JPH0629239B2 - Thiocholine derivative and its use - Google Patents

Description

Detailed Description of the Invention a. TECHNICAL FIELD The present invention relates to a novel thiocholine derivative and its use, and more specifically, a novel thiocholine that can be used for measuring the activity of pseudocholinesterase (acylcholine acylhydrolase EC 3.1.1.8). The present invention relates to a derivative, a method for measuring cholinesterase activity using the derivative as a substrate, and a measurement kit.

B. "Prior art" In clinical tests, serum cholinesterase (E
C. Measuring the activity of 3.1.1.8) is useful for diagnosis of liver parenchymal disorders and liver diseases that reflect nutritional status.

Cholinesterase is a type of esterase that is not present in plant tissues, but is present in most animal tissues. Generally, there are two types of cholinesterases that differ in enzymatic activity and physiological function and biodistribution. Of these, one is acetylcholinesterase (E
C. 3.1.1.7) (also known as true cholinesterase), which is abundant in nerve tissues and erythrocyte membranes, and the other is called cholinesterase (EC 3.1.1.8) (also known as pseudocholinesterase), serum It is also found in the liver and pancreas. Pseudocholinesterase is measured by routine clinical tests, but it has been considered to use a substrate that does not react with true cholinesterase when serum is used as a sample, but that reacts specifically with pseudocholinesterase. Came.

Acylcholines such as acetylcholine, butyrylcholine, and benzoylcholine have been conventionally used as substrates for measuring pseudocholinesterase.

However, when benzoylcholine or its derivative, p-hydroxybenzoylcholine or o-toluoylcholine, is used as a substrate, the system is complicated because the coupled enzyme system that generally requires dissolved enzyme is used as the measurement system, and serum There are problems such as interference of various substances in the sample.

On the other hand, there is also known a method in which acetylcholine, propionylthiocholine and butyrylthiocholine which are derivatives of alkylthiocholine are used as substrates, and thiocholine released by the action of pseudocholinesterase is colored with an SH group reagent. However, in this method, when the optimum pH of pseudocholinesterase is 8 to 8.5, the self-hydrolysis of the substrate is so large that it cannot be used. There is also a method in which the SH group released from this alkylthiocholine derivative is colored with DTNB (5,5'-dithiobis- (2-nitrobenzoic acid), but since the measurement sensitivity (specific activity) is too high in this method, There is a drawback that the normal serum sample level pseudocholinesterase activity cannot be measured using a non-colorimeter unless the sample amount is extremely small compared to the reagent amount. When performing an assay using an automated analyzer or manual method that is used, the sample volume can be accurately collected (sample / reaction solution volume =
If it is 1/30 or more), the sensitivity is too high.

C. [Object of the Invention] In view of various problems associated with such conventional techniques, the object of the present invention is to reduce self-hydrolysis at pH 8.0 to 8.5,
Provided is a synthetic substrate for pseudocholinesterase and a method for quantifying pseudocholinesterase, which has a simple indicator reaction system and has sufficient sensitivity in a volume (sample / reaction solution volume = 1/30 or more) capable of easily collecting a sample volume. Especially.

D. [Configuration of the Invention] one aspect of the present invention, the general _{formula: Y-COSCH 2 CH 2 N} + (CH 3) 3 X - (I) [ in the formula, Y has the formula: (Here, R ₁ , R ₂ and R ₃ are the same or different and each represents hydrogen, a hydroxyl group, a halogen, a nitro group, a lower alkyl group, a lower alkoxy group, an acetamino group or a di (lower alkyl) amino group. , R ₁ , R ₂ and R ₃
At least one of the is not hydrogen. ) A substituted phenyl group represented by (Wherein n represents an integer of 2 to 6) or a cycloalkyl group or an alkyl- or alkoxy-substituted derivative thereof, (Wherein R ₄ , R ₅ and R ₆ all represent an alkyl group, or one represents hydrogen and the other two represent an alkyl group), or X ⁻ (CH ₃ ) ₃ N ⁺ CH ₂ CH ₂ SCOR- (V) (wherein R represents a divalent alkyl group). X represents a halogen atom. ] It exists in the novel thiocholine derivative shown by these.

