JPS61227570A - Novel imidazole derivative and method for determination using said derivative as color-developing component - Google Patents

Abstract

NEW MATERIAL:The imidazole derivative of formula I (R<1> is phenyl, alkyl, aralkyl, alkenyl or furyl; R<2> and R<3> are amino-substituted phenyl; R<4> is alkyl- substituted carbamoyl or phenyl-substituted carbamoyl). EXAMPLE:2-( 4-Carboxyphenyl )-3-N-methylcarbamoyl-4, 5-bis(4-diethylaminophe-nyl)imidazole. USE:A color-developing component for the determination of minor component in a bio-specimen such as serum and urine. That is, a color-developing component for the determination of oxidizing substance and a peroxidase-like substance. PREPARATION:The compound of formula I can be produced e.g. by reacting ethanedione of formula II with the aldehyde of formula R<1>-CHO and antimony acetate and reacting the resultant compound of formula III with an alkyl isocyanate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a novel imidazole derivative, and a method for quantifying oxidizing substances and a method for quantifying peroxidase-like substances using the compound as a coloring component.

[Background of the invention]

Biological components 1 Measuring body fluid components such as blood and urine is essential for diagnosing diseases, elucidating pathological conditions, and determining the course of treatment, as their fluctuations are closely related to diseases. For example, cholesterol, triglycerides, glucose, uric acid, phospholipids in the blood,
It is well known that methods for measuring a wide variety of trace components, including bile acids and monoamine oxidase, have been developed and are useful in diagnosing diseases.

Currently, the method for measuring serum components is to use an enzyme that specifically acts on the target component, and if the target component is an enzyme, use an enzyme that acts specifically on the target component. The so-called "enzyme method" is widely used in which a compound is subjected to an enzymatic reaction and the resulting product is measured to determine the amount of the target component. Among them, H2O2-generating enzymes, such as oxidase, are activated to generate H2O2 corresponding to the target component, and this is converted into peroxidase,
Coupled with the development of oxidizable coloring reagents, the method of determining the amount of the target component by introducing the oxidizable coloring reagent, which is a coloring component, into a coloring system and quantifying it colorimetrically is increasing. For example, peroxidase (PO
D) is a method in which an oxidizable coloring reagent is introduced into a coloring system and the amount of the target component is determined by measuring the absorbance of the coloring. A typical example of the oxidizable color reagent used in this method is a combination of 4-7 minoantipyrine and a phenol compound or an N,N-disubstituted aniline compound. Disfiguring color reagent, 3-
Combination reagent of methylbenzothiazolinone hydrazone (MBTH) and aniline compound, 2.2'-7 dinobis(3-ethylbenzothiazoline, -6-sulfonic acid)
(ABTS), triphenylmethane-based leuco dyes, benzidine derivatives, O-tolidine derivatives, diphenylamine derivatives, O-phenylenediamine, and the like. However, all of these conventionally used oxidizable coloring reagents, except for diphenylamine derivatives, have coloring wavelengths that differ from each other. OOnm or less, it is easily affected by serum components such as bilirubin and hemoglobin (when measuring urine components, it is easy to be affected by the effects of pigment bodies in the urine), and it is also susceptible to the effects of combination reagents with 4-aminoantipyrine and triglycerides. With the exception of some phenylmethane-based leuco dyes, all of them have problems such as low chromogen stability. On the other hand, triallylimidazole derivatives of dye precursors (aico dyes) have been disclosed as chromogens that have relatively good chromogen stability and have a coloration wavelength on the relatively long wavelength side (Tokuko Showa). Publication No. 57-5519,
Japanese Patent Publication No. 57-26118, Japanese Patent Publication No. 58-4555
No. 7, US Pat. No. 1,432,7710, etc.).

However, all of these existing triallylimidazole derivatives are colored by one of the seven phenyl groups, and the coloring wavelength is still 7G.
It is below the On layer. Therefore, even these triallylimidazole derivatives cannot be said to be fully satisfactory for use as coloring components in the measurement of trace components in biological samples such as blood and urine.

[Purpose of the invention]

The purpose of the present invention is to develop a novel oxidizable coloring reagent having a maximum absorption wavelength of 700 nm or more and a stable chromogen, which solves the problems of the conventional methods as described above, and to use this compound as a coloring component. By using it as a compound, the influence of colored coexisting substances such as hemoglobin and bilirubin was avoided. The objective is to realize a highly accurate method for measuring trace components in biological samples.

