Anticancer agents, pharmaceutical compositions, and food and beverage compositions

Abstract

To provide a new anticancer drug, and a pharmaceutical composition including the same, and a food and drink composition.SOLUTION: The present invention discloses a compound represented by general formula (1) or a salt thereof. (In the general formula (1), R1, R2, R3, R4, and R5 independently represent a monovalent substituent). The present invention also discloses, for example, a DHRS11 (short chain dehydrogenase / reductase superfamily member 11) inhibitor and / or an AR (androgen receptor) expression inhibitor comprising the compound represented by the general formula (1) or a salt thereof as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anticancer agents, pharmaceutical compositions, and food and beverage compositions. More specifically, it relates to novel compounds, DHRS11 inhibitors, and / or AR expression inhibitors having excellent DHRS11 inhibitory activity and / or AR expression suppressing activity, as well as pharmaceutical compositions and food and beverage compositions using the same. [Background technology]

[0002] Cancer is the leading cause of death worldwide, and the development of new anticancer drugs aimed at preventing and / or treating cancer is urgently needed. In recent years, many anticancer drugs with excellent therapeutic response rates have been developed, but the majority of these are antibody drugs. Antibody drugs are highly effective because they act directly on antigens on the surface of cancer cells, but their high price is a problem due to difficulties in mass production and other reasons. Therefore, many patients cannot choose antibody drug treatment for financial reasons.

[0003] Therefore, there is a need for the development of novel anticancer drugs that are easy to mass-produce and relatively inexpensive. Here, as shown in Patent Document 1, the research group of the present inventors has identified a novel compound that, despite being a small molecule compound, has high inhibitory activity against AKR1C3 (Aldo-Keto Reductase Family 1 Member C3), which is involved in the proliferation and growth of cancer cells.

[0004] Furthermore, in recent years, the diversity of tumors has become clear, and it is becoming apparent that cancer is not simply composed of one type of cell, but rather a heterogeneous population of cells with different properties. Therefore, even in androgen-dependent cancers such as prostate cancer, it is thought that the main enzymes involved in each reaction of androgen synthesis are different. However, to date, there have been very few studies published on DHRS11 (Dehydrogenase / Reductase SDR Family Member 11), one of the enzymes involved in androgen synthesis. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2015-20966 [Overview of the project] [Problems that the invention aims to solve]

[0006] However, research and development on small molecule compounds that can be used as anticancer agents is still insufficient.

[0007] Under these circumstances, the main objective of the present invention is to provide a new anticancer agent, as well as pharmaceutical compositions and food and beverage compositions using the same. [Means for solving the problem]

[0008] In other words, the present invention first provides a compound represented by the following general formula (1) or a salt thereof. [ka] (In general formula (1), R1, R2, R3, R4, and R5 are each independently monovalent substituents.) In the present invention, R3 may be a hydroxyl group or a (C1-C6) alkoxy group. Furthermore, the present invention also provides a DHRS11 (short-chain dehydrogenase / reductase superfamily member 11) inhibitor and / or an AR (androgen receptor) expression inhibitor containing a compound represented by the above general formula (1) or a salt thereof as an active ingredient. The DHRS11 inhibitor and / or the AR expression inhibitor may be used for the prevention and / or treatment of cancer. In this case, the cancer may be prostate cancer, urothelial carcinoma, or breast cancer. In this case, the breast cancer may be triple-negative breast cancer. Furthermore, the present invention also provides pharmaceutical compositions and food and beverage compositions comprising the DHRS11 inhibitor and / or the AR expression inhibitor. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a new anticancer agent, as well as pharmaceutical compositions and food and beverage compositions using the same. The effects described herein are not necessarily limited to those described herein and may include any of the effects described herein. [Brief explanation of the drawing]

[0010] [Figure 1] This diagram shows the various components extracted from the flowers of Carex kobomugi. [Figure 2] This diagram illustrates DHRS11 inhibitory activity. [Figure 3] This figure illustrates the protein expression of AKR1C3 and DHRS11 in various prostate cancer cells. [Figure 4] This figure illustrates the mRNA expression of AKR1C3, DHRS11, and AR in various breast cancer cells. [Figure 5] This figure illustrates the androgen signaling inhibitory effect of compound CK2 in prostate cancer C4-2 cells. A shows the number of viable cells after 48 hours of treatment, B shows the expression of AR downstream signals, C shows AR nuclear translocation, and D shows nuclear AR expression. In B and C, **: p < 0.01 vs DMSO, and ##: p < 0.01 vs adione alone. [Figure 6]It is a figure explaining the effect of suppressing AR expression by compound CK2 in each prostate cancer cell. A shows AR mRNA expression in C4-2 cells, B shows AR mRNA expression in LNCaP cells, C shows AR protein expression in C4-2 cells, D shows AR protein expression in LNCaP cells, E shows mRNA expression of AR and AR V7 in 22Rv1 cells, and F shows protein expression of AR and AR splicing variants in 22Rv1 cells. In A - E, **: p < 0.01 vs DMSO. [Figure 7] It is a figure explaining the suppression of androgen signal by compound CK2 in prostate cancer LNCaP cells. A shows the expression of AR downstream signal, B shows the nuclear AR expression, and C shows the nuclear translocation of AR. In A and C, **: p < 0.01 vs DMSO, and ##: p < 0.01 vs Adione alone. [Figure 8] It is a figure explaining the effect of suppressing AR expression in triple - negative breast cancer MDA - MB - 453 cells. A shows AR protein expression and B shows nuclear translocation of AR. In A, **: p < 0.01 vs DMSO, and ##: p < 0.01 vs Trione alone. [Figure 9] It is a figure explaining the effect of suppressing AR expression by compound CK2. [Figure 10] It is a figure explaining the effect of compound CK2 on androgen signal regulation. [Figure 11] It is a figure explaining the synergistic effect of compound CK2 on capivasertib, an AKT inhibitor.

Modes for Carrying Out the Invention

[0011] Hereinafter, preferred embodiments for carrying out the present invention will be described. The embodiments described below show an example of typical embodiments of the present invention, and the scope of the present invention should not be construed narrowly thereby.

[0012] 1. Compounds represented by the above general formula (1) or salts thereof The compound according to the present invention is a compound represented by the above general formula (1) or a salt thereof. The compounds according to the present invention exhibit inhibitory activity against DHRS11, one of the enzymes involved in androgen synthesis. Furthermore, the compounds also suppress the expression of androgen receptors (ARs). Therefore, the compounds according to the present invention are particularly useful for the prevention and / or treatment of various symptoms or diseases involving androgen signaling.

