JPS6117550A - Novel synthetic substrate - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula I (R is L-leucyl, or gamma- glutemyl; X and Y are halogen) or its salt. EXAMPLE:L-Leucyl-3,5-dichloro-4-hydroxyanilide. USE:A substitute for measuring activity of peptidase such as LAP, gamma-GTP, etc. An aniline derivative shown by the formula II which is isolated by the action of the peptidase contained in a test specimen on a compound shown by the formula I or its salt is made into a color compound, and determined by colorimetry. PREPARATION:The carboxyl group of L-leucine or gamma-carboxyl group of L- glutamic acid and an aniline derivative shown by the formula II are subjected to condensation reaction in peptide synthesis to give a compound shown by the formula I. The condensationd reaction, for example, can be carried out in the presencr of a condensation agent such as DCC, etc. by using L-leucine or L- glutamic acid having protected alpha-amino group and protected alpha-carboxyl group.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an amide compound or a salt thereof represented by the formula (wherein, 几 is an L-leucyl group or a Dr-L-glutamyl group, and X and Y are the same or different, or a halogen atom).

Peptidases have long been known as a general term for enzymes that act on the peptide bonds of peptides and cleave them from the amino terminus to release amino acids or lower peptides. For example, aminopeptidases (hereinafter True B and C11nicatLA
Cystine aminopeptidase, proline aminopeptidase, arginine aminopeptidase 1 Alanine aminopeptidase, γ-glutamyl transpeptidase (γ-GTP), etc. are known. Peptidase (I
AP) and γ-glutamyl transpeptidase (γ-
GTP) is widely distributed in all tissues in the body, is also present in serum, and is known to increase under pathological conditions, making it an important item for measuring enzyme activity in clinical tests. It is an enzyme that exists.

LAP is an enzyme that hydrolyzes L-peptides, particularly L-peptides whose amine terminus is L-leucine or a related amino acid, to liberate L-leucine by hydrolyzing the amine-terminal residue having a free amine group. It is widely distributed in various tissues in the body and is also present in serum, and it is known that the amount of enzyme in serum varies significantly depending on the state of the body.
-2 activity increases in acute hepatitis, liver cancer, metastatic liver cancer, liver cirrhosis, biliary tract disease, etc. One of the diseases that is active there
Measuring the activity of this enzyme is one of the indispensable test methods for diagnosing these diseases.

γ-GTP is an enzyme involved in the metabolism of γ-glutamiρ peptide in vivo, and is an enzyme that hydrolyzes γ-glutar peptide and transfers the γ-glutamyl group to other peptides or amino acids, It is widely distributed in various tissues in the body and is also present in serum. r-GTP is known to fluctuate markedly depending on pathological conditions;
-GTP shows extremely high levels in hepatostatic hepatitis, cryptogenic jaundo, primary liver cancer, metastatic liver cancer, etc. In particular, it shows low values in inactive forms of chronic hepatitis, but high values in active forms. The measurement of serum γ-GTP activity is generally specific to chronic liver diseases, and has a completely different diagnostic significance from the various deviant enzymes that have been used clinically. It has become one of the indispensable testing methods for diagnosing various diseases and understanding their pathological conditions.

A number of methods have been reported for measuring Shihi activity, but most of them are methods for determining the Ruhi activity value by colorimetrically quantifying amine compounds liberated by synthetic substrates. As a synthetic substrate for this purpose, L-leuci/1
One method is to colorimetrically quantify the yellow color of p-nitroanilide produced by the enzymatic action of p-nitroanilide using z-p-nitroanilide. There was a drawback that the two components overlapped, and the measurement was unavoidable due to the effects of serum components, especially bilirubin pigments. Furthermore, L-
One example is a method using leucyl-β-naphthylamide, but in this method, 5-
A dye is formed by coupling ditro-2-aminomethoxybenzenediazotate or the like, or the resulting β-naphthylamine is diazotized with sodium nitrite to form N-(1-su7thi/I/)-ethylene cyanide. This method involves coupling or condensation of p-dimethylaminobenzaldehyde or p-dimethylaminocinnamaldehyde to form a dye, which is then colorimetrically determined, but the reaction process is complicated and requires strict operations. The standard substance, β-7tylamine, is extremely toxic and has recently been shown to cause bladder tumors and cancer, so it has been classified as a carcinogen. It had drawbacks such as requiring particularly strict caution in its use.

