JP2000081392A - Chemiluminescence derivation reagent compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve detection due to the chemiluminescence derivation of carboxylic acid as a physiological active substance and further aldehyde or the like by using a chemiluminescence derivation reagent compound that is expressed by a specific expression. SOLUTION: Catecholamine metabolic product, prostaglandin, and its related substances (all carboxylic acid) out of physiological substances are allowed to react with a chemiluminescence derivation reagent compound that is expressed by an expression I or II with luminor skeleton, thus detecting the dynamic state of the substances in real time. In the expression, R indicates -NHNH2, -CH=N-NH2, or -CONHNH2, and A indicates hydrocarbon chain that may be through a hetero atom. The hydrocarbon chain may be partially or entirely in an annular form or may form a ring along with benzene ring to be connected. Also, m is either 0 or 1. The reagent compound can be manufactured by appropriately adopting various kinds of synthesis means.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent compound for inducing chemiluminescence. More specifically, the invention of this application relates to a new chemiluminescence-inducing reagent compound useful for detecting a carboxylic acid bioactive substance and the like.

[0002]

2. Description of the Related Art In recent years, a chemiluminescence detection method, which is expected to have higher sensitivity than a fluorescence method, has attracted attention. The chemiluminescence detection method is a method for detecting light emitted when a luminescent molecule (fluorescent molecule) is brought into an excited state by a chemical reaction and returns to a ground state therefrom. Therefore, the chemiluminescence detection method can perform measurement with higher sensitivity based on the reduction of the background. Then, the inventors of this application have developed a chemiluminescent derivatization reagent for 5-hydroxyindoles and catecholamines for the purpose of further increasing the sensitivity.

[0003] In the HPLC chemiluminescence detection method, there are a system which reacts itself and becomes a chemiluminescent species, and a luminescence system in which each is present separately. The former includes luminol derivatives, dioxetanes, acridinium esters, lophine and the like. In particular, chemiluminescent substances such as luminol and isoluminol are widely known, and are also called luminol-type chemiluminescence. In the latter, a chemiluminescence reaction using a peroxalate derivative is well known.

In general, the luminescence generated from a chemiluminescent substance largely depends on the fluorescence quantum yield (fluorescence intensity) of an excited molecule generated by the reaction. Therefore, it is necessary to develop a chemiluminescent substance having a stronger fluorescence than luminol or isoluminol as one measure for increasing the sensitivity. From such a viewpoint, the inventors have already derivatized reagents for α-keto acids and α-dicarbonyl compounds and aldehydes having quinoxalinone and quinoxaline as luminescent nuclei as compounds having better chemiluminescence properties than luminol. Has developed a reagent that specifically reacts with amines using a naphthalene skeleton as a light emitting nucleus, and has newly derivatized chemiluminescent derivatization with the aim of analyzing 5-hydroxyindoles and catecholamines with high sensitivity and high selectivity. The reagent 6-aminomethylphthalazine-1,4 (2H, 3H) -dione; (6-AMP) was developed.

This reagent is a chemiluminescent reagent having a cyclic phthalhydrazide structure (a luminol-type chemiluminescent reagent) having an aromatic methylamine structure and, like benzylamine, a potassium ferricyanide, as exemplified in the following formula. Below, it can selectively react with 5-hydroxyindoles and catecholamines in a weak alkaline solution to lead to a strong fluorescent substance. Further, this derivative reacts with hydrogen peroxide in the presence of potassium ferricyanide under strong alkalinity to emit chemiluminescence.

[0006]

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However, while 6-AMP is effective for hydroxyindoles for chemiluminescence analysis of catecholamines and the like, on the other hand, for analysis of physiologically active substances, particularly for kinetic analysis in a living body, catecholamines are used. It has been desired to develop another type of chemiluminescence-inducing reagent in which metabolites specifically react with carboxylic acids as related substances to prostaglandins.

However, the analysis of the kinetics of a physiologically active substance is extremely important for elucidating its physiological role. In the study of Alzheimer's disease and the like, the behavior of the physiologically active substance in the brain and the drug body Kinetic analysis has been actively performed using the microdialysis method, but sensitivity and selectivity are not sufficient and detection takes time, so it is not a satisfactory method for real-time kinetic analysis. Because there was no.

In view of the circumstances described above, the invention of this application focuses on catecholamine metabolites, prostaglandins and related substances (both of which are carboxylic acids) among physiologically active substances. It is an object of the present invention to provide a new chemiluminescence-inducing reagent compound which can detect the dynamics of these substances in real time by reacting with a chemiluminescent derivatization reagent having a skeleton.

[0010]

Means for Solving the Problems According to the invention of this application, the following general formula (1) or general formula (2) has been proposed to solve the above problems.

