WO2008047655A1

WO2008047655A1 - Urea derivative for use in quantification of hydrogen peroxide

Info

Publication number: WO2008047655A1
Application number: PCT/JP2007/069799
Authority: WO
Inventors: Katsuya Noguchi; Eiji Watanabe; Munetaka Ishiyama
Original assignee: Dojindo Laboratories
Priority date: 2006-10-16
Filing date: 2007-10-11
Publication date: 2008-04-24
Also published as: JP2010001217A

Abstract

Disclosed is a novel substance which has excellent solubility and stability in an aqueous solution corresponding to a biological liquid sample and can be suitably used as a clinical reagent for quantifying hydrogen peroxide in the presence of a peroxidase. Specifically disclosed is an urea derivative represented by the formula (I) [wherein R1 represents an atomic group represented by the formula (II); R2 and R3 independently represent a 4-disubstituted aminoaryl group, provided the aryl group in R2 and the aryl group in R3 may be linked to each other through a sulfur or oxygen atom, and at least one site in the aryl group in each of R2 and R3 may be substituted by a sulfonate group; n represents an integer of 1 to 4; and X represents a hydrogen, sodium or potassium atom].

Description

Specification

Urea derivatives used for the determination of hydrogen peroxide

Technical field

The present invention relates to a novel compound having a urea derivative structure, and is particularly suitable for use as a color reagent for quantifying hydrogen peroxide in the presence of peroxidase in clinical tests. It relates to new compounds.

Background art

[0002] In the living body, several reactions are observed in which a substrate is oxidized by the action of various oxidases, and hydrogen peroxide (H 2 O 2) is generated at that time. Examples include uric acid oxidation by uricase and cholesterol oxidation by cholesterol oxidase. Therefore, quantification of the substrate by quantifying the amount of hydrogen peroxide produced is an important means in clinical tests, and for this purpose, it reacts with hydrogen peroxide in the presence of peroxidase to give color. These compounds are used as clinical reagents.

[0003] A representative example of a color reagent for quantifying hydrogen peroxide in the presence of peroxidase is a compound having a urea derivative structure. For example, Japanese Patent Publication No. 60-33479 (Patent Document 1) discloses a compound having a structure in which two substituted phenyl groups are bonded to urea. Furthermore, Japanese Patent Publication No. 7-121901 (Patent Document 2) proposes a compound having a urea derivative structure that has improved properties such as solubility in water compared to the compound described in Patent Document 1 above. Has been. The compound described in Patent Document 2 has improved water solubility compared to the compound of Patent Document 1. It is still insufficient for use in clinical tests, and is not stable in aqueous solution. The problem remains.

Patent Document 1: Japanese Patent Publication No. 60-33479

Patent Document 2: Japanese Patent Publication No. 7-121901

Disclosure of the invention

Problems to be solved by the invention

[0004] The purpose of the present invention is to achieve solubility and stability in aqueous solutions corresponding to biological fluid samples. It is to provide a new substance that is excellent in use and suitable for use as a clinical reagent for the determination of hydrogen peroxide in the presence of peroxidase.

Means for solving the problem

[0005] As a result of repeated research, the present inventor succeeded in synthesizing a compound that can achieve the above-mentioned object, and derived the present invention.

That is, the present invention is a color reagent that comprises a urea derivative represented by the following general formula (I) and the urea derivative and is used for the determination of hydrogen peroxide in the presence of peroxidase.

[0006] [Chemical 1]

R2- N- R ³

[0007] In formula (I), R ¹ represents an atomic group represented by the following general formula (R), and R ² and R ³ each independently represents a 4-disubstituted aminoaryl group. The aryl groups of R ² and R ³ may be bonded to each other via a sulfur atom or an oxygen atom, and at least one of the aryl groups of R ² and R ^{3 is} an S sulfonic acid group. It ’s replaced!

[0008] [Chemical 2]

II H

_{~ CH 2 C - N - (} CH 2) "- S OaX formula ([pi), n represents an integer of 1 to 4, X represents a hydrogen atom, sodium atom or potassium atom.

The invention's effect

[0009] The compound of the present invention (urea derivative) has very high solubility in water, and also has very good stability in an aqueous solution. Therefore, the compounds of the present invention are used in clinical tests at high concentrations for the determination of hydrogen peroxide in the presence of peroxidase. It is particularly useful as a color reagent.

Brief Description of Drawings

FIG. 1 shows a reaction scheme for synthesizing an example of a compound of the present invention (urea derivative).

FIG. 2A shows a calibration curve when hydrogen peroxide is quantified in the presence of peroxidase using the compound of the present invention and a comparative compound.

FIG. 2B shows a calibration curve when hydrogen peroxide is quantified in the presence of peroxidase using the compound of the present invention and a comparative compound.

