JP2009084374A - Oxidation coloring compound, reagent composition, and test implement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide oxidation coloring compounds, a reagent composition and a test implement. <P>SOLUTION: The oxidation coloring compounds are represented by formula (1), wherein R<SP>1</SP>and R<SP>2</SP>are each independently a 1-5C straight or branched chain alkyl group, and R<SP>3</SP>-R<SP>7</SP>are each independently a substituent containing an alkyl group having at least one hydroxyl group. Alternatively, salts of those are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an oxidative coloring compound, a reagent composition, and a test device for measuring biological components.

  Today, colorimetric analysis using enzymes is widely used as a method for measuring biological components in body fluids such as blood and urine. These methods are incorporated in measurement kits, automatic analyzers, dry chemistry test devices, and the like, and are used in many daily clinical tests (see, for example, Patent Document 1).

  As one type of measurement method, hydrogen peroxide generated by the action of a specific oxidase on the substance to be measured is further converted into active oxygen by the action of peroxidase. There is a method for color determination.

  Examples of the enzyme used herein include glucose oxidase for measuring glucose, cholesterol oxidase for measuring cholesterol, uricase for measuring uric acid, pyruvate oxidase for measuring pyruvic acid, and the like. Since these enzymes specifically oxidize only the substrate in the specimen, it is possible to quantitate by restricting only the measurement object from the specimen containing various components.

  Further, as the coloring reagent, a dye or a dye precursor (chromogen) that changes its absorption wavelength characteristic, absorption intensity, and the like when oxidized with active oxygen is used. That is, the amount of the substance to be measured can be quantified by measuring the color that changes according to the amount of active oxygen. Some of these dyes and chromogens function by only one molecule, and others function by coupling two different molecules.

  Examples of those that function in one molecule include leuco dyes. Specific compounds include benzidine, o-tolidine, o-dianisidine, 2,2′-amino-bis (3-ethylbenzothiazolinone-6-sulfonic acid (ABTS), bis- (4-diethylamino)- 2-sulfophenylmethane (BSPM), bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA), 10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine ( MCDP), 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), bis [4- (N-alkyl-N-sulfopropyl) -2,6-dimethylphenyl] methane (Bis-MAPS), N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ′, 4 ″ ′-triaminotriphenyl Tan (TMP), and the like.

  A representative one that functions in two molecules includes one obtained by oxidatively coupling a coupler and a Trinder reagent (see, for example, Non-Patent Documents 1 and 2).

  As the coupler, 4-aminoantipyrine (4-AA), vanillin diamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), sulfonated methylbenzthiazolinone hydrazone (SMBTH), aminodiphenylamine or a derivative thereof is used. .

  As the Trinder reagent, a phenol derivative or an aniline derivative is used. Phenol derivatives include phenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 3,5-dichlorophenol, 2,4-dibromophenol, 2,6,4-trichlorophenol, 2 , 6,4-tribromophenol, 3,5-dichloro-2-hydroxybenzenesulfonic acid, 3-hydroxy-2,4,6-triiodobenzoyl acid, and the like. Examples of aniline derivatives include N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N-ethyl-N- (3-methylphenyl) -3-acetylethylenediamine (EMAE), N-ethyl. -N- (3-methylphenyl) -N-succinylethylenediamine (EMSE), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N- (2-carboxyethyl) ) -N-ethyl-3-methylaniline (CEMB), N, N-bis- (4-sulfobutyl) -3-methylaniline (TODB), N-ethyl-N- (2-succinylaminoethyl) -3- Methylaniline (ESET), N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline (ADPS), N- (2-hydroxy 3-sulfopropyl) -3,5-dimethoxyaniline (HSDA), N-ethyl-N- (2-hydroxy-3-sulfopropi) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (2 -Hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), etc. Is mentioned.

Here, in view of storage stability, a dye that functions in two molecules tends to be better than a dye that functions in one molecule. Of the dyes that function in two molecules, the most stable 4-aminoantipyrine and methylbenzthiazolinone hydrazone among the above are often used as couplers. Many more advantageous aniline derivatives are used.
JP 2004-223115 A Trider, P.A. , Ann. Clin. Biochem. , 6, 24, 1969 Barham, D.C. and Trinder, P .; , Analyst (London), 97, 142, 1972

  However, in order to increase the value of a product using the above coloring principle, it is necessary to further improve the measurement accuracy or reduce the deterioration over time. In addition, a detection system with higher sensitivity than the existing one is required for detection of a trace amount of components. For these purposes, development of higher-performance coloring reagents is desired.

  That is, an object of the present invention is to provide an oxidative coloring compound, a reagent composition, and a test device that have improved performance as compared with the prior art.

  As a result of various studies to solve such problems, the present inventors have found a novel coupler compound having high performance, a reagent composition using the same, and a test device.

  That is, the present invention provides an oxidation coloring compound or a salt thereof having high performance shown in the following (1) to (11), and a reagent composition and a test device using the same.

  (1) The following formula (1):

However, R ¹ and R ² are each independently a linear or branched alkyl group having 1 to 5 carbon atoms,
At least one or more of R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently a substituent selected from the following formulas (2) to (8), and the rest are hydrogen An atom,
Or an oxidative coloring compound or a salt thereof.

  Following formula (2):

  Following formula (3):

  Following formula (4):

  Following formula (5):

  Following formula (6):

  Following formula (7):

  Following formula (8):

However, R ⁸ is a linear, branched or cyclic alkyl group having at least one hydroxyl group,
R ⁹ is a linear, branched or cyclic alkyl group having at least one hydroxyl group or a hydrogen atom,
At this time, R ⁸ and R ⁹ may be the same or different.

(2) The oxidative coloring compound or a salt thereof according to (1), wherein R ¹ and R ² are methyl groups.

(3) R ⁸ is a linear or branched alkyl group having 2 to 6 carbon atoms having 1 to 5 hydroxyl groups, and R ⁹ is a hydrogen atom, or
The oxidative coloring according to (1) or (2), wherein R ⁸ and R ⁹ are each independently a C 2-6 linear or branched alkyl group having 1 to 5 hydroxyl groups. Compounds or salts thereof.

(4) R ⁸ is a 2,3-dihydroxy-n-propyl group, a bis (hydroxymethyl) methyl group or a 2,3,4,5,6-pentahydroxy-n-hexyl group, and R ⁹ is A hydrogen atom, or
R ⁸ and R ⁹ are each independently a 2,3-dihydroxy-n-propyl group, a bis (hydroxymethyl) methyl group or a 2,3,4,5,6-pentahydroxy-n-hexyl group. , (1) to (3) any one of the oxidation coloring compounds or salts thereof.

(5) R ⁵ is selected from the group consisting of formulas (2) to (8),
The oxidative coloring compound or a salt thereof according to any one of (1) to (4), wherein R ³ , R ⁴ , R ⁶ and R ⁷ are hydrogen atoms.

(6) The oxidative coloring compound or a salt thereof according to any one of (1) to (5);
A Trinder reagent,
An oxidase that acts selectively on the measurement object;
Peroxidase,
A reagent composition comprising:

  (7) The Trinder reagent is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline or N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5 -Reagent composition as described in (6) which is a dimethylaniline.

(8) The measurement object is glucose,
The reagent composition according to (6) or (7), wherein the oxidase is glucose oxidase.

