KR20170052393A - Indoloterpenoid compound, preparing method and use thereof - Google Patents

Abstract

Indolacterephenoid compounds, methods for producing them, and uses thereof. Because the compounds have anti-viral activity, particularly anti-coronavirus activity, they can be used to prevent or treat coronavirus infectious diseases or to disinfect coronaviruses.

Description

INDOLTORPENOID COMPOUND, PREPARING METHOD AND USE THEREOF,

Indolacterephenoid compounds, their production methods and uses.

Physiologically active substances derived from microorganisms were generally the source of antibacterial, antifungal, antiviral, or anticancer drugs, and they have been developed as new drugs for the treatment of various diseases including these and have become templates for the development of new drugs.

Coronavirus (Coronavirus) was first discovered in 1937 in chickens and then through animals such as dogs, pigs, and birds, and was found in humans in 1965. Coronavirus is known to cause colds, upper respiratory tract infections, respiratory illnesses, diarrhea in children and other bowel diseases when infected to humans. Severe acute respiratory syndrome (SARS) is a new or mutated coronavirus-related disease. It was prevalent in China between November 2002 and June 2003, with 8,096 infections and 774 deaths. The Middle East respiratory syndrome (MERS) was first detected in Saudi Arabia in 2012 and confirmed in Jordan, Qatar, United Arab Emirates and Kuwait, according to the World Health Organization, according to May 15, 2014, 572 Of these, 173 were reported to have died. In the case of animals such as dogs and pigs, a vaccine against coronavirus already exists, but the new coronavirus vaccine that occurred in 2003 is undeveloped, and there is no effective treatment.

Therefore, there is a need to search for novel compounds that have new antimicrobial activity and select new microorganisms.

Indoleesterpenoid compounds, isomers, derivatives or salts thereof.

An isomer thereof, a derivative thereof, or a salt thereof.

Indoleesterophenoid compounds, isomers, derivatives or salts thereof.

The present invention provides a composition for an anti-virus comprising an indolorerepenoid compound, an isomer thereof, a derivative, or a salt thereof.

There is provided a method of preventing or treating a coronavirus infection disease using a pharmaceutical composition comprising an indolorerepenoid compound, an isomer thereof, a derivative thereof, or a pharmaceutically acceptable salt thereof.

There is provided a method for disinfecting coronavirus using a composition for disinfection comprising an indolorerepenoid compound, an isomer thereof, a derivative, or a salt thereof.

One aspect provides a compound represented by Formula 1, Formula 2, or a combination thereof, an isomer, derivative, or salt thereof:

[Formula 1]

; 또는

; or

[Formula 2]

.

.

Another aspect provides a Streptomyces sp. HK18 strain producing a compound represented by the above formula 1, formula 2, or a combination thereof, an isomer thereof, a derivative thereof, or a salt thereof.

Another aspect provides a method for producing a compound represented by the above formula 1, formula 2, or a combination thereof, an isomer thereof, a derivative, or a salt thereof.

Another aspect provides a composition for an anti-virus comprising a compound represented by Formula 1, Formula 2, or a combination thereof, an isomer thereof, a derivative, or a salt thereof.

Another aspect is a method for preventing or treating a coronavirus infection disease using a pharmaceutical composition comprising a compound represented by Formula 1, Formula 2, or a combination thereof, an isomer thereof, a derivative, or a pharmaceutically acceptable salt thereof .

Another aspect provides a method for disinfecting coronavirus using a disinfecting composition comprising a compound represented by the above formula 1, formula 2, or a combination thereof, an isomer thereof, a derivative, or a salt thereof.

Since the indoloresterpenoid compound, its isomer, derivative or salt has an anti-viral activity, particularly an anti-coronavirus activity, the compound, an isomer, a derivative or a salt thereof may be used for the prevention or treatment of a coronavirus infectious disease, It can be used for disinfection of virus.

Fig. 1 is a photograph showing a strain of Streptomyces sp. HK18 cultured in a solid medium.
FIG. 2 is a graph showing the results of real-time PCR amplification of mRNA of PEDV GP2 spike when PEDV-infected cells were cultured in the presence of Compound 2 or Compound 3. FIG.
3A to 3C are graphs showing the results of amplification of GP2 spike mRNA, GP6 nucleocapsid mRNA, and GP5 membrane mRNA, respectively, by real-time PCR method.
FIG. 4A is an image obtained by immunoblotting GP6 nucleocapsid and GP2 spike protein of PEDV infected with VERO cells, FIG. 4B is a graph showing the protein intensity level according to the concentration of Compound 2 by measuring the band intensity from the immunoblotted image FIG.
5 is an image obtained by immunofluorescence staining of a nucleocapsid protein of PEDV infected with VERO cells.

One aspect provides a compound, isomer, derivative, or salt thereof, represented by Formula 1, Formula 2, or a combination thereof:

[Formula 1]

; 또는

; or

[Formula 2]

.

.

The compound represented by the formula 1, the formula 2, or a combination thereof is an indolorerpenoid compound. The compound represented by Formula 1, Formula 2, or a combination thereof is a Xiamycin derivative. The giamycin derivative may be a compound in which a carbazole core nucleus and sesquiterpene are combined. The sesquiterpene may be bicyclic sesquiterpene.

In the formula 1, R ₁ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, and the substituted alkyl group is a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group , A C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ cycloalkyl group, a C ₆ -C ₃₀ aryl group, a C ₆ -C ₃₀ aryloxy group, -C (= O) R _a , -C (= O) OR _a , -OCO (OR _a ), -C = N (where R _{a is a} C ₆ -C ₃₀ heteroaryl group, a hydroxy group, a halogen group, a cyano group, ), -SR _a , -S (= O) R _a , -S (= O) ₂ R _a , or a combination thereof, wherein R _a is hydrogen, a C ₁ -C ₁₀ alkyl group, or C ₆ -C ₂₀ aryl group.

In the above formula _{1, R 2, R 3,} R 4, R 5, and R ₆ are, each independently, a hydrogen, a hydroxy group, a halogen group, a cyano group, -C (= O) R _b with each other, -C (= O) _{oR b, -OCO (oR a)} , -C = N (R b), -SR b, -S (= O) R b, -S (= O) 2 R b, a substituted or unsubstituted C ₁ - alkynyl of C ₂₀ alkyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ -C ₂₀ ring of a, C ₂ - for C ₂₀ alkylene oxide group, a substituted or unsubstituted C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted C ₆ -C ₃₀ aryloxy A substituted or unsubstituted C ₆ -C ₃₀ heteroaryl group, or a combination thereof, wherein R _b may be hydrogen, a C ₁ -C ₁₀ alkyl group, or a C ₆ -C ₂₀ aryl group.

In the formula 2, R ₇ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, and the substituted alkyl group is a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group , A C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ cycloalkyl group, a C ₆ -C ₃₀ aryl group, a C ₆ -C ₃₀ aryloxy group, a heteroaryl group, a hydroxy group, a halogen group, a cyano group, a _{c 6 -C 30, -C (=} O) R c, -C (= O) OR c, -OCO (OR c), -C = N (R c ), -SR _c , -S (= O) R _c , -S (= O) ₂ R _c , or a combination thereof, wherein R _c is hydrogen, a C ₁ -C ₁₀ alkyl group, C ₆ -C ₂₀ aryl group.

