CN116284135A - Preparation method and application of anti-coronavirus nucleoside compound - Google Patents
Preparation method and application of anti-coronavirus nucleoside compound Download PDFInfo
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- CN116284135A CN116284135A CN202310488947.0A CN202310488947A CN116284135A CN 116284135 A CN116284135 A CN 116284135A CN 202310488947 A CN202310488947 A CN 202310488947A CN 116284135 A CN116284135 A CN 116284135A
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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 marketed control drug Mo Nupi Lavir (Monspiravir), and has the value of further research.
Description
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: coronavir) belong to the order of the genus Coronaviridae, the family of coronaviridae, are a class of RNA viruses with a envelope and a linear single positive strand genome, and are a large class of viruses which are widely present in nature. This virus is seen under electron microscopy as coronally peripheral coronal, and is therefore known as 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.
Novel coronavirus infection covd-19 potential targets fall into three general classes, structural proteins (structure Protein) 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-like protease (Papain-like protease), NSP12 (RDRP, RNA-dependent RNA polymerase), helicase, NSP13 (RNA Helicase), NSP14 (guard-N7 methyl transferase), NSP15 (uri dylate-specific endoribonuclease), NSP16 (2' -O-methyl transferase ), and other coronavirus therapy related targets mainly ACE2 (angiotensin converting enzyme 2).
The novel coronavirus therapeutic drugs mainly comprise three types, which have different emphasis directions and applicable people: the small molecule antiviral drug can be used for patients with mild to moderate symptoms, and the neutralizing antibody is mainly used for patients with mild to moderate symptoms, and the immunoregulatory drug is basically used for patients with severe symptoms. 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 small molecule drugs available worldwide that act on novel coronavirus polymerases (RdRp) and include adefovir (Remdesivir), mo Nupi ravir (molnupirvir), alfudine, henna true biotechnology limited, and deuterium hydrobromide remide (VV 116), of the company gide sciences. 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; after the addition of concentrated sulfuric acid was completed, stirring was carried out at room temperature overnight; 2.34g of sodium bicarbonate solid is added to adjust the pH to be more than 7, diatomite is used for filtering, a filter cake is washed by methanol, and the filtrate is concentrated to obtain colorless oily compound B which is 6.31g in total;
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, and 22.8g of benzyl bromide is added dropwise at 0 ℃; after the completion of the benzyl bromide addition, stirring overnight at room temperature; pouring an ice water solution of saturated ammonia chloride, and extracting with ethyl acetate for three times; the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and chromatographed on a EA/pe=20/1-10/1 column to give 9.74g of compound C as a colorless transparent oil;
Step 3 ：
；
to 30ml of acetic acid solution containing 2g of compound C at 0℃were added dropwise 0.2ml of sulfuric acid; after the dripping is completed, the reaction solution is heated to 80 ℃ to react for 6 hours; concentrating under reduced pressure to 5-6ml of solvent 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 of 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 added dropwise under the protection of nitrogen, and the mixture is reacted 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 under reduced pressure, and performing PE/EA=20/1 column chromatography to obtain 787mg of colorless transparent oily compound E;
Step 5 ：
；
to a solution of 504mg of Compound F in 5ml of dry tetrahydrofuran under nitrogen protection, 514mg of TMSCl was added; after the addition, the reaction is carried out for 20 minutes at room temperature, then the reaction solution is cooled to-78 ℃,5.38ml of BuLi is added into the reaction solution in a dropwise manner, 1 hour later, 0 ℃ of compound 5 solution is added in a dropwise manner, the compound 5 solution comprises 900mg of compound E and 5ml of dry tetrahydrofuran (the total amount of the compound E is far more than 900mg after a plurality of batches are made), only 900mg is selected for carrying out the test, and the professional understanding of the patent is not influenced); stirring at-78 ℃ for reaction for 1 hour after the dripping is completed; heating to 0 ℃, and then adding saturated ammonia chloride for quenching; ethyl acetate, washing the organic phase with saturated brine, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and performing 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 75 mL dry dichloromethane solution, cooled to-78 ℃, 204mgTfOH is added dropwise and stirred for 10min; then, 317mg of TMSOTF is added dropwise into the reaction solution at the temperature of minus 78 ℃ for reaction for 30min; 269mg of TMSCN is slowly added at the temperature of minus 78 ℃, stirred for 2 hours at the temperature of minus 78 ℃, 241mg of triethylamine reaction liquid is dropwise added, and the temperature is slowly raised to the room temperature; 514mg of solid sodium bicarbonate and 2ml of water are sequentially added and stirred for 10min, the organic phase is extracted by methylene dichloride, the organic phase is washed by saturated saline, and the organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography PE/EA=1/1 to obtain 288mg of pale yellow solid compound H;
