Compound, reagent for measuring enzyme activity of amino acid oxidase, and method for measuring enzyme activity of amino acid oxidase

Abstract

To provide: novel compounds that can emit sufficient fluorescence after an enzymatic reaction and that make it possible to evaluate the enzymatic activity of a D-amino acid oxidase or an L-amino acid oxidase in a one-step reaction without using a complicated two-step reaction; a reagent for measuring the enzymatic activity of an amino acid oxidase; and a method of measuring the enzymatic activity of an amino acid oxidase.SOLUTION: Provided are compounds represented by either (S)-2-amino-4-(2-amino-5-(methylthio)phenyl)-4-oxobutanoic acid or (R)-2-amino-4-(2-amino-5-(methylthio)phenyl)-4-oxobutanoic acid; a reagent for measuring the enzymatic activity of an amino acid oxidase, the reagent being characterized by containing any combination of the compounds; and a method of measuring the enzymatic activity of an amino acid oxidase using the reagent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a compound suitable for measuring the enzyme activity of an amino acid oxidase, a reagent for measuring the enzyme activity of an amino acid oxidase, and a method for measuring the enzyme activity of an amino acid oxidase. [Background technology]

[0002] D-amino acid oxidase (hereinafter sometimes referred to as "DAO") and L-amino acid oxidase (hereinafter sometimes referred to as "LAO") are both important enzymes. DAO has been suggested to be related to schizophrenia and amyotrophic lateral sclerosis (ALS), and DAO inhibitors are being developed as treatments for these diseases. Since snake venom LAO selectively induces cell death in cancer cells, new anticancer therapies using LAO are being researched. Therefore, there is a strong demand for probe molecules that can be used to study the activity of DAO and LAO and to search for enzyme activity inhibitors.

[0003] Conventionally, a two-step reaction method has been used to measure the activity of these enzymes. In the first step, D-amino acids or L-amino acids are reacted with the enzymes. In the second step, hydrogen peroxide generated in the first step is reacted with peroxidase and a color- or fluorogenic substrate. The resulting color or fluorescence can be used to measure the enzyme activity of DAO or LAO. However, this method is complicated, and because it involves an enzymatic reaction using peroxidase, it is not possible to evaluate the inhibitory activity against LAO or DAO in the case of substances that inhibit peroxidase activity or compounds that are decomposed by peroxidase, and it is therefore not possible to screen new enzyme activity inhibitors. In addition, because peroxidase does not enter cells, there is also the problem that it cannot be applied to intracellular enzyme activity imaging research.

[0004] It has been reported that the use of kynurenine (hereinafter sometimes referred to as "KYN") produces the fluorescent substance kynurenic acid by reacting with LAO or DAO (see, for example, Non-Patent Document 1). According to the report, the enzyme activity of LAO or DAO can be measured in a one-step enzyme reaction (see the following formula). [ka]

[0005] However, since the kynurenic acid is weakly fluorescent, it is necessary to add zinc ions to form a complex with strong fluorescence. Although it is possible to add zinc ions to form a complex and obtain strong fluorescence, the concentration of zinc in cells is not sufficient to form a fluorescent complex, which is an obstacle to application to intracellular enzyme activity imaging research, as in the case of the two-step reaction.

[0006] Therefore, there is a strong demand for the development of a probe molecule that can emit sufficient fluorescence after the enzymatic reaction and can evaluate the enzymatic activity of LAO or DAO in a one-step reaction without using a complicated two-step reaction. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Song ZY, Ogaya T, Ishii K, Ichiba H, Iizuka H, ​​Fukushima T. J Health Sci 2010; 56, 341-346. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to solve the above-mentioned conventional problems and achieve the following objects: That is, the present invention aims to provide a novel compound that can emit sufficient fluorescence after an enzymatic reaction and that can evaluate the enzymatic activity of D-amino acid oxidase or L-amino acid oxidase in a one-step reaction without using a complicated two-step reaction, a reagent for measuring the enzymatic activity of amino acid oxidase, and a method for measuring the enzymatic activity of amino acid oxidase. [Means for solving the problem]

[0009] As a result of intensive research conducted by the inventors to achieve the above-mentioned object, they discovered that by using a compound represented by structural formula (1) or a compound represented by structural formula (2), the activity of these enzymes can be detected with sufficient fluorescence intensity by a one-step enzymatic reaction with D-amino acid oxidase or L-amino acid oxidase.

