JPH09316040A - New substrate compound and determination of cholinesterase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new substrate compound that has in its chemical structure such a chromophoric group that contact of the substrate compound with cholinesterase liberates a chromophoric component to form a chromophoric substance, and is useful as a cholinesterase substrate. SOLUTION: This substrate compound comprises a compound of the formula (A<1> and A<2> are each O or S; R' is, as R<1> -AH, an aromatic heterocyclic hydroxy or thiol compound; R<2> is a 2-4C carbon chain; X<-> is an anion), for example, 3-(4-nitrophenoxycarbonyl)propionylcholine, etc. This substrate compound is obtained preferably, for example, by heating a dicarboxylic acid and 2- bromoethyl alcohol on an organic nonpolar solvent, such as benzene, for 1-6hr under reflux condition, adding thionyl chloride to the thus obtained precursor 1 (bromoethoxydicarbonylcarboxylic acid), heating the mixture under reflux condition, removing the excess of thionyl chloride, add an ethereal solution of an aromatic hydrocarbon having a chromophric group and pyrimidine, and introducing triethylamine into the thus obtained precursor 2.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel compound and a method for measuring cholinesterase activity using the novel compound as a substrate.

[0002]

2. Description of the Related Art Cholinesterase in human serum (plasma) is a pseudocholinesterase (pseudochol).
inesterase; strain name: Acylcholin
e acylhydrolase; EC 3.1.1.
It is called 8) and is distinguished from the true cholinesterase involved in the nervous system. Since its biosynthesis is carried out in the liver and released into the blood, measuring cholinesterase in the blood causes liver function, physical condition during the use of anticholinesterase agents, organophosphate poisoning, nephrotic syndrome, or hyperthyroidism. It is an important measurement item in the field of clinical diagnosis because it can obtain useful indexes for diagnosis and treatment.

As a method for measuring cholinesterase,
A method has been used for a long time, in which acetylcholine is used as a substrate to lead the generated acetic acid to the color development of phenolphthalein, and this is displayed as a pH change unit. Thereafter, a method of connecting this acetic acid to an acetate kinase, a phosphoenolpyruvate kinase, and a NADH-lactate dehydrogenase system, or benzoylcholine, or choline produced using toluylcholine as a substrate is treated with choline oxidase and peroxidase, Enzymatic assay methods have been developed in which an enzyme is combined with a detection system, such as a method that leads to oxidative color development of a dye by hydrogen peroxide, or a method that combines 4-hydroxybenzoylcholine with oxygenase and NADH. On the other hand, since thiocholine ester is also easily hydrolyzed by cholinesterase, the -SH group of thiocholine formed from a substrate such as acetylthiocholine, butyrylthiocholine or propionylthiocholine is
A method of tracing the yellow color of free 2-nitro-5-mercaptobenzoic acid through substitution with 5'-dithio-bis (2-nitrobenzoic acid) is also widely used.

[0004]

However, in the conventional measurement method, the color development method of phenolphthalein has insufficient measurement sensitivity, and the enzymatic measurement method in which an enzyme is combined with the detection system is stable in the enzyme used. There was a problem with properties and specificity, and it was necessary to pay attention to maintain the performance of the reagent. Further, also in the color development method using thiocholine, the reaction solution and the sample are likely to be interfered with by other coexisting thiol compounds and redox substances, and like the enzymatic assay method, there is a problem in terms of maintaining the performance of the reagent. is there. That is, in the conventional method, an aromatic or aliphatic carboxylic acid or choline, or thiocholine, which is produced depending on the substrate used, is introduced into an enzymatic coloring system, or 5,5′-dithio-bis (2
-Nitrobenzoic acid) is limited to lead to a color system of free 2-nitro-5-mercaptobenzoic acid through substitution with ascorbic acid in the indicator reaction system,
Due to the interference of reducing substances such as bilirubin or glutathione and other thiols, various measurement methods have been used in the situation where no fundamental improvement is made. Therefore, in order to solve these problems, the present inventors have designed and synthesized a novel substrate substance for measuring cholinesterase activity containing a chromogenic substituent in the structure, and using this novel substrate substance. The present invention has been completed by earnestly examining a method for measuring cholinesterase activity.

[0005]

According to the present invention, there is provided a compound represented by the general formula (I):

Embedded image R ¹ —A ¹ —OC—R ² —CO—A ² — (CH ₂ ) ₂ —N ⁺ (CH ₃ ) ₃ · X ^— (I) [wherein A ¹ and A ² are Each the same or different,
O or S, R ¹ represents an aromatic or aromatic heterocyclic hydroxy compound or a thiol compound as R ¹ -A ¹ H, and R ² represents a saturated or unsaturated substituted or unsubstituted a carbon chain of 2 to 4 carbon atoms, and X ^-
Is an anion]. The present invention also relates to a method for measuring cholinesterase activity, which comprises using the compound represented by the general formula (I) as a substrate substance.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
In the general formula (I), the group R ¹ is a substituted or unsubstituted aromatic group or aromatic heterocyclic ring group, and is preferably a reaction pH range (weakly acidic to alkaline, when measuring the activity of cholinesterase, Specifically, at pH 5 to 10, particularly pH 7 to 9.5), "R ¹ -A ¹ H" released from the compound represented by the general formula (I) is a phenoxide derivative or a phenylmercaptide derivative. Alternatively, it is not particularly limited as long as it induces a quinoid derivative and has a coloration that is spectroscopically distinguished. R ¹ -A ¹ H
Is more preferably a substituted or unsubstituted phenol,
Substituted or unsubstituted naphthol, substituted or unsubstituted hydroxyindole, substituted or unsubstituted hydroxyphenoxazone, substituted or unsubstituted hydroxyphenoxazone-N-oxide, substituted or unsubstituted fluorescein, substituted or unsubstituted hydroxyphenothiazone,
It is a substituted or unsubstituted thiophenol, or a substituted or unsubstituted thionaphthol.

