KR102442047B1 - Fatty acid glyceride compound containing sulfate and amine group, preparing method and use thereof - Google Patents

Abstract

Provided are a fatty acid glyceride compound containing sulfate and an amine group, a strain producing the same, a method for producing the same, and uses thereof. Since the fatty acid glyceride compound has antibacterial activity, in particular, antibacterial activity against vancomycin-resistant bacteria, it can be used for sterilizing or bacteriostatic bacteria or preventing or treating infectious diseases caused or caused by bacteria.

Description

Fatty acid glyceride compound containing sulfate and amine group, production method and use thereof

It relates to a fatty acid glyceride compound containing sulfate and an amine group, a method for its production, and its antibacterial use.

In natural product science, microorganisms related to or symbiotic with insects have been considered as a noteworthy resource for drug discovery over the past 20 years, and research is being conducted actively. Bacterial secondary metabolites have been considered as molecular mediators in insect symbiotic systems based on the study of insect-microbe interactions (Pishchany et al., PNAS, 2018, 115(40):10124-10129; Chevrette et al. ., Nature Communications, 2019, vol.10, Article No. 516). For example, ants of Apterostigma dentigerum defend themselves by utilizing anti-fungal substances produced by symbiotic Pseudonocardia strains (Oh et al., Nat. Chem. Biol. , 2009, 5(6):391-393). Therefore, natural products produced by microorganisms associated with or symbiotic with insects can serve as a chemical basis for the development of new antibiotics.

The cow dung beetle's life history is closely related to feces, so it can be exposed to various microorganisms. Since there may be pathogens that threaten the life of the cow dung beetle, there is a possibility that microorganisms symbiotic with the cow dung beetle will produce substances that inhibit the pathogen.

Therefore, there is a need to search for novel compounds that inhibit the growth of bacteria from microorganisms symbiotic with dung beetles.

A fatty acid glyceride compound containing a sulfate and an amine group, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is provided.

Provided is a bacterium that produces a fatty acid glyceride compound containing a sulfate and an amine group, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

A method for producing a fatty acid glyceride compound containing a sulfate and an amine group, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is provided.

Provided is an antibacterial composition comprising a fatty acid glyceride compound containing sulfate and an amine group, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

Provided is a method for sterilizing or bacteriostatic bacteria using a fatty acid glyceride compound containing sulfate and an amine group, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

Provided is a method for preventing or treating an infectious disease caused by or caused by bacteria using a fatty acid glyceride compound containing a sulfate and an amine group, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

One aspect provides a compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:

[Formula 1]

.

.

In Formula 1, A may be a saturated or unsaturated hydrocarbon chain. A is a substituted or unsubstituted C ₁ to C ₃₀ alkyl group, a substituted or unsubstituted C ₂ to C ₃₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₃₀ alkynyl group, or a combination thereof. can A may be a C ₁ to C ₃₀ alkyl group substituted (ie, branched) with a C ₁ to C ₁₀ alkyl group. A may be a C ₁₃ alkyl group substituted with a methyl group.

In Formula 1, X is each independently a substituted or unsubstituted C ₁ to C ₁₀ alkyl group, a substituted or unsubstituted C ₂ to C ₁₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₁₀ alkynyl group , or a combination thereof. X may be substituted with a hydroxyl group or a C ₁ to C ₁₀ hydroxyalkyl group. X may be a propyl group substituted with a hydroxyl group or an ethyl group substituted with a hydroxymethyl group (-CH ₂ -OH).

In Formula 1, Y is a substituted or unsubstituted C ₁ to C ₁₀ alkyl group, a substituted or unsubstituted C ₂ to C ₁₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₁₀ alkynyl group, or these It may be selected from a combination of Y may be an ethyl group.

In Formula 1, Z may be a substituted or unsubstituted amine group. Z may be an amine group substituted with a C ₁ to C ₁₀ alkyl group. Z may be a methylamine group.

The compound may be referred to as a glyceride compound or a fatty acid glyceride compound. The term “glyceride” refers to fatty acid esters of glycerol. A glyceride compound is a monoglyceride in which one molecule of fatty acid is ester-bonded to one molecule of glycerol, diglyceride in which two molecules of fatty acid are ester-bonded to one molecule of glycerol, or triglyceride in which three molecules of fatty acid are ester-bonded to one molecule of glycerol. can be The compound may be a monoglyceride in which one molecule of glycerol and one molecule of fatty acid are ester-bonded. The compound may be a fatty acid glyceride compound containing sulfate and an amine group.

