Cyclopeptide compounds containing piperazine acids, methods for their production and uses thereof

Abstract

Provided are novel peptide compounds containing piperazine acid and stereoisomers, solvates, or pharmaceutically acceptable salts thereof, methods for producing the same, and uses thereof. The peptide compounds and stereoisomers, solvates, or pharmaceutically acceptable salts thereof exhibit anticancer activity, antimetastatic activity, and activity for inhibiting the growth of drug-resistant tumors, and thus can be used for preventing or treating various cancers or cancer metastasis.

Description

Technical Field

[0001] This disclosure relates to a cyclic peptide compound containing piperazine acid (hexahydropyridazine-3-carboxylic acid), its production method, and its anticancer uses. Background Technology

[0002] Colorectal cancer is a disease caused by untreated polyps on the colonic mucosa due to aging, which can lead to ulceration and bleeding. It ranks second in incidence among all cancers, and its incidence is rapidly increasing in South Korea due to increasingly Westernized diets and improved nutrition. However, commonly used anticancer drugs for colorectal cancer treatment, such as irinotecan, 5-fluorouracil (5-FU), oxaliplatin, and capecitabine, not only cause serious side effects such as bone marrow suppression, neurotoxicity, and diarrhea, but also lead to drug resistance with long-term use. In recent years, cocktail therapy has been used, combining existing anticancer drugs for colorectal cancer treatment. However, this therapy also causes serious side effects and develops resistance with long-term use or cancer recurrence, thus limiting its use. Therefore, the demand for colorectal cancer treatments with novel mechanisms of action is increasing.

[0003] Piperazic acid is a rare amino acid that is biosynthesized from L-ornithine, a precursor. Since its initial discovery in 1959, piperazine has been found in various naturally derived peptides (Nat. Prod. Rep. 2019, vol. 36, no. 12, pp. 1628-1653).

[0004] Therefore, there is a need to find new compounds that include piperazine nucleus and have excellent anti-cancer effects. Summary of the Invention

[0005] Purpose of the invention

[0006] A peptide compound comprising a piperazine core, its stereoisomer, solvate, or pharmaceutical salt is provided.

[0007] A bacterium is provided for producing a peptide compound comprising a piperazine nucleus, a stereoisomer of the peptide compound, a solvate, or a pharmaceutical salt.

[0008] A method is provided for producing a peptide compound comprising a piperazine core, a stereoisomer of the peptide compound, a solvate, or a pharmaceutical salt.

[0009] A pharmaceutical composition is provided for the prevention or treatment of cancer or cancer metastasis, comprising: a peptide compound including a piperazine core, a stereoisomer of the peptide compound, a solvate, or a pharmaceutically acceptable salt.

[0010] A method for preventing or treating cancer or cancer metastasis is provided, which utilizes a peptide compound comprising a piperazine nucleus, a stereoisomer of the peptide compound, a solvate, or a pharmaceutical salt, said peptide compound comprising a piperazine nucleus.

[0011] Technical solution

[0012] Provided a peptide compound represented by chemical formula 1, its stereoisomer, solvate, or pharmaceutical salt.

[0013] [Chemical Formula 1]

[0014]

[0015] In Chemical Formula 1, Y can be piperazine acid. Piperazine acid is also known as hexahydropyridazine-3-carboxylic acid. The carboxyl group of the piperazine acid can be linked to another amino acid via a peptide bond. The piperazine ring of the piperazine acid can be linked to another amino acid via a peptide bond. In Chemical Formula 1, X1 can be an amino acid selected from the group consisting of serine, threonine, cysteine, and tyrosine. In the peptide bond of the amino acid, N can be unsubstituted or replaced by C1 to C2. 20 Alkyl substitution. The C1 to C1... 20 The alkyl group can be, for example, methyl. X1 can be Ser or N-methyl-Ser in which the N in the peptide bond of Ser is replaced by a methyl group.

[0016] In the chemical formula 1, X2 can be an amino acid selected from the group consisting of leucine (Leu), glycine (Gly), alanine (Ala), valine (Val), isoleucine (Ile), and methionine (Met). X2 can be leucine (Leu).

[0017] In chemical formula 1, X3 can be an amino acid of phenylalanine (Phe) or tryptophan (Trp). The nitrogen in the peptide bond of the amino acid may be unsubstituted or replaced by a C1 to C2 bond. 20 Alkyl groups are substituted. The C1 to C1... 20 The alkyl group can be, for example, methyl. The X3 can be Phe or N-methyl-Phe in which the N in the peptide bond of Phe is replaced by a methyl group.

[0018] In the chemical formula 1, X4 can be an amino acid selected from the group consisting of alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), and methionine (Met). X4 can be Ala.

[0019] In chemical formula 1, X5 can be an amino acid selected from the group consisting of valine, glycine, alanine, leucine, isoleucine, and methionine. In the peptide bond of the amino acid, N may be unsubstituted or replaced by C1 to C2. 20 Alkyl groups are substituted. The C1 to C1... 20 The alkyl group can be, for example, methyl. The X5 can be Val or an N-methyl-Val in which the N in the peptide bond of Val is replaced by a methyl group.

[0020] In the chemical formula 1, X6 can be a β-amino acid or aspartic acid (Asp). A β-amino acid is a compound with a carbon atom sandwiched between a carboxyl group and an amino group; it refers to an amino acid on the β-carbon that is attached to a carboxyl group. The β-amino acid can be β-Ala or β-Leu. β-Ala can be biosynthesized from aspartic acid by aspartate 1-decarboxylase.

