Antioxidative stress agent

Abstract

Provided are previously unknown novel uses of 5-deazaflavin derivatives and related compounds.　Provided are: an antioxidative stress agent containing, as an active ingredient, a 5-deazaflavin compound represented by general formula (I), a pyridodipyrimidine compound represented by general formula (II), a deazaflavinotestosterone compound represented by general formula (III), or a deazaflavinocholesterol compound represented by general formula (IV); and a cosmetic, a quasi-drug, and a pharmaceutical each containing any of the compounds.

Description

Antioxidant stress agents

[0001] This invention relates to antioxidant stress agents and cosmetics, quasi-drugs, pharmaceuticals, etc., that utilize the same.

[0002] Mitochondria, which are present in almost all cells, are not only involved in energy production, but also in Ca 2+ Mitochondria are important organelles involved in regulating concentration and reducing oxidative stress. In recent years, it has become clear that mitochondrial dysfunction is a contributing factor to chronic inflammation and is related to the development of various diseases, such as aging, cancer, dementia, mental illnesses like depression, and diabetes. Research is underway on new therapies aimed at extending human healthy lifespan by reactivating dysfunctional mitochondria.

[0003] β-Nicotinamide mononucleotide (β-NMN) is a coenzyme nicotinamide adenine dinucleotide (NAD + β-NMN is a precursor to NAD+, which is essential for cellular activity and is present in all cells (Figure 1(a)). Typically, β-NMN is synthesized in the body from substances such as vitamin B3. However, its production decreases with age. As a result, NAD+ production decreases, further leading to damage to the cell nucleus and mitochondrial activity. β-NMN is converted from NAD + By generating NAD, neuroprotective and anti-aging effects can be expected. + β-NMN acts as a cofactor for various processes in mitochondria, as a sirtuin known as a longevity gene, and as a poly[ADP-ribose] polymerase involved in DNA repair (Non-Patent Documents 1 and 2). Furthermore, β-NMN has beneficial effects on the central nervous system (CNS), such as enhancing mitochondrial function in the brain (Non-Patent Document 3), preventing β-amyloid oligomer-induced dementia (Non-Patent Document 4), and delaying astrocyte-mediated motor neuron cell death (Non-Patent Document 5). Although β-NMN is found in foods such as green and yellow vegetables and fruits, the amount is often insufficient. Given the difficulty in obtaining sufficient amounts from food, there is a need for safe and easy-to-take pharmaceuticals or supplements that can replace food.

[0004] Extensive research has been conducted on the synthesis and pharmacological effects of compounds containing a flavin skeleton. Derivatives of 5-deazaflavin (pyrimido[4,5-b]quinoline-2,4(3H,10H)-diones) are characterized by a unique chemical structure obtained by substituting the N at position 5 of the flavin skeleton with CH (Figure 1(b)), and NAD + These derivatives exhibit similar redox functions. These derivatives activate intracellular ATP production and sirtuin gene function. A representative 5-deazaflavin, TND1128 (10-ethyl-3-methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-dione), is known to have efficient auto-redox activity (Non-Patent Literature 6, 7). Furthermore, it has been reported that TND1128 promotes ATP production in cultured astrocytes (Non-Patent Literature 8), promotes dendritic elongation and branching in cultured nerve cells (Non-Patent Literature 9), and exhibits protective effects in the mouse brain (Non-Patent Literature 10).

[0005] However, there have been very few studies to date that have investigated the neuroprotective effects of 5-deazaflavin other than TND1128. Aiming for application in the treatment of neuropsychiatric disorders such as Alzheimer's disease and depression, various 5-deazaflavin derivatives and related compounds with enhanced lipophilicity have been synthesized to improve their penetration into the central nervous system (Non-Patent Literature 8). Screening of seven of these derivatives for their neurite extension effects revealed four related compounds that significantly promote dendritic extension and branching in cultured nerve cells: pyridodipyrimidine (TNPP0819) (Non-Patent Literature 11), testosterone-deazaflavin hybrid (TNAD3028) (Non-Patent Literature 12), and cholesterol-deazaflavin hybrids (TNCD2611 and TNCD2618) (Non-Patent Literature 13) (Figure 1(d)) (Non-Patent Literature 8).

