JPH069566A - Chemiluminescent compound and assay method using the same - Google Patents

Abstract

PURPOSE:To obtain a new chemiluminescent compound useful as a labeled compound in the chemiluminescence analysis and highly stable in neutral aqueous solution. CONSTITUTION:The compound is an acridinium compound expressed by formula I [R1 is a group which does not prevent chemiluminescence; R2 and R3 are (fluorinated) alkyl; X and Y are O or S; A<-> is an anion which does not prevent chemiluminescence]. E.g. 2'-trifluoromethylphenyl-N-methyl-1-methylacridinium-9- carboxylate fluorosulfonate. A compound of formula I is obtained by esterification of a compound of formula II with a compound of formula III and bringing the ester to a quarternary ammonium salt.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel chemiluminescent compound useful as a labeling substance in a chemiluminescent analysis method, a luminescent complex to which the chemiluminescent compound is bound, and an assay using the chemiluminescent compound. Concerning the law.

[0002]

2. Description of the Related Art The application of chemiluminescent compounds to analytical techniques has been expanding more and more in recent years due to their high sensitivity. Luminol, acridinium, stabilized dioxetane, peroxalate, and the like have high emission yields and are being put to practical use in various fields as reagents for high-sensitivity analysis. Among them, an acridinium compound has a detection sensitivity of 10 ⁻¹⁸ mol level and can be easily bound to various ligand molecules, and thus has attracted attention as a safe labeling substance instead of a radioisotope.

Particularly in the field of clinical examinations, there is an increasing demand for a ligand assay utilizing an antigen / antibody reaction or complementary binding of nucleic acids, and high precision and speedy measurement by an automatic analyzer is desired. On the other hand, since the acridinium compound emits flash-type light emission in seconds, it is a reagent that can sufficiently meet such requirements in the field of clinical examination.

N-methylacridinium-9-carboxylic acid aryl ester chemically changes into excited N-methylacridone by hydrogen peroxide under alkaline conditions and emits light. (Prog. Org. Chem., 8, 231-277, 1973). afterwards,
It was first applied by Woodhead et al. As a labeled substance for immunoassay (Clin. Chem., 29, 1474-1479, 1983). The acridinium ester synthesized by Woodhead et al. For labeling was compound 22 shown in FIG. 3, which was unstable in a solvent system (pH neutral aqueous solution) usually used for immunoassay.

As a means for solving the problem of instability of this acridinium-9-carboxylic acid aryl ester,
The introduction of a methyl group at two ortho positions of a phenoxy ring which constitutes a part of an ester component was disclosed by Seiyon et al. (JP-A-63-101368). Similar effects are also shown by map couplers etc.
Issue). The stability was improved by using the labeling compound having an orthodimethyl group introduced into the phenoxy ring (FIG. 3, compound 21).

[0006]

However, with the improvement in accuracy or speed of the analytical method including the apparatus, it is desired to develop a more stable and high-performance labeled compound.
An object of the present invention is to provide a novel chemiluminescent compound having high stability.

Another object of the present invention is to provide a highly stable luminescent complex prepared from a novel chemiluminescent compound. A further object of the present invention is to provide an assay method using a novel chemiluminescent compound.

[0008]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that acridinium-
By introducing an alkyl group into the peri position (1 position) of the acridine ring and the ortho position of one of the phenoxy rings in the 9-carboxylic acid phenol ester, the stability is remarkably improved without impairing the light emitting property. And completed the present invention.

The present invention has the general formula (I)

[0010]

[Chemical 2]

(Wherein R ₁ is a group that does not interfere with chemiluminescence, R ₁ is
₂ and R ₃ are each an optionally fluorinated alkyl, X and Y are each O or S, and A ⁻ is an anion that does not interfere with chemiluminescence. ), And an acridinium compound or an isomer thereof in which any position of the acridine ring or phenyl ring (excluding the ortho position of the phenyl ring) may be substituted with a group that does not interfere with chemiluminescence [hereinafter, referred to as chemical Light-emitting compound (I)].

The present invention also relates to a luminescent complex comprising a biologically active substance and a chemiluminescent compound (I) bound thereto.

In the present invention, (a) a sample is mixed with a binding substance that is labeled with a chemiluminescent compound (I) and that specifically binds to the test substance in the sample. A complex with a binding substance is formed, (b) the complex and the free labeled binding substance are separated, and (c) the chemiluminescence of the chemiluminescent compound (I) bound to the complex is detected. And (d) an assay method for the test substance in the sample (hereinafter referred to as assay method 1), which comprises determining the amount of the test substance in the sample from the detected luminescence amount.

The present invention further provides (a) a sample, (i) a binding substance which specifically binds to a test substance in the sample, and
(ii) mixing with a test substance labeled with a chemiluminescent compound (I) to form a complex of the test substance and a binding substance,
(B) separating the complex from the free labeled analyte,
(C) Chemiluminescent compound (I) bound to the complex
(D) determining the amount of the test substance in the sample from the detected luminescence amount (d), and assaying the test substance in the sample (hereinafter referred to as assay method 2).

As used herein, the term "biologically active substance" means an antigen (including hapten), an antibody, a protein, a peptide,
It is meant to include nucleic acids, hormones, drugs, drug metabolites and the like.

As used herein, the term "group that does not interfere with chemiluminescence" means a group that does not interfere with the generation of effective chemiluminescence in chemiluminescence analysis. Specifically, the group that does not interfere with chemiluminescence represented by R ₁ includes alkyl, alkenyl, alkynyl, aryl or aralkyl, or a reactive functional group for labeling. One or more hydrogen atoms in the alkyl, alkenyl, alkynyl, aryl and aralkyl may be optionally substituted by a suitable substituent. Suitable substituents here are those selected from nitro, halogen, amino, protected amino, alkoxy, aryloxy, hydroxy, protected hydroxy and the like. Further, one or more carbon atoms in the alkyl, alkenyl, alkynyl, aryl and aralkyl may be optionally substituted with a hetero atom or carbonyl. Here, the hetero atom is one selected from nitrogen, phosphorus, sulfur and oxygen.

The alkyl, alkenyl and alkynyl have 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms, and the aryl and aralkyl have 6 to 20 carbon atoms, preferably 6 to carbon atoms. Twelve.
Examples of the aryl include phenyl and naphthyl, and examples of the aralkyl include benzyl. Preferably R ₁ is alkyl having 1 to 4 carbons.