Other aspects of the present invention have the general _{formula: Y'-COSCH 2 CH 2 N} + (CH 3) 3 X - (I ') [ wherein, Y' has the formula: (Here, R ′ ₁ , R ′ ₂ and R ′ ₃ are the same or different and each represents hydrogen, hydroxyl group, halogen, nitro group, lower alkyl group, lower alkyl group, acetamino group or di (lower alkyl) amino group. A phenyl group represented by (Where n is as defined above) or a cycloalkyl group or an alkyl- or alkoxy-substituted derivative thereof, (Wherein R ₄ , R ₅ and R ₆ have the same meanings as defined above), or X ⁻ (CH ₃ ) ₃ N ⁺ CH ₂ CH ₂ SCOR- (V) (wherein R has the same meaning as defined above.) X has the same meaning as above. ] The method for quantifying pseudocholinesterase characterized by quantifying thiocholine or an aromatic or aliphatic compound released by the action of pseudocholinesterase using the thiocholine derivative represented by the above, and a pseudocholinesterase containing the thiocholine derivative Quantitative kit.

In the general formula, halogen is fluorine, chlorine, bromine or iodine. Further, the lower alkyl group and the lower alkoxy group mean an alkyl group and an alkoxy group having 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms, and the alkyl group contained in the alkyl group and the alkoxy group is either linear or branched. May be The alkyl or alkoxy which is present as a substituent in the cycloalkyl group (III), if desired, preferably has 1 to 4 carbon atoms.

Examples of the thiocholine derivative (I) in which Y is a substituted phenyl group (II) include 4-nitrobenzoylthiocholine halide, 3-nitrobenzoylthiocholine halide, 2-nitrobenzoylthiocholine halide and 4-chlorobenzoylthiocholine. Halide, 3-chlorobenzoylthiocholine halide, 2-chlorobenzoylthiocholine halide, 4-methoxybenzoylthiocholine halide, 3-
Methoxybenzoylthiocholine halide, 2-methoxybenzoylthiocholine halide, 2,4-dimethoxybenzoylthiocholine halide, 2,3-dimethoxybenzoylthiocholine halide, 4-methylbenzoylthiocholine halide, 3-methylbenzoylthiocholine halide, 2-methylbenzoyl thiocholine halide, 4-t
-Butylbenzoylthiocholine halide, 3-t-butylbenzoylthiocholine halide, 2-t-butylbenzoylthiocholine halide, 4-hydroxybenzoylthiocholine halide, 3-hydroxybenzoylthiocholine halide, 2-hydroxybenzoylthiocholine halide , 4-acetamidobenzoylthiocholine halide, 3-asthoamidobenzoylthiocholine halide,
2-acetamido benzoyl thiocholine halide etc. are mentioned. Particularly, a derivative having a substituent at the ortho position is advantageous because it has low self-hydrolyzability and high reactivity.

The thiocholine derivative of the present invention in which Y is a group (III) to (V)
Examples of (I) include pivaloylthiocholine halide, isobutylthiocholine halide, methylpropionylthiocholine halide, dimethylpropionylthiocholine halide, ethylpropionylthiocholine halide, cyclopropanecarbonylthiocholine halide, cyclobutanecarbonylthiocholine halide, Cyclopentanecarbonylthiocholine halide, cyclohexanecarbonylthiocholine halide, cycloheptanecarbonylthiocholine halide, methylcyclopropanecarbonylthiocholine halide, dimethylcyclopropanecarbonylthiocholine halide, ethylcyclopropanecarbonylthiocholine halide, methoxycyclopropanecarbonylthiocholine Halide, dimethoxycyclopropanecarbonylthiocholine halide Diethylcyclopropanecarbonylthiocholine halide, ethoxycyclopropanecarbonylthiocholine halide, diethoxycyclopropanecarbonylthiocholine halide, methylcyclobutanecarbonylthiocholine halide, dimethylcyclobutanecarbonylthiocholine halide, methoxycyclobutanecarbonylthiocholine halide, dimethoxycyclobutanecarbonylthio Choline halide, ethylcyclobutanecarbonylthiocholine halide, ethoxycyclobutanecarbonylthiocholine halide, methylcyclopentanecarbonylthiocholine halide, dimethylcyclopentanecarbonylthiocholine halide, methoxycyclopentanecarbonylthiocholine halide, dimethoxycyclopentanecarbonylthiocholine halide, Tylcyclopentanecarbonylthiocholine halide, ethoxycyclopentanecarbonylthiocholine halide, methylcyclohexanecarbonylthiocholine halide, dimethylcyclohexanecarbonylthiocholine halide, methoxycyclohexanecarbonylthiocholine halide, dimethoxycyclohexanecarbonylthiocholine halide, ethylcyclohexanethiocholine halide, Ethoxycyclohexanecarbonylthiocholine halide, methylcycloheptanecarbonylthiocholine halide, dimethylcycloheptanecarbonylthiocholine halide, methoxycycloheptanecarbonylthiocholine halide, dimethoxycloheptanecarbonylthiocholine halide, ethylcycloheptanecarbonylthiocholine halide, ethoxysilane Examples include cloheptane carbonylthiocholine halide and succinylthiocholine halide.

