JP2002020382A - 1,2-dioxetane derivative having bicyclononylidene group - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new 1,2-dioxetane derivative useful as a reagent for chemiluminescent immunoassay(CLIA). SOLUTION: 1,2-Dioxetane derivative of the formula (1) (wherein, R1 is H, a halogen or a 1-3C alkyl; R2 is a hydroxyl-substituted 2-4C alkoxyl or a hydroxyl-substituted 2-4C alkylthio; R3 is H, a halogen, a 1-3C alkyl or a 1-3C alkoxyl; and R4 is 3-t-butyldimethylsilyloxy, phosphate or hydroxyl).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel 1,2-dioxetane derivative. The 1,2-dioxetane derivative of the present invention can be used as a chemiluminescent reagent in a chemiluminescent immunoassay (CLIA).

[0002]

2. Description of the Related Art Conventionally, a large number of 1,2-dioxetane derivatives have been synthesized, and among them, a 1,2-dioxetane derivative having an adamantylidene group is known to be particularly useful as a chemiluminescent substrate. For example,
No. 21918 and Japanese Patent Publication No. 7-31201). Chemiluminescence detection immunoassay is a chemiluminescence immunoassay (CLIA) using a chemiluminescent compound for labeling.
Chemiluminescent Enzyme Immunoassay (CLEIA) that uses a chemiluminescent reaction to detect the enzyme activity using an enzyme and an enzyme for labeling
Whether to use a reagent for CLIA or a reagent for CLEIA as a medical diagnostic agent depends on the policy of the manufacturer of the diagnostic system including the diagnostic device, but this adamantylidene 1,2-dioxetane The compound is a luminescent reagent for CLEIA, not a reagent for CLIA (Japanese Patent Publication No. 5-21918, Japanese Patent Publication No. 7-31).
No. 201).

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel 1,2-dioxetane compound capable of direct chemiluminescence immunoassay (CLIA).

[0004]

The inventor of the present invention has made intensive studies to achieve the above object, and as a result, the expression (1)

Embedded image (Wherein R ¹ is hydrogen, halogen, a C ₁ -C ₃ alkyl group, R ² is a hydroxyl-substituted C ₂ -C ₄ alkoxy group, a hydroxyl-substituted C ₂ -C ₄ alkylthio group, and R ³ is hydrogen,
A halogen, a C ₁ -C ₃ alkyl group, a C ₁ -C ₃ alkoxy group, and R ⁴ is a 3-t-butyldimethylsilyloxy group, a phosphate group, or a hydroxyl group. 1, 2)
A dioxetane derivative has been found and the present invention has been completed.

[0005] The 1,2-formula represented by the above formula (1) of the present invention.
The dioxetane derivative can be produced according to the following reaction formula.

Embedded image

In the first step, a cyclic alkyl ketone derivative represented by the formula (2) is reacted with an ester derivative or a thioester derivative represented by the formula (3) to form a tetrasubstituted alkene represented by the formula (4). It is for producing derivatives.
The reaction must be performed in the presence of a titanium halide, and titanium tetrachloride is preferably used. It is desirable to form a reduced state by using lithium aluminum hydride or the like as a reducing agent and triethylamine or the like as a base, and to provide the reaction. The reaction can be performed in an organic ether such as tetrahydrofuran. Although the reaction proceeds at 0 to 100 ° C., it is preferable to perform the reaction under reflux of tetrahydrofuran from the viewpoint of operation and reactivity.

In the second step, a compound such as tetrabutylammonium fluoride is allowed to act on the compound represented by formula (4) to carry out a deprotection reaction to produce a compound represented by formula (5). It is.

In the third step, phosphoryl chloride or the like is allowed to act on the compound represented by the formula (5) in the presence of a base such as pyridine, and the compound is taken out in the form of an alkali metal salt or ammonium salt and represented by the formula (6). To produce a compound. The reaction is desirably performed under ice cooling.

In the fourth step, a tetrasubstituted alkene derivative represented by the formula (4), (5) or (6) is reacted with singlet oxygen to form a compound represented by the formula (1). This is for producing a 2-dioxetane derivative. The reaction with singlet oxygen is carried out with dichloroethane, dichloromethane, methanol, ethanol and the like in the case of the compounds of the formulas (4) and (5), and with hydrated ethanol and hydrated methanol in the case of the compound of the formula (6). It can be achieved by dissolving and irradiating visible light under an oxygen atmosphere in the presence of a photosensitizer such as rose bengal, methylene blue, tetraphenylporphine and the like. This reaction is desirably performed at -80 ° C to 0 ° C.