[0006] Incidentally, the inventors of this invention have also found that TND1128 and 5-deazaflavin other than TND1128 activate intracellular ATP production, and have filed a patent application (Patent Document 1).

[0007] However, 5-deazaflavin derivatives and related compounds may have various other efficacy properties besides those mentioned above, but these have not been sufficiently investigated.

[0008] Patent No. 6717989

[0009] Brazill JM, Li C, Zhu Y, Zhai RG. NMNAT: It's an NAD+ synthase… It's a chaperone… It's a neuroprotector. Curr Opin Genet Dev. 2017; 44:156-162. doi:10.1016 / j.gde.2017.03.014.Mills KF, Yoshida S, Stein LR, Grozio A, Kubota S, Sasaki Y, Redpath P, Migaud ME, Apte RS, Uchida K, Yoshino J, Imai SI. Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice. Cell Metab. 2016; 24(6):795-806. doi: 10.1016 / j.cmet.2016.09.013.Long AN, Owens K, Schlappal AE, Kristian T, Fishman PS, Schuh RA. Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model. BMC Neurol. 2015; 15:19. doi: 10.1186 / s12883-015-0272-x.Wang X, Hu X, Yang Y, Takata T, Sakurai T. Nicotinamide mononucleotide protects against β-amyloid oligomer-induced cognitive impairment and neuronal death. Brain Res. 2016; 1643:1-9. doi: 10.1016 / j.brainres.2016.04.060.Harlan BA, Pehar M, Sharma DR, Beeson G, Beeson CC, Vargas MR. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1). J Biol Chem. 2016; 291(20):10836-46. doi: 10.1074 / jbc.M115.698779.Nagamatsu T, Hashiguchi H, Yoneda F. A new, general, and convenient synthesis of 5-deazaflavins (5-deazaisoalloxazines) and bis-(5-deazaflavin-10-yl) alkanes. J Chem Soc Perkin Trans 1. 1984; 561-565, doi: 10.1039 / P19840000561.Kuroda K, Nagamatsu T, Yanada R, Yoneda F. Autorecycling system for reduction of carbonyl compounds to alcohols by 1.5-Dihydro-5-deazaflavins. J Chem Soc Perkin Trans 1. 1993; 547-550, doi:10.1039 / P19930000547.Nagamatsu T, Akaike N. Use of Coenzyme Factor for Activation of ATP Production PCT / JP 2019003860 (WO2019 / 151516A1; US20200246340A1).Katsurabayashi S, Oyabu K, Kubota K, et al.The novel mitochondria activator, 10-ethyl-3-methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-dione (TND1128), promotes the development of hippocampal neuronal morphology. Biochem Biopys Res Comm. 2021; 560:146e151. doi:10.1016 / j.bbrc.2021.04.132.Takahashi N, Akaike N, Nagamatsu T, Uchino H, Kudo Y. Effects of TND1128 (a 5-deazaflavin derivative), with self-redox ability, as a mitochondria activator on the mouse brain slice and its comparison with β-NMN. J Pharmacol Sci. 2023; 151(2):93-109. doi:10.1016 / j.jphs.2022.11.005Nagamatsu T, Yamato H, Ono M, Takarada S, Yoneda F. Autorecycling oxidation of alcohols catalysed by pyridodipyrimidines as an NAD(P)+ model. J. Chem. Soc. Perkin Trans, 1, 1992; 2101-2109.Nagamatsu T, Yamada H, Shiromoto K. New synthesis and biologically active molecular design of deazapteridine-steroid hybrid compounds. Heterocycles, 2004; 63(1): 9 - 8.

[0010] Therefore, the object of the present invention is to provide new applications for previously unknown 5-deazaflavin derivatives and related compounds.

[0011] As a result of intensive studies to solve the above problems, the present inventors have found that 5-deazaflavin derivatives and related compounds have a protective effect against oxidative stress, and have completed the present invention.

