JPH10182688A - Hydroxylalkylpyridine derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound consisting of a specific hydroxyalkylpyridine derivative, useful as a substrate for the measurement of an N-acetylglucosaminidase (NAG) activity excellent in sensitivity and stability, and used for an early diagnostic rea gent of a renal disease. SOLUTION: This new hydroxyalkylpyridine derivative is expressed by formula I (G is a reductive terminal bonding at a β-position and a hexosamine residue having an amino group boded with an acyl group) (e.g.; 3-hydroxymethyl-2- pyridyl-N-acetyl-1-thio-β-D-glucosaminide), and used as a substrate for the measurement of glucosaminidase activity for an early diagnostic reagent of a renal disease. The compound is obtained by reacting a hydroxyalkylpridinethiol derivative expressed by formula II with 2-acetamide-3,4,6-tri-0-acetyl-2-deoxy-α-D- glucopyranosyl chloride, etc., and then performing a deacylation reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to N-acetyl-β
Novel hydroxyalkyl useful as a substrate for activity measurement of -D-glucosaminidase (hereinafter abbreviated as NAG), N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-hexosaminidase and the like. The present invention relates to a pyridine derivative and a method for measuring NAG activity using the same.

[0002]

2. Description of the Related Art NAG is one of glycolytic enzymes present in lysosomes of various cells, and is particularly contained in a large amount in the proximal tubule epithelial cells of the kidney, and enters into the urine due to damage to the renal tubule. Due to its release, its activity in urine is an important indicator of kidney disease. Urinary NAG activity is known to increase due to acute renal failure, chronic renal failure, nephrotic syndrome, glomerulonephritis, diabetic nephropathy and renal damage caused by drugs, and is an early diagnostic index for these renal diseases . Urinary NAG activity also plays an important role as an index for early diagnosis of rejection after kidney transplantation. That is, measuring NAG activity has a very important significance from a clinical point of view.

[0003] Conventionally, various methods have been developed as a method for measuring NAG activity. Basically, compounds released by the action of NAG are detected using a fluorescence detector or an ultraviolet-visible absorbance meter. A method for determining NAG activity based on the method is generally employed. As the measuring method in the field of clinical examination at present, an initial velocity measuring method using a synthetic substrate is mainly used.

The following is a typical example of a conventional method for measuring NAG activity. 4-methylumbelliferyl-N-acetyl-β-D
A method of measuring the fluorescence intensity of 4-methylumbelliferone released by an enzymatic reaction with NAG using glucosaminide as a substrate [Japanese Patent Laid-Open No. 1-265897]. Using p-nitrophenyl-N-acetyl-β-D-glucosaminide as a substrate, p-nitrophenol released by an enzymatic reaction with NAG is made alkaline to develop a color, which is then colorimetrically determined [Clin. Ch
em. , 27, 1180 (1981)]. Using sodio-m-cresol sulfophthalenyl-N-acetyl-β-D-glucosaminide as a substrate, NAG
M-cresolsulfophthalein released by the enzymatic reaction according to the above method is made alkaline by colorimetric determination [Clin. Chem. , 29,17
13 (1983), JP-B-63-7196]. A method for directly colorimetrically determining chlorophenol red released by an enzyme reaction with NAG using sodio-3,3'-dichlorophenolsulfophthalenyl-N-acetyl-β-D-glucosaminide as a substrate [Japanese Patent Laid-Open No. Sho 63]
-309199]. 2-chloro-4-nitrophenyl-N-acetyl-β
A method of directly colorimetrically determining 2-chloro-4-nitrophenol released by an enzymatic reaction with NAG using -D-glucosaminide as a substrate [Japanese Patent Application Laid-Open No. Sho 62-48399]. 2-fluoro-4-nitrophenyl-N-acetyl-
Using β-D-glucosaminide as a substrate, a method for directly colorimetrically determining 2-fluoro-4-nitrophenol released by an enzyme reaction with NAG [Japanese Patent Publication No. 5-73398]
And Japanese Patent Publication No. 5-55517]. Using p-nitrophenyl-N-acetyl-β-D-glucosaminide as a substrate, oxidase (N-acetylglucosamine) is added to N-acetylglucosamine released by an enzyme reaction with NAG.
Acetyl glucosamine oxidase (NAGOD) is acted on, and the generated hydrogen peroxide is converted to peroxidase (PO
D) A method of performing colorimetric determination by reacting with a color developing agent in the presence [instrument / reagent, 13, 887 (1990)]. 6-Methyl-2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide (6-MPT-NAGS) is used as a substrate to be released by an enzyme reaction with NAG.
Method for quantifying -methyl-2-pyridinethiol by determining its absorbance at a specific wavelength [
No. 59083]

[0005] However, each of these methods has many problems in terms of the water solubility of the substrate, the measurement sensitivity, the stability of the solution, and the like. That is, for example, among the measurement methods described above,
The measurement method of (1) has a problem that a special measuring device such as a fluorescence intensity meter is required. And, the chromogen released by the enzymatic reaction has a high pKa,
At the optimal pH of AG, measurement sensitivity sufficient to perform colorimetric quantification cannot be obtained, so that colorimetric quantification must be performed as alkaline after the reaction is stopped, and it cannot be used in the rate assay that is currently dominant. That is, this measurement method is not suitable for rapid multiple sample processing.

