m,1H),4.00‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):548.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.59(d,J＝8.2Hz,1H),7.39‐7.21(m,5H),6.42(s,1H),5.69(d,J＝8.2Hz,1H),5.10–5.04(s,1H),4.58‐4.43(m,2H),4.21‐4.18(m,1H),4.00‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H)。LCMS‐ESI(m/z):553.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.61(s,1H),7.39‐7.21(m,5H),6.42(s,1H),5.06–5.01(m,1H),4.59‐4.43(m,2H),4.20‐4.18(m,1H),4.00‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.06Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.16(s,1H),7.39‐7.21(m,5H),6.42(s,1H),5.71(s,1H),5.06–5.01(m,1H),4.58‐4.43(m,2H),4.20‐4.18(m,1H),4.00‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.39‐7.21(m,5H),6.42(s,1H),5.06–5.01(m,1H),4.59‐4.43(m,2H),4.20‐4.18(m,1H),4.01‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):549.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.60(d,J＝8.2Hz,1H),7.39‐7.21(m,4H),6.42(s,1H),5.69(d,J＝8.2Hz,1H),5.06–5.02(m,1H),4.58‐4.43(m,2H),4.20‐4.18(m,1H),4.00‐3.93(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):548.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.60(d,J＝8.2Hz,1H),6.42(s,1H),5.69(d,J＝8.2Hz,1H),5.08–5.03(m,1H),4.58‐4.43(m,2H),4.20‐4.18(m,1H),4.01‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.06Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):552.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.14(s,1H),7.60(d,J＝8.2Hz,1H),7.39‐7.21(s,2H),6.42(s,1H),5.69(d,J＝8.2Hz,1H),5.04–5.01(m,1H),4.58‐4.43(m,2H),4.20‐4.19(m,1H),4.00‐3.92(m,1H),3.77‐3.69(m,1H),1.59(s,3H),1.37(d,J＝7.0Hz,3H),1.24(m,6H)。LCMS‐ESI(m/z):550.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.44(d,J＝8.2Hz,1H),7.43‐7.19(m,5H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),4.53‐4.39(m,2H),4.15‐4.14(m,1H),3.96‐3.82(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(s,6H)。LCMS‐ESI(m/z):548.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.07(s,1H),7.44(d,J＝8.2Hz,1H),7.43‐7.19(m,5H),6.40(s,1H),5.57(d,J＝8.2Hz,1H),5.10–5.06(s,1H),4.53‐4.39(m,2H),4.17‐4.15(m,1H),4.00‐3.84(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H)。LCMS‐ESI(m/z):553.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.44(s,1H),7.42‐7.19(m,5H),6.40(s,1H),5.06–5.01(m,1H),4.53‐4.39(m,2H),4.17‐4.14(m,1H),3.96‐3.82(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(d,J＝6.3Hz,6H)。LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.08(s,1H),7.43‐7.19(m,5H),6.41(s,1H),5.56(s,1H),5.06–5.02(m,1H),4.53‐4.39(m,2H),4.17‐4.14(m,1H),3.96‐3.82(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(d,J＝6.3Hz,6H)。LCMS‐ESI(m/z):548.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.12(s,1H),7.43‐7.19(m,5H),6.40(s,1H),5.06–5.01(m,1H),4.53‐4.39(m,2H),4.17‐4.15(m,1H),3.96‐3.82(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(d,J＝6.3Hz,6H)。LCMS‐ESI(m/z):549.1(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.43(d,J＝8.2Hz,1H),7.43‐7.20(m,4H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.02(m,1H),4.53‐4.39(m,2H),4.17‐4.15(m,1H),3.96‐3.82(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(d,J＝6.3Hz,6H)。LCMS‐ESI(m/z):548.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.43(d,J＝8.2Hz,1H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.09–5.04(m,1H),4.53‐4.39(m,2H),4.17‐4.14(m,1H),3.96‐3.83(m,1H),3.75‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(d,J＝6.3Hz,6H)。LCMS‐ESI(m/z):552.2(M+H)⁺。

¹H NMR(400MHz,CDCl₃)δ8.06(s,1H),7.44(d,J＝8.2Hz,1H),7.43‐7.20(s,2H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.06–5.02(m,1H),4.53‐4.39(m,2H),4.17‐4.16(m,1H),3.96‐3.82(m,1H),3.74‐3.66(m,2H),1.51(s,3H),1.38(d,J＝7.04Hz,3H),1.25(d,J＝6.3Hz,6H)。LCMS‐ESI(m/z):550.2(M+H)⁺。

example 17

17.1.1- ((2R,3R,4R,5R) -4- ((tert-butyldimethylsilyl) oxy) -3-chloro-5- (hydroxymethyl) -3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (53a)

To nucleoside compound 18a (5.3g,19.21mmol) were added pyridine (30mL) and dichloromethane (30mL), and the solution was cooled to 0 ℃. To the solution was added 4, 4' -dimethoxytrityl chloride (7.16g, 21.14mmol), and the mixture was stirred at 0 ℃ overnight. Methanol (5mL) was added to quench the reaction, the reaction was concentrated to dryness under reduced pressure, and ethyl acetate (300mL) and water (30mL) were added to the residue. The organic layer was washed with brine (40mL) and dried over sodium sulfate. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (1 OOmL). Imidazole (3.92g, 57.5mmol) and tert-butyldimethylsilyl chloride (4.34g, 28.8mmol) were added to the solution. After the reaction mixture was stirred at room temperature overnight, methanol (5mL) was added, the mixture was stirred for ten minutes, the solvent was removed under reduced pressure, and ethyl acetate (300mL) and water (50mL) were added to the residue. The organic layer was separated, dried over sodium sulfate and reduced pressureThe solvent was evaporated. The residue was purified by column chromatography (0-40% ethyl acetate in hexane) to give 5 '-oxo-dimethoxytrityl-3' -oxo-tert-butyldimethylsilyl intermediate product. A solution of 5% trichloroacetic acid in dichloromethane (80mL) was added and the mixture stirred at room temperature for 2 hours, then water (10mL) was added and stirring continued at room temperature for 1 hour, methanol (5mL) was added slowly and the solution stirred at room temperature for l hours. The pH of the solution was adjusted to 7 with ammonia. The organic layer was separated, dried over sodium sulfate, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (0-5% methanol in dichloromethane) to give the product 53a (4.11 g) as a white solid in 55% yield over three steps.¹H NMR(400MHz,Methanol‐d₄)δ8.33(d,J＝8.2Hz,1H),6.35(s,1H),5.70(d,J＝8.1Hz,1H),4.20(d,J＝8.8Hz,1H),4.02–3.99(m,2H),3.75(dd,J＝12.5,1.8Hz,1H),1.51(s,3H),0.95(s,9H),0.18(s,3H),0.15(s,3H)；LCMS‐ESI⁺(m/z):391.4(M+H)⁺.

(2S,3R,4R,5R) -3- ((tert-butyldimethylsilyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-carboxylic acid (54a)

To compound 53a (694mg,1.78mmol), bisacetoxyiodobenzene (1.38 g, 4.3mmol) and 2,2, 6, 6-tetramethyl-1-piperidinyloxy (69mg,0.43mmol) were added acetonitrile (4mL) and water (4mL), the mixture stirred at room temperature for 2h until the reaction was complete, the solution was cooled to 0 deg.C, 0.5N potassium hydroxide solution (30mL) was added, back-extracted with dichloromethane (30mL X3), the aqueous solution was neutralized to pH 2 with 2N HCl, filtered, washed with water and dried to give product 54a (590mg) as a white solid in 85% yield. LCMS-ESI⁺(m/z):405.3(M+H)⁺。

4.3.1- ((2R,3R,4R,5R) -4- ((tert-butyldimethylsilyl) oxy) -3-chloro-5- (hydroxymethyl-d₂) -3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4- (1H,3H) -dione (55a)

A mixture of compound 54a (530mg, 1.31mmol) and THF (50mL) was cooled to 0 deg.C and methyl chloroformate (0.44mL) was slowly added dropwise with addition ofTriethylamine (0.3mL, 2.2mmol) was then added dropwise and the mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 ℃ and sodium deuteroborohydride (0.35g) was added in one portion, followed by slowly dropping heavy water at 0 ℃ until the sodium deuteroborohydride was dissolved. The reaction mixture was reacted at 0 ℃ until completion, the solvent was evaporated under reduced pressure, ethyl acetate (300mL) and water (50mL) were added to the residue, the organic layer was washed with a 1N hydrochloric acid solution (10mL), a saturated sodium bicarbonate solution (20mL) and a saturated saline (30mL), respectively, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (0-50% ethyl acetate in hexane) to give a white solid product 55a (0.35g) in 68% yield.¹H NMR(400MHz,Methanol‐d₄)δ8.33(d,J＝8.1Hz,1H),6.35(s,1H),4.19(d,J＝8.8Hz,1H),4.00(d,J＝8.8Hz,1H),1.51(s,3H),0.95(s,9H),0.18(s,3H),0.15(s,3H)；LCMS‐ESI⁺(m/z):393.4(M+H)⁺.

4.4. Isopropyl ((S) - ((((2R, 3R,4R,5R) -3- ((tert-butyldimethylsilyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -L-alanine ester (56a)

To a 10mL reaction tube were added nucleoside 55a (78.6mg,0.20mmol) and 1.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (0.6mL of a 1M solution in THF, 0.6mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 3(145mg, 0.32mmol) in 1mL THF at 0 ℃. The resulting clear reaction solution was warmed to 35 ℃ and stirred for 1 day. Adding saturated NH₄Cl (3mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (60 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with water (10mL), saturated NaHCO₃(2 × 1OmL), brine (1OmL) and over Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-5% methanol in dichloromethane) to give the product 56a (81mg) as a white solid in a yield of 61%).¹HNMR(400MHz,CDCl₃)δ8.81(brs,1H),7.70(d,J＝8.1Hz,1H),7.36‐7.32(m,2H),7.24‐7.17(m,3H),6.42(s,1H),5.67(d,J＝8.1Hz,1H),5.03–4.99(m,1H),4.19‐4.16(m,1H),4.02‐3.96(m,3H),1.51(s,3H),1.37(d,J＝6.0Hz,3H),1.24(d,J＝6.2Hz,6H),0.94(s,9H),0.16(s,3H),0.15(s,3H)；³¹P NMR(162MHz,CDCl₃)δ2.13；LCMS‐ESI⁺(m/z):662.5(M+H)⁺.

4.5. Isopropyl ((S) - ((((2R, 3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -L-alanine ester (VI-1 a1)

A pre-cooled 90% aqueous trifluoroacetic acid solution (4mL) was added to compound 56a (81mg, 0.122mmol) at 0 deg.C, and the mixture was stirred at room temperature overnight to completion. The solvent was evaporated under reduced pressure, the residue was cooled to 0 ℃, neutralized with sodium hydrogencarbonate, and the mixture was extracted with ethyl acetate and water. The combined organic layers were dried over sodium sulfate and the resulting residue was concentrated and purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give the product VI-1 a1(39.5mg) as a white solid in 59% yield.¹H NMR(400MHz,CDCl₃)δ8.87(brs,1H),7.61(d,J＝8.2Hz,1H),7.39‐7.35(m,2H),7.24‐7.21(m,3H),6.43(s,1H),5.69(d,J＝8.2Hz,1H),5.05–5.01(m,1H),4.20‐4.17(m,1H),4.00‐3.92(m,3H),1.59(s,3H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.22；LCMS‐ESI⁺(m/z):548.5(M+H)⁺.

