JPH0411537B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is based on the formula (In the formula, R is an L-leucyl group, and X and Y are the same or different and represent a halogen atom) or a salt thereof.

[Conventional technology]

Peptidase has long been known as a general term for enzymes that act on the peptide bonds of peptides and cleave them from the amino terminus, releasing amino acids or lower peptides. For example, leucine aminopeptidase (True in clinical chemistry)
(also referred to as LAP) and allyl amitase (also referred to as Clinical LAP in clinical chemistry, hereinafter sometimes referred to as
Aminopeptidases (hereinafter referred to as AA) such as
True LAP and Clinical LAP are collectively referred to as LAP), cystine aminopeptidase, proline aminopeptidase, arginine aminopeptidase, alanine aminopeptidase, and γ-glutamyl transpeptidase (γ-GTP). There is.

Among the enzymes mentioned above, leucine aminopeptidase (LAP) in particular is widely distributed in all tissues in the body, is also present in serum, and is known to increase under pathological conditions. These enzymes are the most important enzyme activity measurement items.

LAP is an L-peptide, especially the amino terminus is L-
L, which is leucine or a related amino acid
- An enzyme that hydrolyzes the amino terminal residue of a peptide to release L-leucine, and is widely distributed in various tissues in the body. This enzyme is also present in serum, and the amount of the enzyme in serum is known to vary significantly depending on the state of the body. increases. Therefore, LAP activity is one of the indicators of such diseases.
Measuring this enzyme activity has become one of the indispensable testing methods for diagnosing these diseases.

Many methods for measuring LAP activity have been reported. Most of them are methods for determining LAP activity values by colorimetrically quantifying amine compounds liberated by LAP from synthetic substrates, using L-leucyl-p-nitroanilide as the synthetic substrate, One example is a method of colorimetrically quantifying the yellow color of P-nitroaniline produced by the enzymatic action of LAP. However, there is a drawback that the wavelength of this colorimetric determination overlaps with the coloring wavelength of the synthetic substrate, and it is also unavoidable to be influenced by serum components, especially bilirubin pigments. Furthermore, there is a method using L-leucyl 7β-naphthylamide as a synthetic substrate,
In this method, the β-naphthylamine produced has 5-
Coupling with nitro-2-aminotoxybenzenediazotate etc. to form a dye, or
Alternatively, the resulting β-naphthylamine is diazotized with sodium nitrite and coupled to N-(1-naphthyl)-ethylene cyanide, or p-dimethylaminobenzaldehyde or p-
A method has been used in which dimethylaminocinnamaldehyde is condensed to form a dye, which is then colorimetrically determined. These methods have complicated reaction processes and require strict operations, which are not only inconvenient as testing methods, but also the standard substance, β-naphthylamine, is extremely toxic, and in recent years bladder tumors have become cancerous. Because it has become clear that it causes carcinogenesis, it has the disadvantage of requiring particularly strict precautions when used as a carcinogen.

[Problem that the invention seeks to solve]

The present inventors have discovered that the conventional
As a result of intensive research to improve the method for measuring LAP activity, we discovered a new synthetic substrate that has excellent properties for measuring LAP activity, and completed the present invention.

The present invention is a novel amide compound [ ] or a salt thereof that is used as a peptidase and a synthetic substrate.

[Means for solving problems]

According to the present invention, in order to solve the above problems, the formula (wherein R is an L-leucyl group, and X and Y are the same or different and represent a halogen atom) or a salt thereof.

The amide compound [ ] used as a synthetic substrate for peptidase in the present invention can be produced by a conventional method for peptide synthesis. That is, L
-Carboxyl group of leucine and aniline derivative [] (wherein X and Y have the same meanings as above).

In the above condensation reaction, it is preferable to protect the functional group that should not participate in the reaction, that is, the α-amino group of L-leucine. As the protecting group for the α-amino group, an α-amino protecting group commonly used in peptide synthesis is used. For example, t-butyloxycarbonyl, t-amyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, adamantatyloxycarbonyl, p-methoxyhenzyloxycarbonyl, phthaloyl, o-nitrophenylthio group, etc. can be mentioned.

Examples of the aniline derivative [] used in the above condensation reaction include 3,5-dibromo-4-hydroxyaniline, 3,5-dichloro-hydroxyaniline, and 3-chloro-5-bromo-4-hydroxyaniline. It will be done.

