Compound, enzyme activity assay agent, enzyme activity assay kit, and method for measuring enzyme activity

Abstract

To provide a novel compound capable of excellently evaluating the enzyme activity of an amino acid oxidative enzyme, an agent for measuring the enzyme activity of the amino-acid oxidative enzyme, a kit for measuring the enzyme activity of the amino-acid oxidative enzyme, and a method for measuring the enzyme activity of the amino-acid oxidative enzyme.SOLUTION: The present embodiment is a compound represented by formula (1) or a salt or solvate thereof. (R1-R5 are each independently a hydrogen atom or a C1-C4 alkyl group.)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, a kit 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 enzyme. 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.

[0004] 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.

[0005] On the other hand, Non-Patent Document 1 reports that the use of kynurenine (hereinafter sometimes referred to as "KYN") produces the fluorescent substance kynurenic acid by reaction with LAO or DAO. According to the report, the enzyme activity of LAO or DAO can be measured in a one-step enzyme reaction.

[0006] 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.

[0007] Therefore, there is a need for the development of a probe molecule that can emit sufficient fluorescence after an enzyme reaction and can evaluate the enzyme activity of LAO or DAO in a one-step reaction without using a complicated two-step reaction. Patent Document 1 discloses, as such a probe molecule, a compound (MeS-D-KYN or MeS-L-KYN) characterized by being represented by either of the following structural formulas (1) and (2). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2023-22662 A [Non-patent literature]

[0009] [Non-Patent Document 1] Song Y, Ogaya T, Ishii K, Ichiba H, Iizuka H, ​​Fukushima TJ Health Sci 2010; 56,341-346. Summary of the Invention [Problem to be solved by the invention]

[0010] As described above, Patent Document 1 discloses a compound represented by a given structural formula (1) or (2) (MeS-D-KYN or MeS-L-KYN) as a probe molecule capable of evaluating the enzymatic activity of LAO or DAO in a one-step reaction. However, in consideration of application to research on the enzymatic activity of LAO or DAO, research on enzyme inhibitors, or research on enzyme activity imaging, there is a demand for the development of a novel compound with even superior detection sensitivity.

[0011] Therefore, an object of the present invention is to provide a novel compound capable of effectively evaluating the enzymatic activity of amino acid oxidase (D-amino acid oxidase or L-amino acid oxidase), a reagent for measuring the enzymatic activity of amino acid oxidase, a kit 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]

[0012] As a result of intensive research conducted by the inventors in order to achieve the above-mentioned object, they discovered that the enzyme activity of amino acid oxidase can be detected with high fluorescence intensity by using a compound represented by formula (1) or a salt or solvate thereof, and thus arrived at the present invention.

[0013] Thus, an embodiment of the present invention is as follows. (1) A compound represented by the following formula (1) or a salt or solvate thereof: [ka] (In formula (1), R1 to R5 are each independently a hydrogen atom or a C1 to C4 alkyl group). (2) The compound according to (1), or a salt or solvate thereof, represented by any one of the following formulas (1-D) and (1-L): [ka] (In formulas (1-D) and (1-L), R1 to R5 each independently represent a hydrogen atom or a C1 to C4 alkyl group). (3) The compound according to (1), or a salt or solvate thereof, represented by any one of the following formulas: [ka] (4) A reagent for measuring the enzyme activity of amino acid oxidase, comprising the compound according to any one of (1) to (3) or a salt or solvate thereof. (5) A kit for measuring the enzyme activity of amino acid oxidase, comprising at least the compound according to any one of (1) to (3) or a salt or solvate thereof, or the reagent for measuring the enzyme activity of amino acid oxidase according to (4). (6) A method for measuring the enzyme activity of an amino acid oxidase, comprising measuring the enzyme activity of the amino acid oxidase using the compound according to any one of (1) to (3) or a salt or solvate thereof, the reagent for measuring the enzyme activity of an amino acid oxidase according to (4), or the kit for measuring the enzyme activity of an amino acid oxidase according to (5). (7) The method according to (6), which is used in screening for amino acid oxidase. (8) The method according to (6), which is used in screening for an inhibitor of the enzyme activity of amino acid oxidase. (9) A compound represented by the following formula (2), or a salt or solvate thereof: [ka] (In formula (2), R1 to R5 are each independently a hydrogen atom or a C1 to C4 alkyl group). (10) The compound according to (9), or a salt or solvate thereof, represented by any one of the following formulas: [ka] (11) A method for measuring the enzyme activity of an amino acid oxidase, comprising detecting fluorescence of the compound according to (9) or (10) or a salt or solvate thereof. (12) The method according to (11), which is used in screening for amino acid oxidase. (13) The method according to (11), which is used in screening for an inhibitor of the enzyme activity of amino acid oxidase. Effect of the Invention

