antioxidant compounds

Abstract

The present invention addresses the problem of providing a compound having antioxidant ability. The problem is solved by a compound represented by general formula (1) or a solvate thereof. [In the formula, R1 and R2 are the same or different and represent an alkyl group having 1-4 carbon atoms.]

Description

[Technical Field]

[0001] The present invention relates to antioxidant compounds and the like. [Background technology]

[0002] It is known that radicals generated by various factors in the body oxidize lipids, proteins, etc., causing various disorders (oxidative stress disorders), such as corneal disorders, dry eye, and ischemic disorders.

[0003] On the other hand, although many substances with antioxidant properties, such as glutathione and vitamins, have been reported, the current situation is that not all substances with antioxidant properties can necessarily exert an improving effect on oxidative stress-related disorders. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2022-528727 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a compound having antioxidant activity. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted extensive research and have found that the above problems can be solved by a compound represented by the general formula (1) described below or a solvate thereof. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Section 1. General formula (1):

[0008] [ka] [In the formula: R 1 and R 2 are the same or different and represent an alkyl group having 1 to 4 carbon atoms. A compound represented by the formula (I) or a solvate thereof.

[0009] Section 2.R 1 and R 2 and R are both methyl groups, or a solvate thereof.

[0010] Item 3. An antioxidant comprising the compound according to Item 1 or 2 or a solvate thereof.

[0011] Item 3A. Use of the compound according to item 1 or 2 or a solvate thereof as an antioxidant.

[0012] Item 3B. Use of the compound or solvate thereof according to item 1 or 2 for the production of an antioxidant.

[0013] Item 3C. A method for suppressing oxidative stress, comprising applying the compound according to item 1 or 2 or a solvate thereof to a subject.

[0014] Item 3D. The compound or solvate thereof according to item 1 or 2 for use as an antioxidant.

[0015] Item 4. The antioxidant according to Item 3, for use in radical scavenging.

[0016] Item 5. The antioxidant according to Item 3 for use in preventing or ameliorating oxidative stress disorders.

[0017] Item 6. An agent for preventing or ameliorating oxidative stress-induced disorders, comprising the compound according to Item 1 or 2 or a solvate thereof.

[0018] Item 6A. Use of the compound according to item 1 or 2 or a solvate thereof as an agent for preventing or ameliorating oxidative stress-induced disorders.

[0019] Item 6B. Use of the compound or solvate thereof according to item 1 or 2 for producing an agent for preventing or ameliorating oxidative stress-induced disorders.

[0020] Item 6C: A method for preventing or ameliorating an oxidative stress disorder, which comprises applying the compound according to item 1 or 2 or a solvate thereof to a subject.

[0021] Item 6D. The compound or solvate thereof according to Item 1 or 2 for use as an agent for preventing or ameliorating oxidative stress-induced disorders.

[0022] Item 7. The prophylactic or ameliorating agent according to Item 6, wherein the oxidative stress-related disorder is at least one selected from the group consisting of corneal disorder, dry eye, and ischemic disorder.

[0023] Item 8. An external preparation containing the compound or solvate thereof according to Item 1 or 2.

[0024] Item 9. The topical preparation according to Item 8, which is an eye drop. [Effects of the Invention]

[0025] According to the present invention, a compound having antioxidant activity can be provided. [Brief explanation of the drawings]