In the general formula (I), the substituted or unsubstituted phenyl group R ¹ is preferably the general formula (1):

Embedded image [In the formula, R ¹¹ represents a hydrogen atom, a halogen atom, a nitro group,
Lower alkyl group having 1 to 4 carbon atoms, sulfoalkyl group having 1 to 4 carbon atoms, hydroxysulfoalkyl group having 1 to 4 carbon atoms, lower alkoxy group having 1 to 4 carbon atoms, lower alkanoyl group having 2 to 5 carbon atoms , A carboxyalkyl group having 2 to 5 carbon atoms, an amino group, an acid amide group having 2 to 5 carbon atoms, and 1 carbon atom
To an amino group mono- or di-substituted with a lower alkyl group of 4 to 4, an acid amide consisting of an amine moiety mono- or di-substituted with a lower alkyl group of 1 to 4 carbons and an acid moiety of 2 to 5 carbons A group, a hydroxyl group, a carboxyl group, a sulfo group, an unsubstituted phenyl group or a substituted or unsubstituted nitrophenylazo group, and n is an integer of 1 to 5,
When n is 2 to 5, R ¹¹ may be the same or different, and at least one of R ¹¹ is a nitro group, a lower alkanoyl group having 2 to 5 carbon atoms, or a substituted or non-substituted one. It is a substituted nitrophenylazo group, and R ¹¹ is represented by the general formula (1a):

Embedded image (In the formula, R ¹¹¹ is a halogen atom, a nitro group, or a carbon number 1
~ 4 lower alkyl group, C1-4 sulfoalkyl group, C1-4 hydroxysulfoalkyl group, C1-4 lower alkoxy group, C2-5 lower alkanoyl group, carbon number 2-5 carboxyalkyl group, amino group, C2-C5 acid amide group, amino group mono- or di-substituted with C1-C4 lower alkyl group, C1-C4 lower alkyl group An acid amide group consisting of a mono- or di-substituted amine moiety and an acid moiety having 2 to 5 carbon atoms, a hydroxyl group, a carboxyl group, a sulfo group,
It is an unsubstituted phenyl group or a substituted or unsubstituted nitrophenylazo group, m is an integer of 1 to 5, and m is 2
And R ¹¹¹ may be the same or different, and at least one of R ¹¹¹ is a nitro group, a lower alkanoyl group having 2 to 5 carbon atoms, or a substituted or unsubstituted nitrophenylazo group. The group represented by the formula (1) can be a group represented by the formula (1).

Further, a preferred substituted or unsubstituted phenyl group R ¹ is the same as the above-mentioned general formula (1), in which R ¹¹ is the formula (1b):

Embedded image [Wherein R ¹² and R ¹³ are the same or different,
Hydrogen atom, halogen atom, nitro group, lower alkyl group having 1 to 4 carbon atoms, sulfoalkyl group having 1 to 4 carbon atoms, hydroxysulfoalkyl group having 1 to 4 carbon atoms, 1 to 4 carbon atoms
Lower alkoxy group, lower alkanoyl group having 2 to 5 carbon atoms, carboxyalkyl group having 2 to 5 carbon atoms, amino group,
An acid amide group having 2 to 5 carbon atoms, an amino group mono-substituted or di-substituted with a lower alkyl group having 1 to 4 carbons, and an amine moiety mono-substituted or di-substituted with a lower alkyl group having 1 to 4 carbons; An acid amide group consisting of an acid moiety having 2 to 5 carbon atoms, a hydroxyl group, a carboxyl group, a sulfo group, the above formula (1
a) is a substituted phenyl group, an unsubstituted phenyl group or a substituted phenyl or unsubstituted nitrophenylazo group, B is CO or SO ₂ , p and q are the same or different, and are 1 to 3 And when p or q is 2 or 3, R ¹² and R ¹³ may be the same or different, respectively]] or a phenolphthalein or phenolsulfonephthalein derivative Can be

In the general formula (I), the substituted or unsubstituted naphthyl group R ¹ is preferably the general formula (2
a):

[Chemical 6] Or general formula (2b):

[Chemical 7] [In the above formula, R ²¹ represents a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group having 1 to 4 carbon atoms, a sulfoalkyl group having 1 to 4 carbon atoms, a hydroxysulfoalkyl group having 1 to 4 carbon atoms, carbon Lower alkoxy group having 1 to 4 carbon atoms, 2 to
5 lower alkanoyl group, C 2-5 carboxyalkyl group, amino group, C 2-5 acid amide group, amino group mono- or di-substituted with C 1-4 lower alkyl group, carbon An acid amide group consisting of an amine moiety mono- or di-substituted by a lower alkyl group having 1 to 4 and an acid moiety having 2 to 5 carbon atoms, a hydroxyl group, a carboxyl group,
A sulfo group, an unsubstituted phenyl group or a substituted phenyl or nitrophenylazo group, r is an integer of 1 to 7, and when r is 2 to 7, R ²¹ may be the same or different. Well, in the case of the general formula (2a), at least one of the 2- to 8-positions respectively, or in the case of the general formula (2b), the 1-position, or 3 to 8 positions, respectively.
At least one of the positions has a nitro group and a carbon number of 2 to
5 is a lower alkanoyl group, or a substituted or unsubstituted nitrophenylazo group R ²¹ is present].

In the general formula (I), the substituted or unsubstituted indolyl group R ¹ is preferably the general formula (3):

Embedded image [In the formula, R ³¹ is a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, or a halogen atom, and R ³² is a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group having 1 to 4 carbon atoms, Sulfoalkyl group having 1 to 4 carbon atoms, hydroxy sulfoalkyl group having 1 to 4 carbon atoms, lower alkoxy group having 1 to 4 carbon atoms, lower alkanoyl group having 2 to 5 carbon atoms, carboxyalkyl group having 2 to 5 carbon atoms , Amino group, carbon number 2
5 acid amide group, amino group mono- or di-substituted with lower alkyl group having 1 to 4 carbons, amine moiety mono-substituted or di-substituted with lower alkyl group having 1 to 4 carbons and 2 to 2 carbon atoms An acid amide group consisting of the acid moiety of 5, a hydroxyl group, a carboxyl group, a sulfo group, a substituted phenyl group represented by the above formula (1a), or an unsubstituted phenyl group, and s
Is an integer of 1 to 4, but when s is 2 to 4, R ³²
May be the same or different from each other].

