JPH0324061A - Novel oxidizable color reagent - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R<1> is aryl sulfonyl, alkyl sulfonyl, carboxyl or alkoxycarbonyl containable of substituting group; R<2> to R<4> are aryl containable of substituting group with a proviso that at least one is substituted phenyl having NH2 containable of OH or substituting group in p-position to imidazole ring). EXAMPLE:1-(p-toluene sulfonylaminocarbonyl)-2-(3,5-dimethoxy-4- hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole. USE:An oxidizable color reagent. PREPARATION:A compound expressed by formula II is reacted with a compound expressed by formula III and ammonium acetate, etc., in acidic solvent to obtain a compound expressed by formula IV, then said compound is reacted with a compound expressed by formula V in halohydrocarbon solvent to afford the compound expressed by formula I.

Description

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

[Background of the Invention] Measuring biological components, such as body fluids such as blood and urine, is useful for diagnosing diseases, elucidating pathological conditions, and determining the course of treatment, as their fluctuations are closely related to diseases. So, it becomes essential. 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 serum content is to use an enzyme that specifically acts on the target protein when it is something other than an enzyme, and to use its substrate when the target protein is an enzyme. The so-called 11-enzyme method, in which enzymatic reactions are carried out using the compounds to be used, and the resulting products are measured to determine the desired amount, is generally widely used.Among these, H202
A production enzyme, such as oxidase, is activated to produce H202 corresponding to a certain purpose, and this is converted into peroxidase,
Along with the development of oxidizable coloring reagents, the method of determining the amount of target components by introducing the coloring system into a coloring system using an oxidizable coloring reagent and quantifying the color development has increased along with the development of oxidizable coloring reagents. It's starting to happen. For example, H202 generated by a combination of cholesterol-cholesterol oxidase, triglyceride lipoprotein lipase-glycerol oxidase, uric acid monouricase, etc. is introduced into a coloring system using peroxidase (POD) and an oxidizable coloring reagent.
This is a method to determine the desired amount by measuring the absorbance of the color. Typical oxidizable color reagents used in this method include 4-aminoantipyrine and phenolic compounds or N
, an oxidizable color reagent in combination with N-disubstituted aniline compound, a combination reagent in 3-methyl-2-penzothiazolinone hydrazone (MBTH) and aniline compound, 2,2'-azinobis( Examples include 3-ethylbenzothiazolinone-6-sulfonic acid), triphenylmethane-based leuco dyes, diphenylamine derivatives, benzidine derivatives, 0-tolidine derivatives, O-phenylenediamine, and the like. However, most of these conventionally used oxidizable coloring reagents, excluding diphenylamine derivatives, have a coloring wavelength of 800 ns or less, and are susceptible to certain serum components such as bilirubin and hemoglobin (
When measuring urine components, it is easily influenced by the pigment bodies in the urine. )
In addition, with the exception of combination reagents with 4-aminoantipyrine and some triphenylmethane-based leuco dyes, all of them have problems such as low chromogen stability. On the other hand, triarylimidazole derivatives of dye precursors (leuco dyes) have been disclosed as chromogens that have relatively good chromogen stability and color development wavelengths on the relatively long wavelength side (especially Publication No. 57-5519, Special Publication No. 57-2
8118, JP 58-4557, JP 61-174287, JP 61-227570
(U.S. Pat. No. 3,297,710, etc.).

However, even with these existing triarylimidazole derivatives, when measuring trace amounts in biological samples such as serum using reagents containing these as coloring components, it is difficult to detect coexisting substances in biological samples. There is a problem that color development sensitivity is reduced due to the influence of , or in other words, color development is hindered by the influence of coexisting substances in biological body fluids. Therefore, even with these existing triarylimidazole derivatives, it cannot be said that their color development is fully satisfactory in the measurement of trace components in biological samples such as serum and urine.