[0012] That is, the present invention is the following invention. [1] An antioxidant stress agent containing a 5-deazaflavin compound represented by the following general formula (I) as an active ingredient. (In the formula, R １ represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group, and R ２ represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group, and R ３ and R ４ represent a hydrogen atom, a lower alkyl group, a halogen atom, a hydroxyl group, a nitro group, a cyano group, a lower alkoxy group, a phenyl-substituted lower alkoxy, a lower alkylamino group, a phenyl-substituted lower alkylamino group, or a lower alkylsulfonyl group.) [2] An antioxidant stress agent containing a pyridodipyrimidine compound represented by the following general formula (II) as an active ingredient. (In the formula, R １ and R ３ represent a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group, and R ２ represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group.) [3] An antioxidant stress agent containing a deazaflavin testosterone compound represented by the following general formula (III) as an active ingredient. (In the formula, R １R represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group substituted with one of a halogen atom, a lower alkyl group, or a lower alkoxy group. ２ (This represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group.) [4] An antioxidant stress agent comprising a deazaflavinocholesterol compound represented by the following general formula (IV) as an active ingredient. (In the formula, R １ R represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group substituted with one of a halogen atom, a lower alkyl group, or a lower alkoxy group. ２ (This refers to an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group.) A cosmetic product containing the antioxidant stress agent described in any one of [5] [1] to [4]. A quasi-drug or pharmaceutical product containing the antioxidant stress agent described in any one of [6] [1] to [4].

[0013] The antioxidant stress agent of the present invention contains 5-deazaflavin derivatives or related compounds as active ingredients and has a cell-protective effect against oxidative stress. For example, it can prevent and improve skin aging (spots, dullness, wrinkles, sagging, etc.), promote skin metabolism such as skin turnover, improve skin quality such as skin firmness and softness, whiten the skin, and prevent and improve diseases such as glaucoma, cataracts, cancer, diabetes, Alzheimer's disease, cerebral infarction, Parkinson's disease, rheumatoid arthritis, myocardial infarction, heart failure, and arteriosclerosis, which are said to be more likely to occur due to increased oxidative stress.

[0014] This figure shows the compounds used in the test. This figure shows the cell viability in cell proliferation of normal cells treated with TND1128 or β-NMN. This figure shows the cell viability in cell proliferation of normal cells treated with TNPP0819, TNAD3028, TNCD2611, or TNCD2618. This figure shows the cell viability in cell proliferation of oxidative stress-induced cells treated with TND1128 or β-NMN. This figure shows the cell viability in cell proliferation of oxidative stress-induced cells treated with TNPP0819, TNAD3028, TNCD2611, or TNCD2618.

[0015] The antioxidant stress agent of the present invention contains as an active ingredient a 5-deazaflavin compound represented by general formula (I), a pyridodipyrimidine compound represented by general formula (II), a deazaflavinotestosterone compound represented by general formula (III), or a deazaflavinocholesterol compound represented by general formula (IV). These compounds can be used to protect against oxidative stress, and preferably to protect against cell damage caused by hydrogen peroxide.

[0016] The 5-deazaflavin compound represented by general formula (I) is represented by the following formula.

[0017] In the above formula, R １ R represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group. ２ R represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of a lower alkyl group or a lower alkoxy group, and a lower alkyldisubstituted phenyl group. ３ and R ４ This represents a hydrogen atom, a lower alkyl group, a halogen atom, a hydroxyl group, a nitro group, a cyano group, a lower alkoxy group, a phenyl-substituted lower alkoxy group, a lower alkylamino group, a phenyl-substituted lower alkylamino group, or a lower alkylsulfonyl group.

[0018] Among the 5-deazaflavin compounds represented by the above general formula (I), R１ is a methyl group, R ２ is an ethyl group, R ３ is a hydrogen atom, R ４ is preferably a hydrogen atom (TND1128). This TND1128 is managed by Chemteglas Co., Ltd. as TND1128JP and can be obtained from Chemteglas Co., Ltd. under the trade name such as "5-DEAZAFLAVIN REDOX".

[0019] The pyridodipyrimidine compound represented by the general formula (II) is represented by the following formula. <000009C> In the above formula, R １ and R ３ represent a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group, and R ２ represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group.

