Application of meroterpenoids in preparation of anti-coronavirus drugs

Abstract

The invention discloses an application of a meroterpenoid compound in preparation of an anti-coronavirus drug. Specifically, the invention relates to a compound shown as a formula I or pharmaceutically acceptable salt thereof. The invention finds that the compound can effectively inhibit the replication of beta coronavirus HCoV-OC43 in vitro, and the IC50 (half maximal inhibitory concentration) to the HCoV-OC43 is 3.61 mu g / ml, so that the compound has anti-coronavirus activity and can be used for treating infection caused by the coronavirus. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a heteroterpenoid compound and a pharmaceutically acceptable salt thereof and a composition containing the compound or the pharmaceutically acceptable salt thereof in the preparation of an anti-coronavirus drug. Background Art

[0002] Coronavirus (CoV) is widely present in nature. It is a class of RNA viruses 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] At present, there are 7 types of coronaviruses known to infect humans, namely human coronavirus 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. Coronaviruses are transmitted in various ways, mainly including droplet transmission, contact transmission and air transmission. Its incubation period is generally 2-14 days, and patients may be contagious during the incubation period, which greatly increases the difficulty of epidemic prevention and control. The symptoms of diseases caused by coronaviruses vary in severity, ranging from mild respiratory infections to severe pneumonia, acute respiratory distress syndrome, and may even lead to death. Different types of coronaviruses have different effects on human health.

[0004] Meroterpenoids refer to natural products that are partly derived from terpenes in terms of biogenic origin. They are unique molecules formed by the combination of terpenes and non-terpenoid biosynthetic pathways and are widely found in plants, fungi and bacteria. Meroterpenoid compounds have diverse structures and are numerous. The differences in the groups of meroterpenoid compounds give this type of compound a variety of biological activities, such as antibacterial, inhibition of tumor cell proliferation and anti-inflammatory. Berkeleyacetal C is a meroterpenoid compound first discovered by Stierle et al. from the fermentation product of a fungus Penicillium isolated from Berkeley Pit Lake. The structural formula is shown in Formula I. Subsequently, our research group also isolated berkeleyacetal C from the fermentation product of Alternaria sp. isolated from Tibetan soil. So far, there has been no report on the use of this compound as a drug for the preparation of anti-coronavirus.

[0005] Summary of the invention

[0006] The purpose of the present invention is to provide a new use of a heteroterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-coronavirus drug. Pharmacodynamic experiments have confirmed that the compound can effectively inhibit the replication of the beta group coronavirus HCoV-OC43 in an in vitro cell model and has the potential to be used in the preparation of an anti-coronavirus drug.

[0007] The heteroterpenoid compound has a structural formula as 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 the 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 a coronavirus inhibitor.

[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.

[0016] Specifically, the coronavirus is selected from at least one of human coronavirus 2019-nCoV, HCoV-229E, HCoV-OC43, SARS-CoV and MERS-CoV.

[0017] In the above application, "pharmaceutically acceptable salts of the compounds of Formula I" refer to salts that are suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable effect / risk ratio within the scope of reliable medical judgment. Pharmaceutically acceptable salts of the compounds of Formula I are well known in the art, including but not limited to sodium salts, potassium salts, calcium salts, hydrochlorides, nitrates, sulfates, bisulfates, phosphates, hydrogen phosphates, acetates, oxalates, lactates, citrates, tartrates, and maleates.

[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 pharmaceutically acceptable carrier material may also be added when preparing the drug.

[0020] The 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 make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems.

[0021] In order to prepare the unit dosage form 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, white clay, 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 dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, 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. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare isotonic injections, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. can also be added. In addition, if necessary, colorants, preservatives, spices, flavoring agents, sweeteners or other materials can also 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, etc.; cavity administration, such as rectal and vaginal; respiratory tract administration, such as nasal cavity; mucosal administration.

[0022] The present invention also provides a medicine or a pharmaceutical composition, the active ingredient of which is the compound shown in Formula I or a pharmaceutically acceptable salt thereof.

[0023] The drug or pharmaceutical composition has at least one of the following effects:

[0024] 1) Treatment of diseases caused by coronavirus or coronavirus infection;

[0025] 2) Preventing diseases caused by coronavirus or coronavirus infection;

[0026] 3) Suppress coronavirus.

