Medium composition for in vitro fertilization and / or in vitro culture of aged oocytes and method for in vitro fertilization and / or in vitro culture using same

Abstract

The present invention relates to a medium composition for in vitro fertilization and / or in vitro culture comprising a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof, and a method using same. The compound or composition suppresses reactive oxygen species and lipid peroxidation in ovarian granulosa cells to preventing cytotoxicity and mitochondrial dysfunction caused by apoptosis and ferroptosis, leading to an improvement in the quality of aged oocytes, and to increase the developmental rate and blastocyst formation rate of pre-implantation embryos, thereby improving the efficiency of in vitro fertilization. In addition, treatment with the medium composition during the vitrification process of in vitro–fertilized embryos improves re-expansion and survival rates, thereby reducing structural and metabolic damage. Accordingly, the present invention can be advantageously applied to an assisted reproductive technology for treating infertility and subfertility, and in vitro fertilization and / or in vitro culture for improving the propagation efficiency of livestock.

Description

Composition of a medium for in vitro fertilization and / or in vitro culture of aging oocytes and a method for in vitro fertilization and / or in vitro culture using the same

[0001] The present invention relates to a medium composition for in vitro fertilization and / or in vitro culture of aging oocytes comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof, and a method for in vitro fertilization and / or in vitro culture using the same.

[0002]

[0003] Over the past few decades, advancements in assisted reproductive technology (ART) have provided infertile couples with the opportunity to conceive, but clinical success rates in older women remain low. As maternal age increases, the quality of eggs and embryos deteriorates significantly, leading to a decrease in implantation rates and pregnancy success rates. Against this backdrop, there is an urgent need to develop new approaches that can improve the quality of eggs and embryos in older women and enhance clinical reproductive outcomes.

[0004] The decline in female fertility is closely linked not only to a decrease in the number of eggs but also to a decline in their quality. In particular, egg quality is directly connected to embryonic developmental potential, and mitochondrial function within the egg plays a key role in various physiological processes, such as cellular energy metabolism, maintenance of oxidative homeostasis, and intracellular signal transduction. However, with increasing age, mitochondrial function gradually declines, leading to the overproduction of reactive oxygen species (ROS), increased lipid peroxidation, and the breakdown of iron homeostasis. There are increasing reports that these changes, along with intracellular oxidative damage, induce ferroptosis—a new form of apoptosis—thereby hindering the survival and development of eggs and embryos.

[0005] Ferroptosis is a form of regulated cell death induced by iron-dependent lipid peroxidation, distinct from traditional apoptosis. In this process, the inactivation or inhibition of glutathione peroxidase 4 (GPX4) acts as a major molecular trigger, resulting in a rapid accumulation of ROS, loss of mitochondrial membrane potential (ΔΨm), and reduced ATP production. Ovarian granulosa cells and oocytes are vulnerable to this ferroptosis, which is identified as a cause of reduced oocyte developmental capacity and embryonic development rates, particularly in older women.

[0006] To date, various anti-ferroptosis agents, including antioxidants and iron chelators, have been studied. For example, representative ferroptosis inhibitors such as ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) have been reported, and antioxidants such as resveratrol, coenzyme Q10, and mitoquinone have also been reported to have effects that alleviate intracellular oxidative stress.

[0007] However, these existing agents have not been able to fundamentally improve the decline in mitochondrial function or the inhibition of embryonic development in ovarian granulosa, and have shown only limited effects, particularly in the development of oocytes derived from older individuals and the restoration of embryonic developmental capacity. Therefore, there is a need to discover new substances that can directly improve embryonic development and blastocyst formation while inhibiting intracellular oxidative stress and lipid peroxidation.

[0008] [Prior Art Literature]

[0009] [Non-patent literature]

[0010] (Non-patent Document 1) Dixon SJ, Lemberg KM, Lamprecht MR, et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012 May 25;149(5):1060-72.

[0011] (Non-patent document 2) Udagawa O, Ishihara T, Maeda M, et al.Mitochondrial dynamics and physiology during oocyte maturation and early embryonic development.Int J Dev Biol. 2014;58(3-5):211-217.

[0012]

[0013] Accordingly, the inventors investigated the effects of ferroptosis on maintaining the function of ovarian granulosa cells and embryonic development, along with the inhibition of reactive oxygen species (ROS) and lipid peroxidation, and on improving the re-expansion rate and survival rate of in vitro produced embryos after vitrification and thawing. As a result, the inventors confirmed that the compound represented by Chemical Formula 1 according to the present invention exhibits excellent effects in preserving mitochondrial function and promoting embryonic development, thereby completing the present invention.

[0014] Accordingly, the object of the present invention is to provide a medium composition for in vitro fertilization and / or in vitro culture for enhancing the in vitro fertilization efficiency of an egg cell, comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] In addition, another object of the present invention is to provide a method for in vitro fertilization and / or in vitro culture using a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof.

[0016] In addition, another objective of the present invention is to provide a use for the compound of Formula 1 or a pharmaceutically acceptable salt thereof for the in vitro fertilization and / or in vitro culture of oocytes.

[0017]

[0018] The present invention provides a medium composition for in vitro fertilization and / or in vitro culture for enhancing the in vitro fertilization efficiency of an egg cell, comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0019] [Chemical Formula 1]

[0020]

[0021] In the above formula,

[0022] n is an integer from 1 to 3, and

[0023] m is 0 or 1, and

[0024] A represents phenyl, and

[0025] R 1 It is hydrogen, or C1-C6-alkyl, and

[0026] R 2 represents hydrogen, a halogen, or a C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 Represents, where p is an integer from 0 to 3, and R 7 represents hydrogen or a C1-C3-alkyl group, and R 8 C 1- C3-alkylpiperidinyl, or C 1- It represents a C3-alkylsulfonyl group,

[0027] R 3-(CH2) represents hydrogen, a halogen, a C1-C6-alkyl or phenyl, or is a 5- to 6-membered ring in which the heterocycle comprises one or two heteroatoms selected from S, N, and O atoms. p - Represents a heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, and

[0028] R 4 is a halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 -(CH2)⁻¹, a heterocycle comprising one or two heteroatoms selected from S, N, and O atoms, and a 5 to 6-membered ring p -heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 ...is as defined above, and the heterocycle can be substituted with one or more oxo (=O) substituents, and

[0029] R 5 is hydrogen, or C1-C6-alkyl, and

[0030] R 6 represents a C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle, or heterocyclil-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring comprising 1 to 3 heteroatoms selected from S, N, and O atoms, and R 6 C1-C6-alkylamine, hydroxy-C 1- It can be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

[0031] In addition, the present invention

[0032] (a) A step for preparing a mature egg of a mammal;

[0033] (b) performing in vitro fertilization by adding the mature oocytes and sperm to a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof; and / or

[0034] (c) A method for in vitro fertilization of a mammal, comprising the step of culturing the fertilized egg produced in step (b) in the medium composition.

[0035] In addition, the present invention

[0036] A step of culturing a fertilized egg of a mammal in a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof; or

[0037] A step of thawing the cultured developmental body in a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof after vitrification;

[0038] A method for in vitro culture including

[0039]

[0040] According to the composition or method of the present invention, the compound of Formula 1 or its salt inhibits the accumulation of reactive oxygen species (ROS) and lipid peroxidation in ovarian granulosa cells, thereby effectively suppressing cytotoxicity and mitochondrial dysfunction induced by ferroptosis, which maintains the quality and developmental potential of the oocyte, and significantly improves the efficiency of in vitro fertilization of aged oocytes. In addition, the composition according to the present invention improves embryonic developmental capacity by promoting the development of pre-implantation embryos and increasing the blastocyst formation rate. Furthermore, it can reduce losses during the embryo preservation process by increasing the re-expansion rate and survival rate during the thawing stage of the vitrification-thawing process of in vitro produced embryos. In particular, it was confirmed that cellular physiological functions such as the recovery of mitochondrial membrane potential (ΔΨm), increased ATP production, and promotion of estradiol (E2) synthesis are improved.

[0041] Therefore, the composition of the present invention can be utilized not only to improve the decline in fertility caused by aging but also as a culture medium additive that inhibits damage during the thawing stage of the vitrification-thawing process of the embryo. This increases the success rate of in vitro fertilization and in vitro embryo production technologies, and contributes to the treatment of infertility and subfertility and the improvement of livestock breeding efficiency.

[0042]

[0043] Figure 1a shows the viability of cells treated for 24 hours with various concentrations of Compound 1 as an anti-ferroptosis agent.

[0044] Figure 1b shows the 24-hour viability of cells treated with RSL3 alone or in combination with Compound 1.

[0045] Figure 1c is a comparative analysis of cell viability (C) in RSL3 (0.3 μM) treated cells with Compound 1, resveratrol, mitoquinone, coenzyme Q10, ferrostatin-1, and reproxtatin-1.

[0046] Figure 1d is a comparative analysis of LDH release (D) in RSL3 (0.3 μM) treated cells of Compound 1, resveratrol, mitoquinone, coenzyme Q10, ferrostatin-1, and reproxtatin-1.

[0047] Figure 1e shows the effects of Compound 1 and other antioxidants / anti-ferroptosis agents on cytoplasmic ROS in RSL3 (0.3 μM) treated cells.

[0048] Figure 1f shows the effects of Compound 1 and other antioxidants / anti-ferroptosis agents on mitochondrial ROS in RSL3 (0.3 μM) treated cells.

[0049] Figure 1g shows the effects of Compound 1 and other antioxidants / anti-ferroptosis agents on cytoplasmic lipid peroxidation in RSL3 (0.3 μM) treated cells.

[0050] Figure 1h shows the effects of Compound 1 and other antioxidants / anti-ferroptosis agents on mitochondrial lipid peroxidation in RSL3 (0.3 μM) treated cells.

