KR20210116151A - Composition for preventing or treating coronavirus infection comprising steroidal derivatives comprising lactone ring - Google Patents

Abstract

The present invention relates to the antiviral use of a lactone ring-containing steroid derivative against coronavirus. Particularly, the present invention relates to: an antiviral pharmaceutical composition against coronavirus; a pharmaceutical composition for preventing or treating coronavirus infection diseases; and a health-aid food composition and feed composition for preventing or alleviating coronavirus infection diseases, including a lactone ring-containing steroid derivative as an active ingredient. According to the present invention, a compound represented by chemical formula I or a pharmaceutically or sitologically acceptable salt thereof shows an effect of inhibiting infection with coronavirus, such as Middle East respiratory syndrome-related coronavirus (MERS-CoV), Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), and thus can be used for developing an antiviral agent for preventing or treating such viral diseases.

Description

A composition for preventing or treating coronavirus infection comprising a steroid-based derivative comprising a lactone ring {Composition for preventing or treating coronavirus infection comprising steroidal derivatives comprising lactone ring}

The present invention relates to the antiviral use of a steroid-based derivative containing a lactone ring against coronavirus, and specifically, the present invention relates to a steroid-based derivative containing a lactone ring or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutical composition for antiviral against coronavirus, including; A pharmaceutical composition for the prevention or treatment of a coronavirus infectious disease; And it relates to a health functional food composition and feed composition for preventing or improving coronavirus infectious diseases.

Coronavirus was first discovered in chickens in 1937, followed by animals such as dogs, pigs, and birds, and then in humans in 1965. During a total solar eclipse, when the sun's photosphere is obscured by the moon, it is given this name because its appearance is similar to the corona phenomenon, which is a phenomenon that glows white around the moon.

Coronaviruses are known to mainly cause pneumonia and enteritis in humans and animals, and are also known to occasionally cause nervous system infections and hepatitis. Coronavirus belongs to the coronaviridae and is a positive sense RNA virus with a spherical outer membrane, about 100-120 nm in size. Coronavirus consists of a total of five structural proteins, including the outermost spike protein (S), hemagglutinin-esterase (HE) protein, transmembrane (M) protein, small membrane (E) protein, and nucleocapsid (N) protein. Among them, the spike protein acts as a ligand that binds to the cell receptor and induces fusion between the host cell and the virus, and is known as the most mutable protein.

So far, coronavirus has been recognized as a pathogen that rarely infects humans and mainly infects animals such as dogs, pigs, and cattle. It is one of several viruses that cause respiratory symptoms even when infecting humans, and only causes a simple cold or intestinal disease such as diarrhea, which is not a high risk for children. However, as the causative agent of severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which has caused hundreds of deaths and thousands of patients worldwide, is known to be a novel (mutated) coronavirus. began to attract attention.

SARS is an infectious disease that originated in Guangdong Province in southern China in November 2002 and spread to the world through Hong Kong. am. The path of propagation is not yet fully elucidated, but it is speculated that it is propagated by fine particles of solid or liquid floating in the atmosphere. The SARS epidemic lasted until July 2003, and in about 7 months, a total of 8,096 infections were reported in 32 countries, and 774 of them were known to have died.

MERS was first discovered in Saudi Arabia in 2012, and it presents severe respiratory symptoms such as high fever, cough and shortness of breath. In severe cases, it is accompanied by complications, leading to death. Although the exact source and route of infection have not been identified, it has been reported that infection is highly likely to be transmitted through contact with camels in the Middle East and transmission through close human contact is possible. The cases were mainly concentrated in the Middle East, but since May 2015, more than 100 cases have been reported across Korea.