[0014] According to the present invention, it is possible to provide a novel compound capable of effectively evaluating the enzymatic activity of an amino acid oxidase, a reagent for measuring the enzymatic activity of an amino acid oxidase, a kit for measuring the enzymatic activity of an amino acid oxidase, and a method for measuring the enzymatic activity of an amino acid oxidase. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a graph showing the fluorescence quantum yields measured for Example compounds 3a to 3d and a comparative compound (MeS-KYNA). [Diagram 2] FIG. 2 is a graph showing the relative fluorescence intensity of compound 3b of the example. [Diagram 3] FIG. 3 is a graph showing the relationship between DAO or LAO concentration (enzyme activity) and fluorescence intensity when Example Compound 3b was used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] (compound) The compound according to this embodiment is a compound represented by the following formula (1), or a salt or solvate thereof: [ka] (In formula (1), R1 to R5 are each independently a hydrogen atom or a C1 to C4 alkyl group).

[0017] The compound according to this embodiment can be used as a fluorescent probe for the enzyme activity of amino acid oxidase, and can be suitably used for detecting and measuring the enzyme activity. The compound according to this embodiment can be suitably used as an enzyme activity measuring reagent for measuring the enzyme activity of amino acid oxidase. The compound according to this embodiment may be a racemate or an enantiomer (D-form or L-form).

[0018] The compound according to the present embodiment may exist in the form of a salt. The salt is not particularly limited, but may be, for example, a sulfate, a hydrochloride, a nitrate, a phosphate, a carbonate, an acetate, a propionate, a maleate, a malonate, a benzoate, a succinate, a sodium salt, or a potassium salt.

[0019] The compounds according to the present invention may exist in solvated and non-solvated forms. The term "solvate" refers to a physical association of the compounds according to the present invention with one or more solvent molecules, for example, by hydrogen bonding. The solvent molecules in a solvate may exist in an ordered and / or non-ordered arrangement. A solvate may contain either stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses both solution-phase and isolable solvates. The compounds according to the present invention may form solvates with water (i.e., hydrates) or common organic solvents. Methods of solvation are generally known in the art.

[0020] In formula (1), the C1-C4 alkyl group means an alkyl group having 1 to 4 carbon atoms, and the alkyl group may be linear or branched. Examples of the C1-C4 alkyl group include a methyl group, an ethyl group, a propyl group, and a butyl group.

[0021] The compound according to this embodiment is preferably a compound represented by any one of the following formulas (1-D) and (1-L), or a salt or solvate thereof: [ka] (In formulas (1-D) and (1-L), R1 to R5 each independently represent a hydrogen atom or a C1 to C4 alkyl group).

[0022] In formulae (1-D) and (1-L), the C1-C4 alkyl group means an alkyl group having 1 to 4 carbon atoms, and the alkyl group may be linear or branched. Examples of the C1-C4 alkyl group include a methyl group, an ethyl group, a propyl group, and a butyl group.

[0023] The compound represented by formula (1-D) or (1-L) is an optically active substance. In this specification, the term "optically active substance" is used to mean non-racemic. The optically active substance according to this embodiment is a D-form represented by formula (1-D) or an L-form represented by formula (1-L), and the optical isomer ratio of either the D-form or the L-form is preferably 90% or more. By using an optically active substance as the compound according to this embodiment, the enzyme activity of DAO or LAO can be directly detected and measured, thereby improving the detection sensitivity.

[0024] The compound according to this embodiment is more preferably a compound represented by any one of the following formulae, or a salt or solvate thereof: [ka] TIFF2024178850000009.tif145167

[0025] The compound according to this embodiment is a compound represented by the following formula (2), or a salt or solvate thereof: [ka] (In formula (2), R1 to R5 are each independently a hydrogen atom or a C1 to C4 alkyl group).

[0026] The compound according to the present embodiment can be used as a fluorescent substance to be detected in the measurement of the enzyme activity of amino acid oxidase, and can be used effectively for the detection and measurement of the enzyme activity. In addition, since the compound has a strong fluorescence intensity, it can also be used for labeling substances such as substances in the body.

[0027] In formula (2), the C1-C4 alkyl group means an alkyl group having 1 to 4 carbon atoms, and the alkyl group may be linear or branched. Examples of the C1-C4 alkyl group include a methyl group, an ethyl group, a propyl group, and a butyl group.

[0028] The compound according to this embodiment is preferably a compound represented by any one of the following formulae, or a salt or solvate thereof: [ka]

[0029] The method for producing the compound according to the present embodiment 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.

[0030] The method described in the section of [Examples] below is one example. In addition, the reaction conditions such as reaction temperature and reaction time in chemical synthesis, the compounds used and their amounts, the solvent, the purification method, etc. are not particularly limited and can be appropriately selected depending on the purpose.

[0031] Whether the obtained compound has a structure represented by formula (1) or formula (2) can be confirmed by various analytical methods selected appropriately. There is no particular limitation on the analytical method, and it can be appropriately selected according to the purpose, for example, mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, carbon-13 nuclear magnetic resonance spectroscopy, elemental analysis, etc. The analytical method may be used alone or in combination with two or more. Although there may be some errors in the measured values ​​by each of the above analytical methods, a person skilled in the art can easily identify that the compound has a structure represented by formula (1) or formula (2).

