Anticancer drug

Abstract

To provide a novel compound that strongly inhibits formation of a cancer stem cell culture medium (XF culture medium) cultivation CSC (cancer stem cell sphere).SOLUTION: A novel specific compound and a composition containing the compound are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a compound that exhibits anticancer activity, and an anticancer agent containing the compound. More specifically, the present disclosure relates to a novel compound that has inhibitory activity against cancer cells (particularly cancer stem cells), and an anticancer agent containing the compound. [Background technology]

[0002] Cancer stem cells (CSCs) are thought to be a major factor in cancer recurrence. Unlike conventional cancer cells, cancer stem cells are known to possess 1) self-renewal and pluripotency, 2) high tumorigenicity, and 3) drug resistance. Even when conventional anticancer drugs kill conventional cancer cells, the remaining cancer stem cells regenerate tumors. Therefore, it is believed that tumor regeneration by cancer stem cells that remain after conventional anticancer drug treatment is the cause of cancer recurrence. Given this background, active research focusing on cancer stem cells is currently being conducted, but no effective therapeutic agents against cancer stem cells have yet been developed (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Saygin, C. et al. Cell Stem Cell, 24, 25, 2019 [Non-patent document 2] Yan, X. et al. Biomaterials, 198, 167, 2019 [Non-patent document 3] Ikeda, H. et al. J. Antibiot., 75, 671, 2022 Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, the present inventors conducted research with the aim of obtaining new drug seeds for cancer stem cells.

[0005] Many assay systems comparing two-dimensional and three-dimensional culture have been reported as a strategy for drug discovery against cancer stem cells (Non-Patent Document 2). Therefore, we first considered constructing a new screening system and constructed a unique drug screening system characterized by comparing three-dimensional cultures of the same cancer cells in different media (Non-Patent Document 3). More specifically, we obtained cell masses called spheres by three-dimensionally culturing the human colon cancer cell line HT29 in either standard media (RPMI1640 medium) or cancer stem cell media (3D Tumorsphere Medium XF medium). We found that cancer stem cell (CSC) spheres obtained using cancer stem cell media (XF medium) exhibited elevated expression of cancer stem cell markers such as Nanog, Oct4, and Sox2 compared to standard two-dimensional (2D) and three-dimensional (3D) cultures, confirming their high cancer stemness. Furthermore, the existing anticancer drugs camptothecin and paclitaxel showed inhibitory effects on the formation of spheres cultured in standard medium (RPMI medium), but did not show inhibitory effects on the formation of spheres cultured in medium for cancer stem cells (XF medium) (Non-Patent Document 3).

[0006] Therefore, we thought that if we could find a compound that exhibits an inhibitory effect on the formation of CSC spheres cultured not only in standard medium (RPMI medium) but also in medium for cancer stem cells (XF medium), it could become a particularly effective therapeutic agent against cancer stem cells, and we continued our research to find such a compound. [Means for solving the problem]

[0007] The present inventors discovered a novel compound that strongly inhibits the formation of CSC spheres cultured in a medium for cancer stem cells (XF medium), and have further improved it.

[0008] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. formula:

[0009] [ka] (In the formula, R 1 , R 2 , R 3 each independently represents a lower alkyl group, R a , R b each independently represents a protecting group for an H or OH group, R c , R d are independent of each other, R c represents a protecting group for an H or OH group, or -OR c indicates =O, R d represents a protecting group for an H or OH group, or -OR d indicates =O, R 456 is H, or the formula:

[0010] [ka] [Wherein: A double line consisting of a dotted line and a solid line represents a single bond or a double bond. R 4 , R 5 , R 6 are independent of each other, R 4 is H, OR 4p OR 4p represents a lower alkyl group optionally substituted by 4p represents a protecting group for an H or OH group, or -OR 4p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 4p may denote =O, R 5 is H, OR 5p OR 5p represents a lower alkyl group optionally substituted by 5prepresents a protecting group for an H or OH group, or -OR 5p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 5p may denote =O, R 6 is H, OR 6p OR 6p represents a lower alkyl group optionally substituted by 6p represents a protecting group for an H or OH group, or -OR 6p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 6p may represent =O. represents a group represented by R 456 is the group and the double line consisting of a dotted line and a solid line represents a single bond, OR c R c is off, R 4 and the carbon atom to which OR is bonded c The O in the R 6 the carbon atom to which OR is attached c the carbon atom to which OR is attached d and the carbon atom to which OR is bonded. c and the carbon atom to which OR is bonded d may form a ring together with carbon atoms bonded to both carbon atoms to which R c indicates H, and R d indicates H and R 4 and the carbon atom to which OR is bonded c If O and are not bonded, OR c R c and OR d R d is removed, OR c O and OR d The O and Z are bonded via -Z- to form OR c the carbon atom to which OR is attached d and the carbon atom to which OR is bonded. c and the carbon atom to which OR is bonded d may form a ring together with the carbon atoms bonded to both of the carbon atoms to which Z is bonded, wherein Z is a lower alkylene group. A compound represented by the formula: Section 2. formula:

[0011] [ka] (In the formula, R 4 and R 5 teeth, R 4 indicates H and R 5 OR 5p represents a lower alkyl group optionally substituted with R 4 OR 4p represents a lower alkyl group optionally substituted with R 5 indicates H, R 6 is OR 6p represents a lower alkyl group optionally substituted by R 1 , R 2 , R 3 , R a , R b , R c , R d , R 4p , R 5p , R 6p is the same as above.) Item 1. The compound according to item 1, represented by the formula: Section 3. formula:

[0012] [ka] (In the formula, R 1 , R 2 , R 3 , R a , R b , R 4 , R 5 , R 6 Item 3. The compound according to item 2, represented by the formula: wherein Z is as defined above. Section 4. formula:

[0013] [ka] (In the formula, R 4 is OR 4p indicates, R 5 is OR 5p represents a lower alkyl group optionally substituted by R 6 indicates H, R 1 , R 2 , R 3 , R a , R b , R d , R 4p , R 5p is the same as above.) Item 1. The compound according to item 1, represented by the formula: Section 5. A pharmaceutical composition comprising the compound according to any one of items 1 to 4. Section 6. Item 5. An anti-cancer composition containing the compound according to any one of Items 1 to 4. Section 7. 5. An anti-cancer stem cell composition comprising the compound according to claim 1. [Effects of the Invention]

[0014] A novel compound capable of potently inhibiting cancer stem cell sphere formation is provided, providing a novel anticancer agent that is effective in cancer treatment. DETAILED DESCRIPTION OF THE INVENTION

[0015] Each embodiment of the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, novel compounds exhibiting anticancer activity and anticancer agents containing such compounds, and the present disclosure includes all that is disclosed herein and that can be recognized by a person skilled in the art.

[0016] The novel compounds encompassed by the present disclosure have the formula:

[0017] [ka] (In the formula, R 1 , R 2 , R 3 each independently represents a lower alkyl group, R a , R b each independently represents a protecting group for an H or OH group, R c , R d are independent of each other, R c represents a protecting group for an H or OH group, or -OR c indicates =O, R d represents a protecting group for an H or OH group, or -OR d indicates =O, R 456 is H, or the formula:

[0018] [ka] [Wherein: A double line consisting of a dotted line and a solid line represents a single bond or a double bond. R 4 , R 5 , R 6 are independent of each other, R 4 is H, OR 4p OR 4p represents a lower alkyl group optionally substituted by 4p represents a protecting group for an H or OH group, or -OR 4p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 4p may denote =O, R 5 is H, OR 5p OR 5p represents a lower alkyl group optionally substituted by 5p represents a protecting group for an H or OH group, or -OR 5p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 5p may denote =O, R 6is H, OR 6p OR 6p represents a lower alkyl group optionally substituted by 6p represents a protecting group for an H or OH group, or -OR 6p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 6p may represent =O. represents a group represented by R 456 is the group and the double line consisting of a dotted line and a solid line represents a single bond, OR c R c is off, R 4 and the carbon atom to which OR is bonded c The O in the R 6 the carbon atom to which OR is attached c the carbon atom to which OR is attached d and the carbon atom to which OR is bonded. c and the carbon atom to which OR is bonded d may form a ring together with carbon atoms bonded to both carbon atoms to which R c indicates H, and R d indicates H and R 4 and the carbon atom to which OR is bonded c If O and are not bonded, OR c R c and OR d R d is removed, OR c O and OR d The O and Z are bonded via -Z- to form OR c the carbon atom to which OR is attached d and the carbon atom to which OR is bonded. c and the carbon atom to which OR is bonded d may form a ring together with the carbon atoms bonded to both of the carbon atoms to which Z is bonded, and Z is a lower alkylene group. This compound may be referred to as the compound of the present disclosure.

[0019] In the present disclosure, the lower alkyl group refers to an alkyl group having 1 to 6 (1, 2, 3, 4, 5, or 6) carbon atoms, with an alkyl group having 1 to 4 carbon atoms being preferred. The lower alkyl group may be linear or branched. More specific examples include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a sec-pentyl group, a 3-pentyl group, a tert-pentyl group, an n-hexyl group, an isohexyl group, a sec-hexyl group, a 3-hexyl group, and a tert-hexyl group. A methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a sec-butyl group are more preferred.

[0020] In the present disclosure, the protecting group for the OH group is R p The state in which the OH group is protected by a protecting group is sometimes referred to as OR. p The protective group for the OH group (R p ) include an acetyl group, a benzyl group, a p-methoxyphenylbenzyl group, a methoxymethyl group, a trimethylsilyl group, a triethylsilyl group, a t-butyl group, a t-butyldimethylsilyl group, a benzoyl group, a trityl group, and the like, with an acetyl group being particularly preferred.

[0021] R 1 , R 2 , R 3 are preferably each independently (the same or different) an alkyl group having 1 to 4 carbon atoms. 1 indicates a methyl group, and R 2 and R 3 are the same or different and more preferably represent an alkyl group having 1 to 4 carbon atoms, and R 1 indicates a methyl group, and R 2 represents an isobutyl group, and R 3 More preferably, represents an alkyl group having 1 to 4 carbon atoms.

[0022] R a , R b As described above, each independently (the same or different) represents a protecting group for an H or OH group (R p) is shown.

[0023] Also, R c , R d are each independent (the same or different) as described above.

[0024] R c is a protecting group for H or OH group (R p ) or alternatively, R c For -OR c may indicate =O, i.e., OR c The carbon atom to which is bonded is C ORc Then, C ORc -OR c is C ORc =O may also be used.

[0025] R d is a protecting group for H or OH group (R p ) or alternatively, R d For -OR d may indicate =O, i.e., OR d The carbon atom to which is bonded is C ORd Then, C ORd -OR d is C ORd =O may also be used.

[0026] R 456 is, as described above, H (hydrogen atom) or a group represented by the formula:

[0027] [ka] R represents a group represented by 456 When represents the group, it is particularly represented by the following formula:

[0028] [ka]

[0029] R 4 As mentioned above, H (hydrogen atom), OR 4p OR 4prepresents a lower alkyl group optionally substituted with, preferably H or OR 4p Also, R 4p is a protecting group for H or OH group (R p ) or alternatively, R 4p For -OR 4p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 4p may indicate =O, i.e., OR 4p The carbon atom to which is bonded is C OR4p Then, C OR4p -OR 4p is C OR4p =O may also be used.

