CN116332996A - Anti-coronavirus compound, preparation method and application thereof - Google Patents

Anti-coronavirus compound, preparation method and application thereof Download PDF

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CN116332996A
CN116332996A CN202310488977.1A CN202310488977A CN116332996A CN 116332996 A CN116332996 A CN 116332996A CN 202310488977 A CN202310488977 A CN 202310488977A CN 116332996 A CN116332996 A CN 116332996A
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compound
reaction
coronavirus
solution
under
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***
钟南山
鲍甫义
步志高
耿志远
金大鹏
叶长青
史训龙
吴军
纪勇
周明
陈锋
何君
肖佐
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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 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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides an anti-coronavirus compound, a preparation method and application thereof, wherein the molecular formula of the anti-coronavirus compound is C 28 H 37 N 6 O 8 P, molecular weight 688.67, the structural formula of the anti-coronavirus compound is:
Figure ZY_1
and/or
Figure ZY_2
. The anti-coronavirus compound can effectively inhibit the activity of coronavirus, can be used as a substitute medicine of Mo Nupi Lawei, and has smaller cytotoxicity on the premise of equivalent antiviral activity of Mo Nupi Lawei.

Description

Anti-coronavirus compound, preparation method and application thereof
Technical Field
The invention relates to an anti-coronavirus compound, a preparation method and application thereof, belonging to the technical field of pharmaceutical chemistry.
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.
The novel potential targets for coronavirus pneumonia covd-19 are classified 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-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 4 new small molecule drugs available worldwide that act on novel coronavirus polymerases (RdRp), including adefovir (Remdesivir) from gilid science, mo Nupi ravir (molnupirvir) developed jointly by moesadong and ridge back, alzvudine from henna real biotechnology, inc, and deuterium hydrobromide remide (VV 116) from Shanghai real biomedical technologies, inc. However, to date, no drug has been able to be a specific drug for new coronaviruses or coronaviruses.
In view of the foregoing, it is desirable to provide an anti-coronavirus compound, and a preparation method and application thereof, so as to solve the above-mentioned problems.
Disclosure of Invention
The invention aims to provide an anti-coronavirus compound with high drug effect and low toxicity, and a preparation method and application thereof.
To achieve the above object, the present invention provides an anti-coronavirus compound having the formula C 28 H 37 N 6 O 8 P, molecular weight 688.67, the structural formula of the anti-coronavirus compound is:
Figure SMS_1
and/or
Figure SMS_2
。
As a further improvement of the present invention, the anti-coronavirus compound is a white solid.
As a further improvement of the present invention, the furanose ring of the anti-coronavirus compound has both D and L configurations.
It is a further object of the present invention to provide a pharmaceutical composition having the above anti-coronavirus compound.
To achieve the above object, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above anti-coronavirus compound, stereoisomer or pharmaceutically acceptable salt thereof in unit dosage form, and a pharmaceutically acceptable carrier or adjuvant.
It is a further object of the present invention to provide the use of the above anti-coronavirus compounds.
To achieve the above object, the present invention provides the use of the above anti-coronavirus compound for the preparation of a medicament for preventing and/or treating coronavirus-related diseases.
As a further improvement of the present invention, the coronaviruses include 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
It is still another object of the present invention to provide a method for producing an anti-coronavirus compound having high efficacy and low toxicity.
In order to achieve the above object, the present invention provides a method for preparing an anti-coronavirus compound, comprising the steps of:
Figure SMS_3
under the protection of nitrogen, the compound 13 is dissolved in a mixed solvent of tetrahydrofuran and water, trifluoroacetic acid is added after cooling, and then the reaction is carried out at room temperature; after the reaction is monitored by thin layer chromatography and LCMS, the reaction is neutralized to pH 7-8 by using saturated sodium bicarbonate solution, concentrated, subjected to C18 reverse phase column and freeze-dried to obtain a target compound 1 and a target compound 2, wherein the target compound 1 and the target compound 2 are chiral compounds.
As a further improvement of the present invention, the preparation method of the compound 13 comprises the following steps:
Figure SMS_4
under the protection of nitrogen, the compound 9 is dissolved in dimethylformamide, a dimethylformamide solution of t-BuMgCl is dripped after cooling for reaction, then a dimethylformamide solution of the compound 12 is dripped for heating for reaction, after the reaction is monitored by thin layer chromatography and LCMS, the reaction solution is poured into a saturated ammonium chloride solution, extraction is carried out by ethyl acetate, and the compound 13 is obtained after drying, concentration and column chromatography of an organic phase, wherein the compound 13 is yellow solid.
As a further improvement of the present invention, the preparation method of the compound 9 comprises the following steps:
step one:
Figure SMS_5
under the protection of nitrogen and under the ice water bath condition, dropwise adding concentrated sulfuric acid into a methanol solution dissolved with a compound 1 for reaction, monitoring the reaction by thin layer chromatography, neutralizing with a saturated sodium bicarbonate solution until the pH value is 7-8, drying, filtering, concentrating, and performing column chromatography to obtain a compound 2, wherein the compound 1 is L-ribose, and the compound 2 is yellow oily matter;
step two:
Figure SMS_6
under the protection of nitrogen and ice water bath, 60% sodium hydrogen is added into the dimethylformamide solution dissolved with the compound 2, and then benzyl bromide is added dropwise; after the completion of the benzyl bromide addition, the reaction was carried out at room temperature overnight. After the reaction is monitored by thin layer chromatography, pouring a reaction solution into ice water solution of saturated ammonia chloride, and extracting by using ethyl acetate, wherein an organic phase is washed, dried, filtered, concentrated and subjected to column chromatography to obtain a compound 3, and the compound 3 is yellow oily matter 3;
step three:
Figure SMS_7
dropwise adding hydrochloric acid into an acetic acid solution dissolved with a compound 3 under the protection of nitrogen, and then carrying out heating reaction; after the reaction is finished, the reaction solution is concentrated under reduced pressure, ethyl acetate and water are used for dissolving after the concentration, and the organic phase is washed, dried, filtered and concentrated to obtain a compound 4;
step four:
Figure SMS_8
concentrating the compound 4, dissolving in dry dimethyl sulfoxide, adding acetic anhydride, reacting at room temperature under the protection of nitrogen until the reaction is completed, concentrating the reaction solution under reduced pressure, dissolving in ethyl acetate, washing, drying, filtering, concentrating under reduced pressure, and performing column chromatography to obtain a compound 5, wherein the compound 5 is yellow oily matter;
step five:
Figure SMS_9
under the protection of nitrogen, adding trimethylchlorosilane into dry tetrahydrofuran solution dissolved with 4-amino-7-bromopyrrolo [2,1-F ] [1,2,4] triazine, reacting at room temperature, cooling the reaction solution to-78 ℃, dropwise adding n-BuLi n-hexane solution into the reaction solution, dropwise adding compound 5 dissolved in dry tetrahydrofuran into the reaction solution for reaction, quenching the reaction after the reaction is finished, adding acetic acid, concentrating under reduced pressure, dissolving with dichloromethane and water, washing an organic phase, drying, filtering, concentrating, and performing column chromatography to obtain compound 6, wherein the compound 6 is yellow solid;
step six:
Figure SMS_10
under the protection of nitrogen, dissolving a compound 6 in a dry dichloromethane solution, cooling, adding trimethylchlorosilane, adding trimethylsilicon triflate into a reaction solution for reaction, adding saturated sodium bicarbonate solution for quenching and stirring after the reaction is completed, separating an organic phase and a water phase, and drying, filtering, concentrating and performing column chromatography on the organic phase to obtain a compound 7, wherein the compound 7 is a yellow solid;
step seven:
Figure SMS_11
under the protection of nitrogen, dissolving the compound 7 in a dichloromethane solution, cooling, dropwise adding an n-hexane solution containing BCl3, then carrying out a cooling reaction, adding methanol for quenching, concentrating under reduced pressure, and separating by a C18 reverse phase column after the reaction is finished to obtain a compound 8, wherein the compound 8 is a light yellow solid;
step eight:
Figure SMS_12
under the protection of nitrogen, dissolving the compound 8 and 2,2' -dimethoxypropane in acetone, adding concentrated sulfuric acid, heating for reaction, monitoring the reaction by thin layer chromatography, neutralizing with sodium bicarbonate to pH 7-8, concentrating, and performing column chromatography to obtain a compound 9, wherein the compound 9 is a yellow solid.
