Use of a naphthoquinone compound in the preparation of anti-coronavirus drugs

Abstract

The present invention discloses the use of a naphthoquinone compound in the preparation of an anti-coronavirus drug. Specifically, the present invention relates to a compound represented by Formula I, or a pharmaceutically acceptable salt thereof. The present invention found that the compound can effectively inhibit the replication of alpha coronavirus HCoV-229E in vitro, and the IC of HCoV-229E is 0. 50 The concentration was 1.62 μg / ml, indicating that the compound has anti-coronavirus activity and can be used to treat infections caused by coronavirus. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a naphthoquinone compound in the preparation of an anti-coronavirus drug. Background Art

[0002] Coronavirus (CoV) is widely present in nature. It is a type of RNA virus with an envelope and a linear single-stranded positive genome. Coronavirus only infects vertebrates and is associated with a variety of diseases in humans and animals. It can cause respiratory, digestive and nervous system diseases in humans and animals.

[0003] Currently, there are seven known coronaviruses that infect humans: human coronaviruses HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1; as well as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The first four coronaviruses cause milder symptoms of the common cold, while the last three can cause severe symptoms and are highly contagious, even leading to fatal viral pneumonia. The novel coronavirus (SARS-CoV-2) undergoes frequent genetic mutations during its spread and transmission, with some variants capable of causing breakthrough infections and a certain rate of reinfection. The currently prevalent variant globally is the Omicron variant. Although evidence from both domestic and international sources suggests that the Omicron variant has reduced pulmonary pathogenicity, primarily presenting clinically with upper respiratory tract infections, its transmissibility and immune evasion abilities are significantly enhanced, significantly reducing the neutralizing effect of some marketed antibody drugs. Therefore, the search for effective anti-coronavirus drugs remains crucial.

[0004] Naphthoquinones are a class of secondary metabolites produced by plants and fungi. They are primarily classified as 1,4-naphthoquinone, 1,2-naphthoquinone, and 2,6-naphthoquinone, depending on the position of the carbonyl group. Naphthoquinone compounds exhibit diverse biological activities, including antiallergic, antibacterial, antifungal, anti-inflammatory, antiplatelet, antiprotozoal, antithrombotic, antiviral, and cytotoxic properties. 6-Ethyl-2,7-dimethoxyjuglone was first discovered by Howe et al. from the fermentation products of the microorganism Henderson ulatoruloidea [Experientia, 1968, 34, 1257]. Our research group also isolated this compound from the fermentation products of the fungus Perenniporia sp., a strain of the weevil larvae [Journal of Natural Products, 2012, 75, 1339-1345]. Its structural formula is shown in Formula I.In addition, Pittayakhajonwut et al. isolated 6-ethyl-2,7-dimethoxyjuglone from the fermentation product of Phaeosphaeria sp. BCC8292 [see, Planta Medica (Medicinal Plants). 2008, 74, 281-286], and reported that the compound has anti-tuberculosis activity; Luo et al. isolated 6-ethyl-2,7-dimethoxyjuglone from the fermentation product of the plant-related fungus Delitschias p. FL1581 [see, Organic Letters (Organic Chemistry Communications). 2014, 16, 5944-5947]; Rivera-Chávez et al. isolated 6-ethyl-2,7-dimethoxyjuglone from the fermentation product of the strain Delitschia sp. [see, Planta Medica (Medicinal Plants). 2018, 85, 62-71], and reported that the compound had an inhibitory effect on the African American prostate cancer cell line E006AA-hT; Bocanegra et al. isolated 6-ethyl-2,7-dimethoxyjuglone from the fermentation product of the Pyrenochaetopsis sp. strain MSX63693 [see, Journal of Natural Products. 2021, 84, 771-778], and reported that the compound had inhibitory activity against the human melanoma cell line MDA-MB-435, the human breast cancer cell line MDA-MB-231, and the human ovarian cancer cell line OVCAR3; Cadelis et al. also isolated 6-ethyl-2,7-dimethoxyjuglone from the fermentation product of the fungus Neofusicoccum australe [see, Molecules. 2021, 26, 1094]. However, there has been no report on the use of this compound as a drug for the preparation of anti-coronavirus drugs.

