JPH01139564A - N-substituted indole ester compound, production thereof, testing composition and testing tool using said compound - Google Patents

Abstract

NEW MATERIAL:An N-substituted indole ester compound shown by formula (R<1> is alkoxy, aralkoxy, aryloxy and alkylsulfonyl which may be replaced with halogen, alkoxy or NO2, or alkylsulfonyl or arylosulfonyl; R<2> is H, halogen, OH, COOH, acylamino, NO<2>, alkoxycarbonyl, alkoxy or amide; X and Y are ordinary protecting group of NH2). EXAMPLE:1-Methoxy-3-[N-(p-toluenesulfonyl)-L-alanyloxy]-indole. USE:A reagent for detecting esterase or protease (e. g., leukocyte). PREPARATION:A compound shown by formula II is condensed with a compound shown by formula III (Z is OH, halogen, alkoxycarboxy or imidazole) to give a compound shown by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an N-substituted indole ester compound, a method for producing the same, a test composition for measuring esterase or protease using the compound, and a test device carrying the same. The N-substituted indole ester compounds provided by the present invention are effectively utilized for detecting esterases or proteases such as leukocytes.

If leukocytes or esterase are contained in the urine or dialysate, it can be assumed that a disease in the kidneys and genitourinary tract or inflammation is progressing at the dialysis site. Therefore, in order to promptly diagnose and treat these diseases, it is important to accurately detect leukocytes or esterase in the urine, dialysate, etc. The N-substituted indole ester compound of the present invention is suitably used as a test reagent for such esterases.

[Conventional technology]

The test device for detecting esterase or protease consists of a carrier impregnated with an ester compound, a diazonium salt compound, a buffer, and an activator.If esterase or protease is present in the sample, the ester compound is hydrolyzed and generates highly reactive hydrogen groups. A compound having the above is produced, and color is developed by coupling this with the diazonium salt compound. As an ester compound, 3-(N-toluenesulfonyl-shi-alanyloxy)-indole (Japanese Patent Publication No. 1983-3
No. 475) and 3-(N-)luenesulfonyl-monoaranyloxy)-5-phenylpyrrole (JP-A-60-26
No. 0558) is known and put into practical use.

[Problem that the invention seeks to solve]

However, although these ester compounds have been put into practical use, they have the disadvantage that the determination time is 120 seconds, which is significantly longer than other urine component detection test strips.

Therefore, an object of the present invention is to provide a novel N-substituted indole ester compound, a method for producing the same, a test composition for measuring esterase or protease using the compound, and a test device supporting the same.

[Means for solving problems]

The present invention is based on the following formula: -% formula () [In the formula, R1 represents an alkoxy group, an aralkoxy group, or an aryloxy group, and is a halogen atom.

It may be substituted with an alkoxy group or a nitro group.

Alternatively, R1 in the formula represents an alkylsulfonyl group or an arylsulfonyl group. R2 represents a hydrogen atom, a halogen atom, a hydroxy group, a carboxyl group, an acylamino group, a nitro group, a carboxyl group, an alkoxycarbonyl group, an alkoxy group, or an amide group. X and Y represent commonly used protecting groups for amino groups. ] It is an N-substituted indole ester compound represented by these.

The present invention also provides a compound of the general formula (2) [wherein R1 represents an alkoxy group, an aralkoxy group, or an aryloxy group, but may be substituted with a halogen atom, an alkoxy group, or a nitro group.

Alternatively, R' in the formula represents an alkylsulfonyl group or an arylsulfonyl group. Rt is a hydrogen atom.

Halogen atoms, hydroxyl groups, carboxyl groups.

Represents an acylamino group, nitro group, carboxyl group, aacoxycarbonyl group, alkoxy group or amide group.

] The compound represented by the general formula (7) [In the formula, Z represents a hydroxy group, a halogen atom, an alkoxycarbonyloxy group, an imidazole group, etc. X,
Y represents a commonly used protecting group for amino groups. Ippukudaiyama Rl (I) [wherein R1 represents an alkoxy group, an aralkoxy group or an aryloxy group, and is a halogen atom].

