JPH05331167A - New optically active derivative of 7-@(3754/24)3-amino-1-pyrrolidinyl)-2,1,3-benzoxadiazole - Google Patents

Abstract

PURPOSE:To provide a new compound useful as a fluorescent labeled reagent for the detection and determination of compound having carboxylic acid group. CONSTITUTION:The compound of formula I [R is SO2N(CH3)2, SO2NH2 or NO2; * mark represents asymmetric center), e.g. (+)-7-(3-amino-1- pyrrolidinyl)-4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole. The compound of formula I can be produced by reacting a 2,1,3-benzoxadiazole derivative of formula II with a 3-aminopyrrolidine derivative of formula III in a solvent (e.g. acetonitrile) at 0-100 deg.C. The compound selectively reacts with carboxylic acid and has high reactivity and, accordingly, all optical isomers can be separated at the same time and detected and determined in high sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to 7- (3-amino-1-pyrrolidinyl) -2,1, which can be used as a fluorescent labeling reagent when detecting and quantifying a compound containing a carboxylic acid group.
The present invention relates to a novel optically active derivative of 3-benzooxadiazole.

[0002]

BACKGROUND ART Conventionally, as a fluorescent labeling reagent for detecting and quantifying carboxylic acid, Br-Mmc [Voelter.
W. J. et al. Chromatogr. , 217, 491
(1981)], Br-Mac [Tsuchiya,
H. J., Chromatogr. , 309, 43 (1
984)], Br-DMEQ [Yamaguchi,
M. Et al. Anal. Sci. , 1,295 (198
5)], Br-MMEQ [Yamaguchi, M .; Chem. Pharm. Bull. , 36, 2263
(1988)], ADAM [Barker, S .; A. Et al. Anal. Biochem. , 107, 116 (19
80)], PDAM [Nimura, N .; Etc. Ana
l. Chem. , 60, 2067 (1988)] and the like are known.

On the other hand, as a fluorescent labeling reagent for separating and quantifying carboxylic acids which may exist in the form of optical isomers,
(-)-1- (4-Dimethylamino-1-naphthyl) ethylamine (DANE) has been reported. The specificity with this reagent is high, and the detection limit is good with subpicomoles, but when separating and quantifying using liquid chromatography, after removing unreacted fluorescent reagents and condensing agents by extraction in advance, Must be done in normal phase,
The measurement operation is complicated.

[0004]

DISCLOSURE OF THE INVENTION The inventors of the present invention have conducted synthesis and screening in order to obtain a compound having high reactivity selectively with carboxylic acid and capable of discriminating optical isomers.
(+)-7- (3-Amino-1-pyrrolidinyl)-or (-)-7- (3-Amino-1-pyrrolidinyl)-
A compound in which a dimethylaminosulfonyl group, an aminosulfonyl group or a nitro group is introduced at the 4-position of 2,1,3-benzoxadiazole is extremely excellent as a fluorescent reagent for carboxylic acid and is an unreacted fluorescent reagent. It was found that the optical isomers containing a carboxylic acid group can be separated and quantified with high sensitivity without being affected by or the condensing agent. The present invention has been made based on such findings.

That is, the present invention has the general formula [I]

[Chemical 6] [In the formula, R is SO ₂ N (CH ₃ ) ₂ , SO ₂ NH ₂ , or NO ₂ , and * means an asymmetric center. ] 7- (3-amino-1-pyrrolidinyl) -2 represented by
The present invention provides a novel optically active derivative of 1,3-benzooxadiazole, a method for producing the same, and a fluorescent labeling reagent comprising the compound.

The compound represented by the above formula [I] according to the present invention is a novel compound which has not been published in the literature, and its production method is, for example, 7-fluoro-
2,1,3-benzoxadiazole and 3S (-)-3
A compound of the present invention, (+)-7- (3-amino-1-pyrrolidinyl)-, by reacting with -aminopyrrolidine or 3R (+)-3-aminopyrrolidine.
Alternatively, a derivative of (−)-7- (3-amino-1-pyrrolidinyl) -2,1,3-benzoxadiazole can be obtained.

[0007]

[Reaction formula]

[Chemical 7] [Wherein, R and * have the same meaning as described above]

Examples of the solvent which can be used in the reaction represented by the above formula include inert organic solvents such as benzene, tetrahydrofuran, diethyl ether, acetone, acetonitrile, dioxane, chloroform, dichloromethane, ethyl acetate and the like. The above reaction can be carried out usually at a temperature of 0-100 ° C,
The preferred temperature is 5-30 ° C. The time required for this reaction varies depending on the reaction temperature, the compound used in the reaction, the solvent, etc., but is usually 5 minutes to 12 hours, preferably 15 minutes to 5 hours, and is appropriately selected within this range.

