Pharmaceutical composition for ovarian anti-aging and its use

Abstract

The present invention relates to a pharmaceutical composition for ovarian anti-aging comprising a compound of Chemical Formula 1 or a pharma- ceutical acceptable salt thereof as an active ingredient, a method for ovarian anti-aging comprising the same, and a pharmaceutical composition for preventing or treating diseases associated with ovarian aging or dysfunction. The compound of Chemical Formula 1 can restore the function of aged ovaries and inhibit ovarian aging, and may be usefully used for preventing or treating diseases associated with ovarian aging or dysfunction.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for ovarian anti-aging, comprising a compound of Chemical Formula 1 or a pharma- ceutically acceptable salt thereof, a pharmaceutical composition for preventing or treating diseases associated with ovarian aging or dysfunction, and a method for ovarian anti-aging. [Background technology]

[0002] Currently, the rate of female infertility is increasing as women age at reproductive age, and ovarian function plays an important role in successful pregnancy outcomes. However, it is known that there is no cure for infertility associated with aging ovarian dysfunction, which is manifested by the decline in the competence of oocytes and embryos (Cimadomo et al. 2018).

[0003] Follicle development is a very complex process regulated by various factors during the gonadotropin-independent and dependent stages, and the quality of oocytes is related to the function of somatic cells in the ovaries, such as granulosa cells and cumulus cells. Among older women attempting in vitro fertilization (IVF), those who have had successful normal births may show different morphological features of granulosa cells and cumulus cells during follicular development than those who have failed to conceive. Therefore, in order to restore ovarian function, not only follicle development and oocytes, but also the functions of cumulus cells and granulosa cells are important.

[0004] To date, the mechanism of ovarian aging and the definitive factors behind infertility associated with maternal aging have not been elucidated. In recent years, however, several cellular and molecular characteristics have been identified as a result of the progression of aging, including telomere attrition, genomic instability, reduced self-cannibalism, stem cell depletion, and mitochondrial dysfunction.

[0005] As a result of medical advances and improved economic standards, we have entered an aging society, and the number of women trying to become pregnant at a later age is increasing. However, ovarian function gradually declines with age, causing infertility and difficulty in pregnancy, and ovarian diseases related to aging are on the rise.

[0006] Many kinds of anti-aging agents have been developed to prevent the progression of aging and rejuvenate the body, but the development of a therapeutic agent that can restore aged ovaries or inhibit ovarian aging is still limited. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Patent Publication WO2009 / 025478 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a substance for restoring the function of aged ovaries and inhibiting ovarian aging.

[0009] Therefore, an object of the present invention is to provide a pharmaceutical composition for ovarian anti-aging comprising a compound of Chemical Formula 1 or a pharma- ceutical acceptable salt thereof as an active ingredient, and a method for ovarian anti-aging using the same.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases associated with ovarian aging or dysfunction, which contains the above-mentioned ingredient, and a method for preventing or treating diseases associated with ovarian aging or dysfunction using the above-mentioned ingredient. [Means for solving the problem]

[0011] The present inventors have confirmed that the compound of Chemical Formula 1 or a pharma- ceutically acceptable salt thereof can restore the function of aging ovaries, and have thus completed the present invention.

[0012] Therefore, the present invention provides a pharmaceutical composition for ovarian anti-aging, comprising the compound of Chemical Formula 1 or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0013] [ka]

[0014] In the above formula, n is an integer from 1 to 3, m is 0 or 1; A represents phenyl; R 1 is hydrogen or C1-C6-alkyl, R 2 represents hydrogen, halogen or C1-C6-alkoxy, or -C1-C6-alkylene-OH, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)R 7 or -NHC(O)R 7 where p is an integer from 0 to 3; R 7 represents hydrogen or C1-C3-alkyl, R 8 represents C1-C3-alkylpiperidinyl or C1-C3-alkylsulfonyl, R 3 represents hydrogen, halogen, C1-C6-alkyl or phenyl, or the heterocycle contains 1 or 2 heteroatoms selected from S, N and O atoms and is a 5- to 6-membered ring -(CH2) p -heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, R4 is halogen, C1-C6-alkyl, -C1-C6-alkylene-OH, -O-phenyl, -(CH2) p CO2R 7 The heterocycle contains 1 or 2 heteroatoms selected from S, N and O atoms and is a 5- to 6-membered ring -(CH2) p -heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3; R 7 is as defined above, R 5 is hydrogen or C1-C6-alkyl, R 6represents C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle or -C1-C6-alkylene-heterocycle, where heterocycle is a 3-8 membered ring containing 1-3 heteroatoms selected from S, N and O atoms; R 6 may be substituted by C1-C6-alkylamine, -C1-C6-alkylene-OH, or C1-C6-alkylsulfonyl.

[0015] The present invention further provides a method for anti-aging of the ovaries, comprising administering a compound of Formula 1, or a pharma- ceutically acceptable salt thereof, to an individual in need thereof.