The compound includes a derivative of the compound represented by the formula (1). The term "derivative" refers to a compound obtained by substituting a part of the structure of the compound with another atom or group of atoms.

The compound represented by Formula 1 may be a compound represented by Formula 2 or Formula 3:

[Formula 2]

; 및

; and

[Formula 3]

.

.

The term “hydrocarbon” refers to an organic compound composed of carbon and hydrogen. The hydrocarbon may be an aliphatic saturated hydrocarbon, an aliphatic unsaturated hydrocarbon, an alicyclic hydrocarbon, or an aromatic hydrocarbon. The saturated hydrocarbon refers to a hydrocarbon composed of a single bond. The unsaturated hydrocarbon refers to a hydrocarbon containing one or more double bonds or triple bonds.

The term "substitution" in the above "unsubstituted" or "substituted" refers to introduced instead of a hydrogen atom when a derivative is formed by substituting one or more hydrogen atoms in an organic compound with another atomic group, and the substituent refers to an introduced atomic group . The substituent is a halogen atom, a hydroxyl group, a nitro group, a cyano group, an amine group, an amidino group, an acetamino group, hydrazine, hydrazone, a carboxyl group, a sulfonyl group, a sulfamoyl group, a sulfonic acid group, phosphoric acid, C ₁ to C ₁₀ Alkyl group, C ₁ To C ₁₀ Alkoxy group, C ₂ To C ₁₀ Alkenyl group, C ₂ To C ₁₀ Alkynyl group, C ₃ To C ₁₀ Cycloalkyl group, C ₆ To C ₁₀ Aryl group, C It may be a ₆ to C ₁₀ heteroaryl group, a C ₆ to C ₂₀ arylalkyl group, a C ₆ to C ₂₀ heteroarylalkyl group, or a combination thereof.

The term "halogen" atom refers to an atom belonging to group 7 of the periodic table. Halogen atoms include fluorine, bromine, chlorine, iodine and the like.

The term “alkyl” refers to a fully saturated branched or unbranched (or straight-chain or linear) hydrocarbon. The alkyl is C ₁ to C ₃₀ , C ₁ to C ₂₀ , C ₁ to C ₁₅ , C ₁ to C ₁₀ , or C ₁ to C ₅ may be an alkyl group. The alkyl is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, iso-amyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, or n-heptyl.

The term “alkenyl” refers to a branched or unbranched hydrocarbon having one or more carbon-carbon double bonds. The alkenyl group may be a C ₂ to C ₃₀ , C ₂ to C ₂₀ , C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ alkenyl group. The alkenyl group is, for example, vinyl, allyl, butenyl, isopropenyl, or isobutenyl.

The term "alkynyl" refers to a branched or unbranched hydrocarbon having at least one carbon-carbon triple bond. The alkynyl group may be a C ₂ to C ₃₀ , C ₂ to C ₂₀ , C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ alkynyl group. The alkynyl is, for example, ethynyl, butynyl, isobutynyl, or isopropynyl.

The term “amine” refers to a functional group in which one or more hydrogens are substituted with an alkyl or aromatic ring in ammonia (NH ₃ ). The amine group may be, for example, an amino group (—NH ₂ ) and an alkylamine group.

The term "hydroxy" refers to a -OH functional group (hydroxyl group). The hydroxyalkyl group may be an alkyl group substituted with a hydroxy group.

The term “alkoxy” refers to an alkyl single bonded to an oxygen atom. Said alkoxy is, for example, methoxy, ethoxy, or butoxy.

The term "isomer" in the term "stereoisomer" refers to a compound that has the same molecular formula but does not have the same spatial arrangement or connection mode of constituent atoms in the bonsai. Isomers include, for example, structural isomers, and stereoisomers. The stereoisomer may be a diasteromer or an enantiomer. Enantiomers refer to isomers that do not overlap their mirror images, such as the relationship between the left and right hands, and are also called optical isomers. Enantiomers are divided into R (Rectus: clockwise) and S (Sinister: counterclockwise) when 4 or more substituents differ from each other at the chiral central carbon. Diastereomers refer to stereoisomers that are not in a mirror image relationship, and are isomers caused by different spatial arrangement of atoms. The diastereomers may be divided into cis-trans isomers and conformational isomers or conformers.