[0032] (Reagent for measuring the enzyme activity of amino acid oxidase) The reagent for measuring the enzyme activity of amino acid oxidase of this embodiment contains at least the compound according to this embodiment (the compound of formula (1)). The reagent for measuring the enzyme activity of this embodiment may further contain an additive, if necessary, in addition to the compound according to this embodiment.

[0033] As shown in the section of [Examples] below, the compound represented by formula (1) can be used to measure the activity of amino acid oxidase. In addition, the compound represented by formula (1-D) can be used to measure the activity of D-amino acid oxidase (EC number: 1.4.3.3), and the compound represented by formula (1-L) can be used to measure the activity of L-amino acid oxidase (EC number: 1.4.3.2). The species from which DAO and LAO are derived are not particularly limited and can be appropriately selected depending on the purpose. In addition, DAO and LAO may be obtained from natural products or may be synthesized by biosynthesis or the like.

[0034] The compound of formula (1-D) of this embodiment reacts with D-amino acid oxidase, and the compound of formula (1-L) reacts with L-amino acid oxidase to produce a fluorescent compound (fluorescent substance). The activity of each enzyme can be evaluated by measuring the fluorescence. The compound of this embodiment (compound represented by formula (1)) can be a precursor of a fluorescent substance (compound represented by formula (2)).

[0035] The reagent for measuring enzyme activity according to this embodiment may be essentially composed of the compound according to this embodiment, or may further contain an additive in addition to the compound according to this embodiment.

[0036] In this specification, "consisting essentially of" preferably means that everything other than the compound according to this embodiment is an impurity, and specifically, it preferably means that the content of the compound according to this embodiment in the reagent is 95% by mass or more, preferably means that it is 96% by mass or more, preferably means that it is 97% by mass or more, preferably means that it is 98% by mass or more, preferably means that it is 99% by mass or more, and preferably means that it is 99.5% by mass or more.

[0037] The additives are not particularly limited and can be appropriately selected according to the purpose. Examples of the additives include stabilizers. The additives may be used alone or in combination of two or more.

[0038] According to the reagent for measuring enzyme activity of this embodiment, the enzyme activity of amino acid oxidase can be measured quickly, easily and efficiently without using a complicated two-step reaction.

[0039] (Amino acid oxidase enzyme activity measurement kit) The enzyme activity measuring kit of the present embodiment for amino acid oxidase contains at least the compound of the present embodiment (the compound represented by formula (1)) or the enzyme activity measuring reagent of the present embodiment. The enzyme activity measuring kit of the present embodiment may contain other materials as necessary, in addition to the compound of the present embodiment or the enzyme activity measuring reagent. The other materials refer to materials necessary for enzyme activity measurement, and examples thereof include solvents, solubilizing agents, vitamins, coenzymes, pH adjusters, buffers, radical scavengers, and isotonic agents. The other components may be used alone or in combination of two or more.

[0040] In the enzyme activity measuring kit according to the present embodiment, the compound according to the present embodiment or the enzyme activity measuring reagent according to the present embodiment and other materials are preferably stored in separate containers and mixed at the time of use. The other materials may also be stored in separate containers, or at least two of them may be mixed and placed in the same container.

[0041] According to the kit for measuring enzyme activity of this embodiment, the enzyme activity of amino acid oxidase can be measured quickly, easily and efficiently without using a complicated two-step reaction.

[0042] (Method for measuring enzyme activity of amino acid oxidase) The method for measuring the enzyme activity of an amino acid oxidase according to the present embodiment includes at least a step of measuring the enzyme activity of an amino acid oxidase using a compound according to the present embodiment (a compound represented by formula (1)), a reagent for measuring the enzyme activity according to the present embodiment, or a kit for measuring the enzyme activity according to the present embodiment. The method for measuring the enzyme activity according to the present embodiment may further include other steps as necessary.

[0043] The measurement step in this embodiment is a step of measuring the enzyme activity of amino acid oxidase using the compound according to this embodiment.

[0044] The composition of the reaction solution in the measurement step is not particularly limited and can be appropriately selected depending on the purpose.

[0045] For example, when measuring the enzyme activity of D-amino acid oxidase, the reaction solution may contain D-amino acid oxidase, flavin adenine dinucleotide, bovine serum albumin, glutathione, and the compound according to the present embodiment. When measuring the enzyme activity of L-amino acid oxidase, the reaction solution may contain L-amino acid oxidase, bovine serum albumin, glutathione, and the compound according to the present embodiment.

[0046] The amount of each component in the reaction solution is not particularly limited and can be appropriately selected depending on the purpose.

[0047] The reaction conditions are not particularly limited and can be appropriately selected depending on the enzyme activity of the amino acid oxidase, and examples include 37° C. and 60 minutes.

[0048] 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, for example, using a known device at an excitation wavelength of 340 nm and a fluorescence wavelength of 418 nm.