Likewise, we also prepared each of the following 5' -deuterated nucleoside compounds:

¹H NMR(400MHz,CDCl₃)δ8.75(brs,1H),7.65(s,1H),7.41‐7.36(m,2H),7.24‐7.20(m,3H),6.43(s,1H),5.71(s,1H),5.04–5.00(m,1H),4.22‐4.18(m,1H),4.01‐3.91(m,3H),1.57(s,3H),1.38(d,J＝6.0Hz,3H),1.26(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.27；LCMS‐ESI⁺(m/z):549.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.66(brs,1H),7.41‐7.36(m,2H),7.26‐7.22(m,3H),6.40(s,1H),5.04–5.00(m,1H),4.23‐4.18(m,1H),4.00‐3.93(m,3H),1.58(s,3H),1.35(d,J＝6.0Hz,3H),1.26(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.25；LCMS‐ESI⁺(m/z):550.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.76(brs,1H),7.37‐7.34(m,2H),7.25‐7.22(m,3H),6.41(s,1H),5.68(s,1H),5.06–5.01(m,1H),4.22‐4.16(m,1H),4.01‐3.91(m,3H),1.57(s,3H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.29；LCMS‐ESI⁺(m/z):549.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.82(brs,1H),7.63(d,J＝8.0Hz,1H),7.39‐7.33(m,2H),7.25‐7.21(m,3H),6.42(s,1H),5.70(d,J＝8.2Hz,1H),4.20‐4.15(m,1H),4.01‐3.93(m,3H),1.57(s,3H),1.34(d,J＝6.0Hz,3H),1.26(s,6H)；³¹P NMR(162MHz,CDCl₃)δ3.28；LCMS‐ESI⁺(m/z):549.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.21(brs,1H),7.58(d,J＝8.2Hz,1H),7.37‐7.33(m,2H),7.25‐7.21(m,3H),6.43(s,1H),5.67(d,J＝8.0Hz,1H),5.01(s,1H),4.23‐4.16(m,1H),4.01‐3.93(m,3H),1.57(s,3H),1.35(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.20；LCMS‐ESI⁺(m/z):554.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.11(brs,1H),7.59(d,J＝8.0Hz,1H),7.37‐7.31(m,2H),7.25‐7.20(m,3H),6.42(s,1H),5.65(d,J＝8.0Hz,1H),4.22‐4.17(m,1H),4.01‐3.91(m,3H),1.58(s,3H),1.36(d,J＝6.2Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.35；LCMS‐ESI⁺(m/z):555.4(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.55(brs,1H),7.63(d,J＝8.2Hz,1H),6.42(s,1H),5.70(d,J＝8.0Hz,1H),5.04–5.01(m,1H),4.21‐4.18(m,1H),4.03‐3.91(m,3H),1.57(s,3H),1.37(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.35；LCMS‐ESI⁺(m/z):553.2(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.95(brs,1H),7.63(d,J＝8.2Hz,1H),7.37(s,2H),6.43(s,1H),5.68(d,J＝8.2Hz,1H),5.04–5.01(m,1H),4.21‐4.17(m,1H),4.03‐3.93(m,3H),1.58(s,3H),1.36(d,J＝6.0Hz,3H),1.24(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.29；LCMS‐ESI⁺(m/z):551.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.05(brs,1H),7.62(d,J＝8.2Hz,1H),7.42‐7.23(m,4H),6.42(s,1H),5.67(d,J＝8.2Hz,1H),5.04–5.00(m,1H),4.22‐4.18(m,1H),4.01‐3.92(m,3H),1.58(s,3H),1.36(d,J＝6.0Hz,3H),1.26(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.31；LCMS‐ESI⁺(m/z):549.2(M+H)⁺.

17.6. isopropyl ((R) - ((((2R, 3R,4R,5R) -3- ((tert-butyldimethylsilyl) oxy) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -D-alanine ester (56b)

To a 10mL reaction tube were added nucleoside 55b (30mg,0.078mmol) and 0.5mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (0.25mL of a 1M solution in THF, 0.25mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 6(60mg, 0.132mmol) in 1mL THF at 0 ℃. The resulting clear reaction solution was warmed to 35 ℃ and stirred for 1 day. Adding saturated NH₄Cl (3mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (60 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (10 mL). The combined organic layers were washed with water (10mL), saturated NaHCO₃(2 × 1OmL), brine (1OmL) and over Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-5% methanol in dichloromethane) to give product 56b (33.5mg, yield 65%) as a white solid.¹HNMR(400MHz,Methanol‐d₄)δ7.79(d,J＝8.1Hz,1H),7.41‐7.37(m,2H),7.29‐7.19(m,3H),6.40(s,1H),5.75(d,J＝8.1Hz,1H),4.97–4.93(m,1H),4.19‐4.16(m,1H),4.09–4.66(m,1H),3.89‐3.85(m,1H),1.51(s,3H),1.21(d,J＝7.2Hz,3H),1.21(d,J＝6.5Hz,3H),1.19(d,J＝6.6Hz,3H),0.95(s,9H),0.16(s,3H),0.12(s,3H)；³¹P NMR(162MHz,CDCl₃)δ3.04；LCMS‐ESI⁺(m/z):662.4(M+H)⁺.

17.7. Isopropyl ((R) - (((2R,3R,4R,5R) -4-chloro-5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy-d₂) (phenoxy) phosphoryl) -D-alanine ester (VI-1 b2)

A pre-cooled 90% aqueous trifluoroacetic acid solution (2mL) was added to compound 56b (33.5mg, 0.05mmol) at 0 deg.C, and the mixture was stirred at room temperature overnight to completion. The solvent was evaporated under reduced pressure, the residue was cooled to 0 ℃, neutralized with sodium hydrogencarbonate, and the mixture was extracted with ethyl acetate and water. The combined organic layers were dried over sodium sulfate and the resulting residue was concentrated and purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give the product VI-1 b2(16.1mg) as a white solid in 58% yield.¹H NMR(400MHz,CDCl₃)δ8.87(brs,1H),7.48(d,J＝8.2Hz,1H),7.38‐7.33(m,2H),7.25‐7.18(m,3H),6.40(s,1H),5.56(d,J＝8.2Hz,1H),5.05–5.00(m,1H),4.18‐4.14(m,1H),4.06‐3.86(m,3H),1.50(s,3H),1.37(d,J＝6.0Hz,3H),1.24(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS‐ESI⁺(m/z):548.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.98(brs,1H),7.49(s,1H),7.39‐7.33(m,2H),7.24‐7.16(m,3H),6.39(s,1H),5.03–4.99(m,1H),4.18‐4.13(m,1H),4.05‐3.87(m,3H),1.50(s,3H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.92；LCMS‐ESI⁺(m/z):549.4(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.39‐7.34(m,2H),7.23‐7.16(m,3H),6.40(s,1H),5.04–4.99(m,1H),4.17‐4.13(m,1H),4.06‐3.88(m,3H),1.51(s,3H),1.35(d,J＝6.0Hz,3H),1.23(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.95；LCMS‐ESI⁺(m/z):550.4(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ9.12(brs,1H),7.40‐7.34(m,2H),7.24‐7.18(m,3H),6.39(s,1H),5.57(s,1H),5.04–5.00(m,1H),4.18‐4.13(m,1H),4.06‐3.87(m,3H),1.51(s,3H),1.36(d,J＝6.0Hz,3H),1.24(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.97；LCMS‐ESI⁺(m/z):549.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.95(brs,1H),7.49(d,J＝8.2Hz,1H),7.37‐7.33(m,2H),7.24‐7.18(m,3H),6.39(s,1H),5.58(d,J＝8.0Hz,1H),4.18‐4.13(m,1H),4.06‐3.85(m,3H),1.51(s,3H),1.36(d,J＝6.0Hz,3H),1.25(s,6H)；³¹P NMR(162MHz,CDCl₃)δ3.90；LCMS‐ESI⁺(m/z):549.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.83(brs,1H),7.49(d,J＝8.0Hz,1H),7.39‐7.33(m,2H),7.24‐7.19(m,3H),6.39(s,1H),5.58(d,J＝8.2Hz,1H),5.01(m,1H),4.19‐4.14(m,1H),4.05‐3.87(m,3H),1.50(s,3H),1.36(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.95；LCMS‐ESI⁺(m/z):554.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.94(brs,1H),7.49(d,J＝8.0Hz,1H),7.40‐7.34(m,2H),7.24‐7.18(m,3H),6.39(s,1H),5.59(d,J＝8.2Hz,1H),4.18‐4.14(m,1H),4.06‐3.88(m,3H),1.51(s,3H),1.37(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ3.90；LCMS‐ESI⁺(m/z):555.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.81(brs,1H),7.49(d,J＝8.2Hz,1H),6.40(s,1H),5.57(d,J＝8.0Hz,1H),5.05–5.01(m,1H),4.18‐4.13(m,1H),4.05‐3.87(m,3H),1.51(s,3H),1.36(d,J＝6.0Hz,3H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.94；LCMS‐ESI⁺(m/z):553.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.98(brs,1H),7.50(d,J＝8.2Hz,1H),7.38(s,2H),6.39(s,1H),5.57(d,J＝8.0Hz,1H),5.05–5.01(m,1H),4.19‐4.14(m,1H),4.06‐3.85(m,3H),1.51(s,3H),1.36(d,J＝6.0Hz,3H),1.24(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.90；LCMS‐ESI⁺(m/z):551.3(M+H)⁺.

¹H NMR(400MHz,CDCl₃)δ8.93(brs,1H),7.49(d,J＝8.2Hz,1H),7.39‐7.18(m,4H),6.39(s,1H),5.57(d,J＝8.2Hz,1H),5.05–5.01(m,1H),4.18‐4.13(m,1H),4.06‐3.85(m,3H),1.51(s,3H),1.36(d,J＝6.0Hz,3H),1.24(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.97；LCMS‐ESI⁺(m/z):549.3(M+H)⁺.

17.8.1- ((2R,3R,4R,5R) -3-chloro-4-hydroxy-5- (hydroxymethyl-d)₂) -3-methyltetrahydrofuran-2-yl) pyrimidine-2, 4(1H,3H) -dione (57a)

A solution of compound 55a (51mg, 0.13mmol) in THF (10mL) was cooled to 0 ℃, tetrabutylammonium fluoride (0.1mL) was added dropwise, the mixture was stirred at room temperature for 2h until the reaction was complete, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (0-10% methanol in dichloromethane) to give product 57a (29.7mg) as a white solid in 82% yield.¹H NMR(400MHz,CD₃OD)δ8.35(d,J＝8.2Hz,1H),6.36(s,1H),5.70(d,J＝8.2Hz,1H),4.19(d,1H),4.00(d,1H),1.50(s,3H),LCMS‐ESI(m/z):279.1(M+H)⁺.

By the same method, the following compounds were prepared:

¹H NMR(400MHz,CD₃OD)δ8.33(d,J＝8.2Hz,1H),6.37(s,1H),4.19(d,1H),4.01(d,1H),1.50(s,3H)；LCMS‐ESI(m/z):280.2(M+H)⁺.

¹H NMR(400MHz,CD₃OD)δ6.36(s,1H),5.70(d,J＝8.2Hz,1H),4.19(d,1H),4.00(d,1H),1.51(s,3H)；LCMS‐ESI(m/z):280.1(M+H)⁺.

¹H NMR(400MHz,CD₃OD)δ6.36(s,1H),4.19(d,1H),4.00(d,1H),1.50(s,3H),LCMS‐ESI⁺(m/z):281.2(M+H)⁺.

example 18

((R) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine isopropyl ester (X-I-01 b)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 6(725mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (50 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (4OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-I-01 b (303mg) as a white solid in 55% yield.¹H NMR(400MHz,CDCl₃)δ9.82(s,1H),7.36–7.30(m,3H),7.25‐7.21(m,2H),7.19‐7.12(m,1H),6.39(s,1H),5.58(d,J＝8.0Hz,1H),5.09–4.95(m,1H),4.55‐4.45(m,2H),4.41–4.33(m,1H),4.29(brs,1H),4.15–4.09(m,1H),3.97–3.77(m,2H),1.39‐1.26(m,6H),1.25(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.92；LCMS‐ESI⁺(m/z):546.2(M+H)⁺.