[0010] The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows. <1> The compound is characterized by being represented by either of the following structural formulas (1) and (2). [ka] In the above structural formulas (1) and (2), "Me" represents a methyl group. <2> The present invention provides a reagent for measuring the enzyme activity of amino acid oxidase, comprising a compound represented by the following structural formula (1), a compound represented by the following structural formula (2), or a combination of these compounds. [ka] In the above structural formulas (1) and (2), "Me" represents a methyl group. <3> The method for measuring the enzyme activity of amino acid oxidase comprises measuring the enzyme activity of amino acid oxidase using any one of a compound represented by the following structural formula (1), a compound represented by the following structural formula (2), and a combination of these compounds. [ka] In the above structural formulas (1) and (2), "Me" represents a methyl group. <4> The above-mentioned method is used in screening for amino acid oxidases with high enzymatic activity. <3> This is a method according to the present invention. <5> The above-mentioned compound is used in screening for an inhibitor of the enzyme activity of amino acid oxidase. <3> This is a method according to the present invention. Effect of the Invention

[0011] According to the present invention, it is possible to solve the above-mentioned problems in the conventional art and achieve the above-mentioned object, and it is possible to provide a novel compound that is capable of emitting sufficient fluorescence after an enzymatic reaction and that enables evaluation of the enzymatic activity of D-amino acid oxidase or L-amino acid oxidase in a one-step reaction without using a complicated two-step reaction, a reagent for measuring the enzymatic activity of amino acid oxidase, and a method for measuring the enzymatic activity of amino acid oxidase. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a graph showing the results of fluorescence intensity at the maximum excitation fluorescence wavelength of each kynurenic acid ethyl ester in Test Example 1. [Figure 2A] FIG. 2A shows the results of DAO activity measurement in Test Example 2. [Figure 2B] FIG. 2B is a graph showing the results of measuring LAO activity in Test Example 2. [Figure 2C] FIG. 2C is a diagram showing an example of an excitation fluorescence spectrum. [Figure 2D] FIG. 2D is FIG. 1 showing the measurement results of LC-UV and LC-MS in Test Example 2. [Figure 2E] FIG. 2E is FIG. 2 showing the measurement results of LC-UV and LC-MS in Test Example 2. [Figure 3A] FIG. 3A shows the results of DAO activity measurement in Test Example 3. [Figure 3B] FIG. 3B is a graph showing the results of LAO activity measurement in Test Example 3. [Figure 3C] FIG. 3C is a diagram showing the state of the supernatant used in the fluorescence measurement in Test Example 3. [Figure 4A] FIG. 4A is a diagram showing the results when MPC was used in Test Example 4. [Figure 4B] FIG. 4B is a graph showing the results when CBIO was used in Test Example 4. [Figure 4C] FIG. 4C shows the results when βCl-L-Ala was used in Test Example 4. [Figure 4D] FIG. 4D shows the results when Anth was used in Test Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] (compound) The compound of the present invention is a compound represented by the following structural formula (1) (hereinafter, sometimes referred to as "MeS-D-KYN" or "5-MeS-D-KYN") or a compound represented by the following structural formula (2) (hereinafter, sometimes referred to as "MeS-L-KYN" or "5-MeS-L-KYN"). Each of the above compounds may also be simply referred to as a fluorescent probe. [ka] In the above structural formulas (1) and (2), "Me" represents a methyl group.

[0014] The method for producing the compound represented by the structural formula (1) or (2) is not particularly limited, and a known chemical synthesis method can be appropriately selected. For example, the compound can be produced by the method described in the section [Examples] below. The method described in the section of [Examples] below is merely an example. In addition, the reaction conditions such as reaction temperature and reaction time in the chemical synthesis, the compounds to be used and their amounts, the solvent, the purification method, etc. are not particularly limited and can be appropriately selected depending on the purpose.

[0015] Whether the obtained compound has a structure represented by the structural formula (1) or structural formula (2) can be confirmed by various analytical methods selected appropriately. The analytical method is not particularly limited and can be appropriately selected according to the purpose, and examples thereof include mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, carbon-13 nuclear magnetic resonance spectroscopy, elemental analysis, and the like. The analytical methods may be used alone or in combination of two or more. Although there may be some errors in the measured values ​​by each of the analytical methods, a person skilled in the art can easily identify that the compound has a structure represented by the structural formula (1) or structural formula (2).

[0016] The use of the compound represented by the structural formula (1) or (2) is not particularly limited and can be appropriately selected depending on the purpose, but can be suitably used as a reagent for measuring the enzyme activity of amino acid oxidase.

[0017] (Reagent for measuring the enzyme activity of amino acid oxidase) The reagent for measuring the enzyme activity of amino acid oxidase of the present invention contains at least any one of a compound represented by structural formula (1) of the present invention, a compound represented by structural formula (2), and a combination of these compounds, and further contains other components as necessary.

[0018] <A compound represented by structural formula (1), a compound represented by structural formula (2), or a combination of these compounds> The compound represented by the structural formula (1) and the compound represented by the structural formula (2) are the compound represented by the structural formula (1) and the compound represented by the structural formula (2) of the present invention described above. The reagent for measuring the enzymatic activity of amino acid oxidase may be in an embodiment that contains only the compound represented by structural formula (1) among the compounds, or may be in an embodiment that contains only the compound represented by structural formula (2), or may be in an embodiment that contains both the compound represented by structural formula (1) and the compound represented by structural formula (2) (a combination of the compound represented by structural formula (1) and the compound represented by structural formula (2)). The total amount of the compound represented by the structural formula (1) or (2) in the reagent for measuring the enzymatic activity of an amino acid oxidase is not particularly limited and may be appropriately selected depending on the purpose. The reagent for measuring the enzymatic activity of an amino acid oxidase may consist of only any one of the compound represented by the structural formula (1), the compound represented by the structural formula (2), and a combination of these compounds.

