JPH0656819A - Trioxane derivative and method for chemiluminescence - Google Patents

Abstract

PURPOSE:To provide a new trioxane derivative, having a functional group bondable to a protein, nucleic acid, etc., and high luminous quantity and utilizable as a detecting agent for various diagnostic agents, various luminous probes, emergency light sources, etc. CONSTITUTION:The trioxane derivative of formula I (R<1> is halogen, 1-10C alkyl, 1-10C alkoxy, carboxyl, OH, sulfonic acid cyano, amino or nitro; R<2> is H, 1-3C alkyl or trialkylsilyl; R<3> and R<4> are each a 1-3C alkyl; (n) is 0-4], e.g. 5-(2- tbutyldimethylsilyloxycarbonylanilino)-3-isopropyl-6,6-dimethyl-1,2,4- trioxane. This compound of formula I is obtained by reacting an aniline derivative of formula II with an aldehyde of formula III and oxygen. The trioxane derivative of formula I is capable of producing the chemiluminescence by mixing thereof with a base. Furthermore, these properties are utilized to combine this trioxane derivative with the base to afford a kit product.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel trioxane derivative, its production method and chemiluminescence method. The derivative of the present invention is used as a detecting agent for various diagnostic agents, various luminescent probes, an emergency light source, and the like.

[0002]

2. Description of the Related Art Chemiluminescence refers to a phenomenon in which an atom or molecule is excited by energy generated by a chemical reaction to emit light. To date, compounds having various skeletons have been developed as chemiluminescent compounds, and acridinium ester, oxalate ester, etc. are known ("Bioluminescence and chemiluminescence" edited by Kazuhiro Imai, published by Hirokawa Shoten. ,
75-150, 1989). These compounds are used as a detecting agent for diagnostic agents or as a light source for emergencies.

Well known are, for example, reactions with luminol, lucigenin (bis (N-methylacridinium nitrate)), lophine (2,4,5-triphenylimidazole) or gallic acid, which are famous for bloodstain identification. Has been.

Under alkaline conditions, they emit light in the presence of hydrogen peroxide and a metal such as Fe. By detecting the light generated here with a photomultiplier tube, the atoms or molecules involved in the reaction can be quantified. Many substances such as vitamin B ₁₂ and glucose have been measured.

Further, as another conventional technique, there is known a light emitting system in which an active intermediate produced by a reaction between an oxalate ester and hydrogen peroxide excites a fluorescent substance to emit light. A method for quantifying hydrogen peroxide generated from glucose by the action of glucose oxidase by this conventional technique and measuring the original glucose amount has been established.

On the other hand, 5- (9 having a trioxane skeleton
It is known that -anthrylamino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane also has chemiluminescent ability. (JCS Chem. Comm., 180, 1
976)

However, such conventional trioxane compounds have a problem that they cannot be used as a detection agent or a probe because they do not have a functional group for modifying proteins, nucleic acids and the like. Therefore, under the present circumstances, there is a strong demand for the development of trioxane having a functional group capable of modifying proteins, nucleic acids and the like.

[0008]

The problem to be solved by the present invention is to provide a novel trioxane derivative having a functional group capable of binding to a protein, a nucleic acid or the like and having a high light emission amount.

Another object of the present invention is to provide a method for producing a novel trioxane derivative having a functional group capable of binding to proteins, nucleic acids and the like and having a high luminescence amount.

A further problem to be solved by the present invention is to provide a chemiluminescence method characterized in that the novel trioxane derivative of the present invention is reacted with a base to cause chemiluminescence.

Furthermore, the problem to be solved by the present invention is to provide a kit comprising a combination of the novel trioxane derivative of the present invention and a base, that is, a so-called "kit product".

Still another problem to be solved by the present invention is to establish a method for quantifying atoms or molecules involved in the reaction by utilizing the chemiluminescence reaction of the novel trioxane derivative of the present invention. To expand the use of derivatives.

[0013]

According to the present invention, the general formula (1)

[0014]

[Chemical 7]

(In the formula, R ¹ is a halogen atom, and has 1 to 1 carbon atoms.
10 represents an alkyl group or an alkoxy group, a carboxyl group, a hydroxyl group, a sulfonic acid group, a cyano group, an amino group, a nitro group, and R ² represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a trialkylsilyl group. , R ³ and R ⁴ are the same or different and each represents an alkyl group having 1 to 3 carbon atoms. n shows the integer of 0-4. ), A trioxane derivative (hereinafter referred to as trioxane 1) is provided.

