JPH05229993A - Triphenylmethane derivative - Google Patents

Abstract

PURPOSE:To provide a new triphenylmethane derivative containing plural p- aminophenyl groups and useful as a reagent for the determination of an oxidation enzyme such as peroxidase and bilirubin oxidase as well as hydrogen peroxide in high sensitivity. CONSTITUTION:The objective triphenylmethane derivative useful as a reagent for the determination of hydrogen peroxide, oxidation enzyme, etc., and expressed by formula III (R<3>, R<4> and R<7> are same as R<1>, R<2> and R<6>) can be produced by reacting an aniline derivative of formula I [R<1> is H or (substituted) alkyl; R<2> is (substituted) alkyl; R<2> is H, alkyl, alkoxy, OH or sulfonic acid group]](e.g. N-ethyl-N-sulfopropylaniline sodium salt) with a benzaldehyde derivative of formula II [R<5> is H, halogen, alkyl or group of the formula N(R<9>)R<10> (R<9> is H or alkyl; R<10> is alkyl); R<8> is same as R] [e.g. 4-(N,N-diethylamino) benzaldehyde] in the presence of sulfuric acid at 100-120 deg.C for 4hr.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention has the general formula

[Chemical 2] (In the formula, R ¹ and R ³ are a hydrogen atom or a substituted or unsubstituted alkyl group, R ² and R ⁴ are a substituted or unsubstituted alkyl group, and R ⁵ is a hydrogen atom, a halogen atom, an alkyl group, or —N (R ⁹ ) R ¹⁰ is a group represented by the formula, wherein R ⁹ is a hydrogen atom or a substituted or unsubstituted alkyl group, R ¹⁰ is a substituted or unsubstituted alkyl group, R ⁶ ,
R ⁷ and R ⁸ are a hydrogen atom, an alkyl group, an alkoxy group, a hydroxyl group or a sulfonic acid group. ) Relating to a triphenylmethane derivative. The triphenylmethane derivative represented by the general formula (I) is a compound that can be used as a coloring dye for measurement of hydrogen peroxide and the like.

[0002]

2. Description of the Related Art Triphenylmethanes develop color when they coexist when decomposing hydrogen peroxide by peroxidase (hereinafter referred to as POD). The amount of hydrogen peroxide or POD activity can be measured by measuring the degree of color development. However, the colored dye cannot be used for the measurement with good reproducibility of trace components such as low molecular extinction coefficient, low water solubility, and precipitation of the dye in solution. To solve this problem, Trinder reagent (K.
Tamaoku; Anal. chim. Acta. ;
136 , 121-127 (1982)), and has been used, for example, for quantitative determination of hydrogen peroxide in the living body or measurement of POD activity.

[0003]

However, although the problem of reproducibility has been solved to some extent by using the Trinder reagent, it is a fact that higher sensitivity is required in analysis in the field of clinical examination. is there. Trinder's reagent was not satisfactory for this purpose, and the appearance of effective compounds had long been expected.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted diligent research to find a color-forming compound capable of obtaining highly sensitive and stable measurement results, and as a result, the triphenylmethane derivative represented by the general formula (I) was identified. The present invention has been completed by discovering that the compound can sufficiently meet the purpose. The present invention is a triphenylmethane derivative represented by the general formula (I).

The compound of the present invention is a compound having triphenylmethane as a main skeleton. In the triphenylmethane derivative represented by the general formula (I), R ¹ and R
³ is a hydrogen atom or a substituted or unsubstituted alkyl group. The alkyl group is an alkyl group having 1 to 4 carbon atoms, and examples thereof include a methyl group, an ethyl group, a propyl group, a butyl group, and a sec-butyl group. Examples of the substituent for the alkyl group include a hydroxyl group and a sulfonic acid group. Further, the sulfonic acid group is a salt with an alkali metal such as sodium and potassium, an alkaline earth metal such as calcium, magnesium and barium. Can also be formed.

