Phosphamide ester nucleoside drug compound as well as synthesis method and drug application thereof
By adopting the phosphoramidate prodrug strategy in nucleoside antiviral drugs, the design and synthesis of new phosphoramidate nucleoside compounds has been solved, and the problems of difficulty in entering cells and slow activation process caused by the carrying of negative electrons in the internal environment of existing drugs have been solved, and the stability and bioavailability of drugs have been improved.
Patent Information
- Application Number
- CN202510174062.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-18
- Publication Date
- 2025-05-23
AI Technical Summary
Existing nucleoside antiviral drugs carry negative electrons in the internal environment, enter cells and rely on endocytosis of the cell membrane. The activation process is slow, the targeting effect is weak and may cause cytotoxicity.
Through the phosphoramidate prodrug strategy, a new phosphoramidate nucleoside compound was designed and synthesized, and phosphorus oxychloride and N-(4-hydroxyphenyl)acetamide were used as raw materials. After multiple reactions such as bromination, hydrolysis, overprotecting group, carbonyl reduction and glycosylation, bioactive compounds were obtained.
It effectively avoids dependence on nucleoside kinases, resists metabolic inactivation of phosphate hydrolase, enhances the stability of the drug, and improves bioavailability and therapeutic effects.
Smart Images
Figure CN120025389A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of organic chemistry, and specifically relates to a preparation method of phosphoramidate nucleoside compounds and a synthesis method and pharmaceutical application thereof. Background Art
[0002] The phosphoramidate prodrug strategy refers to the use of amino acid esters and phenol derivatives to mask the negative charge of phosphate to generate a phosphoramidate structure. This prodrug strategy is also the most successful strategy in the application of nucleoside antiviral drugs so far.
[0003] In the phosphoramidate prodrug strategy, the negative charge of the phosphate group is masked by the aromatic group and the amino acid ester group to be electrically neutral, and then enters the cell through the cell membrane. In the cell, the amino acid ester is first hydrolyzed by esterase to generate a carboxyl group, and then the carboxyl group loses a hydrogen to form an oxygen anion. The oxygen anion attacks the phosphate group by nucleophilic addition, causing the aromatic group to fall and generate an unstable five-membered ring. Then the five-membered ring is hydrolyzed to open the ring to generate an aminophosphoroester metabolite, and finally the PN bond is broken by the action of phosphoramidase (histidine triad nucleotide binding protein 1, HINT-1) to generate an active monophosphate nucleoside drug molecule. Sofosbuvir and tenofovir alafenamide were developed based on this strategy.
[0004] Nucleoside prodrugs designed using the phosphoramidate prodrug strategy have three sites that can be modified: (1) modification of the amino acid ester side chain: the configuration of the amino acid side chain directly affects the activity of the drug. Experiments have shown that the drug activity of L-configured amino acids is far superior to that of D-configured amino acids; (2) modification of the amino acid ester group: the introduction of primary alkanes, secondary alkanes or benzyl groups into the ester group makes it easier to be hydrolyzed by esterases; (3) modification of the aromatic group: the introduction of strong electron-withdrawing groups or halogens into the aromatic benzene ring not only facilitates the departure of the aromatic group, but also facilitates the nucleophilic attack of the carboxyl group on the phosphate ester to generate a five-membered ring intermediate and release the aminophosphoryl metabolite intermediate.
[0005] At present, nucleoside antiviral drugs have become the mainstream of small molecule broad-spectrum antiviral drugs. As more and more nucleoside antiviral drugs are born and used in practice, some of its shortcomings are gradually revealed. First, because nucleoside antiviral drugs carry negative electrons in the body environment, their entry into cells mainly depends on the endocytosis of the cell membrane, and their concentration is low at the pathogen; secondly, the rate-determining step for nucleoside antiviral drugs to enter the cell and become active triphosphate metabolites is that they are activated by nucleoside kinase to monophosphate intermediates, which is a relatively slow process and slows down their onset of action; finally, because most cells in the human body have nucleoside kinases, nucleoside antiviral drugs have weak targeting effects and may even produce cytotoxicity.