[0049] The other steps are not particularly limited and can be appropriately selected depending on the purpose.

[0050] The method for measuring the enzyme activity of amino acid oxidase according to the present embodiment can also be performed in combination with other known methods.

[0051] According to the enzyme activity measurement method of the present embodiment, the enzyme activity of amino acid oxidase can be measured quickly, easily and efficiently without using a complicated two-step reaction. In addition, the activities of D-amino acid oxidase and L-amino acid oxidase can be measured selectively. In addition, since the fluorescence wavelength generated by the enzyme reaction is in the visible region, it is also applicable to fluorescence imaging research.

[0052] The enzyme activity measuring method according to this embodiment can be suitably used, for example, in screening of amino acid oxidase, particularly amino acid oxidase having high enzyme activity, and screening of enzyme activity inhibitors of amino acid oxidase.

[0053] Screening of amino acid oxidases includes, for example, screening of L-amino acid oxidases with high activity that can be used in anticancer therapy. Screening can be performed by adding the L-amino acid oxidase to be measured for activity to the above-mentioned reaction solution and carrying out a reaction. The L-amino acid oxidase to be measured for activity may be derived from a natural product or may be synthesized by biosynthesis or the like.

[0054] Screening for inhibitors of the enzyme activity of amino acid oxidase can be, for example, screening for substances with high inhibitory activity (hereinafter, sometimes referred to as "candidate substances") that can be used as therapeutic agents for schizophrenia, ALS, etc. Screening can be performed by adding the candidate substance to the above-mentioned reaction solution and carrying out a reaction. There are no particular limitations on the candidate substance, and it 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

[0055] Test examples and production examples of this embodiment will be described below, but the present invention is not limited to these test examples and production examples.

[0056] [Experimental Example 1: Search for highly fluorescent kynurenic acid compounds] We searched for kynurenic acid derivatives that emit stronger fluorescence.

[0057] (Synthesis of phenyl-substituted kynurenic acid compounds) Scheme S1: Synthetic route to phenyl-substituted kynurenic acid compounds (fluorophores) [ka]

[0058] Compound 1: Ethyl 6-bromokynurenate [ka]

[0059] Diethyl acetylenedicarboxylate (40.1 mmol) was slowly added to a solution of 4-bromoaniline (39.7 mmol) in MeOH (100 mL) and the mixture was stirred at 65° C. for 0.5 h. The solvent was removed under reduced pressure. The residue was redissolved in diphenyl ether (100 mL) and heated at 250° C. for 1 h. The solution was cooled to room temperature and precipitated by dilution with hexane (200 mL). The precipitate was filtered, washed with hexane, and recrystallized from hot ethanol to give compound 1 as a grey crystalline solid (53% yield).

[0060] m / z 295.99402 (calced for [M+H] 295.99223); mp 246-251℃; 1H-NMR (400 MHz, DMSO-D6) δ 12.21 (s, 1H, OH), 8.14 (d, J = 2.1 Hz, 1H, ArH), 7.90-7.83 (m, 2H, ArH), 6.68 (s, 1H, ArH), 4.41 (q, J = 7.1 Hz, 2H, OCH2CH3), 1.35 (t, J = 7.1 Hz, 3H, OCH2CH3); 13C-NMR (101 MHz, DMSO-D6) δ 176.3, 161.9, 138.9, 138.1, 135.3, 127.2, 126.8, 122.1, 116.9, 110.4, 62.7, 13.9.

[0061] Compound 2: 6-Bromokynurenic acid (Br-KYNA) [ka]

[0062] Compound 1 (10.0 mmol) and potassium hydroxide (45.0 mmol) were dissolved in 50% water / ethanol (40 mL) and stirred at room temperature for 2 hours. The solution was acidified with 2 M hydrochloric acid to precipitate compound 2. The precipitate was filtered, washed with water, and dried under reduced pressure to give a pale orange solid (yield 89%).

[0063] m / z 267.96456 (calced for [M+H] 267.96093); mp 283-286℃; 1H-NMR (400 MHz, DMSO-D6) δ 12.16 (s, 1H, OH), 8.14 (d, J = 2.3 Hz, 1H, ArH), 7.91 (d, J = 8.9 Hz, 1H, ArH), 7.86-7.83 (m, 1H, ArH), 6.65 (s, 1H, ArH); 13C-NMR (101 MHz, 5% NaOD-D2O) δ 174.9, 172.8, 156.7, 147.4, 132.5, 129.4, 127.4, 125.4, 116.9, 107.1.

[0064] Compound 3a: 6-Phenylkynurenic acid (Ph-KYNA) [ka]

[0065] Compound 2 (Br-KYNA) (1.02 mmol), phenylboronic acid (1.14 mmol), and potassium carbonate (2.05 mmol) were dissolved in water (50 mL). A catalytic amount (approximately 5 mg) of palladium acetate was added and stirred for 3 h. The solution was acidified with 1 M hydrochloric acid, and the resulting precipitate was filtered, washed with water, and dried under reduced pressure to give a white solid (yield 96%).

