KR20230040961A - Composition for preventing or treating coronavirus infection comprising Tetrandrine or its analogues - Google Patents

Abstract

The present invention relates to the antiviral use of fangchinoline and cepharanthine, or tetrandrine or an analog thereof against coronaviruses and, more specifically, to a pharmaceutical composition for antiviral use against coronavirus, which contains tetrandrine or an analog thereof or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutical composition for preventing or treating coronavirus infectious diseases, and a functional health food composition and a feed composition for preventing or treating coronavirus infectious diseases. The fangchinoline and cepharanthine, or tetrandrine or an analog thereof according to the present invention have the effect of inhibiting the infection of coronaviruses such as MERS-coronavirus, SARS-coronavirus, SARS-coronavirus-2, and human-coronavirus to be able to be used in developing an antiviral agent for preventing or treating the virus diseases.

Description

Composition for preventing or treating coronavirus infection comprising tetrandrine or its analogues

The present invention relates to the antiviral use of tetrandrin or analogues thereof, pancinolin and cepharantin, against coronavirus. Specifically, the present invention relates to tetrandrin or analogues thereof or pharmaceutically acceptable salts thereof An antiviral pharmaceutical composition for coronavirus comprising as an active ingredient; A pharmaceutical composition for preventing or treating coronavirus infectious diseases; 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, then in animals such as dogs, pigs and birds, and then in humans in 1965. This name was given because it resembles the corona phenomenon, a phenomenon in which the sun's photosphere is covered by the moon during a total solar eclipse.

Coronaviruses are known to cause mainly pneumonia and enteritis in humans and animals, and occasionally to cause nervous system infections and hepatitis. Coronavirus is a positive sense RNA virus with a size of about 100-120 nm, belonging to the Coronaviridae family and having a spherical outer membrane. Coronavirus consists of a total of five structural proteins: 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 to be the most mutated protein.

Until now, coronaviruses have been recognized as pathogens that rarely infect humans and mainly infect animals such as dogs, pigs, and cattle. Even when it infects humans, it is one of many viruses that cause respiratory symptoms, causing simple colds or causing intestinal diseases such as diarrhea, which are not very dangerous to children. However, the causative agent of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19), which have caused hundreds of deaths and thousands of patients worldwide, 19), as the causative agent was known to be a novel (mutant) coronavirus, it gradually began to attract attention.

SARS is an infectious disease that occurred in Guangdong Province in southern China in November 2002 and spread to the world via Hong Kong. am. The propagation route has not yet been fully elucidated, but it is speculated that it is propagated by fine particles of solid or liquid floating in the atmosphere. SARS was prevalent until July 2003, and it is known that a total of 8,096 infected people occurred in 32 countries for about 7 months, and 774 of them died.

MERS was first discovered in Saudi Arabia in 2012, and presents with severe respiratory symptoms such as high fever, cough, and difficulty breathing. Although the exact source and route of infection have not been identified, it has been reported that the possibility of infection through contact with camels in the Middle East is high and transmission through close contact between people is possible. Patients mainly occurred in the Middle East, but since May 2015, more than 100 infected people have occurred 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 has spread worldwide, resulting in a progressive pandemic. As of 7 May 2020, more than 3.75 million cases have been reported in 187 countries, 263 thousand deaths have occurred, and 1.24 million have recovered. 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 cytokine storm. The time from onset of symptoms to onset is generally about 5 days but can be between 2 and 14 days. The virus spreads from person to person mainly during close contact, sometimes through droplets from coughing, sneezing and talking. It is most contagious during the first 3 days after symptom onset and can be transmitted before symptoms appear or even at the end of the disease. The standard method for diagnosing it uses real-time reverse transcription polymerase chain reaction (rRT-PCR) from a nasopharyngeal swab.