[0013] In the above general formula (1), R1, R2, R3, R4, and R5 are each independently monovalent substituents. Monovalent substituents include hydrogen atoms, halogen atoms, hydroxyl groups, carboxyl groups, cyano groups, nitro groups, sulfanyl groups, amino groups, acyl groups, (C1-C6) alkyl groups, halo(C1-C6) alkyl groups, (C3-C6) cycloalkyl groups, halo(C3-C6) cycloalkyl groups, (C3-C6) cycloalkyl(C1-C6) alkyl groups, halo(C3-C6) cycloalkyl(C1-C6) alkyl groups, (C1-C6) alkylcarbonyl groups, halo(C1-C6) alkylcarbonyl groups, (C3-C6) cycloalkyl(C1-C6) alkylcarbonyl groups, (C1-C6) alkoxy groups, halo(C1-C6) alkoxy groups, (C3-C6) cycloalkyl(C1-C6) alkoxy groups, halo(C3-C6) Examples include chloroalkyl(C1-C6)alkoxy groups, (C1-C6)alkoxy(C1-C6)alkyl groups, halo(C1-C6)alkoxy(C1-C6)alkyl groups, (C3-C6)cycloalkyl(C1-C6)alkoxy(C1-C6)alkyl groups, halo(C3-C6)cycloalkyl(C1-C6)alkoxy(C1-C6)alkyl groups, (C2-C6)alkenyl groups, (C2-C6)alkynyl groups, (C1-C6)alkoxycarbonyl groups, halo(C1-C6)alkoxycarbonyl groups, hydroxy(C1-C6)alkyl groups, (C1-C6)alkylthio groups, (C1-C6)alkylsulfinyl groups, (C1-C6)alkylsulfonyl groups, halo(C1-C6)alkylthio groups, halo(C1-C6)alkylsulfinyl groups, and halo(C1-C6)alkylsulfonyl groups.

[0014] Examples of halogen atoms include fluorine, chlorine, bromine, and iodine. Examples of acyl groups include formyl, acetyl, malonyl, and benzoyl groups.

[0015] Examples of (C1-C6) alkyl groups include linear or branched alkyl groups with 1 to 6 carbon atoms, such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, secondary butyl group, tertiary butyl group, n-pentyl group, isopentyl group, tertiary pentyl group, neopentyl group, 2,3-dimethylpropyl group, 1-ethylpropyl group, 1-methylbutyl group, 2-methylbutyl group, n-hexyl group, isohexyl group, 2-hexyl group, 3-hexyl group, 2-methylpentyl group, 3-methylpentyl group, 1,1,2-trimethylpropyl group, and 3,3-dimethylbutyl group.

[0016] Examples of (C3-C6) cycloalkyl groups include cyclic alkyl groups with 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0017] Examples of (C1-C6) alkylcarbonyl groups include methylcarbonyl group, ethylcarbonyl group, n-propylcarbonyl group, isopropylcarbonyl group, n-butylcarbonyl group, isobutylcarbonyl group, secondary butylcarbonyl group, tert-butylcarbonyl group, n-pentylcarbonyl group, isopentylcarbonyl group, tert-pentylcarbonyl group, neopentylcarbonyl group, and 2,3-dimethylpropylcarbonyl This refers to linear or branched alkylcarbonyl groups with 1 to 6 carbon atoms, such as 1-ethylpropylcarbonyl group, 1-methylbutylcarbonyl group, 2-methylbutylcarbonyl group, n-hexylcarbonyl group, isohexylcarbonyl group, 2-hexylcarbonyl group, 3-hexylcarbonyl group, 2-methylpentylcarbonyl group, 3-methylpentylcarbonyl group, 1,1,2-trimethylpropylcarbonyl group, and 3,3-dimethylbutylcarbonyl group.

[0018] Examples of (C1-C6) alkoxy groups include linear or branched alkoxy groups with 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, secondary butoxy, tertiary butoxy, n-pentyloxy, isopentyloxy, tertiary pentyloxy, neopentyloxy, 2,3-dimethylpropyloxy, 1-ethylpropyloxy, 1-methylbutyloxy, n-hexyloxy, isohexyloxy, and 1,1,2-trimethylpropyloxy.

[0019] Examples of (C2-C6) alkenyl groups include linear or branched alkenyl groups with 2 to 6 carbon atoms, such as ethenyl (vinyl) groups, propenyl groups, butenyl groups, pentenyl groups, and hexenyl groups.

[0020] Examples of (C2-C6) alkynyl groups include linear or branched alkynyl groups with 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, pentynyl, and hexynyl groups.

[0021] Examples of (C1-C6) alkoxycarbonyl groups include carbonyl groups to which linear or branched alkoxy groups having 1 to 6 carbon atoms are bonded, such as methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, isopropoxycarbonyl group, n-butoxycarbonyl group, secondary butoxycarbonyl group, tertiary butoxycarbonyl group, n-pentyloxycarbonyl group, isopentyloxycarbonyl group, tertiary pentyloxycarbonyl group, neopentyloxycarbonyl group, 2,3-dimethylpropyloxycarbonyl group, 1-ethylpropyloxycarbonyl group, 1-methylbutyloxycarbonyl group, n-hexyloxycarbonyl group, isohexyloxycarbonyl group, and 1,1,2-trimethylpropyloxycarbonyl group.

[0022] Examples of hydroxy(C1-C6)alkyl groups include linear or branched alkyl groups with 1 to 6 carbon atoms substituted with one or more hydroxyl groups, such as hydroxymethyl, hydroxyethyl, hydroxypropyl, and dihydroxypropyl groups.

[0023] Examples of (C1-C6) alkylthio groups include linear or branched alkylthio groups with 1 to 6 carbon atoms, such as methylthio group, ethylthio group, n-propylthio group, isopropylthio group, n-butylthio group, secondary butylthio group, tertiary butylthio group, n-pentylthio group, isopentylthio group, tertiary pentylthio group, neopentylthio group, 2,3-dimethylpropylthio group, 1-ethylpropylthio group, 1-methylbutylthio group, n-hexylthio group, isohexylthio group, and 1,1,2-trimethylpropylthio group.

[0024] Examples of (C1-C6) alkylsulfinyl groups include linear or branched alkylsulfinyl groups with 1 to 6 carbon atoms, such as methylsulfinyl group, ethylsulfinyl group, n-propylsulfinyl group, isopropylsulfinyl group, n-butylsulfinyl group, secondary butylsulfinyl group, tert-butylsulfinyl group, n-pentylsulfinyl group, isopentylsulfinyl group, tert-pentylsulfinyl group, neopentylsulfinyl group, 2,3-dimethylpropylsulfinyl group, 1-ethylpropylsulfinyl group, 1-methylbutylsulfinyl group, n-hexylsulfinyl group, isohexylsulfinyl group, and 1,1,2-trimethylpropylsulfinyl group.

[0025] Examples of (C1-C6) alkylsulfonyl groups include linear or branched alkylsulfonyl groups with 1 to 6 carbon atoms, such as methylsulfonyl group, ethylsulfonyl group, n-propylsulfonyl group, isopropylsulfonyl group, n-butylsulfonyl group, secondary butylsulfonyl group, tert-butylsulfonyl group, n-pentylsulfonyl group, isopentylsulfonyl group, tert-pentylsulfonyl group, neopentylsulfonyl group, 2,3-dimethylpropylsulfonyl group, 1-ethylpropylsulfonyl group, 1-methylbutylsulfonyl group, n-hexylsulfonyl group, isohexylsulfonyl group, and 1,1,2-trimethylpropylsulfonyl group.

