JPS62103060A - Thiocholine derivative and use thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula I [Y is substituted phenyl of formula II (R1-R3 are H, halogen, nitro, lower alkyl, etc.), cycloalkyl, etc., of formula III (n is 2-6), alkyl of formula IV (all of R4-R6 are alkyl or one of them is H and the other two are alkyl, etc.) or group of formula V (R is bivalent alkyl; X is halogen)]. EXAMPLE:4-Nitrobenzoylthiocholine halide. USE:A substrate for the determination of pseudocholinesterase activity. It exhibits little self-hydrolysis at 8-8.5pH which is optimum pH of cholinesterase, is applicable to hand method and an automatic analyzer at a volume suitable for the correct collection of a specimen and enables the accurate determination of the enzymatic activity over a wide range. PREPARATION:The compound of formula I can be produced by reacting an acid chloride of formula Y-COCl with dimethylaminoethanethiol at room temperature and reacting the reaction product with a methyl halide.

Description

DETAILED DESCRIPTION OF THE INVENTION A. "Field of Industrial Application" The present invention relates to novel diocholine derivatives and their uses, and more specifically to the use of pseudocholinesterase (acylcholine acylhydrolase EC, 3°1.1.8). The present invention relates to a novel diocholine derivative that can be used for activity measurement, a method for measuring cholinesterase activity using the derivative as a substrate, and a kit for measurement.

Oral, ``Prior Art'' In clinical tests, serum cholinesterase (EC
, 3, 1, 1, 8) is useful for diagnosing 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 their enzymatic activity and physiological function as well as their distribution in the body. One of these is acetylcholinesterase (EC,
EC, 3,1,1,7) (also known as true cholinesterase), which is present in large amounts in nervous tissues and red blood cell membranes, and the other is called cholinesterase (EC, 3,1,1,8) (also known as pseudocholinesterase), which is present in serum. It is present in large amounts in the liver and pancreas. Among these, pseudocholinesterase is measured in routine clinical tests, but it is being considered to use a substrate that does not react with true cholinesterase but reacts specifically with pseudocholinesterase when serum is used as a specimen. It's here.

As substrates for measuring pseudocholinesterase, acercholines such as acetylcholine, butyrylcholine, and benzoylcholine have been used.

However, when benzoylcholine or its derivatives p-hydroquinhendiylcholine or 0-toluoylcholine are used as substrates, the measurement system is generally a coupled enzyme system that requires dissolved enzymes, making the system complex. There are problems such as interference from various substances in serum samples.

On the other hand, a method is also known in which thiocholine, which is liberated by the action of pseudocholinesterase, is colored with S) [base reagent] using acedelthiocholine, propionylthiocholine, and butyrylthiocholine, which are derivatives of acedelthiocholine, as substrates. There is. However, in this method, the optimum pLI of pseudocholinesterase, 8 to 85, has the drawback that the self-deicing of the substrate increases significantly, making it unusable. In addition, free ZuroS from this acedelthiocholine derivative
There is also a method of coloring the H group with DTNB (5,5°-nothiobis-(2-nitrobenzoic acid)), but this method has too high measurement sensitivity (specific activity), so the amount of sample must be extremely small compared to the amount of reagent. The disadvantage is that it is not possible to measure pseudocholinesterase activity at the level of a normal serum sample using a colorimeter without special equipment.Especially with the automatic analyzers and manual methods used in recent clinical chemistry tests. When testing, the problem is that the sensitivity is too high in a volume that allows accurate sample collection (specimen/reaction liquid volume - I/30 or more).

C. [Object of the Invention] In view of the various problems associated with the prior art, the object of the present invention is to provide a method in which self-hydrolysis is extremely small in pl-R8,O~8,5, the indicating reaction system is simple, It is also an object of the present invention to provide a synthetic substrate for pseudocholinesterase and a method for quantifying pseudocholinesterase, which have a volume that allows for easy collection of the sample (amount of sample/reaction liquid - 1/30 or more) and has sufficient sensitivity.

2. [Structure of the Invention] One gist of the present invention is the general formula Y-CO5CHtC1(tN”(CH*)3X-(1)
[Wherein, Y is the formula.