[0006] In view of the above shortcomings of the prior art, the present invention designs and synthesizes a series of novel phosphoramidate nucleoside drug prodrugs through the phosphoramidate prodrug strategy, which effectively avoids the dependence of such drugs on nucleoside kinases, resists the metabolic inactivation of phosphohydrolases, and enhances their stability. It repairs the defect that such drugs are not easy to act on target cells due to their ionic characteristics, improves bioavailability, and greatly improves the therapeutic effect. Summary of the invention
[0007] In order to overcome the above technical defects, the present invention provides a novel phosphoramidate nucleoside compound, and studies its synthesis method and active application of the compound. Using phosphorus oxychloride and N-(4-hydroxyphenyl)acetamide as raw materials, a multi-step continuous reaction of bromination, hydrolysis, protection group addition, carbonyl reduction and glycosylation is adopted to obtain a phosphoramidate nucleoside compound containing biological activity. The novel phosphoramidate nucleoside drug prodrug effectively avoids the dependence of this type of drug on nucleoside kinase, resists the metabolic inactivation of phosphohydrolase, enhances its stability, repairs the defect that this type of drug is not easy to act on target cells due to its ionic characteristics, and improves bioavailability.
[0008] The phosphoramidate nucleoside compound of the present invention has the following general structural formula:
[0009]
[0010] Wherein: the glycoside is selected from (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, 5-fluoro-2'-deoxyurea nucleoside, cytarabine, ribavirin, cytidine, stavudine, 2',3'-O-isopropylcytidine, 5-bromocytidine, gemcitabine, zidovudine or lamivudine, and the 5'-hydroxyl group in the glycoside is connected to the phosphorus atom.
[0011] The present invention also provides a method for synthesizing the novel phosphoramidate nucleoside drug prodrug, comprising the following steps: using phosphorus oxychloride 1 and N-(4-hydroxyphenyl)acetamide 2 as raw materials, reacting in an organic solvent in the presence of an organic base to obtain an intermediate 3; reacting intermediate 3 with L-alanine isopropyl ester hydrochloride 4 in an organic solvent in the presence of an organic base to obtain an intermediate 5; reacting intermediate 5 with pentafluorophenol 6 in an organic solvent in the presence of an organic base to obtain an intermediate 7; and reacting intermediate 7 with glycoside 8 in an organic solvent in the presence of a catalyst to obtain a product 9.
[0012] The reaction equation is shown as follows:
[0013]
[0014] Wherein: the glycoside is selected from (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, 5-fluoro-2'-deoxyurea nucleoside, cytarabine, ribavirin, cytidine, stavudine, 2',3'-O-isopropylcytidine, 5-bromocytidine, gemcitabine, zidovudine or lamivudine, and the 5'-hydroxyl group in the glycoside is connected to the phosphorus atom.
[0015] Furthermore, in the first step of the above technical scheme, the organic solvent is selected from dichloromethane, the reaction temperature is selected from -78°C to -60°C, the reaction is carried out in an inert gas, and the molar ratio of compound 1, compound 2 and the organic base is 1:1:1.
[0016] Furthermore, in the second step of the above technical scheme, the organic solvent is selected from dichloromethane, chloroform or 1,2-dichloroethane; the reaction temperature is selected from -78°C to 30°C, the reaction is carried out in an inert gas, and the molar ratio of intermediate 3, L-alanine isopropyl hydrochloride and organic base is 1:1:2.
[0017] Furthermore, in the second step of the above technical scheme, the source of L-alanine isopropyl hydrochloride 4 is: using L-alanine and isopropanol as raw materials, reacting in an organic solvent in the presence of a catalyst thionyl chloride to obtain compound 4.
[0018] Furthermore, in the third step of the above technical scheme, the organic solvent is selected from dichloromethane, chloroform or 1,2-dichloroethane; the reaction temperature is selected from 0-30°C, the reaction is carried out in an inert gas, and the molar ratio of intermediate 5, pentafluorophenol and organic base is 1:0.8:2.
[0019] Furthermore, in the fourth step of the above technical scheme, the organic solvent is selected from DMF and DMSO; the reaction temperature is selected from 0-40°C, the reaction is carried out in an inert gas, and the molar ratio of intermediate 7, glycoside 8 and catalyst is 2:1:2.