[Structure of the invention]

The present invention is based on the following general formula (1) [In the formula, R1 is a lower alkyl group or a lower alkoxy group.

Halogen atom, nitro group, amino group which may have a substituent, cyano group, -503)1 group or -503M'
group (3M1 represents an alkali metal ion or ammonium ion), -COOH group or -C00
Phenyl group which may have a substituent such as M2 group (1M2 represents an alkali metal ion or ammonium ion), alkyl M which may have a substituent, M
Aralkyl group which may have a substituent.

Represents an alkenyl group or a furyl group, and R2 and R3 are each independently a substituted phenyl group substituted with a 7-mino group which may have a substituent at least at either the ortho or para position. and R4 represents an alkyl-substituted carbamoyl group or a phenyl-substituted carbamoyl group which may have a substituent. Quantification of oxidizing substances and peroxidase-like substances using the imidazole derivative and the compound as a coloring component. It is the law.

In other words, the present invention has demonstrated that all of the imidazole derivatives represented by the above general formula CI) have coloration wavelengths on the long wavelength side of 700 nm or more, and are extremely stable as chromogens. The present invention was completed after discovering that the above object of the present invention can be achieved by using it as a coloring component in the measurement of trace components in biological samples such as serum and urine. It is something that

In the imidazole derivative of the present invention represented by general formula (I), examples of the substituent of the phenyl group represented by R1 which may have a substituent include.

Lower alkyl group having 1 to 5 carbon atoms such as methyl group, ethyl group, propyl group, butyl group, pentyl group (can be linear or monobranched) 1 For example, methoxy group, ethoxy group,
A lower alkoxy group having 1 to 4 carbon atoms such as a propoxy group or a butoxy group (either linear or monobranched is acceptable), for example, a phenoxy group.

7-aryloxy group such as methylphenoxy group, halogen such as chlorine, bromine, fluorine, iodine, nitro group, ami7 group, lower alkyl group such as methyl group, ethyl group, propyl group, butyl group, or for example -c2u4on group , -C3HθO
H group, -C2H4NHSO2 (:H3 group.

-C2H4NHCOCH3 group, -c2H4so3H group (
or -c2n4so3Na group, etc.), -C3H6S0
3H group (or -C3H6SO3Na group, etc.).

Cyano group, -5038 group or -503M1 group (however,
Ml represents an alkali metal ion such as sodium, potassium, lithium or ammonium ion, ), -(:
00H group or -cooM group (however, H2 is sodium.

(representing an alkali metal ion such as potassium, lithium, or ammonium ion), lower flukoxycarbonyl groups such as methoxycarbonyl group, ethoxycarbonyl group, impropoxycarbonyl group, sulfonyl group such as methylsulfonyl group, ethylsulfonyl group, etc. Examples of the alkyl group include a kylsulfonyl group, but do not include a hydroxyl group, and the alkyl group represented by R1, which may have a substituent, is a methyl group.

Ethyl group, propyl group, butyl group, hexyl group.

Examples include alkyl groups having 1 to 10 carbon atoms such as octyl group and decyl group, which may be linear or monobranched, or 1M
Substituents include hydroxyl group, halogen atom (chlorine, bromine, etc.),
Examples include a cyano group, a nitro group, a carboxyl group, a sulfone group, etc., and examples of the 7-ralkyl group of the aralkyl group which may have an l substituent include a benzyl group, a phenethyl group, a phenylpropyl group, etc. Substituents include lower alkyl groups such as methyl, ethyl and propyl groups, lower alkoxy groups such as methoxy, ethoxy and propoxy groups, halogens such as chlorine and bromine, carboxyl groups, sulfone groups and nitro groups. Examples of the alkenyl group include a vinyl group, a propenyl group, and a butenyl group. Examples of substituents for the substituted amine group in the substituted phenyl group represented by R2 and R3, which is substituted with an amino group which may have a substituent at least at either the ortho position or the para position, include: Lower alkyl groups such as methyl group, ethyl group, propyl group, butyl group, -C2H4OH group.

-03M60H group, -C2H4NH302CH3 group,
-C2H4NHCOCI3 groups.

-C2H45O3) 1 group (or -C2H4SO3Na group, etc.).

-C3HBSO3) 1 group (or -C3HBSO3Na group, etc.).