As a further improvement of the present invention, the preparation method of the compound 12 comprises the following steps:
step one:
Figure SMS_13
under the protection of nitrogen, dissolving a compound 10 and 2-ethyl-1-butanol in toluene, adding p-toluenesulfonic acid, heating, carrying out a reaction after water diversion by a water diversion device, carrying out a thin layer chromatography and LCMS detection reaction, concentrating, neutralizing to pH 7-8 by using a saturated sodium bicarbonate solution, extracting by using dichloromethane, and drying, concentrating and carrying out column chromatography on an organic phase to obtain a compound 11, wherein the compound 10 is 2-methylalanine, and the compound 11 is a white solid;
step two:
Figure SMS_14
under the protection of nitrogen, phosphorus oxychloride and p-toluenesulfonate of a compound 11 are dissolved in dichloromethane, triethylamine is added dropwise for reaction after cooling, then a dichloromethane solution of triethylamine and ethyl o-hydroxybenzoate is added for reaction, and then triethylamine and p-nitrophenol are added for reaction. After completion of the reaction by thin layer chromatography and LCMS, compound 12 was obtained after concentration and C18 reverse phase column, wherein compound 11 was 2-methylalanine 2-ethylbutyl ester and compound 12 was a yellow oil.
The beneficial effects of the invention are as follows: the invention provides an anti-coronavirus compound, a preparation method and application thereof, wherein the anti-coronavirus compound can effectively inhibit the activity of coronavirus, can be used as a substitute drug of Mo Nupi Lawei, and has smaller cytotoxicity on the premise of equivalent antiviral activity of Mo Nupi Lawei.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of an anti-coronavirus compound of the present invention.
FIG. 2 is a LCMS spectrum of an anti-coronavirus compound of the present invention.
FIG. 3 is a nuclear magnetic resonance spectrum of the target compound 1.
FIG. 4 is a nuclear magnetic resonance spectrum of the target compound 2.
FIG. 5 is a nuclear magnetic resonance spectrum of Compound 13.
FIG. 6 is a nuclear magnetic resonance spectrum of Compound 2.
FIG. 7 is a nuclear magnetic resonance spectrum of Compound 3.
FIG. 8 is a nuclear magnetic resonance spectrum of Compound 5.
FIG. 9 is a nuclear magnetic resonance spectrum of Compound 6.
FIG. 10 is a nuclear magnetic resonance spectrum of Compound 7.
FIG. 11 is a nuclear magnetic resonance spectrum of Compound 8.
FIG. 12 is a nuclear magnetic resonance spectrum of Compound 9.
FIG. 13 is a nuclear magnetic resonance spectrum of Compound 11.
FIG. 14 is a nuclear magnetic resonance spectrum of Compound 12.
Description of the embodiments
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
In this case, in order to avoid obscuring the present invention due to unnecessary details, only the structures and/or processing steps closely related to the aspects of the present invention are shown in the drawings, and other details not greatly related to the present invention are omitted.
In addition, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Referring to the figure, the invention provides an anti-coronavirus compound with a molecular formula of C 28 H 37 N 6 O 8 P, molecular weight 688.67, the structural formula of the anti-coronavirus compound is:
Figure SMS_15
and/or
Figure SMS_16
。
Further, the anti-coronavirus compound is a chiral compound, including a target compound 1 and a target compound 2. That is, the anti-coronavirus compound has one carbon atom with both R and S configurations, or, alternatively, has a furanose ring with both D and L configurations.
Further, the anti-coronavirus compound is a white solid and can be formulated into various preparation forms well known in the art, such as tablets, capsules, granules, pills, oral liquids, injections, according to a specific administration mode.
The invention further provides a pharmaceutical composition with the anti-coronavirus compound. The pharmaceutical composition comprises a therapeutically effective amount of the above anti-coronavirus compound, stereoisomer or pharmaceutically acceptable salt thereof in unit dosage form, and a pharmaceutically acceptable carrier or adjuvant.
The invention further provides the use of the above anti-coronavirus compounds. The use of the anti-coronavirus compound for the preparation of a medicament for the prophylaxis and/or treatment of coronavirus-related diseases.
Further, the coronaviruses include 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV. Wherein 2019-nCoV can induce novel coronavirus pneumonia COVID-19, SARS-CoV can induce severe acute respiratory syndrome, and MERS-CoV can induce middle east respiratory syndrome.
Further, the anti-coronavirus compound is also useful for preparing a medicament for preventing and/or treating a variant strain of coronavirus.
The invention further provides a preparation method of the anti-coronavirus compound with high drug effect and low toxicity, and the preparation method has mild conditions, is easy to synthesize and is suitable for industrial production.
In order to achieve the above object, the present invention provides a method for preparing an anti-coronavirus compound, comprising the steps of:
Figure SMS_17
under the protection of nitrogen, the compound 13 is dissolved in a mixed solvent of Tetrahydrofuran (THF) and water, trifluoroacetic acid is added after cooling, and then the reaction is carried out at room temperature; after the reaction is monitored by Thin Layer Chromatography (TLC) and LCMS, the reaction is neutralized to pH 7-8 by using saturated sodium bicarbonate solution, concentrated, subjected to C18 reverse phase column and freeze-dried to obtain the target compound 1 and the target compound 2, wherein the target compound 1 and the target compound 2 are chiral compounds, and the target compound 1 and the target compound 2 are white solids.