[0026] In this invention, "halo" means "halogen atom," and refers to a fluorine atom, chlorine atom, bromine atom, or iodine atom. Furthermore, at positions where hydrogen atoms of (C1-C6) alkyl groups, (C3-C6) cycloalkyl groups, (C1-C6) alkylcarbonyl groups, and (C1-C6) alkoxycarbonyl groups can be substituted, the hydrogen atoms may be substituted by one or more halogen atoms, and if there are two or more halogen atoms to be substituted, the halogen atoms may be the same or different. Substituents substituted with one or more halogen atoms are represented as halo(C1-C6)alkyl groups, halo(C3-C6)cycloalkyl groups, halo(C1-C6)alkylcarbonyl groups, and halo(C1-C6)alkoxycarbonyl groups, respectively.

[0027] In the present invention, expressions such as "(C1-C6)", "(C2-C6)", and "(C3-C6)" indicate the range of carbon atoms in each substituent. Furthermore, groups to which the substituents are linked can also be described using the above definition. For example, in the case of (C1-C6)alkoxy(C1-C6)alkyl group, it indicates that a linear or branched alkoxy group having 1 to 6 carbon atoms is bonded to a linear or branched alkyl group having 1 to 6 carbon atoms.

[0028] In the present invention, among these, it is particularly preferable that R3 be a hydroxyl group or a (C1-C6) alkoxy group.

[0029] In this invention, "salt" refers to, for example, a pharmaceutically acceptable salt, which is an addition salt of an inorganic or organic acid or base that possesses the biological efficacy of the parent compound (salt-free compound) and is biologically non-toxic or has low biological toxicity. Examples of such salts include inorganic acid addition salts with hydrochloric acid, sulfuric acid, etc.; organic acid addition salts with formic acid, acetic acid, trifluoroacetic acid, tartaric acid, etc.; alkali metal salts with sodium, potassium, etc.; alkaline earth metal salts with calcium, magnesium, etc.; and organic amine salts with methylamine, ethylamine, diethanolamine, etc. Furthermore, the above examples should not be used to restrict the interpretation of "pharmaceutically acceptable salts." In other words, "pharmaceutically acceptable salts" should be interpreted broadly and encompass a wide range of salts.

[0030] The compounds according to the present invention can be prepared by conventionally known synthesis methods. Alternatively, as shown in the examples described later, they may be extracted and isolated from natural products. As shown in the general formula (1) above, the compounds according to the present invention are low-molecular-weight compounds and have the advantage of being able to be manufactured relatively inexpensively. Therefore, they are easy to chemically synthesize and can withstand mass production, which will lead to improved treatment opportunities for patients and a reduction in medical costs.

[0031] 2. DHRS11 inhibitors and / or AR expression suppressors The DHRS11 inhibitor and / or AR expression inhibitor according to the present invention (hereinafter also simply referred to as "the agent according to the present invention") contains at least one compound represented by the above general formula (1) or a salt thereof as an active ingredient.

[0032] In this invention, "DHRS11 inhibitor" means an agent that inhibits DHRS11. Also, "AR expression inhibitor" means an agent that suppresses the expression of AR. The agent according to the present invention can be used as a preventive and / or therapeutic agent for various symptoms or diseases involving DHRS11 and / or AR. It can also be used as a research reagent, as well as a diagnostic reagent, etc.

[0033] The DHRS11 inhibitor according to the present invention suppresses androgen synthesis by inhibiting DHRS11, and can therefore be used for purposes such as inhibiting the proliferation of androgen-dependent cancer cells. Furthermore, the AR expression inhibitor according to the present invention can enhance the anticancer activity of, for example, protein kinase B (AKT) inhibitors by suppressing AR expression. Therefore, the agents according to the present invention can be used for the prevention and / or treatment of cancers involving androgen signaling.

[0034] In this invention, "cancer" is interpreted broadly and used interchangeably with "malignant tumor." Furthermore, before a pathological diagnosis is confirmed, that is, before it is determined whether the tumor is benign or malignant, it may comprehensively include benign tumors, borderline benign-malignant lesions, and malignant tumors. Generally, cancers are named after the organ or tissue from which they originate. Examples include tongue cancer, gum cancer, pharyngeal cancer, maxillary cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, stomach cancer, small intestine cancer, large intestine cancer, rectal cancer, liver cancer, biliary tract cancer, gallbladder cancer, pancreatic cancer, lung cancer, breast cancer, thyroid cancer, adrenal cancer, pituitary tumor, pineal gland tumor, uterine cancer, ovarian cancer, vaginal cancer, kidney cancer, prostate cancer, urothelial carcinoma, retinoblastoma, conjunctival cancer, neuroblastoma, glioma, glioblastoma, skin cancer, medulloblastoma, leukemia, malignant lymphoma, testicular tumor, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, liposarcoma, chondrosarcoma, and Ewing's sarcoma. Furthermore, cancers are further classified according to the characteristics of the organ in which they originate, such as upper, middle, and lower pharyngeal cancer, upper, middle, and lower esophageal cancer, gastric cardia cancer, gastric pyloric cancer, cervical cancer, and uterine cancer. However, these are not limiting and can be included in the description of "cancer" in this invention.

[0035] In this invention, "prevention" also means preventing or delaying the onset (including recurrence) of symptoms or diseases in the subject, or reducing the risk of developing symptoms or diseases in the subject. Furthermore, "treatment" also means alleviating (reducing the severity of) symptoms or accompanying symptoms in the subject, or preventing or delaying the worsening of symptoms.

[0036] The agent according to the present invention is preferably used for the prevention and / or treatment of prostate cancer, urothelial carcinoma, or breast cancer, which have been reported to be involved with androgen signaling. "Urothelial carcinoma" is a general term for tumors arising from the urothelium and includes bladder cancer, renal pelvis cancer, ureteral cancer, and urethral cancer.

[0037] Furthermore, breast cancer is classified into several subtypes according to its characteristics, but the agent according to the present invention is particularly useful against triple-negative breast cancer (TNBC). Triple-negative breast cancer is a refractory cancer due to the lack of therapeutic targets, as it lacks three receptors that can be used as therapeutic targets for breast cancer: ER (estrogen receptor), PgR (progesterone receptor), and HER2 (human epidermal receptor 2). It is known to have a poorer prognosis compared to other types of breast cancer.

[0038] The agent according to the present invention may consist only of the compound represented by the above general formula (1) or a salt thereof, which is the active ingredient, or it may contain the active ingredient and any other ingredients.

[0039] The formulation of the agent according to the present invention can be carried out by conventionally known methods. When formulation, other formulation-acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, surfactants, lubricants, diluents, coatings, sugar coatings, flavoring / deodorizing agents, emulsifiers, solubilizers, dispersants, pH adjusters, isotonic agents, solubilizers, fragrances, colorants, solubilizers, physiological saline, etc.) may be included.

[0040] The dosage form of the agent according to the present invention is not particularly limited. Examples of dosage forms include tablets, powders, granules, capsules, syrups, liquids, suspensions, emulsions, jellies, injections, topical preparations, inhalants, nasal drops, eye drops, suppositories, and the like.

