JPS6010025B2 - Water-soluble diaminodiphenylmethane derivatives and their applications - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a water-soluble diaminodiphenylmethane derivative represented by the general formula (1) and its salts (
The present invention relates to a composition for quantifying a peroxide substance comprising a compound of the present invention (hereinafter abbreviated as "compound of the present invention") and a compound of the present invention as a dye-forming substance.

All of the above compounds of the present invention are novel and useful substances that have not yet been published in the literature, and are particularly useful as pigment-forming substances when quantifying hydrogen peroxide in coexistence with peroxidase or a substance with a similar catalytic action. It is.

Clinical testing methods using enzyme reagents are extremely selective analytical methods due to the substrate specificity of enzymes, and have rapidly become popular in recent years because they can easily quantify various biological components without being affected by complex biological matrices. It is becoming popular.

For example, glucose, uric acid, cholesterol, etc. in body fluids are decomposed by enzymes such as glucose oxidase, uricase, and cholesterol oxidase, producing corresponding amounts of hydrogen peroxide. Therefore, various in-vivo components can be measured by quantifying this hydrogen peroxide. Conventionally, methods for quantifying hydrogen peroxide produced by such a mechanism are often based on oxidative condensation color development, and for example, the following pigment-forming substances have been reported. m Aniline conductor (N,N-dimethylaniline; toluidine; N?N-diethyl-m-toluidine, etc.) [
2} Phenyl diamine derivatives and diamines such as penzidine and dianisidine '3' Phenol derivatives and naphthol derivatives '4} 0 Icotriphenylmethanes (such as icomalachite green and leucophenolphthalein) (5) 2,6- Dichlorophenol Indophenol leuco form Among the various substances above, leukotriphenylmethanes and 2,6-dichlorophenol indophenol are used alone, but aniline derivatives, phenylenediamine derivatives, phenol derivatives It is used in conjunction with a condensed color forming agent such as 4-aminoantipyrine and 3-methyl-2-penzothiazolinone hydrazone (hereinafter abbreviated as "MBTH").

The optimal axis conditions for the action of the enzyme differ depending on the type of enzyme that oxidizes and decomposes the target substance to be measured. For example, the optimal conditions for glucose oxidase are pH 5.0, pH 8.5 for uricase, and pH 8.5 for cholesterol oxidase.
The pH is 6.5 to 8.0, and the pH is generally distributed in the range of 5 to 9. However, the above-mentioned pigment-forming substances, especially monoamines and diamines, have poor solubility in water under conditions of pH 5 to 9. Therefore, measures are taken such as adding a surfactant to solubilize and disperse it, or adding an organic solvent to dissolve it. However, there is a concern that the coexistence of surfactants and organic solvents may deactivate the enzyme. Furthermore, aqueous solutions of these known dye-forming substances have poor solution stability and have the problem of concentration changes over time. The inventors
As a result of extensive research to find a pigment-forming substance that undergoes oxidative condensation with 4-aminoantipyrine and MBTH and has particularly good solubility in water, we have discovered the compound of the present invention represented by the general formula (1).

For example, when the compound of the present invention and MBTH are subjected to oxidative condensation using peroxidase and hydrogen peroxide, the following (0
) The present invention was completed by discovering that a blue pigment represented by the formula can be quantitatively formed and maintains excellent color stability.
By the way, the conventionally used phenol, N,
-Diethylaniline has a maximum absorption wavelength of the condensation product with 4-aminoantipyrine in the vicinity of 500 to 52 m, but on the other hand, the absorption caused by bilirubin contained in body fluids and hemolysis extends from the ultraviolet region. Since it is located over a wide range of around 50 m, there is a risk of positive errors in the measured values.

However, the compound of the present invention represented by the general formula (1) has the great advantage that the maximum absorption wavelength of the condensation product with M old TH is mostly at 56 m or more, so it can avoid the effects of pirirubin and hemolysis. There is. Furthermore, when the compound of the present invention and M old TH are mixed and dissolved in a pH 7 buffer solution and peroxidase and hydrogen peroxide are added, the molar extinction coefficient at the maximum absorption wavelength is 5.0×1. Produces a pigment of 5 to 6.0 x 1 pi.