The above-mentioned "reactive functional group for labeling" means a reactive group capable of binding to an amino group, a carboxyl group, a mercapto group, or another functional group usually contained in biologically active substances. As a specific example, -RW or -W [In the formula, R represents alkylene or arylene, and W
Is

[0019]

[Chemical 3]

(Where R is_FourIs alkyl, aryl or
Aralkyl, R_FiveIs alkylene or arylene, X is
Represents a group selected from (which is a halogen)]
The group is shown. Where alkyl, aryl and aral
Kill the above R ₁Is the same as the definition in. See again
Kirylene and arylene have 1 to 20 carbon atoms, preferably
1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms
It Preferably R is methylene, ethylene, propylene,
Or ortho-, meta- or para-phenylene
It Halogen is chlorine, bromine, iodine or fluorine
And preferably chlorine or bromine.

R ₂ and R ₃ are each optionally fluorinated alkyl having 1 to 4 carbon atoms. The fluorinated alkyl may be perfluoroalkyl in which all hydrogen atoms of the alkyl are fluorine-substituted, or may be one in which a part of the hydrogen atoms is fluorine-substituted, and is preferably perfluoroalkyl.

Preferably R ₂ and R ₃ are each methyl or trifluoromethyl.

X and Y are each O or S, preferably both X and Y are O. Any position of the acridine ring or the phenyl ring (except the ortho position of the phenyl ring) may be substituted with a group that does not prevent chemiluminescence. Here, the “group which does not interfere with chemiluminescence” includes alkyl, alkenyl, alkynyl, aryl or aralkyl defined as the group represented by R ₁ , or a reactive functional group for labeling, nitro, Substituents selected from halogen (eg chlorine, bromine, iodine, fluorine), alkoxy, carboxy, protected carboxy, alkoxycarbonyl, amino, protected amino, hydroxy and protected hydroxy are included. Preferably, an alkyl or haloalkyl having 1 to 4 carbon atoms, nitro,
Halogen or alkoxy having 1 to 4 carbon atoms, more preferably methyl, trifluoromethyl, nitro,
Halogen and methoxy.

[0024] A ^- In the "anion which does not interfere with the chemiluminescence" represented, sulfate, halo sulfonate (e.g., fluorosulfonate), alkyl sulfonates,
Included are haloalkyl sulfonates (eg, fluoroalkyl sulfonates, etc.), aryl sulfonates, haloborates, haloacetates, halophosphates, phosphates, halides and the like.

In the chemiluminescent compound (I), the "reactive functional group for labeling" is introduced into R ₁ or any position of the acridine ring or the phenyl ring (excluding the ortho position of the phenyl ring). To be done. Preferably, the reactive functional group is introduced at the meta or para position of R ₁ or the phenyl ring.

In the present invention, the isomers of general formula (I) are those of general formula (II) in which the acridine ring moiety is a phenanthridine ring.

[0027]

[Chemical 4]

The compounds represented by the formulas (wherein each symbol has the same meaning as defined above) are included.

Suitable as the chemiluminescent compound of the present invention are those represented by the above formula (I) in which R ₁ is alkyl having 1 to 4 carbon atoms, R ₂ and R ₃ are respectively alkyl or perfluoro having 1 to 4 carbon atoms. Alkyl, X and Y are both O
Is a compound in which the meta or para position of the phenyl ring is substituted with a reactive functional group, and more preferably,
R ₁ is methyl, R ₂ and R ₃ are each methyl or trifluoromethyl, and the phenyl ring has a meta or para position substituted with an N-succinimidyloxycarbonyl group.

The luminescent complex of the present invention comprises an antigen (including a hapten), an antibody, a protein, a peptide, a nucleic acid, a hormone, a drug via a reactive functional group for labeling the chemiluminescent compound (I). It is formed by covalently bonding to various biologically active substances such as drug metabolites.

The acridinium compound of the present invention can be produced according to the following synthetic route I.

[0032]

[Chemical 5]

(Wherein R ₂ , R ₃ and A ⁻ have the same meanings as described above) 1-alkyl-substituted acridine-9-carboxylic acid (III) or its reactive derivative is converted into 2-alkyl-substituted phenol (I
The acridine-9-carboxylic acid phenol ester compound (V) is obtained by esterification with V) by a known ester synthesis method. The acridinium compound (I) is produced by subjecting the ester compound (V) to quaternary chloride according to a conventional method.

1-Alkyl-substituted acridine-9-carboxylic acid which is a synthetic intermediate of the acridinium compound of the present invention
(III) can be produced according to the following synthetic route II.

[0035]

[Chemical 6]

(In the formula, R ₂ and R ₃ have the same meanings as described above, and Y is halogen such as chlorine, bromine, iodine and fluorine, preferably chlorine.) Compound (VI) and Compound (VII) And N- (3
The -substituted phenyl) -anthranilic acid (VIII) is prepared. The anthranilic acid compound (VIII) is reacted with phosphorus oxychloride to produce 1-alkyl-substituted acridone (IX). The acridone compound (IX) is the corresponding halide
It is converted to cyanide form (XI) via (X). The cyano group of the cyanide compound (XI) is hydrolyzed to give 1-alkyl-substituted acridine-9-carboxylic acid (III).

The labeling acridinium compound is 1
-Alkyl-substituted acridine-9-carboxylic acid (III) or its reactive derivative is represented by the following formula

[0038]

[Chemical 7]

(In the formula, R ₂ has the same meaning as described above, and R ₅
Represents a reactive functional group for labeling) and is obtained by ester-bonding with a substituted phenol represented by.

Further, 1-alkyl-substituted acridine-9-
The carboxylic acid (III) or its reactive derivative is represented by the following formula

[0041]

[Chemical 8]

(Wherein R ₂ has the same meaning as defined above and Z represents a carboxy-protecting group), after ester-bonding with the substituted phenol, the carboxyl-protecting group is removed, and a suitable compound such as A reactive functional group may be introduced onto the phenyl ring by using N-hydroxysuccinimide or the like. To introduce a reactive functional group at the R ₁ position, see
The method disclosed in 63-112564 may be used.

The luminescent complex composed of the chemiluminescent compound (I) and the biologically active substance may be prepared by a conventional method appropriately selected depending on the kind of the reactive functional group. For example, a luminescent compound of the present invention having an N-succinimidyloxycarbonyl group is mixed with a biologically active substance to be labeled in a buffer solution, and the mixture is mixed at room temperature for several minutes to several tens of minutes (usually 1
It is prepared by leaving it to stand for 5 to 30 minutes.

The assay method of the present invention is characterized by using a luminescent complex labeled with a chemiluminescent compound (I) as a tracer. The assay method of the present invention can be applied to any known assay method as long as the assay method uses a biological specific binding reaction. Here, the biological specific binding reaction means an antigen / antibody reaction, complementary binding of nucleic acid, hormone and its binding protein, drug and its binding protein,
It includes a binding reaction between an enzyme and its substrate.

