JPH09278766A - Novel fluorescent labeling agent, 4-(n-hydrazinoformylmethyl-n-methyl) amino-7-n,n-dimethylaminosulfonyl-2,1,3-benzoxadiazole - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel fluorescent labeling agent which is useful as a fluorescent labeling agent capable of quantitatively detecting carboxylic acids with high sensitivity required in the fields of medicine, pharmacy, agriculture and clinical chemistry. SOLUTION: This compound is 4-(n-hydrazinoformylmethyl-N-methyl) amino-7- N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole represented by the formula (Me is methyl). The objective compound is prepared by allowing hydrazine to react with 4-(N-chloroformylmethyl) amino-7-N,N-dimethylaminosulfonyl-2,1,3- benzoxadiazole in an organic solvent at -50-100 deg.C, particularly 0-50 deg.C.

Description

Detailed Description of the Invention

[0001]

The present invention relates to 4- (N-hydrazinoformylmethyl-N) which reacts quantitatively with carboxyl groups.
-Methyl) amino-7-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole, which is required in the fields of medicine, pharmacy, agriculture, clinical chemistry, and other fields. It is used for highly sensitive detection of existing carboxylic acids.

[0002]

BACKGROUND ART Physiologically active substances having a carboxyl group, such as fatty acids, bile acids, and prostaglandins, and pharmaceuticals having a carboxyl group, play an important role in living organisms, and it is necessary to accurately determine their abundance. Are required in the fields of medicine, pharmacy, agriculture, clinical chemistry and other fields. Most prostaglandins and pharmaceuticals have a strong physiological action even in a very small amount, and their abundance in the living body is extremely small. Therefore, a highly sensitive quantitative method is desired. In recent years, fluorescence detection-liquid chromatography (hereinafter, HPLC) having high sensitivity and selectivity has been widely used as a means for detecting and quantifying a trace component. Fluorescent labeling of a substance to be analyzed has been carried out in order to obtain higher sensitivity and selectivity, and some excellent fluorescent labeling reagents have been reported. For example, under mild conditions,
2- (4-hydrazinocarbonylphenyl) -4,5-diphenylimidazole (hereinafter, referred to as a fluorescent labeling reagent having a hydrazinoformyl group that reacts with a carboxyl group)
HCPI) [J. Chromatogr. , 619 , 1
(1993)], 6,7-dimethoxy-1-methyl-2
(1H) -Quinazolinone-3-propionylcarboxylic acid hydrazide (hereinafter, DMEQ-hydrazide) [Anal
yst, 115 , 1363 (1990)], 4- (5,
6-dimethoxy-2-benzimidazoyl) benzohydrazide (hereinafter, DMBI-hydrazide) [Anal. S
ci. , 8 , 889 (1992)]. All of these reagents can detect and quantify carboxylic acids with high sensitivity. However, the excitation and fluorescence wavelengths of the fluorescent label are λex335 nm and λe, respectively.
m455nm (HCPI), λex360nm, λem
435 nm (DMEQ-hydrazide), λex347n
m, λem 498 nm (DMBI-hydrazide), which is a relatively short wavelength. Therefore, when quantifying carboxylic acid, there is a problem that it is easily affected by impurities.

[0003]

The inventors of the present invention have conducted intensive studies to solve the above problems, and as a result, the compound of the present invention is an excellent fluorescent labeling reagent for separating and quantifying carboxylic acids by HPLC. This led to the completion of the present invention.

[0004]

That is, the present invention provides the following formula 1

[0005]

Embedded image

4- (N-hydrazinoformylmethyl-N-methyl) amino-7-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (hereinafter DBD-CO- Hz). The compound represented by the above formula 1 according to the present invention is a novel compound which has not been published in the literature, and its production method is, for example, 4- (N-chloroformylmethyl) amino-
7-N, N-dimethylaminosulfonyl-2,1,3-
Benzoxadiazole (hereinafter DBD-COCl)
It can be easily produced from [JP-A-7-238075] and hydrazine.