[Purpose of the Invention] The present invention has been made in view of the above-mentioned situation, and aims to produce a pigment that has almost no decrease in color sensitivity due to serum concentration, has high measurement sensitivity, and has an absorption maximum on the long wavelength side. The object of the present invention is to provide a novel triarylimidazole derivative that can be used as a coloring agent, and a highly accurate method for measuring oxidizing substances and peroxidase-like substances using the compound as a coloring agent. [Structure of the Invention] The present invention is based on the general formula [I] ■ C=O l NH 1 R1 (wherein Rl is an arylsulfonyl group which may have a substituent, or an arylsulfonyl group which may have a substituent) -R2 R3 and R4 each independently represent an aryl group which may have a substituent.However, among R2 R3 and R4, at least one represents a substituted phenyl group having a hydroxy group or an amino group which may have a substituent at the P-position relative to the imidazole ring, and oxidation using the compound as a coloring component. This invention provides a method for quantifying a chemical substance, and a method for quantifying a peroxidase-like substance using the compound as a coloring agent. That is, the present inventors are in the process of intensive research to solve the above-mentioned problems of various oxidizable coloring reagents widely used in the so-called "enzyme method." 5-
Triarylimidazole (However, at least one of the three aryl groups is a Wt-substituted phenyl group having a hydroxy group or an amino group which may have a substituent at the p-position with respect to the imidazole ring. ) When a carbamoyl group having an arylsulfonyl group, an alkylsulfonyl group, a carboxyl group, or an alkoxycarbonyl group is further introduced into the N atom at the 1-position of The present invention was completed based on the discovery that the phenomenon in which color development is inhibited by coexisting substances in biological samples can be minimized. In the triarylimidazole derivative of the present invention represented by the general formula [11], R1 is an arylsulfonyl derivative which may have a substituent, and examples of the substituent include a methoxy group, an ethoxy group, a propoxy group, etc. Lower alkoxy groups such as iodine, bromine, @
Examples include fluorine, halogen atoms such as fluorine, and amino groups.
In addition, examples of the alkyl group of the alkylsulfonyl group which may have a substituent include a lower alkyl group having 1 to 4 carbon atoms (linear, branched, ) can be mentioned, and rI1
Examples of the substituent include lower alkoxy groups such as methoxy group, ethoxy group, and propoxy group, hydroxy group, and hydroxyethoxy group. In addition, examples of the alkoxy group of the alkoxycarbonyl group include carbon atoms of 1 to 4, such as methoxy group, ethoxy group, propoxy group, butoxy group, etc.
Lower alkoxy group (can be either linear or branched)
can be mentioned.

In addition, examples of Rl include carboxyl groups. Examples of the aryl group of the aryl group represented by R2, R3, and R4, which may have a substituent, include phenyl group, tolyl group, ethylphenyl group, naphthyl group, methylnaphthyl group, etc. is, for example, a lower alkoxy group having 1 to 4 carbon atoms such as a methoxy group, an ethoxy group, a propoxy group, a butoxy group (either linear or branched), an alkylcarbonyl group [the alkyl of the alkylcarbonyl group] Examples of the group include methyl group, ethyl group,
Examples include lower alkyl groups having 1 to 4 carbon atoms (either linear or branched) such as probyl group and butyl group. Co, arylcarbonyl optionally having substituents 1&[
Examples of the aryl group of the arylcarbonyl group include phenyl group, tolyl group, ethylphenyl group, naphthyl group,
Examples of substituents on these aryl groups include lower alkoxy groups having 1 to 4 carbon atoms (linear, branched, etc.) such as methoxy, ethoxy, propoxy, and butoxy groups. (Anything is acceptable), for example, iodine,
Examples include halogen atoms such as bromine, @, and fluorine, and a-substance groups. ], an amino group which may have a substituent [lI
! Examples of substituents include lower alkyl groups having 1 to 4 carbon atoms (either linear or branched) such as methyl, ethyl, probyl, butyl, hydroxymethyl, hydroxyethyl, etc. groups, hydroxyalkyl groups such as hydroxyprobyl groups, e.g. carboxymethyl groups. Examples include carboxyalkyl groups such as carboxyethyl group and carboxyprobyl group, such as sulfoethyl group. These R2, R3 and R4 may be the same or different, but at least one of R2, R3 and R4 has a hydroxy group or a substituent at the P position with respect to the imidazole ring. It must be a substituted penyl group with an optional amino group. The triarylimidazole derivative of the present invention represented by the general formula [I] can be easily synthesized, for example, as follows. That is, for example, a compound represented by the general formula [■ KO 11 R'-CH [■] (in the formula, R4 is the same as above) (hereinafter abbreviated as compound ■),
A compound represented by the general formula [II1] (in the formula, R2 and R3 are the same as above) (hereinafter abbreviated as compound ■) and ammonium #acid (or ammonia, ammonium carbonate, etc.) A known method of reacting in an acidic solvent (EF Silver
Smith, U. S. Patent 3,297,
710, 1987) to form the general formula [rV
] (in the formula, R2, R3 and R4 are the same as above) (hereinafter abbreviated as compound 2).