Examples of test substances that can be measured by the assay method of the present invention include various biologically active substances such as antigens (including haptens), antibodies, proteins, peptides, nucleic acids, hormones, drugs, and drug metabolites. To be Examples of combinations of the test substance and the binding substance include an antigen and an antibody, a nucleic acid and a complementary polynucleotide, an enzyme and its substrate, a hormone and its binding protein, a drug and its binding protein, and a tumor marker and its antibody. Examples of such test substances include TSH (thyroid stimulating hormone), hCG (placental gonadotropin), CR
P (C-reactive protein), ASO (anti-streptolysin O), AFP (α-fetoprotein), IgG, Ig
A, IgM, IgD, IgE, CEA (carcinoembryonic antigen), transferrin, ferritin, fibrin, fibrinogen degradation product, haptoglobin, α ₁ -antitrypsin, α ₁ -acid glycoprotein, α ₂ -macroglobulin, β _2- Microglobulin etc. can be mentioned.

In the assay method of the present invention, the separation of the complex and the free form (B / F separation) may be carried out by a method usually used in the field of immunoassay. The solid phase method of immobilizing a binding substance on an insoluble carrier such as a polystyrene test tube or well, glass beads, polystyrene latex beads, sepharose, and polyacrylic is advantageous in terms of simplicity.

The chemiluminescence may be detected by a known means. Light emission occurs under alkaline conditions, for example by adding an oxidizing agent such as hydrogen peroxide. The amount of emitted light may be measured with an appropriate measuring device such as a luminometer. Needless to say, instead of measuring the chemiluminescence of the labeled compound bound to the complex, the labeled compound bound to the separated free form may be measured.

A preferred embodiment of the present invention will be described below by taking an immunoassay method utilizing an antigen-antibody reaction as an example. The test substance may be either an antigen or an antibody, but the method for measuring the antigen will be described for convenience.

A preferred embodiment of Assay Method 1 (non-competitive method) is the sandwich method. That is, it is an assay method for an antigen in a sample, which comprises the following steps. (A) a sample is incubated with an immobilized antibody against the antigen in the sample (hereinafter referred to as an immobilized antibody) to form a complex between the immobilized antibody and the antigen, and (b) then a chemiluminescent compound An antibody against an antigen (hereinafter referred to as a labeled antibody) labeled with (I) is added and incubated to form a sandwich consisting of an immobilized antibody, an antigen and a labeled antibody, and (c) the sandwich and a free labeled antibody. Are separated, (d) the chemiluminescent compound (I) bound to the sandwich is detected, and (e) the amount of the antigen in the sample is determined from the detected luminescence amount. This method can be applied to the measurement of an antigen having two or more antibody binding sites.

A preferred embodiment of Assay Method 2 (competitive method) is
It is an assay method for an antigen in a sample, which comprises the following steps. (A) the sample is incubated with (i) the immobilized antibody and (ii) the chemiluminescent compound (I) -labeled antigen (hereinafter referred to as labeled antigen) to form a complex of the antigen and the antibody, (B) separating the complex from free labeled antigen,
(C) Chemiluminescent compound (I) bound to the complex
Chemiluminescence is detected, and (d) the amount of antigen in the sample is determined from the detected amount of luminescence.

[0051]

The present invention will be described in more detail by the following examples. In Reference Examples 1 and 2, 1,3-dimethyl substituted products of acridine-9-carboxylic acid, which is an intermediate for synthesizing the acridinium ester of the present invention, and 1-
A method for producing a methyl-substituted product will be described. In Examples 1 to 4,
The acridinium ester of the present invention (compounds 12, 13,
16 and 17) prepared in Reference Example, acridine-9-
A method for producing from a carboxylic acid and a 2-substituted phenol is described. In Examples 5 to 7, labeling acridinium ester (compounds 18 to 20), which is a preferred embodiment of the present invention, is used.
The manufacturing method of is described. Example 8 includes anti-CEA mouse I
A method for labeling gG with an acridinium ester of the present invention is described. Example 9 describes the chemiluminescence measurement of the labeled antibody prepared in Example 8. Example 10 shows an example in which the labeled antibody of the present invention is applied to an immunoassay method.

Test Example 1 describes a test comparing the chemiluminescent property and stability of the chemiluminescent compound of the present invention with the comparative compound. Test Examples 2 and 3 describe tests in which the stability of the labeled product of the present invention was compared with that of the comparative labeled product.

2'6'-Dimethyl-4 'as a comparative control
The synthesis of-(N-succinimidyloxycarbonyl) -phenyl-N-methylacridinium-9-carboxylate methyl sulfonate (Compound 21) is carried out by the method described in Japanese Patent Laid-Open No. 63-101368 of Seiyon et al. , Also 4 '-(2-succinimidylcarboxyethyl)-
Phenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 22) was synthesized according to the method of Woodhead et al. (Methods in Enzymology, 133, 372-374).
The chemical structures of compounds 1-22 are shown in FIGS.

Reference Example 1 (1) Synthesis of N- (3,5-dimethylphenyl) -anthranilic acid o-Chlorobenzoic acid (50 mmole, 7.83 g), m-
Xylidine (100mmole, 12.12g), potassium carbonate (60mmole, 8.3g), copper powder (2.5g),
Isoamyl alcohol (20 ml) was mixed and refluxed for 3 hours. After cooling, 10% aqueous potassium carbonate solution (75 ml) and ethyl acetate (100 ml) were added, and the copper powder was removed by filtration. The organic layer of the filtrate was washed with a 10% aqueous solution of potassium carbonate and 2N-hydrochloric acid and then dried over anhydrous sodium sulfate. The crystals obtained by evaporation to dryness were recrystallized from ethyl acetate. (7.24 g, 60% yield)

(2) Synthesis of 1,3-dimethylacridone N- (3,5-dimethylphenyl) -anthranilic acid (25.3 mmole, 6.1 g) was added to phosphorus oxychloride (25
ml) was added and refluxed for 1 minute. 5% -on the obtained crystals.
Aqueous ammonia (1.5 liter) was added, the mixture was filtered, washed with chloroform (100 ml) and water (200 ml), and then dried under reduced pressure. (5.4 g, yield 88.4%)

(3) 1,3-Dimethylacridine-9-
Synthesis of chloride 1,3-Dimethylacridone (17.9 mmole, 4.0
Phosphorus oxychloride (50 ml) was added to g) and the mixture was refluxed for 5 hours. After cooling, 5% aqueous ammonia (1.3 liter) and chloroform (250 ml) were added, and the chloroform layer was dried over anhydrous sodium sulfate. After evaporating the solvent to dryness,
Purified by silica gel chromatography (eluent, 33% -ethyl acetate / hexane). (4.1 g, 94% yield)

