Compound or salt thereof and method for producing same, composition, hydrogen peroxide remover, and cytotoxicity inhibitor

Abstract

Provided are: a novel ergothioneine derivative and a method for producing the same; a composition containing the derivative; a hydrogen peroxide remover; and a cytotoxicity inhibitor.　Provided is a compound represented by formula (1) or a salt thereof.

Description

Compound or salt thereof, method for producing the same, composition, hydrogen peroxide remover, and cytotoxicity inhibitor

[0001] The present invention relates to a novel ergothioneine derivative and a method for producing the same, a composition containing the derivative, a hydrogen peroxide scavenger, a cytotoxicity inhibitor, and the like.

[0002] L-ergothioneine (sometimes referred to as "EGT" in this specification) is a type of sulfur-containing amino acid, and is known to have a variety of physiological activities.

[0003] On the other hand, compositions having a hydrogen peroxide-removing effect have been known to date, such as antioxidant functional water containing fine bubbles (see, for example, Patent Document 1). Also, compositions having a cytotoxicity-inhibiting effect have been known, such as an intracellular thioredoxin system activator containing hinokitiol (see, for example, Patent Document 2).

[0004] However, there are a wide variety of applications and targets for which hydrogen peroxide removal and cytotoxicity suppression are required, and there is a need for the development of new hydrogen peroxide removers and cytotoxicity suppressors.

[0005] JP 2008-156320 A JP 2020-103100 A

[0006] In light of the above circumstances, an object of the present invention is to provide a novel ergothioneine derivative, a method for producing the same, a composition containing the derivative, a hydrogen peroxide scavenger, a cytotoxicity inhibitor, and the like.

[0007] As a result of intensive investigations to solve the above-mentioned problems, the present inventors have succeeded in creating the following novel ergothioneine derivative (a compound represented by the following formula 1 or a salt thereof), and have found that the above-mentioned object can be achieved by using the above-mentioned compound, thereby completing the present invention.

[0008] That is, the present invention provides the following compounds or salts thereof (hereinafter, sometimes referred to as "compounds" or "compounds, etc.").

[0009] Item 1. A compound represented by the following formula (1) or a salt thereof:

[0010] The present invention also provides the following compositions.

[0011] Item 2. A composition comprising the compound according to Item 1 or a salt thereof.

[0012] The present invention also provides the following hydrogen peroxide remover.

[0013] Item 3. A hydrogen peroxide scavenger comprising the compound according to Item 1 or a salt thereof.

[0014] Item 4. The hydrogen peroxide scavenger according to Item 3, wherein the compound or a salt thereof is used at a concentration of 0.04 μM or more.

[0015] The present invention also provides the following cytotoxicity inhibitors.

[0016] Item 5. A cytotoxicity inhibitor comprising the compound or salt thereof according to Item 1, which is used in the presence of hydrogen peroxide.

[0017] The present invention also relates to the following production methods.

[0018] Item 6. A method for producing a compound represented by the following formula (1) or a salt thereof, comprising step (1) of heat-treating an aqueous solution of L-ergothioneine at 60°C or higher for 24 hours or longer:

[0019] The compound of the present invention or a salt thereof, or a composition containing the compound or salt thereof can be suitably used, for example, as a hydrogen peroxide scavenger or a cytotoxicity inhibitor.

[0020] Furthermore, the hydrogen peroxide remover of the present invention can exhibit, for example, the effect of suitably removing hydrogen peroxide.

[0021] Furthermore, the cytotoxicity inhibitor of the present invention can exert an effect of reducing cytotoxicity caused by hydrogen peroxide in a culture medium, for example.

[0022] Furthermore, by using the method for producing a compound of the present invention, the above compound or a salt thereof can be easily obtained.

[0023] FIG. 1 is a graph showing the results of HPLC analysis after 96 hours of heat treatment in Example 1. FIG. 2 is a graph showing the results of liquid chromatography high-resolution mass spectrometry (LC-HRMS) measurement in Example 1. FIG. 3 is a graph showing the results of evaluation of the amount of remaining hydrogen peroxide in Example 2. FIG. 4 is a table showing the results of evaluation of the amount of remaining hydrogen peroxide in Example 2. FIG. 5 is a photograph showing the results of evaluation of the cytotoxicity of hydrogen peroxide against HEK293 cells in Example 3. FIG. 6 is a graph showing the results of evaluation of the cytotoxicity of hydrogen peroxide against HEK293 cells in Example 3. FIG. 7 is a graph showing the results of evaluation of the cytotoxicity of hydrogen peroxide against HEK293 cells in Example 3. FIG. 8 is a photograph showing the results of evaluation of the cytotoxicity of hydrogen peroxide against HEK293 cells in Example 3.

