Preparation method and application of anti-coronavirus nucleoside compounds

Abstract

The present invention discloses a preparation method and application of an anti-coronavirus nucleoside compound. The structural formula of the anti-coronavirus compound g1-AA is:. The results of the efficacy test show that under the current high-toxicity background, the test sample still has a certain antiviral activity at a concentration of 0.04uM, which is equivalent to the antiviral activity of the listed control drug Molnupiravir, and is worthy of further research.

Description

Technical Field

[0001] The invention belongs to the technical field of antiviral compound synthesis, and particularly relates to a preparation method and application of an anti-coronavirus nucleoside compound. Background Art

[0002] Coronavirus (English name: Coronavirus) belongs to the order Nidovirales, family Coronaviridae, genus Coronavirus. It is a type of RNA virus with an envelope and a linear single-stranded positive genome. It is a large class of viruses that are widely present in nature. This virus can be seen under an electron microscope with a crown-like outer crown, so it is called Coronavirus (Coronaviridae). In 1975, the Virus Nomenclature Committee officially named the Coronaviridae. It includes rhinovirus, B814 virus, 229E virus, OC43 strain, and those that can infect humans and cause serious respiratory diseases, such as Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS, SARS is a variant of coronavirus and is the pathogen that causes atypical pneumonia) and the new coronavirus (SARS-CoV-2), etc. The symptoms of infection can range from the common cold to severe lung infection.

[0003] Potential targets of COVID-19 are divided into three categories: structural proteins, non-structural proteins and other coronavirus treatment-related targets. Structural proteins include spike protein (Surface Glycoprotein, Spike Protein), E protein (Envelope protein, E Protein), M protein (membrane protein) and N protein (Nucleocapsid Phosphoprotein, N protein). Non-structural proteins include replicase polyprotein 1ab, 3C-like protease, papain-like protease, NSP12 (RDRP, RNA-dependent RNA polymerase), Helicase, Nsp13 (RNA helicase), Nsp14 (Guanine-N7methyltransferase), Nsp15 (Uridylate-specific endoribonuclease), Nsp16 (2'-O-methyl transferase, 2'-O-methyltransferase). Other coronavirus treatment-related targets are mainly ACE2 (angiotensin-converting enzyme 2).

[0004] There are three main types of drugs for the treatment of the new coronavirus, each with different focuses and applicable populations: small molecule antiviral drugs can be used for patients with mild, moderate and severe symptoms, neutralizing antibodies are mainly used for patients with mild and moderate symptoms, and immunomodulatory drugs are basically only used for patients with severe symptoms. At present, according to different mechanisms of action, the new coronavirus small molecule antiviral drugs mainly include RNA polymerase inhibitors, 3CL protease inhibitors, etc. There are only three new coronavirus treatment small molecule drugs approved worldwide that act on the new coronavirus polymerase (RdRp), including Gilead Sciences' Remdesivir, Molnupiravir jointly developed by Merck and Ridgeback, Azvudine from Henan Real Biotechnology Co., Ltd., and Deuterium Hydrobromide Remidewei (VV116) from Shanghai Wangshi Biopharmaceutical Technology Co., Ltd. However, so far, no drug has become a specific drug for the new coronavirus or coronavirus.

[0005] In summary, there is an urgent need in the art to develop more effective inhibitors for inhibiting coronavirus replication for use in related diseases caused by coronavirus infection.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] The object of the present invention is to provide a nucleoside compound for resisting coronavirus, thereby overcoming the defects in the above-mentioned prior art.

[0008] To achieve the above object, the present invention provides an anti-coronavirus nucleoside compound, the structural formula of the anti-coronavirus compound is:

[0009] In formula (I):

[0010]

[0011] Furthermore, in the anti-coronavirus nucleoside compounds, the carbon atom connected to the single bond represented by the wavy line has two configurations, R and S, or the furanose ring has two configurations, D / L.

[0012] A pharmaceutical composition comprising the anti-coronavirus compound as described above or a pharmaceutically acceptable salt, crystalline hydrate or solvate thereof or a prodrug thereof or a combination thereof with other compounds.