On the other hand, many methods for measuring γ-GTP activity have been reported, but most of them are based on synthetic substrates. A method for determining the yellow color of p-nitroaniline produced by the action of a γ-GTPO enzyme using r-L-glutamyl-p-nitroanilide as a synthetic substrate at 410 nm. However, in order to avoid the influence of serum components, especially bilirubin pigments, sample blanks must be precisely measured for each sample, making it difficult to obtain accurate measured values. there were. In addition, p-nitroaniline produced is converted into p-dimethylaminocinnamaldehyde,
Another method is to condense it with an aldehyde compound such as p-dimethylaminobenzaldehyde and measure the red color on the longer wavelength side, but this method has a large effect on temperature on color development sensitivity and has problems in reproducibility of measured values. In addition, a method was proposed in which the resulting p-nitroaniline was diazotized and condensed with 3,5-xylenol to measure the resulting red color, but this method had the drawback of requiring many reaction steps and lacking in simplicity. Examples of these methods include methods using γ-L-glutamyl-β-naphthylamide;
- A method for colorimetric determination of β-naphthylamine liberated by GTP as a diazonium salt, a method for colorimetric determination by developing color with 3-methyl-2-benzothiazolinone hydrazone and an oxidizing agent, or a method for colorimetric determination using the aldehyde compound as a color former. There is a method of condensation and colorimetric determination, but as above, β
-Naphthylamide 79 It has been generally pointed out that it is carcinogenic, and it lacks practicality in that it is complicated to operate and requires a long time for measurement.

The present inventors have investigated conventional stores and γ-GT having such drawbacks.
As a result of intensive research to improve the P activity measurement method, II.
The present invention was completed by discovering a new synthetic substrate that is excellent in measuring AP and γ-GTP activities and has psychotropic properties.

The present invention is a novel amide compound mt or a salt thereof as a synthetic substrate for peptidase.

The amide compound III) used as a synthetic substrate for peptidase in the present invention can be produced by a conventional method for peptide synthesis. That is, the product is obtained by a condensation reaction between the carboxyl group of L-leucine or the γ-cassiboxyl group of L-glutamic acid and aniline [1-body [:U] (wherein, X and Y have the same meanings as above). It will be done.

In the above condensation reaction, it is preferable to protect in advance functional groups that should not participate in the reaction, ie, the α-amino group of L-leucine, the α-7 amino group of L-glutamic acid, and the α-carboxyl group. As the protecting group for the α-amino group, an α-amino protecting group commonly used in peptide synthesis is used. Examples include t-butyloxycarbonyl, t-amyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, adamanemethyloxycarbonyl, p-methoxybenzyloxycarbonyl, phthaloyl, O-nitrophenylthio group, and the like. It is preferable to protect the α-carboxyl group of L-glutamic acid with methyl ester, ethyl ester, t-butyl ester, benzyl ester, p-nitrobenzyl ester, p-methoxybenzyl ester, etc.; It is particularly preferred to protect with a protecting group that can be removed in one step together with the protecting group for the amino group. For example, an α-amine group is protected with a benzyl oxycarbonyl group, and an α-carboxyl group is protected with a benzyl ester.

Examples of the aniline derivative Cl0) used in the above condensation reaction include 3,5-dibromo-4-hydroxyaniline, 3
．． Examples include 5-dichloro-4-hydroxyaniline, 3-riμ-5-promo-4-hydroxyaniline, and the like.

In the above condensation reaction, the carboxyl group of L-leucine with a protected α-amine group or the r-carboxyl group of 7tL-glutamic acid with a protected α-amino group and α-carboxyl group is converted into an acid salt, an acid anhydride, and the like. , acid azides, acid imidazolides, activated esters such as activated acyl derivatives such as cyan methyl ester, p-nitrophenylnisf+, 2°4-dinitropher ester, N-hydroxysuccinimide ester, N-hydroxy 7-thallimide ester, etc. It can be converted and reacted with an aniline derivative [■], or it can be reacted with a carbodiimide, such as Watersorp carbodiimide hydrochloride [1-ethyl-3-(3
-dimethylaminopropyl)carbodiimide hydrochloride, N
, N'-dicyclohexylcarbodiimide, N, N'-
This is carried out by reacting the above-mentioned protected L-leucine or L-glutamic acid with the aniline derivative (II) in the presence of a condensing agent such as carbonylimidazole, inoxacillium salt, or Woodward's reagent.

In the above condensation reaction, an inert organic solvent is usually used, such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrof2/, dioxane,
This is carried out by adding approximately equal amounts of both in a solvent such as benzene, chloroform, dichloromethane, dichloroethane, etc., and reacting at room temperature or lower temperature. The above reaction process can be traced using thin layer chromatography (such as silica gel).
TLC), high performance liquid chromatography (HPLC)
Since the reaction can be traced using methods such as the following, the reaction can be appropriately terminated after waiting for the disappearance of any of the starting materials.