Furthermore, in the general formula (I), the group R ¹ substituted or unsubstituted Fenokisazoniru group R ¹ or an N- oxide thereof is preferably the general formula (4):

Embedded image [In the formula, R ⁴¹ represents a hydrogen atom, a halogen atom, a nitro group,
Lower alkyl group having 1 to 4 carbon atoms, sulfoalkyl group having 1 to 4 carbon atoms, hydroxysulfoalkyl group having 1 to 4 carbon atoms, lower alkoxy group having 1 to 4 carbon atoms, lower alkanoyl group having 2 to 5 carbon atoms , A carboxyalkyl group having 2 to 5 carbon atoms, an amino group, an acid amide group having 2 to 5 carbon atoms, and 1 carbon atom
To an amino group mono- or di-substituted with a lower alkyl group of 4 to 4, an acid amide consisting of an amine moiety mono- or di-substituted with a lower alkyl group of 1 to 4 carbons and an acid moiety of 2 to 5 carbons A group, a hydroxyl group, a carboxyl group, a sulfo group, or an unsubstituted phenyl group, and E is N or N → O
(N-oxide) and t is an integer of 1 to 6,
and when t is 2 to 6, R ⁴¹ may be the same or different.].

Further, in the above general formula (I), a substituted or unsubstituted fluorescein compound (R ¹ -A ¹ H)
Is preferably of the general formula (5):

Embedded image [In the formula, R ⁵¹ , R ^52, and R ⁵³ are the same or different, and each is a hydrogen atom, a halogen atom, a nitro group, or a carbon number of 1 to 1.
A lower alkyl group having 4 carbon atoms, a sulfoalkyl group having 1 to 4 carbon atoms, a hydroxysulfoalkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, a lower alkanoyl group having 2 to 5 carbon atoms, and 2 carbon atoms A carboxyalkyl group having 5 to 5 amino groups, an acid amide group having 2 to 5 carbon atoms, an amino group mono- or di-substituted with a lower alkyl group having 1 to 4 carbon atoms, and a lower alkyl group having 1 to 4 carbon atoms An acid amide group consisting of a mono-substituted or di-substituted amine portion and an acid portion having 2 to 5 carbon atoms, a hydroxyl group, a carboxyl group, a sulfo group,
Or an unsubstituted phenyl group, u and v are integers of 1 to 3, w is an integer of 1 to 4, but when u or v is 2 or 3, and when w is 2 to 4 R ⁵¹ , R ^52, and R ⁵³ may be the same or different.]. 3'-OH or 6'-OH of this substituted or unsubstituted fluorescein compound is
This corresponds to -A ¹ H in the general formula (I).

Further, in the above general formula (I), a substituted or unsubstituted hydroxyphenothiazone compound (R ^1-
A ¹ H) is preferably represented by the general formula (6):

Embedded image [In the formula, R ⁶¹ and R ⁶² are a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group having 1 to 4 carbon atoms, and 1 to 4 carbon atoms.
Sulfoalkyl group, C1-4 hydroxysulfoalkyl group, C1-4 lower alkoxy group, C2-5 lower alkanoyl group, C2-5 carboxyalkyl group, amino group, carbon An acid amide group of the number 2 to 5,
Amino group mono-substituted or di-substituted with a lower alkyl group having 1 to 4 carbon atoms, amine moiety mono-substituted or di-substituted with a lower alkyl group having 1 to 4 carbon atoms and 2 to 5 carbon atoms
An acid amide group, a hydroxyl group, a carboxyl group, a sulfo group, or an unsubstituted phenyl group consisting of the acid moiety of
Are the same or different and each is an integer of 1 to 3, but when x or y is 2 or 3, R ⁶¹ and R ⁶² are
Which may be the same or different from each other]. 11-OH of this substituted hydroxyphenothiazone compound is -A ¹ H of the general formula (I).
Is equivalent to

[0014] Anion X ^- is is not particularly limited, the anion is preferably not significantly interfere with cholinesterase activity. Specifically, for example, a halide ion, a hydrogen sulfate ion, an alkyl sulfate ion (for example, methyl sulfate ion), an alkanesulfonate ion (for example, methanesulfonate ion), or a carboxylate ion (for example, acetate ion) is used. Can be mentioned.
In addition, a divalent or higher organic acid ion (for example, an oxalate ion, a citrate ion, a succinate ion or a succinate ion which forms a salt with two or more cation moiety molecules excluding the anion X ⁻ in the formula (I)) Fumarate ion) or an inorganic acid ion (for example, sulfate ion or phosphate ion).

In the present specification, the halogen atom is a chlorine atom, a bromine atom, an iodine atom or a fluorine atom. The lower alkyl group having 1 to 4 carbon atoms includes a linear or branched lower alkyl group, for example, a methyl group,
Examples thereof include an ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, and t-butyl group. The lower alkoxy group having 1 to 4 carbon atoms includes a linear or branched lower alkoxy group, for example, a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, An isobutoxy group, a s-butoxy group, or a t-butoxy group can be mentioned. The sulfoalkyl group having 1 to 4 carbon atoms or the hydroxysulfoalkyl group is a linear or branched lower alkyl group having 1 to 4 carbon atoms or hydroxyalkyl group substituted with a sulfo group. The C2-C5 lower alkanoyl group is an acyl group derived from a C2-C5 linear or branched lower alkanoic acid, and includes, for example, acetyl group, propionyl group, butyryl group, isobutyryl group. Group, valeryl group, or isovaleryl group.

The carboxyalkyl group having 2 to 5 carbon atoms is
The above-mentioned linear or branched lower alkyl group having 1 to 4 carbon atoms substituted with a carboxyl group. 2-5 carbon atoms
The acid amide group of is an amide group of a linear or branched lower alkanoic acid having 2 to 5 carbon atoms. The amino group mono-substituted with a lower alkyl group having 1 to 4 carbon atoms is an amino group mono-substituted with a linear or branched lower alkyl group having 1 to 4 carbon atoms. The amino group di-substituted by a lower alkyl group having 1 to 4 carbon atoms is an amino group di-substituted by the same or different lower alkyl group having 1 to 4 carbon atoms which is linear or branched. . The acid amide group consisting of an amine moiety mono-substituted or di-substituted with a lower alkyl group having 1 to 4 carbon atoms and an acid moiety having 2 to 5 carbon atoms is a straight chain or branched chain having 1 to 4 carbon atoms as described above. An amine moiety mono-substituted with a chain-like lower alkyl group or the above-mentioned carbon number of 1 to 4
Acid amide group consisting of a linear or branched amine moiety di-substituted by the same or different lower alkyl group and a linear or branched lower alkanoic acid moiety having 2 to 5 carbon atoms Is.