(= O) R _d , -C (= O) R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ independently represent hydrogen, a hydroxyl group, a halogen group, a cyano group, _{oR d, -OCO (oR d)} , -C = N (R d), -SR d, -S (= O) R d, -S (= O) 2 R d, a substituted or unsubstituted C ₁ - alkynyl of C ₂₀ alkyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ -C ₂₀ ring of a, C ₂ - for C ₂₀ alkylene oxide group, a substituted or unsubstituted C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted C ₆ -C ₃₀ aryloxy A substituted or unsubstituted C ₆ -C ₃₀ heteroaryl group, or a combination thereof, wherein R _d may be hydrogen, a C ₁ -C ₁₀ alkyl group, or a C ₆ -C ₂₀ aryl group.

The compound represented by Formula 1, Formula 2, or a combination thereof may be a compound represented by Formula 3, Formula 4, or a combination thereof:

[Formula 3]

; 또는

; or

[Formula 4]

.

.

In the formula 3, each R ₁₃ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, and the substituted alkyl group is a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group , A C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ cycloalkyl group, a C ₆ -C ₃₀ aryl group, a C ₆ -C ₃₀ aryloxy group, A C ₆ -C ₃₀ heteroaryl group, a hydroxy group, a halogen group, a cyano group, or a combination thereof.

R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ are independently selected from the group consisting of hydrogen, a hydroxyl group, a halogen group, a cyano group, a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, An unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide A substituted or unsubstituted C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted C ₆ -C ₃₀ aryloxy group, a substituted or unsubstituted A C ₆ -C ₃₀ heteroaryl group, or a combination thereof.

In the formula 4, R ₁₉ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, and the substituted alkyl group is a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group , A C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ cycloalkyl group, a C ₆ -C ₃₀ aryl group, a C ₆ -C ₃₀ aryloxy group, A C ₆ -C ₃₀ heteroaryl group, a hydroxy group, a halogen group, a cyano group, or a combination thereof.

In the formula 4, R ₂₀ , R ₂₁ , R ₂₂ , R ₂₃ and R ₂₄ independently represent hydrogen, a hydroxyl group, a halogen group, a cyano group, a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, An unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide A substituted or unsubstituted C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted C ₆ -C ₃₀ aryloxy group, a substituted or unsubstituted A C ₆ -C ₃₀ heteroaryl group, or a combination thereof.

The compound represented by Formula 1, Formula 2, or a combination thereof may be a compound represented by Formula 5, Formula 6, or a combination thereof:

[Formula 5]

; 또는

; or

[Formula 6]

.

.

In the formula 5, R ₂₅ is hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, and the substituted alkyl group is a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, an aryloxy C ₂ -C ₂₀ alkylene oxide group, C ₃ -C ₃₀ cycloalkyl group, C ₆ -C ₃₀ aryl group, C ₆ -C ₃₀ of, C ₆ - A C ₃₀ heteroaryl group, a hydroxy group, a halogen group, a cyano group, or a combination thereof.

In the formula 6, R ₂₆ is hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, and the substituted alkyl group is a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, an aryloxy C ₂ -C ₂₀ alkylene oxide group, C ₃ -C ₃₀ cycloalkyl group, C ₆ -C ₃₀ aryl group, C ₆ -C ₃₀ of, C ₆ - A C ₃₀ heteroaryl group, a hydroxy group, a halogen group, a cyano group, or a combination thereof.

Among the compounds of formula 5, the compound wherein R ₂₅ is hydrogen is named Xiamycin C. The compound of formula 5 wherein R ₂₅ is methyl is named Xiamycin D. Among the compounds of the formula 6, the compound wherein R ₂₆ is methyl is named as Xiamycin E.

The C ₁ -C ₂₀ alkyl group may be, for example, a C ₁ -C ₁₅ alkyl group, a C ₁ -C ₁₀ alkyl group, a C ₁ -C ₅ alkyl group, a C ₁ -C ₄ alkyl group, a methyl, , Butyl, or isobutyl. The C ₂ -C ₂₀ alkenyl group may be an alkenyl group having C ₂ to C ₂₀ , C ₅ to C ₂₀ , C ₁₀ to C ₂₀ , or C ₁₀ to C ₁₅ . The C ₂ -C ₂₀ alkynyl group may be an alkynyl group of C ₂ to C ₂₀ , C ₅ to C ₂₀ , C ₁₀ to C ₂₀ , or C ₁₀ to C ₁₅ .

The halogen group may be -Cl, -F, -Br, or -I.

"Substitution" in the above "substituted" means that a hydrogen atom in the organic compound is substituted for another atomic group to form a derivative to be introduced instead of a hydrogen atom, and "substituent" means an introduced atomic group. Substituents can include, for example, a hydroxy group, a halogen atom, C ₁ to C An alkyl group of _C20, aryl group of C ₂ to C ₂₀ alkenyl groups, C ₂ to C ₂₀ alkynyl group, C ₁ to C ₂₀ heterocycloalkyl group, C ₆ to C ₂₀ aryl group, C ₆ to C ₂₀ of the, C ₆ to C ₂₀ heteroaryl group, or C ₆ to C ₂₀ heteroarylalkyl group, a nitro group, a cyano group, an amino group, an amidino group, hydrazine, hydrazone, a carboxyl group or a salt thereof, a sulfonic acid group or a salt thereof, phosphoric acid or his of It can be a salt.

The term "isomer" refers to a compound that has the same molecular formula but does not have the same linking or spatial arrangement of constituent atoms in the bonsai. Isomers include, for example, structural isomers, and stereoisomers.

The term " derivative "refers to a compound obtained by substituting a part of the structure of the above compound with another atom or an atomic group.

The term "salt" refers to an addition salt of an inorganic acid salt, an organic acid salt, or a metal salt of a compound. The salt may be a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The inorganic acid salt may be a hydrochloride, a bromate, a phosphate, a sulfate, or a disulfide. The organic acid salt may be at least one selected from the group consisting of a formate, a acetate, an acetate, a propionate, a lactate, an oxalate, a tartrate, a malate, a maleate, a citrate, a fumarate, a bezylate, a camylate, an edisylate, trichloroacetic acid, But are not limited to, acetic acid salts, benzoic acid salts, gluconic acid salts, methanesulfonic acid salts, glycolic acid salts, succinic acid salts, 4-toluenesulfonic acid salts, Sulfonic acid, or aspartate. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.

Another aspect provides a Streptomyces sp. HK18 strain (accession number: KCTC12940BP) which produces a compound represented by the above formula 1, formula 2, or a combination thereof, an isomer thereof, a derivative thereof, or a salt thereof.

The compounds, isomers, derivatives, and salts represented by the above formula 1, formula 2, or combinations thereof are as described above.

The strain includes its variants. The mutant may be, for example, a mutant caused by a natural mutation or an anthropogenic mutation. Anthropogenic mutations can be caused by chemical mutagens such as ultraviolet light, physical mutagens, or base compounds.

The strain includes a spore, a bacterium, or a culture thereof.

The strain may be isolated or derived from salt water or salt earth.

In another aspect, there is provided a method for culturing Streptomyces sp. HK18 strain (Accession No. KCTC12940BP); And a compound represented by Formula 1, Formula 2, or a combination thereof, which comprises separating a compound represented by Formula 1, Formula 2, or a combination thereof, an isomer thereof, Derivatives, or salts thereof.

The compounds, isomers, derivatives, salts, and strains represented by Formula 1, Formula 2, or a combination thereof are as described above.

The method comprises culturing Streptomyces sp. HK18 strain (accession number: KCTC12940BP).

The step of culturing may be to culture the strain in a liquid medium or a solid medium. The medium may comprise artificial sea salt. The medium may include, for example, glucose, rice, starch, dextrin, starch, molasses, animal oil, or vegetable oil as a carbon source. The nitrogen source may include, for example, yeast extract, peptone, wheat bran, soybean meal, wheat, malt, cottonseed, fish, cones triticale, juice, ammonium sulfate, sodium nitrate, or urea. The culture medium may contain, if necessary, inorganic salts which promote salt formation, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, or other ions. The medium may include, for example, artificial sea salt, yeast extract, malt extract, or glucose.