[0021] In the chemical formula 1, X7 can be an amino acid selected from the group consisting of alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), and methionine (Met). In the peptide bond of the amino acid, N may be unsubstituted or replaced by C1 to C2. 20 Alkyl groups are substituted. The C1 to C1... 20 The alkyl group can be, for example, methyl. The X7 can be Ala or N-methyl-Ala in which the N in the peptide bond of Ala is replaced by a methyl group.

[0022] In the chemical formula 1, X8 can be an amino acid selected from the group consisting of isoleucine (Ile), valine (Val), glycine (Gly), alanine (Ala), leucine (Leu), and methionine (Met). X8 can be either Ile or Val.

[0023] In the chemical formula 1, X9 may be an amino acid of phenylalanine (Phe) or tryptophan (Trp). In the peptide bond of the amino acid, N may be unsubstituted or replaced by C1 to C2. 20 Alkyl groups are substituted. The C1 to C1... 20The alkyl group can be, for example, methyl. X9 can be N-methyl-Phe in which the N in the peptide bond of Phe is replaced by a methyl group, or N-methyl-Trp in which the N in the peptide bond of Trp is replaced by a methyl group.

[0024] The term "substitution" in the context of "unsubstituted" or "substituted" refers to the introduction of a substituent hydrogen atom when one or more hydrogen atoms in an organic compound are replaced by another atomic group to form a derivative. The substituent group refers to the atomic group that is introduced.

[0025] The term "alkyl" refers to a fully saturated branched or unbranched (or straight-chain or linear) hydrocarbon. The alkyl group can be C1 to C2. 20 C1 to C 15 C1 to C 10 Or C1 to C5 alkyl groups. For example, the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, isopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, or n-heptyl.

[0026] The term "peptide compound" refers to compounds that include peptides. A peptide is a compound consisting of two or more amino acids linked by peptide bonds between a carboxyl group and an amino group. Depending on the number of amino acids, peptides are called dipeptides, tripeptides, tetrapeptides, etc. Peptides with about 10 or fewer peptide bonds are called oligopeptides, and peptides with more peptide bonds are called polypeptides.

[0027] The peptide compound is a compound in which the two ends are linked to form a ring. The peptide compound may be referred to as a cyclic peptide compound.

[0028] The peptide compound may be a compound having a sequence identity of more than or equal to about 99%, more than or equal to about 95%, more than or equal to about 90%, more than or equal to about 80%, more than or equal to about 70%, more than or equal to about 60%, or more than or equal to about 50% with the compound represented by Formula 1. In the peptide compound, the carboxyl, amino, or side chain (R group) of each amino acid may be a halogen atom, hydroxyl group, nitro group, cyano group, amino group, amido group, acetamido group, hydrazine group, hydrazone group, carboxyl group, sulfonyl group, aminosulfonyl group, sulfonic acid group, phosphate group, C1 to C5 alkyl group, C1 to C5 alkoxy group, C2 to C5 alkenyl group, C1 to C5 alkynyl group, C3 to C5 alkenyl group, C2 to C5 alkenyl group, C3 to C5 alkenyl group, C1 ... 10 cycloalkyl, C6 to C 10 Aryl, C6 to C10 heteroaryl, C6 to C 20 Aryl alkyl, C6 to C 20 Substituted with heteroaryl groups, or combinations thereof.

[0029] The peptide compound represented by chemical formula 1 can be represented by any one of chemical formulas 2 to 6:

[0030] [Chemical Formula 2]

[0031]

[0032] [Chemical Formula 3]

[0033]

[0034] [Chemical Formula 4]

[0035]

[0036] [Chemical Formula 5]

[0037] as well as

[0038] [Chemical Formula 6]

[0039]

[0040] The term "stereoisomer" refers to a compound with the same molecular formula but different atomic arrangements or spatial configurations. For example, isomers include structural isomers and stereoisomers. Stereoisomers can be either diastereomers or enantiomers. Enantiomers are mirror images of each other, also known as optical isomers. When the four or more substituents of the chiral central carbon are different, enantiomers are classified as R (rectus: clockwise) and S (sinister: counterclockwise). Diastereomers are non-mirror image stereoisomers, which can be classified as cis-trans isomers due to differences in atomic spatial arrangement.

[0041] The term "solvate" refers to a compound that is solvated in an organic or inorganic solvent. For example, a sovate is a hydrate.

[0042] The term "salt" refers to the inorganic and organic acid addition salt of a compound. The pharmaceutical salt can be one that does not cause severe irritation to the organism to which the compound is applied, and does not impair the biological activity and physical properties of the compound. The inorganic acid salt can be a hydrochloride, bromate, phosphate, sulfate, or disulfide. The organic acid salt can be a formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, benzenesulfonate, camphorsulfonate, ethanedisulfonate, trichloroacetate, trifluoroacetate, benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspartate. The metal salt can be a calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

[0043] On the other hand, a Streptomyces sp. PC5 strain (accession number: KCTC14117BP) is provided.

[0044] The strain can produce peptide compounds, stereoisomers, solvates, or pharmaceutical salts according to one aspect.

[0045] The strain includes its variants. For example, variants can be those caused by natural mutations or artificial mutations.

[0046] Artificial mutations can be generated through physical mutagenesis such as ultraviolet light or chemical mutagens such as alkaline compounds.

[0047] The strains include spores, cells, or cultures of the strain.

