WO2025178157A1 - Novel oxazolomycin compound, production method thereof, and use thereof - Google Patents

Abstract

Provided are a novel oxazolomycin compound, a stereoisomer, solvate, or pharmaceutically acceptable salt thereof, a use thereof, and a production method thereof. The compound has anticancer activity and thus the compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof can be used for preventing or treating cancer.

Description

Novel oxazolomycin compounds, methods for producing them, and uses thereof

This patent application relates to a novel oxazolomycin compound, a method for producing the same, and uses thereof. This patent application claims priority to Republic of Korea Patent Application No. 10-2024-026030, filed with the Korean Intellectual Property Office on February 22, 2024, the disclosure of which is incorporated herein by reference.

Cancer, or malignant tumors, are a long-standing enemy of humanity, and despite the constant advancement of medicine, they are the most common cause of death in modern times. Cancer is a disease in which cells ignore the normal cycle of cell death and proliferate uncontrollably, disrupting bodily functions. Because cancer has diverse causes, symptoms, and pathogenesis vary greatly depending on the organ in which it develops, complete cure is extremely difficult. Furthermore, depending on the progression of the cancer, metastasis (the spread of cancer cells to other organs through bodily fluids like blood or lymph) can occur, making even treatment deemed successful the risk of recurrence undeniable. While mortality rates for diseases like rabies and Ebola hemorrhagic fever are higher than cancer, cancer is a terrifying disease because it is so common, yet it can be contracted at any time and in any place, and it is impossible to completely prevent it.

While a variety of anticancer drugs and treatments have been developed for cancer, effectively targeting all the various types of cancer cells that arise in various parts of the body remains a challenge. To address this, new types of anticancer drugs are constantly being developed, and their necessity for a complete cure is growing. Recently, research is underway to develop substances with anticancer activity derived from microorganisms.

Therefore, it is necessary to select microorganisms that produce useful physiologically active substances and to explore novel compounds with anticancer activity from them.

Provided are novel oxazolomycin compounds, stereoisomers, solvates, or pharmaceutically acceptable salts thereof.

A strain of the genus Streptomyces producing a compound or a stereoisomer thereof is provided.

A method for producing the above compound or a stereoisomer thereof is provided.

A pharmaceutical composition for preventing or treating cancer is provided, comprising the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

A method for preventing or treating cancer using the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is provided.

The present invention provides use of the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for preventing or treating cancer.

Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein will be readily apparent and inferred by those skilled in the technical field of this application or similar technical fields, and therefore, their description will be omitted.

Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

One aspect provides a compound represented by the following chemical formula Ⅰ, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

[Chemical Formula I]

In the above chemical formula Ⅰ, R is hydrogen, deuterium, a halogen atom, a hydroxy group, a nitro group, a cyano group, an amino group, an amidino group, hydrazine, a hydrazone, a carboxyl group and its salts, a sulfonyl group, a sulfamoyl group, a sulfonic acid group and its salts, phosphoric acid and its salts, a substituted or unsubstituted _{C 1 to} C ₂₀ alkyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkynyl group, a substituted or unsubstituted _C 3 to C ₂₀ cycloalkyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkoxy, a substituted or unsubstituted C ₁ to C 20 heteroalkyl group, a substituted or unsubstituted C ₃ to C ₂₀ heterocyclic group, a substituted or unsubstituted C ₆ to C ₂₀ aryl group, a substituted or unsubstituted C ₆ to A C ₂₀ arylalkyl group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryl group, a substituted or unsubstituted C ₇ to C ₂₀ heteroarylalkyl group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryloxy group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryloxyalkyl group, and a substituted or unsubstituted C ₆ to C ₂₀ heteroarylalkyl group.

In the above chemical formula I, the numbering of carbon atoms follows the IUPAC nomenclature. For example, R is a substituent attached to carbon 25.

In one specific example, in the above chemical formula Ⅰ, R may be selected from a hydroxy group, a substituted or unsubstituted C ₁ to C ₂₀ alkyl group, or a substituted or unsubstituted C ₁ to C ₂₀ alkoxy.