[0030] R 5 As mentioned above, H, OR 5p OR 5p represents a lower alkyl group which may be substituted with R 5p is a protecting group for H or OH group (R p ) or alternatively, R 5p For -OR 5p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 5p may indicate =O, i.e., OR 5p The carbon atom to which is bonded is C OR5p Then, C OR5p -OR 5p is C OR5p =O may also be used.

[0031] R 6 As mentioned above, H, OR 6p OR 6p represents a lower alkyl group which may be substituted with R 6p is a protecting group for H or OH group (R p ) or alternatively, R 6p For -OR 6p -OR when the carbon atom to which it is attached does not form a carbon-carbon double bond 6p may indicate =O, i.e., OR 6p The carbon atom to which is bonded is C OR6p Then, C OR6p -OR6p is C OR6p =O may also be used.

[0032] Note that OR (where R is R 4p , R 5p , or R 6p As the lower alkyl group (having 1 to 6 carbon atoms) which may be substituted with (representing the group represented by the formula (I)), an alkyl group having 1 to 4 carbon atoms which may be substituted with OR is preferred. When the lower alkyl group is substituted with OR, it is preferably substituted with one or two ORs, and more preferably with one OR. An alkyl group having 1 to 4 carbon atoms which may be substituted with one OR is particularly preferred. More specific examples of the lower alkyl group include -CH2-OR, -CH(CH3)-OR, -C(CH3)2-OR, -CH2-CH2-OR, -CH2-CH(CH3)-OR, -CH2-C(CH3)2-OR, -CH(CH3)-CH(CH3)-OR, -CH(CH3)-C(CH3)2-OR, -CH2-CH2-CH2-OR, -CH(CH3)-CH2-CH2-OR, -CH2-CH(CH3)-CH2-OR, and the like.

[0033] R 456 is the group and the double line consisting of a dotted line and a solid line represents a single bond, OR c R c is off, R 4 and the carbon atom to which OR is bonded c The O in the R 6 the carbon atom to which OR is attached c the carbon atom to which OR is attached d and the carbon atom to which OR is bonded. c and the carbon atom to which OR is bonded d A ring may be formed together with the carbon atoms bonded to both carbon atoms to which R is bonded. When such a ring is formed, R 4 is preferably H or OH, more preferably OH.

[0034] Also, R c indicates H, and R d indicates H and R 4and the carbon atom to which OR is bonded c If O and are not bonded, OR c R c and OR d R d is removed, OR c O and OR d The O and Z are bonded via -Z- to form OR c the carbon atom to which OR is attached d and the carbon atom to which OR is bonded. c and the carbon atom to which OR is bonded d may form a ring together with the carbon atoms bonded to both of the carbon atoms to which Z is bonded, wherein Z is a lower alkylene group.

[0035] In the present disclosure, the lower alkylene group refers to an alkylene group having 1 to 6 (1, 2, 3, 4, 5, or 6) carbon atoms, preferably an alkylene group having 1 to 4 carbon atoms. The lower alkylene group may be linear or branched. For example, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2-CH2-, -CH2-CH(CH3)-, -CH2-C(CH3)2-, -CH(CH3)-CH(CH3)-, -CH(CH3)-C(CH3)2-, -CH2-CH2-CH2-, -CH(CH3)-CH2-CH2-, -CH2-CH(CH3)-CH2-, -C(CH3)2-CH2-CH2-, -CH2-C(CH3)2-CH2-, -CH(CH3)-CH(CH3)-CH2-, - Examples thereof include CH(CH3)2-CH(CH3)-CH2-, -CH(CH3)-C(CH3)2-CH2-, -CH(CH3)-CH(CH3)-CH(CH3)-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-CH2-, and the like, and preferred examples thereof include a methylene group, an ethylene group, an n-propylene group, an isopropylene group, a tetramethylene group, a dimethylmethylene group, a dimethylethylene group, and the like. When -Z- represents a lower alkylene group represented by these formulas, the left side of the formula is OR c This is the O side.

[0036] In the compounds of the present disclosure, at least R a and Rb are preferably the same (particularly preferably H or an acetyl group), and R a , R b , R c , R d are all the same (particularly preferably H or acetyl group), or a and R b are the same (particularly preferably H or an acetyl group) and OR c O and OR d It is more preferred that the O in the formula (I) is bonded to the O in the formula (I) via -Z- to form the ring.

[0037] Among the compounds of the present disclosure, one example of a preferred compound is a compound represented by the following formula:

[0038] [ka] Among the compounds represented by the formula, During the ceremony, R 4 and R 5 teeth, R 4 indicates H and R 5 OR 5p represents a lower alkyl group optionally substituted with R 4 OR 4p represents a lower alkyl group optionally substituted with R 5 indicates H, R 6 is OR 6p represents a lower alkyl group optionally substituted by R 1 , R 2 , R 3 , R a , R b , R c , R d , R 4p , R 5p , R 6p is the same as above, More preferred examples include compounds in which:

[0039] Although not particularly limited, R 4 indicates H and R 5 OR 5p When R represents a lower alkyl group which may be substituted with 6a preferably represents an alkyl group having 1 to 4 carbon atoms which may be substituted with OH, and in this case, R 5 and R 6 may be the same or different. 4 indicates H and R 5 and R 6 More preferably, are the same or different and represent an alkyl group having 1 to 4 carbon atoms.