The reaction product obtained in this way can be treated with a non-hydrophilic organic solvent, with or without distilling off the reaction solvent.
For example, it can be collected by dissolving it in chloroform, dichloromethane, ethyl acetate, butyl acetate, methyl in butyl ketone, benzene, diethyl ether, etc., washing with acidic water and alkaline water, and then distilling off the solvent. If further purification is required, it can be purified by recrystallization with a suitable recrystallization solvent or by column chromatography using an adsorbent such as silica gel, activated alumina, or adsorption resin.

The protecting group of the resulting reaction product is then removed, and this removal is carried out using a protective group removal method in peptide chemistry. For example, when the α-7 amino protecting group is a t-butyloxycarbonyl group, a method using a 2N hydrogen chloride acetic acid solution, trifluoroacetic acid, formic acid, etc. This may be carried out by a method using a catalytic reduction using a carbon catalyst or a method using an acetic acid solution of hydrobromic acid. When the protecting group for the α-carboxyl group of L-glutamic acid is a benzyl ester, catalytic reduction using a palladium-carbon catalyst may be used.

In order to collect the amide compound thus obtained from the t-reaction solution, first neutralize it if the protecting group is removed by acid decomposition, or remove the catalyst if it is done by catalytic reduction. After washing with acidic water and alkaline water, the solvent was washed with acidic water and alkaline water in a solvent such as loloform, dichloromethane, dichloroethane, ethyl acetate, butyl acetate, methyl isobutyl ketone, diethyl ether, etc. It can be collected by distillation. If further purification is required, recrystallization with a suitable recrystallization solvent or silica gel, activated alumina, adsorption resin, etc.
It can be purified by column chromatography using an adsorbent such as

This amide compound (1) may be used as a salt with an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, or phosphoric acid, or with an organic acid such as formic acid, acetic acid, propionic acid, malic acid, citric acid, tartaric acid, or oxalic acid. Able to form salts Next, a method for measuring peptidase activity using the present amide compound CI) as a reference will be described. In this measurement method, the above-mentioned amide compound (1) or its salt is combined with peptidase contained in the test solution, IIAPt or γ-
A = IJ derivative (I) released by the action of GTP
In order to quantify f), a chemical coloring method is used to derive a coloring compound and the colorimetric determination is carried out.

The above chemical coloring method involves oxidative condensation with an aniline derivative l) in the coexistence of Kabutsu to form a coloring compound.

It is preferable to carry out colorimetric determination. The Kabutu to be used may be any aromatic compound as long as it forms a color-forming compound through oxidative condensation with the aniline derivative CI[], but among these, phenolic compounds are preferred as diagonal aromatic compounds. , aminophenol compounds, aniline compounds, and naphthol compounds. Examples of phenol ρ-based compounds include phenol,
Salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, 2.6-dihydroxybenzoic acid, methyl salicylate, o-cresol, m-freseau/', p-cresol, 0-ethylphenol, m-ethylphenol, 2 .3-xylenol, 2.4-xylenol, 2
．． 5-xylenol, 3,5-xylenol, 2.6-
xylenol, O-methoxyphenol, m-methoxyphenol, p-methoxyphenol, 2.6-simethoxyphenol, 0-chlorophenol, m-chlorophenol, p-chlorophenol, 2.4-') chlorophene/-/ l/, 2. s-dichlorophenol, O
-7” romophenol, m-bromophenol, p-7
"Romophenol, 2,4-dibromophenol, 2.
6-dibromophenol, 2-methyl-6-chlorophenol, 2-chloro-5-j'f-phenol, 〇-carboxymethylphenol, 2-hydroxy-4-aminoethylphenol, etc. are listed, and aminophenol-based Examples of compounds include 4-chloro-2-aminophenol, N,N'-diethyl-m-aminephenol, 4-chloro-2-aminophenol,
-Methyl-2-aminophenol, 5-amino-2-hydroxybenzoic acid, 2-amino-3-hydroxybenzoic acid, 0-7 minophenol, m-aminephenol, p
Examples of aniline compounds include aniline, 0-toluidine, m-toluidine, p-toluidine, N-methylaniline, N-ethylaniline, N,N-dimethylaniline, N, N-diethylaniline, N,N-dimethyl-o-)luidine, N
, N-dimethyl-p-)luidine, N,N-diethe/I
/-0-)luidine, NsN-diethyl-m-)luidine, N,N-diethyl-p-)luidine, 0-chloro7niline, m-chloroaniline, m-bromoaniline,
Anthranilic acid, 3-aminobenzoic acid, p-dimethylaminobenzoic acid, p-chloro-0-toluidi/, 3-amino-4-methylbenzoic acid, m-phenylenediamiy1N
, N-dimethy/l/-Ill-phenylenediamine, 2
-Methyl-m-phenylenediamine, 4-methyl-〇-
Phenylenediamine, 4-methyl-m-phenylenediamine, 2-chloro-m-phenylenediamine, 3-chloroo-0-toluidine, 2-methoxy-5-chloroaniline, 0-ethylaniline, 2.5-jethoxy Aniline, N-ethyl-N-hydroxyethylaniline SN-ethyl-N-hydroxyethyl A/-m-) Louisinna E-
Examples of naphthol compounds include α-naphthol, β-su7tol, 2-carboxy-1-naphthol, 4-chloro-1-su7tol, 1-hydroxy-2-naphthoic acid 1.1 -Naphthol-2-sulfonic acid, 1-naphthol-3-sulfonic acid, 1-naphthol-
Examples include 4-sulfonic acid, 2-naphtho-)v-5-sulfonic acid, and 2-naphthol-3,6-disulfonic acid.The above oxidative condensation is carried out at a pH within the range of about 4 to 12. By doing so, the color reaction can be completed quantitatively.
To maintain the above pH, use a buffer or As the alkaline aqueous solution, carbonate buffer, phosphate buffer, borate buffer, alkali hydroxide bath solution, etc. can be effectively used.