According to the present invention, the general formula (2)

[0017]

[Chemical 8]

(In the formula, R ¹ is a halogen atom or a carbon number of 1 to
10 represents an alkyl group or an alkoxy group, a carboxyl group, a hydroxyl group, a sulfonic acid group, a cyano group, an amino group, a nitro group, and R ² represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a trialkylsilyl group. , R ³ and R ⁴ are the same or different and each represents an alkyl group having 1 to 3 carbon atoms. n shows the integer of 0-4. ) And an aniline derivative (hereinafter referred to as aniline 2) represented by the general formula (3)

[0019]

[Chemical 9]

(Wherein R ³ and R ⁴ are the same or different,
Each represents an alkyl group having 1 to 3 carbon atoms. ) Represented by the formula (hereinafter referred to as "aldehyde 3") and oxygen are reacted with each other, the general formula (1)

[0021]

[Chemical 10]

There is provided a process for producing a trioxane derivative represented by the formula: wherein R ¹ , R ² , R ³ , R ⁴ and n have the same meaning as defined above.

Further, according to the present invention, there is provided a chemiluminescent method characterized by reacting the trioxane derivative represented by the above general formula (1) with a base.

The present invention will be described in more detail below.

The trioxane derivative of the present invention is trioxane 1 represented by the above general formula (1). In the above trioxane 1, R ¹ is a halogen atom such as fluorine, chlorine, bromine or iodine, or an alkyl group having 1 to 10 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl. ,
Alternatively, it represents an alkoxy group such as methoxy, ethoxy or propoxy, a carboxyl group, a hydroxyl group, a sulfonic acid group, a cyano group, an amino group or a nitro group. R ² represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms such as methyl, ethyl and propyl, and a trialkylsilyloxy group such as trimethylsilyl, triethylsilyl, isopropyldimethylsilyl and t-butyldimethylsilyl. R ³ and R ⁴ are the same or different and each represents an alkyl group having 1 to 3 carbon atoms such as methyl, ethyl and propyl. n shows the integer of 0-4.

Specific examples of the trioxane 1 include, for example:

5- (2-t-butyldimethylsilyloxycarbonylanilino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane, 5- (3-t-butyldimethylsilyloxycarbonylani) Rhino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane, 5-
(4-t-Butyldimethylsiloxycarbonylanilino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane, 5- (2-carboxyanilino) -3-isopropyl-6,6- Dimethyl-1,2,4-trioxane,
5- (3-carboxyanilino) -3-isopropyl-
6,6-Dimethyl-1,2,4-trioxane, 5- (4-carboxyanilino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane, 5- (4-carboxy-3
-Chloroanilino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane, 5- (4-carboxy-3
-Methylanilino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane, 5- (4-carboxy-3
-Methoxyanilino) -3-isopropyl-6,6-dimethyl-1,2,4-trioxane, 5- (4-carboxy-
3-carboxyanilino) -3-isopropyl-6,6-
Dimethyl-1,2,4-trioxane, 5- (4-carboxy-3-hydroxyanilino) -3-isopropyl-6,
6-dimethyl-1,2,4-trioxane, 5- (4-carboxy-3-sulfoanilino) -3-isopropyl-6,
6-dimethyl-1,2,4-trioxane, 5- (4-carboxy-3-cyanoanilino) -3-isopropyl-6,
6-dimethyl-1,2,4-trioxane, 5- (4-carboxy-3-aminoanilino) -3-isopropyl-6,
6-dimethyl-1,2,4-trioxane, 5- (2-carboxy-5-nitroanilino) -3-isopropyl-6,
6-dimethyl-1,2,4-trioxane

Preferred examples include:

The method for producing the trioxane derivative of the present invention is to react the specific aniline 2 with the specific aldehyde 3 and oxygen to produce the trioxane 1 represented by the general formula (1).