(Here, R,, R7 and R3 are the same or older, hydrogen, halogen, nitro group, lower alkyl bracket, lower alkoxy group, acetamino group, di(lower alkyl)amino group or L(OOCCI-1, 0-group.However, at least one of R1, R2 and R3 is not hydrogen.) Substituted phenyl group represented by (where n represents an integer from 2 to 6) an alkyl radical or its alkyl or alkokene-substituted derivative, R5-C-(IV) li na (where na, rts and R8 all represent an alkyl group, or one represents hydrogen and the other two represent an alkyl or X-(CHs)*N" Cl-1tCHtSCOR(V)
(Here, R represents a divalent alkyl group.) X represents a halogen atom. ] The novel thiocholine derivatives are as follows.

Another gist of the present invention is the general formula: % formula % () [wherein, Yo is the formula: , a nitro group, a lower alkyl group, a lower alkoxy group, an acetamino group, a di(lower alkyl)amino group, or a HOOCCH!O- group. A cycloalkyl group or its alkyl or alkoxy substituted derivative represented by (herein, R4, R6 and R11 have the same meanings as above)
A branched alkyl group represented by X-(CH3)3N"CH,CHtSCOR-(V)
Here, R has the same meaning as above. . ) represents a group represented by X has the same meaning as above. ] A method for quantifying pseudocholinesterase, which is characterized in that thiocholine or an aromatic or aliphatic compound liberated by the action of pseudocholinesterase is determined using a thiocholine derivative represented by the following as a substrate, and a pseudocholinesterase containing the thiocholine derivative. quantification kit.

In the general formula, halogen is fluorine, chlorine, bromine, or iodine. Further, lower alkyl group and lower alkoxy group mean an alkyl group and alkoxy group having 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms, and the alkyl group included in the alkyl group and alkoxy group may be either linear or branched. It may be. In addition, if desired, a cycloalkyl group (I[[)
Alkyl or alkoxy present as a substituent in
Preferably, it has 1 to 4 carbon atoms.

Thiocholine derivatives in which Y is a substituted phenyl group (II) (
Examples of 1) include 4-nitrobenzoylthiocholine halide, 3-nitrobenzoylthiocholine halide, 2
-Nitrobenzoylthiocholine halide, 4-chlorobenzoylthiocholine halide, 3-chlorobenzoylthiocholine halide, 2-chlorobenzoylthiocholine halide, 4-methoxybenzoyldiocholine halide,
3-methoxybenzoyldiocholine halide, 2-methoxybenzoyldiocholine halide, 2,4-dimethoxybenzoylthiocholine halide, 2,3-dimethoxybenzoylthiocholine halide, 4-methylbenzoylthiocholine halide, 3-methylbenzoylthiocholine halide halide, 2-methylbenzoylthiocholine 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 -acetamidobenzoylthiocholine halide, 2-acetamidobenzoylthiocholine halide, etc. In particular, derivatives having a substituent at the ortho position are advantageous because they have low self-decomposing properties and high reactivity.

Examples of the thiocholine derivatives (1) of the present invention in which Y is a group (1) to (V) include bivaloyldiocholine halide, isobutyryldiocholine halide, methylpropionylthiocholine halide, dimethylpropionylthiocholine halide. , ethylpropionylthiocholine halide,
Cyclopropanecarbonylthiocholine halide, cyclobutanecarbonylthiocholine halide, cyclopentanecarbonylthiocholine halide, cyclohexanecarbonylthiocholine halide, cyclohebutanecarbonylthiocholine halide, methylcyclopropanecarbonylthiocholine halide, dimethylcyclopropanecarbonylthiocholine halide, Ethylcyclopropanecarbonylthiocholine halide, methoxycyclopropanecarbonylthiocholine halide, dimethoxycyclopropanecarbonylthiocholine halide, diethylcyclopropanecarbonylthiocholine halide, ethoxycyclopropanecarbonylthiocholine halide, jetoxycyclopropanecarbonylthiocholine halide, methyl Cyclobutanecarbonylthiocholine halide, dimethylcyclobutanecarbonylthiocholine halide, methoxycyclobutanecarbonylthiocholine halide, dimethinecyclobutanecarbonylthiocholine halide, ethylcyclobutanecarbonylthiocholine halide, ethoxycyclobutanecarbonylthiocholine halide, methoxycyclobutanecarbonylthiocholine halide Nylthiocholine halide, dimethylcyclopenkune carbonylthiocholine halide, methoxycyclopentanecarbonylthiocholine halide, dimethoxycyclopentanecarbonylthiocholine halide, ethylcyclopentane luponyldiocholine halide, ethoxycyclopentane luponyldiocholine halide, Methylcyclobutanecarbonylthiocholine halide, dimethylcyclohexanecarbonylthiocholine halide, methoxycyclohexanecarbonylthiocholine halide, dimethoxycyclohexanecarbonylthiocholine halide, ethylcyclohexanethiocholine halide, ethoxycyclohexanecarbonylthiocholine halide, methylcyclohexanecarbonylthiocholine halide Halide, dimethylcyclohebutanecarbonylthiocholine halide, methoxycyclohebutanecarbonylthiocholine halide, dimethoxycyclohebutanecarbonylthiocholine halide, ethylcyclohebutanecarbonylthiocholine halide, ethoxycyclohebutanecarbonylthiocholine halide,
Examples include succinylthiocholine halide.