[0021] Among the pyridodipyrimidine compounds represented by the above general formula (II), those in which R １ is a phenyl group, R ２ is a chlorophenyl group, and R ３ is a phenyl group are preferred (TNPP0819).

[0022] The deazaf lavin testoster one compound represented by the general formula (III) is represented by the following formula.

[0023] In the above formula, R １ represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group substituted with one of a halogen atom, a lower alkyl group or a lower alkoxy group, and R ２ It should be noted that there may be an error in the original text where <000009C> is likely a misrepresentation. It is translated as

[0020] in the English version based on the context. If this is not what you intended, please check and correct the original text.This represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of the lower alkyl or lower alkoxy groups, or a lower alkyldisubstituted phenyl group.

[0024] Among the deazaflavinotestosterone compounds represented by the above general formula (III), R １ is a methyl group, R ２ A methyl group is preferred (TNAD3028).

[0025] The deazaflavinocholesterol compound represented by general formula (IV) is represented by the following formula.

[0026] In the formula, R １ R represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group substituted with one of a halogen atom, a lower alkyl group, or a lower alkoxy group. ２ This represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of the lower alkyl or lower alkoxy groups, or a lower alkyldisubstituted phenyl group.

[0027] Among the deazaflavinocholesterol compounds represented by the above general formula (IV), R １ is a methyl group, R ２ Those with a dimethylphenyl group (TNCD2611) and R １ is a phenyl group, R ２ A dimethylphenyl group (TNCD2618) is preferred.

[0028] The 5-deazaflavin compound represented by general formula (I), the pyridodipyrimidine compound represented by general formula (II), the deazaflavinotestosterone compound represented by general formula (III), or the deazaflavinocholesterol compound represented by general formula (IV) described above can be synthesized based on the literature and manufacturing examples described in Tables 1 to 4 of Japanese Patent No. 6717989 (Patent Document 1). Whether these compounds have been synthesized can be confirmed by performing instrumental analysis such as NMR in accordance with the description in Patent No. 6717989.

[0029] The antioxidant stress agent of the present invention has a cytoprotective effect against oxidative stress, preferably against oxidative stress such as hydrogen peroxide, and can be used in the same way as conventional antioxidant stress agents, except that it contains a 5-deazaflavin compound represented by general formula (I), a pyridodipyrimidine compound represented by general formula (II), a deazaflavinotestosterone compound represented by general formula (III), or a deazaflavinocholesterol compound represented by general formula (IV) as an active ingredient. The cytoprotective effect against oxidative stress referred to here means that the survival rate of cells damaged by hydrogen peroxide is higher, preferably significantly higher, than when the above compounds are not added. The cytoprotective effect of the antioxidant stress agent of the present invention can be obtained not only against hydrogen peroxide as described above, but also against reactive oxygen species such as superoxide anion radicals (commonly known as superoxide ions), hydroxyl radicals, hydrogen peroxide (peroxide ions), singlet oxygen, and other oxidative substances taken in from outside the body.

[0030] In particular, the antioxidant stress agent of the present invention has a cell-protective effect against oxidative stress, and can therefore, for example, prevent and improve skin aging (spots, dullness, wrinkles, sagging, etc.), promote skin metabolism such as skin turnover, improve skin texture such as firmness and softness, and whiten the skin.

[0031] When using the antioxidant stress agent of the present invention for the above-mentioned applications, it may be used alone, but it is preferable to include it in cosmetics. The content of the antioxidant stress agent of the present invention in cosmetics is not particularly limited, but for example, it is 0.0001 to 99.99% by mass.

[0032] Examples of such cosmetics, though not limited to them, include lotions, emulsions, facial washes, cleansers, serums, creams, foundations, eyebrow pencils, mascaras, eyeshadows, eyeliners, lipsticks, lip glosses, blushes, face powders, and nail polishes. Furthermore, the ingredients used in these cosmetics may be those that are known and appropriate.