The aniline derivative used in the present invention is aniline 2 represented by the above general formula (2). Specific examples of the aniline 2 include

2-aminobenzoic acid-t-butyldimethylsilyl, 3-aminobenzoic acid-t-butyldimethylsilyl, 4-aminobenzoic acid-t-butyldimethylsilyl,
4-amino-3-chlorobenzoic acid-t-butyldimethylsilyl, 4-amino-3-methylbenzoic acid-t-butyldimethylsilyl, 4-amino-3-methoxybenzoic acid-
t-Butyldimethylsilyl, 4-amino-3-t-butyldimethylsilyloxycarbonylbenzoate-t-butyldimethylsilyl, 4-amino-3-t-butyldimethylsilyloxybenzoate-t-butyldimethylsilyl, 4 −
Amino-3-sulfobenzoic acid-t-butyldimethylsilyl, 4-amino-3-cyanobenzoic acid-t-butyldimethylsilyl, 4-amino-3-aminobenzoic acid-t-butyldimethylsilyl, 2-amino- 4-nitrobenzoic acid-t-butyldimethylsilyl

Preferred examples include:

The aldehyde used in the present invention is aldehyde 3 represented by the above general formula (3). Specific examples of the aldehyde 3 include:

2-methylpropanal, 2-methylbutanal, 2-methylpentanal, 2-ethylpropanal, 2-ethylbutanal, 2-ethylpentanal, 2-propylpropanal, 2-propylbutanal , 2-propylpentanal

Preferred examples include:

In the present invention, when the aniline 2 represented by the general formula (2) is reacted with the aldehyde 3 represented by the general formula (3), the charged molar ratio of the aniline 2 and the aldehyde 3 is 1 : 2-1: 100
It is preferably 0, and particularly preferably 1: 3 to 1: 100. If the molar ratio is less than 2, the yield of the trioxane derivative formed tends to decrease, and if it exceeds 1000, unreacted aldehyde remains after the reaction is completed, making it difficult to isolate the target product. It is not preferable.
The reaction can be carried out at normal pressure, and the reaction temperature is generally -20 to 150 ° C, preferably -10 to 1
It is preferably in the range of 00 ° C. The reaction temperature is −
If the temperature is lower than 20 ° C, the reaction time tends to be long, and conversely 1
If the temperature exceeds 50 ° C, the reaction operation becomes difficult, which is not preferable.

In the production method of the present invention, the desired trioxane 1 can be obtained by reacting the aniline 2 with the aldehyde 3 and oxygen under the various reaction conditions, but the reaction is smoother. It is desirable to use a solvent to do this. As the solvent, various solvents can be used as long as they are inert under the reaction conditions and can be easily separated from the produced trioxane derivative. Particularly, pentane, hexane, heptane, diethyl ether, dimethoxyethane, tetrahydrofuran, benzene, toluene, xylene, carbon tetrachloride or a mixed solvent thereof is suitable. The amount of the solvent used is not particularly limited, but is preferably 0.5 times to 100 times based on the total weight of the starting materials.

In the production method of the present invention, the desired trioxane 1 can be obtained by reacting the aniline 2, the aldehyde 3 and oxygen, but the oxygen concentration is preferably 5 to 100%. . This reaction is carried out for a time sufficient to produce trioxane 1. The reaction time is determined depending on the conditions such as reaction temperature, reaction pressure, and the amount ratio of starting materials, and can be usually in the range of 1 hour to 14 days, but it is practically 10 hours to 7 days. It is desirable to set the conditions as follows.

The reaction product obtained by the production method of the present invention can be purified by a known method.

In the chemiluminescence method of the present invention, chemiluminescence can be obtained by mixing trioxane 1 and a base. Preferable examples of the base include sodium methoxide, potassium t-butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium hydrogen carbonate and tetrabutylammonium hydroxide.

In the chemiluminescence method of the present invention, trioxane 1 is used.
Although chemiluminescence can be obtained by mixing a base with a base, chemiluminescence can also be obtained by further mixing with another organic solvent. The organic solvent is preferably a polar solvent such as dimethyl sulfoxide, dimethylformamide, HMPA, dioxane, acetonitrile, methanol, ethanol, t-butanol or nitromethane.

[0042]

INDUSTRIAL APPLICABILITY The trioxane 1 of the present invention is a chemiluminescent substance having a substituent capable of binding to proteins, nucleic acids and the like and having a high luminescence amount, as a detecting agent for various diagnostic agents, various luminescent probes, an emergency light source, etc. Can be used as In addition, the production method of the present invention makes it possible to easily produce trioxane 1 in a short time with a high yield. Furthermore, the chemiluminescence method of the present invention can be applied to the above various uses. Furthermore, according to the kit product of the present invention, the trioxane 1 of the present invention and a base are in situ (in sit
u) By mixing, chemiluminescence can be achieved.