[0048] Another method provides a method for preparing a peptide compound, its stereoisomers, solvates, or pharmaceutical salts according to one aspect, comprising: culturing a Streptomyces PC5 strain (accession number: KCTC14117BP); and separating the peptide compound, its stereoisomers, solvates, or pharmaceutical salts according to one aspect from the culture medium. The Streptomyces PC5 strain, peptide compound, its stereoisomers, solvates, or pharmaceutical salts are as described above.

[0049] The method includes culturing Streptomyces PC5 strain (accession number: KCTC14117BP). The culturing step can be performed in a liquid or solid medium. For example, the medium may include glucose, starch syrup, dextrin, starch, molasses, animal oil, or vegetable oil as a carbon source. For example, the medium may include wheat bran, soybean meal, wheat, malt, cottonseed meal, fish scrap, corn steep liquor, meat broth, yeast extract, malt extract, ammonium sulfate, sodium nitrate, or urea as a nitrogen source.

[0050] Culture can be carried out under aerobic conditions by shaking or static incubation. For example, the culture temperature can be 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. For example, the culture time can be 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 from about 1 day to about 1 week.

[0051] The method includes separating a peptide compound represented by chemical formula 1, its stereoisomer, solvate, or pharmaceutical salt from a culture medium.

[0052] Separation steps may include concentrating the culture medium, centrifuging, filtering, or chromatography. For example, depending on the stationary phase, chromatography may be column chromatography, planar chromatography, paper chromatography, or thin-layer chromatography. For example, depending on the physical properties of the mobile phase, chromatography may be gas chromatography, liquid chromatography, or affinity chromatography. For example, liquid chromatography may be high-performance liquid chromatography (HPLC). For example, depending on the separation method, chromatography may be ion-exchange chromatography or size exclusion chromatography.

[0053] For example, chromatography can be normal phase chromatography or reversed phase chromatography.

[0054] On the other hand, a pharmaceutical composition is provided for the prevention or treatment of cancer or cancer metastasis, and includes a peptide compound according to one aspect, a stereoisomer of the peptide compound, a solvate, or a pharmaceutical salt.

[0055] Peptide compounds, stereoisomers, solvates, and pharmaceutical salts are as described above.

[0056] The cancer can be solid or non-solid. For example, solid cancer refers to tumors occurring in organs such as the liver, lungs, breast, and skin. Non-solid cancers are cancers that occur in the bloodstream and are also known as blood cancers. Examples include intrahepatic bile duct cancer, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, mycosis fungoides, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell tumors, male breast cancer, brain tumors, pituitary adenomas, multiple myeloma, gallbladder cancer, bile duct cancer, colorectal cancer, retinoblastoma, choroidal melanoma, periampullary cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain tumors, pediatric lymphoma, and childhood leukemia. Small bowel cancer, meningioma, esophageal cancer, glioma, neuroblastoma, renal ureteral cancer, kidney cancer, malignant soft tissue tumors, malignant bone tumors, malignant lymphoma, malignant mesothelioma, malignant melanoma, ocular tumors, vulvar cancer, urethral cancer, cancer of unknown primary origin, gastric lymphoma, gastric cancer, gastric carcinoid, gastrointestinal stromal tumor, nephroblastoma, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational choriocarcinoma, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic brain tumors, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord tumors, acoustic neuroma, pancreatic cancer, salivary gland cancer, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, laryngeal cancer, or combinations thereof. For example, the cancers mentioned can be lung cancer, colorectal cancer, breast cancer, liver cancer, stomach cancer, or leukemia. Metastasis refers to the spread of cancer cells to other tissues far from the primary site. Metastasis can be epithelial-mesenchymal transition (EMT).

[0057] Peptide compounds represented by Formula 1, their stereoisomers, solvates, or pharmaceutical salts can be used to regulate the cell cycle of cancer cells, induce apoptosis, inhibit metastasis, or combinations thereof. Cell cycle regulation of cancer cells can be G2 / M phase arrest or sub-G1 phase accumulation. The cancer can be resistant to anticancer drugs (anticancer agents). The term "anticancer drug resistance" refers to chemical substances used to treat malignant tumors, such as shrinking, inhibiting, and removing malignant tumors. For example, the anticancer drug can be alkylating agents, antimetabolites, natural substances, hormones and their antagonists, targeted therapies, or combinations thereof. For example, the anticancer drug is fluorouracil (5-FU), irinotecan, etoposide, gemcitabine, and paclitaxel. The term "drug resistance" refers to the extremely low sensitivity to anticancer drug therapy, resulting in no improvement, relief, reduction, or treatment of cancer symptoms. Anticancer drug resistance can manifest in two ways: cancer cells may be resistant to a specific anticancer drug from the outset, or they may not initially show resistance but undergo changes due to prolonged treatment, thus losing sensitivity to the drug and acquiring resistance.

[0058] The term "prevention" refers to all actions that suppress or delay the onset of a disease by applying a composition. The term "treatment" refers to all actions that improve or beneficially alter the symptoms of a disease by applying a composition.

[0059] The pharmaceutical composition may further comprise a known active ingredient with anticancer activity. The known active ingredient with anticancer activity may be an anticancer drug. The anticancer drug may be 5-fluorouracil, irinotecan, etoposide, oxaliplatin, leucovorin, capecitabine, or a combination thereof. The compound represented by Formula 1, its stereoisomers, solvates, or pharmaceutical salts, as well as the anticancer drug, may be a single composition or a single composition for simultaneous or sequential administration.

[0060] The pharmaceutical composition may further include a carrier, excipients, or diluents. For example, the carrier, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil.