Isolation and purification of the desired product from the reaction mixture can be easily carried out by a conventional method. For example, extraction with an organic solvent such as benzene, dichloromethane, chloroform, ethyl acetate, or various chromatography using silica gel, activated carbon, ion exchange resin, dextran crosslinked polymer, styrene or acrylic ester porous polymer, etc. Isolation,
It can be purified.

Starting material 4-nitro-, 4-aminosulfonyl-, or 4-dimethylaminosulfonyl-7
-Fluoro-2,1,3-benzoxadiazole [I
I] is a known compound, for example, the method of Nunno et al. [J. Chem. Soc. (C) 1433, 197
0], Imai et al. [Anal. Biochem.
128,471 (1983)] and Toyo'oka.
Et al. [Anal. Chem. 56,2461 (19
84)]. The other reactant, 3S (−)-3-aminopyrrolidine or 3R (+)-3-aminopyrrolidine [III], is described in JP. Appl. 89-380
It can be obtained by the method described in JP-A-60.

The compound according to the present invention selectively reacts with an activated carboxylic acid in the presence of a condensing agent or the like to form a substance exhibiting remarkable fluorescence. Therefore, by utilizing this, after reacting the optical isomer carboxylic acid in the sample and the compound according to the present invention, the resulting fluorescent substance is separated by liquid chromatography, and the optical isomer in the sample is determined by measurement. The carboxylic acids can be detected separately, and the quantification can be performed using a calibration curve prepared using a standard substance.

As the condensing agent, dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), diethylphosphorocyanidate (DEPC), diphenylphosphonyl azide (DPPA), 2 , 2'-dipyridyl disulfide / triphenylphosphine, 2-chloro-1-methylpyridinium iodide / triethylamine and the like are used.

The preferred conditions for quantifying the optical isomer of carboxylic acid using the compound of the present invention as a fluorescent labeling reagent are as follows. 1) The reaction between the compound of the present invention and carboxylic acid is carried out in the presence of a condensing agent. 2) In this case, acetonitrile, dimethylformamide, dimethylsulfoxide, ethanol, methanol, preferably acetonitrile, dimethylformamide are used as the reaction solvent. 3) The reaction temperature is 15 ° C. or higher, preferably 2
It is 0-60 degreeC. 4) The reaction time is 5 minutes to 6 hours, preferably 30 minutes to 2 hours.

5) Separation of the fluorescent derivative obtained by combining with the optical isomer carboxylic acid is carried out by liquid chromatography, thin layer chromatography, filter paper chromatography or capillary electrophoresis, preferably liquid chromatography. Graph method,
Performed by capillary electrophoresis, the column used in that case may be any of reversed phase system such as octadecyl-, octyl-, cyanopropyl-, aminopropyl-, hydroxypropyl-, silica gel-column, normal phase system, preferably octadecyl column. , Octyl column is effective. Further, as the elution solvent, water, methanol, ethanol, acetonitrile, acetone, diethyl ether,
Chloroform, dichloromethane, ethyl acetate, tetrahydrofuran, benzene, hexane, etc., preferably water, acetonitrile, methanol or a mixed solution thereof is used.

6) The excitation wavelength of the detector for exciting the fluorescent derivative and measuring the generated fluorescence intensity is 440-50.
0 nm, preferably 460-480 nm, the fluorescence wavelength is 510-620 nm, preferably 530-600 n.
m. The features of the compound according to the present invention are listed below.

1) Since it selectively reacts with carboxylic acid and has high reactivity, all of the optical isomers can be separated at the same time and can be detected and quantified with high sensitivity, which is an excellent fluorescent labeling reagent. Is extremely useful as

2) By selectively using the (+) form or (-) form of this compound, the elution position of the optical isomer carboxylic acid [(+) form or (-) form] can be reversed, and a trace amount is mixed. It is possible to elute one of the optical isomers that are present in the chromatogram earlier than the other optical isomer that is present in a large amount. It is possible to accurately measure one of a very small amount of optical isomers.

