KR102301151B1 - Peptide compound, a preparing method thereof, and a use thereof - Google Patents

Abstract

Novel peptide compounds, stereoisomers, solvates, or pharmaceutically acceptable salts thereof, methods for their production, and uses thereof are provided. Since the peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof has a cell autophagy inhibitory activity, it can be used to prevent or treat various types of cancer. It can also be used to inhibit autophagy of cells in vitro.

Description

Peptide compound, its production method, and its use {Peptide compound, a preparing method thereof, and a use thereof}

It relates to novel peptide compounds, methods for their production, and uses thereof.

Physiologically active substances derived from microorganisms have generally been a source of antibacterial, antifungal, and anticancer drugs, and have been developed as new drugs for the treatment of various diseases including these, or as a template for new drug development. Anticancer agents derived from bacteria include, for example, doxorubicin, bleomycin, mithramycin, neocarzinostatin, pentostatin, and epothilone. As such, the study of bioactive substances derived from bacteria is very important in the development of antibacterial agents, antifungal agents, and anticancer agents.

Cellular autophagy is a mechanism by which cells naturally decompose unnecessary or non-functional components of cells. Autophagy functions to provide energy necessary for cells to survive by destroying or recycling elements that make up cells. In relation to disease, autophagy has been observed to enhance cell survival with appropriate adaptation and response to stress. Conversely, it has been confirmed that cell death and disease enhancement are also present. In particular, in relation to cancer development, it is possible to prevent the generation of cancer cells by decomposing damaged cells through autophagy, but it may also potentially contribute to the growth of cancer cells by allowing abnormal cells to avoid apoptosis. It is known to act as a tumor suppressor in the early stage of tumorigenesis and as a tumor promoter in the late stage of tumor progression (Cheng, Y. et al., Pharmacol. Rev. 2013 Aug 13, 65(4):1162-1197). Therefore, there is a high possibility that a substance that inhibits cell autophagy has an anticancer effect or complements the anticancer effect of other anticancer agents or has a synergistic effect.

Therefore, there is a need to screen for novel compounds that modulate cellular autophagy from microorganisms.

Novel peptide compounds, stereoisomers, solvates, or pharmaceutically acceptable salts thereof are provided.

A method for producing the compound is provided.

It provides a pharmaceutical composition for preventing or treating cancer comprising the compound.

Provided is a composition for inhibiting autophagy of cells in vitro comprising the compound.

A method for preventing or treating cancer using the compound is provided.

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

[Equation 1]

.

.

In Formula 1, R ¹ may be hydrogen, a hydroxyl group, a carbonyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkyl group, or a substituted or unsubstituted C ₁ to C ₂₀ alkoxy group. R ¹ is, for example, a hydroxyl group.

In Formula 1, R ² is hydrogen, a hydroxyl group, a halogen group, a cyano group, -C(=O)R _a , -C(=O)OR _a , -OCO(OR _a ), -C=N(R _{a )} ), -SR _a , -S(=O)R _a , -S(=O) ₂ R _a , -PR _a , A substituted or unsubstituted C ₁ to C ₂₀ alkyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ to C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkynyl group, C ₂ to C ₂₀ alkylene oxide group, substituted or unsubstituted C ₃ to C ₃₀ cycloalkyl group, substituted or unsubstituted C ₆ to C ₃₀ aryl group, substituted or unsubstituted It may be a C ₆ to _{C 30} aryloxy group, a substituted or unsubstituted C ₆ to C ₃₀ heteroaryl group, or a combination thereof. R _a means a substituent, and may be hydrogen, a C ₁ to C ₁₀ alkyl group, or a C ₆ to C ₂₀ aryl group. The R ² _{may be a C 1} to C ₂₀ alkyl group substituted with a substituted or unsubstituted C ₆ to C _{30 aryl group.} R ² is, for example, a substituted or unsubstituted benzyl group.