As another human pathogen, human coronavirus is known to cause the common cold. Of these, human coronavirus-OC43 is the most common human coronavirus and is known to cause upper respiratory tract infection, and according to a recent study, many children are infected with human coronavirus as early as 2 years of age. It seroconverts, and human coronavirus-OC43 was found to be the most common. Humancoronavirus-OC43 infection has been shown to be involved in the development of severe respiratory diseases in children or the elderly, and has been confirmed to cause severe respiratory diseases in children (Pediatr. Infect. Dis. J., 32(4):325 -329).

As mentioned earlier, humancoronavirus-OC43, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 are all known variant coronaviruses belonging to the genus Beta coronavirus. Thus, humancoronavirus-OC43, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 share some similar characteristics in clinical symptoms, pathogenesis, 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, while MERS-coronavirus is It belongs to lineage 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). Furthermore, humancoronavirus-OC43 belongs to strain D formed by recombination between line B and line C.

Humancoronavirus-OC43, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 are all believed to have originated from bats, which are the primary carrier hosts of various coronaviruses (Antiviral Res., 101:45 -56), but the exact route of infection has not yet been fully elucidated. On the other hand, cross-species transmission of the virus to palm civet and dromedary camel increased the likelihood of zoonotic infection to humans (Nat. Rev. Microbiol., 14:523-534), hospitals. Infection is considered a major cause of human-to-human MERS-coronavirus and SARS-coronavirus infection (BMC Med., 13:1-12).

On the other hand, tetrandrin, panchinolin, and cepharantin are all compounds having similar chemical structures extracted, isolated, and identified from plants used for food or herbal medicine. However, the preventive, therapeutic or ameliorative effect of coronaviruses, particularly human coronavirus-OC43, MERS-coronavirus, SARS-coronavirus, and/or SARS-coronavirus-2 infectious diseases of tetrandrin and its analogs is Nothing is known.

Since the antiviruses developed so far exhibit severe side effects, great care is required in their application. These treatments are not effective and side effects are also appearing. Therefore, there is an increasing need for the development of new novel coronavirus agents with excellent infection suppression effect and low toxicity for preventing and treating the outbreak of coronavirus.

Under this background, the present inventors continued their efforts to develop novel coronavirus therapeutics, and as a result, tetrandrin and its analogs, pancinolin and cepharantin, isolated from spp. The present invention was completed by confirming that there is an excellent effect of inhibiting the proliferation of coronavirus-OC43, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2.

One object of the present invention is to provide an antiviral pharmaceutical composition for coronavirus containing tetrandrin, an analogue thereof or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating coronavirus infectious diseases comprising tetrandrin, an analogue thereof or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a health functional food composition for preventing or improving coronavirus infectious diseases comprising tetrandrin, an analogue thereof, or a food-acceptable salt thereof.

Another object of the present invention is to provide a feed composition for preventing or improving coronavirus infectious diseases comprising tetrandrin, an analogue thereof, or a food-acceptable salt thereof.

One embodiment of the present invention for achieving the above object provides an antiviral pharmaceutical composition for coronavirus comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

In Formula 1,

는

또는

이고,

Is

or

ego,

R ₁ is methyl, R ₂ is hydrogen or methyl;

R ₁ and R ₂ together with the oxygen atom to which they are attached form a 5-membered heterocycle;

R ₃ to R ₆ are each independently hydrogen or methoxy;

R ₃ and R ₅ or R ₄ and R ₆ are connected to each other to form an -O- bridge.

Another embodiment of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating coronavirus infectious diseases 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 inhibitory effect on coronavirus, it is useful for preventing or treating infectious diseases caused by coronavirus through a method comprising administering it to a subject suspected of having a coronavirus infection. can be utilized

The coronaviruses include human coronavirus-OC43 (HCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome). -related coronavirus; SARS-CoV), or severe acute respiratory syndrome-related coronavirus 2; SARS-CoV-2.

For example, the compound represented by Chemical Formula 1 may be tetrandrin, panchinolin or cepharantin, but is not limited thereto.

