Use of a chromone compound in preparation of a drug for resisting coronavirus

Abstract

The application discloses application of a chromone compound in preparation of a drug for resisting coronavirus. Specifically, the application relates to a compound shown in formula I, or a pharmaceutically acceptable salt thereof. The application finds that the compound can effectively inhibit replication of alpha coronavirus HCoV-229E in vitro, the IC 50 of the compound to HCoV-229E is 0.24 ug / ml, which shows that the compound has anti-coronavirus activity and can be used for treating infection caused by coronavirus.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of a chromone compound in the preparation of anti-coronavirus drugs. Background Technology

[0002] Coronaviruses (CoV) are widespread in nature. They are a class of enveloped RNA viruses with a linear single-stranded positive-sense genome. Coronaviruses infect only vertebrates and are associated with a variety of diseases in humans and animals, causing respiratory, digestive, and nervous system diseases in both.

[0003] Previously, six coronaviruses were known to infect humans: human coronavirus HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1; SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus); and MERS-CoV (Middle East Respiratory Syndrome Coronavirus). SARS-CoV-2 is currently the seventh known coronavirus capable of infecting humans. Of these coronaviruses, the first four cause mild symptoms similar to the common cold, while the latter three can lead to severe symptoms, are highly contagious, and can even cause fatal viral pneumonia. Finding effective drugs against coronaviruses remains crucial.

[0004] Chromones are a class of secondary metabolites widely found in plants and fungi, characterized by a benzopyranone-containing core in their molecules. These compounds exhibit a wide range of biological activities, including anti-inflammatory, antibacterial, antioxidant, and antitumor effects. The structural diversity of the type, number, and position of substituents attached to the core is particularly important for the physical, chemical, and biological properties of both natural and synthetic derivatives. Lachnochromonin is a chromone compound discovered by our research group from the fermentation products of the *Lachnum virgineum* strain, and its structural formula is shown in Formula I. Patent publication CN102442985 A discloses that lachnochromonin has the activity of inhibiting the reproductive growth of tumor cells, and patent publication US 9221811 B2 discloses that a series of chromone derivatives including lachnochromonin have osteogenic effects. Chu et al. also reported that lachnochromonin inhibits tumor cell growth and promotes apoptosis through JAK / STAT3 signaling [see Cellular Signalling. 2023, 106, 110592]. Currently, there are no reports of this compound being used as a drug for the preparation of anti-coronavirus drugs.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide a novel use of chromone compounds in the preparation of anti-coronavirus drugs. Pharmacodynamic studies have confirmed that this compound effectively inhibits the replication of group α coronavirus HCoV-229E in in vitro cell models, demonstrating its potential for use in the preparation of anti-coronavirus drugs.

[0007] The structural formula of the chromone compounds is shown in Formula I.

[0008]

[0009] The applications of the compound of Formula I or its pharmaceutically acceptable salt provided by this invention are as follows (a) and / or (b) and / or (c):

[0010] (a) The use of the compound shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of products for treating diseases caused by coronaviruses or coronavirus infection;

[0011] (b) The use of the compound shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of products for the prevention of disease caused by coronavirus or coronavirus infection;

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

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

[0014] The coronavirus inhibitor is able to suppress the replication of the coronavirus.

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

[0016] In the above applications, "pharmaceutically acceptable salts of the compounds represented by Formula I" refers to salts that, within the scope of reliable medical judgment, are suitable for contact with human and lower animal tissues without causing excessive toxicity, irritation, allergic reactions, etc., and that are commensurate with a reasonable effect / risk ratio. Pharmaceutically acceptable salts of the compounds represented by Formula I are well known in the art and include, but are not limited to, sodium salts, potassium salts, calcium salts, hydrochloride salts, nitrates, sulfates, bisulfates, phosphates, hydrogen phosphates, acetates, oxalates, lactates, citrates, tartrates, and maleates.