Optionally substituted with an alkoxy group or a nitro group,
Alternatively, R' in the formula represents an alkylsulfonyl group or an arylsulfonyl group. R2 represents a hydrogen atom, a halogen atom, a hydroxy group, a carboxyl group, an acylamino group, a nitro group, a carboxyl group, an alkoxycarbonyl group, an alkoxy group, or an amide group. X and Y represent commonly used protecting groups for amino groups. ] Provides a method for producing an N-i substituted indole ester compound represented by the following.

Furthermore, according to the present invention, the N-W substituted ifhindole ester compound and the general formula [wherein R3 represents an alkyl group, an alkoxy group, a halogen atom or a hydrogen atom, and R4 represents an alkylene group, m,
n represents the same or different integers of 1 or 2.3, and Z represents a stabilizing anion. Further, A represents R:I alkylamine or an oxygen atom. ] A test composition for measuring esterase or protease containing a diazonium salt compound represented by the following is provided.

Furthermore, according to the present invention, there is provided a test device for measuring esterase or protease, which comprises the composition supported on a carrier.

The N-substituted indole ester compound of formula (Il) of the present invention is produced by the route shown in the diagram below.

C) 121□ R' That is, N-1 substituted dicarboxylic acid (6) is ring-closed by heating with acetic anhydride in the presence of sodium acetate.
-9-substituted-3-acetoxyindole ■ was obtained, and N-substituted indoxyl (I[
) is obtained. N-substituted indoxyl (penalized by the above formula (2
) By condensing with the carboxylic acid derivative shown in
An N-substituted indole compound (1) is obtained. Preferably carboxylic acid compound (2) and N-substituted indoxyl (5
) in methylene chloride with a dehydrating agent such as N,N'-dicyclohexylcarbodiimide and a reaction aid such as 4-dimethylaminopyridine.

As mentioned above, the N-substituted indole ester compound [11] of the present invention is used as a substrate in the measurement of esterase or protease, and is particularly advantageously used in the detection of leukocytes in urine and dialysate.

Such a test device for measuring esterase or protease contains the N-substituted indole ester compound (I) of the present invention, a diazonium salt compound, and if necessary, a buffer, an activator,
It consists of a carrier impregnated with a composition consisting of stabilizers, wetting agents and solvents.

Typical diazonium salt compounds include C, N,
N'-his(3-tomexyphenyl)piperazine-4
',4'-didiazonium bistetrafluoroporate or 0,0'-bis(3-chlorophenyl)ethylene glycol-4Z 4+-didiazonium bistetrafluoroborate.

The diazonium salt compound is, for example, represented by the following formula [wherein R5 represents an alkyl group, an alkoxy group, a halogen atom or a hydrogen atom, and m and n represent the same or different integers of 1 or 2.3. It is produced by dissolving or suspending the amino compound represented by ] in a cold mineral acid aqueous solution and treating it with sodium nitrite. Preferably, a 6N aqueous hydrogen chloride solution containing the above amino compound is dissolved or suspended in a suitable mineral acid, and an aqueous sodium nitrite solution is added to this solution for reaction. Further generated diazonium salt solution 35% at O℃
React with tetrafluoroboric acid for several hours. After the reaction is complete, the desired product is collected from the reaction mixture in a conventional manner.

For example, the desired product can be obtained by collecting the generated precipitate by filtration and drying it.

Buffers are used to keep the pi value constant on the test device, such as tris-(hydroxymethyl)-aminomethane buffers, phosphate buffers, borate buffers, barbiturate buffers or amino acid buffers. It is preferable that the pif value can be maintained in the range of 6 to 10 when the test device is immersed in the sample. This composition can be used by dropping it directly into a test liquid such as urine, or by dropping a test liquid such as urine into this composition.

Furthermore, the test device for measuring esterase or protease of the present invention has the above composition supported on a carrier. Suitable carriers include filter paper, glass fibers, and nonwoven fabrics made of plastic materials, as long as they do not dissolve or react with solvents and absorb the above composition.