R ² and R ⁴ are substituted or unsubstituted alkyl groups, and examples thereof include the same substituted or unsubstituted alkyl groups as the above R ¹ and R ³ . R ⁵ is a hydrogen atom, a halogen atom, an alkyl group or —N (R ⁹ ) R
It is a group represented by ¹⁰ . Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. Examples of the alkyl group include the same alkyl groups as the above R ¹ and R ³ . -N
(R ⁹ ) In the group represented by R ¹⁰ , R ⁹ is the same hydrogen atom as R ¹ and R ³ or a substituted or unsubstituted alkyl group, and R ¹⁰ is the same as R ¹ and R ³ A substituted or unsubstituted alkyl group can be mentioned, respectively.

R ⁶ , R ⁷ and R ⁸ are a hydrogen atom, an alkyl group, an alkoxy group, a hydroxyl group or a sulfonic acid group.
Examples of the alkyl group and the sulfonic acid group include R ¹ and R
^The same alkyl group and sulfonic acid group as ³ can be mentioned respectively. The alkoxy group is an alkoxy group having 1 to 4 carbon atoms, for example, a methoxy group, an ethoxy group,
Propoxy group, isopropoxy group, butoxy group, isobutoxy group, sec-butoxy group and the like can be mentioned.

Examples of the triphenylmethane derivative represented by the general formula (I) of the present invention include bis [4- (N-
Ethyl-N- (3-sulfopropyl) amino-2-methoxyphenyl)] [4- (N, N-diethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N- (3-sulfo) Propyl) aminophenyl)]
[4- (N, N-diethylamino) phenyl] methane
Disodium salt, bis [4- (N-ethyl-N- (2-
Hydroxy-3-sulfopropyl) amino-2-methylphenyl)] [4- (N, N-diethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N- (3-sulfopropyl) amino] 2-Methoxyphenyl)] [4- (N, N-dimethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-
N- (3-sulfopropyl) aminophenyl)] [4-
(N, N-Dimethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N- (2-hydroxy-3-sulfopropyl) amino-2-methylphenyl)] [4- (N, N-Dimethylamino) phenyl] methane disodium salt, bis [4- (N-ethyl-N-
(3-Sulfopropyl) aminophenyl)] (4-chlorophenyl) methane disodium salt, bis [4- (N
-Ethyl-N- (3-sulfopropyl) aminophenyl)] (4-hydroxyphenyl) methane disodium salt, bis [4- (N-ethyl-N- (3-sulfopropyl) aminophenyl)] (2- Examples thereof include sulfophenyl) methane trisodium salt.

The triphenylmethane derivative represented by the general formula (I) of the present invention has the general formula

[Chemical 3] (In the formula, R ¹ , R ² and R ⁶ are the same as above.)

[Chemical 4] (In the formula, R ⁵ and R ⁸ are the same as above.) And can be produced by reacting with a benzaldehyde derivative. The aniline derivative represented by the general formula (II) and the benzaldehyde derivative represented by the general formula (III) are industrially easily available compounds.

The reaction between the aniline derivative represented by the general formula (II) and the benzaldehyde derivative represented by the general formula (III) is carried out in the presence of an acid to give the general formula (I) in good yield. ) Can be derived to the triphenylmethane derivative represented by. The acid used in the reaction is sulfuric acid, hydrochloric acid, nitric acid or the like, and the use of sulfuric acid is particularly preferable since the desired product can be obtained in good yield. The reaction proceeds smoothly by selecting the range of 50 to 200 ° C.

The above-mentioned general formula (I) obtained as described above
The triphenylmethane derivative represented by can be used for the quantification of hydrogen peroxide as shown in Reference Example, but in addition, quantification of oxidases such as peroxidase and bilirubin oxidase, or quantification of compounds having oxidative power. ,
It can also be used for the quantification of peroxyboric acid or the activity measurement of oxidases that generate hydrogen peroxide, such as glucose oxidase, uricase, cholesterol oxidase, glycerol oxidase, xanthine oxidase, and nicotine adenine-nucleotide oxidase.