The term of the present invention, "tetrandrine (tetrandrine)" is a compound having a structure of formula (2), it can be isolated and identified from the stephania tetrandra (粉防己), specifically, from the extract of the root of the stephania tetrandra. The spinneret is a herbaceous perennial vine of the Menispermaceae family, native to China and Taiwan. It grows on a short, woody caudex and grows to a height of about 3 m. Its roots are also used as herbal medicine. For example, it has a pungent and bitter taste, and is used in Chinese medicine to relieve pain and promote diuresis by removing wind and moisture.

[Formula 2]

The term of the present invention, "fangchinoline" is a compound having a structure represented by the following Chemical Formula 3, and can be isolated and identified from Cocculus orbiculatus , specifically, an extract of the stem of a dang vine. The dangling vine is a plant of the genus Cocculus vine, and is a deciduous broad-leaved tree that is also called dangdangi, dangling vine, yongrin, and togo. In Korea, it is mainly found in sunny places at the foot of mountains, fields, and crevices between stones, and is distributed in the southern part of Hwanghae-do. The fruit is edible and has a sour and bitter taste.

[Formula 3]

The term of the present invention, "cepharanthine" is a compound having a structure represented by the following Chemical Formula 4, and can be isolated and identified from Stephania japonica, specifically, an extract from the root of Stephania japonica. The hambak is a plant of the genus Stephania bird's sand vine, and its origin is Korea. grows in It is a vine without thorns, and is also called hambagi, hambagi vine, and hambaeki. The roots and leaves are called Cheongeumdeung (千金藤) and are used medicinally.

[Formula 4]

The composition containing the compound represented by Formula 1 according to the present invention, for example, tetrandrin, panchinolin or cepharantin, may include derivatives within a range expected by some skilled in the art, and the same effect in the present invention are included without limitation. These compounds can be obtained from extracts containing them, but are not limited thereto, and can also be synthesized and used by known methods.

The term of the present invention, "HCoV-OC43" refers to the genus Betacoronavirus, subfamily Coronavirinae, family Coronavirinae, order Nidovirales ) Betacoronavirus is a subspecies of one enveloped positive-stranded RNA virus. The human coronavirus-OC43 genotype is mainly D-type, which occurs by recombination. Human coronavirus-OC43 is known to cause the common cold. In addition, it can cause severe lower respiratory tract infections, including pneumonia, in infants, the elderly, and immunocompromised individuals such as those undergoing chemical or HIV-AIDS carriers.

The term of the present invention, "MERS-CoV" is an ssRNA virus belonging to the genus beta coronavirus newly discovered on September 24, 2012, and is a 5'-replicase-structural protein (spikeenvelope-membrane- nucleocapsid) -poly(A)-3'[5'-ORF1a/b-S-E-M-N-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 were 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 Enveloped Beta Coronavirus. Its entire genome consists of 29,727 nucleotides, and it is the largest genome among RNA viruses known to date. The genome of SARS-coronavirus is known to have 11 open reading frames (ORFs) and encode 23 proteins. It was found that the main structural proteins of SARS-coronavirus are nucleocapsid (N), spike (S), membrane (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 to 50% compared to other coronaviruses. According to the phylogenetic classification according to the antigenicity of the coronavirus, SARS coronavirus was found to have a high affinity with group Ⅱ among groups Ⅰ, Ⅱ and Ⅲ.