[0066] m / z 266.07989 (calced for [M+H] 266.08172); mp 271-273℃; 1H-NMR (400 MHz, 5% NaOD-D2O) δ 7.98 (s, 1H, ArH), 7.48 (s, 2H, ArH), 7.32 (d, J = 7.3 13C-NMR (101 MHz, 5% NaOD-D2O) δ 175.0, 174.0, 156.4, 148.2, 140.0, 135.5, 129.1, 128.6, 128.1, 127.5, 126.8, 126.3, 120.7, 106.9.

[0067] Compound 3b: 6-(2-Methylphenyl)kynurenic acid (2-MePh-KYNA) [ka]

[0068] Compound 2 (Br-KYNA) (2.00 mmol), 2-methylphenylboronic acid (2.19 mmol), and potassium carbonate (10.6 mmol) were dissolved in water (50 mL). Palladium acetate (10.3 μmol) was added to this solution, and the mixture was reacted under heating and reflux for 1 hour. The resulting reaction solution was acidified with 1 M HCl, and the precipitate was filtered, washed with water, and dried under reduced pressure to obtain a white solid (yield 85%).

[0069] m / z 280.09846 (calced for [M+H] 280.09737); mp 255-259℃; 1H-NMR (400 MHz, DMSO-D6) δ 12.06 (s, 1H, OH), 8.01 (d, J = 8.7 Hz, 1H, ArH), 7.96 13C-NMR (101 MHz, DMSO-D6) δ 177.6, 163.8, 140.4, 139.8, 139.0, 136.8, 134.8, 133.5, 130.5, 129.7, 127.6, 126.1, 125.6, 124.3, 119.6, 109.8, 20.2.

[0070] Compound 3c: 6-(4-Methylphenyl)kynurenic acid (4-MePh-KYNA) [ka]

[0071] Compound 2 (Br-KYNA) (1.00 mmol), 4-methylphenylboronic acid (1.32 mmol), and potassium carbonate (2.34 mmol) were dissolved in water (50 mL). A catalytic amount (approximately 5 mg) of palladium acetate was added to the solution, which was then stirred for 1 h. The resulting solution was acidified with 1 M HCl, and the precipitate was filtered, washed with water, and dried under reduced pressure to give a white solid (100% yield).

[0072] m / z 280.09906 (calced for [M+H] 280.09737); mp 274-278℃; 1H-NMR (400 MHz, DMSO-D6) δ 12.03 (s, 1H, OH), 8.24 (t, J = 1.1 Hz, 1H, ArH), 7.99 13C-NMR (101 MHz, 10% METHAOL-D4 / PYRIDINE-D5) δ 179.8, 165.5, 148.9, 142.1, 139.9, 137.4, 131.4, 130.0, 127.0, 127.0, 124.0, 123.1, 120.0, 110.7, 20.7.

[0073] Compound 3d: 6-(3,5-Dimethylphenyl)kynurenic acid (3,5-diMePh-KYNA) [ka]

[0074] Compound 2 (Br-KYNA) (0.996 mmol), 3,5-dimethylphenylboronic acid (1.04 mmol) and potassium carbonate (2.76 mmol) were dissolved in water (50 mL). A catalytic amount (approximately 5 mg) of palladium acetate was added to the solution, which was stirred under reflux for 1 h and then acidified with 1 M HCl. The precipitate was filtered, washed with water and dried under reduced pressure to give a pale yellow solid (yield 89%).

[0075] m / z 294.11384 (calced for [M+H] 294.11302); mp 273-275℃; 1H-NMR (400 MHz, DMSO-D6) δ 12.06 (s, 1H, OH), 8.27 (t, J = 1.4 Hz, 1H, ArH), 8.01 13C-NMR (101 MHz, DMSO-D6) δ 163.7, 139.1, 138.1, 135.8, 131.2, 129.1, 124.5, 121.7, 21.0

[0076] [evaluation] (Measurement of fluorescence quantum yield of phenyl-substituted kynurenic acid compounds) 10 μL of each phenyl-substituted kynurenic acid compound solution (compounds 3a to 3d) (DMSO solution, 10 mM) was diluted with PBS to 10 μM to prepare a sample solution. These solutions were measured to obtain the fluorescence quantum yield (Φ KYNA ) to the fluorescence quantum yield (Φ Quinine ) was calculated as the relative fluorescence quantum yield.

[0077] The formula used to calculate the relative fluorescence quantum yield is as follows: Φ Quinine The value was calculated as 0.55.

[0078]

number

[0079] The results of measuring the fluorescence quantum yield for each of the compounds 3a to 3d are shown in Figure 1. For comparison, the results of measuring the fluorescence quantum yield for the compound (MeS-KYNA) represented by the following formula are also shown in Figure 1. The compound used was synthesized by the method described in Patent Document 1 (JP Patent Publication 2023-22662 A). [ka]

[0080] As shown in FIG. 1, the fluorescence quantum yields of the phenyl-substituted kynurenic acid compounds 3a to 3d were higher than that of MeS-KYNA.