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

General formula: % formula % [In the formula, Y has the same meaning as above. ] The acid chloride represented by the formula and dimethylaminoethanethiol are each dissolved in a suitable solvent (for example, diethyl ether), and one is slowly added dropwise to the other while stirring, and crystals are precipitated at room temperature. The chloride is liberated from the crystals with an alkaline solution (e.g., sodium carbonate solution), extracted with a suitable solvent (e.g., dimethyl ether), and after dry filtration, methyl halide is added to the obtained product for reaction. Get choline halide.

In the method for measuring pseudocholinesterase of the present invention, thiocholine derivatives (
Thiocholine is liberated from I゛), developed with an S L1 group reagent, quantified using an ordinary spectrophotometer or various automatic analyzers, and the amount of thiocholine produced is determined in a constant manner. Be able to measure cholinesterase activity.

As the SH group reagent, all conventionally used reagents can be used; for example, D i "N 13 (5.

5°-dithiobis-(2-nitrobenzoic acid) can be shown. When DTNB is used as the S H group reagent, T
NB (2-nitro-5-mercaptobenzoic acid) is produced and develops color.

The method for quantifying pseudocholinesterase of the present invention is particularly useful in the field of clinical testing, where analysis is performed using the kinetic (K1netic) method using various automatic analyzers, or the reaction is stopped using a pseudocholinesterase inhibitor (for example, neostigmine). , it can be applied when analyzing using the end point method.

C Example] Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these.

Reference Example Preparation of benzoylthiocholine iodite; -nomedylaminoethandiol hydrochloride [(C)l 3)zNcHt
3.5 g of cHtsH·HCρ is dissolved in a solution of 3 g of sodium carbonate in 15 zC of water.

This is extracted with noethyl ether 150x and the extract is dried over sodium sulfate overnight. The sodium sulfate is removed by filtration, 3.69 g of benzoyl chloride is added dropwise to the filtrate with stirring, and the precipitated crystals are collected by filtration. Yield 4g
.

Of this, 0.59 is added to 10 m of water containing 0.39 sodium carbonate.
The solution was dissolved in Q solution and extracted with 75 kg of diethyl ether (!), and the extract was dried over sodium sulfate overnight. The sodium sulfate was filtered off, and the total volume of the diethyl ether solution was diluted with 50 m
Concentrate to Q, add 0.42 g of methyl iodide, and continue stirring at room temperature. When crystals begin to precipitate and completely disappear (for 2 days), stop stirring and filter to obtain crystals. These crystals were recrystallized from water to obtain 0.59 of the desired benzoylthiocholine iodite. Yield 71.4%. Melting point: 257°C. This is the Journal of No American
Chemical Society (J, Am, Chcm, S
oc,.

R, R, Reushow et 'al, listed at Kimi, +765 (+935).
The melting point (M, P = 257°C) was consistent with that reported by ).

Example 1 Preparation of 2,3-dimethoxyhenzoylthiocholine iodide 2,3-dimethoxybenzoic acid 17.8y (0,15m
Thionyl chloride 1IzQ was added to 1IzQ of thionyl chloride, and after the reaction, excess thionyl chloride was removed using an aspirator to obtain 7.6 g of crystalline acid chloride+. Yield 87.7%.

To a diethyl ether solution of dimethylaminoethanethiol obtained in the same manner as in the reference example, 3.69 (0,0175 mol) of the obtained acid chloride was added, and the precipitated crystals were collected by filtration and dried under reduced pressure. Yield: 4439 o, of which 3
, 1 g was dissolved in a solution of 1 liter of sodium carbonate and 2 g of water at 50 rpQ, extracted with 150 yQ of noethyl ether, and the extract was dried over sodium sulfate overnight. After that, the entire diethyl ether solution was set as roomQ, and methyl iodide l
x ( 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 obtain 2,3-nomethoxybenzoylthiocholine iodide. Melting point 184-18
5°co Infrared absorption spectrum and NM of this compound
The R spectrum is shown in FIGS. 1 and 2.

