JPS6058226B2 - Anthroylnitrile, its production method and fluorescent reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to anthroylnitrile, a process for its preparation, and a fluorescent reagent.

Liquid chromatography, especially high-performance liquid chromatography, has recently been frequently used to analyze trace components in living organisms.

However, there are no detectors that can detect compounds containing alcoholic hydroxyl groups with high sensitivity using high performance liquid chromatography (hereinafter referred to as HPLC), and therefore attempts have been made to convert them into strong ultraviolet absorbing substances or fluorescent substances. There is. However, as far as we know, no prelabeling agent with excellent reactivity, sensitivity, and stability has been found. This invention was made to solve this problem. That is, according to this invention, carbonyl nitrile (-
It has been found that a compound having a COCN) group is excellent in various respects as a fluorescent reagent for an alcoholic hydroxyl group with low reactivity.

Thus, according to the present invention, general formula (1) [wherein, X1
and any one of X2 is carbonyl nitrile (-CO
CN) group, and the others represent hydrogen atoms.

] is provided.

The specific compound name is 1 or 9-anthroylnitrile. All of these compounds are compounds described at the end of the literature. The compound of general formula (1) belongs to acyl nitrile, and generally the corresponding halide is cupric cyanide,
Method of reacting sodium cyanide [K. E. KOe
nig&W. P. WeberNTetrahedrOn
Lett. ,? 2275 (1974)].

However, the compound of general formula (1) can be obtained with high purity and high yield by reacting the corresponding halide with trialkylsilyl cyanide in an anhydrous organic solvent, adding a metal iodide as a catalyst if necessary. It was found that it was possible to obtain Thus, according to one aspect of the present invention, the corresponding general formula (■) [wherein either one of Y1 and Y2 is a carbonyl halide (-COX; in the formula, X represents a halogen atom) group Others indicate hydrogen atoms.

There is provided a method for producing a compound of general formula (1), which comprises reacting an anthroyl halide represented by the formula (1) with a trialkylsilyl cyanide in an anhydrous organic solvent. When this method is used, the product can be obtained with high purity as described above, and is therefore suitable for use as an analytical reagent. The compound of general formula (■) used in the above method can be obtained by, for example, introducing an alkoxycarbonyl group into the corresponding anthroyl bromide by a Grignard reaction, hydrolyzing it with an alkali metal alcoholate, and then using a halogenating agent such as oxalyl chloride. It can be obtained by processing with

As the trialkylsilyl cyanide, it is preferable to use trimethylsilyl cyanide or triethylsilyl cyanide.

Further, as a reaction solvent, a conventional organic solvent (for example, methylene chloride, chloroform, dimethylformamide, tetrahydrofuran, etc.) in a reaction using a silylating agent can be used. These organic solvents are of course used in an anhydrous state. It is usually desirable to carry out this reaction by adding a metal iodide (for example, zinc iodide) as a catalyst.

In addition, specific examples of the amount of each reactant to be used, reaction time, reaction temperature, etc. are explained using the following synthesis examples. However, these various conditions may be changed as appropriate. The compound of general formula (1) thus obtained can be used as a fluorescent reagent.

Thus, one aspect of the present invention is to provide a fluorescent reagent comprising a compound of general formula (1) as an active ingredient. Although this active ingredient can be used alone, it is preferably dissolved in an aprotic organic solvent to form a reagent.

In particular, when the compound of general formula (1) is used as a fluorescent reagent, the presence of an organic base brings about good results in the reaction with the test sample, so it is preferable to add an organic base to the reagent. Specific examples of aprotic organic solvents include chloroform, acetonitrile, benzene, ethyl acetate,
Examples include tetrahydrofuran and acetone.

Among these, acetonitrile is the best. Examples of organic bases include tertiary alkylamines such as triethylamine, trimethylamine, and quinuclidine. The ratio of the aprotic organic solvent to the organic base is not particularly limited, but is usually about 100:0.1.

Further, the concentration of the compound of general formula (1) in these mixtures is not particularly limited, but is, for example, 0.1 to 0.5% by weight.
It is. The fluorescent reagent according to the present invention reacts specifically with primary alcoholic hydroxyl groups, secondary alcoholic hydroxyl groups, and phenolic hydroxyl groups.

However, secondary hydroxyl groups with steric hindrance (for example, 11β-1 position, 17β-1 position of steroids, and axial hydroxyl groups) and
It hardly reacts with alcoholic hydroxyl groups. Particularly, when 1-anthroylnitrile is used among the compounds of this invention, it has the highest reactivity and a high esterification rate with a hydroxyl group can be obtained in a short time, and is therefore most preferred as a fluorescent reagent. Specific examples of these are illustrated in the Examples below. It is desirable to use the fluorescent reagent in an excess amount relative to the test sample to cause a sufficient reaction.

However, it has been found that even if an excessive amount of the fluorescent reagent is not removed after the reaction, the reagent itself does not exhibit fluorescence and has no effect on the fluorescent analysis of the test sample. For actual measurements,
The ester may be reacted with a test sample in advance, subjected to high performance liquid chromatography, and the fluorescence of the resulting ester detected using a fluorophotometer.