[0016] The present invention further aims to provide a pharmaceutical composition for preventing or treating diseases associated with ovarian aging, comprising the compound of Chemical Formula 1 or a pharma- ceutically acceptable salt thereof. Effect of the Invention

[0017] According to the composition or method of the present invention, when the compound of Chemical Formula 1 is administered, it is possible to restore the function of aged ovaries and inhibit ovarian aging by increasing the functional activity of ovaries such as oocyte increase and follicle development, increasing the expression of specific genes for ovarian aging recovery, etc. Therefore, the compound of Chemical Formula 1 of the present invention can be usefully used for diseases related to ovarian dysfunction due to aging. [Brief description of the drawings]

[0018] [Figure 1] Figure 1a is a histological image of mouse ovarian tissue stained with hematoxylin and eosin after treatment with the compound of the Example and hormonal stimulation in Experimental Example 1. Figures 1b and 1c are an image and a graph showing the number of oocytes at the germinal vesicle (GV) stage with and without treatment with the compound of the Example in Experimental Example 1. [Diagram 2] FIG. 2 is a table image showing the effect of treatment with or without the compound of the embodiment on the development of crystal pronuclear cell (2PN) embryos according to Experimental Example 2. [Diagram 3]3a is a graph showing the results of next-generation sequencing (NGS) performed according to Experimental Example 3. FIG. 3b is a graph showing gene expression levels related to tumor necrosis factor superfamily cytokine production according to Experimental Example 3. [Figure 4] 4a and 4b are images and quantification graphs showing the results of gene-specific RT-PCR (Reverse Transcription-Polymerase Chain Reaction) analysis in Experimental Example 4.

[0019] (Best Mode for Carrying Out the Invention) The present invention will be described in detail below.

[0020] Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific description described below.

[0021] When a part is said to "comprise" certain elements, this means that it may further comprise other elements, rather than excluding other elements, unless specifically stated to the contrary.

[0022] The present invention provides a composition for ovarian anti-aging comprising a compound of Chemical Formula 1 or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0023] [ka]

[0024] In the above formula, n is an integer from 1 to 3, m is 0 or 1; A represents phenyl; R1 is hydrogen or C1-C6-alkyl, R 2 represents hydrogen, halogen or C1-C6-alkoxy, or -C1-C6-alkylene-OH, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)R 7 or -NHC(O)R 7 where p is an integer from 0 to 3; R 7 represents hydrogen or C1-C3-alkyl, R 8 represents C1-C3-alkylpiperidinyl or C1-C3-alkylsulfonyl, R 3 represents hydrogen, halogen, C1-C6-alkyl or phenyl, or the heterocycle contains 1 or 2 heteroatoms selected from S, N and O atoms and is a 5- to 6-membered ring -(CH2) p -heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, R4 is halogen, C1-C6-alkyl, -C1-C6-alkylene-OH, -O-phenyl, -(CH2) p CO2R 7 The heterocycle contains 1 or 2 heteroatoms selected from S, N and O atoms and is a 5- to 6-membered ring -(CH2) p -heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3; R 7 is as defined above, R 5 is hydrogen or C1-C6-alkyl, R 6 represents C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle or -C1-C6-alkylene-heterocycle, where heterocycle is a 3-8 membered ring containing 1-3 heteroatoms selected from S, N and O atoms; R 6may be substituted by C1-C6-alkylamine, -C1-C6-alkylene-OH, or C1-C6-alkylsulfonyl.

[0025] In the present invention, the compound of Chemical Formula 1 was administered to aged ovaries, and it was confirmed that the ovaries were restored by the compound of Chemical Formula 1, thereby restoring the function of the aged ovaries. The present invention has thus clarified a novel use of the compound of Chemical Formula 1.

[0026] The compound of the present invention represented by Chemical Formula 1 may be used in the form of a pharma- ceutically acceptable salt. In particular, the pharma- ceutically acceptable salt may be an acid addition salt formed with a free acid. Here, the acid addition salt may be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. Such pharma- ceutically acceptable salt types may include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, and the like.

[0027] The compositions of the present invention may include not only the compounds of formula 1, pharma- ceutically acceptable salts thereof, but also all salts, isomers, hydrates and / or solvates thereof which may be prepared by conventional methods.

[0028] As used herein, the term "isomer" may refer to a compound of the present invention or a salt thereof that has the same chemical or molecular formula but is structurally or stereochemically different. Such isomers include structural isomers such as tautomers, isomers such as R or S isomers having asymmetric carbon centers, geometric isomers (trans, cis), and enantiomers. All of these isomers and mixtures thereof are also included within the scope of the present invention.

[0029] In the present specification, the term "hydrate" may refer to the compound of the present invention or a salt thereof, which contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate of the compound of the present invention represented by Chemical Formula 1 may contain a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate may contain 1 equivalent or more, preferably 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound of the present invention represented by Chemical Formula 1, its isomer, or a pharma- ceutically acceptable salt thereof from water or a solvent containing water.

[0030] As used herein, a "solvate" may refer to a compound of the present invention or a salt thereof that contains a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents in this regard include solvents that are volatile, non-toxic, and / or suitable for administration to humans.

[0031] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon radical. An alkyl may be a "saturated alkyl" that does not contain an alkenyl or alkynyl moiety, or an "unsaturated alkyl" that contains at least one alkenyl or alkynyl moiety, and may have from 1 to 20 carbon atoms, unless otherwise defined. Alkyl, alkenyl, and alkynyl may refer to straight or branched chain acyclic hydrocarbons.

[0032] The term "alkylene" refers to a hydrocarbon divalent group formed by further forming an alkyl radical, examples of which include, but are not limited to, methylene, ethylene, propylene, butylene, isobutylene, and the like.

[0033] The term "alkoxy", unless otherwise defined, means alkyl-oxy having from 1 to 10 carbon atoms.