COVID-19 is an infectious disease caused by SARS-coronavirus-2, a disease that was first identified in Wuhan, the capital of China's Hubei province, at the end of 2019 and spread worldwide, resulting in a progressive pandemic. As of May 7, 2020, more than 3.75 million cases were reported in 187 countries, with 263,000 deaths and 1.24 million recoveries. Common symptoms are fever, cough, fatigue, shortness of breath and loss of smell and taste. In most cases, symptoms are mild, but some progress to viral pneumonia, multi-organ failure, and a cytokine storm. The time from onset of symptoms to onset is usually about 5 days, but can be between 2 and 14 days. The virus is spread from person to person primarily during close contact, and sometimes through droplets that occur when coughing, sneezing and talking. It is most contagious during the first 3 days after onset of symptoms and can be spread before symptoms appear or even late in the disease. A standard method for diagnosing it is using real-time reverse transcription polymerase chain reaction (rRT-PCR) from a nasopharyngeal swab.

As mentioned earlier, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 are all known variant coronaviruses belonging to the genus Beta coronavirus. Thus, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 share some similar characteristics in clinical symptoms, etiology and infection. In the phylogenetic tree, SARS-coronavirus, and SARS-coronavirus-2 belong to lineage B, which includes bat SARS-like coronaviruses and other bat-derived coronaviruses, and MERS-coronaviruses belongs to strain C, which includes bat-derived coronaviruses (Trends Microbiol 24:490-502; https://nextstrain.org/groups/blab/sars-like-cov?f_virus_type=SARS-CoV-2).

All MERS-coronaviruses are thought to be derived from bats, which are the primary carriers of various coronaviruses (Antiviral Res., 101:45-56), and the exact route of infection has not yet been fully elucidated. On the other hand, interspecies transmission of the virus to palm civet and dromedary camel increased the likelihood of zoonotic infection in humans (Nat. Rev. Microbiol., 14:523-534), and hospital My infection is believed to be the main cause of human-to-human MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 infections (BMC Med., 13:1-12).

On the other hand, gitoxygenin and the like are compounds having a similar chemical structure extracted, isolated and identified from plants that are mostly used for food or used as herbal medicines. However, the prevention, treatment or improvement effect of the steroid-based derivative compound containing the lactone ring on coronavirus, in particular, MERS-coronavirus and/or SARS-coronavirus infection is not known.

Since the antivirals developed to date show severe side effects, much attention is required in their application. These treatments are not effective and side effects are also present. Therefore, there is an increasing need for the development of excellent novel coronavirus agents with excellent anti-infection effects and low toxicity for preventing and treating coronavirus outbreaks.

Under this background, the present inventors, as a result of continuing efforts to develop novel coronavirus therapeutic agents, each separated from a plant extract or a steroid system containing a series of lactone rings such as gitoxygenin chemically synthesized by mimicking it By confirming that the derivative has an excellent effect of inhibiting the proliferation of MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2, the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for antiviral against coronavirus comprising a steroid-based derivative comprising a lactone ring or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a coronavirus infection comprising a steroid-based derivative containing a lactone ring or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a health functional food composition for the prevention or improvement of coronavirus-infected diseases, comprising a steroid-based derivative containing a lactone ring or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a feed composition for the prevention or improvement of a coronavirus infection disease comprising a steroid-based derivative comprising a lactone ring or a pharmaceutically acceptable salt thereof.

One embodiment of the present invention for achieving the above object provides a pharmaceutical composition for antiviral against coronavirus comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula I]

In the above formula (I),

또는

이거나,R ₁ is hydrogen, R ₂ is -OH, -O(C=O)CH ₃ ,

or

this,

R ₁ and R ₂ together form =O,

R ₃ is hydrogen or —OH,

R ₄ is —OH, R ₅ is hydrogen, or R ₄ and R ₅ are joined together to form an epoxide ring,

R ₆ is hydrogen, -OH or -O(C=O)CH ₃ ,

또는

임.R ₇ is

or

Lim.

Another embodiment of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating a coronavirus infection disease comprising the compound represented by Formula I or a pharmaceutically acceptable salt thereof as an active ingredient.

Specifically, since the pharmaceutical composition of the present invention exhibits an effect of inhibiting coronavirus, it is useful for preventing or treating infectious diseases caused by coronavirus through a method comprising administering it to an individual suspected of being infected with coronavirus. can be utilized.