), and then the general formula [V]R'-N=C=O
The imidazole derivative of the present invention can be obtained by reacting an isocyanate derivative (hereinafter abbreviated as compound V) represented by [V] (wherein Rl is the same as above). More specifically, compound (1), 0.2 to 1 mole, preferably 0.5 to 0.6 mole of compound (2) per mole of compound U, and 1 to 20 mole of compound (2) per mole of compound U. Preferably 2 to 10 moles of #
After reacting with ammonium acid, etc. in an acidic solvent such as acetic acid or propionic acid at 100 to 150°C, if necessary under reflux, for 2 to 5 hours, WI preparation is performed by a conventional method to obtain compound (1).
Next, Compound (1) and 1 to 10 mol, preferably 1 to 3 mol of Compound V per 1 mol of Compound (2) are mixed in a halogenated hydrocarbon solvent such as chloroform, dichloromethane, dichloroethane, trichloroethane, etc. After reacting at 0 to 30°C for 1 to 72 hours, the triarylimidazole derivative of the present invention can be obtained by purification by a conventional method.

As the compound (2) used in the production of the triarylimidazole derivative of the present invention, it is sufficient to use generally commercially available phenyl aldehyde derivatives and naphthyl aldehyde derivatives. Since it can be easily obtained by formylation using a conventional method, the product obtained in this way may also be used. Compound (1) can be easily obtained by subjecting the corresponding phenyl derivative or/and naphthyl derivative to oxalyl chloride in a Friedel-Crach reaction, so it is sufficient to use the compound obtained in this manner. *For Compound V, it is sufficient to use a generally commercially available isocyanate derivative, but since it can be easily obtained by the Hofmann rearrangement reaction using the corresponding carboxylic acid amide as a raw material, the compound V obtained in this way can be used. Good too.

The triarylimidazole derivative of the present invention is extremely stable in a loose liquid in the presence of water or a surfactant, but when it is oxidized with an oxidizing agent, for example, with hydrogen peroxide in the presence of peroxidase, the coloring becomes stable. Quantitatively forms pigments with excellent properties.

Moreover, the triarylimidazole derivative of the present invention is
Compared to cases in which color is developed using a similar method using triarylimidazole derivatives, color development is almost never inhibited by components in biological body fluids such as serum and urine. Moreover, its molecular extinction coefficient is equal to or higher than that of the dye produced from the triarylimidazole derivative of DJ-. Therefore, the triarylimidazole derivative of the present invention can be effectively used as a coloring agent in the determination of oxidizing substances and peroxidase-like substances, but in particular, it can be used as a coloring agent for hydrogen peroxide produced by an enzymatic reaction in the presence of peroxidase. It can be effectively used as a coloring agent in the quantification of a trace amount in a biological sample by introducing it into a system and colorimetrically quantifying its coloration.

That is, the method for quantifying oxidizing substances of the present invention is a method for quantifying a trace amount of hydrogen peroxide in a biological sample, which is performed by acting on a substrate or a substance produced by an enzymatic reaction with an oxidizing enzyme and quantifying the produced hydrogen peroxide. It can be used particularly effectively as

Examples of trace components in biological samples that can be measured by the quantitative method of the present invention include cholesterol, glucose, glycerin, triglyceride, free fatty acids, uric acid, phospholipids, bile acids, monoamine oxidase, guanase, cholinesterase, etc. The method is not limited to these, but any biological substance that can be measured can be quantified by quantifying hydrogen peroxide produced by an enzymatic reaction. The quantitative method of the present invention is carried out in accordance with a known enzyme method (using an H202-generating enzyme) except that the triarylimidazole derivative of the present invention is used as a coloring agent (oxidizable color reagent). That's enough.