(4) 1,3-Dimethylacridine-9-
Synthesis of cyanide 1,3-Dimethylacridine-9-chloride (17 mmol
e, 4.1 g), potassium cyanide (25.5 mmole,
1.66 g) and dimethylformamide (17 ml) were mixed and refluxed for 2 hours. After cooling, water (300 ml) and chloroform (100 ml) were added, and the chloroform layer was mixed with water (10 ml).
After washing with 0 ml), it was dried over anhydrous sodium sulfate. After the solvent was evaporated to dryness, the residue was purified by silica gel chromatography (eluent, methylene chloride). (1.22 g, yield 31%) IR (KBr): 2218 cm ^-1

(5) 1,3-dimethylacridine-9-
Synthesis of carboxylic acid 1,3-dimethylacridine-9-cyanide (1.05
g, 4.5 mmole) and 15 ml of concentrated sulfuric acid were added and heated at 100 ° C. for 2 hours. After cooling to -10 ° C, sodium nitrite (3.1 g / 15 ml of water) was gradually added over about 30 minutes.
After stirring for 15 minutes, the mixture was heated at 100 ° C. for 30 minutes. After cooling, it was added to ice-cooled water (120 ml), and the obtained crystals were dissolved in a minimum amount of 3N-sodium hydroxide. PH with 6N-hydrochloric acid
After setting the value to 3.0, the precipitated crystal was washed with water and dried under reduced pressure. (0.76 g, yield 67%)

Reference Example 2 (1) Synthesis of N- (3-methylphenyl) -anthranilic acid o-Chlorobenzoic acid (7.83 g, 50 mmole), m-
Toluidine (10.7 g, 100 mmole), potassium carbonate (8.3 g, 60 mmole), copper powder (2.5 g) and isoamyl alcohol (20 ml) were mixed and refluxed for 3 hours. After cooling, 10% aqueous potassium carbonate solution (75 ml) and ethyl acetate (100 ml) were added, and the copper powder was removed by filtration. The organic layer of the filtrate was washed with a 10% aqueous solution of potassium carbonate and 2N-hydrochloric acid and then dried over anhydrous sodium sulfate. The crude crystals obtained by evaporation to dryness were recrystallized from ethanol. (7.1 g, yield 12.5%)

(2) Synthesis of methylacridine-9-chloride N- (3-methylphenyl) -anthranilic acid (5.7
Phosphorus oxychloride (25 ml) was added to 4 g and 25.3 mmol, and the mixture was refluxed for 10 minutes. After cooling, 5% -aqueous ammonia (1.6 liter) and chloroform (100 ml) were added, and the chloroform layer was dried over anhydrous sodium sulfate. After the solvent was evaporated to dryness, the residue was purified by silica gel chromatography (eluent, 33% -ethyl acetate / hexane). (5.07 g, yield 92%)

(3) Synthesis of 1-methylacridine-9-cyanide Methylacridine-9-chloride (4.1 g, 18.9)
mmole), potassium cyanide (1.85 g, 28.4 mm)
ole), dibenzo-18-crown-6 (0.2 g,
0.55 mmole) and dimethylformamide (19 ml) were mixed and refluxed for 2 hours. After cooling, the mixture was added to water (300 ml) and extracted with ethyl acetate (100 ml, 6 times). The organic layers were collected, washed with water (100 ml, 3 times), and dried over anhydrous sodium sulfate. After the solvent was evaporated to dryness, the residue was purified by silica gel chromatography (methylene chloride). (1.6
6 g, yield 40%) IR (KBr): 2218 cm ^-1

The obtained methylacridine-9-cyanide was confirmed to be a 1-methyl substitution product by 400 MHz NMR. Aromatic ring proton signal is 7.48 ~
Seven hydrogen atoms appeared in the range of 8.45 ppm. Among them, coupling with adjacent hydrogen of about 0.022 ppm (doublet) is observed in 4 hydrogen, so it can be concluded that it is a 1-methyl form. By the way, if there is a methyl group at the 3-position, a similar signal must appear 5 hydrogens. FIG. 6 shows the NMR spectrum.

(4) Synthesis of 1-methylacridine-9-carboxylic acid 1-methylacridine-9-cyanide (1.44 g,
33 ml of concentrated sulfuric acid was added to 6.6 mmole) and heated at 100 ° C. for 2 hours. After cooling to -10 ° C, sodium sulfite (4.5 g, 33 ml of water) was added gradually over about 30 minutes. After stirring for 15 minutes, the mixture was heated at 100 ° C. for 30 minutes. After cooling, it was added to ice-cooled water (250 ml), and the obtained crystals were dissolved in a minimum amount of 3N-sodium hydroxide. The crystals obtained were adjusted to pH 3.0 with 6N-hydrochloric acid, washed with water, and dried under reduced pressure. (0.87 g, yield 56%)

Example 1 (1) 2'-methyl-5'-nitrophenyl-1,3-
Synthesis of dimethylacridine-9-carboxylate To 1,3-dimethylacridine-9-carboxylic acid (120 mg, 0.54 mmole) obtained in Reference Example 1 was added thionyl chloride (2 ml) and dimethylformamide (1 drop) for 10 minutes. Refluxed. After removing thionyl chloride under reduced pressure, 2-methyl-5-nitrophenol (132 mg, 0.6 mmole) and 4-dimethylaminopyridine (20 mg, 0.16 mmole) dissolved in anhydrous pyridine (4 ml) were added, and 4 Reflux for hours. After cooling, pyridine was removed by azeotropic distillation with benzene, and silica gel chromatography (eluent, 5%-
Purified with ethyl acetate / methylene chloride). (66m
g, yield 30%)

(2) 2'-methyl-5'-nitrophenyl-N-methyl-1,3-dimethylacridinium-9
-Carboxylate fluorosulfonate (compound 1
Synthesis of 3) 2-Methyl-5-nitrophenyl-1,3-dimethylacridine-9-carboxylate (50 mg, 0.129
mmole) was dissolved in 2.3 ml of anhydrous methylene chloride, methyl fluorosulfonate (103 μl, 1.29 mmole) was added, and the mixture was stirred at room temperature for 20 hours. Anhydrous ether (5 ml)
Was added to obtain crystals. The crystals obtained were recrystallized from a mixture of acetonitrile and ether. (50 mg, yield 78
%) FAB-MS: M ⁺ 401

Example 2 In the same manner as in Example 1, from 1-methylacridine-9-carboxylic acid obtained in Reference Example 2 to 2'-methyl-5'-nitrophenyl-N-methyl-1-methylacridyi. N-
9-carboxylate fluorosulfonate (Compound 12) was synthesized. FAB-MS: M ⁺ 387

The following acridinium ester was synthesized by the same method. Example 3 2'-Trifluoromethylphenyl-N-methyl-1-
Methyl acridinium-9-carboxylate fluorosulphonate (compound 16) FAB-MS: M ⁺ 396 Example 4 2'-trifluoromethylphenyl-N-methyl-1,
3-dimethylacridinium-9-carboxylate
Fluorosulfonate (Compound 17) FAB-MS: M ⁺ 410