[0005] Summary of the Invention

[0006] The present invention aims to provide a novel use of a naphthoquinone compound in the preparation of anti-coronavirus drugs. Pharmacodynamic studies have confirmed that the compound can effectively inhibit the replication of the alpha coronavirus HCoV-229E in an in vitro cell model, suggesting its potential for use in the preparation of anti-coronavirus drugs.

[0007] The structural formula of the naphthoquinone compound is shown in Formula I:

[0008]

[0009] The application of the compound of formula I or a pharmaceutically acceptable salt thereof provided by the present invention is the following (a) and / or (b) and / or (c):

[0010] (a) Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a product for treating a disease caused by a coronavirus or a coronavirus infection;

[0011] (b) Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a product for preventing a disease caused by a coronavirus or a coronavirus infection;

[0012] (c) Use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of coronavirus inhibitors.

[0013] The product may be a drug or a pharmaceutical preparation.

[0014] The coronavirus inhibitor is capable of inhibiting the replication of coronavirus.

[0015] The coronavirus may be an alpha coronavirus and / or a beta coronavirus, specifically at least one selected from human coronavirus 2019-nCoV, HCoV-229E, HCoV-OC43, SARS-CoV and MERS-CoV.

[0016] In the above applications, a "pharmaceutically acceptable salt of the compound of Formula I" means a salt that is suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, or allergic reaction, and is commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts of the compound of Formula I are well known in the art and include, but are not limited to, sodium salts, potassium salts, calcium salts, hydrochlorides, nitrates, sulfates, bisulfates, phosphates, hydrogenphosphates, acetates, oxalates, lactates, citrates, tartrates, and maleates.

[0017] In the above applications, when preparing drugs or pharmaceutical preparations, the compound represented by formula I or a pharmaceutically acceptable salt thereof can be used as one of the active ingredients or as the only active ingredient.

[0018] In the above applications, when preparing drugs or pharmaceutical preparations, the compound represented by Formula I or a pharmaceutically acceptable salt thereof can be used as one of the active ingredients or as the only active ingredient.

[0019] In the above applications, a carrier material may also be added when preparing the drug.

[0020] Carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to prepare a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. The formulations can be conventional preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems. In order to prepare unit dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparation. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection; cavity administration, such as rectal and vaginal administration; respiratory administration, such as nasal cavity administration; and mucosal administration.

[0021] The present invention also provides a medicine or pharmaceutical composition, the active ingredient of which is the compound represented by Formula I or a pharmaceutically acceptable salt thereof.

[0022] The drug or pharmaceutical composition has at least one of the following effects:

[0023] 1) Treatment of diseases caused by coronavirus or coronavirus infection;

[0024] 2) Preventing diseases caused by coronavirus or coronavirus infection;

[0025] 3) Suppress coronavirus.

[0026] The above-mentioned medicine or pharmaceutical composition can be prepared into dosage forms such as solution, tablet, capsule or injection according to conventional methods known to those skilled in the art.

[0027] When using the compound of formula I or a pharmaceutically acceptable salt thereof provided by the present invention to prevent and / or treat infection caused by coronavirus, an effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof is administered to the subject.

[0028] The dosage and method of administration of the compounds of this invention depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, potency of the compound, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 100 mg / kg body weight / day, with the optimal dosage being between 0.1 and 10 mg / kg body weight / day.

[0029] In the present invention, the term "effective amount" refers to a dose that can achieve the treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0030] In the present invention, the term "subject" may refer to a patient or other animal that receives the composition of the present invention to treat, prevent, alleviate and / or relieve the diseases or conditions described in the present invention, particularly mammals, such as humans, dogs, monkeys, cows, horses, etc.

[0031] In the present invention, the disease caused by the coronavirus may be a respiratory system infection and / or a digestive system infection.

