JPH0429981A - Piperazinylquinazoline derivative and fluorescent labeling reagent containing the same - Google Patents

Abstract

NEW MATERIAL:A compound of formula I (R<1>, R<2> are H, methoxy, ethoxy, or together forms a methyleneoxy; (n) is 1-3; X is halogen). EXAMPLE:4-Amino-2-(4-chloroacetylpiperazin-1-yl)-6,7-dimethoxyquinazol ine. USE:A fluorescent labeling reagent capable of being applied to the quantitative analysis for fatty acids, amino acids, bile acid, catecholamine, phenols, alcohols, etc. The compound of formula I has excellent stability as the reagent and a labeling reaction ca quickly be carried out. PREPARATION:4-Amino-6,7-dimethoxy-2-(1-piperazinyl) quinazoline of formula II is made to react with fluoroacetyl chloride of formula: XCO(CH2)nX, etc., at room temperature.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel piperazinylquinazoline derivative useful as a fluorescent labeling reagent and the use of a fluorescent Rahelization reagent containing the same.

[Conventional technology]

Many fluorescent Rahel reagents have been developed and used for the purpose of quantifying trace amounts of fatty acids, sterols, amino acids, bile acids, catecholamines, phenols, alcohols, and the like.

For example, ADAM (9-anthryldiazomethane) and 4-bromomethyl-7-medoxycoumarin are used as fatty acids, and 4-diazomethyl-7-medoxycoumarin and 1-anthroylnitrile are known as alcohols. Therefore, it is necessary to select a reagent according to the purpose from among a large number of fluorescent Rahelization reagents depending on each test compound. However, each reagent generally has a short shelf life. It may not be suitable for the purpose because it has disadvantages such as poor performance, the need for preparation before use, the long time required for the labeling reaction, poor separation ability of each test compound, and the inability to detect at the desired concentration. The development of new Rahelization reagents with specific requirements is being considered.

[Problem to be solved by the invention]

An object of the present invention is to develop and provide a reagent that has good separation ability and stability, and can be used for many target compounds in the separation and quantification of test compounds.

[Means to solve the problem]

During research on the synthesis of piperazinylquinazoline derivatives, the present inventors discovered that by introducing a chloroacetyl group or a similar bromoacetyl group into the piperazine skeleton of this compound, ADAM (9- The present invention was completed based on the discovery that the present invention can lead to a more stable and strongly fluorescent derivative than anthryldiazomethane (anthryldiazomethane) reagent, 4-bromomethyl-7-medoxycoumarin, etc.

That is, the present invention relates to the general formula CI) (wherein R1 and R2 are each hydrogen, methoxy, ethoxy or taken together methylenedioxy; n is 1.
2 or 3, and X represents a halogen atom. ) or a fluorescent labeling reagent containing the same.

The compound of the present invention [■] is produced according to the following reaction formula.

(wherein R1 and R2 are each hydrogen, methoxy, ethoxy or taken together methylenedioxy; n is 1.
2 or 3, and X represents a halogen atom. ) That is, 4-amino-6,7-simethoxy 2(1-piperazinyl)quinazoline (II) and chloroacetyl chloride are reacted at room temperature, purified, and 4-amino-2-
(4-chloroacetylpiperazin-1-yl)-6,7
-Simethoxyquinazoline [Ia] is obtained.

More specific examples of the piperazinylquinazoline derivatives include the following.

4-amino-2-(4-bromoacetylpiperazine-1
-yl) Quinazoline 4-Amino-2-(4-chloropropionylpiperazin-1-yl)-6,7-methylenedioxyquinazoline 4-Amino-2-(4-bromoacetylpiperazine-1
-yl)-6,7-Simethoxyquinazoline 4-Amino-2-(4-chloropropionylpiperazin-1-yl)-6,7-Simethoxyquinazoline 4-Amino-2-(4-bromopropionylpiperazin-1 -yl)-6,7-simethoxyquinazoline 4-amino-2-(4-chlorobutyrylpiperazine-1
-yl L-6.7-Simethoxyquinazoline The fluorescent labeling reaction of the present invention is stable at room temperature, and its solution in dimedyl sulfoxide (DMSO) is also stable. Therefore, even drugs that are difficult to fluorescently label can be easily fluorescently labeled by reacting them at high temperatures.

The piperazinylquinazoline derivative of the present invention is a carboxylic acid,
Because it reacts with amino groups and phenolic hydroxyl groups and can be fluorescently labeled, it is widely used for many pharmaceuticals and their metabolites, fatty acids, sterols, amino acids, bile acids, catecholamines, phenols, etc., and alcohols. I can think of things that can be done.