Preferably, compound 13 (550 mg,0.755 mmol,1.0 eq) is dissolved in a mixed solvent (5.5 mL, THF/H 2 O=10: 1) Cooling to 0 ℃, adding 4.5 mL trifluoroacetic acid, and then reacting at room temperature for 24 hours. The reaction progress was monitored by Thin Layer Chromatography (TLC) and LCMS. Among them, the developing agent of TLC was Dichloromethane (DCM) and methanol (MeOH), and DCM: meoh=10: 1, detection was carried out at UV254 nm. After the reaction is completed, the mixture is neutralized to pH 7-8 by saturated sodium bicarbonate solution, concentrated and passed through a C18 reverse phase column (MeCN/H 2 O0-100%), and freeze-drying to obtain a mixture of the target compound 1 and the target compound 2, wherein the mixture is a white solid.
In this example, the weight of the mixture was 385 mg. In other embodiments, the weight of the resulting mixture may be different due to different dosages of the compound employed, and the like.
Preferably, in this example, the anti-coronavirus compound obtained by the reaction is a mixture of the target compound 1 and the target compound 2, named β -L-1-cyano-2-methylrayleigh drug, having a molecular weight of 689.3 and a nuclear magnetic data of:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.35 – 8.01 (d,J= 57.5 Hz, 3H), 7.98 – 7.94 (s, 1H), 7.77 – 7.71 (d,J= 7.7 Hz, 1H), 7.58 – 7.45 (m, 2H), 7.30 – 7.23 (m, 1H), 6.97 – 6.93 (s, 1H), 6.89 – 6.84 (dd,J= 7.5, 3.4 Hz, 1H), 5.83 – 5.76 (d,J= 9.5 Hz, 1H), 4.68 – 4.63 (d,J= 4.9 Hz, 1H), 4.32 – 4.23 (qd,J= 7.0, 3.5 Hz, 4H), 4.23 – 4.14 (dt,J= 10.5, 5.4 Hz, 1H), 4.00 – 3.92 (m, 1H), 3.89 – 3.85 (dt,J= 5.4, 2.4 Hz, 2H), 1.42 – 1.36 (m, 1H), 1.34 – 1.30 (d,J= 3.3 Hz, 6H), 1.30 – 1.20 (qd,J= 8.7, 7.9, 4.7 Hz, 8H), 0.82 – 0.76 (tt,J= 7.6, 2.1 Hz, 6H)。
the nuclear magnetic resonance spectrum and LCMS spectrum of the mixture are shown in FIG. 1 and FIG. 2.
The mixture is separated to obtain the target compound 1 and the target compound 2.
The molecular weight of the target compound 1 is 689.3, and the nuclear magnetic data are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.01 – 7.83 (s, 3H), 7.77 – 7.70 (d,J= 7.5 Hz, 1H), 7.56 – 7.45 (m, 2H), 7.30 – 7.21 (m, 1H), 6.92 – 6.87 (d,J= 4.5 Hz, 1H), 6.87 – 6.82 (d,J= 4.5 Hz, 1H), 6.34 – 6.28 (d,J= 6.0 Hz, 1H), 5.81 – 5.74 (d,J= 9.5 Hz, 1H), 5.42 – 5.31 (s, 1H), 4.70 – 4.63 (m, 1H), 4.35 – 4.13 (m, 5H), 3.98 – 3.92 (t,J= 5.8 Hz, 1H), 3.92 – 3.83 (m, 2H), 1.45 – 1.36 (p,J= 6.2 Hz, 1H), 1.36 – 1.19 (m, 12H), 0.84 – 0.75 (td,J= 7.5, 2.4 Hz, 6H)。
the nuclear magnetic data of the target compound 2 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.01 – 7.79 (s, 3H), 7.77 – 7.71 (dd,J= 7.8, 1.5 Hz, 1H), 7.58 – 7.50 (ddd,J= 8.8, 7.3, 1.8 Hz, 1H), 7.50 – 7.44 (d,J= 8.3 Hz, 1H), 7.31 – 7.23 (t,J= 7.4 Hz, 1H), 6.91 – 6.86 (d,J= 4.5 Hz, 1H), 6.86 – 6.80 (d,J= 4.5 Hz, 1H), 6.34 – 6.28 (d,J= 6.1 Hz, 1H), 5.81 – 5.74 (d,J= 9.5 Hz, 1H), 5.40 – 5.34 (d,J= 5.7 Hz, 1H), 4.71 – 4.64 (m, 1H), 4.34 – 4.21 (q,J= 7.1 Hz, 4H), 4.21 – 4.12 (dd,J= 10.8, 5.6 Hz, 1H), 4.01 – 3.93 (q,J= 5.6 Hz, 1H), 3.92 – 3.84 (m, 2H), 1.45 – 1.36 (p,J= 6.2 Hz, 1H), 1.35 – 1.18 (m, 13H), 0.83 – 0.74 (td,J= 7.5, 1.6 Hz, 6H)。
the nuclear magnetic patterns of the target compound 1 and the target compound 2 are shown in fig. 3 and 4.
It is to be understood that the compound 13 required for preparing the anti-coronavirus compound may be obtained by self-preparation or by purchase, etc., and the present invention is not limited thereto.
Further, in this example, compound 13 was prepared by self-development. Wherein, the preparation method of the compound 13 comprises the following steps:
Figure SMS_18
under the protection of nitrogen, the compound 9 is dissolved in dimethylformamide, a dimethylformamide solution of t-BuMgCl is dripped after cooling for reaction, then a dimethylformamide solution of the compound 12 is dripped for heating for reaction, after the reaction is monitored by thin layer chromatography and LCMS, the reaction solution is poured into a saturated ammonium chloride solution, extraction is carried out by ethyl acetate, and the compound 13 is obtained after drying, concentration and column chromatography of an organic phase, wherein the compound 13 is yellow solid.
Preferably, compound 9 (500 mg,1.51 mmol,1.0 eq) is dissolved in 6 mL dry Dimethylformamide (DMF), cooled to 0deg.C, 1M t-BuMgCl in DMF (3.0 mL,3.0 mmol,2.0 eq) is added dropwise, then reacted at 0deg.C for 30 minutes, then compound 12 (1.62 g,3.02 mmol,2.0 eq) in DMF is added dropwise, and finally heated to 50deg.C for 2 hours. After TLC (DCM: meoh=10:1, UV254 nm) and LCMS monitoring the reaction was completed, the reaction was poured into saturated ammonium chloride solution, extracted three times with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and column chromatographed (MeOH/DCM 0-20%) to give compound 13 as a yellow solid with a molecular weight of 729.3, in this example 600 mg by weight.