[0041] The agent according to the present invention contains an amount of the active ingredient necessary to achieve the effects of the present invention (i.e., a therapeutically effective amount). The effective amount in the DHRS11 inhibitor according to the present invention generally varies depending on the dosage form, but an effective amount that can achieve the desired dose (for example, within the range of 0.01% to about 100% by weight) is set as appropriate. In addition, the target is usually humans, but it also includes mammals other than humans, such as pet animals such as dogs and cats, livestock such as cattle, sheep, pigs and goats, and experimental animals such as mice, rats, guinea pigs, hamsters, rabbits and monkeys.

[0042] The agent according to the present invention is administered to the target subject by oral or parenteral administration (e.g., intravenous, intra-arterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, percutaneous, nasal, or mucosal injection, depending on its dosage form). These administration routes are not mutually exclusive, and two or more routes may be used in combination. Furthermore, local administration is also possible instead of systemic administration, and a drug delivery system (DDS) may be used.

[0043] 3. Pharmaceutical Compositions The pharmaceutical composition according to the present invention comprises at least the agent according to the present invention.

[0044] The pharmaceutical composition according to the present invention may contain, in addition to the active ingredient of the present invention, other active ingredients such as anticancer agents, or any other optional ingredients. In this invention, "anticancer agent" refers to a drug that exhibits preventive and / or therapeutic effects against cancer, which is the target disease or pathological condition. Furthermore, it is a broad concept that includes not only existing anticancer agents, but also anticancer agents currently under development and those to be developed in the future.

[0045] The pharmaceutical composition according to the present invention can be formulated by conventionally known methods. When formulating, for example, other pharmaceutically acceptable components as described above may be included. The dosage form when formulating the pharmaceutical composition according to the present invention is not particularly limited, similar to the DHRS11 inhibitor according to the present invention. Furthermore, when used in combination with other active ingredients such as anticancer agents, it may be used as a combination product with anticancer agents, etc.

[0046] The pharmaceutical composition according to the present invention preferably contains an amount of active ingredient that can be expected to have therapeutic and / or preventive effects. The amount of active ingredient can be determined considering the medical condition, health status, age, sex, weight, etc., of the person in whom it will be used.

[0047] The pharmaceutical composition according to the present invention can be administered orally or parenterally (for example, intravenous, intra-arterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, percutaneous, nasal, or mucosal injection, depending on its dosage form) This applies to the target population. Note that these administration routes are not mutually exclusive, and two or more can be used in combination. Furthermore, local administration is also possible instead of systemic administration, and drug delivery systems (DDS) may be used.

[0048] The dosage of the pharmaceutical composition according to the present invention may vary depending on the symptoms, health condition, age, sex, weight, etc., but can be appropriately determined by those skilled in the art. The administration schedule can also be appropriately determined by those skilled in the art, for example, once to several times a day, once every two days, or once every three days. When setting the administration schedule, the symptoms of the target and the duration of effect of the active ingredient can be taken into consideration.

[0049] 4. Food and drink composition The food and beverage composition according to the present invention comprises at least the agent according to the present invention.

[0050] Examples of food and beverage compositions according to the present invention include general foods (e.g., grains, vegetables, meat, various processed foods, etc.), confectionery (e.g., cookies, biscuits, jelly, candy, gummies, etc.), beverages (e.g., milk, soft drinks, alcoholic beverages, etc.), nutritional supplements (e.g., supplements, energy drinks, etc.), and food additives. In the case of nutritional supplements or food additives, they can be provided in the form of powder, granules, tablets, paste, liquid, etc. Providing the agent according to the present invention in the form of a food and beverage composition makes it easy to take the agent on a daily basis or continuously.

[0051] The food and beverage composition according to the present invention preferably contains an amount of active ingredient that can be expected to have preventive and / or therapeutic effects. Furthermore, the amount of active ingredient can be determined considering the medical condition, health status, age, sex, weight, etc., of the person in whom it will be used. [Examples]

[0052] The present invention will be described in more detail below based on examples. The embodiments described below are merely examples of typical embodiments of the present invention, and this should not be interpreted as narrowing the scope of the present invention.

[0053] <Experimental Example 1> Figure 1 shows eight components isolated and identified from the flowers of Carex kobomugi. In this experimental example 1, the inhibitory activity of these eight components on DHRS11, which is involved in androgen synthesis, was investigated.

[0054] The dehydrogenase activity of DHRS11 was measured spectroscopically (Ex. 340 nm, Em. 455 nm) by measuring the rate of NADPH degradation in the following reaction system. The standard reaction system consisted of 2.0 mL of 0.1 M potassium phosphate buffer (pH 7.4), 0.1 mM NADPH, 80 μM diacetyl, and the enzyme. One unit (U) of enzyme activity was defined as the amount of enzyme that produces 1 μmol of NADPH per minute at 25 °C. IC50 of the inhibitor 50The values ​​were calculated from the inhibition rates when five different concentrations of inhibitors were added to the standard reaction system. These inhibition constants are expressed as the mean ± standard deviation of at least three measurements.

[0055] Furthermore, since compound CK2 in Figure 1 contains three hydroxyl groups, the partially methylated derivatives CK2A and CK2B (see Figure 2) were evaluated in the same manner.

[0056] From the results of this experimental example 1, only compound CK2 produced an IC50 of 0.350 μM. 50 The compound showed a value indicating strong DHRS11 inhibitory activity. Furthermore, when derivatives CK2A and CK2B, which are partially methylated derivatives of compound CK2, were evaluated, neither compound showed inhibitory activity.

[0057] <Experimental Example 2> In this experimental example 2, a novel organic synthesis was performed to conduct a detailed evaluation of compound CK2.

[0058] [ka]

[0059] Aldehyde I (1.96 g, 12 mmol) and acetophenone II (2.10 g, 10 mmol) were dissolved in ethanol (40 mL) by heating and stirring at 60 °C. Then, 4 M NaOH aqueous solution (5 mL) was added and the mixture was reacted overnight under reflux conditions. The reaction solution was allowed to cool at room temperature and concentrated. The residue was diluted with water (20 mL) and extracted with ethyl acetate (30 mL x 3). The organic layer was dried over sodium sulfate, filtered, and concentrated. The resulting crude product was recrystallized from ethyl acetate / hexane to obtain the target compound III in 88% yield (3.12 g). 1 H NMR (400 MHz, CDCl3) d 7.68-7.39 (m, 4H), 6.93-6.87 (m, 4H), 5.99 (s, 1H), 3.95 (s, 3H), 2.90 (s, 3H), 3.85 (s, 3H), 2.04 (s, 3H).

[0060] [ka]

[0061] Compound III (354 mg, 1.0 mmol) and iodine (0.1 mmol, 25.6 mg) were heated and stirred in DMSO at 120 °C for 14 hours. Then, 10 mL of aqueous sodium thiosulfate solution was added, followed by extraction with ethyl acetate (30 mL x 3), washing with saline solution, drying over sodium sulfate, filtration, and concentration. The crude product was isolated by silica gel column chromatography (CHCl3) to obtain the target compound IV in 82% yield (288 mg). 1 H NMR (400 MHz, CDCl3) d 7.55 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 16.0 Hz, 1H), 6.94 (d, J = 8.6 Hz, 2H), 6.60 (d, J = 16.0 Hz, 1H), 6.40 (s, 1H), 6.14 (s, 1H), 3.99 (s, 3H), 3.96 (s, 3H), 3.86 (s, 3H), 2.32 (S, 3H).