In addition, when a steroid is used as a test sample, the detection sensitivity is 10 pg (cortisol) in the normal phase system of high performance liquid chromatography and 50 pg (lithocholic acid) in the reverse phase system, which is far superior to conventional methods. Further, the fluorescence wavelength was 470nTrL (maximum excitation wavelength 370r17T1.) when 1-anthroylnitrile was used, and 460r17TL (maximum excitation wavelength 360r1rn) when 9-anthroylnitrile was used. Thus, the fluorescent reagent according to the present invention is particularly useful for detecting and quantifying various steroids that are trace components in living organisms. Synthesis Example 1 9-Anthroylnitrile nianthracene-9-carboxylic acid (1 g) was mixed and stirred in anhydrous methylene chloride (15 ml), oxalyl chloride (2 ml) was added thereto, and the solution was heated under reflux for 1 hour. It was concentrated under reduced pressure.

The residue was dissolved in anhydrous methylene chloride (15 ml) and catalytic amounts of zinc iodide and trimethylsilyl cyanide (2 ml) were added.
was added and stirred at room temperature for 2 hours. After concentrating the reaction solution under reduced pressure,
The residue was recrystallized from a mixture of n-hexane/methylene chloride to obtain the title compound (800Tn9) as orange needles. The physical properties of the obtained compound were as follows.

Melting point: 143-144 is CIR (chloroform) γMa
x: 2240 (C=N) and 1670 (C=0) Cm-
1MSm/z: 231 (M and 205 ([M-CN]
Synthesis Example 2 1-Anthroylnitrile anthracene-9-carboxylic acid
The same operation as in Synthesis Example 1 was carried out except that carboxylic acid was used, to obtain the title compound (850 mg) as orange needle-like crystals.

The physical properties of the obtained compound were as follows.

Melting point: 164-165 is C-IR (chloroform) γM
ax: 2240 (C3N) and 1675 (C=0)C7
7! -1MSm/z: 231 (M+) and 205 ([M
-CN]5) Elemental analysis: Actual value Calculated value (Cl6
H9NO) C83. O783. Example of IO analysis Each steroid (1 μy) as shown in Table 1 was used as a test sample.
), which were then treated with triethylamine (4). 1
wt%) in acetonitrile solution.

To this was added 100% each of 9-anthroylnitrile or 1-anthroylnitrile prepared according to the above synthesis example.
py was added and the reaction was carried out at 60° C., and the time until the esterification reaction was completed or the esterification rate after 1 hour was measured. In addition,
In the measurement, the resulting ester was separated by high-performance liquid chromatography, and 460r1rn or 470r1Tr1. This was done by measuring the fluorescence wavelength.

The high performance liquid chromatography device and measurement conditions used are as follows. Equipment: Waters 6000A (manufactured by Waters Association, USA) Detector: 650-1
0LC Fluorophotometer (1) (manufactured by Tate Seisakusho) column Niμ Porasil (1 foot cut 14 inches; mobile phase, hexane/ethyl acetate) and μ Bondervac Cl8 (1 foot x 1'
4 inches; mobile phase, 0.3% phosphate buffer/ethanol) The measurement results are shown in Table 1.

In the table, e represents equatorial, Qe represents quasi-equatorial, a represents axial, Prim represents primary, and Phen represents phenolic hydroxyl group.

Also, the * mark is the result when quinuclidine was used instead of triethylamine. As shown in Table 1, 9-anthroylnitrile (4) and 1-anthroylnitrile (B
), it was found that the reaction with the primary alcoholic hydroxyl group was completed in a short time.

Claims (1)

[Claims] 1 General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula,
Either one of ^1 and X^2 is carbonyl nitrile (
-COCN) group, and the others represent hydrogen atoms. ] Anthroylnitrile. 2. The anthroylnitrile according to claim 1, which is 1-anthroylnitrile or 9-anthroylnitrile. 3 General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula,
Either one of ^1 and X^2 is carbonyl nitrile (
-COCN) group, and the others represent hydrogen atoms. ] A fluorescent reagent containing anthroylnitrile as an active ingredient. 4. The fluorescent reagent according to claim 3, wherein the active ingredient is 1-anthrylnitrile or 9-anthrylnitrile. 5. The fluorescent reagent according to claim 3 or 4, wherein the active ingredient is dissolved in an aprotic organic solvent. 6. The fluorescent reagent according to any one of claims 3 to 5, wherein the active ingredient is dissolved in an aprotic organic solvent and an organic base. 7. The fluorescent reagent according to claim 6, wherein the aprotic organic solvent is acetonitrile and the organic base is triethylamine. 8 General formula (II): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) [In the formula, Y^
Either one of 1 and Y^2 is carbonyl halide (-
COX (in the formula, X represents a halogen atom) group, and the others represent a hydrogen atom. ] The corresponding general formula (I) obtained by reacting anthroyl halide with trialkylsilylyanide in an anhydrous organic solvent: ▲Mathematical formulas, chemical formulas, tables, etc.▼(I
) [In the formula, either one of X^1 and X^2 is a carbonyl nitrile (-COCN) group, and the others represent a hydrogen atom. ] A method for producing androl nitrile, which comprises obtaining androl nitrile represented by: 9. The production method according to claim 8, which is carried out by adding a metal iodide as a catalyst. 10. The manufacturing method according to claim 8 or 9, wherein the trialkylsilylyanide is trimethylsilylianide.