JPH0324060A - Novel oxidizable color reagent - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R<1> is H, alkyl or aryl containable of substituting group or OH; 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-(aminothiocarbonyl)-2-(4-hydroxyphenyl)-4,5-bis(4-diethylami no-2- methylphenyl)imidazole. USE:An oxidizable color reagent used to diagnosis of disease, etc. 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, thus further saponified, as necessary, 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 fluid components such as blood and urine, is important in diagnosing diseases, elucidating pathological conditions, and determining the course of treatment, as their fluctuations are significantly related to diseases. , has become essential. For example, methods have been developed to measure a wide variety of trace components in the blood, including cholesterol, triglycerides, glucose, uric acid, phospholipids, bile acids, monoamine oxidase, etc., and these methods are useful in diagnosing diseases. As is well known.

Currently, when the serum component is measured using something other than an enzyme, an enzyme that specifically acts on the target component is used, and when the target component is an enzyme, its substrate is used. The so-called "enzyme method" is widely used, in which an enzyme reaction is performed using each desired compound, and the resulting product is measured to determine the amount of the target component. Among them, H202
A production enzyme, such as oxidase, is activated to produce H202, which corresponds to the desired substance, and this is introduced into a coloring system using peroxidase and an oxidizable coloring reagent, which is a coloring component, and the coloration is measured colorimetrically. With the development of oxidizable coloring reagents, the number of methods for determining the amount of target components is increasing. For example, peroxidase (
POD) is a method for determining the target component amount by introducing an oxidizable coloring reagent into a coloring system and measuring the absorbance of the coloring. Typical oxidizable coloring reagents used as coloring components in this method include:
4-aminoantipyrine and phenolic compound or N,
An oxidizable color-forming K agent in combination with an N-disubstituted aniline compound, a combination reagent in which 3-methyl-2-penzothiazolinone hydrazone (MBTH) and an aniline compound, 2,2'-azinobis( 3-Ethylbenzothiazolinone-8-sulfone #), triphenylmethane leuco dye, diphenyla millet derivative, benzidine derivative, 0
-tolidine derivatives, 0-phenylenedia chestnut, and the like.

However, most of these conventionally used oxidizable coloring reagents, excluding diphenylamine derivatives, have a coloring wavelength of 80Onl1 or less, and bilirubin, bilirubin,
It is easily affected by serum components such as hemoglobin (when measuring urine components, it is easily affected by the pigment bodies in the urine), and it is also susceptible to the effects of combination reagents with 4-arinoantipyrine and triphenylmethane-based leuco dyes. With a few exceptions, all of them have problems such as low chromogen stability. On the other hand, triarylimidazole derivatives of pre-dye lipids (leuco dyes) have been disclosed as chromogens that have relatively good chromogen stability and color development wavelengths on the relatively long wavelength side ( Special Publication No. 57-5519, Special Publication No. 57-2
6118, JP 58-4557, JP 61-174267, JP 61-227570
(U.S. Pat. No. 3,297,710, etc.). However, even with these existing triarylimidazole derivatives, when measuring the amount of color components in biological samples such as serum using reagents containing these as coloring components,
There is a problem in that color sensitivity is reduced due to the influence of coexisting substances in the biological sample, or in other words, color development is hindered due to the influence of coexisting substances in the biological body fluid. Therefore, even these triarylimidazole derivatives cannot be said to be fully satisfactory as coloring components in the measurement of trace components in biological samples such as serum and urine. [Object of the Invention] The present invention has been made in view of the above-mentioned circumstances, and is a novel dye that produces a pigment that has almost no decrease in color sensitivity due to serum components, has high measurement sensitivity, and has an absorption maximum on the long wavelength side. An object of the present invention is to provide a highly accurate method for measuring oxidizing substances and peroxidase-like substances using triarylimidazole derivatives and the compounds as coloring components.

[Structure of the Invention] The present invention is based on the general formula [I S C= S 1 NH $ Rl C where Rl is a hydrogen atom, an alkyl group which may have a substituent, and a W1 substituent. R2, R3 and R4 each independently represent an aryl group which may have a substituent.

However, at least one of R2, R3 and R4 represents a 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. ), a method for quantifying an oxidizing substance using the compound as a color-forming component, and a method for quantifying a peroxidase-like substance using the compound as a color-forming component.