[0034] The term "cycloalkyl" means, unless otherwise defined, a saturated aliphatic 3-10 membered ring. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0035] The term "heterocycle", unless otherwise defined, refers to a 3-10 membered ring, preferably a 4-8 membered ring, more preferably a 5-6 membered ring, that contains 1-3 heteroatoms selected from the group consisting of N, O and S, may be fused with benzo or C3-C8 cycloalkyl, is saturated or contains 1 or 2 double bonds. It may also be used in combination with the term "heterocyclyl". Examples of heterocycles include, but are not limited to, pyrroline, pyrrolidine, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyran, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran, etc.

[0036] In addition, unless otherwise defined, the terms and abbreviations used in this specification may be interpreted as having the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0037] In one embodiment of the present invention, in the compound of formula 1, R 3 represents hydrogen, halogen, or phenyl, or the heterocycle is morpholino, piperazinonyl, -(CH2) p -heterocycle, where p is an integer from 0 to 1, provided that when m is 0, R 3 may be phenyl.

[0038] In one embodiment of the present invention, in the compound of formula 1, R 4 is halogen, C1-C3-alkyl, -C1-C3-alkylene-OH, -O-phenyl, -(CH2) p CO2-ethyl, the heterocycle is thiomorpholino, morpholino, piperazinonyl, or pyrrolidinyl -(CH2) p- heterocycle, or proline-N-carbonyl, where p may be an integer from 0 to 1.

[0039] In one embodiment of the present invention, in the compound of formula 1, R 5 is hydrogen or C1-C3-alkyl, R 6 represents C1-C3-alkyl, C3-C6-cycloalkyl, heterocycle or -C1-C3-alkylene-heterocycle, where heterocycle is tetrahydro-2H-pyran or piperidinyl, R 6 When is a heterocycle or -C3-C6-alkylene-heterocycle, it may be substituted by C1-C6-alkylamine, -C1-C6-alkylene-OH, or C1-C6-alkylsulfonyl.

[0040] In the present invention, examples of the compound of Chemical Formula 1 include compounds 1 to 32 listed in Table 1 below, or pharma- ceutically acceptable salts thereof.

[0041] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0042] In a preferred embodiment of the present invention, the compound of Chemical Formula 1 may be a compound of Chemical Formula 2 below.

[0043] [ka]

[0044] In the present invention, the term "ovarian anti-aging" refers to restoring the function of the ovary, which has lost function due to ovarian aging, to a normal ovary or a similar level, or is a comprehensive meaning of inhibiting the progression of ovarian aging.

[0045] In the present invention, the term "follicle" refers to the aggregate of cells found in the ovary that contains the oocyte, where female hormones are produced and secreted.

[0046] In the present invention, the term "follicle formation" refers to the process by which ovarian follicles containing (immature) oocytes mature or any stage thereof, including any stage in the progression from primordial follicles to preovulatory follicles and / or from immature oocytes to ovum.

[0047] In the present invention, the term "oocyte" refers to a cell that can mature into an egg by meiosis.

[0048] In the present invention, the term "cumulus cell" refers to a granular cell that surrounds an oocyte and provides it with nutrients.

[0049] In the present invention, the term "old age" or "aging" refers to the manifestation of a natural aging phenomenon in which function declines over time or due to internal / external stimuli (eg, inflammation).

[0050] In the present invention, the term "ovarian function" is meant to collectively refer to, for example, the production of reproductive hormones, the maintenance of adequate levels of follicle stimulating hormone or follicle production, ovulation of follicles, formation of the corpus luteum, the maintenance of adequate levels of follicles and oocytes, and ovarian histology (e.g., size and tissue health).

[0051] In the present invention, the term "loss of ovarian function" means the loss of functions of the ovaries for pregnancy, such as ovulation, implantation, and fertilization, or the loss or reduction of ovarian functions known in the art, such as a decrease in reproductive hormones secreted from the ovaries.

[0052] In the present invention, the term "restoration of ovarian function" refers to the restoration of ovarian function damaged by external factors or internal factors (especially aging, etc.), and the degree of restoration may be the ovaries before they are damaged by aging or other factors or a similar degree, or may indicate a therapeutic agent or an improved degree compared to before administration of the therapeutic agent. Here, restoration of damaged ovarian function means restoration to about 30% or more, 40% or more, or 50% or more of normal ovaries.

[0053] In the present invention, "ovarian anti-aging" collectively means not only delaying ovarian aging, but also restoring the function of aged ovaries or extending reproductive potential or ovarian function, and alleviating ovarian inflammatory responses, and treating ovarian aging.

[0054] Aging ovaries exhibit several phenotypic defects, such as the development of oxidative stress, ovarian inflammation, decreased steroid production, and hair follicle development, which may result in loss of fertility due to a reduced number of follicles and poor oocyte quality.

[0055] According to one embodiment of the present invention, the anti-aging of the ovaries may improve or restore the function of the aging-prone ovaries having the above-mentioned problems.

[0056] In the examples of the present invention, it was confirmed that treatment with the compound of Chemical Formula 1 increased the total number of follicles including preantral follicles and anterior follicles, improved follicular development, increased the number of oocytes, and increased the rate of embryo development before implantation.

[0057] Therefore, in the present invention, ovarian anti-aging may be embodied as at least one selected from the group consisting of an increase in the number of follicles, an increase in the number of oocytes, and an increase in the rate of pre-implantation embryo development.