In the above condensation reaction, the carboxyl group of L-leucine with a protected α-amino group is converted into an acid halide, an acid anhydride, an acid azide, an acid imidazolide, an active ester, such as cyanomethyl ester, p-nitrophel ester, 2, 4-dinitropher ester, N-hydroxysuccinimide ester, N
- converted into activated acyl derivatives such as hydroxyphthalimide esters and reacted with aniline derivatives [] or carbodiimides, such as Watersorp carbodiimide hydrochloride [1-
In the presence of a condensing agent such as ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-carbonylimidazole, isoxazolium salt, e.g. Woodward's reagent, the above This is carried out by reacting protected L-leucine with an aniline derivative [].

In the above condensation reaction, inert organic solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, dioxane, benzene, chloroform,
In solvents such as dichloromethane and dichloroethane,
This is carried out by adding approximately equal amounts of both and reacting at room temperature or lower temperature. The above reaction process can be performed using thin layer chromatography (TLC) such as silica gel or high performance liquid chromatography (HPL).
Since the reaction can be traced by such methods, the reaction can be appropriately terminated when some of the starting materials disappear.

The reaction product thus obtained can be treated with non-hydrophilic organic solvents, such as chloroform, dichloromethane, ethyl acetate, butyl acetate, methyl isobutyl ketone, benzene, with or without distilling off the reaction solvent. It can be collected by dissolving it in diethyl ether or the like, washing it 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.

Next, the protective group of the resulting reaction product is removed, and this removal is carried out using a protective group removal method in peptide chemistry. For example α−
When the amino protecting group is a t-butyloxycarbonyl group, a method using a 2N hydrogen chloride acetic acid solution, trifluoroacetic acid, formic acid, etc., and when it is a henzyloxycarbonyl group, a method using palladium-
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.

In order to collect the amide compound [] obtained in this way from the reaction solution, first, if the protecting group is removed by acid decomposition, it is neutralized, and if it is by catalytic reduction, the catalyst is removed. , non-hydrophilic organic solvent,
For example, it can be collected by washing with acidic water and alkaline water in a solvent such as chloroform, dichloromethane, dichloroethane, ethyl acetate, butyl acetate, methyl isobutyl ketone, benzene, or diethyl ether, and then distilling off the solvent. If further purification is necessary, 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.

This amide compound [ ] may be used as a salt with an inorganic acid such as hydrochloric acid, sulfuric acid, acetic acid, or phosphoric acid, or a salt with an organic acid such as formic acid, acetic acid, propionic acid, malic acid, citric acid, tartaric acid, or oxalic acid. can be formed.

Next, a method for measuring peptidase activity using the present amide compound [] will be described as a reference. In this measurement method, a chemical coloring method is used to quantify the peptidase contained in the test solution of the above-mentioned amide compound [] or its salt, particularly the aniline derivative [] released by the action of LAP. It is used for colorimetric determination.

As for the above-mentioned chemical coloring method, it is preferable to carry out oxidative condensation with the aniline derivative [ ] in the presence of a coupler to form a coloring compound, which is then subjected to colorimetric determination. The coupler to be used may be any aromatic compound as long as it forms a color-forming compound through oxidative condensation with the aniline derivative [ ], but typical aromatic compounds include phenolic compounds, Examples include aminophenol compounds, aniline compounds, and naphthol compounds. Examples of phenolic compounds include phenol, salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, 2,6-hydroxybenzoic acid, methyl salicylate, o-cresol, m-cresol, p-cresol, o-ethyl phenol, m
-ethylphenol, 2,3-xylenol,
2,4-xylenol, 2,5-xylenol,
3,5-xylenol, 2,6-xylenol,
o-methoxyphenol, m-methoxyphenol, p-methoxyphenol, 2,-dimethoxyphenol, o-chlorophenol, m-chlorophenol, p-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, , o-bromophenol, m-bromophenol, p-bromophenol, 2,4-dibromophenol, 2,6-dibromophenol, 2-
Methyl-6-chlorophenol, 2-chloro-5
-methylphenol, o-carboxymethylphenol, 2-hydroxy-4-aminoethylphenol, and the like. Examples of aminophenol compounds include 4-chloro-2-aminophenol, N,N'-dimethyl-m-aminophenol, 4-methyl-2-aminophenol, and 5-chloro-2-aminophenol.
Amino-2-hydroxybenzoic acid, 2-amino-
Examples include 3-hydroxybenzoic acid, o-aminophenol, m-aminophenol, and p-aminophenol. Examples of aniline compounds include aniline, o-toluidine, m-toluidine, p-toluidine, N-methylaniline, N-
Ethylaniline, N,N-dimethylaniline,
N,N-diethylaniline, N,N-dimethyl-
o-toluidine, N,N-dimethyl-p-toluidine, N,N-diethyl-o-toluidine, N,
N-diethyl-m-toluidine, N,N-diethyl-p-toluidine, o-chloroaniline, m-
Chloroaniline, m-bromoaniline, anthranilic acid, 3-aminobenzoic acid, p-dimethylaminobenzoic acid, p-chloro-o-toluidine, 3-
Amino-4-methylbenzoic acid, m-phenylenediamine, N,N-dimethyl-m-phenylenediamine, 2-methyl-m-phenylenediamine,
4-Methyl-o-phenylenediamine, 4-methyl-m-phenylenediamine, 2-chloro-m-
Phenylenediamine, 3-chloro-o-toluidine, 2-methoxy-5-chloroalidine, o-ethylaniline, 2,5-diethoxyaniline, N
-ethyl-N-hydroxyethyl aerin, N-
Examples include ethyl N-hydroxyethyl-m-toluidine. Examples of naphthol compounds include α-naphthol, β-naphthol, 2-carboxy-1-naphthol, 4-chloro-1-naphthol, 1-hydroxy-2-naphthoic acid, 1-naphthol-2-sulfonic acid, -Naphthol-3
-sulfonic acid, 1-naphthol-4-sulfonic acid, 2-naphthol-6-sulfonic acid, 2-naphthol-3,6-disulfonic acid, and the like.