[0019] As described in the Examples section below, the compound represented by the structural formula (1) can be used to measure the activity of D-amino acid oxidase (EC number: 1.4.3.3), and the compound represented by the structural formula (2) can be used to measure the activity of L-amino acid oxidase (EC number: 1.4.3.2). The species from which the DAO and LAO are derived is not particularly limited and can be appropriately selected depending on the purpose. The DAO and LAO may be obtained from natural products or may be synthesized by biosynthesis or the like.

[0020] The compound represented by the structural formula (1) reacts with D-amino acid oxidase, and the compound represented by the structural formula (2) reacts with L-amino acid oxidase to produce a fluorescent compound. The activity of each enzyme can be evaluated by measuring the fluorescence.

[0021] <Other ingredients> The other components are not particularly limited as long as they do not impair the effects of the present invention and can be appropriately selected depending on the purpose, and examples thereof include a solvent for dissolving the compound represented by any one of the structural formulas (1) and (2). These may be used alone or in combination of two or more. The solvent is not particularly limited and can be appropriately selected depending on the purpose. For example, 0.1 M Tris-HCl buffer (pH 8.3) is included. The amount of the other components in the reagent for measuring the enzyme activity of amino acid oxidase is not particularly limited and can be appropriately selected depending on the purpose.

[0022] The reagent for measuring the enzyme activity of amino acid oxidase may be in a form in which the compound represented by structural formula (1), the compound represented by structural formula (2), or any combination of these compounds, and the other components as necessary, are contained in the same container, or may be in a form in which they are divided into separate containers and mixed at the time of use.

[0023] According to the reagent for measuring the enzyme activity of amino acid oxidase of the present invention, the enzyme activity of amino acid oxidase can be measured quickly and simply without using a complicated two-step reaction. In addition, the activity of D-amino acid oxidase and L-amino acid oxidase can be selectively measured. In addition, since the fluorescence wavelength generated by the enzyme reaction is in the visible region, it is also applicable to fluorescence imaging research.

[0024] (Method for measuring enzyme activity of amino acid oxidase) The method for measuring the enzyme activity of amino acid oxidase of the present invention includes at least a measurement step, and may further include other steps as necessary.

[0025] <Measurement process> The measurement step is a step of measuring the enzyme activity of amino acid oxidase using any one of a compound represented by structural formula (1) of the present invention, a compound represented by structural formula (2), and a combination of these compounds.

[0026] In the measurement step, any one of the compounds represented by structural formula (1) of the present invention, the compounds represented by structural formula (2), and a combination of these compounds is reacted with amino acid oxidase.

[0027] The composition of the reaction solution in the above reaction is not particularly limited and can be appropriately selected depending on the purpose. For example, when measuring the enzyme activity of D-amino acid oxidase, the reaction solution can contain D-amino acid oxidase, flavin adenine dinucleotide, bovine serum albumin, glutathione, and the compound represented by the structural formula (1). When the enzyme activity of L-amino acid oxidase is to be measured, the reaction solution may contain L-amino acid oxidase, bovine serum albumin, glutathione, and the compound represented by the structural formula (2). The amount of each component in the reaction solution is not particularly limited and can be appropriately selected depending on the purpose.

[0028] The reaction conditions for the reaction solution are not particularly limited and can be appropriately selected depending on the enzyme activity of the amino acid oxidase, and examples thereof include 37° C. and 60 minutes.

[0029] The enzyme activity of amino acid oxidase can be measured by measuring the fluorescence of the reaction solution after the reaction. The fluorescence can be measured using a known device at an excitation wavelength of 364 nm and a fluorescence wavelength of 450 nm.

[0030] <Other processes> The other steps are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the purpose.

[0031] The method for measuring the enzyme activity of amino acid oxidase of the present invention can also be performed in combination with other known methods.

[0032] According to the method for measuring the enzyme activity of amino acid oxidase of the present invention, the enzyme activity of amino acid oxidase can be measured quickly and simply without using a complicated two-step reaction. In addition, the activities of D-amino acid oxidase and L-amino acid oxidase can be selectively measured. In addition, since the fluorescence wavelength generated by the enzyme reaction is in the visible region, it is also applicable to fluorescence imaging research.

[0033] The method for measuring the enzyme activity of amino acid oxidase of the present invention can be suitably used, for example, in screening for amino acid oxidases with high enzyme activity, and screening for inhibitors of the enzyme activity of amino acid oxidase.

[0034] An example of the screening for amino acid oxidases with high enzymatic activity is screening for L-amino acid oxidases with high activity that can be used in anti-cancer therapy. The above screening can be carried out by adding the L-amino acid oxidase whose activity is to be measured to the above-mentioned reaction solution and carrying out the reaction. The L-amino acid oxidase whose activity is to be measured may be derived from a natural product, or may be synthesized by biosynthesis or the like.

