JPS5930699B2 - Production method of new azobenzylamine compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing novel azobenzylamine compounds.

The azobenzylamine compound obtained by the method of the present invention can be an excellent substrate for measuring the activity of monoamine oxidase contained in biological samples, and provides a simpler and more accurate measurement method than the conventional complicated measurement method. It is possible.

In other words, the measurement of monoamine oxidase is an important clinical test tool for predicting liver cirrhosis, and the conventional method involves performing an enzymatic reaction using benzylamine as a substrate, extracting the generated benzaldehyde with a solvent, and measuring the ultraviolet absorption spectrum. or by reacting with a suitable aldehyde reagent,
The amount of benzaldehyde produced was known by colorimetry and the activity of monoamine oxidase was determined, but the reblank value increased due to autoxidation of benzylamine, and benzylamine had poor reactivity as a substrate.
Conventional methods for measuring monoamine oxidase are complicated and unsatisfactory, such as requiring a long time for the enzymatic reaction and problems with the addition of aldehyde detection reagents and the stability of color development.

The present inventors have developed a diazonium compound obtained from aminobenzylamine or an aminobenzylamine protected with a group that can easily remove the benzylamino group, and N-N
The present invention was completed based on the discovery that an azobenzylamine compound obtained by coupling with -dimethylaniline has high substrate compatibility with monoamine oxidase and can be a useful substrate for quantifying the enzyme.

That is, as raw materials for the diazonium compound according to the present invention, aminobenzylamine or aminobenzylamine protected with a group that can easily remove the benzylamino group, such as m- and p-aminobenzylamine, N-(
Aminobenzyl) phthalimide, tertiary-butoxycarboaminomethylaniline, trichloroacetaminomethylaniline, etc. can be effectively used as one of the reactive components, and N-N, which is the other reactive component, can be converted into a diazonium compound by a known method. -coupling with dimethylaniline.

If the benzylamino group is protected, the protecting group is removed according to a conventional method.

Using the novel azobenzylamine compound according to the present invention, the activity of monoamine oxidase can be measured, for example, by the following method.

That is, a substrate buffer is prepared by dissolving an azobenzylamine compound in an O,IM tris(hydroxymethyl)aminomethane-hydrochloric acid buffer. Add substrate buffer 2 to centrifuge tube.
, Oml was taken and heated at 37°C for 3 minutes, and the serum was 0.0ml.
Add 3 oz. and incubate for 2 hours in a constant temperature water bath at 37°C. Add 4 ml of cyclohexane to the reaction solution, and shake and extract with a pipe mixer for 30 seconds. 3000r. p. The activity of monoamine oxidase is determined by centrifuging at m for 10 minutes, removing the upper layer (extraction layer), and measuring the absorbance at an appropriately selected wavelength in the visible region of the azobenzaldehyde compound produced by the enzyme reaction. .

As described above, when the azobenzylamine compound obtained by the method of the present invention is used as a substrate, the reactivity as a substrate is higher than that of conventional products. Moreover, since the amount of biological samples can be measured in a small amount and only 10 volumes can be measured using the conventional method, the amount of biological samples to be collected here can be smaller than that of the patient, and therefore the pain to be caused to the patient can be reduced.

What should be noted here is that the azobenzaldehyde compound produced by the enzymatic reaction is completely removed from the acidic reaction solution using a water-insoluble organic solvent after the enzymatic reaction is completed and an acid is added to stop the enzymatic reaction. Because it has the characteristic color of azo compounds, it can be immediately measured in the visible region, and the operation is extremely simple compared to conventional methods, and it can be done using a colorimeter or a colorimeter that is normally used in a laboratory. It can be easily measured with a spectrophotometer.

As described above, the novel azobenzylamine compound according to the present invention can be an excellent substrate for monoamine oxidase that has never been seen before, and can be used for colorimetric determination in living organisms by simply measuring the solvent extraction and colorimetry. It is a useful compound in clinical diagnostics in that it can measure the activity of monoamine oxidase in a sample, and it has a great potential to contribute to clinical medicine.

An example is shown below.

Reference Example 5.07 ml of N-(3-aminobenzyl)phthalimide is dissolved in a solution of 6 y of hydrochloric acid and 300 ml of water, and a solution of 1.4 y of sodium nitrite dissolved in 50 ml of water is added while stirring while cooling.