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 1' enzymatic method. 5-
Triarylimidazole (However, among the three aryl groups, at least one is a substituted phenyl group having a hydroxy group or an aξ group which may have a substituent at the P position with respect to the imidazole ring) When a thiocarbamoyl group, which may further have a substituent, is introduced into the N atom at position 1 of The present inventors have discovered that the phenomenon in which color development is inhibited by coexisting substances can be minimized, and the present invention has been completed.

In the triarylimidazole derivative of the present invention represented by the general formula [■], examples of the alkyl group of the alkyl group represented by R1 that may have a substituent include a methyl group, an ethyl group, a proyl group, Butyl group etc. with 1 or more carbon atoms
4 lower alkyl group (can be either linear or branched)
Examples of substituents include lower alkoxy groups such as methoxy, ethoxy, and propoxy groups, hydroxy groups, hydroxyethoxy groups, and carboxyl groups. Further, examples of the aryl group of the aryl group which may have a substituent include phenyl group, tolyl group, ethyl phenyl group, naphthyl group, methylnaphthyl group, etc., and examples of the substituent include methoxy group, ethoxy groups, lower alkoxy groups such as proboxyl groups, e.g. iodine,
Examples include halogen atoms such as bromine, @, and fluorine, ξ-no groups, and sulfo 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), alkylcarbonyl JG [alkylcarbonyl group] Examples of the alkyl group include lower alkyl X& (which may be linear or branched) having 1 to 4 carbon atoms, such as methyl, ethyl, probyl, and butyl. Arylcarbonyl group which may have a substituent [Aryl groups of the arylcarbonyl group include, for example, phenyl group, tolyl group, ethylphenyl group, naphthyl group, methylnaphthyl group, etc. Substituents include, for example, lower alkoxy groups having 1 to 4 carbon atoms (either linear or branched) such as methoxy, ethoxy, propoxy, butoxy, etc., such as iodine, bromine, etc. , halogen atoms such as chlorine and fluorine, and amino groups. ], an amino group [R
Examples of substituents include lower alkyl groups having 1 to 4 carbon atoms (either linear or branched) such as methyl group, ethyl group, probyl group, butyl group, etc., such as hydroxyethyl group, hydroxyl group, etc. A hydroxyalkyl group such as a probyl group, for example a carboxyalkyl group such as a carboxymethyl group, a carboxyethyl group, a carboxyprobyl group, a sulfoalkyl group such as a sulfoethyl group, a sulfobutopyl group,
Examples include 2-hydroxy-3-sulfoprobyl group. ]. These R2, R3 and R4 may be the same or different from each other, but at least one of R2, R3 and R4 has p to the imidazole ring.
It is required to be a substituted phenyl group having a hydroxy group or an amino group which may have a substituent at that position.

The triarylimidazole derivative of the present invention represented by the general formula [11] can be easily synthesized, for example, as follows.

That is, for example, the general formula [■1 R4-CH [11 (in the formula, R4
is the same as above. ,) (hereinafter abbreviated as compound ■),
A compound represented by the general formula [I[[] (in the formula, R2 and R3 are the same as above) (hereinafter abbreviated as compound ■) and ammonium acetate (or ammonia, ammonium carbonate, etc.) are acidified. A known method of reacting in a solvent (EF Silvers
+wit. h. υ． S. Patent. 3,297,
710, 1967) to form the general formula [IV
] (in the formula, R2, R3 and R4 are the same as above) (hereinafter abbreviated as compound 2).
), and then the general formula [V]Rl-N=C=S
isothiocyanate derivative represented by ' [V] (in the formula, R is the same as above) (hereinafter referred to as compound
It is abbreviated as ) and, if necessary, further saponification to obtain the triarylimidazole derivative of the present invention.