Step 7 ：
；
2.5g of Compound H are dissolved in 20ml of dry dichloromethane under the protection of nitrogen, cooled to-78℃and 1M BCl is added dropwise 3 16.91ml of n-hexane solution; heating to-40 ℃ and stirring for 2 hours, after the reaction is completed, dropwise adding 3.85g of methanol, 4.5g of triethylamine and 7.7g of methanol at-78 ℃, heating to room temperature, and rotationally drying the reaction liquid to obtain a crude product; pulping the crude product with n-hexane, pouring off supernatant, and repeating pulping and pouring steps for 3 times; adding 20mL methanol to heat to 45 ℃, adding water according to the volume ratio of 1 to 1 of methanol, rotationally evaporating part of the solution at 45 ℃, cooling to room temperature, filtering, washing a filter cake with a little methanol, and drying the filter cake 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 added dropwise at room temperature and stirred for 30min, and 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; concentrating the reaction solution, adding water and ethyl acetate to separate an organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and performing column chromatography (DCM/MEOH=50/1-20/1) to obtain 617mg of a white solid compound J;
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 water is separated by a water separator for reaction for 24 hours; concentrating the reaction solution after the reaction is finished, adding diethyl ether, pulping and filtering, washing a filter cake with diethyl ether, and drying to obtain a white solid compound M;
Step 10 ：
5.76g of compound M was dissolved in 100mL dry dichloromethane under nitrogen protection, 3.38g of compound N was added at 0℃and stirred for 45min, 3.57g of triethylamine (1.38 g, 13.69 mmol,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, then 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 a 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, an organic phase is separated, 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 ：
；
4.22. 4.22g of Compound 13 was dissolved in 100ml of formic acid at room temperature, stirred overnight at room temperature, and the reaction concentrated on a column (DCM/MeOH=20/1) 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, 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. Chromatographic column: daicel CHIRALPAK is 30 mm in volume from IB 250 to IB 10 mu m, and mobile phase A: n-hexane, mobile phase B: isopropyl alcohol, 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 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; after the addition of concentrated sulfuric acid was completed, stirring was carried out at room temperature overnight; 2.34g of sodium bicarbonate solid is added to adjust the pH to be more than 7, diatomite is used for filtering, a filter cake is washed by methanol, and the filtrate is concentrated to obtain colorless oily compound B which is 6.31g in total;
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, and 22.8g of benzyl bromide is added dropwise at 0 ℃; after the completion of the benzyl bromide addition, stirring overnight at room temperature; pouring an ice water solution of saturated ammonia chloride, and extracting with ethyl acetate for three times; the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and chromatographed on a EA/pe=20/1-10/1 column to give 9.74g of compound C as a colorless transparent oil;
Step 3 ：
;
to 30ml of acetic acid solution containing 2g of compound C at 0℃were added dropwise 0.2ml of sulfuric acid; after the dripping is completed, the reaction solution is heated to 80 ℃ to react for 6 hours; concentrating under reduced pressure to 5-6ml of solvent 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 of 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 added dropwise under the protection of nitrogen, and the mixture is reacted 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 under reduced pressure, and performing PE/EA=20/1 column chromatography to obtain 787mg of colorless transparent oily compound E;
Step 5 ：
;
