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CN116284135B - Preparation method and application of anti-coronavirus nucleoside compounds - Google Patents

Preparation method and application of anti-coronavirus nucleoside compounds Download PDF

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CN116284135B
CN116284135B CN202310488947.0A CN202310488947A CN116284135B CN 116284135 B CN116284135 B CN 116284135B CN 202310488947 A CN202310488947 A CN 202310488947A CN 116284135 B CN116284135 B CN 116284135B
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CN116284135A (en
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鲍甫义
翟虎渠
钟南山
薛金华
胡豫
袁仁涛
马洁
周亚弟
霍立茹
鲍纯谦
许慧巍
吴军
陈锋
潘永利
杨梦琪
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Guoqing Biomedical Shanghai Co ltd
Guoqing Future Co ltd
Nanjing Mingshiheng Venture Capital Co ltd
Nanjing Yiyuan Biomedical Research Institute Co ltd
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Guoqing Future Co ltd
Nanjing Mingshiheng Venture Capital Co ltd
Nanjing Yiyuan Biomedical Research Institute Co ltd
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    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
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Abstract

The invention discloses a preparation method and application of an anti-coronavirus nucleoside compound, wherein the structural formula of the anti-coronavirus compound g1-AA is as follows: . The result of the drug effect test shows that under the current high toxicity attack background, the test sample still has certain antiviral activity at the concentration of 0.04uM, which is equivalent to the antiviral activity of the control drug Mo Nupi Lawei (Molnupiravir) on the market, and has the value of further research.

Description

Preparation method and application of anti-coronavirus nucleoside compound
Technical Field
The invention belongs to the technical field of synthesis of antiviral compounds, and particularly relates to a preparation method and application of an anti-coronavirus nucleoside compound.
Background
Coronaviruses (English name: coronavirus) belong to the genus coronaviridae, the order of the genus Coronaviridae, are RNA viruses with envelope and linear single positive strand genome, and are a large class of viruses widely existing in nature. This virus is visible under electron microscopy as coronal peripheral coronavirus and is therefore called coronavirus (Coronaviridae). In 1975, the virus naming committee formally named coronaviridae. Including rhinoviruses, B814 viruses, 229E viruses, OC43 strains, and infections that can infect humans and cause severe respiratory diseases, such as the Middle East Respiratory Syndrome (MERS), severe acute respiratory syndrome (SARS, a variant of coronavirus, a causative agent of atypical pneumonia), and novel coronaviruses (SARS-CoV-2), among others, with symptoms of infection ranging from common cold to severe pulmonary infections.
The novel potential targets for coronavirus pneumonia COVID-19 are divided into three major classes, structural proteins including Spike Protein (Surface Glycoprotein, spike Protein), E Protein (E Protein), M Protein (membrane Protein) and N Protein (Nucleocapsid Phosphoprotein, N Protein), and non-structural proteins including replicase polyprotein 1ab, 3C-like protease (3C-like protease), papain (Papain-like protease), NSP12 (RDRP, RNA-dependent RNA polymerase), helicase, NSP13 (RNA helicase )、Nsp14(Guanine-N7 methyltransferase)、Nsp15(Uridylate-specific endoribonuclease)、Nsp16(2'-O-methyl transferase,2'-O- methyltransferase), and other coronavirus treatment-related targets mainly ACE2 (angiotensin converting enzyme 2).
The novel coronavirus therapeutic medicine mainly comprises three types, and has different emphasis directions and applicable groups, wherein the small molecule antiviral medicine can be used for treating patients suffering from mild and moderate diseases, and the neutralizing antibody can be used for treating patients suffering from mild and moderate diseases, and the immunoregulation medicine is basically used for patients suffering from severe diseases. At present, according to different action mechanisms, the novel small crown molecule antiviral drugs mainly comprise RNA polymerase inhibitors, 3CL protease inhibitors and the like. There are only 3 new classes of crown therapeutic small molecule drugs available worldwide that act on novel coronavirus polymerases (RdRp), including adefovir (REMDESIVIR) from gilid science, mo Nupi rad (Molnupiravir) co-developed by moesadong and Ridgeback, alzheimers from henna real biotechnology, and deuterium remidervier hydrobromide (VV 116) from shanghai wang real biomedical, inc. However, to date, no drug has been able to be a specific drug for new coronaviruses or coronaviruses.
In view of the foregoing, there is a strong need in the art to develop more potent inhibitors directed to inhibiting coronavirus replication for use in diseases associated with coronavirus infection.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
The present invention aims to provide a nucleoside compound against coronavirus, which overcomes the above-mentioned drawbacks of the prior art.