The thiocholine derivative (I) of the present invention can be synthesized, for example, as follows.

General formula: Y-COC1 [In formula, Y is a same meaning as the above. ] The acid chloride represented by the formula and dimethylaminoethanethiol are each dissolved in a suitable solvent (for example, diethyl ether), and either one is slowly added dropwise to the other while stirring to precipitate crystals at room temperature. The crystals are freed of chloride with an alkaline solution (eg, sodium carbonate solution), extracted with a suitable solvent (eg, dimethyl ether), dried, filtered, and reacted with methyl halide to the resulting product. Get Colin Halide.

In the method for measuring pseudocholinesterase of the present invention, thiocholine is released from the thiocholine derivative (I ′) by the action of pseudocholinesterase, and the color is developed by the SH group reagent, which is then analyzed using a conventional spectrophotometer or various automatic analyzers. The activity of pseudocholinesterase can be quantitatively measured by quantitatively measuring the amount of thiocholine produced.

As the SH group reagent, any of those conventionally used can be used, and DTNB (5,5′-dithiobis- (2-nitrobenzoic acid)) can be shown as an example.
When TNB is used as the SH group reagent, TNB (2-nitro-5-mercaptobenzoic acid) is produced and color is developed.

Pseudocholinesterase quantification method of the present invention, especially in the field of clinical examination using various automatic analyzers Kinetic analysis (Kinetic) method, or an inhibitor of pseudocholinesterase (for example, neostigmine) to stop the reaction, It can be applied when analyzing by the end point method.

EXAMPLES Next, the present invention will be specifically described by showing examples, but the present invention is not limited to these.

Reference Example benzoyl butyrylthiocholine iodide of preparation: - dimethylaminoethanethiol hydrochloride _{[(CH 3) 2 NCH 2} SH ·
HCl] 3.5 g is dissolved in a solution of 3 g of sodium carbonate in 15 ml of water. It is extracted with 150 ml of diethyl ether and the extract is dried over sodium sulphate overnight. Sodium sulfate is filtered off, 3.6 g of benzoyl chloride chloride is added dropwise to the filtrate with stirring, and the precipitated crystals are collected by filtration. Yield 4g.

0.5 g of this is added with 10 g of water containing 0.3 g of sodium carbonate
Dissolve in the solution and extract with 75 ml of diethyl ether,
The extract is dried over sodium sulfate overnight. Sodium sulfate was filtered off, the total volume of the diethyl ether solution was concentrated to 50 ml, 0.42 g of methyl iodide was added, and stirring was continued at room temperature. When the precipitation of crystals occurs and the precipitation completely disappears (2 days), the stirring is stopped and the crystals are obtained by filtration. The crystals were recrystallized from water to obtain 0.5 g of the desired benzoylthiocholine iodide. Yield 71.4%. Melting point 257
° C. This is the Journal of the American Chemical Society (J. Am. Chem. Soc., 60 , 1765 (1
R. Royshaw et al. (RR)
This was in agreement with the melting point (MP = 257 ° C) reported by Reushow et al).

Example 1 Preparation of 2,3-dimethoxybenzoylthiocholine iodide: -2,3-dimethoxybenzoic acid 17.8 g (0.15 mo
Thionyl chloride (11 ml) was added to (l), and after the reaction, excess thionyl chloride was removed by an aspirator to obtain 17.6 g of crystalline acid chloride. Yield 87.7%.

3.6 g (0.0175 mol) of the obtained acid chloride was added to a diethyl ether solution of dimethylaminoethanethiol obtained in the same manner as in the above Reference Example, and the precipitated crystals were collected by filtration and dried under reduced pressure. Yield 4.3g. 3.1 of them
g was dissolved in a solution of 1.2 g of sodium carbonate in 50 ml of water,
It was extracted with 150 ml of diethyl ether, and the extract was dried overnight with sodium sulfate. Thereafter, the total volume of the diethyl ether solution was adjusted to 100 ml, 1 ml of methyl iodide was added, stirring was continued for 2 days at room temperature, the precipitated crystals were collected by filtration, and the crystals were recrystallized from water to give 2,3-dimethoxybenzoylthiocholine iodide. Got Id. Melting point 184-185 [deg.] C. The infrared absorption spectrum and the NMR spectrum of this compound are
Shown in Figures and FIG.

Example 2 Water Solubility and Specificity of Substrates Various thiocholine derivatives were prepared, and their water solubility, stability in aqueous solution, and reactivity with human serum were examined by the following method.