[0026] [Figure 1] NMR (in CDCl3) data for 3'5'-dimethoxyacetophenone (2) is shown below. [Figure 2] NMR (in CDCl3) data for 3'5'-dimethoxyacetophenone (2) is shown below. [Figure 3] NMR (in CDCl3) data for 3'5'-dimethoxyacetophenone (2) is shown below. [Figure 4] NMR (in CDCl3) data of compound 5 is shown below. [Figure 5] NMR (in CDCl3) data of compound 5 is shown below. [Figure 6]NMR (in CDCl3) data of compound 5 is shown below. [Figure 7] NMR (in CDCl3) data of compound X is shown below. [Figure 8] NMR (in CDCl3) data of compound X is shown below. [Figure 9] NMR (in CDCl3) data of compound X is shown below. [Figure 10] FD-MS data of compound X is shown below. [Figure 11] 1 shows the results of measuring the reaction rate constant of the hydroxyl radical scavenging action. [Figure 12] The results of measuring the reaction rate constant of the tyrosyl radical scavenging effect are shown below. Kedv indicates the reaction rate constant of edaravone. [Figure 13] 1 shows the results of measuring the reaction rate constant of the nitric oxide radical scavenging action. [Figure 14] The results of measuring the reaction rate constant of the DPPH radical scavenging effect are shown below. Kedv indicates the reaction rate constant of edaravone. [Figure 15] 1 shows the measurement results of the reaction rate constant of the singlet oxygen radical scavenging action. [Figure 16] The corneal fluorescein scores are shown. "Non-smoke" indicates the group (n=6) that was not exposed to cigarette smoke or given eye drops; "PBS (smoke)" indicates the group (n=6) that was exposed to cigarette smoke and given PBS eye drops; and "0.45 mM Compound X (smoke)" indicates the group (n=6) that was exposed to cigarette smoke and given an aqueous solution of Compound X eye drops. Each column shows the average value. [Figure 17] Modified Tarlov's score (MTS) is shown. Saline indicates the saline administration group (n = 8), Edaravone indicates the edaravone administration group (n = 8), and Compound X indicates the Compound X administration group (n = 5). Each plot shows the data for each sample, and the graph line indicates the average value. [Figure 18]This shows the results of 1H-NMR measurements from a stability test. The left side of the graph shows the number of days since dissolution in heavy water. The percentage shown above the graph indicates the ratio of the area under the curve of the peak on Day 5 to the area under the curve of the peak on Day 0. [Figure 19] The graph shows the measurement results of the reaction rate constant of hydroxyl radicals. In the figure, Substance X means compound X. [Figure 20] The graph shows the measurement results of the reaction rate constant of superoxide anion. In the figure, Substance X means compound X. [Figure 21] The graph shows the measurement results of the reaction rate constant of the tert-butoxyl radical. In the figure, Substance X means Compound X. [Figure 22] The graph shows the measurement results of the reaction rate constant of the tert-butylperoxyl radical. In the graph, Substance X means Compound X. [Figure 23] The results of measuring the reaction rate constant of ascorbyl radicals are shown. In the figure, Substance X means compound X. [Figure 24] The graph shows the measurement results of the reaction rate constant of singlet oxygen. In the figure, Substance X means compound X. [Figure 25] The graph shows the measurement results of the reaction rate constant of nitric oxide radicals. In the figure, Substance X means Compound X. [Figure 26] The graph shows the measurement results of the reaction rate constant of DPPH. In the figure, Substance X means Compound X. [Figure 27] The graph shows the measurement results of the reaction rate constant of tyrosyl radicals. In the figure, Substance X means compound X. [Figure 28] 1 shows a comparison of the reaction rate constants of compound X and edaravone. [Figure 29] The results of quantifying malondialdehyde, an oxidation indicator, using the thiobarbituric acid reactive substance method are shown. Edaravone refers to the edaravone administration group, and Substance X refers to the compound X administration group. In the figure, Substance X means compound X. [Figure 30] The results of measuring cell proliferation are shown. The horizontal axis indicates the time elapsed since the start of culture. The legend indicates the final concentration of compound X in the medium. DETAILED DESCRIPTION OF THE INVENTION

[0027] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0028] 1.Compound In one aspect, the present invention provides a compound represented by general formula (1):

[0029] [ka] [In the formula: R 1 and R 2 are the same or different and represent an alkyl group having 1 to 4 carbon atoms. or a solvate thereof (sometimes referred to as "the compound of the present invention" in this specification). This will be explained below.

[0030] R 1 or R 2 The alkyl group represented by the formula (I) includes any of linear, branched, and cyclic alkyl groups. The alkyl group is preferably linear or branched, and linear is particularly preferred. The alkyl group (when linear or branched) has 1 to 4 carbon atoms. The number of carbon atoms is preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, and a sec-butyl group.

[0031] R 1 and R 2 Preferably, the alkyl groups represented by R 1 and R 2The alkyl groups represented by the following formula (I) preferably have 1 to 3 carbon atoms, more preferably 1 to 2 carbon atoms, and particularly preferably 1 carbon atom (methyl group).

[0032] In a particularly preferred embodiment of the present invention, the compound represented by general formula (1) has formula (1A):

[0033] [ka] It is a compound represented by the formula:

[0034] The compound represented by the general formula (1) includes stereoisomers and optical isomers, and these are not particularly limited.

[0035] Examples of solvents that constitute solvates include water and pharmaceutically acceptable organic solvents (eg, ethanol, glycerol, acetic acid, etc.).

