JPH0745477B2 - Imidazole derivative and measuring method using the same as a coloring component - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel imidazole derivative, a method for quantifying an oxidizing substance and a method for quantifying a peroxidase-like substance using the compound as a color-forming component.

[Background of the Invention]

The measurement of biological components, such as body fluid components such as blood and urine, is essential for diagnosing diseases, elucidating the pathological condition, and determining the course of treatment, because the fluctuations are greatly related to diseases. Has become. For example, cholesterol in blood, triglycerite, glucose, uric acid, phospholipids,
It is well known that assay methods for very various kinds of trace components such as bile acid and monoamine oxidase have been developed and are useful for diagnosis of diseases.

Currently, as a method for measuring serum components, when it is something other than an enzyme, an enzyme that acts specifically on the target component is used, and when the target component is an enzyme, it should be the substrate The so-called "enzyme method", in which a compound is used to carry out an enzyme reaction with each other, and the resulting product is measured to determine the amount of the target component, is generally widespread. Among them, H ₂ O ₂ producing enzyme, for example, oxidase is activated to produce H ₂ O ₂ corresponding to the target component, and this is converted into a coloring system by using peroxidase and an oxidizable coloring reagent which is a coloring component. The number of methods for obtaining the amount of a target component by colorimetrically quantifying the color development is increasing together with the development of an oxidizable color reagent. For example, cholesterol - leads to H ₂ O ₂ generated by a combination of such uricase, peroxidase (POD), the coloring system using an oxidizable color reagent, - cholesterol oxidase, triglyceride - lipoprotein lipase - glycerol oxidase, uric acid This is a method of obtaining the amount of the target component by measuring the absorbance of the color.
A typical example of an oxidizable color reagent that is a color-forming component used in this method is a combination of 4-aminoantipyrine and a phenol compound or an N, N-disubstituted aniline compound. Color reagent, combination reagent of 3-methylbenzothiazolinone hydrazine (MBTH) and aniline compound, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), triphenylmethane Examples thereof include leuco dyes, benzidine derivatives, o-tolidine derivatives, diphenylamine derivatives and o-phenylenediamine. However, most of these conventionally used oxidizable color reagents, except for diphenylamine derivatives, have a color wavelength of 600 nm or less and are easily affected by serum components such as bilirubin and hemoglobin (urinary urine). It is easily affected by urinary plastids when measuring the components), and the stability of the chromogen is low except for some reagents in combination with 4-aminoantipyrine and some of the triphenylmethane-based leuco dyes. Has the problem of. on the other hand,
A triallylimidazole derivative of a dye precursor (leuco dye) is disclosed as a chromogen having a relatively good chromogen stability and a coloring wavelength on the relatively long wavelength side. (Japanese Patent Publication No. 57-5519, Japanese Patent Publication No. 57-26118, Japanese Patent Publication No. 58-45
No. 557, U.S. Pat. No. 3297710, etc.) These imidazole derivatives all have a coloration wavelength of 600 nm or more on the relatively long wavelength side and have relatively high sensitivity, but the stability of the chromogen is It cannot always be said that it is sufficiently satisfactory, and those having high sensitivity still have a problem in stability of the chromogen.

[Object of the Invention]

The object of the present invention is to develop an imidazole derivative having a maximum absorption wavelength on the long wavelength side of 600 nm or more at the time of coloring, high sensitivity, and extremely stable chromogen, and an oxidizing substance and peroxidase using the compound. The purpose of the present invention is to provide a highly accurate quantification method for such substances.

[Structure of Invention]

 In order to achieve the above object, the present invention has the following configuration.

The following general formula [I] [Wherein R ¹ , R ² and R ³ are each an organic group, and ^one of R ¹ , R ² and R ³ is an aryl group in which either the ortho position or the para position is substituted with a hydroxyl group. Wherein one represents an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent, and the remaining one represents the ortho position. Or an aryl group in which either the para position is substituted with a hydroxyl group, or an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent. Represents a group or a benzyl group. R ⁴ represents an optionally substituted phenyl group, an alkenyl group, an acyl group, an alkyl-substituted carbamoyl group, or an optionally substituted phenyl-substituted carbamoyl group. ] The imidazole derivative shown by these, and the quantification method of an oxidizing substance and a peroxidase-like substance which use this compound as a coloring component.