The above oxidative condensation is carried out in the presence of an oxidizing agent capable of oxidative condensation of the aniline derivative [■] and the coupler. Examples of such oxidizing agents include halogen-based oxidizing agents such as periodate, chloramine T, and pan chlorate, peroxide-based oxidizing agents such as persulfate, hydrogen peroxide, etc. Sodium periodate is a suitable example.

The color-forming compound produced by the oxidative condensation of the aniline derivative Cl0) and the above coupler has a maximum absorption wavelength widely distributed from about 550 to 770 nm depending on the type of Kab2-.
Usually, it is a colored dye that has maximum absorption in the range of 570 to 680 nm, and its color development is extremely sensitive and stable.
LAP + r
- Gives extremely good results in measuring peptidase activity such as GTP.

Another method for color-quantifying the above-mentioned aniline derivative (If) is a method in which a pentacyanoiron complex salt is treated with a peroxide to prepare a coloring solution and color is developed. Examples of the above-mentioned peroxides include sodium periodate, potassium periodate, potassium permanganate, and hydrogen peroxide, with hydrogen peroxide being the most preferred example. The above-mentioned iron complex salt is a bicarbonate (for example, bicarbonate).A more stable iron complex salt reagent can be obtained by blending IJium, lithium bicarbonate, potassium bicarbonate, etc. with low molecular weight dextran. Also,
When these compounded reagents are freeze-dried, the iron complex salt becomes very stable, and when making a kit as a reaction reagent for coloring, it is desirable to use a freeze-dried reagent. When carried out, it is preferable to proceed with the reaction on the acidic side. The usable pH is preferably 3 to 7, most preferably 4 to 5.5. A weakly acidic buffer is usually used to maintain the above pH, and lactate, citrate, oxalate and other buffers are particularly preferred.

Buffer concentrations may be used in the range of 0.01 to IM, but concentrations in the range of 0.05 to 0.4M are particularly preferred.

The color-forming compound formed by the reaction between the above iron complex salt and the aniline derivative (If) has a maximum absorption wavelength around 700 nm, and has a stable color tone, making it suitable for measuring the activity of peptidases such as LAP and γ-GTP. .

In addition, sodium benthacyanoacopheria) Na
ICFe(CN)a ``HtO) may be reacted with the liberated aniline derivative (It) to form a coloring compound, and the amount of the coloring compound may be measured colorimetrically to measure peptidase activity such as LAP or γGTP. .

Furthermore, aniline derivatives (I [ji) Peptidase activity such as LAP or γ-GTP may be measured by colorimetrically quantifying [).

Next, the measurement of LAP activity will be explained in more detail.

To measure the activity of LAP, first, L-leucyl-3,5-dihalogeno-4-hydroxyanilide, which is a synthetic substrate of LAP, is reacted with oxygen in the test solution to liberate the aniline derivative [11]. let As the test liquid, serum t-o, oi to 5- is used. The above enzymatic reaction usually only needs to be carried out at around 37°C for 5 minutes or more.
Since the optimum pH for this reaction is in the range of 6.5 to 8.0, the pH ft may be appropriately set within this range. As a buffer solution for maintaining pH, phosphate 1. Buffers such as barbital, borate, trishydroxymethylaminomethane are used.