[0020] Furthermore, in the first to third steps of the above technical scheme, the organic base is selected from triethylamine, diisopropylethylamine or pyridine; in the fourth step, the catalyst is selected from tert-butylmagnesium chloride.
[0021] The present invention also provides the use of the novel phosphoramidate nucleoside drug prodrug in the preparation of antiviral drugs.
[0022] Furthermore, in the above technical solution, the antiviral agent is selected from HepG2 / 2.2.15 cells which can stably and sustainably express HBV virus.
[0023] The present invention also provides a pharmaceutical composition containing the aforementioned phosphoramidate nucleoside drug prodrug.
[0024] Advantageous Effects of the Invention
[0025] 1. The present invention adopts a one-pot synthesis, the reaction raw materials are easily available, the synthesis steps are simple, and the reaction conditions are mild.
[0026] 2. The present invention uses the phosphoramidate prodrug strategy to design and synthesize a series of novel phosphoramidate nucleoside compounds, which effectively avoids the dependence of such drugs on nucleoside kinases, resists the metabolic inactivation of phosphohydrolases, and enhances their stability. This repairs the defect that such drugs are not easy to act on target cells due to their ionic characteristics, improves bioavailability, and greatly improves the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The biological activity test steps of the novel phosphoramidate nucleoside compound in Example 4;
[0028] Figure 2 is the relative expression of HBV DNA in HepG2 / 2.2.15 cells by the novel phosphoramidate nucleoside compound in Example 4;
[0029] Figure 3 EC values for inhibiting DNA replication calculated by the detection of HBV activity in HepG2 / 2.2.15 cells with different concentrations of novel phosphoramidate nucleoside compounds in Example 4 50 ;
[0030] Figure 4 CC was calculated by using MTT colorimetric method to detect the cytotoxicity of the novel phosphoramidate nucleoside compounds at different concentrations in Example 4. 50 . Specific embodiments
[0031] The present invention will be further described below by specific examples. These embodiments should be understood to be only used to illustrate the present invention and not to limit the scope of protection of the present invention. After reading the content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.
[0032] Example 1
[0033]
[0034] In anhydrous, nitrogen environment, add 200 mL of ultra-dry dichloromethane to a 500 mL two-necked bottle with three-way, place it in a -78°C cold trap, wait until the solution temperature drops to -78°C, add acetaminophenol 2 (15.12 g, 100 mmol) and ultra-dry triethylamine (13.90 mL, 100 mmol) to the solution, then add phosphorus oxychloride 1 (9.32 mL, 100 mmol) dropwise, then react at -78°C for 3.5 hours, and monitor the reaction by TLC. After the reaction of N-(4-hydroxyphenyl)acetamide is complete, add L-alanine isopropyl ester hydrochloride 4 (16.75 g, 100 mmol) to the above 3 reaction solution, stir at -78°C for 15 minutes, add ultra-dry triethylamine (27.80 mL, 200 mmol) and continue stirring for 1 hour, then move the reaction solution to room temperature and stir for 1.5 hours. The reaction was placed in an ice-water bath, and the reaction solution was cooled to 0°C. Super dry triethylamine (27.80 mL, 200 mmol) was first added to the reaction solution 5, and then pentafluorophenol 6 (9.43 mL, 90 mmol) was added dropwise in small amounts several times, and then the reaction solution was moved to room temperature and stirred overnight. TLC