A method for preparing an anti-coronavirus nucleoside compound and its application

Abstract

This invention discloses a method for preparing an anti-coronavirus nucleoside compound and its application, the structural formula of which is: ; the efficacy test results show that under the current high concentration background, the test sample still has certain antiviral activity at a concentration of 0.04 μM, which is comparable to the antiviral activity of the marketed control drug monopiravir, and has the value for further research.

Description

Technical Field

[0001] This invention belongs to the field of antiviral compound research and development technology, and specifically relates to a method for preparing an anti-coronavirus nucleoside compound and its application. Background Technology

[0002] Coronaviruses belong to the order Nidovirales, family Coronaviridae, and genus Coronavirus. They are enveloped RNA viruses with a linear, single-stranded, positive-sense genome and are a large, widespread family of viruses in nature. Under an electron microscope, these viruses appear as a crown-like structure, hence the name coronavirus (Coronaviridae). In 1975, the Virus Nomenclature Committee officially named the Coronaviridae family. This family includes rhinoviruses, B814 virus, 229E virus, OC43 strain, and viruses that infect humans and cause severe respiratory illnesses, such as Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS, a variant of coronavirus that causes atypical pneumonia), and the novel coronavirus (SARS-CoV-2). Symptoms can range from the common cold to severe lung infections.

[0003] Potential targets for COVID-19 are categorized into three main groups: structural proteins, non-structural proteins, and other coronavirus-related therapeutic targets. Structural proteins include spike protein (Surface Glycoprotein, Spike Protein), Envelope protein (E Protein), Membrane protein (M Protein), and 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-N7 methyltransferase), Nsp15 (Uridylate-specific endoribonuclease), and Nsp16 (2'-O-methyl transferase). Other coronavirus-related therapeutic targets are primarily ACE2 (angiotensin-converting enzyme 2).

[0004] There are three main types of drugs for treating COVID-19, each with different focuses and target populations: small molecule antiviral drugs are used for mild, moderate, and severe cases; neutralizing antibodies are mainly used for mild to moderate cases; and immunomodulatory drugs are primarily used only for severe cases. Currently, based on their mechanisms of action, small molecule antiviral drugs for COVID-19 mainly include RNA polymerase inhibitors and 3CL protease inhibitors. Globally, only three small molecule drugs targeting the novel coronavirus polymerase (RdRp) have been approved for COVID-19 treatment: Remdesivir from Gilead Sciences, Molnupiravir (co-developed by Merck and Ridgeback), Azvudine from Henan Zhenshi Biotechnology Co., Ltd., and Deuterium Remdesivir hydrobromide (VV116) from Shanghai Wangshi Biopharmaceutical Co., Ltd. However, to date, no drug has been identified as a specific cure for COVID-19 or any coronavirus.

[0005] In summary, there is an urgent need in this field to develop more effective inhibitors that suppress coronavirus replication for use in diseases caused by coronavirus infection.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-coronavirus nucleoside compound, thereby overcoming the deficiencies in the prior art.

[0008] To achieve the above objectives, the present invention provides a nucleoside compound for resisting coronaviruses, the structural formula of which is:

[0009] In formula (I): .

[0010] Further preferred, the carbon atoms connected to the single bonds represented by the wavy lines have two configurations: R and S, or the furanose ring has two configurations: D and L.

[0011] A pharmaceutical composition comprising the aforementioned anticoronavirus compound or a pharmaceutically acceptable salt, crystalline hydrate or solvate thereof or a prodrug thereof or a combination thereof with other compounds.

[0012] Further preferred, the carbon atoms connected to the single bonds represented by the wavy lines have two configurations: R and S, or the furanose ring has two configurations: D and L.

[0013] More preferably, the composition is used in the preparation of an anti-coronavirus drug, wherein the anti-coronavirus drug is an anti-2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.

[0014] A method for synthesizing a nucleoside compound that is effective against coronaviruses, comprising the following steps:

[0015] Step 1:

[0016] ;

[0017] 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 was completed, the mixture was stirred overnight at room temperature. 2.34 g of sodium bicarbonate solid 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 total of 6.31 g of colorless oily compound B.