The amounts of the N-substituted indole ester compound (1) and other reagents used in the test composition and test device are not particularly important and are determined as appropriate.

That is, an amount sufficient to react with the esterase or protease to be detected and cause a color reaction is selected.

Next, the present invention will be explained in more detail with reference to Examples and Test Examples.

Example 1 1-Methoxy-3-(N-(p-)luenesulfonyl)
-L-alanyloxyindole N-hydroxyphenylglycine-2-carboxylic acid 2.0 g (9,47
+*5ol) to D M F 10d1.32g (33,
0 gmol) of 60% sodium hydride, 5.38 g (
37.9 mmol) of methyl iodide was added, and the mixture was stirred and reacted at room temperature for 2 hours in a N2 stream. 10% sodium hydroxide (3Id) was added to the reaction mixture, and the mixture was further stirred at 0°C for 30 minutes, made acidic by adding 1% hydrochloric acid, and extracted with ether 200. The ether layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain N-methoxyphenylglycine.
1.24 g of 2-carboxylic acid was obtained.

3.0 g (13.32 mmol) in 2-5d acetic anhydride
The above compound and 2.0 g of sodium acetate were added and heated under reflux for 4 hours. The reaction mixture was poured into ice water, and the precipitated crude crystals were collected by filtration, washed with water, and then dried under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1.62 g of 1-methoxy-2-acetoxyindole.

3.0 g dissolved in 20 d methanol (14.6 ms
+ol) of the above acetoxyindole was placed in a flask for 3 days, and N2 was bubbled into the reaction solution over the inlet tube. While continuing ventilation, the reaction solution was cooled to -8"C, and a deoxygenated 10% potassium hydroxide aqueous solution 18 was added from the dropping funnel, and the reaction was stirred at -8°C for 1 hour, followed by deoxygenation. Add 30 d of citric acid aqueous solution (2N) and further cool for 30 d at 0°C.
Continue stirring for minutes. The reaction mixture was extracted with deoxygenated chloroform, and the chloroform layer was dried over anhydrous magnesium sulfate and evaporated to dryness to give 2.18 g of crude 1-methoxyindoxyl.
I got g.

2.0 g (12.26 mmol) of crude 1-methoxyindoxyl and 6.0 g (24.7 mmol) of N-p-
Tor: r-7s JLt Honyl-L-alanine was dissolved in 15 d of deoxygenated methylene chloride, and 977 μm (8,0
mmol) of dimethylaminopyridine and 4.95 g (2
4.0 mmol) of dicyclohexylcarbodiimide was added thereto, and the mixture was stirred and reacted at room temperature for 10 hours in a N2 stream. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography to obtain 1-methoxy-3-((N-p-toluenesulfonyl)-L-alanyloxy-indole4.
33 g (11.1 mmol) was obtained.

NMR (δppm, CDCj23) 6.62-7.85 (m, 98), 5.70 (m,
18), 4.22 (m, LH).

3.64 (s, 3) 1), 2.46 (s, 3H), 1.
66(d, J=6tlz, 38)IR(shicm-',
CH(/!:+) 1760 Example 2 1-benzyloxy-3-(N-(p-)luenesulfonyl)-1-alanyloxy)-5-bromoindole (Ts is the same as Example 1) 3.0 g (7,89 mnol) of 4-bromo N-hydroxyphenylglycine-2-carboxylic acid was dissolved in 1 relative DMF.
1.26 g (31.6 mmol) of 60% sodium hydride and 3.50 g (27.6 mmol) of benzyl chloride were added thereto, and the reaction was stirred at room temperature for 5 hours. Cool the reaction mixture to OoC and add 3% sodium hydroxide 3d
After adding and stirring for another 30 minutes, acidified with 1% hydrochloric acid,
Extracted with ether 200 d. The ether layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 2.7 g of 4-bromo N-benzyloxyphenylglycine-2-carboxylic acid.
I got it.