FIG. 3A shows the stability in aqueous solutions measured for the compounds of the present invention and comparative compounds.

FIG. 3B shows the stability in aqueous solutions measured for the compounds of the present invention and comparative compounds.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The compound serving as the color reagent for quantifying hydrogen peroxide described in Patent Document 2 is the terminal of the atomic group represented by R ¹ in the formula (I) representing the urea derivative of the present invention. It is considered that a carboxyl group, an arylsulfonyl group, or a sulfoaryl group was present in the water to improve the water solubility. In particular, Patent Document 2 discloses a compound represented by the following formulas (C 1) and (C 2), for example, as being extremely easily soluble in water.

[0012] [Chemical 3]

[0013] [Chemical 4]

[0014] These compounds are sufficiently satisfactory in the solubility in water and the stability in aqueous solution as a color reagent used at high concentration to quantify hydrogen peroxide in the presence of peroxidase in clinical examinations. It shouldn't have been.

On the other hand, the compound of the present invention (urea derivative) has a specific atomic group as R ¹ of the formula (I), that is, an atomic group represented by the formula (Π). In particular, water solubility is higher than the compounds of the above formulas (C 1) and (C 2), and stability in aqueous solution is also good. Yes (see examples below).

[0016] In the formula (Π) that represents R ^{1 of the} formula (I) representing the urea derivative of the present invention, n is a force that is an integer of;! To 4, preferably 2. In the formula (Π), X represents a hydrogen atom, a sodium atom or a potassium atom, preferably a sodium atom. Further, R ² and R ³ in formula (I) each independently represent a 4-disubstituted aminoaryl group, and the aryl groups of R ² and R ³ are bonded to each other via a sulfur atom or an oxygen atom. Also good. Furthermore, this embodiment, which may be substituted with at least one S sulfonic acid group in the aryl group of R ² and R ³ , includes the case where a sulfonic acid group is bonded to the substituted amino group. Here, a phenyl group is preferred as an aryl group. As an example of the urea derivative of the present invention particularly preferred for constituting a color reagent represented by the formula (I) and used for the determination of hydrogen peroxide in the presence of peroxidase, the following formula ( Examples thereof include, but are not limited to, compounds represented by III) or (IV).

[0017] [Chemical 5]

[0018] [Chemical 6]

[0019] The urea derivative of the present invention represented by the formula (I) can be synthesized by the synthesis method found by the present inventors. That is, as illustrated in FIG. 1, a compound in which R ² is CH C OONa in the formula (I) is used as a raw material (therefore, this raw material compound is a compound of the formula (C 1) or the formula (C 2). (This corresponds to the target compound of Patent Document 2), and this is reacted with an aminoalkanesulfonic acid such as taurine (2-aminoethanesulfonic acid) using a carpositimide such as dicyclohexylcarpositimide as a dehydrating condensing agent. (See examples below)

In order to show the features of the present invention more specifically, examples are described below. The present invention is not limited to these examples.

Example 1

[0020] Synthesis of Ν, Ν-bis (4-dimethylaminophenyl) N '(sulfoethyl) aminocarbonylmethylurea, Na salt “the present compound (111)”

According to the reaction formula shown in (A) of FIG. 1, the compound of the present invention of the formula (III) was synthesized as follows. To 2.0 g of the compound of formula (C 1) was added 0.1N HCllOml and concentrated. 20ml THF to concentrate 3 ml of water and 3.27 g of dicyclohexyl carpositimide were added and stirred in an ice bath. After stirring for 30 minutes, 0.662 g of taurine and 0.444 g of sodium bicarbonate were dissolved in 3 ml of water and added to the reaction solution. -After stirring at room temperature, 2.18 g of dicyclohexyl carpositimide was further added. After 3 hours, stirring was stopped and the reaction solution was filtered and concentrated. After adding 20 ml of water to the concentrate and filtering, the filtrate was concentrated to obtain 2.61 g of a pale blue-white solid, which was subjected to column purification with chloroform: methanol = 9: 1-7: 3 to obtain 1.73 g of a white powder. The suspension was washed with 100 ml of black mouth form, filtered, and the filtered material was dried under reduced pressure to obtain 1.66 g of a white powder.

[Identification data]

TLC (silica gel, butanol: acetic acid: pyridine: water = 4: 1: 1: 2): Rf = 0.45.

NMR (D 0): δ ppm 2 · 82 (12Η,

s, -N- (CH)), 3.08 ppm (2H, d, — CH—CH—SO Na), 3.61 ppm (2H, d, — CH— CH—SO

Na), 3.76 ppm (2H, s, -NH_CH -CO-), 6.92-7.27 ppm (8H, m, aromatic H).

MS: [M-Na] = 462.