The term “solvate” refers to a compound solvated in an organic or inorganic solvent. The solvate is, for example, a hydrate.

The term "salt" refers to inorganic and organic acid addition salts of compounds. 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 hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salt is formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl salt, trichloroacetic acid, trifluoroacetate , benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspartate It may be an acid. 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 compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof. Brevibacillus brevis PTH23 strain (Accession No.: KCTC 14263BP) is provided.

Brevibacillus sp. Bacteria are Gram-positive bacteria of the Paenibacillaceae family. Brevibacillus brevis is a gram-positive, aerobic, and spore-forming bacillus found in soil, air, water, and decay.

The strain may produce a compound represented by Formula 1 according to an aspect, a stereoisomer, a solvate, or a pharmaceutically acceptable salt.

The strain includes variants thereof. A variant may be, for example, a variant caused by natural mutation or artificial mutation. Artificial mutations may be caused by physical mutagens such as ultraviolet rays, or chemical mutagens such as basic compounds.

The strain includes spores, cells, or cultures of the strain.

Another aspect is Brevibacillus brevis ( Brevibacillus brevis ) culturing the PTH23 strain (Accession Number: KCTC 14263BP); and

Comprising the step of isolating the compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof from the culture of the strain,

Provided is a method for producing a compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

Brevibacillus brevis ( Brevibacillus brevis ) PTH23 strain, the compound represented by Formula 1, stereoisomers, solvates, and pharmaceutically acceptable salts are as described above.

The method includes culturing the Brevibacillus brevis PTH23 strain (Accession No.: KCTC 14263BP). The culturing step may be culturing the strain in a liquid medium or a solid medium. The medium may contain, for example, glucose, starch syrup, dextrin, starch, molasses, animal oil, or vegetable oil as a carbon source. The medium may contain, for example, bran, soybean meal, wheat, malt, cottonseed meal, fish meal, cornstarch, broth, yeast extract, malt extract, ammonium sulfate, sodium nitrate, or urea as a nitrogen source.

The culture may be cultured while shaking or standing still under aerobic conditions. The incubation temperature may be, for example, from about 20°C to about 40°C, from about 25°C to about 37°C, from about 28°C to about 35°C, or about 30°C. The incubation time may be, for example, from about 1 day to about 4 months, from about 1 day to about 2 months, from about 1 day to about 6 weeks, from about 1 day to about 1 month, from about 1 day to about 2 weeks, or about 1 day. to about 1 week.

The method includes isolating the compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof from the culture solution.

Separation may include performing concentration, centrifugation, filtration, or chromatography on the culture solution. The chromatography may be, for example, column chromatography, planar chromatography, paper chromatography or thin film chromatography depending on the type of stationary phase. The chromatography may be, for example, gas chromatography, liquid chromatography, or affinity chromatography, depending on the physical properties of the mobile phase. The liquid chromatography may be, for example, high performance liquid chromatography (HPLC). The chromatography may be, for example, ion exchange chromatography, size-exclusion chromatography, depending on the separation method. The chromatography may be, for example, normal phase chromatography or reverse phase chromatography.

Another aspect provides an antibacterial composition comprising a compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

The composition may have antimicrobial activity against microorganisms. The composition may have antimicrobial activity against, for example, pathogenic or non-pathogenic microorganisms. The composition may have antibacterial activity against gram-positive bacteria.

The composition may have antibacterial activity against antibiotic-resistant bacteria. The term “antibiotic resistance” refers to drug resistance that can survive exposure of microorganisms to antibiotics, and antibiotic-resistant bacteria refers to bacteria having antibiotic resistance. The composition may have antibacterial activity against vancomycin-resistant bacteria. The vancomycin-resistant bacterium may be Vancomycin-resistant Enterococcus (VRE).

The composition may have antibacterial activity against bacteria of Enterococcus sp., Bacillus sp., or Staphylococcus sp. The composition may have antibacterial activity against bacteria of Enterococcus faecium, Bacillus thuringiensis, or Staphylococcus aureus.

The composition may further include a known active ingredient having antimicrobial activity against microorganisms.

The composition may be a pharmaceutical composition, a composition for disinfection, a composition for feed, or a combination thereof. The pharmaceutical composition includes pharmaceuticals and quasi-drugs.