[0012]

The present invention will be described in more detail with reference to the following examples and reference examples. . Example 1 Bis [4- (N-ethyl-N- (3-sulfopropyl)
Aminophenyl)] [4- (N, N-diethylamino)
Phenyl] methane disodium salt production

[Chemical 5]

2.65 g (10 mM) of N-ethyl-N-sulfopropylaniline sodium salt (ALPS),
4- (N, N-diethylamino) benzaldehyde 0.
Put 885 g (5 mM) in a 100 ml eggplant flask,
15 ml of 40% sulfuric acid was added thereto, and the eggplant flask was immersed in an oil bath heated to 100 to 120 ° C. and refluxed for 4 hours. After completion of the reaction, the eggplant flask was cooled to room temperature, and further neutralized with 4N-sodium hydroxide aqueous solution while cooling with ice water. This reaction product was transferred to a separating funnel, unreacted diethylaminobenzaldehyde was removed with chloroform, and the washed aqueous layer was concentrated with an evaporator to dryness. Subsequently, this dried product was dissolved in 10 mM hydrochloric acid and purified using C18 gel. The target substance was eluted with a gradient using methanol containing 10 mM hydrochloric acid. The obtained purified product was neutralized with an aqueous sodium hydroxide solution to obtain 0.995 g (yield 24%) of the title compound.

Melting point: 230 ° C. or higher (decomposition) NMR (δ, CD ₃ OD): 7.28 to 7.02 (12
H, m), 5.50 (1H, brs), 3.54-3.
47 (12H, m), 2.87 to 2.83 (4H, t,
J = 7.0 Hz), 2.04 to 1.99 (4H, m),
1.15 to 1.10 (12H, t, J = 7.1Hz)

Example 2 Bis [4- (N-ethyl-N- (3-sulfopropyl)]
Aminophenyl)] [4- (N, N-dimethylamino)
Phenyl] methane disodium salt production

[Chemical 6]

2.65 g (10 mM) of N-ethyl-N-sulfopropylaniline sodium salt (ALPS),
4- (N, N-dimethylamino) benzaldehyde 0.
Put 885 g (5 mM) in a 100 ml eggplant flask,
15 ml of 40% sulfuric acid was added thereto, and the eggplant flask was immersed in an oil bath heated to 100 to 120 ° C. and refluxed for 4 hours. After completion of the reaction, the eggplant flask was cooled to room temperature, and further neutralized with 4N-sodium hydroxide aqueous solution while cooling with ice water. This reaction product was transferred to a separating funnel, unreacted dimethylaminobenzaldehyde was removed with chloroform, and the washed aqueous layer was concentrated with an evaporator to dryness. Subsequently, this dried product was dissolved in 10 mM hydrochloric acid and purified using C18 gel. The target substance was eluted with a gradient using methanol containing 10 mM hydrochloric acid. The obtained purified product was neutralized with an aqueous sodium hydroxide solution to obtain 0.89 g (yield 26%) of the title compound.

Melting point: 230 ° C. or higher (decomposition) NMR (δ, CD ₃ OD): 6.96 to 6.73 (12
H, m), 5.27 (1H, brs), 3.43-3.
41 (8H, m), 2.88 (6H, s), 2.877
~ 2.81 (4H, t, J = 7.5Hz), 2.07 ~
1.97 (4H, m), 1.14 to 1.10 (6H,
t, J = 7.1 Hz)

Example 3 Bis [4- (N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) amino-2-methylphenyl)] [4
Preparation of-(N, N-diethylamino) phenyl] methane disodium salt

[Chemical 7]

N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -m-toluidine sodium salt (TO
OS) 3.33 g (10 mM) and 4- (N, N-diethylamino) benzaldehyde 0.885 g (5 mM) were placed in a 100 ml eggplant flask, and 40% sulfuric acid 15
ml was added, and the eggplant flask was immersed in an oil bath heated to 100 to 120 ° C. and refluxed for 4 hours. After completion of the reaction, the eggplant flask was cooled to room temperature, and further neutralized with 4N-sodium hydroxide aqueous solution while cooling with ice water. This reaction product was transferred to a separating funnel, unreacted diethylaminobenzaldehyde was removed with chloroform, and the washed aqueous layer was concentrated with an evaporator to dryness. Subsequently, this dried product was dissolved in 10 mM hydrochloric acid and purified using C18 gel. The target substance was eluted with a gradient using methanol containing 10 mM hydrochloric acid. The obtained purified product was neutralized with an aqueous sodium hydroxide solution to give 0.89 g of the title compound (yield 28
%) Was obtained.