[0062] [ka]

[0063] A dichloromethane solution of compound IV (564 mg, 1.6 mmol) was cooled to -78 °C, and BBr3 (1 M in DCM, 6.4 mmol) was added dropwise. The mixture was allowed to react for 1 hour. Then, a sodium bicarbonate aqueous solution (30 mL) was added. The solution was extracted with dichloromethane (40 mL x 3), dried with sodium sulfate, filtered, and concentrated. The crude product was isolated and prepared by silica gel column chromatography (CHCl3:MeOH = 100:1), and the target compound V was obtained in 74% yield (400 mg). 1H NMR (400 MHz, CDCl3) d 7.59-7.53 (m, 3H), 6.95 (d, J = 9.2 Hz, 2H), 6.65 (d, J = 16.0 Hz, 1H), 6.40 (s, 1H), 6.14 (s, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 2.92 (S, 3H).

[0064] [ka]

[0065] Compound V (67.6 mg, 0.2 mmol) and BBr3 (1 M in DCM, 0.4 mmol) were dissolved in dichloromethane. The mixture was allowed to dissolve. The mixture was then stirred and allowed to react overnight at room temperature. After the reaction was complete, the reaction solution was concentrated. The residue was purified using VLC (CHCl3:MeOH = 9:1) to obtain the target product VI (=compound CK2) in 68% yield (44.6 mg). 1 H NMR (500 MHz, DMSO-d6) d 12.85 (s, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 16.2 Hz, 1H), 6.97 (d, J = 16.2 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.29 (s, 1H), 6.28 (s, 1H), 2.21 (s, 3H).

[0066] <Experimental Example 3> In this experimental example 3, the expression of 17β-hydroxysteroid dehydrogenases (17β-HSDs), DHRS11 and AKR1C3, in each prostate cancer cell line (LNCaP, C4-2, and 22Rv1) was confirmed by Western blotting.

[0067] [Western blot method] Each prostate cancer cell line (LNCaP, C4-2, and 22Rv1) was cultured in a carbon dioxide incubator at 37°C and 5% CO2. RPMI1640, containing 5% (v / v) FBS, 100 U / mL penicillin-G potassium, 100 μg / mL streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0), was used as the growth medium. Each cell was cultured at 1 × 10⁶ cells. 6 Cells were seeded in a 6 cm dish using the cells / dish method. After 24 hours, the growth medium was replaced with growth medium containing 2% FBS, and the compound was added at a final concentration of 10 μM. After 24 hours of culture, the cells were harvested using a cell scraper with DPBS and subjected to Western blotting as follows: The cell pellet was suspended in Urea buffer containing 8 M Urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na2H2PO4, and the cell membranes were disrupted by sonication. The cell lysates were centrifuged (15,000 xg, 15 minutes, 4 °C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using a 12.5% ​​polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 1% BSA, and then sequentially reacted with a primary antibody against DHRS11 or AKR1C3 and a horseradish peroxidase-labeled secondary antibody. Antibody-reactive proteins were detected by chemiluminescence using the ECL-enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).

[0068] The results of Experiment Example 3 show that 17β-hydroxysteroid deactivation occurs in the androgen metabolic pathway. When the expression levels of two enzymes that catalyze hydrogenase reactions, AKR1C3 and DHRS11, were evaluated, AKR1C3 was highly expressed only in 22Rv1 cells, while DHRS11 was highly expressed in LNCaP cells, C4-2 cells, and 22Rv1 cells. It was found that the expression was observed in all cells.

[0069] <Experimental Example 4> In this experimental example 4, the mRNA expression of AR and two types of 17β-HSD (DHRS11 and AKR1C3) in breast cancer cell lines (MCF7, MDA-MB-231, and MDA-MB-453) was confirmed by semi-quantitative PCR.

[0070] [Semi-quantitative PCR method] Total RNA was isolated using TRI reagent. This total RNA was incubated at 37°C for 30 minutes and 98°C for 5 minutes using the ReverTra Ace qPCR RT Kit, and reverse transcription was performed to prepare single-stranded complementary DNA (cDNA). The prepared cDNA (1 μg) was used as a template for semi-quantitative PCR using Quick Taq HS DyeMix DNA polymerase (Toyobo Co., Ltd.). PCR was performed using a CFX96 Deep Well Real-Time System (BIO RAD, Hercules, CA, USA) under the following temperature conditions: heat treatment at 95°C / 30 seconds, followed by 40 cycles of heat treatment at 95°C / 15 seconds and 55°C / 1 minute. The amplified PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and detected under ultraviolet (UV) irradiation. Human β-actin cDNA was amplified as an internal standard. The sequences of AKR1C3, DHRS11, AR, and β-actin specific primers are shown in Table 1 below.

[0071] [Table 1]

[0072] The results of Experimental Example 4 showed that AKR1C3 expression in the three breast cancer cell lines was low, even compared to LNCaP cells, which have low AKR1C3 expression levels among prostate cancer cell lines. In particular, it was found to be below the detection limit in MDA-MB-453 cells. On the other hand, DHRS11 expression was observed in all breast cancer cell lines. Furthermore, AR was expressed in MCF7 cells and MDA-MB-453 cells, but its expression level was low in MDA-MB-231 cells.

[0073] Therefore, prostate cancer cells LNCaP and C4-2, and three types of breast cancer cells (MCF7, MDA-MB-231) In prostate cancer cells (MDA-MB-453), DHRS11 functions as the primary 17β-HSD, while in prostate cancer cells 22Rv1, both AKR1C3 and DHRS11 enzymes are suggested to function as 17β-HSDs.

[0074] <Experimental Example 5> DHRS11 is known to catalyze the reduction reaction from 5α-androstane-3,17-dione (Adione) to 5α-dihydrotestosterone (DHT). In Experimental Example 5, the effect of the compound CK2 on androgen signaling was investigated using the prostate cancer cell line C4-2, which expresses both DHRS11 and AR.

[0075] [Measurement of viable cell count] 2 × 10⁶ C4-2 cells suspended in growth medium were placed in a 96-well multiplate. 4Cells were seeded in 200 μL portions and cultured overnight in a CO2 incubator. The culture medium was changed to one containing antibiotics and 2% FBS, the sample was added to the medium, and the cells were cultured for a further 24 hours. As a control group, cells treated with dimethyl sulfoxide (DMSO) were prepared. Next, the culture medium was changed to one without serum or phenol red, 40 μM resazurin was added, and the cells were cultured at 37°C for 2-4 hours. The absorbance at wavelengths of 570 nm and 600 nm was then measured using a microplate reader Model 680 (BIO RAD). Cell viability (%) was calculated using the following formula (1).

[0076]

number

[0077] [Quantitative PCR method] Total RNA was isolated using TRI reagent. This total RNA was incubated at 37°C for 30 minutes and at 98°C for 5 minutes using the ReverTra Ace qPCR RT Kit, and reverse transcription was performed to prepare single-stranded complementary DNA (cDNA). The prepared cDNA (1 μg) was used as a template for quantitative PCR using THUNDERBIRD SYBR qPCR Mix reagent (Toyobo Co., Ltd.) and specific primers. A CFX96 Deep Well Real-Time System (BIO RAD Inc.) was used for quantitative PCR, and heat treatment was performed under the following temperature conditions: 94°C / 5 minutes, followed by denaturation at 94°C / 30 seconds, annealing at 55°C / 30 seconds, and extension at 72°C / 1 minute, each cycle being considered one unit. The sequences of the specific primers for PSA, TMPRSS2, and β-actin are listed in Table 2 below.