In the above oxidative condensation, the coloring reaction is quantitatively completed by carrying out the reaction at a pH within the range of about 4 to 12. As the buffer or alkaline aqueous solution for maintaining the above-mentioned pH, carbonate buffer, phosphate buffer, borate buffer, alkali hydroxide aqueous 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, hypozincate, peracid compound-based oxidizing agents such as persulfate, hydrogen peroxide, etc. Sodium metaperiodate is a suitable example.

The color-forming compound produced by the oxidative condensation of the aniline derivative [] and the above coupler has a maximum absorption wavelength widely distributed in the range of approximately 550 to 770 nm depending on the type of coupler, but is usually a colored pigment that has maximum absorption in the range of 570 to 680 nm. The color development is extremely sensitive and stable, and there is almost no change due to temperature, and it is not easily affected by contaminants such as bilirubin in biological samples, so there is almost no positive error. Gives extremely good results in measuring peptidase activity such as LAP.

Another method for color-quantifying the above-mentioned aniline derivatives is a method in which a pentacyanoiron complex salt is treated with a peroxide to prepare and generate a coloring liquid. Examples of the above-mentioned peroxide include periodate nanolium salt, periodate potassium salt, potassium permanganate, hydrogen peroxide, and the like, and hydrogen peroxide is the most preferred example. When the above-mentioned iron complex salt is mixed with a bicarbonate such as sodium bicarbonate, lithium bicarbonate, potassium bicarbonate, etc., and low molecular weight dextran, a more stable iron complex reagent can be obtained. Also,
Since the iron complex of these compounded reagents is greatly stabilized by freeze-drying, it is desirable to use a connected dry reagent when forming a kit as a coloring reaction reagent.

When carrying out a color reaction using the above iron complex salt, it is preferable to proceed with the reaction on the acidic side. The pH that can be used 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, and oxalate buffers are particularly preferred. Buffer concentrations may be used in the range 0.01-10M, with concentrations in the range 0.05-0.4 being particularly preferred.
The color-forming compound formed by the reaction between the above iron complex salt and the aniline derivative [] has a maximum absorption wavelength.
It is distributed around 700 nm and has a stable color length, making it suitable for measuring peptidase activity such as LAP.

Furthermore, sodium pentacyanoacofeliato Na ₂ [Fe(CN) ₅ .H ₂ O] was reacted with the free aniline derivative [ ] to form a color-forming compound, and the amount of the color-forming compound was determined colorimetrically. LAP
Peptidase activity measurement may also be performed.

Furthermore, aniline derivatives can be obtained by applying various known chemical coloring methods, such as methods for colorimetrically quantifying aromatic amines, such as diazo coupling methods and methods for quantifying Schiff's base produced by reacting aldehyde compounds. By colorimetrically quantifying
Peptidase activity measurement such as LAP may also be performed.