Melting point: 230 ° C. or higher (decomposition) NMR (δ, CD ₃ OD): 7.19 to 6.47 (10
H, m), 5.58 (1H, brs), 4.32 (2
H, brs), 3.63 to 3.30 (14H, m),
3.07 to 2.90 (4H, m), 2.13 (6H, b
rs), 1.20 to 1.10 (12H, m)

Example 4 Preparation of bis [4- [N- (3-sulfopropyl)] aminophenyl] [4- (N, N-dimethylamino) phenyl] methane disodium salt

[Chemical 8]

2.4 g (10 mM) of N-sulfopropylaniline sodium salt (HALPS), 4- (N, N)
-Dimethylamino) benzaldehyde 0.885 g (5
(mM) was placed in a 100 ml eggplant flask and 40%
Sulfuric acid (15 ml) was added and the eggplant flask was heated to 100-12.
It was immersed in an oil bath heated to 0 ° C. and refluxed for 4 hours. After completion of the reaction, the eggplant flask was cooled to room temperature, and further neutralized with 4N-sodium hydroxide aqueous solution while cooling with ice water. This reaction product was transferred to a separating funnel, unreacted dimethylaminobenzaldehyde was removed with chloroform, and the washed aqueous layer was concentrated with an evaporator to dryness. Subsequently, this dried product was dissolved in 10 mM hydrochloric acid and purified using C18 gel. The target substance was eluted with a gradient using methanol containing 10 mM hydrochloric acid. The obtained purified product is neutralized with an aqueous sodium hydroxide solution to give 0.62 g of the title compound.
(Yield 19%) was obtained.

Melting point: 230 ° C. or higher (decomposition) NMR (δ, CD ₃ OD): 6.98 to 6.76 (12
H, m), 5.29 (1H, brs), 3.35-3.
27 (6H, m), 2.94 to 2.89 (10H,
m), 2.16 to 2.05 (4H, m)

Example 5 Preparation of bis [4- [N- (2-hydroxy-3-sulfopropyl)] aminophenyl] [4- (N, N-diethylamino) phenyl] methane disodium salt

[Chemical 9]

N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) aniline sodium salt 2.8 g (1
(0 mM), 4- (N, N-diethylamino) benzaldehyde 0.885 g (5 mM) were placed in a 100 ml eggplant flask, 15% of 40% sulfuric acid was added thereto, and the eggplant flask was immersed in an oil bath heated to 100 to 120 ° C. Refluxed for 4 hours. After completion of the reaction, the eggplant flask was cooled to room temperature, and further neutralized with 4N-sodium hydroxide aqueous solution while cooling with ice water. Transfer the reaction to a separatory funnel,
Unreacted diethylaminobenzaldehyde was removed with chloroform, and the washed aqueous layer was concentrated with an evaporator and dried. Subsequently, this dried product was dissolved in 10 mM hydrochloric acid and purified using C18 gel. Elution of target substance is 10 mM
It was performed with a gradient using methanol containing hydrochloric acid.
The obtained purified product was neutralized with an aqueous sodium hydroxide solution to obtain 0.89 g (yield 25%) of the title compound.

