JPH0625146B2 - Novel 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, triglyceride, 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. A method for obtaining the amount of a target component by conducting a colorimetric quantification of this is being increased along with the development of an oxidizable color reagent. For example, H ₂ O ₂ generated by a combination of cholesterol-cholesterol oxidase, triglyceride-lipoprotein lipase-glycerol oxidase, uric acid-uricase, etc. is converted into a coloring system using peroxidase (POD) and an oxidizable color reagent. It is a method of obtaining the amount of a target component by deriving and 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. Sex color reagent, combination reagent of 3-methylbenzothiazolinone hydrazone (MBTH) and aniline compound, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),
Triphenylmethane leuco dye, benzidine derivative,
Examples thereof include o-tolidine derivative, diphenylamine derivative, o-phenylenediamine and the like. However, all of these conventionally used oxidizable color reagents have a coloring wavelength of 700 n except for diphenylamine derivatives.
It is less than m and is easily affected by serum components such as bilirubin and hemoglobin (it is easily affected by urinary plastids when measuring urinary components), and is a combination reagent with 4-aminoantipyrine or triphenylmethane Except for some leuco dyes, all have problems such as low stability of chromogen. On the other hand, a triallylimidazole derivative of a dye precursor (leuco dye) is disclosed as a chromogen having relatively good stability of the chromogen and having a coloration wavelength on the relatively long wavelength side (JP-B-57). -5519, Japanese Patent Publication No. 57-261
18, JP-A-58-45557, US Pat. No. 3,297,710, etc.).

However, these existing triallylimidazole derivatives each have an -OH group in one of the phenyl groups, The coloration wavelength is 700 nm or less. Therefore, it cannot be said that even these triallylimidazole derivatives are still sufficiently satisfactory to be used as color-forming components in the measurement of trace components in biological samples such as blood and urine.

[Object of the Invention]

The object of the present invention is to solve the problems of the conventional methods as described above, to develop a novel oxidizable color reagent having a maximum absorption wavelength of 700 nm or more, and a stable chromogen, and to develop the compound as a coloring component. It is intended to realize a highly accurate method for measuring a trace component in a biological sample by avoiding the influence of colored coexisting substances such as hemoglobin and bilirubin.

[Structure of Invention]

The present invention has the following general formula [I] [Wherein R ¹ is a lower alkyl group, a lower alkoxy group, a halogen atom, a nitro group, an amino group which may have a substituent, a —SO ₃ H group or a —SO ₃ M ¹ group (provided that M ¹ Represents an alkali metal ion or ammonium ion), -COOH
Group or a —COOM ² group (wherein M ² represents an alkali metal ion or an ammonium ion) represents a phenyl group or an aralkyl group which may have as a substituent,
R ² and R ³ each independently represent a substituted phenyl group substituted with an amino group which may have a substituent in at least the ortho position or the para position, and R ⁴ represents an alkyl-substituted carbamoyl group. Or a phenyl-substituted carbamoyl group which may have a substituent. ] 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.

That is, the present inventors have found that the imidazole derivative represented by the above general formula [I] has a coloring wavelength on the long wavelength side of 700 nm or more and is extremely stable as a chromogen. Was found for the first time, and by using this as a color-forming component in the measurement of a trace component in a biological sample such as serum or urine, the inventors have found that the above-mentioned object of the present invention can be achieved and completed the present invention. It is a thing.