monitored the reaction. After the reaction was complete, the reaction system was placed in an ice-water bath to cool, and 150 mL of saturated ammonium chloride solution was quickly added dropwise while stirring to quench, and the organic phase was separated. The aqueous phase was extracted with dichloromethane (50 mL*3), and the organic phases were combined and washed with saturated ammonium chloride solution (200 mL*2), purified water (200 mL*3), and saturated sodium chloride solution (200 mL*2). After washing, the filter funnel was filtered, and the solid dichloromethane was rinsed 3 times (30 mL*3). The filtrate was concentrated under reduced pressure and separated by silica gel column chromatography. The eluent was petroleum ether / ethyl acetate / dichloromethane (15:1:1-3:1:1). The product was 7, a white solid (33.71 g), with a total yield of 73.4% over three steps. 1 H NMR (400 MHz, DMSO-d 6 )δ10.02(s,1H),7.57(d,J=9.0Hz,2H),7.14(dd,J=9.0,1.3Hz,2H),6.80(dd,J=14.1,9.9Hz,1H),4 .95-4.77(m,1H),3.98-3.80(m,1H),2.03(s,3H),1.26(dd,J=7.1,1.1Hz,3H),1.15(dd,J=6.3,1.5H z,6H). 13 C NMR (101 MHz, DMSO-d 6)δ173.0(d,J=6.0Hz),168.6,163.3,150.9,146.3(d,J=7.0Hz),136.5,120.6(d,J=4.0Hz),120.4,68.5,5 0.2, 49.1, 26.0, 24.3, 21.8 (d, J = 3.0Hz), 20.3 (d, J = 6.0Hz), 18.0, 17.4 (d, J = 25.0Hz), -4.1 (d, J = 31.0Hz). 19 F NMR (377 MHz, DMSO-d 6 )δ-153.66(d,J=21.6Hz),-160.41(t,J=23.4Hz),-163.15(t,J=22.1Hz),-163.21. 31 P NMR (162 MHz, DMSO-d 6 )δ0.64.HRMS(ESI)m / z:[M+Na] + Calcd for C 20 H 20 F 5 N 2 O 6 PNa 533.0871; Found 533.0870.
[0035] Example 2
[0036]
[0037] The 10mL Schlenk tube was evacuated 3 times on a double row, heated with a baking gun, and a small magnetic particle was placed in the aerated state while it was hot, and the small magnetic particle was dried with residual heat, and then evacuated 3 times, and the aeration was maintained for 1 hour. Compound 7 (0.4mmol) and glycoside 8 (0.2mmol) were placed in the Schlenk tube under aeration, and then ultra-dry DMF (2.5mL) was added, stirred evenly, and the aeration state was maintained for 2 hours. It was moved to an ice-water bath, and when the temperature of the solution dropped, 0.4mL of tert-butylmagnesium chloride (1M in THF, 0.4m mol) was slowly added to the solution under aeration, and the lid was covered and stirred in an ice-water bath for half an hour, and then moved to a 40℃ sand bath for reaction for 3.5 days. Add 3 mL of ethyl acetate and 3 mL of saturated ammonium chloride solution to the Schlenk tube for quenching, separate the organic phase, extract the aqueous phase with ethyl acetate (10 mL*3), combine the organic phases, wash with saturated ammonium chloride solution (20 mL), purified water (20 mL*2), and saturated sodium chloride solution (20 mL), dry over anhydrous sodium sulfate, concentrate the organic phase under reduced pressure, separate by silica gel column chromatography, eluent is dichloromethane / methanol (100-20 / 1), to obtain product C-4. 1H NMR (400 MHz, DMSO-d 6 )δ9.94(s,1H),7.72(d,J=7.5Hz,1H),7.53(d,J=9.0Hz,2H),7.26(d,J=26.5Hz,2H),7.17-7.07( m,2H),6.23(t,J=5.6Hz,1H),6.04(dd,J=13.3,10.1Hz,1H),5.73(d,J=7.5Hz,1H),5.35(dd,J=5. 1,3.6Hz,1H),4.90-480(m,1H),4.33-4.17(m,2H),3.85-3.70(m,1H),3.40(dd,J=11.6,5.3Hz,2 H),3.04(dd,J=11.5,6.0Hz,1H),2.02(s,3H),1.21(d,J=7.1Hz,3H),1.15(dd,J=6.3,1.6Hz,6H). 13 C NMR (101 MHz, DMSO-d 6 )δ173.1(d,J=5.0Hz),168.6,166.1,155.0,146.3(d,J=7.0Hz),141.2,136.5,120.6(d,J=5.0Hz),120 .5,94.7,87.4,82.1(d,J=9.0Hz),68.5,67.1(d,J=5.0Hz),50.2,36.2,24.4,21.9,20.2(d,J=6.0Hz). 31 P NMR (162 MHz, DMSO-d 6 )δ3.54.HRMS(ESI)m / z:[M+Na] + Calcd for C 22 H 30 N 5 O 8 PSNa 5578.1444; Found578.1443.