In order to achieve the above object, the present invention provides an anti-coronavirus nucleoside compound having the structural formula:
In formula (I):
Further, the carbon atoms connected by single bonds represented by wavy lines of the anti-coronavirus nucleoside compound have two configurations of R and S, or the furanose ring has two configurations of D/L.
A pharmaceutical composition comprising an anti-coronavirus compound or a pharmaceutically acceptable salt, crystalline hydrate or solvate thereof or prodrug thereof, or a combination thereof with other compounds as described hereinbefore.
Further, anti-coronavirus drugs are drugs against 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
A method for synthesizing anti-coronavirus nucleoside compounds comprises the following steps:
Step 1:
Dropwise adding 0.6ml of concentrated sulfuric acid into 100ml of dry methanol solution dissolved with 5g of compound A under the protection of nitrogen and ice water bath conditions, stirring overnight at room temperature after the dropwise addition of the concentrated sulfuric acid is finished, adding 2.34g of sodium bicarbonate solid to adjust the pH to be more than 7, filtering with kieselguhr, washing a filter cake with methanol, and concentrating the filtrate to obtain a total of 6.31g of colorless oily compound B;
Step 2:
Under the protection of nitrogen and ice water bath, 33.3mmol of compound B is dissolved in 100ml of dry DMF solution, 6.66g of 60% sodium hydrogen is added, the mixture is stirred for 10min at room temperature, 22.8g of benzyl bromide is added dropwise at 0 ℃, the mixture is stirred overnight at room temperature after the benzyl bromide is added dropwise, an ice water solution of saturated ammonia chloride is poured, the mixture is extracted three times by ethyl acetate, an organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, filtered, concentrated, and EA/PE=20/1-10/1 column chromatography is carried out to obtain 9.74g of colorless transparent oily compound C;
Step 3:
Dropwise adding 0.2ml sulfuric acid into 30ml acetic acid solution dissolved with 2g of compound C at 0 ℃, heating the reaction solution to 80 ℃ for reaction for 6 hours after the dropwise adding is finished, concentrating to 5-6ml solvent under reduced pressure after the reaction is finished, dissolving the solvent with ethyl acetate and water after concentrating, washing an organic phase with sodium carbonate solid until the pH is more than 7, washing the organic phase with saturated saline solution, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and carrying out PE/EA=5/1 column chromatography to obtain 1.05g colorless transparent oily compound D;
Step 4:
1.05g of compound D is dissolved in 10ml of dry DMSO, 1.27g of acetic anhydride is dripped under the protection of nitrogen and reacted overnight at room temperature, the reaction solution is poured into ice water, extracted by ethyl acetate, the organic phase is washed by saturated salt water, and the organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure, PE/EA=20/1 column chromatography is carried out to obtain 787mg of colorless transparent oily compound E;
Step 5:
Under the protection of nitrogen, 514mg TMSCl is added into 5ml dry tetrahydrofuran solution dissolved with 504mg compound F, after the addition, the reaction is carried out for 20 minutes at room temperature, then the reaction solution is cooled to-78 ℃,5.38ml BuLi is added into the reaction solution in a dropwise manner, after 1 hour, 0 ℃ compound 5 solution is added into the reaction solution in a dropwise manner, the compound 5 solution comprises 900mg compound E and 5ml dry tetrahydrofuran (the total amount of the compound E is far more than 900mg after a plurality of batches is made), only 900mg is selected for small test, the professional cannot understand the patent number is not influenced), after the dropwise addition, the reaction is carried out for 1 hour at-78 ℃, saturated ammonia chloride is added for quenching after the temperature is increased to 0 ℃, the organic phase is extracted by ethyl acetate, the organic phase is washed by saturated salt water, and dried by anhydrous sodium sulfate, filtered, decompressed and concentrated by column chromatography (PE/EA=1/1-1/2) to obtain compound G (375 mg, brown solid);
Step 6:
Under the protection of nitrogen, 375mg of compound G is dissolved in 75mL of dry dichloromethane solution, cooled to-78 ℃, 204mgTfOH is added dropwise and stirred for 10min, 317mg of TMSOTF is then added dropwise to the reaction solution at-78 ℃ for reaction for 30min, 269mg of TMSCN is slowly added at-78 ℃, stirring is carried out for 2H at-78 ℃, 241mg of triethylamine reaction solution is added dropwise and slowly warmed to room temperature, 514mg of solid sodium bicarbonate and 2mL of water are sequentially added and stirred for 10min, the dichloromethane is used for extracting an organic phase, the organic phase is washed by saturated saline, and the organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure and subjected to column chromatography PE/EA=1/1 to obtain 288mg of pale yellow solid compound H;
Step 7:
2.5g of compound H is dissolved in 20mL of dry dichloromethane solution under the protection of nitrogen, the solution is cooled to-78 ℃, 16.91mL of 1M normal hexane solution of BCl 3 is added dropwise, the temperature is raised to-40 ℃ and stirred for 2 hours, after the reaction is completed, 3.85g of methanol, 4.5g of triethylamine and 7.7g of methanol are added dropwise at-78 ℃, the reaction is heated to room temperature and then the reaction solution is dried in a rotating way to obtain a crude product, the crude product is added with normal hexane for pulping, the supernatant is poured off, the pulping and pouring steps are repeated for 3 times, 20mL of methanol is added for heating to 45 ℃, water is added according to the volume ratio of 1 to 1 of methanol, part of solution is distilled off in a rotating way at 45 ℃ and cooled to room temperature for filtering, a little methanol is used for washing filter cakes, and the filter cakes are dried to obtain 686mg of white solid compound I;
Step 8:
Under the protection of nitrogen, 686 compound I and 1.18g of 2, 2-dimethoxy propane are dissolved in 10ml of acetone, 0.17ml of concentrated sulfuric acid is dripped at room temperature and stirred for 30min, the mixture is heated to 45 ℃ and stirred for 30min, 694mg of sodium bicarbonate solid and 680mg of water are added at room temperature and stirred for 15min, the organic phase is separated by adding water and ethyl acetate after the reaction solution is concentrated, and 617mg of white solid compound J is obtained by drying the organic phase with anhydrous sodium sulfate, filtering, decompressing and concentrating and performing column chromatography (DCM/MEOH=50/1-20/1);
Step 9:
Under the protection of nitrogen, 10g of compound K and 19.82g of compound L are dissolved in 300ml of toluene, 18.45g of p-toluenesulfonic acid monohydrate is added, the mixture is heated to 135 ℃ and is separated by a water separator for reaction for 24 hours, the reaction liquid is concentrated after the reaction is finished, diethyl ether is added for pulping and filtering, and filter cakes are washed by diethyl ether and dried to obtain a white solid compound M;
Step 10:
5.76g of Compound M was dissolved in 100mL of dry dichloromethane under nitrogen atmosphere, 3.38g of Compound N was added at 0℃and the mixture was stirred for 45min, 3.57g of triethylamine (1.38 g,13.69mmol,2.1 eq) was added dropwise and reacted for three hours, 2.23g of p-nitrophenol was added at 0℃and 1.62g of triethylamine was added dropwise and reacted overnight at room temperature. Washing the reaction solution with water, separating out an organic phase, washing the organic phase with saturated saline, drying the organic phase with anhydrous sodium sulfate, filtering to remove the anhydrous sodium sulfate, concentrating the filtrate, and passing through a column PE/EA=5/1-4/1 to obtain 4.92g of colorless transparent oily liquid compound O;
Step 11:
617mg of compound O, 951mg of compound P and 177mg of magnesium chloride are mixed in 100ml of dry acetonitrile, stirred for 10min at 50 ℃ under the protection of N2, 601mg of N, N-isopropylethylamine is added, stirring is continued for 20min, cooling is carried out to room temperature, ethyl acetate is added for dilution, 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated saline solution are sequentially added for washing, and the separated organic phase is dried and concentrated by anhydrous sodium sulfate, and column chromatography (DCM/MEOH=40/1-20/1) is carried out to obtain 731mg of white solid compound Q;
Step 12:
4.22g of Compound 13 was dissolved in 100ml of formic acid at room temperature, stirred overnight at room temperature, and the reaction concentrated to give 1.66g of Compound g1-A as a white solid (DCM/MeOH=20/1);
Step 13:
300mg of compound g1-a was dissolved in 10ml of dry tetrahydrofuran, 128mg of n, n-dimethylformamide dimethyl acetal was added at room temperature, stirred overnight at room temperature, and the reaction mixture was concentrated to dryness (DCM/meoh=20/1) to give compound R (274 mg, white solid);
Step 14:
274mg of compound R is dissolved in 5ml of dry dichloromethane, 145mg of triethylamine, 25mg of 4-dimethylaminopyridine DMAP and 161mg of compound S are sequentially added under the protection of nitrogen and the condition of room temperature, the reaction solution is stirred for 1h at room temperature and quenched by adding methanol, the reaction solution is stirred for 30min at room temperature and then concentrated by a column (PE-EA) to obtain compound T (290 mg, colorless transparent oil),
Step 15:
290Mg of compound T is dissolved in 10ml of trifluoroacetic acid, stirred overnight at room temperature, ethyl acetate is added after the reaction solution is concentrated, saturated sodium bicarbonate solution is added to adjust the pH to 7, the organic phase is separated and washed with saturated saline solution, dried over anhydrous sodium sulfate and filtered, and the filtrate is concentrated and then passed through a column (DCM/MeOH=20/1) to obtain compound T (237 mg, white solid);
chiral resolution is carried out on the compound g1-AA to obtain the target compound. Chromatography column Daicel IB 250 x 30mm,10 μm, mobile phase A: n-hexane, mobile phase B: isopropanol, detection wavelength: 254nm/214nm, flow rate: 25mL/min, isocratic elution procedure: mobile phase A: mobile phase B=70:30 (V/V).