Human serum or purified water (0.2 ml) was added to the first reagent (2.5 ml) having the following composition, and the mixture was heated at 37 ° C. for 5 minutes, and then the second reagent (0.2 ml) was added.
5 ml was added, and the change in absorbance (ΔE / min.) Per minute at 405 nm was measured.

First reagent Trishydroxymethyl-240 mmol / l Aminomethane 5,5'-dithiobis-0.3 mmol / l (2-nitrobenzoic acid) maleic acid Appropriate amount The above components are dissolved in purified water to adjust the pH to 8.0.

Second reagent 6 mmol / l aqueous solution of various thiocholine derivatives The sparingly soluble compound was dissolved in 25% ethyl alcohol aqueous solution.

As a result, as shown in Table 1, benzoylthiocholine ester and 2,3-dimethoxythiocholine ester were water-soluble, stable in aqueous solution (small reagent blank), and reactive (absorbance per minute was 0). 0.03 or more)
Was excellent in terms of.

Example 3 The following reagents and samples were prepared, and 0.1 ml of each sample was added to 2.4 ml of the first reagent, and after heating at 30 ° C for 5 minutes, 0.5 ml of the second reagent was added and 405 nm at 30 ° C. The change in the absorbance per minute (ΔE / min) was measured to examine the reactivity of the sample.

As a result, as shown in Table 2, it was confirmed that benzoylthiocholine iodide and 2,3-dimethoxybenzoylthiocholine iodide did not react with true cholinesterase but specifically with pseudocholinesterase.

First reagent Trishydroxymethyl 12.1g (100mM) Aminomethane 5,5'-dithiobis 118.9mg (0.3mM) (2-Nitrobenzoic acid) Maleic acid Appropriate amount Purified water 1 (pH8.2) Second reagent (A) Benzoyl Thiocholine 2.107g (6mM) Iodide Purified Water 1 Second Reagent (B) 2,3-Dimethoxybenzoyl 2.455g (6mM) Thiocholine Iodide Purified Water 1 Specimen (1) Pseudocholinesterase 13U / ml (Sigma) Specimen (2) True cholinesterase 13U / ml (Sigma) Example 4 Trishydroxymethylaminomethane (100 mmol /
l), 5,5'-diotibis (2-nitrobenzoic acid (0.3
(mmol / l) dissolved in purified water, maleic acid pH 7.0-
Adjust to 9.0 to prepare various solutions. This solution 2.4
0.1 ml of pseudocholinesterase (manufactured by Sigma) (adjusted to 13 U / ml by butyrylthiocholine method) was added to ml, and the mixture was heated at 30 ° C for 5 minutes, and then 2,3-dimethylbenzoylthiocholine iodide or benzoylthio was added. 0.5 ml of a 6 mmol / l aqueous solution of choline iodide was added and the rate of increase in absorbance at 405 nm was measured to examine the reactivity at each pH.

As a result, as shown in FIG. 3, 2,3-dimethylbenzoylthiocholine iodide was optimal at pH 8.0 to 8.5, and the blank reaction (self-hydrolysis) was 10 mAb / min or less and was a suitable substrate for cholinesterase measurement. I found out.

Example 5 Trishydroxymethylaminomethane 100 mmol / l,
5,5'-dithiobis- (2-nitrobenzoic acid 0.3 mm
Dissolve ol / l in purified water and adjust to pH 8.2 with maleic acid. To 2.4 ml of this solution, 0.1 ml of pseudocholinesterase (manufactured by Sigma) (adjusted to 13 U / ml by butyrylthiocholine method) was added, and 2,3-dimethylbenzoylthiocholine iodide concentration was 0.06 to 18 mmol / l. Substrate solutions were added to examine the Km value of pseudocholinesterase for this substrate. As a result, as shown in FIG. 4, the Km value is extremely small near 1 _× 10 ⁻⁴ M,
It was found that at 1 mmol / l or more, 90% or more of the maximum activity was shown and the blank reaction (self-hydrolysis) was small, so that it could be used even at a concentration of 1 mmol / l as a substrate for cholinesterase measurement, and was found to be useful.

Example 6 Trishydroxymethylaminomethane 240 mmol / l,
5,5'-dithiobis- (2-nitrobenzoic acid) 0.3
An aqueous solution of pH 8.2 containing mmol / l (pH correction was carried out with maleic acid) was used as the first reagent, 0.1 ml of human serum sample was added to 2.4 ml of the first reagent, and the mixture was kept in a constant temperature bath at 30 ° C for 5 minutes. After preheating, 0.5 ml of an aqueous solution of 2,4-dimethoxybenzoylthiocholine iodide 6 mmol / l was added and reacted, and then 1
The change in absorbance (ΔA) per minute was measured at 405 nm. The reagent blank is similarly obtained by using purified water as the sample (ΔB). The reaction formula is as follows.