In Formula 1, R ³ may be a substituted or unsubstituted C ₁ to C ₂₀ alkyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkenyl group, or a substituted or unsubstituted C ₁ to C ₂₀ alkynyl group. R ³ is a substituted or unsubstituted C ₁ to C ₂₀ alkyl group and may be a branched alkyl group. The R ³ is, for example, a secondary butyl (sec-butyl) group or an isopropyl (isopropyl) group.

In Formula 1, R ⁴ is hydrogen, a hydroxyl group, a halogen group, a cyano group, -C(=O)R _a , -C(=O)OR _a , -OCO(OR _a ), -C=N(R _{a )} ), -SR _a , -S(=O)R _a , -S(=O) ₂ R _a , -PR _a , A substituted or unsubstituted C ₁ to C ₂₀ alkyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkoxy group, a substituted or unsubstituted C ₂ to C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkynyl group, C ₂ to C ₂₀ alkylene oxide group, substituted or unsubstituted C ₃ to C ₃₀ cycloalkyl group, substituted or unsubstituted C ₆ to C ₃₀ aryl group, substituted or unsubstituted It may be a C ₆ to _{C 30} aryloxy group, a substituted or unsubstituted C ₆ to C ₃₀ heteroaryl group, or a combination thereof. R _a means a substituent, and may be hydrogen, a C ₁ to C ₁₀ alkyl group, or a C ₆ to C ₂₀ aryl group. R ⁴ _{may be a C 1} to C ₂₀ alkyl group substituted with a substituted or unsubstituted C ₆ to C _{30 aryl group.} R ⁴ is, for example, a substituted or unsubstituted benzyl group.

In Formula 1, R ⁵ may be a substituted or unsubstituted C ₁ to C ₂₀ alkyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkenyl group, or a substituted or unsubstituted C ₁ to C ₂₀ alkynyl group. R ⁵ is a substituted or unsubstituted C ₁ to C ₂₀ alkyl group and may be a straight-chain or branched alkyl group. R ⁵ is, for example, a propyl group or an isobutyl group.

In Formula 1, when R ⁵ is branched alkyl, R ³ may be an isopropyl group.

The term "substituted" in the above "substituted or unsubstituted" 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 substituents are halogen atom, hydroxyl group, nitro group, cyano group, amino group, amidino group, acetamino group, hydrazine, hydrazone, carboxyl group, sulfonyl group, sulfamoyl group, sulfonic acid group, phosphoric acid, C ₁ to C ₅ of alkyl group, C ₁ to C ₅ alkoxy group, C ₂ to C ₅ alkenyl group, C ₂ to C ₅ alkynyl group, C ₃ to C ₁₀ cycloalkyl group, C ₆ to C ₁₀ aryl group, C ₆ to C ₁₀ heteroaryl group, C ₆ to C ₂₀ arylalkyl group, C ₆ to C ₂₀ heteroarylalkyl group, or a combination thereof.

The term "hydroxy" refers to a -OH functional group (hydroxyl group).

The term "carbonyl" denotes a functional group consisting of one carbon atom double bonded to one oxygen atom, that is, C=O.

The term “alkyl” refers to a fully saturated branched or unbranched (or straight-chain or linear) hydrocarbon. The alkyl is 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 “alkoxy” refers to an alkyl single bonded to an oxygen atom. Said alkoxy is, for example, methoxy, ethoxy, or butoxy.

The term “alkenyl” refers to a branched or unbranched hydrocarbon having one or more carbon-carbon double bonds. The C ₂ to C ₂₀ alkenyl group may be a 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 C ₂ to C ₂₀ alkynyl group may be a 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 “halogen” atom includes fluorine, bromine, chlorine, iodine, and the like.

The term "cyano" refers to a functional group consisting of a triple bond between a carbon atom and a nitrogen atom.

The term "alkylene oxide" refers to a cyclic group consisting of one oxygen atom and two or more carbon atoms.

The term “cycloalkyl” refers to a saturated or partially unsaturated non-aromatic monocyclic, bicyclic or tricyclic hydrocarbon group.