[0027] 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.

[0028] When the compound of formula I or a pharmaceutically acceptable salt thereof provided by the present invention is used 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's organism.

[0029] The dosage and method of use of the compounds of the present invention depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, activity strength of the compound, time of administration, metabolic rate, severity of the disease, 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.

[0030] In the present invention, the term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0031] 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.

[0032] In the present invention, the disease caused by the coronavirus may be a respiratory system infection and / or a digestive system infection.

[0033] 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.

[0034] In the present invention, the diseases caused by the coronavirus generally include viral pneumonia, severe acute respiratory syndrome, etc.

[0035] In the present invention, the coronavirus infection usually causes diseases such as viral pneumonia and severe acute respiratory syndrome.

[0036] The present invention selects the beta group coronavirus HCoV-OC43 to explore the possibility of using the heteroterpenoid compound shown in the above formula I in the preparation of anti-coronavirus drugs. Through experimental research, it is found that the compound can significantly inhibit the replication of the beta group coronavirus HCoV-OC43 in vitro and has the potential to be used in the preparation of anti-coronavirus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The NMR of the compound shown in Formula I 1 H-NMR spectrum.

[0038] Figure 2 The NMR of the compound shown in Formula I 13 C-NMR spectrum. DETAILED DESCRIPTION

[0039] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0040] The coronavirus HCoV-OC43 used in the examples is the human coronavirus OC43 strain (Human coronavirus OC43 ( VR-1558 TM )); Literature: McIntoshK, BeckerWB, ChanockRM. Growth in suckling-mouse brain of "IBV-like" viruses from patients with upper respiratory tractdisease[J]. Proc Natl Acad Sci USA, 1967, 58: 2268-2273. PubMed: 4298953.

[0041] The compound of formula I used in the examples was prepared and isolated in our laboratory (for the isolation method, see the reference: Journal of Natural Products. 2007, 70, 1820-1823), and is a light yellow powder with a purity greater than 98%. The structural confirmation data are shown in Table 1.

[0042] Table 1. Hydrogen spectra of compounds represented by formula I ( 1 H NMR) and carbon spectroscopy ( 13 C NMR data

[0043]

[0044]

[0045] a Tested at 600 MHz with CDCl3 as solvent.

[0046] b Tested at 150 MHz with CDCl3 as solvent.

[0047] The H NMR spectrum and C NMR spectrum of the compound represented by the above formula I are the same as the data of the compound berkeleyacetal C in "The berkeleyacetals, three meroterpenes from a deep water acid mine waste Penicillium" reported in the literature [see Journal of Natural Products. 2007, 70, 1820-1823].

[0048] Example 1. Detection of the in vitro anti-HCoV-OC43 activity of the compound of formula I

[0049] 1. Purpose of the experiment

[0050] 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 inhibitory concentration (IC50) of the compound against coronavirus (HCoV-OC43) in H460 cells. 50 ) and SI. Ribavirin (RBV) was used as a positive control drug.

[0051] The experiments were conducted in the BSL-2 biosafety laboratory of the Virus Laboratory of Jiangsu Food and Drug Vocational and Technical College.

[0052] 2. Materials

[0053] Test product:

[0054] The compound of formula I was prepared and isolated in our laboratory (the isolation method is described in the literature: Journal of Natural Products (Natural Products Magazine). 2007, 70, 1820-1823), and is a light yellow powder with a purity greater than 98%; the structural identification spectrum is shown in Figure 1 , 2 ;

[0055] 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.

[0056] cell

[0057] The human lung cancer H460 cells were subcultured and preserved by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. They were cultured in DMEM or 1640 medium containing 10% inactivated fetal bovine serum and 1% double antibiotics (penicillin and streptomycin) at 37° C. in a 5% CO 2 incubator and subcultured every 2-3 days.

[0058] strain

[0059] HCoV-OC43 was passaged in H460 cells and stored in a -80°C refrigerator.

[0060] 3. Experimental Methods

[0061] Cell culture

[0062] Take H460 cells as an example: add 3 ml of 0.25% Trypsin-EDTA (trypsin cell digestion solution) to the culture flask full of H460 cells, digest at 37°C for 1-2 minutes, discard the digestion solution, add culture solution and pipette, subculture at 1:4, subculture once every 2-3 days, prepare 200,000 cells per ml when seeding, inoculate 96-well cell culture plates, 0.1 ml per well, culture overnight at 37°C, 5% CO2, and conduct experiments after the cells grow into a monolayer.