[0038] Example 3
[0039] A series of compounds were synthesized using the same method as above, and the structural formula is as follows:
[0040]
[0041] Example 4
[0042] This example is a test process and result of the inhibition of HBV virus replication in cells by the compounds described in the previous examples. The materials and consumables listed in this example can be obtained from commercial channels unless otherwise specified, and the cells and viruses are from the cell and microbial resource library of CTCC or other relevant institutions. The experimental method of this example is a standard molecular biology, cell biology or virology operation procedure, which can be easily understood and operated by researchers in the field.
[0043] The specific steps are as follows:
[0044] 1. Cells:
[0045] HepG2 / 2.2.15 (HBV ayw inserted, D subtype, 3182 bp, stable expression), culture conditions: DMEM+10% FBS+1% (strep++pen+)+380 μg / ml G418+0.2% L-Glutamine+0.2% non-essential amino acids.
[0046] Note: HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome. The majority of HBV DNA appears to be present in the form of covalently closed circular (ccc) and nicked recircularized forms, as well as incomplete copies of the genome.
[0047] 2. Main reagents:
[0048] DMEM (Gibco, cat:C11995500BT);
[0049] Fetal bovine serum FBS (Gibco, cat: 10270-106);
[0050] Double Pen-Strep (10,000U / mL) (M&C gene biotechnology, cat: G2723M3);
[0051] Anti-HBV positive drugs: 3TC (lamivudine; gift from Academician Chang Junbiao);
[0052] Tguide Smart Universal Genomic DNA Extraction Kit (TIANGEN, mat: 4995051)
[0053] 3. Experimental steps:
[0054] Step 1: Figure 1 As shown, the initial screening of compounds inhibiting HBV activity:
[0055] Two control groups were set up in the experiment, namely: ① Positive drug group: adding 3TC with a final concentration of 50 μM; ② Virus control group: adding only DMSO without compound. HepG2 / 2.2.15 cells, 1x105 cells / well, 12-well plate, 750 μL / well. After 12 - 16 h when the cells were completely adherent, 750 μL of DMEM and 1.5 μL of the compound to be tested (5 mM) were added, and the final concentration of the compound was 5 μM. On the 4th day of culture at 37 °C, 250 μL of the culture medium containing the compound to be tested at 5 μM was supplemented. On the 6th day of culture at 37 °C, 250 μL of the culture medium containing the compound to be tested at 5 μM was supplemented. On the 8th day of culture at 37 °C, the cells and the culture supernatant were collected. The DNA of the samples was extracted by an automatic nucleic acid extractor, and the inhibition rate of the compound on HBV virus was determined by qPCR (real-time fluorescence quantitative polymerase chain reaction). Calculate the EC for inhibiting virus replication 50 . It can be seen from Figure 3 that the EC of compound C-4 50 = 21.36 μM.
[0056] Supplementary: qPCR detection:
[0057] Primer: F Primer: 5’-CCTAGTAGTCAGTTATGTCAAC-3’
[0058] R Primer: 5’-TCTATAAGCTGGAGGAGTGCGA-3’
[0059] Amplified gene: C gene
[0060] Amplification conditions: 95 °C, 10 min;
[0061] 95 °C, 15 s; 60 °C, 30 s; 72 °C, 30 s × 40
[0062] Step 2. Detection of cytotoxicity by MTT colorimetric method: 5 × 10 4 cells were inoculated into a 96-well cell culture plate containing 100 μL of cell culture medium; 100 μL of the compound solution was added; there were 3 replicates for each concentration; at the same time, a control without compound was set; after 72 h of cell culture, 100 μL of the supernatant was discarded, 20 μL of MTT (5 mg / mL) was added, and the cells were cultured at 37 °C for 4 h; centrifuged, 100 μL of the supernatant was discarded, 100 μL of DMSO was added, and it was shaken in the dark at room temperature for 15 minutes until the blue formazen was dissolved; the OD595 was detected by an enzyme-linked immunosorbent assay reader, with OD630 as the reference wavelength, and the cell survival rate was calculated, and the toxicity CC of the compound was calculated 50 . It can be seen from Figure 4 that the CC of compound C-4 50 = 419.3 μM.