to a solution of 504mg of Compound F in 5ml of dry tetrahydrofuran under nitrogen protection, 514mg of TMSCl was added; after the addition, the reaction is carried out for 20 minutes at room temperature, then the reaction solution is cooled to-78 ℃,5.38ml of BuLi is added into the reaction solution in a dropwise manner, 1 hour later, 0 ℃ of compound 5 solution is added in a dropwise manner, the compound 5 solution comprises 900mg of compound E and 5ml of dry tetrahydrofuran (the total amount of the compound E is far more than 900mg after a plurality of batches are made), only 900mg is selected for carrying out the test, and the professional understanding of the patent is not influenced); stirring at-78 ℃ for reaction for 1 hour after the dripping is completed; heating to 0 ℃, and then adding saturated ammonia chloride for quenching; ethyl acetate, washing the organic phase with saturated brine, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and performing 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 75 mL dry dichloromethane solution, cooled to-78 ℃, 204mgTfOH is added dropwise and stirred for 10min; then, 317mg of TMSOTF is added dropwise into the reaction solution at the temperature of minus 78 ℃ for reaction for 30min; 269mg of TMSCN is slowly added at the temperature of minus 78 ℃, stirred for 2 hours at the temperature of minus 78 ℃, 241mg of triethylamine reaction liquid is dropwise added, and the temperature is slowly raised to the room temperature; 514mg of solid sodium bicarbonate and 2ml of water are sequentially added and stirred for 10min, the organic phase is extracted by methylene dichloride, the organic phase is washed by saturated saline, and the organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography PE/EA=1/1 to obtain 288mg of pale yellow solid compound H;
Step 7 ：
;
2.5g of Compound H are dissolved in 20ml of dry dichloromethane under the protection of nitrogen, cooled to-78℃and 1M BCl is added dropwise 3 16.91ml of n-hexane solution; heating to-40 ℃ and stirring for 2 hours, after the reaction is completed, dropwise adding 3.85g of methanol, 4.5g of triethylamine and 7.7g of methanol at-78 ℃, heating to room temperature, and rotationally drying the reaction liquid to obtain a crude product; pulping the crude product with n-hexane, pouring off supernatant, and repeating pulping and pouring steps for 3 times; adding 20mL methanol to heat to 45 ℃, adding water according to the volume ratio of 1 to 1 of methanol, rotationally evaporating part of the solution at 45 ℃, cooling to room temperature, filtering, washing a filter cake with a little methanol, and drying the filter cake 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 added dropwise at room temperature and stirred for 30min, and 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; concentrating the reaction solution, adding water and ethyl acetate to separate an organic phase, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and performing column chromatography (DCM/MEOH=50/1-20/1) to obtain 617mg of a white solid compound J;
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 water is separated by a water separator for reaction for 24 hours; concentrating the reaction solution after the reaction is finished, adding diethyl ether, pulping and filtering, washing a filter cake with diethyl ether, and drying to obtain a white solid compound M;
Step 10 ：
;
5.76g of compound M was dissolved in 100mL dry dichloromethane under nitrogen protection, 3.38g of compound N was added at 0℃and stirred for 45min, 3.57g of triethylamine (1.38 g, 13.69 mmol,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, then 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 a 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, an organic phase is separated, 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 ：
;
4.22. 4.22g of Compound 13 was dissolved in 100ml of formic acid at room temperature, stirred overnight at room temperature, and the reaction concentrated on a column (DCM/MeOH=20/1) 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, 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. Chromatographic column: daicel CHIRALPAK is 30 mm in volume from IB 250 to IB 10 mu m, and mobile phase A: n-hexane, mobile phase B: isopropyl alcohol, 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 Lavir (available from Jiangsu ai Kang Shengwu pharmaceutical research Co., ltd.), bel7402 cells (supplied by the antiviral drug laboratory at the university of double denier pharmaceutical Co., ltd.), 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 were seeded in 96-well plates 4 Well), culture 24 h. 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. 37 ℃,5% CO 2 After 48 and h incubation, 15. Mu.L of MTT solution at a concentration of 5 mg/mL was added to each well and incubation was continued for 4 h. The supernatant was aspirated, 100 μl DMSO was added to each well, and the OD value was determined by shaking at low speed, 490 nm, 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 were seeded in 96-well plates 4 Well), culture 24 h. The supernatant was aspirated and 10 was added to the drug test group and the virus control group -2 HCoV virus solution 100. Mu.L/well, 37℃and 5% CO 2 Incubator adsorbs 5 h. 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. 37 ℃,5% CO 2 After incubation of 72 h in incubator, the MTT assay was used to determine cell activity (methods described above) and cell viability (average OD of drug group-blank OD)/(average OD value of normal control group-blank OD value) ×100%.