Examples of substituents other than the amino group or substituted amine group in R2 and R3 include, but are not limited to, a methyl group, an ethyl group, and a propyl group.

Examples include lower alkyl groups such as butyl groups, lower alkoxy groups such as methoxy, ethoxy, and propoxy groups, halogen atoms such as chlorine, bromine, fluorine, and iodine, nitro groups, cyano groups, carboxyl groups, and sulfone groups. , hydroxyl groups are not included.

Examples of the alkyl-substituted carbamoyl group represented by R4 include 1, for example, N-methylcarbamoyl group, N-ethylcarbamoyl group, N-isopropylcarbamoyl, 1, N, N
- Lower alkyl-substituted carbamoyl groups such as -dimethylcarbamoyl group, N,N-diethylcarbamoyl group, N,N-diisopropylcarbamoyl group, etc., and substituents of the phenyl-substituted carbamoyl group which may have a substituent include: Examples include lower alkyl groups such as methyl, ethyl, and propyl groups, lower alkoxy groups such as methoxy and ethoxy groups, and halogen atoms such as chlorine and bromine. A carbamoyl group substituted with a phenyl group is preferably used.

It goes without saying that an N-furkyl-N-phenyl-carbamoyl group can also be used in the same manner.

The imidazole derivative of the present invention represented by the general formula (1) is, for example, in the general formula [E], x2 represents another substituent), and by oxidation, the dye has the following structure. generate.

, xl represents another substituent), and x2 represents another substituent), a dye having the following structure is produced by oxidation.

That is, in any case, the alkyl-substituted carbamoyl group represented by R4 or the phenyl-substituted carbamoyl group which may have an H substituent is removed from the imidazole group during the melting reaction and oxidized with hydrogen peroxide, for example. In case R
It becomes NHCOOH, which is completely different from the pigment produced by oxidation. That is, the alkyl-substituted carbamoyl group or the phenyl-substituted carbamoyl group which may have a substituent represented by R4 is only involved in stabilizing one chromogen (stabilizing the blank) and has no effect on color development. I won't give it.

Any of the dyes represented by (■) or ([) above, which are produced by oxidation of the imidazole conductor of the present invention represented by general formula (I), are different from those in existing triallylimidazole derivatives. 00, the coloring wavelength thereof is further shifted to the longer wavelength side than that of the dye produced by the dye, and the maximum absorption wavelength is 700 nm or more in all cases.

Table 1 shows some specific examples of the compounds of the present invention represented by the general formula (1) and their maximum absorption wavelengths at the time of color development, but the compounds of the present invention are not limited to these.

The compound of the present invention represented by general formula (I) can be easily synthesized by a known method. For example, Or
ganic 5intheses Vol, 5.111
Ethanedione represented by formula (IV) was synthesized according to the method described on page 1973.

(In the formula, R2 and R3 are the same as above.) Then, this is converted into R-C)10 (R' is the same as above) aldehydes and acetic acid, for example, according to the method described in US Pat. This is because the imidazole derivative represented by formula (V) can be obtained by reacting with ammonium in an acetic acid solvent by heating (refluxing if necessary) for several hours.

(In the formula, R1, R2, and R3 are the same as above.) The obtained imidazole derivative is mixed with alkyl isocyanate (or phenyl isocyanate) in the presence of a suitable solvent at room temperature or the boiling point of the solvent for several hours to more than ten hours. If the reaction is carried out, the target product can be obtained in good yield.

If necessary, a purified product can be easily obtained by purifying this by an appropriate purification method such as recrystallization or column chromatography.

The imidazole derivative of the present invention can be effectively used as a coloring component in the determination of oxidizing substances and peroxidase-like substances. It can be particularly effectively used as a coloring component in quantifying trace components in biological samples by colorimetrically quantifying coloration.

That is, 1. The method for quantifying oxidizing substances of the present invention uses a substrate.

Alternatively, it can be particularly effectively used as a method for quantifying a substrate or enzyme activity in a biological sample by allowing an oxidizing enzyme to act on a substance produced by an enzymatic reaction and quantifying the produced hydrogen peroxide.

Examples of trace components in biological samples that can be measured by the method of the present invention include cholesterol, glucose, glycerin, triglycerides, free fatty acids, uric acid, phospholipids, bile acids, monoamine oxidase, guanase, cholinesterase, etc. Although not limited to these, all biological components that can be measured can be quantified by quantifying hydrogen peroxide produced by an enzymatic reaction.