3.0 g (7.89 nuwol) of the above compound and 1.5 g of sodium acetate were added to 20 d of acetic anhydride, and the mixture was heated under reflux for 5 hours. The reaction mixture was poured into ice water, and the precipitated crude crystals were collected by filtration, cooled with water, and then dried under reduced pressure. The residue was purified by silica gel column chromatography to give 1.26% of 1-benzyloxy-3-acetoxy-5-bromoindole.
I got g.

3.0 g (8.32 m
mol) of the above acetoxyindole was placed in a 30 flask, and N2 was bubbled into the reaction solution over the inlet tube. While continuing ventilation, the reaction solution was cooled to -8°C, 14 d of deoxygenated 10% potassium hydroxide aqueous solution was added from the dropping funnel, and the reaction was stirred at -8°C for 1 hour, followed by deoxygenated citric acid aqueous solution. Add 25 d of (2N) and continue stirring for an additional 30 minutes at OoC. The reaction mixture was extracted with deoxygenated chloroform, and the chloroform layer was dried over anhydrous magnesium sulfate and then dried under reduced pressure to obtain 2.15 g of crude 1-benzyloxy-5-bromo indoxyl.

2.0 g (6.29 mmol) of the above kindoxil,
3.05 g (12.6 mmol) of N-p-)luenesulfonyl-L-alanine was dissolved in deoxygenated methylene chloride 12 and cooled to 61 L [ng (5.0 mmol) of dimethylaminopyridine and 2.60 g ( 12.6 mmol
) dicyclohexylcarbodiimide was added, and the mixture was stirred and reacted at room temperature in a N2 stream for 10 hours. After concentrating the reaction mixture under reduced pressure, it was purified by silica gel column chromatography.
-benzyloxy-3-(N-(p-)luenesulfonyl)-L-alanyloxy]-5-bromoindole 3
．． 22 g (5.93 mmol) are obtained.

NMR (δppm, CDCj2s) 6.72-7.
96 (n+, 13B), 5.68 (m, 1M), 5.1
8 (s, 2H).

4.22 (m, IH), 2.44 (s, 38), 1.5
8(d, J=6Hz, 3H) IR(cm-', CHC
j23) 1760 Example 3 l-(p-)toluenesulfonyl)-3-(N-(p-toluenesulfonyl)-L-alanyloxy]-indole I T, (T is the same as Example 1) 20d anhydride 3.0 g (8,59 ml) of N-(p-toluenesulfonyl)-phenylglycine-2-carboxylic acid and 1.5 g of sodium acetate were added to acetic acid and heated under reflux for 5 hours.The reaction mixture was placed in ice water. The precipitated crude crystals were collected by filtration, washed with water, and dried under reduced pressure.This was purified by silica gel column chromatography to obtain 1.37 g of 1-(p-)luenesulfonyl)-3-acetoxyindole.

3.0 g (9,11a+) dissolved in methanol of 20 relatives
mol) of the above compound was placed in a 30 flask and placed on the introduction tube.
Bubble N2 into the reaction solution. While continuing ventilation, -
Cool the reaction solution to 8°C, add 15ml of deoxygenated 10% potassium hydroxide aqueous solution from the dropping funnel, and cool to -8°C.
After reacting with stirring for 1 hour, deoxygenated citric acid solution (
Add 25d of 2N) and continue stirring at o'c for another 30 minutes. The reaction mixture is extracted with deoxygenated chloroform, and the chloroform layer is dried over anhydrous magnesium sulfate and evaporated to dryness to obtain 2.34 g of crude 1-(p-)luenesulfonyl)-indoxyl.

2, Og (6,96 mmol) of crude 1-(p-toluenesulfonyl)-indoxyl and 3.38 g (13
, 9 mmol) of N-(p-toluenesulfonyl)-L
-Alanine was dissolved in 12 m of deoxygenated methylene chloride, and under cooling, 610 mg (5.0 mn+ol) of dimethylaminopyridine and 2.87 g (13.9 mmol) of dicyclohexylcarbodiimide were added, and the mixture was heated for 10 min at room temperature in a stream of N2.
The reaction was stirred for hours. After concentrating the reaction mixture under reduced pressure, it was purified by silica gel column chromatography to give 3.11 g (
6,07 mmol) of 1-(p-toluenesulfonyl)
-3-(N-(p-)luenesulfonyl)-L-alanyloxy)-indole was obtained.