[0032] The respiratory system infection is a respiratory tract infection and / or a lung infection; the respiratory tract infection may be nasopharyngitis, rhinitis, pharyngitis, tracheitis and / or bronchitis; the lung infection may be pneumonia; and the digestive system infection may be diarrhea.

[0033] In the present invention, the diseases caused by the coronavirus generally include viral pneumonia, severe acute respiratory syndrome, etc.

[0034] In the present invention, the coronavirus infection usually causes diseases such as viral pneumonia and severe acute respiratory syndrome.

[0035] The present invention selects α-group coronavirus HCoV-229E to explore the possibility of using the naphthoquinone compound represented by the above formula I in the preparation of anti-coronavirus drugs. Through experimental studies, it was found that the compound can significantly inhibit the replication of α-group coronavirus HCoV-229E in vitro and has the potential to be used in the preparation of anti-coronavirus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The nuclear magnetic resonance of the compound shown in formula I 1 H-NMR spectrum.

[0037] Figure 2 The nuclear magnetic resonance of the compound shown in formula I 13 C-NMR spectrum. DETAILED DESCRIPTION

[0038] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be appreciated by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. In the examples, if no specific conditions are specified, the conditions are followed according to conventional conditions or manufacturer recommendations. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional products.

[0039] The coronavirus HCoV-229E used in the embodiment is human coronavirus 229E strain (Human coronavirus 229E ( VR-740 TM )); Literature: Hamre D, Procknow JJ. A new virus isolated from the human respiratory tract. Proc. Soc. Exp. Biol. Med. 121: 190-193, 1966. PubMed: 4285768.

[0040] The compound of formula I used in the examples was prepared and isolated in this laboratory (for the isolation method, see the reference: Journal of Natural Products (Natural Products Magazine) 2012, 75, 1339-1345), and is orange needle-shaped crystals with a purity greater than 98%. The structural confirmation data are shown in Table 1.

[0041] Table 1. Hydrogen spectrum of the compound represented by formula I ( 1 H NMR) and carbon spectroscopy ( 13 C NMR data

[0042]

[0043]

[0044] a Tested at 400 MHz with CDCl3 as solvent.

[0045] b Tested at 400 MHz with CDCl3 as solvent.

[0046] Example 1: Detection of the in vitro anti-HCoV-229E activity of the compound of formula I

[0047] 1. Purpose of the experiment

[0048] The in vitro anti-coronavirus efficacy of the compound of formula I was studied, and the cytopathic effect (CPE) assay was used to determine the half-maximal inhibitory concentration (IC50) of the compound against coronavirus (HCoV-229E) in Huh7 cells. 50 ) and SI. Ribavirin (RBV) was used as a positive control drug.

[0049] The experiments were conducted in the BSL-2 biosafety laboratory of the Virology Department, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences (Room 329, Laboratory Building).

[0050] 2. Materials

[0051] Test sample:

[0052] The compound of formula I was prepared and isolated in our laboratory (for the isolation method, see the reference: Journal of Natural Products (Natural Products Magazine). 2012, 75, 1339-1345), and is orange needle-shaped crystals with a purity greater than 98%; the structural identification pattern is shown in Figure 1 、 2 ;

[0053] The positive control drug ribavirin injection (RBV) was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd. with a specification of 100 mg / ml. It was diluted to the required concentration when used and stored in a refrigerator at 4°C.

[0054] Cells: Huh7 human hepatoma cells were cultured at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, and cultured in DMEM or 1640 medium supplemented with 10% inactivated fetal bovine serum and 1% double-antibiotics (penicillin and streptomycin) at 37°C in a 5% CO2 incubator. They were passaged every 2-3 days.

[0055] Strain: HCoV-229E was passaged in Huh7 cells and stored in a -80°C freezer.