The coronavirus is Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome-related coronavirus (SARS-CoV), or severe acute respiratory syndrome coronavirus -2 (Severe acute respiratory syndrome-related coronavirus 2; SARS-CoV-2).

For example, the compound of Formula I may be a compound represented by Formulas 1 to 11, but is not limited thereto.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

.

.

The compound having the structure of Formula 1 is a cardiac glycoside compound of a plant-derived steroid (phydtosteroid) family called digitalis purpurea called digitalis purpurea. It can be isolated and identified from an extract, specifically, a root extract thereof. It has a structure and effect similar to digoxin, which is known to treat heart disease, but unlike digoxin that is removed through the kidneys, it is removed from the liver, so it can be used for patients with kidney problems. Due to its toxicity, side effects such as anorexia, nausea, vomiting, diarrhea, confusion, visual disturbance and cardiac arrhythmias may be exhibited, but the anti-digoxin antibody fragment used for the treatment of digoxin toxicity is also effective in the toxicity of digitoxin. The digitalis is a perennial plant native to Europe and is cultivated for ornamental and medicinal purposes. It has been known since ancient times that the component of the flower is effective for heart disease, so it is also called heart grass or heart centella.

The compound having the structure of Formula 2 is a steroid produced from the hydrolysis of the cardiac glycoside gitoxin that occurs in Digitalis lanata, 16β-substituted digitoxygenin. , specifically 16β-hydroxydigitoxygenin. It belongs to the class of cardioactive steroids, cardenolides, which share a common structural property with structurally unsaturated gamma-lactones. It is an aglycone of the glycoside Lanatoside B (Lanatoside B).

The compound having the structure of Formula 3 is gitoxygenin diacetate as It is a derivative in which acetate is substituted at the hydroxyl site at positions 3 and 16 of gitoxygenin. It is known that the cytotoxicity of cardiac glycosides in colon cancer.

The compounds having the structures of Chemical Formulas 4 to 9 are cardiac glycosides, which are major constituents of Bufonis Venenum, also known as toad venum, a traditional herbal medicine derived from glandular body secretions of toad (Bufo bufo gargarizans Cantor) skin. as Bufalin, Bufotalin, Resibufogenin, Cinobufagin, Cinobufotalin, and Telocinobufagin, respectively. Seomsu is effective for detoxification and pain relief, so it has been used in traditional oriental medicine for swelling of the throat, sore throat, and cancer. Lead and bufadienolide compounds are also known to have antitumor, analgesic, anti-inflammatory and immunoactive effects.

The compound having the structure of Chemical Formula 10 is a cardiac glycoside from Woolly Foxglove aka Digitalis lanata , called gitoxin, which is being studied for application to potential heart disease similar to digoxin. The gitoxin acts as an inhibitor of sodium and potassium ion channels to interfere with APTase activity, and is used for cancer treatment as a growth inhibitory agent. In addition, it is used as a starting material for the synthesis of a gitoxin derivative having activity as a cardiac glycoside.

The compound having the structure of Formula 11 is a derivative compound prepared by oxidizing the hydroxy group at the 7th position in the compound of Formula 6 above.

A composition comprising a compound represented by Formula I according to the present invention, for example, a compound represented by Formulas 1 to 11, may include derivatives within a range expected by those skilled in the art from these compounds, and has the same effect in the present invention. included without limitation. These compounds may be obtained from an extract containing them, but are not limited thereto, and may be synthesized and used by a known method.

The term of the present invention, "MERS-coronavirus (MERS-CoV)" is an ssRNA virus belonging to the genus beta-coronavirus newly discovered on September 24, 2012, 5'-replicate-structural protein (spikeenvelope-membrane- nucleocapsid)-poly(A)-3'[5'-ORF1a/bSEMN-poly(A)]. The MERS-Coronavirus genome is phylogenetically classified into clade A and clade B. Early MERS cases were clade A (EMC/2012 and Jordan-N3/2012), and newly reported cases are genetically distinct. Individual clade B (Emerging Infectious Diseases, Vol. 20, No. 6, June 2014).