The nuclear magnetic data of compound 13 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.05 – 7.85 (s, 3H), 7.76 – 7.69 (d,J= 7.8 Hz, 1H), 7.53 – 7.48 (m, 1H), 7.46 – 7.41 (d,J= 8.3 Hz, 1H), 7.30 – 7.22 (t,J= 7.1 Hz, 1H), 6.92 – 6.88 (dd,J= 4.6, 1.9 Hz, 1H), 6.87 – 6.83 (t,J= 4.9 Hz, 1H), 5.87 – 5.78 (dd,J= 12.8, 9.5 Hz, 1H), 5.43 – 5.31 (dd,J= 22.5, 6.5 Hz, 1H), 5.02 – 4.90 (m, 1H), 4.59 – 4.50 (d,J= 3.2 Hz, 1H), 4.29 – 4.20 (dd,J= 7.1, 4.0 Hz, 2H), 4.20 – 4.14 (t,J= 6.3 Hz, 2H), 4.12 – 4.06 (d,J= 5.2 Hz, 1H), 3.89 – 3.84 (dd,J= 5.8, 3.3 Hz, 2H), 1.67 – 1.61 (s, 3H), 1.38 – 1.33 (s, 4H), 1.33 – 1.28 (d,J= 3.2 Hz, 7H), 1.28 – 1.22 (d,J= 1.7 Hz, 7H), 0.82 – 0.75 (dd,J= 7.3, 1.1 Hz, 6H)。
the nuclear magnetic pattern of compound 13 is shown in FIG. 5.
It is to be understood that the compound 9 and the compound 12 required for the preparation of the compound 13 may be obtained by self-preparation or by purchase, etc., and the present invention is not limited thereto.
In this example, both compound 9 and compound 12 were self-developed.
Further, the preparation method of the compound 9 comprises the following steps:
step one:
Figure SMS_19
under the protection of nitrogen and ice water bath, dropwise adding concentrated sulfuric acid into a methanol solution dissolved with a compound 1 for reaction, monitoring the reaction by thin layer chromatography, neutralizing with a saturated sodium bicarbonate solution until the pH value is 7-8, drying, filtering, concentrating, and performing column chromatography to obtain a compound 2, wherein the compound 1 is L-ribose, and the compound 2 is yellow oily matter.
Preferably, concentrated sulfuric acid (3.0 mL,57.09 mmol,0.33 eq) is added dropwise to a solution of L-ribose (26 g,0.173 mol,1.0 eq) in dry methanol (260 mL) under nitrogen protection and ice water bath. After the completion of the dropwise addition of concentrated sulfuric acid, the reaction was carried out at room temperature for 5 hours. After TLC (PE: ea=5:1, uv254 nm) monitoring the reaction was completed, it was neutralized to pH 7-8 with saturated sodium bicarbonate solution, dried, filtered, concentrated, and column chromatographed (EA/PE 0-50%) to give compound 2 as a yellow oil having a molecular weight of 332.1, in this example 29.65 and g.
The nuclear magnetic data of compound 2 are:
1 H NMR (400 MHz, Methanol-d 4 ) δ 4.77 – 4.72 (m, 1H), 4.06 – 4.00 (m, 1H), 4.00 – 3.95 (dd,J= 12.4, 3.8 Hz, 1H), 3.95 – 3.91 (m, 1H), 3.75 – 3.69 (dd,J= 11.7, 3.5 Hz, 1H), 3.57 – 3.51 (dd,J= 11.8, 6.4 Hz, 1H), 3.37 – 3.34 (s, 3H)。
the nuclear magnetic pattern of compound 2 is shown in FIG. 6.
Step two:
Figure SMS_20
under the protection of nitrogen and ice water bath, 60% sodium hydrogen is added into the dimethylformamide solution dissolved with the compound 2, and then benzyl bromide is added dropwise; after the completion of the benzyl bromide addition, the reaction was carried out at room temperature overnight. After the reaction is monitored by thin layer chromatography, pouring the reaction liquid into ice water solution of saturated ammonia chloride, extracting by using ethyl acetate, wherein the organic phase is washed, dried, filtered, concentrated and subjected to column chromatography to obtain a compound 3, and the compound 3 is yellow oily matter 3.
In a solution of compound 2 (26 g, 0.158 mol,1.0 eq) in dry DMF (260 mL) under nitrogen protection and ice-water bath, 60% sodium hydrogen (31.6 g,0.79 mmol,5.0 eq) was added in three portions, followed by the dropwise addition of benzyl bromide (108.01 g, 0.632 mol,4.0 eq). After the completion of the benzyl bromide addition, the reaction was carried out at room temperature overnight. After completion of the reaction, monitored by TLC (PE: ea=5:1, UV254 nm), the reaction mixture was poured into an ice water solution of saturated ammonia chloride and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed (EA/PE 0-30%) to give compound 3 as yellow oil 3 having a molecular weight of 452.34, in this case 43.29 g.
The nuclear magnetic data of compound 3 are:
1 H NMR (400 MHz, Chloroform-d) δ 7.42 – 7.22 (m, 15H), 4.95 – 4.90 (s, 1H), 4.71 – 4.60 (q,J= 11.9 Hz, 2H), 4.59 – 4.52 (m, 3H), 4.49 – 4.42 (d,J= 11.8 Hz, 1H), 4.39 – 4.32 (m, 1H), 4.06 – 3.99 (dd,J= 7.1, 4.7 Hz, 1H), 3.88 – 3.82 (m, 1H), 3.65 – 3.59 (dd,J= 10.6, 3.7 Hz, 1H), 3.56 – 3.49 (dd,J= 10.6, 5.8 Hz, 1H)。
the nuclear magnetic pattern of compound 3 is shown in FIG. 7.
Step three:
Figure SMS_21
/>
dropwise adding hydrochloric acid into an acetic acid solution dissolved with a compound 3 under the protection of nitrogen, and then carrying out heating reaction; after the reaction, the reaction solution was concentrated under reduced pressure, and dissolved in ethyl acetate and water, and the organic phase was washed, dried, filtered, and concentrated to give compound 4.
Preferably, 1M hydrochloric acid (109.6 mL,109.6 mmol,1.1 eq) is added dropwise to a solution of compound 3 (43.29 g, 99.62 mmol,1.0 eq) in acetic acid (430 mL) under nitrogen protection. After completion of the dropwise addition, the reaction solution was heated to 80℃and reacted for 4 hours. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, and dissolved in ethyl acetate and water, and the organic phase was washed with sodium hydrogencarbonate solution, saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude compound 4, which was used directly in the next step.
Step four:
Figure SMS_22
concentrating the compound 4, dissolving in dry dimethyl sulfoxide (DMSO), adding acetic anhydride, reacting at room temperature under the protection of nitrogen until the reaction is completed, concentrating the reaction solution under reduced pressure, dissolving in ethyl acetate, washing, drying, filtering, concentrating under reduced pressure, and performing column chromatography to obtain the compound 5, wherein the compound 5 is yellow oily matter.