The term of the present invention, "SARS-CoV-2" is a virus strain that causes respiratory disease, coronavirus disease 2019 (COVID-19). 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. It is contagious in humans, and the World Health Organization (WHO) has declared an ongoing pandemic of COVID-19 a Public Health Emergency of International Concern (PHEIC). . SARS-CoV-2 is taxonomically considered to be a strain of SARS-CoV, has zoonotic origins, and has close genetic similarities to bat coronaviruses. The SARS-CoV-2 is mainly spread 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 enter human cells. enter

In the present invention, a pharmaceutical composition for preventing or treating coronavirus infectious diseases comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient is human coronavirus-OC43, MERS-coronavirus, SARS - It is characterized by inhibiting coronavirus, and SARS-coronavirus-2. Specifically, in one embodiment of the present invention, tetrandrin, panchinolin or cepharantin inhibits the infection of human coronavirus-OC43, MERS-coronavirus, SARS-coronavirus, and SARS-coronavirus-2 confirmed and compared with the positive control group lopinavir (Figs. 1 to 6).

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bonded by electrostatic attraction, and is usually combined with metal salts and organic bases. 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.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine It can be a salt with; 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 include 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; A salt with a basic amino acid may be a salt with arginine, lysine, ornithine, and the like; A salt with an acidic amino acid may be a salt with aspartic acid or glutamic acid.

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

The term "infection" refers to a state in which a pathogenic microorganism invades the body of a host organism and grows and proliferates.

The term of the present invention, "prevention" means any action that suppresses or delays the onset of infectious diseases caused by coronavirus by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities that improve or beneficially change the symptoms of suspected or affected individuals of coronavirus infectious diseases by the administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, and the carrier 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, gum acacia, alginate, gelatin, and 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 compositions may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively, The form of the carrier may include various irregular carriers, microspheres, nanofibers, and the like.

When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

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

Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The content of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof included 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 preventing or improving coronavirus infectious diseases comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

At this time, the description of the "compound represented by Formula 1", "coronavirus", "infectious disease" and "prevention" is as described above.

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

Since the compound represented by Formula 1 according to the present invention, for example, tetrandrin, panchinolin or cepharantin, exhibits an excellent inhibitory effect on coronavirus, it can be included in a food composition for the purpose of preventing or improving infectious diseases caused by coronavirus. It can be, and since the food composition can be consumed on a daily basis, a high effect on preventing or improving infection by coronavirus can be expected.

As used herein, the term "improvement" refers to all actions that at least reduce the parameters related to the condition to be treated by administering the health functional food composition according to the present invention, for example, the degree of symptoms.

The term of the present invention, "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Act No. 6727 on Health Functional Food, and 'functional' refers to the structure of the human body. and to obtain useful effects for health uses such as regulating nutrients with respect to functions or physiological actions, and the like. 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 food refers to food for the purpose of supplementing health. In some cases, the terms of health functional food, health food, and health supplement food can be mixed.

The compound represented by Formula 1 or a food chemically acceptable salt thereof of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. 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, bactericides, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, reinforcing agents, emulsifiers, thickeners, coating agents, gum base agents, foam inhibitors, solvents, and improvers. can 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 the formulation is recognized as food. The composition for food of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time using food as a raw material, and has excellent portability, so the present invention Of the foods can be consumed as supplements to enhance the effect of preventing or improving infectious diseases caused by coronavirus.

The compound represented by Formula 1 of the present invention or a food-acceptable salt thereof may be included in a food composition in various weight percent if it can exhibit an effect of preventing or improving infectious diseases caused by coronavirus. Specifically, it may be included in 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the food composition, but is not limited thereto. In the case of long-term intake for health and hygiene purposes, the content below the above range may be included, 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 coronavirus infectious diseases comprising the compound represented by Formula 1 or a food-acceptable salt thereof.

At this time, the description of the "compound represented by Formula 1", "food acceptable salt", "coronavirus", "infectious disease", "prevention" and "improvement" is as described above.

Since the compound represented by Formula 1 or a food-acceptable salt thereof according to the present invention 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, and the feed Since the composition can be consumed by animals on a daily basis, a high effect can be expected for the prevention or improvement of infectious diseases caused by coronavirus.

As used herein, the term "feed" refers to any natural or artificial diet, meal, etc., or component of said meal, intended for or suitable for consumption by animals.