[0036] The compounds of the present invention can be synthesized, for example, by a method including the following steps.

[0037] [ka] [In the formula: R 1 and R 2 is the same as above.]

[0038] Compound A and compound B may be commercially available or may be synthesized according to known methods.

[0039] In view of yield, ease of synthesis, etc., the amount of compound B used is usually preferably 0.5 to 2 mol, more preferably 0.7 to 1.5 mol, per 1 mol of compound A.

[0040] Step I is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, but examples thereof include tetrahydrofuran, etc. The solvent may be used alone or in combination.

[0041] Step I is preferably carried out in the presence of titanocene dichloride and zinc powder. From the viewpoints of yield, ease of synthesis, etc., the amount of titanocene dichloride used is usually preferably 2 to 5 moles per mole of Compound A. From the viewpoints of yield, ease of synthesis, etc., the amount of zinc powder used is usually preferably 5 to 15 moles per mole of Compound A.

[0042] The reaction in step I can be carried out under heating, at room temperature, or under cooling, and can usually be carried out at a temperature of 0 to 120° C. The reaction temperature is particularly preferably 50 to 70° C. The reaction time is not particularly limited, and can usually be 30 minutes to 60 hours.

[0043] Step II is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, but examples thereof include dichloromethane. The solvent may be used alone or in combination.

[0044] Step II is preferably carried out in the presence of boron tribromide. The amount of boron tribromide used is usually preferably 5 to 15 moles per mole of Compound C, from the viewpoints of yield, ease of synthesis, etc.

[0045] The reaction in step II can be carried out under heating, at room temperature, or under cooling, and is usually carried out at a temperature of -50 to 50° C. The reaction temperature is particularly preferably -35 to 25° C. The reaction time is not particularly limited, and is usually 30 minutes to 60 hours.

[0046] The progress of the reactions in Step I and Step II can be monitored by a conventional method such as chromatography. After the reaction is completed, the solvent is removed by distillation, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. The structure of the product can also be confirmed by elemental analysis, MS (ESI-MS) analysis, IR analysis, etc. 1 H-NMR, 13 It can be identified by C-NMR or the like.

[0047] The solvate can be obtained according to or in accordance with a known method.

[0048] 2.Applications The compounds of the present invention have radical scavenging activity and are therefore useful as active ingredients in antioxidants. The compounds of the present invention have an inhibitory effect on oxidative stress-induced disorders and are therefore useful as active ingredients in agents for preventing or ameliorating oxidative stress disorders. The compounds of the present invention have a certain level of water solubility and a certain level of lipid solubility, which allows the antioxidant activity of the compounds of the present invention to be more effectively expressed. Furthermore, the compounds of the present invention are easy to formulate, and the compounds of the present invention can be suitably used, for example, as topical agents (particularly, aqueous solutions), especially as eye drops. In this specification, the above-mentioned various agents may be collectively referred to as "agents of the present invention."

[0049] The compounds of the present invention have the advantages of being highly stable and being easily synthesized, making them suitable for the above-mentioned applications.

[0050] 3',4'-Dimethoxyacetophenone, the raw material for isomers of the present compound, is unstable because its OH groups are adjacent, and 2',4'-dimethoxyacetophenone, the raw material for isomers of the present compound, has one OH group at the o-position, making it difficult to synthesize isomers of the present compound using these.

[0051] As used herein, "amelioration" refers to improvement or alleviation of a symptom or condition, prevention or delay of worsening of a symptom or condition, or reversal, prevention or delay of progression of a symptom or condition.

[0052] The target radicals for the radical scavenging action are not particularly limited, but include, for example, nitric oxide radical, hydroxyl radical, tyrosyl radical, peroxide radical, alkyloxy radical, alkylperoxy radical, methyl radical, etc., and preferably include nitric oxide radical, hydroxyl radical, tyrosyl radical, etc. In particular, from the viewpoint of exhibiting a more powerful radical scavenging action than edaravone, an existing antioxidant, it is preferable that the target radical includes nitric oxide radical. The target radicals may be one type alone or two or more types in combination.