The term "aryl", used alone or in combination, refers to an aromatic system comprising one or more rings, including groups in which the aromatic ring is fused to one or more carbon rings. The C ₆ to C ₃₀ aryl group may be a C ₆ to C ₁₅ , or C ₆ to C _{10 aryl group.} The aryl is, for example, phenyl, naphthyl, or tetrahydronaphthyl.

The term "aryloxy" refers to an aryl group single bonded to an oxygen atom.

The term "heteroaryl" refers to a monocyclic or bicyclic organic compound containing one or more heteroatoms selected from the group consisting of N, O, P and S, and the remaining ring atoms are carbon. say The heteroaryl group may include 1 to 5 heteroatoms, and 5 to 10 ring members. The S or N may be oxidized to have several oxidation states.

The term "peptide compound" refers to a compound comprising a peptide or modified peptide derivative compound. A peptide is a compound in which two or more amino acids are linked by a peptide bond between a carboxyl group and an amino group. Depending on the number of constituent amino acids, they are called dipeptides, tripeptides, tetrapeptides, etc., and those with about 10 or less peptide bonds are called oligopeptides, and those with multiple peptide bonds are called oligopeptides. It is called a polypeptide. The modified peptide derivative compound refers to a compound including a compound in which two or more amino acids or amino acid derivatives are linked by a peptide bond. The amino acid refers to an organic compound containing an amine group and a carboxyl group. The amino acid derivative may be a compound in which a functional group of an amino acid is substituted with a substituent. The substituents are the same as described above. The amino acid derivative is, for example, phenylalaninol, phenylalanine substituted with butanoic acid, and phenylalanine substituted with 3-methylbutanoic acid.

The peptide compound may be a compound composed of phenylalanine-isoleucine-phenylalaninol in which the N-terminus is substituted with butanoic acid. The peptide compound may be a compound consisting of phenylalanine-valine-phenylalinol in which the N-terminus is substituted with 3-methylbutanoic acid.

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

[Equation 2]

; 및

; and

[Equation 3]

.

.

The peptide compound may be a compound represented by Formula 4 or Formula 5:

[Equation 4]

(액시디필라미드 A); 및

(acidipilamide A); and

[Equation 5]

(액시디필라미드 B).

(acidipyramid B).

"Isomer" of the term "stereoisomer" refers to a compound in which the molecular formula is the same, but the connection mode or spatial arrangement of the constituent atoms in the bonsai is not the same. Isomers include, for example, structural isomers, and stereoisomers. The stereoisomer may be a diasteromer or an enantiomer. An enantiomer refers to an isomer that does not overlap the mirror image as in the relationship between the left hand and the right hand, and is also called an optical isomer. 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 can be divided into cis-trans isomers due to the spatial arrangement of atoms.

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 the 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 is Streptacidiphilus rugosus ( Streptacidiphilus rugosus ) culturing the strain; And it provides a method for producing a peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, comprising the step of isolating the peptide compound according to an aspect from the culture solution.

The peptide compound, stereoisomer, solvate, and pharmaceutically acceptable salt are as described above.

The method comprises culturing a Streptacidophilus lugosus strain.

The Streptacidophilus lugosus strain may be a Streptacidophilus lugosus species AM-16 strain.

The culturing step may be culturing the strain in a liquid medium or a solid medium. The medium may contain, for example, glucose, rice, starch syrup, dextrin, starch, molasses, animal oil, or vegetable oil as a carbon source. These may include, for example, yeast extract, beef extract, peptone, bran, soybean meal, wheat, malt, cottonseed meal, fish meal, cornstarch, broth, ammonium sulfate, sodium nitrate or urea as a nitrogen source. If necessary, the medium may contain salt, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, or other inorganic salts that promote ion formation. The medium includes, for example, yeast extract, bovine organ extract, peptone, and glucose.

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 37°C, from about 25°C to about 30°C, or 27°C. The culture time is, for example, 1 to 4 days, 1 to 8 days, 1 to 2 weeks, 1 to 4 weeks, 1 to 2 months, 1 week to 2 months, 2 weeks to 2 months, 1 month. to 2 months, or 6 weeks.

The method comprises isolating the peptide compound from the culture medium.