[0040] In addition, although not particularly limited, R 4 OR 4p represents a lower alkyl group optionally substituted with R 5 If indicates H, then R 6 preferably represents an alkyl group having 1 to 4 carbon atoms which may be substituted with OH, and in this case, R 4 and R 6 may be the same or different. 5 indicates H and R 4 and R 6 More preferably, are the same or different and represent an alkyl group having 1 to 4 carbon atoms substituted with OH.

[0041] In addition, in the compounds of the present disclosure, particularly OR c O and OR d and O are bonded via -Z- to form the ring, the formula is as follows: In the following formula, it is more preferable that the double line consisting of a dotted line and a solid line represents a double bond.

[0042] [ka] Also among the compounds of the present disclosure, R 456 is the expression:

[0043] [ka] The double line consisting of a dotted line and a solid line represents a single bond, and R 4 and the carbon atom to which OR is bonded c When the O is directly bonded to form the ring, the formula is as follows:

[0044] [ka] Among the compounds represented by the formula, R 4 represents H or OH, and R 5 is OR 5p represents a lower alkyl group optionally substituted with R 6 indicates H, and R 1 , R 2 , R 3 , R a , R b , R c , R d , R 5p , and Z is the same as above, are more preferred.

[0045] Although not particularly limited, specific examples of compounds that are preferred as compounds of the present disclosure are summarized below.

[0046] [ka] TIFF2025121722000015.tif178170TIFF2025121722000016.tif189170TIFF2025121722000017.tif193170

[0047] The compounds of the present disclosure are Streptomyces actinomycetes, Streptomyces The compound can be obtained by culturing sp. K240, or by using the compound thus obtained as a raw material and modifying it by known methods or methods that can be easily derived from known methods. Streptomycessp. K240 has been internationally deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD: 2-5-8 Kazusa Kamatari, Kisarazu, Chiba Prefecture) (accession number NITE BP-04045).

[0048] The compounds of the present disclosure may be, for example: Streptomyces sp. K240 can be cultured using a known medium and conditions used for fermentation production, and the compound can be isolated and collected from the resulting culture by known methods such as solvent extraction and chromatography (preferably in combination). More specifically, the compound of the present disclosure can be obtained, for example, by Streptomyces The compound is produced by aerobic shaking culture of Bacillus sp. K240 in a liquid medium. Any synthetic or natural medium containing an appropriate carbon source, nitrogen source, and inorganic salts can be used for the growth of the strain. Vitamins or other nutrients may be added to the medium as needed. For efficient production of the compound of the present disclosure, it is preferable to add a rich carbon source and a buffer to the medium.

[0049] Preferred carbon sources include glucose, as well as maltose, fructose, sucrose, starch, glycerol, mannitol, animal oil, vegetable oil, etc. These can be used alone or in combination. Nitrogen sources include yeast extract, cottonseed meal, soybean flour, meat extract, peptone, corn steep liquor, etc., which can be used alone or in combination. Inorganic salts include calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, various phosphates, and metal salts, which can be added alone or in combination as needed. When using inorganic salts, calcium carbonate is preferred. Antifoaming agents, etc. can also be added as needed.

[0050] The culture conditions are: StreptomycesThe culture conditions can be appropriately changed within the range that allows growth of the strain K240 and production of the compound of the present invention. For example, the culture temperature is preferably 27 to 30°C, and the initial pH of the medium is preferably near neutral. The culture time is, for example, about 4 to 7 days. These various culture conditions can be appropriately changed, for example, using the production efficiency of the compound of the present disclosure as an indicator.

[0051] The compounds of the present disclosure can be recovered from either the culture medium (particularly the filtrate of the culture medium) after cultivation or from the bacterial cells. To separate and collect the compounds of the present disclosure, known means used to obtain microbial metabolic products can be appropriately utilized. Examples of such means include various ion exchange resins, nonionic adsorption resins, gel filtration chromatography, column chromatography using silica gel or alumina, thin layer chromatography, high performance liquid chromatography using normal-phase or reverse-phase columns, vacuum concentration, freeze-drying, and the like. Purification is possible by using these means alone, in appropriate combination, or repeatedly.

[0052] When using the compounds of the present disclosure, they can be used as solids or dissolved in a solvent (e.g., organic solvent or aqueous organic solvent). It is preferable to use them dissolved in a solvent. Examples of organic solvents include alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol (PEG)), dimethyl sulfoxide (DMSO), and the like. These organic solvents can also be used in combination with water (i.e., as aqueous organic solvents). More specifically, they can be used by mixing them with, for example, water, saline, glucose, or an isotonic solution containing other adjuvants.

[0053] As described above, the compounds of the present disclosure can potently inhibit the sphere formation of cancer stem cells. Therefore, compositions containing the compounds of the present disclosure can be preferably used as pharmaceutical compositions. Furthermore, compositions containing the compounds of the present disclosure can also be preferably used as anti-cancer compositions, and in particular, because they potently inhibit cancer stem cells, they can also be used as anti-cancer stem cell agents. They can be preferably used to suppress the proliferation of cancer stem cells. They can also be preferably used to inhibit the formation of cancer stem cell clusters (spheres).

[0054] It is also preferable to use the composition of the present disclosure in combination with a known anticancer drug.It is expected that the compound of the present disclosure inhibits cancer stem cells that cannot be inhibited by known anticancer drugs, thereby exerting a more powerful anticancer effect.In addition, a composition containing the compound of the present disclosure may be referred to as the composition of the present disclosure.

[0055] The amount of the compound of the present disclosure contained in the composition of the present disclosure is not particularly limited as long as it is within a range in which the effect is exhibited, and may be, for example, 0.01 to 100% by mass, or may be, for example, 0.1 to 99% by mass.