In one specific example, in the chemical formula I, R may be hydrogen.

In the above chemical formula I, the geometric structure of the double bond portion between carbons 10 to 15 and carbons 15 to 18 can be changed between E or Z structures.

In the above chemical formula Ⅰ, the bond between the amide portion and carbon 14 can be changed to a structure in which it is broken.

The compound represented by the above chemical formula Ⅰ may be a methyl-oxazolomycin compound.

The term “Oxazolomycin” refers to a compound having a spiro-β-lactone-γ-lactam moiety and an oxazole ring, which is a mixture of peptide and polyketide.

The above “methyl-oxazolomycin” may refer to an oxazolomycin compound having a methyl group bonded to carbon 1.

The compound represented by the above chemical formula Ⅰ can have the spiro-β-lactone-γ-lactam moiety of oxazolomycin transformed into a γ-lactam ring.

The compound represented by the above chemical formula Ⅰ may be a compound represented by chemical formula Ⅱ.

[Chemical Formula II]

The compound represented by the above chemical formula Ⅱ may be methyl-oxazolomycin A.

The compound represented by the above chemical formula Ⅰ may be a compound represented by chemical formula Ⅲ.

[Chemical Formula III]

The compound represented by the above chemical formula Ⅱ may be methyl-oxazolomycin B.

The above methyl-oxazolomycin A and methyl-oxazolomycin B are diastereomers.

The term “substitution” refers to the introduction of a substituted hydrogen atom in an organic compound to form a derivative by replacing one or more hydrogen atoms with another atomic group, and “substituent” refers to the introduced atomic group. The substituent is, for example, a halogen atom, a C ₁ to C ₂₀ alkyl group substituted with a halogen atom (e.g., CCF ₃ , CHCF ₂ , CH ₂ F, CCl ₃ , etc.), a C ₁ to C ₂₀ alkoxy, a C ₂ to C ₂₀ alkoxyalkyl, a hydroxy group, a nitro group, a cyano group, an amino group, an amidino group, a hydrazine, a hydrazone, a carboxyl group or a salt thereof, a sulfonyl group, a sulfamoyl group, a sulfonic acid group or a salt thereof, a phosphoric acid or a salt thereof, or a C ₁ to C ₂₀ alkyl group, a C ₂ to C ₂₀ alkenyl group, a C ₂ to C ₂₀ alkynyl group, a C ₁ to C ₂₀ heteroalkyl group, a C ₆ to C ₂₀ aryl group, a C ₆ to C ₂₀ arylalkyl group, a C ₆ to C ₂₀ It may be a heteroaryl group, a C ₇ to C ₂₀ heteroarylalkyl group, a C ₆ to C ₂₀ heteroaryloxy group, and a C ₆ to C ₂₀ heteroaryloxyalkyl group or a C ₆ to C ₂₀ heteroarylalkyl group.

The term “halogen” atoms include fluorine, bromine, chlorine, iodine, etc.

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

The terms “alkoxy” and “aryloxy” refer to an alkyl or aryl group, respectively, bonded to an oxygen atom.

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

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

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

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

The term “heterocyclic group” refers to a non-aromatic organic compound that is a group (ring) containing one or more heteroatoms selected from N, O, P and S, and the remaining ring atoms are carbon. The heterocyclic group is, for example, tetrahydropyran or tetrahydropyridine.

The term "aryl", used alone or in combination, refers to an aromatic system comprising one or more rings, and also includes groups in which an aromatic ring is fused to one or more carbon rings. The C ₆ -C ₃₀ aryl group may be an aryl group having a carbon atom of C ₆ to C ₁₅ , or a carbon atom of C ₆ to C ₁₀ . The aryl group is, for example, phenyl, naphthyl, or tetrahydronaphthyl.

The term “heteroaryl group” refers to a monocyclic or bicyclic organic compound containing one or more heteroatoms selected from the group consisting of N, O, P, and S, with the remaining ring atoms being carbon. The heteroaryl group may contain 1 to 5 heteroatoms and may contain 5 to 10 ring members. The S or N may be oxidized to have multiple oxidation states.