[0011]

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(Wherein R is -NHNH ₂ , -CH = N
Indicates -NH ₂ or -CONHNH _2, A represents an optionally substituted hydrocarbon chain optionally via a heteroatom, benzene The hydrocarbon chain may be a partially or entirely circular, to bind It may form a ring with the ring. M is 0 or 1. ) Is provided.

Further, the invention of this application relates to the above compound wherein m is 0, wherein m is 1 and A is-(CH ₂ ) _n -or the following formula:

[0014]

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Wherein n is a number from 1 to 4.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The invention of this application has the features described above. However, the reagent compounds of the present invention represented by the above formulas (1) and (2) are denoted by symbols R, A and m. For example, the following are specifically exemplified as corresponding to the above-mentioned rules.

[0017]

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From the above example, it is understood that the above-mentioned code is
_{Is, -NHNH 2, -CH = N-} NH 2 or -CO,
A represents NHNH ₂ , wherein A is a hydrocarbon chain which may be via a hetero atom, and this hydrocarbon chain may be partially or entirely cyclic, or may form a ring together with a benzene ring to which A is bonded. It can be seen that they may be formed and m is 0 or 1.

When m is 1, A is equal to-(C
H ₂ ) _n − or as described above.

[0020]

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[0021] Here, n is a number from 1 to 4. All of the above are useful as luminol-type chemiluminescence-inducing reagents, and the kinetics of carboxylate bioactive substances such as catecholamine metabolites and prostaglandins can be understood in real time, thus preventing and treating brain diseases. It can be expected to be widely used for research on pharmacology and pharmacological action.

The reagent compound of the present invention can be produced by appropriately employing various conventionally known synthetic means. Next, specific examples of the synthesis together with the emission characteristics will be described in Examples.

[0023]

EXAMPLES (Example 1) The following reaction formula

[0024]

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Thus, PMH of the exemplified compound was produced. That is, 4-formyl-N was added to 5 ml of ethanol.
-Phenylphthalimide 0.05 g and hydrazine 10
0 μl was added, and the mixture was heated and refluxed for 30 minutes after dissolution. The precipitated crystals (PMH) were collected by filtration.

[0026]

[Table 1]

(Example 2) The following reaction formula

[0028]

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In accordance with the above, 6-HMP of the above exemplified compound was produced. That is, first, compound I was synthesized according to the method of Isida et al.
Add 0.05 g and 100 μl of hydrazine.
Heated to reflux for 0 minutes. The precipitated crystals (6-HMP) were collected by filtration.

[0030]

[Table 2]

Example 3 According to the following reaction formula, Exemplified compound 6-HP was produced.

[0032]

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That is, first, 1.2 g of 4-aminophthalimide was added to 3.2% hydrochloric acid (prepared with ethanol).
dissolved in water (dissolved by sonication but not completely dissolved; the remaining large lump was crushed finely with a stir bar) and sodium nitrite dissolved in a small amount of water (1 ml).
55 g was added with slow stirring (at this time, the reagent was sufficiently cooled in a water bath and operated at 0-5 ° C.). The resulting orange solution is quickly poured into a solution of 5.5 g of sodium sulfite and 10 g of sodium carbonate in 80 ml of water (the solution is sufficiently cooled, then at room temperature). Ten minutes later, 15 ml of concentrated hydrochloric acid was added, and the mixture was refluxed on a water bath (100 ° C.) for 30 minutes. After the heat was sufficiently cooled, the mixture was allowed to stand at 0 ° C. overnight, and the resulting crystals (compound II) 1.1
7 g (yield: 57%) was collected by filtration.

MS (FAB) m / z = 278 (M-1)
^- , 256 (M-23, M-Na, Basepea)
k) ^- the resulting compound II1.45g and concentrated hydrochloric acid 10ml and refluxed for 3 minutes on a water bath (100 ° C.). After the solution was sufficiently cooled, it was neutralized with 10% aqueous ammonia. 0.71 g (yield: 77%) of yellow crystals (compound III) generated at this time was collected by filtration.

Mp: 267 ° C. MS (FAB) m / z = 178 (M + 1, Base p)
eak) ⁺ Then, 1 ml of hydrazine was added to 0.1 g of compound III, and the mixture was heated at 100 ° C for 2 hours. Thereafter, the mixture was dried to dryness with nitrogen, and 0.1 g of white crystals (6-HP) (yield: 91%).
Was isolated.

[0036]

[Table 3]

Example 4 An exemplary compound BETH was produced according to the following reaction formula.

[0038]

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First, IPO was synthesized according to the method of Isida et al. Next, 0.1 g of this IPO and 0.08 g of β-alanine methyl ester were dissolved in 5 ml of DMSO,
For 20 minutes. After cooling, 0.15 g of crystals (compound IV) (yield: 107%) was isolated by distillation under reduced pressure. Then 0.15 g of this compound IV and 100 μl of hydrazine
Was dissolved in 10 ml of ethanol and heated under reflux for 2 hours.
After cooling, the precipitated crystals (BETH) 0.1 g (yield: 67)
%) Was filtered off.