Melting point: 230 ° C. or higher (decomposition) NMR (δ, CD ₃ OD): 7.07 to 6.74 (12
H, m), 5.27 (1H, brs), 4.32 (2
H, brs), 3.50 to 3.26 (14H, m),
3.05 to 2.89 (4H, m), 1.13 to 1.09
(12H, t, J = 7.0Hz)

Example 6 Bis [4- (N-ethyl-N- (3-sulfopropyl)]
Aminophenyl)] (4-chlorophenyl) methane disodium salt production

[Chemical 10]

2.65 g (10 mM) of N-ethyl-N-sulfopropylaniline (ALPS) and 0.75 g (5 mM) of p-chlorobenzaldehyde were placed in a 100 ml eggplant flask, and 15 ml of 40% sulfuric acid was added thereto. The flask was immersed in an oil bath heated to 100 to 120 ° C. and refluxed for 4 hours. After completion of the reaction, the eggplant flask was cooled to room temperature, and further neutralized with 4N-sodium hydroxide aqueous solution while cooling with ice water. The reaction product was transferred to a separating funnel, unreacted p-chlorobenzaldehyde was removed with chloroform, and the washed aqueous layer was concentrated with an evaporator to dryness. Subsequently, this dried product was dissolved in 10 mM hydrochloric acid and purified using C18 gel. Elution of target substance is 10 mM
It was performed with a gradient using methanol containing hydrochloric acid.
The obtained purified product was neutralized with an aqueous sodium hydroxide solution to obtain 0.43 g (yield 13%) of the title compound.

Melting point: 230 ° C. or higher (decomposition) NMR (δ, CD ₃ OD): 7.24 to 6.68 (12)
H, m), 5.30 (1H, brs), 3.42-3.
36 (8H, m), 2.87-2.82 (4H, t, J
= 7.5 Hz), 2.08 to 1.98 (4H, t),
1.14-1.10 (6H, t, J = 7.0Hz)

Example 7 Bis [4- (N-ethyl-N- (3-sulfopropyl)]
Aminophenyl)] (2-sulfophenyl) methane trisodium salt production

[Chemical 11]

2.65 g (10 mM) of N-ethyl-N-sulfopropylaniline sodium salt (ALPS),
1.1 g (5 mM) of o-sulfobenzaldehyde was added to 10
Place in a 0 ml eggplant flask and add 10 ml of 40% sulfuric acid.
Was added, and the eggplant flask was immersed in an oil bath heated to 100 to 120 ° C. and refluxed for 4 hours. After completion of the reaction, cool the eggplant flask to room temperature, then cool with ice water to 4N.
Neutralized with aqueous sodium hydroxide solution. This reaction product was transferred to a separating funnel, unreacted o-sulfobenzaldehyde was removed with chloroform, and the washed aqueous layer was concentrated with an evaporator to dryness. Subsequently, this dried product was added to 10 mM
It was dissolved in hydrochloric acid and purified using C18 gel. The target substance was eluted with a gradient using methanol containing 10 mM hydrochloric acid. The obtained purified product was neutralized with an aqueous sodium hydroxide solution to obtain 0.62 g (yield 17%) of the title compound.

Melting point: 230 ° C. or higher (decomposition) NMR (δ, CD ₃ OD): 8.01 to 6.57 (13)
H, m), 3.72 to 3.30 (8H, m), 2.86.
-2.81 (4H, m), 2.08-2.00 (4H,
m), 1.15 to 1.08 (6H, m)

Example 8 Preparation of tri [4-N, N-di (3-sulfopropyl) aminophenyl] methane hexasodium salt

[Chemical 12]

Tris (aminophenyl) methane (TAP
M) 2.9 g (10 mM) and propane sultone 15 g
(120 mM) was added to a 100 ml eggplant-shaped flask, and this was reacted for 5 hours while heating and stirring on an oil bath at 130 ° C. After completion of the reaction, the eggplant flask was cooled to room temperature and further neutralized with a 4N-sodium hydroxide aqueous solution. The reaction product was transferred to a separating funnel, unreacted tris (aminophenyl) methane was removed with ethyl acetate, and the washed aqueous phase was concentrated with an evaporator. Subsequently, hydrochloric acid was added to the dried product to adjust the pH to 2, and C1 equilibrated with 10 mM hydrochloric acid was used.
Purified on a column with 8 silica. Elution of target material is 1
Performed on a gradient with 0 mM hydrochloric acid / methanol. The obtained purified product was neutralized with an aqueous sodium hydroxide solution to obtain 410 mg (yield 4%) of the title compound.