As used herein, the term "SARS-CoV" is an ssRNA virus belonging to the genus Beta-coronavirus with an envelope. The entire genome consists of 29,727 nucleotides, and it is the virus with the largest genome among RNA viruses known to date. The SARS-coronavirus genome has 11 open reading frames (ORFs) and is known to encode 23 types of proteins. It has been found that the major structural proteins of SARS-coronavirus are nucleocapsid (N), spike (S), membrane (M), and small envelope (E) proteins.

As a result of sequencing of these proteins, it was found that SARS-coronavirus has a very low sequence homology of about 40-50% compared to other coronaviruses. According to the phylogenetic classification according to the antigenicity of the coronavirus, the SARS coronavirus was found to be highly related to group II among groups I, II and III.

As used herein, the term "SARS-CoV-2" is a virus strain that causes coronavirus disease 2019 (COVID-19), a respiratory disease. As known by the National Institutes of Health (NIH), it is the successor of SARS-CoV. SARS-CoV-2 is a positive-sense single-stranded RNA virus. Contagious in humans, the World Health Organization (WHO) has declared the ongoing pandemic of COVID-19 as a Public Health Emergency of International Concern (PHEIC). . SARS-CoV-2 is a taxonomic strain of SARS-CoV, has zoonotic origins, and is considered to have close genetic similarity to bat coronavirus. The SARS-CoV-2 is mainly transmitted through close contact between people and/or through droplets generated during coughing or sneezing, and mainly binds to receptor angiotensin converting enzyme 2 (ACE2) to bind to human cells. enter

In the present invention, a pharmaceutical composition for the prevention or treatment of a coronavirus infection disease comprising the compound represented by Formula I or a pharmaceutically acceptable salt thereof as an active ingredient is MERS-Coronavirus, SARS-Coronavirus, and SARS. - Characterized in inhibiting coronavirus-2. Specifically, in one embodiment of the present invention, the steroid-based derivative compound containing 11 kinds of lactone rings confirmed the ability to inhibit infection of MERS-Coronavirus, SARS-Coronavirus, and SARS-Coronavirus-2, compared to the positive control lopinavir ( FIGS. 2 to 4 ).

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a form that can be used pharmaceutically among salts, which are substances in which cations and anions are bonded by electrostatic attraction. salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine salts with, etc.; salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; Salts with organic acids may be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; Salts with basic amino acids may be salts with arginine, lysine, ornithine and the like; The salt with an acidic amino acid may be a salt with aspartic acid, glutamic acid, or the like.

As used herein, the term "infectious disease" refers to a disease caused by infection.

As used herein, the term “infection” refers to a state in which pathogenic microorganisms invade the body of a host organism and develop and multiply.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of an infectious disease caused by a coronavirus by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any action in which the symptoms of a suspected and onset individual of a coronavirus infection are improved or beneficially changed by administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, which may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid), poly-L-lactic acid, mineral oil, and the like.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The content of the compound represented by Formula I or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition of the present invention is not particularly limited.

Another embodiment of the present invention for achieving the above object provides a health functional food composition for the prevention or improvement of coronavirus infection disease comprising the compound represented by Formula I or a pharmaceutically acceptable salt thereof.

In this case, the descriptions of the "compound represented by Formula I", "coronavirus", "infectious disease" and "prevention" are the same as described above.

In addition, the "food pharmaceutically acceptable salt" herein is as defined in the above "pharmaceutically acceptable salt".

Compounds represented by Formula I according to the present invention, for example, Compounds 1 to 11 represented by Formulas 1 to 11, exhibit an excellent coronavirus inhibitory effect, and thus are used in food compositions for the purpose of preventing or improving infectious diseases caused by coronavirus. It may be included, and since it is possible to consume the food composition on a daily basis, a high effect can be expected for the prevention or improvement of infection caused by coronavirus.