[0033] Furthermore, because the antioxidant stress agent of the present invention has a cell-protective effect against oxidative stress, it can prevent and improve diseases that are thought to be more susceptible to increased oxidative stress, such as glaucoma, cataracts, cancer, diabetes, Alzheimer's disease, Huntington's disease, cerebral infarction, Parkinson's disease, rheumatoid arthritis, myocardial infarction, heart failure, and arteriosclerosis. In particular, because the antioxidant stress agent of the present invention crosses the blood-brain barrier, it is preferable for preventing and improving brain-related diseases such as Alzheimer's disease, Huntington's disease, cerebral infarction, and Parkinson's disease.

[0034] When using the antioxidant stress agent of the present invention for the above-mentioned applications, it may be used alone, but it is preferable to include it in quasi-drugs or pharmaceuticals. The content of the antioxidant stress agent of the present invention in quasi-drugs and pharmaceuticals is not particularly limited, but for example, it is 0.0001 to 100% by mass. Note that some countries do not have a category equivalent to quasi-drugs. In such cases, it goes without saying that the category appropriate to that country will be applied.

[0035] Such quasi-drugs and pharmaceuticals are not particularly limited, but examples include oral preparations such as tablets, granules, pills, capsules, powders, liquids, suspensions, emulsions, syrups, elixirs, and extracts, and parenteral preparations such as injections, liquids, suppositories, ointments, patches, and lotions. Furthermore, the ingredients used in these quasi-drugs and pharmaceuticals may be those that are known as appropriate. In addition, the method of administration of these quasi-drugs and pharmaceuticals may be set as appropriate according to the purpose and dosage form.

[0036] Furthermore, the antioxidant stress agent of the present invention can be incorporated into, for example, foods and beverages such as supplements, drinks, and health foods; pet supplies such as pet food; various miscellaneous goods such as supporters, toothbrushes, dental floss, shoe insoles, and mouthpieces; reagents for measuring antioxidant stress; industrial raw materials for stabilizing reactions for the production of enzymes and other substances; food raw materials; cosmetic raw materials; pharmaceutical raw materials; and various other raw materials. When the antioxidant stress agent of the present invention is incorporated into foods and beverages such as health foods or pet supplies such as pet food, it can impart an antioxidant stress effect. This effect can be described on the product packaging and instructions, on the box, or in flyers, advertisements, etc.

[0037] The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited in any way to these examples.

[0038] Example 1 Measurement of antioxidant stress of 5-deazaflavin derivatives and related compounds: (1) Method and materials TND1128 and four related compounds (TNPP0819, TNAD3028, TNCD2611 and TNCD2618) (Figure 1) were synthesized as follows. β-NMN was purchased from Sigma-Aldrich (St. Louis, USA).

[0039] TND1128 (10-ethyl-3-methylpyrimido[4,5-b]quinoline-2,4(3H,10H)-dione) (10-ethyl-3-methyl-5-deazaflavin) was synthesized according to the method described in document (4) (F. Yoneda, Y. Sakuma, S. Mizumoto, and R. Ito, J. Chem. Soc., Perkin Trans. 1, 1805-1808 (1976)) in Japanese Patent No. 6717989. The successful synthesis of this compound was confirmed by examining the properties of the crystals and performing NMR measurements.

[0040] TNPP0819 (10-(4-chlorophenyl)-3,7-diphenylpyrido[2,3-d:6,5-d']dipyrimidine-2,4,6,8(1H,3H,7H,10H)-tetraone) was synthesized according to the method described in document (15) of Japanese Patent No. 6717989 (T. Nagamatsu, H. Yamato, M. ONO, S. Takarada, and F. Yoneda, J. Chem. Soc., Perkin Trans. 1, 2101-2109 (1992)). The successful synthesis of this compound was confirmed by examining the properties of the crystals and performing NMR measurements.

[0041] TNAD3028 (5'-deaza-17b-hydroxy-3',8'-dimethylandrost-2,4-dieno[2,3-g]pteridine-2',4'(3'H,8'H)-dione) was synthesized according to the methods described in references (20) and (21) of Japanese Patent No. 6717989 (T. Nagamatsu, H. Yamada, and K. Shiromoto, Heterocycles, 63 9-16 (2004) and Japanese Patent Application Publication No. 2005-104868). The successful synthesis of this compound was confirmed by crystal properties, NMR measurements, etc.