The culture may be culturing under aerobic conditions with shaking or standing. The incubation temperature may be, for example, about 20 째 C to about 40 째 C, about 25 째 C to about 37 째 C, about 28 째 C to about 35 째 C, or about 30 째 C. The incubation time may be, for example, from 1 day to 2 months, from 1 day to 6 weeks, from 1 day to 1 month, from 1 day to 2 weeks, or from 1 day to 1 week.

The method includes isolating a compound, an isomer, derivative, or salt thereof represented by Formula 1, Formula 2, or a combination thereof from a culture.

Among the cultures, a compound represented by the formula 1, formula 2, or a combination thereof, isomers, derivatives or salts thereof can be isolated from Streptomyces sp. HK18 strain. A compound represented by Formula 1, Formula 2, or a combination thereof, an isomer thereof, a derivative thereof, or a pharmaceutically acceptable salt thereof may be isolated from a culture solution containing no Streptomyces sp. HK18 strain among the cultures.

The step of separating the compound represented by the formula 1, the formula 2, or a combination thereof, the isomer thereof, the derivative or the pharmaceutically acceptable salt from the culture may be carried out by concentrating the culture, centrifuging, Or a combination thereof. For example, the culture can be extracted with ethyl acetate, water, or a combination thereof. The resulting concentrate can be fractionated by chromatography to yield six fractions depending on the polarity. The chromatography may be reversed phase chromatography using, for example, water, methanol, dichloromethane, or a combination thereof as a mobile phase. The fractions eluted with water / methanol having a volume ratio of 20% of the obtained fractions can be separated by performing high performance liquid chromatography (HPLC). The HPLC can be carried out using a reversed-phase semi-preparative HPLC column under gradient conditions of water / acetonitrile. The concentration gradient may be, for example, from 70% (v / v) water / acetonitrile to 40% (v / v) water / acetonitrile.

Another aspect provides a composition for anti-virus comprising a compound represented by Formula 1, Formula 2, or a combination thereof, an isomer thereof, a derivative, or a salt thereof.

The compounds, isomers, derivatives, and salts represented by the above formula 1, formula 2, or combinations thereof are as described above.

The composition may have activity to inhibit viruses. In the above "anti-virus", the virus may be coronavirus. The coronavirus may be selected from the group consisting of porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), avian infectious bronchitis virus (IBV), transmissible gastroenteritis virus gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or a combination thereof.

The composition may be a pharmaceutical or disinfecting composition.

The composition may be a pharmaceutical composition for preventing or treating a coronavirus infection disease. The coronavirus infectious disease may be coronaviral enterocolitis, coronaviral diarrhea, severe acute respiratory syndrome coronavirus (SARS), Middle East respiratory syndrome (MERS), or a combination thereof.

The term "prophylactic " refers to any act that inhibits coronavirus infectious disease or delays its development by administration of the composition. The term "treatment" refers to any action that improves or alleviates the symptoms of a coronavirus infection disease by the administration of the composition. The pharmaceutical composition may further comprise known active ingredients having anticancer activity.

The pharmaceutical composition may further comprise known active ingredients having antiviral activity. The active ingredient may be selected from the group consisting of Oseltamivir, Zanamivir, Peramivir, ribavirin, azauridin, amantadine, rimantadine, valacyclo Valaciclovir, interferon, immunoglobulin preparation, or a combination thereof.

The pharmaceutical composition may further comprise a carrier, an excipient or a diluent. The carrier, excipient or diluent may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition may have an oral or parenteral dosage form. The pharmaceutical compositions can be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

In the above pharmaceutical composition, the solid preparation for oral administration may be a tablet, a pill, a powder, a granule, or a capsule. The solid preparation may further contain an excipient or a lubricant. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. The lubricant may also be magnesium stearate, or talc. In the above pharmaceutical composition, the liquid preparation for oral administration may be a suspension, a solution, an emulsion, or a syrup. The liquid formulation may comprise water, or liquid paraffin. The liquid preparations may contain excipients such as wetting agents, sweetening agents, perfumes, or preservatives. In the above pharmaceutical composition, the preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an oil, a lyophilized or a left-over preparation.

The pharmaceutical composition may comprise an effective amount of the compound, an isomer, a derivative, or a pharmaceutically acceptable salt thereof. The effective amount may vary depending on factors such as the severity of the disease, the age, body weight, health, sex, sensitivity of the patient to the drug, administration time, route and rate of excretion, duration of treatment, And other factors well known in the medical arts. Wherein said effective amount is from about 10 mg to about 10 g, from about 100 mg to about 8 g, from about 250 mg to about 6 g, from about 500 mg to about 4 g, from about 500 mg to about 2 g, From about 500 mg to about 1 g. The pharmaceutical composition may be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes. The dosage of the pharmaceutical composition may be, for example, from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, or from about 0.1 mg / kg to about 1 mg / kg, and may be administered once a day, once a day, or once a day.

The composition may be a disinfecting composition. The term "disinfection" means losing or killing the activity of a pathogenic microorganism or a hospital virus. Loss of activity includes inhibiting infection, survival, proliferation, propagation, or a combination thereof, of a pathogenic microorganism or a hospital virus. The disinfecting composition may be formulated as a disinfectant or a detergent. The composition for disinfection may be commercialized as an anti-corona virus disinfectant for direct spraying on animal-related facilities and animals, and may be manufactured as an anti-corona virus disinfectant for the cleaning of hands and utensils at animal- And as a detergent. The composition for disinfecting may further contain known components required for a disinfectant or a detergent. The composition for disinfecting may further comprise known additives which can be added. The additive may be ethanol or isopropyl alcohol. The additive may be contained in the composition in an amount of 1.0 wt% to 80.0 wt%, preferably 10.0 wt% to 50.0 wt%.

The composition may be a composition for food or a composition for feed addition.

The food composition may be prepared by encapsulating, pulverizing, or suspending a compound represented by the formula 1, formula 2, or a combination thereof, an isomer thereof, a derivative thereof, or a salt thereof as a functional food or adding it to various foods . Such foods are, for example, beverages, vitamin complexes, and health supplement foods.

The composition for feed addition may further comprise a known additive which can be added for killing the virus according to the preparation. The feed additive composition may be in the form of a high concentrate, powder or granule. The feed additive composition may further comprise any protein-containing organic kernel meal commonly used to meet the dietary needs of an animal. The feed composition may be added to an animal feed by dipping, spraying or mixing.

Another aspect is a method of treating a coronavirus infectious disease comprising administering to a subject a pharmaceutical composition comprising a compound represented by Formula 1, Formula 2, or a combination thereof, an isomer, derivative, or pharmaceutically acceptable salt thereof, Prevention or treatment of cancer.

The compounds, isomers, derivatives, salts, pharmaceutical compositions, prophylactic, therapeutic, and pharmaceutical compositions represented by Formula 1, Formula 2, or a combination thereof are as described above.

The subject may be a mammal, including a mouse, rat, dog, pig, cat, cow, horse, monkey, camel, and human.

The preferred dosage of a compound represented by the above formula 1, formula 2, or a combination thereof, an isomer thereof, a derivative thereof, or a pharmaceutically acceptable salt thereof depends on the condition and weight of the patient, the degree of disease, the drug form, But may be suitably selected by those skilled in the art. However, the compound is for example in the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / Once a day, once a day, once a day, or once a day.