[0063] Anti-HCoV-OC43 activity assay (CPE method)

[0064] The experiment was carried out in H460 cells, with 1×10 4 Each well was inoculated in a 96-well plate, and after overnight culture, 100 μl of HCoV-OC43 virus solution (100 TCID 50 ) was used to infect H460 cells in a 96-well plate. The drug to be tested was diluted with maintenance solution and administered simultaneously with infection and 2 hours after infection for determination. The drug to be tested was diluted three times with 8 doses of samples for experiment. Two parallel wells were set for each dose. A drug-free virus control group was also set up. The cytopathic effect was observed under a microscope as an indicator. The cell death ratio was 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 survived). When the lesion of the virus control group reached 4+, the results were observed and recorded, and the half-inhibitory concentration of the drug on the virus (the formula is as follows) and the selection index (SI=TC 50 / IC 50 ).

[0065]

[0066] 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

[0067] Cytotoxicity assay (CPE method)

[0068] The cells were counted at 1.5×10 4 100 cells / well were inoculated in a 96-well plate. After overnight culture, maintenance solution containing the drug to be tested was added. The drug to be tested was diluted three-fold to 8 doses for the experiment and culture was continued. 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:

[0069]

[0070] 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

[0071] 4. Experimental Results

[0072] Inhibitory effects of drugs on HCoV-OC43 in H460 cells

[0073] As shown in Table 2, the IC values ​​of the compound of formula I against the HCoV-OC43 strain were determined by CPE method. 50 The IC value of RBV against HCoV-OC43 was 3.61 μg / ml, and the selection index SI was 20.57. 50 It was 4.95 μg / ml, and the selection index SI was 17.18.

[0074] Table 2. Inhibitory effects of compounds on HCoV-OC43 in H460 cells (IC 50 )(CPE Method)

[0075]

[0076]

[0077] 5. Conclusion

[0078] Under the experimental conditions, the compound of formula I has an inhibitory effect on the HCoV-OC43 strain; RBV has an inhibitory effect on the HCoV-OC43 strain, and the anti-coronavirus HCoV-OC43 activity of RBV is comparable to the results in the literature and before this experiment, indicating that the experimental system is established.

[0079] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof. The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

Claims

1. Use of the compound of 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 product for treating a disease caused by a coronavirus or a coronavirus infection; (b) Use of the 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; (c) Use of the compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a coronavirus inhibitor; 2. The use according to claim 1, characterized in that: The product is a drug or a drug preparation.

3. The use according to claim 1 or 2, characterized in that: The coronavirus inhibitor is capable of inhibiting the replication of coronavirus.

4. The use according to any one of claims 1 to 3, characterized in that: The coronavirus is an alpha coronavirus and / or a beta coronavirus.

5. The use according to claim 4, characterized in that: The coronavirus is selected from at least one of human coronavirus 2019-nCoV, HCoV-229E, HCoV-OC43, SARS-CoV and MERS-CoV.

6. A drug or pharmaceutical composition, the active ingredient of which is a compound of formula I or a pharmaceutically acceptable salt thereof; The drug or pharmaceutical composition has at least one of the following effects: 1) Treatment of diseases caused by coronavirus or coronavirus infection; 2) Preventing diseases caused by coronavirus or coronavirus infection; 3) Suppress coronavirus.

7. The drug or pharmaceutical composition according to claim 6, characterized in that: The coronavirus inhibitor is capable of inhibiting the replication of coronavirus.

8. The drug or pharmaceutical composition according to claim 6 or 7, characterized in that: The coronavirus is an alpha coronavirus and / or a beta coronavirus.

9. The drug or pharmaceutical composition according to any one of claims 6 to 8, characterized in that: The coronavirus is selected from at least one of human coronavirus 2019-nCoV, HCoV-229E, HCoV-OC43, SARS-CoV and MERS-CoV.

10. The drug or pharmaceutical composition according to any one of claims 6 to 9, characterized in that: The medicine or pharmaceutical composition is in any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

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