The concentration of the triarylimidazole derivative of the present invention used as a coloring agent is not particularly limited, but is usually several microns.
mo1/1 or more, preferably 50 to 100 μmoJ/l
A concentration range of is used. Oxidase used as an enzyme to generate hydrogen peroxide, enzymes used for other purposes, and substrates and other substances involved in enzyme reactions in the quantitative determination of biological substances by the quantitative method of the present invention. The type and amount to be used may be selected as appropriate depending on the substance to be measured, in accordance with a known method for quantifying biological substances using oxidizable coloring reagents. Furthermore, the peroxidase or peroxidase-like substance used in the quantitative determination of hydrogen peroxide according to the present invention is not particularly limited in its origin, and one type of peroxidase or peroxidase-like substance obtained from plants, animals, and microorganisms is used. Alternatively, two or more types may be used in combination if necessary. Further, the amount used is appropriately determined depending on the purpose and is not particularly limited.

The determination of a certain amount of a living body by the determination method of the present invention is usually performed at PH4
．． It is carried out at a pH of 0 to 10.0, preferably 6.0 to 8.0. As for the buffering agent used. Phosphate, -N,
Examples include, but are not limited to, mild mM commonly used in this field, such as Good's buffer such as N'-bis(2-ethanesulfonic acid) (PIPES).

The triarylimidazole derivative of the present invention can be effectively used for quantifying oxidizing substances such as hydrogen peroxide and periodic acid, but it can also be used in combination with hydrogen peroxide to quantify peroxidase-like substances. is also possible. Examples of peroxidase-like substances include hemoglobin and other heme compounds in addition to peroxidase itself.

That is, the triarylimidazole derivatives of the present invention can be applied, for example, to enzyme immunoassay using peroxidase as a labeling compound, and can also be used to oxidize hemoglobin in serum with hydrogen peroxide or sodium periodate. It can also be effectively used in measurements using sexual substances. Furthermore, the triarylimidazole derivative of the present invention can be used in the field of so-called dry chemistry, in which specific components in samples such as biological body fluids are measured using, for example, test strips obtained by impregnating and drying absorbent carriers such as filter paper with reaction reagents. It can also be used effectively. The present invention will be explained in more detail with reference to Examples and Reference Examples below, but the present invention is not limited by these in any way. [Example Example 1. 1-(p- }luenesulfonylaminocarbonyl)-2-(3.5-dimethoxy-4-hydroxyphenyl)-4.5-bis(4-diethylaminophenyl)imidazole (the compound of the present invention [2)] Synthesis (1) Synthesis of 1,2-his(4-diethylaminophenyl)ethane-1,2-dione or aluminum chloride (anhydrous) 11.6. carbon disulfide 6
0II11 was added thereto, and 30 g of N,N-diethylaniline was added dropwise thereto under water cooling. Next, oxalyl chloride log was added dropwise to this at 5° C. or lower, and the reaction was stirred for 1 hour.

After the reaction was completed, 100 ml of water and 200 ml of chloroform were poured into the reaction solution, and the resulting chloroform layer was washed with 2N hydrochloric acid, dried, and concentrated. The residual solution was recrystallized from ethyl acetate to give yellow 1,2-bis(4-diethylaminophenyl)ethane-1,2-dione7. I got Og. (2) 2-(3,5-dimethoxy4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)
1.2-bis(4-diethylaminophenyl)ethane-1,2-dione obtained by synthesis of imidazole (1)7. Og, 3.8 g of syringaldehyde (Tokyo Kaoru II) and 4 g of ammonium acetate were reacted in 100 g of #acid under reflux for 5 hours.

