JPS63190863A - Gamma-l-glutamyl-p-aminoanilide derivative and measuring method of gamma-gtp by use thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (A is lower alkylene; R<1> and R<2> are lower alkyls). EXAMPLE:gamma-L-glutamyl-o-methyl-p-(N-ethyl-N-beta-hydroxyethylamino) anilide. USE:A substrate for measuring gamma-GTP active value. The compound has high reactivity of gamma-GTP as a substrate and sufficient solubility and can be measured in a measuring wavelength having longer wavelength area than 410nm. PREPARATION:N-Protected-gamma-L-glutamic anhydride is reacted with a p- phenylenediamine derivative expressed by formula II in a solvent such as chloroform, etc., as necessary in the presence of an acid at 20-100 deg.C and a protecting group such as phthalyl group, etc., is removed from the reaction product to provide the compound expressed by formula I.

Description

[Detailed Description of the Invention] [Industrial Field] The present invention relates to T-glutamyltransferase (hereinafter referred to as
The present invention relates to a method for measuring γ-GTP).
- γ-GTP is a biological enzyme that is an important indicator in liver function tests, and r-ic'rp activity measurement is an extremely frequently measured measurement item.

[Prior art and its drawbacks]

Conventionally, r-GTP activity was measured using γ-L- as a substrate.
Glutamyl p-nitroanilide has been used. However, this substrate is extremely difficult to dissolve in a buffer for carrying out an enzyme reaction, and therefore requires the use of a solubilizing agent. In addition, since the amount of p-nitroaniline produced by the enzymatic reaction is measured by absorbance at a wavelength of 410n+w,
It has the disadvantage that the influence of bilirupin, a substance that exists in serum components and has absorption in the same wavelength range, is unavoidable.

[Purpose of the invention]

In view of these drawbacks, the present inventors conducted intensive research and found that γ-L
-glutamyl-p-aminoanilide derivative CI) or a salt thereof, which eliminates all the above-mentioned drawbacks and which can be used as a substrate for r-
We have found a material that has extremely remarkable effects, such as high GTP reactivity and sufficient solubility, and the ability to measure at wavelengths longer than 410 nm.

[Structure of the invention]

The present invention is based on the general formula (where A represents a lower alkylene group, R1 and R
Each z represents a lower alkyl group.
-L-glutamyl-p-aminoanilide derivative or a salt thereof and a method for measuring γ-〇TPO, which is characterized by using the same as a substrate.

Examples of the γ-L-glutamyl-p-aminoanilide derivative CI) or a salt thereof of the present invention include r-L-glutamyl-p-methyl-p-(N-ethyl-N-hydroxyethylamino)anilide, γ-L- Examples include glutamyl-ethiethi p-(N-ethyl-N-hydroxypropylamino)anilide or salts thereof.

The γ-L-glutamyl-p-aminoanilide derivative (1) or a salt thereof of the present invention can usually be easily produced, for example, by the following method.

That is, for example, N-protected-γ-L-glutamic anhydride such as N-phthalyl-γ-L-glutamic anhydride and the general formula (wherein A represents a lower alkylene group, R1 and R
Each z represents a lower alkyl group. ) denoted by p
- reacting with phenylenediamine derivative CI+),
An N-protected-γ-L-glutamyl-p-aminoanilide derivative such as an N-phthalyl-γ-L-glutamyl-p-aminoanilide derivative is obtained, and then a protective group such as a phthalyl group is removed by a method known per se, for example, a conventional method. γ- of the present invention
An L-glutamyl-p-aminoanilide derivative (r) or a salt thereof is obtained.

Examples of the lower alkylene group according to the present invention include lower alkylene groups having 1 to 5 carbon atoms such as methylene group, ethylene group, and propylene group, and examples of the lower alkyl group include methyl group, ethyl group, propyl group, etc. Examples include lower alkyl groups having 1 to 5 carbon atoms. R1 is preferably a methyl group or an ethyl group.

In the first reaction, it is preferable to use the N-protected-γ-L-glutamic anhydride in an equivalent amount or in slightly excess amount relative to the p-phenylenediamine derivative C11), and if necessary, use a solvent such as chloroform, dichloroethane, etc. , if necessary, in the presence of an organic acid such as acetic acid, and the reaction is usually carried out at 20-100'C. After completion of the reaction, the N-protected-r-L-glutamyl-p-aminoanilide derivative is separated according to a conventional method. , then, for example, hydrazine is reacted, optionally in the presence of a solvent. Hydrazine may be used as it is, as a hydrate, or as a salt thereof, and it is usually convenient to use the hydrate. This reaction usually uses water or a solvent, but any solvent may be used as long as it does not affect the reaction. For example, water-soluble solvents such as alcohols such as methyl alcohol and ethyl alcohol, and ethers such as tetrahydrofuran and dioxane can be used. preferable.