The separating may include concentration, centrifugation, filtration, or chromatography of the culture solution. For example, the culture solution may be extracted with ethyl acetate, water, or a combination thereof. The obtained concentrate can be fractionated by high performance liquid chromatography (HPLC). The HPLC may be performed using a reverse phase semipreparative HPLC column using 30% to 60% acetonitrile aqueous solution as a mobile phase. The isolated peptide compound can be obtained with a purity of at least about 80%, about 90%, or about 99%.

Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising the peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to an aspect.

The peptide compound, stereoisomer, solvate, and pharmaceutically acceptable salt are as described above.

The peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof may inhibit autophagy activity of cancer cells. The peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof may promote the death of tumor cells by inhibiting autophagy. Accordingly, the peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof may be used as an anticancer agent.

The cancer is, for example, intrahepatic cholangiocarcinoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, mycosis fungoides, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, basal cell carcinoma ; Non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, juvenile brain tumor, juvenile lymphoma, juvenile leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, neuroblastoma, pelvic ureteric cancer, renal cancer, malignant soft tissue tumor, malignant bone tumor, malignancy Lymphoma, malignant mesothelioma, malignant melanoma, eye tumor, vulvar cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoma, gastrointestinal stromal tumor, Wilms tumor, breast cancer, sarcoma, penile cancer, pharyngeal cancer, pregnancy villi Disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic brain tumor, rectal cancer, rectal carcinoma, vaginal cancer, spinal cord tumor, acoustic schwannoma, pancreatic cancer, salivary gland cancer, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung It may be squamous cell carcinoma, skin cancer, anal cancer, laryngeal cancer, or a combination thereof. The cancer may be, for example, breast cancer, colorectal cancer, adenocarcinoma, or lung cancer.

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 act of improving or beneficially changing the symptoms of a disease by administering the composition.

The pharmaceutical composition may include a peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition may further include a known active ingredient having anticancer activity.

The pharmaceutical composition may further include a carrier, excipient or diluent. Carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline 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 oral 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 individual, 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, 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 compositions may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

Another aspect provides a composition for inhibiting autophagy of cells in vitro, comprising the peptide compound, stereoisomer, solvate, or salt thereof according to the aspect. Another aspect provides a method of inhibiting autophagy of a cell by adding the peptide compound, stereoisomer, solvate, or salt thereof according to the aspect to a cell in vitro.

The peptide compounds, stereoisomers, solvates, salts, and autophagy are as described above.

The cell may be a cancer cell. The cell may be a cervical cancer cell, for example a HeLa cell.

Another aspect is to prevent or treat cancer comprising administering to an individual a pharmaceutical composition for preventing or treating cancer comprising a peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to an aspect provide a way

The peptide compound, stereoisomer, solvate, pharmaceutically acceptable salt, cancer, prophylaxis, treatment, and pharmaceutical composition are as described above.

The subject may be a mammal, such as a human, mouse, rat, cow, horse, pig, dog, monkey, sheep, goat or cat. The subject may be suffering from, or likely to have, cancer.

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 severity of the 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 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 on an adult basis. can be The administration may be administered once a day, multiple times a day, or once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks to once a year.

Since the novel peptide compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof has a cell autophagy inhibitory activity, it can be used to prevent or treat various types of cancer. It can also be used to inhibit autophagy of cells in vitro.

1 is a photograph of the culture medium of Streptacidiphilus rugosus (Streptacidiphilus rugosus) AM-16 strain.
Figures 2a to 2c are images of immunoblotting when HeLa cells were treated with an aciddipilamide compound for 4 hours (Figure 2a), 8 hours (Figure 2b), and 12 hours (Figure 2c), Figure 2d is a graph showing the hydrolysis level (relative fluorescence unit (RFU)) of suc-LLVY-AMC according to the treatment time of the axidipilamide compound.
3 is an image obtained by immunoblotting when the axidipilamide compound and bafilomycin A1 are co-treated or when the axidipilamide compound is treated in the condition of amino acid deficiency.