The pharmaceutical composition may further include a carrier, excipient or diluent. Carriers, excipients and diluents can be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, unknown. vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid formulation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid formulation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid formulation may be a suspension, an internal solution, an emulsion, or a syrup. The liquid formulation may contain water or liquid paraffin. The liquid formulation may contain excipients, for example, wetting agents, sweetening agents, perfuming agents, or preservatives. In the pharmaceutical composition, the preparation for parenteral administration may be a sterile aqueous solution, non-aqueous solution, suspension, emulsion, freeze-dried or suppository. Non-aqueous solvents or suspending agents may include vegetable oils or esters. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin, or glycerogelatin.

The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the subject, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, the compound, an isomer, derivative, solvate, or pharmaceutically acceptable salt thereof may be, for example, from about 0.0001 mg/kg to about 100 mg/kg, or from about 0.001 mg/kg to about 100 mg/kg. The amount can be administered in divided doses from 1 to 24 times a day, 1 to 7 times per 2 days to 1 week, or 1 to 24 times per 1 month to 12 months. In the pharmaceutical composition, the compound, isomer, derivative, solvate, or pharmaceutically acceptable salt thereof is present in an amount of from about 0.0001% to about 10% by weight, or from about 0.001% to about 1% by weight, based on the total weight of the composition. may be included.

The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. The composition may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

The disinfecting composition may be a disinfectant cleaner, shower foam, mouthwash, wet tissue, hand sanitizer, humidifier filler, mask, ointment, or filter filler.

The composition for feed may be added to the feed of mammals or insects other than humans. For example, the composition for feed may be added to silkworm feed or mulberry leaves through application, spraying, coating, immersion, or a combination thereof.

Another aspect comprises the step of applying, spraying, coating, immersing, or a combination thereof in vitro with the compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof , to provide a method for sterilizing or bacteriostatic bacteria.

The term “applying” refers to the application of the composition to the surface of an object. The term "spray" refers to ejecting the composition. The term "coating" refers to coating the surface of an object with a thin film of the composition. The term “immersion” refers to immersion of an object in the composition.

The term “sterilization” refers to the action of killing microorganisms. The term "bacteriostatic" refers to the action of inhibiting the growth or proliferation of microorganisms.

The method may be performed by applying, spraying, coating, immersing, or a combination thereof, a compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof to silkworm.

Another aspect is a method of preventing or treating an infectious disease caused or caused by bacteria, comprising administering to an individual a compound represented by Formula 1, a stereoisomer, a derivative, or a pharmaceutically acceptable salt thereof to provide.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat or cat. The subject may be an insect, such as a silkworm. The subject may be suffering from, or highly likely to suffer from, a bacterial infection or symptoms associated therewith.

The term "prevention" refers to any action that suppresses or delays the onset of a disease by administration of a composition. The term “treatment” refers to any action in which the symptoms of a disease are ameliorated or beneficially changed by administration of the composition.

Infectious diseases caused or caused by bacteria are, for example, caused by or caused by bacteria of the genus Enterococcus sp., Bacillus sp., or Staphylococcus sp. It could be an infectious disease causing it. The disease may be, for example, meningitis, endocarditis, suppurative arthritis, acne, boils, folliculitis, impetigo, cellulitis, food poisoning, osteomyelitis, otitis media, cystitis, sepsis, pneumonia, diarrhea, or a combination thereof. have. The disease may be, for example, sotto disease of silkworms.

The method may further comprise administering an antibiotic to the subject. The antibiotic may be administered to the subject simultaneously, separately, or sequentially with the compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. The pharmaceutical composition may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. The dosage 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/kg, on an adult basis. can be The administration may be administered once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

The novel sulfate and amine group-containing fatty acid glyceride compounds, stereoisomers, solvates, or pharmaceutically acceptable salts thereof have antibacterial activity, in particular, antibacterial activity against vancomycin-resistant bacteria, thereby sterilizing or bacteriostatic or bactericidal It can be used to prevent or treat infectious diseases caused by or caused by.

1 is a photograph of the culture medium of Brevibacillus brevis ( Brevibacillus brevis ) PTH23 strain.

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Isolation of Renjimycin and Confirmation of Structure

1-1. Isolation of Renjimycin-producing Strain

Renjimycin A and Renjimycin B production strains Brevibacillus brevis ( Brevibacillus brevis ) PTH23 strain was isolated from that subjected to heat shock at 55 ° C. to secretions of Renji beetle ( Onthophagus lenzii ). PTH23 strain is actinomycete isolation solid medium (2 g sodium caseinate, 0.1 g asparagine, 4 g sodium propionate (sodium propionate), 0.5 g dipotassium phosphate (dipotassium phosphate) ), 0.1 g of magnesium sulfate, 0.001 g of ferrous sulfate, and 15 g of agar powder/1 L of sterile water) (FIG. 1).