  (9) A test device in which the reagent composition according to any one of (6) to (8) is held on a carrier.

  (10) The test device according to (9), wherein the carrier is a polymer film.

  (11) The test device according to (10), wherein the polymer film is made of polysulfone or polyethersulfone.

  The coloring reagent compound according to the present invention or a salt thereof (hereinafter, also simply referred to as “compound according to the present invention”) is superior to the conventional reagent in the following points (a) to (c). Further, when used as a reagent composition using the compound according to the present invention, and particularly as a test device held on a carrier, the effect is most remarkable.

(A) High solubility Since the compound according to the present invention is highly water-soluble, it is quickly dissolved and homogenized in a sample such as blood, particularly when held on a carrier, and a rapid color reaction occurs. . Since the compound according to the present invention has high solubility, a reagent solution having a higher concentration than that of a conventional reagent can be prepared. When used on a carrier, it is possible to increase the concentration of the coating solution and hold more reagent on the carrier.

(A) High sensitivity The dye compound produced by the color reaction also has high solubility and affinity in the specimen and improves development in the carrier, so that the color development on the carrier surface is vivid and uniform. It will be expensive. As a result, highly sensitive and stable measurement is possible.

(C) Excellent stability over time Since the compound according to the present invention has improved hydrophilicity and high molecular weight, the loss of reagent due to sublimation during storage observed in 4-aminoantipyrine can be suppressed. . In addition, since the present compound has little influence on the color intensity due to the change in reagent concentration, even when the compound is deteriorated with time, the measurement value is less affected as long as a sufficient amount is present for measurement. Furthermore, the hydrophilic functional group of the present compound increases the adsorptivity to the carrier and improves the stability of the compound in the carrier. In view of the above, it is possible to reduce deterioration with time and extend the effective period.

  Hereinafter, several preferred embodiments of the oxidative coloring compound or salt thereof of the present invention will be described in detail.

<First Embodiment>
1st Embodiment of this invention is related with the oxidation coloring compound or its salt represented by following formula (1).

    Following formula (1):

However, R ¹ and R ² are each independently a linear or branched alkyl group having 1 to 5 carbon atoms,
At least one or more of R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently a substituent selected from the following formulas (2) to (8), and the rest are hydrogen An atom,
An oxidative coloring compound represented by or a salt thereof,
Following formula (2):

  Following formula (3):

  Following formula (4):

  Following formula (5):

  Following formula (6):

  Following formula (7):

  Following formula (8):

However, R ⁸ is a linear, branched or cyclic alkyl group having at least one hydroxyl group,
R ⁹ is a linear, branched or cyclic alkyl group having at least one hydroxyl group or a hydrogen atom,
At this time, R ⁸ and R ⁹ may be the same or different.

Here, in the formula (1), the alkyl group representing R ¹ and R ² is a substituent having 1 to 5 carbon atoms composed of hydrocarbon, and has a straight chain structure. It may contain a chain structure. At this time, R ¹ and R ² may be the same or different. Examples of the linear or branched alkyl group having 1 to 5 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and isopentyl. Group, and neopentyl group. Of these, considering solubility, a methyl group, an ethyl group, and a propyl group are more preferable, and a methyl group is more preferable. That is, it is particularly preferable that R ¹ and R ² are methyl groups.

In the above formula (1), at least one of R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ is independently a substituent selected from formula (2) to formula (8). And the remainder is a hydrogen atom. That is, R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ may be the same or different. “Remaining hydrogen atoms” means, for example, when R ³ and R ⁵ are substituents of formula (2) and formula (8), respectively, R ⁴ , R ⁶ and R ⁷ is a hydrogen atom.

In the formulas (2) to (8), R ⁸ is a linear, branched or cyclic alkyl group having at least one hydroxyl group, and R ⁹ is a straight chain having at least one hydroxyl group. A chain, a branched or cyclic alkyl group or a hydrogen atom, wherein R ⁸ and R ⁹ may be the same or different, each having at least one hydroxyl group. A chain, branched or cyclic alkyl group (hereinafter also simply referred to as “alkyl group having a hydroxyl group”) includes an ether bond, sulfide bond, amino bond, ester bond, amide bond, and urethane bond. May be. Moreover, some of the hydroxyl groups in the “alkyl group having a hydroxyl group” may be substituted with alkyl, acyl, alkyloxycarbonyl or the like.

In addition, “an alkyl group having a hydroxyl group may include an ether bond, a sulfide bond, an amino bond, an ester bond, an amide bond, or a urethane bond” means that any adjacent carbon in alkylene is included. It means that the above bond is introduced between them. For example, “containing an ether bond in an alkyl group having a hydroxyl group” means that “—CH ₂ —O—CH ₂ —” exists in the alkyl group.

  2-20 are preferable, as for carbon number of the alkyl group in the said "alkyl group which has a hydroxyl group", 2-6 are more preferable, and 3-6 are especially preferable. 1-19 are preferable, as for the number of the hydroxyl groups in "an alkyl group which has a hydroxyl group", 1-5 are more preferable, and 2-5 are especially preferable. A larger ratio of the hydroxyl group to the carbon number contributes to the improvement of hydrophilicity.

  Examples of the alkyl group having 2 to 20 carbon atoms include ethyl group, propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, Examples include hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, 2-ethylhexyl, tetradecyl, octadecyl, icosyl and the like.

That is, R ⁸ is a linear or branched alkyl group having 2 to 6 carbon atoms having 1 to 5 hydroxyl groups, and R ⁹ is a hydrogen atom, or
R ⁸ and R ⁹ are preferably each independently a linear or branched alkyl group having 2 to 6 carbon atoms and having 1 to 5 hydroxyl groups.

  More specifically, a 2-hydroxyethyl group, 2-hydroxy-n-propyl group, 3-hydroxy-n-propyl group, 2,3-dihydroxy-n-propyl group, bis (hydroxymethyl) methyl group, 3 , 4-dihydroxy-n-butyl group, 2,4-dihydroxy-n-butyl group, 2,3,4-trihydroxy-n-butyl group, 1-methyl-2,3-dihydroxy-n-propyl group, 2,2-bis (hydroxymethyl) ethyl group, {2,2-bis (hydroxymethyl) -2-hydroxy} ethyl group, {3,3-bis (hydroxymethyl) -3-hydroxy} propyl group, 2, 3,4,5-tetrahydroxy-n-pentyl group, 4,2-dihydroxy-3- (hydroxymethyl) -n-butyl group, 2,3,4,5,6-pentahydroxy-n-hex Group, (2,3-dihydroxy-n-propyloxy) ethyl group, 2- (2,3-dihydroxy-n-propyloxy) ethyl group, 2- (2,3,4-trihydroxy-n-butyloxy) ) Ethyl group, 2- {bis (hydroxymethyl) methyloxy} ethyl group, 2,3-dihydroxy-4-methoxy-n-butyl group, 2-methoxy-3,4-dihydroxy-n-butyl group, 2- Examples include {N, N-bis (2-bis (2-hydroxymethyl) ethyl) amino} ethyl, 2- {N, N-bis (2,3-dihydroxypropyl) amino} ethyl and the like. Among these, 2,3-dihydroxy-n-propyl group, bis (hydroxymethyl) methyl group, 2,3,4-trihydroxy-n-butyl group, 2,3,4,5-tetrahydroxy-n-pentyl Group, 2,3,4,5,6-pentahydroxy-n-hexyl group and the like. These compounds can be suitably used from the viewpoint of improving hydrophilicity.