The same method synthesizes X-I' -01 c,¹H NMR(400MHz,CDCl₃)δ9.88(s,1H),7.35–7.30(m,3H),7.25‐7.22(m,2H),7.19‐7.13(m,1H),6.42(s,1H),5.68(d,J＝8.0Hz,1H),5.08–4.95(m,1H),4.54‐4.45(m,2H),4.41–4.33(m,1H),4.26(brs,1H),4.16–4.09(m,1H),3.97–3.78(m,2H),1.36‐1.26(m,6H),1.24(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.88；LCMS‐ESI⁺(m/z):546.2(M+H)⁺.

example 19

((S) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine isopropyl ester (X-I-01 c)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 6' (725mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (50 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-I-01 c (311mg) as a white solid in a yield of 57%.¹H NMR(400MHz,CDCl₃)δ9.77(s,1H),7.39–7.30(m,3H),7.28‐7.22(m,2H),7.20‐7.15(m,1H),6.40(d,J＝19.0Hz,1H),5.53(d,J＝8.0Hz,1H),5.05–4.92(m,1H),4.53‐4.48(m,2H),4.42–4.34(m,1H),4.22(brs,1H),4.16–4.08(m,1H),3.96–3.75(m,2H),1.37‐1.26(m,6H),1.24(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.62；LCMS‐ESI⁺(m/z):546.2(M+H)⁺.

Example 20

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) -1-isopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-alanine ester (X-I-02 b)

Synthesis of X-I-02' b

The amino acid Boc-Ala-OH (43mg, 225. mu. mol) was dissolved in 0.5mL of 1, 4-dioxane in a reaction tube, cooled to-5 ℃, DCC (46.5mg, 225. mu. mol) was added with stirring, the reaction was continued at room temperature for 30min, then cooled to-5 ℃ followed by the addition of 0.5mL of 1, 4-dioxane solution of X-I-01 b (81.9mg,0.15mmol), triethylamine (24uL,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) in sequence, the reaction was allowed to react overnight at room temperature, after the reaction was completed, poured into 10mL of water, extracted 3 times with 15mL of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and after solvent spin-drying, the solid intermediate X-I-02' b,65.5mg, yield 61%, was isolated by silica gel column chromatography.¹H NMR(400MHz,CD₃OD)δ7.69(d,J＝8.1Hz,1H),7.40–7.35(m,2H),7.29‐7.24(m,2H),7.22‐7.16(m,1H),6.36(s,1H),5.75(d,J＝8.1Hz,1H),5.55–5.41(m,1H),4.98–4.91(m,1H),4.56‐4.51(m,1H),4.38–4.23(m,2H),4.26‐4.16(m,1H),3.98–3.89(m,1H),1.49(s,9H),1.42‐1.21(m,15H)；³¹P NMR(162MHz,CDCl₃)δ3.62；LCMS‐ESI⁺(m/z):717.3[M+1]⁺。

Synthesis of X-I-02 b

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-I-02' b (60mg,84umol) under stirring, reacting at room temperature for 1 hr, pouring into 10mL water, extracting with 35mL ethyl acetate for 3 times, combining organic layers, extracting with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-I-02 b,41.5mg, in 80% yield.¹H NMR(400MHz,CD₃OD)δ7.50(d,J＝8.1Hz,1H),7.43–7.38(m,2H),7.29‐7.15(m,3H),6.41(s,1H),5.65(d,J＝8.1Hz,1H),5.48–5.37(m,1H),4.96–4.90(m,1H),4.55‐4.51(m,2H),4.38–4.23(m,2H),3.98–3.87(m,1H),1.60‐1.21(m,15H)；³¹P NMR(162MHz,CDCl₃)δ3.35；LCMS‐ESI⁺(m/z):617.2[M+1]⁺。

The same method synthesizes X-I' -02 c,¹H NMR(400MHz,CD₃OD)δ7.52(d,J＝8.1Hz,1H),7.39–7.36(m,2H),7.29‐7.19(m,3H),6.43(s,1H),5.68(d,J＝8.1Hz,1H),5.49–5.38(m,1H),4.97–4.91(m,1H),4.55‐4.51(m,2H),4.38–4.23(m,2H),3.99–3.88(m,1H),1.60‐1.23(m,15H)；³¹P NMR(162MHz,CDCl₃)δ3.32；LCMS‐ESI⁺(m/z):617.2[M+1]⁺。

example 21

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) - ((S) -1-isopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-alanine ester (X-I-02 c)

Synthesis of X-I-02' c

The amino acid Boc-Ala-OH (43mg, 225. mu. mol) was dissolved in 0.5mL of 1, 4-dioxane in a reaction tube, cooled to-5 ℃, DCC (46.5mg, 225. mu. mol) was added with stirring, the reaction was continued at room temperature for 30min, then cooled to-5 ℃ followed by the addition of 0.5mL of 1, 4-dioxane solution of X-I-01 c (81.9mg,0.15mmol), triethylamine (24uL,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) in sequence, the reaction was allowed to proceed overnight at room temperature, after the reaction was completed, poured into 10mL of water, extracted three times with 15mL of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, the solvent was dried, and separated by silica gel column chromatography to give the solid intermediate X-I-02' c, 65.8mg, with a yield of 61.2%.

¹H NMR(400MHz,CD₃OD)δ7.62(d,J＝8.1Hz,1H),7.45–7.35(m,2H),7.29‐7.24(m,2H),7.21‐7.16(m,1H),6.40(s,1H),5.78(d,J＝8.1Hz,1H),5.55–5.39(m,1H),4.96–4.93(m,1H),4.55‐4.52(m,1H),4.38–4.25(m,2H),4.21‐4.15(m,1H),3.95–3.87(m,1H),1.45(s,9H),1.42‐1.21(m,15H)；³¹P NMR(162MHz,CDCl₃)δ3.57；LCMS‐ESI⁺(m/z):717.3[M+1]⁺。

Synthesis of X-I-02 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-I-02' c (61mg,85umol) under stirring, reacting at room temperature for 1 hr, pouring into 10mL water, extracting with 35mL ethyl acetate for 3 times, combining organic layers, extracting with water, saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-I-02 c, 45.6mg, yield 87%.¹H NMR(400MHz,CD₃OD)δ7.55(d,J＝8.1Hz,1H),7.44–7.37(m,2H),7.28‐7.16(m,3H),6.39(s,1H),5.66(d,J＝8.1Hz,1H),5.50–5.41(m,1H),4.96–4.93(m,1H),4.57‐4.51(m,2H),4.39–4.23(m,2H),3.98–3.88(m,1H),1.60‐1.20(m,15H)；³¹P NMR(162MHz,CDCl₃)δ3.39；LCMS‐ESI⁺(m/z):601.4[M+1]⁺。

Example 22

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) -1-isopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-I-05 b)

Synthesis of X-I-05' b

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 μmol) is dissolved by 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 μmol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of X-I-01 b (81.9mg,0.15mmol), triethylamine (24uL,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 10mL of water, extracted by 35mL of ethyl acetate for 3 times, organic layers are combined, dried by anhydrous sodium sulfate, and solvent is dried by spin-drying, and then separated by silica gel column chromatography to obtain a solid intermediate X-I-05' b, 69.3mg and the yield is 62%.¹H NMR(400MHz,CD₃OD)δ7.63(d,J＝8.1Hz,1H),7.42–7.35(m,2H),7.29‐7.24(m,2H),7.21‐7.16(m,1H),6.36(s,1H),5.79(d,J＝8.1Hz,1H),5.51–5.39(m,1H),4.98–4.91(m,1H),4.55‐4.50(m,1H),4.39–4.22(m,2H),4.09‐4.03(m,1H),3.98–3.88(m,1H),2.20–2.12(m,1H),1.47(s,9H),1.39‐1.33(m,6H),1.22(d,J＝6.0Hz,6H),0.99(d,J＝6.3Hz,3H)0.98(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ2.82；LCMS‐ESI⁺(m/z):745.3[M+1]⁺。

Synthesis of X-I-05 b

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-I-05' b (67mg,90umol) under stirring, reacting at room temperature for 1 hr, pouring into 10mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried, and then separated by silica gel column chromatography to give the product X-I-05 b, 49.3mg, 85% yield.¹H NMR(400MHz,CD₃OD)δ7.55(d,J＝8.0Hz,1H),7.45‐7.35(m,2H),7.31‐7.25(m,3H),6.43(s,1H),5.76(d,J＝8.1Hz,1H),5.51–5.36(m,1H),5.12‐4.99，(m,1H)，4.52‐4.44(m,2H),4.38–4.32(m,1H),4.19–4.13(m,1H),3.98–3.88(m,1H),2.47–2.31(m,1H),1.45‐1.33(m,6H),1.26(d,J＝6.0Hz,3H),1.20(d,J＝6.2Hz,3H),1.11(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.28；LCMS‐ESI⁺(m/z):645.3(M+H)⁺.

The same method synthesizes X-I' -05 c,¹H NMR(400MHz,CD₃OD)δ7.57(d,J＝8.0Hz,1H),7.40‐7.33(m,2H),7.31‐7.21(m,3H),6.45(s,1H),5.79(d,J＝8.1Hz,1H),5.52–5.38(m,1H),5.13‐4.99，(m,1H)，4.54‐4.45(m,2H),4.39–4.34(m,1H),4.19–4.14(m,1H),3.98–3.89(m,1H),2.47–2.32(m,1H),1.45‐1.34(m,6H),1.27(d,J＝6.0Hz,3H),1.21(d,J＝6.2Hz,3H),1.13(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.25；LCMS‐ESI⁺(m/z):645.3(M+H)⁺.

example 23

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- ((((S) - (((R) -1-isopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-I-05 c)

Synthesis of X-I-05' c

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 mu mol) is dissolved by 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 mu mol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of X-I-01 c (81.9mg,0.15mmol), triethylamine (24uL,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 15mL of water, extracted by 35mL of ethyl acetate for three times, organic layers are combined, dried by anhydrous sodium sulfate, solvent is dried by spinning, and then separated by silica gel column chromatography to obtain a solid intermediate X-I-05' c, 72.6mg and the yield is 65%.¹H NMR(400MHz,CD₃OD)δ7.68(d,J＝8.1Hz,1H),7.43–7.34(m,2H),7.31‐7.24(m,2H),7.22‐7.18(m,1H),6.44(s,1H),5.75(d,J＝8.0Hz,1H),5.49–5.37(m,1H),4.98–4.92(m,1H),4.55‐4.51(m,1H),4.37–4.26(m,2H),4.09‐4.01(m,1H),3.99–3.88(m,1H),2.21–2.11(m,1H),1.43(s,9H),1.38‐1.30(m,6H),1.21(d,J＝6.2Hz,6H),0.98(d,J＝6.1Hz,3H)0.97(d,J＝6.1Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ2.92；LCMS‐ESI⁺(m/z):745.3[M+1]⁺。

Step 2 Synthesis of X-I-05 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-I-05' c (67mg,90umol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried, and then separated by silica gel column chromatography to give the product X-I-05 c, 51.1mg, in 88% yield.¹H NMR(400MHz,CD₃OD)δ7.65(d,J＝8.1Hz,1H),7.45‐7.38(m,2H),7.33‐7.22(m,3H),6.41(s,1H),5.71(d,J＝8.0Hz,1H),5.49–5.39(m,1H),5.09‐4.95，(m,1H)，4.55‐4.48(m,2H),4.41–4.34(m,1H),4.19–4.11(m,1H),3.98–3.86(m,1H),2.49–2.36(m,1H),1.43‐1.33(m,6H),1.23(d,J＝6.1Hz,3H),1.22(d,J＝6.2Hz,3H),1.15(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ2.95；LCMS‐ESI⁺(m/z):645.2(M+H)⁺.