Therefore, as a measuring method applicable to a rate assay, a method using a substrate having a reduced pKa of a chromogen moiety and a measuring method using a substrate have been developed.
At the optimum pH of NAG of 4.5 to 5.0, the dissociation (that is, color development) of the chromogen generated by the enzymatic reaction is insufficient, so that the absorbance fluctuates even with slight pH fluctuation in the solution. However, there is a problem that a measurement error easily occurs and the long-term stability of the liquid after dissolving the substrate is poor.

[0007] In the above methods, the maximum absorption wavelength of the released chromogen is around 400 nm.
There is also a problem that it is easily affected by biological components such as bilirubin. Is applicable to rate assays, but since it is a measurement system that generates hydrogen peroxide using an oxidase, it can be used for reducing substances such as bilirubin and other coexisting substances contained in biological components. It has a problem that it is easily affected and, like the above-mentioned substrates, the stability after dissolution of the substrate is poor.

[0008] On the other hand, a measuring method has been developed to solve these problems, but the stability of the substrate used in this method has been improved in the short term, but the long-term stability after dissolving the substrate. In consideration of the above, the tendency to decompose in an acidic to alkaline region was observed, and thus was not always satisfactory.

[0009]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above situation, and uses a hydroxyalkylpyridine derivative which is excellent in sensitivity and stability and is useful as a substrate for measuring NAG activity, and using the same as a substrate. An object of the present invention is to provide a method for measuring NAG activity and a reagent therefor.

[0010]

The present invention provides a compound represented by the general formula [1]:

[0011]

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(Wherein G represents a hexosamine residue having a β-bond at the reducing end and an acyl group bonded to an amino group, and R represents a hydroxyalkyl group). It is an invention of a derivative and a method for measuring NAG activity, wherein the derivative is used as a substrate. That is, the present inventors have conducted intensive studies on synthetic substrates that can be effectively used in the measurement of NAG activity. As a result, if the hydroxyalkylpyridine derivative represented by the general formula [1] is used as a substrate, the problems described above will occur. The inventors have found that the points can be solved, and have completed the present invention.

In the general formula [1], the acyl group represented by G in the hexosamine residue having an acyl group bonded to an amino group is derived from an aliphatic carboxylic acid or an aliphatic hydroxycarboxylic acid. And acyl groups derived from aliphatic aminocarboxylic acids and aromatic carboxylic acids.
Here, as the aliphatic carboxylic acid, for example, having 2 to 2 carbon atoms
8, preferably those having 2 to 4 carbon atoms, specifically, for example, acetic acid, propionic acid, butyric acid, isobutyric acid and the like. The aliphatic hydroxycarboxylic acid may be any carboxylic acid having a hydroxyl group, but is preferably, for example, a carboxylic acid having 1 to 7 carbon atoms, more preferably a carboxylic acid having a hydroxyalkyl group having 1 to 4 carbon atoms. Examples include acids, specifically, carboxylic acids having a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, a hydroxybutyl group, and the like. The aliphatic amino carboxylic acid may be any carboxylic acid having an amino group, preferably a so-called amino acid, and more preferably an essential amino acid such as methionine, leucine, glycine, and alanine. Amino acids. Further, examples of the aromatic carboxylic acid include benzoic acid.

The hexosamine residue in the hexosamine residue represented by G in which an acyl group is bonded to an amino group includes, for example, a glucosamine residue and a galactosamine residue. Preferred specific examples of the hexosamine residue having an acyl group bonded to an amino group include, for example, N-
Acetylglucosamine, N-acetylgalactosamine,
N-acylhexosamines such as N-propionylglucosamine, N-propionylgalactosamine, N-benzoylglucosamine, N-benzoylgalactosamine, etc., from which the hydroxyl group at the 1-position has been eliminated (residues).

The hydroxyalkyl group represented by R may be linear or branched, and is preferably a hydroxyalkyl group having 1 to 7 carbon atoms, more preferably 1 to 3 carbon atoms. Specifically, for example, a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxyethyl group, a 1-hydroxypropyl group, a 2-hydroxypropyl group, a 3-hydroxypropyl group, a 1-methyl-1-hydroxyethyl group, And a methyl-2-hydroxyethyl group.

The pyridine ring in the general formula [1] may further have a substituent, and preferred substituents include, for example, a halogen atom, an alkyl group and an alkoxy group. Here, examples of the halogen atom include chlorine, bromine, iodine and the like. The alkyl group may be straight-chain or branched, and preferably has 1 to 3 carbon atoms. Specifically, for example, a methyl group, an ethyl group, n
-Propyl group, isopropyl group and the like. The alkoxy group may be linear or branched and includes, for example, those having 1 to 3 carbon atoms. Specific examples include a methoxy group, an ethoxy group, an n-propoxy group, and an isopropoxy group.

Preferred specific examples of the hydroxyalkylpyridine derivative of the present invention include the following compounds. 3-hydroxymethyl-2-pyridyl-N
-Acetyl-1-thio-β-D-glucosaminide, 4-
Hydroxymethyl-2-pyridyl-N-acetyl-1-
Thio-β-D-glucosaminide, 5-hydroxymethyl-2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide, 6-hydroxymethyl-2-pyridyl-
N-acetyl-1-thio-β-D-glucosaminide, 5
-(1-hydroxyethyl) -2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide, 5-hydroxymethyl-4-methyl-2-pyridyl-N-acetyl-1-thio-β-D -Glucosaminide, 5-hydroxymethyl-3-methyl-2-pyridyl-N-acetyl-1
Thio-β-D-glucosaminide, a compound in which glucosaminide of the above compound is replaced with galactosaminide, and / or an acetyl group bonded to an amino group is, for example, a propionyl group, a butyryl group, a benzoyl group, a methionine residue, a leucine residue. , An alanine residue, a glycine residue and the like.