As used herein, the term “improvement” refers to any action that at least reduces a parameter related to a condition to be treated by administration of the dietary supplement composition according to the present invention, for example, the degree of symptoms.

As used herein, the term "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the structure of the human body. and regulating nutrients for function or obtaining useful effects for health applications such as physiological action. On the other hand, health food refers to food that has an active health maintenance or promotion effect compared to general food, and health supplement refers to food for the purpose of health supplementation. can be mixed.

The compound represented by the formula (I) of the present invention or a pharmaceutically acceptable salt thereof may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, fungicides, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, emulsifiers, thickeners, filming agents, gum base agents, foam inhibitors, solvents, and improving agents. may include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and is excellent in portability, so the present invention Foods of can be consumed as a supplement to enhance the effect of prevention or improvement of infectious diseases caused by coronavirus.

The compound represented by Formula I of the present invention or a pharmaceutically acceptable salt thereof may be included in various weight % in the food composition if it can exhibit the effect of preventing or improving infectious diseases caused by coronavirus. Specifically, it may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. When ingested for a long period of time for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another embodiment of the present invention for achieving the above object provides a feed composition for preventing or improving a coronavirus infection disease comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof.

In this case, the descriptions of the "compound represented by Formula I", "food pharmaceutically acceptable salt", "coronavirus", "infectious disease", "prevention" and "improvement" are as described above.

Since the compound represented by Formula I according to the present invention or a pharmaceutically acceptable salt thereof exhibits an excellent coronavirus inhibitory effect, it may be included in a feed composition for the purpose of preventing or improving infectious diseases caused by coronavirus, the feed Since the composition can be routinely ingested by animals, a high effect can be expected for the prevention or improvement of infectious diseases caused by coronavirus.

As used herein, the term "feed" means any natural or artificial diet, meal, etc., or a component of said meal, intended for or suitable for eating, ingestion, and digestion by an animal.

The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton or food. These may be used alone or in mixture of two or more.

The compound represented by Formula I of the present invention, or a pharmaceutically or pharmaceutically acceptable salt thereof, has an effect of inhibiting infection of coronaviruses such as MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2. Therefore, it can be used to develop an antiviral agent for preventing or treating the viral disease.

1 is a view showing the chemical structural formulas of the compounds of the present invention, specifically, compounds represented by Formulas 1 to 11.
2 is a Middle East respiratory syndrome-related coronavirus (MERS-CoV) and a severe acute respiratory syndrome-related coronavirus (SARS-CoV) of the compounds represented by Formulas 1 to 6, respectively. ) is a diagram showing the dose-dependent antiviral activity against.
3 is a Middle East respiratory syndrome-related coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus of Lopinavir as a compound and a control group represented by Chemical Formulas 7 to 11, respectively; A diagram showing dose-dependent antiviral activity against respiratory syndrome-related coronavirus; SARS-CoV).
4 is a diagram showing the dose-dependent antiviral activity of the compounds represented by Formulas 1 to 10, respectively, against severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2).

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Preparation Example 1: Preparation of 11 compounds

Compounds 1 to 11 of the present invention, represented by Formulas 1 to 11, were dissolved in DMSO and stored at -80° C. as a stock solution at a concentration of 20 mM, and diluted to a desired concentration using an FBS-free medium during the experiment. .