[0058] According to one embodiment of the present invention, the compound of Chemical Formula 1 can increase the expression of at least one gene selected from the group consisting of intracellular longevity-related genes, steroidogenesis-related genes, primordial follicle factors, and functional factors of ovary.

[0059] The primordial follicle-related factor can improve the survival of primordial follicles, regulate the proliferation of stromal cells and the differentiation of granulosa cells due to the development of anterior antral follicles, and may be at least one gene selected from the group consisting of Nanog, Oct3 / 4, and p63, which are genes associated with the development of follicular follicles.

[0060] The longevity-related gene is a gene associated with the extension of survival of ovarian-related cells, and may be at least one selected from the group consisting of SOD2 and SIRT1.

[0061] The steroidogenesis-related gene may be at least one selected from the group consisting of Edn2, Tbxa2r, Oxtr and Adra1d.

[0062] The ovarian function-related factor may be at least one selected from the group consisting of Lhcgr, AMH, GDF9, BMP15, and Kitl.

[0063] It was confirmed in this example that the expression level of the gene was increased by treatment with the compound of Chemical Formula 1.

[0064] The compound of formula 1 according to the present invention may be used to prevent, improve, alleviate or treat diseases associated with ovarian aging or dysfunction by inhibiting follicle formation and ovarian aging or restoring the function of aged ovaries.

[0065] Therefore, the present invention provides a pharmaceutical composition for preventing or treating diseases associated with ovarian aging or dysfunction, comprising the compound of Formula 1 or a pharma- ceutically acceptable salt thereof.

[0066] [ka]

[0067] In the above formula, R 1 ~R 6 , A, n and m are as defined above.

[0068] According to one embodiment of the present invention, the disease associated with ovarian aging or dysfunction may be at least one selected from the group consisting of premature ovarian failure, infertility, difficulty in pregnancy, miscarriage, ovarian cyst, ovarian teratoma, ovarian endometrioma, polycystic ovary syndrome, menopause, menopausal symptoms, ovarian cancer, and oophoritis, but is not limited thereto, and is not limited thereto as long as it is a disease that develops due to ovarian aging or dysfunction.

[0069] The term "menopausal symptoms" refers to symptoms and disorders occurring in women before, during and after menopause due to ovarian aging, hormonal changes and / or other biological processes.

[0070] Examples of the menopausal symptoms include, but are not limited to, facial flushing, sweating, sweating during sleep, sleep disorders, dry skin, vaginal dryness, vaginal atrophy, lower urethral atrophy, pain during intercourse, vaginitis, cystitis, pain during urination, urgent urination, concentration disorder, memory disorder, anxiety, depression, fatigue, irritability, decreased libido, muscle pain, joint pain, and osteoporosis.

[0071] The term "premature ovarian failure" refers to the cessation of ovarian function before the age of 40. Causes of premature ovarian failure include, but are not limited to, chemotherapy, radiation therapy, autoimmune disease, thyroid disease, diabetes, and surgically induced menopause (e.g., hysterectomy or oophorectomy).

[0072] The present invention further provides a pharmaceutical composition for preventing or suppressing delayed or early menopause, comprising the compound of formula 1 or a pharma- ceutically acceptable salt thereof.

[0073] The compound of formula 1 or a pharma- ceutically acceptable salt thereof according to the present invention may further be used to protect the ovaries.

[0074] "Protection of the ovary" further refers to protection against any injury or trauma (e.g., engraftment or injury) to the ovary. "Protection of the ovary" refers to protecting the function of the ovary at an appropriate level. As the ovary ages, the possibility of injury or trauma occurring increases, so treatment with the compound of Chemical Formula 1 can protect the ovary from said injury or trauma.

[0075] The present invention further provides a method for anti-aging of the ovaries, comprising administering a compound of Formula 1, or a pharma- ceutically acceptable salt thereof, to an individual in need thereof.

[0076] [ka]

[0077] In the above formula, R 1 ~R 6 , A, n and m are as defined above.

[0078] In the ovarian anti-aging method, the ovary means an aged ovary, and treatment of the aged ovary with the compound of Chemical Formula 1 restores the function of the aged ovary to the normal ovarian level.

[0079] The present invention may also provide a method for preventing or treating a disease associated with ovarian aging or dysfunction, comprising administering a compound of Chemical Formula 1 to an individual in need thereof.

[0080] In the ovarian anti-aging method and the preventive or therapeutic method, the contents of the pharmaceutical composition for ovarian anti-aging may be applied mutatis mutandis, unless otherwise specified.

[0081] The present invention may also provide a use of the compound of Formula 1 or a pharma- ceutical acceptable salt thereof in anti-aging of the ovary, and a use of the compound of Formula 1 or a pharma-ceutical acceptable salt thereof in the prevention or treatment of diseases associated with ovarian aging or dysfunction.

[0082] In the above uses, unless otherwise specified, the contents of the above pharmaceutical composition for ovarian anti-aging may be applied mutatis mutandis.

[0083] As used herein, "treatment" means halting or slowing the progression of a disease when used in a subject who shows symptoms of onset, and "prevention" means halting or slowing the onset of disease when used in a subject who does not show symptoms of onset but is at high risk of such onset.

[0084] In the present invention, the pharmaceutical composition may contain a pharma- ceutically acceptable carrier together with the compound of the present invention, if necessary.

[0085] The compound of formula 1 according to the present invention may be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, it is prepared using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant, etc.