Compound 4 was concentrated and dissolved in 220 mL dry DMSO, acetic anhydride (132 mL, 1.045 mol, 1.5 eq) was added and reacted overnight at room temperature under nitrogen protection. After the reaction, the reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate, washed with water, sodium bicarbonate solution, saturated brine, dried, filtered, concentrated under reduced pressure, and subjected to column chromatography (EA/PE 0 to 100%, UV254 nm) to give Compound 5. Compound 5 was a yellow oil with a molecular weight of 436.3, in this example 33.63 g by weight.
The nuclear magnetic data of compound 5 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 7.42 – 7.18 (m, 15H), 4.77 – 4.55 (m, 6H), 4.51 – 4.46 (s, 2H), 4.33 – 4.27 (d,J= 5.4 Hz, 1H), 3.70 – 3.65 (d,J= 3.4 Hz, 2H)。
the nuclear magnetic pattern of compound 5 is shown in FIG. 8.
Step five:
Figure SMS_23
under the protection of nitrogen, adding trimethylchlorosilane (TMSCl) into dry tetrahydrofuran solution dissolved with 4-amino-7-bromopyrrolo [2,1-F ] [1,2,4] triazine, reacting at room temperature, cooling the reaction solution to-78 ℃, dropwise adding n-BuLi n-hexane solution into the reaction solution, dropwise adding compound 5 dissolved in dry tetrahydrofuran into the reaction solution for reaction, quenching the reaction solution after the reaction is completed, concentrating the solution under reduced pressure, dissolving the solution with dichloromethane and water, washing an organic phase, drying, filtering, concentrating the solution, and performing column chromatography to obtain compound 6, wherein the compound 6 is yellow solid.
Preferably, TMSCl (19.52 g,179.67 mmol,2.2 eq) is added to a solution of 4-amino-7-bromopyrrolo [2,1-F ] [1,2,4] triazine (17.4 g,81.67 mmol,1.0 eq) in dry tetrahydrofuran (175 mL) under nitrogen. After the addition was completed, the reaction was carried out at room temperature for 20 minutes, and then the reaction solution was cooled to-78℃and an n-BuLi n-hexane solution of 1.6. 1.6M was added dropwise to the reaction solution for 10 minutes, and Compound 5 (34.18 g,81.67 mol,1.0 eq) was dissolved in dry tetrahydrofuran and added dropwise to the reaction solution. After completion of the dropwise addition, the reaction was allowed to react at-78℃for 4 hours. After the reaction, acetic acid was added to quench and reacted at-78 ℃ for 10 minutes, concentrated under reduced pressure, and after the concentration, the organic phase was dissolved in methylene chloride and water, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatographed (ethyl acetate/petroleum ether 0 to 100%, UV254 nm) to give compound 6 as a yellow solid having a molecular weight of 553.3, in this example, 7.36 g by weight.
The nuclear magnetic data of compound 6 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.12 – 8.00 (s, 2H), 8.00 – 7.96 (s, 1H), 7.79 – 7.76 (s, 2H), 7.74 – 7.61 (d,J= 22.5 Hz, 4H), 7.60 – 7.57 (dd,J= 2.6, 1.6 Hz, 2H), 7.37 – 7.22 (m, 13H), 7.18 – 7.11 (m, 3H), 7.02 – 6.97 (dd,J= 7.4, 2.2 Hz, 2H), 6.95 – 6.92 (d,J= 4.8 Hz, 1H), 6.87 – 6.83 (dd,J= 4.3, 1.6 Hz, 2H), 6.62 – 6.57 (dd,J= 4.3, 2.6 Hz, 2H), 5.42 – 5.35 (d,J= 6.0 Hz, 1H), 5.07 – 5.01 (d,J= 5.2 Hz, 1H), 4.60 – 4.54 (dt,J= 11.7, 2.5 Hz, 2H), 4.48 – 4.43 (m, 3H), 3.95 – 3.90 (dd,J= 6.0, 4.4 Hz, 1H), 3.72 – 3.66 (dd,J= 10.1, 3.5 Hz, 1H), 3.51 – 3.43 (dd,J= 10.1, 6.4 Hz, 1H)。
the nuclear magnetic pattern of compound 6 is shown in FIG. 9.
Step six:
Figure SMS_24
under the protection of nitrogen, the compound 6 is dissolved in a dry dichloromethane solution, after cooling, trimethylchlorosilane (TMSCN) is added, trimethylsilicon triflate (TMSOTF) is added into a reaction liquid for reaction, after the reaction is completed, saturated sodium bicarbonate solution is added for quenching and stirring, after an organic phase and an aqueous phase are separated, the organic phase is dried, filtered, concentrated and subjected to column chromatography to obtain the compound 7, and the compound 7 is yellow solid.
Preferably, compound 6 (7.75 g,14.02 mmol,1.0 eq) is dissolved in a solution of dry dichloromethane (75 mL) under nitrogen, cooled to-78 ℃, and TMSCN (5.38 g,54.23 mmol,3.87 eq) is added. After the addition was completed, the reaction was carried out for 10 minutes, and then TMSOTF (11.5 mL,63.09 mmol,4.5 eq) was added to the reaction mixture to carry out the reaction for 2 hours. After the reaction was completed, saturated sodium bicarbonate solution was added to quench and stir for 10 minutes, the organic phase was separated, the aqueous phase was extracted three times with methylene chloride, and the combined organic phases were dried over anhydrous magnesium sulfate, filtered, concentrated, and column chromatographed (methanol/methylene chloride 0 to 20%, UV254 nm) to give compound 7 as a yellow solid having a molecular weight of 562.3, which in this example was 2.90 g by weight.
The nuclear magnetic data for compound 7 were:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.01 – 7.82 (d,J= 19.5 Hz, 3H), 7.37 – 7.22 (m, 15H), 6.88 – 6.85 (d,J= 4.6 Hz, 1H), 6.77 – 6.74 (d,J= 4.6 Hz, 1H), 4.92 – 4.89 (d,J= 5.0 Hz, 1H), 4.87 – 4.82 (d,J= 11.7 Hz, 1H), 4.79 – 4.74 (d,J= 11.7 Hz, 1H), 4.56 – 4.51 (dd,J= 9.0, 3.9 Hz, 2H), 4.51 – 4.48 (d,J= 3.7 Hz, 2H), 4.14 – 4.09 (m, 1H), 3.72 – 3.66 (dd,J= 11.1, 3.6 Hz, 1H), 3.61 – 3.56 (dd,J= 11.0, 4.7 Hz, 1H)。
the nuclear magnetic pattern of compound 7 is shown in FIG. 10.
Step seven:
Figure SMS_25
under the protection of nitrogen, the compound 7 is dissolved in dichloromethane solution, after cooling, normal hexane solution containing BCl3 is added dropwise, then cooling reaction is carried out, after the reaction is completed, methanol is added for quenching, decompression concentration and C18 reverse phase column separation are carried out, and then the compound 8 is obtained, wherein the compound 8 is a light yellow solid.
