JPH0912915A - New coupler compound - Google Patents

Abstract

PURPOSE: To obtain a new coupler compound by oxidatively condensing a hydrogen donor, coloring in a higher wavelength and in absorbance than 4- aminopyrine, having high absorbance, suitable for microanalysis of hydrogen peroxide useful in clinical chemical test, etc. CONSTITUTION: This new coupler compound comprises and aminodiphenyl-based compound of formula I (R<1> to R<9> are each an alkyl, sulfo group, an alkoxy, a halogen, carboxyl, etc.; R<2> and R<5> may be bonded as they are or through a methylene chain; R<10> is a 1-5C alkyl which may contain sulfo group, carboxyl group, hydroxyl group, etc.). A compound of formula II among the compound of formula I is treated with a compound of formula III as a hydrogen donor in the presence of a peroxidase by hydrogen peroxide to form an oxidative condensate of formula IV and colored. The colored substance has the maximum absorption wavelength in the vicinity of 750nm.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel coupler compound, and in particular, in the microanalysis of hydrogen peroxide used in clinical chemistry tests and the like, in the presence of hydrogen peroxide and peroxidase, it is oxidative with a hydrogen donor such as TOOS. The present invention relates to a novel coupler that can be coupled to generate a dye and can be measured with higher sensitivity than before.

[0002]

2. Description of the Related Art At present, most of the enzymatic analysis methods are used for the detection of various substances in biological components such as blood and urine. In particular, various oxidases such as glucose oxidase, cholesterol oxidase and uricase are used. The method used is also widely applied to daily inspection.

Hydrogen peroxide produced by oxidase is
It can be easily quantified by reacting with an oxidative chromogenic substrate in the presence of catalase or peroxidase.

These enzymatic analysis methods are more specific than the measurement methods utilizing chemical reactions, and since the reaction is completed under mild conditions, they can be easily automated, It is expected that it will become more popular in the future.

At present, a method called the Trinder method is often used in this analysis method, and a method of oxidatively condensing a hydrogen donor and a coupler with hydrogen peroxide in the presence of peroxidase to form a dye is often used. It is used.

In clinical chemistry tests, it is inevitable to increase the detection sensitivity because of the use of a valuable sample called blood and the measurement of trace components. Therefore, a method that can measure with higher sensitivity than the method currently used is required.

Most of the wavelengths of dyes produced by these known methods are 500 nm to 600 nm, but the absorption produced by bilirubin and hemolysis contained in body fluids is in a wide range from the ultraviolet region to around 530 nm. , There may be a positive error during measurement,
There is a demand for a method of developing color in a higher wavelength region.

Further, since the reagent is used in the state of an aqueous solution at the time of actual measurement, stability of the reagent in a solution is required.

At present, many kinds of hydrogen donors such as phenol, TOOS, DAOS are used, but 4-aminoantipyrine is used almost everywhere as a coupler, and other reagents are used. It is rarely used.

[0010]

The object of the present invention is, as a coupler used in the conventional Trinder method, to oxidatively condense with a hydrogen donor and to develop color at a wavelength higher than that of 4-aminoantipyrine both in wavelength and absorbance, and in the absorbance. To provide high couplers.

[0011]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the novel coupler obtained is an aminodiphenyl compound represented by the following general formula (1).

[0012]

Embedded image (1) (In the formula, R ^{1 to} R ⁹ are an alkyl group, a sulfonic acid group, an alkoxyl group, a halogen group, a carboxyl group, or an alkyl group which may have a substituent, and R ² and R ⁵ are the same. or a methylene chain which may be bonded via a .R ¹⁰ represents a sulfonic acid group, a carboxyl group, a hydroxyl group or an amino group having optionally also an alkyl group having 1 to 5 carbon atoms.)

These compounds undergo an oxidative condensation reaction with a hydrogen donor in the same manner as conventional couplers.

For example, the following general formula (2) is

Embedded image (2) With the general formula (3), which is a hydrogen donor,

Embedded image (3) Hydrogen peroxide in the presence of peroxidase forms an oxidative condensate as represented by general formula (4) to develop color.

Embedded image (4)

This chromophore has a maximum absorption wavelength near 750 nm, unlike a chromophore using 4-aminoantipyrine. This wavelength region is extremely advantageous in that it is not affected by the biological matrix, and a laser can be used as a light source for this measurement wavelength, and high sensitivity measurement is expected.

The molar absorption coefficient at the maximum absorption wavelength is 2 to 3 times that of the conventional method, which is also advantageous in terms of high sensitivity.

The compound represented by the general formula (1) is
The following general formula (5)

[Chemical 6] (5) (wherein R ^{1 to} R ¹⁰ have the same meaning as in the general formula (1) above) can be synthesized by reacting sodium nitrite and zinc dust under acidic conditions.