In the present invention, for example, a drug is extracted from 50 uj2 of plasma or serum, a DMSO solution of a piperazinylquinazoline derivative is added to the obtained drug in the presence of a base or in a basic solvent, and the mixture is incubated at room temperature to 90°C. By reacting for 5 to 30 minutes, the target drug can be rapidly fluorescently labeled.

Fluorescently labeled compounds are subjected to thin layer chromatography,
It is separated from the reagent blank using high performance liquid chromatography and detected quantitatively.

The fluorescently labeled derivative according to the present invention has excellent fluorescence intensity, and therefore can be measured up to 50 pg.

The present invention will be explained below with reference to Examples.

(2) Example of Claim 1 [Example 1] 5 g of 4-amino-67-simethoxy-2-(1-piperazinyl)quinazoline (II) was added to Kolben, 3.6 g of triethylamine, and 153 g of tetrahydrofuran (THF).
ml, suspend, and stir with a stirrer under water cooling.

Meanwhile, chloracetyl chloride 2 in 15 ml of THF
．． Dissolve 7g in 'F4' and drop into colchen. After stirring for 30 minutes, the mixture is further stirred at room temperature for 4 hours. After stirring, the precipitate was collected by filtration, washed with water, and recrystallized with dimethylformamide to obtain 4-amino-2-(4-chloroacedelpiperazin-1-yl)-6,7-simethoxyquinazoline (Ia). (yield 70%, melting point (decomposition point) 270°C).

MASS:M" 365.m/z 316.m/z 2
BB, m/z 259m/z 233. m/z 205
．． m/z 123NMR: 'tl-NMR (DMSO-
d6) δ: 3.52 (411, bs, piperazine-
11), 3.79 (311,3, OCI+3).

3.83 (311,3, OCI+3), 3.70 (4
11, bs, piperazine-11).

4.43 (2t1.s, COCl1zCI), 6.76
(111, s, 8r-11).

7.19 (2t1.bs, Nl1z), 7.43 (
111,s, Ar-tl)IR: νmax(KBr
) cm-': 3480, 3350.3250 (Nllz
).

3000, 2900, 2840 (C11),
1650 (C-0) [Example 2] Similarly to Example 1, from (II) and chlorobutyryl chloride, 4-amino-2-(4-chlorobutyrylpiperazin-1-yl)-6,7- Cymethoxyquinazoline [Ib
) (yield 65%, melting point 191'c) is obtained.

2. Example of Claim 2 [Example 3] Acetone solution of mixture of free fatty acids 5 Q /71! After fractionating and distilling off the solvent, (I a ) 2. 0mg/m
l DMSO solution and potassium carbonate 0.2 mB/mu D
Add 0.1 ml of MSO solution to each and heat at 90°C115
The fluorescent labeling reaction was performed by heating for a minute. After cooling, add 0.8 ml of acetonitrile, take 10μ2 of it, and HP
Analysis was performed by LC (high performance liquid chromatography).

HPLC conditions were as follows: Waters Model 510 equipped with a YMC-back A-314 column (6φ x 300 mm, Koyamamura Kagaku Kenkyusho). The mobile phase was acetonitrile:water:acetic acid:PIC@B-8-75:25:
1. : 1, the column temperature is room temperature, the flow rate is 1,
7 m1/min, and fluorescence detection was performed using Fluorescence Spectromonitor RF-530 (@Shimadzu Corporation) at an excitation wavelength of 340 nm and a fluorescence wavelength of 384 nm. Table 1 shows each fatty acid and its retention time (Rt value).

(Left below) Table 1 Rt values of fluorescent derivatives of each fatty acid in HPLC [Example 4] Take 50μi of SD male and female rat serum, add 5ml of a mixture of chloroform-methanol-2=1, and determine the total lipid was extracted. After taking 2d of the extract and concentrating it to dryness, add 0.5ml of chloroform! Dissolved in a simple column (Ana
lytichem 1internationa1 B
Adsorb onto OND [LUT■] and elute with methanol to obtain eluate 4d. After concentrating this eluate, 2%
Add potassium hydroxide-ethanol solution 0.57 to 80
``Phospholipids were hydrolyzed into free fatty acids by heating with Cl2O for minutes.