Drawings
FIGS. 1.1-1.3 show chiral liquid phase diagrams of the products g 1-AA;
FIGS. 2.1 and 2.2 are nuclear magnetic patterns of two configurations of the product g 1-AA;
fig. 3-15 are nuclear magnetic patterns of the products of each sub-step in the synthesis process.
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
An anti-coronavirus nucleoside compound, the structural formula of the anti-coronavirus compound is as follows:
In formula (I):
Further, the carbon atoms connected by single bonds represented by wavy lines of the anti-coronavirus nucleoside compound have two configurations of R and S, or the furanose ring has two configurations of D/L.
A pharmaceutical composition comprising an anti-coronavirus compound or a pharmaceutically acceptable salt, crystalline hydrate or solvate thereof or prodrug thereof, or a combination thereof with other compounds as described hereinbefore.
Further, anti-coronavirus drugs are drugs against 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
A method for synthesizing anti-coronavirus nucleoside compounds comprises the following steps:
Step 1:
Dropwise adding 0.6ml of concentrated sulfuric acid into 100ml of dry methanol solution dissolved with 5g of compound A under the protection of nitrogen and ice water bath conditions, stirring overnight at room temperature after the dropwise addition of the concentrated sulfuric acid is finished, adding 2.34g of sodium bicarbonate solid to adjust the pH to be more than 7, filtering with kieselguhr, washing a filter cake with methanol, and concentrating the filtrate to obtain a total of 6.31g of colorless oily compound B;
Step 2:
Under the protection of nitrogen and ice water bath, 33.3mmol of compound B is dissolved in 100ml of dry DMF solution, 6.66g of 60% sodium hydrogen is added, the mixture is stirred for 10min at room temperature, 22.8g of benzyl bromide is added dropwise at 0 ℃, the mixture is stirred overnight at room temperature after the benzyl bromide is added dropwise, an ice water solution of saturated ammonia chloride is poured, the mixture is extracted three times by ethyl acetate, an organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, filtered, concentrated, and EA/PE=20/1-10/1 column chromatography is carried out to obtain 9.74g of colorless transparent oily compound C;
Step 3:
Dropwise adding 0.2ml sulfuric acid into 30ml acetic acid solution dissolved with 2g of compound C at 0 ℃, heating the reaction solution to 80 ℃ for reaction for 6 hours after the dropwise adding is finished, concentrating to 5-6ml solvent under reduced pressure after the reaction is finished, dissolving the solvent with ethyl acetate and water after concentrating, washing an organic phase with sodium carbonate solid until the pH is more than 7, washing the organic phase with saturated saline solution, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and carrying out PE/EA=5/1 column chromatography to obtain 1.05g colorless transparent oily compound D;
Step 4:
1.05g of compound D is dissolved in 10ml of dry DMSO, 1.27g of acetic anhydride is dripped under the protection of nitrogen and reacted overnight at room temperature, the reaction solution is poured into ice water, extracted by ethyl acetate, the organic phase is washed by saturated salt water, and the organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure, PE/EA=20/1 column chromatography is carried out to obtain 787mg of colorless transparent oily compound E;
Step 5:
Under the protection of nitrogen, 514mg TMSCl is added into 5ml dry tetrahydrofuran solution dissolved with 504mg compound F, after the addition, the reaction is carried out for 20 minutes at room temperature, then the reaction solution is cooled to-78 ℃,5.38ml BuLi is added into the reaction solution in a dropwise manner, after 1 hour, 0 ℃ compound 5 solution is added into the reaction solution in a dropwise manner, the compound 5 solution comprises 900mg compound E and 5ml dry tetrahydrofuran (the total amount of the compound E is far more than 900mg after a plurality of batches is made), only 900mg is selected for small test, the professional cannot understand the patent number is not influenced), after the dropwise addition, the reaction is carried out for 1 hour at-78 ℃, saturated ammonia chloride is added for quenching after the temperature is increased to 0 ℃, the organic phase is extracted by ethyl acetate, the organic phase is washed by saturated salt water, and dried by anhydrous sodium sulfate, filtered, decompressed and concentrated by column chromatography (PE/EA=1/1-1/2) to obtain compound G (375 mg, brown solid);
Step 6:
Under the protection of nitrogen, 375mg of compound G is dissolved in 75mL of dry dichloromethane solution, cooled to-78 ℃, 204mgTfOH is added dropwise and stirred for 10min, 317mg of TMSOTF is then added dropwise to the reaction solution at-78 ℃ for reaction for 30min, 269mg of TMSCN is slowly added at-78 ℃, stirring is carried out for 2H at-78 ℃, 241mg of triethylamine reaction solution is added dropwise and slowly warmed to room temperature, 514mg of solid sodium bicarbonate and 2mL of water are sequentially added and stirred for 10min, the dichloromethane is used for extracting an organic phase, the organic phase is washed by saturated saline, and the organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure and subjected to column chromatography PE/EA=1/1 to obtain 288mg of pale yellow solid compound H;