The compound represented by the above formula 1, formula 2, or a combination thereof, an isomer thereof, a derivative, or a pharmaceutically acceptable salt thereof may be administered alone or in combination with another pharmaceutically active compound.

The pharmaceutical composition may be administered to the individual via various routes. The method of administration may be oral or parenteral administration. The method of administration may be, for example, oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, percutaneous, topical, intraocular, or intradermal.

Another aspect relates to a method of spraying, applying, cleaning, spraying, spraying, spraying, or a combination thereof, which comprises applying a disinfecting composition comprising a compound represented by Formula 1, Formula 2, or a combination thereof, an isomer thereof, A method for disinfecting coronavirus comprising the steps of:

The compounds, isomers, derivatives, salts, and disinfecting compositions represented by the above-mentioned Formula 1, Formula 2, or a combination thereof are as described above.

The method may sterilize the coronavirus by spraying, spraying or spraying the composition for disinfection, applying it to the surface of the article, or rinsing the article or body.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.

Example  One. Of indolol-terfenoid  detach

One. Streptomyces  genus( Streptomyces species ) HK18  Isolation of strain

In order to screen for strains producing antiviral activity, the strain HK18 was isolated from the tiller surface soil collected from the tidal flat of Shinan Toppan Salt Farming Association, 445-9 Shinsei-myeon, Shinan-myeon, Shinan-gun, Jeollanam-do, Whole genome of HK18 strain was isolated and 16S ribosomal DNA was cloned by polymerase chain reaction (PCR) and the nucleic acid sequence was analyzed (SEQ ID NO: 1, GenBank Accession No. LC060261.1).

Nucleic acid sequence analysis results, the strain HK18 genus Streptomyces (Streptomyces The strain was named HK18 strain of the genus Sreptomyces and the strain was deposited on October 23, 2015 in the Korean Collection for Type Cultures.

2. Streptomyces sp. HK18  Culture of strain

Streptomyces sp. Strain HK18 was inoculated into sterilized YEME solid medium (10 g of malt extract, 4 g of yeast extract, 2 g of glucose, 33 g of artificial sea salt, and 18 g of agar per 1 L of water) Lt; RTI ID = 0.0 > 30 C < / RTI > for several days. FIG. 1 shows a photograph of the culture of Streptomyces sp. HK18 strain in a solid medium.

From the primary cultured solid medium, strain HK18 was cultured in 50 ml of A1 + YPM liquid medium (10 g of starch per liter, 6 g of yeast, 4 g of peptone, 4 g of mannitol, and 33 g of artificial sea Salt) and secondary cultured at about 30 째 C for 2 days while shaking at 200 rpm.

10 ml of the second cultured culture was inoculated into 1 L of the A1 + YPM liquid medium and cultured for 3 days at about 30 DEG C for 5 days while shaking at 180 rpm.

3. Giamycin  Separation and purification

HK18 cultures were obtained, which were cultured as described in Example 1.2.

Separatory funnel (capacity 3 L) was fixed with a mounting ring on the stand, and 1 L of HK18 culture and 1 L of ethyl acetate (purified gold Co.) were injected. The inlet was closed with a stopper, the mixture was stirred for 30 seconds, and then allowed to stand for 3 minutes so as to be divided into an ethyl acetate layer and a water layer. The water layer was removed from the culture and 100 g of Anhydrous Sodium Sulfate (purified water) was added to the ethyl acetate layer to remove residual water in the culture. The resulting ethyl acetate layer was filtered on filter paper (Advantec), transferred to a round flask and concentrated using a concentrator. The remaining water layer was again extracted with 1 L of ethyl acetate. This procedure was repeated to obtain about 7 g crude extract in a total of 60 L of HK18 culture.

The obtained crude extract was divided into six fractions using reverse phase chromatography (Sepak, C ₁₈ resin 20 g, reversed-phase). The eluate from the six fractions was used from 20% water reduction from 80% (v / v) water / methanol and the last fraction was fractionated with methanol / dichloromethane 50% (v / v) Fractions (200 ml each) were obtained.

Fraction 4 of 20% (v / v) water / methanol fraction was analyzed by liquid chromatogram-mass spectrometry and hydrogen nuclear magnetic resonance spectroscopy. The composition of the fractions was analyzed using LC / MS coupled with Agilent Technologies' 1200 series LC and 6130 series mass spectrometer. The mass spectrum was measured using a Q-High resolution ESI mass spectrometer from Thermo Scientific and expressed in terms of mass / charge (m / z). It was confirmed by liquid chromatography-mass spectrometry and hydrogen nuclear magnetic resonance spectroscopy analysis that the culture of the strain contained new secondary metabolites.

The fraction 4, which had been concentrated by vacuum drying, was dissolved in 2 ml of methanol (manufactured by Daqing K.K.) and the lysate was dissolved in a mixture of 70% (v / v) water / acetonitrile to 40% (v / v) water / acetonitrile (C ₁₈ reversed-phase semi-preparative HPLC column, particle diameter 5 탆, 250 x 10 mm (length x inner diameter), elution rate 2 ml / min, UV detector) under the concentration gradient of Kromasil ) For about 70 minutes. Thereby, 7.0 mg of Xiamycin C (Compound 1), 5.0 mg of Giamycin D (Compound 2) and 2.0 mg of Zieacin D (Compound 2), respectively, at an elution time of about 31 minutes, about 38 minutes, Methylene < / RTI > E (compound 3). In addition, 14.0 mg and 2.0 mg of the previously known giamycin A (compound 4) and chlorogiamycin (compound 5) were obtained at an elution time of about 48 minutes and about 53 minutes, respectively.

A gyamycin A methyl ester derivative was synthesized from the gyamycin A obtained. Specifically, 8 mg of the obtained zymesicin A was dissolved in 2 ml of anhydrous methanol. Trimethylsilyldiazomethane (TMS) in 200 μl of a 2 M diethyl ether solution was added to the reaction solution, Incubation was carried out with stirring for 2 hours. The reaction was then run on a reversed-phase semi-preparative HPLC column (C18 reversed-phase semi-preparative HPLC column, gradient elution with gradient of acetonitrile to 100% (v / v) from water / acetonitrile 50% The particles were separated with a particle diameter of 5 탆, 250 x 10 mm (length x inner diameter), elution rate of 2 ml / min, UV detector) (Kromasil) for about 60 minutes. Approximately 7 mg of giamycin A methyl ester derivative (Compound 6) was obtained at an elution time of about 32 minutes.

The chemical structures of giamycin C to E, giamycin A, giamycin A methyl ester derivatives, and chlorogiamycin are shown below.

[Giamycin C and D]

.

.

In this chemical structure, giamycin C is R ₁ is H (compound 1) and giamycin D is R ₁ is CH ₃ (compound 2).

[Giamycin E]

.

.

The compound of the above formula is giamycin E (compound 3).

[Giamycin A and its methyl ester derivative]

.

.

In this chemical structure, giamycin A is R ₁ is H (compound 4), and giamycin A methyl ester derivative is R ₁ is CH ₃ (compound 6).

[Chlorogiamycin]

.

.

The compound of the above formula is chlorogiamycin (Compound 5).

4. Physicochemical Characterization of Compounds 1 to 3

Compounds 1 to 3 obtained as described in Example 1. 3 were in a pale yellow oily state, stable at room temperature, and well dissolved in methanol, which is a medium organic solvent.