[0056] 3. Experimental Methods

[0057] Cell culture

[0058] Taking Huh7 cells as an example: add 3 ml of 0.25% Trypsin-EDTA (trypsin cell digestion solution) to a culture flask full of Huh7 cells, digest at 37°C for 1-2 minutes, discard the digestion solution, add culture solution and pipette, subculture at a ratio of 1:4, subculture once every 2-3 days, and prepare 200,000 cells per ml when seeding the plate. Inoculate 96-well cell culture plates with 0.1 ml per well, culture at 37°C, 5% CO2 overnight, and perform experiments after the cells grow into a monolayer.

[0059] Anti-HCoV-229E activity assay (CPE method)

[0060] The experiment was performed in passaged Huh7 cells, with 1×10 4 Each well was inoculated into a 96-well plate, and after overnight culture, 100 μl of HCoV-229E virus solution (100 TCID 50 ) Infect Huh7 cells in 96-well plates, and the drug to be tested is diluted with maintenance solution. The drug to be tested is tested at the same time as infection and at 2 hours after infection. The drug to be tested is diluted three times into 8 doses of samples for experiment. Two parallel wells are set for each dose. At the same time, a virus control group without drug is set up. The cytopathic effect is observed under a microscope as an indicator. The cell death ratio is marked as 4+ (cell death ratio 75% to 100%), 3+ (cell death ratio 50% to 75%), 2+ (cell death ratio 25% to 50%), 1+ (cell death ratio 0 to 25%), and 0+ (all cells survive). When the lesion of the virus control group reaches 4+, the results are observed, recorded, and the half-maximal inhibitory concentration of the drug on the virus is calculated using the Reed-Muench method (the formula is as follows) and the selection index (SI=TC 50 / IC 50 ).

[0061]

[0062] Where: A = drug concentration at which cumulative inhibition rate is less than 50%, B = inhibition rate at which cumulative inhibition rate is greater than 50%, C = inhibition rate at which cumulative inhibition rate is less than 50%, D = log dilution factor

[0063] Cytotoxicity assay (CPE method)

[0064] The cells were counted at 1.5×10 4 Cells were seeded in 96-well plates. After overnight culture, maintenance solution containing the drug to be tested was added. The drug to be tested was diluted threefold to eight doses and cultured again. The toxicity of the drug to the cells was observed under an inverted microscope 2 days after administration, and the half-toxic concentration (TC) was calculated using the Reed-Muench method. 50 , the calculation formula is as follows:

[0065]

[0066] Where: A = drug concentration at which cumulative inhibition rate is less than 50%, B = inhibition rate at which cumulative inhibition rate is greater than 50%, C = inhibition rate at which cumulative inhibition rate is less than 50%, D = log dilution factor

[0067] 4. Experimental Results

[0068] Inhibitory effects of drugs on HCoV-229E in Huh7 cells

[0069] As shown in Table 2, the IC values of the compound of formula I against HCoV-229E strain were determined by CPE method. 50 The IC of RBV against HCoV-229E was 1.62 μg / ml, and the selection index SI was 11.84; 50 It was 4.81 μg / ml, and the selection index SI was 19.23.

[0070] Table 2. Inhibitory effects of compounds on HCoV-229E in Huh7 cells (IC 50 (CPE Law)

[0071]

[0072]

[0073] 5. Conclusion

[0074] Under the experimental conditions, the compound of formula I has an inhibitory effect on the HCoV-229E strain; RBV has an inhibitory effect on the HCoV-229E strain, and the anti-coronavirus HCoV-229E activity of RBV is comparable to the results in the literature and previous experiments, indicating that the experimental system is established.

[0075] Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that, based on all disclosed teachings, various modifications and substitutions may be made to those details, and such variations are within the scope of the present invention. The full scope of the present invention is set forth in the appended claims and any equivalents thereof. The present invention is further illustrated below with reference to specific examples, but the present invention is not limited to these examples. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials described can be obtained from publicly available commercial sources.

Claims

1. Use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof, wherein the use is the following (a) and / or (b) and / or (c): (a) Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease caused by a coronavirus or a coronavirus infection; (b) Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing a disease caused by a coronavirus or a coronavirus infection; (c) Use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a coronavirus inhibitor; Formula I The coronavirus is selected from human coronavirus HCoV-229E.

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