[0017] In the above applications, when preparing drugs or pharmaceutical preparations, the compound shown in Formula I or its pharmaceutically acceptable salt may be used as one of the active ingredients or as the sole active ingredient.

[0018] In the above applications, when preparing drugs or pharmaceutical preparations, the compound shown in Formula I or its pharmaceutically acceptable salt may be used as one of the active ingredients or as the sole active ingredient.

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

[0020] Carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose 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, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, 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, alginate, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added.In addition, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary. The above dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; via cavities, such as rectal and vaginal; via the respiratory tract, such as nasal; and via mucosal administration.

[0021] The present invention also provides a drug or pharmaceutical composition wherein the active ingredient is a compound of formula I or a pharmaceutically acceptable salt thereof.

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

[0023] 1) To treat diseases caused by coronaviruses or coronavirus infection;

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

[0025] 3) Inhibit coronavirus.

[0026] The above-mentioned drugs or drug compositions can be prepared into dosage forms such as solutions, tablets, capsules or injections 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 infections caused by coronaviruses, an effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof is administered to the subject organism.

[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 condition, nutritional status, the activity intensity of the compound, the time of administration, the metabolic rate, the severity of the illness, 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 this invention, the term "effective dose" refers to a dose that can achieve treatment, prevention, reduction, and / or relief of the disease or condition described in this invention in a subject.

[0030] In this invention, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the composition of this invention to treat, prevent, reduce and / or alleviate the disease or condition described in this invention.

[0031] In this invention, the diseases caused by the coronavirus may be respiratory infections and / or digestive system infections.

[0032] The respiratory 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; the digestive system infection may be diarrhea.

[0033] In this invention, the diseases caused by coronaviruses typically include viral pneumonia, severe acute respiratory syndrome, etc.

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

[0035] This invention selects α-group coronavirus HCoV-229E to explore the possibility of using the chromone compounds shown in 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. Attached Figure Description

[0036] Figure 1 Nuclear magnetic resonance of the compound shown in Formula I 1 H-NMR spectrum.

[0037] Figure 2 Nuclear magnetic resonance of the compound shown in Formula I 13 C-NMR spectrum. Detailed Implementation

[0038] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0039] The coronavirus HCoV-229E used in the examples is the human coronavirus 229E strain. 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] Example 1: Detection of the in vitro anti-HCoV-229E activity of compound I

[0041] 1. Experimental Objective

[0042] To investigate the in vitro anti-coronavirus efficacy of compound I, a cytopathic effect (CPE) assay will be used to determine the half-maximal inhibitory concentration (IC50) of the compound against coronavirus (HCoV-229E) in Huh7 cells. 50 Ribavirin (RBV) was used as a positive control.

[0043] The experiment was conducted in the BSL-2 biosafety laboratory (Room 329, Experimental Building) of the Virus Laboratory, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

[0044] 2. Materials

[0045] Test sample:

[0046] The compound of formula I was prepared and separated in our laboratory (the separation method is disclosed in patent CN 102442985 A). It is an amorphous powder with a purity greater than 98%. The structural confirmation data are shown in Table 1.

[0047] Table 1. Proton NMR spectra of the compounds shown in Formula I ( 1 H NMR and carbon spectroscopy 13 C NMR data

[0048]

[0049]

[0050] a Tested at 400MHz using DMSO-d6 as solvent.

[0051] b Tested at 100MHz using DMSO-d6 as solvent.

[0052] 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 before use and stored at 4°C.

[0053] Cells: Human liver cancer cells Huh7 were passaged and preserved by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, and cultured in DMEM or 1640 medium containing 10% inactivated fetal bovine serum and 1% penicillin and streptomycin in a 37°C, 5% CO2 incubator, and passaged every 2-3 days.

[0054] The strain HCoV-229E was passaged in Huh7 cells and stored at -80°C.