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

As used herein, the term "feed" refers to any natural or artificial diet, meal, etc., or component of said meal, intended for or suitable for consumption by animals.

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

Tetrandrin and its analogs, panchinolin and cepharantin, of the present invention inhibit infection of coronaviruses such as MERS-coronavirus, SARS-coronavirus, SARS-coronavirus-2, and human-coronavirus Since it shows an effect, it can be used in the development of antiviral agents for preventing or treating the above viral diseases.

Figure 1 shows (A) chemical structural formulas of the compounds of the present invention, tetrandrin, pancinolin, and cephalanthin, (B) schematic diagrams of human coronavirus-OC43 infection in MRC-5 cells and treatment with the compounds, ( C) It is a graph showing the human coronavirus-OC43 (human coronavirus-OC43; HCoV-OC43) inhibitory efficacy according to the concentration of these compounds and (D) the inhibitory concentration (IC ₅₀ ) curve calculated therefrom.
2 is a graph showing dose-dependent and time-dependent antiviral activities of tetrandrin, panchinolin, and cepharantin against human coronavirus-OC43. MRC-5 cells were infected with human coronavirus-OC43, but not treated with each compound at the indicated concentrations, or MRC-5 cells were pre-treated with human coronavirus-OC43 for 24 hours, co-treated, or after 24 hours- (A) shows the experimental design for the treatment time of the compound on MRC-5 cells, and (B) shows the cell viability by MTS-based assay at 4 days after infection.
Figure 3 shows a time course assay of tetrandrin, fanchinolin and cepharantin. MRC-5 cells were incubated with humancoronavirus-OC43 at 5 μM of each compound 24 hours (pre 24), 12 hours (pre 12), 6 hours (pre 6), and 3 hours (pre 3) before infection and Treatment was administered concurrently (during infection, co), and 12 hours (post 12-96), 24 hours (post 24-96), and 48 hours (post 48-96) post-infection. (A) is a schematic diagram of the overall time-of-additional assay (blue line indicates the administration period), and (B) is the cell viability by MTS-based assay at 4 days after infection. indicates
Figure 4 shows Middle East respiratory syndrome-related coronaviruses according to the concentrations of (A) tetrandrin, (B) pancinolin, (C) cepharantin and (D) Lopinavir as a control. coronavirus; MERS-CoV) is a graph showing the inhibitory efficacy.
Figure 5: Severe acute respiratory syndrome-related coronavirus of (A) tetrandrin, (B) pancinolin, (C) cepharantin and (D) Lopinavir as control; It is a graph showing dose-dependent antiviral activity against SARS-CoV).
Figure 6 shows the severe acute respiratory syndrome-related coronavirus-2 of (A) tetrandrin, (B) pancinolin, (C) cepharantin and (D) Lopinavir as a control. It is a graph showing dose-dependent antiviral activity against coronavirus 2; SARS-CoV-2).

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

Preparation Example 1: Preparation of compounds

Tetrandrin, panchinolin, and cepharantin were dissolved in DMSO and stored at -80°C as stock solutions 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 Human Coronavirus-OC43 Infection

Example 1-1: Cell line and virus preparation

MRC-5 cells used in the present invention were purchased from the American Type Culture Collection (ATCC, CCL-81; Manassas, VA). Cells were attached and cultured in minimal essential medium (MEM, Corning) containing 10% heat-inactivated fetal bovine serum (FBS, WelGENE), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco). did Cells were seeded at a concentration of 1.5×10 ⁴ cells in a 96-well plate (Thermo) or 8×10 ⁴ cells in a 24-well plate (Corning) and cultured at 37° C. under 5% carbon dioxide. Human coronavirus (hCo-V, strain; OC43) was purchased from ATCC. HumanCoronavirus-OC43 (10 ¹⁰ copies/mL) was MEM with 2% heat inactivated FBS, penicillin (100 U/mL)/streptomycin (100 μg/mL) at 90% confluency with MRC- 5 at 33° C. under 5% carbon dioxide. When 50% confluency was reached, the supernatant was withdrawn and filtered through a 0.22 μm syringe filter. The filtered supernatant was aliquoted and frozen at -80°C until use.