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

Human serum or purified water 02πQ was added to the first reagent 2 and 5 wQ having the following composition, and after heating at 37°C for 5 minutes, the second reagent U 0 and 5 w12 was added, and 1 at 405 nm.
The absorbance change per minute (ΔE/min) was measured.

First reagent trishydroquine 240 mmol/Q aminomethane 5.5°-notiobis-0.3 mmol/Q.

(2-nitrobenzoic acid) Maleic acid suitable The above components were dissolved in purified water and adjusted to pH 8.0.

Second reagent Aqueous solution of various thiocholine derivatives 6 mmol#! Cone-soluble compounds were dissolved using a 25% aqueous solution of alcohol.

As a result, as shown in Table 1, benzoylthiocholine ester and 2,3-dimethquine thiocholine ester are water-soluble and stable in aqueous solution (reagent blank is small).
It was excellent in terms of reactivity (absorbance of 0.03 L or more per minute).

Example 3 The following reagents and specimens were prepared, and the first reagent was 2°4 m (l
Add 0.1 mQ (!) of each sample to the sample, heat at 30°C for 5 minutes, add 0.5 mQ of the second reagent, and incubate at 30°C for 40 min.
Absorbance change per minute at 5 nm (ΔE/mi
n,) was measured to examine the reactivity of the specimen.

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

First reagent trishydroxymethyl 12.19 (loOmM
) Aminomethane 5.5'-dithiobis 118.9m? (0,3
mM) (2-nitrobenzoic acid) Maleic acid Titun purified water 1g (PH8,2) Second reagent (A) Benzoylthiocholine 2.1071079 (6
Iodite purified water IQ Second reagent (B) 2.3-dimethoxybenzoyl 2.4559 (6mM)
Thiocholine iodite purified water lσ Specimen (1) Pseudocholinesterase 13U/xg (Sigma) Specimen (2) Truecholinesterase 13U/zQ.

(Sigma) Table 2 Example 4 Trishydroxymethylaminomethane (100 mmol
d), 5,5°-dithiobis(2-nitrobenzoic acid (0
, 3 mmol/12) in purified water and adjusted to p+-17, 0 to 9.0 with maleic acid to prepare various solutions. Add 2.41 grams of this solution to 0.00 grams of pseudocholinesterase (manufactured by Sigma) (adjusted to 13 U/mQ using the butyrylthiocholine method). Add lx & and heat at 30℃ for 5 minutes, 2.3
A 6 mmol/12 aqueous solution of -dimethylbenzoylthiocholine iodite or benzoylthiocholine iodite at 0.5 mQ was added, and the rate of increase in absorbance at 405 nm was measured to examine reactivity at each pH.

As a result, as shown in Figure 3, 2,3-dimethylbenzoylthiocholine iodite is optimal at pH 8.0 to 8.5, and the blank reaction (self-decomposition) is also 10 mAb/mi.
It was found that it is a suitable substrate for cholinesterase measurement at n or less.

Example 5 Trishydroxymethylaminomethane 100nunol
/ Q, 5, 5°-dithiobis-(2-nitrobenzoic acid 0,3 mmol/ (manufactured by Sigma) (13U/z (adjusted to 2 using the butyrylthiocholine method) O, 1x (7) was added, and a substrate solution of 2,3-dimethylbenzoylthiocholine iodite concentration 0.06~l 8mmol/(!) was added. In addition, we investigated the Km value of pseudocholinesterase for this substrate. As a result, as shown in Figure 4, the Kra value was extremely small near lXl0-'M, and 1
At mmol/12 or more, it shows more than 90% of the maximum activity,
Since the blank reaction (self-decomposition) was also small, it was found to be useful as a substrate for cholinesterase measurement even at a concentration of 1 mmol/12.

Example 6 Trishydroxymethylaminomethane 240 mmol/
12.5.5'-dithiobis-(2-nitrobenzoic acid)
An aqueous solution of pH 8.2 containing 0.3 mmol/12 (pH correction was performed with maleic acid) was used as the first reagent,
First reagent 2.4 tp (l to human direct sample 0, 1 R (
After prewarming for 5 minutes in a 30°C constant temperature bath, 2.
4-dimethylbenzoylthiocholine iodite 6 mm
After adding and reacting aqueous tL05mQ of ol/σ, 1
Absorbance change per minute (ΔA) was measured at 405 nm.