A specific substituted phenol (R ¹ -OH),
And examples of the substituted thiophenol (R ¹ -SH) include:
2-nitrophenol, 3-nitrophenol, 4-nitrophenol, 3,4-dinitrophenol, 2-chloro-4-nitrophenol, 2-nitro-4-chlorophenol, 2,4-dinitrophenol, 2,5 -Dinitrophenol, 2,6-dichloro-4-nitrophenol, 2,6-dibromo-4-nitrophenol,
2,3,6-trichloro-4-nitrophenol, 2-
Methyl-4-nitrophenol, 2,6-dimethyl-4
-Nitrophenol, 2-methoxy-4-nitrophenol, 2,6-dimethoxy-4-nitrophenol, 2
-Nitro-6-methylphenol, 2,6-dichloro-
4-acetylphenol, 4- (4-nitrophenylazo) phenol, 4- (2-chloro-4-nitrophenylazo) phenol, 4- (4-nitrophenylazo)
-2-methyl-4-nitrophenol, or 4-
(4-nitrophenylazo) -2-methylphenol,
Or, a substituted thiophenol corresponding to the above-mentioned substituted phenol, further phenolphthalein, phenolsulfonephthalein, bromophenol blue, or chlorophenol red can be mentioned.

In addition, substituted hydroxyphenoxazone (R
Examples of ^1- OH) include 7-hydroxyphenoxazone, 4-methyl-7-hydroxyphenoxazone, or 8-chloro-4-methyl-7-hydroxyphenoxazone, which may be substituted naphthol. (R ¹ -OH)
Examples of the substituted thionaphthol (R ¹ -SH) include 4-nitro-1-naphthol, 6-nitro-2-naphthol, 2-chloro-4-nitro-1-naphthol,
Examples thereof include 2-methyl-4-nitro-1-naphthol, 1,3-dichloro-6-nitro-2-naphthol, and substituted thionaphthol corresponding to the substituted naphthol. Further, examples of substituted hydroxyindole (R ¹ -OH), can be mentioned 5-bromo-4-chloro-3-hydroxyindole. Furthermore, examples of the substituted fluorescein (R ¹ —OH) include:
4,5,6,7-tetrachlorofluorescein, or 4,5,6,7-tetrachloro-2 ', 4', 5 ',
7'-Tetraiodofluorescein (Rose Bengal)
And the like, and examples of the substituted hydroxyphenothiazone (R ¹ —OH) include 6-bromo-hydroxyphenothiazone, 1,9-dimethyl-hydroxyphenothiazone, and the like.

Preferred R in the general formula (I)
² is a divalent substituted or unsubstituted saturated or unsaturated hydrocarbon group, which hydrocarbon group is represented by the general formula (I)
Is a group derived from a cyclic acid anhydride of a dicarboxylic acid formed together with two —CO— groups adjacent to R ² and —O—. The substituent of R ² is a halogen atom,
A lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, an amino group, an amino group mono- or di-substituted with a lower alkyl group having 1 to 4 carbon atoms, or a hydroxyl group, It can have the same or different 1 to 6 substituents. Preferred R ² is ^two adjacent-
Substituted or unsubstituted alkylene group having 2 to 4 carbon atoms between CO- groups (for example, ethylene group, trimethylene group, tetramethylene group, propylene group, or ethylethylene group) or alkenylene group (for example, vinylene group, or It is a propenylene group).

[0020] Another preferred R ² of the general formula (I) wherein R number of carbon atoms in the shortest carbon chain between ² two -CO- group adjacent to the substituted or unsubstituted 2-4 cyclopentylene in A group, a cyclopentenylene group, a cyclohexylene group, a cyclohexynylene group or an arylene group. Here, the constituent carbon atoms of the carbon chain of R ² are simultaneously a substituted or unsubstituted cycloalkyl ring or cycloalkenyl ring having 5 to 6 carbon atoms or a substituted or unsubstituted 6 to 6 carbon atoms.
When the ring-constituting carbon atom of 10 aryl rings, there are two types of carbon chains between two —CO— groups adjacent to R ² in the general formula (I). Therefore, the "shortest carbon chain" means the carbon chain having the smaller number of carbons of the two kinds of carbon chains.

R ² as a cyclopentylene group, a cyclopentenylene group, a cyclohexylene group or a cyclohexenylene group is, for example, 1,2- or 1,3-cyclopentylene group, 1,2- or A 1,3-cyclohexylene group, a 1,2- or 1,3-cyclopentenylene group, or a 1,2- or 1,3-cyclohexenylene group can be mentioned. The unsaturated bond portion of the cycloalkenyl moiety may be contained in the shortest carbon chain mentioned above or in the other carbon chain. R ² as an arylene group preferably has 6 to 10 carbon atoms.
Examples of the arylene group include 1,2-phenylene group, 1,2-naphthylene group, 2,3-naphthylene group, and 1,8-naphthylene group. R ² as an arylene group may have one or more ring carbon atoms replaced with a heteroatom (eg, a nitrogen atom, an oxygen atom or a sulfur atom).

Still another preferred R ² is (1) in which some (2 or 3) of the constituent carbon atoms of the carbon chain of R ² are simultaneously substituted or unsubstituted with 5 to 6 carbon atoms. A cycloalkyl ring, a cycloalkenyl ring or a substituted or unsubstituted aryl ring having 6 to 10 carbon atoms [one or more ring carbon atoms of the aryl ring is replaced by a hetero atom (for example, nitrogen atom, oxygen atom or sulfur atom)] Which is a ring-constituting carbon atom, and (2) another part (1 or 2) of the constituent carbon atoms of the carbon chain of R ² is
A methylene group (—CH ₂ —) or an ethylene group (—CH ₂ CH ₂ —) interposed between a ring-constituting carbon atom of the cycloalkyl ring, cycloalkenyl ring or aryl ring and one or two —CO— groups. ) Or a vinylene group (-CH = CH
-). -OC-R ² -CO in this case
The -group is, for example, 2 (or 3) -carbonylcyclopentylacetyl group, 2 (or 3) -carbonylcyclopentenylacetyl group, 2 (or 3) -carbonylcyclohexylacetyl group, 2 (or 3) -carbonylmethylcyclopentylacetyl group. Group, 2 (or 3) -carbonylmethylcyclohexylacetyl group, 2-carbonylphenylacetyl group, 1-carbonylmethylnaphthyl-2 (or 8) -carbonyl group, 2-carbonylmethylnaphthyl-3-carbonyl group, 2-carbonyl It is a methylphenylacetyl group, a 1-carbonylmethylnaphthyl-2 (or 8) -acetyl group, a 2-carbonylmethylnaphthyl-3-acetyl group, or a 2,3-pyridylene group.