[0024] Hereinafter, embodiments of the present invention will be described in detail.

[0025] [Compound or Salt Thereof] The compound of the present invention or a salt thereof (hereinafter also referred to as "compound (1)") is represented by the following formula 1.

[0026] The above compound is a type of oxidized dimer of L-ergothioneine.

[0027] The salts of the above compounds may be intramolecular salts in these structures, and may contain other counter cations and / or counter anions.

[0028] The salt of the compound may be, for example, a pharmacologically or physiologically acceptable salt. The salt is not particularly limited, and specific examples include organic acid salts, inorganic acid salts, organic bases, and inorganic bases. Examples of organic acid salts include monocarboxylic acid salts such as acetate, trifluoroacetate, butyrate, palmitate, and stearate; polycarboxylic acid salts such as fumarate, maleate, succinate, and malonate; oxycarboxylic acid salts such as lactate, tartrate, and citrate; and organic sulfonates such as toluenesulfonates such as methanesulfonate and tosylate. Examples of inorganic acid salts include hydrochloride, sulfate, nitrate, hydrobromide, and phosphate. Examples of salts with organic bases include salts with organic amines such as methylamine, triethylamine, triethanolamine, diethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, picoline, and ethylenediamine. Examples of salts with inorganic bases include ammonium salts; salts with alkali metals such as sodium or potassium, alkaline earth metals such as calcium or magnesium, and metals such as aluminum. These salts may be used alone or in any combination of two or more. The above salts may include solvates or hydrates of the salts.

[0029] The compound (1) can be obtained by known methods such as synthesis, extraction, and fermentation.

[0030] Compound (1) can be synthesized, for example, by appropriately oxidizing L-ergothioneine, or by subjecting L-ergothioneine to a high-temperature or long-term heat treatment, followed by isolation and purification by high-performance liquid chromatography or the like.

[0031] More specifically, for example, L-ergothioneine can be synthesized by heating an aqueous solution of L-ergothioneine under basic conditions, and it is preferable to use a method including step (1) of heat-treating the solution at 60°C or higher for 24 hours or longer. It is even more preferable to use a method including step (1) of heat-treating the solution at 60°C or higher and pH 9-11 for 24 hours or longer. Furthermore, by performing step (1) at, for example, 80°C or higher, it is possible to synthesize an L-ergothioneine solution having a concentration of, for example, 1500 mM as an aqueous solution. The concentration of the aqueous L-ergothioneine solution is preferably, for example, 1 mM or higher, more preferably 10 mM or higher, and even more preferably 100 mM or higher, depending on the intended use and application. The concentration of the aqueous L-ergothioneine solution can be, for example, 2 M or lower, 1.8 M or lower, 1.5 M or lower, or 1.3 M or lower, depending on the intended use and application.

[0032] [Composition] The composition of the present invention contains the above-mentioned compound (1). The compound (1) may be contained alone or in combination of two or more kinds.

[0033] In the composition of the present invention, the content of the compound (1) is appropriately adjusted depending on the use of the composition, the types and contents of other components, and the like, and is not limited thereto. For example, the content can be 0.000001% by mass or more, 0.0000015% by mass or more, 0.000005% by mass or more, 0.00001% by mass or more, 0.00005% by mass or more, 0.0001% by mass or more, 0.0005% by mass or more, 0.001% by mass or more, and the like, relative to the total amount of the composition. The content of compound (1) can be, for example, 99.999% by mass or less, 99.9% by mass or less, 99.5% by mass or less, 99% by mass or less, 98.5% by mass or less, 98% by mass or less, 80% by mass or less, and examples thereof include 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, etc. In another embodiment, for example, when prepared as a liquid composition or liquid formulation, the content of compound (1) can be, for example, 80% by mass or less, based on the total amount of the composition, and examples thereof include 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, etc.

[0034] In addition, the above composition can further contain various known components and additives depending on the purpose of the composition and the types of other components. Examples of the above components and additives include excipients, lubricants, binders, and disintegrants; and for liquid preparations, solvents, solubilizers, emulsifiers, emulsion stabilizers, thickeners, moisturizers, suspending agents, isotonicity agents, buffers, soothing agents, preservatives, antioxidants, colorants, sweeteners, and flavors. These may be used alone or in combination of two or more.