[0051] Figure 1i shows the effects of Compound 1 and other antioxidants / anti-ferroptosis agents on mitochondrial iron accumulation in RSL3 (0.3 μM) treated cells.

[0052] Figure 2a shows the effects of RSL3 and Compound 1 on mitochondrial morphology and ΔΨm in KGN cells, including (AD) phase-contrast microscopy images of the control group, RSL3-treated group, RSL3+Compound 1 combined treatment group, and Compound 1-alone treatment group, and (EH) confocal microscopy images under the same conditions.

[0053] Figure 2b confirms the quantification of red fluorescence intensity representing ΔΨm of RSL3 and Compound 1 in KGN cells.

[0054] Figure 3a shows the oxygen consumption rate (OCR) measured by evaluating mitochondrial respiratory parameters via Seahorse XF analysis under RSL3 (3 μM) and RSL3+ Compound 1 (3 μM) treatment conditions to confirm the alleviating effect of Compound 1 on RSL3-induced mitochondrial dysfunction in KGN cells.

[0055] Figure 3b shows the measurement of basal respiration by evaluating mitochondrial respiration parameters via Seahorse XF assay under RSL3 (3 μM) and RSL3+ Compound 1 (3 μM) treatment conditions to confirm the alleviating effect of Compound 1 on RSL3-induced mitochondrial dysfunction in KGN cells.

[0056] Figure 3c shows the measurement of reserve respiratory capacity (SRC) by evaluating mitochondrial respiratory parameters via Seahorse XF analysis under RSL3 (3 μM) and RSL3+ Compound 1 (3 μM) treatment conditions to confirm the alleviating effect of Compound 1 on RSL3-induced mitochondrial dysfunction in KGN cells.

[0057] Figure 3d shows the measurement of proton leakage by evaluating mitochondrial respiration parameters via Seahorse XF analysis under RSL3 (3 μM) and RSL3+ Compound 1 (3 μM) treatment conditions to confirm the alleviating effect of Compound 1 on RSL3-induced mitochondrial dysfunction in KGN cells.

[0058] Figure 3e shows the measurement of ATP production by evaluating mitochondrial respiration parameters via Seahorse XF assay under RSL3 (3 μM) and RSL3+ Compound 1 (3 μM) treatment conditions to confirm the alleviating effect of Compound 2 on RSL3-induced mitochondrial dysfunction in KGN cells.

[0059] Figure 4 shows the results of quantifying E2 levels by ELISA in the culture supernatant after treating KGN cells with control, RSL3 (3 μM), and RSL3+Compound 1 (3 μM) conditions, as well as the restorative effect of Compound 1 on E2 production impaired by RSL3 in KGN cells.

[0060] Figure 5a shows the effects of RSL3 and Compound 1 on mitochondrial morphology and ΔΨm in pre-implantation embryos, with (AD) phase-contrast microscopy images of the control group, RSL3-treated group, RSL3+Compound 1 combined-treatment group, and Compound 1-alone treated group, and (EH) confocal microscopy images under the same conditions.

[0061] Figure 5b confirms the quantification of MitoSpy Orange fluorescence intensity representing ΔΨm.

[0062] Figure 6a shows the effect of Compound 1 on embryonic development rate in young (8-9 weeks old) and old (60-70 weeks old) mice, and shows time-lapse images of embryos cultured under control and Compound 1 treatment conditions.

[0063] Figure 6b confirms the quantification of the embryonic development rate.

[0064] Figure 7 is a graph quantifying confocal microscopy images of pre-implantation embryos cultured under control and Compound 1 treatment conditions, showing the effect of Compound 1 on ΔΨm of pre-implantation embryos derived from young and old mice.

[0065] Figure 8a shows the ratios classified according to each developmental stage of the bovine blastocyst after the vitrification-thawing process.

[0066] Figure 8b shows the diameter of the blastocyst in its early and expanded phases after the vitrification-thawing process of a bovine blastocyst.

[0067] Figure 9 shows the vitrification-thawing process of bovine blastocysts and the blastocyst survival rate.

[0068] Figure 10 confirms the quantification of H2DCF-DA fluorescence intensity representing ROS generated during the vitrification-thawing process of bovine blastocysts.

[0069] Figure 11 confirms the quantification of JC-1 fluorescence intensity representing ΔΨm.

[0070] Figure 12 shows the length of aggregated F-actin measured during the vitrification-thawing process of bovine blastocysts.

[0071]

[0072] The present invention will be described in detail below.

[0073] Meanwhile, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the invention. Furthermore, the scope of the invention is not to be limited by the specific descriptions provided below.

[0074] When a part is said to "include" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather may include additional components.

[0075] The present invention provides a medium composition for in vitro fertilization and / or in vitro culture for enhancing the in vitro fertilization rate of an egg, comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0076] [Chemical Formula 1]

[0077]

[0078] In the above formula,

[0079] n is an integer from 1 to 3, and

[0080] m is 0 or 1, and

[0081] A represents phenyl, and

[0082] R 1 It is hydrogen, or C1-C6-alkyl, and

[0083] R 2 represents hydrogen, a halogen, or a C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 Represents, where p is an integer from 0 to 3, and R 7 represents hydrogen or a C1-C3-alkyl group, and R 8 C 1-C3-alkylpiperidinyl, or C 1- It represents a C3-alkylsulfonyl group,

[0084] R 3 -(CH2) represents hydrogen, a halogen, a C1-C6-alkyl or phenyl, or is a 5- to 6-membered ring in which the heterocycle comprises one or two heteroatoms selected from S, N, and O atoms. p - Represents a heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, and

[0085] R 4 is a halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 -(CH2)⁻¹, a heterocycle comprising one or two heteroatoms selected from S, N, and O atoms, and a 5 to 6-membered ring p -heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 ...is as defined above, and the heterocycle can be substituted with one or more oxo (=O) substituents, and

[0086] R 5 is hydrogen, or C1-C6-alkyl, and

[0087] R 6 represents a C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle, or heterocyclil-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring comprising 1 to 3 heteroatoms selected from S, N, and O atoms, and R 6 C1-C6-alkylamine, hydroxy-C 1- It can be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

[0088] The compound of Chemical Formula 1 is a compound disclosed in International Patent Publication WO2009-025478 and is a substance known to exhibit preventive, therapeutic, and improving effects on cell necrosis and related diseases.

[0089] The present invention confirmed that by treating ovarian granulosa cells and in vitro fertilization / in vitro production embryos with the compound of Formula 1, cell function is maintained through the inhibition of reactive oxygen species (ROS) accumulation, lipid peroxidation, and ferroptosis induced by aging and oxidative stress, as well as the restoration of mitochondrial membrane potential (ΔΨm), increased ATP production, and promotion of estradiol (E2) synthesis. Furthermore, the compound showed effects of improving the quality of aging eggs, promoting the development of pre-implantation embryos and blastocyst formation, and enhancing the vitrification and re-expansion rate and survival rate of in vitro production embryos after thawing. Accordingly, the present invention identifies a novel use of the compound of Formula 1 and relates to a culture medium composition for in vitro fertilization and / or in vitro culture to enhance the efficiency of in vitro fertilization of eggs or embryos, and a method for in vitro fertilization and / or in vitro culture using the same.

[0090] The compound of Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt thereof. In particular, the pharmaceutically acceptable salt may be an acid addition salt formed by a free acid. Here, the acid addition salt may be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromide, hydroiodide, nitrous acid, phosphoric 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, etc., 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. These types of pharmaceutically acceptable salts may include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chloride, bromides, iodides, fluorides, acetates, propionates, etc.

[0091] The composition of the present invention may include the compound of Formula 1 and its pharmaceutically acceptable salt, as well as all salts, isomers, hydrates, and / or solvates that can be prepared by conventional methods.

[0092] In this specification, "isomer" may refer to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but differs structurally or stereochemically. Such isomers include structural isomers such as tautomers, R or S isomers having an asymmetric carbon center, geometric isomers (trans, cis), and enantiomers. All of these isomers and mixtures thereof are also included within the scope of the present invention.

[0093] In this specification, "hydrate" may mean a compound of the present invention or a salt thereof containing stoichiometric or non-stoichiometric amounts of water bound by non-covalent intermolecular forces. A hydrate of the compound represented by Formula 1 of the present invention may contain stoichiometric or non-stoichiometric amounts of water bound by non-covalent intermolecular forces. The hydrate may contain at least 1 equivalent, preferably 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound represented by Formula 1 of the present invention, its isomers, or pharmaceutically acceptable salts thereof from water or a solvent containing water.

[0094] In this specification, "solvate" may mean a compound of the present invention or a salt thereof comprising stoichiometric or non-stoichiometric amounts of solvent bonded by non-covalent intermolecular forces. Preferred solvents thereof include volatile, non-toxic, and / or solvents suitable for administration to humans.

[0095] In this specification, the term 'alkyl' means an aliphatic hydrocarbon radical. An alkyl may be a “saturated alkyl” that does not contain an alkenyl or alkynyl group, or an “unsaturated alkyl” that contains at least one alkenyl or alkynyl group, and may have 1 to 20 carbon atoms unless otherwise defined.

[0096] The term 'alkoxy' means an alkyl-oxy having 1 to 10 carbon atoms unless otherwise defined.

[0097] The term 'cycloalkyl' means a saturated aliphatic 3- to 10-membered ring unless otherwise defined. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0098] Unless otherwise defined, the term 'heterocycle' refers to a 3- to 10-membered ring, preferably a 4- to 8-membered ring, more preferably a 5- to 6-membered ring, comprising 1 to 3 heteroatoms selected from the group consisting of N, O, and S, which can be fused with a benzo or C3-C8 cycloalkyl, and which is saturated or contains 1 or 2 double bonds. Additionally, the term 'heterocyclil' may be used interchangeably. Examples of heterocycles include, but are not limited to, pyrroline, pyrrolidine, imidazoline, imidazolidine, pyrazolin, pyrazolidine, pyran, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran, etc.