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 axidipilamide and confirmation of structure

1-1. Isolation of acidipilamide-producing strains

The AM-16 strain, an acidophilic soil bacterium, was isolated from acidified pine forest soil. Various types of isolation media were used for isolating the strain, and the AM-16 strain was acidified to pH 4.5 on modified Bennett solid medium (1 g yeast extract, 2 g beef extract, 1 g peptone, 10 g glucose, 20 g of agar powder/1 L of sterile distilled water (acidified with HCl).

Based on the 16S rDNA sequencing results, it was confirmed that the corresponding strain was a strain of a new species that had not been previously identified (FIG. 1). This strain was named Streptacidiphilus rugosus species AM-16 strain.

1-2. Culture of Streptacidophilus lugosus AM-16 strain

Streptacidophilus lugosus sp. AM-16 strain acidified to pH 4.5 on modified Bennett solid medium (1 g yeast extract, 2 g beef extract, 1 g peptone, 10 g glucose, 20 g agar powder / 1 L of sterile distilled water (acidified with HCl)) and incubated at 30° C. for about 4 weeks.

The spores of the cultured AM-16 strain were cultured in a 150 mL volume of pH 4.5 acidified modified Bennett broth (1 g yeast extract, 2 g beef extract, 1 g peptone, 10 g glucose/1 L sterile distilled water). (acidified with HCl)) and incubated for 4 days at a temperature of 30° C. while shaking at 200 rpm. Then, 10 mL of the culture medium was inoculated in 1 L of pH4.5 acidified modified Bennett liquid medium, and incubated at 30° C. for 4 days while shaking at 170 rpm.

1-3. Isolation and purification of acidipilamide

The AM-16 strain cultured in Example 1-2 was organically extracted using ethyl acetate (EtOAc). Specifically, 1 L of AM-16 strain culture solution and 1 L of EtOAc (Daejeong Chemical Co., Ltd.) were put into a separatory funnel mounted on a stand, the stopper was closed, and the primary extraction was performed by shaking up and down, left and right for 1 minute. After that, it was mounted on a stand again and the water layer and EtOAc layer were completely divided, 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 stored separately in a clean flask, and anhydrous sodium sulfate was added to remove excess water. The dehydrated EtOAc layer was transferred back to a 3 L round flask, and then dried under reduced pressure using a vacuum dryer. A total of 72 L strains were cultured to obtain about 10 g of a crude extract.

The obtained crude extract was dissolved in a sufficient amount of methanol (MeOH), and solid impurities were removed using a syringe filter (ADVANTEC, 25HP045AN). The purification process was performed using semi-preparative HPLC. For purification, a reversed-phase column (YMC-Triart C ₁₈ 250 × 10 mm) is used, and as a separation solvent, 30% (v/v) to 60% (v/v) acetonitrile (ACN)/aqueous solution ( 0.1% (v/v) formic acid) was used under gradient conditions (flow rate: 2 mL/min, detection: UV 230 nm) for 40 minutes. To confirm the composition of the fraction, LC/MS was used in which Agilent technologies' 1200 series LC and 6130 series mass spectrometer were connected. Under these conditions, it was confirmed through LC/MS analysis that the new substances, axidipilamides A and B, were simultaneously eluted about 38 minutes after the HPLC injection.

In order to purify acidipilamides A and B into their respective pure substances, an additional separation experiment was performed by modifying the separation conditions. The second purification condition uses the same reverse-phase HPLC column, and isocractic conditions (flow rate: 2 mL/min, detection: methanol/aqueous 70% (v/v) (0.1% (v/v) formic acid) solvent) UV 230 nm). After HPLC injection, pure axidipilamide A was eluted at about 31 minutes and pure axidipilamide B at about 34 minutes, respectively. Through repeated experiments, it was possible to obtain about 8 mg of axidipilamide A and 9 mg of axidipilamide B from 10 g of the extract.

1-4. Physicochemical Characterization of Acidipilamide

(1) Confirmation of physicochemical properties of acidipilamide

The structure of acidipilamide was 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 MeOD-d ₃ was used as the solvent.