The produced aniline derivative CI[) is condensed with an oxidizing agent such as sodium metaperiodate under alkaline conditions in the presence of a coupler such as Ctrx and p-xylenol to form a color-forming compound. Alternatively, a color reagent obtained by oxidizing pentacyanoaminoferroate with an oxidizing agent such as hydrogen peroxide may be used to develop color, and colorimetric determination may be performed.

Next, the measurement of r-GTP activity will be explained in more detail. When measuring the activity of γ-σP, first γ-G
r-L-glutamyl'-3,5-, a synthetic substrate for TP
γ- in the test solution to dihalogeno-4-hydroxyanilide.
GTP is subjected to an enzymatic reaction to liberate the aniline derivative (If). As the test solution, serum is used within the range of 0.01 to 5-5. The above enzymatic reaction may be carried out usually at around 37°C for 5 minutes or more. The optimum pH for this reaction is 7.5-9
, 0, so the pH can be set appropriately within this range. For the 0 reaction, pH 7, which contains an appropriate amount of an amino acid or peptide, such as glycylglycine, as a receptor.
γ-GTP in the sample by reacting in a buffer solution of 5 to 9.0
The a=IJ derivative [II] produced in proportion to the activity may be quantified. As a buffer for maintaining pH, a buffer such as phosphate, barbital, borate, carbonate, triethanolamine, glycine, trishydroxymethylaminomethane, etc. is used.

In quantifying the produced aniline derivative (II), a color-forming compound may be derived using a chemical coloring method similar to that used in measuring the activity of IIAP, and the aniline derivative (II) may be determined colorimetrically.

As mentioned above, the measurement method using the synthetic substrate of the present invention has simple reaction steps, so it can be used to quickly and accurately measure LAP,
Not only can peptidase activity values such as γ-GTP be measured, but the color-forming compounds produced are usually 5
Since it has maximum absorption around 50 to 750 nm, it is a highly accurate method for measuring peptidase activity, regardless of the influence of impurities in biological samples.

Next, the present invention will be specifically explained with reference to Examples and Reference Examples, but the present invention is not limited thereto in any way.

The abbreviations described in Examples and Reference Examples have the following meanings: 0 Leu ; L-leucyl r-Glu : γ-L-glutamyl BOC; t-butyloxycarbonyl O8u : N
-Hydroxysuccinimide ester AcOH; Acetic acid Example I L-leucyl-3,5-dichloro-4-hydroxyanilide hydrochloride 2,6-dichloro-4-7 minophenol 1.789 (
10 mmol) and 1.26 g of sodium bicarbonate (
15 mmol) was dissolved in 25 d of water, and 3.28 t of BOC-Leu -O8u was added thereto while cooling to 0 to 5°C.
A solution of (10 mmol) in dioxane (25 m2) was added under stirring. After stirring overnight at room temperature, dioxane was distilled off under reduced pressure at a temperature below 30°C. The residue was dissolved in 200ml of ethyl acetate, saturated aqueous sodium hydrogen carbonate solution, water, IN hydrochloric acid,
It was washed three times with saturated saline and then with 50% of each solution. The ethyl acetate layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain 2.96 t of BOC-L-leucyl-3,5-dichloro-4-hydroxyanilide. Next, this
'2 Dissolve in N-HCL/AcOH15,
After stirring for 2 hours at room temperature, dry diethyl ρ ether 100
m/l was added to crystallize and decanted twice with dry ether. The obtained crystals were dried under reduced pressure to obtain L-leucyl-3,5-dichloro-4-hydroxyanilide hydrochloride. Yield: IB9 # (Yield 57.7)
Molecular formula: C1, H□, N, 0. C1, HCl fusion
Point; 127-133°C (decomposition) Silica gel thin layer chromatography (TLC): Rf =
0.63 [: n-butanol-acetic acid-water (4:1
:1):IIR Char) (KBr): Example 2 from Figure 1 2 L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride 2,6-dibromo-4-aminophenol 4.90 t
(18,3 mm/I/) and sodium bicarbonate 1
．． 68f (20 mmol) was dissolved in water 70-, and BOC-Leu-O8u was added to this while cooling to 0-5°C.
6D1t (18,3 mmA/) of dioxane (70 m
) The solution was lowered under stirring. After stirring overnight at room temperature, 30
Dioxane was distilled off under reduced pressure at a temperature below .degree. C., and the 7'Co residue was dissolved in 400 ml of ethyl acetate and washed three times with 100 d each of saturated aqueous sodium bicarbonate solution, water, IN hydrochloric acid, and saturated brine in this order. After drying the ethyl acetate layer over anhydrous magnesium sulfate, it was concentrated under reduced pressure to obtain BOC-L-leucyl-3,
5-dibromo-4-hydroxyanilide 5,8t was obtained. Next, this was dissolved in 2N -HC1/AcOH30-, stirred at room temperature for 2 hours, and then crystallized by adding dry ether to obtain L-leucyl 3゜5-dibromo-4-hydroxyanilide hydrochloride. .