Example 24

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) -1-isopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl isobutyrate (X-I-22 b)

Isobutyric acid (20mg, 225. mu. mol) was mixed with 0.5mL of 1, 4-dioxane in a reaction tube, the temperature was reduced to-5 ℃, DCC (46.5mg, 225. mu. mol) was added with stirring, the reaction was carried out at room temperature for 30min, the temperature was further reduced to-5 ℃, then X-I-01 b (81.9mg,0.15mmol) of 0.5mL of 1, 4-dioxane solution, triethylamine (24uL,0.18mmol), a catalytic amount of 4-Dimethylaminopyridine (DMAP) was added in this order, the reaction was allowed to react overnight at room temperature, after the reaction was completed, the mixture was poured into 15mL of water, extracted three times with 35mL of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was spin-dried, the solid product X-I-22 b was isolated by silica gel column chromatography, 51.7mg, yield 56%.¹H NMR(400MHz,CD₃OD)δ7.55(d,J＝8.0,1H),7.43‐7.35(m,2H),7.32‐7.21(m,3H),6.41(s,1H),5.73(d,J＝8.1Hz,1H),5.48–5.33(m,1H),5.01‐4.92(m,1H)，4.55‐4.49(m,2H),4.21–4.16(m,1H),3.98–3.88(m,1H),2.49–2.36(m,1H),1.43–1.35(m,6H),1.23(d,J＝6.2Hz,3H),1.21(d,J＝6.2Hz,3H),1.12(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.29；LCMS‐ESI⁺(m/z):616.3(M+H)⁺.

The same method synthesizes X-I' -22 c,¹H NMR(400MHz,CD₃OD)δ7.59(d,J＝8.0,1H),7.40‐7.34(m,2H),7.32‐7.20(m,3H),6.44(s,1H),5.75(d,J＝8.1Hz,1H),5.49–5.35(m,1H),5.02‐4.92(m,1H)，4.56‐4.47(m,2H),4.22–4.16(m,1H),3.99–3.89(m,1H),2.49–2.37(m,1H),1.43–1.36(m,6H),1.24(d,J＝6.2Hz,3H),1.22(d,J＝6.2Hz,3H),1.13(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.22；LCMS‐ESI⁺(m/z):616.3(M+H)⁺.

example 25

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((S) - (((R) -1-isopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl isobutyrate (X-I-22 c)

Isobutyric acid (20mg, 225. mu. mol) was mixed with 0.5mL1, 4-dioxane in a reaction tube, cooled to-5 ℃ and DCC (46.5mg, 225. mu. mol) was added with stirring, the reaction was continued at room temperature for 30min, then cooled to-5 ℃ and then X-I-01 c (81.9mg,0.15mmol) of 0.5mL1, 4-dioxane solution, triethylamine (24uL,0.18mmol) and a catalytic amount of 4-Dimethylaminopyridine (DMAP) were added in order, the reaction was allowed to react overnight at room temperature, after the reaction was completed, poured into 15mL water, extracted three times with 35mL ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, the solvent was dried by spin-drying, and the solid product X-I-22 c, 54.1mg, was isolated by silica gel column chromatography, 58.6% yield.¹H NMR(400MHz,CD₃OD)δ7.58(d,J＝8.0Hz,1H),7.46‐7.36(m,2H),7.35‐7.23(m,3H),6.41(s,1H),5.73(d,J＝8.0Hz,1H),5.47–5.36(m,1H),5.08‐4.96(m,1H)，4.55‐4.45(m,2H),4.18–4.12(m,1H),3.98–3.85(m,1H),2.49–2.35(m,1H),1.45–1.37(m,6H),1.23(d,J＝6.2Hz,3H),1.22(d,J＝6.0Hz,3H),1.19(d,J＝6.1Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.21；LCMS‐ESI⁺(m/z):616.4(M+H)⁺.

Example 26

((R) - ([1, 1' -biphenyl ] -4-yloxy) (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) D-isopropyl propionate (X-I-23 b)

26.1((R) - ([1,1 'biphenyl ] -4-yloxy) (pentafluorophenoxy) phosphoryl) -D-alanine isopropyl ester (58) and ((S) - ([1, 1' biphenyl ] -4-yloxy) (pentafluorophenoxy) phosphoryl) -D-alanine isopropyl ester (59)

Phosphorus oxychloride (9g,58.7mmol) and dichloromethane (200mL) were added to the reaction flask, cooled to-70 deg.C, and a solution of p-phenylphenol 57(l 0g, 58.7mmol) and triethylamine (6g,58.7mmol) in dichloromethane (50mL) was added dropwise slowly, and after the addition was complete, the temperature was allowed to rise slowly to room temperature, and the reaction was allowed to proceed overnight. Cooling the mixture to 0 deg.C, adding D-alanine isopropyl ester hydrochloride (9g, 53.1mmol), cooling to-70 deg.C, adding triethylamine (13.4g,130mmol) in dichloromethane (60mL), heating to 0 deg.C, reacting for 3 hours, adding pentafluorophenol (l0g,53mmol) and triethylamine (8g, 77.6mmol) in dichloromethane (100mL) dropwise to the solution, stirring at 0 deg.C for 1 hour, heating to room temperature, stirring overnight, adding 150mL dichloromethane and 120mL water, separating out the organic phase, drying with anhydrous sodium sulfate, concentrating under reduced pressure, separating the residue with silica gel column (0-30% ethyl acetate/hexane) to obtain 13.6 g white solid, recrystallizing the solid with 10% t-butyl methyl ether/hexane to obtain white solid 58(5.3 g), separating the mother liquor with silica gel column (50% ethyl acetate/hexane) to obtain product 59(3.7 g) and 58(0.8 g), 58 and 59 both had a purity greater than 99%.

58：¹H NMR(400MHz,CDCl₃)δ(ppm):7.78–7.70(m,4H),7.50–7.33(m,5H),5.16–4.97(m,1H),4.26–4.08(m,1H),3.97–3.93(m,1H),1.45(d,J＝7.0Hz,3H),1.27(d,J＝5.9Hz,3H),1.26(d,J＝5.9Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐2.65.

59：¹H NMR(400MHz,CDCl₃)δ(ppm):7.75–7.68(m,4H),7.48–7.32(m,5H),5.20–4.98(m,1H),4.22–4.05(m,1H),3.96–3.91(m,1H),1.47(d,J＝7.0Hz,3H),1.26(d,J＝6.2Hz,3H),1.25(d,J＝6.2Hz,3H).³¹P NMR(162MHz,CDCl₃)δ‐2.05.

Synthesis of X-I-23 b

To a 10mL reaction tube were added nucleoside 18a (138.3mg,0.5mmol) and 3.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Magnesium tert-butylchloride Grignard reagent (1.5mL of 1M THF, 1.5mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 48(846mg, 1.6mmol) in 3mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (150 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-I-23 b (171mg) as a white solid in 55% yield.¹H NMR(400MHz,CD₃OD)δ7.79–7.68(m,4H),7.55‐7.36(m,6H),6.41(s,1H),5.69(d,J＝8.0Hz,1H),5.01–4.91(m,1H),4.58‐4.52(m,1H),4.47–4.35(m,1H),4.18–4.07(m,1H),3.96–3.71(m,2H),1.43‐1.26(m,6H),1.25(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ3.75；LCMS‐ESI⁺(m/z):622.3(M+H)⁺.

Example 27

((S) - ([1, 1' -biphenyl ] -4-yloxy) (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) D-isopropyl propionate (X-I-23 c)

To a 10mL reaction tube were added nucleoside 18a (138.3mg,0.5mmol) and 3.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Magnesium tert-butylchloride Grignard reagent (1.5mL of 1M THF, 1.5mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 59(846mg, 1.6mmol) in 3mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (150 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-I-23 c (180.4mg) as a white solid in a yield of 58%.¹H NMR(400MHz,CD₃OD)δ7.75–7.67(m,4H),7.55‐7.41(m,6H),6.38(s,1H),5.76(d,J＝8.0Hz,1H),5.02–4.96(m,1H),4.55‐4.50(m,1H),4.45–4.35(m,1H),4.17–4.07(m,1H),3.96–3.77(m,2H),1.40‐1.27(m,6H),1.23(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ2.78；LCMS‐ESI⁺(m/z):622.2(M+H)⁺.

Example 28

(2R,3R,4R,5R) -2- ((((R) - ([1, 1' -biphenyl ] -4-yloxy) ((R) -1-isopropyl-1-oxoprop-2-yl) amino) phosphoryl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-4-methyltetrahydrofuran-3-yl) L-alanine ester (X-I-25 b)

Synthesis of X-I-25' b

Boc-Val-OH (49mg, 225. mu. mol) is dissolved in 0.5mL of 1, 4-dioxane in a reaction tube, the temperature is reduced to-5 ℃, DCC (46.5mg, 225. mu. mol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of X-I-23 b (93.3mg,0.15mmol), triethylamine (24. mu.L, 0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, the reaction is finished and poured into 10mL of water, the mixture is extracted for 3 times by 35mL of ethyl acetate, organic layers are combined, dried by anhydrous sodium sulfate, solvent is dried by spin drying, and then a silica gel column chromatography is used for separation to obtain a solid intermediate X-I-25' b, 61.6mg and the yield is 50.¹H NMR(400MHz,CD₃OD)δ7.78–7.61(m,4H),7.58‐7.30(m,6H),6.38(s,1H),5.75(d,J＝8.0Hz,1H),5.47–5.35(m,1H),4.95–4.91(m,1H),4.58‐4.51(m,1H),4.37–4.25(m,2H),4.11‐4.03(m,1H),3.96–3.85(m,1H),2.19–2.12(m,1H),1.45(s,9H),1.38‐1.30(m,6H),1.25(d,J＝6.0Hz,6H),0.98(d,J＝6.2Hz,3H)0.97(d,J＝6.1Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ2.90；LCMS‐ESI⁺(m/z):821.4[M+1]⁺。

Synthesis of X-I-25 b

4M HCl 1, 4-dioxane (2mL) was added to the reaction tube and the temperature was reduced toAdding intermediate X-I-25' b (70mg, 85. mu. mol) at 0 deg.C under stirring, reacting at room temperature for 1 hr, pouring into 10mL of water, extracting with 35mL of ethyl acetate for three times, combining the organic layers, extracting with water, saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried, and then separated by silica gel column chromatography to give the product X-I-25 b, 53.3mg, 87% yield.¹H NMR(400MHz,CD₃OD)δ7.81–7.68(m,4H),7.56‐7.32(m,6H),6.37(s,1H),5.79(d,J＝8.0Hz,1H),5.50–5.33(m,1H),5.01‐4.96，(m,1H)，4.53‐4.47(m,2H),4.38–4.33(m,1H),4.19–4.11(m,1H),3.93–3.82(m,1H),2.46–2.33(m,1H),1.45‐1.36(m,6H),1.25(d,J＝6.2Hz,3H),1.23(d,J＝6.0Hz,3H),1.12(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.35；LCMS‐ESI⁺(m/z):721.3(M+H)⁺.