The constituent carbon atoms of the carbon chain of R ² are
When it is a ring-constituting carbon atom of a substituted or unsubstituted cycloalkenyl ring having 5 to 6 carbon atoms, the unsaturated bond portion of the cycloalkenylene ring may or may not be contained in the shortest carbon chain. It does not have to be.

The novel substrate compound of the present invention can be prepared by a method known per se. A representative example of the preparation process of the compound of the present invention is shown in FIG. R ¹ , R ² and A ^{2 in} FIG. 1 have the same meanings as described above. More specifically, a dicarboxylic acid anhydride containing a group R ² and 2-bromoethanol (or 2-bromoethylmercaptan) are heated under reflux in a nonpolar organic solvent such as benzene for 1 to 6 hours, After cooling, aqueous sodium carbonate solution is added, the aqueous phase is separated, washed with ether, acidified with hydrochloric acid, and extracted with ether. The ether is washed with saturated brine, dried over anhydrous sodium sulfate, and dried under reduced pressure to give bromoethoxycarbonylcarboxylic acid (or bromoethylthiocarbonylcarboxylic acid) [corresponding to precursor (1) in FIG. 1]. Examples of the dicarboxylic acid anhydride containing a group R ² used herein include aliphatic dicarboxylic acids such as succinic acid, glutaric acid, adipic acid, maleic acid, itaconic acid, and citraconic acid, and further camphoric acid and cyclohexane-1. ，
Cyclic acid anhydrides such as 2 (or 1,3) -dicarboxylic acid, phthalic acid, naphthalene-1,8 (1,2,2,3) -dicarboxylic acid, or quinoline 2,3- (or 3,4-) , 4,5-, 5,6-, 6,7-, or 7,8-) dicarboxylic acid, or a halogen atom, a lower alkyl group, a lower alkoxyl group, an amino group as a substituent on these basic skeletons, Examples thereof include derivatives having a lower alkyl-substituted amino group, a hydroxyl group, and / or a phenyl group.

The above-mentioned carboxylic acid derivative [precursor (1)]
Thionyl chloride is added to the mixture and heated under reflux for 5 minutes to 1 hour. After cooling, benzene is added and the mixture is concentrated under reduced pressure to remove thionyl chloride. Aromatic hydrocarbon compounds (R ¹ -O
H) and an ether solution of pyridine are added and the mixture is heated under reflux for 10 minutes to 1 hour. Examples of the chromogenic aromatic hydrocarbon compound (R ¹ —OH) used here include 2-nitrophenol, 3-nitrophenol, 4-nitrophenol,
3,4-dinitrophenol, 2-chloro-4-nitrophenol, 2-nitro-4-chlorophenol, 2,
4-dinitrophenol, 2,5-dinitrophenol, 2,6-dichloro-4-nitrophenol, 2,6
-Dibromo-4-nitrophenol, 2,3,6-trichloro-4-nitrophenol, 2-methyl-4-nitrophenol, 2,6-dimethyl-4-nitrophenol, 2-methoxy-4-nitrophenol, 2,6-dimethoxy-4-nitrophenol, 2-nitro-6-methylphenol, 2,6-dichloro-4-acetylphenol, 4- (4-nitrophenylazo) phenol,
4- (2-chloro-4-nitrophenylazo) phenol, 4- (4-nitrophenylazo) -2-methyl-4
-Nitrophenol, 4- (4-nitrophenylazo)
2-Methylphenol, 7-hydroxyphenoxazone, 7-hydroxy-4-methylphenoxazone, 4-nitro-1-naphthol and the like can be mentioned.
After cooling, the reaction product was added to hydrochloric acid, extracted with ether, washed with water, a saturated aqueous solution of sodium hydrogencarbonate, and saturated brine in this order, and dried over anhydrous sodium sulfate,
After drying under reduced pressure, the resulting oily substance is purified by silica gel column chromatography to obtain an aromatic compound ester of bromoethoxycarbonylcarboxylic acid (or an aromatic compound ester of bromoethylthiocarbonylcarboxylic acid) [precursor (2 Equivalent to)). A gas of trimethylamine is introduced into an anhydrous benzene solution of this ester compound [precursor (2)] under stirring for 1 to 5 days. The precipitate formed is filtered off, washed with benzene and dried under reduced pressure to obtain a solid, which is recrystallized from acetone and dried under reduced pressure to give a new substrate compound for measuring cholinesterase activity, a chromogenic aroma A choline derivative (or a thiocholine derivative) having a group can be obtained (corresponding to the “substrate of the present invention” in FIG. 1).