Specific examples of the compound of the present invention represented by the general formula (1) thus synthesized are shown in Table 1, and the melting point, elemental analysis value and TOOS with hydrogen peroxide at pH 7.4 of the compound. The maximum absorption wavelength λ _max (nm) of the coupling dye is shown in Table 2.

[0019]

[Table 1]

[0020]

[Table 2]

[0021]

EXAMPLES Next, the production method of the compound of the present invention (Examples 1 to 3) and the quantification method using the same (Examples 4 and 5) will be described in more detail with reference to Examples. Is not limited to the following examples as long as the gist thereof is not exceeded.

Example 1 Preparation of Compound (A) 4.6 g of N-methyldiphenylamine was dissolved in 20 ml of hydrochloric acid, cooled to 0 ° C. in an ice bath while stirring, and sodium nitrite was stirred at 5 ° C. or lower. Aqueous solution (2g / 5m
1 deionized water) was added dropwise. After dropping, add 1 at below 5 ℃.
Stirring is continued for an hour, and then 100 ml of pure water and 100 m of hydrochloric acid
and 1 were added. Zinc powder 5 while stirring at 10 ° C or lower
g was added and stirring was continued for 1 hour. After that, adjust the pH to 8 with a 1N aqueous sodium hydroxide solution, and add 200 ml of ethyl acetate.
And extracted. The extract was dried over magnesium sulfate and concentrated, and the target substance was separated on a silica gel column using methyl chloride as an eluent. The separated fractions were concentrated and dissolved in 500 ml of ether, and then hydrochloric acid gas was blown into the solution while stirring, and the precipitated hydrochloride was collected by filtration and dried to obtain 2.8 g of the desired product. The yield was 47.5%.

TLC (silica gel, methylene chloride): R
f = 0.15. ¹ H-NMR (CD ₃₀ D) σppm (TMS): 3.
31 (s, 3H), 6.91 to 7.40 (m, 9H). IR (cm ^-1 ): 2904, 2592, 1602, 15
16, 1508, 1350, 1252, 1134, 75
0.

Example 2 Production of Compound (E) 3, N-methyldiphenylamine (3.9 g) was added to hydrochloric acid (20 m).
It was dissolved in 1 and cooled to 0 ° C. in an ice bath while stirring.
An aqueous sodium nitrite solution (1.6 g / 3 ml pure water) was added dropwise with stirring at 5 ° C or lower, and when the addition was completed, stirring was continued at 5 ° C or lower for 1 hour, and then 80 ml of pure water and 80 ml of hydrochloric acid were added, While stirring below 10 ° C,
Zinc dust (4 g) was added and stirring was continued for 1 hour. After that, the pH was adjusted to 8 with a 1N aqueous sodium hydroxide solution, and ethyl acetate 2
00 ml was added for extraction. The extract was dried over magnesium sulfate and concentrated, and then methyl chloride was used as an eluent to separate the desired product on a silica gel column. The separated fractions were concentrated, dissolved in 500 ml of ether and then stirred.
Hydrochloric acid gas was blown in, and the precipitated hydrochloride was collected by filtration and dried to obtain 1.7 g of the desired product. Yield 34.6%.

TLC (silica gel, methylene chloride): R
f = 0.22. ¹ H-NMR (CD ₃ OD) σppm (TMS):
2.30 (s, 3H), 3.29 (s, 3H), 6.7
2 to 7.39 (m, 9H). IR (cm ^-1 ): 2910, 2592, 1602, 15
22, 1350, 1244, 1115, 792, 72
6.

Example 3 Preparation of compound (J) 5 g of N-sulfopropylcarbazole monosodium salt
Is dissolved in 20 ml of hydrochloric acid and stirred in an ice bath at 0 ° C.
And cooled. Aqueous sodium nitrite solution (1.5 g / 3 ml pure water) was added dropwise with stirring at 5 ° C or lower, and when the addition was completed, stirring was continued at 5 ° C or lower for 1 hour, and then pure water 5
0 ml and 50 ml of hydrochloric acid were added. While further stirring at 10 ° C. or lower, 5 g of zinc dust was added and stirring was continued for 1 hour. After that, adjust to pH 8 with 1N sodium hydroxide solution,
The zinc hydroxide precipitate was collected by filtration. The filtrate is concentrated,
Chloroform / methanol = 8: 2 was used as an eluent, and the target product was separated on a silica gel column. The separated fractions were concentrated, recrystallized with a water / methanol solution, and the product collected by filtration was dried to obtain 1.4 g of the desired product. Yield 26.7%.