Comparative Examples The following comparative compounds (Compounds 1 to 11, 14 and 15) were synthesized in the same manner as in Example 1. Phenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 1, FAB-M
S: M ⁺ 314) 2'-methyl-5'-nitrophenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 2, FAB-MS: M ⁺ 373) 2'-methyl-4'- (Benzyloxycarbonyl)-
Phenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 3, FAB-M
S: M ⁺ 462) 2'6'-dimethylphenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 4, FAB-MS: M ⁺ 342) 2'6'-dimethyl-4 '-( Benzyloxycarbonyl) -phenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 5, FA
B-MS: M ⁺ 476) 2'6'-dimethyl-4'-nitrophenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 6, FAB-MS: M ⁺ 387) 2'6 '. -Dibromo-4'-methylphenyl-N-methylacridinium-9-carboxylate fluorosulfonate (Compound 7, FAB-MS: M ⁺ 486)

2'4'6'-triiodophenyl-N-
Methyl acridinium-9-carboxylate fluor
Rosulfonate (Compound 8, FAB-MS: M⁺69
2) Phenyl-N-methyl-1-methylacridinium-9
-Carboxylate fluorosulfonate (compound 9,
FAB-MS: M⁺328) Phenyl-N-methyl-1,3-dimethylacridiniu
Mu-9-carboxylate fluorosulfonate
Compound 10, FAB-MS: M⁺342) 4'-acetoxyphenyl-N-methyl-1,3-dime
Cilacridinium-9-carboxylate fluoro
Sulfonate (Compound 11, FAB-MS: M ⁺38
4) 2'-nitrophenyl-N-methyl-1,3-dimethyl
Acridinium-9-carboxylate fluorosulfur
Honate (Compound 14, FAB-MS: M⁺387) 2-Bromophenyl-N-methyl-1-methylacridi
Nium-9-carboxylate fluorosulfonate
(Compound 15, FAB-MS: M⁺407)

Example 5 (1) Synthesis of 4-hydroxy-3-methylbenzoic acid 4-hydroxy-3-methylbenzaldehyde (2.7
2g, 20mmole) to silver nitrate (6g, 35mmole), sodium hydroxide (7.2g, 180mmole), water 40ml
Was added and refluxed for 6 hours. After cooling, 2N hydrochloric acid (150m
l), the crystals were filtered, washed with water and dried under reduced pressure (2.
(37 g, yield 78%)

(2) Synthesis of 4-hydroxy-3-methylbenzoic acid benzyl ester 4-hydroxy-3-methylbenzoic acid potassium salt (2.
54 g, 13.4 mmole) was dissolved in hexamethylphosphoric triamide (30 ml) by heating. After cooling, benzyl chloride (12.3 ml) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was added to 2N-hydrochloric acid (250 ml), and ethyl acetate (1
(00 ml, 3 times). After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The obtained crude crystals were subjected to silica gel chromatography (eluent, 3% -ethyl acetate /
It was purified by (methyl chloride). (2.18 g, yield 67
%)

(3) Synthesis of 2'-methyl-4 '-(benzyloxycarbonyl) -phenyl-1,3-dimethylacridine-9-carboxylate 1,3-Dimethylacridine-9-carboxylic acid (161
mg, 0.64 mmole) was dissolved in anhydrous pyridine (5.3 ml) and cooled to -23 ° C. 4-Toluenesulfonyl chloride (366 mg, 1.92 mmole / anhydrous pyridine, 2.6
ml) was added, and the mixture was stirred at -23 ° C for 15 minutes. 4-Hydroxy-3-methylbenzoic acid benzyl ester (124m
g, 0.51 mmole / anhydrous pyridine, 2.6 ml) was added, and the mixture was stirred at room temperature for 15 hours, and then water (1.1 ml),
And benzene (5 ml) were added, and the solvent was evaporated under reduced pressure.
The obtained crude crystals were purified by silica gel chromatography (eluent, 3% -ethyl acetate / chloroform). (60 mg, 20% yield)

(4) Synthesis of 2'-methyl-4'-carboxyphenyl-1,3-dimethylacridine-9-carboxylate 2'-methyl-4 '-(benzyloxycarbonyl)-
Phenyl-1,3-dimethylacridine-9-carboxylate (60 mg, 0.126 mmole) was added to acetic acid (1.1
ml) and dissolved by heating. 48% -HBr / acetic acid (0.28
ml) was added and the mixture was stirred at 100 ° C. for 3 hours. After cooling, the reaction solution was added to water (3.5 ml), and the obtained crystals were filtered and washed with water. (40 mg, yield 82%)

(5) Synthesis of 2'-methyl-4 '-(N-succinimidyloxycarbonyl) -phenyl-1,3-dimethylacridine-9-carboxylate 2'-methyl-4'-carboxyphenyl -1,3-Dimethylacridine-9-carboxylate (40 mg,
0.12mmole) was added to anhydrous dimethylformamide (2.6
ml) and cooled to -10 ° C. Dicyclohexylcarbodiimide (44mg, 0.21mmole) and 4-dimethylaminopyridine (5mg, 0.04mmole) dissolved in anhydrous dimethylformamide (0.8ml) were added,
The mixture was stirred at -10 ° C for 10 minutes. N-Hydroxysuccinimide (18 mg, 0.156 mmol / anhydrous dimethylformamide, 0.8 ml) was added, and the mixture was stirred at room temperature for 40 hours. After the solvent was distilled off under reduced pressure, the residue was dissolved in methylene chloride and the precipitated dicyclohexylurea was removed by filtration (this operation was repeated 3 times). The crude crystals obtained by distilling methylene chloride under reduced pressure were subjected to silica gel chromatography (eluent, 25% -ethyl acetate / chloroform).
Purified by. (36 mg, 72% yield)

(6) 2'-Methyl-4 '-(N-succinimidyloxycarbonyl) -phenyl-N-methyl-1,3-dimethylacridinium-9-carboxylate fluorosulfonate (Compound 18) Synthesis of 2'-methyl-4 '-(N-succinimidyloxycarbonyl) -phenyl-1,3-dimethylacridine-
9-carboxylate- (36mg, 0.075mmole)
Was dissolved in 2.8 ml of anhydrous methylene chloride and methyl fluorosulfonate (0.058 ml, 0.75 mmole) was added.
The mixture was stirred at room temperature for 20 hours. Anhydrous ether (3 ml) was added to obtain crystals. The crystals obtained were recrystallized from a mixture of acetonitrile and ether. (29 mg, 65% yield) FAB-MS: M ⁺ 497 Melting point: 189-191 ° C (decomposition)