In the present invention, all of the imidazole derivatives represented by the above general formula [I] have a coloring wavelength on the long wavelength side of 600 nm or more, and have a group represented by R ⁴ at N at the 3-position of the imidazole ring. According to the present inventor, the present invention makes it extremely stable as a chromogen regardless of the coloration sensitivity, and by using this as a color-forming component in the quantification of oxidizing substances and peroxidase-like substances, it is possible to perform more accurate measurement. Have found and completed.

In the imidazole derivative of the present invention represented by the general formula [I], an aryl group represented by R ¹ , R ² and R ³ in which either the ortho position or the para position is substituted with a hydroxyl group, and the ortho position Or, as the substituent other than the ortho position or the para position of the aryl group substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent, either of the para positions is, for example, a methyl group or an ethyl group. Group, propyl group, butyl group, pentyl group and other lower alkyl groups having 1 to 5 carbon atoms (either linear or branched), such as methoxy group, ethoxy group, propoxy group, butoxy group, etc. A lower alkoxy group having 1 to 4 carbon atoms (which may be linear or branched), for example, an aryloxy group such as a phenoxy group, a methylphenoxy group, a carboxyphenoxy group or a chlorophenoxy group. Chlorine, bromine, fluorine, halogen atom iodine, etc., hydroxyl group, a nitro group, an amino group, for example a methyl group, an ethyl group, a propyl group, a lower alkyl group, or for example -C ₂ H ₄ OH group or butyl group, -C ₃ H ₆ OH group, -C ₂ H ₄ NHSO ₂ CH
₃ groups, -C ₂ H ₄ NHCOCH ₃ group, -C ₂ H ₄ SO ₃ H group (or -C ₂ H ₄ SO ₃ Na group, etc.), -C ₃ H ₆ SO ₃ H group (or -C ₃ H ₆ SO ₃ Na group, etc.), An amino group substituted with a substituted lower alkyl group or the like, for example, a lower alkoxycarbonyl group such as a methoxycarbonyl group, an ethoxycarbonyl group, an isopropoxycarbonyl group,
Examples thereof include sulfonyl groups, for example, lower alkylsulfonyl groups such as methylsulfonyl group and ethylsulfonyl group, but are not limited thereto. Also, R ¹ , R ² and R
Examples of the alkoxy group which is the substituent at the ortho position or the para position in ³ include a methoxy group, an ethoxy group, a propoxy group, a butoxy group and the like, and an aryloxy group includes a phenoxy group such as a methylphenoxy group, Substituted phenoxy groups such as carboxyphenoxy group, sulfophenoxy group, chlorophenoxy group and the like can be mentioned. Examples of the substituent of the amino group which may have a substituent include a methyl group, an ethyl group, a propyl group and a butyl group. Lower alkyl group such as -C ₂ H ₄ OH group, -C ₃ H ₆ OH group, -C ₂ H ₄ NHSO ₂ CH ₃ group,-
C ₂ H ₄ NHCOCH ₃ group, -C ₂ H ₄ SO ₃ H group (or -C ₂ H ₄ SO ₃ Na group), -C
₃ H ₆ SO ₃ H group (or —C ₃ H ₆ SO ₃ Na group), And a substituted lower alkyl group thereof.