Example 1: Confirmation of inhibition of MERS-coronavirus infection

Example 1-1: Cell line and virus preparation

Vero cells used in the present invention were purchased from the American Type Culture Collection (ATCC, CCL-81; Manassas, VA), and were used, L-glutamine and 1 × antibiotic-antimycotic (Antibiotic-Antimycotic, Gibco/Thermo Fisher) Scientific, Waltham, MA) containing Dulbecco's modified Eagle's medium (DMEM; Welgene, Gyeongsan, Korea) and incubated for 24 hours at 37°C under 5% carbon dioxide. After 24 hours, the compounds 1 to 11 of the present invention dissolved in DMSO were added by concentration, and then infected with MERS-coronavirus. As the MERS-coronavirus, a Korean isolate (MERS-CoV/KOR/KNIH/002_05_2015, Genbank accession no. KT029139.1 [Kim et al., 2015 doi:10.1128/genomeA.00787-15]) was used for Korean disease management Headquarters, provided by the National Institutes of Health, were proliferated in vero cells according to the method presented in the prior literature (Kim et al., 2016 doi:10.1093/cid/ciw239). All experiments using MERS-Coronavirus were performed at the Pasteur Institute in Korea in compliance with the National Institutes of Health's enhanced Biosafety Level 3 (BL-3) containment procedures approved by the Korea Centers for Disease Control and Prevention.

Example 1-2: Preparation of reagents

Lopinavir (LPV, GP6351) was purchased from Glentham Life Science (UK). The anti-MERS-coronavirus spike antibody used as the primary antibody was obtained from Sino Biological Inc. (Beijing, China), Alexa Fluor 488 goat anti-rabbit IgG, a secondary antibody, and Hoechst 33342, a nuclear chromosome, were purchased from MolecularProbes/ Thermo Fisher Scientific (Waltham, MA). In addition, 32% paraformaldehyde (PFA) aqueous solution and normal goat serum were purchased from Electron Microscopy Sciences (Hatfield, PA) and Vector Laboratories, Inc. (Burlingame, CA), respectively.

Example 1-3: MERS-Coronavirus infection inhibition confirmation using immunofluorescence assay

MERS-coronavirus infection inhibitory ability of the compounds 1 to 11 of the present invention was confirmed using an immunofluorescence assay.

More specifically, the black 384-well microplate ^{was inoculated with 1.2×10 4} Vero cells per well, treated with L-glutamine and 1× antibiotic-antifungal agent, and cultured at 37° C., 5% carbon dioxide condition for 24 hours. After 24 hours, the compounds 1 to 11 of the present invention dissolved in DMSO were added by concentration and then infected with MERS-coronavirus. 24 hours after infection, the infection was fixed with PFA, stained using Alexa Fluor 488 and Hoechst 33342, and the stained cells were imaged with a fluorescence imaging system at 20× magnification to confirm the inhibition of infection. In addition, lopinavir was used as a positive control.

As a result, as can be seen in FIGS. 2 and 3 , the IC ₅₀ representing the 50% inhibitory concentration of the compounds 1 to 11 of the present invention was 14.7 to 7367 nM, and the CC ₅₀ representing 50% cytotoxicity was >25 μM. or >50 μM. _{In addition, the IC 50} representing the 50% inhibitory concentration of lopinavir as a control group was 1629 nM, and the CC ₅₀ representing 50% cytotoxicity was measured to be >50 μM.

Through this, it was confirmed that the compounds 1 to 11 of the present invention exhibit the same or better MERS-coronavirus infection inhibitory effect compared to the positive control.

Example 2: Confirmation of SARS-coronavirus infection inhibition

Example 2-1: Cell line and virus preparation

VeroE6 cells used in the present invention were purchased from American Type Culture Collection (ATCC, CCL-81, CRL-1586; Manassas, VA) and used, 10% heat-inactivated fetal bovine serum and 1× antibiotic- It was put in DMEM containing antifungal agent and cultured at 37°C under 5% carbon dioxide. SARS-coronavirus HK39849 was provided by Dr. Malik Parris of the University of Hong Kong and propagated in Vero E6 cells according to the method presented in Peiris JS et al., 2003 doi: 10.1016/s0140-6736(03)13077-2 (PMID: 12711465). did it All experiments using SARS-coronavirus were performed at the Pasteur Institute in Korea, which complied with the National Institutes of Health's enhanced Biosafety Level 3 (BL-3) containment procedures approved by the Korea Centers for Disease Control and Prevention.

