Antioxidant compound

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

antioxidant compounds

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

[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.

[0004] Special table 2022-528727 publication

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

[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] Item 1. General formula (1):

[0008] [In the formula: R 1 and R 2 and are the same or different and represent an alkyl group having 1 to 4 carbon atoms.] or a solvate thereof.

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

[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, which comprises applying the compound or solvate thereof according to Item 1 or 2 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-induced 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 or solvate thereof according to Item 1 or 2 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-induced 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.

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

[0026] NMR (in CDCl3) data for 3'5'-dimethoxyacetophenone (2) is shown. NMR (in CDCl3) data for 3'5'-dimethoxyacetophenone (2) is shown. NMR (in CDCl3) data for 3'5'-dimethoxyacetophenone (2) is shown. NMR (in CDCl3) data for compound 5 is shown. NMR (in CDCl3) data for compound 5 is shown. NMR (in CDCl3) data for compound X is shown. NMR (in CDCl3) data for compound X is shown. NMR (in CDCl3) data for compound X is shown. FD-MS data for compound X is shown. Measurement results of the reaction rate constant for hydroxyl radical scavenging activity are shown. Measurement results of the reaction rate constant for tyrosyl radical scavenging activity are shown. k edv indicates the reaction rate constant of edaravone. The measurement results of the reaction rate constant of nitric oxide radical scavenging action are shown. The measurement results of the reaction rate constant of DPPH radical scavenging action are shown. k edv indicates the reaction rate constant of edaravone. The results of measuring the reaction rate constant of the singlet oxygen radical scavenging effect are shown. The corneal fluorescein score is shown. Non-smoke indicates the group (n = 6) that was not exposed to cigarette smoke and did not receive eye drops, PBS (smoke) indicates the group (n = 6) that was exposed to cigarette smoke and received PBS eye drops, and 0.45mM Compound X (smoke) indicates the group (n = 6) that was exposed to cigarette smoke and received an eye drop of Compound X aqueous solution. Each column indicates the average value. 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. In the stability test 1H-NMR measurement results are shown. The left side of the graph shows the number of days since dissolution in heavy water. The ratio 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. Measurement results of the reaction rate constant of the hydroxyl radical are shown. In the graph, Substance X refers to Compound X. Measurement results of the reaction rate constant of the superoxide anion are shown. In the graph, Substance X refers to Compound X. Measurement results of the reaction rate constant of the tert-butoxyl radical are shown. In the graph, Substance X refers to Compound X. Measurement results of the reaction rate constant of the tert-butylperoxyl radical are shown. In the graph, Substance X refers to Compound X. Measurement results of the reaction rate constant of the ascorbyl radical are shown. In the graph, Substance X refers to Compound X. Measurement results of the reaction rate constant of singlet oxygen are shown. In the graph, Substance X refers to Compound X. Measurement results of the reaction rate constant of the nitric oxide radical are shown. In the graph, Substance X refers to Compound X. Measurement results of the reaction rate constant of DPPH are shown. In the figure, Substance X means compound X. This shows the results of measuring the reaction rate constant of tyrosyl radicals. In the figure, Substance X means compound X. This shows a comparison of the reaction rate constants of compound X and edaravone. This shows the results of quantifying malondialdehyde, an oxidation indicator, using the thiobarbituric acid reactive substance method. Edaravone represents the edaravone-administered group, and Substance X represents the compound X-administered group. In the figure, Substance X represents compound X. This shows the results of measuring cell proliferation. The horizontal axis shows the time elapsed since the start of culture. The legend indicates the final concentration of compound X in the medium.

[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 the general formula (1):

[0029] [In the formula: R 1 and R 2and 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), which 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 2 The alkyl groups represented by the 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) is represented by formula (1A):

[0033] 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 the solvent that constitutes the solvate 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] [In the formula: R1 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 moles, more preferably 0.7 to 1.5 moles, per mole 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 conventional methods such as chromatography. After the reaction is completed, the solvent is removed by distillation, and the product can be isolated and purified by conventional methods such as chromatography and 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. Uses 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), and in particular, 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 caused by radicals or oxidative stress in the living body.Examples of the oxidative stress disorder include corneal disorder, dry eye, ischemic disorder (for example, 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, cancer, etc. 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 in vitro (for example, by adding to a culture 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 may be used in any mammalian animal, including, but not limited to, humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, including, but not limited to, blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), 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.