Step 7:
2.5g of compound H is dissolved in 20mL of dry dichloromethane solution under the protection of nitrogen, the solution is cooled to-78 ℃, 16.91mL of 1M normal hexane solution of BCl 3 is added dropwise, the temperature is raised to-40 ℃ and stirred for 2 hours, after the reaction is completed, 3.85g of methanol, 4.5g of triethylamine and 7.7g of methanol are added dropwise at-78 ℃, the reaction is heated to room temperature and then the reaction solution is dried in a rotating way to obtain a crude product, the crude product is added with normal hexane for pulping, the supernatant is poured off, the pulping and pouring steps are repeated for 3 times, 20mL of methanol is added for heating to 45 ℃, water is added according to the volume ratio of 1 to 1 of methanol, part of solution is distilled off in a rotating way at 45 ℃ and cooled to room temperature for filtering, a little methanol is used for washing filter cakes, and the filter cakes are dried to obtain 686mg of white solid compound I;
Step 8:
Under the protection of nitrogen, 686 compound I and 1.18g of 2, 2-dimethoxy propane are dissolved in 10ml of acetone, 0.17ml of concentrated sulfuric acid is dripped at room temperature and stirred for 30min, the mixture is heated to 45 ℃ and stirred for 30min, 694mg of sodium bicarbonate solid and 680mg of water are added at room temperature and stirred for 15min, the organic phase is separated by adding water and ethyl acetate after the reaction solution is concentrated, and 617mg of white solid compound J is obtained by drying the organic phase with anhydrous sodium sulfate, filtering, decompressing and concentrating and performing column chromatography (DCM/MEOH=50/1-20/1);
Step 9:
Under the protection of nitrogen, 10g of compound K and 19.82g of compound L are dissolved in 300ml of toluene, 18.45g of p-toluenesulfonic acid monohydrate is added, the mixture is heated to 135 ℃ and is separated by a water separator for reaction for 24 hours, the reaction liquid is concentrated after the reaction is finished, diethyl ether is added for pulping and filtering, and filter cakes are washed by diethyl ether and dried to obtain a white solid compound M;
Step 10:
5.76g of Compound M was dissolved in 100mL of dry dichloromethane under nitrogen atmosphere, 3.38g of Compound N was added at 0℃and the mixture was stirred for 45min, 3.57g of triethylamine (1.38 g,13.69mmol,2.1 eq) was added dropwise and reacted for three hours, 2.23g of p-nitrophenol was added at 0℃and 1.62g of triethylamine was added dropwise and reacted overnight at room temperature. Washing the reaction solution with water, separating out an organic phase, washing the organic phase with saturated saline, drying the organic phase with anhydrous sodium sulfate, filtering to remove the anhydrous sodium sulfate, concentrating the filtrate, and passing through a column PE/EA=5/1-4/1 to obtain 4.92g of colorless transparent oily liquid compound O;
Step 11:
617mg of compound O, 951mg of compound P and 177mg of magnesium chloride are mixed in 100ml of dry acetonitrile, stirred for 10min at 50 ℃ under the protection of N2, 601mg of N, N-isopropylethylamine is added, stirring is continued for 20min, cooling is carried out to room temperature, ethyl acetate is added for dilution, 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated saline solution are sequentially added for washing, and the separated organic phase is dried and concentrated by anhydrous sodium sulfate, and column chromatography (DCM/MEOH=40/1-20/1) is carried out to obtain 731mg of white solid compound Q;
Step 12:
4.22g of Compound 13 was dissolved in 100ml of formic acid at room temperature, stirred overnight at room temperature, and the reaction concentrated to give 1.66g of Compound g1-A as a white solid (DCM/MeOH=20/1);
Step 13:
300mg of compound g1-a was dissolved in 10ml of dry tetrahydrofuran, 128mg of n, n-dimethylformamide dimethyl acetal was added at room temperature, stirred overnight at room temperature, and the reaction mixture was concentrated to dryness (DCM/meoh=20/1) to give compound R (274 mg, white solid);
Step 14:
274mg of compound R is dissolved in 5ml of dry dichloromethane, 145mg of triethylamine, 25mg of 4-dimethylaminopyridine DMAP and 161mg of compound S are sequentially added under the protection of nitrogen and the condition of room temperature, the reaction solution is stirred for 1h at room temperature and quenched by adding methanol, the reaction solution is stirred for 30min at room temperature and then concentrated by a column (PE-EA) to obtain compound T (290 mg, colorless transparent oil),
Step 15:
290Mg of compound T is dissolved in 10ml of trifluoroacetic acid, stirred overnight at room temperature, ethyl acetate is added after the reaction solution is concentrated, saturated sodium bicarbonate solution is added to adjust the pH to 7, the organic phase is separated and washed with saturated saline solution, dried over anhydrous sodium sulfate and filtered, and the filtrate is concentrated and then passed through a column (DCM/MeOH=20/1) to obtain compound T (237 mg, white solid);
chiral resolution is carried out on the compound g1-AA to obtain the target compound. Chromatography column Daicel IB 250 x 30mm,10 μm, mobile phase a: n-hexane, mobile phase B: isopropanol, detection wavelength 254nm/214nm, flow rate 25mL/min, isocratic elution procedure:
mobile phase a mobile phase b=70:30 (V/V).