  Furthermore, 2,3-dihydroxy-n-propyl group, bis (hydroxymethyl) methyl group, 2,3,4,5,6-pentahydroxy-n-hexyl group is from the viewpoint of improving hydrophilicity and reducing production costs. Therefore, it can be used more suitably.

That is, R ⁸ is 2,3-dihydroxy-n-propyl group, bis (hydroxymethyl) methyl group or 2,3,4,5,6-pentahydroxy-n-hexyl group, and R ⁹ is hydrogen Is an atom, or
R ⁸ and R ⁹ are each independently a 2,3-dihydroxy-n-propyl group, a bis (hydroxymethyl) methyl group or a 2,3,4,5,6-pentahydroxy-n-hexyl group. And preferred.

Further, it is preferable that at least one or more of R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently a substituent selected from the formulas (2) to (8). From the viewpoint of interference with the reaction, R ⁵ is selected from the group consisting of formulas (2) to (8), and R ³ , R ⁴ , R ⁶ and R ⁷ are each a hydrogen atom. preferable.

The oxidative coloring compound according to the present invention may be in the form of a salt. That is, when the oxidative coloring compound according to the present invention contains an ionic functional group, these functional groups may be in a free state or may form a salt. The partner that forms the salt may be an inorganic compound or an organic compound. For example, inorganic bases such as sodium, potassium and calcium, inorganic acids such as fluorine, chlorine and bromine, organic bases such as ammonia and alkylamines, organic acids such as carbonic acid, formic acid, acetic acid, citric acid and paratoluenesulfonic acid, Alternatively, amphoteric compounds such as amino acids may be used. As an example of forming a salt with an inorganic acid as a salt-forming partner, a salt is formed with an amino group at the 4-position, such as “—NH ₄ ⁺ Cl ⁻ ”.

  Next, the manufacturing method of the compound based on this invention is demonstrated.

  In the production of the compound according to the present invention, a method is used in which a compound having a reactive substituent introduced into the benzene ring of 4-aminoantipyrine is bonded to a hydroxyl group-containing compound having a reactive substituent introduced.

[Production of a compound in which a reactive substituent is introduced into 4-aminoantipyrine]
Although the manufacturing method of the compound which introduce | transduced the reactive substituent into 4-aminoantipyrine is not restrict | limited, following Chemical formula (9);

A compound represented by formula (hereinafter also referred to as “compound (9)”), and the following chemical formula (10):

It is preferable to have the process (process (A)) with which the compound (henceforth "compound (10)") represented by these is made to react. In the chemical formula (9), R ¹ is the same as the definition of the chemical formula (1), and thus the description thereof is omitted here. In the chemical formula (10), R ^{13 to} R ¹⁷ are each independently a hydrogen atom, amino group, carboxyl group, halogencarbonyl group, sulfonic acid group, hydroxyl group, thiol group, halogen group, or tosyl group. In this case, at least one of R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ and R ¹⁷ is an amino group, a carboxyl group, a halogencarbonyl group, a sulfonic acid group, a hydroxyl group, a thiol group, a halogen group, or tosyl. Group and the rest are hydrogen atoms.

In the step (A) in which the compound (9) and the compound (10) are reacted, the mixing ratio of the compounds (9) and (10) is not particularly limited. Preferably, the compounds (9) and (10) are mixed in equimolar amounts, or the compound (9) is mixed slightly more with the compound (10). In the latter case, the compound (9) is preferably mixed at a ratio of about 0.8 to 2 mol, more preferably about 1 to 1.2 mol, relative to 1 mol of the compound (10). In addition, the reaction conditions of the compound (9) and the compound (10) are not particularly limited as long as these reactions proceed, but for example, the reaction temperature is preferably 20 to 200 ° C., more preferably 80 to The temperature is 150 ° C., and the reaction time is preferably 0.5 to 5 hours, more preferably 1 to 1.5 hours. In addition, the reaction between the compound (9) and the compound (10) may be performed in the absence of a solvent or in a solvent. Examples of the solvent that can be used in the latter case include alcohols such as methanol, ethanol, and propanol; ethers such as tetrahydrofuran (THF), diethyl ether, and dioxane; solvents such as pyridine, dimethylformamide, and dimethyl sulfoxide. Of these, THF is preferred. After the reaction between the compound (9) and the compound (10), the product can be solidified by adding the reaction mixture into an appropriate solvent, whereby the starting compound can be separated and recovered. Incidentally, the reaction product obtained in the step (A) has a structure of the following Formula R ² in the following chemical formula (11) is a hydrogen atom (H) (11).

  Next, by alkylating the reaction product obtained in the step (A) [step (B)], the following chemical formula (11):

(Hereinafter also referred to as “compound (11)”).

  In the step (B), the alkylation can be performed using an alkylating agent such as an alkyl halide such as methyl iodide or ethyl iodide. Here, the alkylating agent is not limited to the above-mentioned alkyl halide, and other known alkylating agents can be used in the same manner. This reaction may be performed using an appropriate solvent or base, and is performed at room temperature or under heating. More specifically, as the solvent that can be used, the same solvents as listed in the step (A) can be used, and among these, methanol and THF are preferable. As the base, triethylamine (TEA), diisopropylethylamine (DIEA), 4- (dimethylamino) pyridine and the like can be used, and among these, TEA is preferable.

  In the step (B), the amount of the alkylating agent used is not particularly limited as long as it can sufficiently alkylate the reaction product obtained in the step (A), but the alkylating agent was obtained in the step (A). Preferably it is 0.5-5 mol with respect to 1 mol of reaction products, More preferably, it is 1-2 mol. Although the reaction conditions are not particularly limited, the alkylation reaction of the reaction product obtained in the step (A) is preferably performed at a temperature of 40 to 200 ° C, more preferably 100 to 140 ° C, and preferably 1 to 24. It is performed for a time, more preferably for 10 to 20 hours.

  Next, using the compound (11) obtained in the step (B) as a starting material, a nitrosation reaction, followed by a reduction reaction subsequent to the nitrosation reaction [step (C)].

  In the step (C), the nitrosation reaction may be performed in the absence of a solvent, but is preferably performed in a solvent. More specifically, the nitrosation reaction is carried out using hydrochloric acid, nitric acid (preferably hydrochloric acid) or the like using water, an organic acid, or a mixture of both as a solvent, preferably at pH 1-5, more preferably at pH 1-2. After adjusting to, it is done by adding sodium nitrite. Examples of the organic acid include acetic acid, propionic acid, butyric acid, formic acid, lactic acid, succinic acid, and citric acid, and acetic acid is preferable. The amount of sodium nitrite is not particularly limited, but is usually used in an equimolar or excess amount with respect to the starting material, preferably 1.0 to 4.0 equivalents of the starting material, more preferably 1.1 to Used in 1.5 equivalents.

  In the step (C), the reaction conditions for the nitrosation reaction are not particularly limited as long as the nitrosation reaction can sufficiently proceed. Specifically, the reaction temperature is preferably −20 ° C. to 40 ° C., more preferably 0 to 25 ° C., and the reaction time is preferably 5 seconds to 40 minutes, more preferably 10 seconds to 15 minutes. . Since the product obtained by this nitrosation reaction is precipitated, it can be easily separated and recovered from the reaction system.