[0081] Example 2: Synthesis of phenyl-substituted kynurenine compounds In Example 1, 2-MePh-D,L-KYN (2-MePh-D-KYN or 2-MePh-L-KYN) as a compound according to the present embodiment, which is a precursor of the kynurenic acid derivative of compound 3b that exhibited the strongest fluorescence, was synthesized by the following method.

[0082] (Synthesis of 2-MePh-D,L-KYN) Scheme S2: Synthesis of 2-MePh-D,L-KYN [ka]

[0083] Compound 4: 1-(2-Amino-5-bromophenyl)-2-chloroethan-1-one [ka]

[0084] To a solution of 4-bromoaniline (66.3 mmol) in toluene (72 mL), boron trichloride (approximately 1 M, dichloromethane solution, 72 mL), anhydrous aluminum chloride (75.1 mmol) and chloroacetonitrile (5 mL) were added at room temperature. The mixture was then heated at reflux temperature under an argon atmosphere for 16 hours. After the reaction solution was cooled to room temperature, 0.5 M hydrochloric acid (100 mL) was added. The target compound was extracted into chloroform (200 mL x 3). Next, the organic layer was dried over anhydrous sodium sulfate, and the drying agent was filtered off, after which the solvent was distilled off. Toluene (50 mL) and hexane (200 mL) were added to the residue to solidify it. The supernatant was discarded, and the remaining solid was dried under reduced pressure to obtain compound 4 as a brown solid (yield 14%).

[0085] m / z 247.95136 (calced for [M+H] 247.94778); mp 129-132℃; 1H-NMR (400 MHz, CHLOROFORM-D) δ 7.72 (d, J = 2.3 Hz, 1H, ArH), 7.36 (dd, J = 8.8, 2.2 Hz, 1H, ArH), 6.60 (d, J = 8.9 Hz, 1H, ArH), 6.35 (s, 2H, NH2), 4.64 (s, 2H, CH2); 13C-NMR (101 MHz, CHLOROFORM-D) δ 191.6, 149.9, 137.9, 132.7, 119.3, 116.2, 106.8, 46.3.

[0086] Compound 5: 2-Acetamido-4-(2-amino-5-bromophenyl)-4-oxobutanoic acid [ka]

[0087] Compound 4 (37.1 mmol), diethyl acetamidomalonate (38.0 mmol), sodium ethoxide (77.6 mmol), sodium iodide (3.96 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (3 mL) were dissolved in ethanol (100 mL). The solution was stirred under reflux for 3 hours, transferred to a separatory funnel, saturated sodium bicarbonate water (100 mL) was added, and extracted into chloroform (100 mL x 3). The organic layer was dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the solvent was evaporated under reduced pressure. The residue was roughly purified by chromatography (silica gel, 50% ethyl acetate AcOEt / hexane), and the crude intermediate and sodium hydroxide (103 mmol) were dissolved in water (100 mL). The mixture was heated to reflux for 30 minutes, then glacial acetic acid (10 mL) was added, and the mixture was heated to reflux for another 1.5 hours. After the reaction, the mixture was transferred to a separatory funnel and extracted with ethyl acetate (100 mL x 4). The organic layer was dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the solvent was evaporated under reduced pressure. The residue was purified by chromatography (ODS column, acetic acid / water / methanol [0.1:50:50]) and further recrystallized from hot water to obtain compound 5 (yield 9.5%).

[0088] m / z 329.01680 (calced for [M+H] 329.01369); mp 177-180℃; 1H-NMR (400 MHz, DMSO-D6) δ 12.59 (s, 1H, CO2H), 8.11 (d, J = 8.0 Hz, 1H, ArH), 7.84 (d, J = 2.3 Hz, 1H, ArH), 7.36 (dd, J = 8.9, 2.3 Hz, 1H, ArH), 7.32 (s, 2H, NH2), 6.75 (d, J = 8.9 Hz, 1H, NHAc), 4.70-4.65 (m, 1H, α-CH2), 3.35-3.33 (m, 2H, β-CH2), 1.81 (s, 3H, Ac); 13C-NMR (101 MHz, DMSO-D6) δ 197.5, 173.1, 169.1, 150.2, 136.8, 132.9, 119.4, 117.5, 104.6, 48.0, 40.3, 22.4.

[0089] Compound 6: 2-Acetamido-4-(4-amino-2'-methyl-[1,1'-biphenyl]-3-yl)-4-oxobutanoic acid [ka]

[0090] Compound 5 (2.09 mmol), 2-methylphenylboronic acid (2.24 mmol) and potassium carbonate (4.20 mmol) were dissolved in 50% water / methanol (20 mL), and a catalytic amount (approximately 5 mg) of palladium acetate was added to the solution. The mixture was stirred at room temperature for 1 h and at reflux temperature for an additional 2 h. The reaction solution was adjusted to an acidic pH with hydrochloric acid and purified by ODS chromatography column (water / methanol [1:1] as mobile phase). After column, the target fraction was concentrated to obtain compound 6 as a white solid (yield 59%).