[0013] Furthermore, anti-coronavirus drugs are drugs against 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

[0014] A method for synthesizing an anti-coronavirus nucleoside compound comprises the following steps:

[0015] Step 1:

[0016] Under nitrogen protection and ice-water bath conditions, 0.6 ml of concentrated sulfuric acid was added dropwise to 100 ml of dry methanol solution containing 5 g of compound A; after the addition of concentrated sulfuric acid, the mixture was stirred overnight at room temperature; 2.34 g of solid sodium bicarbonate was added to adjust the pH to > 7, the mixture was filtered through diatomaceous earth, the filter cake was washed with methanol, and the filtrate was concentrated to obtain a colorless oily compound B totaling 6.31 g;

[0017] Step 2:

[0018] Under nitrogen protection and ice-water bath conditions, 33.3mmol of compound B was dissolved in 100ml of dry DMF solution, 6.66g of 60% sodium hydrogen was added and stirred at room temperature for 10min, and 22.8g of benzyl bromide was added dropwise at 0℃; after the addition of benzyl bromide was completed, it was stirred at room temperature overnight; saturated ammonium chloride ice water solution was poured into it, and extracted with ethyl acetate three times; the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and chromatographed by EA / PE=20 / 1-10 / 1 column to obtain 9.74g of colorless transparent oily compound C;

[0019] Step 3:

[0020] At 0°C, 0.2 ml of sulfuric acid was added dropwise to 30 ml of acetic acid solution containing 2 g of compound C; after the addition was completed, the reaction solution was heated to 80°C for 6 hours; after the reaction was completed, it was concentrated under reduced pressure to 5-6 ml of solvent, and after concentration, it was dissolved with ethyl acetate and water, and the organic phase was washed with solid sodium carbonate until the pH was greater than 7, and the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and PE / EA=5 / 1 column chromatography to obtain 1.05 g of colorless and transparent oily compound D;

[0021] Step 4:

[0022] 1.05 g of compound D was dissolved in 10 ml of dry DMSO. Under nitrogen protection, 1.27 g of acetic anhydride was added dropwise and reacted at room temperature overnight. The reaction solution was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and chromatographed by PE / EA=20 / 1 column to obtain 787 mg of colorless transparent oily compound E.

[0023] Step 5:

[0024] Under nitrogen protection, 514 mg of TMSCl was added to a 5 ml dry tetrahydrofuran solution containing 504 mg of compound F; after addition, the reaction was allowed to react at room temperature for 20 minutes, then the reaction solution was cooled to -78 ° C, 5.38 ml of BuLi was added dropwise to the reaction solution, and after 1 hour, a 0 ° C solution of compound 5 was added dropwise, the solution of compound 5 containing 900 mg of compound E and 5 ml of dry tetrahydrofuran (compound E has been made in several batches, the total amount is far more than 900 mg, here only 900 mg was selected for a small test, which will not affect the understanding of professionals of this patent.); after the addition was completed, the reaction was stirred at -78 ° C for 1 hour; after heating to 0 ° C, saturated ammonium chloride was added to quench; the organic phase was extracted with ethyl acetate, the organic phase was washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography (PE / EA=1 / 1-1 / 2) was performed to obtain compound G (375 mg, brown solid);

[0025] Step 6:

[0026] Under nitrogen protection, 375 mg of compound G was dissolved in 75 mL of dry dichloromethane solution, cooled to -78 ° C, 204 mg of TfOH was added dropwise and stirred for 10 min; then 317 mg of TMSOTf was added dropwise to the reaction solution at -78 ° C, and the reaction was reacted for 30 min; 269 mg of TMSCN was slowly added at -78 ° C, and stirred at -78 ° C for 2 h, 241 mg of triethylamine was added dropwise, and the reaction solution was slowly heated to room temperature; 514 mg of solid sodium bicarbonate and 2 ml of water were added in sequence and stirred for 10 min, and the organic phase was extracted with dichloromethane, and the organic phase was washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography PE / EA=1 / 1 to obtain 288 mg of light yellow solid compound H;

[0027] Step 7:

[0028] Under nitrogen protection, 2.5 g of compound H was dissolved in 20 ml of dry dichloromethane solution, cooled to -78 °C, and 1 M BCl was added dropwise. 3 16.91 ml of n-hexane solution; heated to -40°C and stirred for 2 hours. After the reaction was completed, 3.85 g of methanol, 4.5 g of triethylamine and 7.7 g of methanol were added dropwise at -78°C. After the reaction was heated to room temperature, the reaction solution was dried by rotary drying to obtain a crude product; the crude product was pulped with n-hexane and the supernatant was poured off. The pulping and pouring steps were repeated 3 times; 20 mL of methanol was added and heated to 45°C, and water was added in a volume ratio of 1:1 with methanol. Part of the solution was evaporated at 45°C and cooled to room temperature for filtration. The filter cake was washed with a little methanol. The filter cake was dried to obtain 686 mg of white solid compound I;