The hydroxyalkylpyridine derivative of the present invention is sufficiently soluble in water as a substrate for measuring NAG activity, since it is dissolved in water in an amount of 200 mM or more. When the aqueous solution is used, a wide pH range, for example, pH 4.5 to pH 1
When it is in the range of 0, it is stable for a long time, and particularly in the range of pH 7 to pH 10, it can be extremely stable for a long time. Furthermore, since this derivative does not have an absorption wavelength of 320 nm or more, it has a property of not affecting the absorbance change measurement derived from the hydroxyalkylpyridine thiol derivative (maximum absorption wavelength: 320 to 360 nm) released from this derivative. I have. In addition, among the compounds represented by the general formula [1], N
A suitable substrate for measuring AG activity is NAG
Is preferably 0.5 to 5.0 mM, more preferably 1.0 to 3.5 mM, and still more preferably 1.5.
It is preferably about 1.7 mM.

The hydroxyalkylpyridine derivative of the present invention has the general formula [2]

[0020]

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(In the formula, R represents a hydroxyalkyl group.) The compound can be easily synthesized from a hydroxyalkylpyridinethiol derivative represented by the formula: That is, the compound represented by the general formula [2] has a group capable of reacting with an SH group at the 1-position (eg, a halogen such as -Cl, -Br, -I, etc.), and the amino group corresponds to the target compound. A hexosamine residue modified with an acyl group and the hydroxyl group of which may be optionally protected, for example, 2-acetamide-
3,4,6-tri-O-acetyl-2-deoxy-α-
D-glucopyranosyl chloride (for example, Org. S
ynth. 46 , 1 (1966)) and an alkaline aqueous solution in the presence of a phase transfer catalyst such as tetra-n-butylammonium bromide. And the like, and the resulting S-glycosylated form is subjected to deacylation of the hydroxyl group with a metal alkoxide such as sodium methoxide to give the compound of the present invention. Further, instead of the above glycosylation reaction, the compound of the present invention may be synthesized by a direct or indirect ordinary glycosidic bond formation reaction.

The hydroxyalkylpyridinethiol derivative represented by the general formula [2], which is a raw material for synthesizing the hydroxyalkylpyridine derivative of the present invention, can be synthesized, for example, by the following method. That is, first, the general formula [3]

[0023]

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(Wherein, R ¹ represents a substituent having a carbonyl group, and X represents a halogen atom such as Cl, Br, I), for example, LiAlH ₄ or N 2
reduction by a conventional method using a reducing agent ABH ₄ such as 2-halogeno - a hydroxyalkyl pyridine derivatives.

Next, the obtained 2-halogeno-hydroxyalkylpyridine derivative is thiolated by a conventional method using a general thiolating agent such as NaSH to give a hydroxyalkyl represented by the general formula [2]. A pyridinethiol derivative is obtained. Examples of the enzyme whose activity can be measured using the compound represented by the general formula [1] as a substrate include NAG, N-acetyl-β-D-galactosaminidase, N-acetyl-β-D-hexosaminidase and the like. And preferably NAG, N-acetyl-β-
D-hexosaminidase, more preferably NAG.

By reacting NAG with a hydroxyalkylpyridine derivative represented by the general formula [1], a hydroxyalkylpyridinethiol derivative represented by the following general formula [2] is released. General formula [2]

[0027]

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(In the formula, R is the same as described above.) The hydroxyalkylpyridinethiol derivative released from the hydroxyalkylpyridine derivative of the present invention by an enzyme reaction such as NAG exhibits a maximum absorption in the range of 320 nm to 360 nm. For example, an ultraviolet region (320 nm or more) not affected by bilirubin, hemoglobin, or the like.
380 nm). In particular, for example, 3-hydroxymethyl-2-pyridinethiol (maximum absorption wavelength 341 nm) and 4-hydroxymethyl-2-pyridinethiol (maximum absorption wavelength 339 nm) in which a hydroxyalkyl group is substituted at the 3- or 4-position of the pyridine ring are exemplified. Since the maximum absorption wavelength is almost the same as the set wavelength (340 nm) of an automatic analyzer that is currently mainstream in the clinical examination field, the measurement wavelength can be set to the peak of the maximum absorption wavelength. That is, when the hydroxyalkyl derivative of the present invention is used as a substrate for measuring the activity of NAG or the like, errors and variations in measured values caused by analytical models and measurement conditions in the activity of NAG or the like can be minimized.

The hydroxyalkylpyridine derivative, which is the compound of the present invention, has a low reagent blank rise over the entire pH range, and particularly when stored in a solution having a pH of 7 or more, raises the reagent blank over a long term of at least 3 months or more. Is very useful as a substrate for measuring NAG activity. It is also desirable to use the hydroxyalkylpyridine derivative of the present invention as a substrate for a reagent for measuring NAG activity by a two-part method, particularly because the storage stability in a solution state increases.

The method for measuring NAG activity of the present invention using a hydroxyalkylpyridine derivative represented by the general formula [1] has a known initial rate except that the hydroxyalkylpyridine derivative represented by the general formula [1] is used as a substrate. It suffices to measure according to the law. Specifically, for example, the following method is included.