Yield 2) Of (yield 32.8 cm) Molecular formula C1, HtiN, 0tBr, "HCl (41
a54) Silica gel thin layer chromatography (TLC)
:R, f=0.65 [: n-butanol:acetic acid:water=4:1:1) Melting point: 131-134°C (decomposition) IR char) (KBr method); Example 3 γ-L-glutamyl-3.5-dichloro-4-hydroxyanilide N,N-7 talloylu L-glutamic anhydride 5.16
t (20 mmol) and 3.56 # (20 mmol) of 4-amino-2,6-dichlorophenol were dissolved in dioxane 50- and stirred at 60° C. for 2 hours.

Dioxane was distilled off under reduced pressure, and a methanol (50d) solution of hydrazine-Hittlede 1.5- was added to the residue, and the mixture was allowed to stand at room temperature for 2 days. Methanol was distilled off under reduced pressure, water was added to the residue, and the pH was adjusted to 3 with 05N-HCt to precipitate γ-L-glutamyl-3゜5-dichloro-4-hydroxyani IJ)' 3.96 # I got it.

Yield 3.96 t (yield 64.5 t) Molecular formula C
1□H1, N20. C.L.

Melting point: 214-217°C (decomposition) Silicage # TLC; nf = Q, 49 (n-butanol:acetic acid:water = 4:1:1) IR char) (KBr method); example
4 γ-L-glutamyl-3.5-dibromo-4-hydroxyanilide N, N-7 talloyol L-glutamic anhydride 2.18
t (8,4 mm/I/) and 4-amino-2,
2.26 t (8.4 mmol) of 6-dipromophenol was dissolved in 20-dioxane and stirred at 60°C for 1.5 hours. The dioxane was distilled off under reduced pressure, and the residue was diluted with 0.7-hydrazine-Hitler. A methanol (20d) solution was added, the mixture was left at room temperature for 2 days, the methanol was removed under reduced pressure, water was added to the residue, the pH was adjusted to 3 with 0.5 N HCl, and the precipitated γ-L- Glutamyl-3゜5
2.13 t of -dibromo-4-hydroxyanilide was obtained.

Yield 2.13 # (Yield 64.0) Molecular formula C
,,H,,N,0. Br.

Melting point: 191-194°C (decomposed) [Measured according to the Japanese Pharmacopoeia General Test Method Melting Point Determination section: 199-200°C (decomposed) and 7t.

Silicage # TLC: Rf = 0.53 (butano)v: acetic acid: water = 4:1:1) IR char) (KBr method); reference example shown in Figure 4 I L-leucyl-3,5 - Numbness activity measurement using dichloro-4-hydroxyanilide, sodium metaperiodate, '9-xylenol - Leucyl 3. s-dichloro-4-hydroxyanilide hydrochloride was dissolved in 0.1M phosphate buffer (pH 7.0) to prepare a substrate solution (5 mM) (to this substrate solution 1-, patient serum (236G- LAP in R units)
20 μtt was added, mixed well, and reacted at 37° C. for 20 minutes. After the reaction, 2mM sodium metaperiodate, 1
An oxidizing reagent solution 3 consisting of a 0.2 N ωH solution containing 0 mM p-xylenol was added and reacted to develop color.

Next, when the absorbance of the coloring liquid was measured at a wavelength of 585 nm, 00Dl+811 = 0.18 was obtained.

In other words, the synthetic substrate of the present invention was shown to act well against LAP, which is clinically important.

Reference example 2 L-loin) L/-3*5-dibromo-4-hydroxyanilide, sodium metaperiodate, p-xyleno-
Measurement of LAP activity using A/-Leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride t-0,1M
Dissolved in phosphate buffer (p) (7, O) to make a substrate solution (5 mM) o To this substrate solution 1-, 20 μt of patient serum (250 G-B units of LAP) was added and mixed well, After 0 reaction for 20 minutes at ℃, 2ff1
Oxidizing reagent solution 3- consisting of a 0.2 N-KOH solution containing M sodium metaperiodate and 10 mM p-xylenol was added to develop color.