Example 29

(2R,3R,4R,5R) -2- ((((S) - ([1, 1' -biphenyl ] -4-yloxy) (((R) -1-isopropyl-1-oxoprop-2-yl) amino) phosphoryl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-4-methyltetrahydrofuran-3-yl) L-alanine ester (X-I-25 c)

Synthesis of X-I-25' c

Boc-Val-OH (49mg, 225. mu. mol) is dissolved in 0.5mL of 1, 4-dioxane in a reaction tube, the temperature is reduced to-5 ℃, DCC (46.5mg, 225. mu. mol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of X-I-23 c (93.3mg,0.15mmol), triethylamine (24. mu.L, 0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, the mixture is poured into 10mL of water after the reaction is finished, extracted with 35mL of ethyl acetate for 3 times, organic layers are combined, and dried by anhydrous sodium sulfateAfter the solvent was dried by spinning, the solid intermediate X-I-25' c, 69mg, yield 56% was obtained by silica gel column chromatography.¹H NMR(400MHz,CD₃OD)δ7.72–7.61(m,4H),7.54‐7.31(m,6H),6.35(d,1H),5.75(d,J＝8.0Hz,1H),5.45–5.36(m,1H),4.95–4.91(m,1H),4.55‐4.51(m,1H),4.38–4.23(m,2H),4.08‐4.00(m,1H),3.97–3.85(m,1H),2.22–2.11(m,1H),1.45(s,9H),1.39‐1.31(m,6H),1.24(d,J＝6.1Hz,6H),0.99(d,J＝6.2Hz,3H)0.98(d,J＝6.1Hz,3H)；³¹P NMR(162MHz,CDCl₃)δ2.68；LCMS‐ESI⁺(m/z):821.3[M+1]⁺。

Synthesis of X-I-25 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-I-25' c (70mg, 85. mu. mol) under stirring, reacting at room temperature for 1 hr, pouring into 10mL water, extracting with 35mL ethyl acetate for three times, combining the organic layers, adding water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried, and then separated by silica gel column chromatography to give the product X-I-25 c, 52.1mg, 85% yield.¹H NMR(400MHz,CD₃OD)δ7.79–7.63(m,4H),7.55‐7.31(m,6H),6.37(s,1H),5.79(d,J＝8.0Hz,1H),5.53–5.33(m,1H),5.02‐4.97，(m,1H)，4.56‐4.45(m,2H),4.39–4.31(m,1H),4.19–4.11(m,1H),3.95–3.83(m,1H),2.45–2.32(m,1H),1.42‐1.35(m,6H),1.25(d,J＝6.0Hz,3H),1.23(d,J＝6.0Hz,3H),1.15(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.22；LCMS‐ESI⁺(m/z):721.2(M+H)⁺。

Example 30

((R) - (4-Cyclopropylphenoxy) (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) phosphoryl) -D-alanine isopropyl ester (X-I-27 b)

((R) - (4-Cyclopropylphenoxy) (pentafluorophenoxy) phosphoryl) -D-alanine isopropyl ester (61) and ((S) - (4-Cyclopropylphenoxy) (pentafluorophenoxy) phosphoryl) -D-alanine isopropyl ester (62)

Phosphorus oxychloride (9g,58.7mmol) and dichloromethane (200mL) were added to the reaction flask, the mixture was cooled to-70 ℃ and a solution of p-cyclopropylphenol 60(7.88g,58.7mmol) and triethylamine (6g,58.7mmol) in dichloromethane (50mL) was added dropwise slowly, after the addition was complete, the temperature was raised slowly to room temperature and the reaction was allowed to proceed overnight. Cooling the mixture to 0 deg.C, adding D-alanine isopropyl ester hydrochloride (9g, 53.1mmol), cooling to-70 deg.C, adding triethylamine (13.4g,130mmol) in dichloromethane 60mL dropwise, heating to 0 deg.C after adding, reacting for 3 hours, adding pentafluorophenol (l0g,53mmol) and triethylamine (8g, 77.6mmol) in dichloromethane 100mL dropwise to the solution, stirring at 0 deg.C for 1 hour, heating to room temperature, stirring overnight, adding 150mL dichloromethane and 120mL water, separating out the organic phase, drying with anhydrous sodium sulfate, concentrating under reduced pressure, separating the residue with silica gel column (0-30% ethyl acetate/hexane) to obtain 11.2 g of white solid, recrystallizing the solid with 10% t-butyl methyl ether/hexane to obtain 61(3.8g), separating the mother liquor with silica gel column (50% ethyl acetate/hexane) to obtain 62(3.1g) and 61(0.6g), both 61 and 62 are greater than 99% pure.

61：¹H NMR(400MHz,CDCl₃)δ(ppm):7.45(d,2H),7.33(d,2H),5.16–4.97(m,1H),4.16–4.08(m,1H),3.97(brs,1H),1.88–1.78(m,1H),1.61–1.43(m,2H),1.33–1.22(m,11H).³¹P NMR(162MHz,CDCl₃)δ‐2.85.

62：¹H NMR(400MHz,CDCl₃)δ(ppm):7.44(d,2H),7.35(d,2H),5.13–4.96(m,1H),4.01–3.90(m,1H),3.87(brs,1H),1.86–1.75(m,1H),1.63–1.46(m,2H),1.35–1.23(m,11H).³¹P NMR(162MHz,CDCl₃)δ‐2.15.

Synthesis of X-I-27 b

To a 10mL reaction tube were added nucleoside 18a (138.3mg,0.5mmol) and 3.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (1.5mL of 1M THF, 1.5mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 61(790mg, 1.6mmol) in 3mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (150 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-I-27 b (167mg) as a white solid in a yield of 57%.¹H NMR(400MHz,CD₃OD)δ7.50–7.38(m,3H),7.33(m,2H),6.39(s,1H),5.67(d,J＝8.0Hz,1H),5.02–4.93(m,1H),4.55‐4.50(m,1H),4.47–4.38(m,1H),4.19–4.10(m,1H),3.96–3.75(m,2H),1.88–1.73(m,1H),1.61–1.47(m,2H),1.40–1.23(m,14H)；³¹P NMR(162MHz,CDCl₃)δ3.69；LCMS‐ESI⁺(m/z):586.2(M+H)⁺.

Example 31

((S) - (4-Cyclopropylphenoxy) (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) phosphoryl) -D-alanine isopropyl ester (X-I-27 c)

To a 10mL reaction tube were added nucleoside 18a (138.3mg,0.5mmol) and 3.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (1.5mL of 1M THF, 1.5mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 62(790mg, 1.6mmol) in 3mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (150 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-I-27 c (143.6mg) as a white solid in 49% yield.¹H NMR(400MHz,CD₃OD)δ7.48–7.36(m,3H),7.31(d,2H),6.45(s,1H),5.65(d,J＝8.0Hz,1H),5.05–4.96(m,1H),4.54‐4.51(m,1H),4.46–4.38(m,1H),4.15–4.08(m,1H),3.96–3.78(m,2H),1.88–1.77(m,1H),1.63–1.43(m,2H),1.40‐1.21(m,14H)；³¹P NMR(162MHz,CDCl₃)δ3.29；LCMS‐ESI⁺(m/z):586.3(M+H)⁺.

Example 32

(2R,3R,4R,5R) -2- ((((R) - (4-Cyclopropylphenoxy)) (((R) -1-isopropyl-1-oxoprop-2-yl) amino) phosphoryl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-4-methyltetrahydrofuran-3-yl-L-alanine ester (X-I-29 b)

Synthesis of X-I-29' b

The amino acid Boc-Val-OH (49mg, 225. mu. mol) was dissolved in 0.5mL of 1, 4-dioxane in a reaction tubeHydrolyzing, cooling to-5 ℃, adding DCC (46.5mg,225 mu mol) under stirring, reacting for 30min at room temperature, continuously cooling to-5 ℃, then sequentially adding X-I-27 b (87.9mg,0.15mmol) in 0.5mL of 1, 4-dioxane solution, triethylamine (24 mu L,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP), reacting overnight at room temperature, pouring into 10mL of water after the reaction is finished, extracting for 3 times with 15mL of ethyl acetate, combining organic layers, drying with anhydrous sodium sulfate, spin-drying the solvent, and separating by silica gel column chromatography to obtain a solid intermediate X-I-29' b, 67.1mg and the yield of 57%.¹H NMR(400MHz,CD₃OD)δ7.49–7.37(m,3H),7.33(d,2H),6.39(s,1H),5.75(d,J＝8.0Hz,1H),5.47–5.38(m,1H),4.97–4.92(m,1H),4.53‐4.50(m,1H),4.39–4.29(m,2H),4.08‐4.03(m,1H),3.96–3.88(m,1H),2.20–2.10(m,1H),1.85–1.73(m,1H),1.61–1.51(m,2H),1.47(s,9H),1.44–1.38(m,6H),1.34–0.96(m,14H)；³¹P NMR(162MHz,CDCl₃)δ2.95；LCMS‐ESI⁺(m/z):785.3[M+1]⁺。

Synthesis of X-I-29 b

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-I-29' b (70mg,89 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried, and then separated by silica gel column chromatography to give the product X-I-29 b, 49.4mg, in 81% yield.¹H NMR(400MHz,CD₃OD)δ7.48–7.36(m,3H),7.35(d,2H),6.33(s,1H),5.75(d,J＝7.8Hz,1H),5.43–5.34(m,1H),4.97–4.93(m,1H),4.55‐4.47(m,1H),4.39–4.28(m,2H),4.06‐4.00(m,1H),3.95–3.85(m,1H),2.23–2.12(m,1H),1.87–1.76(m,1H),1.60–1.51(m,2H),1.46–1.36(m,6H),1.33–0.93(m,14H)；³¹P NMR(162MHz,CD₃OD)δ3.38；LCMS‐ESI⁺(m/z):685.3(M+H)⁺.

Example 33

(2R,3R,4R,5R) -2- ((((S) - (4-Cyclopropylphenoxy)) (((R) -1-isopropyl-1-oxoprop-2-yl) amino) phosphoryl) oxy) methyl) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-4-methyltetrahydrofuran-3-yl-L-alanine ester (X-I-29 c)

Synthesis of X-I-29' c

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 μmol) is dissolved by 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 μmol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of X-I-27 c (87.9mg,0.15mmol), triethylamine (24 μ L,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 15mL of water, extracted by 35mL of ethyl acetate for 3 times, organic layers are combined, dried by anhydrous sodium sulfate, and solvent is dried by spin-drying, and then separated by silica gel column chromatography to obtain a solid intermediate X-I-29' c, 65.9mg and the yield is 56%.¹H NMR(400MHz,CD₃OD)δ7.50–7.38(m,3H),7.34(d,2H),6.37(s,1H),5.75(d,J＝8.2Hz,1H),5.43–5.35(m,1H),4.96–4.91(m,1H),4.57‐4.51(m,1H),4.37–4.27(m,2H),4.13‐4.06(m,1H),3.93–3.87(m,1H),2.21–2.10(m,1H),1.89–1.77(m,1H),1.65–1.55(m,2H),1.45(s,9H),1.45–1.38(m,6H),1.36–0.97(m,14H)；³¹P NMR(162MHz,CDCl₃)δ2.69；LCMS‐ESI⁺(m/z):785.3[M+1]⁺。

Synthesis of X-I-29 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-I-29' c (70mg,89 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washing with brineAfter drying over anhydrous sodium sulfate and spin-drying the solvent, the product X-I-29 c was obtained in a yield of 83% by chromatography on silica gel column, 60.6 mg.¹H NMR(400MHz,CD₃OD)δ7.45–7.38(m,3H),7.36(d,2H),6.35(d,1H),5.73(d,J＝8.1Hz,1H),5.45–5.32(m,1H),4.97–4.92(m,1H),4.54‐4.47(m,1H),4.39–4.26(m,2H),4.08‐4.00(m,1H),3.96–3.82(m,1H),2.22–2.12(m,1H),1.88–1.76(m,1H),1.63–1.50(m,2H),1.48–1.38(m,6H),1.36–0.90(m,14H)；³¹P NMR(162MHz,CD₃OD)δ3.21；LCMS‐ESI⁺(m/z):685.3(M+H)⁺.