The term "isomer" in "stereoisomer" refers to compounds that have the same molecular formula but different connection methods or spatial arrangements of the constituent atoms within the molecule. Isomers include, for example, structural isomers and stereoisomers. The stereoisomers can be diastereomers or enantiomers. Enantiomers are isomers that do not overlap with their mirror images, like the relationship between left and right hands, and are also called optical isomers. Enantiomers are distinguished as R (Rectus: clockwise) and S (Sinister: counterclockwise) when four or more substituents are different on the chiral center carbon. Diastereoisomers are stereoisomers that are not mirror images, and can be divided into cis-trans isomers that are created by different spatial arrangements of atoms.

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

The term "salt" refers to inorganic and organic acid addition salts of a compound. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The inorganic acid salt may be a hydrochloride, a bromate, a phosphate, a sulfate, or a disulfate. The organic acid salt may be a formate, an acetate, an acetate, a propionate, a lactate, an oxalate, a tartrate, a malate, a maleate, a citrate, a fumarate, a besylate, a camsylate, an edisyl salt, a trichloroacetic acid, a trifluoroacetic acid, a benzoate, a gluconate, a methanesulfonate, a glycolate, a succinate, a 4-toluenesulfonate, a galacturonate, an embronate, a glutamate, a methanesulfonic acid, an ethanesulfonic acid, a benzenesulfonic acid, a p-toluenesulfonic acid, or an aspartate. The above metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.

Another aspect provides a strain of Streptomyces sp. AH05 (accession number: KCTC15725BP) producing a compound or a stereoisomer thereof according to one aspect.

In one specific example, the 16S rRNA base sequence of the Streptomyces sp. AH05 strain (accession number: KCTC15725BP) may be the base sequence represented by SEQ ID NO: 1. The strain may have a 16S rRNA gene having a sequence identity of 98% or more, 99% or more, 99.5% or more, or 99.5% or more with SEQ ID NO: 1.

In one embodiment, a Streptomyces sp. AH05 strain having a 16rRNA gene including a base sequence represented by sequence number 1 was isolated and extracted from tidal flat sediments of Anmyeondo Island through analysis and identification using a molecular genetic method based on 16rRNA sequence search, and a compound was obtained.

The above strain includes its variants. The variants may be, for example, those resulting from natural mutations or artificial mutations. Artificial mutations may be caused by physical mutagens, such as ultraviolet light, or chemical mutagens, such as basic compounds.

The above strain includes spores, mycelia, or cultures of the strain.

The above strain may be isolated or derived from the sediment of Anmyeondo tidal flat.

Another aspect is the step of culturing Streptomyces sp. AH05 strain (Accession number: KCTC15725BP); and

A method for producing a compound according to one aspect or a stereoisomer thereof is provided, comprising a step of isolating the compound according to one aspect or a stereoisomer thereof from a culture of the strain.

The above method includes a step of culturing Streptomyces sp. AH05 strain (Accession number: KCTC15725BP).

The step of culturing the above Streptomyces sp. AH05 strain (Accession No.: KCTC15725BP) may be culturing the strain in a liquid medium or a solid medium. The medium may include, as a carbon source, for example, glucose, corn syrup, dextrin, starch, molasses, animal oil, or vegetable oil. The medium may include, as a nitrogen source, for example, wheat bran, soybean meal, wheat, malt, cottonseed meal, fish meal, corn syrup, meat juice, yeast extract, ammonium sulfate, sodium nitrate, or urea.

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

The method comprises a step of isolating a compound represented by any one of Chemical Formulas I to III or a stereoisomer thereof from a culture of the strain.