More specifically, compound (1), 0.2 to 1 mole, preferably 0.5 to 0.6 mole of compound (2) per mole of compound (2), and 1 to 20 mole of compound (2) per mole of compound (2) , preferably 2 to 10 moles of ammonium #acid, etc., to acetic acid,
After reacting for 2 to 5 hours in an acidic solvent such as propionic acid at 100 to 150°C under reflux if necessary, compound After reacting 1 to 10 mol, preferably 1 to 3 mol, of compound (2) in a halogenated hydrocarbon solvent such as chloroform, dichloromethane, dichloroethane, trichloroethane at 0 to 30°C for 1 to 72 hours, The triarylimidazole derivative of the present invention can be obtained by purifying it by a conventional method (if necessary, by further saponifying it and then purifying it by a conventional method). As the compound (2) used in the production of the triarylimidazole derivatives of the present invention, it is sufficient to use generally commercially available phenylaldehyde derivatives and naphthylaldehyde derivatives, but the corresponding phenyl derivatives or naphthyl derivatives can be prepared by conventional methods such as Vilsmeier reaction. It can also be easily obtained by formylation according to the method, so 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 and oxalyl chloride to a Friedel-Crafts reaction, so it is sufficient to use the compound obtained in this manner. Compound (1) can be obtained by using generally commercially available isothiocyanate derivatives, but since it can be easily obtained by the Hofmann rearrangement reaction using the corresponding thiocarboxylic acid amide as a raw material, it is also possible to use the compound obtained in this way. good. The triarylimidazole derivative of the present invention is extremely stable in an RI solution containing water or a surfactant;
When this is oxidized with an oxidizing agent, for example with hydrogen peroxide in the presence of peroxidase, a dye with excellent color stability is quantitatively formed. Moreover, the triaryl-based dazole derivative of the present invention is said to be more likely to inhibit color development due to the presence of substances in biological body fluids such as serum and urine, compared to cases in which color development is achieved using a similar method using known triarylimidazole derivatives. It has the property that almost no phenomena occur,
Moreover, its molecular extinction coefficient is equal to or higher than that of dyes produced from known triarylimidazole derivatives. Therefore, the triarylimidazole 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 effectively used as a coloring component in the determination of trace components in biological samples by colorimetrically quantifying its coloration. That is, the method for quantifying oxidizing substances of the present invention is particularly suitable as a method for measuring trace components in biological samples, which is carried out by quantifying filtered hydrogen oxide produced by the action of an oxidizing enzyme on a substrate or a substance produced by an enzymatic reaction. It can be used effectively. Trace components in biological samples that can be measured by the method of the present invention include, for example, cholesterol, glucose, glycerin, triglycerides, free fatty acids, uric acid, phospholipids, bile acids, monoamine oxidase, guanase,
Examples include, but are not limited to, cholinesterase, and all biological components that can be measured can be quantified by quantifying the filtered hydrogen oxide produced by enzymatic reactions. The measurement method of the present invention is carried out in accordance with a known enzymatic method (using an H202-generating enzyme) except that the triaryl ξ dazole derivative of the present invention is used as a coloring agent (oxidizable coloring reagent). It's enough if you do it. 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 μmol/l
A concentration range of is used.

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, according to a known method for quantifying biological components using an oxidizable coloring reagent. 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. Or 2 if necessary
It is used in combination of more than one species. Further, the amount used is determined as appropriate depending on the purpose and is not particularly limited.

Quantification of biological components by the method of the present invention is usually performed at a pH of 4.0.
~10.0. It is preferably carried out at a pH of 6.0 to 8.0. The laxatives used include phosphate, citrate, porium W1@, charcoal Wi salt, acetate, tris(hydroxymethyl)aminomethane, piperazine-N,N-bis(2
- Ethanesulfone a ) (PIPES) etc. Good (
Examples include, but are not limited to, buffers commonly used in this field, such as the buffer of J. Good.

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 peroxidase itself as well as hemoglobin and other heme compounds.

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 reaction reagents into absorbent carriers such as filter paper and drying them. It can also be used effectively.

EXAMPLES The present invention will be described in more detail below with reference to Examples and Reference Examples, but the present invention is not limited thereto.