Example 6 (1) Synthesis of 2'-methyl-4 '-(benzyloxycarbonyl) -phenyl-1-methylacridine-9-carboxylate 1-Methylacridine-9-carboxylic acid (240 mg, 1
mmole) was dissolved in anhydrous pyridine (8 ml) and cooled to -23 ° C. 4-Toluenesulfonyl chloride (570 mg, 3
mmole / anhydrous pyridine, 4 ml) was added and the mixture was stirred at -23 ° C for 15 minutes. 4-Hydroxy-3-methylbenzoic acid benzyl ester (193 mg, 0.8 mmole / anhydrous pyridine 4 ml) was added, and the mixture was stirred at room temperature for 25 hours. Water (2 ml) and benzene (10 ml) were added, and the solvent was evaporated under reduced pressure.
The obtained crude crystals were purified by silica gel chromatography (eluent, 2% -ethyl acetate / chloroform). (129 mg, 28% yield)

(2) Synthesis of 2'-methyl-4'-carboxyphenyl-1-methylacridine-9-carboxylate 2'-methyl-4 '-(benzyloxycarbonyl)-
Phenyl-1-methylacridine-9-carboxylate (110 mg, 0.24 mmole) was dissolved in acetic acid (2.1 ml) by heating. 48% -HBr / acetic acid (0.53 ml) was added, and the mixture was stirred at 100 ° C for 3 hr. After cooling, the reaction solution was added to water (7 ml), and the obtained crystals were filtered, washed with water, and dried under reduced pressure. (76 mg, yield 85%)

(3) Synthesis of 2'-methyl-4 '-(N-succinimidyloxycarbonyl) -phenyl-1-methylacridine-9-carboxylate 2'-methyl-4'-carboxyphenyl-1 -Methyl acridine-9-carboxylate (74 mg, 0.2 mm
ole) was dissolved in anhydrous dimethylformamide (5 ml),
Cooled to -10 ° C. Dichlorohexylcarbodiimide (73 mg, 0.35 mmole) and 4-dimethylaminopyridine (10 mg, 0.08 mmole) dissolved in anhydrous dimethylformamide (1.2 ml) were added, and the mixture was stirred at -10 ° C for 10 minutes. N-hydroxysuccinimide (3
0 mg, 0.26 mmol / anhydrous dimethylformamide
2 ml) was added and the mixture was stirred at room temperature for 64 hours. After the solvent was distilled off under reduced pressure, the residue was dissolved in methylene chloride and the precipitated dicyclohexylurea was removed by filtration (this operation was repeated 3 times). The crude crystals obtained by distilling methylene chloride under reduced pressure were subjected to silica gel chromatography (eluent, 15
% -Ethyl acetate / chloroform). (5
5 mg, 59% yield)

(4) 2'-methyl-4 '(N-succinimidyloxycarbonyl) -phenyl-N-methyl-
Synthesis of 1-methylacridinium-9-carboxylate fluorosulfonate (Compound 19) 2'-methyl-4 '(N-succinimidyloxycarbonyl) -phenyl-1-methylacridine-9-carboxylate (26 mg , 0.055 mmole) was dissolved in anhydrous methyl chloride (2 ml), methyl fluorosulfonate (0.042 ml, 0.55 mmole) was added, and the mixture was stirred at room temperature for 37 hours. Add anhydrous ether (2.5 ml),
Crystals were obtained. The crystals obtained were recrystallized from a mixture of acetonitrile and ether. (14 mg, yield 44%) FAB-MS: M ⁺ 483 Melting point: 185-188 ° C (decomposition)

Example 7 (1) Synthesis of 3-amino-5-nitro-p-toluic acid 3,5-dinitro-p-toluic acid (2.26 g, 10)
mmole) was dissolved in ethanol (50 ml), platinum-carbon (110 mg) and cyclohexene (6 ml) were added, and the mixture was refluxed for 12 hours. The platinum-carbon was removed by filtration and the filtrate was evaporated to dryness under reduced pressure. The obtained crude crystals were purified by silica gel chromatography [dry column, eluent, methylene chloride / ethyl acetate / acetic acid (10: 10: 1)]. (1.4 g, yield 71.4%) Melting point: 211-213 ° C (decomposition)

(2) 3-hydroxy-4-methyl-5-
Synthesis of nitrobenzoic acid 3-amino-5-nitro-p-toluic acid (1.26
g, 6.4 mmole) was dissolved in 76% sulfuric acid (22 ml) by heating. After cooling with ice, sodium nitrite (0.92 g / 3.5 water)
ml) was gradually added over about 1 hour. After stirring for 1 hour under ice cooling, the mixture was stirred at 100 ° C. for 2 hours. After cooling, the reaction solution was added to water (28 ml), the obtained crystals were separated by filtration, washed with water, and dried under reduced pressure. (0.9 g, 71% yield) Melting point: 212-213 ° C

(3) 3-hydroxy-4-methyl-5-
Synthesis of nitrobenzoic acid benzyl ester 3-Hydroxy-4-methyl-5-nitrobenzoic acid potassium salt (0.84 g, 3.6 mmole) was dissolved in hexamethylphosphoric triamide (8 ml) and benzyl chloride (3. 3 ml) was added and the mixture was stirred at room temperature for 6 hours. The reaction solution was transferred to 2N-hydrochloric acid (60 ml), and ethyl acetate (50 ml, 2 ml) was added.
Times). After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure (toluene azeotrope, 20 ml × 3). The obtained oil is subjected to silica gel chromatography (eluate,
50% -ethyl acetate / hexane).
(0.75 g, yield 72.5%)

(4) 2'-methyl-3'-nitro-5 '
-(Benzyloxycarbonyl) -phenyl-1,3-
Synthesis of dimethyl acridine-9-carboxylate 1,3-Dimethyl acridine-9-carboxylic acid (251
mg, 1 mmole) was dissolved in anhydrous pyridine (8.3 ml),
Cooled to -23 ° C. 4-toluenesulfonyl chloride (5
72 mg, 3 mmole / anhydrous pyridine, 4 ml) was added, and -2
Stir for 15 minutes at 3 ° C. 3-Hydroxy-4-methyl-5-nitrobenzoic acid benzyl ester (253 mg,
0.88 mmole / anhydrous pyridine, 4 ml) was added, and the mixture was stirred at room temperature for 15 hours. Water (1.7 ml) and benzene (8 ml)
Was added and the solvent was distilled off under reduced pressure. The crude crystals obtained are
Purified by silica gel chromatography (eluent, 6% -ethyl acetate / chloroform). (134 mg, yield 25%)