The structures of compounds 1 to 3 were determined by nuclear magnetic resonance spectrum, infrared and ultraviolet spectroscopic data, and high resolution mass spectrometry data. Nuclear magnetic resonance spectrum ( ¹ H NMR, ¹³ C NMR) was performed by Bruker's 600 MHz or 500 MHz NMR, and the solvent used was methanol-d ₄ . The mass spectrum was measured using a JMS-700 FAB mass spectrometer from JEOL GmbH and expressed in the form of mass / charge (m / z). The infrared spectrum was measured using a Thermo NICOLET iS10 spectrometer. The ultraviolet spectrum was measured using a Lambda 35 UV / VIS spectrometer from Perkin Elmer.

Tables 1 and 2 show the structural position designations of the compounds 1 to 3 by nuclear magnetic resonance spectroscopy.

[Giamycin C (Compound 1)]

(1) Molecular formula: C ₂₃ H ₂₅ NO ₄

(2) Molecular weight: 379

(3) Color: Pale yellow

(4) Infrared absorption band (ZnSe): 3679, 2966, 1671, 1361, 1052 cm ^-1

(5) ¹ H-NMR (MeOD, 500 MHz): See Table 1

(6) ¹³ C-NMR (MeOD, 125 MHz): See Table 1

[Giamycin D (Compound 2)]

(1) Molecular formula: C ₂₄ H ₂₇ NO ₄

(2) Molecular weight: 393

(3) Color: Pale yellow

(4) Infrared absorption band (ZnSe): 3680, 2965, 1674, 1346, 1243, 1054 cm ^-1

(5) ¹ H-NMR (MeOD, 500 MHz): See Table 1

(6) ¹³ C-NMR (MeOD, 125 MHz): See Table 1

[Giamycin E (Compound 3)]

(1) Molecular formula: C ₂₄ H ₂₅ NO ₄

(2) Molecular weight: 391

(3) Color: Pale yellow

(4) Infrared absorption band (ZnSe): 3342, 2927, 1599, 1365, 1249, 1088 cm ^-1

(5) ¹ H-NMR (MeOD, 600 MHz): See Table 1

(6) ¹³ C-NMR (MeOD, 150 MHz): See Table 1

The chemical shift values of the nuclear magnetic resonance spectra of giamycin C to E (compounds 1 to 3) location Giamycin C (compound 1) ^a Giamycin D (Compound 2) ^a Giamycin E (compound 3) ^b d _C , type d _H, (J in Hz) mult d _C , type d _H, (J in Hz) mult d _C , type d _H, (J in Hz) mult 1-N - - - - - - 2 140.2, C 140.2, C 139.5, C 3 124.1, C 124.2, C 129.9, C 4 124.4, C 124.4, C 123.6, C 5 120.8, CH 8.00, d (8.0) 120.9, CH 8.01, d (8.0) 122.2, CH 8.15, d (8.0) 6 119.4, CH 7.10, dd (8.0, 8.0) 119.5, CH 7.11, dd (8.0, 8.0) 120.3, CH 7.20, dd (8.0, 8.0) 7 126.4, CH 7.31, dd (8.0, 8.0) 126.5, CH 7.32, dd (8.0, 8.0) 128.6, CH 7.45, dd (8.0, 8.0) 8 111.6, CH 7.38, d (8.0) 111.6, CH 7.38, d (8.0) 112.1, CH 7.46, d (8.0) 9 142.4, C 142.4, C 143.9, C 10 116.3, CH 7.94, s 116.4, CH 7.94, s 116.0, CH 8.14, s 11 141.7, C 141.3, C 147.4, C 12 39.0, C 39.0, C 38.7, C 13 39.2, CH ₂ 2.62, ddd (13.0, 3.5, 3.5)
1.70, m 39.1, CH ₂ 2.64, ddd (13.0, 3.5, 3.5)
1.70, m 38.1, CH ₂ 2.74, m
1.95, m 14 28.5, CH ₂ 1.89, m 28.5, CH ₂ 1.92, m 28.1, CH ₂ 1.98, m 15 76.3, CH 4.08, dd (8.0, 6.5) 76.1, CH 4.07, dd (9.5, 6.5) 75.9, CH 4.11, m 16 54.8, C 55.3, C 54.7, C 17 46.1, CH 2.20, dd (12.0, 2.0) 46.3, CH 2.18, dd (13.0, 1.5) 46.8, CH 2.63, dd (14.0, 3.5) 18 33.2, CH ₂ 1.97, m
1.93, m 33.2, CH ₂ 2.00, m
1.68, m 38.6, CH ₂ 2.97, dd (18.0, 14.0)
2.19, dd (18.0, 3.5) 19 72.2, CH 4.91, dd (10.0, 7.5) 72.0, CH 4.85, m 200.7, C 20 137.6, C 137.3, C 129.2, C 21 110.1, CH 7.58, s 110.1, CH 7.58, s 110.6, CH 8.06, s 22 26.8, CH ₃ 1.38, s 26.8, CH ₃ 1.38, s 24.8, CH ₃ 1.37, s 23 11.9, CH ₃ 1.26, s 11.4, CH ₃ 1.28, s 10.9, CH ₃ 1.35, s 24 182.0, C 179.4, C 178.5, C 25 52.7, CH ₃ 3.74, s 52.8, CH ₃ 3.72, s

a : ¹ H 500 MHz, ¹³ C 125 MHz. b : ¹ H 600 MHz, ¹³ C 150 MHz.

The basic skeleton of giamycin C to E was confirmed to be an indolosesquiterpenoid derivative having carbazole and sesquiterpene bound thereto.

Example  2. Identification of antiviral activity of compounds 1 to 6

1. Recovery effects of the compounds 1 to 6 on cell lesions

The cytopathic effect reduction assay (CPE reduction) assay, which evaluates the degree of recovery of cells infected with porcine epidemic diarrhea virus (PEDV) assay. CPEs represent morphological changes due to viral infection, and degenerative changes in cell shape are associated with infection of viruses (Semin. Neurol., 1999, vol.19, p.113-127; Virol. Methods, 2002, vol. 106, p. 71-79).

Specifically, 1xlO ⁵ cells / ml of VERO cells (ATCC CCR-81, ATCC, USA) were inoculated in a 96-well microplate at 190 μl per well. The inoculated cells were incubated with 10% fetal bovine serum (FBS), 1% (w / v) PS (penicillin and streptomycin) and L-glutamine (L-glutamine, DMEM culture medium at 37 ° C and 5% CO ₂ for one day. The cultured cells were washed with PBS (phosphate buffer saline) solution.

PEDV (Central Vaccine Research Institute, Daejeon, Korea) was inoculated into a medium (DMEM (HyClone, USA), 0.5 (50% Tissue Culture Infective Dose: TCID ₅₀ ) / 100 ml / (Sigma-Aldrich, USA) and 1 mg / ml trypsin (Gibco, USA)) to prepare PEDV-infected medium. Was added to the washing medium of 100 ㎕ PEDV infection the cells, the cells were incubated for 2 hours in a 37 ℃ and 5% CO ₂ condition. The cells were then washed with PBS solution. Compounds 1 to 6, which were step-diluted from the initial concentration of 100 μM, were added to the washed cells, and the cells were cultured in DMEM medium containing L-glutamine (2 ml based on 500 ml culture medium) ₂ < / RTI > conditions for about 2 days. As a positive control, 10 μM 6-azauridin (Sigma-Aldrich, USA) was added instead of the compounds 1 to 6, and cells without any compounds were used as a negative control.

Two days after cell culture, virus-induced damage was detected in the negative control and MTT assay was performed to determine the viability of living VERO cells. Living cells are transformed with light blue MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) into dark blue formazan by respiratory enzymes in mitochondria However, dead cells can not transform MTT. MTT (Sigma-Aldrich) at 2 mg / ml was added to the cultured cells, and the concentration of formazan produced at an absorbance of 550 nm was measured to measure the cytotoxicity of the compounds 1 to 6 and PEDV. The concentration of compounds without VERO cytotoxicity by compounds was calculated and the difference in denaturation of VERO cells caused by the infected PEDV virus was measured to confirm the CPE effect depending on the compound concentration.