TLC (silica gel, chloroform / methanol = 8.2): Rf = 0.45. ¹ H-NMR (D ₂ O) σppm (TMS): 1.7
2 to 2.01 (m, 2H), 3.62 (t, 2H),
7.02-8.17 (m, 7H). IR (cm ^-1 ): 2945, 2877, 1606, 15
02, 1469, 1345, 1280, 1148, 75
5,730.

Example 4 Preparation of Calibration Curve Using Hydrogen Peroxide Standard Solution Four preparations were prepared by dissolving 500 units of peroxidase and 3.31 mg of TOOS in 100 ml of 50 mM phosphate buffer of pH 7.4, each containing compound (A). 2.3
4 mg, (E) 3.28 mg, (J) 3.26 mg
Was dissolved to obtain three kinds of color developing solutions. Test tubes a to f were provided for each color developing solution, and the color developing solution and the hydrogen peroxide standard solution were taken in these test tubes as follows.

[0029]

After immersing each test tube in a 37 ° C. constant temperature bath for 10 minutes, the absorbance at each maximum absorption wavelength was measured using the reagent blind test as a control. Figure 1 shows the calibration curve prepared from the obtained values.
Shown in

From FIG. 1, it can be seen that the calibration curve shows linearity for each color forming reagent in the concentration range of 1 to 50 μm. From the above results, it is clear that all the compounds of the present invention have an effect as a coupler in the determination of a peroxide substance.

Regarding Compound A, which has the highest sensitivity in this measurement, specific examples of quantification are shown below.
Of course, this is merely an example, and the present invention is not limited thereto.

Example 5 Determination of glucose in serum by Compound A 500 ml of glucose oxidase, 500 units of peroxidase, 3.31 mg of TOOS and 2.34 mg of Compound A were dissolved in 1 ml of 50 mM phosphate buffer solution (pH 7.4) to prepare a coloring solution. .

I) Preparation of Calibration Curve 200 mg of glucose dissolved in 100 ml of pure water is used as a glucose standard solution, which is appropriately diluted and used in the experiment.

The following reagents were taken in test tubes a to d.

[0036]

Each test tube was immersed in a constant temperature bath at 37 ° C. for 10 minutes, and then the absorbance at 748.2 nm was measured using a reagent blind test as a control. The calibration curve prepared from the obtained values is shown in FIG.

As a result, the glucose concentration was 0 to 200 mg.
Good linearity was exhibited in the range of / dl.

II) Quantification of Glucose in Serum 10 ml of deproteinized serum was placed in a test tube, 0.1 ml of the above coloring solution was added, and the mixture was immersed in a constant temperature bath at 37 ° C. for 10 minutes. The results of measuring the absorbance at 748.2 nm and comparing the concentration of glucose in serum with the calibration curve were as follows. It is shown in Table 3.

[0040]

[Table 3]

[0041]

By using the coupler compound of the present invention as a coupler for the Trinder method, the detection sensitivity is improved by a factor of 2 to 3 and the required sample amount is small. Further, since the measurement wavelength is on the long wavelength side, it is possible to avoid a measurement error due to hemolysis or the like. Also, a laser can be used as a light source, and higher sensitivity measurement is expected.

[Brief description of the drawings]

FIG. 1 is a calibration curve with respect to the hydrogen peroxide concentration of the oxidative condensates of reagents A, E, J and TOOS, in which the vertical axis represents the absorbance at the maximum absorption wavelength and the horizontal axis represents the hydrogen peroxide concentration. However,
The symbols ●, ○, and ■ represent compounds A, E, and J, respectively.

FIG. 2 is a calibration curve for glucose concentration when Reagent A is used, in which the vertical axis is the absorbance at 748.2 nm and the horizontal axis is the glucose concentration.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07C 307/02 7419-4H C07C 307/02 309/24 7419-4H 309/24 309/29 7419- 4H 309/29 C07D 209/88 C07D 209/88 219/08 219/08 G01N 31/00 G01N 31/00 M 31/22 122 31/22 122 33/52 33/52 C // C12Q 1/28 7823− 4B C12Q 1/28

Claims (1)

[Claims] 1. The following general formula (1): (1) (In the formula, R ^{1 to} R ⁹ are an alkyl group, a sulfonic acid group, an alkoxyl group, a halogen group, a carboxyl group, or an alkyl group which may have a substituent, and R ² and R ⁵ are the same. or it may be bonded via a methylene chain .R ¹⁰ is a sulfonic acid group, a carboxyl group, an amino diphenyl represented by also have a hydroxyl group or an amino group represents an alkyl group having 1 to 5 carbon atoms.) A new coupler compound consisting of a series of compounds.