[0035] Examples of screening for inhibitors of the enzyme activity of amino acid oxidase include screening for substances with high inhibitory activity (hereinafter sometimes referred to as "candidate substances") that can be used as therapeutic agents for schizophrenia, ALS, and the like. The above screening can be carried out by adding the candidate substance to the above reaction solution and carrying out the reaction. The candidate substance is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be derived from a natural product, or may be synthesized by chemical synthesis, biosynthesis, or the like. EXAMPLES

[0036] Test examples and production examples of the present invention are described below, but the present invention is not limited to these test examples and production examples.

[0037] (Test Example 1: Search for highly fluorescent kynurenic acid derivatives) We searched for kynurenic acid derivatives that emit stronger fluorescence.

[0038] <Synthesis of kynurenic acid derivatives> Using a substituted aniline represented by the following chemical formula 1 as a starting material, a kynurenic acid derivative represented by the following chemical formula 2 (kynurenic acid ethyl ester derivative) was synthesized. [ka]

[0039] Specifically, substituted aniline (chemical formula 1) and diethyl acetylenedicarboxylate were dissolved in methanol and refluxed at 80°C for 4 hours. After the reaction, the reaction solvent was distilled off, and the mixture was redissolved in 20 mL of diphenyl ether and heated at 220°C for another 4 hours. When the reaction solution was cooled to room temperature, a kynurenic acid derivative (chemical formula 2) precipitated. The precipitated solid was filtered, washed with n-hexane, and vacuum dried.

[0040] The kynurenic acid derivatives synthesized above and represented by Chemical Formula 2 are compounds represented by the following Chemical Formulae 2a to 2h. Identification data for the kynurenic acid derivatives synthesized together with the raw material substituted aniline are shown below.

[0041] <Chemical formula 2a: Ethyl 4-hydroxyquinoline-2-carboxylate> [ka] Substituted aniline raw material: Aniline (R=H) Identification data of the compound of formula 2a: 1H-NMR(400MHz, DMSO-D6) δ 12.03(s, 1H, OH), 8.07(dd, J=8.1, 1.3Hz, 1H, ArH), 7.93(d, J=8.5 Hz, 1H, ArH), 7.72-7.68(m, 1H, ArH), 7.38-7.34(m, 1H, ArH), 6.63(s, 1H, ArH), 4.41(q, J=7.1Hz, 2H, OCH 2 CH 3 ), 1.36(t, J=7.1Hz, 3H, OCH 2 CH 3 ), m / z [M+H] 218.0725 (calcd. 218.0738)

[0042] <Chemical formula 2b: Ethyl 6-acetyl-4-hydroxyquinoline-2-carboxylate> [ka] Raw material replacement アニリン: 4-アミノアセトフェノン(R=COCH 3 ) The chemical formula 2b is the same as the compound: 1H-NMR(400MHz, DMSO-D6) δ 12.29(s, 1H, OH), 8.63(d, J=2.1Hz, 1H, ArH), 8.19(dd, J=8.8, 2.1Hz, 1H, ArH), 8.00(d, J=8.8Hz, 1H, ArH), 6.70(d, J=1.7Hz, 1H, ArH), 4.42 (q, J=7.1Hz, 2H, OCH 2 CH 3 ), 2.64(s, 3H, COCH 3 ), 1.36(t, J=7.1Hz, 3H, OCH 2 CH 3 ), m / z [M+H] 260.0856 (calcd. 260.0845)

[0043] <Chemical formula 2c:Ethyl 4-hydroxy-6-methylquinoline-2-carboxylate>

change

[0044] <Chemical formula 2d:Ethyl 4-hydroxy-6-fluoroquinoline-2-carboxylate> [ka] Substituted aniline starting material: 4-fluoroaniline (R=F) Identification data for the compound of formula 2d: 1H-NMR(400MHz, DMSO-D6) δ 12.25(s, 1H, OH), 8.02(dd, J=9.2, 4.8Hz, 1H, ArH), 7.71(dd, J=9.3, 2.8Hz, 1H, ArH), 7.66-7.61(m, 1H, ArH), 4.41(q, J=7.1Hz, 2H, OCH 2 CH 3 ), 1.35(t, J=7.1Hz, 3H, OCH 2 CH 3 ), m / z [M+H] 236.0648 (calcd. 236.0645)

[0045] <Chemical formula 2e: Ethyl 6-(dimethylamino)-4-hydroxyquinoline-2-carboxylate> [ka] Substituted aniline raw material: N,N-dimethyl-p-phenylenediamine (R=N(CH 3 ) 2 ) Identification data for compound of formula 2e: 1H-NMR(400MHz, DMSO-D6) δ 11.92(s, 1H, OH), 7.84(d, J=9.2Hz, 1H, ArH), 7.35(d, J=8.8Hz, 1H, ArH), 7.13(s, 1H, ArH), 6.52(s, 1H, ArH), 4.42-4.36(q, J=7.1Hz, 2H, OCH 2 CH 3 ), 2.97(s, 6H, N(CH3 ) 2 ), 1.35(t, J=7.1Hz, 3H, OCH 2 CH 3 ), m / z [M+H] 261.1200 (calcd. 261.1161)