[0029] Step 8:

[0030] Under nitrogen protection, 686g of compound I and 1.18g of 2,2-dimethoxypropane were dissolved in 10ml of acetone, 0.17ml of concentrated sulfuric acid was added dropwise at room temperature and stirred for 30min, heated to 45°C and stirred for 30min; 694mg of solid sodium bicarbonate and 680mg of water were added at room temperature and stirred for 15min; the reaction solution was concentrated and water and ethyl acetate were added to separate the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography (DCM / MEOH=50 / 1-20 / 1) was performed to obtain 617mg of white solid compound J;

[0031] Step 9:

[0032] Under nitrogen protection, 10 g of compound K and 19.82 g of compound L were dissolved in 300 ml of toluene, 18.45 g of p-toluenesulfonic acid monohydrate was added, the mixture was heated to 135°C and separated by a water separator, and the reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated, ether was added to make pulp and filtered, the filter cake was washed with ether and dried to obtain a white solid compound M.

[0033] Step 10:

[0034]

[0035] Under nitrogen protection, 5.76g of compound M was dissolved in 100mL of dry dichloromethane, 3.38g of compound N was added at 0°C, and 3.57g of triethylamine (1.38g, 13.69mmol, 2.1eq) was added dropwise at 0°C for 45min, and then reacted for three hours. 2.23g of p-nitrophenol was added at 0°C, and then 1.62g of triethylamine was added dropwise at room temperature for overnight reaction. The reaction solution was washed with water, and the organic phase was separated. The organic phase was washed with saturated brine, then dried with anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated and passed through a column PE / EA=5 / 1-4 / 1 to obtain 4.92g of colorless, transparent, oily liquid compound O;

[0036] Step 11:

[0037]

[0038] 617 mg of compound O, 951 mg of compound P and 177 mg of magnesium chloride were mixed in 100 ml of dry acetonitrile, stirred at 50°C for 10 min under N2 protection, 601 mg of N,N-isopropylethylamine was added and stirring was continued for 20 min, cooled to room temperature and diluted with ethyl acetate, washed with 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated saline solution, separated the organic phase, dried over anhydrous sodium sulfate, filtered, concentrated and column chromatographed (DCM / MEOH=40 / 1-20 / 1) to obtain 731 mg of white solid compound Q;

[0039] Step 12:

[0040]

[0041] At room temperature, 4.22 g of compound 13 was dissolved in 100 ml of formic acid and stirred overnight at room temperature. The reaction solution was concentrated and passed through a column (DCM / MeOH=20 / 1) to obtain 1.66 g of white solid compound g1-A;

[0042] Step 13:

[0043]

[0044] 300 mg of compound g1-A was dissolved in 10 ml of dry tetrahydrofuran, and 128 mg of N,N-dimethylformamide dimethyl acetal was added at room temperature. The mixture was stirred overnight at room temperature. The reaction solution was concentrated and filtered through a column (DCM / MeOH=20 / 1) to obtain compound R (274 mg, white solid);

[0045] Step 14:

[0046]

[0047] 274 mg of compound R was dissolved in 5 ml of dry dichloromethane. Under nitrogen protection and room temperature, 145 mg of triethylamine, 25 mg of 4-dimethylaminopyridine DMAP, and 161 mg of compound S were added in sequence. The reaction solution was stirred at room temperature for 1 h and methanol was added to quench. The reaction solution was stirred at room temperature for 30 min and then concentrated and passed through a column (PE→EA) to obtain compound T (290 mg, colorless and transparent oil).

[0048] Step 15:

[0049]

[0050] 290 mg of compound T was dissolved in 10 ml of trifluoroacetic acid and stirred at room temperature overnight. After the reaction solution was concentrated, ethyl acetate was added. Saturated sodium bicarbonate solution was added to adjust the pH to 7. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and passed through a column (DCM / MeOH=20 / 1) to obtain compound T (237 mg, white solid);

[0051] Compound g1-AA was chiral resolved to obtain the target compound. Chromatographic column: Daicel IB 250*30mm, 10μm, mobile phase A: n-hexane, mobile phase B: isopropanol, detection wavelength: 254nm / 214nm, flow rate: 25mL / min, isocratic elution program: mobile phase A: mobile phase B = 70:30 (V / V). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1.1-Figure 1.3 This is the chiral liquid phase spectrum of the product g1-AA;

[0053] Figure 2.1 and Figure 2.2 The NMR spectra of two configurations of product g1-AA;

[0054] Figure 3-Figure 15 These are the NMR spectra of the products of each step in the synthesis process. DETAILED DESCRIPTION

[0055] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0056] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0057] An anti-coronavirus nucleoside compound, the structural formula of the anti-coronavirus compound is:

[0058] In formula (I):

[0059]

[0060] Furthermore, in the anti-coronavirus nucleoside compounds, the carbon atom connected to the single bond represented by the wavy line has two configurations, R and S, or the furanose ring has two configurations, D / L.