Examples of the phenyl group which may have a substituent represented by R ⁴ include a phenyl group, a methylphenyl group, a chlorophenyl group, a carboxyphenyl group and a sulfophenyl group, and an alkenyl group may be, for example, vinyl. Group, propenyl group, butenyl group and the like, and examples of the acyl group include acetyl group, propionyl group, butyryl group, valeryl group, benzoyl group, toluoyl group, salicyloyl group, phthaloyl group and the like. Examples of the alkyl-substituted carbamoyl group include N-methylcarbamoyl group, N-ethylcarbamoyl group, N-isopropylcarbamoyl group, N, N-dimethylcarbamoyl group, N, N
-Diethylcarbamoyl group, lower alkyl-substituted carbamoyl group such as N, N-diisopropylcarbamoyl group, and the like. Examples of the substituent of the phenyl-substituted carbamoyl group which may have a substituent include, for example, methyl group, ethyl group and propyl group. Groups such as lower alkyl groups, for example, lower alkoxy groups such as methoxy and ethoxy groups, halogen atoms such as chlorine, bromine, fluorine and iodine, and the like, and phenyl groups which may have these substituents. Alternatively, a di-substituted carbamoyl group is used. Needless to say, an N-alkyl-N-phenylcarbamoyl group can be used as well.

The imidazole derivative of the present invention represented by the general formula [I] reacts with hydrogen peroxide in the presence of peroxidase, for example, as follows.

That is, for example, in the general formula [I], Represents an amino group which may have a substituent. ), (However, R ⁷ represents an alkyl group or a phenyl group which may have a substituent.) It represents a phenyl group which may have a substituent or a substituent. ), Furthermore, for example If R ⁴ = -CH = CH ₂ , That is, when R ⁴ is an alkyl-substituted carbamoyl group or a phenyl-substituted carbamoyl group which may have a substituent, these groups are separated from the imidazole group during the oxidation reaction and oxidized with, for example, hydrogen peroxide. In some cases, it becomes RNHCOOH, which is completely different from the dye produced by oxidation. That is, the alkyl-substituted carbamoyl group represented by R ⁴ or the phenyl-substituted carbamoyl group which may have a substituent is involved only in the stabilization of the chromogen (stabilization of the blank) and has no influence on the color development. Don't give. On the other hand, in the case where R ⁴ is a phenyl group which may have a substituent, an alkenyl group, or an acyl group, at least in the hydrogen peroxide-peroxidase-based oxidation reaction, it is not separated from the imidazole group and is as it is. It remains, and serves to stabilize the chromogen and double the sensitivity per H ₂ O ₂ .

Table 1 shows some specific examples of the compound of the present invention represented by the general formula [I], the maximum absorption wavelength and the molecular extinction coefficient ε at the time of coloring.
(H ₂ O ₂ basis) and chromogen stability (blank stability) are shown, but it goes without saying that the compound of the present invention is not limited thereto.

* Blank stability Dissolve POD 2000U / l and each leuco body 300μmol / l in 50mM phosphate buffer (pH7.0) to make a test solution, and compare the absorbance of this test solution with that after 24 hours at 25 ℃. When the difference is 0.100 or more, it is −, when it is 0.050 to 0.100, it is ±,
When it was 0.050 or less, it was regarded as +.

As is clear from Table 1, R ⁴ (= hydrogen) of the existing triallylimidazole derivative shown in Comparative Examples is substituted with various substituents (alkyl-substituted carbamoyl group and optionally substituted phenyl) according to the present invention. (Excluding carbamoyl group)
By replacing with, the sensitivity per H ₂ O ₂ is doubled.

The compound of the present invention represented by the general formula [I] can be easily synthesized by a known method. That is, for example, Orga
nic Syntheses Vol. 5, page 111, according to the method described in 1973, to synthesize ethanedione represented by the formula [II], (In the formula, R ² and R ³ are the same as the above.) Then, the aldehydes R ¹ -CHO (R ¹ is the same as the above) according to the method described in, for example, US Pat. No. 3297710. And ammonium acetate and a reaction for several hours in an acetic acid solvent (reflux if necessary) to obtain an imidazole derivative represented by the formula [III], (In the formula, R ¹ , R ² and R ³ are the same as above.) The obtained imidazole derivative is treated with an alkyl isocyanate (or phenyl isocyanate, an acyl halide, etc.) at room temperature to the boiling point of the solvent. ) For several hours to several tens of hours, the target product can be obtained in good yield. If necessary, a purified product can be easily obtained by purifying it by an appropriate purification method such as recrystallization or column chromatography.