From the change in absorbance per minute obtained as described above, the measured value of this method was obtained by the following formula.

Looking at the correlation when the benzoylcholine method (Katayama Chemical Co., Ltd.) is used as the reference method, the correlation coefficient r = 0.
A good result of 999 was obtained (see Table 3).

Also, instead of human serum samples, high value pseudocholinesterase (manufactured by Sigma) (3 by butyrylthiocholine method)
0 IU / ml) was diluted in 10 steps, and a calibration curve with the activity value was calculated by the following formulas. As shown in FIG. 6, a high value of 150 IU / (30-50 IU / normal sample) was obtained.
) Maintained linearity.

Relationship between measurement values of this method and benzoylcholine method ΔA: Change in absorbance of the sample at 405 nm at 30 ° C. per minute ΔB: Change in absorbance of purified water at 405 nm at 30 ° C. per minute 3.0: Total amount of reagent and sample 0.1: Sample amount 13. 3: Molecular extinction coefficient of thio-nitrobenzoic acid at 405 nm Example 7 Preparation of isobutyrylthiocholine iodide: -50 g of dimethylaminoethanethiol hydrochloride was liberated with an aqueous sodium carbonate solution and extracted with methylene chloride at pH 10 or above. Then, vacuum distillation was carried out to obtain 30 g of dimethylaminoethanethiol (yield 80.9%, boiling point 40 ° C./1
2mmHg). Of these, 3.5 g and isobutyryl chloride 3.5 g were mixed in a diethyl ether 150 ml solution,
The precipitated crystals were collected by filtration. Further, this was dissolved in an aqueous solution of sodium carbonate (10%), adjusted to pH 10 or more, extracted with diethyl ether, and the extract was dried over magnesium sulfate. Then, diethyl ether was added to a total volume of 350 ml, methyl iodide 5 ml was added thereto, and the mixture was stirred at room temperature. The precipitated crystals were collected by filtration and recrystallized from water to obtain 9.9 g of isobutyrylthiocholine iodide. Yield 9
4.6%. Melting point 157-158 [deg.] C.

The infrared absorption spectrum and the NMR spectrum of the obtained compound are shown in FIG. 7 and FIG. 8, respectively.

Example 8 Preparation of cyclohexanecarbonylthiocholine iodide: -Dimethylaminoethanethiol prepared in Example 7.
5g and cyclohexane carbonyl chloride 4.8g
Was mixed in 150 ml of diethyl ether, and the precipitated crystals were collected by filtration. Further, this was dissolved in a sodium carbonate (10%) aqueous solution, adjusted to pH 10 or more, extracted with diethyl ether, and dried with magnesium sulfate. Then, the total volume was adjusted to 350 ml with diethyl ether, 5 ml of methyl iodide was added, and the mixture was stirred at room temperature. The precipitated crystals were collected by filtration and recrystallized from water to obtain 7 g of cyclohexanecarbonylthiocholine iodide. Yield 59.4%. Melting point 159-1
60 ° C.

The infrared absorption spectrum and NMR spectrum of the obtained compound are shown in FIGS. 9 and 10.

Example 9 Water Solubility and Specificity of Substrates Various thiocholine derivatives were prepared, and their water solubility, stability in aqueous solution, and reactivity with human serum were examined by the following method.

Mix 2.5 ml of the first reagent of the following composition with 0.2 ml of the second reagent, add 0.05 ml of the sample or purified water, and mix at 5 ℃ at 37 ℃.
After heating for 1 minute, 0.5 ml of the third reagent was added and the change in absorbance (ΔE / min) per minute at 405 nm was measured.

First reagent Trishydroxymethylamino-methane 325 mmol / l maleic acid Appropriate amount The above components were dissolved in purified water and adjusted to 37 ° C. and pH 7.5.

Second reagent 5,5′-dithiobis (2-nitro-benzoic acid) aqueous solution 0.65 mmol / l Third reagent various thiocholine derivative aqueous solution 3.25 mmol / l As a result, as shown in Table 4, cyclohexanecarbonylthiocholine Iodide and isobutylthiocholine iodide were particularly excellent in terms of stability of the reactive reagent blank.