[0099] Other terms and abbreviations used in this specification may be interpreted in the sense commonly understood by those skilled in the art to which the present invention pertains, unless otherwise defined.

[0100] In one embodiment of the present invention, in a compound of Formula 1,

[0101] R 3 -(CH2) represents hydrogen, a halogen, or a phenyl, or the heterocycle is a morpholino or piperazinonyl p - Represents a heterocycle, where p is an integer from 0 to 1, provided that when m is 0, R 3 It can be phenyl.

[0102] In one embodiment of the present invention, in a compound of Formula 1,

[0103] R 4 is a halogen, C1-C3-alkyl, hydroxy-C 1- C3-alkyl, -O-phenyl, -(CH2)p -(CH2) ethyl CO2, heterocycle of which is thiomorpholino, morpholino, piperazinonyl, or pyrrolidineyl p - It is a heterocycle, or proline-N-carbonyl, where p can be an integer from 0 to 1. R 4 In this case, the heterocycle can be substituted with one or more oxo (=O) substituents.

[0104] In one embodiment of the present invention, in a compound of Formula 1,

[0105] R 5 is hydrogen, or C1-C3-alkyl, and

[0106] R 6 represents a C1-C3-alkyl, C3-C6-cycloalkyl, heterocyclic, or heterocyclil-C1-C3-alkyl, where the heterocyclic is tetrahydro-2H-pyran or piperidine, and R 6 In the case of this heterocyclic or heterocyclil-C1-C3-alkyl, C1-C6-alkylamine, hydroxy-C 1- It can be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

[0107] In the present invention, examples of compounds of Formula 1 include compounds 1 to 33 listed in Table 1 below or pharmaceutically acceptable salts thereof.

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] In a preferred embodiment of the present invention, the compound of Formula 1 may be a compound of Formula 2 below.

[0117] [Chemical Formula 2]

[0118]

[0119]

[0120] In the present invention, "in vitro fertilization (IVF)" refers to a series of laboratory processes in which an egg and sperm are fertilized and cultured in a laboratory incubator under conditions similar to the environment of the uterus, as a method distinct from fertilization within the body. The culture medium composition of the present invention is a culture medium composition optimized for such in vitro fertilization, specifically for the in vitro fertilization of aged eggs and sperm.

[0121] Furthermore, "in vitro culture (IVC)" refers to the process of culturing fertilized eggs produced after the aforementioned in vitro fertilization in a medium outside the body to form embryos and blastocysts through the in vitro culture of fertilized eggs. Moreover, it refers to in vitro culture in a broad sense that includes the process of preserving cultured cells or developmental bodies by vitrification, and re-culturing them after thawing. In other words, the in vitro culture may include vitrification and thawing processes.

[0122] Accordingly, the culture medium composition of the present invention is a culture medium composition optimized for in vitro culture in this broad sense, specifically for the entire process of culture, vitrification, thawing, and reculture of fertilized eggs produced after in vitro fertilization of aged oocytes and sperm.

[0123] As used herein, “vitrification” refers to a process in which cells, fertilized eggs, or developmental bodies are rapidly cooled under ultra-low temperature conditions so that water inside and outside the cell is fixed in an amorphous (vitreous) state without freezing in crystalline form. This prevents the formation of ice crystals that damage cell structures and can increase the survival rate of germ cells, such as embryos and eggs, during long-term preservation.

[0124] As used in this specification, “warming or thawing” means a process of restoring the water inside and outside the cell from an amorphous (free) state to a liquid state by gradually warming a cell, fertilized egg, or developmental body preserved in a vitrified state using a medium or solution at an appropriate temperature.

[0125] The thawing process is an important step that minimizes damage caused by rapid osmotic pressure changes or recrystallization of cells, and restores the viability and developmental capacity of preserved germ cells or developmental bodies.

[0126] As used in this specification, the term “developing cell mass” (or “developmental body”) includes all cellular stages formed and developed through the in vitro culture of a fertilized egg; specifically, it is a concept encompassing the fertilized egg, dividing embryo, morula, blastocyst, embryoid body, etc., during in vitro culture.

[0127] Additionally, the term “developmental body” in this specification is interpreted to include a cell or structure that is re-cultured after vitrification and thawing as needed.

[0128] In the present invention, the culture medium composition may include a compound of Formula 1 at a concentration of 0.001 μM to 30 μM, specifically a compound of Formula 1 at a concentration of 0.005 to 25 μM, and more specifically a compound of Formula 1 at a concentration of 0.01 to 15 μM, but is not limited thereto.

[0129] In the present invention, the medium composition may be added to an in vitro culture medium. The in vitro culture medium refers to a substance that enables cell growth and proliferation outside the body by including elements essential for cell growth and proliferation, such as carbon sources, amino acids, various nutrients, serum, growth factors, and inorganic salts. Specifically, any basic medium used for culturing in vitro fertilization or in vitro fertilized embryos of mammals may be used without limitation. Depending on the type of in vitro fertilization or in vitro fertilized embryo, a medium known to a person skilled in the art may be appropriately selected and used. Examples of the carbon source include glucose, sodium pyruvate, calcium lactate, or sodium lactate, but are not limited thereto.

[0130] Examples of the above inorganic salts include sodium chloride (NaCl), potassium chloride (KCl), or sodium bicarbonate (NaHCO3), but are not limited thereto.

[0131] In one embodiment of the present invention, DMEM / F-12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) or a medium was used as the in vitro culture medium. In addition to this medium, commercial media such as Seahorse XF Base Medium may also be used, but are not limited thereto.

[0132] In the present invention, the mammal may be, for example, livestock such as humans, pigs, cattle, goats, sheep, and horses, pets, experimental animals such as mice, rats, and guinea pigs, but is not limited thereto.

[0133] In a specific embodiment of the present invention, the inventors prepared a medium for in vitro fertilization and / or in vitro culture comprising a compound of Formula 1.

[0134] In addition, the inventors confirmed that when embryos fertilized through mating in an aged mouse model were cultured in vitro using the above medium, the rates of embryonic division and blastocyst formation significantly increased.

[0135] In addition, the inventors confirmed that when an in vitro produced embryo is vitrified and then thawed and cultured using a medium for in vitro fertilization and in vitro culture containing the compound of Formula 1, the re-expansion rate and survival rate of the embryo are improved.

[0136] Accordingly, the compound of Formula 1 of the present invention is suitable for in vitro fertilization and / or in vitro culture of aged eggs, as well as for improving the viability of thawed embryos, and can ultimately contribute to pregnancy rates and implantation rates by improving the quality of in vitro cultured embryos.

[0137] In the present invention, the compound of Formula 1 can inhibit the accumulation of reactive oxygen species (ROS), reduce lipid peroxidation, restore mitochondrial membrane potential (ΔΨm), increase ATP production, and promote estradiol (E2) synthesis.

[0138] In the present invention, the compound of Formula 1 can maintain and increase the expression of GPX4, a ferroptosis-related factor, in ovarian granulosa cells, suppress cytotoxicity caused by ROS and lipid peroxidation, and improve cell viability.

[0139] In the present invention, the compound of Formula 1 can promote the development of pre-implantation embryos, improve the blastocyst formation rate, and increase the re-expansion rate and survival rate of in vitro produced embryos after thawing.

[0140] Accordingly, the compound of Formula 1 according to the present invention inhibits the functional decline caused by oxidative stress and ferroptosis of eggs and embryos, thereby restoring the quality of eggs and embryo developmental ability to a normal range and exhibiting the effect of improving in vitro fertilization efficiency.

[0141] According to a specific embodiment of the present invention, the compound of Formula 1 (Compound 1) inhibited cytotoxicity and mitochondrial dysfunction induced by ferroptosis in ovarian granulosa cells. In addition, the compound reduced the accumulation of reactive oxygen species (ROS) and lipid peroxidation, restored the mitochondrial membrane potential (ΔΨm), increased ATP production, and promoted the synthesis of estradiol (E2). Through these actions, the in vitro culture of aged oocytes was improved, and the development rate of pre-implantation embryos and the blastocyst formation rate were significantly increased. Furthermore, the re-expansion rate and survival rate of in vitro produced embryos after thawing were also improved, confirming the effect of reducing damage during the embryo preservation process. Therefore, the compound of Formula 1 of the present invention has been proven to be an effective active ingredient capable of improving the quality and developmental capacity of oocytes and embryos.

[0142] In addition, the present invention may include the use of a compound of Formula 1 or a pharmaceutically acceptable salt thereof for the in vitro fertilization and / or in vitro culture of an egg.

[0143] The compound of Formula 1 or its pharmaceutically acceptable salt and other terms used herein have the meanings previously defined or described in this specification.

[0144] In addition, the present invention

[0145] 1) The stage of preparing mature mammalian eggs;

[0146] 2) a step of adding the mature oocyte and sperm from step 1) to a culture medium composition comprising a compound represented by Chemical Formula 1 or a salt thereof to perform in vitro fertilization; and / or

[0147] 3) A method for in vitro fertilization of a mammalian in vitro fertilized egg is provided, comprising the step of culturing the in vitro fertilized egg modified in step 2) in a medium composition containing a compound represented by Chemical Formula 1 or a salt thereof.

[0148] In the present invention, the mature oocyte of a mammal in step 1) may be prepared by a method comprising, but is not limited to, the following steps:

[0149] a) a step of collecting mammalian eggs; and

[0150] b) A step of in vitro maturation (IVM) of the above oocyte.

[0151] In the present invention, "in vitro maturation (IVM)" refers to a method of inducing oocytes collected prior to in vitro fertilization to undergo meiosis and reach metaphase II. The oocytes refer to cells that have differentiated from oocytes and whose cellular activity has ceased, and the term "egg" in the present invention may be considered to refer to cells substantially identical to the "oocytes."