[0018] Step 2:

[0019] ;

[0020] Under nitrogen protection and an ice-water bath, 33.3 mmol of compound B was dissolved in 100 ml of dry DMF solution, 6.66 g of 60% sodium hydroxide was added and stirred at room temperature for 10 min, and 22.8 g of benzyl bromide was added dropwise at 0 °C; after the addition of benzyl bromide was completed, the mixture was stirred overnight at room temperature; the solution was poured into an ice-water solution of saturated ammonium chloride and extracted three times with ethyl acetate; the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (EA / PE = 20 / 1 - 10 / 1) to give 9.74 g of colorless transparent oily compound C;

[0021] Step 3:

[0022] ;

[0023] At 0℃, 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 complete, the reaction solution was heated to 80℃ and reacted for 6 hours. After the reaction was completed, the solution was concentrated under reduced pressure to 5-6 ml of solvent. The concentrated solution was dissolved in ethyl acetate and water. The organic phase was washed with sodium carbonate solid until pH > 7. The organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (PE / EA = 5 / 1) to obtain 1.05 g of colorless transparent oily compound D.

[0024] Step 4:

[0025] ;

[0026] 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 the reaction was carried out overnight at room temperature. The reaction solution was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to PE / EA=20 / 1 column chromatography to obtain 787 mg of colorless transparent oily compound E.

[0027] Step 5:

[0028] ;

[0029] Under nitrogen protection, 514 mg of TMSCl was added to 5 ml of dry tetrahydrofuran solution containing 504 mg of compound F. After the addition, the reaction was allowed to proceed at room temperature for 20 minutes. Then, the reaction solution was cooled to -78°C, and 5.38 ml of BuLi was added dropwise to the reaction solution. One hour later, a solution of compound 5 containing 900 mg of compound E and 5 ml of dry tetrahydrofuran was added dropwise at 0°C. After the addition was completed, the reaction was stirred at -78°C for 1 hour. The reaction was then quenched by adding saturated ammonium chloride after the temperature was raised to 0°C. The organic phase was extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and precipitated by PE / EA = 1 / 1-1 / 2 column chromatography to obtain 375 mg of brown solid compound G.

[0030] Step 6:

[0031] ;

[0032] Under nitrogen protection, 375 mg of compound G was dissolved in 10 ml of dry dichloromethane solution, cooled to -78 °C, and 204 mg of TfOH was added dropwise while stirring for 10 min. Then, 317 mg of TMSOTf was added dropwise to the reaction solution at -78 °C and reacted for 30 min. 269 mg of TMSCN was slowly added at -78 °C and stirred for 2 h at -78 °C. 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. The organic phase was extracted with dichloromethane, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and precipitated by PE / EA=1 / 1 column chromatography to obtain 288 mg of light yellow solid compound H.

[0033] Step 7:

[0034] ;

[0035] 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 1 M BCl3 n-hexane solution was added dropwise. The mixture was heated 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. The reaction mixture was heated to room temperature and then dried by rotary evaporation to obtain crude product. The crude product was slurried with n-hexane, and the supernatant was discarded. The slurrying and decanting steps were repeated 3 times. Methanol (20 mL) was added and heated to 45 °C. Water was added in a 1:1 volume ratio with methanol. Part of the solution was evaporated by rotary evaporation at 45 °C and cooled to room temperature for filtration. The filter cake was washed with a small amount of methanol and dried to obtain 686 mg of white solid compound I.

[0036] Step 8:

[0037] ;

[0038] Under nitrogen protection, compound I (686) 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. The mixture was then heated to 45 °C and stirred for 30 min. 694 mg of sodium bicarbonate solid and 680 mg of water were added at room temperature and stirred for 15 min. After concentrating the reaction solution, 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 subjected to column chromatography (DCM / MEOH = 50 / 1-20 / 1) to obtain 617 mg of white solid compound J.

[0039] Step 9:

[0040] ;

[0041] Under nitrogen protection, 10 g of compound K and 19.82 g of compound L were dissolved in 300 ml of toluene, and 18.45 g of p-toluenesulfonic acid monohydrate was added. The mixture was heated to 135 °C and water was separated using a water separator. The reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated, slurry was added with diethyl ether, filtered, and the filter cake was washed with diethyl ether and dried to obtain a white solid compound M.