NMR (δppm, CDCj!z) 6.86-8.
08 (m, 13H), 5.50 (m, 18), 4.70
(m.

IH), 2.48 (s, 3H), 2.46 (s, 3■)
, 1.64(d,3H)IR(sew-'-,CHCj
! 3) 1750 Example 4 1-Methylsulfonyl-3-(N-(p-toluenesulfonyl)-L-alanyloxy]-indole So, -CH.

(T is the same as in Example 1) 3.0 g (10.9 mn+ol) of N-methylsulfonylphenylglycine-2-carboxylic acid and 1.7 g of sodium acetate were added to 20 d of acetic anhydride and heated under reflux for 5 hours. .

The reaction mixture was poured into ice water, and the precipitated crude crystals were collected by filtration, washed with water, and dried under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 1.26 g of 1-methylsulfonyl-3-acetoxiindole.

3.0 g (11.8 m+) dissolved in 20 d of methanol
*ol) The above compound was placed in a 30 flask, and N2 was bubbled into the reaction solution over the inlet tube. While continuing ventilation,
Cool the reaction solution to -8°C, add 16d of deoxygenated 10% potassium hydroxide aqueous solution from the dropping funnel, and cool to -8°C.
After reacting with stirring for 1 hour, deoxygenated citric acid aqueous solution (
Add 25 d of 2N) and continue stirring at 0°C for an additional 30 minutes. The reaction mixture was extracted with deoxygenated chloroform,
The chloroform layer was dried over anhydrous magnesium sulfate and then dried under reduced pressure to obtain 2.17 g of 1-methylsulfonylindoxyl.

2.0 g (9.47 mmol) of crude 1-methylsulfonylindoxyl and 4.60 g (18.9 mmol) of N-P-tolu Isulfonyl-L-alanine were dissolved in 15 Ildl of deoxygenated methylene chloride and cooled. 733 m
g (6.0 mmol) of dimethylaminopyridine and 3.
90 g (18.9 mmol) of dicyclohexylcarbodiimide was added, and the reaction was stirred at room temperature in a N2 stream for 10 hours. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography to give 3.05 g (6.99 mmol).
1-methylsulfonyl-3-(N-(p-)luenesulfonyl)-L-alanyloxy]-indole is obtained.

NMR (δppm, CDC13) 6.80-7.98 (n+, 9B), 5.62 (m, I
H) +4.18 (m, IH).

2.84 (s, 31ri), 2.44 (s, 3H),
1.64(d,3H)IR(shicm-',CHCj!s
) 1750 Example 5 [Composition Production Example 1] Solution ■ 0, LM Tris-HCj2 buffer
100d and solution (1) ethanol/decanol 98:2 100d were mixed to obtain a test composition for measuring esterase or protease according to the present invention.

Example 6 [Production Example 2 of Composition] Solution IO, IM Tris-H (F! Buffer
100d and solution■ Acetone/decanol 98:2 100m
j! were mixed to obtain a test composition for measuring esterase or protease according to the present invention.

Example 7 [Test paper production example 1] Solution ■ 0, IM Tris-HCj! buffer solution
100d solution (1) is dissolved in ethanol/1-decanol 98:2100d.

A test paper according to the present invention was obtained by impregnating a filter paper with solution (1) and drying it at 60°C for 50 minutes, then impregnating it with solution (2) and drying it at 40°C for 20 minutes.

Example 8 [Test paper production example 2] Solution ■ 0, IM Tris-H (J!1,1 street liquid
100 - Dissolve solution ■ in acetone/decanol 98:2 LooIRft.

A test paper according to the present invention was obtained by impregnating a filter paper with Solution I and drying it at 60°C for 50 minutes, then impregnating it with Solution II and drying it at 40°C for 20 minutes.

[Test example]

The test strip obtained in Example 7.8 above is soaked in urine for 1 second. The color development of the test paper is visually read over time.