[0040]

[Table 4]

Example 5 According to the following reaction formula, Exemplified compound 5-HP was produced according to the method of Drew et al.

[0042]

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Example 6 The luminol-type compound emits chemiluminescence by reaction with hydrogen peroxide.
As exemplified in Examples 5 to 5, the reagent compounds of the present invention obtained as pure crystals were evaluated for their own chemiluminescence in comparison with the aforementioned 6-AMP as a known luminol-type chemiluminescence reagent. did.

The method for this was as follows. 100 μl of a 2 × 10 ⁻⁸ M (dimethyl sulfoxide) solution of each reagent was placed in a polyethylene tube, and 50 mM
100 μl of hydrogen peroxide and 100 μl of a 30 μM potassium ferricyanide (2 M sodium hydroxide) solution are added in sequence using an automatic injector. Immediately after injection, the resulting chemiluminescence is measured for 30 seconds with a Lumiphotometer. The light emission amount for 0 to 30 seconds was integrated.

[0045]

[Table 5]

It can be seen that all the compounds show a stronger luminescence than 6-AMP, and that BETH in particular has the largest luminescence. Chemiluminescence was also confirmed in sodium hydroxide solution and in the presence of potassium ferricyanide. Example 7 Evaluation of synthesized chemiluminescent reagents as derivatizing reagents for carboxylic acids was examined. In the evaluation, lauric acid was used as a representative of carboxylic acids.

The derivatization is performed by the R group of the reagent compound of the present invention, that is, the terminal amino group (-NH ₂ ) of -NH-NH ₂ , -CH = N-NH ₂ or -CO-NH-NH ₂
Based on the reaction of the amide bond with the carboxyl group of the carboxylic acid. The derivatization reaction is based on the following procedure.

That is, an aqueous solution of lauric acid (test solution)
To 100 μl, 50 μl of a 4.9 mM solution of each reagent in dimethylformamide, 50 μl of 2M 1-ethyl-3- (dimethylaminopropyl) carbodiimide (EDC) and 50 μl of 10% pyridine are added and reacted at room temperature for 15 minutes. 10 μl of this reaction solution is injected into a high performance liquid chromatograph (HPLC) -chemiluminescence detection system.

The HPLC-chemiluminescence detection system is HP
It consists of an LC separation method and a chemiluminescence detection method. The chemiluminescent derivative separated and eluted by the former is mixed with each solution of hydrogen peroxide and potassium ferricyanide by two mixing devices. The resulting chemiluminescence is based on detecting the latter. The resulting chromatograms for each reagent are shown in FIG. FIG.
As is clearer, a single peak based on lauric acid was observed for each of the reagents. This indicates that all the synthesized reagents selectively react with lauric acid to form a chemiluminescent derivative.

The peak areas on the chromatograms obtained from the reagents were compared. The results can be shown, for example, in Table 6.

[0051]

[Table 6]

Example 8 The calibration curve of myristic acid and palmitoleic acid of the conventional compound BETH was examined. As a result, it was found that the linearity passing through the origin was at least 33 pmol per injection volume (20 μl). In addition, when five standard mixed solutions of two kinds of fatty acids were measured at the same time, the number of peak height variations was within 3%.

The detection limits (S / N = 3) of myristic acid and palmitoleic acid were 59.4 fmol and 70.5 fmol, respectively, per injection volume (20 μl), indicating high sensitivity. In addition, application to other carboxylic acids was attempted, and the detection limit (S / N = 3) and the retention time were examined.

Table 7 shows the results.

[0055]

[Table 7]

Further, when the solution was applied to n-nonylaldehyde (20 μM), which is an aldehyde, a derivative peak with BETH was observed at 7.5 minutes. The detection limit (S / N = 3) is 12 per injection volume (20 μl).
2.0 fmol.

[0057]

As described in detail above, the invention of this application provides a new reagent compound which enables detection of carboxylic acids as physiologically active substances, and furthermore, aldehydes and the like by chemiluminescence induction. .

[Brief description of the drawings]

FIG. 1 is a diagram showing a chromatogram of a reagent-derived derivative of lauric acid as an example.

Claims (3)

[Claims] 1. The following general formula (1) or (2): (R in the formula, -NHNH _2, shows a -CH = N-NH ₂ or -CONHNH _2, A represents an even or hydrocarbon chain optionally via a heteroatom, the hydrocarbon chain may include one that A part or the whole thereof may be cyclic, or may form a ring together with a benzene ring to be bonded, and m is 0 or 1.) 2. The compound according to claim 1, wherein m is 0. 3. m is 1 and A is — (CH ₂ ) _n — or the following formula: The compound according to claim 1, wherein n is any number of 1 to 4.