JPH07188224A - New optically active (r)-(+)-and (s)-(-)-4-(2-chloroformylpyrrolidin-1-yl)-7-nitro-2,1,3-benzoxaziazole - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as a fluorescent labeling reagent quantitatively reacting with a hydroxyl group or an amino group. CONSTITUTION:Optically active (R)-(+)- and (S)-(-)-4-(2-chloroformylpyrrolidin-1- yl)-7-nitro-2,1,3-benzoxaziazole is represented by formula I (* is an asymmetric carbon atom). The compound of formula I can be synthesized by reacting oxalyl chloride, phosphorus pentoxide, phosphorus trichloride, etc., with optically active 4-(2-carboxypyrrolidin-1-yl)-7-nitro-2,1,3-benzoxaziazole of formula II. This fluorescent labeling reagent is useful for separation and quantitative analysis of an optical isomer of an alcohol or an amine, required in the fields belonging to medicine, pharmacy, agriculture, clinical chemistry and other fields. The compound of formula I quickly reacts and bonds to an amine in the presence of an acid scavenger and slowly reacts and bonds to an alcohol in the presence of an acid scavenger. This properties are utilized for quantitative analysis.

Description

Detailed Description of the Invention

[0001]

The present invention relates to (R)-(+)-and (S)-(-)-4- which can be used as a fluorescent labeling reagent which reacts quantitatively with a hydroxyl group or an amino group.
The present invention relates to (2-chloroformylpyrrolidin-1-yl) -7-nitro-2,1,3-benzoxadiazole, which is required in the fields of medical, pharmaceutical, agricultural, clinical chemistry and other fields. It is used for separation and quantification of the optical isomers of alcohols or amines.

[0002]

BACKGROUND OF THE INVENTION Many physiologically active substances such as pharmaceuticals have compounds having an asymmetric center, and compounds having these asymmetric centers have optical isomers. Especially optically active alcohol,
Amines are major constituents of bioactive substances. The physiological activities of these optical isomers may differ greatly or their behavior in vivo may differ. Therefore, separating these optical isomers and quantifying them with high sensitivity and reproducibility is an important analytical task. There are several known methods for separating and quantifying optical isomers of optically active alcohols or amines. Examples thereof include NMR method using a shift reagent, optical rotation dispersion method, chromatography using an optically active stationary phase, chromatography using an optically active mobile phase, and chromatography by a diastereomer method. Among these methods, the diastereomer chromatography is excellent in the ability to separate and detect optical isomers, and the following compounds have been reported as optically active derivatization reagents with high detection sensitivity. . (+)-Or (-)-α for separation and quantification by gas chromatography (GC)
-Trifluoromethyl-α-methoxyphenylacetyl chloride, a fluorescent labeling reagent (-)-1- for separation and quantification by liquid chromatography (hereinafter HPLC)
(1-Naphthyl) ethyl isocyanate [Sasak
i, K. J. et al. Chromatogr. , 585 , 11
7 (1991)], (+)-or (-)-2-methyl-1,1'-binaphthalene-2'-carbonyl cyanide [Goto, J. et al. Et al. Anal. Sci. , 6 , 261
(1990)] and the like.

[0003]

However, the GC method cannot be used as a versatile method because it cannot be used for the separation and quantification of thermally unstable compounds. Also, HP
The diastereomer method using the above-mentioned fluorescent labeling reagent by LC has high detection sensitivity, but the reaction conditions for producing diastereomer are relatively harsh. Therefore, it cannot be used for derivatization of thermally unstable compounds. In addition, since the generated derivative has a short excitation and fluorescence wavelength, it has a problem that it is easily affected by impurities.

The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, the compounds of the present invention were excellent for separating and quantifying optically active alcohols or optical isomers of amines by HPLC. The inventors have found that it is a fluorescent labeling reagent and completed the present invention.