  Next, in the step (C), a reduction reaction is performed following the nitrosation reaction. Here, the reduction reaction may be performed in the absence of a solvent, but is preferably performed in a solvent. The solvent that can be used in the reduction reaction is not particularly limited as long as it can dissolve the nitrosation reaction product obtained by the nitrosation reaction. For example, water; alcohols such as methanol, ethanol, propanol; acetic acid, propionic acid And organic acids such as butyric acid, formic acid, lactic acid, succinic acid and citric acid. These solvents may be used alone or in the form of a mixture of two or more. The reducing agent is not particularly limited, but metals such as zinc and reducing inorganic salts such as sodium hydrosulfite are used. The reaction solution is neutral or basic or acidic by adding sodium hydroxide or hydrochloric acid. It is used after adjusting. The pH of the reaction solution is preferably adjusted to 1 to 12, more preferably 1 to 5.

  After the reduction reaction, the product is recovered by adding an appropriate solvent, precipitating and filtering, or extracting with an appropriate organic solvent. The nitrosation reaction and the reduction reaction may be continuously performed in one pot without taking out the product of the nitrosation reaction. The recovered product may have a higher purity by recrystallization or column chromatography purification.

[Reaction of a compound having a reactive functional group introduced into 4-aminoantipyrine and a hydroxyl group-containing compound having a reactive substituent introduced]
As the hydroxyl group-containing compound into which a reactive substituent has been introduced, a commercially available compound or a synthesized compound may be used.

  Examples of reactive substituents introduced into the hydroxyl group-containing compound include, for example, amino groups, carboxylic acid groups, halogenated carboxylic acid groups, sulfonic acid groups, hydroxyl groups, thiol groups, halogen groups, and tosyl groups. It is done.

  In this reaction, a combination of reactive substituents is appropriately selected. In the case of an amino group and a carboxylic acid group, an amide bond can be formed using an appropriate dehydration condensing agent. That is, the bond in Formula (3) or Formula (7) can be formed.

  In the case of a hydroxyl group and a carboxylic acid group, an ester bond can be formed using an appropriate dehydration condensing agent. That is, the bond in formula (6) can be formed.

  In the case of an amino group and a sulfonic acid group, it can be formed with a sulfonic acid amide using an appropriate dehydration condensing agent. That is, the bond in formula (8) can be formed.

  As the dehydrating condensing agent, carbodiimides and the like can be used. Alternatively, a halogenated acid can be produced and then reacted with an amine or a hydroxyl group for condensation.

  In the reaction of an amino group, a hydroxyl group or a thiol group with a halogen group or a tosyl group, a substituted amine, an ether bond or a thioether bond can be formed, respectively. That is, the bond in Formula (2), Formula (4), and Formula (5) can be formed.

  The production of the compound according to the present invention will be described in more detail.

  For example, 4-amino-1- [4-{(2,3-dihydroxy-n-propyl) amino} phenyl] -2,3-dimethyl, which is an example of a compound having a substituent represented by the formula (2) -5-pyrazolone is composed of 4-amino-1- (4-aminophenyl) -2,3-dimethyl-5-pyrazolone, 2,2-dimethyl-1,3-dioxolane-4-methane bromide and diisopropylethylamine It is obtained by heating to 80 ° C. in dimethylformamide in the presence and deprotecting the product with acid.

  4-amino-1- [4-{(2,3-dihydroxy-n-propyl) carbonylamino} phenyl] -2,3-dimethyl-, which is an example of a compound having a substituent represented by the formula (3) 5-pyrazolone is 4-amino-1- (4-aminophenyl) -2,3-dimethyl-5-pyrazolone and 2,2-dimethyl-1,3-dioxolane-4-methanecarboxylic acid. It is obtained by reacting in methanol at room temperature in the presence of (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride and deprotecting the product with acid.

  4-amino-1- [4-{(2,3-dihydroxy-n-propyl) oxy} phenyl] -2,3-dimethyl-5, which is an example of a compound having a substituent represented by the formula (4) -Pyrazolone is 4-amino-1- (4-hydroxyphenyl) -2,3-dimethyl-5-pyrazolone and 2,2-dimethyl-1,3-dioxolane-4-methane bromide in the presence of sodium hydride It is obtained by heating under reflux in tetrahydrofuran and deprotecting the product with acid.

  4-amino-1- [4-{(2,3-dihydroxy-n-propyl) thio} phenyl] -2,3-dimethyl-5, which is an example of a compound having a substituent represented by the formula (5) -Pyrazolone is 4-amino-1- (4-mercaptophenyl) -2,3-dimethyl-5-pyrazolone and 2,2-dimethyl-1,3-dioxolane-4-methane bromide in the presence of diisopropylethylamine. Obtained by heating to 80 ° C. in dimethylformamide and deprotecting the product with acid.

  4-amino-1- [4-{(2,3-dihydroxy-n-propyl) oxycarbonyl} phenyl] -2,3-dimethyl-, which is an example of a compound having a substituent represented by the formula (6) 5-Pyrazolone is 4-amino-1- (4-carboxyphenyl) -2,3-dimethyl-5-pyrazolone and 2,2-dimethyl-1,3-dioxolane-4-methanol. , 6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride in the presence of methanol at room temperature and the product is deprotected with acid.

  4-amino-1- [4-{(2,3-dihydroxy-n-propyl) aminocarbonyl} phenyl] -2,3-dimethyl-, which is an example of a compound having a substituent represented by the formula (7) 5-Pyrazolone is 4-amino-1- (4-carboxyphenyl) -2,3-dimethyl-5-pyrazolone and 2,2-dimethyl-1,3-dioxolane-4-methanamine. , 6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride in tetrahydrofuran at room temperature and the product is deprotected with acid.

  4-amino-1- [4-{(2,3-dihydroxy-n-propyl) aminosulfo} phenyl] -2,3-dimethyl-5, which is an example of a compound having a substituent represented by the formula (8) -Pyrazolone is 4-amino-1- (4-sulfonylphenyl) -2,3-dimethyl-5-pyrazolone and 2,2-dimethyl-1,3-dioxolane-4-methanamine. It is obtained by reacting in methanol at room temperature in the presence of 6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride and deprotecting the product with acid.

  In the production method according to the present invention described above, if necessary, a compound in which a reactive substituent is introduced into 4-aminoantipyrine and a hydroxyl group-containing compound in which a reactive substituent is introduced are bonded. A suitable protecting group may be introduced before the reaction step (hereinafter, also simply referred to as “reaction step”), followed by a step of deprotecting.

  Examples of the protecting group include acetals, ketals, acyl groups, ether groups and the like for the hydroxyl group. Examples of amino groups include carbamates and amides. Examples of carboxylic acid include methyl ester and ethyl ester.

  In the process of extracting and purifying the compound according to the present invention from the mixture (reaction compound) generated in the above reaction step, methods such as extraction, precipitation, recrystallization, column chromatography, gel filtration and the like can be used.

  As described above, the production method of the compound according to the present invention is not particularly limited, and the production methods as described above can be applied in the same manner, appropriately modified, or appropriately combined.