The novel substrate compound thus obtained is basically an asymmetric dicarboxylic acid ester of a chromogenic substituted aromatic group and choline or thiocholine. Examples of specific compounds include 3- (4-nitrophenoxycarbonyl) propionylcholine, 4- (4-nitrophenoxycarbonyl) butyrylcholine, 4- (4-nitrophenoxycarbonyl) -4,4-dimethylbutyrylcholine. ,
4- (4-nitrophenoxycarbonyl) -3,3-dimethylbutyrylcholine, 2- (4-nitrophenoxycarbonyl) benzoylcholine, 3- (3,4-dinitrophenoxycarbonyl) propionylcholine, 4-
(3,4-dinitrophenoxycarbonyl) butyrylcholine, 4- (3,4-dinitrophenoxycarbonyl)
-4,4-Dimethylbutyrylcholine, 4- (3,4-dinitrophenoxycarbonyl) -3,3-dimethylbutyrylcholine, 2- (3,4-dinitrophenoxycarbonyl) benzoylcholine, 3- (2-chloro- 4-nitrophenoxycarbonyl) propionylcholine, 4-
(2-chloro-4-nitrophenoxycarbonyl) butyrylcholine, 4- (2-chloro-4-nitrophenoxycarbonyl) -4,4-dimethylbutyrylcholine, 4-
(2-chloro-4-nitrophenoxycarbonyl)-
3,3-dimethylbutyrylcholine, 2- (2-chloro-
4-nitrophenoxycarbonyl) benzoylcholine,
2- (2-chloro-4-nitrophenoxycarbonyl)
Cyclohexanecarbonylcholine, 2- (4-nitrophenoxycarbonyl) cyclohexanecarbonylcholine, 2- (4-nitrophenoxycarbonyl) naphthalene-1-carbonylcholine, 3- (4-nitrophenoxycarbonyl) acrylcholine, or 4-nitrophenoxy Carbonyl methacrylcholine etc. can be mentioned. Also included are compounds in which the choline structure of each of the above compounds is replaced with thiocholine.

The compounds according to the invention liberate an amount of chromogenic substituted aromatic compound which, when contacted with cholinesterase, is proportional to cholinesterase activity. Therefore, since the cholinesterase activity can be directly measured by monitoring the chromophoric compound, it is useful as a substrate substance for cholinesterase activity measurement. Conventional substrates either lead acetic acid or choline produced by contacting the substrate with cholinesterase to a coloring system enzymatically, or 5,5'-dithio-bis (2-nitrobenzoic acid)
It leads to the coloring system of 2-nitro-5-mercaptobenzoic acid which is released through the substitution with, and since it is not a direct coloring method, it not only decomposes the substrate itself but also deactivates the enzyme and changes it to another coloring system. There are many factors that contribute to the inaccuracy of the measurement, such as the deterioration of the composition that leads, and the cholinesterase activity value may not be obtained accurately. On the other hand, the compound of the present invention has a remarkable effect that these problems can be solved at once. That is, when the compound according to the present invention is used as a substrate substance for measuring cholinesterase activity, a substituted phenoxide or the like which can be directly detected by cholinesterase is produced, so that cholinesterase activity can be measured accurately.

The cholinesterase activity in a biological fluid, particularly serum or plasma, can be measured using the above-mentioned novel compound. That is, by contacting the compound of the present invention with cholinesterase as a substrate substance, a chromogenic substituted phenoxide, etc., a dicarboxylic acid anhydride and choline (or thiocholine) are produced, and this chromogenic substituted phenoxide, etc. is optically detected. By doing so, if you perform operations and calculations in advance, such as creating a calibration curve using a cholinesterase standard or determining the amount of change in absorbance per activity unit, the cholinesterase activity contained in the test sample is quantitatively determined. be able to.

More specifically, for example, a test sample such as serum is treated with a conventionally known buffer solution having a pH of 7 to 10 (eg Tris buffer solution, phosphate buffer solution, borate buffer solution, Good buffer solution, etc.). It is appropriately diluted and maintained at 25 to 40 ° C., and a conventionally known buffer solution (for example, citrate buffer solution, phosphate buffer solution, acetate buffer solution) having a pH of 5 to 10 containing the substrate of the present invention of 0.05 to 100 mM is present therein. , Tris buffer, borate buffer, Good's buffer, etc.) and reacted at a wavelength of 400 to 500 n.
The cholinesterase activity can be accurately determined by spectroscopically detecting the change in absorbance at m and determining the amount of change in absorbance per unit time (so-called rate assay method).

At this time, in order to promote the dissociation of the chromogenic substituted phenoxide, etc., α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or a basic functional group (eg, diethylaminoethyl, dimethyl Quaternary ammonium salts such as aminoethyl or trimethylammonioethyl)
It is also possible to allow the introduction of cyclodextrins. In addition, when preparing these reagents as a kit, various surfactants, preservatives, chelating agents and the like which have been conventionally used for the purpose of preservability and avoiding the influence of interfering substances in the sample are appropriately added. The present invention also includes use.

[0031]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. In addition, each organic solvent and each compound used in each of the following examples are commercially available reagent special grade products. Example 1: 3- (4-nitrophenoxycarbonyl) p
Synthesis of Ropionylcholine A benzene (40 ml) solution of 10.0 g (100 mmol) of succinic anhydride and 12.5 g (100 mmol) of 2-bromoethanol was heated under reflux for 4 hours. After cooling, the reaction solution was added with an aqueous sodium carbonate solution (sodium carbonate 14.8 g
Is added to 400 ml of purified water), stirred to separate the aqueous phase, and the aqueous phase is washed with ether, and then 1
The mixture was acidified with 0% hydrochloric acid and extracted with 300 ml of ether. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and further dried under vacuum to give the precursor (1) [1]. FIG. 1] was obtained to obtain 16.1 g (yield 71.1%) of a white solid product of 3- (2-bromoethoxycarbonyl) propionic acid.

Subsequently, at room temperature, 14.8 g (66 mm) of the above-mentioned 3- (2-bromoethoxycarbonyl) propionic acid.
1 drop of dimethylformamide and thionyl chloride 7.
0 ml (99 mmol) was added, the mixture was stirred for 15 minutes, and then heated under reflux for 15 minutes. After cooling, dry benzene 70m
1 was added and concentrated under reduced pressure to remove excess thionyl chloride. 9.2 g (66 m) of 4-nitrophenol was added to this residue.
mol) and a solution of 6.5 ml (80 mmol) of anhydrous pyridine in 70 ml of anhydrous ether were added with stirring, and the mixture was reacted at room temperature for 30 minutes and further heated under reflux for 60 minutes. After cooling, the reaction mixture was added to 350 ml of cold 10% hydrochloric acid and extracted with 700 ml of ether, and the extract was washed with water, saturated aqueous sodium hydrogen carbonate solution and saturated brine in that order, and dried over anhydrous sodium sulfate. Then, it was concentrated under reduced pressure to obtain an oily substance. This was subjected to silica gel column chromatography (hexane: ethyl acetate = 2: 1, Rf value = 0.
39), which is the precursor (2) [see FIG. 1].
(2-Bromoethoxycarbonyl) propionic acid 4-nitrophenyl ester (18.2 g, yield 81%) was obtained.