Example 34

((R) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine heptadeuteroisopropyl ester (X-II-01 b)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Magnesium tert-butylchloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorous reagent 11h (736mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-II-01 b as a white solid in 63% yield.¹H NMR(400MHz,CDCl₃)δ9.85(brs,1H),7.43–7.35(m,3H),7.28‐7.21(m,2H),7.19‐7.13(m,1H),6.38(s,1H),5.53(d,J＝8.0Hz,1H),5.09–4.97(m,1H),4.53‐4.45(m,2H),4.43–4.32(m,1H),4.27(brs,1H),3.95–3.78(m,2H),1.39‐1.27(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.65；LCMS‐ESI⁺(m/z):553.2(M+H)⁺.

The same method synthesizes X-II' -01 c,¹H NMR(400MHz,CDCl₃)δ9.89(brs,1H),7.49–7.36(m,3H),7.28‐7.22(m,2H),7.20‐7.14(m,1H),6.42(s,1H),5.61(d,J＝8.0Hz,1H),5.09–4.98(m,1H),4.54‐4.45(m,2H),4.43–4.33(m,1H),4.28(brs,1H),3.97–3.79(m,2H),1.39‐1.24(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.60；LCMS‐ESI⁺(m/z):553.2(M+H)⁺.

example 35

((S) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine heptadeuteroisopropyl ester (X-II-01 c)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of the phosphorus reagent 11 h' (736mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL).The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product X-II-01 c (276.5mg) as a white solid in 50% yield.¹H NMR(400MHz,CDCl₃)δ9.72(brs,1H),7.40–7.32(m,3H),7.26‐7.21(m,2H),7.20‐7.13(m,1H),6.34(s,1H),5.57(d,J＝8.0Hz,1H),5.04–4.92(m,1H),4.51‐4.46(m,2H),4.45–4.32(m,1H),4.24(brs,1H),3.93–3.75(m,2H),1.39‐1.31(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.52；LCMS‐ESI⁺(m/z):553.2(M+H)⁺.

Example 36

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) -1-heptadeuteroisopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-II-03 b)

Synthesis of X-II-03' b

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 mu mol) is dissolved by 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 mu mol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of X-II-01 b (83mg,0.15mmol), triethylamine (24 mu L,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 15mL of water, extracted for 3 times by 35mL of ethyl acetate, organic layers are combined, dried by anhydrous sodium sulfate and solvent is dried by spin drying, and then separated by silica gel column chromatography to obtain a solid intermediate X-II-03' b, 68.8mg and the yield is 61%.¹H NMR(400MHz,CD₃OD)δ7.63(d,J＝8.0Hz,1H),7.43–7.35(m,2H),7.29‐7.24(m,2H),7.21‐7.15(m,1H),6.38(d,1H),5.78(d,J＝8.0Hz,1H),5.42–5.31(m,1H),4.98–4.91(m,1H),4.55‐4.50(m,1H),4.39–4.23(m,2H),3.99–3.86(m,1H),2.17–2.11(m,1H),1.46(s,9H),1.38‐1.31(m,6H),1.24(d,J＝6.1Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ2.81；LCMS‐ESI⁺(m/z):752.3[M+1]⁺。

Synthesis of X-II-03 b

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-II-03' b (70mg,93 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-II-03 b, 51.5mg, in 85% yield.¹H NMR(400MHz,CD₃OD)δ7.53(d,J＝8.0Hz,1H),7.43‐7.37(m,2H),7.31‐7.20(m,3H),6.38(d,1H),5.75(d,J＝8.1Hz,1H),5.48–5.34(m,1H),5.03‐4.92，(m,1H)，4.54‐4.49(m,2H),4.39–4.33(m,1H),3.97–3.87(m,1H),2.47–2.34(m,1H),1.45‐1.37(m,6H),1.19(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CD₃OD)δ3.38；LCMS‐ESI⁺(m/z):652.3(M+H)⁺.

Example 37

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((S) - ((R) -1-heptadeuteroisopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-II-03 c)

Synthesis of X-II-03' c

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 mu mol) is dissolved by 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 mu mol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of X-II-01 c (83mg,0.15mmol), triethylamine (24 mu L,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 15mL of water, extracted for 3 times by 35mL of ethyl acetate, organic layers are combined, dried by anhydrous sodium sulfate and solvent is dried by spin drying, and then separated by silica gel column chromatography to obtain a solid intermediate X-II-03' c, 65.4mg and the yield is 58%.¹H NMR(400MHz,CD₃OD)δ7.65(d,J＝8.0Hz,1H),7.46–7.35(m,2H),7.27‐7.24(m,2H),7.22‐7.15(m,1H),6.39(s,1H),5.70(d,J＝8.0Hz,1H),5.47–5.32(m,1H),4.96–4.90(m,1H),4.56‐4.51(m,1H),4.39–4.25(m,2H),3.98–3.86(m,1H),2.16–2.11(m,1H),1.46(s,9H),1.38‐1.31(m,6H),1.25(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ2.93；LCMS‐ESI⁺(m/z):752.4[M+1]⁺。

Synthesis of X-II-03 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-II-03' c (68mg, 90.4. mu. mol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water, saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried, and then separated by silica gel column chromatography to give the product X-II-03 c, 47.7mg, in 81% yield.¹H NMR(400MHz,CD₃OD)δ7.65(d,J＝8.0Hz,1H),7.41‐7.36(m,2H),7.32‐7.21(m,3H),6.35(s,1H),5.66(d,J＝8.2Hz,1H),5.42–5.33(m,1H),5.01‐4.93，(m,1H)，4.55‐4.49(m,2H),4.39–4.32(m,1H),3.97–3.86(m,1H),2.47–2.36(m,1H),1.43‐1.32(m,6H),1.25(d,J＝6.2Hz,3H),1.22(d,J＝6.1Hz,3H)；³¹P NMR(162MHz,CD₃OD)δ2.91；LCMS‐ESI⁺(m/z):652.2(M+H)⁺.

Example 38

((R) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine L hexadeuteroisopropyl ester (IX-2 b2)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 11a (735mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product IX-2 b2(292mg) as a white solid in 53% yield.¹H NMR(400MHz,CDCl₃)δ9.77(brs,1H),7.45–7.31(m,3H),7.28‐7.21(m,2H),7.18‐7.13(m,1H),6.38(s,1H),5.54(d,J＝8.0Hz,1H),5.05–4.93(m,1H),4.54‐4.45(m,2H),4.45–4.33(m,1H),4.27(brs,1H),4.19–4.07(m,1H),3.96–3.78(m,2H),1.39‐1.25(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.60；LCMS‐ESI⁺(m/z):552.2(M+H)⁺.

IX-2 a1 was synthesized in a similar manner,¹H NMR(400MHz,CDCl₃)δ9.79(brs,1H),7.42–7.30(m,3H),7.28‐7.20(m,2H),7.18‐7.14(m,1H),6.43(s,1H),5.66(d,J＝8.0Hz,1H),5.05–4.96(m,1H),4.55‐4.46(m,2H),4.45–4.34(m,1H),4.29(brs,1H),4.19–4.09(m,1H),3.98–3.79(m,2H),1.39‐1.24(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.53；LCMS‐ESI⁺(m/z):552.2(M+H)⁺.

example 39

((S) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine hexadeuteroisopropyl ester (IX-2 b1)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 11 a' (735mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product IX-2 b1(279.3mg) as a white solid in 50.6% yield.¹H NMR(400MHz,CDCl₃)δ9.78(brs,1H),7.45–7.32(m,3H),7.29‐7.21(m,2H),7.19‐7.12(m,1H),6.42(s,1H),5.52(d,J＝8.1Hz,1H),5.01–4.92(m,1H),4.55‐4.46(m,2H),4.43–4.32(m,1H),4.31(brs,1H),4.18–4.02(m,1H),3.95–3.78(m,2H),1.38‐1.30(m,6H)；³¹P NMR(162MHz,CDCl₃)δ3.56；LCMS‐ESI⁺(m/z):551.3(M+H)⁺.

Example 40

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) -1-hexadeuteroisopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-III-03 b)

Synthesis of X-III-03' b

The amino acid Boc-Val-OH (49mg, 225. mu. mol) was dissolved in 0.5mL of 1, 4-dioxane in a reaction tube, the temperature was reduced to-5 ℃, DCC (46.5mg, 225. mu. mol) was added with stirring, the reaction was continued at room temperature for 30min, the temperature was further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of IX-2 b2(113mg,0.15mmol), triethylamine (24. mu.L, 0.18mmol), a catalytic amount of 4-Dimethylaminopyridine (DMAP) was added in order, the reaction was allowed to proceed overnight at room temperature, after the reaction was completed, the mixture was poured into 15mL of water, extracted with 35mL of ethyl acetate for 3 times, the organic layers were combined, dried over anhydrous sodium sulfate, the solvent was spin-dried, and then separated by silica gel column chromatography to obtain solid intermediate X-III-03' b, 70mg, 62% yield.¹H NMR(400MHz,CD₃OD)δ7.63(d,J＝8.2Hz,1H),7.40–7.33(m,2H),7.27‐7.21(m,2H),7.18‐7.13(m,1H),6.39(s,1H),5.73(d,J＝8.1Hz,1H),5.45–5.31(m,1H),4.98–4.91(m,1H),4.52‐4.49(m,1H),4.36–4.22(m,2H),4.17–4.08(m,1H),3.96–3.88(m,1H),2.19–2.11(m,1H),1.45(s,9H),1.39‐1.31(m,6H),1.26(d,J＝6.0Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ2.68；LCMS‐ESI⁺(m/z):751.2[M+1]⁺。

Synthesis of X-III-03 b

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-III-03' b (70mg,93 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-III-03 b, 51.5mg, with a yield of 85%.¹H NMR(400MHz,CD₃OD)δ7.59(d,J＝8.1Hz,1H),7.44‐7.36(m,2H),7.33‐7.22(m,3H),6.36(s,1H),5.77(d,J＝8.0Hz,1H),5.44–5.33(m,1H),5.02‐4.97，(m,1H)，4.55‐4.47(m,2H),4.38–4.34(m,1H),4.12–4.07(m,1H),3.96–3.85(m,1H),2.43–2.32(m,1H),1.47‐1.36(m,6H),1.23(d,J＝6.0Hz,6H)；³¹PNMR(162MHz,CD₃OD)δ3.33；LCMS‐ESI⁺(m/z):651.3(M+H)⁺.

EXAMPLE 41

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((S) - ((R) -1-hexadeuteroisopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-III-03 c)

Synthesis of X-III-03' c

Dissolving the amino acid Boc-Val-OH (49mg,225 mu mol) in 0.5mL of 1, 4-dioxane in a reaction tube, cooling to-5 ℃, adding DCC (46.5mg,225 mu mol) under stirring, reacting at room temperature for 30min, further cooling to-5 ℃, then sequentially adding 0.5mL of 1, 4-dioxane solution of IX-2 b1(113mg,0.15mmol), triethylamine (24 mu L,0.18mmol) and a catalytic amount of 4-Dimethylaminopyridine (DMAP), reacting at room temperature overnight, pouring into 15mL of water after the reaction is finished, and addingThe mixture was extracted 3 times with 35mL of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was dried by evaporation, and then separated by silica gel column chromatography to obtain solid intermediate X-III-03' c (70 mg), yield 62%.¹H NMR(400MHz,CD₃OD)δ7.69(brd,J＝8.1Hz,1H),7.44–7.31(m,2H),7.29‐7.24(m,2H),7.22‐7.16(m,1H),6.36(s,1H),5.76(d,J＝8.0Hz,1H),5.47–5.33(m,1H),4.96–4.92(m,1H),4.57‐4.49(m,1H),4.37–4.25(m,2H),4.12–4.07(m,1H),3.96–3.88(m,1H),2.16–2.10(m,1H),1.46(s,9H),1.36‐1.30(m,6H),1.24(d,J＝6.2Hz,6H)；³¹P NMR(162MHz,CDCl₃)δ2.99；LCMS‐ESI⁺(m/z):751.4[M+1]⁺。

Synthesis of X-III-03 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-III-03' c (70mg,93 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, followed by separation by silica gel column chromatography to give the product X-III-03 c, 52mg, with a yield of 86%.¹H NMR(400MHz,CD₃OD)δ7.54(d,J＝8.0Hz,1H),7.45‐7.39(m,2H),7.31‐7.21(m,3H),6.42(s,1H),5.68(d,J＝8.0Hz,1H),5.47–5.35(m,1H),5.03‐4.94，(m,1H)，4.55‐4.47(m,2H),4.39–4.33(m,1H),4.19–4.09(m,1H),3.98–3.87(m,1H),2.44–2.36(m,1H),1.40‐1.31(m,6H),1.25(d,J＝6.1Hz,3H),1.24(d,J＝6.0Hz,3H)；³¹P NMR(162MHz,CD₃OD)δ2.99；LCMS‐ESI⁺(m/z):651.2(M+H)⁺.