After completion of the reaction, 600+ Al of water was poured into the reaction solution, the resulting viscous residual liquid was separated, and this viscous residual liquid was purified by silica gel column chromatography (elution solvent: mixed solvent of chloroform and methanol), 2-(3. 1.8 g of 5-dimethoxy-4-hydroxyphenyl)-4.5-bis(4-diethylaminophenyl)imidazole was obtained. (
3) I-(P- }ruenesulfonylaminocarbonyl)-2-(3.5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole (compound of the present invention [2] ) or 0.8 g of 2-(3.5-dimethoxy4-hydroxyphenyl)-4.5-bis(4-diethylaminophenyl)imidazole obtained in (2) was added to 20+*l of chloroform.
1 g of P-toluenesulfonyl isocyanate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the solution, and the mixture was reacted with stirring at room temperature for 5 hours. After completion of the reaction, methanol 5III1 was injected into the reaction solution and purified by excess isocyanate P-chromatography (elution solvent: mixed solvent of #ethyl acid and n-hexane). 1-(p-}luenesulfonylaminocarbonyl)-2-(3,5-dimethoxy-4-
0.7 g of dark green crystals of (hydroxyphenyl)-4,5-bis(4-diethylaminophenyl) iyodazole were obtained.

mp: 85°C.

IR: 3400cm-'(OH), 2800~3
000cm-'(CH), 1720cm-'(C=0)
, 1600cm-' (Aromatic CH), 15
00 cm-'(Aro+*atic Cl).

Elemental analysis value (CiJ4sN,06S) Calculated value (%): C 65.74, H 6.37
, N 9.84, actual value (%>:C 85.34
．． H 6.10. N9.72. Example 2. 1,2-bis(4-diethylaminophenyl)ethane-l,2-dione and N,N-jethyl-2,6-dimethoxy-4-photylaminophenyl)imidazole were synthesized and processed. 1-(p-Toluenesulfonylaminocarbonyl)-2-(3.5-dimethoxy-4-jethylaminophenyl)-4.5-bis(4-jethylaminophenyl)imidazole (compound of the present invention [4 ]) 0.
8g was obtained.

Example 3.1-(Ethoxycarbonylaminocarbonyl)-2-(3.5-dimethoxy-4-hydroxyphenyl)-4.5-bis(4-diethylaminophenyl)idazole (Compound of the Invention [5 ]) Synthesis of 2-(3.5-dimethoxy4-hydroxyphenyl)-4.5-bis(4-diethylaminophenyl)imidazole o. obtained in (2) of Example 1. 0-7 g of ethoxycarbonyl isocyanate (Aldrich Co., Ltd. 11) was added to a solution of Ag in 20 ml of chloroform.
The reaction was allowed to proceed at room temperature for 5 hours with stirring. After the reaction was completed, 5 ml of methanol was added to the reaction solution to decompose excess ethoxycarbonyl isocyanate, and then a mixed solvent of ethyl and n-hexane was added under reduced pressure. 0.6 g of green crystals of 1-(ethoxycarbonylaminocarbonyl)-2-(3.5-dimethoxy-4-hydroxyphenyl)-4.5-bis(4-diethylaminophenyl)imidazole was obtained.

mp: 80°C.

IR: 3400cm-' (Oil). 2800~3
000cn-'(CH). 1735cm-'(C-0)
, 1720c "'(C=O), 1600cm-'(Ar
omatic CIIL 1500c+a-'(Aro
matic CH).

Elemental analysis value (C351143NsOa) Calculated value (Me)
: C 6B. 75, H 6.88, N 11.
12. Actual value (%): C 66.09, H 6
．． 60, N 10.80. Example 4. 2-(3,5-dimethoxy4-hydroxyphenyl)
-4,5-bis(4-diethylaminophenyl)imidazolenylsulfonylaminocarbonyl)-2-(3
．． 5-dimethoxy-4-hydroxyphenyl)-4.5
-bis(4-diethylaminophenyl)imidazole (
Compound of the present invention [1 compound] 1.2. I got it. Example 5. Measurement of various properties of the compound of the present invention (1) Measurement of molecular extinction coefficient and absorption maximum (λwax) (coloring solution) Sodium buffer (
p}17.0), the compound of the present invention was added to 0. A coloring solution was prepared by dissolving 5mM and peroxidase to a ratio of 4/IJ+el. (Procedure) 20 μJ of 1 mM hydrogen peroxide solution was added to 3 ml of a coloring solution prepared using a predetermined compound of the present invention, mixed well, and then heated at 37° C. for 5 minutes. The absorption curve of this reaction solution was measured, and the absorbance Es at λ+nax and λ+tax was determined. In addition, as a control when calculating the absorption curve and λwax,
20 μl of purified water was added to 3 ml of each coloring solution, mixed well, and then heated at 37° C. for 5 minutes before use.