[0042] Step 10:

[0043] ;

[0044] Under nitrogen protection, 5.76 g of compound M was dissolved in 100 mL of dry dichloromethane. 3.38 g of compound N was added at 0 °C, and the mixture was stirred for 45 min. Then, 3.57 g of triethylamine (1.38 g, 13.69 mmol, 2.1 eq) was added dropwise, and the reaction proceeded for three hours. 2.23 g of p-nitrophenol was added at 0 °C, followed by the dropwise addition of 1.62 g of triethylamine, and the reaction proceeded overnight at room temperature. The reaction solution was washed with water, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove the anhydrous sodium sulfate. The filtrate was concentrated, and column chromatography (PE / EA = 5 / 1-4 / 1) yielded 4.92 g of a colorless, transparent, oily liquid compound O.

[0045] Step 11:

[0046] ;

[0047] 617 mg of compound O, 951 mg of compound P, and 177 mg of magnesium chloride were mixed in 100 ml of dry acetonitrile and 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. The mixture was cooled to room temperature and diluted with ethyl acetate. It was washed successively with 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated saline solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 40:1-20 / 1) to give compound Q (731 mg white solid).

[0048] Step 12:

[0049] ;

[0050] 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 passed through a column (DCM / MeOH=20 / 1) to give compound g1-A (1.66 g, white solid).

[0051] Compound g1-A was chirally resolved to yield two compounds with single configurations. (Chromatographic column: DaicelCHIRALPAK® 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: mobile phase A: mobile phase B = 60:40 (V / V)).

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The efficacy test results showed that, under the current high concentration background, the test sample still had certain antiviral activity at a concentration of 0.04 uM, which was comparable to the antiviral activity of the marketed control drug Molnupiravir, and was worthy of further research. Attached Figure Description

[0054] Figures 1.1-1.3 The image shows the chiral liquid phase spectrum of product g1-A.

[0055] Figure 2 shows the NMR spectrum of product g1-A;

[0056] Figure 3 This is a schematic diagram of the synthetic route of the present invention;

[0057] Figures 4-11 The NMR spectra of the products from each step of the synthesis are shown. Detailed Implementation

[0058] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0059] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0060] A nucleoside compound that fights coronaviruses has the following structural formula:

[0061] In formula (I): The wavy line indicates that the carbon atom connected to the single bond has two configurations, R and S, or that the furanose ring has two configurations, D and L.

[0062] A pharmaceutical composition comprising a compound of the following formula or a pharmaceutically acceptable salt thereof: an anticoronavirus compound or a pharmaceutically acceptable salt thereof as claimed in claim 1, a crystalline hydrate thereof or a solvation thereof or a prodrug thereof or a combination thereof with other compounds.

[0063] Anti-coronavirus drugs are those effective against 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV. Coronaviruses are selected from: 2019-nCoV (causing COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome), and MERS-CoV (causing Middle East respiratory syndrome).

[0064] A method for synthesizing a nucleoside compound that is effective against coronaviruses, comprising the following steps:

[0065] Step 1:

[0066] ;

[0067] 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 was completed, the mixture was stirred overnight at room temperature. 2.34 g of sodium bicarbonate solid 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 total of 6.31 g of colorless oily compound B.

[0068] Step 2:

[0069] ;

[0070] Under nitrogen protection and an ice-water bath, 33.3 mmol of compound B was dissolved in 100 ml of dry DMF solution, 6.66 g of 60% sodium hydroxide was added and stirred at room temperature for 10 min, and 22.8 g of benzyl bromide was added dropwise at 0 °C; after the addition of benzyl bromide was completed, the mixture was stirred overnight at room temperature; the solution was poured into an ice-water solution of saturated ammonium chloride and extracted three times with ethyl acetate; the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (EA / PE = 20 / 1 - 10 / 1) to give 9.74 g of colorless transparent oily compound C;

[0071] Step 3:

[0072] ;

[0073] At 0℃, 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 complete, the reaction solution was heated to 80℃ and reacted for 6 hours. After the reaction was completed, the solution was concentrated under reduced pressure to 5-6 ml of solvent. The concentrated solution was dissolved in ethyl acetate and water. The organic phase was washed with sodium carbonate solid until pH > 7. The organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (PE / EA = 5 / 1) to obtain 1.05 g of colorless transparent oily compound D.