Based on the results of 16S rDNA sequencing, the strain was identified as the genus Brevibacillus . This strain was named Brevibacillus brevis PTH23 strain, and the strain was deposited with the Korea Research Institute of Bioscience and Biotechnology on August 06, 2020 (Accession No.: KCTC 14263BP).

1-2. Culture of Brevibacillus brevis PTH23 strain

Brevibacillus brevis PTH23 strain was spread on sterile YEME solid medium (4 g yeast extract, 10 g malt extract, 4 g glucose, and 18 g agar/1 L distilled water) and plated at about 4 Weekly cultured.

The spores of the cultured PC5 strain were inoculated into a 50 mL volume of YEME liquid medium (4 g of yeast extract, 10 g of malt extract, and 4 g of glucose/1 L of distilled water), and stirred at 30 °C while shaking at 170 rpm. It was incubated for about 3 days at the temperature.

1-3. Isolation and purification of Renjimycin

A culture of the Brevibacillus brevis PTH23 strain cultured in Example 1-2 was obtained.

1 L of the cultured PTH23 strain and about 2.5 L of ethyl acetate (EtOAc, Daejung Hwa Gold Co., Ltd.) were put into a separatory funnel mounted on a stand, and then the stopper was closed and shaken up, down, left and right for 3 minutes to perform primary extraction. After that, it was mounted on a stand again to completely separate the water layer and the EtOAc layer, and then the valve of the separatory funnel was opened to remove the water layer. Fresh EtOAc was added to the aqueous layer, and repeated extraction was performed in the same manner. The EtOAc layer was separately stored in a clean flask, and anhydrous sodium sulfate (Anhydrous Sodium Sulfate) (Daejeong Chemical Co., Ltd.) was added to remove the water remaining in the culture, and foreign substances were removed by passing through several layers of clean gauze. . The EtOAc layer from which the water was removed was transferred back to a 3 L round flask, and then dried under reduced pressure using a vacuum dryer. By culturing a total of 60 L of the strain, about 5 g of crude extract was obtained as a result of extraction.

In order to purify renjimycin A and renjimycin B from the PTH23 strain extract, reverse phase fractionation was performed using an open column primarily. An open column filled with C ₁₈ resin was stabilized with methanol (MeOH) 20% (v/v) / H ₂ O 80% (v/v), and the extract adsorbed to Celite 545 was packed thereon. The extracts were fractionated with 20% (v/v), 40% (v/v), 60% (v/v), 80% (v/v), and 100% (v/v) methanol solvents, respectively. An 80% (v/v) fraction containing Renjimycin A and Renjimycin B was transferred to a 3 L round flask and dried under reduced pressure.

To obtain pure renjimycin A and renjimycin B from the dried 80% (v/v) methanol fraction, it was purified by high-performance liquid chromatography (HPLC).

Conditions: using a reversed-phase column (C ₁₈ YMC-Pack ODS-A S-5 μm 250 × 10.0 mm), 90% (v/v) methanol (MeOH)/aqueous solution gradient conditions (flow rate: 2 mL/min, Detection: RI (refractive index)) was separated and purified for about 30 minutes. Renjimycin A was identified in the approximately 18-minute fraction, and renjimycin B was identified in the approximately 16-minute fraction.

This process was repeated to obtain about 10 mg of renjimycin A and about 8 mg of renjimycin B from about 5 g of the crude extract.

1-4. Confirmation of chemical structure of Renjimycin

The structures of renjimycin A and renjimycin B were confirmed based on 1D and 2D nuclear magnetic resonance (NMR) spectra. Nuclear magnetic resonance spectra ( ¹ H NMR, ¹³ C NMR) were 500 MHz NMR of Bruker, and CDCl ₃ -d ₁ was used as the solvent.

Table 1 shows the structural positioning of renjimycin A and renjimycin B by nuclear magnetic resonance spectra.