The structural positioning of axidipilamides A and B by nuclear magnetic resonance spectra are shown in Tables 1 and 2, respectively.

[acidiphilamide A]

(1) Molecular formula: C ₂₈ H ₄₀ N ₃ O ₄

(2) molecular weight: 481

(3) Color: White

(4) ¹ H-NMR (DMSO- d ₆ , 600 MHz): see Table 1

(5) ¹³ C-NMR (DMSO- d ₆ , 150 MHz): see Table 1

location δ _c type δ _H mult ( J in Hz) 1a 62.4 CH ₂ 3.32 m 1b 3.24 m 1-OH OH 4.79 br s 2 52.1 CH 3.92 m 2-NH NH 7.84 d (8.5) 3a 36.3 CH ₂ 2.87 dd (14.0, 5.5) 3b 2.61 dd (14.0, 8.5) 4 139.0 C 5, 9 129.0 2CH 7.20 d (7.0) 6, 8 128.1 2CH 7.23 dd(7.0, 7.0) 7 125.8 CH 7.14 dd(7.0, 7.0) 10 170.3 C 11 57.0 CH 4.10 dd (9.0, 8.0) 11-NH NH 7.82 d (9.0) 12 36.9 CH 1.64 m 13a 24.2 CH ₂ 1.37 m 13b 1.0 m 14 11.0 CH ₃ 0.78 t (7.5) 15 15.2 CH ₃ 0.74 d (7.0) 16 171.1 C 17 53.6 CH 4.54 ddd (10.0, 8.5, 4.0) 17-NH NH 8.07 d (8.5) 18a 37.2 CH ₂ 2.93 dd (14.0, 4.0) 18b CH 2.70 dd (14.0, 10.0) 19 138.0 C 20, 24 129.1 2CH 7.24 d (7.0) 21, 23 127.9 2CH 7.22 dd(7.0, 7.0) 22 126.1 CH 7.16 dd(7.0, 7.0) 25 172.0 C 26 28.3 CH ₂ 1.98 td (7.5, 2.0) 27 18.6 CH ₂ 1.37 m 28 13.4 CH ₃ 0.68 t (7.5)

[acidiphilamide B]

(1) Molecular formula: C ₂₈ H ₄₀ N ₃ O ₄

(2) molecular weight: 481

(3) Color: White

(4) ¹ H-NMR (DMSO- d ₆ , 600 MHz): see Table 2

(5) ¹³ C-NMR (DMSO- d ₆ , 150 MHz): see Table 2

location δ _c type δ _H mult ( J in Hz) 1a 62.4 CH2 3.32 m 1b 3.24 m 1-OH OH 4.80 br s 2 52.2 CH 3.92 m 2-NH NH 7.83 d (8.5) 3a 36.4 CH2 2.86 dd (14.0, 5.5) 3b 2.61 dd (14.0, 8.5) 4 139.1 C 5, 9 129.1 2CH 7.20 d (7.0) 6, 8 128.1 2CH 7.23 dd(7.0, 7.0) 7 125.9 CH 7.14 dd(7.0, 7.0) 10 170.3 C 11 57.8 CH 4.09 dd (9.0, 8.0) 11-NH NH 7.81 d (9.0) 12 30.9 CH 1.89 m 13 19.1 CH3 0.78 d (7.0) 14 18.1 CH3 0.78 d (7.0) 15 171.3 C 16 53.7 CH 4.56 ddd (10.0, 8.5, 4.0) 16-NH NH 8.07 d (8.5) 17a 37.1 CH2 2.94 dd (14.0, 4.0) 17b 2.71 dd (14.0, 10.0) 18 138.2 C 19, 23 129.1 2CH 7.25 d (7.0) 20, 22 127.9 2CH 7.23 dd(7.0, 7.0) 21 125.9 CH 7.15 dd(7.0, 7.0) 24 171.5 C 25 44.5 CH2 1.88 d (6.5) 26 25.6 CH 1.81 m 27 22.2 CH3 0.73 d (7.0) 28 22.1 CH3 0.65 d (7.0)

(2) Confirmation of the three-dimensional structure of acid dipilamide

In order to identify the absolute three-dimensional structure of axidipilamide A, Marfey's method was used (Fujii, K. et al., Anal. Chem. 1997, 69, 5146-5151).