The subsequent reaction is carried out using an equivalent or slightly excess amount of hydrazine to the N-protected-γ-L-glutamyl-p-aminoanilide derivative at 60°C or lower, preferably 10 to 30°C. After the reaction, the desired product is prepared according to a conventional method. The desired salt can be obtained by isolating and, if necessary, neutralizing with acid or alkali.

The novel γ-L-glutamyl-p-aminoanilide derivative (1) or a salt thereof of the present invention is used as a substrate for measuring r-GTP activity, and is usually at an optimum pH of 8.0 to 8.0.
r-L-glutamyl-p, which has been widely used as a substrate for conventional γ-GTP activity measurement.
-Nitroanilide has extremely low solubility in the above weakly alkaline pH range, for example 0.05M at pH 8.0.
To make a solution of 0.01 to 0,015M, which is the required amount of substrate, by simply dissolving 0.09% (0,003M) in borate buffer, dissolve it as a hydrochloride, then dilute with buffer.
Either it is necessary to maintain the required temperature as a supersaturated solution during the measurement time, or it is necessary to assist dissolution with a surfactant, etc., and in either case, it is not satisfactory.

However, the γ-L-glutamyl-p-aminoanilide derivative (1) or a salt thereof, which is the novel substrate of the present invention, has excellent solubility. For example, under the above r-cTp activity measurement conditions, γ-L-glutamyl-methymethyp-(N
-Ethyl-1N-hydroxyethylamino)anilide can be made into an 8.7% (0.28M) solution and the required temperature can be easily met.

As an example, the solubility of γ-L-glutamyl-p-(N,N-diethylamino)anilide is 1 under the same conditions as above.
．． 3% (0,045M), which shows how excellent the solubility of the γ-L-glutamyl-p-aminoanilide derivative (1) or its salt of the present invention is.

In addition, it has excellent solubility during industrial production, so
In producing a reagent formulation, the γ-L-glutamyl-p-aminoanilide derivative (1) or its salt of the present invention can be handled in a concentrated state; for example, in producing a lyophilized formulation, it can be lyophilized from a concentrated solution. Therefore, the amount of evaporated water is small, which is extremely advantageous from the viewpoint of energy saving.

The novel γ-L-glutamyl-p-aminoanilide derivative (1) or a salt thereof of the present invention has excellent substrate reactivity with γ-GTP.

One example is the γ-L-glutamyl p-
The substrate affinity (substrate reactivity) for r-GTP of the aminoanilide derivative CI) or its salt is different from that of γ-L-glutamyl-p-nitroanilide and γ-L-glutamyl-p-nitroanilide, which have been widely used in conventional amounts.
-L-glutamyl-p-(N,N-dimethylamino)
Table 1 shows some examples of its superior properties compared to anilide, T-L-glutamyl p-(N,N-diethylamino)anilide, and the like.

Table 1 Substrate reactivity The r-L-glutamyl-p-aminoanilide derivative (1) of the present invention or a salt thereof is used as a substrate to be freely produced by enzymatic reaction of γ-GTP to measure the γ-GTP activity value. To quantify the p-phenylenediamine derivative (I[), conventionally known methods of colorimetrically fixing aromatic amines, such as the diazo cuff-pulling method or the method of quantifying the Schiff base produced by reacting with aldehydes, can be used. Although applicable, the method of quantifying colored compounds produced by oxidation is the most preferred method in terms of operational simplicity, rapidity, and accuracy of measured values. That is, p-phenylenediamine derivatives ([3] can be directly oxidized to produce a colored compound or oxidatively condensed to produce a colored compound, such as phenol, m-acetylaminophenol, p-chlorophenol, butyl hydroxy Phenols such as anisole, 1-naphthol-2-sulfonic acid, 4-chloro-1-naphthol-2-sulfonic acid, 8-
It is oxidized with metaperiodate, hydrogen peroxide and an oxidizing agent such as peroxidase or red blood salt in the presence of naphthols such as oxyquinoline to form a colored compound, which is then subjected to colorimetric determination.

Phenols that can be used in addition to the above include, for example, 2,4-dichloro-3-methylphenol, 2-chloro-3-methyl-4-methoxycarbonylmethoxyphenol, and 2-chloro-3-methyl-4-carboxymethoxyphenol. Phenol, 2゜4-dichloro-3-methyl-6-benzamidophenol, 2,4-dibromo-3-methylphenol, 2-propionamidophenol, 2-propionamido-5-methylphenol, 2-phenyldichloromethyl- 4-chlorophenol, 2-methyl-
4-methanesulfonylamidophenol, 2-hydroxybenzamide, 2-acetylaminophenol, 2.
5-dichlorophenol, orthocresol, metacresol, 2-chlorophenol, 3-chlorophenol, 2-methoxyphenol, (2-23)3-methoxyphenol, 3-acetylamino-5-methylphenol, 1-hydroxy- N-methyl-2-naphthamide,
1-Hydroxy-N-phenyl-2-naphthamide.