[0152] In the present invention, “mature oocyte” refers to an oocyte that has reached the MII stage (oocyte at metaphase II) by completing meiosis I and releasing the first polar body, and includes both naturally ovulated oocytes and oocytes that have reached the maturation stage through in vitro maturation. Additionally, it may be obtained from mammals that have maintained reproductive capacity and may also include oocytes that have undergone vitrification and thawing processes.

[0153] In the present invention, “aged oocyte” refers to an oocyte in a stage where normal reproductive capacity is reduced, and includes oocytes in which mitochondrial function decline, increased reactive oxygen species (ROS), cell membrane function decline, and developmental capacity decrease are exhibited due to, for example, physiological aging or in vitro aging.

[0154] The present invention also,

[0155] A step of culturing a fertilized egg of a mammal in a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof; or

[0156] A step of thawing the cultured developmental body in a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof after vitrification;

[0157] It includes an in vitro culture method that includes...

[0158] The compound of Formula 1 or its pharmaceutically acceptable salt and other terms used herein have the meanings previously defined or described in this specification.

[0159] The in vitro fertilization and / or in vitro culture method of the present invention can improve the development of pre-implantation embryos, embryonic division, and blastocyst formation rates by using a compound of Formula 1 or a pharmaceutically acceptable salt thereof that improves the embryonic development ability of aged oocytes, or by using a medium composition for in vitro fertilization and / or in vitro culture that includes a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and can also reduce cell stress occurring during the vitrification / thawing process and increase post-warming survival and developmental ability.

[0160]

[0161] The numerical values ​​described in this specification above should be interpreted to include equivalent values ​​unless otherwise specified.

[0162]

[0163] The present invention will be explained in more detail below through embodiments according to the present invention, but the scope of the present invention is not limited by the embodiments presented below.

[0164]

[0165] Examples

[0166] Reference Example: Abbreviation

[0167] ΔΨm: Mitochondrial membrane potential, 2PN: Two-pronuclear, ANOVA: Analysis of variance, ART: Assisted reproductive technology, BDF1: Hybrid mouse strain (C57BL / 6 × DBA / 2), CCK: Cell Counting Kit, DMEM / F-12: Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12, E2: Estradiol, FACS: Fluorescence-activated cell sorting, FBS: Fetal bovine serum, GC: Granulosa cell, GPX4: Glutathione peroxidase 4 peroxidase 4), GSH: glutathione, hCG: human chorionic gonadotropin, KGN: human granulosa-like tumor cell line, LDH: lactate dehydrogenase, OCR: oxygen consumption rate, PMSG: pregnant mare serum gonadotropin, ROS: reactive oxygen species, RSL3: Ras-selective lethal 3, SEM: standard error of the mean,SRC: spare respiratory capacity

[0168]

[0169] [Experimental Method]

[0170] In this example, as a representative example of the compound of Formula 1, (5-[(1,1-deoxydo-4-thiomorphorinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indole-7-amine) (hereinafter referred to as 'Example Compound 1' or 'Compound 1') was used.

[0171]

[0172] (1) KGN cell preparation

[0173] Immortalized human ovarian granulosa cells (KGN cells) were cultured in DMEM / F-12 supplemented with 10% fetal bovine serum (FBS; 16000-044; Gibco, CA, USA) and 1% penicillin / streptomycin (Penicillin / Streptomycin; 15140122, Gibco) according to a standard cell culture protocol.

[0174] Total 5 × 10 5 Canine cells were inoculated into cell culture dishes (90 × 20 mm; SPL-20100; SPL, Pocheon, Korea) and stored in an incubator containing 5% CO₂ at 37°C. Cells were subcultured every 3-4 days whenever they reached 70-80% confluency. At each subculture, the culture medium was replaced on the 2nd day after inoculation.

[0175]

[0176] (2) Cytotoxicity analysis

[0177] KGN cells were seeded into 96-well plates at a density of 5×10³ cells per well and stored in an incubator for 2 days. Afterward, the cells were treated for 24 hours with Compound 1 at various concentrations (1, 2.5, 3, 5, 7, 10, 25, 30, 50, 70, and 100 μM) added to DMEM / F-12 containing 10% FBS.

[0178] After 24 hours following treatment with Compound 1 and / or RSL3, 10 μL of Cell Counting Kit (CCK)-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours to evaluate cell viability. Absorbance was measured at 450 nm using a microplate reader (Epoch Biotech, CA, USA). Additionally, the efficacy of the compounds was compared using the CCK-8 Assay Kit (CK04; DOJINDO, Kumamoto, Japan).

[0179] The CCK-8 test was performed to determine the cytotoxic concentration range of Compound 1 in KGN cells. Additionally, it was used to evaluate the protective effect of Compound 1 against ferroptosis induced by RSL3. RSL3 is a well-known ferroptosis inducer targeting GPX4 and was used to model ferroptotic apoptosis. Preliminary screening results showed that a concentration of RSL3 at 3 μM induced potent and reproducible cytotoxicity in KGN cells, so this concentration was selected. RSL3 was chosen because it exhibited superior efficacy in inducing ferroptosis-associated apoptosis compared to other ferroptosis inducers (e.g., MMRI62 and temsolololamide) and allowed for the greatest recovery upon co-treatment with Compound 1. All experimental conditions included an untreated control group to serve as a baseline for comparing cell viability and mitochondrial function.

[0180]

[0181] (3) Lactate dehydrogenase (LDH) test

[0182] The Cytotoxicity LDH Assay Kit (CK-12-20, DOJINDO) was used according to the manufacturer's protocol. To briefly explain, KGN cells were seeded into a 96-well plate at a density of 1.5 × 10⁴ cells per well and incubated for 24 hours, after which Compound 1, resveratrol (S1396; Selleckchem, Houston, TX, USA), mitoquinone (89950; Cayman Chemical, Ann Arbor, MI, USA), coenzyme Q10 (11506, Cayman Chemical), ferrostatin-1 (S7243, Selleckchem), and reprostatin-1 (S7699, Selleckchem) were treated for 20 minutes at concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. Subsequently, cells were exposed to 0.3 μM RSL3 (S8155, Selleckchem) for 24 hours. Afterward, the supernatant of the cell culture medium was transferred to a new 96-well plate, the working solution of the test kit was added, and the plates were incubated at room temperature for 15 minutes. Finally, the stop solution was added. LDH release was determined by measuring absorbance at 490 nm using a SpectraMax iD3 multimode microplate reader (Molecular Devices, CA, USA). LDH release was not measured in cells treated only with Compound 1, as Compound 1 was not expected to induce cytotoxicity and LDH release under these conditions was negligibly low in preliminary experiments. An untreated control group was included to evaluate the baseline LDH release of untreated KGN cells.

[0183]

[0184] (4) Feroptosis inhibition test

[0185] To evaluate the potential of Compound 1 to inhibit ferroptosis, KGN cells were treated with RSL3, a well-known GPX4 inhibitor used to induce ferroptotic apoptosis, at various concentrations (1, 2, and 3 μM). Cells were pre-treated with Compound 1 (1, 2, 3, and 5 μM) for 10 minutes, followed by co-treatment with RSL3 and Compound 1. After 24 hours of incubation, cell viability was assessed using the CCK-8 assay to determine the Compound 1 concentration that effectively inhibits ferroptosis. An untreated control group was included to evaluate baseline LDH release and ferroptotic damage. This study design follows a previously reported protocol [Y. It was based on Fan, Y. Zhang, K. Shi, S. Cheng, D. Pei, X. Shu, Identification of a group of bisbenzylisoquinoline (BBIQ) compounds as ferroptosis inhibitors, Cell Death Dis 13(11) (2022) 1000.]

[0186]

[0187] (5) KGN cell culture and Compound 1 / RSL3 treatment

[0188] KGN cells are 5 × 10⁶ per well in a 6-well plate. 5Cells were inoculated at a certain density and stored in an incubator for 24 hours. Cells were treated with Compound 1 (10 μM; Mitoimmune Therapeutics), resveratrol (50 μM, Selleckchem), mitoquinone (1 μM, Cayman Chemical), coenzyme Q10 (10 μM; Sigma-Aldrich, St. Louis, MO, USA), ferrostatin-1 (2 μM, Selleckchem), or reproxtatin-1 (2 μM, Selleckchem) for 20 minutes, followed by exposure to RSL3 (1 μM, MedChemExpress) for 24 hours. An untreated control group was included for comparison with all compound-treated groups.

[0189]

[0190] (6) Flow cytometry analysis (FACS)

[0191] Cytoplasmic ROS, mitochondrial ROS, cytoplasmic lipid peroxidation, mitochondrial lipid peroxidation, and mitochondrial iron were measured using a BD FACSLyric™ instrument (BD Bioscience, Franklin Lakes, NJ, USA) with BODIPY 581 / 591 C11 (D3861, Invitrogen), MitoSOX (M36008, Invitrogen), CM-H2DCFDA (C6827, Invitrogen), MitoPerOx (AB146820; Abcam, Cambridge, UK), and Mito-FerroGreen (M489, DOJINDO), respectively. Briefly, KGN cells were 1×10 5 Dogs were inoculated, fluorescent staining was performed for 20 minutes, washed with phosphate-buffered saline, and resuspended in the buffer for FACS analysis. This was performed according to the fluorescent stain manufacturer's protocol. ROS, lipid peroxidation, and iron levels were presented as relative values ​​compared to the control group.