Next, the absorbance of the coloring solution was measured at a wavelength of 585 nm, and the result was O, D, -0,22. In other words, the synthetic substrate of the present invention has a good effect on LAP, which is clinically important. It is shown below.

Reference Example 3 r-GTP activity measurement using γ-L-glutamyl-3,5-dichloro-4-hydroxyanilide, sodium metaperiodate, and p-xylenol r-L-glutamyl-3,5-dichloro-4- Hydroxyanilide (5mM
) and glycyl glycy (100mM) at 50%
mM) in Lis-HCl buffer (pH 8,0) to make a substrate solution.90 To this substrate solution 05-, patient serum (130
Add 10μA of r-GTP (mU/-) and mix well.
The reaction was carried out at 37°C for 20 minutes.

After reaction, 2mM sodium metaperiodate, 10mMp
An oxidizing reagent solution 2td consisting of a 0.2 N -KOH solution containing -xylenol was added to develop color.

Next, when the absorbance of the coloring liquid was measured at a wavelength of 585 nm, it was found to be 0. D, ,11=0.112 was obtained.

In other words, the synthetic substrate of the present invention was shown to act well on γ-GTP, which is clinically important.

Reference Example 4 γ-QTP activity measurement using γ-L-glutamyl-3,5-dibromo-4-hydroxyanilide, sodium metaperiodate, and p-xylenol γ-L-glutamyl-
3,5-dibromo-4=hydroxyanilide (5mM
) and glycyl glyci7 (100mM) at 50%
0.5 mM) was dissolved in Lis-HCl buffer (pH 8, O) to make a substrate solution. Patient serum (13
0mU/-r-GTP) 10μt1i? After adding, mixing well and reacting at 37°C for 20 minutes,
Oxidizing reagent solution 2- consisting of a 0.2 N KOH solution containing 2rrIM sodium metaperiodate and 10 mM p-xylenol was added to develop color.

Next, when the absorbance of the coloring liquid was measured at a wavelength of 585 nm, it was found to be 0. D.1. l=0.140 was obtained.

In other words, the synthetic substrate of the present invention was shown to have a good effect on γ-σP, which is considered to be clinically important.

Reference Example 5 LAP activity measurement using L-leuci 3,5-dibromo-4-hydroxyanilide, sodium metaperiodate, and 2-chloro-5-methylpheno-fiv.・Dissolve hydrochloride in 0.1M phosphate buffer (pH 7,0) to make a substrate solution (5 mM) o To this substrate solution 1-, patient serum (250 G-R units of LAP) 2
0 μt was added, mixed well, and reacted at 37° C. for 20 minutes. After the reaction, 2mM sodium metaperiodate, 5 r
nWi 2- Oxidizing reagent solution 3- consisting of 9,2 N -K, QH bath solution containing chloro-5-methylphenol
was added to develop color.

Next, when the absorbance of the coloring liquid was measured at a wavelength of 635 nm, O, D, , = 0.33- was obtained f
C.

That is, the synthetic substrate of the present invention was shown to act well on LAP.

Reference example 6 γ-L-glutamyl-3,5-dibromo-4-hydroxyanilide, sodium metaperiodate, 2-chloro-
γ-GTP activity measurement using 5-methylphenol γ-L-glutami/I/-3,5-dibromo-4-hydroxyanilide (s mM) and glycylglycine (100 mM ffi 50 mM) in lithium-HCl buffer (p
H8,0) to prepare a substrate solution. This substrate solution 0
．． 5- to patient serum (130 mU/- r-GTP
) 10'ltt was added, mixed well, and reacted at 37°C for 20 minutes.

After reaction, 2 fans of sodium metaperiodate, 5ff1M2
-0,2N containing chloro-5-methylphenol
-Add an oxidizing reagent w1.2- consisting of KOHi solution to develop color at 7'C.

Next, when the absorbance of the coloring solution was measured at a wavelength of 635 nm, Dos = 0.214 was obtained.

That is, the synthetic substrate of the present invention was shown to have a good effect on γ-GTP. Measurement of LAP activity using Eloto Sodium pentaphenoaminoferro
-Dissolve in 0.3-hydrogen peroxide (60 sd) and add 20 d of sodium carbonate solution. Next, 4 ft of Dextran T 10 (manufactured by 7 Almasia) was added to prepare a coloring stock solution. Reaction termination/coloring stock solution 1- was prepared by adding 50 ml of 0.2M citric acid buffer (pH 4,5) containing 1-salt and 0.5% Tween 80.