(5) 2'-methyl-3'-nitro-5 '
-Carboxyphenyl-1,3-dimethylacridine-
Synthesis of 9-carboxylate 2'-methyl-3'-nitro-5 '-(benzyloxycarbonyl) -phenyl-1,3-dimethylacridine-9-carboxylate (134 mg, 0.26 mmole)
Was dissolved in acetic acid (2.3 ml) by heating. 48% -HBr /
Acetic acid (0.58 ml) was added, and the mixture was stirred at 100 ° C for 3 hr. After cooling, the reaction solution was added to water (14 ml), and the obtained crystals were filtered and washed with water. (103 mg, 93% yield)

(6) 2'-methyl-3'-nitro-5 '
Synthesis of-(N-succinimidyloxycarbonyl) -phenyl-1,3-dimethylacridine-9-carboxylate 2'-methyl-3'-nitro-5'-carboxyphenyl-1,3-dimethylacridine- 9-Carboxylate (102 mg, 0.24 mmole) was added to anhydrous dimethylformamide (6 ml) and cooled to -10 ° C. Dicyclohexylcarbodiimide (88mg, 0.42mmole)
And 4-dimethylaminopyridine (10 mg, 0.08 mmol
A solution of e) dissolved in anhydrous dimethylformamide (2 ml) was added, and the mixture was stirred at -10 ° C for 10 minutes. N-Hydroxysuccinimide (41 mg, 0.36 mmole / anhydrous dimethylaformamide, 2 ml) was added, and the mixture was stirred at room temperature for 25 hours. After the solvent was distilled off under reduced pressure, the residue was dissolved in methylene chloride and the precipitated dicyclohexylurea was removed by filtration (this operation was repeated 3 times). The crude crystals obtained by distilling methylene chloride under reduced pressure were purified by silica gel chromatography (eluent, 25% -ethyl acetate / chloroform). (63 mg, yield 49%)

(7) 2'-methyl-3'-nitro-5 '
-(N-succinimidyloxycarbonyl) -phenyl-N-methyl-1,3-dimethylacridinium-9
-Carboxylate fluorosulfonate (compound 2
Synthesis of 0) 2'-methyl-3'-nitro-5 '-(N-succinimidyloxycarbonyl) -phenyl-1,3-dimethylacridine-9-carboxylate (53 mg, 0.1
mmole) was dissolved in anhydrous methylene chloride (4 ml), methyl fluorosulfonate (0.08 ml, 1 mmole) was added, and the mixture was stirred at room temperature for 45 hours. The crystals formed are filtered off,
It was washed with anhydrous ether. (50 mg, yield 78%) FAB-MS: M ⁺ 542 (see FIG. 7) Melting point: 176-178 ° C. (decomposition)

Test Example 1 Chemiluminescence of Acridinium Ester and Stability in Neutral Aqueous Solution Synthesized acridinium ester (Compounds 1 to 17) was dissolved in dimethylformamide to contain 0.1% bovine serum albumin. The solution was diluted with 0.1 M phosphate buffer (pH 7.0) to give a 5 × 10 ⁻⁹ M solution. Each diluted test solution (20 μl) contained 1.2% -hydrogen peroxide at 0.
Mix with 4N-nitric acid (40 μl) and after 30 seconds 0.25
The amount of chemiluminescence was measured by shooting N-sodium hydroxide (150 μl). The luminescence amount of the diluted preparation of each compound was measured at the time of preparation and at 37 ° C. (1 day, 4 days, 7 days). The measurement was performed by using a luminometer LC-1000 manufactured by Nichine Medical Science Instruments Co., Ltd. and integrating the light emission amount for 5 seconds. The results are shown in Table 1.

[0088]

[Table 1]

Regarding the chemiluminescence, it was recognized that the presence of the substituents hardly hinders the luminescence except for compound 4. Regarding stability in a neutral aqueous solution, an acridinium ester stabilized by introducing an orthodimethyl group into a phenyl ring (compounds 4 to
It was found that the compound of the present invention is extremely stable as compared with 6). It should be noted that an acridinium ester having an alkyl introduced into only one ortho-position of the phenyl ring (compounds 2 and 3) and only the 1-position of the acridine ring (compounds 9 to 11), and substitution of the ortho-position of the phenyl ring with a group other than alkyl The group-substituted acridinium ester (Compounds 7, 8, 14 and 15) did not provide a sufficient stabilizing effect. From these results, it is concluded that it is essential to introduce an alkyl group at both the 1-position of the acridine ring and one of the ortho-positions of the phenyl ring in order to obtain high stability.

Example 8 Labeling of antibody protein with acridium ester Anti-CEA mouse monoclonal antibody IgG (450 μm
g) in 0.1 M-phosphate buffer (pH 8.1),
Acridinium ester for labeling (compounds 18, 19, 2
0, 21) was dissolved in dimethylformamide to a concentration of 5 mM, and 9 μl of the solution was added. After leaving it for 30 minutes at room temperature, 1M-glycine buffer (pH 8.0) was added to 100
μl was added and the mixture was allowed to stand for another 10 minutes. P this in advance
The protein-containing fractions were collected by passing through a Sephadex G-25 column that had been equilibrated with BS (20 mM-phosphate buffer containing 0.15 M sodium chloride, pH 7.0). The acridinium ester bound to IgG is labeled 18, 19, 20, 21 (compounds 18, 1 respectively).
(Prepared from 9, 20, 21) was about 4 molecules per IgG molecule. The compound 22 was labeled according to the method of Woodhead et al. (Methods in Enzymology, Vol. 133, pp. 374-378). in this case,
The number of acridinium esters bound to IgG was about 3 molecules per IgG molecule. (Labeled body 22)

Example 9 Measurement of chemiluminescence of labeled body Labeled bodies 19, 20, 21 and 22 prepared in Example 8
Each was diluted with PBS containing 0.1% bovine serum albumin to make 10-fold dilution series. Each dilution series (20μ
l) 0.1N nitric acid containing 0.3% hydrogen peroxide (1)
The amount of chemiluminescence was measured by adding 50 μl) and, after 1 minute, shot 0.25 N-sodium hydroxide (150 μl). The results are shown in Fig. 4. Labeled substances 19 and 20 labeled with the compounds 19 and 20 of the present invention were capable of quantitative luminescence detection, indicating that the structure based on the present invention is preferable.

Test Example 2 Stability of Labeled Body Labeled body 18, 19, 20, 21, 2 prepared in Example 8
2 PBS containing 0.1% -bovine serum albumin
And diluted so that the amount of luminescence was about 20,000 counts per 20 μl. Keep each dilution at 37 ° C,
The daily change in the amount of luminescence was examined. The results are shown in Table 2.

[0093]

[Table 2]

When heated at 37 ° C. for 7 days, the labeled product 21 in which an orthodimer group was introduced into the phenyl ring showed a decrease in the amount of luminescence up to 75%, while the labeled products 18 to 2 of the present invention.
At 0, the light emission amount of 85% or more was retained.