[0053] The oxidative stress disorder is not particularly limited as long as it is a disorder caused by radicals or oxidative stress in a living body. Examples of the oxidative stress disorder include corneal disorder, dry eye, ischemic disorder (e.g., spinal cord ischemia, cerebral infarction, myocardial infarction, etc.), age spots, wrinkles, gray hair, cataract, diabetes, ulcer, stomatitis, periodontal disease, arteriosclerosis, hypertension, sensitivity to cold, numbness, myocardial infarction, cerebral hemorrhage, chronic kidney disease (CKD), gastric ulcer, intestinal ulcer, inflammatory bowel disease (ulcerative colitis, Crohn's disease, etc.), atopic dermatitis, burns, frostbite, rough skin, collagen disease, Alzheimer's dementia, Parkinson's disease, Behçet's disease, Kawasaki disease, rheumatoid arthritis, Raynaud's disease, hay fever, rhinitis, age-related macular degeneration, fatty liver (particularly non-alcoholic steatohepatitis: NASH), chronic hepatitis (viral hepatitis, etc.), chronic obstructive pulmonary disease, and cancer. Of these, corneal disorders, dry eyes, and ischemic disorders are preferred.

[0054] The compound of the present invention and the agent of the present invention can be used in the form of a composition as needed, such as a medicine, a reagent, a food additive, or a food composition (including health foods, health-promoting agents, and nutritional supplements (supplements, etc.)).

[0055] The agent of the present invention is not particularly limited as long as it contains the compound of the present invention, and may further contain other components as necessary.The other components are not particularly limited as long as they are pharmaceutically acceptable components.The other components include not only components with pharmacological effects but also additives.The additives include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0056] The mode of use of the compound of the present invention and the agent of the present invention is not particularly limited. The compound of the present invention and the agent of the present invention can be used, for example, in vitro (for example, by adding to the medium of cultured cells) or in vivo (for example, by administering to / ingesting from an animal).

[0057] The compounds and agents of the present invention are not particularly limited to mammals, and examples thereof include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Cells include animal cells. The types of cells are also not particularly limited, and examples thereof include blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, ova), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0058] The agent of the present invention may be in any dosage form, for example, oral preparations (tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquid preparations (including drinks, suspensions, syrups), jellies), injectable preparations (for example, drip injections (for example, intravenous drip preparations, etc.), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (for example, ointments, poultices, lotions, suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, etc.).

[0059] The route of administration of the agent of the present invention is not particularly limited as long as the desired effect can be obtained, and examples thereof include enteral administration such as oral administration, tube feeding, and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

[0060] When the agent of the present invention is used as a food composition, examples thereof include liquid, gel or solid foods, such as beverages such as juice, soft drinks, tea, soup, and soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, powdered milk for infants, cake mix, powdered or liquid dairy products, bread, and cookies.

[0061] The content of the active ingredient in the agent of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0062] The dosage of the compound or agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is generally 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in the case of oral administration, and 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in the case of parenteral administration. The dosage can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc. [Example]

[0063] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0064] Example 1. Synthesis of Compound X Compound X was synthesized according to the following scheme.

[0065] [ka]

[0066] <Synthesis of Example 1-1. 3',5'-Dimethoxyacetophenone (2)> A 1 L three-neck flask equipped with a stirrer and thermometer was charged with 3',5'-dihydroxyacetophenone (1) (24.15 g, 158.72 mmol, 1.0 eq) and distilled water (120.75 mL). While stirring at room temperature, 10 wt% aqueous NaOH (317.44 mL, 793.60 mmol, 3.0 eq) was added and the mixture was stirred at room temperature until the exotherm subsided (24.3 °C → 27.0 °C, black liquid). Dimethyl sulfate (90.16 g, 714.24 mmol, 2.5 eq) was then added (25.4 °C → 42.2 °C, brown liquid). After 1 h (1 h after the start of the reaction), LC analysis confirmed the disappearance of the starting materials, and peaks believed to represent the reaction intermediate (RT = 3.9 min) and the target product (RT = 6.9 min) were observed. Stirring was continued. After 2 hours (2 hours after the start of the reaction), LC analysis confirmed that the reaction had stopped. Therefore, 10 wt% NaOH aqueous solution (31.74 mL, 79.36 mmol, 0.5 eq) and dimethyl sulfate (10.02 g, 79.36 mmol, 0.5 eq) were added, and stirring was continued. After 1 hour (4 hours after the start of the reaction), LC analysis confirmed that 18.5% of the reaction intermediate (RT = 3.9 min) remained. The reaction temperature was changed to 40 °C, and stirring was continued. After 1 hour (5.5 hours after the start of the reaction), LC analysis showed that the amount of the reaction intermediate (RT = 3.9 min) remained almost unchanged. Therefore, dimethyl sulfate (20.04 g, 158.72 mmol, 1.0 eq) was added, and stirring was continued. After 1 hour (6.5 hours after the start of the reaction), LC analysis confirmed that the concentration of the reaction intermediate (RT = 3.9 min) had decreased to 5.82%, so the reaction was quenched by pouring into ice water (1500 mL). The mixture was then stirred overnight in an ice bath, but no solid precipitated. Therefore, ethyl acetate (500 mL) was added to the reaction solution, and the aqueous layer was separated. The remaining organic layer was washed with distilled water (300 mL × 2). After drying over anhydrous magnesium sulfate, the desiccant was filtered off, and the filtrate was concentrated. 27.7 g of crude 3',5'-dimethoxyacetophenone (2) was obtained as a brown liquid.The crude product was purified by silica gel column chromatography (using 110.8 g of 60N, eluent: toluene). The fraction was concentrated and then vacuum dried at 40 °C for 1 hour to obtain 25.34 g of 3',5'-dimethoxyacetophenone (2) as a yellow oil (yield 88.6%, LC purity 97.8%). NMR (in CDCl3) data are shown in Figures 1 to 3.

