JPS5930698B2 - 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. Alternatively, the amount of benzaldehyde produced was known by reacting with an appropriate aldehyde reagent and colorimetrically determined, and the activity of monoamine oxidase was determined, but the reblank value increased due to autoxidation of benzylamine.
Conventional methods for measuring monoamine oxidase are complicated and unsatisfactory, such as benzylamine having poor reactivity as a substrate, requiring a long time for enzymatic reaction, and problems with the addition of aldehyde detection reagents and the stability of color development. It was hot.

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.
．． After heating 0ml at 37℃ for 3 minutes, remove serum 0,
Add 3ml and incubate for 2 hours in a constant temperature water bath at 37°C. Add 4d 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.

Examples are shown below.

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

After completing the diazotization, N-N-dimethylaniline 2
．． A solution consisting of 6y, 5 ml of hydrochloric acid, and 50 ml of water was added, and the mixture was stirred for 1 hour while adding an aqueous sodium acetate solution so that the reaction solution remained 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)
Benzyl)phthalimide 74f (yield 97%) 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.

Claims (1)

[Claims] 1. Obtained by a known method of dissolving aminobenzylamine protected with a group that can easily remove the benzylamino group, that is, a phthaloyl group, or suspending it in the form of a salt in a dilute inorganic acid, and adding sodium nitrite and reacting. 4-(aminomethylphenylazo)-N・N- is a novel azobenzylamine compound characterized by coupling a diazonium compound and N・N-dimethylaniline.
Method for producing dimethylaniline.