(I) Method 1 For example, a biological sample such as serum, blood, urine, etc.
A reagent solution containing a hydroxyalkylpyridine derivative represented by the general formula [1] and an appropriate buffer, which has been incubated at -50 ° C, preferably 30-40 ° C (pH is usually 3.0-7.0, preferably Is 4.0 to 6.5) and 20
The reaction is carried out at ５０50 ° C., preferably 30-40 ° C. NA
The amount of increase in the hydroxyalkylpyridine thiol derivative produced by the action of G is measured as an amount of change in absorbance per unit time at 320 to 380 nm using an appropriate measuring device such as a spectrophotometer. By converting the obtained change in absorbance into units using the molecular extinction coefficient of the hydroxyalkylpyridine thiol derivative, the N
AG activity can be determined. The sample solution used here is a pH solution containing a hydroxyalkylpyridine derivative represented by the general formula [1] and an appropriate buffer.
1 It is preferably prepared by mixing a reagent solution having a pH of 7 to 9 with a reagent solution containing a suitable buffer (the pH is usually about 3.0 to 7.0, preferably about 4.0 to 6.5). It may be something.

(Ii) Method 2 A sample derived from a living body is mixed with a first reagent solution containing an appropriate buffer (pH is usually 3.0 to 7.0, preferably about 4.0 to 6.5). 20 to 50 ° C., preferably 30 to 40 ° C.
After incubation for an appropriate time at
A hydroxyalkylpyridine derivative represented by the general formula [1], which has been incubated at -50 ° C, preferably 30-40 ° C, is mixed with a second reagent solution containing an appropriate buffer, and the mixture is mixed at 20-50 ° C, preferably 30 ° C. React at ４０40 ° C. The increase in the amount of the hydroxyalkylpyridine thiol derivative generated by the action of NAG is measured as an amount of change in absorbance per unit time at 320 to 380 nm using a suitable measuring device such as a spectrophotometer. The NAG activity in the sample can be determined by converting the obtained change in absorbance into a unit using the molecular absorption coefficient of the hydroxyalkylpyridine thiol derivative.

In the above method, the pH of the second reagent solution and the concentration of the buffering agent are adjusted to be 3.0 to 7.0, preferably 4.0 to 6, when mixed with the first reagent solution. The pH of the hydroxyalkylpyridine derivative of the present invention is from 7 to 11, preferably from 7 to 11, considering the stability of the hydroxyalkylpyridine derivative of the present invention in an aqueous solution.
It is desirable to set it in the range of -9.

In the above methods (i) and (ii),
The concentration of the hydroxyalkylpyridine derivative of the present invention used as a substrate may be any concentration at which the activity of NAG can be measured, and is not particularly limited.
The concentration at the time of reaction with G may be appropriately selected from the range of 0.1 to 500 mM, preferably 1 to 50 mM.

The buffer used in the reagent solution in the above methods (i) and (ii) is not particularly limited as long as it is usually used in this field. For example, Good's buffer, citric acid Acid salts, borates and phosphates are preferred. The concentration of the buffer used is not particularly limited as long as it is a concentration at which the activity of NAG can be measured, but the concentration at the time of measuring the NAG activity is 1 to 1000 mM, preferably 10 to 500 mM.
May be appropriately selected from the range. In addition, the above (i) and
In the reagent solution used in (ii), if necessary, a solubilizer, a preservative, a stabilizer, a surfactant and the like usually used in this field may be appropriately selected and contained. May be appropriately selected from the range usually used in this field.

The reagent for measuring NAG activity of the present invention comprises the hydroxyalkylpyridine derivative of the present invention as a substrate. More specifically, a one-pack method reagent comprising a hydroxyalkylpyridine derivative and a buffer, a first reagent comprising a buffer, and a second reagent comprising a hydroxyalkylpyridine derivative and a buffer And a reagent for a two-pack method, and the like. The preferred embodiments and specific examples of the components of the reagent of the present invention are as described above. Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0037]

【Example】

Example 1 3-hydroxymethyl-2-pyridyl-N-acetyl-
1-thio-β-D-glucosaminide [IUPAC name: 3
-Hydroxymethyl-2-pyridinyl 2- (acetylamino) -2-deoxy-1-thio-β-D-glucopyranoside Abbreviated as compound [1]. ]

(1) 2-chloronicotinic acid (Wako Pure Chemical Industries, Ltd.)
10.52 ml of ethyl chlorocarbonate was added to a toluene solution (1 L) in which 15.76 g (100 mmol) of triethylamine and 15.32 ml (110 mmol) of triethylamine were dissolved.
Was added and the mixture was stirred and reacted at room temperature for 1 hour. The precipitated crystals were removed by filtration, and the brown oil obtained by distilling off toluene under reduced pressure was dissolved in tetrahydrofuran. This was previously dissolved in tetrahydrofuran in 4.17 g of lithium aluminum hydride (11.
0 mmol) was added dropwise to the solution which had been purged with nitrogen and cooled to -78 ° C. After the dropwise addition, the mixture was stirred and reacted at the same temperature for 2 hours. The reaction was stopped with water (30 ml), and the temperature was adjusted to room temperature.
N-NaOH (200 ml) was added, and the mixture was extracted with diethyl ether. By evaporating the solvent from the extract under reduced pressure and crystallizing the obtained brown oil from hexane,
2-chloro-3-hydroxymethylpyridine 11.91
g (75% yield). Melting point: 58-59 [deg.] C. Elemental analysis: C ₆ H ₆ ClNO, observed (%); C: 50.24, H: 4.11, N: 9.74. Calculated value (%); C: 50.19, H: 4.21, N: 9.76.