Preferably, compound 7 (2.8 g,4.99 mmol,1.0 eq) is dissolved in a dry dichloromethane (30 mL) solution under nitrogen protection, cooled to-20 ℃, and 1m BCl3 in n-hexane (29.81 mL,29.81 mmol,6.0 eq) is added dropwise. After the addition was completed, the mixture was cooled to-78℃and reacted for 2 hours. After the reaction was completed, the mixture was quenched with methanol at-78 ℃, concentrated under reduced pressure, and separated by a C18 reverse phase column (acetonitrile/water 0 to 100%, UV254 nm) to give compound 8 as a pale yellow solid, the weight of which in this example was 0.99. 0.99 g.
The nuclear magnetic data of compound 8 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.04 – 7.80 (d,J= 5.2 Hz, 3H), 6.94 – 6.86 (m, 2H), 6.14 – 6.06 (d,J= 6.3 Hz, 1H), 5.22 – 5.15 (d,J= 5.3 Hz, 1H), 4.95 – 4.88 (dd,J= 6.3, 5.2 Hz, 1H), 4.67 – 4.62 (dd,J= 6.3, 5.1 Hz, 1H), 4.08 – 4.03 (m, 1H), 3.98 – 3.93 (q,J= 5.3 Hz, 1H), 3.68 – 3.60 (ddd,J= 12.2, 5.2, 3.4 Hz, 1H), 3.54 – 3.47 (ddd,J= 12.2, 6.3, 4.5 Hz, 1H)。
the nuclear magnetic pattern of compound 8 is shown in FIG. 11.
Step eight:
Figure SMS_26
/>
under the protection of nitrogen, dissolving the compound 8 and 2,2' -dimethoxypropane in acetone, adding concentrated sulfuric acid, heating for reaction, monitoring the reaction by thin layer chromatography, neutralizing with sodium bicarbonate to pH 7-8, concentrating, and performing column chromatography to obtain a compound 9, wherein the compound 9 is a yellow solid.
Preferably, compound 8 (500 mg,1.72 mmol,1.0 eq) and 2,2' -dimethoxypropane (0.95 mL,7.74 mmol,4.5 eq) are dissolved in acetone (10 mL) under nitrogen protection, concentrated sulfuric acid (0.12 mL, 2.24 mmol,1.3 eq) is added and heated to 50 ℃ for reaction for 1 hour. After TLC (DCM: meoh=10:1, UV254 nm) monitoring the reaction was completed, neutralized to pH 7-8 with sodium bicarbonate, concentrated and column chromatographed (MeOH/DCM 0-20%) to give compound 9 as a yellow solid with a molecular weight of 332.2, in this example 520. 520 mg.
The nuclear magnetic data of compound 9 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.06 – 7.82 (d,J= 12.9 Hz, 3H), 6.93 – 6.87 (m, 2H), 5.40 – 5.34 (d,J= 6.6 Hz, 1H), 5.03 – 4.98 (t,J= 5.7 Hz, 1H), 4.91 – 4.86 (dd,J= 6.6, 3.1 Hz, 1H), 4.33 – 4.28 (td,J= 5.3, 3.0 Hz, 1H), 3.59 – 3.45 (dtd,J= 17.4, 11.6, 5.6 Hz, 2H), 1.68 – 1.57 (s, 3H), 1.41 – 1.32 (s, 3H)。
the nuclear magnetic pattern of compound 9 is shown in FIG. 12.
Further, the preparation method of the compound 12 comprises the following steps:
step one:
Figure SMS_27
under the protection of nitrogen, dissolving a compound 10 (2-methylalanine) and 2-ethyl-1-butanol in toluene, adding p-toluenesulfonic acid, heating, carrying out water diversion reaction by a water diversion device, concentrating after the reaction is detected by thin layer chromatography and LCMS, neutralizing to pH 7-8 by using saturated sodium bicarbonate solution, extracting by using dichloromethane, and drying, concentrating and carrying out column chromatography on an organic phase to obtain a compound 11, wherein the compound 10 is 2-methylalanine, and the compound 11 is white solid.
Preferably, 2-methylalanine (20 g, 194 mmol,1.0 eq) and 2-ethyl-1-butanol (29.7 g, 291 mmol, 1.5 eq) are dissolved in toluene (200 mL) under nitrogen protection, p-toluenesulfonic acid (37 g, 213.4 mmol,1.1 eq) is added, heated to 130 ℃ and water is separated by a water separator for 16 hours. After completion of the reaction by TLC (DCM: meOH=10:1, UVL254 nm/I2) and LCMS, the reaction was concentrated, neutralized to pH 7-8 with saturated sodium bicarbonate solution, extracted three times with dichloromethane, the organic phase was dried over anhydrous sodium sulfate and concentrated to give the crude product. Crude column chromatography (MeOH/DCM 0-20%) gives compound 11. This compound 11 was a white solid with a molecular weight of 188.3, a weight of 28 g in this example, and a yield of 40.2%.
The nuclear magnetic data of compound 11 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 7.54 – 7.47 (m, 2H), 7.13 (d,J= 8.0 Hz, 2H), 4.05 (d,J= 5.5 Hz, 2H), 2.30 (s, 3H), 1.52 (p,J= 6.1 Hz, 1H), 1.38 (s, 6H), 1.35 – 1.27 (m, 4H), 0.85 (dt,J= 13.2, 7.4 Hz, 6H)。
the nuclear magnetic pattern of compound 11 is shown in FIG. 13.
Step two:
Figure SMS_28
under the protection of nitrogen, phosphorus oxychloride and p-toluenesulfonate of a compound 11 are dissolved in dichloromethane, triethylamine is added dropwise for reaction after cooling, then a dichloromethane solution of triethylamine and ethyl o-hydroxybenzoate is added for reaction, and then triethylamine and p-nitrophenol are added for reaction. After completion of the reaction by thin layer chromatography and LCMS, compound 12 was obtained after concentration and C18 reverse phase column, wherein compound 11 was 2-methylalanine 2-ethylbutyl ester and compound 12 was a yellow oil.
Phosphorus oxychloride (1.0 g,6.52 mmol,1.0 eq) and 2-methylalanine 2-ethylbutyl p-toluenesulfonate (2.46 g,6.85 mmol,1.05 eq) are dissolved in dry dichloromethane (10 mL) under the protection of nitrogen, cooled to-70 ℃, triethylamine (1.38 g, 13.69 mmol,2.1 eq) is added dropwise for three hours, a dichloromethane solution of triethylamine (0.69 g,6.85 mmol,1.05 eq) and ethyl o-hydroxybenzoate (1.14 g,6.85 mmol,1.05 eq) is added for three hours, and triethylamine (1.50 g,14.62 mmol,1.05 eq) and p-nitrophenol (2.03 g,14.62 mmol,1.05 eq) are added for reaction overnight at room temperature. After TLC (PE: ea=3:1, uv254 nm) and LCMS monitoring the reaction was complete, concentrated and passed through a C18 reverse phase column (MeCN/H2O 0-100%) to give compound 12 as a yellow oil with a molecular weight of 537.17, in this example 1.49 g by weight, yield 42.6%.