3) It has high stability in a solution state and maintains stability at room temperature for one week or more. 4) The fluorescent derivative bound to the carboxylic acid gives a stable fluorescent intensity independent of pH. 5) The above fluorescent derivative has high stability and keeps the stability in the refrigerator for one week or more. 6) The above-mentioned fluorescent derivative has the maximum wavelength of excitation and fluorescence in the long wavelength region, so that the optical isomer carboxylic acid can be obtained not only by the usual fluorescence detection method but also by the chemiluminescence detection method or the laser fluorescence detection method. It is possible to highly sensitively separate and detect either the (+) form or the (-) form of.

The compound according to the present invention can be applied not only to the qualitative and quantitative analyzes of individual optical isomer carboxylic acids which are present in an ultratrace amount due to the characteristics as described above, but also to proteins containing a wide range of carboxylic acids. Various methods for quantifying peptides, studying the function of carboxylic acids in enzymes, separating and detecting optical isomer carboxylic acids in living organisms such as cells, membranes, tissues, or for studying the relationship between the structure and function of biological components It can be applied to the quantification of carboxylic acids in secretory fluids and other clinical samples, and further based on these, metabolism, clinical analysis, basic technology for their automation, biochemistry, physiology, and basic and clinical medical research. It can be applied in a very wide range, and so on.

Among the compounds of the present invention, (+)-7-
(3-Amino-1-pyrrolidinyl) -4- (N, N-dimethylaminosulfonyl) -2,1,3-benzoxadiazole (hereinafter abbreviated as [(+)-DBD-APy]), (+) -7- (3-amino-1-pyrrolidinyl)
-4- (aminosulfonyl) -2,1,3-benzoxadiazole (hereinafter abbreviated as [(+)-ABD-APy]) and (+)-7- (3-amino-1-pyrrolidinyl) -4 -Nitro-2,1,3-benzoxadiazole (hereinafter abbreviated as [(+)-NBD-APy]) is taken as a representative example, and an optical isomer carboxylic acid [(+) form or (-) form] is obtained. The following is an example of an experiment conducted on the reactivity and the resolution (Rs) of the produced fluorescent derivative by liquid chromatography.

Experimental Example 1 Reactivity of (+)-DBD-APy with (+)-Naproxen 1. Preparation of Fluorescent Labeling Reagent (+)-DBD-APy 3.1 mg
It was dissolved in acetonitrile so that 2. Preparation of test sample 2.3 mg of (+)-naproxen was dissolved in acetonitrile so that the total amount was 10 ml. Next, 0.2 ml of the solution was collected, and acetonitrile was added to this to make the total amount 100 ml.

3. Preparation of Condensation Agent Test Solution 2.2 mg of 2,2′-dipyridyl disulfide and 2.6 mg of triphenylphosphine were dissolved in acetonitrile so that the total amount was 1.0 ml.

4. 0.5 ml of the sample prepared in 2 above
Of the condensate reagent solution prepared in the above 3
ml and 0.2 ml of the fluorescent labeling reagent prepared in 1 above were added, and the whole was allowed to stand at room temperature for 15, 30, 45, 60, 90, 1
After standing for 20, 180, and 240 minutes, a fixed amount of each of the reaction solutions at each of these times was taken and separated by liquid chromatography under the conditions shown below, and fluorescence detection was performed. The result is shown in FIG.

Equipment: Shimadzu LC-9A liquid chromatograph Column: Inertosil ODS-2 (150 × 4.6 m)
m, i. d. , 5 μm) Eluent: water / acetonitrile (55/45) Injection volume: 10 μl Flow rate: 1.0 ml / min Fluorescence detector: Shimadzu RF-550 Detection wavelength: excitation wavelength 470 nm, fluorescence wavelength 580n
m

As is apparent from FIG. 1 below, it was found that the reaction reached the maximum after 1 to 1.5 hours, and thereafter showed a substantially constant fluorescence intensity (yield).

[Experimental Example 2] Examination of the degree of separation (Rs) of the fluorescent derivative produced by the reaction of the fluorescent labeling reagent with (+)-carboxylic acid or (-)-carboxylic acid. Preparation of Fluorescent Labeling Reagent (A) 15.6 mg of (+)-DBD-APy was dissolved in acetonitrile so that the total amount was 5 ml. (B) 15.6 mg of (-)-DBD-APy was dissolved in acetonitrile so that the total amount was 5 ml. (C) (+)-ABD-APy (14.2 mg) was dissolved in acetonitrile so that the total amount became 5 ml. (D) (+)-NBD-APy (12.5 mg) was dissolved in acetonitrile so that the total amount was 5 ml.