The separation step may include concentrating, centrifuging, filtering, or performing chromatography on the culture medium. The chromatography may be, for example, column chromatography, planar chromatography, paper chromatography, or thin-layer chromatography, depending on the type of stationary phase. The chromatography may be, for example, gas chromatography, liquid chromatography, or affinity chromatography, depending on the physical properties of the mobile phase. The liquid chromatography may be, for example, high-performance liquid chromatography (HPLC). The chromatography may be, for example, ion exchange chromatography or size-exclusion chromatography, depending on the separation method. The chromatography may be, for example, normal phase chromatography or reverse phase chromatography.

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

The cancer described above can be either solid or non-solid. Solid cancers refer to tumors that develop in organs such as the liver, lungs, breasts, or skin. Non-solid cancers originate in the blood and are also called hematologic cancers.

In one specific example, the cancer may be breast cancer.

The above breast cancer is classified into types based on the presence (positive) or absence (negative) of estrogen receptors (ER), progesterone receptors (PR), and epidermal growth factor receptors (HER2). The group that expresses ER and PR but not HER2 accounts for the largest proportion of breast cancers. Estrogen binds to estrogen receptors, and the homodimerized estrogen receptors induce the transcription of various growth factors, such as cyclin D1, c-myc, and CDK4, in the nucleus.

In one specific example, the breast cancer may be HER2 negative breast cancer.

In one embodiment, the compound according to one aspect has selective anticancer activity against ER+, PR+, and HER2- breast cancer cell lines and can be used as an effective ingredient for treating and preventing HER2-negative breast cancer.

The term "prevention" refers to any action that suppresses or delays the onset of a disease by administering a composition. The term "treatment" refers to any action that improves or beneficially alters the symptoms of a disease by administering a composition.

The pharmaceutical composition may further comprise a known active ingredient having anticancer activity. The known active ingredient having anticancer activity may be an anticancer agent. The anticancer agent may be 5-fluorouracil, irinotecan, etoposide, oxaliplatin, leucovorin, capecitabine, or a combination thereof. The compound represented by Chemical Formula I, a stereoisomer, solvate, or pharmaceutically acceptable salt thereof, and the anticancer agent may be a single or separate composition for simultaneous or sequential administration.

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

The above pharmaceutical compositions may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. When formulating, the compositions may be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid preparation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the liquid preparation for oral administration may be a suspension, an oral solution, an emulsion, or a syrup. The liquid preparation may include water or liquid paraffin. The liquid preparation may include an excipient such as a wetting agent, a sweetener, a flavoring agent, or a preservative. In the above pharmaceutical composition, the preparation for parenteral administration may be a sterile aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized product, or suppository. The non-aqueous solvent or suspension may contain a vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, Tween 61, cocoa butter, laurin butter, or glycerogelatin.

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

Administration may be oral or parenteral. For example, the route of administration may be oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal. The composition may be administered systemically or locally, and may be administered alone or in combination with other pharmaceutically active compounds.

Another aspect provides a health functional food for preventing or improving cancer, comprising a compound according to one aspect, a stereoisomer, a solvate, or a salt thereof.

The term "improvement" includes any action that improves or benefits the symptoms of a disease.

The above health functional food can be used as a functional food or added to various foods by formulating the compound represented by Chemical Formula Ⅰ, its stereoisomer, solvate, or salt into an encapsulated form, powdered form, or suspension. The above foods include, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, functional foods, and health foods.

Another aspect provides a method of preventing or treating cancer, comprising administering to a subject a compound according to one aspect, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be diagnosed with cancer or may be at a high risk of being diagnosed with cancer.

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

The preferred dosage of the pharmaceutical composition may vary depending on the patient's condition and weight, the extent of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. For example, 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 for adults. The administration may be administered once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks, or once a year.

Another aspect provides the use of the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for preventing or treating cancer.

The term "include" in this specification is used to indicate that other components may be added and/or interposed, rather than to the exclusion of other components, unless specifically stated otherwise.

Duplicate content is omitted in consideration of the complexity of this specification, and terms not otherwise defined herein have the meanings commonly used in the technical field to which the present invention belongs.

Provided are novel oxazolomycin compounds, stereoisomers, solvates, or pharmaceutically acceptable salts thereof, uses, and methods for producing the same. Since the compounds have anticancer activity, the compounds, stereoisomers, solvates, or pharmaceutically acceptable salts thereof can be used to prevent or treat cancer.