In quantifying biological components by the method of the present invention, oxidases (oxidases) used as enzymes to generate hydrogen peroxide, enzymes used for other purposes, and types of substrates and other substances involved in enzyme reactions and the amount used may be appropriately selected according to the substance to be measured in accordance with a known method for quantifying biological components using an oxidizable coloring reagent. The origin of peroxidase used in

There is no particular limitation on the origin, and one or, if necessary, a combination of two or more peroxidases or peroxidase-like substances of plant, animal, or microbial origin may be used. Further, the amount used is appropriately determined depending on the purpose and is not particularly limited.

Quantitation of biological components by the method of the present invention is usually performed using pi44
．． 0-1G, 0. More preferably, PHe is carried out at o~8.0. Buffers used include phosphate, citrate, borates, carbonates, Tris buffer, Gud(
Examples include, but are not limited to, Goad's II buffer.

The imidazole derivative of the present invention can be effectively used for the determination of oxidizing substances such as hydrogen peroxide (1), but it is also possible to quantify peroxidase-like substances by combining it with hydrogen peroxide.

Examples of peroxidase-like substances include hemoglobin and other heme compounds in addition to peroxidase itself.

That is, the imidazole derivative of the present invention can be applied, for example, to enzyme immunoassay using peroxidase as a labeling compound, and hemoglobin in serum can be detected by oxidizing substances such as hydrogen peroxide or sodium perborate. It can also be effectively used when measuring using.

Examples are given below, but the present invention is not limited in any way by these Examples.

〔Example〕

Example 1 Synthesis of 2-(4-carboxyphenyl)-3-N-methylcarbamoyl-4,5-bis(4-diethylaminophenyl)imidazole [compound (2) of the present invention] (i) 1,2-bis (4-diethylaminophenyl)
Synthesis of ethane-1,2-dione 201 carbon disulfide was added to 6.7 g of anhydrous aluminum chloride, and 20 g of N and diethylaniline were added dropwise under water cooling.

Further, while stirring and cooling with ice, add oxalyl chloride.1.
5g of water was added dropwise and stirred for 80 minutes.After the reaction, 5g of water was added dropwise.
0 ml and 100 s 1 of chloroform were added, and the resulting chloroform layer was concentrated under reduced pressure to precipitate crystals. The precipitated crystals were collected and recrystallized from ethyl acetate to obtain 5.0 g of a yellow target product.

(ii) 2-(4-carboxyphenyl)-4
, 1.5 g of 1,2-bis (4-diethylaminophenyl) ethane-1,2-dione obtained in synthesis (i) of 5-bis (4-diethylaminophenyl) imidazole and 0.7 g of p-carpoxybenzaldehyde, 5 g of ammonium acetate was reacted in 30 ml of acetic acid by heating under reflux for 2 hours. After cooling, 80 ml of wood was added and the mixture was cooled with water and neutralized with aqueous ammonia to precipitate crystals. The precipitated crystals were collected, washed with water, dried, and then treated with hexane to obtain 1.1 g of a white target compound.

(iii) 2-(4-carboxyphenyl)-3-N-
2-(4-carboxyphenyl)-4, obtained in synthesis (ii) of methylcarbamoyl-4,5-bis(4-diethylaminophenyl)imidazole,
1.1 g of 5-bis(4-diethyl7minophenyl)imidazole was dissolved in 501 chloroform, and 511 methyl isocyanate was added with stirring, and the mixture was left to stand at room temperature for 12 hours.Next, 501 methanol was added to stop the reaction, and the mixture was concentrated.

The concentrate was purified using a silica gel column using chloroform as an eluent to obtain 980 layers of the desired product (grain-colored powder).

NMR(CD(:13.TMS) pHllll:
1.2 (12H,t.

CH3-C-), 1.9 (L H,s, -C-
N-C), 2.7 (3H.

■ s, -N-0 勺), 3.8 (8H, q, C
-C station-), 7.1-7.7 (12H, broa
d, 7z nyl hydrogen), 11.8 (IH, s.