[Lenzimycin A]

(1) Molecular formula: C ₂₁ H ₄₄ NO ₇ S

(2) molecular weight: 453

(3) Color: Transparent

(4) ¹ H-NMR (CDCl ₃ -d ₁ , 800 MHz): see Table 1

(5) ¹³ C-NMR (CDCl ₃ -d ₁ , 200 MHz): see Table 1

[Lenzimycin B]

(1) Molecular formula: C ₂₁ H ₄₄ NO ₇ S

(2) molecular weight: 453

(3) Color: Transparent

(4) ¹ H-NMR (CDCl ₃ -d ₁ , 850 MHz): see Table 1

(5) ¹³ C-NMR (CDCl ₃ -d ₁ , 212.5 MHz): see Table 1

location Renjimycin A Renjimycin B δ _C , category δ _H , mult ( J in Hz) δ _C , category δ _H , mult ( J in Hz) One 173.8 C 173.3 C 2 34.1 CH ₂ 2.30 t (7.7) 34.3 CH ₂ 2.30 t (7.0) 3 24.9 CH ₂ 1.60 m 24.9 CH ₂ 1.60 m 4-10 30.0-27.1 CH ₂ 1.31-1.23 m 30.0-27.1 CH ₂ 1.31-1.23 m 11 36.6 CH ₂ 1.27, 1.07 m 36.6 CH ₂ 1.27, 1.07 m 12 34.4 CH 1.28 m 34.4 CH 1.28 m 12-Me 19.2 CH ₃ 0.83 t (6.5) 19.2 CH ₃ 0.84 t (6.5) 13 29.5 CH ₂ 1.25, 1.12 m 29.5 CH ₂ 1.25, 1.12 m 14 11.4 CH ₃ 0.84 t (7.2) 11.4 CH ₃ 0.85 t (7.2) One' 64.5 CH ₂ 4.11 m 59.8 CH ₂ 3.73 d (5.3) 2' 69.1 CH 3.98 m 72.5 CH 4.97 m 3' 68.1 CH ₂ 4.00, 3.90 m 63.4 CH ₂ 4.08 m One" 61.6 CH ₂ 4.19 m 61.2 CH ₂ 4.20 m 2" 49.7 CH ₂ 3.15 m 49.9 CH ₂ 3.14 m 2"-NH 9.64 br s 9.73 br s 2"-N-Me 33.6 CH ₃ 2.70 s 33.7 CH ₃ 2.71 s

Chemical structural formulas of Renjimycin A and Renjimycin B analyzed from the nuclear magnetic resonance spectral data and the three-dimensional structure analysis result are shown below.

[Rengemycin A]

[renjimycin B]

1-5. Confirmation of antibacterial activity of Renjimycin

In order to measure the bacterial growth inhibitory effect of Renjimycin A and Renjimycin B, Muller Hinton I medium (BBL, Sparks, MD, USA) was used for plate medium dilution.

As each quality control (QC) strain, Escherichia coli (ATCC 25922), Enterococcus faecalis ( Enterococcus faecalis ATCC 29212), Enterococcus faecium ( Enterococcus faecium ATCC 19434), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ATCC 27853 ), Staphylococcus aureus ATCC 29213 ), Salmonella enterica ( ATCC 14028 ), and Klebsiella pneumoniae ATCC 10031 ) were used. As control antibiotics, ampicillin and norfloxacin were used, and the minimum inhibitory concentration was measured by diluting the sample to a concentration of 0.25 μg/mL to 128 μg/mL in the medium.

Table 2 shows the results of measuring the antibacterial activity of renjimycin A and renjimycin B, and control samples, ampicillin and norproxacin, against the strains to be searched.