[0061] The pharmaceutical composition can be formulated according to conventional methods in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, or in the form of external preparations, suppositories, or sterile injections. When formulated, commonly used fillers, expanders, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients can be used.

[0062] In the pharmaceutical composition, the solid dosage form for oral administration may be a tablet, pill, powder, granule, or capsule. The solid dosage form may further include excipients. For example, excipients may be starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, the solid dosage form may further include magnesium stearate or lubricants such as talc. In the pharmaceutical composition, the liquid dosage form for oral administration may be a suspension, oral solution, emulsion, or syrup. The liquid dosage form may include water or liquid paraffin. The liquid dosage form may include excipients, such as wetting agents, sweeteners, flavoring agents, or protective agents. In the pharmaceutical composition, the formulation for parenteral administration may be a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or / and a suppository. Non-aqueous solvents or suspensions may include vegetable oils or esters. For example, vegetable oils may be propylene glycol, polyethylene glycol, or olive oil. For example, esters may be ethyl oleate. The base of suppositories can be Wipedsol, polyethylene glycol (Macrogol), Tween 61, cocoa butter, lauryl ester, or glycerin gelatin.

[0063] The pharmaceutical composition may include an effective amount of the compound represented by Formula 1, its stereoisomers, solvates, or pharmaceutical salts. The term "effective amount" refers to an amount sufficient to produce a preventive or therapeutic effect when administered to an individual requiring prevention or treatment. Those skilled in the art can appropriately select the effective amount based on the selected cells or individual. Preferred dosages of the pharmaceutical composition vary depending on the individual's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. However, for example, the compound, its stereoisomers, solvates, or pharmaceutical salts can be administered at amounts from about 0.0001 mg / kg to about 100 mg / kg, or from about 0.001 mg / kg to about 100 mg / kg, once to 24 times daily, once to 7 times within 2 days or 1 week, or once to 24 times within 1 month to 12 months. In the pharmaceutical composition, the compound, its stereoisomer, solvate, or pharmaceutical salt may be in an amount of about 0.0001% by weight to about 10% by weight, or about 0.001% by weight to about 1% by weight, relative to the total weight of the composition.

[0064] The method of administration can be oral or parenteral. For example, the methods of administration can be oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, local, intranasal, intratracheal, or intradermal. The composition can be administered systemically or locally, and can be administered alone or in combination with other pharmaceutically active compounds.

[0065] On the other hand, a functional food is provided for the prevention or improvement of bacterial infections or any related symptoms caused by Mycobacterium spp., comprising a compound, its stereoisomer, solvate, or pharmaceutical salt according to one aspect. The compound, its stereoisomer, solvate, pharmaceutical salt, bacterial infection disease caused by Mycobacterium spp., or any related symptoms, and prevention are as described above.

[0066] The term "improvement" includes all actions that result in or benefit from the symptoms of a disease.

[0067] The functional foods can be formulated into capsules, powders, or suspensions by means of peptide compounds represented by Formula 1, their stereoisomers, solvates, or salts, and can then be used as functional foods or added to various foods. For example, the foods can be meat, sausages, bread, chocolate, candy, snacks, biscuits, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin supplements, functional foods, and health foods.

[0068] On the other hand, a method for preventing or treating cancer or cancer metastasis is provided, which includes administering a pharmaceutical composition according to one aspect to an individual.

[0069] The pharmaceutical composition, cancer, cancer metastasis, prevention, and treatment are as described above.

[0070] The individual may be a mammal, such as a human, mouse, rat, cow, horse, pig, dog, monkey, sheep, goat, or cat. The individual may have cancer or have symptoms associated with cancer metastasis or be at risk of developing cancer.

[0071] The method may further include administering an anticancer drug to the individual. The anticancer drug may be administered to the individual simultaneously, separately, or sequentially with a compound represented by Formula 1, its stereoisomers, solvates, or pharmaceutical salts.

[0072] The administration method can be oral or parenteral. For example, the administration method can be oral, percutaneous, subcutaneous, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, local, intranasal, intratracheal, or intradermal. The pharmaceutical composition can be administered systemically or locally, and can be administered alone or in combination with other pharmaceutically active compounds.

[0073] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, severity of illness, form of medicine, route of administration, and duration of administration, but may be appropriately selected by those skilled in the art. For example, for adults, the dosage may be 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. The administration may be once daily, multiple times daily, once weekly, once every two weeks, once every three weeks, or once every four weeks to once a year.

[0074] Beneficial effects

[0075] Novel peptide compounds containing piperazine, their stereoisomers, solvates, or pharmaceutical salts have anticancer, antimetastatic, and inhibitory activities against the growth of drug-resistant tumors, and therefore can be used to prevent or treat various types of cancer or cancer metastasis. Attached Figure Description

[0076] Figure 1 This is a diagram showing the solid culture medium used to culture Streptomyces sp. PC5 strain.

[0077] Figure 2aThis is a graph showing the cell cycle of the colorectal cancer cell line HCT116 as detected by flow cytometry based on lenziamide A concentration (μM). Figure 2b This is a graph showing the cell cycle distribution (%) of HCT116 based on dung benzamide A concentration (μM).

[0078] Figure 3a This is a flow cytometry plot showing the results of Annexin V / Propidium iodide analysis. Figure 3b This is a diagram showing cell states based on dung beetle amide A concentration.

[0079] Figure 4a This shows the tumor volume (mm²) based on the amount of dung benzamide A applied. 3 (The image) Figure 4b This is a graph showing the body weight (g) of an xenotransplant animal model. Figure 4c This is a graph showing the final tumor weight (g) based on the amount of dung benzamide A applied.