After completing the diazotization, N-N-dimethylaniline 2
．． A solution consisting of 5 ml of 6y1 hydrochloric acid and 50 ml of water was added, and the mixture was stirred for 1 hour while adding an aqueous sodium acetate solution to keep the reaction liquid yellow-green. Finally, add a sodium carbonate aqueous solution to make the liquid alkali and filter it out to form yellow crystals of N.
-(3-(4-N-N-dimethylaminophenylazo)
7.47 (yield 97%) of benzyl)phthalimide is obtained. The crystals obtained above are heated under reflux together with dilute methanol and hydrazine hydroxide, and then hydrochloric acid is added and the mixture is heated under reflux.

After the reaction, the mixture is concentrated under reduced pressure, water is added, and phthalhydrazide is filtered off. The filtrate was adjusted to pH 2 with an aqueous sodium carbonate solution, and then the soluble components were extracted and removed with cyclohexane. The separated aqueous layer is brought to a pH of 4 or higher with an aqueous sodium carbonate solution, and extracted and separated with chloroform. The chloroform layer is dehydrated with anhydrous sodium sulfate and then concentrated under reduced pressure. Dissolve the concentrated residue in acetone, add hydrochloric acid, and dry under reduced pressure. Mpl44~145℃(d
) of 4-(3'-aminomethylphenylazo)-N-N-dimethylaniline dihydrochloride 3.5y (
yield of 57%). Elemental analysis: Cl5Hl8N4・2
HC1 Example 1 26v of N-(4-aminobenzyl)phthalimide is suspended in a solution of 185ml of ethanol and 7.57ml of 90% hydrazine hydrate, and heated to reflux with stirring.

After 3 hours, 30 ml of hydrochloric acid was added and the mixture was further heated under reflux for 1.5 hours.

The reaction mixture is cooled to room temperature and the precipitated phthalhydrazide is stripped off. After concentrating the filtrate to dryness under reduced pressure, add 150ml of water.
Dissolve and decolorize using activated carbon. The f solution was again concentrated to dryness and dried under heat to obtain 18.2y of slightly yellow 4-aminobenzylamine [raw tuna salt] (yield: 91%). Mp
3OO℃ (d) or more. Accurately weigh 0.27 of this product and 500 ml of pure water.
ml and titrated with N/10 sodium monohydroxide normal solution, the pH was 6.6 and PHLO. The neutralization inflection point was found at 8, and the content was determined to be 101.0%.

Although this product can be recrystallized with a small amount of water, it can be used in the next diazo coupling reaction without purification. 20 ml of hydrochloric acid and 100 ml of water were added to 10.07 g of the 4-aminobenzylamine salt obtained in this way, and dissolved therein, and then 100 y of ice was added and cooled with stirring.

Keep the internal temperature at 0°C and add 3.7y of sodium nitrite to 20ml of water.
A solution dissolved in m1 is added to complete diazotization. A solution consisting of 6.47 mL of N-N-dimethylaniline, 10 mL of hydrochloric acid, and 100 mL of water is added to the obtained diazonium salt solution, and the mixture is stirred for 1 hour while adding an aqueous sodium acetate solution to maintain the yellow-green color of the reaction solution. Finally, an aqueous solution of sodium carbonate is added to make the mixture alkaline, and the mixture is washed with water to obtain 11.67 yellow crystals of 4(4'-aminomethylphenylazo)-N-N dimethylaniline. Dissolve this crystal in 50 ml of acetone, add 10 ml of hydrochloric acid, and concentrate under reduced pressure to obtain a blue-purple 4-
(4'-aminomethylphenylazo)N-N-dimethylaniline dihydrochloride 12.27 (yield 71%) is obtained. Elemental analysis Cl5Hl8N4.2HC1 Example 2 3-Aminobenzylamine dihydrochloride was obtained by the same reaction as in Example 1 except that N-(3-aminobenzyl)phthalimide was used as a raw material, and then the same reaction as in Example 1 was carried out. 4-(3'-Aminomethylphenylazo)-N.N-dimethylaniline dihydrochloride was obtained by a diazo coupling reaction.

Claims (1)

[Claims] 1. 4-(aminomethylphenylazo)-N/N-dimethylaniline, a novel azobenzylamine compound characterized by coupling a diazonium compound obtained from aminobenzylamine with N/N-dimethylaniline. Production method.