In the imidazole derivative of the present invention represented by the general formula [I], the substituent of the phenyl group which may have a substituent represented by R ¹ is, for example, a methyl group, an ethyl group, a propyl group, Butyl group, pentyl group, etc. 1 to 5 carbon atoms
Lower alkyl groups (both linear and branched) such as methoxy, ethoxy, propoxy, butoxy, etc. lower alkoxy groups having 1 to 4 carbon atoms (linear or branched) Any of the above.) Chlorine, bromine, fluorine,
Halogen atom such as iodine, nitro group, amino group, lower alkyl group such as methyl group, ethyl group, propyl group, butyl group or the like, or -C ₂ H ₄ OH 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.), (Or Etc.) etc., an amino group substituted with a substituted lower alkyl group etc.,
-SO ₃ H group or -SO ₃ M ¹ group (provided that M ¹ represents an alkali metal ion such as sodium, potassium or lithium or ammonium ion), -COOH group or -COOM ² group (provided that
M ² represents an alkali metal ion such as sodium, potassium or lithium, or an ammonium ion. ) And the like, but does not include hydroxyl groups. Examples of the aralkyl group represented by R ¹ include benzyl group, phenethyl group, phenylpropyl group and the like. R ² ,
The substituent of the substituted amino group in the substituted phenyl group represented by R ³ which is substituted with an amino group which may have a substituent in at least the ortho position or the para position is, for example, methyl. Group, lower alkyl group such as ethyl group, propyl group, butyl group, -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, etc.), -C ₃ H ₆ SO ₃ H group (or -C ₃ H ₆ SO ₃ Na group, etc.), (Or Groups, etc.), but not limited thereto. The substituents other than the amino group or the substituted amino group in R ² and R ³ include, for example, lower alkyl groups such as methyl group, ethyl group, propyl group and butyl group, methoxy group, ethoxy group, propoxy group. Examples thereof include lower alkoxy groups such as, halogen atoms such as chlorine, bromine, fluorine, iodine, etc., nitro groups, cyano groups, carboxyl groups, sulfone groups and the like, but hydroxyl groups are not included therein. Examples of the alkyl-substituted carbamoyl group represented by R ⁴ include N-methylcarbamoyl group, N-ethylcarbamoyl group, N-isopropylcarbamoyl group,
Examples include lower alkyl-substituted carbamoyl groups such as N, N-dimethylcarbamoyl group, N, N-diethylcarbamoyl group, N, N-diisopropylcarbamoyl group, etc. Substituents of phenyl-substituted carbamoyl group which may have a substituent Examples thereof include lower alkyl groups such as methyl group, ethyl group and propyl group, lower alkoxy groups such as methoxy group and ethoxy group, halogen atoms such as chlorine and bromine, and the like. A carbamoyl group substituted with an optionally substituted phenyl group is used. Needless to say, an N-alkyl-N-phenyl-carbamoyl group can be used as well.

The imidazole derivative of the present invention represented by the general formula [I] is, for example, in the general formula [I]: (However, Represents an amino group which may have a substituent,
X ¹ represents another substituent. ) (However, Represents an amino group which may have a substituent,
X ² represents another substituent. ), A dye having the following structure is formed by oxidation.

Further, in the general formula [I], (However, Represents an amino group which may have a substituent, and X ³ represents another substituent. ) (However, Represents an amino group which may have a substituent, and X ¹ represents another substituent. ) (However, Represents an amino group which may have a substituent, and X ² represents another substituent. ), A dye having the following structure is formed by oxidation.

That is, in any case, the alkyl-substituted carbamoyl group represented by R ⁴ or the phenyl-substituted carbamoyl group which may have a substituent is removed from the imidazole group during the oxidation reaction and is oxidized by, for example, hydrogen peroxide. RN if done
It becomes HCOOH, 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 (blank stabilization) and has no influence on the color development. Don't give.

The dyes represented by the above [II] or [III], which are produced by the oxidation of the imidazole derivative of the present invention represented by the general formula [I], are the same as existing triallylimidazole derivatives. But The coloration wavelength of the resulting dye shifts to a longer wavelength side than that of the dye, and the maximum absorption wavelength is 700 nm or more.

Table 1 shows some specific examples of the compound of the present invention represented by the general formula [I] and the maximum absorption wavelength at the time of coloring, but the compound of the present invention is not limited thereto.

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 the ethanedione represented by the formula [IV], (In the formula, R ² and R ³ are the same as the above.) Then, according to the method described in, for example, US Pat. No. 3,297,710, aldehydes such as R ¹ -CHO (R ¹ is the same as above) and When the reaction is carried out with ammonium acetate in an acetic acid solvent for several hours while heating (reflux if necessary), an imidazole derivative represented by the formula [V] can be obtained. (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) at room temperature to the boiling point of the solvent for several hours to several hours. If you react for more than 10 hours,
The desired product is 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.