The antiviral activity of each compound as compared to the negative control was represented by EC ₅₀ (half maximal effective concentration) in Table 2 below, and the cytotoxicity of the compound itself by MTT analysis was expressed by CC ₅₀ (50% cytotoxic concentration). The EC ₅₀ was calculated by MTT analysis of virus-induced apoptosis in cells treated with virus and compound, and the CC ₅₀ was calculated by MTT analysis of the compound itself due to the compound alone in the compound-treated cells. The selectivities (SI, CC ₅₀ / EC ₅₀ ) values showing the difference between the cytotoxicity of the compound and the therapeutic concentration were calculated to show the antiviral effect of the compounds 1 to 6.

compound EC ₅₀ (μM) CC ₅₀ (μM) SI  ( CC ₅₀ / EC ₅₀ ) Compound 1 11.49 + 1.36 76.90 ± 3.29  6.75 + - 0.57 Compound 2 0.93 + 0.07 56.03 + - 3.45 60.31 + - 0.89 Compound 3 2.89 ± 0.36 98.74 1.52 34.63 + - 3.32 Compound 4 20.14 + - 4.04 138.12 ± 14.69  7.00 + - 0.81 Compound 5 12.76 ± 1.96 162.08 + - 10.25 12.89 ± 1.00 Compound 6 5.69 ± 0.10 103.15 ± 12.90 18.09 ± 1.98 6-azauridine 4.48 0.25 57.26 + 9.58 12.70 ± 1.34

As shown in Table 2, infection PEDV viability (EC ₅₀₎ of VERO cells is lower than the value of the EC _50-aza uridine when added to compound 2 or compound 3. Particularly, Compound 2 had an EC ₅₀ of 0.93 + 0.07 μM, so Compound 2 was found to be more excellent in antiviral activity than Azuridine (EC ₅₀ : 4.48 ± 0.25 μM). In addition, aza uridine cytotoxicity (CC ₅₀₎ it was 57.26 ± 9.58 μM and SI values are 12.70 ± 1.34, and, CC ₅₀ of compound 2 is 56.03 ± 3.45 μM and SI values of 60.31 and ± 0.89, Compound 3 CC ₅₀ Was 2.89 ± 0.36 μM and the SI value was 34.63 ± 3.32. Therefore, it was confirmed that the compounds 2 and 3 have higher antiviral activity and safety than aziridine.

2. By compounds 2 and 3 PEDV of GP6 Nucleocapsid , GP2  Spike and GP5  Membrane mRNA  Suppression of expression

PEDV has been reported to require the GP2 spike, GP5 membrane and GP6 nucleocapsid proteins during the proliferation (Virus Genes, 2012, vol.44, p.167-175) . Thus, it was confirmed whether or not the compounds 2 and 3 in which the antiviral activity was confirmed in Example 2.1 change mRNA expression of the GP2 spike protein for PEDV replication.

As described in Example 2.1, the culture grown to approximately 90% on a 6-well plate the VERO cell line, and was added to 0.01 MOI PEDV of the VERO cells cultured for 2 hours in a 37 ℃ and 5% CO ₂ conditions (MOI: multiplicity of infections with the number of viruses and the number of susceptible cells). Then, the cell culture solution was removed to remove the virus, and the cells were washed with PBS. The washed cells were supplemented with 2 μM or 5 μM of Compound 2, or DMEM medium containing 2 μM or 5 μM of Compound 3, respectively, and cultured at 37 ° C. and 5% CO ₂ for about 24 hours. Cells to which 6-azuridine was added as a positive control were used, and cells without any compounds were used as a negative control.

The cultured cells were then harvested and RNA was obtained from the cells using TRIzol RNA isolation reagent (Fisher Scientific). CDNA was synthesized from the RNA obtained using a HiSenScript ™ RH (-) cDNA synthesis kit (iNtRON Biotechnology, Korea) containing a random primer.

CDNA was amplified by real-time PCR using Maxima SYBR Green qPCR master mix (Thermo Scientific) and the primer set of SEQ ID NOs: 2 and 3 shown below and the results are shown in Fig. 2 (y axis: relative Expression level, **: p < 0.01, ***: p < 0.001). The beta-actin mRNA was amplified using the primer sets of SEQ ID NOS: 8 and 9, and the results were used to standardize real-time PCR results.

Primer set for GP2 spike mRNA amplification;

Forward primer: 5'-GCTAGTGGCGTTCATGGTATTTT-3 '(SEQ ID NO: 2) and

Reverse primer: 5'-ACGGCTCTTGCGAAATGC-3 '(SEQ ID NO: 3).

Primer set for beta actin mRNA amplification;

Forward primer: 5'-AGCGGGAATCGTGCGTGACA-3 '(SEQ ID NO: 8) and

Reverse primer: 5'-GTGGACTTGGGAGAGGACTGG-3 '(SEQ ID NO: 9).

As shown in Figure 2, both compounds 2 and 3 reduced GP2 spike mRMA expression at a concentration of 2 [mu] M. In particular, 2 μM of compound 2 decreased mRMA expression levels compared to 5 μM 6-azuridine. Therefore, it was confirmed that the compounds 2 and 3 had remarkable antiviral activity than the positive control 6-azuridine.

In addition, it was confirmed whether Compound 2 changes mRNA expression of GP2 spike, GP6 nucleocapsid, and GP5 membrane required for PEDV replication

1 μM to 10 μM of Compound 2 was added to PEDV-infected cells in the same manner as described above, and cDNA was prepared from the cells. And amplified by real-time PCR as described above. A primer set for amplifying GP2 spike mRNA of SEQ ID NOS: 2 and 3 was used, and the following primer sets were used for amplifying mRNA of GP6 nucleocapsid mRNA and GP5 membrane. The beta-actin mRNA was amplified using the primer sets of SEQ ID NOS: 8 and 9, and the results were used to standardize real-time PCR results.

A primer set for GP6 nucleocapsid mRNA amplification;

Forward primer: 5'-GAAAATCCTGACAGGCATAAGCA-3 '(SEQ ID NO: 4) and

Reverse primer: 5'-TTGCCGCTGTTGTCAGACTT-3 '(SEQ ID NO: 5).

GP5 Membrane is a set of primers for amplifying mRNA;

Forward primer: 5'-TTTGACGCATGGGCTAGCT-3 '(SEQ ID NO: 6) and

Reverse primer: 5'-GCAAGCCATAAGGATGCTGAA-3 '(SEQ ID NO: 7).

The real-time amplification results of GP2 spike mRNA, GP6 nucleocapsid mRNA, and GP5 membrane mRNA are shown in FIGS. 3a to 3c, respectively (y axis: relative expression level *: p <0.05, **: p <0.01, ***: p < 0.001).

As shown in FIGS. 3A to 3C, Compound 2 showed concentration-dependent expression of GP2 spike, GP6 nucleocapsid, and GP5 membrane, in particular Compound 2, decreased expression of GP2 spike and GP6 nucleocapsid .

3. Compound 2 GP6 Nucleocapsid  And P2  Suppress protein expression of spike

Whether Compound 2 changes the expression of GP6 nucleocapsid and GP2 spike protein for PEDV replication was confirmed by immunoblotting method.