2. Preparation of test sample (A) 2.3 mg of (+)-naproxen or (−)-naproxen was dissolved in acetonitrile so that the total amount was 10 ml. Then 0.2m each
l was taken, acetonitrile was added, and the total amount was 100 ml.
And (B) (+)-ibuprofen or (-)-ibuprofen, 10.3 mg of each was dissolved in acetonitrile so that the total amount was 10 ml. Next, 0.2 ml of each was taken, acetonitrile was added, and the total amount was adjusted to 1
It was set to 00 ml. (C) (+)-loxoprofen or (-)-loxoprofen, 12.3 mg each, total amount 10 ml
It was dissolved in acetonitrile so that Next, 0.2 ml of each was collected and acetonitrile was added to make the total amount 100 ml.

3. Preparation of condensing agent reagent solution 22.0 mg of 2,2′-dipyridyl disulfide and 26.2 mg of triphenylphosphine in a total amount of 10 ml
It was dissolved in acetonitrile so that 4. 0.5 for each of the samples prepared in 2 above
aliquots of the condensing agent test solution prepared in the above 3
3 ml and 0.2 ml of the fluorescent labeling reagent prepared in 1 above
Was added, and the whole was reacted at room temperature for 1 hour, and then the reaction solutions were separated by liquid chromatography as in [Experimental Example 1], and fluorescence was detected. The elution time of the peak of the fluorescently labeled optical isomer carboxylic acid was measured, and the resolution (Rs) was calculated according to the following formula. The results are shown in Table 1.

Equipment: Shimadzu LC-9A liquid chromatograph Column: Inertosil ODS-2 (150 × 4.6 m)
m, i. d. , 5 μm) Eluent: water / acetonitrile (60/40) when (+)-NBD-APy and naproxen were reacted; water / acetonitrile (62) when (+)-ABD-APy and naproxen were reacted. / 38); (+)-DBD-APy or (-)-DBD-APy
And naproxen were reacted with water / acetonitrile (55/45); (ten) -DBD-APy or (-)-DBD-APy.
And ibuprofen were reacted with water / acetonitrile (45/55); (+)-DBD-APy or (-)-DBD-APy.
When reacting loxoprofen with water, acetonitrile / water (60/40)

Injection volume: 10 μl Flow rate: 1.0 ml / min Fluorescence detector: Shimadzu RF-550 Detection wavelength: Excitation wavelength 470 nm, Fluorescence wavelength 580 n
m [when (+)-ABD-APy, (+)-DBD-APy or (-)-DBD-APy was used]; excitation wavelength 470 nm, fluorescence wavelength 540 nm [(+)-NBD-APy was used. _{when] Rs = 2 (t R2 -t} R1) / W 1 + W 2

[0031] _{t R1, t R2:} each was fluorescent labeled (+) - carboxylic acid and (-) - W _{1, W 2} representing each elution time of carboxylic acids: each was fluorescent labeled (+ ) -Carboxylic acid and (-)-representing each peak width of carboxylic acid

[0032]

[Table 1]

The fluorescent derivative produced by the reaction of the compound according to the present invention and a carboxylic acid has a long excitation wavelength and a long fluorescence wavelength. Therefore, when handling a biological sample,
Excitation wavelength and fluorescence wavelength are relatively short wavelength range (400 nm
It is not easily affected by the biological components described below), and reproducible data can be obtained when measuring trace substances.
It can also be noted as one of the excellent characteristics that the complicated pretreatment operation of previously removing unreacted fluorescent reagents, condensing agents, etc. is unnecessary. Furthermore, it can be separated and detected in a reverse phase system, and as is clear from Table 1, Rs
The value is 1.2 or more, which shows that the compound of the present invention has extremely excellent ability to separate and identify optical isomer carboxylic acids. The detection limit is about 10 femtomoles, which is highly sensitive. Next, the synthetic method of the compound of the present invention will be explained by examples.