[0086] Oral solid preparations include tablets, pills, powders, granules, capsules, lozenges, etc., and such solid preparations are prepared by mixing one or more compounds of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium, stylate, and talc are also used. Oral liquid preparations include suspensions, liquids, emulsions, and syrups, and may contain various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents.

[0087] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. As suppository bases, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. may be used.

[0088] In addition, the effective dosage of the compound of the present invention represented by Chemical Formula 1 for the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and is generally about 0.001-100 mg / kg / day, preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, the dosage is generally 0.07-7000 mg / day, preferably 0.7-2500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

[0089] The term "individual" as used herein means, but is not limited to, humans and vertebrates such as mammals, including livestock, and birds, which can be ameliorated, prevented, or treated for inflammatory diseases by administration of the pharmaceutical composition according to the present invention.

[0090] As used herein, "administration" means introducing a given substance into a human or animal by any suitable method, and the route of administration of the prophylactic or therapeutic composition according to the present invention may be oral or parenteral administration via any common route as long as it can reach the target tissue.

[0091] The numerical values ​​described in this specification should be interpreted as including an equivalent range unless otherwise specified.

[0092] The present invention will be described in detail below with reference to the following experimental examples. However, the following experimental examples are merely for illustrating the present invention, and the contents of the present invention are not limited by the following experimental examples. Furthermore, these experimental examples are merely intended to aid in understanding the present invention, and are not intended to limit the scope of the present invention in any sense. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0093] Materials and Preparation In this example, (5-[(1,1-dioxide-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine) (hereinafter referred to as "Example compound" or "Compound 1")) was used as a representative example of the compound of Chemical Formula 1.

[0094] In this experiment, young mice (7-9 weeks, n=15) were used as the control group, and old mice (55-60 weeks, n=30) were used as the experimental group. In the figures, experimental results for young mice are shown as "young group," and experimental results for old mice are shown as "old group."

[0095] <Collection of fertilized pronuclear cells> Aged mice were treated with the example compounds to stimulate endogenous hormones, and 75 IU Pregnant Mare Serum Gonadotropin (PMSG: RP1782725000; BioVendor, Czech Republic) was injected 12 hours later. The control group was administered 75 IU PMSG only. Female mice were superovulated by intraperitoneal hormone injection at 48-hour intervals. The hormones administered were PMSG 7.5 IU and Human chorionic gonadotropin (hCG: 668900221; LG Chem, Seoul, Korea) 7.5 IU. After the second hormone treatment, the female mice were mated with male mice (2:1 or 1:1). Fertilized pronuclear cells (2PN) were obtained 24 hours after hCG injection.

[0096] <Collection of Embryo Vesicles (GV), Oocytes and Embryos> Female mice (young mice: 7-8 weeks old; old mice: 40 weeks old or older) were superovulated by intraperitoneal injection of Pregnant Mare Serum Gonadotropin (PMSG; G4527, Sigma Aldrich; 7.5 IU). Germ vesicle (GV) oocytes were collected from the ovaries 48 hours after PMSG injection. For fertilized eggs, female mice were injected with PMSG and injected with hCG 48 hours later, followed by overnight placement with BDF1 stud male mice aged approximately 10-12 weeks. The next day, the plugs of the female mice were examined, and zygotes and 2-cell embryos were collected from the oviducts 18-20 hours and 36 hours after hCG injection, respectively.

[0097] Experimental Example 1. Confirmation of increase in oocytes and follicle development by treatment with the compound of the embodiment Experiment 1 evaluated the effect of treatment with an example compound on the ovaries of mice stimulated with PMSG in vivo. Optical microscopy imaging was performed on the ovaries of young mice, treated with an example compound, and untreated old mice.

[0098] Each sample was fixed with 4% paraformaldehyde for 30 min at room temperature, the fixed tissue was washed with PBS, and embedded in paraffin with a section thickness of 5 μm. For histological analysis with hematoxylin and eosin staining, the samples were deparaffinized in xylene from 100% to 75% per 10 min, and rehydrated using 100%, 95%, 80%, and 75% EtOH per 10 min at room temperature, where they were immunofluorescently stained with hematoxylin and eosin. Antigens were regenerated by boiling for 20 min in an autoclave container (Nalgene Jars) with 0.01 M citrate buffer (pH 6.0).

[0099] Figure 1a shows histological images of mouse ovarian tissue stained with hematoxylin and eosin after hormonal stimulation with or without treatment with the compound of the present invention. It can be seen that the number of total follicles (indicated by arrows), including preantral and anterior follicles, is significantly higher in the compound-treated ovaries than in the control ovaries.

[0100] After induction of superovulation, the number of germinal vesicle (GV) oocytes in the ovaries of old and young mice treated with the example compound was evaluated, and the results are shown in Figure 1b and Figure 1c.

[0101] The number of GV oocytes was measured by collecting mouse eggs (young mouse eggs: 15; old mouse eggs: 30; old mouse eggs treated with the example compound: 30) and physically chopping them to collect follicles. GV oocytes in the follicles were then isolated using a stereomicroscope and counted to measure the number of cells.

[0102] FIG. 1b is an optical microscope image, and FIG. 1c is a graph showing the number of GV oocytes. In FIGS. 1b and 1c, CTL means the control group not treated with the example compound.

[0103] Figures 1b and 1c show that at the germ sac (GV) stage, the total number of oocytes collected from aged ovaries treated with the example compounds was significantly higher than that from aged ovaries not treated with the example compounds as a control group.