[0056] The compositions of the present disclosure can be used in various forms other than pharmaceutical compositions, and can be used in a variety of fields, such as medicines, reagents, cosmetics, food additives, food compositions (including health foods, health enhancers, and nutritional supplements (supplements, etc.)).

[0057] The composition of the present disclosure can be used either in vitro or in vivo. The target cells are preferably cancer cells (particularly cancer stem cells). The tissue from which the cells are derived is not particularly limited, and examples include epithelial tissue, connective tissue, muscle tissue, and nerve tissue. More specifically, digestive tissue is preferred.

[0058] Cancers to which the compositions of the present disclosure can be applied are not particularly limited, and examples include lung cancer, stomach cancer, liver cancer, esophageal cancer, pancreatic cancer, colon cancer, colorectal cancer, biliary tract cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, breast cancer, prostate cancer, testicular cancer, skin cancer, leukemia, bone tumor, osteosarcoma, soft tissue tumor, multiple myeloma, malignant lymphoma, pharyngeal cancer, head and neck cancer, pediatric cancer, etc. Of these, colon cancer is particularly preferred, although not limited thereto.

[0059] The organisms to which the composition of the present disclosure is applied are not particularly limited, and examples thereof include humans as well as mammals, more specifically various mammals such as monkeys, mice, rats, dogs, cats, and rabbits.

[0060] The compositions of the present disclosure may be in a dosage form appropriate for the intended use (in vitro or in vivo) or application (e.g., administration, ingestion, inoculation, etc.).

[0061] When the composition of the present disclosure is intended for pharmaceutical use, it may take the form of oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquid preparations (including drinks, suspensions, syrups), and jellies; or parenteral preparations such as injectable preparations (for example, drip injections (for example, intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (for example, ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

[0062] Other forms include, for example, tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including suspensions and syrups), jellies, etc. For food compositions, examples include liquid, gel, or solid foods such as juice, soft drinks, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, powdered milk for infants, cake mixes, dairy products (for example, powder, liquid, gel, solid, etc.), bread, and confectioneries (for example, cookies, etc.).

[0063] The composition of the present disclosure may contain any additive in addition to the compound of the present disclosure. The additive is not particularly limited as long as it is a pharmaceutically acceptable component, and examples thereof include a base, carrier, solvent, dispersant, emulsifier, buffer, stabilizer, excipient, binder, disintegrant, lubricant, thickener, moisturizer, colorant, flavoring, chelating agent, etc.

[0064] The administration route of the composition of the present disclosure can be appropriately set, and examples thereof include topical administration, enteral administration, parenteral administration, etc. More specific administration routes include epicutaneous administration, enema administration, oral administration, intravenous administration, intramuscular administration, subcutaneous administration, intraosseous administration, transmucosal administration, intraperitoneal administration, etc.

[0065] The application form and effective dosage of the composition of the present disclosure can be appropriately determined depending on various conditions such as the subject, administration route, dosage form, condition of the subject, and the doctor's judgment, and is not limited thereto, but for example, 1 μg to 1000 mg can be administered per dose to an adult weighing 60 kg. The number of doses can be, for example, 1 to 3 times per day.

[0066] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0067] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0068] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0069] Example 1. Production, isolation, and purification of compound 1 Streptomyces K240 was cultured on agar medium (0.40% starch, 0.40% yeast extract, 1.0% malt extract, 2.0% agar, pH 7.2) at 30°C for 4 days. The cells were inoculated into KG liquid medium (2.5% glucose, 1.5% soybean meal, 0.20% yeast extract, 0.40% CaCO3, pH 7.2) and precultured at 30°C for 2 days. Subsequently, 2% of the preculture was inoculated into KG medium (100 mL each) and cultured at 30°C for 5 days to prepare a culture medium (total 3 L).

[0070] Acetone was added to the culture medium to disrupt the cells, and the cells were removed by filtration. The pH was adjusted to 9.0, and then ethyl acetate was added and extracted. The extract was concentrated (451.0 mg), and n-hexane was added and centrifuged. The n-hexane-insoluble fraction (206.6 mg) was fractionated by silica gel chromatography using a chloroform-methanol system. The chloroform / methanol (100:2-100:3) elution fraction (26.0 mg) was fractionated by silica gel chromatography using a toluene-acetone system. The resulting toluene / acetone (2:1) elution fraction (5.7 mg) was purified by reverse-phase HPLC (Develosil C30, 45% MeCN) to obtain 0.4 mg of compound 1 of the present invention. The physicochemical properties of compound 1 of the present invention are shown below.

[0071] Color and appearance: White powder Molecular formula:C 23 H 36 O7 High resolution ESI MS: m / z 447.2339 ([M+Na] + C 23 H 36 O7Na, D 1.4 mmu). UV(MeOH):λ max nm (e) 218 (6,050), 242 (sh), 290 (4,282), 332 (1,573). IR (neat):ν max 3389, 2959, 2930, 2869, 1709, 1621, 1460, 1417, 1375, 1269, 1240, 1214, 1159, 1065, 975, 875 cm -1 . NMR (500 MHz): Data measured in CD3OD are shown in Table 1.

[0072] [ka]

[0073] [Table 1]

[0074] Example 2. Production, isolation, and purification of compound 2 Streptomyces K240 was cultured on ISP-2 agar medium at 30°C for 4 days. The cells were inoculated into T liquid medium (5.0% glucose, 0.40% yeast extract, 0.25% CaCO3) and precultured at 30°C for 2 days. Next, 2% of the preculture was inoculated into T medium (100 mL each) and cultured at 30°C for 4 days to prepare a culture medium (3 L in total).