[0080] Figure 5a This shows the tumor volume (mm²) in an animal model of xenograft of drug-resistant cancer cell lines, based on the administration of dung benzamide A, 5-FU, or a combination thereof. 3 (The image is missing from the original text.) Figure 5b This is a graph showing the body weight (g) of an animal model of a drug-resistant cancer cell line xenograft.

[0081] Figure 6a This is a graph showing the expression of metastatic biomarkers in metastatic colorectal cancer cell lines based on dung benzamide A administration, as confirmed by immunoblotting analysis. Figure 6b This shows the tumor volume (mm²) based on the amount of dung benzamide A administered in a xenograft animal model of metastatic colorectal cancer cell lines. 3 (The image) Figure 6c This is a graph showing the body weight (g) of an animal model of a metastatic colorectal cancer cell line xenograft. Detailed Implementation

[0082] The present invention is further described in detail through the following embodiments. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0083] Example 1. Isolation of lenziamide and confirmation of its structure

[0084] 1-1. Isolation of dung beetle amide-producing strains

[0085] Streptomyces sp. strain PC5, the lenziamide-producing strain, is a strain isolated from the exoskeleton of the dung beetle (Onthophagus lenzii). PC5 strain was isolated from actinomycete isolation solid medium (each 1L of sterile water contains 2g sodium caseinate, 0.1g asparagine, 4g sodium propionate, 0.5g dipotassium hydrogen phosphate, 0.1g magnesium sulfate, 0.001g ferrous sulfate, and 15g agar powder) Figure 1 Based on the 16S rDNA sequence analysis, the strain was identified as belonging to the genus *Streptomyces*. The strain was named *Streptomyces* PC5 strain and was deposited on January 29, 2020, at the Korean Center for Type Culture Collection (KCTC), Korea Research Institute of Biotechnology (KRIBB) (accession number: KCTC14117BP).

[0086] 1-2. Culture of Streptomyces PC5 strain

[0087] Streptomyces PC5 strain was spread on sterile yeast extract-malt extract (YEME) solid medium (4g yeast extract, 10g malt extract, 4g glucose and 18g agar per 1L distilled water) and cultured at about 30°C for about 4 weeks.

[0088] After culturing, PC5 spores were inoculated into 50 mL of YEME liquid medium (4 g yeast extract, 10 g malt extract, and 4 g glucose per 1 L distilled water), shaken at 200 rpm, and cultured at 30°C for approximately 8 days. Then, 5 mL of the culture was inoculated into 200 mL of modified YEME liquid medium (4 g yeast extract, 10 g malt extract, 4 g glucose, and 1 g humic acid per 1 L distilled water), and cultured at 170 rpm and 30°C for approximately 5 days.

[0089] 1-3. Separation and purification of dung beetylamide

[0090] Cultures of the PC5 strain obtained through Examples 1-2 were obtained.

[0091] In a separatory funnel placed on a support, 1 L of PC5 strain culture medium (terminated) and approximately 1.5 L of ethyl acetate (EtOAC, Daejung Hwageum Co., Ltd.) were added. The funnel was sealed and agitated for 3 minutes to perform one extraction. The funnel was then returned to the support, and after complete separation of the aqueous and EtOAC layers, the valve of the separatory funnel was opened to remove the aqueous layer. Fresh EtOAC was added to the aqueous layer, and the extraction was repeated in the same manner. The EtOAC layer was separately stored in a clean flask, and anhydrous sodium sulfate (Daejung Hwageum Co., Ltd.) was added to remove any remaining water from the culture medium. The mixture was then filtered through several layers of clean gauze to remove impurities. The dehydrated EtOAC layer was transferred back to a 3 L round-bottom flask and dried under reduced pressure using a desiccator. After co-culturing 150 L of the strain, approximately 5 g of crude extract was obtained.

[0092] To purify dung beetylamine from the PC5 strain extract, reversed-phase fractionation was first performed using an open column. The column was stabilized with C40 using 20% ​​(v / v) methanol (MeOH) / 80% (v / v) H2O. 18 Following the open column of the resin, the extract adsorbed on Celite 545 was packed onto it. The extract was fractionated with 20% (v / v), 40% (v / v), 60% (v / v), 80% (v / v), and 100% (v / v) methanol solvents, respectively. The 80% (v / v) and 100% (v / v) fractions of dung beetyl amide A were transferred to 3 L round flasks and dried under reduced pressure.

[0093] To obtain pure dung beetle amide A from the dried 80% (v / v) and 100% (v / v) methanol fractions, HPLC (high-performance liquid chromatography) was performed, followed by further purification in the following three steps: (1) Using a reversed-phase column (C10 ... 18 YMC-Pack ODS-A (S-5μm 250×10.0mm) was eluted for approximately 50 min using a concentration gradient of acetonitrile (ACN) / water solution containing 0.1% (v / v) formic acid, from 50% (v / v) to 80% (v / v) (flow rate: 2 mL / min, detection: UV 210 nm). Dung benzimidamides A, B, C, D, and E were identified in fractions at approximately 31 min, 26 min, 28 min, and 23 min, respectively.

[0094] (A-2) Using the inverting column (C 18Further fractionation was performed on the fraction dried under reduced pressure for 31 minutes using YMC-Pack ODS-A (S-5μm 250×10.0mm) and under isocratic conditions (flow rate: 2 mL / min, detection: UV 230 nm) with the addition of 0.1% (v / v) formic acid in 76% (v / v) methanol / water solution. Dung benzimidamide A was confirmed in the fraction dried for approximately 28 minutes.