[0192]

[0193] (7) Analysis of mitochondrial function in KGN cells

[0194] The mitochondrial membrane potential (ΔΨm) and morphology of KGN cells were analyzed using confocal live imaging. To evaluate ΔΨm, KGN cells were cultured in glass-bottom confocal dishes for 2 days, followed by treatment with RSL3 or RSL3 and Compound 1 for 24 hours. Subsequently, cells were stained with 250 nM MitoSpy Orange CMTMRos (424803; BioLegend, San Diego, CA, USA), a dye specifically localized to mitochondria depending on ΔΨm. Nuclei were stained with 5 μg / mL Hoechst®33342 (H1399; Thermo Fisher Life Technologies, Waltham, MA, USA) for 5 minutes at 37°C. Live images were acquired using an LSM880 microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with an Airyscan META system inside a sealed live chamber. The intensity of MitoSpy Orange CMTMRos staining representing ΔΨm was quantified using ImageJ software (National Institutes of Health, USA).

[0195]

[0196] (8) Oxygen consumption rate (OCR) measurement

[0197] OCR was measured using a Seahorse XFp Analyzer (Agilent, CA, USA). A total of 75,000 cells were seeded into Seahorse XFp Cell Culture Miniplates and cultured for 48 hours. Subsequently, the cells were treated with 3 μM Compound 1 for 10 minutes, followed by treatment with 3 μM RSL3 and 3 μM Compound 1 together for 4 hours. Afterward, the culture medium was replaced with Seahorse XF Base Medium, which contains 2 mM L-glutamine, 5.5 mM D-glucose, and 1 mM sodium pyruvate according to the composition of DMEM. OCR was measured for 2 minutes following a 3-minute mixing. Subsequently, the cells were treated with 1 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone / antimicin A. Basal OCR was normalized based on protein concentrations determined by the bicinchoninic acid assay. Three samples of KGN cells were analyzed, and values ​​were normalized using the manufacturer's Multi-File Seahorse XF Cell Mito Stress Test Report Generator to calculate basal respiration rate, ATP production, positive leakage, and binding efficiency. Stacked vertical bar graphs of mitochondrial profile values ​​were generated using GraphPad Prism version 10.4.1 software.

[0198]

[0199] (9) Evaluation of E2 production in KGN cells

[0200] KGN cells are 5 × 10⁶ per well in a 96-well plate. 3Canine cells were inoculated at a density and allowed to adhere for 48 hours prior to treatment. Cells were cultured in DMEM / F-12 supplemented with 10% FBS and 1% penicillin-streptomycin under 5% CO₂ conditions at 37°C. After the acclimatization period, cells were treated for 4 hours with the following: DMEM / F-12 alone (control), 3 μM RSL3, and a combination of 3 μM RSL3 and 3 μM Compound 1. Subsequently, the cell culture supernatant was collected and E2 levels were analyzed immediately using a commercial ELISA kit (Estradiol ELISA, #ESD31-K01) according to the manufacturer's instructions. Optical density values ​​at 450 nm were measured using a SpectraMax Plus 384 microplate reader (Molecular Devices). Results were normalized and expressed in pg / mL.

[0201]

[0202] (10) Collection of two-cell embryos after mouse mating

[0203] Young female (8–9 weeks old) and male (8–14 weeks old) hybrid B6D2F1 (BDF1) mice were purchased from Oriental Bio (Seongnam, Korea). Five female mice were used in each experimental replicate, and the experiment was repeated seven times independently using different animals. Older female mice (60–70 weeks old) and male mice (12–14 weeks old) were used for mating. All procedures related to animal care and breeding were performed in accordance with the guidelines of the Committee on Animal Care and Use (IACUC) at CHA University (IACUC Approval No.: ACUC240081) and were approved.

[0204] In young female mice, follicular growth was promoted by intraperitoneal injection of 8.0 IU of end-pregnancy serum gonadotropin (PMSG; RP1782725000; BioVendor, Brno, Czech Republic). After 48 hours, superovulation was induced by intraperitoneal injection of 8.0 IU of human chorionic gonadotropin (hCG; 668900221; LG Chem, Seoul, Korea). In aged female mice, 12.5 IU each of PMSG and hCG was injected intraperitoneally according to the same schedule. After the second hormone injection, each female was paired with a male in a 1:1 ratio for mating. Two-cell embryos were collected 36 hours after hCG administration.

[0205] To confirm the success of mating, the presence of a vaginal plug was checked the morning after mating. Females with positive vaginal plugs were sacrificed, and their fallopian tubes were resected. Binary (2PN) embryos were recovered from the ampullary region of the fallopian tubes using a micro glass pipette under a stereomicroscope in M2 medium. The recovered binary (2PN) embryos were subsequently used for culture and analysis.

[0206]

[0207] (11) Compound 1 treatment of 2PN embryos

[0208] 2PN embryos were RSL3 (HY-100218A; MCE, CA, USA) and Compound 1 (C 24 H 29The embryos were treated with N3O3S (Patent No. KR20080080519; donated by MitoImmune Therapeutics Inc.). The optimal concentration for treating 2PN embryos was determined experimentally. Embryos were cultured in individual wells at 37°C and 5% CO₂ using a time-lapse system incubator (CNC Biotech, Suwon, Korea). The culture medium was EmbryoMax®KSOM Mouse Embryo Media (1X), Liquid, with 1 / 2 Amino Acids & Phenol Red (MR-121-D, Sigma-Aldrich). Images were acquired every 5 minutes. Embryonic development rate and cleavage time were determined. Embryo quality was evaluated based on cleavage rate and blastocyst formation rate.

[0209]

[0210] (12) Analysis of mitochondrial function in embryos

[0211] To evaluate ΔΨm, 4-cell and 8-cell embryos were stained with 250 nM MitoSpy Orange CMTMRos (424803, BioLegend), which specifically localizes to mitochondria depending on ΔΨm. Nuclei were stained with 5 μg / mL Hoechst®33342 (H1399, Thermo Fisher Life Technologies) for 5 minutes at 37°C. The stained embryos were transferred to a drop of culture medium on a confocal glass-bottom dish. Live images of the embryos were acquired in a sealed live chamber using an LSM880 microscope (Carl Zeiss AG) equipped with the Airyscan META system.

[0212]

[0213] (13) Obtaining a bovine blastocyst through in vitro fertilization

[0214] Bovine ovaries were collected from a local slaughterhouse, 75 μg / mL of penicillin G sodium salt was added to 0.9% saline solution, transported to the laboratory, and stored at 36-38℃.

[0215] Cumulus-oocyte complexes (COCs) were aspirated from follicles with a diameter of 3–6 mm using a 10 mL disposable syringe equipped with an 18-gauge needle. Approximately 15 COCs were placed in a 60 mm dish, paraffin oil was added, and the mixture was cultured for 22 hours under 5% CO2 and 38.5°C conditions. The oocyte maturation medium was prepared by adding 0.2 mM sodium pyruvate, 0.6 mM cysteine, 10 IU / mL PMSG, 10 IU / mL hCG, 10 ng / mL EGF, 25 μg / mL gentamicin, 25 μM β-mercaptoethanol, 10% FBS (Gibco-BRL), and 1 μg / mL estradiol-β to Medium 199 (Gibco-BRL, Grand Island, NY, USA). After in vitro maturation (IVM), 15 oocytes were placed in 50 μL of fertilization medium (Fert-TALP) at a rate of 2 x 10⁶ 6Fertilization was performed using vitrified-thawed sperm at a concentration of cells / mL. 0.2 mM sodium pyruvate, 25 μg / mL gentamicin, and 0.6% bovine serum albumin (BSA) were added to the fertilization medium. Additionally, 10 μg / mL heparin, 80 μM penicillamine, 4 μM hypotaurine, and 2 μM epinephrine (PHE) were added upon sperm injection. After 22 hours of fertilization, the oocytes were vortexed to separate the cumulus membranes and transferred to Charles Rosekrans amino acid (CR1-aa) medium containing 0.4 mM sodium pyruvate, 1 mM-4 glutamine, 0.3 mg / mL glutathione, 25 μg / mL gentamicin, and 0.3% BSA for in vitro culture (IVC). After culturing for 2 days, cleaved embryos were further cultured for 5 days at 38.5°C and 5% CO2 in 50 μL of CR1-aa medium supplemented with 0.4 mM sodium pyruvate, 1 mM glutamine, 0.3 mg / mL glutathione, 25 μg / mL gentamicin, and 10% FBS to obtain blastocysts.

[0216]

[0217] (14) Vitrification-thawing process and measurement of blastocyst re-expansion and survival rate

[0218] The blastocysts were transferred to an equilibrated solution (ES) containing 7.5% ethylene glycol and 7.5% dimethyl sulfoxide, and then placed in phosphate-buffered saline (PBS; Gibco-BRL) containing 20% ​​FBS for 5 minutes. The blastocysts were then transferred to a vitrification solution (VS) containing 20% ​​FBS in PBS containing 15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), and 0.5 M sucrose. After 25 seconds, they were placed in a cryotop and immediately immersed in liquid nitrogen. The entire process from exposure to VS-5 to immersion in liquid nitrogen took less than 1 minute. Vitrified blastocysts were thawed by immersing them in a Cryotop thaw solution (1.0 M sucrose in PBS containing 20% ​​FBS) for 1 minute, followed by thawing in a dilution solution (0.5 M sucrose in PBS containing 20% ​​FBS) for 3 minutes. Then, they were treated with an additional dilution solution (0.25 M sucrose in PBS containing 20% ​​FBS) at room temperature for 5 minutes, followed by incubation in wash medium (PBS containing 20% ​​FBS) for 5 minutes. The viability of vitrified-thawed blastocysts was measured based on the re-expansion rate after recovery in culture medium for 24 hours. Blastocysts were divided into four groups (Non-treated, WARM, VITR, VITR-WARM) based on whether they were treated with Compound 1 (1 μM) during the vitrification and / or thawing steps.