Example 1-2: Confirmation of Inhibition of Human Coronavirus-OC43 Infection Using MTS Assay

In order to confirm the ability of tetrandrin, panchinolin, and cepharantin of the present invention to inhibit human coronavirus-OC43 infection, it was confirmed using the MTS assay.

More specifically, 1.5×10 ⁴ MRC-5 cells per well were seeded in a 96-well tissue culture plate. The next day, they were infected with human coronavirus-OC43, and the MRC-5 cells were treated by serially diluting each compound at a concentration of 10 mM by 2 times. After culturing for 4 days, the cell viability of each group was measured by MTS assay.

As a result, as can be seen in Figure 1, the IC ₅₀ representing 50% inhibitory concentration of tetrandrin, panchinolin and cepharantin were 321.7 nM, 956.7 nM, and 790.7 M, respectively, indicating 50% cytotoxicity. CC ₅₀ were all measured to be >10 μM.

Through this, it was confirmed that the tetrandrin, panchinolin, and cepharantin of the present invention exhibit excellent infection inhibitory effects of human coronavirus-OC43.

Example 1-3: Assay according to addition time

A 96-well tissue culture plate was seeded with 1.5×10 ⁴ MRC-5 cells per well. The next day, compounds at concentrations of 0.2, 1, and 5 mM were added to MRC-5 cells (pre-treatment). After 24 hours, the compounds were washed off and the cells were infected with humancoronavirus-OC43 at a concentration of 1×10 ^-3 for 4 days. Then, the compound was added to MRC-5 cells during infection (co-treatment) or added 24 hours after infection (post-treatment). Additionally, compounds at a concentration of 5 μM were added to MRC-5 cells at 24 h, 12 h, 6 h and 3 h before infection (pre-treatment). Compounds were added to MRC-5 cells during infection (co-treatment) or 12, 24, and 48 hours after infection (post-treatment). After infection for 4 days, cell viability for each group was determined by MTS assay, and the results are shown in FIG. 2 .

As shown in FIG. 2, it was confirmed that the case of pre-treatment or co-treatment for human coronavirus-OC43 infection in all three compounds showed a superior infection inhibitory effect compared to the case of post-treatment.

Example 1-4: MTS assay for cell viability

Cytotoxicity was measured using a CellTiter ^96® AQueous One Solution cell proliferation assay (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Specifically, the medium was recovered from the cultured cells and 2% heat inactivated FBS, 90 μL MEM containing penicillin (100 U/mL)/streptomycin (100 μg/mL) and 10 μL of MTS reagent (Promega Corporation, Madison, WI, USA) was added to each well. After culturing for 2 hours at 37° C. under 5% carbon dioxide humid conditions, cell viability was calculated by measuring the optical density at 490 nm using a GloMax ^® Discover microplate reader (Promega Corporation), and the results are shown in FIG. 3 showed up

As shown in Figure 3, when not infected with the virus, even if treated at any time and / or for any time, the survival rate was maintained as high as in the non-infected non-treated group, which indicates that the compounds according to the present invention, tetrandrin, and fungi This indicates that both Nolin and Cepharantin do not exhibit cytotoxicity. On the other hand, in the case of the virus-infected group, although there is some difference, in all three compounds of the present invention, regardless of the administration time and / or total administration time, compared to the untreated group, tetrandrin, panchinolin, and cepharantin treatment The survival rate was improved in the group, indicating that these compounds are all effective in preventing and/or treating human coronavirus-OC43 infection. Specifically, in the case of tetrandrin and fancinolin administration groups, from 24 hours before infection to the co-treatment group, the survival rate was very high, similar to that of the non-infected group, and then the survival rate increased as the time from infection to compound administration increased. showed a slight decreasing trend. This indicates that the reduction in survival rate due to human coronavirus-OC43 infection can be inhibited by the treatment of the compounds, and a very good prevention effect of human coronavirus-OC43 infection can be obtained by treating the compounds prior to infection. On the other hand, in the case of Cepharantin, although the survival rate decreased slightly over time in the case of pre-treatment and post-treatment, it still showed excellent prevention and / or treatment effects of human coronavirus-OC43 infection, and 24 hours of infection It was confirmed that a particularly excellent effect was exhibited when treated before or simultaneously with infection.