The above-mentioned 3- (2-bromoethoxycarbonyl) propionic acid 4-nitrophenyl ester 15.0
Into a solution of g (43.3 mmol) in 80 ml of anhydrous benzene, a gas of trimethylamine (generated by heating a 30% trimethylamine aqueous solution) which had been dried through a sodium hydroxide tube was introduced under stirring for 3 days. The generated precipitate was filtered off, washed with 100 ml of benzene, dried under reduced pressure at room temperature to obtain a solid, which was recrystallized from acetone using activated carbon and further dried under reduced pressure to give 3- (4-nitrophenoxy). Carbonyl) propionylcholine bromide (hereinafter, also referred to as ChE-I) was obtained as an almost white crystalline powder (7.67 g, total yield 43.7%). This product is extremely water soluble and hygroscopic. The result of elemental analysis of the product is C ₁₅ H ₂₁ N ₂ O ₆ Br (molecular weight: 4
05.25), and the results of ¹ H-NMR signal attribution are shown in Table 1.

Example 2: 4- (4-nitrophenoxyca)
Synthesis of Lubonyl) butyrylcholine Instead of succinic anhydride in Example 1, glutaric anhydride 1
4-by repeating the synthetic procedure described in Example 1 above, except using 1.4 g (100 mmol).
14.2 g (total yield 34%) of almost white crystalline powder, which was a bromide of (4-nitrophenoxycarbonyl) butyrylcholine (hereinafter, also referred to as ChE-II), was obtained. This product is extremely water soluble and hygroscopic. The result of elemental analysis of the product was C ₁₆ H ₂₃ N ₂ O ₆ Br (molecular weight:
419.27), and the results of ¹ H-NMR signal assignment are shown in Table 1.

Example 3: 4- (4-nitrophenoxyca)
Synthesis of Rubonyl) -4,4-dimethylbutyrylcholine As described in Example 1, except that 14.2 g (100 mmol) of 2,2-dimethylglutaric anhydride was used instead of the succinic anhydride of Example 1. 4- (4-nitrophenoxycarbonyl)-by repeating the synthetic procedure
Bromide of 4,4-dimethylbutyrylcholine (hereinafter referred to as Ch
8.5 g (total yield 19%) of a white solid substance which is E-III) was obtained. The result of elemental analysis of this product is C ₁₈ H
₂₇ N ₂ O ₆ Br (molecular weight: 447.33) and ¹ H
The results of NMR signal assignment are shown in Table 1.

Example 4: 4- (4-nitrophenoxyca)
Synthesis of Lubonyl) -3,3-dimethylbutyrylcholine The procedure described in Example 1 except that 14.2 g (100 mmol) of 3,3-dimethylglutaric anhydride was used instead of the succinic anhydride of Example 1. 4- (4-nitrophenoxycarbonyl)-by repeating the synthetic procedure
Bromide of 3,3-dimethylbutyrylcholine (hereinafter referred to as Ch
4.5 g of white solid (also called E-IV) (total yield 1
0%). The elemental analysis result of this product is C ₁₈ H ₂₇
N ₂ O ₆ Br (molecular weight: 447.33) and ¹ H-
The results of NMR signal assignment are shown in Table 1.

[0037]

Table 1 Results of ¹ H-NMR signal assignment ChE-I ChE-II ChE-III ChE-IV ¹ H-NMR in D ₂ O in D ₂ O in D ₂ O DMSO-d ₆ in N (CH ₃ ) ₃ 3.21s 3.16s 3.16s 3.16s N-CH ₂ 3.74t 3.68t J = 4.6 3.70broad 3.68t O-CH ₂ 4.57t 4.41-4.51m 4.47broad 4.48t 2-CH ₂ 2.96t 2.45t J = 7.3 2.46t 2.58s 3-CH ₂ 3.01t 1.85tt J = 7.3 1.98t 4-CH ₂ 2.57t J = 7.3 2.76s C (CH ₃ ) ₂ 1.32s 1.18s o-H 7.33-7.43m 7.11d J = 8.9 7.45d 7.47m m-H 8.29-8.39m 7.98d J = 8.9 8.32d 8.34m

Example 5: Measurement of cholinesterase activity (1) Cholinesterase standard sample A physiological saline solution containing 0.1% bovine serum albumin containing human plasma-derived cholinesterase (Boehringer Mannheim Co .; butyrylcholine as a substrate and containing about 100 U per vial). What was dissolved in 5 ml and further diluted 10 times with physiological saline containing 0.1% bovine serum albumin at the time of use was used as a cholinesterase standard sample. (2) Reagent 1 A 100 mM Tris buffer solution having a pH of 7.5 was prepared as the reagent (1). (3) Reagent 2 Synthetic substrate (ChE-I, prepared in Examples 1 to 4)
5 mM of ChE-II, ChE-III, or ChE-IV)
A 20 mM-2-morpholinoethanesulfonic acid buffer solution (MES buffer solution) containing pH 5.0 was prepared as a reagent (2). (4) Operation An H-7150 type automatic analyzer (Hitachi, Ltd.) was used to measure cholinesterase activity. The parameters at the time of measurement are as follows. Input item Input RATE-A 40-50 POINT Sa VOLUME 10 μl R ¹ VOLUME 320 μl R ² VOLUME 80 μl WAVELENGTH 700-405 nm k-FACTOR 10000 The measuring instrument follows the above parameters and 10 μl of cholinesterase standard sample and reagent (1) ( 320 μl) was dispensed and mixed, and after a constant constant temperature time (37 ° C., 5 minutes),
Reagent (2) (80 μl) was added and mixed to start the reaction. The change in absorbance (time course) at this time is shown in FIG. In the symbols in FIG. 2,-●-is ChE-I and -Δ.
-Is ChE-II,-□-is ChE-III,-○-is Ch
It represents E-IV. When a reagent containing the substrate of the present invention was added to the cholinesterase standard sample, an increase in the absorbance derived from 4-nitrophenol produced from the substrate of the present invention due to the reaction between the cholinesterase and the substrate of the present invention was confirmed.