Note that a reagent blank is similarly performed using purified water as the specimen (ΔB). The reaction formula is as follows.

511C11, CIl, N"(CIl3)31-] From the absorbance change per minute determined as described above, the measured value of Honbo was determined using the f formula.

Looking at the correlation when the hendiylcholine 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).

In addition, instead of human serum samples, high-value pseudocholinesterase (manufactured by Sigma) (butyrylthiocholine method)
01tJ/ff12) was diluted in 10 stages, and the activity value was calculated using the formula below to find the test m line.As shown in Figure 6, a high value of +501 U/12 (3
Linearity was maintained from 0 to 50 r U/12).

Table 3 Relationship between measured values of Honbo and benzoylcholine method 13.3 x
O,1 Pseudocholinesterase activity (IU#) ΔA of the specimen
: Absorbance change per minute of sample at 405 nm at 30°C ΔB : Absorbance change per minute at 405 nm of purified water at 30°C 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 Preparation of physobutyrylthiocholine iodide Niedimethylaminoethanethiol hydrochloride 509 was liberated with an aqueous sodium carbonate solution, adjusted to pH 10 or higher, extracted with methylene chloride, and reduced pressure. Distillation was performed to obtain 30 g of dimethylaminoethanethiol (yield 80.9%, boiling point 40°C/I
2 mmHg). Of these, 3.59 g and 35 g of isobutyryl chloride were mixed in a diethyl ether 15011I2 solution, and the precipitated crystals were collected by filtration. Further, this was dissolved in an aqueous sodium carbonate (10%) solution, the pHI was adjusted to 0 or higher, extracted with diethyl ether, and the extract was dried over magnesium sulfate. Then, add diethyl ether to the total volume of 350+12, and add methyl iodide 5IIIQ to this.
was added and stirred at room temperature. The precipitated crystals were collected by filtration and recrystallized from water to obtain 9.9 g of isobutyrylthiocholine iodide. Yield 94.6%. Melting point 157-158
℃.

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

Example 8 Preparation of cyclohexanecarbonylthiocholine iodite Dimethylaminoethanethiol prepared in Example 73.
5 g and 4.89 cyclohexane carbonyl chloride
were mixed in diethyl ether 150R12, and the precipitated crystals were collected by filtration. Furthermore, this was mixed with sodium carbonate (10%
) Dissolved in an aqueous solution to a concentration of I) I-110 or higher, extracted with diethyl ether, and dried over magnesium sulfate. Thereafter, the entire volume was diluted with diethyl ether (350+), methyl iodide (5j+) was added, and the mixture was stirred at room temperature. .Yield 59.4
%. Melting point: 159-160°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 substrate Six types of thiocholine derivatives were prepared, and their water solubility, stability in aqueous solution, and reactivity to human blood were examined by the following method.

2.5 ml of the first reagent with the following composition (0.2 ml of the second reagent per liter)
1 was mixed, 0.0!5 mQ of the specimen or purified water was added, and after heating at 37°C for 5 minutes, 0.51 of the third reagent was added.
Absorbance change per minute at 405 nm (ΔE/m
1n) was measured.

First reagent trishydroquine medylaminomethane 325 mmol/1
2-maleic acid An appropriate amount of the above components was dissolved in purified water, and the solution was adjusted to 37° C. and pH 7.5.

Second reagent 5.5°-dithiobis(2-nitrobenzoic acid) aqueous solution 0, 65 mmol/
Q Uragomachi father species thiocholine derivative aqueous solution 3, 25 mmol/
Q As a result, as shown in Table 4, cyclohexanecarbonylthiocholine iodide and isobutyrylthiocholine iodide were particularly excellent in terms of stability of the reactive reagent blank.

Example IO Prepare the following reagents and specimens, mix 2°5 mQ of the first reagent and 0.2 vr (l) of the second reagent, add 0.05 m12 of the specimen or purified water, and heat at 37°C for 5 minutes. , add 0.5 MC of third reagent (A) or (B), and incubate at 37°C for 4 hours.
Absorbance change per minute at 05 nm (ΔE/m1
n) was measured to examine the reactivity of the specimen.