[0104] Experimental Example 2. Preimplantation embryo development with treatment with example compounds Experiment 2 evaluated the effect of fertilized pronuclear cell (2PN) embryo development on the ovaries of aged mice treated with example compounds.

[0105] 27 pronuclear cells from aged mice (n=15) treated with the Example compound and 62 pronuclear cells from aged mice (n=15) not treated with the Example compound as a control group were cultured. Then, the number of cells corresponding to each stage of 2-cell, 4-cell, 8-cell, morula, early blastocyst and late blastocyst was observed and measured under a microscope and expressed as a ratio.

[0106] As shown in FIG. 2, the development rate of the embryos treated with the compound of the embodiment was 20% higher than that of the conventional control group in terms of morula, early blastocyst, and late blastocyst. Specifically, the morula rate was 55.56% in the embryos of the old mice treated with the compound of the embodiment. However, the control group had a rate of 37.10%, which was quite low. The early blastocyst rate was higher in the embryos treated with the compound of the embodiment (51.85%) than in the embryos of the control group (35.48%), and finally, the late blastocyst rate was also significantly higher in the embryos of the old mice treated with the compound of the embodiment (51.85%) than in the embryos of the control group (35.48%).

[0107] Experimental Example 3. Confirmation of gene expression in the ovary <Confirmation of gene expression profile by next-generation sequencing> The major changes in gene expression due to the treatment with the example compounds were confirmed by next generation sequencing (NGS).

[0108] mRNA sequencing was performed according to references [1, 2]. RNA was extracted using TRIzol (TRIzol® 15596026, Thermo Fisher) solution. Sequencing libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) according to the manufacturer's manufacturing method. Paired-end 101bp sequencing was performed using the Illumina platform (NovaSeq 6000, Illumina). Two experimental results were generated for each group.

[0109] Paired end reads data were trimmed using Trimmomatic (Bolger et al., 2014) according to the following criteria: 1st trimming: base quality<3, window size=4, mean quality=15; 2nd trimming: min length=36bp. Trimmed reads were mapped to the UCSC genome database using the HISAT2 program (Kim et al., 2019; Nat Biotechnol 37,907-915,2019). Then, quantification was performed using StringTie (Pertea et al., 2015; Nat Biotechnol.2015 Mar;33(3):290-295) and gene / transcript-based (NCBI Annotation Release 108) methods.

[0110] Differentially expressed gene analysis was performed using DESeq2 with the following criteria: baseMean counts>14, false discover rate (FDR)<0.1, and log2FoldChange>1. Gene set enrichment analysis (GSEA) was performed using the clusterProfiler packages in R / Bioconductor [3] to analyze Gene Ontology (GO; biological process, cellular component, and molecular function) and canonical pathways (REACTOME, WikiPathway [4]).

[0111] Figure 3a shows the results of NGS analysis of RNA purified from the ovaries of a young group (Young, YNG; 10 weeks; n = 2), an old group (Old; 55 weeks; n = 2), and an old group injected with an example compound (Old + compound1; 55 weeks; n = 2).

[0112] In Figure 3a, the differentially expressed genes (DEGs) such as differences between groups were represented in a Venn diagram format. There were 1,214 differentially expressed genes (DEGs) between the old group and the young group, and 1,021 DEGs between the old group and the old group treated with the example compound. It was confirmed that there were 267 common DEGs among these DEGs. Among the common DEGs, it was confirmed that there were quite a few gene ontology (GO) related to the regulation of the immune system (enrich).

[0113] Also, DEGs (n = 38) related to tumor necrosis factor superfamily cytokine production among the GO related to immune system regulation were confirmed. As genes that increased in the old group and were down-regulated by the example compound, Fcer1g, Slamf9, Clec4a3, Ptpn6, Tyrobp, Adipoq, Bcl3, Trem2, and Sash3 were confirmed.

[0114] <Confirmation of changes in the expression of immune system regulatory genes by RT-qPCR> In this experiment, the changes in the expression of immune system regulatory genes (genes related to tumor necrosis factor superfamily cytokine production) by treatment with the example compound in the aging model were quantitatively evaluated using RT-qPCR (Real-time quantitative PCR).

[0115] Based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathway, primers for up-regulated genes and down-regulated genes were selected.

[0116] RT-qPCR was performed on each ovary using the Trizol protocol, and total RNA was extracted from the lysate using the RNeasy Mini Kit (Qiagen Ltd.). The extracted total RNA was then reverse transcribed to synthesize cDNA using the Superscript kit (Invitrogen) containing random hexamers.

[0117] Gene expression was confirmed using primers and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, US) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, US). The primer sequences are shown in Table 2.

[0118] [Table 2]

[0119] A comparative threshold cycle (CT) method was used in which the accumulated PCR products for each gene examined were normalized to the reference gene beta-actin, which was repeated three times.

[0120] The PCR products were loaded onto a 2% agarose gel and analyzed by Safeview TM The gels were stained with 100% ribosomal RNA (Biological Materials, Richmond, Canada), the gels were visualized under UV illumination using a gel documentation system (WSE-6100 LuminoGraph; ATTO, Tokyo, Japan), and the product bands were analyzed through intensity ratio analysis using ImageJ software.