Next, LAP activity measurement will be explained in more detail. To measure the activity of LAP, first, L-leucyl-3,5, which is a synthetic substrate of LAP,
-Aniline derivatives are produced by enzymatically reacting LAP in the test solution with dihalogeno-4-hydroxyanilide []
release. Serum is used as the test solution in a range of 0.01 to 5 ml. The above enzymatic reaction may normally be carried out at around 37°C for 5 minutes or more. Optimum of this reaction
Since the PH is in the range of 6.5 to 8.0, the PH can be set appropriately within this range. As a buffer for maintaining pH, a buffer such as phosphate, barbital, borate, trishydroxymethylaminomethane, etc. is used.

To quantify the generated aniline derivative [], oxidative condensation is performed with an oxidizing agent such as sodium metaperiodate under alkaline conditions in the presence of a coupler such as p-xylenol to form a color-forming compound, and colorimetry is performed. It may be quantitatively determined, or it may be determined colorimetrically by developing a color using a coloring reagent obtained by oxidizing pentacyanoaminoferroate with an oxidizing agent such as hydrogen peroxide.

As mentioned above, the measurement method using the synthetic substrate of the present invention not only enables quick and accurate measurement of peptidase activity values such as LAP because each reaction step is simple, but also allows for the rapid and accurate measurement of peptidase activity values such as LAP. Since it has maximum absorption around 550 to 750 nm, it is less affected by contaminants in biological samples and is a highly accurate method for measuring peptidase activity.

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.

In addition, the abbreviations described in Examples and Reference Examples have the following meanings.

Leu; L-leucyl BOC; t-butyloxycarbonyl OSu; N-hydroxysuccinimide ester AcOH; acetic acid, Example 1 L-leucyl-3,5-dichloro-4-hydroxyanilide hydrochloride 2,6-dichloro- 4-Aminophenol 1.78
g (10 mmol) and sodium bicarbonate 1.26
Dissolve g (15 mmol) in 25 ml of water and add
While cooling to 5℃, add 3.28 g of BOC-Leu-OSu (10
A solution of 2 mmol) in dioxane (25 ml) was added dropwise with stirring. After stirring overnight at room temperature, dioxane was distilled off under reduced pressure at a temperature below 30°C. Add 200ml of ethyl acetate to the residue.
Dissolved in saturated aqueous sodium bicarbonate solution, water,
It was washed three times with 50 ml each of 1N hydrochloric acid and saturated saline, in that order. After drying the ethyl acetate layer over anhydrous magnesium sulfate, it was concentrated under reduced pressure to obtain BOC-L-leucyl-3,5-dichloro-4-hydroxyanilide.
2.96g was obtained. Next, 3 this was mixed with 2N-Hcl/AcOH
The mixture was dissolved in 15 ml and stirred at room temperature for 2 hours, then crystallized by adding 100 ml of dry diethyl ether 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: 1.89 (yield 57.7%) Molecular formula: C ₁₂ H ₁₆ O ₂ Cl ₂ .HCl Melting point: 127-133°C (decomposition) Silica gel thin layer chromatography (TLC); Rf
= 0.63 [n-butanol-acetic acid-water (4:1:
1)] IR chart (KBr method); as shown in Figure 1 Example 2 L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride 2,6-dichloro-4-aminophenol 4.90
g (18.3 mmol) and sodium bicarbonate
Dissolve 1.68 g (20 mmol) in 70 ml of water, cool it to 0 to 5°C, and add 6.01 g of BOC-Leu-OSu.
A solution of (18.3 mmol) in dioxane (70 ml) was added dropwise with 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 ethyl acetate.
Dissolve in 400ml of saturated sodium bicarbonate aqueous solution,
It was washed three times with 100 ml each of water, 1N hydrochloric acid, and saturated saline in that order. The ethyl acetate layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure to obtain 5.8 g of BOC-L-leucyl-3,5-dibromo-4-hydroxyanilide. Next, this was mixed with 2N−Hcl/
After dissolving in 30 ml of ACOH and stirring at room temperature for 2 hours, dry ether was added for crystallization to obtain L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride.

Yield: 2.50g (yield 32.8%) Molecular formula: C ₁₂ H ₁₆ O ₂ Br ₂ .HCl (416.54) Silica gel thin layer chromatography (TLC); Rf = 0.65
[n-butanol-acetic acid-water = 4:1:1)] Melting point: 131-134°C (decomposition) IR chart (KBr method); as shown in Figure 2 Reference example 1 L-leucyl-3,5- Dichloro-4-hydroxyanilide, sodium metaperiodate, p
- LAP activity measurement using xylenol L-leucyl-3,5-dichloro-4-hydroxyanilide hydrochloride was added to 0.1M phosphate buffer (PH
7.0) to prepare a substrate solution (5mM). Add patient serum (236 G-R units) to 1 ml of this substrate solution.
LAP) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 0.2N containing 2mM sodium metaperiodate, 10mM p-xylenol
3 ml of an oxidizing reagent solution consisting of -KOH solution was added to react and color was developed.