A preferred compound synthesized by the above reaction is 4- (3-t-butyldimethylsilyloxyphenyl) -4- (4-hydroxybutyloxy) spiro [1,2-dioxetane-3,9'-bicyclo [ 3.
3.1] Nonane], 4- (3-hydroxyphenyl)-
4- (4-hydroxybutyloxy) spiro [1,2-
Dioxetane-3,9'-bicyclo [3.3.1] nonane], 4- (3-phosphatephenyl) -4- (4-
Hydroxybutyloxy) spiro [1,2-dioxetane-3,9'-bicyclo [3.3.1] nonane], disodium salt, 4- (3-tert-butyldimethylsilyloxy-4-methoxyphenyl)- 4- (4-hydroxybutyloxy) spiro [1,2-dioxetane-3,9 '
-Bicyclo [3.3.1] nonane], 4- (3-t-butyldimethylsilyloxy-4-methylphenyl) -4
-(4-hydroxybutyloxyspiro [1,2-dioxetane-3,9'-bicyclo [3.3.1] nonane;

These 1,2-dioxetane compounds can be used for chemiluminescence immunoassay (chemiluminescence immunoassay). As a tracer in an immunoassay, these chemiluminescent 1,2-dioxetane compounds are used for labeling an antibody or an antigen, and after immunoreaction by a competitive method or an immunometric method, B (antigen-antibody conjugate) / F ( After separation, the chemiluminescent label adsorbed mainly on the solid phase is detected by a chemiluminescence reaction. Separation of labeled antigen-antibody conjugate (or labeled antibody-antigen conjugate) [complex] from free labeled antigen (or free labeled antibody) [free body] (B / F separation) is an immunoassay field. May be performed by a method usually used. The solid phase method of immobilizing a binding substance on an insoluble carrier such as a polystyrene test tube or well, sepharose, or polyacrylamide gel is advantageous in terms of simplicity. Chemiluminescence is detected by using, as a labeling compound, a binding substance that specifically binds to the test substance or 1,2 represented by the above formula (1) that is covalently bonded to the test substance via a reactive functional group. A base such as tetra-n-butylammonium fluoride as a trigger when R ⁴ of the dioxetane compound is a 3-t-butyldimethylsilyloxy group or a hydroxyl group, or an alkaline phosphatase when R ⁴ is a phosphate group; May be measured with a suitable measuring instrument such as a luminometer.

In the assay using the 1,2-dioxetane compound represented by the above formula (1), the measurable test substances include antigens (including haptens), antibodies, proteins,
Examples include biologically active substances such as nucleic acids and hormones.
Examples of the combination of the test substance and the binding substance include an antigen and an antibody,
Examples include tumor markers and their antibodies, hormones and their binding proteins, nucleic acids and their complementary polynucleotides, enzymes and their substrates, and drugs and their binding proteins. As an example of such a test substance,
CEA (carcinoembryonic antigen), TSH (thyroid stimulating hormone), hTSH (human thyrotropin), hCG (placental gonadotropin), CRP (C-reactive protein), ASO
(Anti-streptolysin O), AFP (α-fetoprotein), transferrin, ferritin, fibrin,
Haptoglobin, IgG, IgE and the like can be mentioned. If necessary, a protein such as bovine serum albumin, a fluorescent agent such as fluorescein, or a cationic surfactant such as cetyltrimethylammonium bromide may be added to an assay system using the compound represented by the formula (1). Needless to say, it may be added and used as a luminescence intensity enhancing substance).

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

Reference Example 1 Synthesis of 3-t-butyldimethylsilyloxybenzoic acid

Embedded image 3-hydroxybenzoic acid (3 g, 21.7 mmole)
And t-butyldimethylsilyl chloride (8.52 g, 5
6.5 mmole) and imidazole (5.76 g, 8
4.6 mmole) and 4A molecular sieve dehydration D
A mixture with MF (45 ml) was added under a nitrogen atmosphere at room temperature for 1 hour.
Stir for 7 hours. The reaction solution was dissolved in 300 ml of ethyl acetate and transferred to a 1000 ml separating funnel. The ethyl acetate layer was separated and washed twice with 200 ml of 1.2N aqueous hydrochloric acid, twice with 200 ml of 10% K ₂ CO ₃ water, and further three times with 100 ml of distilled water. The ethyl acetate layer was dried over MgSO ₄ and concentrated,
The obtained concentrate was purified by a column packed with 2 kg of silica gel (eluent: 10% ethyl acetate-hexane). A single spot fraction (Rf 0.14) was collected by TLC (thin layer chromatography) to obtain 4.94 g of waxy 3-t-butyldimethylsilyloxybenzoic acid. Yield: 90% Thin plate: HPTLC plate coated with silica gel 60F ₂₅₄ s, manufactured by Merck Co., Ltd. Detection: Drying after development, spotting with a UV lamp, and placing in an iodine tank to develop color. Developing phase: toluene, ethyl acetate (volume ratio = 100: 2)
0)