[0074] Step 4:

[0075] ;

[0076] 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 the reaction was carried out overnight at room temperature. The reaction solution was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography (PE / EA=20 / 1) to give 787 mg of colorless transparent oily compound E.

[0077] Step 5:

[0078] ;

[0079] Under nitrogen protection, 514 mg of TMSCl was added to 5 ml of dry tetrahydrofuran solution containing 504 mg of compound F. After the addition, the reaction was allowed to proceed at room temperature for 20 minutes. Then, the reaction solution was cooled to -78°C, and 5.38 ml of BuLi was added dropwise to the reaction solution. After 1 hour, a solution of compound 5 containing 900 mg of compound E and 5 ml of dry tetrahydrofuran was added dropwise at 0°C. After the addition was completed, the reaction was stirred at -78°C for 1 hour. The reaction was then quenched by adding saturated ammonium chloride after the temperature was raised to 0°C. The organic phase was extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography (PE / EA = 1 / 1-1 / 2) to obtain compound G (375 mg, brown solid).

[0080] Step 6:

[0081] ;

[0082] Under nitrogen protection, 375 mg of compound G was dissolved in 10 ml of dry dichloromethane solution, cooled to -78 °C, and 204 mg of TfOH was added dropwise while stirring for 10 min. Then, 317 mg of TMSOTf was added dropwise to the reaction solution at -78 °C, and the reaction was carried out for 30 min. 269 mg of TMSCN was slowly added at -78 °C, and the mixture was 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 the mixture was stirred for 10 min. The organic phase was extracted with dichloromethane, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography (PE / EA = 1 / 1) to obtain compound H (288 mg of pale yellow solid).

[0083] Step 7:

[0084] ;

[0085] 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 1 M BCl3 n-hexane solution was added dropwise. The mixture was heated 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. The reaction mixture was heated to room temperature and then dried by rotary evaporation to obtain crude product. The crude product was slurried with n-hexane, and the supernatant was discarded. The slurrying and decanting steps were repeated 3 times. Methanol (20 mL) was added and heated to 45 °C. Water was added in a 1:1 volume ratio with methanol. Part of the solution was evaporated by rotary evaporation at 45 °C and cooled to room temperature for filtration. The filter cake was washed with a small amount of methanol and dried to obtain 686 mg of white solid compound I.

[0086] Step 8:

[0087] ;

[0088] Under nitrogen protection, compound I (686) 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. The mixture was then heated to 45 °C and stirred for 30 min. 694 mg of sodium bicarbonate solid and 680 mg of water were added at room temperature and stirred for 15 min. After concentrating the reaction mixture, 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 subjected to column chromatography (DCM / MEOH = 50 / 1-20 / 1) to obtain compound J (617 mg of white solid).

[0089] Step 9:

[0090] ;

[0091] Under nitrogen protection, 10 g of compound K and 19.82 g of compound L were dissolved in 300 ml of toluene, and 18.45 g of p-toluenesulfonic acid monohydrate was added. The mixture was heated to 135 °C and water was separated using a water separator. The reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated, slurry was added with diethyl ether, filtered, and the filter cake was washed with diethyl ether and dried to obtain a white solid compound M.

[0092] Step 10:

[0093] ;

[0094] Under nitrogen protection, 5.76 g of compound M was dissolved in 100 mL of dry dichloromethane. 3.38 g of compound N was added at 0 °C, and the mixture was stirred for 45 min. Then, 3.57 g of triethylamine (1.38 g, 13.69 mmol, 2.1 eq) was added dropwise, and the reaction proceeded for three hours. 2.23 g of p-nitrophenol was added at 0 °C, followed by the dropwise addition of 1.62 g of triethylamine, and the reaction proceeded overnight at room temperature. The reaction solution was washed with water, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered to remove the anhydrous sodium sulfate. The filtrate was concentrated and column chromatography (PE / EA = 5 / 1-4 / 1) to obtain compound O (4.92 g, colorless transparent oily liquid).