Example 1 (+)-7- (3-Amino-1-pyrrolidinyl) -4-
(N, N-dimethylaminosulfonyl) -2,1,3-
Benzoxadiazole (hereinafter [(+)-DBD-AP
y]) and (−)-7- (3-amino-1)
-Pyrrolidinyl) -4- (N, N-dimethylaminosulfonyl) -2,1,3-benzoxadiazole (hereinafter abbreviated as [(-)-DBD-APy])

3S (-)-3-aminopyrrolidine 0.2
Dissolve ml in 20 ml acetonitrile and add 5 ml to this.
4- (N, N-dimethylaminosulfonyl) -dissolved in 20 ml of acetonitrile at 0 ° C in advance
0.1 g of 7-fluoro-2,1,3-benzooxadiazole was added dropwise. After reacting for 30 minutes at room temperature, the solvent was distilled off, 100 ml of 10% hydrochloric acid was added to the residue, and the mixture was extracted with ethyl acetate. Then add 5% to this hydrochloric acid solution
It was made alkaline by adding sodium hydroxide and extracted with ethyl acetate. The ethyl acetate extract phase was washed with a small amount of water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure.
N-Hexane was added to the obtained residue to obtain (+)-DBD.
0.12 g of orange crystals of -APy were obtained (melting point 103-1
06 ° C). Mass spectrum molecular ion peak, 31
1.

[0036] In place of the above 3S (−)-3-aminopyrrolidine, 3
Using 0.2 ml of R (+)-3-aminopyrrolidine, the reaction and treatment were carried out in the same manner as described above to obtain 0.10 g of orange crystals of (-)-DBD-APy (melting point 96-98 ° C). Mass spectrum molecular ion peak, 311.

[0037]

Example 2 (+)-7- (3-Amino-1-pyrrolidinyl) -4-
(Aminosulfonyl) -2,1,3-benzooxadiazole (hereinafter abbreviated as [(+)-ABD-APy])
Synthesis of 3S (-)-3-aminopyrrolidine (0.2 ml) to 20 m
1-acetonitrile, which was previously dissolved in 20 ml of acetonitrile at 5 ° C. 4-
0.1 g of (aminosulfonyl) -7-fluoro-2,1,3-benzoxadiazole was added dropwise. 30 at room temperature
After reacting for 10 minutes, the solvent was distilled off, and 10% was added to the residue.
100 ml of hydrochloric acid was added, and the mixture was extracted with ethyl acetate. Then, 5% sodium hydroxide was added to the hydrochloric acid solution to make it alkaline, and the mixture was extracted with ethyl acetate. The obtained ethyl acetate extract phase was washed with a small amount of water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain 0.13 g of (+)-ABD-APy yellow crystals (melting point 199-201 ° C). .. Mass spectrum molecular ion peak, 283.

[0039]

Example 3 (+)-7- (3-amino-1-pyrrolidinyl) -4-
Synthesis of nitro-2,1,3-benzoxadiazole (hereinafter abbreviated as [(+)-NBD-APy]) 3S (-)-3-aminopyrrolidine (0.2 ml) was added to 20 m.
1-acetonitrile, which was previously dissolved in 20 ml of acetonitrile at 5 ° C. 4-
0.1 g of nitro-7-fluoro-2,1,3-benzoxadiazole was added dropwise. After reacting for 30 minutes at room temperature, the solvent was distilled off, and 100 ml of water was added to the residue,
It was extracted with 400 ml of ethyl acetate. The obtained ethyl acetate extract phase was washed with a small amount of water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain (+)-NBD-APy.
0.10 g of red crystals were obtained (melting point 177-180
C). Mass spectrum molecular ion peak, 249.

[0041]

[Brief description of drawings]

FIG. 1 is one of the compounds according to the present invention, (+)-DB.
It is a graph which shows the reaction time at the time of making D-APy and (+)-naproxen react, and the relationship of fluorescence intensity (yield).

Claims (3)

[Claims] 1. A compound represented by the general formula [I]: (In the formula, R is SO ₂ N (CH ₃ ) ₂ , SO ₂ NH ₂ or NO ₂ , and * means an asymmetric center) 7- (3-amino-1-pyrrolidinyl) -2 , 1, 3
-Benzoxadiazole optically active derivatives. 2. A compound represented by the general formula [II]: (Wherein R is SO ₂ N (CH ₃ ) ₂ or SO ₂ NH ₂ ), and a 2,1,3-benzoxadiazole derivative represented by the formula [III] (In the formula, * means an asymmetric center)
General formula [I] characterized by reacting with an aminopyrrolidine derivative (In the formula, R and * have the same meaning as described above) 7- (3-amino-1-pyrrolidinyl) -2,
Process for producing 1,3-benzoxadiazole optically active derivative. 3. A compound represented by the general formula [I]: (In the formula, R is SO ₂ N (CH ₃ ) ₂ , SO ₂ NH ₂ or NO ₂ , and * means an asymmetric center.) 7- (3-amino-1-pyrrolidinyl)- 2,1,
A fluorescent labeling reagent for carboxylic acid analysis, comprising a 3-benzoxadiazole optically active derivative.