Example 10 The following reagents and samples were prepared, and 2.5 ml of the first reagent was mixed with 0.2 ml of the second reagent to prepare 0.05 ml of the sample or purified water.
Then, after heating at 37 ° C for 5 minutes, 0.5 ml of the third reagent (A) or (B) was added, and the mixture was added to 1 ° C at 405 nm at 37 ° C.
The reactivity of the sample was examined by measuring the change in absorbance (ΔE / min) per minute.

First reagent Trishydroxymethylamino-methane 325 mmol / l maleic acid Appropriate amount The above components were dissolved in purified water and adjusted to 37 ° C. and pH 7.5.

Second reagent 5,5'-dithiobis (2-nitro-benzoic acid) aqueous solution 0.65 mmol / l Third reagent (A) Isobutyrylthiocholine-iodide aqueous solution 3.25 mmol / l (B) Cyclohexanecarbonyl-thiocholine ioda Id aqueous solution 3.25 mmol / l Specimen (1) Pseudocholinesterase 13U / ml (Sigma) Specimen (2) Tru-cholinesterase 13U / ml (Sigma) As a result, as shown in Table 5, isobutyrylthiocholine It was confirmed that iodide and cyclohexanecarbonylthiocholine iodide did not react with crane-cholinesterase but specifically with pseudocholinesterase.

Example 11 Trishydroxymethylaminomethane (100 mmol /
l), 5,5-dithiobis (2-nitrobenzoic acid) was dissolved in purified water (0.25 mmol / l), and the pH was adjusted to 7 with maleic acid.
Various solutions were prepared by adjusting to 0 to 9.0. Using this as the first reagent, add 2.4 ml and human pool serum 0.05 ml,
After heating at 37 ° C for 5 minutes, add 0.5 ml of a 63 mmol / l aqueous solution of isobutyrylthiocholine iodide or cyclohexanecarbonylthiocholine iodide, and add 4
The amount of change in absorbance per minute at 05 nm was determined using the reagent blank as a control, and the reactivity at each pH was examined.

As a result, as shown in FIG. 11, each of the substrates was optimal at around pH 8.0. Blank reaction is pH
It was 0.005 / min or less around 8.0, and it was found to be a substrate suitable for cholinesterase measurement.

Example 12 Concentration of trishydroxymethylaminomethane of 60 mmol / l
To 1200 mmol / l, 5,5-dithiobis (2
-Nitrobenzoic acid) at a concentration of 0.25 mmol / l and adjusted to pH 8.0 with maleic acid to prepare a solution. 0.05 ml of human pool serum was added to 2.6 ml of each of these solutions.
After heating at 7 ° C for 5 minutes, 0.5 ml of a 6.3 mmol / l aqueous solution of isobutyrylthiocholine iodide or cyclohexylcarbonylthiocholine iodide was added, and the change in absorbance per minute at 405 nm was measured. The optimum concentration of the liquid was determined. The final concentration of the reaction under these conditions was defined as the concentration of Tris buffer.

As a result, as shown in FIG. 12, 100 mM of Tris buffer at the time of the final reaction showed the maximum activity for all the substrates.

Example 13 Trishydroxymethylaminomethane (100 mmol / l)
And 5,5'-dithiobis- (2-nitrobenzoic acid)
An aqueous solution of pH 8.0 containing (0.25 mmol / l) (pH is corrected with maleic acid) was used as the first reagent, and 2.6 ml of the first reagent was pseudocholinesterase (Sigma) (13 U / butyrylthiocholine method). (Adjusted to 0.05 ml), add 0.05 ml, isobutyryl thiocholine iodide concentration 0.38-6.3 mm
0.5 ml of each ol / l substrate solution was added to examine the Km value of pseudocholinesterase for this substrate.
As a result, as shown in FIG. 13, the Km value was 6.9 × 10 ^−5.
Extremely small near M and maximum activity of 95 at 1 mmol / l or more
It was found to show more than%.

Example 14 A substrate solution of 0.126 to 3.15 mmol / l was prepared using cyclohexanecarbonylthiocholine iodide instead of the substrate isobutyrylthiocholine iodide used in Example 13, and the same procedure as in Example 13 was performed. The test was conducted. As shown in FIG. 14, the Km value of pseudocholinesterase for cyclohexanecarbonylthiocholine iodide was around 6.9 × 10 ⁻⁵ M, which was almost the same as that of isobutyrylthiocholine iodide.

Example 15 Trishydroxymethylaminomethane 100 mmol / l,
5,5'-dithiobis- (2-nitrobenzoic acid) (0.
An aqueous solution of pH 8.0 containing 25 mmol / l) (pH is corrected with maleic acid) is used as the first reagent, 0.05 ml of human serum sample is added to 2.6 ml of the first reagent, and after heating at 37 ° C. for 5 minutes, Isobutyryl thiocholine iodide 0.62mm as the second reagent
After 0.5 ml of an ol / l aqueous solution was added and reacted, the change in absorbance (ΔA) per minute was measured at 405 nm. As a reagent blank, purified water was used instead of the sample, and the same procedure was carried out (ΔB).