[0095] Step 11:

[0096] ;

[0097] 617 mg of compound O, 951 mg of compound P, and 177 mg of magnesium chloride were mixed in 100 ml of dry acetonitrile and 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. The mixture was cooled to room temperature and diluted with ethyl acetate. It was washed successively with 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated saline solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 40:1-20 / 1) to give compound Q (731 mg white solid).

[0098] Step 12:

[0099] ;

[0100] 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 passed through a column (DCM / MeOH=20 / 1) to give compound g1-A (1.66 g, white solid).

[0101] Compound g1-A was chirally resolved to yield two compounds with single configurations. (Chromatographic column: DaicelCHIRALPAK® 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: mobile phase A: mobile phase B = 60:40 (V / V)).

[0102] Cytotoxicity test steps:

[0103] Monupiravir (purchased from Jiangsu Aikon Biomedical R&D Co., Ltd.), Bel7402 cells (provided by the Antiviral Drug Research Laboratory of the School of Pharmacy, Fudan University), fetal bovine serum (FBS) (produced by Thermo Fisher Scientific), DMEM culture medium (Thermo Fisher Scientific), carbon dioxide incubator (Thermo Fisher Scientific), and real-time PCR (Thermo Fisher Scientific).

[0104] Bel7402 cells (1.5*10⁶ cells) were seeded in 96-well plates. 4 / well), and cultured for 24 h. After removing the culture medium, add 100 μL / well of the test drug solution, with two replicates for each concentration. The normal control group received an equal volume of culture medium. Incubate at 37℃ in a 5% CO2 incubator for 48 h. Then, add 15 µL of 5 mg / mL MTT solution to each well and continue incubation for another 4 h. Remove the supernatant, add 100 µL of DMSO to each well, and dissolve by low-speed shaking. Measure the OD value at 490 nm. Compare the OD value with the normal control group and calculate the cell viability as (mean OD value of the drug group – blank OD value) / (mean OD value of the normal control group – blank OD value) × 100%.

[0105] Steps for testing anti-coronavirus 229E activity:

[0106] Bel7402 cells (2.5*10⁶ cells) were seeded in 96-well plates. 4 / well), incubate for 24 h. Aspirate the supernatant; add 10 [units of liquid name missing] to the drug experimental group and the virus control group. -2 100 μL of HCoV virus solution (229E virus) was added to each well and incubated at 37°C in a 5% CO2 incubator for 5 h. After removing the culture medium, 100 μL of the test drug solution was added to each well in the drug experimental group, with two replicates for each concentration. The normal control group and the virus control group were incubated with an equal volume of maintenance culture medium. After incubation at 37°C in a 5% CO2 incubator for 72 h, cell viability was measured using the MTT assay (method as above), and cell survival rate was calculated as (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] Cytotoxicity assays showed that the test samples at concentrations below 125 μM, in the absence of an infection background, exhibited no significant cytotoxicity and were lower than the control drug.

[0109] The efficacy test results showed that, under the current high challenge conditions, the test sample still had certain antiviral activity at a concentration of 0.04 μM, which was comparable to the antiviral activity of the marketed control drug Molnupiravir, and is worthy of further research.

[0110]

[0111] (Note: Molnupiravir is one of only two small molecule nucleoside inhibitors against COVID-19 that have been approved for marketing in the United States, jointly developed by Merck and Ridgeback. It has also been officially introduced and approved for use in China.)