Example 2: Confirmation of inhibition of MERS-Coronavirus infection

Example 2-1: 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 L-glutamine and 1 × 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 ℃ under 5% carbon dioxide. After 24 hours, the compounds of the present invention, tetrandrin, panchinolin, and cepharantin dissolved in DMSO were added at different concentrations, and then infected with MERS-coronavirus. The MERS-coronavirus is 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]) It was proliferated in vero cells according to the method presented in the preceding literature (Kim et al., 2016 doi: 10.1093/cid/ciw239), provided by the headquarters and the National Institutes of Health. All experiments using MERS-CoV were performed at the Pasteur Institute in Korea, which followed 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-MERS-coronavirus spike antibody used as the primary antibody was manufactured by Sino Biological Inc. (Beijing, China), Alexa Fluor 488 goat anti-rabbit IgG as a secondary antibody and Hoechst 33342 as a nuclear chromosome were purchased from Molecular Probes/ 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 2-3: Confirmation of inhibition of MERS-coronavirus infection using immunofluorescence assay

In order to confirm the ability of tetrandrin, panchinolin and cepharantin of the present invention to inhibit MERS-coronavirus infection, immunofluorescence assay was used.

More specifically, 1.2×10 ⁴ Vero cells were inoculated per well in a black 384-well microplate, treated with L-glutamine and 1Хantibiotic-antimycotic, and cultured at 37°C and 5% carbon dioxide for 24 hours. After 24 hours, tetrandrin, panchinolin, and cepharantin of the present invention dissolved in DMSO were added according to 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 infection inhibition rate. In addition, lopinavir was used as a positive control group.

As a result, as can be seen in FIG. 4, the IC ₅₀ representing 50% inhibitory concentration of tetrandrin, panchinolin, and cepharantin were 8.72 μM, 7.04 μM, and 5.25 μM, respectively, indicating 50% cytotoxicity. CC ₅₀ was measured to be 24.02 μM, 45.81 μM and 12.59 μM (calculated from FIGS. 4(A), 4(B) and 4(C), respectively). In addition, the IC ₅₀ representing 50% inhibitory concentration of lopinavir as a control group was 16.29 μM, and the CC ₅₀ representing 50% cytotoxicity was determined to be >50 μM (calculated from FIG. 4(D)).

Through this, it was confirmed that the tetrandrin, panchinolin, and cepharantin of the present invention showed a significantly superior MERS-CoV infection inhibitory effect compared to the positive control group.

Example 3: Confirmation of inhibition of SARS-coronavirus infection

Example 3-1: Cell line and virus preparation

VeroE6 cells used in the present invention were purchased and used from American Type Culture Collection (ATCC, CCL-81, CRL-1586; Manassas, VA), 10% heat-inactivated fetal bovine serum and 1 × antibiotic- It was placed in DMEM containing an antifungal agent and cultured under 5% carbon dioxide at 37°C. 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) made it All experiments using SARS-coronavirus were conducted at the Pasteur Institute in Korea, which followed 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; 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), the secondary antibody Alexa Fluor 488 goat anti-mouse IgG and the cell nuclear chromosome Hoechst 33342 were purchased from Molecular Probes/ 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 3-3: Confirmation of inhibition of SARS-coronavirus infection using immunofluorescence assay

In order to confirm the SARS-coronavirus inhibitory ability of tetrandrin, panchinolin, and cepharantin of the present invention, it was confirmed using an immunofluorescence assay.