On the other hand, when measured under the same conditions using phenol and 4-aminoantipyrine, the molar extinction coefficient at the maximum absorption wavelength is only about 5.0×1. Therefore, the method for quantifying hydrogen peroxide using the compound of the present invention can be said to be a very sensitive method. Furthermore, the compounds of the present invention have good solubility in buffer solutions of various pH levels, and there is also the advantage that there is no need to add surfactants or organic solvents, and reagent preparation is simple.

Now, the compound represented by the general formula (1) is the following general formula (m
) and (W) (in the formula R1, R2, R3, R4, R5, R
6 has the same meaning as the above general formula (1)) can be synthesized by reacting formalin with the compound represented by formula (1).

Specific examples of the compounds of the present invention represented by the general formula (1) synthesized in this manner are shown in Table 1, as well as their melting points, elemental analysis values, and the maximum of the oxidative condensation dye with M old TH at pH 7.11. Table 2 shows the absorption wavelength max (nm).
Table 2 (Continued) Now, when carrying out the quantitative determination according to the present invention, the pigment-forming substance of the present invention is mixed with
Use one containing a low coloring agent.

Examples of the former (i) include peroxidase and transition metals such as iron ions, and examples of the latter ('o) include MBTH and 4-aminoantipyrine. However, these are merely examples, and the present invention is not limited thereto. Next, examples will be given to explain more specifically the manufacturing method of the compounds of the present invention (Examples 1 to 6) and the quantitative method using the same (Example 7), but the present invention does not go beyond the gist thereof. However, the present invention is not limited to the following examples.

Example 1 Preparation of compound (1) Dissolve 10.3 parts of N-ethyl-N-(2-sulfoethyl)aniline sodium salt in 20 parts of water, add 1.78 parts of 37% formalin, and 0.20 parts of 90% formic acid. additional 350
While maintaining the temperature at 0, N-methylaniline is added and the reaction is carried out.

The obtained crystals were collected in a furnace and recrystallized from methanol to obtain 8.25 g of white di{4-[N-ethyl-N-(2-sulfoethyl)amino]phenyl}methane disodium salt. Yield 77.8% TLC (silica gel, n-butanol saturated with aqueous ammonia) Rf = 0.251 days - NMR
(d6-DMSO) 6 traces (TMS) 1.12 (t, J=
7.8HZ, bait) 2.44-2.79 (m, thin) 2
．． 83~3.41 (m, 4H) 3.64 (S, ga) 6
．． 32 (d, J=8.2HZ, 4H) 6.74 (d,
J=8.2HZ,'H)R(enemy-1)16081263
12.

0 Example 2 Preparation of compound legs 4.89 ml of N-(3-sulfopropyl)aniline sodium salt was dissolved in 10 parts of water and kept at 35q0 to give a concentration of 37%.
A reaction is carried out by adding 0.89% of formalin and 0.10% of 90% formic acid.

The obtained white crystals were collected in a furnace and recrystallized from a mixed solvent of acetone and water to obtain 4.23 g of di{4-[N-(3-sulfopropyl)amino/]phenyl}methane disodium salt. Yield 84.4% TLC (silica gel, n-butanol saturated with aqueous ammonia) Rf = 0.251H-NMR
(d6-DMSO) 6 Yanagi (TMS) 1.60-2.16
(m, 4H)2. Spot ~ 2.76 (m, 4H) 3.02 (
t, J=6,2HZ, Cape) 3.58 (S, Ga) 6.4
7 (d, J=8.2HZ, 4H) 6.90 (d, J=
8.2Hz, 4H) IR (skin-1) 332016101
28311691050 Example 3 Preparation of Compound D Dissolve 5.47 g of N-ethyl-N-(3-sulfopropyl)aniline sodium salt in 10 g of water, 0.89 g of 37% formalin, 0.89 g of 90% formic acid. .10 screams added to 35
N-methylaniline is added and the reaction is carried out while maintaining the temperature at .