Further, a dilution series of the cholinesterase standard sample was prepared, the same operation as above was performed using the sample as the sample, and 180 seconds after the addition of the reagent (2), 120 seconds thereafter, respectively. The change in the absorbance of the substrate-containing reagent of was measured per unit time. The results are shown in FIG.
In addition, in the symbols in FIG. 3, as in the above,-●-is ChE-
I, -Δ- is ChE-II,-□-is ChE-III,-○
-Represents ChE-IV. With respect to the dilution series of cholinesterase standard samples, the amount of change in absorbance per unit time for each substrate shows linearity, and when cholinesterase and each substrate according to the present invention react, 4-nitrophenol is quantitatively produced. Was confirmed. From this result, it was confirmed that the cholinesterase activity in the test sample can be quantitatively measured using the novel substrate substance according to the present invention.

Example 6: Cholinesterase activity in serum
Measurement of sex According to Example 5, control serum (trade name: Serachem; In
STUDENTATION LABORATORY
The cholinesterase activity in Company) was measured. That is, using ChE-I as a substrate, the rest was carried out according to the operation method of Example 5. For comparison, the same test was performed using a conventional cholinesterase assay reagent (trade name: iatrofine Ch-E rate; manufactured by Yatron, substrate substance: butyrylthiocholine). However, the amount of the sample was changed to 3 μl and the measurement wavelength was changed to 600 to 480 nm. Table 2 shows the measurement results obtained by repeating each of these tests 10 times. As shown in Table 2, the compound of the present invention can be used as a substrate for measuring cholinesterase activity, and was able to measure cholinesterase activity in serum with high accuracy and comparable to the conventional one.

[0041]

Table 2 Method of the present invention (IU / ml) Conventional method (IU / ml) 1 5.35 5.59 2 5.40 5.33 3 5.33 5.46 4 5.39 5.42 5 5. 47 5.57 6 5.42 5.32 7 5.36 5.46 8 5.46 5.51 9 5.32 5.20 10 5.30 5.48 Average 5.38 5.43 Standard deviation 0.4 06 0.12 C.I. V. (%) 1.12. 2.21

Example 7: 2- (4-Nitrophenoxyca)
Synthesis of Rubonyl) benzoylcholine Substituting succinic anhydride in Example 1 with phthalic anhydride 13.
By repeating the synthetic procedure described in Example 1 above, except using 6 g (100 mmol), 2- (4
-Nitrophenoxycarbonyl) benzoylcholine bromide almost white crystalline powder 10.2 g (total yield 2
2.5%). This product is easily soluble in water and has hygroscopicity. The result of elemental analysis of this product is C ₁₉ H ₂₁ N ₂
It was O ₆ Br (molecular weight: 453.29). A 20 mM-MES buffer solution having a pH of 5.0 containing 5 mM of the above-mentioned product was prepared and reacted with a cholinesterase standard sample according to Example 5, and the color development derived from 4-nitrophenoxide released was confirmed.

Example 8: 4- (4-nitrophenoxyca)
Synthesis of Lubonyl) propionylthiocholine By repeating the synthetic procedure described in Example 1 above, except that 14.1 g (100 mmol) of 2-bromoethanethiol was used instead of 2-bromoethanol of Example 1, 4 -(4-nitrophenoxycarbonyl)
7.8 g (total yield 18.5%) of pale yellowish white powder which is a bromide of propionylthiocholine was obtained. The product was easily soluble in water. The elemental analysis result of this product is C ₁₅ H
_{It was 21} N ₂ O ₅ SBr (molecular weight: 421.19).
A 20 mM MES buffer solution having a pH of 5.0 containing the above-produced 5 mM was prepared and reacted with a cholinesterase standard sample in accordance with Example 5, and the liberated 4-nitrophenoxide-derived color was confirmed.

[0044]

INDUSTRIAL APPLICABILITY The compound according to the present invention is a substrate of cholinesterase and at the same time contains a chromogenic component in its structure. When it is brought into contact with cholinesterase, the chromogenic component is released to release the chromogenic compound. Can be generated. Moreover, the amount produced is proportional to the activity of the cholinesterase contacted with the compound of the present invention. Therefore, the cholinesterase activity can be directly measured by monitoring the chromophoric compound, which is more accurate than the conventional method using an enzyme or reagent that induces a chromogenic system.

[Brief description of drawings]

FIG. 1 is an explanatory view showing an example of a synthetic process of a novel compound of the present invention.

FIG. 2 is a graph (time course) showing a change in absorbance derived from a chromogenic compound produced when four kinds of novel compounds according to the present invention are brought into contact with a cholinesterase standard reagent.

FIG. 3 is a graph showing changes in absorbance per unit time of chromogenic compounds produced when four novel compounds according to the present invention are brought into contact with a cholinesterase standard reagent dilution series.

Continuation of the front page (72) Inventor Tatsuhiko Yagi 1664-33 Kita, Shizuoka City, Shizuoka Prefecture

Claims (4)

[Claims] 1. General formula (I): embedded image R ¹ —A ¹ —OC—R ² —CO—A ² — (CH ₂ ) ₂ —N ⁺ (CH ₃ ) ₃ · X ⁻ (I) [Wherein A ¹ and A ² are the same or different,
O or S, R ¹ represents an aromatic or aromatic heterocyclic hydroxy compound or a thiol compound as R ¹ -A ¹ H, and R ² represents a saturated or unsaturated substituted or unsubstituted a carbon chain of 2 to 4 carbon atoms, and X ^-
Is an anion]. 2. R ¹ -A ¹ H is substituted or unsubstituted phenol, substituted or unsubstituted naphthol, substituted or unsubstituted hydroxyindole, substituted or unsubstituted hydroxyphenoxazone, substituted or unsubstituted hydroxyphenoxazone- The compound according to claim 1, which is N-oxide, substituted or unsubstituted fluorescein, substituted or unsubstituted hydroxyphenothiazone, substituted or unsubstituted thiophenol, or substituted or unsubstituted thionaphthol. 3. A method for measuring cholinesterase activity, which comprises using the compound represented by the general formula (I) according to claim 1 as a substrate substance. 4. In measuring cholinesterase activity,
Use of the compound represented by the general formula (I) according to claim 1 as a substrate substance.