Next, when the absorbance of the coloring liquid was measured at a wavelength of 585 nm, OD ₅₈₅ =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-leucyl-3,5-dibromo-4-hydroxyanilide, sodium metaperiodate, p-
Measurement of LAP activity using xylenol L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride was dissolved in 0.1M phosphate buffer (PH7.0) to prepare a substrate solution (5mM). This substrate solution 1
to ml. 20 μl of patient serum (250 G-R units of LAP) was added, mixed well, and reacted at 37° C. for 20 minutes.
After reaction, 2mM sodium metaperiodate, 10mM
3 ml of an oxidizing reagent solution consisting of a 0.2N-KOH solution containing Mp-xylenol was added to cause a reaction and color development.

Next, when the absorbance of the coloring solution was measured at a wavelength of 585 nm, OD ₅₈₅ =0.22 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 3 L-leucyl-3,5-dibromo-4-hydroxyanilide, sodium metaperiodate, 2
-using chloro-5-methylphenol
LAP activity measurement L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride was added to 0.1M phosphate buffer (PH
7.0) to prepare a substrate solution (5mM). Add patient serum (250 G-R units) to 1 ml of this substrate solution.
LAP) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 3 ml of an oxidizing reagent solution consisting of a 0.2N KOH solution containing 2mM sodium metaperiodate and 5mM 2-chloro-5-methylphenol was added to react and develop color.

Next, when the absorbance of the coloring solution was measured at 635 nm, OD ₆₃₅ =0.33 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 4 LAP activity measurement using L-leucyl-3,5-dibromo-4-hydroxyanilide, sodium pentacyanoamin ferrote 2 g of sodium pentacyanoamin ferrote
Dissolve in 20ml of water, add 60ml of 0.3% hydrogen peroxide,
An additional 20 ml of 10% sodium bicarbonate solution was added. Next, 4 g of Dextran T10 (manufactured by Pharmacia) was added to prepare a coloring stock solution. Reaction stop/coloring stock solution 1
Contains 1% salt and 0.5% Tween 80 in ml
Adjustments were made by adding 50 ml of 0.2M citrate buffer (PH4.5).

5mM dissolved in 0.1M phosphate buffer (PH7.0)
Add patient serum (250G-
20 μl of R unit LAP) was added, mixed well, and reacted at 37° C. for 20 minutes. After the reaction, 5 ml of reaction stop/coloring solution was added and left at room temperature for 20 minutes to develop color.
Absorbance was measured at 700 nm. As a result, OD ₇₀₀
= 0.12.

Reference Example 5 Measurement of LAP activity using L-leucyl-3,5-dibromo-4-hydroxyanilide and sodium pentacyanoacopheriate L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride was dissolved in 0.1 M phosphorus Support buffer solution (PH
7.0) to prepare a substrate solution (5mM). Add patient serum (250 G-R units) to 1 ml of this substrate solution.
20 μl of LAP) was added, mixed well, and reacted at 37°C for 15 minutes. After the reaction, add 0.2M EDTA・2Na to 30ml of 1%-sodium pentacyanoacopheriate.
4 ml of a solution prepared by adding 90 ml of a (PH11) solution was added, and the mixture was reacted at 37°C for 15 minutes. After reaction coloration,
Absorbance was measured at 730 nm. As a result, OD ₇₃₀
= 0.232.

1%-sodium pentacyanoacopheriate is 1%-sodium nitroprusside Na
[Fe(CH) ₅ ND]-1% sodium carbonate solution was irradiated with ultraviolet rays for 15 minutes.

[Brief explanation of drawings]

Figure 1 shows L-leucyl-3,5-dichloro-4
-IR chart of hydroxyanilide hydrochloride,
Figure 2 shows the IR chart of BOC-L-leucyl-3,5-dibromo-4-hydroxyanilide.

Claims (1)

[Claims] 1 formula (In the formula, R is an L-leucyl group, and X and Y are the same or different and represent a halogen atom) or a salt thereof. 2. The amide compound or salt thereof according to claim 1, wherein R is an L-leucyl group and the halogen atom is a bromine atom or a chlorine atom. 3. The amide compound or salt thereof according to claim 2, which is L-leucyl-3,5-dibromo-4-hydroxyanilide or L-leucyl 3,5-dichloro-4-hydroxyanilide.