JPH062720B2 - Novel diphenylamine derivative - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel diphenylamine derivative that can be used as a color-forming reagent for quantifying oxidative substances and peroxidase-like substances.

“Background of the Invention” Measuring a biological component, for example, a body fluid component such as blood or urine, is associated with a disease, and therefore, in diagnosing a disease, elucidating a pathological condition, and determining a course of treatment. , Has become mandatory. For example, cholesterol in blood, triglyceride, glucose, uric acid, phospholipids,
It is well known that a measuring method for a very large variety of trace components such as bile acid and monoamine oxidase has been developed and is useful for diagnosing 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 allowed to act to produce H ₂ O ₂ corresponding to a target component, and this is led to a coloring system by using peroxidase and an oxidizable coloring reagent which is a coloring component. The number of methods for determining the amount of a target component by colorimetrically quantifying the color is increasing along with the development of a specific oxidative 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 peroxidase (P
OD), it is a method of obtaining the amount of the target component by introducing it into a coloring system using an oxidizable coloring reagent and measuring the absorbance of the coloring. A typical example of an oxidizable color reagent that is a color-forming component used in this method is a combination of 4-aminoantipyrine with a phenol compound or an N, N-disubstituted aniline compound. Color reagent, combination reagent of 3-methylbenzothiazoline hydrazone (MBTH) and aniline compound, 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), triphenylmethane leuco Examples thereof include dyes, benzidine derivatives, o-tolidine derivatives, triallylimidazole derivatives and o-phenylenediamine. However, most of these conventionally used oxidizable coloring reagents have a coloring wavelength of 600 nm or less, and are easily affected by serum components such as bilirubin and hemoglobin (when measuring urinary components, Are susceptible to the plastids of chlorophyll), and except for some of the combination reagents with 4-aminoantipyrine and some of the triphenylmethane-based leuco dyes, all have problems such as low stability of chromogen. . On the other hand, a diphenylamine derivative of a dye precursor (leuco dye) has been disclosed as a chromogen having a relatively stable chromogen and a coloration wavelength relatively on the long wavelength side (JP-A-56- 145352, JP 59-18
No. 2361, Japanese Patent Publication No. 60-33479, etc.).

All of these diphenylamine derivatives have a coloration wavelength of 700 nm or more on the relatively long wavelength side and have relatively high sensitivity, but the stability of the chromogen and the coloration stability are still sufficiently satisfactory. However, they all have the drawback of poor solubility in water.

"Object of the invention" The object of the present invention is that the maximum absorption wavelength at the time of coloring is on the long wavelength side of 700 nm or more, high sensitivity, and stability of the chromogen,
It is intended to provide a water-soluble diphenylamine derivative having excellent coloration stability.

"Structure of Invention" The present invention provides a compound represented by the general formula [I]: [Wherein R ¹ , R ² , R ³ and R ⁴ are each independently a compound represented by the formula C
_m H _{2m + 1} —O—C ₂ H ₄ — (wherein m represents an integer of 1 to 4) or an alkyl group having 1 to 5 carbon atoms. However, at least one of R ^{1 to} R ⁴ represents a group represented by the above C _m H _{2m + 1} —O—C ₂ H ₄ —. Further, R ⁵ , R ⁶ and R ⁷ each independently represent hydrogen, a lower alkyl group or a lower alkoxy group. ] It is a diphenylamine derivative shown by these.

In general, conventional color-developing reagents have poor solubility in water and are often dissolved and dispersed by adding a surfactant. The present inventors have focused on the oxy structure which is a partial structure of this surfactant (particularly nonionic surfactant), and by introducing this oxy structure into the dye compound, the water solubility is improved and the surfactant activity is improved. As a result of repeated studies, it was thought that the hydrophilicity with the agent would increase, and as a result, stabilization of the chromogen (stabilization of the blank) and improvement of coloration stability could be achieved, and as a result of repeated studies, the present invention was completed. It was.

In the diphenylamine derivative of the present invention represented by the general formula [I], the alkyl group represented by R ^{1 to} R ⁴ is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group or the like and has 1 to 5 carbon atoms. The alkyl group may be a straight chain or branched chain.