number strain Accession number note MIC (μg/mL) Renjimycin A Renjimycin B ampicillin norproxacin One Bacillus sp . CCARM 9248 64 8 16 0.12 2 Bacillus cereus CCARM 0002 128 128 8 One 3 Enterococcus faecalis CCARM 5025 VRE ( vanB ) >128 >128 2 2 4 Enterococcus faecium CCARM 5024 VRE ( vanA ) 0.5 One 32 4 5 Staphylococcus aureus CCARM 0205 sensibility 32 64 2 One 6 Staphylococcus aureus CCARM 3855 sensibility >128 >128 ≤0.25 One 7 Staphylococcus aureus CCARM 3089 MDR >128 >128 64 128 8 Escherichia coli CCARM 0230 >128 >128 2 ≤0.03 9 Escherichia coli CCARM 0237 >128 >128 8 0.5 10 Escherichia coli CCARM 0235 >128 >128 4 0.06 11 Pseudomonas aeruginosa CCARM 0219 >128 >128 >128 One 12 Pseudomonas aeruginosa CCARM 0223 >128 >128 >128 0.5 13 Pseudomonas aeruginosa CCARM 0225 >128 >128 0.5 0.25 14 Salmonella Typhimurium CCARM 0240 >128 >128 One 0.06 15 Enterobacter cloacae CCARM 0252 >128 >128 >128 0.06 16 Enterobacter cloacae CCARM 0253 >128 >128 One ≤0.03 17 Klebsiella oxytoca CCARM 0248 >128 >128 >128 ≤0.03 18 Klebsiella aerogenes CCARM 0249 >128 >128 2 0.06 19 Escherichia coli ATCC 25922 QC strain >128 >128 4 0.06 20 Enterococcus faecalis ATCC 29212 QC strain >128 >128 0.5 2 to 8 21 Enterococcus faecium ATCC 19434 QC strain 16 8 0.5 - 22 Pseudomonas aeruginosa ATCC 27853 QC strain >128 >128 >128 1 to 4 ^a
23 Staphylococcus aureus ATCC 29213 QC strain >128 >128 0.13 0.5 to 2 ^a 24 Salmonella enterica ATCC 14028 QC strain 128 128 0.13 - 25 Klebsiella pneumoniae ATCC 10031 QC strain >128 >128 32 -

a: allowable range

VRE: Vancomycin-Resistant Enterococci

MDR: multiple drug resistance

As shown in Table 2, the MIC for the search strain of renjimycin A was 0.5 μg/mL in E. faecium CCARM 5024, Bacillus sp. 64 μg/mL for CCARM 9248 and 32 μg/mL for S. aureus CCARM 0205. For other experimental bacteria, it was 128 μg/mL or more.

The MIC for the search strain of Renjimycin B was 1 μg/mL in E. faecium CCARM 5024, Bacillus sp. 8 μg/mL for CCARM 9248, and 64 μg/mL for S. aureus CCARM 0205. For other experimental bacteria, it was 128 μg/mL or more.

All three types of bacteria with high antibacterial activity of Renjimycin A and Renjimycin B were Gram-positive bacteria. In particular, E. faecium CCARM 5024, which has vancomycin resistance gene van A, and Bacillus thuringiensis , a known insect pathogen, and Bacillus sp. CCARM 9248 showed high antibacterial activity.

Therefore, it was confirmed that renjimycin A and renjimycin B had excellent antibacterial activity against gram-positive bacteria, especially vancomycin antibiotic-resistant bacteria, and that renjimycin A and renjimycin B could be used for prevention or treatment of antibiotic-resistant bacteria. .

Korea Institute of Bioscience and Biotechnology KCTC14263BP 20200806

Claims (17)

A compound represented by the following Formula 2 or Formula 3, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:
[Formula 2]

; and
[Formula 3]

. delete delete delete delete A Brevibacillus brevis PTH23 strain (Accession No.: KCTC 14263BP) that produces the compound of claim 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof. Brevibacillus brevis ( Brevibacillus brevis ) Culturing the PTH23 strain (accession number: KCTC 14263BP); and
It comprises the step of isolating the compound of claim 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof from the culture of the strain,
A method for producing a compound of claim 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof. An antibacterial composition comprising the compound of claim 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof. The composition of claim 8, wherein the composition has antibacterial activity against Gram-positive bacteria. The composition of claim 8, wherein the composition has antibacterial activity against vancomycin-resistant bacteria. The composition of claim 8, wherein the composition has antibacterial activity against bacteria of Enterococcus sp., Bacillus sp., or Staphylococcus sp. The method according to claim 8, wherein the composition is Enterococcus faecium ( Enterococcus faecium ), Bacillus thuringiensis ( Bacillus thuringiensis ), or Staphylococcus aureus ( Staphylococcus aureus ) The composition having antibacterial activity against bacteria. The composition of claim 8, wherein the composition is a pharmaceutical composition. The composition of claim 8, wherein the composition is a composition for disinfection. The composition of claim 8, wherein the composition is a composition for feed. Sterilizing or sterilizing bacteria, comprising the step of applying, spraying, coating, immersing, or a combination thereof in vitro with the compound of claim 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof How to sterilize. A method for preventing or treating an infectious disease caused or caused by bacteria, comprising administering the compound of claim 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof to a mammal or insect other than a human. Way.

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