First reagent Trishydroxymedylaminomethane 325 mmol/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-ammonium, fragrant acid) aqueous solution 0, 65 mmol
/ Q third reagent (A) inbutyrylthiocholine-iodide aqueous solution 3, 25 m'mol/
Q (B) Cyclohexane carbonyl-thiocholine iodite aqueous solution 3, 25 mmol/Q Sample (+) Pseudocholinesterase 13 U/mQ.

(Sigma) Sample (2) Truecholinesterase I 3 U/m (2(
As a result, as shown in Table 5, isobutyrylthiocholine iodide and cyclohexane grouponylthiocholine iodide do not react with true cholinesterase, but specifically react with pseudocholinesterase. was confirmed.

Example 11 Trishydroxymethylaminomethane (100 mmol
/12), 5,5-dithiobis(2-nitrobenzoic acid) is dissolved in purified water at a concentration of 0,25 mmol/&),
Various solutions were prepared by adjusting the pH to 7.0 to 9.0 with maleic acid. Use this as the first reagent and add 2.4 mQ and 0.05 iC of human pool serum, and after heating at 376C for 5 minutes, 63 mmol of isobutyrylthiocholine iodite or cyclohexane phosphonylthiocholine iodite is added.
/Q, aqueous solution 0,5 m (l) respectively, 40
The amount of change in absorbance per minute at 5 nm was determined using a reagent blank as a control, and the reactivity in each of 1) and 11 was examined.

As a result, as shown in FIG. 11, all substrates were optimal at pH around 8 and 0. The blank reaction is p
It was found to be 0.005/min or less around H8,0, and is a suitable substrate for cholinesterase measurement.

Example 12 Trishydroxymethylaminomethane at a concentration of 60III
5.5-dithiobis(2-nitrobenzoic acid) in the range of 200 mmol/f2 from lol/Q to 0.2
Contains at a concentration of 5 mmol/12, pH 8 with maleic acid,
A solution adjusted to 0 was prepared. These various solutions 2, 6
Add 0.05xQ of human pool serum to m(l), and after heating at 37°C for 5 minutes, add 6
．． 3mmol/Q aqueous solution 0.5in! Add 405
The change in absorbance per minute in nm was measured to determine the optimal concentration of the Tris buffer. 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, for all substrates, the Tris buffer at the final reaction time of 100 mM showed the maximum activity.

Example 13 Trishydroxymethylaminomethane (100 mmol
/Q) and pl-(8,
0 aqueous solution (pH corrected with maleic acid) as the first reagent, and the first reagent 2 and 6 mQ were mixed with butoidcholinesterase (manufactured by Nguma) (13 U/m by the butyrylthiocholine method).
Add 0.05fff2 (adjusted to
Add 0 and 5 ttt (l) of the substrate solution of Q, respectively.
The Km value of pseudocholinesterase for this substrate was investigated. As shown in Fig. 13, the result is <Km value is 6,
Extremely small around 9XIO-5M, 1 mmol/
It was found that 95% or more of the maximum activity was exhibited at Q or higher.

Example 14 Cyclohexanecarbonylthiocholine iodide was used in place of the substrate isobutyrylthiocholine iodide used in Example 13, and the amount was 0.126 to 3°15 mmol/1.
A substrate solution of No. 2 was prepared and the same test as in Example 13 was conducted. The Km value of pseudocholinesterase for cyclohexane carbonylthiocholine iodide is the 14th
As shown in the figure, it exhibited a Kffl value near 6°9xlO-'M, which was almost the same as that of isobutyrylthiocholine iodite.

Example 15 Trishydroxymethylaminomethane 100 mmol/
&, 5,5'-dithiobisu(2-nitrobenzoic acid) (0,25 mmol/12) containing an aqueous solution of pH 8.0 (pH corrected with maleic acid) was used as the first reagent, and the first reagent 2, 6 * Add 0.05112 of a human serum sample to Q, and after heating at 37°C for 5 minutes, add isobutyrylthiocholine iodite 0.62 mmol/Q as a second reagent.
After adding an aqueous solution of 0,5*Q and causing a reaction, the absorbance change (ΔA) per minute was measured at 405 nm. A reagent blank was prepared in the same manner using purified water instead of the sample (ΔB).