As R ⁵ , R ⁶ and R ^{7 in the} general formula [I], hydrogen, for example, a methyl group, an ethyl group, a propyl group, a butyl group, an amyl group and the like, having 1 to 5 carbon atoms (straight chain, It may be branched.) For example, an alkoxy group having 1 to 5 carbon atoms such as methoxy group, ethoxy group, propoxy group, butoxy group, amyloxy group (which may be linear or branched). ) And the like.

The compound of the present invention represented by the general formula [I] can be produced by a known method for producing indamine (R. Nietzki, Chemische Berich).
te, 1883, 16 , 464), and a dye can be easily obtained by synthesizing a dye and reducing it.

For example, first, the aniline derivative represented by the general formula [II] is synthesized, and separately, the phenylenediamine derivative represented by the general formula [III] is synthesized.

However, R ^{1 to} R ⁷ in the general formulas [II] and [III] are the same as in the general formula [I].

Then, the compound of [II] and the compound of [III] are oxidatively condensed with periodate in an aqueous solution according to the method for producing indamine to obtain a dye (type). The desired diphenylamine derivative is obtained by reducing this with zinc and hydrochloric acid.

Alternatively, it can be obtained by an Ullmann condensation reaction between a halogenoaniline derivative represented by the general formula [IV] and a phenylenediamine derivative represented by the general formula [III].

However, R ¹ , R ² , and R ⁵ in the general formula [IV] are the same as those in the general formula [I]. X represents a halogen atom.

For example, a compound represented by the general formula [IV] and a compound represented by the general formula [II
I]] a phenylenediamine derivative or an acyl derivative thereof is subjected to a condensation reaction in nitrobenzene under reflux,
The target product or its acyl derivative is obtained. In the case of an acyl derivative, for example, this is deacylated by heating with potassium hydroxide in ethylene glycol to obtain the desired product.

The compound of the present invention is extremely stable in water or an aqueous solution in which a surfactant is dissolved, and has a low so-called blank value. On the other hand, when this is oxidized with hydrogen peroxide in the presence of peroxidase in the presence of peroxidase, a dye having an absorption maximum in the visible deep color to near infrared region and excellent color stability is quantitatively formed according to the following reaction formula. To do. Further, since the compound of the present invention has a structure having a surface-active ability, it has very good solubility in water or an aqueous solution in which a surfactant is dissolved, which is extremely advantageous in the preparation of a coloring reagent solution, preferable.

Table 1 shows the stability of the leuco solution, the stability after coloring, and the maximum absorption wavelength (λ
_max ) and sensitivity (ε), but the compounds of the present invention are not limited to these specific examples.

Regarding the stability of the leuco solution and the stability of the coloring solution in Table 1, AA is extremely stable, A is stable, B is slightly unstable, and C is
Means unstable.

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

That is, a method for quantifying an oxidative substance using the compound of the present invention as a color-developing reagent is a substrate or a substrate in a biological sample, which is obtained by causing an oxidase to act on a substance produced by an enzymatic reaction and quantifying produced hydrogen peroxide. Alternatively, it is particularly effective as a method for quantifying enzyme activity.

As a trace component in a biological sample that can be measured by a quantification method using the compound of the present invention as a coloring reagent, for example, cholesterol, glucose, glycerin, triglyceride, floating fatty acid, uric acid, phospholipid, bile acid, monoamine oxidase, guanase, cholinesterase. However, the present invention is not limited to these, and all the biological components that can be measured by quantifying the hydrogen peroxide generated by the enzymatic reaction can be quantified.

The quantification method using the compound of the present invention as a color-developing reagent is a measurement method by an enzyme method (using H ₂ O ₂ -producing enzyme) known per se except that the diphenylamine derivative of the present invention is used as a color-developing reagent (oxidizable color reagent). It suffices to do this according to.

The concentration of the coloring reagent used is not particularly limited, but a concentration of several μmol / l or more, preferably 50 μmol / 1 to 100 μmol / l is usually used.