[0075] Acetone was added to the culture medium to disrupt the cells, and the cells were removed by filtration. The pH was adjusted to 7.0, and then ethyl acetate was added for extraction. The extract was concentrated (742.5 mg) and fractionated by silica gel chromatography using a chloroform-methanol system. The chloroform / methanol (10:1) elution fraction (141.8 mg) was fractionated by silica gel chromatography using an n-hexane-ethyl acetate system. The resulting n-hexane / ethyl acetate (1:9-0:10) fraction (28.0 mg) was fractionated by reverse-phase HPLC (Cosmosil Cholesterol, 37% MeCN). The resulting crude fraction (1.9 mg) was purified by preparative thin-layer chromatography using ethyl acetate (100%), yielding 1.4 mg of compound 2 of the present invention. The physicochemical properties of compound 2 of the present invention are shown below.

[0076] Color and appearance: White powder Molecular formula:C 24 H 38 O7 High resolution ESI MS: m / z 461.2465 ([M+Na] + C 24 H 38 O7Na, D 4.5 mmu). UV(MeOH):λ max nm (e) 220 (7,813), 240 (sh), 289 (6,784), 333 (2,582). IR (neat):ν max3341, 2958, 2930, 2871, 1619, 1464, 1421, 1381, 1338, 1307, 1295, 1274, 1234, 1213, 1164, 1065, 1039, 1028, 931, 879, 828 cm -1 . NMR (600 MHz): Data measured in CDCl3 are shown in Table 2.

[0077] [ka]

[0078] [Table 2]

[0079] Example 3. Production, isolation, and purification of compound 3 Streptomyces K240 was cultured on ISP-2 agar medium at 30°C for 4 days. The cells were inoculated into T liquid medium (5.0% glucose, 0.40% yeast extract, 0.25% CaCO3) and precultured at 30°C for 2 days. 2% of the preculture was then inoculated into T medium (100 mL each) and cultured at 30°C for 4 days to prepare a culture medium (total 3 L).

[0080] Acetone was added to the culture medium to disrupt the cells, and the cells were removed by filtration. The pH was adjusted to 7.0, and then ethyl acetate was added for extraction. The extract was concentrated (742.5 mg) and fractionated by silica gel chromatography using a chloroform-methanol system. The chloroform / methanol (100:1) elution fraction (209.2 mg) was fractionated by silica gel chromatography using an n-hexane-ethyl acetate system (3:1). The resulting crude fraction (65.8 mg) was fractionated by reverse-phase HPLC (Cosmosil Cholester, 57% MeCN). The resulting crude fraction (1.9 mg) was purified by preparative thin-layer chromatography using a chloroform-methanol system (50:1), yielding 1.2 mg of compound 3 of the present invention. The physicochemical properties of compound 3 of the present invention are shown below.

[0081] Color and appearance: White powder Molecular formula:C 23 H 36 O5 High resolution ESI MS: m / z 415.2421 ([M+Na] + C 23 H 36 O5Na, D 3.4 mmu). UV(MeOH):λ max nm (e) 222 (10,200), 240 (sh), 285 (9,789), 331 (5,279). IR (neat):ν max 3317, 2961, 2952, 1618, 1446, 1420, 1380, 1314, 1232, 1166, 1073, 1039, 980, 906, 803 cm -1 . NMR (600 MHz): Data measured in CDCl3 are shown in Table 3.

[0082] [ka]

[0083] [Table 3]

[0084] Example 4. Production, isolation, and purification of compound 4 Streptomyces K240 was cultured on ISP-2 agar medium at 30°C for 4 days. The cells were inoculated into T liquid medium (5.0% glucose, 0.40% yeast extract, 0.25% CaCO3) and precultured at 30°C for 2 days. 2% of the preculture was then inoculated into T medium (100 mL each) and cultured at 30°C for 4 days to prepare a culture medium (total 3 L).

[0085] Acetone was added to the culture medium to disrupt the cells, and the cells were removed by filtration. The pH was adjusted to 7.0, and then ethyl acetate was added and extracted. The extract was concentrated (742.5 mg) and fractionated by silica gel chromatography using a chloroform-methanol system. The chloroform / methanol (100:1) elution fraction (209.2 mg) was fractionated by silica gel chromatography using an n-hexane-ethyl acetate system (3:1). The resulting crude fraction (65.8 mg) was fractionated by reverse-phase HPLC (Cosmosil Cholesterol, 57% MeCN). The resulting crude fraction (27.6 mg) was purified by preparative thin-layer chromatography using an n-hexane-ethyl acetate system (2:1), yielding 19.8 mg of compound 4 of the present invention. The physicochemical properties of compound 4 of the present invention are shown below.

[0086] Color and appearance: White powder Molecular formula:C 24 H 38 O5 High resolution ESI MS: m / z 429.2609 ([M+Na] + C 24 H 38 O5Na, D 0.2 mmu). UV(MeOH):λ max nm (e) 221 (9,444), 240 (sh), 284 (9,356), 331 (5,215). IR (neat):ν max 3366, 2958, 2932, 2871, 1619, 1464, 1419, 1380, 1336, 1318, 1307, 1272, 1236, 1216, 1163, 1050, 879, 822 cm -1 . NMR (600 MHz): Data measured in CDCl3 are shown in Table 4.

[0087] [ka]

[0088] [Table 4]

[0089] Example 5. Synthesis of derivatives II and III of invention compound 4

[0090] [ka]

[0091] Using invention compound 4 as the starting material, derivatives II and III were obtained in one or two steps. Specifically, invention compound 4 (3.0 mg, 7.37 μmol) was dissolved in dichloromethane (200 μL), 2,2-dimethoxypropane (3.6 mL, 29.3 μmol) and pyridinium p-toluenesulfonate (cat.) were added, and the mixture was stirred at room temperature for 22 hours. The resulting reaction mixture was purified by reverse-phase HPLC (Cosmosil Cholester, 30-100% MeCN) to obtain 2.2 mg of derivative II.