The nuclear magnetic data for compound 12 are:
1 H NMR (400 MHz, DMSO-d 6 ) δ 8.35 – 8.26 (m, 2H), 7.79 – 7.72 (dt,J= 7.7, 1.3 Hz, 1H), 7.67 – 7.58 (ddd,J= 8.9, 7.3, 1.9 Hz, 1H), 7.58 – 7.51 (d,J= 8.7 Hz, 1H), 7.51 – 7.44 (m, 2H), 7.37 – 7.28 (t,J= 7.5 Hz, 1H), 6.60 – 6.53 (d,J= 10.1 Hz, 1H), 4.27 – 4.14 (qt,J= 7.1, 3.6 Hz, 2H), 3.89 – 3.83 (d,J= 5.6 Hz, 2H), 1.45 – 1.38 (d,J= 2.1 Hz, 6H), 1.38 – 1.29 (p,J= 6.3 Hz, 1H), 1.29 – 1.18 (td,J= 7.2, 4.4 Hz, 8H), 0.86 – 0.73 (m, 6H)。
the nuclear magnetic pattern of compound 12 is shown in FIG. 14.
It should be noted that in this embodiment, each compound is prepared by itself, and in other embodiments, each compound may be obtained by purchasing, etc., which is not limited by the present invention. Furthermore, the steps for preparing the above-mentioned compounds are not essential, and the reaction sequence may be added, subtracted or adjusted according to the actual experimental needs, which is not limited by the present invention. In addition, the weight of the resulting compound varies depending on the amount of the compound to be used and the like, and can be adjusted by those skilled in the art according to actual practice.
The invention further provides anti-coronavirus activity and cytotoxicity tests for the coronavirus compounds.
The anti-coronavirus compound (including target compound 1, target compound 2, and a mixture of target compound 1 and target compound 2) prepared in the present invention was used as a test sample, and a commercially available Mo Nupi Lavir (Molnupiravir) was used as a control drug, and applied to a Bel7402 cell line containing coronavirus (supplied by Xiamen university) to perform anti-coronavirus activity and cytotoxicity test. Among them, mo Nupi Lavir (Monnupiravir) is one of the only two approved small molecule nucleoside anti-new coronavirus inhibitors in the United states developed by the company Mitsadone and Ridgeback in the United states, and formally introduced and approved for use in China.
Cytotoxicity test procedure:
the 96-well plates were seeded with Bel7402 cells (1.5×104/well) and cultured 24 h. After the culture solution was aspirated, 100. Mu.L/well of the drug solution to be tested, i.e., the anti-coronavirus compound solution (target compound 1, target compound 2, and a mixture of target compound 1 and target compound 2) and Mo Nupi Lawei solution were added, 2 multiplex wells were set for each concentration, and the normal control group was added with an equal volume of the culture solution. At 37℃with 5% CO 2 After incubating Bel7402 cells 48 h, 15 μl of MTT solution at a concentration of 5 mg/mL was added to each well, and incubation was continued for 4 h. Supernatant was aspirated, 100 μl DMSO was added per well, low speedThe OD value was determined at 490 nm by shaking lysis and cell viability (average OD value in drug group-blank OD value)/(average OD value in normal control group-blank OD value) ×100% was calculated as compared to normal group cells.
Anti-coronavirus activity test procedure:
the 96-well plates were seeded with Bel7402 cells (2.5×104/well) and cultured 24 h. The supernatant was aspirated, and 100. Mu.L/well of 10-2 HCoV virus solution was added to the drug test group and the virus control group at 37℃with 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. After incubation at 37℃in a 5% CO2 incubator for 72 h, the MTT assay measures cell activity (method as above) and calculates the cell viability (average OD value of drug group-blank OD value)/(average OD value of normal control group-blank OD value) ×100%.
The specific results are shown in Table 1.
TABLE 1 anti-coronavirus Activity and cytotoxicity test results
Figure SMS_29
/>
As can be seen from the results in table 1, in the cytotoxicity test, at drug concentrations below 125 μm, the cell viability of the three anti-coronavirus compounds, i.e., the target compound 1, the target compound 2, the mixture of the target compound 1 and the target compound 2 (wherein the ratio of the target compound 1 to the target compound 2 in the mixture is 1:1), was higher than that of the control Mo Nupi pyrad, i.e., the cytotoxicity of the three anti-coronavirus compounds was not apparent, and the toxicity of the three anti-coronavirus compounds was lower than that of the control Mo Nupi pyrad, without an infectious background. Especially, at the concentration of 31.25 mu M, the cell viability of the three anti-coronavirus compounds respectively reaches 96.5%, 99.0% and 97.8%, and the cell activity of the control drug Mo Nupi Lavir is respectively 70.5%, namely, the cell viability of the test sample is higher than that of the control drug Mo Nupi Lavir, that is, the cell viability of the three anti-coronavirus compounds is lower than that of the marketed Mo Nupi Lavir, and the anti-coronavirus compounds have less cytotoxicity and less harm to human bodies.
In the anti-coronavirus activity efficacy test, under the current high-toxicity attack background, three anti-coronavirus compounds still have certain antiviral activity at the concentration of 0.04 mu M, the cell activity is respectively 72.9%, 75.9% and 74.4%, which is higher than 60% of that of a virus control, and the anti-coronavirus activity is equivalent to that of a control drug Mo Nupi Lavir, so that the anti-coronavirus compound has the value of further research, can effectively replace Mo Nupi Lavir, and the preparation method of the three anti-coronavirus compounds is simple, mild and low in cost, and provides a direction for drug development for treating coronaviruses.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. An anti-coronavirus compound, wherein the anti-coronavirus compound has a formula of C 28 H 37 N 6 O 8 P, molecular weight 688.67, the structural formula of the anti-coronavirus compound is:
Figure QLYQS_1
and/or
Figure QLYQS_2
。
2. An anti-coronavirus compound according to claim 1, characterized in that: the anti-coronavirus compound is a white solid.
3. An anti-coronavirus compound according to claim 1, characterized in that: the furanose ring of the anti-coronavirus compound has two configurations of D and L.
4. A pharmaceutical composition comprising a therapeutically effective amount of an anti-coronavirus compound, stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in unit dosage form, and a pharmaceutically acceptable carrier or adjuvant.
5. Use of an anti-coronavirus compound as claimed in any one of claims 1 to 3 for the preparation of a medicament for the prophylaxis and/or treatment of coronavirus-related diseases.
6. The use of an anti-coronavirus compound of claim 5 for the preparation of a medicament for preventing or treating coronavirus-related diseases, wherein said coronavirus comprises 2019-nCoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV and MERS-CoV.