[0121] As shown in Figure 3b, it can be confirmed that the mRNA expression levels of Ptpn6, Fcer1g, Tyrobp, Slamf9, and Clec4a3, which are genes related to tumor necrosis factor superfamily cytokine production, are significantly increased in the old control group. However, it can be confirmed that the expression levels are lower in the old experimental group (Old) treated with the example compound, at the same level as in the young control group (Young).

[0122] Experimental Example 4. Confirmation of gene expression patterns of ovarian function The expression of ovarian function-related genes in the ovaries of aged mice treated with the Example compounds was confirmed by RT-PCR (Reverse Transcription-Polymerase Chain Reaction).

[0123] mRNA was extracted from the oocytes using TRIzol (registered trademark) RNA (15596026, Thermo Fisher Scientific) solution, and reverse transcription-PCR (RT-PCR) was performed. PCR products were analyzed using SafeView TM After staining with 100 mM KOH (Applied Biological Materials, Richmond, Canada), the gels were loaded onto 2% agarose gels. The gels were visualized under UV illumination using a gel documentation system (WSE-6100 LuminoGraph; ATTO, Tokyo, Japan). The intensity ratios of the product bands were analyzed using the ImageJ software program.

[0124] In the ovaries of mice treated with the example compounds, the expression of primordial follicle-related factors Nanog, Oct3 / 4, and p63, and the expression of ovarian function-related factors AMH and BMP15 were analyzed by RT-PCR as described above, and the results are shown in Figure 4.

[0125] In Figures 4a and 4b, "young group" refers to young mice, "old group" refers to old mice, and "old group treated with an example compound" refers to old mice treated with an example compound.

[0126] In Fig. 4a, the upper left box indicates longevity-related genes, the lower left box indicates steroidogenesis-related genes, the upper right box indicates primordial follicle factors, and the lower right box indicates functional factors of ovary. Fig. 4b is a quantification graph showing the band images of each gene in Fig. 4a normalized with beta-actin.

[0127] As shown in Figures 4a and 4b, the mRNA expression of Oct3 / 4, Nanog, p63, Lhcgr, AMH, GDF9, BMP15 and Kitl was significantly higher in the experimental group treated with the example compound than in the control group not treated with the example compound in aged mice.

[0128] In addition, as shown in Figures 4a and 4b, the analysis of longevity-related genes such as SOD2 and SIRT1 confirmed that the expression levels of SOD2 and SIRT1 were higher in old mice treated with the example compounds than in old mice not treated with the example compounds. Furthermore, the expression levels of steroidogenesis-related genes such as Edn2, Tbxa2r, Oxtr and Adra1d in old mice treated with the example compounds were significantly increased compared to the control group old mice not treated with the example compounds.

[0129] [References] [1]Jeffrey A Martin and Zhong Wang(2011)Next-generation transcriptome assembly.Nat Rev Genet.12(10):671-82. [2]Love MI,Huber W,Anders S.(2014)Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol 15:550. [3] Subramanian A, et al. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102:15545-15550. [4]Jassal B, et al.(2020)The reactome pathway knowledgebase.Nucleic Acids Res 48:D498-d503.

Claims

1. Chemical formula 1: 【Chemistry 1】 [In the formula, n is an integer from 1 to 3, m is 0 or 1; A represents phenyl; R １ is hydrogen or C １ -C ６ - alkyl, R ２ is hydrogen, halogen or C １ -C ６ -alkoxy, or -C １ -C ６ -Alkylene-OH, -(CH ２ ) ｐ CO ２ R ７ , -NHR ８ , -N(H)S(O) ２ R ７ or -NHC(O)R ７ represents where p is an integer from 0 to 3, and R ７ is hydrogen or C １ -C ３ - represents alkyl, and R ８ is C １ -C ３ - alkylpiperidinyl, or C １ -C ３ represents alkylsulfonyl, R ３ is hydrogen, halogen, C １ -C ６ -alkyl or phenyl, or the heterocycle contains one or two heteroatoms selected from S, N and O atoms and is a 5- to 6-membered ring -(CH ２ ) ｐ -heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R ３ is phenyl, R ４ is a halogen, C １ -C ６ -alkyl, -C １ -C ６ -Alkylene-OH, -O-phenyl, -(CH ２ ) ｐ CO ２ R ７ , the heterocycle contains one or two heteroatoms selected from S, N and O atoms and is a 5- to 6-membered ring -(CH ２ ) ｐ -heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3; R ７ is as defined above, R ５ is hydrogen or C １ -C ６ - alkyl, R ６ is C １ -C ６ -Alkyl, C ３ -C ６ -cycloalkyl, heterocycle or -C １ -C ６ -alkylene-heterocycle, where the heterocycle is a 3- to 8-membered ring containing 1 to 3 heteroatoms selected from S, N, and O atoms; R ６ is C １ -C ６ - alkylamine, -C １ -C ６ -alkylene-OH, or C １ -C ６ -optionally substituted by alkylsulfonyl] or a pharmaceutically acceptable salt thereof as an active ingredient.