-COOH) Example 2゜2-(3-methoxy-4-diethylaminophenyl)-
Synthesis of 3-N-methylcarbamoyl-4,5-bis(2-methyl-4-diethylaminophenyl)imidazole [Compound (1G) of the present invention] (i) 1,2-bis(2-methyl-4-diethylaminophenyl) ) ethane-1,2-
Synthesis of dione 20 l of carbon disulfide was added to 8.7 g of anhydrous aluminum chloride, and 22 g of 3-methyl-N,N-diethylaniline was added dropwise under ice cooling. Further, 1.5 g of oxalyl chloride was added dropwise while stirring and ice-cooling, and the reaction was stirred for 80 minutes. After the reaction, 50 ml of water and 1 ml of chloroform were added.
001 was added and the mixture was separated for 1 minute, and the obtained chloroform layer was concentrated under reduced pressure to precipitate crystals. The precipitated crystals were recrystallized from pure ethyl acetate to obtain 5.4 g of a yellow target product.

(ii) Synthesis of 2-(3-methoxy-4-diethylaminophenyl)-4,5-bis(2-methyl-4-diethyl7minophenyl)imidazole 1,2- obtained in (i)
1.5 g of bis(2-methyl-4-diethylaminophenyl)ethane-1,2-dione, 1.0 g of 3-methoxy-4-diethylaminobenzaldehyde, and 5 g of ammonium acetate were reacted in 30 ml of acetic acid by heating under reflux for 2 hours. Ta.

After cooling, 80 ml of water was added and the mixture was neutralized with aqueous ammonia under water cooling to precipitate crystals. Kana, wash the precipitated crystals with water,
After drying, it was treated with hexane to obtain 0.8 g of a white target compound.

(iii) Synthesis of 2-(3-methoxy-4-diethylaminophenyl)-3-N-methylcarbamoyl-4゜5-bis(2-methyl-4-diethylaminophenyl)imidazole 2-( obtained in (ii) 0.8 g of 3-methoxy-4-diethyl-7minophenyl)-4,5-bis(2-methyl-4-diethylaminophenyl)imidazole was dissolved in chloroform 5011, and while stirring, 5 liters of methyl isocyanogen was added and the mixture was heated at room temperature. It was left for 12 hours.

Next, methanol 501 was added to stop the reaction, and the mixture was concentrated. The concentrate was purified using a silica gel column using chloroform as an eluent to obtain 570 mg of the target product (white powder).

NMR (COOH3, TMS) pp■: 1.1 (
18H,t.

8.4-7.2 (91, broad, 7 x nylhydrogen) Example 3 Quantification of uric acid (1) Reagent 50+ss+ol/IME S (2-(
N-molyporino)ethanesulfonic acid buffer (p) 18
．． 5) Nitsu'J Case 100 U/I, Pek:t
A-sidase 2,0OOU/I, compound of the present invention (2)
It was prepared by dissolving it to a concentration of 100 g ol/1.

(2) Sample uric acid with distilled water 10.7.5.5.2.5
It was dissolved and prepared to have a concentration of g/dl.

(3 measurement operations) 3.01 of the reagent was added to each sample solution and 50 pl of distilled water, heated at 37°C for 5 minutes, and the absorbance at 740 nm was measured using a blind control.

Figure 1 shows the relationship between uric acid concentration and absorbance.

As is clear from FIG. 1, the calibration curve connecting the absorbance plotted for each uric acid concentration is a straight line passing through the origin, and the calibration curve shows good quantitative properties.

Example 4 Determination of hydrogen peroxide (1) Reagent 50 mmal/I phosphate buffer (pH 7,
Peroxidase (2.0000/l) and compound (2) of the present invention were dissolved in 100 g/l/1.

(2) Sample Commercially available hydrogen peroxide solution was diluted with distilled water to 2.0
．． It was dissolved and prepared to have a concentration of 1.5.1.0.0.5 mmol/l.

(3) 1s constant operation: Add 3.0 servings of reagent to each sample solution and 20p+ volume of distilled water, heat at 37°C for 3 minutes, and heat for 740n.
The absorbance of m was measured using a blind control.

Figure 2 shows the relationship between hydrogen peroxide concentration and absorbance.
As is clear from the figure, the calibration curve connecting the absorbance plotted for each hydrogen peroxide concentration is a straight line passing through the origin, and the calibration curve shows good quantitative properties.

〔Effect of the invention〕

As mentioned above, one novel imidazole derivative of the present invention is
In both cases, the maximum absorption wavelength when coloring is on the long wavelength side, 7Q Onm or more, so when used as a coloring component in quantifying trace components in serum or original biological samples,
It is stabilized as a chromogen by being able to perform measurements without being affected by colored interfering substances that coexist in the sample, and by attaching a substituted carbamoyl group to the N-3 position of the imidazole ring. It has a remarkable effect in that it can be used as an imidazole derivative, and it is a great contribution to this industry.