First, acid hydrolysis was performed to break the amide bond of the substance and divide it into individual amino acids. Hydrolysis was performed by adding 6 N hydrochloric acid to 2 mg of axidipilamide A and incubating at 120° C. for 2 hours. The hydrolysis reaction was terminated and the hydrolyzed material was freeze-dried to completely remove the remaining hydrochloric acid. The material was divided into two vials and the rubbing method was performed. The hydrolysis product was dissolved in 200 μl of 1 N NaHCO ₃ , and 100 μl was added to each vial. Dissolve L-FDAA (N-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide) in acetone solvent to prepare 10 mg/mL of L-FDAA, and D-FDAA (N-( 5-Fluoro-2,4-dinitrophenyl)-D-alanine amide) reagent was prepared in the same manner. 100 μl of L-FDAA solution and 100 μl of D-FDAA solution were added to each vial. The vial was shaken at about 80° C. and allowed to react for about 3 minutes. Thereafter, 100 μl of 2 N hydrochloric acid was added to each vial to perform a neutralization reaction, and 300 μl of 50% (v/v) acetonitrile/aqueous solution was added to each reaction to dilute the reaction. The reaction product was subjected to LC/MS and a Phenomenex C18(2) (Luna, 100 × 4.6 mm, 5 μm) column and reversed-phase gradient conditions (flow rate 0.7 mL/min, UV wavelength 340 nm, 10% (v/v) to 60 % (v/v) acetonitrile/aqueous solution, and formic acid 0.1% (v/v) for more than 50 minutes). As a result of the reaction analysis, phenylalanine, isoleucine, and phenylalanol, which are amino acids constituting axidipilamide A, were detected at 31.4 minutes, 30.5 minutes, and 30.7 minutes in the L-FDAA derivative, and at 34.2 minutes in the D-FDAA derivative, Since it was detected at 34.4 minutes and 33.7 minutes, it was confirmed that the three-dimensional structures were L, L, and S shapes, respectively.

In order to determine the absolute conformation of the beta position of isoleucine, a GITC derivatization reaction was performed. Acid hydrolysis was carried out under the same conditions as in the Mafi reaction to break the amide bond. Then, 100 μl of 1% (w/v) GITC (2,3,4,6-tetra-O-acetyl-β-D-glucopyanosyl isothiocyanate) acetone solution and 200 μl of 6% (v/v) triethyl After adding an amine solution, the reaction was carried out by shaking at 25° C. for about 15 minutes. The reaction was terminated by adding 200 μl of acetic acid to the reaction, and 20 μl of the reaction was analyzed by LC/MS. Analytical conditions were 30% (v/v) to 55% (v/v) acetonitrile/aqueous solution with a Phenomenex C18 Gemini, 250 × 4.6 mm, 5 μm column, a flow rate of 0.3 mL/min, and UV 254 nm under reverse phase gradient conditions. , was analyzed for about 80 minutes under the conditions of 0.1% (v/v) formic acid solvent. The reaction product with GITC attached to L-isoleucine eluted at about 57.15 minutes, the reaction product with GITC attached to standard L-isoleucine eluted at about 57.2 minutes, and the reaction product with GITC attached to standard L-allo-isoleucine eluted at about 56.6 minutes, respectively. Therefore, it was confirmed that the beta position absolute conformational structure of isoleucine of axidipilamide A is in the S form.

In the case of axidipilamide B, the horsefly reaction proceeded through the same process as that of axidipilamide A, and as a result, phenylalanine, valine, and phenylalaninol each have L, L, and S conformational structures. confirmed that. In addition, in the case of axidipilamide B, since valine exists instead of isoleucine, there was no need to elucidate the absolute three-dimensional structure of the beta position, so no additional GITC reaction proceeded.