[0067] Example 1-2: Synthesis of Compound 5 A 1000 mL three-neck flask equipped with a stirrer and thermometer was charged with titanocene dichloride (47.47 g, 190.17 mmol, 2.8 eq), zinc powder (39.30 g, 601.10 mmol, 8.9 eq), and THF (262.3 mL). After purging with nitrogen, the mixture was stirred at 70 °C (reddish-brown solution). Under a nitrogen atmosphere, a mixture of 2 (12.18 g, 67.61 mmol, 1.0 eq), 4-methoxyacetophenone (3) (10.15 g, 67.61 mmol, 1.0 eq), and THF (112.4 mL) was added over 45 min using a dropping funnel (dark green solution). Immediately after addition, LC analysis confirmed the disappearance of the two starting peaks and the appearance of a new peak around 8–9 min. After 1 hour of reaction, the LC peaks showed no change, so the reaction was stopped. The reaction solution was dissolved in ethyl acetate (1900 mL) and washed with 1N HCl (450 mL × 2) and distilled water (500 mL × 4). After drying over anhydrous magnesium sulfate, the desiccant was filtered off, and the filtrate was concentrated. 29.46 g of crude compound 5 was obtained as a yellow liquid. The crude product was purified by silica gel column chromatography (60N 1030.0 g, eluent: hexane:ethyl acetate = 3:1). The fraction was concentrated and dried in vacuo at 40 °C for 1 hour, yielding 8.12 g of compound 5 as a yellow viscous liquid (yield 36.1%, LC purity 97.3%). NMR (in CDCl3) data are shown in Figures 4–6.

[0068] Example 1-3: Synthesis of Compound X A 1000 mL three-neck flask equipped with a stirrer and thermometer was charged with compound 5 (8.12 g, 24.43 mmol, 1.0 eq) and dichloromethane (400 mL). While stirring under a nitrogen atmosphere at -30 °C, a 17% solution of boron tribromide in dichloromethane (196.0 mL, 196.0 mmol, 8.0 eq) was added dropwise (clear liquid → reddish-brown liquid). After the addition, the mixture was stirred under a nitrogen atmosphere at -30 °C for 30 minutes, then at room temperature for an additional 2 hours. Upon returning to room temperature (0.5 hours into the reaction), LC analysis confirmed the disappearance of the peak for compound 5 (RT = 8.0 min) and the appearance of several new peaks around RT = 2.5–4.5 min. LC analysis performed 1 hour after the completion of the dropwise addition (1 hour of reaction) confirmed that the peaks at RT = 3.2 min and 3.6 min were becoming larger. LC analysis performed after another 1 hour (2 hours of reaction) revealed no change in the peak area ratio, so the reaction was stopped by slowly pouring the reaction mixture into ice water (600 mL). Ethyl acetate (1600 mL) was added to the reaction solution, and the aqueous layer was separated. The remaining organic layer was washed with distilled water (500 mL x 2). After drying over anhydrous magnesium sulfate, the desiccant was filtered off, and the filtrate was concentrated. 7.26 g of crude compound X was obtained as a brown liquid. The crude product was purified by silica gel column chromatography (60N 350.0 g used, eluent: hexane:ethyl acetate = 2:3). The fractions were concentrated, yielding 3.44 g of crude compound X as a brown liquid. The column-purified crude product was divided into two aliquots, each of which was purified by medium-pressure preparative chromatography (dichloromethane / methanol system: 100% dichloromethane for 5 min → gradient for 10 min → 95% dichloromethane for 10 min → gradient for 10 min → 90% dichloromethane for 20 min). Compound X (1.15 g) was obtained as a gray powder (yield 17.3%, LC purity 97.8%). NMR (in CDCl3) data are shown in Figures 7-9. FD-MS data are shown in Figure 10.