The reaction equation is as follows: From the change in absorbance per minute obtained as described above, the measured value of this method was obtained by the following formula: ΔA: Change in absorbance of the sample at 405 nm at 30 ° C. per minute ΔB: Change in absorbance of the purified water at 405 nm at 30 ° C. per minute Total amount of reagent sample: 3.15 Sample amount: 0.05 13.3: Molecular extinction coefficient of thio-nitrobenzoic acid at 405 nm.

Looking at the correlation when the benzoylcholine method (Katayama Chemical Co., Ltd.) was used as the reference method, as shown in Table 6 and FIG. 15, a correlation coefficient r = 0.998 was obtained, and good results were obtained.

Also, instead of human serum sample, high value pseudocholinesterase (Sigma) (30 IU by butyrylthiocholine method)
(Adjusted to 1 ml / ml) was diluted in 10 steps, and a calibration curve with the activity value was obtained. As shown in Table 6 and FIG.
The linearity was maintained up to U / (normal specimen 300 to 700 IU /).

Example 16 A procedure similar to that in Example 15 was repeated except that the second reagent used as a substrate was cyclohexanecarbonylthiocholine iodide 0.62.
When it was replaced with an aqueous solution of mmol / l, a correlation coefficient r = 0.998 was shown as shown in Table 7 and FIG. 17, and good results were obtained.

Further, the calibration curve with the activity value similarly performed is 3000 IU / up (normal samples 600 to 150) as shown in FIG.
0 IU /) linearity was maintained.

Example 17 Preparation of Phenylacetylthiocholine iodide: -Dimethylaminoethanethiol prepared in Example 7.
0 g and phenylacetyl chloride were mixed in 150 ml of diethyl ether, and the precipitated crystals were collected by filtration. Furthermore,
This was dissolved in 10% aqueous sodium carbonate solution, adjusted to pH 10 or above, extracted with diethyl ether, and dried over magnesium sulfate. Then, use diethyl ether to bring the total volume to 350.
Then, 3 ml of methyl iodide was added, and the mixture was stirred at room temperature. The precipitated crystals were collected by filtration and recrystallized from water to obtain 6.0 g of phenylacetylthiocholine iodide. Melting point 182-183 [deg.] C., yield 57.7%.

The infrared absorption spectrum and NMR spectrum of the obtained compound are shown in FIGS. 19 and 20.

Example 18 Preparation of Phenylpropionyl Thiocholine Iodide: -Dimethylaminoethanethiol prepared in Example 7.
0 g and 3.3 g of phenylpropionyl chloride were mixed in 150 ml of diethyl ether, and the precipitated crystals were collected by filtration. Further, this was dissolved in a 10% sodium carbonate aqueous solution, adjusted to pH 10 or higher, extracted with diethyl ether, and dried over magnesium sulfate. Then, the total volume was adjusted to 350 ml with diethyl ether, 3 ml of methyl iodide was added, and the mixture was stirred at room temperature. The precipitated crystals were collected by filtration and recrystallized from water to obtain 7.0 g of phenylpropionylthiocholine iodide. Melting point 200-201 ° C. Yield 64.8
%.

The infrared absorption spectrum and the NMR spectrum of the obtained compound are shown in FIGS. 21 and 22.

E. [Effects of the Invention] By using the thiocholine derivative of the present invention as a substrate for measuring cholinesterase activity, self-hydrolysis is less at an optimum pH of cholinesterase of 8.0 to 8.5, and a technique and a capacity for accurately collecting a sample amount. It can be applied to an automatic analyzer with (sample / reaction solution volume = 1/30 or more), and can perform wider-range and more accurate measurement than using conventional substrates such as butyrylcholine.