[0005]

That is, the present invention is based on the following formula

[0006]

[Chemical 2] (However, * represents an asymmetric carbon atom.)

(R)-(+) and (S)-
It relates to (−)-4- (2-chloroformylpyrrolidin-1-yl) -7-nitro-2,1,3-benzoxadiazole. 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, an optically active compound according to the following reaction formula.
By reacting-(2-carboxypyrrolidin-1-yl) -7-nitro-2,1,3-benzoxadiazole with oxalyl chloride, phosphorus pentachloride, phosphorus trichloride, phosgene, triphosgene, thionyl chloride, etc. , Optically active 4- (2-chloroformylpyrrolidin-1-yl) -7-nitro-2,1,3-benzoxadiazole, which is a compound of the present invention, can be obtained.

[0008]

[Chemical 3] (* In the formula has the same meaning as above)

In the above reaction scheme, examples of the solvent that can be used in the reaction I include tetrahydrofuran, acetone, acetonitrile, dioxane, a mixed solution of dimethylformamide and an alkaline aqueous solution. Examples of the solvent that can be used in the reaction II include inert organic solvents such as hexane, benzene, tetrahydrofuran, diethyl ether, acetone, acetonitrile, dioxane, chloroform, dichloromethane, ethyl acetate, dimethylformamide and the like. The above reaction is usually
It can be carried out within the range of 50 to 100 ° C, but is preferably 0 to 50 ° C (reaction I) and -5 to 80 ° C (reaction II). The time required for the reaction varies depending on the reaction temperature, the chlorinating agent, the solvent, etc., but for Reaction I and Reaction II, it is usually selected within the range of 1 minute to 12 hours, preferably 10 minutes to 2 hours. Isolation of the desired product from the reaction mixture,
Purification can be easily performed according to a conventional method. For example, filtration, extraction with an organic solvent such as ether, benzene, dichloromethane, chloroform, ethyl acetate, recrystallization, or silica gel, activated carbon, ion exchange resin, dextran crosslinked polymer, styrene, porous polymer of acrylate ester It can be isolated and purified by performing various kinds of chromatography using, for example.

The compound of the present invention rapidly reacts with amines in the presence of a deoxidizing agent and the like and slowly reacts with alcohols. The reaction product exhibits a marked fluorescence. Therefore, utilizing this, after reacting the optically active amine or optically active alcohol in the sample with the compound according to the present invention, the resulting fluorescent compound is separated by HPLC,
By measuring, the optically active amines or alcohols in the sample can be detected separately, and the quantification can be performed using a calibration curve prepared with a standard substance. Among the compounds of the present invention, (S)-(-)-4-
(2-Chloroformylpyrrolidin-1-yl) -7-nitro-2,1,3-benzoxadiazole [hereinafter (S)-(-)-NBD-Pro-COCl] is taken as a representative example, and the optical activity is shown. Reactivity with alcohols and amines [(R)-and (S) -forms] and resolution of the produced fluorescent derivative by liquid chromatography (R
The example of the experiment performed about s) is shown below. Reference Example 1 (S)-(-)-NBD-Pro-COCl and (R)-
Investigation of reactivity with (-)-2-heptanol 1. Preparation of fluorescent labeling reagent (S)-(-)-NBD-Pro-COCl 3.0 mg
Was dissolved in benzene so that the total amount was 1.0 ml. 2. Preparation of test sample solution 11.6 mg of (R)-(-)-2-heptanol was dissolved in benzene so that the total amount was 10 ml. Then, 0.1 ml was taken from this, and 0.1 ml of a 20% pyridine solution of pyridine prepared separately was added to bring the total volume to 1.0 ml.
And 3. Preparation of reaction stop solution Total amount of 30% methylamine aqueous solution 3.33 ml is 100
It was dissolved in acetonitrile so that the amount became ml. 4. 0.5 ml of the sample solution prepared as in 2 above
0.5m of fluorescent labeled reagent solution that was collected and prepared as in 1 above
1 was added. 15, 30, 45, 60, 90 at 80 ℃,
After standing for 120, 180, and 240 minutes, a fixed amount (0.1 ml) of these reaction solutions was added with the reaction stop solution prepared as described in 3 above to make the total volume 1.0 ml, and then liquid chromatography under the following conditions: They were separated by the method and fluorescence was detected. The results are shown in Table 1. Equipment: Shimadzu LC-10A liquid chromatograph column: Inertocyl ODS-80A (150x
4.6 mm, i, d. , 5 μm) Column temperature: 40 ° C. Eluent: Water / acetonitrile (35/65) Injection volume: 5 μl Flow rate: 1.0 ml / min Fluorescence detector: Shimadzu RF-10A Detection wavelength: Excitation wavelength 470 nm, fluorescence wavelength 540 nm