[0055] 3. Experimental Methods

[0056] Cell culture

[0057] Taking Huh7 cells as an example: Add 3 ml of 0.25% Trypsin-EDTA (trypsin cell digestion solution) to a culture flask confluent with Huh7 cells, digest at 37°C for 1-2 minutes, discard the digestion solution, add culture medium and pipette, passage at a 1:4 ratio, passage once every 2-3 days, prepare a solution of 200,000 cells per ml, seed 0.1 ml into a 96-well cell culture plate, culture at 37°C, 5% CO2 overnight, and conduct experiments after the cells grow into a monolayer.

[0058] Assay for activity against HCoV-229E (CPE method)

[0059] The experiment was conducted in passaged Huh7 cells, with 1 × 10⁶ Huh7 cells per cell line. 4 Inoculate 100 μl of HCoV-229E virus solution (100 TCID50) into each well of a 96-well plate and incubate overnight. 50 Huh7 cells in 96-well plates were infected with the test drug, diluted with maintenance medium. Two administration regimens were used: simultaneous administration with infection and administration 2 hours post-infection. Eight doses of the test drug were diluted three-fold for each test, with two parallel wells for each dose. A virus control group without the drug was also included. Cytopathic effects were observed under a microscope, and cell death rates were categorized as 4+ (75%–100% cell death), 3+ (50%–75% cell death), 2+ (25%–50% cell death), 1+ (0–25% cell death), and 0+ (all cells survived). Results were observed when the virus control group reached 4+ cytopathic effects. The results were recorded, and the Reed-Muench method was used to calculate the half-maximal inhibitory concentration (IC50) and selectivity index (SI = TC) against the virus. 50 / IC 50 ).

[0060]

[0061] Where: A = drug concentration with cumulative inhibition rate < 50%, B = inhibition rate with cumulative inhibition rate > 50%, C = inhibition rate with cumulative inhibition rate < 50%, D = log dilution factor.

[0062] Cytotoxicity assay (CPE method)

[0063] Cells were spaced at 1.5 × 10⁻⁶. 4Cells were seeded per well in 96-well plates and cultured overnight. Maintenance medium containing the test drug was then added. The test drug was diluted three-fold to eight doses for the experiment, and culturing continued. Two days after drug administration, the cytotoxicity of the drug was assessed using an inverted microscope, and the half-maximal toxic concentration (TC) was calculated using the Reed-Muench method. 50 The calculation formula is as follows:

[0064]

[0065] Where: A = drug concentration with cumulative inhibition rate < 50%, B = inhibition rate with cumulative inhibition rate > 50%, C = inhibition rate with cumulative inhibition rate < 50%, D = log dilution factor.

[0066] 4. Experimental Results

[0067] Inhibitory effect of the drug on HCoV-229E in Huh7 cells

[0068] As shown in Table 1, the IC50 of compound I against HCoV-229E strain was determined by the CPE method. 50 The concentration was 0.24 μg / ml, and the selectivity index (SI) was 27; the IC50 of RBV against HCoV-229E was... 50 The concentration was 4.81 μg / ml, and the selectivity index (SI) was 19.23.

[0069] Table 1. Inhibitory effect of compounds on HCoV-229E in Huh7 cells (IC50, 100 mg / kg / 2 ... 50 (CPE method)

[0070]

[0071] 5. Conclusion

[0072] Under the experimental conditions, compound I showed inhibitory activity against HCoV-229E strain; RBV also showed inhibitory activity against HCoV-229E strain, and the anti-coronavirus HCoV-229E activity of RBV was comparable to the results in the literature and previous experiments, indicating that the experimental system was valid.

[0073] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the disclosed teachings, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof. The present invention is further illustrated below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

Claims

1. The use of a compound with the structural formula shown in Formula I or a pharmaceutically acceptable salt thereof, wherein the use is as follows (a) and / or (b) and / or (c): (a) The use of the compound shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases caused by coronaviruses or coronavirus infection; (b) The use of the compound shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention of disease caused by coronavirus or coronavirus infection; (c) Use of the compound shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of coronavirus inhibitors; The coronavirus in question is HCoV-229E.

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