[0091] m / z 341.14693 (calced for [M+H] 341.15013); mp 177-180℃; 1H-NMR (400 MHz, DMSO-D6) δ 12.55 (s, 1H, CO2H), 8.10 (d, J = 7.8 Hz, 1H, AcNH), 7.64 (d, J = 2.1 Hz, 1H, ArH), 7.29-7.24 (m, 4H, ArH), 7.23-7.21 (m, 2H, NH2), 7.21-7.18 (m, 1H, ArH), 6.84 (d, J = 8.7 Hz, 1H, NHAc), 4.74-4.69 (m, 1H, α-CH), 3.43-3.33 (m, 2H, β-CH2), 2.26 (s, 3H, PhCH3), 1.81 (s, 3H, Ac); 13C-NMR (101 MHz, DMSO-D6) δ 198.3, 173.2, 169.1, 150.1, 140.8, 135.2, 134.8, 131.2, 130.3, 129.5, 127.3, 126.8, 126.0, 116.9, 115.9, 48.0, 40.3, 22.4, 20.4.

[0092] 5-(2-Methylphenyl)-D,L-Methylphenyl-D, L-kynurenine)(2-MePh-D,L-KYN)

change

[0093] Compound 6 (1.11 mmol), D-aminoacylase (105 mg), and cobalt chloride hexahydrate (12.7 mg) were dissolved in 50 mL of phosphate buffer (0.1 M, pH 8.4) and stirred at 37 ° C for 185 hours. The resulting solution was then purified by chromatography (ODS, acetic acid / water / methanol, 0.1:50:50) to obtain a fraction containing 2-MePh-D-KYN and the L-isomer of compound 6. The former was concentrated and recrystallized in water to obtain pure 2-MePh-D-KYN as a pale yellow solid (yield 45%). The latter was concentrated to dryness and dissolved in 50 mL of phosphate buffer (0.1 M, pH 8.4), and acylase I (101 mg) and cobalt chloride hexahydrate (12.1 mg) were added and stirred at 45 ° C for 18 hours. It was then purified in the same manner as for 2-MePh-D-KYN to obtain 2-MePh-L-KYN (yield 51%).

[0094] [2-MePh-D-KYN]:m / z 299.13497 (calced for [M+H] 299.13957); mp214-216℃; 1H-NMR (400 MHz, DMSO-D6) δ 7.60 (d, J = 2.1 Hz, 1H, ArH), 7.28-7.19 (m, 7H, ArH and NH2), 6.84 (d, J = 8.7 Hz, 1H, ArH), 3.63 (dd, J = 8.6, 3.5 Hz, 1H, α-CH), 3.52 (dd, J = 17.9, 3.7 Hz, 1H, β-CH2), 3.29 (dd, J = 17.9, 8.5Hz, 1H, β-CH2), 2.25 (s, 3H, CH3); 13C-NMR (101 MHz, DMSO-D6) δ 199.3, 169.2, 150.0, 140.7, 135.1, 134.8, 131.0, 130.3, 129.4, 127.3, 126.7, 126.0, 116.8, 116.1, 49.9, 20.3; [2-MePh-L-KYN]: m / z 299.14318 (calcd for [M+H] 299.13957); m.p. 214 - 215 °C; 1H-NMR (400 MHz, DMSO-D6) δ 7.60 (d, J = 2.1 Hz, 1H, ArH), 7.29 - 7.17 (m, 7H, ArH and NH2), 6.84 (d, J = 8.5 Hz, 1H, ArH), 3.61 (dd, J = 8.6, 3.5 Hz, 1H, α-CH), 3.52 (dd, J = 17.7, 3.5 Hz, 1H, β-CH2), 3.27 (q, J = 8.8 Hz, 1H, β-CH2), 2.25 (s, 3H, CH3); 13C-NMR (101 MHz, DMSO-D6) δ 199.4, 169.1, 150.0, 140.7, 135.1, 134.8, 131.0, 130.3, 129.4, 127.3, 126.7, 126.0, 116.8, 116.1, 49.9, 20.3.

[0095] [Evaluation] (General procedure for the enzyme assay method using the 2-MePh-KYN probe) <DAO assay method> Add FAD solution (10 mM, 10 μL), BSA solution (20 mg / mL, 10 μL), GSH solution (1 mM, 10 μL), and pkDAO solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3), 10 μL) to 450 μL of assay buffer (0.1 M Tris-HCl buffer (pH 8.3), 450 μL) to prepare a mixture, and pre-incubate at 37 °C for 15 minutes. Then, add 2-MePh-D-KYN solution (solvent: 0.1 M Tris-HCl buffer (pH 8.3), 10 mM, 10 μL) to the mixture and incubate at 37 °C for 60 minutes. Heat the resulting solution (at 95 °C for 5 minutes), and remove the precipitated protein by centrifugation (1800×g, 5 minutes). Dilute 0.5 mL of the supernatant with 1.5 mL of water, transfer a certain amount to a 1 cm square quartz cell, and perform fluorescence measurement at an excitation wavelength of 340 nm and an emission wavelength of 418 nm.