A preferred example of naphthols that can be used other than the naphthols mentioned above is 1-hydroxy-2-naphthamide.

The γ-L-glutamyl-p-aminoanilide derivative (1) or a salt thereof of the present invention has excellent reactivity, solubility, etc. as a substrate for measuring γ-GTP activity value, and is colorimetrically capable of accurate and rapid measurement. It can be easily applied to automatic analyzers due to its simplicity.

The present invention will be further explained with reference to Examples below.

Example I 28.4 g of N-fucryl monoglutamic anhydride and 4
-amino-3-methyl-N-(β-hydroxyethyl)
-19.4 g of N-ethylaniline and 100 g of dioxane
- dissolved in triethylamine 15.2. It! Add. Reflux on oil bath for 2 hours. After that, it was concentrated and the residue was mixed with 40J of methanol! Dissolve by heating. Foam water hydrazine 4
0. Add e and leave it to solidify. Stir well and filter with suction. Wash thoroughly with methanol and dry. After extraction with 2N hydrochloric acid and neutralization with an aqueous sodium carbonate solution, the target product is precipitated. After filtering and washing with water, wash with methanol and dry. Purification was repeated by dissolving in hydrochloric acid and neutralizing with an aqueous sodium carbonate solution to obtain 11 g of r-L-glutamyl-〇-methyl-p-(N-ethyl-N-β-hydroxyethylamino)anilide.

Melting point 171-178°C Example 2 28.4 g of N-phthalyl-di-glutamic anhydride and 4-amino-3-methyl-N-hydroxypropyl-N
- Dissolve 20.3 g of ethylaniline in 100.e of dioxane and add 15.2 g of triethylamine. Reflux on oil bath for 2 hours. Thereafter, it is concentrated and the residue is heated and dissolved in 40 methanol. Add 40J of bubble water hydrazine and leave it to solidify. Stir and suction filtrate. Wash thoroughly with methanol and dry. Extract with 2N hydrochloric acid and neutralize with aqueous sodium carbonate to precipitate the target product. Filter, wash with water, then methanol and dry. Purification is as follows: 12 g of r-L-glutamyl-〇-methyl-p-(N-ethyl-N-hydroxypropylamino)anilide by dissolving it in hydrochloric acid and neutralizing it with an aqueous sodium carbonate solution.
was gotten.

Melting point 167-175°C Example 3 (Analysis example) A Reagent (1) Substrate buffer: γ-L-glutamyl-methymethyp-(N-ethyl-N-hydroxyethylamino)anilide 12-/i! ,,Glycylglycine 50mM/j
! , 1-naphthol-2-sulfonic acid 0.2mM/l,
0 at pH 83, containing ethylene glycol, wt%
．． 05M#! Prepare borate buffer.

(2) Oxidizing reagent: A 0.3M #borate buffer solution containing 0.5% potassium ferricyanide and having a pH of 8.3 was prepared.

B Measurement procedure 0.02J of serum sample to 1.0J of substrate buffer! After adding and mixing well, heat in a constant temperature bath at 37°C for 15 minutes.
Then, oxidation test solution 20- was added. Water 0.0 instead of sample
The absorbance at 650 nm was measured using a reagent-blind sample obtained by performing the same procedure as the sample using 2000 as a control.

The γ-GTP activity value was calculated by comparing with a calibration curve prepared in advance according to a conventional method using N-ethyl-N-hydroxyethylamino-3-methyl-4-aminoaniline.

Example 4 (Analysis Example 2) γ-L-glutamyl-0-methyl-p- was used instead of γ-L-glutamyl-0-methymethyp-(N-ethyl-N-hydroxyethylamino)anilide in Analysis Example 1 as a substrate.
Using (N-ethyl-N-hydroxypropylamino)anilide, the absorbance at 650 nm was measured according to Use Example 1, and the r-GTP activity value was calculated.

Patent applicant: Fuji Film Co., Ltd. 1, Indication of the case: Poetry Application No. 22゜6j of 1939, 6th party making the amendment: po “J!7F! Relationship with the case: Patent applicant: 4, Subject of amendment: Description of the specification 5. Contents of amendment to "Detailed Description of the Invention" The description of the detailed description of the invention is amended as shown in the attached sheet.

1) Change “, weight %” on page 14, line 12 of the specification to “1”
Correct to ``% by weight''.

2) Change rpH83J on page 14, line 13 to 'PH8
, 3J.

3) Change “L-guritami” in line 10 of page 15 to “L
-Corrected to glutami.

Claims (2)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [ I ] (In the formula, A represents a lower alkylene group, and R^1 and R^2 each represent a lower alkyl group.) γ -L-glutamyl-p-aminoanilide derivative or salt thereof (2) General formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, A represents a lower alkylene group, and R^1 and R^2 each represent a lower alkyl group.) A method for measuring γ-glutamyltransferase, which comprises using the shown γ-L-glutamyl-p-aminoanilide derivative or a salt thereof as a substrate.