The obtained white crystals were collected in a furnace and recrystallized with a THF-methanol mixed solvent to obtain 4.95% of di{4-[N-ethyl-N-(3-sulfopropyl)amino]phenyl}methane disodium salt. Approximate yield: 3% TLC (silica gel, n-butanol saturated with aqueous ammonia) Rf = 0.
301 days - NMR (d6-DMSO) 6 legs (TMS) 1
, 10 (t, J=7.7HZ, thin) 1.68~2.1
8 (m, 4H) 2.40~2-75 (m, mother H) 2
-81~3-30 (m, 4H) 3,52 (S, group)
6.43 (d, J = 8.2 days 2, rainbow) 6. Machine (d, J
=8.2 days 2, rainbow H) IR (skin-1) 34801602
126111781041 Example 4 Preparation of Compound (G) 3-Hydroxy-N-ethyl-N-(3-sulfopropyl)aniline sodium salt 11.59% dissolved in 20% water, 37% formalin 1.80%, 90% formic acid 0
．． Add 20' of N-methylaniline and allow to react while maintaining the slat.

The resulting precipitate was pulverized and crystallized while being decanted several times with acetone, and the resulting bluish-white crystals were collected in an oven and recrystallized with methanol to give white di{2-hydroxy-4-[N
-Ethyl-N-(3-sulfobropyl)ami/]phenyl}methane disodium salt 6.27 hours are obtained. Yield 5
3.0% TLC (silica gel, n-butanol saturated with aqueous ammonia) Rf = 0.201 days-NMR (d6-
DMSO) 6 legs (TMS) 1.18 (t, J=7.5H
Z, Thin) 1.72-2.29 (m, Cape) 2.36 (
m, thin) 3.08 (t, J: 6.3HZ, cape) 6,
25-6.98 (m, 4H) 7.38 (S, wide) m (
Arc-1) 3500160512 Iso 11751103 Example 5 Preparation of (compound) N-ethyl-N-(3-sulfopropyl) aniline sodium salt 5.46% dissolved in water 10%, 37% formalin 1.80%, 90% Add 0.20' of formic acid and 35℃
While maintaining the temperature, 4.89 g of sodium N-(3-sulfopropyl)aniline salt was dissolved in 1 portion of water and added dropwise to carry out the reaction.

The resulting blue-white crystals were collected in a furnace and recrystallized with methanol.
6.25 g of white 4'-{N'-ethyl-N-(3-sulfopropyl)amino}phenyl-4-{N-(3-sulfopropyl)amino}phenylmethane disodium salt was obtained. Yield 59.0% TLC (silica gel, n-butanol saturated with aqueous ammonia) Rf = 0.301 days -
NMR (d6-DMSO) hexapod (TMS) 1,12 (t
, J=7,6HZ, general) 1,59~2.16 (m, 4H
) 2.36-2.77 (m, thin) 2.80-3.2
5 (m, cape) 3,49 (S, wide) 6.28 to 6,5
6 (m, 4H) 6.60-7.02 (m, spots) IR (skin-1) 33801600129512011025 Example 6 Preparation of compound) 3-Methoxy-N-ethyl-N-(3-sulfopropyl)aniline sodium salt 6. Dissolve 100% of water in 10% of water, 1.80% of 37% formalin, 0.20% of 90% formic acid.
' of N-ethyl-N-(
3-Sulfopropyl) aniline sodium salt 5.47 g was dissolved in 10 g of water and added, and further N-methylaniline was added to carry out the reaction.