Cytotoxicity test procedure:
Mo Nupi Rawei (available from Jiangsu ai Kang Shengwu pharmaceutical research Co., ltd.), bel7402 cells (available from the antiviral pharmaceutical laboratory at the university of double denier), fetal Bovine Serum (FBS) (available from the Semerle Feishmania Biochemical Co., ltd.), DMEM medium (Semerle Feishmania Biochemical Co., ltd.), carbon dioxide incubator (Semerle Feishmania Biochemical Co., ltd.), and fluorescent quantitative PCR (Semerle Feishmania Biochemical Co., ltd.).
Bel7402 cells (1.5×10 4/well) were seeded in 96-well plates and cultured for 24h. The culture solution was aspirated, 100 μl/well of the drug solution to be tested was added, 2 duplicate wells were set for each concentration, and the normal control group was added with an equal volume of culture solution. After incubation for 48h at 37℃in a 5% CO 2 incubator, 15. Mu.L of MTT solution at a concentration of 5mg/mL was added to each well and incubation was continued for 4h. The supernatant was aspirated, 100. Mu.L of DMSO was added to each well, and the OD value was determined by shaking at a low speed and at 490nm, and the cell viability (average OD value of drug group-blank OD value)/(average OD value of normal control group-blank OD value) ×100% was calculated as opposed to normal group cells.
Anti-coronavirus 229E activity test procedure:
Bel7402 cells (2.5×10 4/well) were seeded in 96-well plates and cultured for 24h. The supernatant was aspirated, and the drug experimental group and the virus control group were added with 100. Mu.L/well of 10 -2 HCoV virus solution, and adsorbed in a 37℃5% CO 2 incubator for 5 hours. The culture solution was aspirated, 100 μl/well of the drug solution to be tested was added to the drug test group, 2 duplicate wells were set for each concentration, and the normal control group and the virus control group were added to the equal volume of maintenance culture solution. After incubation for 72h at 37℃in a 5% CO 2 incubator, the MTT assay was used to determine cell activity (method as above) and cell viability (average OD in drug group-blank OD)/(average OD in normal control group-blank OD) ×100% was calculated.
Test conclusion:
the cytotoxicity test shows that the test sample is below 125 mu M, the cytotoxicity is not obvious under the condition of no infection background, and the toxicity is lower than that of the control medicine.
The result of the drug effect test shows that under the current high toxicity attack background, the test sample still has certain antiviral activity at the concentration of 0.04uM, which is equivalent to the antiviral activity of the control drug Mo Nupi Lawei (Molnupiravir) on the market, and has the value of further research.
(Note: mo Nupi Lavir (Molnupiravir) is one of the only two approved small molecule nucleoside anti-novel coronavirus inhibitors in the United states, commonly developed by the company Mitsadone and Ridgeback, and formally introduced and approved for use in China).
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (5)

1. An anti-coronavirus nucleoside compound is characterized in that the structural formula of the anti-coronavirus compound g1-AA is as follows: Wherein the furanose ring has L configuration.
2. A pharmaceutical composition comprising the anti-coronavirus compound g1-AA or a pharmaceutically acceptable salt thereof as claimed in claim 1.