The imidazole derivative of the present invention can be effectively used as a color-forming component in the quantification of oxidative substances and peroxidase-like substances, but especially hydrogen peroxide produced by an enzymatic reaction is led to a chromogenic system in the presence of peroxidase, It can be used particularly effectively as a color-forming component in the quantification of trace components in a biological sample by colorimetrically quantifying coloration.

That is, the method for quantifying an oxidative substance of the present invention is a quantification of a substrate or an enzyme activity in a biological sample, which is performed by causing an oxidase to act on a substrate or a substance produced by an enzymatic reaction, and quantifying produced hydrogen peroxide. It can be used particularly effectively as a method.

As a trace component in a biological sample that can be measured by the method of the present invention, for example, cholesterol, glucose, glycerin, triglyceride, free fatty acid, uric acid, phospholipid,
Examples thereof include bile acid, monoamine oxidase, guanase, cholinesterase, and the like, but not limited to these, and all biological components that can be measured by quantifying hydrogen peroxide produced by an enzymatic reaction can be quantified.

In the determination of biological components by the method of the present invention, oxidase (oxidase) used as an enzyme for producing hydrogen peroxide, enzymes used for other purposes, and types of substrates and other substances involved in the enzymatic reaction And the amount to be used may be appropriately selected according to the substance to be measured in accordance with a method for quantifying biological components known per se using an oxidizable color reagent. Further, the peroxidase used in the determination of hydrogen peroxide according to the present invention is not particularly limited in its origin and origin, and one or two kinds of peroxidase or peroxidase-like substance of plant, animal or microbial origin may be used. The above is used in combination. The amount used is appropriately determined according to the purpose and is not particularly limited.

The quantification of biological components by the method of the present invention is usually pH 4.0-1.
It is carried out at 0.0, more preferably pH 6.0 to 8.0. Examples of the buffer used include phosphate, citrate, borate, carbonate, Tris buffer, Good's buffer and the like, but are not particularly limited thereto.

The imidazole derivative of the present invention can be effectively used for quantifying oxidative substances such as hydrogen peroxide, but it is also possible to quantify a peroxidase-like substance by combining this with hydrogen peroxide. Examples of the peroxidase-like substance include hemoglobin and other heme compounds, in addition to peroxidase itself.

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

Examples will be given below, but the present invention is not limited by these examples.

〔Example〕

Example 1. Synthesis of 2- (4-hydroxy-3,5-dimethoxyphenyl) -3-N-methylcarbamoyl-4,5-bis (4-diethylaminophenyl) imidazole [the present compound (1)] (i ) Synthesis of 1,2-bis (4-diethylaminophenyl) ethane-1,2-dione 20 ml of carbon disulfide was added to 6.7 g of anhydrous aluminum chloride, and 20 g of N, N-diethylaniline was added dropwise under ice cooling. With further stirring and ice-cooling, add 1.5 g of oxalyl chloride dropwise to 60
The reaction was stirred for a minute. After the reaction, 50 ml of water and chloroform
The chloroform layer obtained by adding 100 ml and separating the layers 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) Synthesis of 2- (4-hydroxy-3,5-dimethoxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole 1,2-bis (4-diethylaminophenyl) ethane obtained in (i) -1,2-dione 1.5 g and 4-hydroxy-3,5-
Dimethoxybenzaldehyde 0.8g, ammonium acetate 5g
Was reacted by heating under reflux in 30 ml of acetic acid for 2 hours. After cooling, 60 ml of water was added and neutralized with aqueous ammonia under ice cooling to precipitate crystals. The precipitated crystals are collected, washed with water and dried,
It was treated with hexane to obtain 0.8 g of the target compound having a slightly red color.