Reference Example 2 Synthesis of 4- (3-t-butyldimethylsilyloxy) -hydroxybutylbenzoate

Embedded image 3-t-butyldimethylsilyloxybenzoic acid (3 g,
11.9 mmole) and 1,4-butanediol (5.
9 g, 65.5 mmole), dicyclohexylcarbodiimide (2.7 g, 13.1 mmole) and 4-dimethylaminopyridine (147 mg, 1.2 mmole)
And 4A molecular sieves dehydrated dichloromethane (25
ml) was stirred at room temperature under a nitrogen atmosphere for 20 hours. The reaction solution was diluted with 300 ml of ethyl acetate,
It was transferred to a 0 ml separatory funnel. Ethyl acetate layer is distilled water 15
Once with 0 ml, twice with 150 ml of 10% K ₂ CO ₃ water,
Further, the mixture was washed three times with 100 ml of distilled water. The ethyl acetate layer was dried over MgSO ₄ and then concentrated.
Purification was performed using a column packed with 2 kg of silica gel (eluent: 2% ethyl acetate-hexane). A single spot fraction (Rf 0.21) was collected by TLC (thin layer chromatography), and the oily 4- (3-t
-Butyldimethylsilyloxy) -hydroxybutylbenzoate (2.1 g) was obtained. Yield: 54% Thin layer plate: HPTLC plate coated with silica gel 60F ₂₅₄ s, manufactured by Merck Co., Ltd. Detection: After development, drying is performed, and spots are confirmed by a UV lamp, and then placed in an iodine tank to develop color. Developing phase: hexane, ethyl acetate (volume ratio = 50: 1)

Reference Example 3 4- (3-t-butyldimethylsilyloxyphenyl)
-4- (4-hydroxybutyloxy) methylene-2-
Synthesis of Bicyclo [3.3.1] nonane

Embedded image A well-dried 1000 ml four-necked flask was equipped with a reflux condenser, a 200 ml dropping funnel, and a nitrogen supply tube, and nitrogen was passed during the reaction. First, 200 ml of 4A molecular sieve dehydrated THF was added, and the flask was cooled in an ice bath. 3.29 ml of titanium tetrachloride (30 mmol
e) is added rapidly, and then 30 ml of a 1 M solution of lithium aluminum hydride in tetrahydrofuran (30 mmol)
e) was added dropwise with stirring. After the completion of the dropwise addition, the cooling bath was removed, and the temperature of the mixture was raised to about room temperature. Next, 4.2 ml (30 mmole) of triethylamine was added to the stirred mixture, and the mixture was refluxed for 15 minutes. Then 4
A Molecular sieves 4-dehydrated in 100 ml of THF
1.95 g (6 mmole) of (3-t-butyldimethylsilyloxy) -hydroxybutylbenzoate and 1.66 g of bicyclo [3.3.1] nonan-9-one
(12 mmole) was added to the refluxing mixture at 6
Added dropwise over 0 minutes. Reflux was continued for another 50 minutes, after which the reaction was concentrated and the reaction was washed with 40 mL of ethyl acetate.
Diluted with 0 ml. The ethyl acetate layer was washed with 5% hydrochloric acid in 100
The mixture was washed twice with 200 ml of 10% K ₂ CO ₃ water five times with 200 ml of distilled water and three times with 200 ml of distilled water, dried over MgSO _{4 and} concentrated. The obtained concentrate was purified by a column packed with 1 kg of silica gel (eluent: 1% ethyl acetate-hexane), and a single spot fraction (Rf 0.39) was collected by TLC (thin layer chromatography).
4- (3-t-butyldimethylsilyloxyphenyl) -4- (4-hydroxybutyloxy) methylene-2-bicyclo [3.3.1] nonane in the form of syrup was 0.93
g was obtained. EXACT MASS: 430.29 Yield: 36% Thin layer plate: HPTLC plate coated with silica gel 60F ₂₅₄ s, manufactured by Merck Co., Ltd. Detection: Drying after development, spotting with a UV lamp, and placing in an iodine tank to develop color. . Developing phase: toluene, ethyl acetate (volume ratio = 1: 50)