Figure 1 is a photograph of a medium in which Streptomyces sp. AH05 strain was cultured.

Figure 2a is a diagram showing the level of estrogen receptor expression in the MCF-7 cell line confirmed by Western blot.

Figure 2b is a diagram showing the level of estrogen receptor expression in the T-47D cell line confirmed by Western blot.

Figure 3a is a diagram showing the results of inhibition of estrogen receptor synthesis by cyclohexamide treatment in MCF-7 cell lines.

Figure 3b is a graph showing the half-life of the estrogen receptor after treatment with 0.16 μM methyl-oxazolomycin A in MCF-7 cells. *p<0.05

Figure 4a is a diagram showing the results of inhibition of estrogen receptor degradation by treatment with MG132, a proteasome inhibitor, in MCF-7 cell lines.

Figure 4b is a graph showing the relative intensity of estrogen receptor expression after MG132 treatment in MCF-7 cell lines.

Figure 5a is a diagram showing the results of inhibiting the degradation of estrogen receptors by treatment with MG132, a proteasome inhibitor, in the T-47D cell line.

Figure 5b is a graph showing the relative intensity of estrogen receptor expression after MG132 treatment in the T-47D cell line.

Figure 6a is a diagram showing the results of flow cytometry analysis according to the concentration of methyl-oxazolomycin A in the MCF-7 cell line.

Figure 6b is a graph showing the cell cycle distribution according to the concentration of methyl-oxazolomycin A in the MCF-7 cell line.

Figure 7a is a diagram showing the results of flow cytometry analysis according to the concentration of methyl-oxazolomycin A in the T-47D cell line.

Figure 7b is a graph showing the cell cycle distribution according to the concentration of methyl-oxazolomycin A in the T-47D cell line.

Figure 8a is a diagram showing the level of breast cancer cell growth factor expression in the MCF-7 cell line confirmed by Western blot.

Figure 8b is a diagram showing the expression level of breast cancer cell growth-related factors in the T-47D cell line using Western blot.

The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[Example]

Example 1. Streptomyces genus ( Streptomyces sp.) Isolation of AH05 strain

To isolate the strain from the tidal flat sediment of Anmyeondo in 2019, YEME (Yeast extract-malt extract) medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, 16 g of agar powder, and 28 g of artificial sea salt per 1 L of distilled water) was used (Fig. 1).

Based on the 16s rRNA sequence analysis, the strain was identified as Streptomyces sp. The strain was named Streptomyces sp. AH05 and deposited with the Korea Center for Microbial Resources (KCTC) (Accession No.: KCTC15725BP). The 16s rRNA sequence of the Streptomyces sp. AH05 strain is indicated as sequence number 1.

Example 2. Cultivation and extraction of Streptomyces AH05 strain

After inoculating 50 mL of YEME liquid medium from the solid medium isolated with Streptomyces AH05 strain, primary culture was performed by shaking at 180 rpm and 30℃ for 3 days. Afterwards, 7 mL of the culture was inoculated into 250 mL of sterilized YEME liquid medium and secondary culture was performed under the same conditions for 3 days. 20 mL of the culture was inoculated into 1 L of GLY liquid medium (20 g of glycerol, 10 g of lactose, 5 g of malt extract, 5 g of yeast extract, 1 g of calcium carbonate, 28 g of artificial sea salt per 1 L of distilled water) and tertiary culture was performed at 180 rpm and 30℃ for 3-4 days.

Afterwards, organic extraction was performed by mixing ethyl acetate (EtOAc) corresponding to 1.5 to 2 times the amount of the culture medium. Specifically, the mixed culture medium and EtOAc were sufficiently shaken using a separatory funnel, and the aqueous layer and organic layer were allowed to sufficiently separate (upper layer: organic layer/lower layer: aqueous layer) and then the aqueous layer was removed. After removing the aqueous layer, the remaining moisture in the organic layer was removed with anhydrous sodium sulfate. Thereafter, the ethyl acetate layer in the organic layer was dried under reduced pressure. As a result, a total of 80 L of culture was cultured to obtain 20 g of extract.