  As described above, the compound according to the present invention has high water solubility. Therefore, the reagent composition described later, and when the reagent composition is held on a carrier and used as a test device, it quickly dissolves in a sample such as blood and is homogenized, and a rapid color reaction occurs. It is an excellent one. In addition, when the compound according to the present invention is subjected to a color reaction (colorimetric analysis method), the dye compound produced by the compound has high solubility and affinity for the specimen, and developability in the carrier. Therefore, the color development on the surface of the carrier is vivid and highly uniform, and the measurement can be performed with high sensitivity and stability. Furthermore, since the compound according to the present invention has high solubility, it is possible to prepare a reagent composition liquid (reagent liquid) having a higher concentration than conventional reagents. Then, when the reagent composition containing the compound according to the present invention is supported on a carrier and used as a test device, it becomes possible to increase the concentration of the coating solution and hold more reagent on the carrier. In addition, since the compound according to the present invention is improved in hydrophilicity and has a high molecular weight, loss of the reagent due to sublimation during storage can be suppressed. In addition, since the compound according to the present invention has little influence on the color intensity due to the change in reagent concentration, even when the compound deteriorates and decreases with time, the influence on the measured value is small. Furthermore, the hydrophilic functional group of the compound according to the present invention increases the adsorptivity to the carrier and improves the stability of the compound in the carrier even when used as a test device. By providing the compound according to the present invention, it becomes possible to provide a reagent composition and a test device that are reduced in deterioration over time and have an extended effective period.

Second Embodiment
In the present embodiment, embodiments relating to particularly preferred uses and uses of the compounds according to the present invention will be described. 2nd Embodiment of this invention is a reagent composition containing the compound which concerns on this invention, a Trinder reagent, the oxidase which acts selectively with respect to a measuring object, and peroxidase.

  The Trinder reagent is not particularly limited, and any conventionally known compound can be preferably used. However, among them, N-ethyl-N- (3-sulfopropyl) -3-methylaniline, N-ethyl-N- (3-methylphenyl) -3-acetylethylenediamine, N-ethyl-N- (3-methylphenyl) ) -N-succinylethylenediamine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N- (2-carboxyethyl) -N-ethyl-3-methylaniline, N, N -Bis- (4-sulfobutyl) -3-methylaniline, N-ethyl-N- (2-succinylaminoethyl) -3-methylaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropi) -3 5-Dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl)- 3,5-dimethylaniline is preferred, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-Dimethylaniline is particularly preferable from the viewpoint of color development intensity and wavelength.

  That is, the Trinder reagent is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline or N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5- Dimethylaniline is preferable from the viewpoint of color development intensity and wavelength.

  Examples of the “oxidase that acts selectively on the measurement target” include, for example, when the measurement target is glucose, the oxidase is glucose oxidase, and when the measurement target is cholesterol, the oxidase is cholesterol oxidase. When the measurement object is uric acid, the oxidase is uricase, and when the measurement object is pyruvate, the oxidase is pyruvate oxidase. Since these specifically oxidize only the substrate of the measurement object, only the measurement object can be quantified in a limited manner from a specimen containing various components.

  In the reagent composition according to the present invention, the molar ratio between the compound according to the present invention and the Trinder reagent is preferably 1: 1 to 2: 1, and more preferably 1.3: 1. When the molar ratio between the compound according to the present invention and the Trinder reagent is less than 1: 1, or when the molar ratio between the compound according to the present invention and the Trinder reagent exceeds 2: 1, poor color development may occur. .

  In the reagent composition according to the present invention, the content of the oxidase that selectively acts on the measurement target is preferably 433 k to 2600 kU, more preferably 866 k to 2600 kU with respect to 15 mmol of the coupler. preferable. When the content of the oxidase that selectively acts on the measurement object is less than 433 kU with respect to 15 mmol of the coupler, poor color development may occur. In the present invention, the number of units measured by the 4AA-TOOS method is adopted as the content of oxidase that selectively acts on the measurement object.

  The content of peroxidase in the reagent composition according to the present invention is preferably 166.5 k to 1666 kU, and more preferably 333 k to 999 kU with respect to 15 mmol of coupler. When the content of peroxidase is less than 166.5 kU with respect to 15 mmol of coupler, poor color development may occur. In the present invention, the content of the peroxidase is the number of units measured by the pyrogallol method.

  The reagent composition according to the present invention includes the oxidized color forming compound according to the present invention or a salt thereof, a Trinder reagent, an oxidase that selectively acts on a measurement target, and a peroxidase. That is, components other than the “oxidative coloring compound according to the present invention or a salt thereof, a Trinder reagent, an oxidase that selectively acts on a measurement object, and a peroxidase” do not impair the function as a reagent composition. It may contain a degree. Examples of the component include a pH buffer, an osmotic pressure adjusting agent, a surfactant, a solubilizer, a stabilizer, a protective agent and the like.

  The content of these components is not particularly limited as long as it does not impair the function as a reagent composition, but it is selectively used for the oxidative coloring compound or a salt thereof according to the present invention, the Trinder reagent, and the measurement object. Preferably it is 10-30 mass% with respect to the total mass of the oxidase which acts, and peroxidase, More preferably, 13-20 mass% may be contained.

  The oxidative coloring compound / salt according to the present invention can be used as a coupler suitably used for a colorimetric analysis method using an enzyme, which is a kind of a method for measuring biological components in body fluids such as blood and urine. . That is, the compound according to the present invention is used together with the above-mentioned Trinder reagent, and the compound according to the present invention is converted into active oxygen generated by peroxidase (POD) oxidation of oxygen peroxide generated by enzymatic oxidation of the measurement object. The coupler and the Trinder reagent are colored by oxidative condensation. Therefore, the compound according to the present invention is characterized by the compound itself as described above, but is particularly preferably used as a coupler.

  As described above, since the compound according to the present invention has high water solubility, the reagent composition containing the compound (hereinafter also referred to as “reagent composition according to the present invention”) and the reagent composition held on a carrier are tested. When used as a tool, it is very excellent in that it quickly dissolves and homogenizes in a sample such as blood and a color reaction occurs quickly. In addition, in the color reaction (colorimetric analysis method) using the reagent composition according to the present invention, the dye compound produced thereby has high solubility and affinity for the specimen, and developability within the carrier. As a result, the color development on the surface of the carrier is vivid and highly uniform, and a highly sensitive and stable measurement is possible. Furthermore, since the compound according to the present invention has high solubility, it is possible to prepare a reagent composition liquid (reagent liquid) having a higher concentration than conventional reagents. Then, when the reagent composition is supported on a carrier and used as a test device, it becomes possible to increase the concentration of the coating solution and hold more reagent on the carrier. In addition, since the compound according to the present invention has improved hydrophilicity and high molecular weight, loss of the reagent composition due to sublimation during storage can be suppressed. In addition, since the compound according to the present invention has little influence on the color intensity due to the change in reagent concentration, even when the compound deteriorates and decreases with time, the influence on the measured value is small. Furthermore, the hydrophilic functional group of the compound according to the present invention contained in the reagent composition according to the present invention increases the adsorptivity to the carrier and improves the stability of the compound in the carrier even when used as a test device. Let As described above, by providing the reagent composition according to the present invention, it is possible to provide a test device in which deterioration over time is reduced and the effective period is extended.