[Example] Example 1.1-(aminothiocarbonyl)-2-(4-
Synthesis of (1) 1. , Synthesis of 2-bis(4-diethylamino-2-methylphenyl)ethane-1,2-dione Aluminum chloride (anhydrous) 8.3. carbon disulfide 30
II+1 was added, and to this was added N,N-diethyl-1-toluidine 18.3. was dropped. Next, 5 g of oxalyl chloride was added dropwise to this at 5° C. or lower, and the reaction was stirred for 1 hour. After the reaction is complete, add 50 μl of water and 100 μl of chloroform.
After washing the chloroform layer obtained by separating the layers with 2N hydrochloric acid, it was dried and concentrated. The residue was recrystallized from ethyl acetate to give yellow 1,2-bis(4-diethylamino-2
-Methylphenyl)ethane-1,2-dione (3.5 g) was obtained. (2) Synthesis of 1-(aminothiocarbonyl)-2-(4-hydroxyphenyl)-4,5-bis(4-diethylamino-2-methylphenyl)imidazole (the present compound [1]) or (1 ) 1,2-bis(4-diethylamino-2-
methylphenyl)ethane-1,2-dione 3.5 g, 4
- 1.2 g of hydroxybenzaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) and 2 g of ammonium acetate to 50 g of #acid
The mixture was reacted for 3 hours under reflux. After the reaction was completed, 300 ml of water was poured into the reaction solution, the resulting viscous residue was separated, and this viscous residue was purified by silica gel column chromatography (
Elution solvent: mixed solvent of chloroform and methanol).
2-(4-hydroxyphenyl)-4,5-bis(4
-diethylamino-2-methylphenyl)imidazole (0.9 g) was obtained.

Next, 3 g of ethoxycarbonyl isothiocyanate (manufactured by Aldrich) was added to a solution of this in 20 ml of chloroform, and the mixture was allowed to react under stirring for 24 hours at insect temperature. After the reaction was completed, 5 ml of methanol was added to the reaction solution to decompose excess ethoxycarbonyl isothiocyanate, and then the solvent was distilled off under reduced pressure. The resulting residual liquid was purified by silica gel column chromatography (elution solvent: mixed solvent of ethyl acetate and n-hexane). 1-[
(ethoxycarbonylamino)thiocarbonyl]-2-
(4-hydroxyphenyl)-4,5-bis(4-diethylamino-2-methylphenyl)imidazole was obtained. Furthermore, this was dissolved in 20 ml of methanol, and I
After saponifying the mixture by adding 2-1 N NaOH, the reaction solution was neutralized with INHCI. The reaction solution was concentrated to dryness, and the resulting residue was purified again by silica gel column chromatography (elution eluent*: mixed solvent of #ethyl acid and n-hexane), 1, (aminothiocarbonyl)-2 Light green crystals of -(4-hydroxyphenyl)-4.5-bis(4-diethylamino-2-methylphenyl)imidazole 0.
5g was obtained.

IR: 3400c ``'(OH). 2800-30
00cm-' (Cl{), ■200c+e-' (C=
S), 1600c+u-' (Aromatic Cl
l), 1500cm-' (^romatic CH)
．． Elemental analysis value (C32H39N50S) Calculation a (%): C70.95, H7.26, N12.
93, real Tj! i value (%): C 70.60, H
7.01. N12.55.

Example 2.1-(ethylaminothiocarbonyl)-2-
(3,5-dimethoxy4-hydroxyphenyl)-4
．． Synthesis of 5-bis(4-diethylaminophenyl)imidazole (the compound of the present invention [2]) (1) 1.2-His(4-diethylaminophenyl)
Synthesis of ethane-1,2-dione Aluminum chloride (anhydrous) 5.8g and carbon disulfide 30
II+1 was added, and 15 g of water-cooled 1,000 N,N-diethylaniline was added dropwise thereto. Next, 5 g of oxalyl chloride was added dropwise to the mixture at 5° C. or below, and the reaction was stirred for a working time.