(2) 8.61 g (60 mmol) of 2-chloro-3-hydroxymethylpyridine obtained in (1) and 3.36 g (60 mmol) of sodium hydrogen sulfide were added to 1-methyl-2-pyrrolidone. The reaction was stirred at 140 ° C. for 2 hours. After stirring, the solvent was distilled off under reduced pressure, and water (200 ml) was added.
Was added, and the pH was adjusted to 4.0 with acetic acid, followed by extraction with chloroform. After evaporating the solvent from the extract under reduced pressure, the residue was recrystallized from ethanol to obtain 6.4 g (yield: 67) of 3-hydroxymethyl-2-pyridinethiol as yellow needles.
%). Melting point: 174-175 [deg.] C. Elemental analysis C ₆ H ₇ NOS Found (%); C: 51.14, H: 4.97, N: 9.98. Calculated value (%); C: 51.04, H: 5.00, N: 9.92.

(3) 3-hydroxymethyl obtained in (2)
4.24 g (30 mmol) of 2-pyridinethiol, 1
-Chloro-1-deoxy-2,3,4,6-tetraacetyl-α-D-glucosamine 10.97 g (30 mmol
l) and tetra-n-butylammonium bromide 9.
67 g (30 mmol) was reacted by stirring at room temperature for 1 hour in a mixed solvent of 50 ml of 1N-NaOH and 50 ml of dichloromethane. After completion of the reaction, 100 ml of water and 100 ml of dichloromethane were added to the reaction solution, and the mixture was separated by stirring. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The obtained pale yellow crystals were recrystallized with ethanol to give white flocculent crystals of 3-hydroxymethyl-2-pyridyl-.
2,3,4,6-tetraacetyl-1-thio-β-D-
5.14 g of glucosaminide (36.4% yield) was obtained. Melting point: 172-173C. Elemental analysis _{C 20 H 26 N 2 O 9} S Found (%); C: 51.14, H: 5.44, N: 5.98. Calculated value (%); C: 51.01, H: 5.56, N: 5.95.

(4) 3-Hydroxymethyl obtained in (3)
2-pyridyl-2,3,4,6-tetraacetyl-1-
5.14 g of thio-β-D-glucosaminide (11 mmol
100 ml of methanol was added to 1), and 10 drops of a 28% methanol solution of sodium methoxide were added to the solution, and the mixture was stirred and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was neutralized with acetic acid, and the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from ethanol to give 3.50 g of 3-hydroxymethyl-2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide as white needle crystals (93% yield). (Total yield 17%). Melting point: 190-193C. Elemental analysis _{C 14 H 20 N 2 O 6} S Found (%); C: 48.81, H: 5.97, N: 8.20. Calculated value (%); C: 48.83, H: 5.85, N: 8.13. IR: 1643 cm ^-1 (C = O)

Example 2 4-Hydroxymethyl-2-pyridyl-N-acetyl-
1-thio-β-D-glucosaminide [IUPAC name: 4
-Hydroxymethyl-2-pyridinyl 2- (acetylamino) -2-deoxy-1-thio-β-D-glucopyranoside Abbreviated as compound [2]. ]

Instead of 150 mmol of 2-chloronicotinic acid, 2-chloroisonicotinic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) 2
Using the same reagents as in Example 1 except that 3.63 g (150 mmol) was used, the reaction and post-treatment were carried out in the same manner as in Example 1 to give 4-hydroxymethyl-2-pyridyl-
N-acetyl-1-thio-β-D-glucosaminide 1.
65 g were obtained (total yield 8%). Melting point: 198-199 [deg.] C. Elemental analysis _{C 14 H 20 N 2 O 6} S Found (%); C: 48.77, H: 5.67, N: 8.24. Calculated value (%); C: 48.83, H: 5.85, N: 8.13. IR: 1641 cm ^-1 .

Example 3 5-Hydroxymethyl-2-pyridyl-N-acetyl-
1-thio-β-D-glucosaminide [IUPAC name: 5
-Hydroxymethyl-2-pyridinyl 2- (acetylamino) -2-deoxy-1-thio-β-D-glucopyranoside Abbreviated as compound [3]. ]

6-chloronicotinic acid (a product of Wako Pure Chemical Industries, Ltd.) instead of 2-chloronicotinic acid 150 mmol
Except that 3.63 g (150 mmol) was used, the same reagent as in Example 1 was used, and the reaction and post-treatment were carried out in the same manner as in Example 1 to give 5-hydroxymethyl-2-pyridyl-N
-Acetyl-1-thio-β-D-glucosaminide 5.1
5 g were obtained (total yield 25%). Melting point: 213-214C. Elemental analysis _{C 14 H 20 N 2 O 6} S Found (%); C: 48.80, H: 5.78, N: 8.17. Calculated value (%); C: 48.83, H: 5.85, N: 8.13. IR: 1643 cm ^-1 .