[0043]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Example 1 0.11 g of t-butyldimethylsilyl 4-aminobenzoate
(0.4 mmol) was dissolved in 10 ml of hexane, and 0.95 g (13.1 mmol) of isobutyraldehyde was added. The reaction was carried out in the presence of air for 3 days. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel chromatography. As a result, 5- (4-t-butyldimethylsilyloxycarbonylanilino) -3-isopropyl-6,6-
Dimethyl-1,2,4-trioxane [Structural formula A]

[0045]

[Chemical 11]

0.03 g (0.07 mmol) of was obtained.
The spectrum data is shown below.

¹ H NMR (CDCl ₃ ); δ = 0.35
(S, 6H), 0.9-1.1 (m, 15H), 1.32 (s, 3
H), 1.53 (s.3H) 1.80 (m, 1H), 4.12 (d,
1H, J = 10.4Hz), 4.94 (d, 1H, J = 10.4H)
z), 5.14 (d, 1H, J = 5.6Hz), 6.74 (d, 2)
H, J = 9.6 Hz), 7.90 (d, 2H, J = 9.6 Hz) IR (KBr cm ⁻¹ ); 3400,1750 MS; 410 (M ⁺⁺ 1), 376

Example 2 5- (4-t-butyldimethylsilyloxycarbonylanilino) -3-isopropyl-6,6 obtained in Example 1
A 10 μmol / ml potassium t-butoxide-dimethylsulfoxide solution was added to a dimethylsulfoxide solution of -dimethyl-1,2,4-trioxane to cause chemiluminescence.
The chemiluminescence at this time is measured by a chemiluminescence detector CLD-100 (manufactured by Tohoku Electronics Industry Co., Ltd.) and an instant multi-photometric system I
It was detected with MUC-7000 (manufactured by Otsuka Electronics Co., Ltd.).
The amount of emitted light and the maximum emission wavelength are shown in Table 1. The count number is a relative value of "no unit".

[0049]

[Table 1]

Comparative Example 1 5- (9-anthrylamino) -3-isopropyl-
6,6-Dimethyl-1,2,4-trioxane [Structural Formula B]

[0051]

[Chemical 12]

10 μmo in the dimethylsulfoxide solution of
1 / ml of potassium t-butoxide-dimethylsulfoxide solution was added to cause chemiluminescence. The amount of emitted light and the maximum wavelength of emitted light are also shown in Table 1.

Claims (4)

[Claims] 1. A compound represented by the general formula (1): (In the formula, R ¹ is a halogen atom, an alkyl group or an alkoxy group having 1 to 10 carbon atoms, a carboxyl group, a hydroxyl group,
Indicates a sulfonic acid group, a cyano group, an amino group, a nitro group,
R ² represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a trialkylsilyl group, and R ³ and R ⁴ are the same or different and each represent an alkyl group having 1 to 3 carbon atoms. n is 0
Indicates an integer of ˜4. ) A trioxane derivative represented by: 2. A general formula (2): (In the formula, R ¹ is a halogen atom, an alkyl group or an alkoxy group having 1 to 10 carbon atoms, a carboxyl group, a hydroxyl group,
Indicates a sulfonic acid group, a cyano group, an amino group, a nitro group,
R ² represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a trialkylsilyl group. n shows the integer of 0-4. ) And an aniline derivative represented by the general formula (3): (R ³ and R ⁴ are the same or different and each has 1 carbon
~ 3 alkyl groups are shown. ) And an oxygen are reacted with the general formula (1): (Wherein R ¹ , R ² , R ³ , R ⁴ and n have the same meanings as defined above). 3. A compound represented by the general formula (1): (In the formula, R ¹ is a halogen atom, an alkyl group or an alkoxy group having 1 to 10 carbon atoms, a carboxyl group, a hydroxyl group,
Indicates a sulfonic acid group, a cyano group, an amino group, a nitro group,
R ² represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a trialkylsilyl group, and R ³ and R ⁴ are the same or different and each represent an alkyl group having 1 to 3 carbon atoms. n is 0
Indicates an integer of ˜4. (3) A chemiluminescence method, characterized in that chemiluminescence is obtained by mixing a trioxane derivative represented by (4) with a base. 4. A compound represented by the general formula (1): (In the formula, R ¹ is a halogen atom, an alkyl group or an alkoxy group having 1 to 10 carbon atoms, a carboxyl group, a hydroxyl group,
Indicates a sulfonic acid group, a cyano group, an amino group, a nitro group,
R ² represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or a trialkylsilyl group, and R ³ and R ⁴ are the same or different and each represent an alkyl group having 1 to 3 carbon atoms. n is 0
Indicates an integer of ˜4. ) A kit comprising a combination of a trioxane derivative represented by (4) and a base.