After injecting water into the reaction solution, the target product was extracted with chloroform, and the extract was mixed with 2N1! After washing with acid, it was dried and concentrated. The residue was recrystallized from ethyl acetate to give yellow 1.2-
Bis(4-diethylaminophenyl)ethane-1,2-
3.3 g of dione was obtained. (2) l(ethylaminothiocarbonyl)-2-(3.
5-dimethoxy4-hydroxyphenyl)-4.5-
3.3 g of 1,2-bis(4-diethylaminophenyl)ethane-1,2-sion obtained in synthesis (1) of bis(4-diethylaminophenyl)imidazole (invention compound [2]), syringa Aldehyde (Tokyo Kasei Co., Ltd., 11 g) L-2 g and ammonium #acid 2K were reacted in 50 g of acetic acid under reflux for 3 hours. After the reaction was completed, 300 ml of water was poured into the reaction solution, the resulting viscous residue was separated, and this viscous residue was purified by silica gel column chromatography (elution solvent: a mixed solvent of chloroform and methanol). 0.8 g of 2-(3.5-dimethoxy-4-hydroxyphenyl)-4.5-bis(4-diethylaminophenyl)imidazole was obtained. Next, this was dissolved in 20 ml of chloroform,
Ethyl isothiocyanate (Wako Pure Chemical Industries, Ltd. II)
Log was added and the reaction was allowed to proceed at room temperature for 48 hours with stirring. After the reaction was completed, 30 ml of methanol was injected into the reaction solution to decompose excess ethyl isothiocyanate, and then the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (elution solvent: ethyl acetate and n
- hexane mixed solvent), i(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole o. I got ag. IR: 3400cm-' (OH), 2800~
3000cm-'(CH). 1200cm-' (
C=S), 1800cIm-' (Aromatic C
H), 1500cm-'(^romat.ie CH
). Elemental analysis value (C241{43N503s) Calculated value (%'
): C 59.85, H 9.00. N 1
4.54, actual value (%): C 59.25, H 8
．． 70, N 14.14.

Examples 3-5. 1. Reaction and post-treatment were carried out in the same manner as in Example 1 using 2-bis[4-[bis(2-hydroxyethyl)amino]phenyl]ethane-1,2-dione, syringaldehyde and methylisothiocyanate as raw materials. The compound of the present invention [3]) 1.1 g was obtained. Also,
1,2-bis[4-[N-(2-carboxyethyl)-
1-(phenylaminothiocarbonyl)-2-(4 -Hydroxynaphthyl)-4.5-bis[4-[N-(2-carboxyethyl)-N-ethylaminocophenylcoimidazole (compound of the present invention [4]) 1.0 g was further added to 1.2-bis 1-(P -fluorophenylaminothiocarbonyl)-2-(3.5-dimethoxy4-hydroxyphenyl)-4.5-bis[4-
1.2 g of [N-ethyl-N-(2-sulfoethyl)aminocophenyl]-primary dazole (the compound of the present invention [5]) was obtained. Example 6. Measurement of various properties of the compound of the present invention (1) Measurement of molecular extinction coefficient and absorption maximum (λmax) (coloring solution) 50+mM Piverazine-N,N'-bis(2-ethanesulfone lm) (PIPES) Sodium monohydroxide buffer A coloring solution was prepared by dissolving the compound of the present invention at 0.5 mM and peroxidase at 4 U/ml in a solution (pH 7.0).

(Procedure) 20 μl 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 λaax and λwax was measured.
I asked for

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.
Absorbance at 505 nm obtained by the same procedure using 50+aM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) sodium monohydroxide solution (pH 7.0) containing U/ml. It was determined by the following formula using EOH.

Molecular extinction coefficient = (Es÷Eoo) X 5 XIO3 The results are shown in Table 1. (2) Measurement of the influence of serum precepts Add and mix 20μ1 of normal pool human serum to 3ml of the coloring solution prepared by vR in (1), then add and mix 20μ1 of 1mM hydrogen peroxide solution. Allowed to react for minutes. The absorbance Eef of this reaction solution at λWaX was measured. Also, using purified water instead of normal pool human serum,
A similar operation was performed using the same coloring solution as above to obtain the absorbance Estd. By substituting these values into the following equation, the a value, which is an index to examine the influence of serum precepts, was calculated. a=(Eef÷E std) X 100The influence of serum components was expressed as follows based on the a value. 1: a value is O~3. ±:a [is 3-6. ++: a{+lI is 20 or more.

The results are also shown in Table 1.

In addition, for comparison, the results of measuring various properties of existing triarylimidazole derivatives in the same manner are also shown in the table.