[0219]

[0220] (15) ROS measurement of bovine blastocysts

[0221] ROS levels in blastocysts during the vitrification-thaw process were measured using dichlorodihydrofluorescein diacetate (H2DCF-DA, 2',7'-dichlorodihydrofluorescein diacetate). After the vitrification-thaw process, blastocysts were placed in CR1-aa medium (containing 10% FBS) and washed three times with PBS containing 0.1% polyvinyl alcohol (PVA). The blastocysts were transferred to 1X PBS containing 5 μM H2DCF-DA and treated at 38.5°C for 30 minutes. The intensity of H2DCF-DA was measured using an LSM 800 confocal microscope (Zeiss).

[0222]

[0223] (16) F-actin (Filamentous actin) staining of bovine blastocysts

[0224] Blastodes that had undergone the vitrification-thaw process were washed with PBS containing 0.1% PVA and fixed in PBS containing 3.7% formaldehyde at 38.5°C for 2 hours. Blastodes were infiltrated with 0.5% Triton X-100 at room temperature for 30 minutes, and after washing, incubated with 5 μg / ml Phalloidin-FITC at 38.5°C for 1 hour. After washing, embryos were counterstained with 1.5 μg / ml 4',6-Diamidino-2-Phenylindole (DAPI; Vector Laboratories, Burlingame, USA) for 1 minute and fixed on glass slides. F-actin-stained embryos were imaged using a Zeiss LSM 800 confocal microscope, and the fluorescence intensity of F-actin within the embryos was quantified using ImageJ 1.46r software (NIH).

[0225]

[0226] (17) Statistical analysis

[0227] All experiments were repeated three times. Data were presented as mean ± standard error (SEM). Statistical analysis was performed using SigmaPlot 12.5 and GraphPad Prism version 10.4.1. Two groups were compared using Student's t-test, and two or more groups were compared using Holm-Sidak follow-up test after one-way analysis of variance (ANOVA). Statistical significance was defined as follows: *P<0.05, **P<0.01, ***P<0.001. #P<0.05, ##P<0.01, and ###P<0.001 indicate significance compared to the RSL3 treatment group (vehicle control). All statistical comparisons were performed between relevant experimental groups indicated in each plot (e.g., Control vs. RSL3, RSL3 vs. RSL3 + Compound 1). The exact comparison group was indicated by the horizontal bars in the plots.

[0228]

[0229] Experimental results

[0230] Experimental Example 1: Confirmation of apoptosis protective effect

[0231] KGN cells were incubated for 24 hours in DMEM / F-12 supplemented with 1, 2.5, 3, 5, 7, 10, 25, 30, 50, 70, or 100 μM Compound 1. At concentrations of 30 μM or less, Compound 1 treatment did not reduce cell viability compared to the control group. However, at concentrations exceeding 50 μM, Compound 1 treatment significantly reduced cell viability (Fig. 1a). Next, the restorative effect of Compound 1 on RSL3-treated cells was investigated. RSL3 had a distinct cytotoxic effect, and 1 μM RSL3 treatment reduced cell viability to less than 1%. Compound 1 protected against the reduction in cell viability induced by RSL3 (Fig. 1b). Compound 1 effectively restored cell viability to baseline levels at all tested concentrations (1–5 μM), suggesting therapeutic potential to mitigate ferroptosis-related cytotoxicity. To evaluate the regulatory effects on KGN cells, cell viability and LDH release were measured after Compound 1 treatment and compared with the effects of antioxidants (resveratrol, mitoquinone, coenzyme Q10) and existing antiferroptotic agents such as ferrostatin-1 and reproxtatin-1. Notably, Compound 1 had the lowest EC10 among all tested compounds. 50 It showed a value, suggesting that it has excellent efficacy in inhibiting ferroptosis. A comparative analysis of cell viability and LDH release after exposure to RSL3 (0.3 μM) further confirmed the potent apoptotic protective effect of Compound 1 (Fig. 1c-d).

[0232]

[0233] Experimental Example 2: Confirmation of antioxidant effect under ferroptosis conditions

[0234] Compound 1 effectively alleviated the increase in cytoplasmic and mitochondrial ROS induced by RSL3. Cytoplasmic ROS levels using BODIPY (Fig. 1e) and mitochondrial ROS levels using MitoSox (Fig. 1f) were significantly reduced by Compound 1 compared to resveratrol, mitoquinone, coenzyme Q10, and ferrostatin-1, demonstrating that Compound 1 possesses excellent antioxidant properties.

[0235] In addition, Compound 1 effectively inhibited cytoplasmic lipid peroxidation (Fig. 1g) as measured by CM-H2DCFDA and showed a more pronounced protective effect than other antioxidants. Compound 1 and ferrostatin-1 significantly inhibited mitochondrial lipid peroxidation (Fig. 1h) as evaluated by MitoPerOx and mitochondrial iron accumulation (Fig. 1i) as measured by Mito-FerroGreen, suggesting that they preserve mitochondrial function.

[0236]

[0237] Experimental Example 3: Protection against mitochondrial damage in KGN cells

[0238] Optical microscopy was used to visually evaluate the protective effect of Compound 1 against RSL3-induced mitochondrial damage. This showed that ferroptosis induced by RSL3 resulted in changes in mitochondrial morphology and ΔΨm. Control KGN cells were healthy, elongated, and strongly attached to the culture surface (Fig. 2a A). Treatment with RSL3 resulted in distinct morphological changes, such as cell shrinkage, rounding, and partial detachment, which are consistent with ferroptosis-related damage (Fig. 2a B). These morphological changes were accompanied by mitochondrial damage, namely changes in mitochondrial morphology and a loss of ΔΨm. Among the ferroptosis inducers tested, RSL3 was selected because it possesses the characteristic of consistently inducing such morphological and mitochondrial damage, and because the protective effect of Compound 1 was confirmed in the initial experiments according to the present invention. Co-treatment with Compound 1 (Fig. 2a C) resulted in the recovery of overall cell morphology. However, some cells exhibited a short and irregular morphology similar to the group treated only with RSL3; while this may superficially appear to indicate increased cell density, it is more likely to reflect heterogeneous cell populations at different stages of recovery from ferroptosis-induced damage. Conversely, cells treated only with Compound 1 (D in Fig. 2a) maintained a morphology similar to the control group, and no observable cytotoxicity was observed. MitoSpy™ Orange CMTMRos staining and fluorescence microscopy were performed to evaluate ΔΨm. In control KGN cells, strong red fluorescence indicated that mitochondria were undamaged and in a highly polarized state (E in Fig. 2a). In contrast, RSL3-treated cells showed significantly reduced fluorescence, reflecting a substantial loss of ΔΨm (F in Fig. 2a). However, co-treatment with Compound 1 restored the red fluorescence signal, indicating that ΔΨm was restored (G in Fig. 2a).Cells treated solely with Compound 1 exhibited strong fluorescence similar to that of control cells (H in Fig. 2a). Quantitative analysis of the fluorescence data (Fig. 2b) revealed that RSL3 significantly reduced ΔΨm to approximately 40% of that in control cells (P<0.001). In contrast, co-treatment with Compound 1 restored ΔΨm to approximately 72% of that in control cells, representing a statistically significant improvement compared to cells treated solely with RSL3 (P<0.001). Notably, there was no significant difference in ΔΨm between control cells and cells co-treated with RSL3 and Compound 1, suggesting that Compound 1 effectively restored mitochondrial function to near-normal levels. These results provide further support for the role of Compound 1 in preserving mitochondrial function under ferroptotic stress.

[0239]

[0240] Experimental Example 4: Enhancement of Mitochondrial Oxygen Consumption and ATP Production in KGN Cells

[0241] The effects of Compound 1 on the mitochondrial functional activity of KGN cells were further investigated. Mitochondrial respiration was evaluated by measuring OCR, which is directly associated with oxygen consumption and ATP production in adherent cells. Mitochondrial metabolic status was investigated using an extracellular flux analyzer that simultaneously identifies multiple metabolic profiles, including basal respiration, positive leakage, and extrarespiratory capacity (SRC). OCR was significantly higher in cells treated with both RSL3 and Compound 1 than in cells treated with RSL3 alone (Fig. 3a). Basal respiration was 177.54 pmol / min / cell lower in RSL3-treated cells (9.42 ± 4.12 pmol / min / cell) than in control cells (186.96 ± 40.22 pmol / min / cell, P<0.01) (Fig. 3b). Basal respiration in cells treated with both RSL3 and Compound 1 (235.87 ± 34.65 pmol / min / cell) was 226.45 pmol / min / cell higher than in cells treated with RSL3 only (9.42 ± 4.12 pmol / min / cell) (P<0.01). SRC, an important indicator of mitochondrial reserve capacity and health, was significantly lower in RSL3-treated cells (34.75 ± 7.45%) than in control cells (209.88 ± 45.33%, P<0.01) (Fig. 3c). Co-treatment with Compound 1 significantly improved SRC to 156.11 ± 21.74%, which was higher than in cells treated with RSL3 only (P<0.05). Proton leakage, reflecting mitochondrial membrane integrity and efficiency, was significantly lower in RSL3-treated cells (5.63 ± 1.27 pmol / min / cell) compared to control cells (26.43 ± 7.35 pmol / min / cell, P<0.001), but increased significantly to 41.23 ± 5.92 pmol / min / cell upon co-treatment with Compound 1 (P<0.001 vs. RSL3) (Fig. 3d). ATP production in RSL3-treated cells (4.28 ± 3.Although levels in RSL3-treated cells (15 pmol / min / cell) were significantly lower than in control cells (147.92 ± 37.22 pmol / min / cell, P<0.01), they recovered to 233.06 ± 25.16 pmol / min / cell upon simultaneous treatment with Compound 1, which was significantly higher than in cells treated with RSL3 alone (P <0.001) (Fig. 3e). These results indicate that RSL3 impairs mitochondrial function by reducing mitochondrial respiration, SRC, proton leakage, and ATP production. Compound 1 effectively restores this damage and enhances mitochondrial metabolism and function in KGN cells under ferroptotic stress.