[0063] Step 3: Calculation of the drug antiviral activity selectivity index: Selective Index (SI) = CC 50 / EC 50 .
[0064] 4. Test results:
[0065] Initial screening of anti-HBV activity at a single concentration (5 μM) Figure 2 )
[0066] (Compound concentration: 5 μM)
[0067] (Control drug 3TC concentration: 50μM)
[0068] Conclusion: Under the condition of 5μM concentration, the difference between the expression of HBV DNA of C-4 compound and DMSO control group reached a very significant level. The relative expression of HBV DNA of C-4 compound reached 7.8%, and the inhibitory effect of DNA replication was extremely significant. Figure 2 )
[0069] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A phosphoramidate nucleoside compound, characterized in that: The general structure is as follows: Wherein: the glycoside is selected from (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, 5-fluoro-2'-deoxyurea nucleoside, cytarabine, ribavirin, cytidine, stavudine, 2',3'-O-isopropylcytidine, 5-bromocytidine, gemcitabine, zidovudine or lamivudine, and the 5'-hydroxyl group in the glycoside is connected to the phosphorus atom.
2. The method for synthesizing the phosphoramidate nucleoside compound according to claim 1, characterized in that: The method comprises the following steps: using phosphorus oxychloride 1 and N-(4-hydroxyphenyl)acetamide 2 as raw materials, reacting in an organic solvent in the presence of an organic base to obtain an intermediate 3; reacting the intermediate 3 with L-alanine isopropyl ester hydrochloride 4 in an organic solvent in the presence of an organic base to obtain an intermediate 5; reacting the intermediate 5 with pentafluorophenol 6 in an organic solvent in the presence of an organic base to obtain an intermediate 7; reacting the intermediate 7 with a glycoside 8 in an organic solvent in the presence of a catalyst to obtain a phosphoramidate nucleoside compound 9; the reaction equation is as follows: Wherein: the glycoside is selected from (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, 5-fluoro-2'-deoxyurea nucleoside, cytarabine, ribavirin, cytidine, stavudine, 2',3'-O-isopropylcytidine, 5-bromocytidine, gemcitabine, zidovudine or lamivudine, and the 5'-hydroxyl group in the glycoside is connected to the phosphorus atom.
3. The method for synthesizing the phosphoramidate nucleoside compound according to claim 2, characterized in that: In the first to third steps, the organic base is selected from triethylamine, diisopropylethylamine or pyridine; in the fourth step, the catalyst is selected from tert-butylmagnesium chloride.
4. The method for synthesizing the phosphoramidate nucleoside compound according to claim 3, characterized in that: In the first step, the organic solvent is selected from dichloromethane, chloroform or 1,2-dichloroethane, the reaction temperature is selected from -78°C to -60°C, the reaction is carried out in an inert gas, and the molar ratio of compound 1, compound 2 and the organic base is 1:1:1; in the second step, the organic solvent is selected from dichloromethane, chloroform or 1,2-dichloroethane; the reaction temperature is selected from -78°C to 30°C, the reaction is carried out in an inert gas, and the molar ratio of intermediate 3, L-alanine isopropyl hydrochloride and the organic base is 1:1:
2.
5. The method for synthesizing the phosphoramidate nucleoside compound according to claim 3, characterized in that: In the third step, the organic solvent is selected from dichloromethane, chloroform or 1,2-dichloroethane; the reaction temperature is selected from 0-30°C, the reaction is carried out in an inert gas nitrogen, and the molar ratio of intermediate 5, pentafluorophenol and organic base is 1:0.8:2; in the fourth step, the organic solvent is selected from DMF or DMSO; the reaction temperature is selected from 0-40°C, the reaction is carried out in an inert gas, and the molar ratio of intermediate 7, glycoside and tert-butyl magnesium chloride is 2:1:
2.
6. Use of the phosphoramidate nucleoside compound as claimed in claim 1 in the preparation of antiviral drugs.
7. The use of the phosphoramidate nucleoside compound according to claim 8 in the preparation of antiviral drugs, characterized in that: The antiviral agent is selected from Hep G2 / 2.2.15 cells which can stably and continuously express the HBV virus.
8. A pharmaceutical composition comprising the phosphoramidate nucleoside compound according to claim 1.