Example 42

((R) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine monodeuterated isopropyl ester (IX-1 b2)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 11c (727mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (25mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product IX-1 b2(317mg) as a white solid in 58% yield.¹H NMR(400MHz,CDCl₃)δ9.88(brs,1H),7.45–7.35(m,3H),7.29‐7.23(m,2H),7.20‐7.16(m,1H),6.41(s,1H),5.57(d,J＝8.0Hz,1H),5.02–4.95(m,1H),4.54‐4.45(m,2H),4.42–4.35(m,1H),4.31(brs,1H),3.97–3.78(m,2H),1.41‐1.29(m,12H)；³¹P NMR(162MHz,CDCl₃)δ3.66；LCMS‐ESI⁺(m/z):547.2(M+H)⁺.

IX-1 a1 was synthesized in the same manner,¹H NMR(400MHz,CDCl₃)δ9.93(brs,1H),73–7.36(m,3H),7.32‐7.23(m,2H),7.21‐7.18(m,1H),6.45(s,1H),5.66(d,J＝8.0Hz,1H),5.04–4.96(m,1H),4.55‐4.46(m,2H),4.44–4.37(m,1H),4.31(brs,1H),3.97–3.77(m,2H),1.42‐1.25(m,12H)；³¹P NMR(162MHz,CDCl₃)δ3.61；LCMS‐ESI⁺(m/z):547.2(M+H)⁺.

example 43

((S) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -D-alanine monodeuterated isopropyl ester (IX-1 b1)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorus reagent 11 c' (727mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product IX-1 b1(290mg) as a white solid in 53% yield.¹H NMR(400MHz,CDCl₃)δ9.78(brs,1H),7.44–7.33(m,3H),7.28‐7.23(m,2H),7.21‐7.13(m,1H),6.37(s,1H),5.52(d,J＝8.0Hz,1H),5.03–4.92(m,1H),4.51‐4.41(m,2H),4.47–4.31(m,1H),4.27(brs,1H),3.96–3.77(m,2H),1.40‐1.28(m,12H)；³¹P NMR(162MHz,CDCl₃)δ3.61；LCMS‐ESI⁺(m/z):547.2(M+H)⁺.

Example 44

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) -1-monodeodeuteroisopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-IV-03 b)

Synthesis of X-IV-03' b

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 μmol) is dissolved by 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 μmol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of IX-1 b2(82mg,0.15mmol), triethylamine (24 μ L,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 15mL of water, extracted for 3 times by 35mL of ethyl acetate, organic layers are combined, dried by anhydrous sodium sulfate and solvent is dried by spin drying, and then a silica gel column chromatography is carried out to obtain solid intermediate X-IV-03' b, 66mg and the yield is 59%.¹H NMR(400MHz,CD₃OD)δ7.83(d,J＝8.2Hz,1H),7.43–7.32(m,2H),7.29‐7.23(m,2H),7.21‐7.13(m,1H),6.39(s,1H),5.76(d,J＝8.0Hz,1H),5.42–5.32(m,1H),4.99–4.92(m,1H),4.55‐4.50(m,1H),4.39–4.25(m,2H),3.96–3.85(m,1H),2.19–2.10(m,1H),1.47(s,9H),1.43‐1.22(m,18H)；³¹P NMR(162MHz,CDCl₃)δ2.72；LCMS‐ESI⁺(m/z):746.4[M+1]⁺。

Synthesis of X-IV-03 b

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-IV-03' b (73mg,97.8 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water, saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-IV-03 b, 52.4mg, 83% yield.¹H NMR(400MHz,CD₃OD)δ7.57(d,J＝8.2Hz,1H),7.46‐7.38(m,2H),7.30‐7.23(m,3H),6.39(s,1H),5.75(d,J＝8.0Hz,1H),5.49–5.33(m,1H),5.01‐4.91，(m,1H)，4.53‐4.44(m,2H),4.36–4.32(m,1H),3.98–3.87(m,1H),2.48–2.35(m,1H),1.45‐1.21(m,18H)；³¹P NMR(162MHz,CD₃OD)δ3.48；LCMS‐ESI⁺(m/z):646.3(M+H)⁺.

Example 45

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((S) - ((R) -1-monodeodeuteroisopropyl-1-oxoprop-2-yl) amino) (phenoxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-IV-03 c)

Synthesis of X-IV-03' c

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 μmol) is dissolved in 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 μmol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then IX-1 b1(82mg,0.15mmol) of 0.5mL of 1, 4-dioxane solution, triethylamine (24 μ L,0.18mmol) and a catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 15mL of water, extracted for 3 times by 35mL of ethyl acetate, organic layers are combined, dried by anhydrous sodium sulfate, solvent is dried by spin drying, and then separated by silica gel column chromatography to obtain a solid intermediate X-IV-03' c, 70.8mg and the yield is 63.3%.¹H NMR(400MHz,CD₃OD)δ7.61(d,J＝8.0Hz,1H),7.42–7.33(m,2H),7.29‐7.24(m,2H),7.22‐7.17(m,1H),6.40(s,1H),5.76(d,J＝8.0Hz,1H),5.46–5.35(m,1H),4.95–4.91(m,1H),4.56‐4.51(m,1H),4.38–4.25(m,2H),3.88–3.76(m,1H),2.21–2.12(m,1H),1.45(s,9H),1.39‐1.16(m,18H)；³¹P NMR(162MHz,CDCl₃)δ2.90；LCMS‐ESI⁺(m/z):746.4[M+1]⁺。

Synthesis of X-IV-03 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-IV-03' c (70mg,93.8 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water, saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-IV-03 c, 48.5mg, with a yield of 80%.¹H NMR(400MHz,CD₃OD)δ7.66(d,J＝8.2Hz,1H),7.45‐7.33(m,2H),7.30‐7.21(m,3H),6.44(s,1H),5.70(d,J＝8.0Hz,1H),5.40–5.33(m,1H),5.00‐4.93，(m,1H)，4.55‐4.44(m,2H),4.38–4.32(m,1H),3.99–3.86(m,1H),2.46–2.35(m,1H),1.45‐1.24(m,18H)；³¹P NMR(162MHz,CD₃OD)δ2.97；LCMS‐ESI⁺(m/z):646.2(M+H)⁺.

Example 46

((R) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (pentadeuteroxyphenoxy) phosphoryl) -D-alanine isopropyl ester (IX-4 b2)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorous reagent 11e (733mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). Is divided outThe organic phase, the aqueous layer were extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give the product IX-4 b2(281mg) as a white solid in 51% yield.¹H NMR(400MHz,CDCl₃)δ9.82(brs,1H),7.46(d,J＝8.0Hz,1H),6.37(s,1H),5.55(d,J＝8.0Hz,1H),5.01–4.94(m,1H),4.53‐4.47(m,2H),4.43–4.32(m,1H),4.25(brs,1H),4.18–4.01(m,1H),3.96–3.77(m,2H),1.42‐1.21(m,12H)；³¹P NMR(162MHz,CDCl₃)δ3.79；LCMS‐ESI⁺(m/z):551.2(M+H)⁺.

Example 47

((S) - (((2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy) (pentadeuteroxyphenoxy) phosphoryl) -D-alanine isopropyl ester (IX-4 b1)

To a 10mL reaction tube were added nucleoside 18a (276.7mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. Tert-butylmagnesium chloride Grignard reagent (3.0mL of a 1M solution in THF, 3.0mmol) was added dropwise and the reaction mixture was stirred at O ℃ for 30min, followed by addition of a solution of phosphorous reagent 11 e' (733mg, 1.6mmol) in 5mL THF at 0 ℃. The resulting clear reaction solution was warmed and stirred for 1 day. Adding saturated NH₄Cl (15mL), stirred for 5 min, and the mixture was diluted with ethyl acetate (200 mL). The organic phase was separated and the aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO₃(2X3OmL), brine (3OmL) and Na₂SO₄And (5) drying. The solvent was evaporated under reduced pressure and the residue was chromatographed on silica gel column (0-10% methanol in dichloromethane)) Purification gave product IX-4 b1(331.7mg) as a white solid in 60.2% yield.¹H NMR(400MHz,CDCl₃)δ9.78(brs,1H),7.58(d,J＝8.1Hz,1H),6.34(s,1H),5.59(d,J＝8.1Hz,1H),5.01–4.90(m,1H),4.52‐4.43(m,2H),4.41–4.30(m,1H),4.28(brs,1H),4.21–4.08(m,1H),3.92–3.79(m,2H),1.39‐1.16(m,12H)；³¹P NMR(162MHz,CDCl₃)δ3.61；LCMS‐ESI⁺(m/z):551.2(M+H)⁺.

IX-4 a1 was synthesized in the same manner,¹H NMR(400MHz,CDCl₃)δ9.82(brs,1H),7.58(d,J＝8.1Hz,1H),6.42(s,1H),5.67(d,J＝8.1Hz,1H),5.01–4.92(m,1H),4.52‐4.44(m,2H),4.42–4.32(m,1H),4.29(brs,1H),4.22–4.09(m,1H),3.94–3.79(m,2H),1.39‐1.18(m,12H)；³¹P NMR(162MHz,CDCl₃)δ3.53；LCMS‐ESI⁺(m/z):551.2(M+H)⁺.

example 48

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((R) -1-isopropyl-1-oxoprop-2-yl) amino) (pentadeuteroxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-X-03 b)

Synthesis of X-X-03' b

Dissolving the amino acid Boc-Val-OH (49mg, 225. mu. mol) in 0.5mL1, 4-dioxane in a reaction tube, cooling to-5 ℃, adding DCC (46.5mg, 225. mu. mol) under stirring, reacting at room temperature for 30min, further cooling to-5 ℃, and then sequentially adding 0.5mL1, 4-dioxane solution of IX-4 b2(82.7mg,0.15mmol)Triethylamine (24 μ L,0.18mmol) and a catalytic amount of 4-Dimethylaminopyridine (DMAP) were reacted at room temperature overnight, the reaction was completed, poured into 15mL of water, extracted 3 times with 35mL of ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was dried by spin-drying, separated by silica gel column chromatography to obtain a solid intermediate X-03' b, 73.1mg, yield 65%.¹H NMR(400MHz,CD₃OD)δ7.67(d,J＝8.0Hz,1H),6.39(s,1H),5.73(d,J＝8.2Hz,1H),5.43–5.32(m,1H),4.97–4.91(m,1H),4.56‐4.51(m,1H),4.40–4.24(m,2H),4.19‐4.06(m,1H),3.97–3.85(m,1H),2.19–2.10(m,1H),1.46(s,9H),1.43–0.99(m,18H)；³¹P NMR(162MHz,CDCl₃)δ2.87；LCMS‐ESI⁺(m/z):750.4[M+1]⁺。

Synthesis of X-X-03 b

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-X-03' b (72mg,96 μmol) under stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-X-03 b, 56mg, with a yield of 90%.¹H NMR(400MHz,CD₃OD)δ7.58(d,J＝8.1Hz,1H),6.39(s,1H),5.55(d,J＝8.2Hz,1H),5.48–5.35(m,1H),5.02‐4.92，(m,1H)，4.55‐4.48(m,2H),4.38–4.32(m,1H),4.15–4.10(m,1H),3.93–3.85(m,1H),2.48–2.33(m,1H),1.43‐1.10(m,18H)；³¹P NMR(162MHz,CD₃OD)δ3.34；LCMS‐ESI⁺(m/z):650.3(M+H)⁺.