The reaction formula is as follows: CH3 CHs CC05CHtCHtN”(CHa)sl-
HSCHzCHtN"(CHa)sE-2HSGHzC
H*N"(CH3)5 +-> 2[-5CHyCt
ltN''(CII*)sl +L The actual measurement value was determined by the following formula from the change in absorbance per minute determined as described above: Pseudcholinesterase activity of the specimen (I
ΔA of U/: Absorbance change ΔB per minute at 405 nm at 30°C of the specimen - Purified water at 405 nm at 30°C 1
Absorbance change 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 a reference method, as shown in Table 6 and FIG. 15, a correlation coefficient r=0.998 was obtained, and good results were obtained.

In addition, high-value pseudocholinesterase (Sigma) (3010
/m12) was diluted in 10 steps and a calibration curve with the activity value was calculated, as shown in Table 6 and Figure 16.
Up to 01 U/Q (normal samples 300-7ootu/i2
) Maintained linearity.

Table 6 Table 6 (Continued) Example 16 The same procedure as in Example 15 was carried out except that the second reagent used as the substrate was cyclohexanecarbonylthiocholine iodite 0,6
When the test was carried out using an aqueous solution of 211 Imol/12, as shown in Table 7 and FIG. 17, the correlation coefficient r=0.
998, a good result was obtained.

In addition, the calibration curve with the activity value obtained in the same manner is as shown in Figure 18, up to 30001U/(2 (normal samples 600 to +
5001 U/Q) Maintained linearity.

Table 7 Table 7 (continued) Example 17 Preparation of phenylacetylthiocholine iodide Example 7
3.0 g of dimethylaminoethanethiol prepared above and 3.1 g of phenylacetyl chloride were mixed in 150 ml of diethyl ether, and the precipitated crystals were collected by filtration. Furthermore, this was dissolved in a 10% aqueous sodium carbonate solution, and the pH
10 or higher, extracted with diethyl ether, and dried over magnesium sulfate. Thereafter, the total volume was made up to 350xQ with diethyl ether, 3yQ of methyl iodide was added, and the mixture was stirred at room temperature. The precipitated crystals were collected by filtration, and the crystals were recrystallized from water to obtain phenylacetylthiocholine iodide.

I got 09. Melting point: 182-183°C. Yield 57.7%.

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

Example 18 Co1' of phenylpropionylthiocholineyotite,
Dimethylaminoethanethiol 30 prepared in Example 7
g and 33 g of phenylpropionyl chloride were mixed in diethyl ether 15OIlIQ, and the precipitated crystals were collected by filtration. Further, this was dissolved in a 10% aqueous sodium carbonate solution to give a pti of 0 or more, extracted with diethyl ether, and dried over magnesium sulfate for 1 hour. Then, the total volume was made up to 350 ml (l) with diethyl ether, and methyl iodide
mQ was added and stirred at room temperature. Filter the precipitated crystals,
The crystals were recrystallized from water to obtain 7.0 g of phenylpropionylthiocholineyotite. Melting point 200-201
'C.

Yield 648%.

Infrared absorption spectrum and N M of the obtained compound
(The spectra are shown in FIGS. 21 and 22.

[Effect of the invention] By using the thiocholine derivative of the present invention as a substrate for measuring cholinesterase activity, there is less self-thawing at the optimum pt+s, o~8.5 of cholinesterase, and the method and sample amount can be collected accurately. It can be applied to automatic analyzers with a large volume (analyte/reaction liquid volume = 1/30 or more), and can be measured over a wider range and more accurately than with conventional substrates such as butyrylcholine.

[Brief explanation of drawings]

Figure 1 is an example! FIG. 2 shows the NMR spectrum of the compound produced in Example 1. FIG. 3 shows the relationship between cholinesterase activity and self-ice melting in Example 4 and l) FIG. 4 is a diagram showing the relationship between 2,3-dimethoxybenzoylthiocholine iodite concentration and cholinesterase activity in Example 5. FIG. 5 is a diagram showing the correlation between the method of the present invention and the benzoylcholine method in Example 6. FIG. 6 and FIG. 6 are diagrams showing the linearity of the method of the present invention in Example 6. Figure 7 is an infrared absorption spectrum of the compound produced in Example 7. Figure 8 is an NMR spectrum of the compound produced in Example 7. Figure 9 is an infrared absorption spectrum of the compound produced in Example 8. The figure shows the NMR spectrum of the compound produced in Example 8. Figure 11 shows the relationship between cholinesterase activity and self-ice melting in Example 11 and pH. Figure 12 shows the
Figure 13 shows the Km value of pseudocholinesterase against isobutyrylthiocholine iodite substrate; Figure 14 shows the Km value of pseudocholinesterase against cyclohexanecarbonylthiocholine iodite substrate. Km of
Figure 15 is a diagram showing the correlation between the method of the present invention and the benzoylcholine method of Example I5, Figure 1621 is a diagram showing the relationship between dilution and activity value in Example I5, Figure 17 The figure shows the correlation between the method of the present invention and the benzoylcholine method in Example 16. Figure 18 shows the relationship between dilution and activity value in Example 16. Figures 19 and 20 show the relationship between dilution and activity value in Example 16. , an infrared absorption spectrum and an NMR spectrum of the compound produced in Example 17, and FIGS. 21 and 22 are an infrared absorption spectrum and an NMR spectrum of the compound produced in Example I8.