Example 10 Chemiluminescence immunoassay (1) Preparation of sensitization test tube Anti-CEA goat IgG was added to 0.1 M phosphate buffer (pH).
It was diluted with 7.0) to 30 μg / ml, 300 μl was added to a polystyrene test tube (12 × 75 mm, Nunc Co. maxisorp tube), and the mixture was allowed to stand at 5 ° C. overnight.
Wash twice with PBS containing 0.1% -Tween 20,
Add 1 ml of PBS containing 0.5% -bovine serum albumin,
Further, it was left at 5 ° C. overnight. (2) Dilution of labeled bodies 18 to 22 On the other hand, the labeled bodies 18 to 22 were diluted with PBS containing 0.5% -bovine serum albumin and 0.1% -human serum albumin, and the luminescence amount per 20 μl was 40, Diluted to 000 counts. (3) Measurement After removing the liquid in the sensitization test tube by suction, 0.3 ml of PBS containing 0.5% -bovine serum albumin and 0.1% -human serum albumin was added. Furthermore, 20 μl of CEA standard solution (or test serum) diluted to various concentrations was added, and after stirring, 37 ° C.
Left for 1 hour. 0.1% -PB with Tween 20
It was washed twice with S, diluted marker 18 was added, and the mixture was left at 37 ° C. for 1 hour. After washing 4 times with PBS containing 0.1% -Tween 20, 0.1N-nitric acid containing 3% hydrogen peroxide (3%
00 μl) was added and 1 minute later, 0.25 N-sodium hydroxide (300 μl) was shot, and the chemiluminescence amount was measured (5 seconds integration). The calibration curve of CEA when the labeled body 18 is used is shown in FIG. The detection sensitivity of CEA was 1 ng / ml.

Test Example 3 Stability of chemiluminescence immunoassay The diluted labels 18 to 22 described in Example 10 were left at 25 ° C. and 100 ng / ml of CEA standard solution was measured under the same conditions as in Example 10. The daily change in chemiluminescence was examined.
The results are shown in Table 3.

[0097]

[Table 3]

When CEA was measured using the labeled substances prepared by using the compounds 18, 19 and 20 of the present invention, even if the diluted labeled substances were left at room temperature (25 ° C.) for 3 weeks, a constant luminescence sensitivity was obtained. It was demonstrated that the chemiluminescent compound of the present invention was extremely stable.

[0099]

INDUSTRIAL APPLICABILITY The chemiluminescent compound of the present invention has an alkyl group introduced at the 1-position (peri-position) of the acridine ring and one of the ortho-positions of the phenyl ring. And the stability, especially in a neutral aqueous solution, is extremely high, and the chemiluminescent property is comparable to or higher than that of conventional compounds. It is very useful as a labeling compound in chemiluminescence analysis.

[Brief description of drawings]

FIG. 1 is a diagram showing chemical structures of Compounds 1 to 9.

FIG. 2 is a diagram showing chemical structures of Compounds 10 to 17.

FIG. 3 is a diagram showing chemical structures of compounds 18 to 22.

FIG. 4 is a graph showing the relative amount of emitted light with respect to the concentration of labeled substance in the measurement of Example 9.

5 is a graph showing a calibration curve in the measurement of Example 10. FIG.

FIG. 6 is an NMR spectrum of 1-methylacridine-9-cyanide obtained in Reference Example 2.

FIG. 7 is a FAB-MS spectrum of compound 20 obtained in Example 7.

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[Procedure amendment]

[Submission date] July 2, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

[0019]

[Chemical 3]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

(Where R is_FourIs alkyl, aryl or
Aralkyl, R_FiveIs alkylene or arylene,X ₁
Represents a group selected from
Groups are indicated. Where alkyl, aryl and ara
Rukiru is the R ₁Is the same as the definition in. Again
Rukiren and arylene have 1 to 20 carbon atoms, preferably
Has 1 to 10 carbon atoms, and more preferably 1 to 6 carbon atoms.
It Preferably R is methylene, ethylene, propylene,
Or ortho-, meta- or para-phenylene
It Halogen is chlorine, bromine, iodine or fluorine
And preferably chlorine or bromine.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0033

[Correction method] Change

[Correction content]

( Wherein R ₁ , R ₂ , R ₃ and A ⁻ have the same meanings as described above) 1-Alkyl-substituted acridine-9-carboxylic acid (III) or its reactive derivative is converted into 2-alkyl-substituted phenol. (I
The acridine-9-carboxylic acid phenol ester compound (V) is obtained by esterification with V) by a known ester synthesis method. The acridinium compound (I) is produced by subjecting the ester compound (V) to quaternary chloride according to a conventional method.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

[0035]

[Chemical 6]

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0036

[Correction method] Change

[Correction content]

(In the formula, R ₂ and R ₃ have the same meanings as described above, and Y ₁ is halogen such as chlorine, bromine, iodine or fluorine, preferably chlorine.) Compound (VI) and Compound (VII) ) And N- (3
The -substituted phenyl) -anthranilic acid (VIII) is prepared. The anthranilic acid compound (VIII) is reacted with phosphorus oxychloride to produce 1-alkyl-substituted acridone (IX). The acridone compound (IX) is the corresponding halide
It is converted to cyanide form (XI) via (X). The cyano group of the cyanide compound (XI) is hydrolyzed to give 1-alkyl-substituted acridine-9-carboxylic acid (III).

Claims (4)

[Claims] 1. A compound represented by the general formula (I): (Wherein R ₁ is a group that does not interfere with chemiluminescence, R ₂ and R ₃
Are each optionally fluorinated alkyl, X and Y are each O or S, and A ⁻ is an anion that does not interfere with chemiluminescence. ), And an acridinium compound or an isomer thereof in which any position of the acridine ring or phenyl ring (excluding the ortho position of the phenyl ring) may be substituted with a group that does not interfere with chemiluminescence. 2. A luminescent complex comprising a biologically active substance and the compound according to claim 1 bound thereto. 3. A sample is mixed with a binding substance which is labeled with the compound according to claim 1 and which specifically binds to the test substance in the sample. Forming a complex, (b) separating the complex and a free labeled binding substance, (c) detecting chemiluminescence of the compound bound to the complex, (d) An assay method for a test substance in a sample, which comprises determining the amount of the test substance in the sample from the amount of luminescence detected. 4. A binding substance which specifically binds (a) a sample to (i) a test substance in the sample, and (ii)
By mixing with the test substance labeled with the compound described above to form a complex of the test substance and the binding substance, (b) separating the complex from the free labeled test substance, and (c) the Detecting chemiluminescence of the compound of claim 1 bound to a complex,
(D) An assay method for a test substance in a sample, which comprises determining the amount of the test substance in the sample from the detected luminescence amount.