Example 49

(2R,3R,4R,5R) -5- (2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -4-chloro-2- (((((S) - (((R) -1-isopropyl-1-oxoprop-2-yl) amino) (pentadeuteroxy) phosphoryl) oxy) methyl) -4-methyltetrahydrofuran-3-yl L-valine ester (X-X-03 c)

Synthesis of X-X-03' c

In a reaction tube, the amino acid Boc-Val-OH (49mg,225 μmol) is dissolved by 0.5mL of 1, 4-dioxane, the temperature is reduced to-5 ℃, DCC (46.5mg,225 μmol) is added under stirring, the reaction is carried out for 30min at room temperature, the temperature is further reduced to-5 ℃, then 0.5mL of 1, 4-dioxane solution of IX-4 b1(82.7mg,0.15mmol), triethylamine (24 μ L,0.18mmol) and catalytic amount of 4-Dimethylaminopyridine (DMAP) are sequentially added, the reaction is carried out overnight at room temperature, after the reaction is finished, the mixture is poured into 15mL of water, extracted by 35mL of ethyl acetate for 3 times, organic layers are combined, dried by anhydrous sodium sulfate, and solvent is dried by spin-drying, and then separated by silica gel column chromatography to obtain solid intermediate X-X-03' c, 69mg and the yield is 61%.¹H NMR(400MHz,CD₃OD)δ7.62(d,J＝8.1Hz,1H),6.34(s,1H),5.73(d,J＝8.0Hz,1H),5.48–5.37(m,1H),4.97–4.92(m,1H),4.54‐4.47(m,1H),4.37–4.26(m,2H),4.08‐4.01(m,1H),3.92–3.84(m,1H),2.21–2.11(m,1H),1.44(s,9H),1.38‐0.97(m,18H)；³¹P NMR(162MHz,CDCl₃)δ2.89；LCMS‐ESI⁺(m/z):750.2[M+1]⁺。

Synthesis of X-X-03 c

Adding 4M HCl 1, 4-dioxane (2mL) into a reaction tube, cooling to 0 deg.C, adding intermediate X-X-03' c (72mg,96 μmol) while stirring, reacting at room temperature for 1 hr, pouring into 15mL water, extracting with 35mL ethyl acetate for three times, combining organic layers with water and saturated NaHCO₃Washed with brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin-drying, and then separated by silica gel column chromatography to give the product X-X-03 c, 54.3mg, yield 87%.¹H NMR(400MHz,CD₃OD)δ7.65(d,J＝8.1Hz,1H),6.33(s,1H),5.66(d,J＝8.1Hz,1H),5.45–5.37(m,1H),5.01‐4.91，(m,1H)，4.55‐4.45(m,2H),4.38–4.35(m,1H),4.19–4.11(m,1H),3.97–3.87(m,1H),2.47–2.38(m,1H),1.43‐1.12(m,18H)；³¹P NMR(162MHz,CD₃OD)δ2.90；LCMS‐ESI⁺(m/z):650.2(M+H)⁺.

EXAMPLE 50 preparation of hydrochloride salt of Compound

Sofosbuvir can not be salified under alkaline or acidic conditions, and has poor solubility in neutral water. In the invention, the compound protected by the amino acid ester at the 3' -position can form a salt with acid, so that the solubility of the compound in water can be increased, and the preparation is convenient.

Preparation of compound hydrochloride salt: dissolving the compound (X-I-02 b, X-I-02 c, X-I-05 b, X-I-05 c, X-I-25 b, X-I-25 c, X-I-29 b, X-I-29 c, X-II-03 b, X-II-03 c, X-III-03 b, X-III-03 c, X-IV-03 b, X-IV-03 c, X-X-03 b, X-X-03 c) protected by amino acid ester at the 3' -position with ethyl acetate, dripping 1M HCl ethyl acetate solution at 0 ℃, stirring for 10min, adding cyclohexane, precipitating, washing the precipitate with cyclohexane for three times, removing the solvent under reduced pressure, and drying to obtain a hydrochloride product.

The above-mentioned method for synthesizing a nucleoside D-type phosphoramidate compound can be used for synthesizing a similar nucleoside L-type phosphoramidate compound.

Biological evaluation

1. Detection of antiviral Activity of Compounds of the present invention in HCV replicon (HCVpp) System

HCV replicon assay procedure

General procedure Huh-7 derived cell lines (Zluc) harboring HCV genotype 1b replicon and luciferase reporter genes were supplemented with 10% fetal bovine serum, 2mM GlutaMAX, 1% MEM nonessential amino acids, 100IU/mL penicillin, 100. mu.g/mL streptomycin, and 0.5mg/mL(G418) Dulbecco's Modified Eagle's Medium (DMEM).Zluc cells were transiently transfected with human carboxylesterase 1(CES1) by using a lipid/histone based transfection procedure. 24 and 48 hours after transfection, expression of CES1 was confirmed by Western blotting (Western blot) using anti-CES 1 and anti-tag antibodies. For dose response testing, 7.5xl0³Cells/well, in a volume of 50 μ L, cells were seeded in 96-well plates and at 37 ℃/5% CO₂And (4) incubating. Drug solutions were freshly prepared in Huh-7 medium as 2X stock solutions. 10 additional 5-fold dilutions were prepared from these stocks in DMEM without G418. At least 3 hours after seeding with the Huc cells, drug treatment was started by adding 50 μ Ι _ of drug dilution to the plates in duplicate. The final concentration of the drug ranges from 1OOnM to 0.0000512 nMm. Cells were then incubated at 37 ℃/5% CO₂And (4) incubating. Alternatively, compounds were tested at two concentrations (1OnM and 1 OOnM). In all cases, Huh-7 (which does not carry an HCV replicon) was used as a negative control. Inhibition of HCV replication was measured by quantifying the photons emitted by the singlet oxidation of 5' -fluoroluciferin to oxyfluoroluciferin (oxyiuoroluteciferin) by firefly luciferase after 72 hours of incubation. To this end, the medium was removed from the plate by tapping and 50 microliters of ONE-glo luciferase assay reagent was added to each well. The plate was gently shaken for 3 minutes at room temperature using a 700nm cut-off filter at Victor with a1 second read-out time³Luminescence was measured on a V1420 multiple mark counter (PerkinElmer). EC was calculated from the dose-response curve of the resulting best-fit equation, as determined by Microsoft Excel and XLFit4.1 software₅₀The value is obtained. For cytotoxicity evaluation, Zluc cells were treated with the above compounds, and cell viability was monitored by adding 20 μ L of assay solution to each well using CellTiter-Blue cell viability assay (Promega). The plates were then incubated at 37 ℃/5% CO2 for at least 3 hours. With excitation and emission wavelengths of 560 and 590nm, respectively, at Victor³Fluorescence of the plate was detected in a V1420 MultiMark counter (Perkin Elmer) and CC was determined using Microsoft Excel and XLFit4.1 software₅₀The value is obtained.

The compounds provided in the table below were determined according to the replicon assay described above.

^*Sofosbuvir is prepared according to reference j

2. In vivo PK assay data for the Compounds of the examples

HCV NS5B is an RNA polymerase responsible for HCV viral replication using nucleoside triphosphates as substrates. The compound of the embodiment of the invention is a phosphoramidate nucleotide analogue prodrug which is metabolized in liver cells to generate an active ingredient nucleoside triphosphate analogue, thereby inhibiting the activity of NS5B and the replication of HCV virus. As a prodrug, the ability to produce uridine triphosphate analogs in vivo was directly correlated with inhibitory activity against NS5B, and therefore the inventors performed an in vivo PK assay to test the ability of the compounds of the invention to produce the active drug uridine triphosphate analogs, an in vivo PK assay model being a CD-1 mouse in vivo liver PK assay.

Examples pharmacokinetic experiments of the compounds metabolized in the mouse liver to the active ingredient 2 '-methyl-2' -chlorouridine triphosphate were performed with CD-1 mice. The administration was a single gavage, at a dose of 10 mg/5 ml/kg. The prescription of the preparation of the sofosbuvir is 20 percent of PEG200 and 0.5 percent of sodium carboxymethyl cellulose; the formulation of the compound of the examples was formulated with 30% PEG200 and 0.5% sodium carboxymethylcellulose. The sampling points for the liver samples were 1,2,4, 6, 12, 24 hours after dosing. When sampling, mice were first CO-administered₂Sacrificed, livers washed with ice cold saline through the hepatic portal vein, cut into small pieces of approximately 0.2 grams of liver samples, snap frozen in liquid nitrogen, and stored at-80 ℃.

The liver samples were analyzed by LC-MS/MS for active species nucleoside triphosphates. The Internal Standard (IS) adopts (2 ' R) -2 ' -deoxy-2 ' -methyl-2 ' -chlorouridine triphosphate or (2 ' R) -2 ' -deoxy-2 ' -methyl-2 ' -fluorouridine triphosphate, and uses tandem mass spectrometry in positive ion mode to detect and quantify a sample to be detected on an API5500 type liquid chromatograph-mass spectrometer of the American AB company, so as to measure the exposure amount (2 ' -deoxy-2 ' -Me-2 ' -Cl-U TP AUC) of the mouse liver active drug (2 ' R) -2 ' -deoxy-2 ' -methyl-2 ' -chlorouridine triphosphate.

As can be seen from the table of the test results of epimers prepared in examples, R in the compounds of examples_pThe concentration of the formed compound in the liver cell to generate 2 ' -deoxy-2 ' -Me-2 ' -Cl-U TP is far better than that of S_pThe configurational isomer, too, far exceeds the liver concentration of sofosbuvir at the same dose. The in vitro activity of the compound (D-alanine phosphate prodrug) prepared in all the examples is higher than that of sofosbuvir (L-alanine phosphate prodrug), the in vivo activity of the compound is more remarkable and superior to that of the sofosbuvir, and the compound shows extremely high clinical application value.

The experimental results show that the compound has high-efficiency HCV virus inhibition capacity, and shows remarkable superiority under in-vitro and in-vivo activity evaluation compared with a positive control medicament sofosbuvir.

Although the present invention has been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the present invention without departing from the spirit and scope of the invention. The scope of the invention is not to be limited by the above detailed description but is only limited by the claims.

Claims

1. A nucleoside phosphoramidate compound characterized by: is a compound of the formula:

2. the nucleoside phosphoramidate compound of claim 1 wherein: mixing with pharmaceutically acceptable carrier, diluent or excipient to prepare pharmaceutical preparation suitable for oral or parenteral administration; methods of administration include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes.

3. A pharmaceutical composition comprising the nucleoside phosphoramidate compound of claim 1.

4. The pharmaceutical composition of claim 3, wherein: further comprising an additional therapeutic agent independently selected from the group consisting of: ribavirin, interferon, hepatitis c NS3 protease inhibitors, HCV reverse transcriptase NS5B non-nucleoside inhibitors, HCV reverse transcriptase NS5B nucleoside inhibitors, NS5A inhibitors, and potentiators of NS5A inhibitors, entry inhibitors, cyclosporin immunosuppressants, NS4A antagonists, NS4B inhibitors, cyclophilin inhibitors.

5. Use of a nucleoside phosphoramidate compound according to claim 1 in the manufacture of a medicament against a flaviviridae virus, wherein: the flaviviridae virus is hepatitis c virus.