[0069] Example 2. Evaluation of radical scavenging activity 1 The radical scavenging activity of Compound X was evaluated as follows.

[0070] The scavenging activity of Compound X against multiple free radical species was evaluated using electron spin resonance (ESR), the only method for directly measuring free radicals. Free radicals are highly reactive and unstable, so they were measured as stable spin adducts using a spin trapping agent. The following nine free radicals were targeted: hydroxyl radical; DPPH, nitric oxide radical; tyrosyl radical; and singlet oxygen radical. Free radicals were generated in a test tube, and different concentrations of Compound X were added to evaluate the concentration-dependent scavenging activity. Regression analysis using the Cheng-Prusoff equation yielded the IC 50 The reaction rate constants (k X ), or the ratio of the spin trapping agent to that of the spin trapping agent (k X / k trap ) was estimated.

[0071] The results are shown in Figures 11 to 15. Compound X was found to have the effect of scavenging the above five types of free radicals. Furthermore, when the nitric oxide radical scavenging effect of Compound X was compared with that of edaravone, an existing antioxidant, the reaction rate constant of Compound X was 1.4 times that of edaravone.

[0072] Example 3. Evaluation of corneal damage inhibitory effect Corneal damage was induced by exposing the eyes to smoke (mainstream smoke) produced by cigarette smoking, and the inhibitory effect of Compound X on corneal damage was evaluated. Specifically, the procedure was as follows.

[0073] For rats, exposure of the eyes to tobacco smoke and instillation of an aqueous solution of Compound X (0.45 mM) or Vehicle (PBS) were performed daily for a total of 5 days. The treatment details for each day were as follows. The exposure treatment to tobacco smoke was performed 6 times a day at 30-minute intervals for a total of 6 times per day (from 9:00 to 11:30) with 50 mL of smoke exposure to the eyes. Instillation was performed 4 times a day at 5 μL / eye for both eyes (at 9:00, 12:00, 14:00, and 15:30). On the 6th day, fluorescein (Nippon Alcon Co., Ltd.) 500 mg / 5 mL (0.1 g / mL = 10%) was diluted with physiological saline to 0.5% (0.05 mL + 0.95 mL physiological saline), and the resulting fluorescein solution was instilled at 2 μL / eye. After corneal staining, fluorescence microscope images of the corneas of the eyes were obtained. Based on the images, the Fluorescein score was calculated as follows.

[0074] <Method for calculating the Fluorescein score> The circular corneal staining image is divided into a grid of 9 parts by two vertical and two horizontal lines. Each divided part is scored 1 point if it is stained by 10% or more, and 0 point if it is not stained. The scores of each section are summed to obtain the score. The score ranges from 0 to 9 points.

[0075] The results are shown in Figure 16. It was found that corneal damage was significantly suppressed by instillation of Compound X.

[0076] Example 4. Evaluation of ischemic damage suppression effect The inhibitory effect of Compound X on the spinal cord injury and paralysis associated with ischemia-reperfusion that can occur during spinal cord ischemia was evaluated. Specifically, it was performed as follows.

[0077] The rabbit's femoral artery was exposed, and a Fogarty catheter was inserted 15 cm into the inguinal ligament. The balloon was inflated, and the aorta was occluded for 20 minutes to create a spinal cord ischemia model. Immediately after the aortic occlusion was released, 12 ml of compound X solution (6 mg / kg), 12 ml of edaravone (6 mg / kg), or 12 ml of saline was administered intravenously over 3 minutes. Before the aortic occlusion was released, the compound X solution was dissolved in 2 ml of distilled water, and 10 ml of saline was added to make a total of 12 ml. Neurological evaluation was performed 3 hours after the occlusion was released and daily thereafter for a total of 5 days using the Modified Tarlov's Score (MTS).<Modified Tarlov’s score> 0: No movement of lower limbs 1: slight movement of lower limbs 2: sits with support 3: Sits alone 4: Weak Hop 5: normal hop *MTS = 0 was defined as complete paraplegia.