Example 1 4- (3-t-butyldimethylsilyloxyphenyl)
-4- (4-hydroxybutyloxy) spiro [1,2
-Dioxetane-3,9'-bicyclo [3.3.1] nonane]

Embedded image 200 mg of the tetrasubstituted alkene compound synthesized in Reference Example 3 was dissolved in 100 ml of methylene chloride in a photo-oxidation tube. Next, 5 mg of methylene blue was added as a photosensitizer, and an oxygen injector was connected. Oxygen was slowly passed through the solution and the photochemical reactor was immersed in a stainless steel vat containing dry ice / methanol and kept at about -76 ° C. 400W high pressure sodium vapor lamp while blowing oxygen continuously
Irradiation was carried out for about 40 minutes through industrial water and air instead of the UV cut solution. After the reaction, silica gel 15
g was added to adsorb methylene blue, and the mixture was filtered through a PTFE membrane filter (0.45 μm) and concentrated at room temperature. The obtained concentrate was purified by a column packed with 500 g of silica gel (eluent: 1% ethyl acetate-hexane), and a single spot fraction (Rf 0.36) was collected by TLC (thin layer chromatography).
Syringe-like 4- (3-t-butyldimethylsilyloxyphenyl) -4- (4-hydroxybutyloxy) spiro [1,2-dioxetane-3,9'-bicyclo [3.
3.1] Nonane] was obtained in an amount of 160 mg. EXACT MASS: 462.28 Yield: 74% Thin plate: HPTLC plate coated with silica gel 60F ₂₅₄ s, manufactured by Merck Ltd. Detection: After development, drying is performed, and spots are confirmed with a UV lamp and a fluorescent lamp. It is also placed in an iodine tank to develop color. Developing phase: toluene, ethyl acetate (volume ratio = 1: 50)

Example 2 4- (3-t-butyldimethylsilyloxyphenyl)
-4- (4-hydroxybutyloxy) spiro [1,2
-Dioxetane-3,9'-bicyclo [3. Nonane] chemiluminescence pattern The amount of luminescence of the title 1,2-dioxetane
Auto Lumat LB953 (Berthold Auto Lumat)
LB 953). The title 1,2-dioxetane is D
After dissolving in MSO, 100 μl thereof was placed in a luminometer and the dark count was monitored. When the dark count stabilized, 100 μl of a 1 M tetra-n-butylammonium fluoride in THF solution was added as a luminescence initiator, and the chemiluminescence reaction was monitored for 50 seconds. The calculated specific activity is shown in Table 1, the chemiluminescence pattern is shown in FIG. 1, and the sensitivity is shown in FIG.

[Table 1] 4- (3-t-butyldimethylsilyloxyphenyl) -4- (4-hydroxybutyloxy) spiro [1,2-dioxetane-3,9'-bicyclo [3. Nonan]Quantity Net count / 50 seconds Specific activity  5 × 10^-17moles 21634 4.3 × 10²⁰  10 × 10^-17moles 42317 4.2 × 10²⁰  15 × 10^-17moles 57342 3.8 × 10²⁰

Example 3 4- (3-t-butyldimethylsilyloxyphenyl)
-4- (4-hydroxybutyloxy) spiro [1,2
-Dioxetane-3,9'-bicyclo [3. Nonane] for Chemoluminescence Immunoassay of Human Thyrotropin (hTSH) The following literature was referred to in conducting this experiment using the title 1,2-dioxetane compound. References 1. Clin. Chem., 37 , 1540-1547 (1991A) Clin. Chem., 37 , 1612-1617 (1991B). U. S. Patent 5006309 4. U. S. Patent 5098424