[0061] A pharmaceutical composition comprising the anti-coronavirus compound as described above or a pharmaceutically acceptable salt, crystalline hydrate or solvate thereof or a prodrug thereof or a combination thereof with other compounds.

[0062] Furthermore, anti-coronavirus drugs are drugs against 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

[0063] A method for synthesizing an anti-coronavirus nucleoside compound comprises the following steps:

[0064] Step 1:

[0065] Under nitrogen protection and ice-water bath conditions, 0.6 ml of concentrated sulfuric acid was added dropwise to 100 ml of dry methanol solution containing 5 g of compound A; after the addition of concentrated sulfuric acid, the mixture was stirred overnight at room temperature; 2.34 g of solid sodium bicarbonate was added to adjust the pH to > 7, the mixture was filtered through diatomaceous earth, the filter cake was washed with methanol, and the filtrate was concentrated to obtain a colorless oily compound B totaling 6.31 g;

[0066] Step 2:

[0067] Under nitrogen protection and ice-water bath conditions, 33.3mmol of compound B was dissolved in 100ml of dry DMF solution, 6.66g of 60% sodium hydrogen was added and stirred at room temperature for 10min, and 22.8g of benzyl bromide was added dropwise at 0℃; after the addition of benzyl bromide was completed, it was stirred at room temperature overnight; saturated ammonium chloride ice water solution was poured into it, and extracted with ethyl acetate three times; the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and chromatographed by EA / PE=20 / 1-10 / 1 column to obtain 9.74g of colorless transparent oily compound C;

[0068] Step 3:

[0069] At 0°C, 0.2 ml of sulfuric acid was added dropwise to 30 ml of acetic acid solution containing 2 g of compound C; after the addition was completed, the reaction solution was heated to 80°C for 6 hours; after the reaction was completed, it was concentrated under reduced pressure to 5-6 ml of solvent, and after concentration, it was dissolved with ethyl acetate and water, and the organic phase was washed with solid sodium carbonate until the pH was greater than 7, and the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and PE / EA=5 / 1 column chromatography to obtain 1.05 g of colorless and transparent oily compound D;

[0070] Step 4:

[0071] 1.05 g of compound D was dissolved in 10 ml of dry DMSO. Under nitrogen protection, 1.27 g of acetic anhydride was added dropwise and reacted at room temperature overnight. The reaction solution was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and chromatographed by PE / EA=20 / 1 column to obtain 787 mg of colorless transparent oily compound E.

[0072] Step 5:

[0073] Under nitrogen protection, 514 mg of TMSCl was added to a 5 ml dry tetrahydrofuran solution containing 504 mg of compound F; after addition, the reaction was allowed to react at room temperature for 20 minutes, then the reaction solution was cooled to -78 ° C, 5.38 ml of BuLi was added dropwise to the reaction solution, and after 1 hour, a 0 ° C solution of compound 5 was added dropwise, the solution of compound 5 containing 900 mg of compound E and 5 ml of dry tetrahydrofuran (compound E has been made in several batches, the total amount is far more than 900 mg, here only 900 mg was selected for a small test, which will not affect the understanding of professionals of this patent.); after the addition was completed, the reaction was stirred at -78 ° C for 1 hour; after heating to 0 ° C, saturated ammonium chloride was added to quench; the organic phase was extracted with ethyl acetate, the organic phase was washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography (PE / EA=1 / 1-1 / 2) was performed to obtain compound G (375 mg, brown solid);

[0074] Step 6:

[0075] Under nitrogen protection, 375 mg of compound G was dissolved in 75 mL of dry dichloromethane solution, cooled to -78 ° C, 204 mg of TfOH was added dropwise and stirred for 10 min; then 317 mg of TMSOTf was added dropwise to the reaction solution at -78 ° C, and the reaction was reacted for 30 min; 269 mg of TMSCN was slowly added at -78 ° C, and stirred at -78 ° C for 2 h, 241 mg of triethylamine was added dropwise, and the reaction solution was slowly heated to room temperature; 514 mg of solid sodium bicarbonate and 2 ml of water were added in sequence and stirred for 10 min, and the organic phase was extracted with dichloromethane, and the organic phase was washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography PE / EA=1 / 1 to obtain 288 mg of light yellow solid compound H;

[0076] Step 7:

[0077] Under nitrogen protection, 2.5 g of compound H was dissolved in 20 ml of dry dichloromethane solution, cooled to -78 °C, and 1 M BCl was added dropwise. 316.91 ml of n-hexane solution; heated to -40°C and stirred for 2 hours. After the reaction was completed, 3.85 g of methanol, 4.5 g of triethylamine and 7.7 g of methanol were added dropwise at -78°C. After the reaction was heated to room temperature, the reaction solution was dried by rotary drying to obtain a crude product; the crude product was pulped with n-hexane and the supernatant was poured off. The pulping and pouring steps were repeated 3 times; 20 mL of methanol was added and heated to 45°C, and water was added in a volume ratio of 1:1 with methanol. Part of the solution was evaporated at 45°C and cooled to room temperature for filtration. The filter cake was washed with a little methanol. The filter cake was dried to obtain 686 mg of white solid compound I;

[0078] Step 8:

[0079] Under nitrogen protection, 686g of compound I and 1.18g of 2,2-dimethoxypropane were dissolved in 10ml of acetone, 0.17ml of concentrated sulfuric acid was added dropwise at room temperature and stirred for 30min, heated to 45°C and stirred for 30min; 694mg of solid sodium bicarbonate and 680mg of water were added at room temperature and stirred for 15min; the reaction solution was concentrated and water and ethyl acetate were added to separate the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography (DCM / MEOH=50 / 1-20 / 1) was performed to obtain 617mg of white solid compound J;

[0080] Step 9:

[0081] Under nitrogen protection, 10 g of compound K and 19.82 g of compound L were dissolved in 300 ml of toluene, 18.45 g of p-toluenesulfonic acid monohydrate was added, the mixture was heated to 135°C and separated by a water separator, and the reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated, ether was added to make pulp and filtered, the filter cake was washed with ether and dried to obtain a white solid compound M.

[0082] Step 10:

[0083]

[0084] Under nitrogen protection, 5.76g of compound M was dissolved in 100mL of dry dichloromethane, 3.38g of compound N was added at 0°C, and 3.57g of triethylamine (1.38g, 13.69mmol, 2.1eq) was added dropwise at 0°C for 45min, and then reacted for three hours. 2.23g of p-nitrophenol was added at 0°C, and then 1.62g of triethylamine was added dropwise at room temperature for overnight reaction. The reaction solution was washed with water, and the organic phase was separated. The organic phase was washed with saturated brine, then dried with anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated and passed through a column PE / EA=5 / 1-4 / 1 to obtain 4.92g of colorless, transparent, oily liquid compound O;

[0085] Step 11:

[0086]

[0087] 617 mg of compound O, 951 mg of compound P and 177 mg of magnesium chloride were mixed in 100 ml of dry acetonitrile, stirred at 50°C for 10 min under N2 protection, 601 mg of N,N-isopropylethylamine was added and stirring was continued for 20 min, cooled to room temperature and diluted with ethyl acetate, washed with 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated saline solution, separated the organic phase, dried over anhydrous sodium sulfate, filtered, concentrated and column chromatographed (DCM / MEOH=40 / 1-20 / 1) to obtain 731 mg of white solid compound Q;

[0088] Step 12:

[0089]

[0090] At room temperature, 4.22 g of compound 13 was dissolved in 100 ml of formic acid and stirred overnight at room temperature. The reaction solution was concentrated and passed through a column (DCM / MeOH=20 / 1) to obtain 1.66 g of white solid compound g1-A;

[0091] Step 13:

[0092]

[0093] 300 mg of compound g1-A was dissolved in 10 ml of dry tetrahydrofuran, and 128 mg of N,N-dimethylformamide dimethyl acetal was added at room temperature. The mixture was stirred overnight at room temperature. The reaction solution was concentrated and filtered through a column (DCM / MeOH=20 / 1) to obtain compound R (274 mg, white solid);

[0094] Step 14:

[0095]

[0096] 274 mg of compound R was dissolved in 5 ml of dry dichloromethane. Under nitrogen protection and room temperature, 145 mg of triethylamine, 25 mg of 4-dimethylaminopyridine DMAP, and 161 mg of compound S were added in sequence. The reaction solution was stirred at room temperature for 1 h and methanol was added to quench. The reaction solution was stirred at room temperature for 30 min and then concentrated and passed through a column (PE→EA) to obtain compound T (290 mg, colorless and transparent oil).