3. Use of a composition according to claim 2 for the preparation of an anti-coronavirus drug, said anti-coronavirus drug being a drug against 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
4. A method for synthesizing anti-coronavirus nucleoside compounds is characterized by comprising the following steps:
Step 1 :;
Dropwise adding 0.6ml of concentrated sulfuric acid into 100ml of dry methanol solution dissolved with 5g of compound A under the protection of nitrogen and ice water bath conditions, stirring overnight at room temperature after the dropwise addition of the concentrated sulfuric acid is finished, adding 2.34g of sodium bicarbonate solid to adjust the pH to be more than 7, filtering with kieselguhr, washing a filter cake with methanol, and concentrating the filtrate to obtain a total of 6.31g of colorless oily compound B;
Step 2 :;
Under the protection of nitrogen and ice water bath, 33.3mmol of compound B is dissolved in 100ml of dry DMF solution, 6.66g of 60% sodium hydrogen is added, the mixture is stirred for 10min at room temperature, 22.8g of benzyl bromide is added dropwise at 0 ℃, the mixture is stirred overnight at room temperature after the benzyl bromide is added dropwise, an ice water solution of saturated ammonia chloride is poured, the mixture is extracted three times by ethyl acetate, an organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, filtered, concentrated, and EA/PE=20/1-10/1 column chromatography is carried out to obtain 9.74g of colorless transparent oily compound C;
Step 3 :;
Dropwise adding 0.2ml sulfuric acid into 30ml acetic acid solution dissolved with 2g of compound C at 0 ℃, heating the reaction solution to 80 ℃ for reaction for 6 hours after the dropwise adding is finished, concentrating to 5-6ml solvent under reduced pressure after the reaction is finished, dissolving the solvent with ethyl acetate and water after concentrating, washing an organic phase with sodium carbonate to pH of >7, washing the organic phase with saturated saline solution, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate, and carrying out PE/EA=5/1 column chromatography to obtain 1.05g colorless transparent oily compound D;
Step 4 :;
Dissolving 1.05g of compound D in 10ml of dry DMSO, dropwise adding 1.27g of acetic anhydride under the protection of nitrogen, reacting overnight at room temperature, pouring the reaction solution into ice water, extracting with ethyl acetate, washing an organic phase with saturated salt water, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and performing PE/EA=20/1 column chromatography to obtain 787mg of colorless transparent oily compound E;
Step 5 :;
Under the protection of nitrogen, 514mg TMSCl is added into 5ml dry tetrahydrofuran solution dissolved with 504mg of compound F, after the addition, the reaction is carried out for 20 minutes at room temperature, then the reaction solution is cooled to-78 ℃,5.38ml n-BuLi is dropwise added into the reaction solution, 0 ℃ compound E solution containing 900mg compound E and 5ml dry tetrahydrofuran is dropwise added after 1 hour, the reaction is stirred for 1 hour at-78 ℃ after the dropwise addition, the temperature is raised to 0 ℃ and then saturated ammonia chloride is added for quenching, ethyl acetate is used for extracting an organic phase, the organic phase is washed with saturated salt water, and then dried with anhydrous sodium sulfate, filtered, the filtrate is concentrated under reduced pressure, PE/EA=1/1-1/2 column chromatography is carried out to obtain 375mg, and brown solid compound G is obtained;
Step 6 :;
Under the protection of nitrogen, 375mg of compound G is dissolved in 10ml of dry dichloromethane solution, cooled to-78 ℃, 204mgTfOH is added dropwise and stirred for 10min, 317mg of TMSOTF is then added dropwise to the reaction solution at-78 ℃ for reaction for 30min, 269mg of TMSCN is slowly added at-78 ℃, stirring is carried out for 2H at-78 ℃, 241mg of triethylamine reaction solution is added dropwise and slowly warmed to room temperature, 514mg of solid sodium bicarbonate and 2ml of water are sequentially added and stirred for 10min, the dichloromethane is used for extracting an organic phase, the organic phase is washed by saturated saline, and the organic phase is dried by anhydrous sodium sulfate, filtered, the filtrate is concentrated under reduced pressure, and PE/EA=1/1 column chromatography is carried out to obtain 288mg of pale yellow solid compound H;
Step 7 :;
2.5g of compound H is dissolved in 20ml of dry dichloromethane solution under the protection of nitrogen, the solution is cooled to-78 ℃, 16.91ml of 1M normal hexane solution of BCl 3 is added dropwise, the temperature is raised to-40 ℃ and stirred for 2 hours, after the reaction is completed, 3.85g of methanol, 4.5g of triethylamine and 7.7g of methanol are added dropwise at-78 ℃, the reaction is heated to room temperature and then the reaction solution is dried in a rotating way to obtain a crude product, the crude product is added with normal hexane for pulping, the supernatant is poured off, the process is repeated for 3 times, 20mL methanol is added for heating to 45 ℃, water is added according to the volume ratio of 1 to 1 of methanol, part of the solution is distilled off in a rotating way at 45 ℃ and cooled to room temperature for filtration, a little methanol is used for washing filter cakes, and the filter cakes are dried to obtain 686mg of white solid compound I;