In a method for quantifying a biological component using the compound of the present invention as a coloring reagent, an oxidase (oxidase) used as an enzyme for producing hydrogen peroxide, an enzyme used for other purposes, a substrate involved in an enzymatic reaction, and others The kind and the amount of the substance 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. In addition, the peroxidase used in the quantification of hydrogen peroxide using the compound of the present invention as a color-developing reagent is not particularly limited in its origin and origin, and a peroxidase or a peroxidase-like substance of plant, animal, or microbial origin is one or If necessary, two or more kinds may be used in combination. The amount used is appropriately determined according to the purpose and is not particularly limited.

The quantification of biological components using the compound of the present invention as a coloring reagent is usually carried out at pH 4.0 to 10.0, 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 diphenylamine 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 diphenylamine derivative of the present invention can be applied, for example, as a color reagent in an enzyme immunoassay using peroxidase as a labeling compound, and hemoglobin in serum can be converted to hydrogen peroxide or sodium perborate. It can also be effectively used as a color-developing reagent in the case of measurement using such an oxidizing substance.

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

"Example" Example 1. Synthesis of 4'-bis (butoxyethyl) amino-2-methyl-4-dipentylaminodiphenylamine [Compound (1) of the present invention] 25 g of aniline and 200 g of tris (butoxyethyl) phosphoric acid
Was heated at 170 ° C for 6 hours, washed with 500 ml of 1N sodium hydroxide persalt, desalted, and purified by column chromatography (silica gel, n-hexane) to obtain 51 g of N, N-bis (butoxyethyl) aniline. It was

Separately, 25 g of m-toluidine was dissolved in triamyl phosphate, heated at 170 ° C. for 4 hours, washed with 500 ml of 1N aqueous sodium hydroxide for desalting, and purified by column chromatography (silica gel, n-hexane). N, N-diamyl-m
-I got 40 g of toluidine. 4 g of this N, N-diamyl-m-toluidine was dissolved in 10 liters of a water: methanol = 1: 1 solution,
A coupler solution was prepared. Next, 4 g of N, N-bis (butoxyethyl) aniline was dissolved in 100 ml of 6N hydrochloric acid, and 5 g of sodium nitrite was added on an ice bath for nitrosation. 160 ml of 5N aqueous sodium hydroxide solution was poured into this, extracted with 300 ml of hexane, dried and concentrated, and purified by column chromatography (silica gel, n-hexane). The obtained nitroso derivative was dissolved in 2 liters of methanol to give zinc. 120
g, and reduced with 90 ml of 6N hydrochloric acid to give a phenylenediamine derivative. Remove zinc from this solution, combine with the previously prepared coupler solution, add 34 g of periodic acid thereto,
An oxidative condensation reaction was performed. After the reaction, this is again zinc 35
g, reduced with 50 ml of 6N hydrochloric acid, and then extracted with ethyl acetate. This was purified by silica gel column chromatography (silica gel, n-hexane) to give a pale green oil 1.9 g.
(21% yield from dipentyl toluidine) was obtained.

TLC (silica gel, ethyl acetate: n-hexane = 1: 1
9) R _f = 0.4.

NMR (CDCl ₃ ): δ 0.90 (t, 12H, aliphatic CH ₃ ), 1.12
~ 1.88 (m, 16H, pentyl CH ₂ ), 3.30 ~ 3.72 (m, 8H, buthoxye
thyl CH ₂ ), 6.30-6.91 ppm (m, 7H, aromatic H). [First
Figure] MS (EI): M ⁺ = 553.

IR: ν1050, 3400 cm ^-1 .

Example 2. Synthesis of 4'-bis (buxyethyl) amino-2,2'-diketyl-4-dipentylaminodiphenylamine [Compound of the present invention (2)] The intermediate phenyldiamine and the coupler in the present Example were prepared respectively. This was synthesized according to the production method of the intermediate phenylenediamine and the coupler in Example 1.

Dissolve 1 g of N, N'-bis (butoxyethyl) -3-methyl-p-phenylenediamine and 250 mg of N, N-dipentyl-m-toluidine in 1 liter of water: methanol-1: 1 solution to give 2.3 g of periodic acid. Was added and the reaction was carried out. The following post-treatment is Example 1
The same procedure was followed to obtain 50 mg (yield 8.8%) of a pale yellow oil.