[0095] (A-3) The fraction dried under reduced pressure for 28 minutes was fractionated again under the same conditions as in (A-2) to obtain pure dung beetylamide A. Through repeated experiments, approximately 100 mg of dung beetylamide A was obtained from approximately 5 g of crude extract.

[0096] (B-2) The fraction dried under reduced pressure for approximately 26 minutes was further fractionated for approximately 40 minutes using a cyano column (CN YMC-Pack S-5μm 250×4.6mm) with a concentration gradient of 40% (v / v) to 70% (v / v) methanol / water solution containing 0.1% (v / v) formic acid (flow rate: 0.8 mL / min, detection: UV 230 nm). Pure dung benzimidamide B was obtained in the fraction dried under reduced pressure for approximately 25 minutes.

[0097] (C-2) The fraction dried under reduced pressure for approximately 28 minutes was further fractionated for approximately 40 minutes using a cyano column (CN YMC-Pack S-5μm 250×4.6mm) with a concentration gradient of 40% (v / v) to 70% (v / v) methanol / water solution containing 0.1% (v / v) formic acid (flow rate: 0.8 mL / min, detection: UV 230 nm). Pure dung benzimidamide C was obtained in the fraction dried under reduced pressure for approximately 27 minutes.

[0098] (D-2) The fraction dried under reduced pressure for approximately 23 minutes was further fractionated for approximately 40 minutes using a cyano column (CN YMC-Pack S-5μm 250×4.6mm) with a concentration gradient of 40% (v / v) to 70% (v / v) methanol / water solution containing 0.1% (v / v) formic acid (flow rate: 0.8 mL / min, detection: UV 230 nm). Pure dung benzimidamide D was obtained in the approximately 23-minute fraction, and impure dung benzimidamide E was obtained in the approximately 21-minute fraction.

[0099] (E-3) Using the inverting column (C 18(2) Under isostatic conditions (flow rate: 0.8 mL / min, detection: UV 230 nm) of 73% (v / v) methanol / water solution with added 0.1% (v / v) formic acid, the fraction obtained after vacuum drying for approximately 21 minutes in the process of obtaining pure dung benzimidamide D was further fractionated. Pure dung benzimidamide E was obtained in the fraction of approximately 21 minutes.

[0100] 1-4. Confirm the chemical structure of dung beetle amide.

[0101] The structure of dung beetle amide A was confirmed using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. 1 H NMR, 13 The C NMR was performed using a 500MHz NMR from Bruker, with acetone-d6 as the solvent.

[0102] The structural positions of dung benzimidamide A, as confirmed by nuclear magnetic resonance spectroscopy, are shown in Table 1.

[0103] [Dung beetle amide A]

[0104] (1) Molecular formula: C 57 H 87 N 11 O 11

[0105] (2) Molecular weight: 1101

[0106] (3) Color: Transparent

[0107] (4) 1 ¹H-NMR (acetone-d6, 850 MHz): Refer to Table 1

[0108] (5) 13 C-NMR (acetone-d6, 212.5 MHz): Refer to Table 1

[0109] [Table 1]

[0110]

[0111]

[0112]

[0113] The chemical structure of dung beetle amide A, as analyzed by nuclear magnetic resonance spectroscopy and stereostructure analysis, is shown below.

[0114] [Dung beetle amide A]

[0115]

[0116] In addition, the chemical structural formulas of dung beetle amides B to E are shown below.

[0117] [Dung beetle amide B]

[0118]

[0119] [Dung beetle amide C]

[0120]

[0121] [Dung beetle amide D]

[0122]

[0123] [Dung beetle amide E]

[0124]

[0125] Example 2. Confirmation of the anticancer activity of dung beetle amide

[0126] 2-1. Assess the activity in inhibiting cancer cell growth

[0127] We purchased six cancer cell lines from the Korean Cell Line Bank (Seoul, Korea): lung adenocarcinoma A549, colorectal cancer HCT116, breast cancer MDA-MB-231, liver adenocarcinoma SK-HEP-1, gastric adenocarcinoma SNU638, and chronic myeloid leukemia cells K562; as well as a normal cell line of lung fibroblasts MRC-5.

[0128] The dung beetle amide compound prepared as described in Example 1 was added to the prepared cell line and cultured for approximately 72 hours. Then, the cells were stained with sulforhodamine (SRB), a live-cell staining agent, followed by washing to remove unbound dye. The stained cells were then resuspended in 10 mM Tris (pH 10.0). Absorbance at 515 nm was measured, and cell proliferation was assessed. The half-maximal inhibitory concentration (IC50) was calculated using TableCurve 2D v5.01 software (Systant Software Inc., Richmond, CA, USA) and nonlinear regression analysis. 50 IC50 values. All reagents were purchased from Sigma-Aldrich. The calculated IC50 values ​​will be used to determine the IC50 values. 50 Values ​​(μM) are shown in Table 2 below. Etoposide was used as a positive control group.

[0129] [Table 2]

[0130]

[0131] As shown in Table 2, the dung beetle amide compound effectively inhibits the growth of cancer cells.

[0132] 2-2. The cell cycle regulation effect of dung beetylamine A in colorectal cancer cells

[0133] To confirm the mechanism of action of dung benzimidamide A, which exhibits excellent cell proliferation inhibition, HCT116 colorectal cancer cells were treated with dung benzimidamide A at concentrations of 0 μM, 1.25 μM, 2.5 μM, or 5 μM. The cell cycle regulation effect of dung benzimidamide was confirmed by flow cytometry, and the results are presented below. Figure 2a and Figure 2b .