[0042] TNCD2611 (3'-methyl-8'-(3,4-dimethylphenyl)-5'-deazacholest-2,4-dieno[2,3-g]pteridine-2',4'(3'H,8'H)-dione) and TNCD2618 (8'-(3,4-dimethylphenyl)-3'-phenyl-5'-deazacholest-2,4-dieno[2,3-g]pteridine-2',4'(3'H,8'H)-dione) were synthesized according to the method described in document (22) of Japanese Patent No. 6717989 (ARShrestha, T. Shindo, N. Ashida, and T. Nagamatsu, Bioorg. & Med. Che., 16, 8685-8696 (2008)). The successful synthesis of these compounds was confirmed by crystal properties, NMR measurements, etc.

[0043] (2) Cell Culture: Rat pheochromocytoma (PC12) cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). These cells were cultured in RPMI-1640 medium (Nissui, Tokyo, Japan) supplemented with 5% (v / v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 10% (v / v) equine serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 units / ml penicillin, and 100 μg / ml streptomycin (FUJIFILM Wako, Osaka, Japan). These cells were cultured at 37°C in a 5% carbon dioxide atmosphere until confluence was reached. The medium was changed 2-3 times per week.

[0044] (3) Cell viability assay The effects of TND1129 and related compounds on the cell viability of PC12 cells and hydrogen peroxide-damaged PC12 cells were determined using a colorimetric assay with Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer's instructions. Specifically, PC12 cells were seeded in a 96-well plate and 2 × 10⁶ cells were measured. 4Cells were cultured at a density of cells / well for 24 hours. Subsequently, cells were treated with stepwise concentrations of β-NMN, TND1128, and related compounds (0.01–1.0 μM, dissolved in DMSO) or solvent (DMSO) for 24 hours. CCK-8 reagent was then added, and the absorbance of the resulting formazan at 450 nm was measured using an automated multi-well spectrophotometer (Tecan, Mannedorf, Switzerland). Absorbance values ​​were expressed as a percentage relative to the control.

[0045] Furthermore, the effects of β-NMN, TND1129, and related compounds on reducing hydrogen peroxide-induced cytotoxicity were evaluated. PC12 cells were seeded in 96-well plates, and 2 × 10⁶ cells were used. 4 After culturing at a cell / well density for 24 hours, the cells were treated with β-NMN, TND1128, and related compounds for 2 hours prior to hydrogen peroxide exposure. Subsequently, 20 μM hydrogen peroxide or β-NMN, TND1128, and related compounds were added to the culture medium and cultured for 24 hours. To assess cell viability, the hydrogen peroxide-containing medium was discarded, and cell viability was measured using the CCK-8 assay.

[0046] (4) Statistical Analysis All statistical analyses were performed using GraphPad Prism 8 software (La Jolla, CA, USA). Results were expressed as mean ± standard error (SEM). For multiple comparisons between the treatment group and the control group, one-way ANOVA was performed using post-hoc Tukey's multiple comparison test or Dunnett's multiple comparison test. The criterion for statistical significance was p < 0.05.

[0047] (5) Results <Effects of TND1128 and 5-deazaflavin derivatives on cell proliferation> The effects of TND1128, a representative 5-deazaflavin, and β-NMN on the proliferation of normal cells were investigated (Figure 2). PC12 cells treated with TND1128 for 24 hours showed significant activation of cell proliferation at concentrations of 0.01–1.0 μM (F (5, 158)=8.679, p=0.0193 for 0.01 μM, p=0.0002 for 0.03 μM, p=0.0036 for 0.1 μM, p=0.0071 for 0.3 μM, p<0.0001 for 1.0 μM vs. Control; Figure 2a). On the other hand, NAD + β-NMN, which has been widely recognized for its various effects, significantly activated cell proliferation at concentrations of 0.1–1.0 μM (F (5, 158) = 3.883, p = 0.0432 for 0.1 μM, p = 0.0099 for 0.3 μM, p = 0.0197 for 1.0 μM vs. Control; Figure 2b). These results suggest that TND1128 has a strong NAD effect even at low concentrations. + This suggests that it has a similar effect.