As is clear from the results of the margin table below, the compound of the present invention has a molecular extinction coefficient that is equivalent to or higher than that of dyes produced from known triarylimidazole derivatives, but is It can be seen that this method has the property that when biological body fluids such as serum are used as samples, color development is almost never hindered by components in the body fluids, which was a problem in the past. Example 7. Quantification of hydrogen peroxide (m constant sample solution) 50-M piperazine-N,N'-bis(2-ethane snolephon a) (PIPES) - Sodium hydroxide buffer (pH 7.0) i: The following reagents were dissolved at a prescribed concentration. This was used as the measurement sample solution. Compound of the present invention [2] 0.5% peroxidase 4U/ml (sample) Commercially available hydrogen peroxide solution was appropriately diluted with distilled water to give 0.5% peroxidase.
The samples were 1.0, 1.5, 2.0 and 4.0mM solutions. (#k method) Add 20μl of sample to measurement reagent 3a+1, mix well,
After heating at 7° C. for 5 minutes, absorbance (Es) at 680 nm was measured.

In addition, the same operation was performed using ion-exchanged water instead of the sample, and a blind value (E Bl) was measured.

(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-EBI) obtained for each hydrogen peroxide concentration on the horizontal axis along the vertical axis shows good linearity, and the present invention It can be seen that hydrogen peroxide can be quantitatively measured by the method described above.

Incidentally, in place of the present compound [2], the present compound OF.
[3]. Similar results were obtained when using [4] and [5]. Example 8. Determination of hydrogen peroxide in the presence of serum (measurement reagent solution) Same as Example 7.

(sample) Same as Example 7.

(Procedure) Measurement sample solution 3 + al, normal human serum and ion exchange water 5
After adding 0 μl and mixing well, 20 μl of the sample was added and mixed well, and after heating at 37°C for 5 minutes, the absorbance (Es) of 6BOn+* was measured. In addition, the same operation was performed for 6 seconds using ion exchange water instead of the sample, and a blind value (E 81) was measured.

(Results) The measurement results are shown in Table 2. Table 2 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 9. Quantification of uric acid (lI!I constant test solution) 50mM Viverazine-N,N'-bis(2-ethanesulfone a) (PIPES) Sodium monohydroxide 1 t
fM (pH 7.0) i: A measurement reagent solution was prepared by dissolving the following reagent at a predetermined concentration.

Compound of the present invention [2] 0.05w aperoxidase 411/Il+1 uricase 0.05U/ml (sample) Standard solution containing 10mg/di uric acid and human serum 5
The specimen was used as a sample. (Procedure) Add 20 μl of the sample to 1 part of the measurement sample solution, mix well, and add 37
After heating at ℃ for 5 minutes. The absorbance (Es) at 860 nm was measured. In addition, the same operation was performed using ion-exchanged water instead of the sample to measure blind {1 (FBI).

The uric acid concentration in human serum was calculated according to the following formula.

Uric acid (B/di) = (Es-FBI of human serum) ÷
(Es-EBI of standard solution) The XIO results are shown in Table 3. Reference example 1. Quantification of uric acid Using the same sample as in Example 9, a commercially available kit for uric acid measurement (
Urine wIC-Test Wako, Wako Pure Chemical Industries, Ltd. ilK)
was used to measure uric acid concentration.

The results are also shown in Table 3.

Table 3 (Results) As is clear from Table 3, the measured value of uric acid obtained by the measurement method of Example 9 using the imidazole derivative of the present invention as a coloring component has a good correlation with that of the conventional method using a commercially available kit. It can be seen that it shows.

[Effects of the Invention] As described above, the imidazole derivative of the present invention is extremely stable in a buffer solution containing water or a surfactant.
In addition, the dye produced from this dye has a high molecular extinction coefficient, which means that the measurement sensitivity is high, and it has extremely excellent properties such that color development is almost never hindered by substances contained in biological samples such as serum. This is a great invention that contributes to this industry.

[Brief explanation of the drawing]

Figure 1 shows the calibration curve obtained in Example 7,
66 obtained for each hydrogen peroxide concentration (mM) on the horizontal axis
It connects the points where the absorbance at 0 nm 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 a hydrogen atom, an alkyl group that may have a substituent, or a substituent. R^2, R^3 and R^4 each independently represent an aryl group which may have a substituent, provided that R^2, R^3 and R^4, at least one of which 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. derivative. (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.