The precipitated white crystals were collected in a furnace and recrystallized from methanol to give 2-methoxy-4-{N-ethyl-N-(3-sulfopropyl)amino}phenyl-4-{N'-ethyl-N'-(3-sulfopropyl). ) phenylmethane disodium salt (7.25%) was obtained. Yield 61.5%T
LC (silica gel, n-butanol saturated with aqueous ammonia) Rf = 0.281 days-NMR (d6-DMSO)
6 traces (TMS) 1,10 (t, J: 7.7HZ, thin)
1.66-2.15 (m, cape) 2.39-2.78 (
m, mother H) 2.83-3,33 (m, cape) 3.55
(S, Ga) 4.11 (S, Group) 6.25-7.06
(m, 7H)m(伽-1)1608124412.

61100 and above described the method for producing the compound of the present invention.

Next, we will explain their application to the determination of peroxide substances. Before describing the examples of the quantitative method, we would like to explain i) the coloring reactivity of the coloring reagent using hydrogen peroxide-peroxidase.
We will explain the influence of spots on color development, 5) stability of color development over time, and 1) each item of the calibration curve using hydrogen peroxide standard solution. i Effect of pH on color development 20 peaks of 100% F-oxidase, 40.8% O and di{4-[N-ethyl-N-(3-sulfopropyl)amino] ) phenyl}methane disodium salt (compound D) string. 2 of 9
Dissolve and use as a coloring reagent.

About 5.5% of 30% hydrogen peroxide solution. The hydrogen peroxide standard solution is obtained by diluting the solution to hM concentration and titrating it with permanganic acid to accurately determine the concentration.

Hereinafter, this standard solution is diluted appropriately and used for experiments. test tube a
~i. Take reagents as shown below.

After soaking each test tube in a 370 temperature bath for 10 minutes,
Add 2.0 μl of 1N hydrochloric acid aqueous solution, and measure the absorbance on the armpit skin using a reagent-blind test as a control. Based on the obtained values, the relationship between pH and absorbance is shown in FIG. From Figure I, good coloring can be obtained in the neutral region. Moreover, it was found that the coloring sensitivity of other compounds A to C and E to Q was also maximized in the pH range of 7.0±0.5. This is shown in Table 3. ii. Stability of color development over time. 200 units of peroxidase, MBTH 40. & Yu and Ji {41
N-Ethyl-N-(3-sulfopropyl)amino]phenyl}methane disodium salt (Compound D) 54.2-9
Dissolve and use as a coloring reagent.

Add 20 μl of 2.8 hM hydrogen peroxide standard solution and 1.0 μl of coloring reagent to a test tube, soak it in a 3700 thermostat for 10 minutes, add 2.0 μl of 0.1 N hydrochloric acid aqueous solution, and blind the reagent. Using the test as a control, the change in absorbance over time at 59 m is measured. Figure 2 shows the results. Further, the results for other compounds B, 1, and M conducted under the same conditions are also shown in the same figure. Figure 2 shows that the resulting dye is stable for more than 1 hour.
In addition, other compounds, namely compounds A, C, E~day, J~L
Similar results were obtained for N to Q. iii
Creating a calibration curve using hydrogen peroxide standard solution Add i to test tubes a to f
Take the coloring reagent prepared in i) and the hydrogen peroxide standard solution as follows.

After soaking each test tube in a constant temperature bath of 37oo for 10 minutes,
．． Add 2.0% of 1N hydrochloric acid aqueous solution and use the reagent blind as a control.
Measure the absorbance at 98 nm.

FIG. 3 shows a calibration curve created from the obtained values. It can be seen from FIG. 3 that the calibration curve exhibits linearity in the concentration range of about 3 to 2 M. From the above results, it is clear that all the compounds of the present invention are effective as reagents for quantifying peroxide substances.

Specific examples of quantitative determination of four representative compounds are shown below. Of course, these are merely examples, and the present invention is not limited thereto. Example 7 Determination of glucose in serum using compound D
PH7 10 skin recose oxidase 20 units, peroxidase 200 units, M old TH40.8 evening, di{41 [N-ethyl N-
(3-Sulfopropyl)amino]phenyl}methane disodium salt (compound D) 54.2-9 is dissolved to prepare a coloring reagent.

i Creation of a calibration curve using a glucose standard solution A glucose standard solution is prepared by dissolving 200 o of glucose in 100M water, diluted as appropriate, and used in the experiment.