Melting point: 206.5 ° C. NMR (δ, CD ₃ OD): 6.91 to 6.55 (12
H, m), 5.15 (1H, s), 3.47 to 3.41.
(12H, m), 2.88 to 2.82 (12H, m),
2.08 to 2.03 (12H, m)

Example 9 Preparation of Bis [4- (N, N-di (3-sulfopropyl) aminophenyl)] 4-N-3-sulfopropylphenylmethane pentasodium salt

[Chemical 13]

Tris (aminophenyl) methane (TAP
M) 2.9 g (10 mM) and propane sultone 15 g
(120 mM) was mixed in 30 ml of dimethylformamide, the same reaction as in Example 8 was carried out, and then the product was similarly purified by column chromatography to obtain 560 mg of the title compound (yield 5%).

Melting point: 205.1 ° C. NMR (δ, CD ₃ OD): 6.90-6.55 (12
H, m), 5.13 (1H, s), 3.72 (1H,
s), 3.46 to 3.42 (10H, m), 2.87 to
2.82 (10H, m), 2.07 to 2.03 (10
H, m)

Reference Example Each triphenylmethane derivative 1 obtained in Examples 1 to 9
00 μM solution (Tris-succinate buffer, pH = 7.
0,100 mM, including POD30000U / L) 2 ml
In addition, the test liquid containing hydrogen peroxide of 0 to 2000 μM 10 μm
1 is added and left for 2 minutes in a 37 ° C. thermostat. After 2 minutes, the absorbance of each test solution was measured, and the maximum absorption wavelength and the molecular extinction coefficient converted to the hydrogen peroxide concentration shown in Table 1 were obtained. Also, the absorbance was plotted against the concentration of hydrogen peroxide to obtain FIGS. 1 and 2. For comparison, 4-aminoantipyrine and N-
Ethyl-N-sulfopropylaniline sodium (4-
AP-ALPS) and 4-aminoantipyrine and phenol (4AP-Phenol) are used for the color development, and the light absorbency is also shown in FIG.

[0040]

[Table 1]

[0041]

INDUSTRIAL APPLICABILITY The triphenylmethane derivative represented by the above general formula (I) of the present invention is more effective than conventional oxidative color formers such as Trinder's reagent and 4-aminoantipyrine in the colorimetric method. Molecular extinction coefficient 3-1
0 times, 6-24 times in terms of hydrogen peroxide concentration,
Highly sensitive quantification of hydrogen peroxide, boric acid peroxidation, or activity measurement of peroxidase or bilirubin oxidase, and highly sensitive quantification of substrates using oxidases that produce hydrogen peroxide (glucose oxidase, cholesterol oxidase, uricase) Becomes

[Brief description of drawings]

FIG. 1 is a diagram in which hydrogen peroxide is measured using each compound synthesized in Examples 1 to 6 and the absorbance of a colored dye is measured.

FIG. 2 is a diagram in which hydrogen peroxide was measured using the compounds synthesized in Examples 8 and 9 and the absorbance of a dye that developed a color was measured.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C07C 309/46 9160-4H 309/47 9160-4H

Claims (1)

[Claims] 1. A general formula: Wherein R ¹ and R ³ are hydrogen atoms or substituted or unsubstituted alkyl groups, R ² and R ⁴ are substituted or unsubstituted alkyl groups, and R ⁵ is hydrogen. An atom, a halogen atom, an alkyl group or a group represented by -N (R ⁹ ) R ¹⁰ , wherein R ⁹ is
Hydrogen atom or a substituted or unsubstituted alkyl group, R ¹⁰ is a substituted or unsubstituted alkyl group, R ⁶ , R ⁷ and R ⁸
Is a hydrogen atom, an alkyl group, an alkoxy group, a hydroxyl group or a sulfonic acid group. ).