[0046] <Chemical formula 2f: Ethyl 4-hydroxy-6,7-dimethoxyquinoline-2-carboxylate> [ka] Substituted aniline raw material: 3,4-dimethoxyaniline (R=3,4-OCH 3 ) Identification data for compound of formula 2f: 1H-NMR(400MHz, DMSO-D6) δ 7.43(s, 1H, Ar-H), 7.40(s, 1H, Ar-H), 6.66(s, 1H, Ar-H), 4.38(q, J=7.1Hz, 2H, CH 2 CH 3 ), 3.85(s, 3H, OCH 3 ), 3.84(s, 3H, OCH 3 ), 1.35(t, J=7.1Hz, 3H, CH 2 CH 3 ), m / z [M+H] 278.1100 (calcd. 278.1023)

[0047] <Chemical formula 2g:Ethyl 4-hydroxy-6-methoxyquinoline-2-carboxylate> [ka] Substituted aniline raw material: 4-methoxyaniline (R=OCH 3 ) Identification data for compound of formula 2g: 1H-NMR (400 MHz, DMSO-D6) δ 12.08 (s, 1H, OH), 7.90 (d, J = 9.2 Hz, 1H, ArH), 7.46 (d, J = 3.0 Hz, 1H, ArH), 7.36 (dd, J = 9.2, 3.0 Hz, 1H, ArH), 6.60 (d, J = 1.8 Hz, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, OCH 2 CH 3 ), 3.83 (s, 3H, OCH 3 ), 1.35 (t, J = 7.1 Hz, 3H, OCH 2 CH 3 ), m / z [M + H] 248.1200 (calcd. 248.0917)

[0048] <Chemical formula 2h: Ethyl 4-hydroxy-6-(methylthio)quinoline-2-carboxylate>

Chem.

[0049] The compounds represented by the above chemical formulas 2a to 2h were dissolved in 50 μM methanol, and the fluorescence spectrum at the maximum excitation wavelength and the excitation spectrum at the maximum fluorescence wavelength of each compound (kynurenic acid ethyl ester) shown in Figure 1 were obtained using a Hitachi F-7000 spectrofluorometer. The results of the fluorescence intensity at the maximum excitation fluorescence wavelength of each kynurenic acid ethyl ester are shown in Figure 1. As shown in Figure 1, it was confirmed that the fluorescence intensities of the kynurenic acid derivatives vary, and that the compound represented by chemical formula 2h exhibits the strongest fluorescence intensity.

[0050] (Production Example 1) In Test Example 1, the compound represented by structural formula (1) (5-MeS-D-KYN) or the compound represented by structural formula (2) (5-MeS-L-KYN), which is a precursor of the kynurenic acid derivative represented by chemical formula 2h that showed the strongest fluorescence, was synthesized as follows.

[0051] [ka] A solution of boron trichloride in dichloromethane (ca. 1M, 5mL) was added to a solution of p-methylthioaniline (5.12mmol) in toluene (5mL). Then, anhydrous aluminum chloride (6.84mmol) and chloroacetonitrile (1mL) were added and the mixture was heated to reflux overnight. 1M hydrochloric acid (50mL) was added to the reaction solution cooled to room temperature and the mixture was refluxed for another hour. The reaction solution was transferred to a separatory funnel, and the product was extracted into chloroform (50mL x 2). The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The crude product was purified by column chromatography [silica gel, ethyl acetate / hexane (1:9)] to obtain 1-(2-amino-5-(methylthio)phenyl)-2-chloroethan-1-one as a yellow oil (1.09mmol, 18.7%). The identification data of 1-(2-amino-5-(methylthio)phenyl)-2-chloroethan-1-one was as follows. m / z [M+H] 216.02339 (calcd. 216.02499), 1H-NMR(400 MHz, CHLOROFORM-D) δ 7.68(d, J=2.1Hz, 1H, Ar-H), 7.37(dd, J=8.7, 2.1Hz, 1H, Ar-H), 6.67(d, J=8.7Hz, 1H, Ar-H), 6.34(brs, 2H, NH 2 ), 4.68(s, 2H, CH 2 ), 2.43 (s, 3H, SCH 3 )