[0092] Separately, invention compound 4 (5.0 mg, 12.2 μmol) was dissolved in dichloromethane (200 μL), and 2,2-dimethoxypropane (6.0 mL, 48.9 μmol) and pyridinium paratoluenesulfonate (cat.) were added. The mixture was stirred at room temperature for 20 hours. After the solvent was evaporated, acetic anhydride / pyridine (= 1:1, 200 μL) was added and the mixture was stirred at room temperature for an additional 24 hours. The resulting reaction mixture was quenched with water, diluted with dichloromethane, and washed twice with 0.1 M hydrochloric acid and twice with saturated aqueous sodium bicarbonate. The organic layer was dried over sodium sulfate, filtered, concentrated, and the solvent was evaporated azeotropically with toluene. The residue was purified by silica gel chromatography using n-hexane-ethyl acetate (6:1) to obtain 3.6 mg of derivative III. The physicochemical properties of derivatives II and III are shown below.

[0093] [Derivative II] Color and appearance: Light brown oil Molecular formula:C27 H 42 O5. High resolution ESI MS: m / z 469.2966 ([M+Na] + C 27 H 42 O5Na, D 4.2 mmu). UV(MeOH):λ max nm (e) 219 (4,115), 240 (sh), 289 (3,658), 333 (1,304). IR (neat):ν max 3415, 2985, 2959, 2932, 2871, 1620, 1464, 1420, 1380, 1344, 1317, 1273, 1222, 1161, 1080, 1003, 873, 828 cm -1 .

[0094] [Inducer III] Color and shape: light brown oily Molecular formula: C 31 H 46 O7. High resolution ESI MS: m / z 553.3195 ([M+Na] + C 31 H 46 O7Na, D 5.9 mmu). UV(MeOH):λ max nm (e) 217 (10,877), 250 (9,292), 290 (sh). IR (neat):ν max 2986, 2959, 2931, 2872, 1770, 1687, 1681, 1593, 1464, 1421, 1368, 1223, 1192, 1092, 1039, 902, 860, 813 cm -1 . NMR (600 MHz): Measurement results in CDCl3 are shown in Table 5 and Table 6.

[0095]

change

[0096] [ka]

[0097] [Table 5]

[0098] [Table 6]

[0099] Example 6. Production, isolation, and purification of compound 5 Streptomyces K240 was cultured on ISP-2 agar medium at 30°C for 4 days. The cells were inoculated into T liquid medium (5.0% glucose, 0.40% yeast extract, 0.25% CaCO3) and precultured at 30°C for 2 days. 2% of the preculture was then inoculated into T medium (100 mL each) and cultured at 30°C for 4 days to prepare a culture medium (total 3 L).

[0100] Acetone was added to the culture medium to disrupt the cells, and the cells were removed by filtration. The pH was adjusted to 7.0, and then ethyl acetate was added and extracted. The extract was concentrated (742.5 mg) and fractionated by silica gel chromatography using a chloroform-methanol system. The chloroform / methanol (100:1) elution fraction (209.2 mg) was fractionated by silica gel chromatography using an n-hexane-ethyl acetate system (3:1). The resulting crude fraction (65.8 mg) was fractionated by reverse-phase HPLC (Cosmosil Cholesterol, 57% MeCN). The resulting crude fraction (3.5 mg) was purified by preparative thin-layer chromatography using an n-hexane-ethyl acetate system (2:1), yielding 2.3 mg of compound 5 of the present invention. The physicochemical properties of compound 5 of the present invention are shown below.

[0101] Color and appearance: White powder Molecular formula:C 25 H 40 O5 High resolution ESI MS: m / z 443.2723 ([M+Na] + C 25 H 40 O5Na, D 4.5 mmu). UV(MeOH):λ max nm (e) 219 (8,943), 240 (sh), 288 (6,498), 333 (2,539). IR (neat):ν max 3357, 2957, 2928, 2870, 1620, 1455, 1418, 1381, 1337, 1309, 1275, 1239, 1201, 1162, 1035, 994, 832 cm -1 . NMR (600 MHz): Data measured in CDCl3 are shown in Table 7.

[0102] [ka]

[0103] [Table 7]

[0104] Example 7. Production, isolation, and purification of compound 6 Streptomyces K240 was cultured on ISP-2 agar medium at 28°C for 5 days. The cells were inoculated into A-16 liquid medium (2.0% glucose, 1.0% Pharmamedia, 0.50% CaCO3) and precultured at 28°C for 3 days. 2% of the preculture was then inoculated into A-16 medium (100 mL each) and cultured at 28°C for 7 days to prepare a culture medium (total 3 L).

[0105] Acetone was added to the culture medium to disrupt the cells, and the cells were removed by filtration. The pH was adjusted to 9.0, and then ethyl acetate was added for extraction. The extract was concentrated (516.9 mg) and fractionated by silica gel chromatography using a chloroform-methanol system. The chloroform / methanol (100:2) elution fraction (45.0 mg) was fractionated by silica gel chromatography using an n-hexane-ethyl acetate system (3:2). The resulting crude fraction (19.1 mg) was fractionated by reverse-phase HPLC (Cosmosil Cholesterol, 42% MeCN). The resulting crude fraction (0.8 mg) was purified by preparative thin-layer chromatography using an n-hexane-ethyl acetate system (2:3), yielding 0.7 mg of compound 6 of the present invention. The physicochemical properties of compound 6 of the present invention are shown below.