[0242]

[0243] Experimental Example 5: Increase in E2 production in KGN cells

[0244] To evaluate steroid production activity, E2 production was measured using ELISA. RSL3 treatment significantly reduced E2 levels from the control group (104.2 ± 10.1 pg / mL) to 71.3 ± 8.4 pg / mL (P<0.05). Surprisingly, co-treatment with Compound 1 restored E2 levels to 115.6 ± 9.3 pg / mL, which was significantly higher than cells treated with RSL3 alone (P<0.05) and slightly higher than control cells. Additionally, treatment with Compound 1 alone increased E2 levels to 117.2 ± 7.6 pg / mL, indicating that Compound 1 not only reverses the inhibition of steroid production induced by RSL3 but also enhances basal steroid production in KGN cells (Fig. 4).

[0245]

[0246] Experimental Example 6: Protective effect on mouse-derived embryos and improvement of blastocyst development rate

[0247] The protective effect of Compound 1 against RSL3-induced mitochondrial damage was visually evaluated in embryos using an optical microscope. This revealed changes in mitochondrial morphology caused by RSL3-induced ferroptosis. The morphology of RSL3-treated embryos differed significantly from that of control embryos (AB in Fig. 5a). However, embryos treated with both RSL3 and Compound 1 recovered mitochondrial morphology similar to that of control embryos (C in Fig. 5a). Notably, treatment with Compound 1 alone did not induce morphological abnormalities, indicating non-toxicity to embryos derived from young females (D in Fig. 5a).

[0248] Next, live confocal imaging was performed to measure the ΔΨm of the embryos. RSL3 disrupted the ΔΨm and mitochondrial structural integrity of the embryos (EF in Fig. 5a). However, embryos treated with RSL3 and Compound 1 showed significant recovery of ΔΨm and mitochondrial morphology (G in Fig. 5a). Embryos treated with Compound 1 alone also showed strong mitochondrial staining, suggesting that Compound 1 has a beneficial effect on mitochondrial integrity (H in Fig. 5a). Quantitative analysis of the imaging data (Fig. 5b) showed that Compound 1 restored ΔΨm in RSL3-treated embryos to 65.23% of the control level, demonstrating a strong protective effect against mitochondrial damage caused by RSL3.

[0249] In embryos derived from young females, simultaneous treatment with RSL3 and Compound 1 significantly restored the blastocyst development rate, increasing by 10.09% compared to embryos treated with RSL3 alone. Pre-implantation embryos derived from aged females not treated with Compound 1 failed to develop into blastocysts, whereas 10% of aged female mouse embryos treated with Compound 1 developed to the blastocyst stage. There was no significant difference between untreated embryos derived from young females and Compound 1-treated embryos.

[0250] Time-lapse incubation experiments were performed to evaluate the duration of developmental stages, including the transition from the two-cell stage to the blastocyst stage. Untreated aged female embryos failed to transition to the blastocyst stage, whereas aged mouse embryos treated with Compound 1 successfully developed to this stage (Fig. 6a-b). These results highlight the therapeutic potential of Compound 1 to promote development, particularly in aged female mouse embryos, and suggest potential strategies for improving ART outcomes.

[0251]

[0252] Experimental Example 7: Increase in ΔΨm of mouse-derived embryos

[0253] The functional mitochondrial activity of embryos was analyzed by measuring ΔΨm using MitoSpy Orange CMTMRos. In embryos derived from young mice, Compound 1 treatment significantly increased ΔΨm from 57.3 ± 6.2 to 118.4 ± 8.1 (P<0.001). Similarly, in embryos derived from aged mice, ΔΨm increased from 43.7 ± 8.7 to 83.5 ± 4.9 after Compound 1 treatment (P<0.01) (Fig. 7). These results indicate that Compound 1 improves mitochondrial function in both young and aged oocytes, showing a particularly pronounced recovery effect in embryos derived from aged oocytes. This suggests that Compound 1 may be particularly beneficial in reversing age-related mitochondrial dysfunction.

[0254]

[0255] Experimental Example 8: Improvement in the re-expansion rate of bovine blastocysts during the vitrification-thawing process

[0256] After the vitrification-thawing process of bovine blastocysts, the blastocysts were classified according to their developmental stage and analyzed using an optical microscope to measure their size. It was confirmed that the proportion of expanded blastocysts (Ex, Expanded) during the developmental stage after vitrification-thawing increased in the Compound 1 treatment group (WARM) at 41.4 ± 21.5% compared to other groups (Fig. 8a). On the other hand, the proportion of early blastocysts (E, Early) decreased in the Compound 1 treatment group (WARM) at 8.0 ± 8.5% compared to other groups (Fig. 8a).

[0257] Analysis of blastocyst diameters during the developmental stage following the vitrification-thawing process revealed no significant differences in the initial blastocysts across all groups (Fig. 8b). The diameter of the expanded blastocysts was significantly improved in the Compound 1 treated group (WARM) during the thawing process (264.3 ± 22.9 μm) compared to the non-treated group (233.9 ± 8.4 μm) (p<0.01). In contrast, the diameter of the Compound 1 treated group (VITR) during the vitrification stage was 242.7 ± 15.6 μm, and the diameter of the Compound 1 treated group (VITR-WARM) at all vitrification-thawing stages was 238.0 ± 4.3 μm, showing no difference from the non-treated group.

[0258] These results suggest that treatment with Compound 1 during the thawing stage of vitrification-thawing of blastocysts can enhance embryonic development and re-expansion of blastocysts.

[0259]

[0260] Experimental Example 9: Improvement in the survival rate of bovine blastocysts during the vitrification-thawing process

[0261] To confirm the survival rate of bovine blastocysts during the vitrification-thawing process, an optical microscope was used for analysis (Fig. 9). During the vitrification-thawing process, the survival rate of the Compound 1 treated group (WARM) at the thawing stage was 74.2 ± 7.3%, which was significantly higher than that of the non-treated group (57.3 ± 2.3%) (p<0.01). On the other hand, the Compound 1 treated group (VITR) at the vitrification stage was 56.3 ± 6.7%, and the Compound 1 treated group (WARM-VITR) at all stages of vitrification-thawing was 54.3 ± 5.6%, showing no difference from the non-treated group. These results suggest that Compound 1 can improve the survival rate of embryos during the thawing stage of the vitrification-thawing process of bovine blastocysts.

[0262]

[0263] Experimental Example 10: Antioxidant effect during vitrification-thawing process

[0264] To investigate the oxidative stress generated during the vitrification-thaw process of bovine blastocysts, immunofluorescence staining of H2DCF-DA was performed and analyzed using a confocal microscope (Fig. 10). During the vitrification-thaw process, it was confirmed that intracellular ROS was reduced in the Compound 1-treated groups compared to the untreated group through a decrease in the intensity of H2DCF-DA fluorescence. In particular, it was confirmed that intracellular ROS was most significantly reduced in the Compound 1-treated group (WARM) during the thaw stage. These results suggest that Compound 1 can suppress the oxidative stress generated during the thaw stage of the vitrification-thaw process of bovine blastocysts.

[0265]

[0266] Experimental Example 11: Enhancement of mitochondrial function in bovine blastocysts during vitrification-thawing process

[0267] To investigate the effect of Compound 1 treatment on mitochondrial function during the vitrification-thaw process of bovine blastocysts, immunofluorescence staining of JC-1 was performed and analyzed using a confocal microscope (Fig. 11). During the vitrification-thaw process, the mitochondrial membrane potential of the Compound 1-treated group (WARM) at the thaw stage was significantly increased compared to the untreated group (Non-treated) (p<0.01). These results suggest that Compound 1 can enhance mitochondrial function during the thaw stage of the vitrification-thaw process of bovine blastocysts.

[0268]

[0269] Experimental Example 12: Stabilization of F-actin in bovine blastocysts during vitrification-thawing process

[0270] To investigate the effect of Compound 1 treatment on F-actin aggregation during the vitrification-thawing process of bovine blastocysts, immunofluorescence staining using Phalloidin-FITC was performed and analyzed by confocal microscopy (Fig. 12). During the vitrification-thawing process, the thickness of F-actin was significantly reduced compared to the non-treated group (4.1 μm), with the Compound 1-treated group (WARM) at the thawing stage measuring 2.08 μm and the Compound 1-treated group (VITR) at the vitrification stage measuring 3.56 μm (p<0.001 and p<0.01, respectively). In contrast, the Compound 1-treated group (VITR-WARM) showed no difference at all stages of vitrification-thawing, measuring 3.82 μm. These results suggest that Compound 1 can lead to structural stabilization by inhibiting the aggregation of F-actin during the thawing phase of the vitrification-thawing process of bovine blastocysts.

[0271]

[0272] conclusion

[0273] According to the results of the embodiments of the present invention, the compound of Formula 1 (Compound 1) protected KGN cells from cytotoxicity and mitochondrial dysfunction caused by ferroptosis induction. The compound inhibited the accumulation of reactive oxygen species (ROS) and lipid peroxidation, restored the mitochondrial membrane potential (ΔΨm), and promoted ATP production and estradiol (E2) synthesis. Through these actions, the developmental rate and blastocyst formation rate of embryos derived from aged females were significantly improved.

[0274] The ferroptosis inducer RSL3 used in this example is a potent inhibitor of GPX4 and was confirmed to induce ferroptosis in KGN cells most effectively among several candidate substances (MMRI62, temozolomide, etc.). Through this, the protective effect and applicability of Compound 1 were clearly demonstrated.