After infecting VERO cells with PEDV, as described in Example 2.2, VERO cell lines were grown to about 90% growth on 6-well plates and then PEDV was infected for 2 hours at 0.01 MOI. Then, the cell culture solution was removed to remove the virus, and the cells were washed with PBS. DMEM medium containing 0.5 μM to 5 μM of Compound 2 was added to the washed cells and cultured at 37 ° C. and 5% CO ₂ for about 24 hours. As a negative control, cells without any compounds were used.

Then, the cultured cells were washed twice with PBS, and then 100 쨉 l of cell lysis buffer (50 mM NaF, 0.5% (v / v) NP-40, 1 mM EDTA, 120 mM NaCl, Tris-HCl, pH 7.6) was added to dissolve the cells, and a cell eluate was obtained. The cell extracts were subjected to electrophoresis and incubated with anti-GP6 nucleocapsid antibody (AbFrontier Co., Ltd., Korea), anti-GP2 spike protein antibody (AbFrontier Co., Ltd., Korea) (Abcam, USA) was used for immunoblotting.

FIG. 4A shows the image obtained by immunoblotting, and the graph showing the protein expression level according to the concentration of Compound 2 by measuring the band intensity is shown in FIG. 4B (y-axis: relative intensity relative to negative control, *: p < 0.05, **: p <0.01, #: p <0.05, ##: p <0.01). The band strength was normalized to the band strength of beta-actin.

As shown in FIG. 4A and FIG. 4B, Compound 2 was found to be superior in the anti-PEDV activity because of its excellent inhibitory effect on the expression of GP6 nucleocapsid and GP2 spike protein of PEDV.

4. Compound 2 Intracellular PEDV  Growth inhibition

To determine whether Compound 2 inhibited the growth of PEDV infected with cells, VERO cells were infected with PEDV and then PEDV was detected in the cells.

Specifically, VERO cells were infected with PEDV and the VERO cell line was grown to about 90% on a 6-well plate, as described in Example 2.2, and then PEDV was infected for 2 hours at 0.01 MOI. Then, the cell culture solution was removed to remove the virus, and the cells were washed with PBS. DMEM medium containing 0.5 μM to 10 μM of Compound 2 was added to the washed cells and cultured at 37 ° C. and 5% CO ₂ for about 24 hours. Cells in which 10 μM of 6-azuridine was added as a positive control, PEDV-infected cells as negative controls, and PEDV-infected cells without any compounds were used.

Then, the cultured cells were washed three times with PBS at 4 ° C, and then 4% (v / v) paraformaldehyde solution (Sigma-Aldrich, USA) was added to the cells and the cells were incubated at room temperature for about 30 minutes The cells were fixed by incubation. Immobilized cells were blocked with 1% (w / v) BSA (Sigma-Aldrich, USA) and incubated at room temperature for about 1 hour. An anti-GP6 nucleocapsid monoclonal antibody (AbFrontier Co., Ltd., Korea) was added to the cells, followed by incubation at 4 ° C for about 12 hours. Then, a FITC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch, Inc., USA) as a secondary antibody was added to the cells, and the cells were further treated with 500 nM DAPI solution (Molecular Probes, USA) DNA was stained and mounted (Vectashield, Vector Laboratories Inc., Burlingame, CA, USA) and confirmed by fluorescence microscopy (Olympus ix70 Fluorescence Microscope, Olympus Corporation, Japan). The obtained fluorescence image is shown in Fig. 5 (PEDV untreated: cells not infected with PEDV, PEDV: PEDV infected cells without any compounds).

As shown in FIG. 5, no PEDV was detected in the VERO cells that did not infect the PEDV, and the expression of the nucleocapsid protein of the PEDV was strongly detected in the VERO cells infected with the PEDV. However, the expression of the nucleocapsid protein of PEDV was inhibited by the compound 2-treated VERO cells in a concentration-dependent manner. Thus, Compound 2 was found to be excellent in anti-viral effect.

Korea Biotechnology Research Institute KCTC12940BP 20151023

<110> SEOUL NATIONAL UNIVERSITY R & DB Foundation <120> Indoloterpenoid compound, preparing method and use thereof <130> GN-111552-KR <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 1427 <212> DNA <213> Artificial Sequence <220> <223> Streptomyces sp. HK18 gene for 16S rRNA <400> 1 taggggccag ctctcgctgt gtctcgagcg atgaacctct tccgacggga ttactggcga 60 acgggtgagt aacacgtggg caatctgccc tgcactctgg gacaagccct ggaaacgggg 120 tctaataccg gatacgacca tctcgggcat ccgatggtgg tggaaagctc cggcggtgca 180 ggatgagccc gcggcctatc agcttgttgg tggggtgatg gcctaccaag gcgacgacgg 240 gtagccggcc tgagagggcg accggccaca ctgggactga gacacggccc agactcctac 300 gggaggcagc agtggggaat attgcacaat gggcggaagc ctgatgcagc gacgccgcgt 360 gagggatgac ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg tgagtgacgg 420 tacctgcaga agaagcaccg gctaactacg tgccagcagc cgcggtaata cgtagggtgc 480 gagcgttgtc cggaattatt gggcgtaaag agctcgtagg cggcttgtcg cgtcggatgt 540 gaaagcccgg ggcttaaccc cgggtctgca ttcgataccg gcaggctaga gttcggtagg 600 ggagatcgga attcctggtg tagcggtgaa atgcgcagat atcaggagga acaccggtgg 660 cgaaggcgga tctctgggcc gatactgacg ctgaggagcg aaagcgtggg gagcgaacag 720 gattagatac cctggtagtc cacgccgtaa acgttgggaa ctaggtgtgg gcgacattcc 780 acgtcgtccg tgccgcagct aacgcattaa gttccccgcc tggggagtac ggccgcaagg 840 ctaaaactca aaggaattga cgggggcccg cacaagcggc ggagcatgtg gcttaattcg 900 acgcaacgcg aagaacctta ccaaggcttg acatacaccg gaaagccgta gagatacggc 960 cccccttgtg gtcggtgtac aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt 1020 tgggttaagt cccgcaacga gcgcaaccct tgttctgtgt tgccagcatg cctttcgggg 1080 tgatggggac tcacaggaga ctgccggggt caactcggag gaaggtgggg acgacgtcaa 1140 gtcatcatgc cccttatgtc ttgggctgca cacgtgctac aatggccggt acaatgagct 1200 gcgataccgc gaggtggagc gaatctcaaa aagccggtct cagttcggat tggggtctgc 1260 aactcgaccc catgaagtcg gagtcgctag taatcgcaga tcagcatcgc tgcggtgaat 1320 cccgtcacg cggtggccca acccgctatg tatgcactta caagaagagc gttcacg 1427 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying GP2 Spike mRNA <400> 2 gctagtggcg ttcatggtat ttt 23 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying GP2 Spike mRNA <400> 3 acggctcttg cgaaatgc 18 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying GP6 nucleocapsid mRNA <400> 4 gaaaatcctg acaggcataa gca 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying GP6 nucleocapsid mRNA <400> 5 ttgccgctgt tgtcagactt 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying GP5 membrane mRNA <400> 6 tttgacgcat gggctagct 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying GP5 membrane mRNA <400> 7 gcaagccata aggatgctga a 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying beta actin <400> 8 agcgggaatc gtgcgtgaca 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for amplifying beta actin <400> 9 gtggacttgg gagaggactg g 21

Claims (13)