<Third Embodiment>
As in the second embodiment, the oxidative coloring compound or salt thereof according to the present invention is a reagent composition according to the present invention, which is a combination of an oxidase that acts selectively on a measurement object and a Trinder reagent. It becomes.

  The reagent composition according to the present invention may be used as an analytical composition in a state of being held in a liquid, powder or tablet. In addition to the above, these analytical compositions may contain components such as a pH buffer, an osmotic pressure regulator, a surfactant, a solubilizer, a stabilizer, and a protective agent. The allowable content of this component has been described above and will not be described.

  Moreover, when using it hold | maintaining in a liquid, after mixing with a test substance and making it react, you may measure a transmission light absorbency with a spectrophotometer other than judging the color change visually. Further, the reagent composition according to the present invention may be used as a test device while being held on a carrier.

  As described above, the reagent composition according to the present invention can be used while being held in various objects, but the reagent composition according to the present invention is preferably used as a test device while being held on a carrier.

  Therefore, the third embodiment is a test device in which the reagent composition according to the present invention is held on a carrier.

  When used in a state of being held on a carrier, such as a test tool used in such dry chemistry, in addition to a layer containing these reagent compositions, a measuring layer and a developing layer are provided as long as the functions thereof are not impaired. In addition, a filtration layer, a retention layer and the like may be included. Thus, when using it by hold | maintaining to a support | carrier, after giving a test substance, you may measure a reflected light absorbency with a spectrophotometer besides judging the color change visually. The measured value can be converted into the amount of the measurement object using a calibration curve prepared in advance.

  As the carrier material, porous materials such as paper, fabric, and polymer membrane can be used. In particular, a polymer membrane is preferable in terms of coloring performance.

  The polymer film is a water-insoluble porous body made of a polymer. Polymers include polysulfone, polyethersulfone, cellulose, cellulose acetate, cellulose nitrate, polyacrylonitrile, polyamide, aromatic polyamide, polycarbonate, polyethylene terephthalate, polyimide, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, poly Examples thereof include vinyl chloride and polyvinyl alcohol. Preferably, polysulfone and polyether sulfone are used. These polymers can form a film by using a generally known film forming method. Among these polymer films, polysulfone or polyether sulfone is particularly preferable in terms of color development performance.

  The method of holding the reagent composition according to the present invention on the carrier is not particularly limited. In addition to coating the carrier with a reagent composition solution dissolved in a suitable solvent on the carrier, a matrix precursor containing the reagent composition is used. A known method such as molding to form a test layer can be used.

  Existing couplers such as 4-aminoantipyrine have the lowest solubility among various reagents used in the measurement, and this has been a problem when preparing a coating solution having a high concentration. On the other hand, the compound according to the present invention has a high solubility and can easily produce a coating solution having a high concentration.

  In a test device in which a reagent composition is held on a carrier, after the sample is applied, the reagent composition is first dissolved by the liquid component of the sample and mixed to cause a reaction to produce a dye compound. Quantification is carried out through a step of moving to the reading surface on the opposite side of and measuring the color change. For this reason, the high solubility of the reagent composition is not only advantageous for uniform dissolution, homogenization, and rapid reaction by the specimen, but also the dye produced is excellent in solubility, so that it can move to the reading surface. There is also an advantage that it is smooth and highly uniform. These features contribute to shortening measurement time, improving measurement accuracy, and reducing measurement value variation. Therefore, since the oxidative coloring compound of the present invention or a salt thereof has high solubility, in a test device in which a reagent composition containing the compound is held on a carrier, measurement time is shortened, measurement accuracy is improved, and measurement is performed. It is expected to contribute to the reduction of value variation.

  When a test device in which a reagent containing 4-aminoantipyrine, which is a conventionally known coupler, is held on a carrier is used, there arises a problem that the measured value increases with a decrease in deterioration over time. The cause of the increase in the measured value due to the decrease is that the higher the concentration of the compound, rather than the inhibition of the enzyme reaction or the color reaction, may cause some obstacle to the dissolution and movement of other reagents and the fluidity of the generated dye. It is considered.

  The compound according to the present invention had less influence on the measured value even when the amount was increased or decreased compared to 4-aminoantipyrine, and was able to improve the problems seen with 4-aminoantipyrine. When a test device in which a reagent containing an existing coupler such as 4-aminoantipyrine is held on a certain carrier is used, there is a problem that a change in concentration affects the measured value. If unevenness occurs when coating the bulk carrier, there will be a difference in sensitivity between the test devices cut out and used, or the measured value will be affected if the amount changes due to degradation during storage. Suggests that. The cause is not inhibition of the enzyme reaction or color reaction, but it is thought that the higher the concentration of the compound, the more the dissolution and movement of other reagents and the fluidity of the generated dye will be.

  On the other hand, the compound according to the present invention has less influence on the measured value of concentration compared to 4-aminoantipyrine and can improve these problems.

  When the compound according to the present invention is oxidatively colored in combination with a Trinder reagent, the absorbance increases compared with the case where an existing coupler such as 4-aminoantipyrine is used, and the measurement sensitivity is improved. For example, 4-amino-1- [4-{(2,3,4,5,6-pentahydroxy-n-hexyl) aminocarbonyl} phenyl] -2,3-dimethyl-5-pyrazolone of the present invention, 4 -Amino-1- [4-{(2,3-dihydroxy-n-propyl) aminocarbonyl} phenyl] -2,3-dimethyl-5-pyrazolone, 4-amino-1- [4-{(2,3 -Dihydroxy-n-propyl) carbonylamino} phenyl] -2,3-dimethyl-5-pyrazolone and the like include N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS) and When colored in combination with N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), the slope of the calibration curve is improved and the measurement sensitivity is improved. It can be.

  As described above, since the compound according to the present invention is highly water-soluble, a test device (hereinafter, also simply referred to as “test device according to the present invention”) that holds a reagent composition containing the compound on a carrier is used. Since the reagent composition according to the present invention is quickly dissolved and homogenized in a specimen such as blood and a color reaction occurs rapidly, the test device according to the present invention can be used as a very excellent one.

  In addition, when the test device according to the present invention is used, the dye compound produced in the color reaction of the reagent composition according to the present invention also has high solubility and affinity for the specimen, and developability within the carrier. As a result, the color on the surface of the carrier is vivid and highly uniform. That is, when the test device according to the present invention is used, a highly sensitive and stable measurement can be performed. Furthermore, since the compound according to the present invention has high solubility, it is possible to prepare a reagent composition liquid (reagent liquid) having a higher concentration than conventional reagents. Then, when the reagent composition is supported on a carrier and used as a test device, it becomes possible to increase the concentration of the coating solution and hold more reagent on the carrier.

  In addition, since the compound according to the present invention has improved hydrophilicity and high molecular weight, loss of the reagent composition due to sublimation during storage can be suppressed. In addition, since the compound according to the present invention has little influence on the color intensity due to the change in reagent concentration, even when the compound deteriorates and decreases with time, the influence on the measured value is small. Furthermore, the hydrophilic functional group of the compound according to the present invention contained in the reagent composition according to the present invention enhances the adsorptivity to the carrier. That is, the test device according to the present invention has improved stability of the compound in the carrier.