As a result, in both Examples 7 and 8, it was possible to detect 10 leukocytes/ill urine in a determination time of 90 seconds, and the color of the test paper changed from white to purple.

〔Effect of the invention〕

According to the present invention, novel N-substituted indole ester compounds are provided.

Furthermore, the present invention provides an advantageous method for producing such N-substituted indole ester compounds.

Furthermore, the N-substituted indole ester compounds of the present invention are effectively used for detecting esterases or proteases, particularly leukocytes.

Conventionally known 3-(N-(p-)luenesulfonyl)-L-alanyloxy)indole and 2-methoxy-
It takes 120 seconds to determine the ingredients of the combination of 4-morpholinopenzolediazonium-tetrachlorzincate using a test paper, but the test paper we developed this time takes 90 seconds.
Since the determination can be made in seconds, the present invention provides a test composition and a test device with superior performance.

As described above, the N-substituted indole ester compound (Il) of the present invention has excellent performance as a substrate for measuring esterase or protease.

Claims (1)

[Claims] 1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) [In the formula, R^1 represents an alkoxy group, an aralkoxy group, or an aryloxy group, but a halogen atom, an alkoxy group, or It may be substituted with a nitro group, or R^1 in the formula represents an alkylsulfonyl group or an arylsulfonyl group. R^2 represents a hydrogen atom, a halogen atom, a hydroxy group, a carboxyl group, an acylamino group, a nitro group, a carboxyl group, an alkoxycarbonyl group, an alkoxy group, or an amide group. X and Y represent commonly used protecting groups for amino groups. ] N-substituted indole ester compound represented by these. 2) The compound according to claim 1, wherein R^1 in the above formula (I) is a methoxy group, a benzyloxy group, a p-toluenesulfonyl group, or a methylsulfonyl group. 3) The N-substituted indole ester compound in the above formula (I) is 1-methoxy-3-[N-(p-toluenesulfonyl)-L-alanyloxy]indole, 1-benzyloxy-3-[N-( p-toluenesulfonyl)-
L-alanyloxy]-5-bromoindole, 1-(
A patent claim that is p-toluenesulfonyl)-3-[N-(p-toluenesulfonyl)-L-alanyloxy]indole or 1-methylsulfonyl-3-[N-(p-toluenesulfonyl)-L-alanyloxy]indole A compound according to item 1 of the scope. 4) General formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (II) [In the formula, R^1 represents an alkoxy group, aralkoxy group, or aryloxy group, but it is not a halogen atom, an alkoxy group, or a nitro group. May be replaced. Alternatively, R^1 in the formula represents an alkylsulfonyl group or an arylsulfonyl group. R^2 represents a hydrogen atom, a halogen atom, a hydroxy carboxyl group, an acylamino group, a nitro group, a carboxyl group, an alkoxycarbonyl group, an alkoxy group, or an amide group. ] Compounds represented by the general formula (III) ▲Mathematical formulas, chemical formulas, tables, etc.▼(III) [In the formula, Z represents a hydroxy group, a halogen atom, an alkoxycarbonyloxy group, or an imidazole group. X,
Y represents a commonly used protecting group for amino groups. ] General formula (I) characterized by condensation with the compound represented by ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) It has the same meaning as a thing. ] A method for producing an N-substituted indole ester compound represented by: 5) The N-substituted indole ester compound according to any one of claims 1 to 3 and the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (IV) or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (V) [In the formula, R^3 represents an alkyl group, an alkoxy group, a halogen atom, or a hydrogen atom, and R^4 represents an alkylene group,
m and n are the same or different integers of 1, 2, and 3, and Z
indicates a stabilizing anion. Also, A ▲ has mathematical formulas, chemical formulas, tables, etc. ▼ indicates an alkylamine or an oxygen atom. ] An esterase or protease measurement-like test composition containing a diazonium salt compound represented by: 6) The composition according to claim 5, wherein in the above formula (IV) or (V), R^3 is a methoxy group. 7) A test device for measuring esterase or protease, comprising a carrier supporting the composition according to claim 5 or 6.