[0048] Next, we investigated the effects of 5-deazaflavin compounds other than TND1128 on cell proliferation in normal cells (Figure 3). When PC12 cells were treated with each compound for 24 hours, all compounds significantly increased cell proliferation (TNPP0819: F (5, 158)=18.55, p=0.0018 for 0.01 μM, p<0.0001 for 0.03 μM, p<0.0001 for 0.1 μM, p<0.0001 for 0.3 μM, p<0.0001 for 1.0 μM vs. Control; Figure 3a, TNAD3028: F (5, 158)=7.800, p=0.0053 for 0.03 μM, p=0.0100 for 0.1 μM, p=0.0015 for 0.3 μM, p<0.0001 for 1.0 μM vs. Control; Figure 3b, TNCD2611: F (5, F(5, 158)=6.961, p=0.0041 for 0.01 μM, p=0.0097 for 0.03 μM, p=0.0018 for 0.1 μM, p=0.0008 for 0.3 μM vs. Control; Figure 3c, TNCD2618: F(5, 158)=7.820, p=0.0479 for 0.01 μM, p=0.0251 for 0.03 μM, p<0.0001 for 0.1 μM, p=0.0002 for 0.3 μM vs. Control; Figure 3d). Among these four compounds, TNPP0819 was potent compared to the positive control β-NMN (Figure 3a). These results suggest that 5-deazaflavin compounds have a significant effect on cell proliferation. Furthermore, no significant cytotoxicity was observed in any of the compounds used in this example.

[0049] <Protective Effects of TND1128 and Related Compounds Against Oxidative Stress-Induced Cell Death> To test the effects of 5-deazaflavin compounds against oxidative stress damage, conditions for inducing cell damage were determined (Figure 4). Cell viability significantly decreased when the hydrogen peroxide treatment concentration exceeded 20 μM (F (9, 238) = 156.3, p < 0.0001 for 20-100 μM H2O2 vs. Control; Figure 4a). Since a viability of approximately 50% was observed with treatment at 20-25 μM, the next experiment was conducted at this concentration to induce cell damage.

[0050] Next, the protective effects of TND1128 and four 5-deazaflavin analogs against oxidative stress-induced damage to PC12 cells were tested and compared with the positive control β-NMN. β-NMN was shown to affect cell proliferation at concentrations of 0.1–1.0 mg / ml, while the other compounds, including TND1128, showed effects even at lower concentrations (Figures 2 and 3). Based on these data, the cytoprotective effect of β-NMN could be investigated up to a concentration of 1.0 mg / ml, while the effects of the other compounds could be investigated down to a low dose of 0.3 mg / ml. TND1128 exhibited cytoprotective effects, improving the decrease in cell viability induced by hydrogen peroxide treatment in a bell-type, dose-dependent manner ((F (5, 708)=40.26, p<0.0001 for 20 μM H2O2 (+ 0.05% DMSO) vs. Control, p=0.0313 for 0.01 μM TND1128 + 20 μM H2O2, p=0.0034 for 0.03 μM TND1128 + 20 μM H2O2, p<0.0001 for 0.1 μM TND1128 + 20 μM H2O2, p=0.0029 for 0.3 μM TND1128 + 20 μM H2O2 vs. 20 μM H2O2 (+ 0.05% DMSO)); (Figure 4b). Similarly, β-NMN showed a cytoprotective effect similar to TND1128 (F (4, 613) = 51.91, p < 0.0001 for 20 μM H2O2 (+ 0.05% DMSO) vs. Control, p = 0.0223 for 0.3 μM β-NMN + 20 μM H2O2 vs. 20 μM H2O2 (+ 0.05% DMSO); Figure 4c). However, consistent with the results in Figure 3, TND1128 had a stronger cytoprotective effect than β-NMN.