  As described above, it can be said that the test device according to the present invention has reduced the deterioration with the passage of time and significantly increased the effective period compared with the conventional one.

  Next, the present invention will be described by way of examples.

<Example 1>
Synthesis of [4-amino-1- [4-{(2,3,4,5,6-pentahydroxy-n-hexyl) aminocarbonyl} phenyl] -2,3-dimethyl-5-pyrazolone (abbreviation CP8) ]
-5.0 g of 1- (4-carboxyphenyl) -3-methyl-5-pyrazolone phenylhydrazine-4-carboxylic acid (41799-1A manufactured by Kanto Chemical Co., Ltd.) and ethyl 3-oxobutanoate (A0649 manufactured by Tokyo Chemical Industry Co., Ltd.) 9 g of the mixture was heated and stirred at 120 ° C. for 1 hour. The solid precipitated after standing to cool was collected by filtration, washed with ethanol, and dried to obtain 8.3 g of the desired product.

1- (4-Carboxyphenyl) -2,3-dimethyl-5-pyrazolone methyl ester 3.0 g of pre-reaction product, 3.9 g of methyl iodide (I0060) manufactured by Kanto Chemical Co., and 100 mL of methanol are placed in a high-pressure reaction vessel. And stirred at 120 ° C. for 18 hours. The reaction mixture was evaporated to dryness, dissolved in chloroform, washed with water, and the organic layer was dried over anhydrous sodium sulfate. The organic layer was evaporated to dryness and subjected to silica gel column chromatography using methanol / chloroform (1: 9) as a solvent. The main fraction was concentrated to dryness to obtain 2.2 g of the desired product.

4-amino-1- (4-carboxyphenyl) -2,3-dimethyl-5-pyrazolone methyl ester 2.0 g of the pre-reaction product was dissolved in 25 mL of 0.4N hydrochloric acid, and 40% sodium nitrite under ice-cooling. 1.2 mL of an aqueous solution (37402-00 manufactured by Kanto Chemical Co., Inc.) was added over 2 minutes. Subsequently, the mixture was stirred at room temperature for 10 minutes, and the precipitated solid was collected by filtration, washed and dried. The solid was dissolved in 10 mL of methanol, 2 mL of 4N hydrochloric acid and 1.1 g of zinc powder (48005-00 manufactured by Kanto Chemical Co., Inc.) were added, and the mixture was stirred at 45 ° C. for 10 minutes. After filtration of the reaction mixture, the filtrate was concentrated to dryness and subjected to silica gel column chromatography using methanol / chloroform (1: 9) as a solvent. The main fraction was concentrated and dried to obtain 1.0 g of the desired product.

-0.37 g of 4-amino-1- (4-carboxyphenyl) -2,3-dimethyl-5-pyrazolone pre-reaction product was dissolved in 5 mL of methanol, and 0.25 mL of 4N aqueous sodium hydroxide solution was added at room temperature. The reaction was performed for 30 minutes. The reaction mixture was treated with a strongly acidic cation exchange resin (Aldrich product 216544-250G) and then concentrated to dryness to obtain 0.25 g of the desired product.

4-amino-1- [4-{(2,3,4,5,6-pentahydroxy-n-hexyl) aminocarbonyl} phenyl] -2,3-dimethyl-5-pyrazolone (CP8)
200 mg of the pre-reaction product and 800 mg of penta (O-methoxymethyl) glucamine (in-house synthesized product) are dissolved in 6 mL of THF, and 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4 is dissolved. -265 mg of methylmorpholinium chloride (DMT-MM, manufactured by Kokusan Chemical Co., Ltd. 2522021) was added, and the reaction was performed at room temperature for 1 hour. The reaction mixture was concentrated to dryness, and the main component was isolated by silica gel column chromatography using methanol / chloroform (1: 9) as a solvent. The dried main component was dissolved in 5 mL of methanol, 1.2 mL of 8N hydrochloric acid was added, and the mixture was reacted at room temperature for 1 hour for deprotection. The reaction mixture was concentrated to dryness and dried with a desiccator to obtain 186 mg of the desired product.

<Example 2>
[Synthesis of 4-amino-1- [4-{(2,3-dihydroxy-n-propyl) aminocarbonyl} phenyl] -2,3-dimethyl-5-pyrazolone (abbreviation CP9)]
300 mg of 4-amino-1- (4-carboxyphenyl) -2,3-dimethyl-5-pyrazolone synthesized in Example 1 and 2,2-dimethyl-1,3-dioxolane-4-methanamine (manufactured by Aldrich 483117- 5G) 400 mg is dissolved in 10 mL of methanol, and 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM, manufactured by Kokusan Chemical Co., Ltd. 2522021) 600 mg The mixture was reacted at room temperature for 1 hour, and then reacted at 80 ° C. for 30 minutes. The reaction mixture was concentrated to dryness, and the main component was isolated by silica gel column chromatography using methanol / chloroform (1: 9) as a solvent. The dried main component was dissolved in 3 mL of methanol, 2 mL of 4N hydrochloric acid was added, and the mixture was reacted at room temperature for 30 minutes for deprotection. The reaction mixture was concentrated to dryness and dried with a desiccator to obtain 316 mg of the desired product.

<Example 3>
[Synthesis of 4-amino-1- [4-{(2,3-dihydroxy-n-propyl) carbonylamino} phenyl] -2,3-dimethyl-5-pyrazolone (abbreviation CP10)]
-5.0 g of 1- (4-nitrophenyl) -3-methyl-5-pyrazolone 4-nitrophenylhydrazine (N0230 manufactured by Tokyo Chemical Industry) and 4.7 g of ethyl 3-oxobutanoate (A0649 manufactured by Tokyo Chemical Industry) It was dissolved in 50 mL and heated to reflux for 1.5 hours. The reaction mixture was evaporated to dryness, the residue was suspended in ether, the solid was collected by filtration and dried to obtain 6.0 g of the desired product.

1- (4-Nitrophenyl) -2,3-dimethyl-5-pyrazolone 5.0 g of pre-reaction product, 8.1 g of methyl iodide (I0060 manufactured by Kanto Chemical Co., Inc.) and 100 mL of methanol were placed in a high pressure reaction vessel. The mixture was heated and stirred at ° C for 18 hours. The reaction mixture was evaporated to dryness and purified by silica gel column chromatography using methanol / chloroform (1: 9) as a solvent to obtain 2.7 g of the desired product.
4-amino-1- (4-aminophenyl) -2,3-dimethyl-5-pyrazolone 1.7 g of the pre-reaction product was dissolved in 250 mL of 0.1N hydrochloric acid, and 40% sodium nitrite (37402, manufactured by Kanto Chemical Co., Inc.) -00) 1.5 mL of an aqueous solution was added, and the mixture was stirred at room temperature for 1.5 hours, and the precipitated solid was collected by filtration, washed and dried. The solid was dissolved in 140 mL of methanol, 5 mL of 4N hydrochloric acid and 1.0 g of zinc powder (manufactured by Kanto Chemical Co., Ltd. 48005-00) were added, and the mixture was stirred at ice temperature for 5 minutes and then at room temperature for 10 minutes. After filtration of the reaction mixture, the filtrate was concentrated to dryness and subjected to silica gel column chromatography using methanol / chloroform (1: 9) as a solvent. The main fraction was concentrated and dried to obtain 1.1 g of the desired product.