Example 2

10-"(sulfoechinole) aminocarbonylmethyl- (aminocarbonyl) 1 -3.7-bis (dimethylamino) phenothiazine, Na salt" Synthesis of Compound (1) of the Present Invention

According to the reaction formula shown in FIG. 1 (B), the compound of the present invention of the formula (IV) was synthesized as follows. The compound l.Og of the formula (C2) was charged with 0.1N HCllOml and concentrated. To the concentrate were added THFlOml, 3 ml of water and 2.16 g of dicyclohexyl carpositimide, and the mixture was stirred in an ice bath. After stirring for 30 minutes, 0.328 g of taurine and 0.213 g of sodium bicarbonate were dissolved in 3 ml of water and added to the reaction solution. After 3 hours, stirring was stopped and the reaction solution was filtered and concentrated. The concentrate was purified by chromoform form and dried under reduced pressure to obtain 0.40 g of white powder.

[Identification data]

TLC (silica gel, black mouth form: methanol = 9: 1): Rf = 0.15.

NMR (D Ο): δ ppm 2 · 82 (12 Η, s,-N_ (CH)), 3.12 ppm (2H, d,-CH-CH-SO Na), 3. 70 ppm (2H, d,-CH- CH ^ SO ^ a), 3.80 ppm (2H, s, —NH—CF ^ —CO—), 6.50—7.41 ppm (6H, m, aromatic H).

MS: [M-Na] — = 492. Example 3

[0022] Determination of vortex hydrogen oxide

(Measurement solution)

The compounds (III) and (IV) of the present invention and the compounds (C 1) and (C 2) described in Patent Document 2 for comparison have a concentration of 0 · lmmol / U peroxidase at a concentration of 3.3 u / ml. Thus, it was dissolved in 50 mM MES [2 morpholinoethanesulfonic acid] sodium hydroxide buffer (pH 5.5).

[Sample solution]

Commercially available hydrogen peroxide was diluted with pure water to a concentration of 1 mmol / 1.

〔Measuring method〕

The sample solution was added to 3 ml of the measurement solution 1.5, 3.0, 15 and 301 and heated at 37 ° C for 5 minutes, and then the absorbance at the maximum absorption wavelength was measured.

[Result]

Figures 2A and 2B show the relationship between hydrogen peroxide concentration and absorbance. From FIG. 2A and FIG. 2B, the calibration curve of absorbance at the maximum absorption wavelength plotted against each hydrogen peroxide concentration M) showed quantitativeness. The compounds (III) and (IV) of the present invention showed the same or better sensitivity than the comparative compounds (C 1) and (C 2).

Example 4

[0023] 7k

The compounds (III) and (IV) of the present invention and the comparative compounds (C 1) and (C 2) were each dissolved in pure water at 25 ° C., and the solubility was determined. Table 1 shows the solubilities of the compounds (III) and (IV) of the present invention and the comparative compounds (C 1) and (C 2).

[0024] [Table 1] Solubility in water

[0025] As shown in Table 1, the compounds (III) and (IV) of the present invention are more water-soluble than (C 1) and (C 2), respectively.

Example 5

[0026] Solution stability

[Preservation solution]

The compounds (III) and (IV) of the present invention and the comparative compounds (C 1) and (C 2) were each prepared with 50 mM MES sodium hydroxide buffer (ρΗ5 · 5) so as to be 10 mmol / l. Stored at 40 ° C, protected from light.

[Sample solution]

〔Measuring method〕

Add 2.9 ml of 50 mM MES sodium hydroxide buffer (ρΗ5 · 5), peroxidase aqueous solution (33011 / ½1) 30 1 and 30 1 to the stock solution 30 1, heat at 37 ° C for 5 minutes, and then absorb maximum. Absorbance at wavelength was measured.

[Result]

Figures 3A and 3B show the relationship between elapsed days and absorbance. As shown in FIGS. 3A and 3B, the compounds (III) and (IV) of the present invention have better stability than the comparative compounds (C 1) and (C 2).

Claims

The scope of the claims

A urea derivative represented by the following general formula (I):

[In formula (I), R ¹ represents an atomic group represented by the following general formula (Π), IT and R ³ each independently represents a 4-disubstituted aminoaryl group, R The aryl groups of ² and R ³ may be bonded to each other via a sulfur atom or an oxygen atom, and at least one of the aryl groups of R ² and R ³ is substituted with an S sulfonic acid group. May be. ]

[Chemical 2]

Yes

II H,

~ CH ₂ -C-N-(CH ₂ ) _N -S 0 ₃ X ^UIJ

[In the formula (Π), n represents an integer of 1 to 4, and X represents a hydrogen atom, a sodium atom or a potassium atom. ]

The urea derivative according to claim 1, which is represented by the following formula (III) or formula (IV):

[Chemical 3]

[Chemical 4]

[3] A color reagent comprising the urea derivative according to any one of claims 1 and 2, which is used for the determination of hydrogen peroxide in the presence of peroxidase.