Step 8 :;
Under the protection of nitrogen, 686mg of compound I and 1.18g of 2,2' -dimethoxypropane are dissolved in 10ml of acetone, 0.17ml of concentrated sulfuric acid is dropwise added at room temperature and stirred for 30min, the mixture is heated to 45 ℃ and stirred for 30min, 694mg of sodium bicarbonate solid and 680mg of water are added at room temperature and stirred for 15min, the organic phase is separated by adding water and ethyl acetate after the reaction liquid is concentrated, and 617mg of white solid compound J is obtained by drying the organic phase with anhydrous sodium sulfate, filtering, concentrating under reduced pressure and carrying out column chromatography on DCM/MEOH=50:1-20/1;
Step 9 :;
Under the protection of nitrogen, 10g of compound K and 19.82g of compound L are dissolved in 300ml of toluene, 18.45g of p-toluenesulfonic acid monohydrate is added, the mixture is heated to 135 ℃ and is separated by a water separator for reaction for 24 hours, the reaction liquid is concentrated after the reaction is finished, diethyl ether is added for pulping and filtering, and filter cakes are washed by diethyl ether and dried to obtain a white solid compound M;
Step 10 :
;
Under the protection of nitrogen, 5.76g of compound M is dissolved in 100 mL dry dichloromethane, 3.38g of compound N is added at 0 ℃, 3.57g of triethylamine is added dropwise after stirring for 45min for reaction for three hours, 2.23g of p-nitrophenol is added at 0 ℃, 1.62g of triethylamine is added dropwise for reaction overnight at room temperature, the reaction liquid is washed with water, an organic phase is separated, the organic phase is washed with saturated common salt, then dried with anhydrous sodium sulfate, anhydrous sodium sulfate is removed by filtration, the filtrate is concentrated, and PE/EA=5/1-4/1 is separated by column chromatography to obtain 4.92 g colorless transparent oily liquid compound;
Step 11 :
;
617mg of compound O,951mg of compound P and 177mg of magnesium chloride are mixed in 100ml of dry acetonitrile, stirred for 10min at 50 ℃ under the protection of N 2, 601mg of N, N-isopropylethylamine is added, stirring is continued for 20min, cooling is carried out to room temperature, ethyl acetate is added for dilution, the reaction solution is washed by 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated saline solution, an organic phase is separated, dried and concentrated by anhydrous sodium sulfate, and DCM/MEOH=40:1-20/1 is separated by column chromatography to obtain 731mg of white solid compound Q;
Step 12 :
;
4.22g of Compound Q was dissolved in 100ml of formic acid at room temperature, stirred overnight at room temperature, the reaction concentrated and the DCM/MeOH=20/1 was isolated by column chromatography to give 1.66 g as a white solid, compound g1-A;
Step 13 :
;
300mg of compound g1-A was dissolved in 10ml of dry tetrahydrofuran, 128mg of N, N-dimethylformamide dimethyl acetal was added at room temperature, stirred overnight at room temperature, the reaction mixture was concentrated, and DCM/MeOH=20/1 was separated by column chromatography to give 274mg of compound R as a white solid;
Step 14 :
;
274mg of compound R is dissolved in 5ml of dry acetonitrile, 145mg of triethylamine, 25mg of 4-dimethylaminopyridine DMAP and 161mg of compound S are sequentially added under the protection of nitrogen and the condition of room temperature, the reaction solution is stirred for 1h at room temperature, methanol is added for quenching, the reaction solution is stirred for 30min at room temperature, and then the mixture is concentrated and passes through a column to obtain 290mg of colorless transparent oily compound T;
Step 15 :
;
290mg of compound T is dissolved in 10ml of trifluoroacetic acid, stirred overnight at room temperature, ethyl acetate is added after the reaction solution is concentrated, saturated sodium bicarbonate solution is added to adjust the pH to 7, the separated organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate and filtered, and 237mg of white solid compound T is obtained after the filtrate is concentrated by DCM/MeOH=20/1 column chromatography;
chiral resolution is carried out on the compound g1-AA to obtain the target compound.
5. The method for synthesizing the coronavirus-resistant nucleoside compound according to claim 4, wherein chiral resolution conditions are a chromatographic column of DAICEL CHIRALPAK cubic IB 250 x 30 mm, a mobile phase A of n-hexane, a mobile phase B of isopropanol, a detection wavelength of 254nm/214nm, a flow rate of 25mL/min, and an isocratic elution procedure according to a volume ratio of mobile phase A to mobile phase B=70:30.
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