2. R ３ represents hydrogen, halogen, or phenyl, or the heterocycle is morpholino, piperazinonyl, —(CH ２ ) ｐ -heterocycle, where p is an integer from 0 to 1, provided that when m is 0, R ３ is phenyl, R ４ is a halogen, C １ -C ３ -alkyl, -C １ -C ３ -Alkylene-OH, -O-phenyl, -(CH ２ ) ｐ CO ２ -ethyl, the heterocycle is thiomorpholino, morpholino, piperazinonyl, or pyrrolidinyl -(CH ２ ) p-heterocycle, or proline-N-carbonyl; where p is an integer from 0 to 1, R ５ is hydrogen or C １ -C ３ - alkyl, R ６ is C １ -C ３ -Alkyl, C ３ -C ６ -cycloalkyl, heterocycle or -C １ -C ３ -alkylene-heterocycle, where the heterocycle is tetrahydro-2H-pyran, or piperidinyl, and R ６ is a heterocycle or -C １ -C ３ - alkylene-heterocycle, C １ -C ６ - alkylamine, -C １ -C ６ -alkylene-OH, or C １ -C ６ The pharmaceutical composition for ovarian anti-aging according to claim 1, wherein the compound is substituted with -alkylsulfonyl.

3. 2. The pharmaceutical composition for ovarian anti-aging according to claim 1, wherein the compound of Formula 1 is any one selected from the following compound group: <1> 5-[(1,1-dioxide-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine, <2> ethyl 7-(cyclopentylamino)-2-phenyl-1H-indole-5-carboxylate, <3> (7-(cyclopentylamino)-2-phenyl-1H-indol-5-yl)methanol, <4> 5-chloro-N,1-dimethyl-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine, <5> 4-((7-cyclopentylamino)-2-(3-fluorophenyl)-1H-indol-5-yl)methyl)piperazin-2-one, <6> 4-((2-phenyl-7-(((tetrahydro-2H-pyran-4-yl)methyl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide, <7> 5-chloro-N-(1-methylpiperidin-4-yl)-2-phenyl-1H-indol-7-amine, <8> 5-phenoxy-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine, <9> 5-chloro-3-(morpholinomethyl)-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine, <10> 2-(4-((5-fluoro-2-phenyl-1H-indol-7-yl)amino)piperidin-1-yl)ethan-1-ol, <11> N-(4-(5-chloro-7-(cyclopentylamino)-1H-indol-2-yl)phenyl)methanesulfonamide, <12> 5-chloro-3-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine, <13> 4-((2-(3-fluorophenyl)-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide, <14> 5-chloro-N-cyclopentyl-2-(4-((1-methylpiperidin-4-yl)amino)phenyl)-1H-indol-7-amine, <15> 4-((7-isopentylamino)-2-(4-methoxyphenyl)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide, <16> N-(4-(7-(cyclopentylamino)-5-((1,1-dioxidothiomorpholino)methyl)-1H-indol-2-yl)phenyl)acetamide, <17> 4-((3-bromo-2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide, <18> 4-((5-chloro-2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-3-yl)methyl)piperazin-2-one, <19> 4-((7-(methyl(tetrahydro-2H-pyran-4-yl)amino)-2-phenyl-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide, <20> 5-methyl-N-(1-(methylsulfonyl)piperidin-4-yl)-2-phenyl-1H-indol-7-amine, <21> N-(4-(5-(1,1-dioxidothiomorpholino)-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetamide, <22> 4-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-2-phenyl-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide, <23>N １ -(5-chloro-2-phenyl-1H-indol-7-yl)-N ４ -methylcyclohexane-1,4-diamine, <24> methyl 2-(3-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetate, <25> (2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indole-5-carbonyl)-D-proline, <26> (3-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)methanol, <27> N-cyclopentyl-2-phenyl-5-(2-(pyrrolidin-1-yl)ethyl)-1H-indol-7-amine, <28> methyl 2-(4-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetate, <29> methyl 4-(5-chloro-7-(cyclopentylamino)-1H-indol-2-yl)benzoate, <30> 2-(4-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)ethan-1-ol, <31> 3-bromo-5-(morpholinomethyl)-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine, <32> 4-((3-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide

4. The compound of Chemical Formula 1 is a compound of Chemical Formula 2: 【Chemistry 2】 The pharmaceutical composition for ovarian anti-aging according to claim 1, wherein the compound is

5. The pharmaceutical composition for ovarian anti-aging according to claim 1, wherein the anti-aging of the ovaries is to restore the function of ovaries that have undergone advanced aging.

6. 2. The pharmaceutical composition for ovarian anti-aging according to claim 1, wherein the anti-aging of the ovaries is embodied by at least one selected from the group consisting of an increase in the number of follicles, an increase in the number of oocytes, and an increase in the rate of pre-implantation embryo development.

7. 2. The pharmaceutical composition for ovarian anti-aging according to claim 1, wherein the compound of Formula 1 increases the expression of at least one gene selected from the group consisting of intracellular longevity-related genes SOD2 and SIRT1, steroidogenesis-related genes Edn2, Tbxa2r, Oxtr, and Adra1d, primordial follicle-related factors Nanog, Oct3 / 4, and p63, and ovarian function-related factors Lhcgr, AMH, GDF9, BMP15, and Kitl.

8. Chemical formula 1: 【Transformation 3】 [In the formula, R １ ~R ６ , A, n, and m are the same as defined in the first paragraph. or a pharmaceutically acceptable salt thereof as an active ingredient.

9. 9. The pharmaceutical composition for preventing or treating diseases associated with ovarian aging or dysfunction according to claim 8, wherein the disease associated with ovarian aging or dysfunction is at least one selected from the group consisting of premature ovarian failure, infertility, difficulty in pregnancy, miscarriage, ovarian cyst, ovarian teratoma, ovarian endometrioma, polycystic ovary syndrome, menopause, menopausal symptoms, ovarian cancer, and oophoritis.

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