[0052] [ka] 1-(2-amino-5-(methylthio)phenyl)-2-chloroethan-1-one (1.09mmol) and diethyl acetamidomalonate (1.12mmol) were dissolved in dry dimethylformamide (2mL), sodium ethoxide (1.21mmol) was added thereto, and the mixture was stirred overnight at room temperature under an argon atmosphere. The reaction solution was diluted with ethyl acetate (100mL), transferred to a separatory funnel, and then washed with water (50mL x 2). The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The crude product was purified by column chromatography [silica gel, ethyl acetate / hexane (1:1)] to obtain 2-acetamido-2-(2-(2-amino-5-(methylthio)phenyl)-2-oxoethyl)diethyl malonate as a brown oil (0.482mmol, 44%). The identification data for diethyl 2-acetamido-2-(2-(2-amino-5-(methylthio)phenyl)-2-oxoethyl)malonate was as follows. m / z [M+H] 397.14954 (calcd. 397.14333), 1H-NMR(400MHz, CHLOROFORM-D) δ 7.81(d, J=2.1Hz, 1H, AcNH), 7.33(dd, J=8.6, 2.2Hz, 1H, Ar-H), 7.09(s, 1H, Ar-H), 6.61(d, J=8.5Hz, 1H, Ar-H), 6.25(s, 2H, Ar-NH 2 ), 4.32-4.24(m, 4H, CH 2CH 3 ), 4.23-4.21(m, 2H, COCH 2 ), 2.43(s, 3H, SCH 3 ), 2.01(d, J=2.5Hz, 1H, α-CH), 1.99(s, 3H, Ac-H3), 1.27-1.23(m, 6H, CH 2 CH 3 )

[0053] [ka] 2-Acetamido-2-(2-(2-amino-5-(methylthio)phenyl)-2-oxoethyl)diethylmalonate (0.482mmol) was dissolved in ethanol (99.5, 10mL), 2M potassium hydroxide aqueous solution (10mL) was added thereto, and the mixture was heated under reflux for 3 hours. Glacial acetic acid (4mL) was added to the reaction solution, and the mixture was refluxed for another 0.5 hours. The reaction solution was diluted with ethyl acetate (100mL), transferred to a separatory funnel, and then washed with water (50mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off. The crude product was purified by column chromatography [silica gel, chloroform / methanol (7:3)] to obtain 2-acetamido-4-(2-amino-5-(methylthio)phenyl)-4-oxobutanoic acid as a yellow-brown oil (0.303mmol, 63%). The identifying data for 2-acetamido-4-(2-amino-5-(methylthio)phenyl)-4-oxobutanoic acid was as follows: m / z [M+H] 297.08603 (calcd. 297.09090), 1H-NMR (400MHz, METHANOL-D4) δ 7.70(d, J=2.1Hz, 1H, Ar-H), 7.24(dd, J=8.7, 2.1Hz, 1H, Ar-H), 6.66(d, J=8.7Hz, 1H, Ar-H), 4.81(m, 1H, α-CH), 3.57-3.39(m, 2H, COCH 2 ), 2.33(s, 3H, SCH 3 ), 1.92(d, J=4.8Hz, 3H, Ac-H3)

[0054] [ka] 2-Acetamido-4-(2-amino-5-(methylthio)phenyl)-4-oxobutanoic acid (0.303 mmol) was dissolved in 50 mM phosphate buffer (pH 7.4, 20 mL), and a catalytic amount of cobalt(III) chloride and D-aminoacylase were added thereto, followed by stirring at 37° C. for 40 hours. The reaction solution was concentrated and purified by column chromatography [ODS column, 0.2% acetic acid-acetonitrile / water (1:9) mixture] to obtain a fraction containing (S)-2-acetamido-4-(2-amino-5-(methylthio)phenyl)-4-oxobutanoic acid and 5-MeS-D-KYN as a pale yellow solid (0.0709 mmol, 23%). The identification data of 5-MeS-D-KYN was as follows: m / z [M+H] 255.98409 (calcd. 255.08034), 1H-NMR(400MHz, METHANOL-D4) δ 7.76(d, J=2.1Hz, 1H, Ar-H), 7.32(dd, J=8.7, 2.1Hz, 1H, Ar-H), 6.74(d, J=8.7Hz, 1H, Ar-H), 3.98(dd, J=9.2, 2.5Hz, 1H, CH), 3.68(d, J=2.7Hz, 1H, COCH 2 ), 3.51(m, 1H, COCH 2 ), 2.39(s, 3H, SCH 3 )

[0055] [ka] The fraction containing (S)-2-acetamido-4-(2-amino-5-(methylthio)phenyl)-4-oxobutanoic acid was concentrated and redissolved in 50 mM phosphate buffer (pH 7.4, 20 mL). A catalytic amount of cobalt(III) chloride and Acylase I derived from Aspergillus melleus were added thereto, and the mixture was stirred at 45 °C for 40 hours. The reaction solution was concentrated and purified by column chromatography [ODS column, 0.2% acetic acid-acetonitrile / water (1:9) mixture] to obtain 5-MeS-L-KYN as a pale yellow solid (0.0787 mmol, 26%). The identification data of 5-MeS-L-KYN were as follows. m / z [M+H] 255.98409 (calcd. 255.08034), 1H-NMR(400MHz, METHANOL-D4) δ 7.76(d, J=2.1Hz, 1H, Ar-H), 7.32(dd, J=8.5, 2.5Hz, 1H, Ar-H), 6.74(d, J=8.7Hz, 1H, Ar-H), 3.98(d, J=6.2Hz, 1H, CH), 3.71(d, J=18.1Hz, 1H, COCH 2 ), 3.54-3.49(m, 1H, COCH 2 ), 2.39 (s, 3H, SCH 3 )

[0056] (Test Example 2: Measurement of the activities of DAO and LAO) Using the compound of the present invention produced in Production Example 1, the activities of D- or L-amino acid oxidase were measured.