Example 3. Isolation and purification of methyl-oxazolomycin A and B from extracts of Streptomyces sp. AH05 strain.

Methyl-oxazolomycin A and B were isolated and purified from the extract of Streptomyces sp. AH05 strain of Example 2. Specifically, methanol and Celite were added to the dried extract of Example 2, and the extract was adsorbed onto Celite after drying under reduced pressure. Subsequently, fractionation was performed using solvents prepared in 20%, 40%, 60%, 80%, and 100% (methanol/water) compositions sequentially on an open column packed with ODS (C ₁₈ ) resin. As a result, methyl-oxazolomycin A and B were isolated in the 80% MeOH-H ₂ O fraction, which was confirmed through LC/MS.

Afterwards, the fractions were evaporated under reduced pressure and the 80% methanol fraction was redissolved in methanol and subjected to semi-preparative reversed-phase high-performance liquid chromatography (HPLC) under gradient solvent conditions (40-70% aqueous CH ₃ CN for more than 30 min, flow rate: 2 mL/min, UV detection: 280 nm) and YMC column (250 x 10 mm, C ₁₈ , 5 μm).

As a result, methyl-oxazolomycin A was eluted in 20 minutes, and methyl-oxazolomycin B was eluted in 22 minutes, respectively. When further purification was performed using the same column under 40% acetonitrile solvent conditions, methyl-oxazolomycin A was purified in 44 minutes, and methyl-oxazolomycin B was purified in 46 minutes.

Example 4. Identification of the double bond structure and physicochemical properties of methyl-oxazolomycin A and B.

The double bond structures of methyl-oxazolomycin A and B were determined by ROESY NMR spectral analysis.

The structural and physicochemical properties of methyl-oxazolomycin A and B are as follows.

*Methyl-oxazolomycin A

(1) Molecular formula: C ₃₆ H ₅₁ N ₄ O ₉

(2) Molecular weight: 669

(3) Color: Yellow

(4) ¹ H-NMR (DMSO- d ₆ , 800 MHz): See <Table 1>

(5) ¹³ C-NMR (DMSO- d ₆ , 200 MHz): See <Table 1>

[Table 1]

*Methyl-oxazolomycin B (1) Molecular formula: C ₃₆ H ₅₁ N ₄ O ₉

(2) Molecular weight: 669

(3) Color: Yellow

(4) ¹ H-NMR (DMSO- d ₆ , 800 MHz): See <Table 2>

(5) ¹³ C-NMR (DMSO- d ₆ , 200 MHz): See <Table 2>

[Table 2]

The chemical structures of methyl-oxazolomycin A and B are shown below.

[Methyl-oxazolomycin A]

[Methyl-oxazolomycin B]

[Experimental Example]

Experimental Example 1. Confirmation of the cell growth inhibition ability of methyl-oxazolomycin A and B.

To confirm the cell growth inhibitory activity of methyl-oxazolomycin A and B, a sulforhodamine B (SRB) assay was performed. Specifically, the triple-negative breast cancer (ER-, PR-, HER-) MDA-MB-231 cell line, and the ER+, PR+, HER2- breast cancer MCF-7 cell line and T-47D cell line were prepared. The prepared cells were seeded at a density of 5-7 × 10 ⁴ cells/mL in 96-well culture plates and cultured with methyl-oxazolomycin for 72 hours. After incubation, the plates were fixed with a 10% trichloroacetic acid solution for 30 minutes at 4°C. The fixed cells were stained with a 0.4% SRB solution in 1% acetic acid for 30 minutes at room temperature, dissolved in 10 mM Tris (pH 10.0), and the absorbance was measured at 515 nm. The IC ₅₀ values were calculated through nonlinear regression analysis using TableCurve 2D v5.01 and are shown in Table 3 below. In this experimental example, the anticancer drug etoposide was used as a positive control.