(Iii) 2- (4-hydroxy-3,5-dimethoxyphenyl) -3-N-methylcarbamoyl-4,5-bis (4
Synthesis of -diethylaminophenyl) imidazole 0.8 g of 2- (4-hydroxy-3,5-dimethoxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole obtained in (ii) was dissolved in 50 ml of chloroform and stirred. Below, 5 ml of methyl isocyanate was added, and the mixture was left at room temperature for 12 hours. Then, 50 ml of methanol was added to stop the reaction and then concentrated. The concentrate was purified by a silica gel column using chloroform as an eluent to obtain 760 mg of the desired product (light red powder).

Example 2. Synthesis of 2- (4-hydroxy-3,5-dimethoxyphenyl) -3-acetyl-4,5-bis (4-diethylaminophenyl) imidazole [compound (2) of the present invention] As in Example 1. 2- (4-hydroxy-3,5 obtained in
1.0 g of -dimethoxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole and 0.4 ml of pyridine were dissolved in 200 ml of tetrahydrofuran, 0.2 ml of acetyl chloride was added, and the mixture was stirred and reacted at room temperature for 12 hours. Then 300 ml of ice water
After stopping the reaction by adding 500 ml of chloroform, the chloroform layer was separated and concentrated. The concentrate was purified by a silica gel column using chloroform as an eluent to obtain 800 mg of the desired product (light red powder).

Example 3. 2- (4-Hydroxy-3,5-dimethoxyphenyl) -3-benzoyl-4,5-bis (4-diethylaminophenyl) imidazole [the present compound (3)]
Synthesis of 2- (4-hydroxy-3,5 obtained in the same manner as in Example 1
-Dimethoxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole (1.0 g) and pyridine (0.4 ml) were dissolved in tetrahydrofuran (200 ml) to give benzoyl chloride (0.2 ml).
Was added and the reaction was stirred at room temperature for 12 hours. Next, ice water 300m
After the reaction was stopped by adding l, 500 ml of chloroform was added, and the chloroform layer was separated and concentrated. The concentrate was purified by a silica gel column using chloroform as an eluent to obtain 850 mg of the desired product (light red powder).

_{NMR (CDCl 3, TMS) ppm} : 1.2 (12H, t, C H 3 -C -), 3.6 (8
H, q, C-C H _2- ), 3.9 (6H, s, -O-C H ₃ ), 6.5 to 7.5 (1
5H, broad, phenyl hydrogen), 7.9 (1H, s, -O H) Example 4 Determination of uric acid (1) Test solution 50 mmol / l MES [2- (N-morpholino)
Ethane sulfonate] buffer solution (pH 6.5) uricase 100U /
l, peroxidase 2000 U / l, compound of the present invention (6) 100
It was prepared by dissolving so as to have a concentration of μmol / l.

(2) Sample Uric acid was dissolved in distilled water to a concentration of 10,7.5,5,2.5 mg / dl for preparation.

(3) Measurement operation Test solution for each 50 μl of each sample solution and distilled water
3.0 ml was added, and the mixture was heated at 37 ° C. for 5 minutes and the absorbance at 700 nm was measured using a blind test as a control.

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

Example 5 Determination of hydrogen peroxide (1) Test solution 50 mmol / l phosphate buffer (pH 7.0) 2000 U / l peroxidase, compound of the present invention (2) 100 μmol / l
Was prepared by dissolving so as to have a concentration of.

(2) Sample Dilute commercially available hydrogen peroxide solution with distilled water to 2.0,
It melt | dissolved and prepared it so that it might become 1.5,1.0,0.5 mmol / l.

(3) Measurement operation Test solution for each 20 μl of each sample solution and distilled water
3.0 ml was added, and the mixture was heated at 37 ° C. for 3 minutes and the absorbance at 670 nm was measured using a blind test as a control.

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

〔The invention's effect〕

As described above, the novel imidazole derivative of the present invention is
Most of them have a maximum absorption wavelength at the time of coloring, which is on the long wavelength side of 600 nm or more. Therefore, when this is used as a color-forming component in the determination of trace components in biological samples such as serum and urine,
The point that measurement can be performed without being greatly affected by the colored interfering substance coexisting in the sample, and the addition of the above-mentioned substituent to the nitrogen at the 3-position of the imidazole ring stabilizes the chromogen. In addition, when the substituent is a phenyl group, an alkenyl group or an acyl group which may have a substituent, for example, in an oxidation reaction in a hydrogen peroxide-peroxidase system. Has a remarkable effect in that the sensitivity of color development per hydrogen peroxide is twice as high as that of the existing imidazole derivative having no substituent, and it is a great contribution to the art.