In addition, the molecular extinction coefficient is E obtained using each coloring solution.
s, 50mM each of 4-aminoantivirine and phenol, and 4% peroxidase in place of each coloring solution.
0/al containing 50 pyogenes piperazine-N,N'-bis(
2-Ethanesulfone W! ) (PIPES) Using sodium monohydroxide buffer (pH 7.0) and the absorbance at 505 na obtained by the above procedure, EOH was determined by the following formula.

Molecular extinction coefficient = (Es÷E or+) X 5 X 1
03 results are shown in Table 1.

(2) Measurement of the influence of a certain amount of serum To the coloring solution 3+++1 prepared in (1), 20μl of normal pool human serum was added and mixed, and then 20μl of 1mM hydrogen peroxide was added and mixed, and the mixture was heated at 37℃ for 5 minutes. Allowed to react for minutes. The absorbance Eef of this reaction solution at λnax was measured. In addition, the same operation was performed using purified water instead of normal pool human serum and the same coloring solution as above to obtain the absorbance E std.

By substituting these values into the following equation, the a value, which is an index for examining the influence of serum concentration, was determined.

a = (Eef'- Estd) X too The influence of a certain amount of serum was expressed as follows based on the a value.

12a value is 0-3.

±: a4fl is 3-6. ++: A4m is 20 or more.

The results are also shown in Table 1.

For comparison, Table 1 also shows the results of measurements of various properties of existing triarylidazole derivatives in the same manner.

As is clear from the results in Table 1, the compound of the present invention has a molecular extinction coefficient that is equal to or higher than that of the dye produced from the triarylimidazole derivative shown above, but compared to that of the triarylimidazole derivative shown above. However, when biological body fluids such as serum are used as samples, color development is hindered by components in the body fluids. It can be seen that it has the property that almost no elephants occur.

Example 6. Quantification of hydrogen peroxide (measurement sample solution) Measure the following reagents dissolved at a specified concentration in 50mM biperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) sodium monohydroxide buffer (PH7.0). It was used as a test solution.

Compound of the present invention [2] 0.5mM peroxidase 4 U/+1 (sample) Commercially available hydrogen peroxide solution was appropriately diluted with distilled water to give 0.5,
1.0, 1.5, 2.0 and 4.0+sM solutions were used as samples. (Procedure) Add 20μl of sample to measurement reagent 3+al, mix well,
After heating at 7° C. for 5 minutes, absorbance (Es) at 660 nm was measured.

In addition, the same operation was performed using ion-exchanged water instead of the sample to measure blind {+1 (FBI).

(result) The measurement results are shown in Figure 1.

As is clear from FIG. 1, the calibration curve connecting the points obtained by plotting the absorbance (Es-FBI) obtained for each hydrogen peroxide concentration on the horizontal axis along the vertical axis shows good linearity. It can be seen that hydrogen peroxide can be quantitatively measured by the method described above.

In addition, in place of the present invention compound [2, the present invention compound [1]
．． Similar results were obtained when using [3], [4co, and [5]. Example 7. Quantification of hydrogen peroxide in the presence of serum components (measurement reagent solution) Same as Example 6. (Sample) Same as Example 6.

(Procedure) Add 50μ of normal human serum or ion-exchanged water to 3mL of measurement sample solution.
1 was added and mixed well, then 20 μl of the sample was added and mixed well, and after heating at 37°C for 5 minutes. Absorbance (Es) at 660 nm was measured.