[0096] <LAO assay method> LAO activity was measured in the same manner as the above DAO assay method, except that 2-MePh-L-KYN was used instead of 2-MePh-D-KYN and svLAO was used instead of pkDAO.

[0097] FAD; Flavin adenine dinucleotide BSA; Bovine serum albumin GSH; Glutathione Tris-HCl; Tris(hydroxymethyl)aminomethane hydrochloride pkDAO; D-Amino acid oxidase (derived from pig kidney) svLAO; L-Amino acid oxidase (derived from the Japanese mamushi snake)

[0098] For the obtained fluorescence intensity, the relative values of the relative fluorescence intensities (excitation wavelength 340 nm, emission wavelength 418 nm) of 2-MePh-D-KYN or 2-MePh-L-KYN with respect to the fluorescence intensities (excitation wavelength 364 nm, emission wavelength 450 nm) of the comparative MeS-D-KYN or MeS-L-KYN are shown in Figure 2.

[0099] From the results in Figure 2, 2-MePh-D-KYN and 2-MePh-L-KYN as novel fluorescence probes were each capable of detecting DAO and LAO with sensitivities approximately 60 times and 5 times, respectively, compared to MeS-D-KYN and MeS-L-KYN. Therefore, it was confirmed that the compounds of the present application are excellent in detection sensitivity.

[0100] <Calibration curve preparation and linearity confirmation> Using various concentrations of pkDAO solutions (0.50, 1.0, 2.5, 5.0 U / mL) or svLAO solutions (0.025, 0.050, 0.075, 0.10 U / mL) as sample solutions, fluorescence measurements were performed according to the procedure described in the general procedure of the enzyme assay method using the 2-MePh-KYN probe, and calibration curves were prepared.

[0101] The results are shown in Figure 3. As shown in Figure 3, a high linear relationship was observed between the enzyme activity and the fluorescence intensity (R2 = 0.9987 or 0.9934).

[0102] The upper limit and / or lower limit of the numerical ranges described in this specification can be arbitrarily combined to define a preferred range. For example, the upper limit and lower limit of the numerical range can be arbitrarily combined to define a preferred range, the upper limit of the numerical range can be arbitrarily combined to define a preferred range, and the lower limit of the numerical range can be arbitrarily combined to define a preferred range.

[0103] The claims following this written disclosure are expressly incorporated herein into this written disclosure, with each claim standing on its own as a separate embodiment. The present disclosure includes all instances in which an independent claim is replaced by its dependent claim. Furthermore, any additional embodiments derived from the independent claim and the subsequent dependent claims are also expressly incorporated into this written specification.

Claims

1. A compound represented by the following formula (1), or a salt or solvate thereof: 【Chemistry 1】 (In formula (1), R １ ~R ５ are each independently a hydrogen atom or C １ ~C ４ is an alkyl group).

2. The compound according to claim 1, which is represented by any one of the following formulas (1-D) and (1-L), or a salt or solvate thereof: 【Chemistry 2】 (In formulas (1-D) and (1-L), R １ ~R ５ are each independently a hydrogen atom or C １ ~C ４ is an alkyl group).

3. The compound according to claim 1, which is represented by any one of the following formulas, or a salt or solvate thereof: 【Chemistry 3】 【change】

4. A reagent for measuring the enzyme activity of amino acid oxidase, comprising the compound according to any one of claims 1 to 3, or a salt or solvate thereof.

5. A kit for measuring the enzyme activity of an amino acid oxidase, comprising at least the reagent for measuring the enzyme activity of an amino acid oxidase according to claim 4.

6. A method for measuring the enzyme activity of an amino acid oxidase, comprising measuring the enzyme activity of an amino acid oxidase using the compound according to any one of claims 1 to 3, or a salt or solvate thereof.

7. The method according to claim 6, which is used in screening for amino acid oxidases.

8. The method according to claim 6, which is used in screening for inhibitors of the enzyme activity of amino acid oxidase.

9. A compound represented by the following formula (2), or a salt or solvate thereof: 【Chemistry 4】 (In formula (2), R １ ~R ５ are each independently a hydrogen atom or C １ ~C ４ is an alkyl group).

10. The compound according to claim 9, which is represented by any one of the following formulas, or a salt or solvate thereof: 【Chemistry 5】

11. A method for measuring the enzyme activity of an amino acid oxidase, comprising detecting fluorescence of the compound according to claim 9 or 10, or a salt or solvate thereof.

12. The method according to claim 11, which is used in screening for amino acid oxidases.

13. The method according to claim 11, which is used in screening for inhibitors of the enzyme activity of amino acid oxidase.

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