[0051] Furthermore, tests on four other 5-deazaflavin analogs also showed significant improvements in cell viability in all cases (TNPP0819: F (5, 720)=39.83, p<0.0001 for 20 μM H2O2 (+ 0.05% DMSO) vs. Control, p=0.0018 for 0.1 μM TNPP0819 + 20 μM H2O2, p=0.0073 for 0.3 μM TNPP0819 + 20 μM H2O2 vs. 20 μM H2O2 (+ 0.05% DMSO); Figure 5a, TNAD3028: F (5, 720)=38.86, p<0.0001 for 20 μM H2O2 (+ 0.05% DMSO) vs. Control, p<0.0001 for 0.1 μM TNAD3028 + 20 μM H2O2, p=0.0177 for 0.3 μM TNAD3028 + 20 μM H2O2 vs. 20 μM H2O2 (+ 0.05% DMSO); Figure 5b, TNCD2611: F (5, 720)=45.25, p<0.0001 for 20 μM H2O2 (+ 0.05% DMSO) vs. Control, p=0.0095 for 0.1 μM TNCD2611 + 20 μM H2O2; Figure 5c, TNCD2618: F (5, 720)=42.58, p<0.0001 for 20 μM H2O2 (+ 0.05% DMSO) vs. Control, p=0.0009 for 0.1 μM TNCD2618 + 20 μM H2O2; Figure 5d).

[0052] Based on these results, it was found that TND1128 and other 5-deazaflavin analogs (TNPP0819, TNAD3028, TNCD2611, TNCD2618) have a protective effect against hydrogen peroxide-induced cell damage. Therefore, it was determined that these could be used as active ingredients to create antioxidant stress agents.

[0053] Furthermore, since TND1128 and other 5-deazaflavin analogs share a common nicotinamide skeleton as shown in Figure 1(b), it was found that 5-deazaflavin compounds represented by general formula (I), pyridodipyrimidine compounds represented by general formula (II), deazaflavinotestosterone compounds represented by general formula (III), and deazaflavinocholesterol compounds represented by general formula (IV), all possessing these skeletons, also have an effect of protecting against hydrogen peroxide-induced cell damage. It was found that these can be used as active ingredients to create antioxidant stress agents.

[0054] The antioxidant stress agent of the present invention can be used in cosmetics, quasi-drugs, pharmaceuticals, food and beverages, pet supplies, general merchandise, reagents, raw materials, etc.

Claims

1. An antioxidant stress agent comprising a 5-deazaflavin compound represented by the following general formula (I) as the active ingredient. (In the formula, R １ R represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group. ２ R represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of a lower alkyl group or a lower alkoxy group, and a lower alkyldisubstituted phenyl group. ３ and R ４ (This represents a hydrogen atom, a lower alkyl group, a halogen atom, a hydroxyl group, a nitro group, a cyano group, a lower alkoxy group, a phenyl-substituted lower alkoxy group, a lower alkylamino group, a phenyl-substituted lower alkylamino group, or a lower alkylsulfonyl group.) 2. An antioxidant stress agent containing a pyridodipyrimidine compound represented by the following general formula (II) as the active ingredient. (In the formula, R １ and R ３ R represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group, a halogen atom, or a phenyl group substituted with one of a lower alkyl group or a lower alkoxy group, and a lower alkyldisubstituted phenyl group. ２ This represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of the following: a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group.

3. An antioxidant stress agent containing a deazafravin testosterone compound represented by the following general formula (III) as an active ingredient. (In the formula, R １ represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group, or a lower alkoxy group, and R ２ represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a phenyl group substituted with one of a halogen atom, a lower alkyl group, or a lower alkoxy group, or a lower alkyldisubstituted phenyl group.) 4. An antioxidant stress agent containing a deazaflavinocholesterol compound represented by the following general formula (IV) as the active ingredient. (In the formula, R １ R represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group substituted with one of a halogen atom, a lower alkyl group, or a lower alkoxy group. ２ This represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a halogen atom, or a phenyl group substituted with one of the following: a lower alkyl group or a lower alkoxy group, or a lower alkyldisubstituted phenyl group.

5. A cosmetic product containing an antioxidant stress agent according to any one of claims 1 to 4.

6. A quasi-drug or pharmaceutical product containing the antioxidant stress agent described in any one of claims 1 to 4.

Images