[0078] [Table 2]

[0079] [AR Nuclear Translocation Assessment] Nuclear and cytoplasmic proteins in the cell extract were separated using the LysoPure Nuclear and Cytoplasmic Extractor Kit (WAKO) according to the protocol. The separation of the nuclear and cytoplasmic fractions was confirmed by Western blot analysis using antibodies against histone H1, which is specifically expressed in the nucleus, and α-tubulin, which is specifically expressed in the cytoplasm. Each separated fraction was used for Western blot analysis.

[0080] [Fluorescence immunostaining] C4-2 cells suspended in growth medium were seeded at 2×10 4 cells / 500 μL each in a 24-well multiplate and cultured at 37 °C under 5% CO2 for 24 hours. Then, the growth medium was replaced with a growth medium with a reduced FBS concentration of 2%, and compound CK2 was added. After 2 hours, androgen signaling was induced with 10 nM 4-androstenedione (Adione), and after further culturing for 24 hours, the cells were washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added, and the cells were fixed for 10 minutes. Then, 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added, and the cells were allowed to stand for 10 minutes. 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA was added, and the cells were blocked for 1 hour. After washing twice with DPBS, the cells were incubated overnight at 4 °C in a primary antibody solution (anti-AR antibody) diluted 300:1 in DPBS. After washing twice with PBS, the cells were incubated for 1 hour at room temperature in the dark in an Alexa Fluoro-488-labeled rabbit secondary antibody solution diluted 500:1 in DPBS. After washing twice with PBS, excess moisture was removed, and the cells were mounted on a slide glass using a mounting agent (DAPI fluoromount-G) and covered with a coverslip. The fluorescence immunostained cells were set on a confocal laser microscope LSM700 (Carl Zeiss) for fluorescence observation.

[0081] The results of Experiment Example 5 showed that when compound CK2 was treated with C4-2 cells for 48 hours, a concentration of 10 μM reduced the number of viable cells by approximately 20%. Furthermore, since a 24-hour treatment at 5 μM did not reduce the number of viable cells, subsequent studies used 5 μM CK2 for 24 hours. In addition, when 10 nM adione was used to induce androgen signaling, the expression of PSA and TMPRSS2, which are induced downstream of AR, was enhanced, and this increase in expression was significantly suppressed by pretreatment with compound CK2.

[0082] In addition, when we examined the ratio of AR expression levels in the cytoplasm and nucleus, we found that androgens significantly increased the nucleus / cytoplasmic ratio of AR, and that this ratio was suppressed by the compound CK2. This trend was also confirmed by immunofluorescence staining.

[0083] <Experimental Example 6> In this experimental example 6, we investigated whether AR expression was reduced in other prostate cancer cell lines as well.

[0084] [Quantitative PCR method] Total RNA was isolated using TRI reagent. This total RNA was incubated at 37°C for 30 minutes and at 98°C for 5 minutes using the ReverTra Ace qPCR RT Kit, and reverse transcription was performed to prepare single-stranded complementary DNA (cDNA). The prepared cDNA (1 μg) was used as a template for quantitative PCR using THUNDERBIRD SYBR qPCR Mix reagent (Toyobo Co., Ltd.) and specific primers. A CFX96 Deep Well Real-Time System (BIO RAD Inc.) was used for quantitative PCR, and heat treatment was performed under the following temperature conditions: 94°C / 5 minutes, followed by denaturation at 94°C / 30 seconds, annealing at 55°C / 30 seconds, and extension at 72°C / 1 minute, each cycle. The sequences of the specific primers for AR, AR V7, and β-actin are shown in Table 3 below.

[0085] [Table 3]

[0086] [Western blot method] Each prostate cancer cell line (LNCaP, C4-2, and 22Rv1) was cultured in a carbon dioxide incubator at 37°C and 5% CO2. Growth medium was 5% (v / v) FBS, 100 U / mL penicilli. RPMI1640 containing nG potassium, 100 μg / mL streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0) was used. Each cell was 1 × 10⁶ 6 Cells were seeded in a 6 cm dish using the cells / dish method. After 24 hours, the growth medium was replaced with growth medium containing 2% FBS, and the compound was added at a final concentration of 10 μM. After 24 hours of culture, the cells were harvested using a cell scraper with DPBS and subjected to Western blotting as follows: The cell pellet was suspended in Urea buffer containing 8 M Urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na2H2PO4, and the cell membranes were disrupted by sonication. The cell lysates were centrifuged (15,000 xg, 15 minutes, 4 °C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using a 12.5% ​​polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 1% BSA, and then sequentially reacted with primary antibodies against AR, AR Vs, or AR V7 and horseradish peroxidase-labeled secondary antibodies. Antibody-reactive proteins were detected by chemiluminescence using the ECL-enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).

[0087] The results of Experiment Example 6 showed that compound CK2 dose-dependently reduces mRNA and protein expression in C4-2 cells and LNCaP cells. Furthermore, in 22Rv1 cells, it was found to reduce the expression of AR Vs and ARV7, splicing variants of AR involved in the activation of constitutive androgen signaling.

[0088] <Experimental Example 7> In this experimental example 7, the effect of the compound CK2 on androgen signaling was investigated using the prostate cancer cell line LNCaP.

[0089] [Measurement of viable cell count] LNCaP cells suspended in growth medium were placed in a 96-well multiplate in a 2 × 10⁶ arrangement. 4 Cells were seeded in 200 μL portions and cultured overnight in a CO2 incubator. The culture medium was changed to one containing antibiotics and 2% FBS, the sample was added to the medium, and the cells were cultured for a further 24 hours. As a control group, cells treated with dimethyl sulfoxide (DMSO) were prepared. Next, the culture medium was changed to one without serum or phenol red, 40 μM resazurin was added, and the cells were cultured at 37°C for 2-4 hours. The absorbance at wavelengths of 570 nm and 600 nm was then measured using a microplate reader Model 680 (BIO RAD). Cell viability (%) was calculated using the formula (1) described above.

[0090] [Quantitative PCR method] Total RNA was isolated using TRI reagent. This total RNA was incubated at 37°C for 30 minutes and at 98°C for 5 minutes using the ReverTra Ace qPCR RT Kit, and reverse transcription was performed to prepare single-stranded complementary DNA (cDNA). The prepared cDNA (1 μg) was used as a template for quantitative PCR using THUNDERBIRD SYBR qPCR Mix reagent (Toyobo Co., Ltd.) and specific primers. A CFX96 Deep Well Real-Time System (BIO RAD Inc.) was used for quantitative PCR, and heat treatment was performed under the following temperature conditions: 94°C / 5 minutes, followed by denaturation at 94°C / 30 seconds, annealing at 55°C / 30 seconds, and extension at 72°C / 1 minute, each cycle being considered one unit. The sequences of the specific primers for PSA, TMPRSS2, and β-actin are listed in Table 2 above.