Place reagents in test tubes a to d as shown below.

After soaking each test tube in a 370 temperature bath for 10 minutes, 0.
Add 1N hydrochloric acid aqueous solution 2.0 m
Measure the absorbance at m. A calibration curve created from the obtained values is shown in FIG. ii. Determination of Glucose in Serum Take 20 pieces of deproteinized serum in a test tube, add the above coloring reagent 1.0, and soak it in a constant temperature bath at 37°C for 1 minute, then add 0.1N hydrochloric acid aqueous solution. 2.0' was added and the absorbance at 59 m was measured using a reagent blind test as a control, and compared with the calibration curve to determine the glucose concentration in the serum.

Absorbance 0.411 Glucose concentration 9/d' of 91 Example 8 Determination of glucose in serum using compound 1 di{4-[N
-ethyl-N-(3-sulfo-2-hydroxypropyl)amino]phenyl}methane disodium salt (compound 1
) and the same procedure as in Example 7 to measure the glucose concentration in serum.

Absorbance is measured at a measurement wavelength of 601 mm using a glucose standard solution, and a calibration curve is similarly created. The glucose concentration in the serum is determined from the obtained calibration curve (FIG. 5). Absorbance 0
．． 302 Glucose Concentration 92 Tano's Example 9 Determination of Glucose in Serum by Compound L' {4-[
N-ethyl-N-(3-sulfopropyl)amino]-3
-methylphenyl}methane disodium salt (compound L)
Glucose concentration in serum is measured using the same operation as in Example 7.

Absorbance is measured at a measurement wavelength of 534 nm using a glucose standard solution, and a calibration curve is similarly created. The glucose concentration in the serum is determined from the obtained calibration curve (FIG. 6). Absorbance 0
．． 459 Glucose concentration 9/d' of 90 Example 10 Determination of glucose in serum by compound P di{4-[N
Example using methane disodium salt (compound P) Glucose concentration in serum is measured by the same procedure as in 7.

Using a glucose standard solution, measure the absorbance at different measurement wavelengths and create a calibration curve in the same way. Obtained calibration curve (Figure 7)
Determine the glucose concentration in the serum. Absorbance 0.4
40 Glucose concentration 92mp/

[Brief explanation of the drawings] Figure 1... Effect of pH on color development of compounds, Figure 2...
- Stability of color development over time: However, ▲, 0, △, ● represent compounds B, D, 1, M, respectively, Figure 3... Calibration curve using hydrogen peroxide, Figure 4... Calibration of glucose using compounds Figure 5 shows the calibration curve for glucose using compound 1, Figure 6 shows the calibration curve for glucose using Ihi compound L, and Figure 7 shows the calibration curve for glucose using Ihi compound P, respectively. Box diagram 2 Figure 3 Figure 4 Box S Box diagram 7

Claims (1)

[Claims] 1 General formula ▲ Numerical formula, chemical formula, table, etc. R^2 and R^4 represent a sulfoalkyl group or a hydroxysulfoalkyl group having 2 to 3 carbon atoms;
^5, R^6 represent a substituent selected from the group consisting of a hydrogen atom, a methoxy group, a hydroxyl group, and a lower alkyl group) and salts thereof. 2 General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R^1 and R^3 are selected from the group consisting of a hydrogen atom, a lower alkyl group, and a sulfoalkyl group having 2 to 3 carbon atoms or a hydroxysulfoalkyl group. R^2 and R^4 represent a sulfoalkyl group or a hydroxysulfoalkyl group having 2 to 3 carbon atoms, and R
^5, R^6 represent a substituent selected from the group consisting of a hydrogen atom, a methoxy group, a hydroxyl group, and a lower alkyl group) and its salts as a pigment-forming substance. A composition for quantifying a peroxide substance.