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

As a result, as can be seen in FIG. 5, the IC ₅₀ representing 50% inhibitory concentration of tetrandrin, panchinolin, and cepharantin were 2.57 μM, 3.15 μM, and 1.86 μM, respectively, indicating 50% cytotoxicity. CC ₅₀ was determined to be 14.14 μM, 20.29 μM and 11.73 μM (calculated from FIGS. 5(A), 5(B) and 5(C), respectively). In addition, the IC ₅₀ representing 50% inhibitory concentration of lopinavir as a control group was 16.7 μM, and the CC ₅₀ representing 50% cytotoxicity was determined to be >50 μM (calculated from FIG. 5(D)).

Through this, it was confirmed that the tetrandrin, panchinolin, and cepharantin of the present invention exhibit a significantly superior SARS-coronavirus inhibitory effect compared to the positive control group.

Example 4: Confirmation of inhibition of SARS-coronavirus-2 infection

Example 4-1: 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 used, and 10% heat-inactivated fetal bovine serum and 1 × antibiotic-antimycotic (Gibco) were used. Incubated in the included DMEM and incubated at 37 ℃ 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-CoV-2 were conducted at the Pasteur Institute in Korea, which followed 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 4-2: 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 Molecular Probes. 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 4-3: Confirmation of inhibition of SARS-coronavirus infection using immunofluorescence assay

In order to confirm the SARS-Coronavirus-2 inhibitory ability of tetrandrin, panchinolin, and cepharantin of the present invention, it was confirmed using an immunofluorescence assay.

Ten-point dose-response curves (DRCs) were prepared for each sample. Specifically, 1.2×10 ⁴ Vero cells were seeded per well in a black, 384-well, μClear plate (Greiner Bio-One) containing 2% fetal bovine serum and 1×antibiotic-antimycotic for 24 hours prior to the experiment. cultured in DMEM. Ten-point DRCs were drawn over a 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. Twenty-four 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 the anti-viral activity was positive (mock) and negative (0.5%) in each assay plate. % DMSO) normalized to the following equation using XLfit 4 software or Prism 7 to the control:

.

.

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. 6, the IC ₅₀ representing 50% inhibitory concentration of tetrandrin, panchinolin, and cepharantin were 2.68 μM, 5.17 μM, and 2.18 μM, respectively, indicating 50% cytotoxicity. CC ₅₀ was measured to be 16.47 μM, 28.87 μM and 32.81 μM (calculated from FIGS. 6(A), 6(B) and 6(C), respectively). In addition, the IC ₅₀ representing 50% inhibitory concentration of lopinavir as a control group was 13.00 μM, and the CC ₅₀ representing 50% cytotoxicity was determined to be >50 μM (calculated from FIG. 6(D)).

Through this, it was confirmed that the tetrandrin, panchinolin, and cepharantin of the present invention exhibit a significantly superior SARS-coronavirus-2 inhibitory effect compared to the positive control group.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (5)

An antiviral pharmaceutical composition for coronavirus comprising a compound represented by Formula 3 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 3]

.
The pharmaceutical composition of claim 1, wherein the coronavirus is HCoV-OC43, MERS-CoV, SARS-CoV, or SARS-CoV-2.
A pharmaceutical composition for preventing or treating coronavirus infectious diseases comprising a compound represented by Formula 3 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 3]

.
A health functional food composition for preventing or improving coronavirus infectious diseases comprising a compound represented by Formula 3 or a food-acceptable salt thereof as an active ingredient:
[Formula 3]

.
A feed composition for preventing or improving coronavirus infectious diseases comprising a compound represented by the following formula (3) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 3]

.

Images