A compound represented by Formula 1, Formula 2, or a combination thereof, an isomer thereof, a derivative or a salt thereof:
[Formula 1]

as,
In Equation (1)
R ₁ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
The substituted alkyl group may be substituted with a substituent selected from the group consisting of a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ cycloalkyl group, C ₆ -C ₃₀ aryl group, C ₆ -C ₃₀ aryloxy group of, C ₆ -C ₃₀ heteroaryl group, a hydroxy group, a halogen group, a cyano group, -C (= O) a R _a, - _{C (= O) oR a,} -OCO (oR a), -C = N (R a), -SR a, -S (= O) R a, -S (= O) 2 R a, or a , Wherein R _a is hydrogen, a C ₁ -C ₁₀ alkyl group, or a C ₆ -C ₂₀ aryl group, and
_{R 2, R 3, R 4} , R 5, and R ₆ are, each independently, hydrogen, a hydroxy group, a halogen group, a cyano _{group, -C (= O) R b} , -C (= O) OR b, -OCO _{(oR a), -C = N} (R b), -SR b, -S (= O) R b, -S (= O) alkyl ₂ R _b, a substituted or unsubstituted C ₁ -C _20, A substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a substituted or unsubstituted C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted aryloxy group of C ₆ -C ₃₀ of a, substituted or unsubstituted A substituted C ₆ -C ₃₀ heteroaryl group, or a combination thereof, wherein R _b is hydrogen, a C ₁ -C ₁₀ alkyl group, or a C ₆ -C ₂₀ aryl group; or
[Formula 2]

as,
In the above formula 2,
R ₇ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
The substituted alkyl group may be substituted with a substituent selected from the group consisting of a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ cycloalkyl group, C ₆ -C ₃₀ aryl group, C ₆ -C ₃₀ aryl oxy group, a cyano group, -C (= O) heteroaryl group, a hydroxy group, a halogen group of a _{C 6 -C 30, R c,} - _{c (= O) oR c,} -OCO (oR c), -C = N (R c), -SR c, -S (= O) R c, -S (= O) 2 R c, or a , Wherein R _c is hydrogen, a C ₁ -C ₁₀ alkyl group, or a C ₆ -C ₂₀ aryl group, and
_{R 8, R 9, R 10} , R 11, and R ₁₂ are, each independently, a hydrogen, a hydroxy group, a halogen group, a cyano _{group, -C (= O) R d} , -C (= O) OR d, -OCO each other (OR _d ), -C═N (R _d ), -SR _d , -S (═O) R _d , -S (═O) ₂ R _d , a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, A substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a substituted or unsubstituted C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted aryloxy group of C ₆ -C ₃₀ of a, substituted or unsubstituted A substituted C ₆ -C ₃₀ heteroaryl group, or a combination thereof, wherein R _d is hydrogen, a C ₁ -C ₁₀ alkyl group, or a C ₆ -C ₂₀ aryl group. The compound of claim 1, wherein the compound is represented by Formula 3, Formula 4, or a combination thereof, an isomer, derivative, or salt thereof:
[Formula 3]

as,
In Equation (3)
R ₁₃ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
The substituted alkyl group may be substituted with a substituent selected from the group consisting of a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ is an alkyl group substituted with a cycloalkyl group, C ₆ -C ₃₀ aryl group, an aryloxy group, a heteroaryl group, a hydroxy group, a halogen group, a cyano group, a C ₆ -C _30, or a combination of C ₆ -C _30, And
R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ and R ₁₈ independently of one another are hydrogen, a hydroxyl group, a halogen group, a cyano group, a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted polyalkylene oxide group, a substituted or unsubstituted alkynyl group, a C ₂ -C ₂₀ unsubstituted C ₂ -C ₂₀ hwandoen of C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted aryloxy group unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted C ₆ -C _30, a substituted or unsubstituted C ₆ -C ₃₀ A heteroaryl group, or a combination thereof; or
[Formula 4]

as,
In Equation (4)
R ₁₉ is independently hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
The substituted alkyl group may be substituted with a substituent selected from the group consisting of a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ is an alkyl group substituted with a cycloalkyl group, C ₆ -C ₃₀ aryl group, an aryloxy group, a heteroaryl group, a hydroxy group, a halogen group, a cyano group, a C ₆ -C _30, or a combination of C ₆ -C _30, And
R ₂₀ , R ₂₁ , R ₂₂ , R ₂₃ and R ₂₄ independently of one another are hydrogen, a hydroxyl group, a halogen group, a cyano group, a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ -C ₂₀ alkenyl group, a substituted or unsubstituted polyalkylene oxide group, a substituted or unsubstituted alkynyl group, a C ₂ -C ₂₀ unsubstituted C ₂ -C ₂₀ hwandoen of C ₃ -C ₃₀ cycloalkyl group, a substituted or unsubstituted aryloxy group unsubstituted C ₆ -C ₃₀ aryl group, a substituted or unsubstituted C ₆ -C _30, a substituted or unsubstituted C ₆ -C ₃₀ Heteroaryl group, or a combination thereof. The compound of claim 1, wherein the compound is represented by Formula 5, Formula 6, or a combination thereof, an isomer, derivative, or salt thereof:
[Formula 5]

as,
In the formula 5, R ₂₅ is hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
The substituted alkyl group may be substituted with a substituent selected from the group consisting of a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ cycloalkyl group, C ₆ -C ₃₀ aryl group, C ₆ -C ₃₀ aryloxy group of, C ₆ -C ₃₀ heteroaryl group, a hydroxy group, a halogen group, a cyano group or an alkyl group substituted with a combination thereof; or
[Formula 6]

as,
In the above formula 6, R ₂₆ is hydrogen or a substituted or unsubstituted C ₁ -C ₂₀ alkyl group,
The substituted alkyl group may be substituted with a substituent selected from the group consisting of a C ₁ -C ₂₀ alkoxy group, a C ₂ -C ₂₀ alkenyl group, a C ₂ -C ₂₀ alkynyl group, a C ₂ -C ₂₀ alkylene oxide group, a C ₃ -C ₃₀ a cycloalkyl group, an aryloxy group, an alkyl group substituted with a heteroaryl group, a hydroxy group, a halogen group, a cyano group, or a combination of C ₆ -C ₃₀ in C ₆ -C ₃₀ aryl group, C ₆ -C _30. Streptomyces sp. Strain HK18 (accession number: KCTC12940BP) producing the compound of claim 1, an isomer thereof, a derivative or a salt thereof. Culturing Streptomyces sp. Strain HK18 (accession number: KCTC12940BP); And
A method of producing a compound of claim 1, an isomer thereof, a derivative or a salt thereof, comprising the step of isolating the compound of claim 1, an isomer thereof, a derivative or a salt thereof from said culture. A composition for an anti-virus comprising the compound of claim 1, an isomer thereof, a derivative, or a salt thereof. 7. The composition of claim 6, wherein the virus is coronavirus. The method of claim 7, wherein the coronavirus is selected from the group consisting of porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), avian infectious bronchitis virus (IBV), infectious gastroenteritis coronavirus wherein the composition is selected from the group consisting of transmissible gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) . 7. The composition of claim 6, wherein the composition is for pharmaceutical or disinfection. 10. The composition of claim 9, wherein the composition is a pharmaceutical composition for the prevention or treatment of a coronavirus infection disease. 11. The method of claim 10, wherein the coronavirus infection disease is selected from the group consisting of coronaviral enterocolitis, coronaviral diarrhea, severe acute respiratory syndrome coronavirus (SARS), Middle East respiratory syndrome (MERS) &Lt; / RTI &gt; A method for preventing or treating a coronavirus infection disease comprising administering to a subject a pharmaceutical composition comprising a compound of claim 1, an isomer thereof, a derivative, or a pharmaceutically acceptable salt thereof. A method of disinfecting a coronavirus comprising spraying, applying, cleaning, spraying, spraying, or a combination thereof, of a disinfecting composition comprising a compound of claim 1, an isomer, derivative or salt thereof.

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