[Table 3]

As a result, as shown in Table 3, both methyl-oxazolomycin A and B had no activity in the MDA-MB-231 cell line with IC ₅₀ values of 20 μM or higher. On the other hand, both methyl-oxazolomycin A and B showed strong cell growth inhibition in ER+, PR+, and HER2- breast cancer cell lines. The above results confirmed that both methyl-oxazolomycin A and B have selective anticancer activity in ER+, PR+, and HER2- breast cancer cell lines.

Experimental Example 2. Confirmation of the estrogen receptor inhibition ability of methyl-oxazolomycin A.

To confirm the estrogen receptor inhibitory activity of methyl-oxazolomycin, which exhibits a specific cell growth inhibitory effect on cell lines expressing estrogen receptors. Specifically, MCF-7 and T-47D cell lines were treated with concentrations of 0, 0.02, 0.04, 0.08, and 0.16 μM for 24 h. Then, 2x sample loading buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% β-mercaptoethanol, 50 mM sodium fluoride, and 5 mM sodium orthovanadate) was added to disrupt the cells and incubated at 100°C for 15 minutes. After cooling, the samples were stored at -20°C and thawed at room temperature just before use to perform Western blotting.

As a result, as shown in Fig. 2, it was confirmed that methyl-oxazolomycin A inhibited the expression of estrogen receptors in a concentration-dependent manner in MCF-7 and T-47D cells.

Experimental Example 3. Confirmation of the Estrogen Receptor Degradation Ability of Methyl-Oxazolomycin A

We aimed to investigate the estrogen receptor inhibition mechanism of methyl-oxazolomycin A. First, protein synthesis was inhibited in MCF-7 cells by treatment with 20 μM cyclohexamide. After treatment with 0.16 μM methyl-oxazolomycin A for 0, 1, 2, 4, and 6 hours, estrogen receptor expression was determined by Western blotting. 100% DMSO was used as a control.

As a result, as shown in Fig. 3, it was confirmed that the half-life of the estrogen receptor was reduced by methyl-oxazolomycin A compared to the control group.

Additional experiments were conducted to determine whether the inhibitory effect of methyl-oxazolomycin A on estrogen receptor expression was mediated by lysosomal or proteasomal degradation. MCF-7 and T-47D cell lines were simultaneously treated with methyl-oxazolomycin A and the proteasome inhibitor MG132. A control group treated with methyl-oxazolomycin A alone was used.

As a result, as shown in Figures 4 and 5, it was confirmed that the level of estrogen receptor expression increased in the group simultaneously treated with methyl-oxazolomycin A and the proteasome inhibitor MG132. This revealed that methyl-oxazolomycin A causes the degradation of estrogen receptors through the proteasome.

Experimental Example 4. Confirmation of the cell growth inhibition mechanism of methyl-oxazolomycin A.

The cell growth inhibition ability of methyl-oxazolomycin A due to G0/G1 cell cycle arrest was confirmed. For this, MCF-7 and T-47D cells were seeded at 24 X 10 ⁴ cells/well in 60 mm petri dishes, and the cell cycle was synchronized with 1% FBS medium after 24 hours. After 24 hours, the cell lines were treated with methyl-oxazolomycin A at concentrations of 0, 0.02, 0.04, 0.08, and 0.16 μM. After 24 hours, the cells were collected, stained with propidium iodide (PI), and the cell cycle distribution was analyzed using a flow cytometer.

As a result, as shown in Figures 6 and 7, the G0/G1 distribution increased in a concentration-dependent manner in both MCF-7 and T-47D cell lines.

In addition, we attempted to confirm that it inhibits G0/G1 cell cycle arrest and progression to S cycle through Western blot. Cyclin D1 forms a complex with CDK4 (cyclin-dependent kinase 4) to regulate G1 phase and induce transcription of Rb/E2F. E2F, which forms a complex with Rb, is released when Rb phosphorylation occurs, inducing progression to S phase.