7. A method for preparing an anti-coronavirus compound, which is applied to the anti-coronavirus compound of any one of claims 1 to 3, and comprises the following steps:
Figure QLYQS_3
under the protection of nitrogen, the compound 13 is dissolved in a mixed solvent of tetrahydrofuran and water, trifluoroacetic acid is added after cooling, and then the reaction is carried out at room temperature; after the reaction is monitored by thin layer chromatography and LCMS, the reaction is neutralized to pH 7-8 by using saturated sodium bicarbonate solution, concentrated, subjected to C18 reverse phase column and freeze-dried to obtain a target compound 1 and a target compound 2, wherein the target compound 1 and the target compound 2 are chiral compounds.
8. The method for producing an anti-coronavirus compound as claimed in claim 7, wherein: the preparation method of the compound 13 comprises the following steps:
Figure QLYQS_4
under the protection of nitrogen, the compound 9 is dissolved in dimethylformamide, a dimethylformamide solution of t-BuMgCl is dripped after cooling for reaction, then a dimethylformamide solution of the compound 12 is dripped for heating for reaction, after the reaction is monitored by thin layer chromatography and LCMS, the reaction solution is poured into a saturated ammonium chloride solution, extraction is carried out by ethyl acetate, and the compound 13 is obtained after drying, concentration and column chromatography of an organic phase, wherein the compound 13 is yellow solid.
9. The method for preparing an anti-coronavirus compound according to claim 8, wherein the method for preparing the compound 9 comprises the steps of:
step one:
Figure QLYQS_5
under the protection of nitrogen and under the ice water bath condition, dropwise adding concentrated sulfuric acid into a methanol solution dissolved with a compound 1 for reaction, monitoring the reaction by thin layer chromatography, neutralizing with a saturated sodium bicarbonate solution until the pH value is 7-8, drying, filtering, concentrating, and performing column chromatography to obtain a compound 2, wherein the compound 1 is L-ribose, and the compound 2 is yellow oily matter;
step two:
Figure QLYQS_6
under the protection of nitrogen and ice water bath, 60% sodium hydrogen is added into the dimethylformamide solution dissolved with the compound 2, and then benzyl bromide is added dropwise; after the benzyl bromide is added dropwise, reacting overnight at room temperature; after the reaction is monitored by thin layer chromatography, pouring a reaction solution into ice water solution of saturated ammonia chloride, and extracting by using ethyl acetate, wherein an organic phase is washed, dried, filtered, concentrated and subjected to column chromatography to obtain a compound 3, and the compound 3 is yellow oily matter 3;
step three:
Figure QLYQS_7
dropwise adding hydrochloric acid into an acetic acid solution dissolved with a compound 3 under the protection of nitrogen, and then carrying out heating reaction; after the reaction is finished, the reaction solution is concentrated under reduced pressure, ethyl acetate and water are used for dissolving after the concentration, and the organic phase is washed, dried, filtered and concentrated to obtain a compound 4;
step four:
Figure QLYQS_8
concentrating the compound 4, dissolving in dry dimethyl sulfoxide, adding acetic anhydride, reacting at room temperature under the protection of nitrogen until the reaction is completed, concentrating the reaction solution under reduced pressure, dissolving in ethyl acetate, washing, drying, filtering, concentrating under reduced pressure, and performing column chromatography to obtain a compound 5, wherein the compound 5 is yellow oily matter;
step five:
Figure QLYQS_9
under the protection of nitrogen, adding trimethylchlorosilane into dry tetrahydrofuran solution dissolved with 4-amino-7-bromopyrrolo [2,1-F ] [1,2,4] triazine, reacting at room temperature, cooling the reaction solution to-78 ℃, dropwise adding n-BuLi n-hexane solution into the reaction solution, dropwise adding compound 5 dissolved in dry tetrahydrofuran into the reaction solution for reaction, quenching the reaction after the reaction is finished, adding acetic acid, concentrating under reduced pressure, dissolving with dichloromethane and water, washing an organic phase, drying, filtering, concentrating, and performing column chromatography to obtain compound 6, wherein the compound 6 is yellow solid;
step six:
Figure QLYQS_10
under the protection of nitrogen, dissolving a compound 6 in a dry dichloromethane solution, cooling, adding trimethylchlorosilane, adding trimethylsilicon triflate into a reaction solution for reaction, adding saturated sodium bicarbonate solution for quenching and stirring after the reaction is completed, separating an organic phase and a water phase, and drying, filtering, concentrating and performing column chromatography on the organic phase to obtain a compound 7, wherein the compound 7 is a yellow solid;
step seven:
Figure QLYQS_11
under the protection of nitrogen, dissolving the compound 7 in a dichloromethane solution, cooling, dropwise adding an n-hexane solution containing BCl3, then carrying out a cooling reaction, adding methanol for quenching, concentrating under reduced pressure, and separating by a C18 reverse phase column after the reaction is finished to obtain a compound 8, wherein the compound 8 is a light yellow solid;
step eight:
Figure QLYQS_12
under the protection of nitrogen, dissolving the compound 8 and 2,2' -dimethoxypropane in acetone, adding concentrated sulfuric acid, heating for reaction, monitoring the reaction by thin layer chromatography, neutralizing with sodium bicarbonate to pH 7-8, concentrating, and performing column chromatography to obtain a compound 9, wherein the compound 9 is a yellow solid.
10. The method for producing an anti-coronavirus compound according to claim 8, wherein: the preparation method of the compound 12 comprises the following steps:
step one:
Figure QLYQS_13
under the protection of nitrogen, dissolving a compound 10 and 2-ethyl-1-butanol in toluene, adding p-toluenesulfonic acid, heating, carrying out a reaction after water diversion by a water diversion device, carrying out a thin layer chromatography and LCMS detection reaction, concentrating, neutralizing to pH 7-8 by using a saturated sodium bicarbonate solution, extracting by using dichloromethane, and drying, concentrating and carrying out column chromatography on an organic phase to obtain a compound 11, wherein the compound 10 is 2-methylalanine, and the compound 11 is a white solid;
step two:
Figure QLYQS_14
under the protection of nitrogen, phosphorus oxychloride and p-toluenesulfonate of a compound 11 are dissolved in dichloromethane, triethylamine is dripped for reaction after cooling, then dichloromethane solution of triethylamine and ethyl o-hydroxybenzoate is added for reaction, and then triethylamine and p-nitrophenol are added for reaction; after completion of the reaction by thin layer chromatography and LCMS, compound 12 was obtained after concentration and C18 reverse phase column, wherein compound 11 was 2-methylalanine 2-ethylbutyl ester and compound 12 was a yellow oil.
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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
WO2022217153A2 (en) * 2021-04-09 2022-10-13 Emory University Nucleosides and nucleotides analogs as antiviral agents
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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Publication number Priority date Publication date Assignee Title
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
WO2022217153A2 (en) * 2021-04-09 2022-10-13 Emory University Nucleosides and nucleotides analogs as antiviral agents

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