Human thyrotropin (hTS) coated on carboxylated latex beads by covalent bonds
H) A mouse monoclonal antibody specific to the β subunit was used as a capture antibody. Human thyrotropin (hT
SH) specific affinity chromatographically purified sheep polyclonal antibody was labeled with the title 1,2-dioxetane compound and used as an indicator reagent. Sign operation 2
Implemented in a step process. Activation of the title 1,2-dioxetane compound is achieved by reacting a portion of the 4-hydroxybutyloxy group of the title 1,2-dioxetane compound with phosgene to form a chloroformate derivative. 0.5 mg of T
Dissolve in HF, 500μ of 10% phosgene in toluene
Mix with l. The reaction is performed at room temperature for 3 hours and then concentrated, and the concentrated residue is dissolved in 200 μl of THF. Reaction with affinity chromatographically purified sheep anti-TSH
0.1 M NaHCO titrated to pH 9.0 with OH water
Perform in ₃ waters. The antibody concentration was 1 mg / m in carbonate buffer.
Adjust to l. 2 ml of the antibody solution is placed in a vial, and 50 μl of dioxetane chloroformate in THF is divided into 4 equal portions, and mixed with a magnetic stirrer while adding 50 μl of each aliquot at 30 minute intervals. After the last addition, the sample is left at room temperature for a further 30 minutes. Purification of this chemiluminescent conjugate was performed by Sephadex G-25 column chromatography. React the reaction mixture in the vial with PBS
(20 m of pH 7.0 containing 0.15 M sodium chloride
M phosphate buffer) and Sephadex G equilibrated with
Charge -25 columns (Column Vol. 30ml). The chemiluminescent conjugate is eluted with PBS and 1 ml fractions are collected.
Fractions having an absorbance of 0.1 or more at 280 nm are pooled as chemiluminescent conjugates, and the concentration of the pool is adjusted to an absorbance of 1.5.
Calculated using an extinction coefficient of 10 mg / m
Store at low temperature (5 ° C. ± 3) in the presence of 1 fish fish gelatin. The chemiluminescent conjugate is used at about 1 μg / ml and
Dilute in PBS containing g / ml fish gelatin.

First, at the start (0 minutes), 30 μl of the capture antibody-coated latex beads are dispensed into the incubation well, and the inside of the tray is kept at 37 ° C. After 10 minutes add 100 μl of sample (or calibrator or control) to the wells. The resulting mixture is kept at 37 ° C. for 20 minutes.
Incubate for minutes. Thirty minutes after starting, transfer the reaction mixture to the reaction tray pad with 600 μl of 0.1% Tween 20 in PBS. 40 from start
After 30 minutes, 30 μl of the chemiluminescent conjugate is added to the reaction beads captured on the pad of the reaction tray. Incubate the chemiluminescent conjugate with the beads for 30 minutes. The beads and pad are then washed with two 50 μl aliquots with 100 μl of PBS containing 0.1% Tween 20 (70 min extinction time). After further incubation for 10 minutes, 1M
The reaction is read by adding 100 μl of tetra-n-butylammonium fluoride in THF. The reading of the chemiluminescence output is started immediately and the total photon count for 20 seconds is accumulated and stored in memory. For calibrators, create a standard curve. In the case of control or patient unknowns, the total count over a 20 second reading time is compared to the standard curve and the calculated unknown concentration. Berthold Auto Lumat LB953 is used for the measurement.

[0023]

The 1,2-dioxetane derivative having a bicyclononylidene group of the present invention can be used as a chemiluminescent reagent.
It can be used for chemiluminescence immunoassay (chemiluminescence immunoassay).

[Brief description of the drawings]

FIG. 1 shows DMSO-THF with tetra-n-butylammonium fluoride added as a luminescence initiator.
4- (3-t-butyldimethylsilyloxyphenyl) -4- (4-hydroxybutyloxy) spiro [1,2-dioxetane-3,9'-bicyclo [3.
3.1] Nonane] in a graph.

FIG. 2 shows DMSO-THF with tetra-n-butylammonium fluoride added as a luminescence initiator.
4- (3-t-butyldimethylsilyloxyphenyl) -4- (4-hydroxybutyloxy) spiro [1,2-dioxetane-3,9-bicyclo [3.3.
1] Nonane] is shown in a graph.

Claims (5)

[Claims] (1) Formula (1)A 1,2-dioxetane derivative represented by the formula:¹Is water
Element, halogen, C₁~ C ₃An alkyl group;²Is water
Acid group substitution C₂~ C₄Alkoxy and hydroxyl substituted C ₂~ C
₄An alkylthio group;³Is hydrogen, halogen, C₁
~ C₃Alkyl group, C₁~ C₃An alkoxy group;
⁴Is a 3-t-butyldimethylsilyloxy group, phosphate
Group, hydroxyl group. ). 2. The compound according to claim 1, wherein R ² is a 4-hydroxybutyloxy group in the formula (1). 3. In the formula (1), R ¹ is hydrogen, R ³ is hydrogen, a methoxy group, an ethoxy group, a methyl group, an ethyl group, and R ⁴ is a hydroxyl group or a 3-t-butyldimethylsilyloxy group. 2. The compound according to 1. 4. The compound according to claim ¹ , wherein R ¹ is hydrogen, R ² is a 4-hydroxybutyloxy group, R ³ is hydrogen, and R ⁴ is a 3-t-butyldimethylsilyloxy group in the formula (1). Compound. 5. A method for measuring by chemiluminescence, wherein the compound according to claim 1 is used in an antigen-antibody reaction.