[0035] [Hydrogen Peroxide Removal Agent] The hydrogen peroxide removal agent of the present invention contains the above-mentioned compound (1). Compound (1) may be contained alone or in combination of two or more kinds.

[0036] The hydrogen peroxide remover of the present invention has the above-described configuration, and thus can exhibit, for example, the effect of suitably removing hydrogen peroxide.

[0037] In the hydrogen peroxide remover of the present invention, the content of the compound (1) (when multiple types are present, the total amount thereof) is appropriately adjusted depending on the application of the hydrogen peroxide remover, the types and contents of other components, etc., and is not limited thereto. For example, the content can be 0.000001% by mass or more, 0.0000015% by mass or more, 0.000005% by mass or more, 0.00001% by mass or more, 0.00005% by mass or more, 0.0001% by mass or more, 0.0005% by mass or more, 0.001% by mass or more, and the like, relative to the total amount of the hydrogen peroxide remover. Furthermore, the content of the above compound can be, for example, 99.999% by mass or less, 99.9% by mass or less, 99.5% by mass or less, 99% by mass or less, 98.5% by mass or less, 98% by mass or less, 80% by mass or less, and examples thereof include 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, etc. In another embodiment, for example, when prepared as a liquid hydrogen peroxide remover or a liquid formulation, the content of compound (1) can be, for example, 80% by mass or less, based on the total amount of the hydrogen peroxide remover, and examples thereof include 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, etc.

[0038] When the hydrogen peroxide remover is used in a liquid form, the concentration of compound (1) may be, for example, 0.01 μM to 0.1 μM, 0.02 μM to 0.08 μM, 0.03 μM to 0.06 μM, or 0.04 μM to 0.05 μM, depending on the purpose and application. In addition, for example, compound (1) is preferably used at a concentration of 0.04 μM or more.

[0039] The hydrogen peroxide remover may further contain the above-mentioned various known components and additives, depending on the intended use of the hydrogen peroxide remover and the types of other components. These may be used alone or in combination of two or more.

[0040] [Cytotoxicity Inhibitor] The cytotoxicity inhibitor of the present invention contains the above-mentioned compound (1) and is used in the presence of hydrogen peroxide. Compound (1) may be contained alone or in combination of two or more kinds.

[0041] The cytotoxicity inhibitor of the present invention has the above-mentioned constitution, and can therefore exert an effect of reducing cytotoxicity caused by hydrogen peroxide in a culture medium, for example.

[0042] In the cytotoxicity inhibitor of the present invention, the content of the above compound (1) (the total amount when multiple types are present) is appropriately adjusted depending on the application of the cytotoxicity inhibitor, the types and contents of other components, etc., and is not limited, and can be, for example, 0.000001 mass% or more, 0.000005 mass% or more, 0.00001 mass% or more, 0.00005 mass% or more, 0.0001 mass% or more, 0.0005 mass% or more, 0.001 mass% or more, etc., relative to the total amount of the cytotoxicity inhibitor. The content of compound (1) can be, for example, 99.999% by mass or less, 99.9% by mass or less, 99.5% by mass or less, 99% by mass or less, 98.5% by mass or less, 98% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, etc. In another embodiment, for example, when prepared as a liquid cytotoxicity inhibitor or a liquid formulation, the content of compound (1) can be, for example, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, etc., based on the total amount of the cytotoxicity inhibitor.

[0043] When the cytotoxicity inhibitor is used in a liquid form, the concentration of the compound or a salt thereof may be, for example, 0.01 μM to 10 μM, 0.1 μM to 5 μM, or 1 μM to 3 μM, depending on the purpose and application.

[0044] The cytotoxicity inhibitor may further contain various known components and additives as described above, depending on the intended use of the cytotoxicity inhibitor and the types of other components. These may be used alone or in combination of two or more.

[0045] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0046] Example 1 (Preparation of Compound) A compound represented by the following formula (1) (hereinafter also referred to as "Compound A" or "EGT-X") was prepared by the following method.