[0106] Color and appearance: White powder Molecular formula:C 19 H 30 O5 High resolution ESI MS: m / z 361.1973 ([M+Na] + C 19 H 30 O5Na, D 1.2 mmu). UV (MeOH):λ max nm (e) 221 (8,064), 240 (sh), 291 (8,531), 334 (3,472). IR (neat):ν max 3325, 2956, 2919, 2871, 2848, 1622, 1540, 1465, 1419, 1381, 1310, 1273, 1236, 1217, 1164, 1015, 969, 877 cm -1 . NMR (600 MHz): Data measured in CDCl3 are shown in Table 8

[0107] [ka]

[0108] [Table 8]

[0109] Example 8. Inhibitory activity of compounds 1 to 6 and derivatives II and III on colon cancer stem cell sphere formation The sphere formation inhibitory activity against the human colon cancer-derived cell line HT29 was measured as follows, according to the description in Non-Patent Document 3 (Ikeda, H. et al. J. Antibiot., 75, 671, 2022). HT29 cells were cultured in 3D Tumorsphere Medium XF (PromoCell) on poly(2-hydroxyethyl methacrylate)-coated plates at 37°C for 7 days. The compounds were added to the cells at final concentrations of 1-100 μg / mL in dimethyl sulfoxide (DMSO). After 3 days of culture at 37°C, sphere formation was completely inhibited at concentrations of 10 μg / mL (compound 1), 100 μg / mL (compound 2), 30 μg / mL (compound 3), 10 μg / mL (compound 4), 10 μg / mL (compound 5), 100 μg / mL (compound 6), 10 μg / mL (derivative II), and 30 μg / mL (derivative III). The results are shown in the following table.

[0110] [Table 9]

[0111] These results indicate that compounds 1 to 6 and derivatives II and III are highly effective as inhibitors of colon cancer stem cell sphere formation.

Claims

1. formula: 【Chemical 1】 (In the formula, R １ , R ２ , R ３ each independently represents a lower alkyl group, R ａ , R ｂ each independently represents a protecting group for an H or OH group, R ｃ , R ｄ are independent of each other, R ｃ represents a protecting group for an H or OH group, or -OR ｃ indicates =O, R ｄ represents a protecting group for an H or OH group, or -OR ｄ indicates =O, R ４５６ is H, or the formula: 【Chemistry 2】 [Wherein: A double line consisting of a dotted line and a solid line represents a single bond or a double bond. R ４ , R ５ , R ６ are independent of each other, R ４ is H, OR ４ｐ , or OR ４ｐ represents a lower alkyl group optionally substituted by ４ｐ represents a protecting group for an H or OH group, or -OR ４ｐ When the carbon atom to which is bonded does not form a carbon-carbon double bond, -OR ４ｐ may represent =O, R ５ is H, OR ５ｐ , or OR ５ｐ represents a lower alkyl group optionally substituted by ５ｐ represents a protecting group for an H or OH group, or -OR ５ｐ When the carbon atom to which is bonded does not form a carbon-carbon double bond, -OR ５ｐ may represent =O, R ６ is H, OR ６ｐ , or OR ６ｐ represents a lower alkyl group optionally substituted by ６ｐ represents a protecting group for an H or OH group, or -OR ６ｐ When the carbon atom to which is bonded does not form a carbon-carbon double bond, -OR ６ｐ may represent ═O.] represents a group represented by R ４５６ is the group, and the double line consisting of a dotted line and a solid line represents a single bond, OR ｃ R ｃ is off, R ４ and the carbon atom to which OR ｃ is directly bonded to O in R ６ the carbon atom to which OR is bonded ｃ the carbon atom to which OR is bonded ｄ and the carbon atom to which OR ｃ and the carbon atom to which OR ｄ may form a ring together with carbon atoms bonded to both carbon atoms to which R ｃ indicates H, and R ｄ represents H, and R ４ and the carbon atom to which OR ｃ When O is not bonded to OR ｃ R ｃ and OR ｄ R ｄ is off, OR ｃ O and OR ｄ is bonded to O via -Z-, and ｃ the carbon atom to which OR is bonded ｄ and the carbon atom to which OR ｃ and the carbon atom to which OR ｄ may form a ring together with carbon atoms bonded to both carbon atoms to which Z is bonded, wherein Z is a lower alkylene group. A compound represented by the formula:

2. formula: 【Chemistry 3】 (In the formula, R ４ and R ５ teeth, R ４ indicates H and R ５ is OR ５ｐ represents a lower alkyl group optionally substituted by R ４ is OR ４ｐ represents a lower alkyl group optionally substituted with R ５ indicates H, R ６ is OR ６ｐ represents a lower alkyl group optionally substituted by R １ , R ２ , R ３ , R ａ , R ｂ , R ｃ , R ｄ , R ４ｐ , R ５ｐ , R ６ｐ is the same as above.) The compound according to claim 1, wherein the compound is represented by the formula:

3. formula: 【Chemistry 4】 (In the formula, R １ , R ２ , R ３ , R ａ , R ｂ , R ４ , R ５ , R ６ 3. The compound according to claim 2, wherein Z is the same as above.

4. formula: 【Chemistry 5】 (In the formula, R ４ is OR ４ｐ indicates, R ５ is OR ５ｐ represents a lower alkyl group optionally substituted by R ６ indicates H, R １ , R ２ , R ３ , R ａ , R ｂ , R ｄ , R ４ｐ , R ５ｐ is the same as above.) The compound according to claim 1, wherein the compound is represented by the formula:

5. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4.

6. An anti-cancer composition comprising the compound according to any one of claims 1 to 4.

7. An anti-cancer stem cell composition comprising the compound according to any one of claims 1 to 4.

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