[0078] The MTS for each of the compound X (n = 8), edaravone (n = 8), and saline (n = 8) groups is shown in Figure 17. The incidence of complete paraplegia was 5 / 8 cases (63%) for saline, 4 / 8 cases (50%) for edaravone, and 0 / 8 cases (0%) for compound X, demonstrating that administration of compound X suppressed the incidence of paraplegia after ischemia-reperfusion compared to saline and edaravone.

[0079] Example 5. Stability evaluation Compound X was dissolved in heavy water (1 mM) and left at room temperature for 5 days. It was exposed to room light without any special protection. Every 24 hours, 1 Measurement was performed by H-NMR (AVANCE III-400, BRUKER; water suppression by NOESY pulse sequence; 8 additions).

[0080] The results are shown in Figure 18. Compound X was found to be stable.

[0081] Example 6. Evaluation of radical scavenging activity 2 The radical scavenging activity of Compound X was evaluated as follows.

[0082] The scavenging activity of Compound X against multiple free radical species was evaluated using electron spin resonance (ESR), the only method for directly measuring free radicals. Free radicals are highly reactive and unstable, so they were measured as stable spin adducts using a spin trapping agent. The nine free radicals targeted were the hydroxyl radical, superoxide anion, tert-butoxyl radical, tert-butylperoxyl radical, ascorbyl radical, singlet oxygen, nitric oxide radical, DPPH, and tyrosyl radical. Free radicals were generated in a test tube, and different concentrations of Compound X were added to evaluate the concentration-dependent scavenging activity. IC was calculated by regression analysis using the Cheng-Prusoff equation. 50 The reaction rate constants (k X ), or the ratio of the spin trapping agent to that of the spin trapping agent (k X / k trap ) was estimated.

[0083] The results are shown in Figures 19 to 27. Compound X was found to have the ability to scavenge the above nine types of free radicals. Furthermore, when the superoxide anion and nitric oxide radical scavenging activity of Compound X was compared with that of edaravone, an existing antioxidant, the reaction rate constants of Compound X were 3 and 1.4 times those of edaravone, respectively (Figure 28).

[0084] Example 7. Antioxidant effect on lipids in brain tissue The inhibitory effect of Compound X on lipid oxidation was evaluated as follows.

[0085] Hydrogen peroxide was added to mouse brain tissue homogenized under ice cooling to oxidize the lipids in the brain tissue. Malondialdehyde was quantified as an indicator of oxidation using the thiobarbituric acid reactive substance method. Different concentrations of compound X were added to the homogenized tissue to evaluate the concentration-dependent antioxidative activity.

[0086] The results are shown in Figure 29. Compound X exhibited lipid oxidation inhibitory activity comparable to that of edaravone. At the same concentration, the production of malondialdehyde was significantly suppressed more than that of edaravone.

[0087] Example 8. Cytotoxicity of Compound X The toxicity of Compound X to cultured cells was evaluated as follows.

[0088] Human lung fibroblast MRC-5 cells were cultured for 24 hours with various concentrations of compound X, and cell growth was observed.

[0089] The results are shown in Figure 30. Cell proliferation was not impaired by Compound X up to a final concentration of 100 μM.

Claims

1. General formula (1): 【Chemistry 1】 [In the formula: R 1 and R 2 are the same or different and represent an alkyl group having 1 to 4 carbon atoms. A compound represented by the formula (I) or a solvate thereof.

2. R 1 and R 2 and are both methyl groups, or a solvate thereof, according to claim 1 .

3. An antioxidant comprising the compound according to claim 1 or 2 or a solvate thereof.

4. The antioxidant according to claim 3 for use in radical scavenging.

5. The antioxidant according to claim 3 for use in preventing or ameliorating oxidative stress disorders.

6. 3. A preventive or ameliorating agent for oxidative stress-induced disorders, comprising the compound according to claim 1 or 2 or a solvate thereof.

7. The preventive or ameliorating agent according to claim 6, wherein the oxidative stress-related disorder is at least one selected from the group consisting of corneal disorder, dry eye, and ischemic disorder.

8. An external preparation comprising the compound or solvate thereof according to claim 1 or 2.

9. The external preparation according to claim 8, which is an eye drop.

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