Claims (1)

[Claims] 1. General formula: Y-COSCH_2CH_2^+(CH_3)_3X^
-(I) [Wherein, Y is the formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (Here, R_1, R_2 and R_3 are the same or different, hydrogen, halogen, nitro group, lower Represents an alkyl group, lower alkoxy group, acetamino group, di(lower alkyl)amino group or HOOCCH_2O- group.However, at least one of R_1, R_2 and R_3 is not hydrogen.) A substituted phenyl group represented by the formula ▲ , chemical formulas, tables, etc. ▼ (III) (Here, n represents an integer from 2 to 6) Cycloalkyl group or its alkyl or alkoxy substituted derivatives, ▲ Numerical formulas, chemical formulas, tables, etc. (IV) (Here, R_4, R_5 and R_8 all represent an alkyl group, or one represents hydrogen and the other two represent an alkyl group, or two represent hydrogen and the other represents an alkyl group or an arylalkyl group excluding a methyl group and an ethyl group.) or a branched alkyl group represented by X-(CH_3)_3N^+CH_2CH_2SCOR
-(V) (Here, R represents a divalent alkyl group.)
represents a group represented by X represents a halogen atom. ] Thiocholine derivative shown. 2. General formula: Y'-COSCH_2CH_2N^+(CH_3)_3
X＾−( I ′) [where, Y′ is a formula: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II′) (Here, R′_1, R′_2, and R′_3 are
The same or different hydrogen, halogen, nitro group, lower alkyl group, lower alkoxy group, acetamino group, di(
represents a lower alkyl) amino group or a HOOCCH_2O- group. ) phenyl group represented by ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (III) (where n represents an integer from 2 to 6) cycloalkyl group or its alkyl or alkoxy substituted derivative, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (IV) (Here, R_4, R_5 and R_6 all represent alkyl groups, one represents hydrogen and the other two represent alkyl groups, or two represents hydrogen, and the other represents an alkyl group or arylalkyl group excluding methyl and ethyl groups.) or a branched alkyl group represented by
R-(V) (herein, R represents a divalent alkyl group). X represents a halogen atom. ] A method for quantifying pseudocholinesterase, which comprises using a thiocholine derivative represented by the following as a substrate, and quantifying an aromatic or aliphatic compound similar to thiocholine liberated from the substrate by the action of pseudocholinesterase. 3. General formula: Y'-COSCH_2CH_2N^+(CH_3)_3
X＾−( I ′) [In the formula, Y′ is a formula: ▲ Numerical formula, chemical formula, table, etc. ▼ (II′) (Here, R′_1, R′_2, and R′_3 are the same or, differently, represents hydrogen, halogen, nitro group, lower alkyl group, lower alkoxy group, acetamino group, di(lower alkyl)amino group or HOOCCH_2O- group.) Phenyl group represented by ▲ Numerical formula, chemical formula, table, etc. ▼ (III) (Here, n represents an integer from 2 to 6.) A cycloalkyl group or its alkyl or alkoxy substituted derivative, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (IV) (here In, R_4, R_5 and R_6 all represent an alkyl group, one represents hydrogen and the other two represent an alkyl group, or two represent hydrogen and the other one represents a methyl group and an ethyl group. Represents an alkyl group or arylalkyl group excluding the group.) or a branched alkyl group represented by X^-(CH_3)_3N^+CH_2CH_2SCO
R-(V) (herein, R represents a divalent alkyl group). X represents a halogen atom. ] A kit for quantifying pseudocholinesterase, comprising a thiocholine derivative represented by the following as a substrate.