[0097] Step 15:

[0098]

[0099] 290 mg of compound T was dissolved in 10 ml of trifluoroacetic acid and stirred at room temperature overnight. After the reaction solution was concentrated, ethyl acetate was added. Saturated sodium bicarbonate solution was added to adjust the pH to 7. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and passed through a column (DCM / MeOH=20 / 1) to obtain compound T (237 mg, white solid);

[0100] Compound g1-AA was chiral resolved to obtain the target compound. Chromatographic column: Daicel IB 250*30mm, 10μm, mobile phase A: n-hexane, mobile phase B: isopropanol, detection wavelength: 254nm / 214nm, flow rate: 25mL / min, isocratic elution program:

[0101] Mobile phase A:mobile phase B=70:30 (V / V).

[0102] Cytotoxicity test steps:

[0103] Monoupiravir (purchased from Jiangsu Aikang Biopharmaceutical R&D Co., Ltd.), Bel7402 cells (provided by the Antiviral Drug Research Laboratory, School of Pharmacy, Fudan University), fetal bovine serum (FBS) (produced by Thermo Fisher Scientific), DMEM medium (Thermo Fisher Scientific), carbon dioxide incubator (Thermo Fisher Scientific), and fluorescent quantitative PCR (Thermo Fisher Scientific).

[0104] Bel7402 cells (1.5*10 4 / well) and culture for 24h. Aspirate the culture medium and add 100μL / well of the drug solution to be tested. Set up 2 replicate wells for each concentration, and add an equal volume of culture medium to the normal control group. 37℃, 5% CO 2 After incubation in the incubator for 48 hours, 15 μL of 5 mg / mL MTT solution was added to each well and incubated for another 4 hours. The supernatant was removed by aspiration, 100 μL DMSO was added to each well, and the cells were dissolved by low-speed oscillation. The OD value was measured at 490 nm, and the cell survival rate was calculated relative to the cells in the normal group (mean OD value of the drug group – blank OD value) / (mean OD value of the normal control group – blank OD value) × 100%.

[0105] Anti-coronavirus 229E activity test steps:

[0106] Bel7402 cells (2.5*10 4 / well) and cultured for 24 h. The supernatant was removed by aspiration, and 10 -2 HCoV virus solution 100 μL / well, 37°C, 5% CO 2Incubate for 5 hours. Remove the culture medium, add 100 μL / well of the drug solution to be tested to the drug experimental group, set up 2 replicates for each concentration, and add an equal volume of culture medium to the normal control group and the virus control group. 37°C, 5% CO 2 After incubation in the incubator for 72 h, the cell activity was determined by MTT method (the method is the same as above), and the cell survival rate was calculated (mean OD value of the drug group - blank OD value) / (mean OD value of the normal control group - blank OD value) × 100%.

[0107] Test conclusion:

[0108] The cytotoxicity test showed that the test sample was below 125 μM, and in the absence of an infection background, the cytotoxicity was not obvious, and the toxicity was lower than that of the control drug.

[0109] The results of the efficacy test showed that under the current high-toxicity background, the test sample still had a certain antiviral activity at a concentration of 0.04uM, which was equivalent to the antiviral activity of the marketed control drug Molnupiravir, and was worthy of further research.

[0110]

[0111] (Note: Molnupiravir is one of the only two small molecule nucleoside anti-coronavirus inhibitors approved for marketing in the United States, jointly developed by Merck and Ridgeback, and has been officially introduced and approved for use in China).

[0112] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A nucleoside compound for resisting coronavirus, characterized in that: The structural formula of the anti-coronavirus compound g1-AA is: The furanose ring is in L configuration.

2. A pharmaceutical composition, characterized in that: The composition comprises the anti-coronavirus compound g1-AA or a pharmaceutically acceptable salt thereof according to claim 1.