[Brief description of drawings]

FIG. 1 shows the calibration curve obtained in Example 4, in which the absorbance obtained for each uric acid concentration (mg / dl) on the horizontal axis is plotted along the vertical axis. . FIG. 2 shows the calibration curve obtained in Example 5, in which the absorbance obtained for each hydrogen peroxide concentration (mmol / l) on the horizontal axis is plotted along the vertical axis. Is.

Claims (10)

[Claims] 1. The following general formula [I] [Wherein R ¹ , R ² and R ³ are each an organic group, and ^one of R ¹ , R ² and R ³ is an aryl group in which either the ortho position or the para position is substituted with a hydroxyl group. Wherein one represents an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent, and the remaining one represents the ortho position. Or an aryl group in which either the para position is substituted with a hydroxyl group, or an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent. Represents a group or a benzyl group. R ⁴ represents an optionally substituted phenyl group, an alkenyl group, an acyl group, an alkyl-substituted carbamoyl group, or an optionally substituted phenyl-substituted carbamoyl group. ] The imidazole derivative shown by these. 2. The following general formula [I] [Wherein R ¹ , R ² and R ³ are each an organic group, and ^one of R ¹ , R ² and R ³ is an aryl group in which either the ortho position or the para position is substituted with a hydroxyl group. Wherein one represents an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent, and the remaining one represents the ortho position. Or an aryl group in which either the para position is substituted with a hydroxyl group, or an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent. Represents a group or a benzyl group. R ⁴ represents an optionally substituted phenyl group, an alkenyl group, an acyl group, an alkyl-substituted carbamoyl group, or an optionally substituted phenyl-substituted carbamoyl group. ] The imidazole derivative shown by these is used as a coloring component, The quantification method of the oxidizing substance characterized by the above-mentioned. 3. The quantification method according to claim 2, wherein the oxidizing substance is hydrogen peroxide. 4. The quantification method according to claim 3, wherein the color-developing component is oxidatively developed in the presence of peroxidase to colorimetrically quantify the color development. 5. The quantitative method according to claim 3 or 4, wherein the hydrogen peroxide is hydrogen peroxide produced by an enzymatic reaction. 6. The quantification method according to claim 5, wherein the hydrogen peroxide is hydrogen peroxide produced by an enzymatic reaction in the quantification of a trace component in a biological sample. 7. A method for quantifying a trace component in a biological sample by quantifying a substrate or a hydrogen peroxide produced by reacting an oxidase with a substance produced by an enzymatic reaction to determine a substrate or enzyme activity in the biological sample. The quantitative method according to claim 6, which is quantitative. 8. The following general formula [I] [Wherein R ¹ , R ² and R ³ are each an organic group, and ^one of R ¹ , R ² and R ³ is an aryl group in which either the ortho position or the para position is substituted with a hydroxyl group. Wherein one represents an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent, and the remaining one represents the ortho position. Or an aryl group in which either the para position is substituted with a hydroxyl group, or an aryl group in which either the ortho position or the para position is substituted with an alkoxy group, an aryloxy group or an amino group which may have a substituent. Represents a group or a benzyl group. R ⁴ represents an optionally substituted phenyl group, an alkenyl group, an acyl group, an alkyl-substituted carbamoyl group, or an optionally substituted phenyl-substituted carbamoyl group. ] The quantification method of the peroxidase-like substance characterized by using the imidazole derivative shown by these as a coloring component. 9. The assay method according to claim 8, wherein the peroxidase-like substance is peroxidase. 10. The quantification method according to claim 8, wherein the peroxidase-like substance is hemoglobin or another heme compound.