[0112] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for synthesizing a nucleoside compound that inhibits coronaviruses, characterized in that: Includes the following steps: 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 was completed, the mixture was stirred overnight at room temperature. 2.34 g of sodium bicarbonate solid 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 total of 6.31 g of colorless oily compound B. Step 2: ; Under nitrogen protection and an ice-water bath, 33.3 mmol of compound B was dissolved in 100 ml of dry DMF solution, 6.66 g of 60% sodium hydroxide was added and stirred at room temperature for 10 min, and 22.8 g of benzyl bromide was added dropwise at 0 °C; after the addition of benzyl bromide was completed, the mixture was stirred overnight at room temperature; the solution was poured into an ice-water solution of saturated ammonium chloride and extracted three times with ethyl acetate; the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (EA / PE = 20 / 1 - 10 / 1) to give 9.74 g of colorless transparent oily compound C; Step 3: ; At 0℃, 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 complete, the reaction solution was heated to 80℃ and reacted for 6 hours. After the reaction was completed, the solution was concentrated under reduced pressure to 5-6 ml of solvent. After concentration, the solution was dissolved in ethyl acetate and water. The organic phase was washed with sodium carbonate solid until pH > 7. The organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and column chromatography was performed to obtain 1.05 g of colorless transparent oily compound D with PE-PE / EA = 5 / 1. 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 the reaction was carried out overnight at room temperature. The reaction solution was poured into ice water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to obtain 787 mg of colorless transparent oily compound E (PE-PE / EA = 20 / 1). Step 5: ; Under nitrogen protection, 514 mg of TMSCl was added to 5 ml of dry tetrahydrofuran solution containing 504 mg of compound F. After the addition, the reaction was allowed to proceed at room temperature for 20 minutes. Then, the reaction solution was cooled to -78°C, and 5.38 ml of BuLi was added dropwise to the reaction solution. One hour later, a solution of compound 5 containing 900 mg of compound E and 5 ml of dry tetrahydrofuran was added dropwise at 0°C. After the addition was completed, the reaction was stirred at -78°C for 1 hour. The reaction was then quenched by adding saturated ammonium chloride after the temperature was raised to 0°C. The organic phase was extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography (PE / EA = 1 / 1-1 / 2) 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 and 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 reacted for 30 min. 269 mg of TMSCN was slowly added at -78 °C and stirred for 2 h at -78 °C. 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. The organic phase was extracted with dichloromethane, washed with saturated brine, dried with 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. 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 1 M BCl3 n-hexane solution was added dropwise. The mixture was heated 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. The reaction mixture was heated to room temperature and then dried by rotary evaporation to obtain crude product. The crude product was slurried with n-hexane, and the supernatant was discarded. The slurrying and decanting steps were repeated 3 times. 20 mL of methanol was added and heated to 45 °C. Water was added in a 1:1 volume ratio with methanol. Part of the solution was evaporated by rotary evaporation at 45 °C and cooled to room temperature. The mixture was filtered, and the filter cake was washed with a small amount of methanol. The filter cake was dried to obtain 686 mg of white solid compound I. Step 8: ; Under nitrogen protection, compound I (686) 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. The mixture was then heated to 45 °C and stirred for 30 min. 694 mg of sodium bicarbonate solid and 680 mg of water were added at room temperature and stirred for 15 min. After concentrating the reaction solution, water and ethyl acetate were added, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to 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, and 18.45 g of p-toluenesulfonic acid monohydrate was added. The mixture was heated to 135 °C and water was separated using a water separator. The reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was concentrated, slurry was added with diethyl ether, filtered, and the filter cake was washed with diethyl ether and dried to obtain a white solid compound M. Step 10: ; Under nitrogen protection, 5.76 g of compound M was dissolved in 100 mL of dry dichloromethane. 3.38 g of compound N was added at 0 °C, and after stirring for 45 min, 3.57 g of triethylamine was added dropwise, and the reaction was allowed to proceed for three hours. 2.23 g of p-nitrophenol was added at 0 °C, followed by the dropwise addition of 1.62 g of triethylamine, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was washed with water, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated. Column chromatography with PE / EA = 5 / 1-4 / 1 yielded 4.92 g of a colorless, transparent, oily liquid compound O. 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. The mixture was stirred at 50 °C for 10 min under N2 protection. 601 mg of N,N-isopropylethylamine was added and the mixture was stirred for another 20 min. The mixture was cooled to room temperature and diluted with ethyl acetate. The solution was washed successively with 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated saline solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography (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 column chromatography (DCM / MeOH = 20 / 1) yielded 1.66 g of white solid compound g1-A. Compound g1-A was chirally resolved to yield two compounds with single configurations.

2. The method for synthesizing the anti-coronavirus nucleoside compound according to claim 1, characterized in that: The chiral separation conditions were as follows: 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 volume ratio: mobile phase A: mobile phase B = 60:40.

Images