[0057] <Measurement of the activity of DAO> To 370 μL of 0.1 M Tris-HCl buffer (pH 8.3), 50 μL of 200 μM aqueous flavin adenine dinucleotide solution, 20 μL of 20 mg / mL aqueous bovine serum albumin solution, 20 μL of 1 mM aqueous glutathione solution, and 20 μL of DAO solution (derived from porcine kidney, solvent: 0.1 M Tris-Hcl buffer (pH 8.3), at concentrations of 0.064, 0.16, 0.32, 0.64, 1.6, 3.2, or 6.4 U / mL) prepared at each concentration were mixed and incubated at 37 °C for 20 minutes. Subsequently, 20 μL of 10 mM MeS-D-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)) was added and incubated at 37 °C for 60 minutes. Thereafter, 1.5 mL of methanol was added, and the supernatant obtained by protein removal was subjected to fluorescence measurement (excitation wavelength: 364 nm, fluorescence wavelength: 450 nm).

[0058] The results are shown in Fig. 2A. As shown in Fig. 2A, a high linear relationship was observed between the activity of DAO and the fluorescence intensity (y = 183.82x - 29.921, R 2 = 0.9972).

[0059] <Measurement of LAO activity> To 390 μL of 0.1 M Tris-Hcl buffer (pH 8.3), 20 μL of 20 mg / mL aqueous bovine serum albumin solution, 20 μL of 1 mM aqueous glutathione solution, and 20 μL of LAO solution (derived from Nisidaiyagarahagabi snake, solvent: 0.1 M Tris-HCl buffer (pH 8.3), at concentrations of 0.46, 2.3, 4.6, 11.5, 23, or 46 mU / mL) prepared at each concentration were mixed and incubated at 37 °C for 20 minutes. Subsequently, 20 μL of 10 mM MeS-L-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)) was added and incubated at 37 °C for 60 minutes. Thereafter, 1.5 mL of methanol was added, and the supernatant obtained by protein removal was subjected to fluorescence measurement (excitation wavelength: 364 nm, fluorescence wavelength: 450 nm).

[0060] The results are shown in Fig. 2B. As shown in Fig. 2B, a high linear relationship was observed between the activity of LAO and the fluorescence intensity (y = 20.974x + 6.7469, R 2 = 0.9995).

[0061] FIG. 2C shows an example of an excitation fluorescence spectrum. MeS-KYN emits blue fluorescence (λ 蛍光 It was confirmed that the compound was converted to kynurenic acid, which emits light at 450 nm. [ka]

[0062] After the above-mentioned enzyme reaction, the sample was subjected to fluorescence measurement and then measured by LC-UV and LC-MS (cation electrospray ionization). The measurement conditions were as follows. LCMS equipment: JEOL JMS-100LP “AccuTOF” LC-plus, Agilent 1200 HPLC system Column: XBridge C18 (2.1 x 50 mm, 3.5 μm) Flow rate: 0.3mL / min Column temperature: 40℃ Mobile phase A: 0.05% HCO 2 H in H 2 O Mobile phase B: 0.05% HCO 2 H in CH 3 OH Flow program: Mobile phase B = 3% (0-2 min), 3-100% (2-15 min), 100% (15 min-)

[0063] The results are shown in Figures 2D and 2E. As shown in Figures 2D and 2E, it was confirmed that a fluorescent substance was produced by the enzyme reaction.

[0064] The above results demonstrate that the compounds of the present invention enable fluorescence measurement of the enzyme activity of DAO or LAO.

[0065] (Test Example 3: Selectivity of LAO and DAO) The selectivity of the compound of the present invention produced in Production Example 1 for D- or L-amino acid oxidase was examined.

[0066] <Measurement of DAO Activity> To 370 μL of 0.1 M Tris-HCl buffer (pH 8.3), add 50 μL of 200 μM flavin adenine dinucleotide aqueous solution, 20 μL of 20 mg / mL bovine serum albumin aqueous solution, 20 μL of 1 mM glutathione aqueous solution, and 20 μL of DAO solution (derived from pig kidney, solvent: 0.1 M Tris-Hcl buffer (pH 8.3), 6.4 U / mL). Mix well and incubate at 37 °C for 20 minutes. Then, add 20 μL of 10 mM MeS-D-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)) or 20 μL of 10 mM MeS-L-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)), and incubate at 37 °C for 60 minutes. After that, add 1.5 mL of methanol to remove proteins, and measure the fluorescence of the supernatant (excitation wavelength: 364 nm, emission wavelength: 450 nm).

[0067] <Measurement of LAO Activity> To 390 μL of 0.1 M Tris-Hcl buffer (pH 8.3), add 20 μL of 20 mg / mL bovine serum albumin aqueous solution, 20 μL of 1 mM glutathione aqueous solution, and 20 μL of LAO solution (prepared at each concentration, derived from Naja naja atra, solvent: 0.1 M Tris-HCl buffer (pH 8.3), 46 mU / mL). Mix well and incubate at 37 °C for 20 minutes. Then, add 20 μL of 10 mM MeS-D-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)) or 20 μL of 10 mM MeS-L-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)), and incubate at 37 °C for 60 minutes. After that, add 1.5 mL of methanol to remove proteins, and measure the fluorescence of the supernatant (excitation wavelength: 364 nm, emission wavelength: 450 nm).