TLC (silica gel, ethyl acetate: n-hexane = 1: 1
9) R _f = 0.5.

NMR (CDCl ₃ ): δ 0.90 (t, 12H, aliphatic CH ₃ ), 1.12
~ 1.88 (m, 16H, pentyl CH ₂ ), 2.20 (s, 6H, aromatic C
_{H 3), 3.30~3.75 (m,} 8H, buthoxyethyl CH 2), 6.40~6.85
ppm (m, 7H, aromatic H). [Fig. 2] MS (EI): M ⁺ = 567.

IR: ν1050, 3400 cm ^-1 .

Reference example 1. Determination of hydrogen peroxide [Measurement test] 50 mmol / l PIPES [piperazine-] so that the concentration of the compound of the present invention (2) is 0.5 mmol / l and peroxidase is 4 U / ml.
N, N-bis (2-ethanesulfonic acid)]-sodium hydroxide buffer (pH 7.0) was dissolved.

[Sample solution]

Commercially available hydrogen peroxide solution is deionized water 0.5, 1.0, 1.5, 2.
It was diluted to a concentration of 0,4.0 mmol / l.

〔Measuring method〕

20 μl of the sample solution was added to 3 ml of the measurement reagent solution, and the mixture was heated at 37 ° C. for 5 minutes, and then the absorbance at 755 nm was measured.

〔result〕

 FIG. 3 shows the relationship between hydrogen peroxide concentration and absorbance.

As is clear from Fig. 3, each hydrogen peroxide concentration (mmol / l)
The calibration curve connecting the absorbances plotted in the figure shows good quantification.

Reference example 2. Quantification of hydrogen peroxide in the coexistence of normal human serum [Measurement reagent] The same as in Reference Example 1.

[Sample solution]

 Same as Reference Example 1.

〔Measuring method〕

After adding 50 μl of normal serum or ion-exchanged water to 3 ml of test reagent, add 20 μl of sample solution and warm at 37 ° C for 5 minutes.
Absorbance at 5 nm was measured.

〔result〕

 Table 2 shows the relationship between the hydrogen peroxide concentration and the absorbance.

As is clear from Table 2, human serum has no influence on the absorbance.

Reference example 3. Determination of uric acid [Measurement reagent] 0.5 mmol / l of the present compound (2), 4 U / m of peroxidase
l, uricase to a concentration of 0.05 U / ml, 50 mmol / l
It was dissolved in PIPES-sodium hydroxide buffer (pH 7.0).

[Sample solution]

Standard solutions containing 10 mg / dl uric acid and 5 human sera were used as samples.

〔Measuring method〕

20 μ of the sample solution was added to 3 ml of the measurement reagent solution, and after heating at 37 ° C. for 5 minutes, the absorbance at ₇₅₅ nm (OD ₇₅₅ ) was measured.

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

Further, using a commercially available kit for measuring uric acid [uric acid C-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.], the same human serum was measured and compared with this method. [Comparative Example] [Results] The results are shown in Table 3.

Reference example 4. Measurement of glucose oxidase activity [Measurement reagent] Compound of the present invention (2) 0.05 mmol / l, peroxidase 4 U /
l, glucose 50 mmol / l so that the concentration becomes 5%
It was dissolved in PIPES-sodium hydroxide buffer (pH 7.0).

[Sample solution]

Glucose oxidase activity value is 0.02, 0.04, 0.0 respectively
50mmol / lPI to be 8, 0.12, 0.16, 0.2U / l
A PES-sodium hydroxide buffer solution (solution dissolved in pH 7.0 was prepared.

〔Measuring method〕

After adding 20 μl of the sample solution to 3 ml of the measurement reagent solution, the time change of the absorbance at 755 nm was measured.

〔result〕

 The time course shown in Fig. 4 was obtained.

Reference example 5. Quantification of guanase [Measurement reagent] 1st reagent Compound (2) 0.1 mmol / l, uricase 1.2 U / ml, xanthine oxidase 0.15 U / ml, superoxide dismutase 90 U / ml, ascorbate oxidase 1 U / ml,
Catalase was dissolved in 50 mmol / l phosphate buffer (pH 7.0) so as to have a concentration of 1000 U / ml.