Example 2-2: Reagent Preparation

Lopinavir (LPV; GP6351) was purchased from Glentham Life Science (UK). The anti-SARS-coronavirus spike antibody used as the primary antibody was purchased from Sino Biological Inc. (Beijing, China), and the secondary antibody Alexa Fluor 488 goat anti-mouse IgG and the nuclear chromosome Hoechst 33342 were MolecularProbes/ Thermo Fisher Scientific (Waltham, MA). In addition, 32% PFA aqueous solution and normal goat serum were purchased from Electron Microscopy Sciences (Hatfield, PA) and Vector Laboratories, Inc. (Burlingame, CA), respectively.

Example 2-3: SARS-coronavirus infection inhibition confirmation using immunofluorescence assay

SARS-coronavirus inhibitory ability of the compounds 1 to 11 of the present invention was confirmed using an immunofluorescence assay.

More specifically, a black 384-well microplate ^{was inoculated with 1.2×10 4} Vero E6 cells per well, treated with fetal bovine serum and 1× antibiotic-antifungal agent, and cultured at 37° C., 5% carbon dioxide condition for 24 hours. After 24 hours, the compounds 1 to 11 of the present invention dissolved in 2% DMEM were added by concentration, followed by SARS-coronavirus infection. Twenty-four hours after infection, the infection was fixed with PFA and stained using Alexa Fluor 488 and Hoechst 33342. The stained cells were imaged with a fluorescence imaging system with a magnification of 20× to confirm the infection inhibition rate. In addition, lopinavir was used as a positive control.

As a result, as can be seen in FIGS. 2 and 3 , the IC ₅₀ representing the 50% inhibitory concentration of the compounds 1 to 11 of the present invention was 16 to 5281 nM, and the CC ₅₀ representing 50% cytotoxicity was >25 μM. or >50 μM. _{In addition, the IC 50} representing the 50% inhibitory concentration of lopinavir as a control group was 1670 nM, and the CC ₅₀ representing 50% cytotoxicity was measured to be >50 μM.

Through this, it was confirmed that the compounds 1 to 11 of the present invention exhibit the same or superior SARS-coronavirus inhibitory effect compared to the positive control.

Example 3: Confirmation of SARS-coronavirus-2 infection inhibition

Example 3-1: Cell line and virus preparation

Vero cells used in the present invention were purchased from the American Type Culture Collection (ATCC, CCL-81, Manassas, VA), and 10% heat-inactivated fetal bovine serum and 1× antibiotic-antifungal (Gibco) were used. It was placed in the included DMEM and incubated at 37°C under 5% carbon dioxide. SARS-coronavirus-2 (βCoV/KOR/KCDC03/2020) was provided from the Korea Centers for Disease Control and Prevention (KCDC) and propagated in Vero cells. Viral titers were determined by plaque assay in Vero cells. All experiments using SARS-coronavirus-2 were performed at the Pasteur Institute in Korea, which complied with the National Institutes of Health's enhanced Biosafety Level 3 (BL-3) containment procedures approved by the Korea Centers for Disease Control and Prevention.

Example 3-2: Reagent Preparation

Lopinavir (LPV; S1380) was purchased from SelleckChem (Houston, TX) and dissolved in DMSO for selection. Anti-SARS-coronavirus-2 N protein antibody was purchased from Sino Biological Inc. (Beijing, China). Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody and Hoechst 33342 were purchased from MolecularProbes. In addition, 32% PFA aqueous solution and normal goat serum were purchased from Electron Microscopy Sciences (Hatfield, PA) and Vector Laboratories, Inc. (Burlingame, CA), respectively.

Example 3-3: Confirmation of SARS-coronavirus infection inhibition using immunofluorescence assay

In order to confirm the SARS-coronavirus-2 inhibitory ability of the compounds 1 to 11 of the present invention, it was confirmed using an immunofluorescence assay.