Example 4 6-Hydroxymethyl-2-pyridyl-N-acetyl-
1-thio-β-D-glucosaminide [IUPAC name: 6
-Hydroxymethyl-2-pyridinyl 2- (acetylamino) -2-deoxy-1-thio-β-D-glucopyranoside Abbreviated as compound [4]. ]

6-chloropicolinic acid (manufactured by Wako Pure Chemical Industries, Ltd.) instead of 150 mmol of 2-chloronicotinic acid
Except that 3.63 g (150 mmol) was used, the same reagent as in Example 1 was used, and the reaction and post-treatment were carried out in the same manner as in Example 1 to give 6-hydroxymethyl-2-pyridyl-
N-acetyl-1-thio-β-D-glucosaminide 2.
68 g was obtained (total yield 13%). Melting point: 164-165C. Elemental analysis _{C 14 H 20 N 2 O 6} S Found (%); C: 48.65, H: 5.83, N: 8.24. Calculated value (%); C: 48.83, H: 5.85, N: 8.13. IR: 1644 cm ^-1 (C = O)

Example 5 5- (1-hydroxyethyl) -2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide [IUPA
C name: 5- (1-hydroxyethyl) -2-pyridinyl 2- (acetylamino) -2-deoxy-1-thio-
β-D-glucopyranoside Abbreviated as compound [5]. ]

6-chloronicotinic acid (Wako Pure Chemical Industries, Ltd.)
39.39 g (250 mmol) of toluene (0.5
L), thionyl chloride (36 ml) and N, N-dimethylformamide (1 ml) were added, and the mixture was stirred and reacted at room temperature for 18 hours. Next, the reaction solvent was distilled off under reduced pressure, and the residue was dissolved in chloroform (1.5 L).
5.8 ml) in chloroform (100 ml) and triethylamine (70 ml) in chloroform (150 ml).
ml) was added. After completion of the addition, the mixture was stirred at room temperature for 2 hours. After completion of the reaction, purified water (1 L) was added, and the organic layer was separated and washed.
The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The obtained crude N, N-diethyl-6-chloronicotinamide (49.16 g) was dissolved in tetrahydrofuran (2 L), and after purging with nitrogen, methyllithium (165 ml: 1.4 M diethyl ether solution) was added at -78 ° C. It was dropped. After completion of the dropwise addition, the mixture was stirred and reacted at the same temperature for 1 hour. After stopping the reaction with a saturated ammonium chloride solution, the mixture was cooled to room temperature, and the solvent was distilled off. Purified water (0.5 L) and chloroform (0.5 L) were added to the obtained residue, and the mixture was separated. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off. The obtained residue was purified by recrystallization from ethanol to obtain 24.57 g of 3-acetyl-6-chloropyridine (yield 6).
3%). Melting point: 98-99C. Elemental analysis C ₇ H ₆ ClNO Found (%); C: 55.15, H: 3.83, N: 8.24. Calculated value (%); C: 54.91, H: 3.81, N: 8.83.

23.79 g (150 mmol) of the obtained 3-acetyl-6-chloropyridine was dissolved in tetrahydrofuran, and 6.25 g (165 mmol) of lithium aluminum hydride was added to tetrahydrofuran in advance, and the mixture was purged with nitrogen at −78 ° C. Was added dropwise to the cooled solution. After the dropwise addition, the mixture was stirred and reacted at the same temperature for 2 hours, and water (30 m
After stopping the reaction with 1), the mixture was brought to room temperature, and 1N-NaOH (2
00 ml) and extracted with diethyl ether. The solvent was distilled off from the extract under reduced pressure, and the resulting brown oil was crystallized from hexane to obtain 20.48 g (yield: 85%) of 3- (1-hydroxyethyl) -6-chloropyridine. Then, instead of 8.61 g (60 mmol) of 2-chloro-3-hydroxymethylpyridine, 20.48 g (127.5 mmol) of 3- (1-hydroxyethyl) -6-chloropyridine obtained above was used. Except for using the same reagents as in (2) to (3) of Example 1,
The reaction and post-treatment were carried out in the same manner as in (2) to (3) to give 5-
5.93 g of (1-hydroxyethyl) -2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide was obtained (13% overall yield). Melting point: 201-202 ° C. Elemental analysis: C ₁₅ H ₂₂ N ₂ O ₆ S Found (%); C: 50.55, H: 6.08, N: 7.69. Calculated value (%); C: 50.27, H: 6.19, N: 7.82. IR: 1651 cm ^-1 (C = O)

Experimental Example 1 Examination of pH Stability of Hydroxyalkylpyridine Derivative of the Present Invention Among the conventional products, 6-methyl-stable for the longest time in aqueous solution state
2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide [substrate used in N-assay NAG Nitto Bo (Nitto Bo Medical Co., Ltd.)] (hereinafter abbreviated as 6-MPT-NAGS). ) And 6-hydroxymethyl-2- having a substituent at the same position as the conventional product among the hydroxyalkylpyridine derivatives of the present invention.
The stability in an aqueous solution state with pyridyl-N-acetyl-1-thio-β-D-glucosaminide (compound [4]) is expressed as p
H was compared in the range of 4.5 to 10.

(Operation Procedure) 6-MPT-NAGS and Compound [4] were each dissolved in purified water to prepare a 20 mM substrate stock solution. The same stock solution and 50 mM citrate buffer (pH 4.
5,5.0,5.5,6.0), 50 mM N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid buffer (pH 7.0) or 50 mM borate buffer (p
H8.0, 9.0, 10.0) were mixed at 1: 1 to prepare substrate solutions at each pH, and stored at 10 ° C. for 16 days. For the same substrate solution, 34
The OD value (absorbance) at 0 nm was measured. (Results) OD value immediately after preparation of each substrate solution, O on day 16
Table 1 also shows the D value and the OD value change (ΔOD value) obtained by subtracting the OD value immediately after preparation from the OD value on the 16th day.