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 marketed control drug Mo Nupi Lavir (Monspiravir), and has the value of further research.
(note: mo Nupi Lavir (Monnupiravir) is one of the only two approved small molecule nucleoside anti-new coronavirus inhibitors available in the United states, commonly developed by the company Mitsadode and Ridgeback, inc., 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 (6)
1. An anti-coronavirus nucleoside compound, characterized in that: the structural formula of the anti-coronavirus compound g1-AA is as follows:
。
2. the anti-coronavirus nucleoside compound of claim 1, wherein: the carbon atoms connected by single bonds represented by wavy lines have two configurations of R and S, or the furanose ring has two configurations of D/L.
3. A pharmaceutical composition characterized by: the composition comprising the anti-coronavirus compound g1-AA of claim 1 or a pharmaceutically acceptable salt thereof.
4. Use of a composition according to claim 3 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.
5. A method for synthesizing anti-coronavirus nucleoside compounds is characterized in that: the method 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; after the addition of concentrated sulfuric acid was completed, stirring was carried out at room temperature overnight; 2.34g of sodium bicarbonate solid is added to adjust the pH to be more than 7, diatomite is used for filtering, a filter cake is washed by methanol, and the filtrate is concentrated to obtain colorless oily compound B which is 6.31g in total;
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, and 22.8g of benzyl bromide is added dropwise at 0 ℃; after the completion of the benzyl bromide addition, stirring overnight at room temperature; pouring an ice water solution of saturated ammonia chloride, and extracting with ethyl acetate for three times; the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and chromatographed on a EA/pe=20/1-10/1 column to give 9.74g of compound C as a colorless transparent oil;
Step 3 ：
;
to 30ml of acetic acid solution containing 2g of compound C at 0℃were added dropwise 0.2ml of sulfuric acid; after the dripping is completed, the reaction solution is heated to 80 ℃ to react for 6 hours; concentrating under reduced pressure to 5-6ml of solvent 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 of 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 added dropwise under the protection of nitrogen, and the mixture is reacted 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 under reduced pressure, and performing PE/EA=20/1 column chromatography to obtain 787mg of colorless transparent oily compound E;
Step 5 ：
;
to a solution of 504mg of Compound F in 5ml of dry tetrahydrofuran under nitrogen protection, 514mg of TMSCl was added; after the addition, the reaction was carried out at room temperature for 20 minutes, then the reaction solution was cooled to-78℃and 5.38ml of BuLi was added dropwise to the reaction solution, and after 1 hour, a 0℃solution of Compound 5 was added dropwise, the Compound 5 solution comprising 900mg of Compound E and 5ml of dried tetrahydrofuran; stirring at-78 ℃ for reaction for 1 hour after the dripping is completed; heating to 0 ℃, and then adding saturated ammonia chloride for quenching; the organic phase was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to PE/ea=1/1-1/2 column chromatography to give 375mg of compound G as a brown solid;
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; then, 317mg of TMSOTF is added dropwise into the reaction solution at the temperature of minus 78 ℃ for reaction for 30min; 269mg of TMSCN is slowly added at the temperature of minus 78 ℃, stirred for 2 hours at the temperature of minus 78 ℃, 241mg of triethylamine reaction liquid is dropwise added, and the temperature is slowly raised to the room temperature; 514mg of solid sodium bicarbonate and 2ml of water are sequentially added and stirred for 10min, the organic phase is extracted by methylene dichloride, 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 PE/EA=1/1 column chromatography to obtain 288mg of pale yellow solid compound H;
Step 7 ：
;