[0275] Compound 1 was safe as it did not affect cell viability at concentrations below 30 μM, and it showed superior inhibitory effects compared to existing antioxidants (resveratrol, mitoquinone, coenzyme Q10) and antiferroptosis agents (ferostatin-1, reprostatin-1) by inhibiting two key pathways of ROS accumulation and lipid peroxidation.

[0276] In addition, Compound 1 improved the development of embryos derived from aged mice and did not have a negative effect on the development of embryos in young individuals, confirming its safety and selectivity. Rapid progression to the morula was also enhanced, suggesting that the metabolic activity and quality of the embryos were improved.

[0277] As a result of mitochondrial function analysis, Compound 1 maintained impaired mitochondrial respiratory function under ferroptosis conditions and restored ΔΨm, and activated energy metabolism by improving basal respiratory rate and SRC (Spare Respiratory Capacity).

[0278] In addition, Compound 1 restored and promoted E2 synthesis in granule cells, maintaining steroid production function and stabilizing mitochondrial bioenergetic balance.

[0279] In experiments using bovine blastocysts, a large animal model, Compound 1 also improved embryonic viability and quality during the thawing stage of the vitrification and thawing process. As ΔΨ increased and cytoplasmic ROS and aggregated F-actin decreased, the re-expansion rate and developmental capacity of embryos after thawing were improved.

[0280] In summary, Compound 1 has been demonstrated to be a novel culture medium supplement that can enhance embryonic developmental ability and inhibit cell damage occurring during the thawing phase of in vitro culture and vitrification-thawing processes of aged oocytes and embryos by protecting mitochondrial function through potent anti-ferroptosis action and promoting cellular energy metabolism and steroid production.

Claims

1. A medium composition for in vitro fertilization and / or in vitro culture of mammalian oocytes, comprising a compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] In the above formula, n is an integer from 1 to 3, and m is 0 or 1, and A represents phenyl, and R 1 It is hydrogen, or C1-C6-alkyl, and R 2 represents hydrogen, a halogen, or a C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 Represents, where p is an integer from 0 to 3, and R 7 represents hydrogen or a C1-C3-alkyl group, and R 8 C 1- C3-alkylpiperidinyl, or C 1- It represents a C3-alkylsulfonyl group, R 3 -(CH2) represents hydrogen, a halogen, a C1-C6-alkyl or phenyl, or is a 5- to 6-membered ring in which the heterocycle comprises one or two heteroatoms selected from S, N, and O atoms. p - Represents a heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, and R 4 is a halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 -(CH2)⁻¹, a heterocycle comprising one or two heteroatoms selected from S, N, and O atoms, and a 5 to 6-membered ring p -heterocyclic, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 ...is as defined above, and the heterocycle may be substituted with one or more oxo (=O) substituents, and R 5 is hydrogen, or C1-C6-alkyl, and R 6 represents a C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle, or heterocyclil-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring comprising 1 to 3 heteroatoms selected from S, N, and O atoms, and R 6 C1-C6-alkylamine, hydroxy-C 1- It can be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

2. In Paragraph 1, R 3 -(CH2) represents hydrogen, a halogen, or a phenyl, or the heterocycle is a morpholino or piperazinonyl p - Represents a heterocycle, where p is an integer from 0 to 1, provided that when m is 0, R 3 is phenyl, and R 4 is a halogen, C1-C3-alkyl, hydroxy-C 1- C3-alkyl, -O-phenyl, -(CH2) p -(CH2) ethyl CO2, heterocycle of which is thiomorpholino, morpholino, piperazinonyl, or pyrrolidineyl p - It is a heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 1, and the heterocycle may be substituted with one or more oxo (=O) substituents, and R 5 is hydrogen, or C1-C3-alkyl, and R 6 represents a C1-C3-alkyl, C3-C6-cycloalkyl, heterocyclic, or heterocyclil-C1-C3-alkyl, where the heterocyclic is tetrahydro-2H-pyran or piperidine, and R 6 In the case of this heterocyclic or heterocyclil-C1-C3-alkyl, C1-C6-alkylamine, hydroxy-C 1- A culture medium composition that can be substituted with a C6-alkyl or C1-C6-alkylsulfonyl.

3. In Paragraph 1, A culture medium composition in which the compound of Chemical Formula 1 is the compound of Chemical Formula 2 below. [Chemical Formula 2] 4. In Paragraph 1, A culture medium composition comprising the above active ingredient at a concentration of about 0.001 μM to 30 μM.

5. In Paragraph 1, The above-mentioned in vitro culture is a culture medium composition comprising vitrification and thawing processes.

6. In Paragraph 1, The above-mentioned oocyte is an aged oocyte, and the medium composition.

7. In Paragraph 1, A culture medium composition in which the compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof inhibits cytotoxicity and mitochondrial dysfunction induced by ferroptosis of ovarian granulosa cells.

8. In Paragraph 1, A culture medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof that inhibits the accumulation of reactive oxygen species (ROS); restores mitochondrial membrane potential (ΔΨm); increases ATP production; or promotes the synthesis of estradiol (E2).

9. In Paragraph 1, A medium composition that is used during the thawing step of the vitrification-thawing process of a developmental body to improve the survival rate or development rate of the developmental body after thawing. 10.(a) Step of preparing a mature egg of a mammal; (b) performing in vitro fertilization by adding the mature oocytes and sperm to a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof; and / or (c) a method for in vitro fertilization of a mammal comprising the step of culturing the fertilized egg produced in step (b) in a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above formula, n is an integer from 1 to 3, and m is 0 or 1, and A represents phenyl, and R 1 It is hydrogen, or C1-C6-alkyl, and R 2 represents hydrogen, a halogen, or a C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 Represents, where p is an integer from 0 to 3, and R 7 represents hydrogen or a C1-C3-alkyl group, and R 8 C 1- C3-alkylpiperidinyl, or C 1- It represents a C3-alkylsulfonyl group, R 3 -(CH2) represents hydrogen, a halogen, a C1-C6-alkyl or phenyl, or is a 5- to 6-membered ring in which the heterocycle comprises one or two heteroatoms selected from S, N, and O atoms. p - Represents a heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, and R 4 is a halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 -(CH2)⁻¹, a heterocycle comprising one or two heteroatoms selected from S, N, and O atoms, and a 5 to 6-membered ring p -heterocyclic, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 ...is as defined above, and the heterocycle may be substituted with one or more oxo (=O) substituents, and R 5 is hydrogen, or C1-C6-alkyl, and R 6 represents a C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle, or heterocyclil-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring comprising 1 to 3 heteroatoms selected from S, N, and O atoms, and R 6 C1-C6-alkylamine, hydroxy-C 1- It can be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

11. In Paragraph 10, The above-mentioned egg is an aged egg, method.

12. A step of culturing a fertilized egg of a mammal in a medium composition comprising a compound of the following chemical formula 1 or a pharmaceutically acceptable salt thereof; or A step of thawing the cultured developmental body in a medium composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof after vitrification; An in vitro culture method comprising: [Chemical Formula 1] In the above formula, n is an integer from 1 to 3, and m is 0 or 1, and A represents phenyl, and R 1 It is hydrogen, or C1-C6-alkyl, and R 2 represents hydrogen, a halogen, or a C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 Represents, where p is an integer from 0 to 3, and R 7 represents hydrogen or a C1-C3-alkyl group, and R 8 C 1- C3-alkylpiperidinyl, or C 1- It represents a C3-alkylsulfonyl group, R 3 -(CH2) represents hydrogen, a halogen, a C1-C6-alkyl or phenyl, or is a 5- to 6-membered ring in which the heterocycle comprises one or two heteroatoms selected from S, N, and O atoms. p - Represents a heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, and R 4 is a halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 -(CH2)⁻¹, a heterocycle comprising one or two heteroatoms selected from S, N, and O atoms, and a 5 to 6-membered ring p -heterocyclic, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 ...is as defined above, and the heterocycle may be substituted with one or more oxo (=O) substituents, and R 5 is hydrogen, or C1-C6-alkyl, and R 6 represents a C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle, or heterocyclil-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring comprising 1 to 3 heteroatoms selected from S, N, and O atoms, and R 6 C1-C6-alkylamine, hydroxy-C 1- It can be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

13. In Paragraph 12, The above method is a method for promoting the development of a fertilized egg or enhancing blastocyst formation.

14. In Paragraph 12, A method for increasing culture efficiency or viability.

15. Use of the compound of Chemical Formula 1 below or a pharmaceutically acceptable salt thereof for the in vitro fertilization and / or in vitro culture of oocytes. [Chemical Formula 1] In the above formula, n is an integer from 1 to 3, and m is 0 or 1, and A represents phenyl, and R 1 It is hydrogen, or C1-C6-alkyl, and R 2 represents hydrogen, a halogen, or a C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 Represents, where p is an integer from 0 to 3, and R 7 represents hydrogen or a C1-C3-alkyl group, and R 8 C 1- C3-alkylpiperidinyl, or C 1- It represents a C3-alkylsulfonyl group, R 3 -(CH2) represents hydrogen, a halogen, a C1-C6-alkyl or phenyl, or is a 5- to 6-membered ring in which the heterocycle comprises one or two heteroatoms selected from S, N, and O atoms. p - Represents a heterocycle, where p is an integer from 0 to 3, provided that when m is 0, R 3 is phenyl, and R 4 is a halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 -(CH2)⁻¹, a heterocycle comprising one or two heteroatoms selected from S, N, and O atoms, and a 5 to 6-membered ring p -heterocyclic, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 ...is as defined above, and the heterocycle may be substituted with one or more oxo (=O) substituents, and R 5 is hydrogen, or C1-C6-alkyl, and R 6 represents a C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle, or heterocyclil-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring comprising 1 to 3 heteroatoms selected from S, N, and O atoms, and R 6 C1-C6-alkylamine, hydroxy-C 1- It can be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

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