[Brief explanation of the drawing]

Figure 1 shows the calibration curve obtained in Example 3, which connects the absorbance obtained for each uric acid concentration (g/di) on the horizontal axis and plotted along the vertical axis. be. Figure 2 shows the calibration curve obtained in Example 4, connecting the points obtained by plotting the absorbance obtained for each hydrogen peroxide concentration (1 layer o1/l) on the horizontal axis along the vertical axis. It is something. Patent applicant: Wako Pure Chemical Industries, Ltd. No. 1 concave urine #concentration (ttl/Jl)

Claims (10)

[Claims] (1) The following general formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] [In the formula, R^1 is a lower alkyl group, a lower alkoxy group,
Halogen atom, nitro group, amino group which may have a substituent, cyano group, -SO_3H group or -SO_3M^
1 group (however, M^1 represents an alkali metal ion or ammonium ion), -COOH group or -COOM
A phenyl group that may have a substituent such as ^2 group (where M^2 represents an alkali metal ion or an ammonium ion), an alkyl group that may have a substituent,
Represents an aralkyl group, alkenyl group or furyl group which may have a substituent, and R^2 and R^3 each independently have a substituent at least at either the ortho position or the para position. represents a substituted phenyl group which is optionally substituted with an amino group, and R^4 represents an alkyl-substituted carbamoyl group or a phenyl-substituted carbamoyl group which may have a substituent. ] An imidazole derivative represented by. (2) The following general formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] [In the formula, R^1 is a lower alkyl group, a lower alkoxy group,
Halogen atom, nitro group, amino group which may have a substituent, cyano group, -SO_3H group or -SO_3M^
1 group (however, M^1 represents an alkali metal ion or ammonium ion), -COOH group or -COOM
A phenyl group that may have a substituent such as ^2 group (where M^2 represents an alkali metal ion or an ammonium ion), an alkyl group that may have a substituent,
Represents an aralkyl group, alkenyl group or furyl group which may have a substituent, and R^2 and R^3 each independently have a substituent at least at either the ortho position or the para position. represents a substituted phenyl group which is optionally substituted with an amino group, and R^4 represents an alkyl-substituted carbamoyl group or a phenyl-substituted carbamoyl group which may have a substituent. ] A method for quantifying oxidizing substances, characterized by using an imidazole derivative represented by the following as a coloring component. (3) The quantitative method according to claim 2, wherein the oxidizing substance is hydrogen peroxide. (4) The assay method according to claim 3, wherein the coloring component is oxidized and colored in the presence of peroxidase, and the color development is measured colorimetrically. (5) The quantitative method according to claim 3 or 4, wherein the hydrogen peroxide is hydrogen peroxide produced by an enzymatic reaction. (6) The assay method according to claim 5, wherein the hydrogen peroxide is hydrogen peroxide produced by an enzymatic reaction in quantifying trace components in biological samples. (7) Quantification of trace components in biological samples is performed by quantifying hydrogen peroxide produced by acting oxidizing enzymes on substrates or substances produced by enzymatic reactions. A quantitative method according to claim 6. (8) The following general formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] [In the formula, R^1 is a lower alkyl group, a lower alkoxy group,
Halogen atom, nitro group, amino group which may have a substituent, cyano group, -SO_3H group or -SO_3M^
1 group (however, M^1 represents an alkali metal ion or ammonium ion), -COOH group or -COOM
A phenyl group that may have a substituent such as ^2 group (where M^2 represents an alkali metal ion or an ammonium ion), an alkyl group that may have a substituent,
Represents an aralkyl group, alkenyl group or furyl group which may have a substituent, and R^2 and R^3 each independently have a substituent at least at either the ortho position or the para position. represents a substituted phenyl group which is optionally substituted with an amino group, and R^4 represents an alkyl-substituted carbamoyl group or a phenyl-substituted carbamoyl group which may have a substituent. ] A method for quantifying a peroxidase-like substance, characterized in that an imidazole derivative represented by the following is used as a coloring component. (9) The quantitative method according to claim 8, wherein the peroxidase-like substance is peroxidase. (10) The assay method according to claim 8, wherein the peroxidase-like substance is hemoglobin or other heme compound.