[0091] [AR Nuclear Translocation Assessment] Nuclear and cytoplasmic proteins in cell extracts are found in LysoPure Nuclear and Cytoplasmic Separation was performed using the Extractor Kit (WAKO) according to the protocol. Separation of the nuclear and cytoplasmic fractions was confirmed by Western blotting analysis using antibodies against histone H1, which is specifically expressed in the nucleus, and α-tubulin, which is specifically expressed in the cytoplasm. Each separated fraction was subjected to Western blotting.

[0092] [Fluorescent immunohistochemistry] LNCaP cells suspended in growth medium were placed in a 24-well multiplate in a 2×10⁶ 4Cells were seeded in 500 μL portions and cultured for 24 hours at 37 °C under 5% CO2 conditions. The culture medium was then replaced with growth medium containing 2% FBS, and compound CK2 was added. After 2 hours, androgen signaling was induced with 10 nM 4-androstenedione (Adione), and the cells were cultured for another 24 hours before being washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added, and the cells were fixed for 10 minutes. 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added, and the cells were allowed to stand for 10 minutes. 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA was added, the cells were blocked for 1 hour, washed twice with DPBS, and then incubated overnight at 4 °C in a primary antibody (anti-AR antibody) solution diluted 300:1 in DPBS. After washing twice with PBS, the cells were incubated at room temperature for 1 hour in a rabbit secondary antibody solution labeled with Alexa Fluoro-488, diluted 500:1 in DPBS. After washing twice with PBS and removing excess water, the coverslip was fixed onto a glass slide using a mounting agent (DAPI fluoromount-G). The immunofluorescently stained cells were placed on a confocal laser microscope LSM700 (Carl Zeiss) and observed for fluorescence.

[0093] The results of this experimental example 7 show that the same results as those in experimental example 5, as described above, can be obtained even when using LNCaP cells.

[0094] <Experimental Example 8> DHRS11 is known to catalyze the reduction reaction from 11-Keto-5α-androstenedione (Trione) to 11-keto-5α-dihydrotestosterone (11KDHT). In this experimental example 8, the effect of compound CK2 on androgen signaling was investigated using the triple-negative breast cancer cell line MDA-MB-453.

[0095] [Western blot method] MDA-MB-453 cells were cultured in a carbon dioxide incubator at 37°C and 5% CO2. RPMI1640, containing 5% (v / v) FBS, 100 U / mL penicillin-G potassium, 100 μg / mL streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0), was used as the growth medium. Cells were cultured in 1 × 10⁶ cells. 6 Cells were seeded in a 6 cm dish using the cells / dish method. After 24 hours, the growth medium was replaced with growth medium containing 2% FBS, and the compound was added at a final concentration of 10 μM. After 24 hours of culture, the cells were harvested using a cell scraper with DPBS and subjected to Western blotting as follows: The cell pellet was suspended in Urea buffer containing 8 M Urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na2H2PO4, and the cell membranes were disrupted by sonication. The cell lysates were centrifuged (15,000 xg, 15 minutes, 4 °C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using a 12.5% ​​polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 1% BSA, and then sequentially reacted with a primary antibody (anti-AR antibody) and a horseradish peroxidase-labeled secondary antibody. Antibody-reactive proteins were detected by chemiluminescence using the ECL-enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).

[0096] [Immunofluorescence staining] MDA-MB-453 cells suspended in growth medium were placed in a 24-well multiplate in a 2×10⁶ arrangement. 4Cells were seeded in 500 μL portions and cultured for 24 hours at 37 °C under 5% CO2 conditions. After that, the growth medium containing 5% FBS was replaced with medium containing 2% activated carbon-treated FBS, and compound CK2 was added. After 2 hours, androgen signaling was induced with 10 nM Trione, and after a further 24 hours of culture, the cells were washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added, and the cells were fixed for 10 minutes. 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added, and the cells were allowed to stand for 10 minutes. 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA was added, the cells were blocked for 1 hour, and after washing twice with DPBS, the cells were incubated overnight at 4 °C in a primary antibody (anti-AR antibody or anti-Ki67 antibody) solution diluted 300:1 in DPBS. After washing twice with PBS, the cells were incubated at room temperature for 1 hour in a solution of rabbit secondary antibody labeled with Alexa Fluoro-488 or mouse secondary antibody labeled with Alexa Fluoro-555, diluted 500:1 in DPBS. After washing twice with PBS, excess water was removed, and the coverslip was fixed onto a glass slide using a mounting agent (DAPI fluoromount-G). The immunofluorescently stained cells were placed on a confocal laser microscope LSM700 (Carl Zeiss) and observed for fluorescence.

[0097] The results of this experimental example 8 showed that compound CK2 significantly reduced the expression level of androgen-induced AR protein. Furthermore, immunofluorescence staining revealed a decrease in nuclear AR expression and a decrease in the fluorescence intensity of the proliferation marker Ki67, suggesting that compound CK2 can suppress cancer-drogen signaling even in triple-negative breast cancer cells.

[0098] <Experimental Example 9> In this experimental example 9, we investigated the inhibitory effect of compound CK2 on androgen receptor (AR) expression.

[0099] MDA-MB-453 cells suspended in growth medium were placed in a 96-well multiplate in a 2 × 10⁶ arrangement. 5 Cells were seeded individually and cultured overnight in a CO2 incubator. The culture medium was changed to one containing antibiotics and 2% FBS, and the sample was added to the medium and cultured for a further 6 hours. As a control group, cells supplemented with dimethyl sulfoxide (DMSO) were prepared. Total RNA was isolated using the TRI reagent. This total RNA was reverse transcribed using the ReverTra Ace qPCR RT Kit by incubation at 37 °C for 30 minutes and at 98 °C for 5 minutes to prepare single-stranded complementary DNA (cDNA). The prepared cDNA (1 μg) was used as a template for quantitative PCR using the THUNDERBIRD SYBR qPCR Mix reagent (Toyobo) and specific primers. A CFX96 Deep Well Real-Time System (BIO-RAD) was used for quantitative PCR, and heat treatment was performed under the following temperature conditions. Specifically, one cycle consisted of heat treatment at 94 °C / 5 minutes, denaturation at 94 °C / 30 seconds, annealing at 55 °C / 30 seconds, and extension at 72 °C / 1 minute. The sequences of the specific primers for AR and β-actin are shown in Table 4 below.

[0100] [Table 4]

[0101] Previous studies using prostate cell lines have reported that luteolin, a plant-derived component, inhibits AR expression (anti-inflammatory drug reaction). (Tsui et al., Int J Cancer, 2012;130:2812-2823). However, there are no reports of plant-derived components exhibiting AR expression inhibitory effects in triple-negative breast cancer (TNBC) cells, and no compound has shown stronger activity than luteolin in studies using prostate cancer cell lines. The results of this experimental example 9 show that compound CK2 exhibited AR expression inhibitory effects at lower concentrations than luteolin.

[0102] <Experimental Example 10> In this experimental example 10, we investigated the androgen signaling regulatory effect of compound CK2.