[0068] The results are shown in Figures 3A - 3C. In Figures 3A - 3C, (A) shows the results when MeS-D-KYN was used in the measurement of DAO activity, (B) shows the results when MeS-L-KYN was used in the measurement of DAO activity, (C) shows the results when MeS-D-KYN was used in the measurement of LAO activity, and (D) shows the results when MeS-L-KYN was used in the measurement of LAO activity.

[0069] As shown in FIGS. 3A to 3C, blue fluorescence occurred only when MeS-D-KYN was added to DAO and only when MeS-L-KYN was added to LAO. Therefore, it was confirmed that the compounds of the present invention can clearly distinguish between DAO and LAO.

[0070] (Test Example 4: Application to Screening) It was examined whether the compound of the present invention produced in Production Example 1 can be applied to the screening of enzyme activity inhibitors of DAO or LAO.

[0071] (Examination Using DAO Activity Inhibitor) To 370 μL of 0.1 M Tris-HCl buffer (pH 8.3), 50 μL of 200 μM flavin adenine dinucleotide aqueous solution, 20 μL of 20 mg / mL bovine serum albumin aqueous solution, 20 μL of 1 mM glutathione aqueous solution, DAO solution (derived from pig kidney, solvent: 0.1 M Tris-Hcl buffer (pH 8.3), 6.4 U / mL) 20 μL, and 1 μL of DMSO solution of DAO activity inhibitor (solution concentration: 10, 1.0, 0.1 mM (200, 20, 2 μM at the time of enzyme reaction)) were mixed and incubated at 37° C. for 20 minutes. Then, 20 μL of 10 mM MeS-D-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)) or 10 mM MeS-L-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)) was added and incubated at 37° C. for 60 minutes. Thereafter, 1.5 mL of methanol was added, and the supernatant obtained by removing proteins was measured for fluorescence (excitation wavelength: 364 nm, fluorescence wavelength: 450 nm). The DAO activity inhibitors used were 5-methyl-1H-pyrazole-3-carboxylic acid (MPC) or 6-chlorobenzo[d]isoxazol-3-ol (CBIO). [Chemical formula]

[0072] The results when using MPC are shown in Fig. 4A, and the results when using CBIO are shown in Fig. 4B.

[0073] <Examination using LAO activity inhibitor> To 390 μL of 0.1 M Tris-Hcl buffer (pH 8.3), 20 μL of 20 mg / mL bovine serum albumin aqueous solution, 20 μL of 1 mM glutathione aqueous solution, 20 μL of LAO solution (derived from Nisidaiyagaragara snake, solvent: 0.1 M Tris-HCl buffer (pH 8.3), 46 mU / mL), and 1 μL of DMSO solution of LAO activity inhibitor (solution concentration: 10, 1.0, 0.1 mM (200, 20, 2 μM during enzyme reaction)) were mixed and incubated at 37 °C for 20 minutes. Then, 20 μL of 10 mM MeS-L-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3)) was added and incubated at 37 °C for 60 minutes. Thereafter, 1.5 mL of methanol was added to remove proteins, and the supernatant was measured for fluorescence (excitation wavelength: 364 nm, fluorescence wavelength: 450 nm). The LAO activity inhibitors used were β-chloro-L-alanine (βCl-L-Ala) or anthranilic acid (Anth).

Chemical formula

[0074] The results when using βCl-L-Ala are shown in Fig. 4C, and the results when using Anth are shown in Fig. 4D.

[0075] As shown in Figs. 4A to 4D, by using the compound of the present invention, a decrease in enzyme activity corresponding to the added amount of the enzyme activity inhibitor could be detected. Therefore, it was shown that the compound of the present invention can also be applied to screening.

Claims

1. A compound represented by any one of the following structural formulas (1) and (2): 【Chemistry 1】 In the above structural formulas (1) and (2), "Me" represents a methyl group.

2. A reagent for measuring the enzyme activity of amino acid oxidase, comprising any one of a compound represented by the following structural formula (1), a compound represented by the following structural formula (2), and a combination of these compounds: 【Chemistry 2】 In the above structural formulas (1) and (2), "Me" represents a methyl group.

3. A method for measuring the enzyme activity of amino acid oxidase, comprising measuring the enzyme activity of amino acid oxidase using any one of a compound represented by the following structural formula (1), a compound represented by the following structural formula (2), and a combination of these compounds: 【Chemistry 3】 In the above structural formulas (1) and (2), "Me" represents a methyl group.

4. The method according to claim 3, which is used in screening for amino acid oxidases with high enzymatic activity.

5. The method according to claim 3, which is used in screening for inhibitors of the enzyme activity of amino acid oxidase.

Images