[0053]

[Table 1]

The pH and ΔO prepared based on the results in Table 1
FIG. 1 is a graph showing the relationship between the D values. In the figure,-●
-Indicates the graph obtained for the conventional product 6-MPT-NAGS, and-◆-indicates the graph obtained for the compound [4]. From the results shown in Table 1 and FIG. 1, the hydroxyalkylpyridine derivative of the present invention was
It can be seen that it has better stability in an aqueous solution than 6-MPT-NAGS which is a substrate of AG. In particular, N
Since the stability of AG near the optimum pH is remarkably improved as compared with the conventional product, it is understood that the hydroxyalkylpyridine derivative of the present invention is preferable as a substrate for NAG. Experimental Example 2 Study on long-term storage stability of the compound of the present invention in an aqueous solution state
PH of NAGS and compounds [1] to [4] of the present invention
The stability was compared in the 8.0 solution. A conventional product and a substrate solution (25 mM borate buffer, pH 8.0) of the compounds [1] to [4] prepared in the same manner as in Experimental Example 1 were stored at 10 ° C., immediately after each preparation and after storage. Day, 16 days after storage, 60 days after storage, 90 days after storage at 340 nm
The D value was measured.

(Results) The results are shown in Table 2.

[0056]

[Table 2]

FIG. 2 is a graph showing the relationship between the number of storage days and the OD value, which was created based on the results shown in Table 2. still,
In FIG. 2,-○-indicates the results obtained for the conventional product,-◆-indicates the results obtained for compound [1],-
●-shows the result obtained for compound [2],-△-shows the result obtained for compound [3], and-×-shows the result obtained for compound [4]. As is clear from the results shown in Table 2 and FIG. 2, the hydroxyalkylpyridine derivative of the present invention is different from the conventional 6-MPT-NAGS.
It can be seen that the long-term storage stability in an aqueous solution is remarkably improved as compared to In other words, it can be seen that the hydroxyalkylpyridine derivative of the present invention is suitable as a substrate for measuring a NAG activity for a so-called liquid reagent.

Example 6 Measurement of N-acetylglucosaminidase activity using 3-hydroxymethyl-2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide (compound [1]) as a substrate (sample) 34 samples prepared by appropriately diluting placenta-derived NAG (manufactured by Sigma) with physiological saline were used. (Reagent) First reagent solution 100 mM citrate buffer (pH 4.40, at25
° C). Second reagent solution A borate buffer containing 41.7 mM of compound [1] (p
H8.0).

(Procedure) 1.8 ml of the first reagent solution and 0.1 ml of the sample were mixed, incubated at 37 ° C. for 5 minutes, and 0.6 ml of the second reagent solution was added thereto, and at the same time, the absorbance at 340 nm was measured. The measurement was started and the absorbance was measured every minute for 5 minutes. The amount of change in absorbance per minute (ΔA) was determined from the obtained measured values. The reagent blank (ΔB) was measured by using the same reagent and performing the same operation except that physiological saline was used instead of the sample. The obtained ΔA and ΔB were substituted into the following equation to calculate an N-acetylglucosaminidase activity value. N-acetylglucosaminidase activity of sample (u / L)
= (ΔA−ΔB) × total liquid volume during reaction × 10 ⁶ / molecular extinction coefficient × sample liquid volume ΔA: change in absorbance of sample at 340 nm per minute ΔB: change in absorbance of reagent blank at 340 nm per minute Volume Total liquid volume during reaction: 2.5 (ml) Molecular extinction coefficient: 8354 Sample liquid volume: 0.1 (ml)

Example 7 Measurement of N-acetylglucosaminidase activity using 4-hydroxymethyl-2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide (compound [2]) as a substrate (sample) Same as in Example 6 (Reagent) First reagent solution Same as in Example 6 Second reagent solution Borate buffer (p) containing 41.7 mM of compound [2]
H8.0)

(Operating method) Measurement was performed by the same operating method as in Example 6 except that the above reagent was used, and the obtained ΔA and ΔB were substituted into the following formula to obtain N-acetylglucosaminidase activity. Values were calculated. N-acetylglucosaminidase activity of sample (u / L)
= (ΔA−ΔB) × total liquid volume during reaction × 10 ⁶ / molecular extinction coefficient × sample liquid volume ΔA: change in absorbance of sample at 340 nm per minute ΔB: change in absorbance of reagent blank at 340 nm per minute Volume Total liquid volume during reaction: 2.5 (ml) Molecular extinction coefficient: 8397 Sample liquid volume: 0.1 (ml)

Example 8 Measurement of N-acetylglucosaminidase activity using 5-hydroxymethyl-2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide (compound [3]) (Specimen) Example 6 (Reagent) 1st reagent solution Same as in Example 6 2nd reagent solution Borate buffer containing 41.7 mM of compound [3] (p
H8.0)

(Operating method) Measurement was carried out by the same operating method as in Example 6 except that the above reagent was used, and the obtained ΔA and ΔB were substituted into the following formula to obtain N-acetylglucosaminidase activity. Values were calculated. N-acetylglucosaminidase activity of sample (u / L)
= (ΔA−ΔB) × total liquid volume during reaction × 10 ⁶ / molecular extinction coefficient × sample liquid volume ΔA: change in absorbance of sample at 340 nm per minute ΔB: change in absorbance of reagent blank at 340 nm per minute Volume Total solution volume during reaction: 2.5 (ml) Molecular extinction coefficient: 8049 Sample solution volume: 0.1 (ml)

Reference Example 1 Measurement of N-acetylglucosaminidase activity using a commercially available product Examples 6 and 7 were carried out using a commercially available reagent for measuring N-acetylglucosaminidase activity (N-assay NAG Nitto Bo: Nitto Bo Medical Co., Ltd.). The N-acetylglucosaminidase activity was measured for 34 specimens for which the N-acetylglucosaminidase activity was measured in 8. The measurement operation was performed in accordance with the standard operation method described in the instruction manual attached to the product.