[Brief description of drawings]

1 is an infrared absorption spectrum of the compound prepared in Example 1. FIG. 2 is an NMR spectrum of the compound prepared in Example 1. FIG. 3 is a cholinesterase activity and self-hydrolysis of Example 4. And FIG. 4 is a graph showing the relationship between the concentration of 2,3-dimethoxybenzoylthiocholine iodide and cholinesterase activity in Example 5, and FIG. 5 is the method of the present invention of Example 6. FIG. 6 is a diagram showing the correlation with the benzoylcholine method, and FIG. 6 is a diagram showing the linearity of the method of the present invention in Example 6. FIG. 7 is an infrared absorption spectrum of the compound produced in Example 7. FIG. 8 is an NMR spectrum of the compound produced in Example 7. FIG. 9 is an infrared spectrum of the compound produced in Example 8. Absorption spectrum FIG. 10 is an NMR spectrum of the compound produced in Example 8, FIG. 11 is a diagram showing the relationship between cholinesterase activity and self-hydrolysis and pH in Example 11, and FIG. 12 is Tris. FIG. 13 is a diagram showing the finger proper concentration of the buffer solution, FIG. 13 is a diagram showing the Km value of pseudocholinesterase against an isobutyrylthiocholine iodide substrate, and FIG. 14 is a diagram showing the Km value of pseudocholinesterase against a cyclohexanecarbonylthiocholine iodide substrate. Km
FIG. 15 shows the values, FIG. 15 shows the correlation between the method of the present invention of Example 15 and the benzoylcholine method, and FIG. 16 shows the relationship between the dilution and the activity value in Example 15. FIG. 17 is a diagram showing the correlation between the method of the present invention of Example 16 and the benzoylcholine method, and FIG. 18 is a diagram showing the relationship between the dilution and the activity value in Example 16, FIGS. 19 and 20. Is the infrared absorption spectrum and NMR spectrum of the compound prepared in Example 17, and FIGS. 21 and 22 are the infrared absorption spectrum and NMR spectrum of the compound prepared in Example 18. is there.

Front page continuation (72) Inventor Nagazo Hayashi 1-2-610 Takahata-cho, Nishinomiya-shi, Hyogo

Claims (3)

[Claims] 1. A general _{formula: Y-COSCH 2 CH 2 N} + (CH 3) 3 X - (I) [ in the formula, Y has the formula: (Here, R ₁ , R ₂ and R ₃ are the same or different and each represent hydrogen, hydroxyl group, halogen, nitro group, lower alkyl group, lower alkoxy group, acetamino group or di (lower alkyl) amino group. , R ₁ , R ₂ and R ₃
At least one of the is not hydrogen. ) A substituted phenyl group represented by (Wherein n represents an integer of 2 to 6) or a cycloalkyl group or an alkyl- or alkoxy-substituted derivative thereof, (Wherein R ₄ , R ₅ and R ₆ all represent an alkyl group, or one represents hydrogen and the other two represent an alkyl group), or X ⁻ (CH ₃ ) ₃ N ⁺ CH ₂ CH ₂ SCOR- (V) (wherein R represents a divalent alkyl group). X represents a halogen atom. ] The thiocholine derivative shown by these. 2. A general _{formula: Y'-COSCH 2 CH 2 N} + (CH 3) 3 X - (I ') [ wherein, Y' has the formula: (Here, R ′ ₁ , R ′ ₂ and R ′ ₃ are the same or different and each represents hydrogen, hydroxyl group, halogen, nitro group, lower alkyl group, lower alkoxy group, acetamino group or di (lower alkyl) amino group. A phenyl group represented by (Wherein n represents an integer of 2 to 6) or a cycloalkyl group or an alkyl- or alkoxy-substituted derivative thereof, (Wherein R ₄ , R ₅ and R ₆ all represent an alkyl group, or one represents hydrogen and the other two represent an alkyl group), or X ⁻ (CH ₃ ) ₃ N ⁺ CH ₂ CH ₂ SCOR- (V) (wherein R represents a divalent alkyl group). X represents a halogen atom. ] The method for quantifying pseudocholinesterase, which comprises quantifying thiocholine or an aromatic or aliphatic compound released from the substrate by the action of pseudocholinesterase using the thiocholine derivative represented by 3. A general _{formula: Y'-COSCH 2 CH 2 N} + (CH 3) 3 X - (I ') [ wherein, Y' has the formula: (Here, R ′ ₁ , R ′ ₂ and R ′ ₃ are the same or different and each represents hydrogen, hydroxyl group, halogen, nitro group, lower alkyl group, lower alkoxy group, acetamino group or di (lower alkyl) amino group. A phenyl group represented by (Wherein n represents an integer of 2 to 6) or a cycloalkyl group or an alkyl- or alkoxy-substituted derivative thereof, (Wherein R ₄ , R ₅ and R ₆ all represent an alkyl group, or one represents hydrogen and the other two represent an alkyl group), or X ⁻ (CH ₃ ) ₃ N ⁺ CH ₂ CH ₂ SCOR- (V) (wherein R represents a divalent alkyl group). X represents a halogen atom. ] The pseudocholinesterase quantification kit containing the thiocholine derivative shown by these as a substrate.