Examples of trace components in a biological sample that can be measured by the method of the present invention include cholesterol, glucose, glycerin, triglyceride, free fatty acids, uric acid, phospholipids, bile acids, monoamine oxidase, guanase, and cholinesterase. The present invention is not limited to these, and all biological components that can be measured by quantifying hydrogen peroxide generated 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 The amount and the amount to be used may be appropriately selected according to the substance to be measured in accordance with a known method for quantifying biological components using an oxidizable 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 peroxidase or peroxidase-like substance of plant, animal or microbial origin may be used in one kind or in two kinds if necessary. 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-carboxyphenyl) -3-N-methylcarbamoyl-4,5-bis (4-diethylaminophenyl) imidazole [the present compound (2)] (i) 1,2-bis (4-diethylamino) Synthesis of (phenyl) 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. Further, 1.5 g of oxalyl chloride was added dropwise with stirring under ice-cooling, and the reaction was stirred for 60 minutes. After the reaction, 50 ml of water and 100 ml of chloroform were added, and the chloroform layer obtained by liquid separation 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-carboxyphenyl) -4,5-bis (4-diethylaminophenyl) imidazole 1,2-bis (4-diethylaminophenyl) ethane-1,2-dione obtained in (i) 1.5 g of p-carboxybenzaldehyde 0.7 g of ammonium acetate 5 g of acetic acid 30 m
The mixture was heated under reflux for 2 hours in 1 to react. After cooling, 60 ml of water
When the mixture was added and neutralized with aqueous ammonia under ice cooling, crystals were precipitated. The precipitated crystals were collected, washed with water, dried and treated with hexane to obtain 1.1 g of the white target compound.

(Iii) Synthesis of 2- (4-carboxyphenyl) -3-N-methylcarbamoyl-4,5-bis (4-diethylaminophenyl) imidazole 2- (4-carboxyphenyl) -4, obtained in (ii) 5
-Bis (4-diethylaminophenyl) imidazole 1.
1 g was dissolved in 50 ml of chloroform, 5 ml of methyl isocyanate was added with stirring, 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. Concentrate
Purification with a silica gel column using chloroform as an eluent gave 980 mg of the desired product (almost white powder).

NMR (CDCl ₃ , TMS) ppm: 1.2 (12H, t, CH ₃ -C-), 1.
9 (1H, s, 2.7 (3H, s, -N-CH ₃ ), 3.6 (8H, qC-CH _2- ), 7.1 ~ 7.7 (12H, b
road, phenyl hydrogen), 11.8 (1H, s, -COOH) Example 2. 2- (3-methoxy-4-diethylaminophenyl)-
Synthesis of 3-N-methylcarbamoyl-4,5-bis (2-methyl-4-diethylaminophenyl) imidazole [the present compound (10)] (i) 1,2-bis (2-methyl-4-diethylaminophenyl) ) Synthesis of ethane-1,2-dione 20 ml of carbon disulfide was added to 6.7 g of anhydrous aluminum chloride, and 22 g of 3-methyl-N, N-diethylaniline was added dropwise under ice cooling. While further stirring and cooling with ice, oxalyl chloride
1.5 g was added dropwise, and the reaction was stirred for 60 minutes. After the reaction, water 50m
l and chloroform (100 ml) were added, and the layers were separated and the resulting chloroform layer was concentrated under reduced pressure to precipitate crystals. The precipitated crystals are collected and recrystallized from ethyl acetate to give the yellow target compound 5.4.
g was obtained.

(Ii) Synthesis of 2- (3-methoxy-4-diethylaminophenyl) -4,5-bis (2-methyl-4-diethylaminophenyl) imidazole 1,2-bis (2-methyl-) obtained in (i) 4-diethylaminophenyl) ethane-1,2-dione 1.5 g and 3-
Methoxy-4-diethylaminobenzaldehyde 1.0
g and 5 g of ammonium acetate were heated and refluxed in 30 ml of acetic acid for 2 hours to react. After cooling, 60 ml of water was added and neutralized with aqueous ammonia under ice cooling to precipitate crystals. The precipitated crystals were collected, washed with water, dried and treated with hexane to obtain 0.6 g of the white target compound.

(Iii) Synthesis of 2- (3-methoxy-4-diethylaminophenyl) -3-N-methylcarbamoyl-4,5-bis (2-methyl-4-diethylaminophenyl) imidazole 2- (2-) obtained in (ii) Chloroform 50 m of 0.6 g of 3-methoxy-4-diethylaminophenyl) -4,5-bis (2-methyl-4-diethylaminophenyl) imidazole
It was dissolved in 1 l, 5 ml of methyl isocyanate was added with stirring, and the mixture was left at room temperature for 12 hours. Then, 50 m 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 570 mg of the desired product (almost white powder).

Examples 3 to 10 The compounds of the present invention shown in Table 2 (1) to Table 2 (2) were synthesized by the same method as in Example 1 or Example 2. The structural formula and physical property data are also shown in Tables 2 (1) and 2 (2).

Example 11 Determination of uric acid (1) Reagent 50 mmol / l MES [2- (N-morpholino) ethanesulfonic acid] buffer (pH 6.5) with uricase 100 U /
l, peroxidase 2,000 U / l, compound of the present invention (2) 100 μm
It was prepared by dissolving so as to have a concentration of ol / l.