3. The use of a composition as claimed in claim 2 in the preparation of an anti-coronavirus drug, wherein the anti-coronavirus drug is a drug against 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

4. A method for synthesizing an anti-coronavirus nucleoside compound, characterized in that: The steps include: Step 1 : ; Under nitrogen protection and ice-water bath conditions, 0.6 ml of concentrated sulfuric acid was added dropwise to 100 ml of dry methanol solution containing 5 g of compound A; after the addition of concentrated sulfuric acid, the mixture was stirred overnight at room temperature; 2.34 g of solid sodium bicarbonate was added to adjust the pH to >7, the mixture was filtered through diatomaceous earth, the filter cake was washed with methanol, and the filtrate was concentrated to obtain a colorless oily compound B totaling 6.31 g; Step 2 : ; Under nitrogen protection and ice-water bath conditions, 33.3mmol of compound B was dissolved in 100ml of dry DMF solution, 6.66g of 60% sodium hydrogen was added and stirred at room temperature for 10min, and 22.8g of benzyl bromide was added dropwise at 0℃; after the addition of benzyl bromide was completed, it was stirred at room temperature overnight; saturated ammonium chloride ice water solution was poured into it, and extracted with ethyl acetate three times; the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and chromatographed by EA / PE=20 / 1-10 / 1 column to obtain 9.74g of colorless transparent oily compound C; Step 3 : ; At 0°C, 0.2 ml of sulfuric acid was added dropwise to 30 ml of acetic acid solution containing 2 g of compound C. After the addition was completed, the reaction solution was heated to 80°C for 6 hours. After the reaction was completed, the solution was concentrated under reduced pressure to 5-6 ml of solvent, and then dissolved with ethyl acetate and water. The organic phase was washed with sodium carbonate until the pH was greater than 7. The organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated. 1.05 g of colorless and transparent oily compound D was obtained by PE / EA=5 / 1 column chromatography. Step 4 : ; 1.05 g of compound D was dissolved in 10 ml of dry DMSO. Under nitrogen protection, 1.27 g of acetic anhydride was added dropwise and reacted at room temperature overnight. The reaction solution was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. 787 mg of colorless and transparent oily compound E was obtained by PE / EA=20 / 1 column chromatography. Step 5 : ; Under nitrogen protection, 514 mg of TMSCl was added to a 5 ml dry tetrahydrofuran solution containing 504 mg of compound F; after addition, the reaction was allowed to react at room temperature for 20 minutes, then the reaction solution was cooled to -78°C, 5.38 ml of n-BuLi was added dropwise to the reaction solution, and 1 hour later, a 0°C compound E solution was added dropwise, the compound E solution containing 900 mg of compound E and 5 ml of dry tetrahydrofuran; after the addition was completed, the reaction was stirred at -78°C for 1 hour; after the temperature was raised to 0°C, saturated ammonium chloride was added to quench; the organic phase was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and PE / EA=1 / 1-1 / 2 column chromatography was performed to obtain 375 mg of brown solid compound G; Step 6 : ; Under nitrogen protection, 375 mg of compound G was dissolved in 10 ml of dry dichloromethane solution, cooled to -78 ° C, 204 mg of TfOH was added dropwise and stirred for 10 min; then 317 mg of TMSOTf was added dropwise to the reaction solution at -78 ° C, and the reaction was reacted for 30 min; 269 mg of TMSCN was slowly added at -78 ° C, and stirred at -78 ° C for 2 h, 241 mg of triethylamine was added dropwise, and the reaction solution was slowly heated to room temperature; 514 mg of solid sodium bicarbonate and 2 ml of water were added sequentially and stirred for 10 min, and the organic phase was extracted with dichloromethane, and the organic phase was washed with saturated brine, and the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and PE / EA=1 / 1 column chromatography was performed to obtain 288 mg of light yellow solid compound H; Step 7 : ; Under nitrogen protection, 2.5 g of compound H was dissolved in 20 ml of dry dichloromethane solution, cooled to -78 ° C, and 16.91 ml of 1M BCl3 n-hexane solution was added dropwise; the temperature was raised to -40 ° C and stirred for 2 h. After the reaction was completed, 3.85 g of methanol, 4.5 g of triethylamine and 7.7 g of methanol were added dropwise at -78 ° C. After the reaction was heated to room temperature, the reaction liquid was rotary dried to obtain a crude product; the crude product was slurried with n-hexane, and the supernatant was poured off, and this was repeated 3 times; 20 mL of methanol was added and heated to 45 ° C, and water was added in a volume ratio