Then, after adjusting the pH to 6 to 7 with 20% acetic acid, 5 mfl'E of chloroborum-n-hebutane (1:1) was added.
In addition, it was extracted by shaking. 4 ml of the organic layer was separated, and after distilling off the solvent, (I a ) 2.0+ng/mQ of DM
SOi solution and potassium carbonate 0, 2 mg/d DM
0.1 m each of SO solution! l added, 90'C115
The fluorescent labeling reaction was performed by heating for a minute. After cooling, add 0.8 ml of acetonitrile. Add 10tt1. was analyzed using HPLC (high performance liquid chromatography). HPLC conditions were the same as in Example 1.

The analysis results are shown in Table 2.

Table 2 Phospholipid fatty acid composition in rat serum [Example 5] Take 50 μm of SD male and female rat serum and add 5 ml of a chloroform-methanol-2:1 mixture. was added to extract total lipids. After 1 mρ of this total lipid was taken and concentrated to dryness, 0.5 mQ of 2% potassium hydroxide-ethanol solution was added and heated at 80'C for 20 minutes to hydrolyze the total lipid and convert it into free fatty acids.

Then, after adjusting the pH to 6 to 7 with 20% acetic acid, 5 mL of chloroform-n-heptane (1:1) was added and extracted by shaking. 4 ml of the organic layer was separated, the solvent was distilled off, and then CI a E 2. 0.1 ml each of a 0 mg/mfl DMSO solution and a 0.2 mg/ml potassium carbonate DMSO solution were added and heated at 90'C for 15 minutes to perform a fluorescent labeling reaction. After cooling, 0.8 d of acetonitrile was added, 10 ul of the solution was taken, and analyzed by HPLC (high performance liquid chromatography).

HPLC conditions are the same as in Example 1.

The analysis results are shown in Table 3.

Table 3 Total lipid fatty acid composition in rat serum [Example 6] 1.6-sihydro-6-oxo-2-(2-isobutoxyanilino)-5-pyrimidinecarboxylic acid (trial symbol M
AR-99) 1ug/me and 1,6-dihydro-6-oxo)-2-(2-pentoxyanilino)-
5-pyrimidinecarboxylic acid (internal standard Y#) 1μg/mp
0.1 mQ fraction of each methanol solution containing ,
Add to this 0.5 methanol containing 0.2% (V/V) acetic acid.
mff1 was added, 0.5 ml of diazomethane solution was added and mixed, and the mixture was allowed to react at room temperature for 30 minutes. After the solvent was distilled off, the concentration of potassium carbonate and 4-amino-2-(4-chloroacetylpiperazin-1-yl)-6,'l-dimethoxyquinazoline (Ia) was 2 mg/mQ. 0.2 ml of the previously prepared DMSO solution was added, and a fluorescent labeling reaction was carried out at 90°C for 30 minutes. After the reaction is complete, the solvent is distilled off, dissolved in methanol at 100μ℃, and the 30μ! was analyzed using summer 1 PLC.

The HPLC conditions were MMC-510 manufactured by Waters.
A Pack A-212 column (6φX15Qmm, ■Yamamura Kagaku Kenkyusho) was used. The mobile phase was acetonitrile:water:acetic acid:PIC@13-s=4 (1:60
;1:0.5, column temperature is room temperature, flow rate is 1
, 5 ml/min, and fluorescence detection was performed using Fluorescence Spectromonitor RF-530 (Shimadzu Corporation) at an excitation wavelength of 340 nm and a fluorescence wavelength of 384 nm. Table 4 shows the retention time (Rt) of each drug used.

Table 4 Retention time values of MAR-99 and internal standard Test compound Retention time value (min) AR-99 6,48 Internal standard 8.15 [Effects of the invention] The present invention provides novel piperazine useful as a fluorescent labeling reagent. A luquinazoline derivative and a fluorescent labeling reagent containing the same, which can be applied to the quantitative analysis of fatty acids, sterols, amino acids, bile acids, catecholamines, phenols, alcohols, etc., and has excellent stability as a reagent. It is useful as a new labeling reagent because the labeling reaction can be carried out in a short time.

Claims (2)

[Claims] (1) General formula [I] ▲Mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, R^1 and R^2 are hydrogen, methoxy, ethoxy, or together methylenedioxy, and n is 1, 2 or 3, and X represents a halogen atom.) A piperazinylquinazoline derivative represented by: (2) General formula [I] ▲Mathematical formulas, chemical formulas, tables, etc.▼[I] (In the formula, R^1 and R^2 are hydrogen, methoxy, ethoxy, or together methylenedioxy, and n is 1, 2 or 3, and X represents a halogen atom.) A fluorescent labeling reagent containing a piperazinylquinazoline derivative represented by the following.