In addition, the same operation was performed using ion-exchanged water instead of the sample, and a blind value (FBI) was measured. (M fruit) The measurement results are shown in Table 2.

Table 2 (margin below) As is clear from Table 2, the dye produced from the imidazole derivative of the present invention is hardly affected by serum components, regardless of the hydrogen peroxide concentration.

Example 8. Quantification of uric acid (measurement sample solution) 50mM Viverazine-N,N'-bis(2-ethanesulfonic acid) (P(PES) Sodium monohydroxide tIW
A measurement reagent solution was prepared by dissolving the following reagents at a predetermined concentration in a solution (PII 7.0).

Compound of the present invention [2 0.05mM peroxidase 41J/ml uricase
Standard solution containing 0.05U/1 (sample) 10n/dl of uric acid and human serum 5
The specimen was used as a sample. (Procedure) Add 20 μl of sample to 3 ml of measurement reagent solution, mix well,
After heating at ℃ for 5 minutes, the absorbance (Es) of 660nl1 was measured.

In addition, blind W (FBI) was measured by performing the same procedure using ion-exchanged water instead of the sample. The uric acid concentration in human serum was calculated according to the following formula.

Urine a (IIlg/di) = (Es- of human serum
FBI)÷(Es-EBI of It semi-liquid)XIO The results are shown in Table 3.

Reference example 1. Quantification of uric acid Using the same sample as in Example 8, a commercially available kit for uric acid measurement (
Uric acid concentration was measured using Urine@C-Test Wako, manufactured by Wako Junkuri Industries, Ltd.

The results are also shown in Table 3.

Table 3 As is clear from Table 3, the imidazole of the present invention
! :) It can be seen that the measured value of uric acid obtained by the measuring method of Example 8 using the derivative as a coloring agent shows a good correlation with that of the conventional method using a commercially available kit.

[Effects of the Invention] As described above, the imidazole derivative of the present invention is extremely stable in a buffer solution in which a surfactant coexists;
In addition, the dye produced from this has a high molecular extinction coefficient, which means that the measurement sensitivity is high, and furthermore, color development is hindered by components contained in biological samples such as serum. It has an extremely excellent property that almost no particles occur, and it is a great invention that contributes to this industry.

[Brief explanation of drawings]

Figure t shows the calibration curve obtained in Example 6,
8 obtained for each hydrogen peroxide concentration (+*M) on the horizontal axis
It connects the points where the absorbance of 60n+* is plotted along the vertical axis.

Claims (10)

[Claims] (1) General formula [I] ▲Mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, R^1 is an arylsulfonyl group that may have a substituent; It represents a good alkylsulfonyl group, carboxyl group or alkoxycarbonyl group, and R^2, R^3 and R^4 each independently represent an aryl group which may have a substituent.However, R^
At least one of 2, R^3 and R^4 represents a substituted phenyl group having a hydroxy group or an amino group which may have a substituent at the p-position relative to the imidazole ring. ) A triarylimidazole derivative represented by (2) A method for quantifying an oxidizing substance, characterized in that the triarylimidazole derivative according to claim 1 is used as a coloring component. (3) The quantitative method according to claim 2, wherein the oxidizing substance is hydrogen peroxide. (4) The quantitative method according to claim 3, wherein the coloring component is oxidized to develop color in the presence of peroxidase, and the color development is determined 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 quantitative method according to claim 5, wherein the hydrogen peroxide is hydrogen peroxide produced by an enzymatic reaction in quantifying trace components in a biological sample. (7) Quantification of trace components in biological samples is the quantification of substrates or enzyme activity in biological samples, which is performed by acting on oxidizing enzymes on substrates or substances produced by enzymatic reactions and quantifying hydrogen peroxide produced. , The quantitative method according to claim 6. (8) A method for quantifying a peroxidase-like substance, which comprises using the triarylimidazole derivative according to claim 1 as a coloring component. (9) The quantitative method according to claim 8, wherein the peroxidase-like substance is peroxidase. (10) The quantitative method according to claim 8, wherein the peroxidase-like substance is hemoglobin or other heme compound.