As a result, as shown in Fig. 8, treatment with methyl-oxazolomycin A decreased the amount of phosphorylated Rb in both MCF-7 and T-47D cell lines, indicating arrest in the G0/G1 cycle.

In summary, the above results showed that methyl-oxazolomycin A and B inhibited the expression of estrogen receptors in breast cancer cells and arrested the cell cycle, thereby inhibiting cell growth.

[Accession number]

Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

Accession number: KCTC15725BP

Date of acceptance: 20231206

Claims (9)

A compound represented by the following chemical formula Ⅰ, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above chemical formula Ⅰ, R is hydrogen, deuterium, a halogen atom, a hydroxy group, a nitro group, a cyano group, an amino group, an amidino group, hydrazine, a hydrazone, a carboxyl group and its salts, a sulfonyl group, a sulfamoyl group, a sulfonic acid group and its salts, phosphoric acid and its salts, a substituted or unsubstituted _{C 1 to} C ₂₀ alkyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkynyl group, a substituted or unsubstituted _C 3 to C ₂₀ cycloalkyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkoxy, a substituted or unsubstituted C ₁ to C 20 heteroalkyl group, a substituted or unsubstituted C ₃ to C ₂₀ heterocyclic group, a substituted or unsubstituted C ₆ to C ₂₀ aryl group, a substituted or unsubstituted C ₆ to A C ₂₀ arylalkyl group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryl group, a substituted or unsubstituted C ₇ to C ₂₀ heteroarylalkyl group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryloxy group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryloxyalkyl group, and a substituted or unsubstituted C ₆ to C ₂₀ heteroarylalkyl group. A compound, a stereoisomer, a solvate, or a _{pharmaceutically acceptable salt thereof, in claim 1, wherein in the chemical formula I, R is selected from a hydroxy group, a substituted or unsubstituted C 1 to} C ₂₀ alkyl group, or a substituted or unsubstituted C 1 to C ₂₀ alkoxy. A compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, wherein in the chemical formula I, R is hydrogen in claim 1. In claim 1, the compound is a compound represented by the following chemical formula II, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: [Chemical Formula II] . In claim 1, the compound is a compound represented by the following chemical formula III, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: [Chemical Formula III] . Streptomyces sp. AH05 strain (Accession number: KCTC15725BP) producing the compound of claim 1 or a stereoisomer thereof. A step of culturing Streptomyces sp. AH05 strain (Accession number: KCTC15725BP); and Comprising a step of isolating a compound represented by the chemical formula I of claim 1 or a stereoisomer thereof from a culture of the above strain. A method for producing a compound represented by chemical formula Ⅰ of claim 1 or a stereoisomer thereof. A pharmaceutical composition for preventing or treating cancer, comprising a compound represented by Chemical Formula Ⅰ, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above chemical formula Ⅰ, R is hydrogen, deuterium, a halogen atom, a hydroxy group, a nitro group, a cyano group, an amino group, an amidino group, hydrazine, a hydrazone, a carboxyl group and its salts, a sulfonyl group, a sulfamoyl group, a sulfonic acid group and its salts, phosphoric acid and its salts, a substituted or unsubstituted _{C 1 to} C ₂₀ alkyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkynyl group, a substituted or unsubstituted _C 3 to C ₂₀ cycloalkyl group, a substituted or unsubstituted C ₁ to C ₂₀ alkoxy, a substituted or unsubstituted C ₁ to C 20 heteroalkyl group, a substituted or unsubstituted C ₃ to C ₂₀ heterocyclic group, a substituted or unsubstituted C ₆ to C ₂₀ aryl group, a substituted or unsubstituted C ₆ to A C ₂₀ arylalkyl group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryl group, a substituted or unsubstituted C ₇ to C ₂₀ heteroarylalkyl group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryloxy group, a substituted or unsubstituted C ₆ to C ₂₀ heteroaryloxyalkyl group, and a substituted or unsubstituted C ₆ to C ₂₀ heteroarylalkyl group. A pharmaceutical composition for preventing or treating cancer, wherein the cancer is breast cancer, according to claim 8.