2.5g of Compound H are dissolved in 20ml of dry dichloromethane under the protection of nitrogen, cooled to-78℃and 1M BCl is added dropwise 3 16.91ml of n-hexane solution; heating to-40 ℃ and stirring for 2 hours, after the reaction is completed, dropwise adding 3.85g of methanol, 4.5g of triethylamine and 7.7g of methanol at-78 ℃, heating to room temperature, and rotationally drying the reaction liquid to obtain a crude product; pulping the crude product with n-hexane, pouring off supernatant, and repeating for 3 times; adding 20mL methanol to heat to 45 ℃, adding water according to the volume ratio of 1 to 1 of methanol, rotationally evaporating part of the solution at 45 ℃, cooling to room temperature, filtering, washing a filter cake with a little methanol, and drying the filter cake 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 added dropwise at room temperature and stirred for 30min, and 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; concentrating the reaction solution, adding water and ethyl acetate to separate an organic phase, drying the organic phase by using anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and obtaining 617mg of white solid compound J by 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 water is separated by a water separator for reaction for 24 hours; concentrating the reaction solution after the reaction is finished, adding diethyl ether, pulping and filtering, washing a filter cake with diethyl ether, and drying to obtain a white solid compound M;
Step 10 ：
;
under the protection of nitrogen, 5.76g of compound M is dissolved in 100mL dry dichloromethane, 3.38g of compound N is added at 0 ℃, 3.57g of triethylamine is added dropwise after stirring for 45min, 1.38 g is added dropwise after 13.69 mmol,2.1 eq for three hours, 2.23g of p-nitrophenol is added at 0 ℃, and 1.62g of triethylamine is added dropwise for reaction 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 a colorless transparent oily liquid compound of 4.92 g;
Step 11 ：
;
617mg of Compound O,951mg of Compound P and 177mg of magnesium chloride are mixed in 100ml of dry acetonitrile, N 2 Stirring at 50deg.C for 10min under protection, adding 601mg of N, N-isopropylethylamine, stirring for 20min, cooling to room temperature, diluting with ethyl acetate, sequentially adding 5% citric acid solution, saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated saline solution, washing, separating out organic phase, drying with anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography DCM/MEOH=40:1-20/1 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 passed over DCM/MeOH=20/1 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 concentrated and passed through a column DCM/MeOH=20/1 to give 274mg of compound R as a 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, 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.
6. The method for synthesizing coronavirus-resistant nucleoside compound according to claim 5, wherein: the chiral resolution conditions are as follows: chromatographic column: daicel CHIRALPAK is 30 mm in volume from IB 250 to IB 10 mu m, and mobile phase A: n-hexane, mobile phase B: isopropyl alcohol, detection wavelength: 254nm/214nm, flow rate: 25mL/min, and the isocratic elution procedure is as follows: mobile phase a: mobile phase b=70:30.
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| WO2022040761A1 (en) * | 2020-08-31 | 2022-03-03 | Starpharma Pty Ltd | Dendrimer-drug conjugate |
| WO2022174779A1 (en) * | 2021-02-19 | 2022-08-25 | 南京赛弗斯医药科技有限公司 | Nucleotide derivative having anti-tumor activity, pharmaceutical composition and use thereof |
| CN115996928A (en) * | 2020-06-24 | 2023-04-21 | 吉利德科学公司 | 1' -cyanonucleoside analogs and uses thereof |
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| CN102711765A (en) * | 2009-11-16 | 2012-10-03 | 加利福尼亚大学董事会 | Kinase inhibitors |
| WO2016069826A1 (en) * | 2014-10-29 | 2016-05-06 | Gilead Sciences, Inc. | Methods for treating filoviridae virus infections |
| CN108285438A (en) * | 2018-01-18 | 2018-07-17 | 上海仁实医药科技有限公司 | A kind of synthesis technology of benzyl ribonolactone |
| CN115996928A (en) * | 2020-06-24 | 2023-04-21 | 吉利德科学公司 | 1' -cyanonucleoside analogs and uses thereof |
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