0.1 M lyf [l concentration K (pH 7,0) K:
fll'M l, ft, 5 rrMI--leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride 1- to patient serum (250 G-R units LAP) 20
μL was added, mixed well, and reacted at 37° C. for an addition period.

After the reaction, reaction stop/coloring solution 5- was added, and the mixture was left at room temperature for 20 minutes to develop color.* The absorbance was measured at 700 nm. As a result, O, D, o=0.21.

Reference Example 8 γ-GTP activity measurement using γ-L-glutamyl-3.5-dibromo-4-hydroxyanilide, sodium pentacyanoaminoferroate (50 mM) 5rrMr dissolved in Lis-HCl buffer (pH 8,0) -L-
Add 20 μt of patient serum (30 mU/- of γ-〇TP) to glutamy/re3.5-dibromo-4-hydroxyanilide, 1100 rn glycylglycine solution 1-, mix well, and react at 37°C for 200 minutes. Ta. After the reaction,
Add the reaction termination/coloring solution 5-gold described in Reference Example 7, leave it at room temperature for 20 minutes, allow the color to develop, and then measure the absorbance at 700 nm.
132 Reference Example 9 L-leucyl-3,5-dibromo-4-human oxyanilide, LAP activity measurement method using sodium pentacyanoacopheriate, leucyl-3,5-dibromo-4-hydroxyanilide, Hydrochloride in 0.1 M phosphate buffer (pH 7,0)
A substrate solution (5mM) was prepared by dissolving it in the following. 20μ of patient serum (250G-R units of LAP) to 1mg of this substrate solution.
t was added, mixed well, and reacted at 37°C for 15 minutes.

After the reaction, add 0.2 M EDTA and 2 Na (pH
11) Solution 4- prepared by adding solution 901R1 was added and reacted at 37°C for 15 minutes. After reaction coloring, 730n
Absorbance was measured at m. As a result, 0. D, O = 0
．． It was 232.

1cs-sodium penxyanoacopheria) fd
A NaCFe
(ON)=ND,)-11bFc was prepared by irradiating a sodium carbonate solution with ultraviolet rays for 15 minutes.

Reference Example 10 γ-GTPO calibration curve using γ-L-glutamyl-3,5-dibromo-4-hydroxyanilide γ-L-glutamyl-3,5-dibromo-4-hydroxyanilide (
5mM) and chrysyl glycy (100mM)
50 mM) was dissolved in Lis-HCl buffer (pH 8.0) to make a substrate solution.
-GTP (74-370 mU/vd) 10
μt was added, mixed well, and reacted at 37° C. for 200 minutes. After the reaction, 2mM sodium metaperiodate, 10mM
An oxidizing reagent solution consisting of a 0,2N KOH solution containing 1b-xylenol was added to the oxidizing reagent solution 2-t- to develop a color.Then, the absorbance of the coloring solution at a wavelength of 585 nm was measured.The results are shown in Figure 5. 7'Co This result showed that serum γ-GTP can be measured well.

[Brief explanation of the drawing]

Figure 1 is an IR chart of L-leucyl 3.5-)chloro-4-hydroxyanilide hydrochloride, Figure 2 is BoC-
IR channel of L-leucyl-3,5-dibromo-4-hydroxyanilide, Figure 3 shows γ-L-glutamyl-3
．． IR chart of 5-dichloro-4-hydroxyanilide, Figure 4 shows γ-L-glutamyl-3,5-dibromo-
IR chart of 4-hydroxyanilide, Figure 5 shows γ-
This is a GTPO calibration curve.

Claims (5)

[Claims] (1) Amides represented by the formula ▲ Numerical formulas, chemical formulas, tables, etc.▼ compound or its salt. (2) The amide compound or its salt according to claim 1, wherein R is an L-leucyl group and the halogen atom is a bromine atom or a chlorine atom. (3) L-leucyl-3,5-dibromo-4-hydroxyanilide or L-leucyl-3,5-dichloro-4
- The amide compound or salt thereof according to claim 2, which is a hydroxyanilide. (4) The amide compound or salt thereof according to claim 1, wherein R is a γ-L-glutamyl group and the halogen atom is a bromine atom or a chlorine atom. (5) γ-L-glutamyl-3,5-dibromo-4-hydroxyanilide or γ-L-glutamyl-3,5-
The amide compound or salt thereof according to claim 4, which is dichloro-4-hydroxyanilide.