[0134] like Figure 2a and Figure 2b As shown, cell cycle arrest in the G2 / M phase increased until 24 hours after treatment with dung benzimidamide A. Subsequently, programmed cell death, i.e., accumulation of cells in the Sub G1 phase, occurred in the presence of high concentrations of dung benzimidamide A. This confirms that dung benzimidamide A induces cell cycle arrest in the G2 / M phase of colorectal cancer cell lines, thereby exhibiting an inhibitory effect on cancer cell proliferation through programmed cell death.

[0135] 2-3. Apoptosis-inducing effect of dung benzamide A on colorectal cancer cells

[0136] To confirm whether the accumulation of cells in the Sub-G1 phase induced by G2 / M phase arrest of dung benzimidamide A leads to apoptosis, HCT116 cells were treated with different concentrations of dung benzimidamide A for 48 hours. The apoptosis-inducing efficacy of dung benzimidamide A was then assessed using the Annexin V / propidium iodide double staining method, which confirms apoptosis. The results of the Annexin V / propidium iodide double staining method are shown below. Figure 3a Cell states based on dung beetle amide A concentration will be shown in... Figure 3b As a cellular state, the percentage (%) of live cells, early apoptosis, late apoptosis, necrosis, and total cell death were calculated.

[0137] like Figure 3a and Figure 3bAs shown, apoptosis increased in a concentration-dependent manner with dung benzimidamide A. Specifically, treatment with 5 μM dung benzimidamide A resulted in a cell distribution of approximately 20.37% early apoptosis and 51.40% late apoptosis. Therefore, it is confirmed that dung benzimidamide A inhibits cancer cell growth by inducing apoptosis.

[0138] 2-4. In vivo inhibitory effect of dung benzimidamide A on colorectal cancer tumor growth

[0139] To evaluate the antitumor efficacy of dung beetle amide A in animal models, xenograft animal models of colorectal cancer cells were prepared.

[0140] The human colorectal cancer cell line HCT116 was used at 5 × 10⁻⁶. 6 The tumor cells were diluted per mL in RPMI medium and then injected subcutaneously at 0.2 mL into the right flank of 5-week-old male BALB / c mice. The tumor was inoculated when the tumor size reached 100 mm. 3 When the tumor size is left or right, it is randomly grouped based on the tumor size.

[0141] For the prepared xenograft animal models, dung benzamide A was administered intravenously at a dose of 10 mg / kg body weight or 30 mg / kg body weight. A mixture of dimethyl sulfoxide, Cremophor, and physiological saline in a ratio of 0.5:0.5:9 was used as a negative control group, and irinotecan was used as a positive control group. Tumor volume (mm) was measured from the day of administration. 3 The body weight (g) and final tumor weight (g) of the xenograft animal model were measured, and the results are presented in [the table / data]. Figures 4a to 4c .

[0142] like Figures 4a to 4c As shown, dung benzimidamide A exhibited a concentration-dependent tumor growth inhibitory effect at both application concentrations (10 mg / kg, 30 mg / kg). Conversely, when toxicity of dung benzimidamide A was assessed by body weight (g) during administration, no significant weight loss was observed.

[0143] 2-5. Growth inhibitory effect of dung benzimidamide A on 5-fluorouracil-resistant colorectal cancer cell lines. To evaluate the efficacy of dung benzimidamide A in drug-resistant cell lines, HCT116-5-FU was prepared against 5-fluorouracil (5-FU), a colorectal cancer cell line that is resistant to 5-fluorouracil (5-FU), which is used as a first-line treatment for colorectal cancer.

[0144] To prepare the HCT116-5-FU resistant cell line, HCT116 cells were treated with 5-fluorouracil three times a week, and only the surviving cells were passaged. The treatment was then repeated for three months by gradually increasing the concentration of 5-fluorouracil. The HCT116-5-FU resistant cell line was constructed by collecting and culturing only the ultimately surviving cells.

[0145] After adding 5-FU, irinotecan, etoposide, and dung benzimidamide A to the non-resistant HCT116 cell line and the HCT116-5-FU resistant cell line, the IC50 was calculated as described in Example 2-1. 50 Value (μM). Calculated based on the cellular IC50 value of the compound. 50 Value (μM) and IC50 of HCT116-5-FU 50 Value relative to the IC of HCT116 50 The values ​​were changed by multiples, and the results are shown in Table 3.

[0146] [Table 3]

[0147]

[0148]

[0149] As shown in Table 3, in addition to 5-FU, the HCT116-5-FU-resistant cell line also exhibited resistance to other drugs used for the treatment of colorectal cancer. However, dung benzamide A effectively inhibited the growth of the HCT116-5-FU-resistant cell line. Therefore, it is confirmed that dung benzamide A effectively inhibits the growth of drug-resistant cancer cells.

[0150] 2-6. Inhibition of growth of drug-resistant tumor cells by dung benzamide A and the efficacy of combined 5-FU administration. To verify the efficacy of dung benzamide A at the animal experimental level, xenograft animal models were constructed using drug-resistant cancer cell lines.

[0151] As shown in Experiment 2-5, the prepared HCT116-5-FU resistant cell line was cultured at 5 × 10⁻⁶ cells / year. 6 The tumor cells were diluted per mL in RPMI medium and then injected subcutaneously at 0.2 mL into the right flank of 5-week-old male BALB / c mice. The tumor was inoculated when the tumor size reached 100 mm. 3 When the tumor size is left or right, it is randomly grouped based on the tumor size.