10-point dose-response curves (DRCs) were generated for each sample. ^{Specifically, inoculate 1.2×10 4} Vero cells per well in a black, 384-well, μClear plate (Greiner Bio-One) and contain 2% fetal bovine serum and 1× antibiotic-antifungal for 24 h prior to the experiment. incubated in DMEM. Ten point DRCs were generated in the range of compound concentrations from 0.05 to 50 μM. For viral infection, plates were transferred to a BSL-3 receiving facility and SARS-coronavirus-2 was added at a multiplicity of infection (MOI) of 0.0125. 24 hours after infection (24 hpi), cells were fixed with 4% PFA and analyzed by immunofluorescence. In order to quantify the cell number and infection ratio, the acquired images were analyzed using in-house software, and anti-viral activity was positive (mock) and negative (0.5) in each assay plate. % DMSO) controls were normalized to the following equation using XLfit 4 software or Prism 7:

.

.

The IC ₅₀ values were calculated from normalized activity data set-optimized curves. All IC ₅₀ and CC ₅₀ values were measured in duplicate, and the quality of each assay was adjusted by Z'-factor and coefficient of variation in percent (%CV).

As a result, as can be seen in FIG. 4, compounds 1 to 11 of the present invention were _{confirmed to have significantly lower values in IC 50} representing 50% inhibitory concentration compared to the positive control (0.0191 to 6.861 μM vs. 12.998 μM, Fig. 4), the CC ₅₀ showing 50% cytotoxicity was high enough with >10 μM (calculated from FIG. 4 ).

Through this, it was confirmed that the compounds 1 to 11 of the present invention exhibit a significantly superior SARS-coronavirus-2 inhibitory effect compared to the positive control.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (6)

A pharmaceutical composition for antiviral against coronavirus comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula I]

In the above formula (I),
R ₁ is hydrogen, R ₂ is -OH, -O(C=O)CH ₃ ,

or

this,
R ₁ and R ₂ together form =O,
R ₃ is hydrogen or —OH,
R ₄ is —OH, R ₅ is hydrogen, or R ₄ and R ₅ are joined together to form an epoxide ring,
R ₆ is hydrogen, -OH or -O(C=O)CH ₃ ,
R ₇ is

or

Lim.
The pharmaceutical composition of claim 1, wherein the coronavirus is at least one selected from the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV-2.
The pharmaceutical composition according to claim 1, wherein the compound of formula I is a compound represented by the following formulas 1 to 11:
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

.
A pharmaceutical composition for preventing or treating a coronavirus infection disease comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula I]

In the above formula (I),
R ₁ is hydrogen, R ₂ is -OH, -O(C=O)CH ₃ ,

or

this,
R ₁ and R ₂ together form =O,
R ₃ is hydrogen or —OH,
R ₄ is —OH, R ₅ is hydrogen, or R ₄ and R ₅ are joined together to form an epoxide ring,
R ₆ is hydrogen, -OH or -O(C=O)CH ₃ ,
R ₇ is

or

Lim.
A health functional food composition for the prevention or improvement of a coronavirus infection disease comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula I]

In the above formula (I),
R ₁ is hydrogen, R ₂ is -OH, -O(C=O)CH ₃ ,

or

this,
R ₁ and R ₂ together form =O,
R ₃ is hydrogen or —OH,
R ₄ is —OH, R ₅ is hydrogen, or R ₄ and R ₅ are joined together to form an epoxide ring,
R ₆ is hydrogen, -OH or -O(C=O)CH ₃ ,
R ₇ is

or

Lim.
A feed composition for preventing or improving a coronavirus infection disease comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula I]

In the above formula (I),
R ₁ is hydrogen, R ₂ is -OH, -O(C=O)CH ₃ ,

or

this,
R ₁ and R ₂ together form =O,
R ₃ is hydrogen or —OH,
R ₄ is —OH, R ₅ is hydrogen, or R ₄ and R ₅ are joined together to form an epoxide ring,
R ₆ is hydrogen, -OH or -O(C=O)CH ₃ ,
R ₇ is

or

Lim.

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