The chemical structural formulas of axidipilamides A (Formula 1) and B (Formula 2) analyzed from the nuclear magnetic resonance spectrum data and the three-dimensional structure analysis results are shown below.

[Formula 1 (acidylamide A)]

[Formula 2 (acidylamide B)]

On the other hand, axidipilamide A and axidipilamide C, which have similar chemical structures to those of aciddipilamide B, were also isolated, and the chemical formulas of axidipilamide C are as follows.

[Formula 3 (acidylamide C)]

Example 2. Confirmation of activity of acidipilamide

2-1. Intracellular autophagy inhibitory activity of axidipilamides A and B

It was confirmed whether axidipilamides A and B inhibit the autophagy activity of cells.

Various concentrations of axidipilamides A, B, and C were added to HeLa cells. The aciddipilamide compound was treated for 4 hours ( FIG. 2A ), 8 hours ( FIG. 2B ), and 12 hours ( FIG. 2C ). As a comparison group, L-isoleucinamide and ecdypilamide C were used. Quantitative changes in LC3-II, a representative marker of autophagy activity, were measured by immunoblotting, and the results are shown in FIGS. 2A, 2B and 2C. For immunoblotting, anti-LC3 antibody (Sigma-Aldrich), anti-Ub antibody (Santa Cruz Biotechnology), anti-α3 antibody (Enzo), and anti-β-actin antibody (Sigma) were used.

As shown in Figs. 2a, 2b and 2c, L-isoleucinamide and exidipyramide C did not change the amount of LC3-II. However, the autophagy change effect of axidipilamide B was found to be very high compared to that of axidipalamide A, especially when treated for 12 hours.

On the other hand, the degree of hydrolysis (relative fluorescence unit: RFU) of the proteasome substrate suc-LLVY-AMC (succinyl-Leu-Leu-Val-Tyr-(amino-4-methylcoumarin)) was confirmed. As a result, it was confirmed that axidipilamides A, B, and C did not affect the activity of the proteasome at all ( FIG. 2D ).

2-2. Identification of the mechanism of inhibition of intracellular autophagy of axidipilamides A and B

As described in Example 2-1, in order to find the cause of the change in intracellular autophagic flux, 200 μM of axidipilamide A or B was added to HeLa cells with bafilo, an inhibitor of the late mechanism of autophagy. Co-treatment with bafilomycin A1 (100 nM) or 200 μM of axidipilamide A or B for 8 hours under amino acid-deficient conditions to activate autophagy (amino acid deficiency, Earle's Balanced Salt Solution (EBSS) medium) treated during Quantitative changes in LC3-II were measured by immunoblotting, and the results are shown in FIG. 3 .

As shown in FIG. 3 , when axidipilamides A and B were co-treated with bafilomycin A1, the amount of LC3-II did not change. On the other hand, when axidipilamides A and B were treated in an amino acid deficiency condition, the amount of LC3-II tended to increase. Therefore, it was confirmed that axidipilamide A and axidipilamide B did not directly increase the activity of the proteasome, but inhibited the latter part of the autophagy mechanism.

Claims (13)

delete delete delete delete delete delete A peptide compound represented by Formula 2 or Formula 3, a stereoisomer, solvate, or pharmaceutically acceptable salt thereof:
[Equation 2]

; and
[Equation 3]

. The method according to claim 7, wherein the peptide compound is a peptide compound represented by Formula 4 or Formula 5, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:
[Equation 4]

; and
[Equation 5]

. Streptacidiphilus rugosus ( Streptacidiphilus rugosus ) Culturing the strain; and
A method for producing the peptide compound of claim 7, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, comprising the step of isolating the peptide compound of claim 7 from the culture solution. A pharmaceutical composition for preventing or treating cancer, comprising the peptide compound of claim 7, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 10, wherein the peptide compound of claim 7, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof inhibits the autophagy activity of cancer cells. A composition for inhibiting autophagy of cells in vitro, comprising the peptide compound of claim 7, a stereoisomer, a solvate, or a salt thereof. delete

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