Second reagent solution The solution was dissolved in 50 mmol / l phosphate buffer (pH 7.2) so that the concentrations of guanine 2.5 mmol / l, NaN ₃ 0.12% and peroxidase 10 U / ml were obtained.

[Sample solution]

Guanase (derived from rabbit liver) has activity values of 2, 4 and 4, respectively.
It was dissolved in 50 mmol / l phosphate buffer (pH 7.2) so as to be 6, 8, 10 U / l.

〔Measuring method〕

1 ml of the first test solution was added to 0.1 ml of the sample solution, and after preliminarily heating at 37 ° C. for 5 minutes, 1 ml of the second test solution was added, and the time change of the absorbance at 755 nm was measured.

〔result〕

 The time course shown in Figure 5 was obtained.

Reference example 6. Quantification of guanase [Measurement reagent] The same as in Reference Example 5.

[Sample solution]

Guanase (derived from rabbit liver) has activity values of 2, 4 and 4, respectively.
It was diluted with normal human serum to be 6, 8, 10 U / l.

〔Measuring method〕

 Same as the reference example.

〔result〕

FIG. 6 shows the relationship between enzyme activity and absorbance change. However,
The blind test was performed using serum without addition of guanase.

"Effects of the Invention" As described above, the novel diphenylamine derivatives of the present invention have a maximum absorption wavelength at the time of color development of 700 nm or more on the long wavelength side, and thus, for example, trace components in biological samples such as serum and urine. When this is used as a color-developing reagent in the quantification of, it is possible to carry out the measurement without being affected by a colored interfering substance coexisting in the sample, and the novel diphenylamine derivative of the present invention is It has a very good solubility in water or an aqueous solution in which a surfactant is dissolved, and has a remarkable effect in that the stability of the chromogen and the coloration stability are remarkably excellent, which contributes to the industry. However, it is a great thing.

[Brief description of drawings]

FIG. 1 shows an NMR chart of 4′-bis (butoxyethyl) amino-2-methyl-4-dipentylaminodiphenylamine obtained in Example 1. FIG. 2 shows an NMR chart of 4′-bis (butoxyethyl) amino-2,2′-dimethyl-4-dipentylaminodiphenylamine obtained in Example 2. FIG. 3 shows the calibration curve obtained in Reference Example 1, in which the absorbance obtained for each hydrogen peroxide concentration (mmol / l) on the horizontal axis is plotted along the vertical axis. Is. FIG. 4 shows the time course obtained for each glucose oxidase activity of Reference Example 4, the horizontal axis represents the reaction time (minutes), and the vertical axis represents the absorbance. FIG. 5 shows the time course obtained for each guanase activity of Reference Example 5, the horizontal axis represents time (minutes), and the vertical axis represents absorbance. FIG. 6 shows the calibration curve obtained in Reference Example 6, in which the amount of change in absorbance per minute obtained for each guanase activity (U / l) on the horizontal axis is plotted along the vertical axis. Are tied together.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yutaka Miki, 6-1, Takada-cho, Amagasaki-shi, Hyogo Prefecture Wako Pure Chemical Industries, Ltd. Osaka Research Institute (72) Ritsuji Hashizume, 6-takada-cho, Amagasaki-shi, Hyogo No. 1 Wako Pure Chemical Industries, Ltd. Osaka Research Institute (72) Inventor Nobuyuki Tokioka 6-1 Takadacho, Amagasaki City, Hyogo Prefecture Wako Pure Chemical Industries, Ltd. Osaka Research Laboratories

Claims (1)

[Claims] 1. A general formula [I]: [Wherein R ¹ , R ² , R ³ and R ⁴ are each independently a compound represented by the formula C
_m H _{2m + 1} —O—C ₂ H ₄ — (wherein m represents an integer of 1 to 4) or an alkyl group having 1 to 5 carbon atoms. However, at least one of R ^{1 to} R ⁴ represents a group represented by the above C _m H _{2m + 1} —O—C ₂ H ₄ —. Further, R ⁵ , R ⁶ and R ⁷ each independently represent hydrogen, a lower alkyl group or a lower alkoxy group. ] The diphenylamine derivative shown by these.