FIGS. 3 to 5 show correlation diagrams between the measured activity values of the samples obtained in Examples 6, 7 and 8 and the measured activity values obtained in Reference Example 1, respectively. The regression line and correlation coefficient (r) obtained by statistically processing these measured values were as follows. 1. X: NAG activity value obtained in Reference Example 1, Y: NAG activity value obtained in Example 6. Regression linear equation: Y = 0.93X−0.24 Correlation coefficient (r): 0.993 2. X: NAG activity value obtained in Reference Example 1, Y: NAG activity value obtained in Example 7. 2. Regression linear equation: Y = 0.90X + 0.47 Correlation coefficient (r): 0.993 X: NAG activity value obtained in Reference Example 1, Y: NAG activity value obtained in Example 8. Regression linear equation: Y = 0.84X + 0.81 Correlation coefficient (r): 0.993 From the above results and the results of FIGS. 3 to 5, NAG using the hydroxyalkylpyridine derivative of the present invention as a substrate
According to the activity measurement method, N having a good correlation with a commercial product
It turns out that an AG activity value is obtained.

Example 9: Examination of linearity NAG (Sigma product) derived from human placenta was treated with physiological saline for 10 minutes.
The serial dilution (dilution ratio 1/10 to 1) was used as a sample, and the reagents of Examples 6 to 8 were used, and the same operation was performed to obtain linearity of the calibration curve of the NAG activity measurement method of the present invention. Study was carried out. FIG. 6 shows the results obtained using the reagent of Example 6.
The results obtained using the reagent of Example 7 are shown in FIG.
The results obtained using the reagents of Example 8 are shown in FIG. As is clear from the results shown in FIGS.
It can be seen that the calibration curve of the G activity measurement method shows good linearity passing through the origin.

[0067]

As described above, the present invention provides a novel hydroxyalkylpyridine derivative, a method for measuring NAG activity using this derivative as a substrate, and a reagent for measuring NAG activity. The hydroxyalkylpyridine derivative of the present invention is excellent in water solubility and less non-enzymatically degraded, and therefore has a very high stability in an aqueous solution over a wide range from acidic to alkaline. PH of solution containing hydroxyalkylpyridine derivative
Is set to 7 or more (preferably pH 7 to 10), which is an extremely excellent property as a substrate for measuring NAG activity, which is stable for at least 3 months or more in an aqueous solution state. In addition, the hydroxyalkylpyridine derivative of the present invention releases a hydroxyalkylpyridinethiol derivative by the action of NAG or the like, and this derivative can be measured at an ultraviolet region wavelength that can avoid the influence of biological components such as bilirubin. Since the maximum absorption wavelength is almost the same as the set wavelength of the automatic analyzer, the NAG activity measurement method and the reagent using the same as a substrate have the effect that they can be easily applied not only to the method used but also to general-purpose automatic analyzers. Play. Therefore, the present invention is a very large invention that contributes to the industry.

[0068]

[Brief description of the drawings]

FIG. 1 shows the change in pH and OD value (Δ) obtained in Experimental Example 1.
6 is a graph showing the relationship with the OD value.

FIG. 2 is a graph showing the relationship between the number of storage days and the OD value obtained in Experimental Example 2.

FIG. 3 shows the N-acetyl-β-D-glucosaminidase (hereinafter referred to as N-acetyl-β-D-glucosaminidase) obtained in Example 6.
Abbreviated as AG. 4) Correlation diagram between the measured activity value and the measured NAG activity value obtained in Reference Example 1. FIG.

FIG. 4 is a correlation diagram between a measured value of NAG activity obtained in Example 7 and a measured value of NAG activity obtained in Reference Example 1.

FIG. 5 is a correlation diagram between the measured value of NAG activity obtained in Example 8 and the measured value of NAG activity obtained in Reference Example 1.

FIG. 6 is a diagram showing the linearity of a calibration curve obtained in Example 9 by the NAG activity measurement method of the present invention.

FIG. 7 is a view showing the linearity of a calibration curve obtained in Example 9 by the NAG activity measurement method of the present invention.

FIG. 8 is a diagram showing the linearity of a calibration curve obtained in Example 9 by the NAG activity measurement method of the present invention.

[0069]

[Brief description of reference numerals]

In FIG. 1,-●-is obtained using a conventional product, 6-methyl-2-pyridyl-N-acetyl-1-thio-β-D-glucosaminide (hereinafter abbreviated as 6-MPT-NAGS). -◆-shows the results obtained using Compound [4], respectively. In FIG. 2, -−- is 6 of the conventional product.
The results obtained using -MPT-NAGS,-◆-indicates the results obtained using compound [1],-●-indicates the results obtained using compound [2],-△- Is the compound [3]
, And-×-indicate the results obtained using compound [4], respectively.

Claims (4)

[Claims] 1. A compound of the general formula [1] (Wherein, G represents a hexosamine residue having a β-bond at the reducing end and an acyl group bonded to an amino group, and R represents a hydroxyalkyl group). 2. The hydroxyalkylpyridine derivative according to claim 1, wherein G is an N-acetylglucosamine residue. 3. The method of claim 1, wherein the hydroxyalkylpyridine derivative according to claim 1 is used as a substrate.
A method for measuring the activity of acetyl-β-D-glucosaminidase. 4. A reagent for measuring the activity of N-acetyl-β-D-glucosaminidase, comprising the hydroxyalkylpyridine derivative according to claim 1.