of 1:1 with methanol, part of the solution was rotary evaporated at 45 ° C, and the solution was cooled to room temperature and filtered. The filter cake was washed with a little methanol, and the filter cake was dried to obtain 686 mg of white solid compound I; Step 8 : ; Under nitrogen protection, 686 mg of compound I and 1.18 g of 2,2'-dimethoxypropane were dissolved in 10 ml of acetone, 0.17 ml of concentrated sulfuric acid was added dropwise at room temperature and stirred for 30 min, heated to 45 °C and stirred for 30 min; 694 mg of solid sodium bicarbonate and 680 mg of water were added at room temperature and stirred for 15 min; the reaction solution was concentrated and water and ethyl acetate were added to separate the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography DCM / MEOH=50:1-20 / 1 to obtain 617 mg of white solid compound J; Step 9 : ; Under nitrogen protection, 10 g of compound K and 19.82 g of compound L were dissolved in 300 ml of toluene, 18.45 g of p-toluenesulfonic acid monohydrate was added, and the mixture was heated to 135 °C and separated by a water separator, and reacted for 24 hours. After the reaction was completed, the reaction solution was concentrated, ether was added to make pulp and filtered, and the filter cake was washed with ether and dried to obtain a white solid compound M. Step 10: ； Under nitrogen protection, 5.76g of compound M was dissolved in 100mL of dry dichloromethane, 3.38g of compound N was added at 0°C, 3.57g of triethylamine was added dropwise after stirring for 45min, and the mixture was reacted for three hours, 2.23g of p-nitrophenol was added at 0°C, and 1.62g of triethylamine was added dropwise to react at room temperature overnight; the reaction solution was washed with water, the organic phase was separated, the organic phase was washed with saturated brine, and then dried with anhydrous sodium sulfate, the anhydrous sodium sulfate was removed by filtration, the filtrate was concentrated, and 4.92g of colorless transparent oily liquid compound was obtained by column chromatography separation of PE / EA=5 / 1-4 / 1; Step 11: ； 617 mg of compound O, 951 mg of compound P and 177 mg of magnesium chloride were mixed in 100 ml of dry acetonitrile, stirred at 50 °C for 10 min under N2 protection, 601 mg of N,N-isopropylethylamine was added and stirred for 20 min, cooled to room temperature and diluted with ethyl acetate, the reaction solution was washed with 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated saline solution, the organic phase was separated, dried, filtered and concentrated with anhydrous sodium sulfate, and separated by column chromatography with DCM / MEOH=40:1-20 / 1 to obtain 731 mg of white solid compound Q; Step 12: ； At room temperature, 4.22 g of compound Q was dissolved in 100 ml of formic acid and stirred overnight at room temperature. The reaction solution was concentrated and separated by column chromatography with DCM / MeOH=20 / 1 to obtain 1.66 g of white solid compound g1-A; Step 13: ； 300 mg of compound g1-A was dissolved in 10 ml of dry tetrahydrofuran, and 128 mg of N,N-dimethylformamide dimethyl acetal was added at room temperature. The mixture was stirred overnight at room temperature. The reaction solution was concentrated and separated by column chromatography with DCM / MeOH=20 / 1 to obtain 274 mg of white solid compound R; Step 14: ； 274 mg of compound R was dissolved in 5 ml of dry acetonitrile. Under nitrogen protection and at room temperature, 145 mg of triethylamine, 25 mg of 4-dimethylaminopyridine DMAP, and 161 mg of compound S were added in sequence. The reaction solution was stirred at room temperature for 1 h, and methanol was added to quench. The reaction solution was stirred at room temperature for 30 min and then concentrated and filtered to obtain 290 mg of colorless and transparent oily compound T. Step 15: ； 290 mg of compound T was dissolved in 10 ml of trifluoroacetic acid and stirred at room temperature overnight. After the reaction solution was concentrated, ethyl acetate was added. Saturated sodium bicarbonate solution was added to adjust the pH to 7. The organic phase was separated and washed with saturated brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and subjected to column chromatography with DCM / MeOH=20 / 1 to obtain 237 mg of white solid compound T; Compound g1-AA was chiral resolved to obtain the target compound.

5. The method for synthesizing the anti-coronavirus nucleoside compound according to claim 4, characterized in that: Chiral separation conditions were as follows: chromatographic column: Daicel CHIRALPAK® IB 250*30 mm, 10µm, mobile phase A: n-hexane, mobile phase B: isopropanol, detection wavelength: 254nm / 214nm, flow rate: 25mL / min, isocratic elution program in volume ratio: mobile phase A: mobile phase B = 70:30.

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