(2) Sample Uric acid was prepared by dissolving it in distilled water to give concentrations of 10,7.5,5,2.5 mg / dl.

(3) Measurement operation 3.0 ml of reagent for each 50 μl of each sample solution and distilled water
Was added and the mixture was heated at 37 ° C. for 5 minutes, and the absorbance at 740 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 12 Quantification of hydrogen peroxide (1) Reagent was prepared by dissolving it in 50 mmol / l phosphate buffer (pH 7.0) so that peroxidase 2,000 U / l and the compound (2) of the present invention 100 μmol / l.

(2) Sample Dilute commercially available hydrogen peroxide solution with distilled water to 2.0, 1.5,
It was prepared by dissolving so as to be 1.0, 0.5 mmol / l.

(3) Measurement operation 3.0 ml of reagent for each 20 μl of each sample solution and distilled water
Was added, and the mixture was heated at 37 ° C. for 3 minutes, and the absorbance at 740 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
In both cases, the maximum absorption wavelength at the time of color development is 700 nm or longer, which is on the long wavelength side. Therefore, when it is used as a color-forming component in the determination of trace components in biological samples such as serum and urine, That the measurement can be performed without being affected by the colored interfering substances coexisting in the
It has a remarkable effect in that it can be an imidazole derivative which is stabilized as a chromogen by adding a substituted carbamoyl group to N at position N, which is a great contribution to the art.

[Brief description of drawings]

FIG. 1 shows the calibration curve obtained in Example 11,
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 12,
The abscissa represents the absorbance obtained for each hydrogen peroxide concentration (mmol / l), and the points plotted along the ordinate are connected.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-61-176572 (JP, A) Chem, Ab, 74 (22) Abstract number 119391W (1971) Chem, Ab, 72 (17) Abstract number 89636U (1970) )

Claims (10)

[Claims] 1. The following general formula [I] [Wherein R ¹ is a lower alkyl group, a lower alkoxy group, a halogen atom, a nitro group, an amino group which may have a substituent, a —SO ₃ H group or a —SO ₃ M ¹ group (provided that M ¹ Represents an alkali metal ion or ammonium ion), -COOH
Group or a —COOM ² group (wherein M ² represents an alkali metal ion or an ammonium ion) represents a phenyl group or an aralkyl group which may have as a substituent,
R ² and R ³ each independently represent a substituted phenyl group substituted with an amino group which may have a substituent in at least the ortho position or the para position, and R ⁴ represents an alkyl-substituted carbamoyl group. Or a phenyl-substituted carbamoyl group which may have a substituent. ] The imidazole derivative shown by these. 2. The following general formula [I] [Wherein R ¹ is a lower alkyl group, a lower alkoxy group, a halogen atom, a nitro group, an amino group which may have a substituent, a —SO ₃ H group or a —SO ₃ M ¹ group (provided that M ¹ Represents an alkali metal ion or ammonium ion), -COOH
Group or a —COOM ² group (wherein M ² represents an alkali metal ion or an ammonium ion) represents a phenyl group or an aralkyl group which may have as a substituent,
R ² and R ³ each independently represent a substituted phenyl group substituted with an amino group which may have a substituent in at least the ortho position or the para position, and R ⁴ represents an alkyl-substituted carbamoyl group. Or a phenyl-substituted carbamoyl group which may have a substituent. ] The quantification method of the oxidizing substance characterized by using the imidazole derivative shown by these as a coloring component. 3. The quantification method according to claim 2, wherein the oxidizing substance is hydrogen peroxide. 4. The quantitative method according to claim 3, wherein the color-forming component is subjected to oxidative coloration 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 is a lower alkyl group, a lower alkoxy group, a halogen atom, a nitro group, an amino group which may have a substituent, a —SO ₃ H group or a —SO ₃ M ¹ group (provided that M ¹ Represents an alkali metal ion or ammonium ion), -COOH
Group or a —COOM ² group (wherein M ² represents an alkali metal ion or an ammonium ion) represents a phenyl group or an aralkyl group which may have as a substituent,
R ² and R ³ each independently represent a substituted phenyl group substituted with an amino group which may have a substituent in at least the ortho position or the para position, and R ⁴ represents an alkyl-substituted carbamoyl group. Or a phenyl-substituted carbamoyl group which may have a substituent. ] The imidazole derivative shown by these is used as a coloring component, The quantitative method of the peroxidase-like substance characterized by the above-mentioned. 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.