[0047] L-Ergothioneine (manufactured by Tetraedron) was dissolved in N-cyclohexyl-3-aminopropanesulfonic acid buffer (pH 10) to prepare 20 mL of a 250 mM ergothioneine solution. After heating at 60°C for 96 hours, HPLC analysis was performed under the following conditions (Figure 1). Apparatus: Shimadzu LC20A (manufactured by Shimadzu Corporation). Column: μBondasphere C18 5 μm 100A, 150 x 3.9 mm I.D. (manufactured by Waters Corporation) Flow rate: 0.5 mL / min, temperature: 30°C Detector: PDA (260 nm) Eluent: distilled water, isocratic Retention time: around 3.3 minutes (L-ergothioneine), around 7.8 minutes (compound A) Apparatus: medium pressure column preparative system EPCLC AI-580S (manufactured by Yamazen Corporation) Column: ODS-C-50C 37 x 300 mm (manufactured by Yamazen Corporation) Flow rate: 6 mL / min, temperature: room temperature Detector: UV (260 nm) Eluent: distilled water, isocratic Retention time: around 20 minutes (L-ergothioneine), around 30 minutes (compound A) Injection amount: 0.5-0.6 mL Fractionation was performed 10 times under the above conditions, and the obtained compound A fractions were collected.

[0048] The obtained fraction was concentrated from about 1000 mL to 30 mL using a rotary evaporator (60°C water bath). Then, it was freeze-dried to obtain 108 mg of compound A as a yellow powder. ２ The mixture was redissolved in 0 and subjected to NMR analysis.

[0049] ( １ H-NMR measurement) １ The results of H-NMR measurement are shown below. １ H-NMR (400MHz, D ２ O) DSS-d ６ was set to 0 ppm. δ 7.00 (s, 1H), 3.91 (m, 2H), 3.33 (m, 2H), 3.29 (s, 9H), 3.24 (s, 9H), 3.14 (m, 2H)

[0050] ( １３ C-NMR measurement) １３ The results of C-NMR measurement are shown below. １３ C-NMR (400MHz, D ２ O) DSS-d ６ was set to 0 ppm. δ 173.49, 172.93, 169.98, 140.47, 136.72, 134.11, 122.56, 117.67, 80.98, 79.37, 54.79, 28.00, 25.65

[0051] (High-resolution mass spectrometry) The results of liquid chromatography-high-resolution mass spectrometry (LC-HRMS) are shown below. (LC-HRMS analysis conditions) Apparatus: Agilent Technologies 6224 TOF LC / MS (Agilent Technologies) Column: μBondasphere C18 5 μm 100A, 150×3.9 mm I.D. (Waters) Flow rate: 0.5 mL / min Temperature: 30°C Eluent: Distilled water, isocratic Ionization conditions: ESI Capillary voltage 3500 V Fragmenter voltage 100 V Retention time: Around 7.8 minutes (Compound A) C １８ H ２８ N ６ O ４ S ２ [M+H] ＋ The theoretical value was 457.1686, whereas the actual measured value was 457.1686.

[0052] Furthermore, in order to confirm whether Compound A has a plurality of thione groups, a reaction test with an S-modifying reagent, phenacyl bromide, was carried out.

[0053] 100 μL of a 32 mg / mL aqueous solution of Compound A and 100 μL of a 15 mg / mL N,N-dimethylformamide solution of phenacyl bromide (Tokyo Chemical Industry Co., Ltd.) were mixed and incubated at room temperature for 10 minutes. The mixture was subjected to liquid chromatography-high resolution mass spectrometry (LC-HRMS) under the following conditions: Apparatus: Agilent Technologies 6224 TOF LC / MS (Agilent Technologies) Column: μBondasphere C18 5 μm 100A, 150 x 3.9 mm I.D. (Waters) Flow rate: 0.5 mL / min, temperature: 30°C Detector: PDA (260 nm) Eluent: 0.1% formic acid (A), acetonitrile (B) gradient Gradient conditions: 0% B (0 min) to 100% B (20 min) Ionization conditions: ESI Capillary voltage 3500 V Fragmenter voltage 100 V

[0054] The results are shown in Figure 2. The main peak detected was one with a retention time of around 7.9 minutes. The results of liquid chromatography-high resolution mass spectrometry (LC-HRMS) of the main peak with a retention time of around 7.9 minutes are shown below. ２６ H ３４ N ６ O ５ S ２ [M+H] ＋ The theoretical value was 575.2105, whereas the actual value was 575.2099, confirming that one phenacyl group was bonded (Figure 2).