[0152] Dung benzimidamide A (10 mg / kg body weight), 5-FU (30 mg / kg body weight), or a combination thereof (10 mg / kg body weight dung benzimidamide A + 30 mg / kg body weight 5-FU) were administered intravenously to xenograft animal models of drug-resistant cancer cell lines. A mixture of dimethyl sulfoxide: Cremophor: saline in a ratio of 0.5:0.5:9 was used as a negative control. Tumor volume (mm²) was measured from the day of administration. 3 The weight (g) of the xenograft animal model and the results are presented in... Figures 5a to 5b .

[0153] like Figure 5a and Figure 5b As shown, when dung benzimidamide A (1) was administered at a dose of 10 mg / kg, it effectively inhibited the growth of drug-resistant tumors without causing a significant decrease in body weight. Furthermore, compared to 5-FU alone, the combined administration of dung benzimidamide A and 5-FU exhibited a synergistic effect. Therefore, this demonstrates that dung benzimidamide A has an in vivo growth-inhibiting effect on drug-resistant cells and exhibits a synergistic effect when combined with anticancer drugs such as 5-FU.

[0154] 2-7. Anti-transfer efficacy of dung benzamide A

[0155] (1) Confirmation of in vitro anti-metastasis efficacy

[0156] It is well known that metastatic cancers typically grow faster than conventional cancers, making them difficult to cure. Colorectal cancer cell line (C-1) was injected into the cecum of experimental mice and left for 6 weeks to artificially induce tumor growth and metastasis. Then, only colorectal cancer cells that had metastasized to the liver (L-2) were isolated and cultured. After applying dung beetyl amide A at doses ranging from 0 μM to 4 μM to both the colorectal cancer cell line (C-1) and the resulting metastatic colorectal cancer cell line (L-2), biomarkers associated with cancer metastasis were identified by RT-PCR, and the results are presented below. Figure 6a .

[0157] like Figure 6a As shown, compared to the colorectal cancer cell line (C-1), metastatic colorectal cancer cells (L-2) exhibited decreased expression of the epithelial biomarker E-cadherin, but increased expression of N-cadherin and vimentin, biomarkers associated with cancer metastasis. In colorectal cancer cells (L-2) that had metastasized to the liver, administration of dung beetylamide A resulted in increased E-cadherin expression and decreased N-cadherin and vimentin expression. Therefore, dung beetylamide A is confirmed to have anti-metastatic efficacy in vitro.

[0158] (2) Confirmed anti-metastasis efficacy in vivo

[0159] As shown in Examples 2-7(1), metastatic colorectal cancer cell line (L-2) was reinjected into the cecum of experimental mice to generate tumors, and dung benzimidamide A was administered at a dose of 5 mg / kg body weight or 15 mg / kg body weight. A mixture of dimethyl sulfoxide: Cremophor: saline in a ratio of 0.5:0.5:9 was used as a negative control. Irinotecan was administered as a positive control at a dose of 10 mg / kg body weight. Tumor volume (mm) was measured from the day of administration. 3 The weight (g) of the xenograft animal model and the results are presented in... Figures 6b to 6c .

[0160] like Figure 6b and Figure 6c As shown, in the 5 mg / kg or 15 mg / kg administration groups of dung benzamide A, tumor metastasis to other organs was effectively inhibited, and tumor growth in the cecum was also inhibited. Therefore, it is confirmed that dung benzamide A also has excellent anti-metastatic efficacy in vivo.

[0161] <Collection Number>

[0162] Preservation Institution: KCTC - Korean Center for Type Cultures

[0163] Collection Number: KCTC14117BP

[0164] Preservation date: 20200129

[0165]

Claims

1. A peptide compound, its stereoisomer, or a pharmaceutical salt, wherein, The peptide compound is represented by any one of chemical formulas 2 to 6: [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] ； [Chemical Formula 5] as well as [Chemical Formula 6] 2. A Streptomyces sp. PC5 strain, accession number: KCTC14117BP, capable of producing the peptide compound, its stereoisomer, or pharmaceutical salt as described in claim 1.

3. A method for preparing the peptide compound of claim 1, its stereoisomers, or pharmaceutical salts, the method comprising culturing Streptomyces PC5 strain, accession number: KCTC14117BP; and Isolate the peptide compound of claim 1, its stereoisomers, or pharmaceutical salts from the culture medium.

4. A pharmaceutical composition for the prevention or treatment of cancer or cancer metastasis, comprising the peptide compound of claim 1, its stereoisomer, or a pharmaceutical salt.

5. The pharmaceutical composition according to claim 4, wherein, The cancers mentioned are selected from the group consisting of lung cancer, colorectal cancer, breast cancer, liver cancer, stomach cancer, and leukemia.

6. The pharmaceutical composition according to claim 4, wherein, The peptide compound of claim 1, its stereoisomers, or pharmaceutical salts are used to regulate the cell cycle of cancer cells, induce apoptosis of cancer cells, inhibit cancer metastasis, or combinations thereof.

7. The pharmaceutical composition according to claim 4, wherein, The cancer has developed resistance to anti-cancer drugs.

8. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition further comprises an anticancer drug.

9. Use of the peptide compound of claim 1 in the preparation of a medicament for treating cancer or cancer metastasis, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, breast cancer, liver cancer, stomach cancer, and leukemia.

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