4-amino-1- [4-{(2,3-dihydroxy-n-propyl) carbonylamino} phenyl] -2,3-dimethyl-5-pyrazolone (CP10)
200 mg of the pre-reaction product and 366 mg of 2,2-dimethyl-1,3-dioxolane-4-methanecarboxylic acid (in-house synthesized product) were dissolved in 10 mL of methanol, and 4- (4,6-dimethoxy-1,3,5 was obtained. -Triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM, manufactured by Kokusan Chemical Co., Ltd. 2522021) 506 mg was added, and the reaction was carried out at 80 ° C. for 30 minutes. The reaction mixture was concentrated to dryness, and the main component was isolated by silica gel column chromatography using methanol / chloroform (1: 9) as a solvent. The dried main component was dissolved in 5 mL of methanol, 0.5 mL of 4N hydrochloric acid was added, and the mixture was reacted at room temperature for 30 minutes for deprotection. The reaction mixture was concentrated to dryness and dried with a desiccator to obtain the desired product (125 mg).

<Example 4>
Using a coating solution having the following composition, coating was performed on an in-house manufactured polyethersulfone film (thickness: 130 μm) by a kiss coating method, followed by drying at 37 ° C. for 18 hours. The membrane was cut into 1 cm squares to obtain test pieces.

  In the evaluation using blood, after fixing the test piece to the reflection spectrophotometer, 1 μL of a specimen was dropped from the upper part of the test film, and the reflected absorbance was measured continuously or at a predetermined time. As a specimen, human blood whose hematocrit value and blood glucose level were adjusted to predetermined values was used.

  FIG. 1 shows 4-aminoantipyrine (4AA), 4-amino-1- {4- (2,3,4,5,6-pentahydroxy-n-hexyl) aminocarbonylphenyl} -2,3- as couplers. Dimethyl-5-pyrazolone (CP8) and 4-amino-1- {4- (2,3-dihydroxy-n-propyl) aminocarbonylphenyl} -2,3-dimethyl-5-pyrazolone (CP9) were used. 4 shows the time variation of the reflected absorbance value when blood having a hematocrit value of 40 and a blood glucose level of 400 mg / dL is applied as a specimen to the test piece prepared by the above method.

  Regarding the absorbance value, it took 15 seconds or longer for 4AA to be compared to reach the maximum value, whereas it took only 10 seconds for CP9 and 5 seconds for CP8, and the measurement time was rapid by using the compound according to the present invention. It was shown that

<Example 5>
Using the test piece prepared in Example 4, a calibration curve was prepared from values measured with hematocrit value of 40 and blood of various glucose concentrations, and quantitative sensitivity was observed (FIG. 2). As the reflection absorbance value, the maximum time value for each test piece was used. In FIG. 2, since the reflection absorption is taken on the x-axis and the blood sugar level is taken on the y-axis, it means that the sensitivity is higher as the inclination is gentler.

  As shown in FIG. 2, 4-amino-1- {4- (2,3,4,5,6-pentahydroxy-n-hexyl) aminocarbonylphenyl} -2,3-dimethyl-5 of the present invention. -Pyrazolone (CP8) and 4-amino-1- {4- (2,3-dihydroxy-n-propyl) aminocarbonylphenyl} -2,3-dimethyl-5-pyrazolone (CP9) are When the color is developed in combination, the slope of the calibration curve is improved compared to when 4-aminoantipyrine (4-AA) is used, indicating that the measurement sensitivity can be significantly improved.

<Example 6>
After the test piece prepared in Example 4 was stored in a light-shielding sealed state at 25 ° C. for a predetermined period, the reflection absorbance value was measured using blood having a hematocrit value of 40 and a blood glucose level of 100 mg / dL as a specimen, The relationship was graphed (FIG. 3). As the reflection absorbance value, the maximum time value for each test piece was used.

  The comparison 4-aminoantipyrine (4-AA) increases in sensitivity with storage, and 4-amino-1- {4- (2,3,4,5,6-pentahydroxy-n-hexyl). ) Aminocarbonylphenyl} -2,3-dimethyl-5-pyrazolone (CP8), and 4-amino-1- {4- (2,3-dihydroxy-n-propyl) aminocarbonylphenyl} -2,3-dimethyl Both -5-pyrazolone (CP9) showed little variation in sensitivity over time, suggesting that the compound according to the present invention is excellent in stability over time.

It is a figure showing the time change of the reflective absorbance value of the compound according to the present invention and 4-AA in Example 4. In Example 5, it is a figure showing the measurement sensitivity of the compound which concerns on this invention, and 4-AA. In Example 6, it is the figure showing the time-dependent stability of the compound which concerns on this invention, and 4-AA.

Claims (11)

Following formula (1):

However, R ¹ and R ² are each independently a linear or branched alkyl group having 1 to 5 carbon atoms,
At least one or more of R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently a substituent selected from the following formulas (2) to (8), and the rest are hydrogen An atom,
Or an oxidative coloring compound or a salt thereof.
Following formula (2):

Following formula (3):

Following formula (4):

Following formula (5):

Following formula (6):

Following formula (7):

Following formula (8):

However, R ⁸ is a linear, branched or cyclic alkyl group having at least one hydroxyl group,
R ⁹ is a linear, branched or cyclic alkyl group having at least one hydroxyl group or a hydrogen atom,
At this time, R ⁸ and R ⁹ may be the same or different. The oxidative coloring compound or a salt thereof according to claim 1, wherein R ¹ and R ² are methyl groups. R ⁸ is a linear or branched alkyl group having 2 to 6 carbon atoms having 1 to 5 hydroxyl groups, and R ⁹ is a hydrogen atom, or
The oxidative coloring compound according to claim 1 or 2, wherein R ⁸ and R ⁹ are each independently a linear or branched alkyl group having 2 to 6 carbon atoms having 1 to 5 hydroxyl groups. These salts. R ⁸ is a 2,3-dihydroxy-n-propyl group, a bis (hydroxymethyl) methyl group or a 2,3,4,5,6-pentahydroxy-n-hexyl group, and R ⁹ is a hydrogen atom Is or
R ⁸ and R ⁹ are each independently a 2,3-dihydroxy-n-propyl group, a bis (hydroxymethyl) methyl group or a 2,3,4,5,6-pentahydroxy-n-hexyl group. The oxidative coloring compound or a salt thereof according to any one of claims 1 to 3. R ⁵ is selected from the group consisting of the above formulas (2) to (8),
The oxidative coloring compound or a salt thereof according to any one of claims 1 to 4, wherein R ³ , R ⁴ , R ⁶ and R ⁷ are hydrogen atoms. The oxidation color developing compound or a salt thereof according to any one of claims 1 to 5,
A Trinder reagent,
An oxidase that acts selectively on the measurement object;
Peroxidase,
A reagent composition comprising:   The Trinder reagent is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline or N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline The reagent composition according to claim 6, wherein The measurement object is glucose;
The reagent composition according to claim 6 or 7, wherein the oxidase is glucose oxidase.   A test device, wherein the reagent composition according to any one of claims 6 to 8 is held on a carrier.   The test device according to claim 9, wherein the carrier is a polymer film.   The test device according to claim 10, wherein the polymer film is made of polysulfone or polyethersulfone.

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