[0055] Example 2 (Evaluation of Hydrogen Peroxide Removal) The obtained compound A (EGT-X) was diluted in 2-fold increments starting from 10 μM with the buffer provided with the kit (OxiSelect hydrogen peroxide assay kit (CELL BIOLABS)), and 25 μL of each was added to a 96-well plate. Hydrogen peroxide was also diluted with the buffer provided with the kit to a concentration of 4 μM, and 25 μL of each was added to the 96-well plate. Furthermore, a blank was prepared under the same conditions except that no hydrogen peroxide was added.

[0056] Next, according to the kit's protocol, 50 μL of the ADHP (10-acetyl-3,7-dihydroxyphenoxazine) and HRP (horseradish peroxidase) mixture included in the kit was added and stirred. After 30 minutes of incubation at room temperature, the fluorescence intensity was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Values ​​were calculated by subtracting the blank value from each measurement value. The percentage values ​​obtained when EGT-X was added were calculated, with the control value (Compound A: 0 μM) taken as 100%, and the results were plotted on graphs (Figures 3 and 4).

[0057] As shown in FIGS. 3 and 4, the amount of residual hydrogen peroxide decreased as the amount of Compound A (EGT-X) added increased.

[0058] Example 3 (Evaluation of Cytotoxicity Inhibition) To evaluate cytotoxicity inhibition, the cytotoxicity of hydrogen peroxide on HEK293 cells was evaluated.

[0059] HEK293 cells 15x10 ４ A suspension of 100 μL of 100 cells / mL (Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS)) was added to a 96-well plate. After 24 hours, 100 μL of DMEM alone or DMEM containing hydrogen peroxide was added to each well to adjust the hydrogen peroxide concentration to 0, 0.001, 0.002, or 0.004%. The cell viability after 24 hours of exposure to hydrogen peroxide was observed. To measure cell viability, 50 μL of a 1:1 mixture of DMEM containing 5% FBS and WST-1 Premix (TAKARA Bio) was added, and the cells were incubated in 5% CO ２ The mixture was incubated in an incubator for 30 minutes. The absorbance at 450 nm was measured. A blank measurement was performed under the same conditions as above, except that no cells were present. The blank value was subtracted from the results above to calculate the cell viability (Figures 5 and 6).

[0060] HEK293 cells 15x10 ４A 100 μL suspension of 100 cells / mL (5% FBS-DMEM) was added to a 96-well plate. After 24 hours, 50 μL each of EGT-X dissolved in DMEM and hydrogen peroxide dissolved in DMEM were added to the plate. The hydrogen peroxide concentration was 0% for the control and 0.0015% for the other samples, and the EGT-X concentration was adjusted to the concentrations shown in the figures (Figures 5 and 6). After further culturing for 24 hours, 50 μL of a 1:1 mixture of DMEM containing 5% FBS and WST-1 Premix (manufactured by TAKARA BIO) was added and the cells were incubated in 5% CO. ２ The mixture was incubated in an incubator for 30 minutes. The absorbance at 450 nm was measured. A blank measurement was performed under the same conditions as above except that no cells were present. The blank value was subtracted from the result to calculate the cell viability.

[0061] The cell viability without hydrogen peroxide or EGT-X (control) was set at 100%, and the results are shown in the graphs (FIGS. 7 and 8).

[0062] As shown in Figures 5 and 6, only a slight decrease in viability was observed at 0.001% (292 μM) hydrogen peroxide, and cells were killed at 0.002% (585 μM). Therefore, we decided to examine the effect of EGT-X on the cytotoxicity of hydrogen peroxide at a hydrogen peroxide concentration of 0.0015%, an intermediate value.

[0063] Furthermore, as shown in FIGS. 7 and 8, the cytotoxicity caused by hydrogen peroxide was suppressed in the presence of 125 μM or more of EGT-X.

Claims

1. A compound represented by the following formula (1) or a salt thereof: 【Chemistry 1】

2. A composition comprising the compound or salt thereof described in claim 1, containing 0.229% or more ergothioneine.

3. A hydrogen peroxide removing agent comprising the compound or a salt thereof according to claim 1, which is used at a concentration of 125 μM or more.

4. A cytotoxicity inhibitor comprising the compound or its salt described in claim 1, which is used in the presence of hydrogen peroxide at a concentration of the compound or its salt of 125 μM or more.

5. 2. A method for producing the compound or a salt thereof according to claim 1, comprising: (1) heat-treating an aqueous solution of L-ergothioneine at 60° C. or higher for 24 hours or longer.

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