[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]

[0066] Example 1-1 Synthesis of 3'5'-dimethoxyacetophenone (2) 3'5'-Dihydroxyacetophenone (1) (24.15 g, 158.72 mmol, 1.0 eq) and distilled water (120.75 mL) were added to a 1 L three-neck flask equipped with a stirrer and thermometer, and while stirring at room temperature, 10 wt% NaOH aqueous solution (317.44 mL, 793.60 mmol, 3.0 eq) was added and stirred at room temperature until the heat generation 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 hour (1 hour 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 increased to 40 °C, and stirring was continued. After 1 h (5.5 h from the start of the reaction), LC analysis showed almost no change in the remaining amount of the reaction intermediate (RT = 3.9 min). Therefore, dimethyl sulfate (20.04 g, 158.72 mmol, 1.0 eq) was added and stirring was continued. After 1 h (6.5 h from the start of the reaction), LC analysis confirmed that the reaction intermediate (RT = 3.9 min) had decreased to 5.82%. The reaction was quenched by pouring into ice water (1500 mL). After stirring overnight in an ice bath, 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 give 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: 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) were added to a 1000 mL three-neck flask equipped with a stirrer and thermometer. 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 peaks of the two starting materials 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 x 2) and distilled water (500 mL x 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 (1030.0 g of 60N silica gel, 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. Compound 5 (8.12 g, 24.43 mmol, 1.0 eq) and dichloromethane (400 mL) were added to a 1000 mL three-neck flask equipped with a stirrer and thermometer. While stirring at -30 °C under a nitrogen atmosphere, a 17% boron tribromide / dichloromethane solution (196.0 mL, 196.0 mmol, 8.0 eq) was added dropwise (transparent liquid → reddish-brown liquid). After the addition, the mixture was stirred at -30 °C under a nitrogen atmosphere for 30 minutes, and then at room temperature for an additional 2 hours. LC analysis was performed upon returning the mixture to room temperature (0.5 hours into the reaction), revealing the disappearance of the peak for compound 5 (RT = 8.0 min) and the appearance of several new peaks around RT = 2.5 to 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 terminated 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 portions, each of which was purified by medium-pressure preparative chromatography (dichloromethane / methanol system: dichloromethane 100%, 5 min → gradient 10 min → dichloromethane 95%, 10 min → gradient 10 min → dichloromethane 90%, 20 min). 1.15 g of compound X 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. Specifically, the evaluation was carried out 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. Because free radicals are highly reactive and unstable, 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 for each free radical (k X ), or the ratio of the spin trapping agent to that (k X / k trap ) was estimated.

[0071] The results are shown in Figures 11 to 15. Compound X was found to have the ability to scavenge the above five types of free radicals. Furthermore, when the nitric oxide radical scavenging activity 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 corneal damage inhibitory effect of Compound X was evaluated. Specifically, the evaluation was carried out as follows.

[0073] Rats were exposed to cigarette smoke and given a 5-day daily instillation of Compound X aqueous solution (0.45 mM) or vehicle (PBS). Each treatment was as follows: For cigarette smoke exposure, 50 mL of smoke was applied to the eyes every 30 minutes, six times a day (9:00 AM to 11:30 AM). Eye instillation was performed at 5 μL per eye, four times a day (9:00 AM, 12:00 PM, 2:00 PM, and 3:30 PM). On day 6, fluorescein (Alcon Japan Co., Ltd.) 500 mg / 5 mL (0.1 g / mL = 10%) was diluted with saline to 0.5% (0.05 mL + 0.95 mL saline), and 2 μL of the resulting fluorescein solution was instilled into each eye. Fluorescence microscopy images of the cornea were then taken. Fluorescein scores were calculated based on the images as follows:

[0074] <How to calculate the Fluorescein score> A circular stained corneal image is divided into nine grid sections with two lines vertically and two lines horizontally. If each section is stained at 10% or more, it is given a score of 1, and if there is no staining, it is given a score of 0. The scores for each section are added together to calculate 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 the effect of suppressing ischemic damage The effect of Compound X to suppress the occurrence of spinal cord damage and paraplegia associated with ischemia-reperfusion, which may occur during spinal cord ischemia, was evaluated. Specifically, the procedure was 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 aortic occlusion, 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 occlusion, 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 occlusion 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. * An MTS of 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. Evaluation of stability 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 from light. 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. Specifically, the evaluation was carried out 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. Because free radicals are highly reactive and unstable, 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. Regression analysis using the Cheng-Prusoff equation yielded the IC 50 The reaction rate constants for each free radical (k X ), or the ratio of the spin trapping agent to that (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 antioxidant effect on lipids of compound X was evaluated. Specifically, the test was carried out 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 its 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, compound X significantly inhibited the production of malondialdehyde more than edaravone.

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

[0088] Human lung fibroblast MRC-5 cells were cultured for 24 hours with various concentrations of compound X added, 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): [In the formula: R 1 and R 2 and R 1 to R 4 are the same or different and each represents an alkyl group having 1 to 4 carbon atoms.] 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 improving oxidative stress disorders.

6. An agent for preventing or improving 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 disorder is at least one selected from the group consisting of corneal disorder, dry eye, and ischemic disorder.

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

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

Images