[0103] MDA-MB-453 cells suspended in growth medium were placed in a 96-well multiplate in a 2 × 10⁶ arrangement. 5 Cells were seeded individually and cultured overnight in a CO2 incubator. The culture medium was changed to one containing antibiotics and 2% FBS, and the sample was added to the medium and cultured for a further 6 hours. As a control group, cells supplemented with dimethyl sulfoxide (DMSO) were prepared. Total RNA was isolated using the TRI reagent. This total RNA was reverse transcribed using the ReverTra Ace qPCR RT Kit by incubation at 37 °C for 30 minutes and at 98 °C for 5 minutes to prepare single-stranded complementary DNA (cDNA). The prepared cDNA (1 μg) was used as a template for quantitative PCR using the THUNDERBIRD SYBR qPCR Mix reagent (Toyobo) and specific primers. A CFX96 Deep Well Real-Time System (BIO-RAD) was used for quantitative PCR, and heat treatment was performed under the following temperature conditions. Specifically, one cycle consisted of heat treatment at 94 °C / 5 minutes, denaturation at 94 °C / 30 seconds, annealing at 55 °C / 30 seconds, and extension at 72 °C / 1 minute. The sequences of the specific primers for AR and β-actin are shown in Table 5 below.

[0104] [Table 5]

[0105] In women, in addition to conventional androgens such as testosterone and dihydrotestosterone, the adrenal gland-derived androgens 11-keto-5α-dihydrotestosterone (Dione) and 11-keto-5α-androstane-3,17-dione (Trione) are produced and are known to activate androgen signaling. The results of this experimental example 10 showed that 10 nM Dione and its precursor 100 nM Trione induced the expression of TMPRSS2 and c-Myc, and that androgen signaling was enhanced in TNBC cells. Compounds CK2 and luteolin significantly reduced the expression levels of both genes, with compound CK2 showing a stronger effect.

[0106] <Experimental Example 11> In this experimental example 11, we investigated the synergistic effect of compound CK2 on the AKT inhibitor capivasertib.

[0107] [A: Measurement of cell viability] MDA-MB-453 cells suspended in growth medium were placed in a 96-well multiplate in a 2 × 10⁶ arrangement. 4 Cells were seeded individually and cultured overnight in a CO2 incubator. The culture medium was changed to one containing antibiotics and 2% FBS, and the sample was added to the medium and cultured for a further 48 hours. As a control group, cells were prepared with dimethyl sulfoxide (DMSO) added. Next, the medium was changed to one without serum or phenol red, 40 μM resazurin was added, and the cells were cultured at 37°C for 2-4 hours. After that, the absorbance at wavelengths of 570 nm and 600 nm was measured using a microplate reader Model 680 (Bio Rad). Cell viability (%) was calculated using the above formula (1).

[0108] [B: Measurement of lactate dehydrogenase (LDH) release] The activity of lactate dehydrogenase (LDH) released into the culture medium was evaluated using the Cytotoxicity LDH Assay Kit-WST (Wako Pure Chemical Industries), and calculated from the absorbance at a wavelength of 490 nm using a microplate reader Model 680 (Bio Rad).

[0109] [C, D: Calculation of synergistic effects using the CompuSyn program] Based on the cell viability in experiment A described above, the synergistic effect of compound CK2 on capivasertib was calculated.

[0110] [E: Confirmation of apoptosis induction using anti-cleavage caspase-3 antibody] Cells suspended in growth medium are placed in a 24-well multiplate in a 2×10⁶ 4 Cells were seeded individually and cultured for 24 hours at 37°C under 5% CO2 conditions. The culture medium was then replaced with growth medium containing 2% FBS, and compound CK2, capivasetib, or both were added. After 48 hours of culture, the cells were washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added, and the cells were fixed for 10 minutes. 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added, and the cells were allowed to stand for 10 minutes. 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA was added, the cells were blocked for 1 hour, and after washing twice with DPBS, the cells were incubated overnight at 4°C in a solution of primary antibody (anti-cleavage caspase 3 antibody) diluted 300:1 in DPBS. After washing twice with PBS, the cells were incubated at room temperature for 1 hour in a rabbit secondary antibody solution labeled with Alexa Fluoro-488, diluted 500:1 in DPBS. After washing twice with PBS and removing excess water, the coverslip was fixed onto a glass slide using a mounting agent (DAPI fluoromount-G). The immunofluorescently stained cells were placed on a confocal laser microscope LSM700 (Carl Zeiss) and observed for fluorescence.

[0111] [F, G: Suppressive effect of compound CK2 on capivasertib-induced AR expression] The experiment was conducted using the same method as in Experiment Example 9 above.

[0112] TNBC (transcutaneously invasive neurotransmitters) are highly malignant, have a poor prognosis, and do not express hormone receptors or HER2, making it impossible to use therapeutic drugs targeting these molecules. Recently, clinical trials of inhibitors of AKT, a kinase involved in cell proliferation and cell death, have been conducted. Therefore, with the aim of effectively utilizing AKT inhibitors, we investigated the effects of combination therapy with AKT inhibitors.

[0113] The results of Experimental Example 11 showed that the combination of capivasertib with 5 μM CK2 significantly reduced cell viability. Furthermore, lactate dehydrogenase release, an indicator of toxicity, also increased. On the other hand, compound CK2 alone had no effect on either. The combination index (CI) was less than 1 in the evaluation of the combined effect using the CompuSyn program, indicating a synergistic effect by compound CK2. Since capivasertib treatment significantly induced AR gene expression, it is possible that capivasertib suppresses cancer cell survival and proliferation by inhibiting AKT signaling, while simultaneously enhancing androgen-dependent cell proliferation by increasing AR expression. The significant decrease in AR gene expression when capivasertib was combined with compound CK2 suggests that compound CK2 exerted its combined effect through an AR expression inhibitory effect. [Industrial applicability]

[0114] The compound represented by the general formula (1) above, which is the active ingredient of the present invention, acts on androgen signaling by inhibiting DHRS11 and / or suppressing AR expression, and is expected to be useful for applications such as anticancer drugs. Furthermore, since it is a low-molecular-weight compound and a natural compound, it has the advantage of being inexpensive to manufacture. Therefore, it is easy to chemically synthesize and can withstand mass production, which will lead to improved treatment opportunities for patients and a reduction in medical costs.

Claims

1. A DHRS11 (short-chain dehydrogenase / reductase superfamily member 11) inhibitor and / or AR (androgen receptor) expression inhibitor containing the following compound or a salt thereof as an active ingredient. 【Chemistry 1】

2. A DHRS11 inhibitor and / or AR expression inhibitor according to claim 1, used for the prevention and / or treatment of cancer.

3. The DHRS11 inhibitor and / or AR expression inhibitor according to claim 2, wherein the cancer is prostate cancer, urothelial carcinoma, or breast cancer.

4. The DHRS11 inhibitor and / or AR expression inhibitor according to claim 3, wherein the breast cancer is triple-negative breast cancer.

5. A pharmaceutical composition comprising a DHRS11 inhibitor and / or an AR expression inhibitor according to any one of claims 1 to 4.

6. A food and beverage composition comprising a DHRS11 inhibitor and / or an AR expression inhibitor according to any one of claims 1 to 4.

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