[0007]

Embedded image

In the above formula 2, examples of the solvent that can be used include organic solvents such as acetonitrile, tetrahydrofuran, dioxane, ethyl acetate, benzene and toluene, or a mixed solvent thereof. This reaction can be carried out usually in the range of -50 to 100 ° C, preferably 0 to 50 ° C. Although the time required for the reaction varies depending on the reaction temperature and the solvent, it is generally selected within the range of 1 minute to 12 hours, preferably 5 minutes to 2 hours. Isolation and purification of the desired product from the reaction mixture can be easily performed according to a conventional method such as extraction with an organic solvent, column purification, and recrystallization.

[0009]

The compound of the present invention is a hydrazinoformyl group for gently reacting with a carboxyl group in the presence of a condensing agent, and 2,1,3-benzoxadiazine having strong fluorescence of long wavelength for both excitation and fluorescence wavelengths. It has an azole skeleton. Also, the reaction product with carboxylic acids shows good separation by HPLC. As an example, the chromatogram of higher fatty acid is shown below to clarify the usefulness of the compound of the present invention, DBD-CO-Hz, as a fluorescent labeling reagent.

[0010]

[Table 1]

The above chromatogram shows 1 in 50 μl of a dimethylformamide solution of arachidonic acid (6), stearic acid (5), palmitic acid (4), myristic acid (3), lauric acid (2) and capric acid (1). -Ethyl-3- (3-dimethylaminopropyl) carbodiimide aqueous solution 50 μl, DBD-CO-Hz dimethylformamide solution 50 μl, 20% pyridine aqueous solution 50 μl
Are added sequentially and left at room temperature for 30 minutes, then 1 μl
It was obtained when injected into the LC. HP at this time
LC conditions are as follows. Pump: Hitachi L-6300 Intelligent Pump Column: Toso-ODS80TM (150 × 4.6 mm, i.d.5 μm) Eluent: A solution; Acetonitrile B solution: Distilled water Gradient elution (10 to 30 minutes); A50% → A100 % 0-10 minutes isocratic elution; A50% 30-45 minutes isocratic elution; A100% Flow rate: 1.0 ml / min Injection volume: 1 μl Fluorescence detector: Hitachi L-1080 fluorescence detector Detection wavelength: Excitation wavelength 440 nm, fluorescence wavelength 550 nm As shown in the above chromatogram, the peak of the fluorescent label of capric acid is sufficiently separated from the interfering peaks caused by DBD-CO-Hz and the reaction solvent, and the fatty acids with relatively few carbon atoms Can detect and quantify with good reproducibility. The compound DBD-CO-Hz of the present invention also reacts with a carbonyl group to form a strongly fluorescent label. Therefore,
It is also useful as a fluorescent labeling reagent for aldehydes and ketones as well as carboxylic acids.

[0012]

The following is a description of preferred embodiments of the present invention, which are for purposes of illustration and not limitation of the invention. It will be apparent to those skilled in the art that variations are possible within the scope of the invention.

Example 1 Synthesis of DBD-CO-Hz 70 mg of DBD-COCl was dissolved in 20 ml of acetonitrile. This solution is added to 50 ml of a methanol solution containing 50 mg of hydrazine hydrate. After stirring at room temperature for 30 minutes, the reaction solvent is distilled off under reduced pressure. The residue was dissolved in dichloromethane, purified by a silica gel column, and then recrystallized to obtain 60 mg of DBD-CO-Hz yellow crystals. The main physical property values of DBD-CO-Hz are as follows. Melting point 165 ° C; ¹ H NMR (deuterated chloroform) δ2.
85 (6H, s), 3.39 (3H, s), 3.94
(2H, s), 4.72 (2H, s), 6.22 (1
H, d), 7.87 (1H, d), 9.19 (1H,
s) ppm; elemental analysis value C40.26, H4.95, N
25.32% (C ₁₁ H ₁₆ N ₆ O ₄ S theoretical value C4
0.24, H4.91, N25.60%)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/533 G01N 33/533

Claims (1)

[Claims] (1) The following structural formula: A new fluorescent labeling agent 4- (N-hydrazinoformylmethyl-N-methyl) amino-7-N, N-dimethylaminosulfonyl-2,1,3-benzoxadiazole.