[0011]

[Table 1]

The reaction was almost completed after 1 to 2 hours, and a slight decrease in fluorescence intensity (peak area) was observed thereafter.

Reference Example 2 (S)-(-)-NBD-Pro-COCl and (R)-
Reactivity with (+)-1- (1-naphthyl) ethylamine
Examination of 1. Preparation of fluorescent standard reagent (S)-(-)-NBD-Pro-COCl 3.0 mg
Was dissolved in benzene so that the total amount was 1.0 ml. 2. Preparation of test sample solution (R)-(+)-1- (1-naphthyl) ethylamine 1
7.1 mg was dissolved in benzene so that the total amount became 10 ml. Then, 0.1 ml was taken from this, and 0.1 ml of a 20% pyridine solution of pyridine prepared separately was added to make the total volume 1.0 ml. 3. Preparation of reaction stop solution Total amount of 30% methylamine aqueous solution 3.33 ml is 100
It was dissolved in acetonitrile so that the amount became ml. 4. 0.5 ml of the sample solution prepared as in 2 above
Fluorescent standard test solution 0.5m
1 was added. 15, 30, 45, 60, 90 at 50 ℃,
After standing for 120, 180, and 240 minutes, a fixed amount (0.1 ml) of these reaction solutions was added with the reaction stop solution prepared as described in 3 above to make the total volume 1.0 ml, and then liquid chromatography under the following conditions: They were separated by the method and fluorescence was detected. The results are shown in Table 2. Equipment: Shimadzu LC-10A liquid chromatograph column: Inertocyl ODS-80A (150x
4.6 mm, i. d. , 5 μm) Column temperature: 40 ° C. Eluent: Water / acetonitrile (55/45) Injection volume: 5 μl Flow rate: 1.0 ml / min Fluorescence detector: Shimadzu RF-10A Detection wavelength: Excitation wavelength 470 nm, fluorescence wavelength 540 nm

[0014]

[Table 2]

The reaction was almost completed after 15 minutes, and a slight decrease in fluorescence intensity (peak area) was observed thereafter.

Reference Example 3 (S)-(-)-NBD-Pro-COCl and (R)-
And reaction with (S) -alcohols or amines
Of the Degree of Separation (Rs) of the Fluorescent Derivative Produced by Reaction with Optically Isomeric Alcohols 1. Preparation of fluorescent labeling reagent (S)-(-)-NBD-Pro-COCl 3.0 mg
Was dissolved in benzene so that the total amount was 1.0 ml. 2. Preparation of test sample solution 11.6 mg of 2-heptanol was dissolved in benzene so that the total amount was 10 ml. Then from now on, 0.1m
1 was taken and 0.1 ml of a benzene solution of 20% pyridine prepared separately was added to make the total volume 1.0 ml. Similarly, test sample solutions were prepared for 2-hexanol, 2-nonanol, and 1-phenylethanol. 3. Preparation of reaction stop solution Total amount of 30% methylamine aqueous solution 3.33 ml is 100
It was dissolved in acetonitrile so that the amount became ml. 4. In 50 μl of the sample solution prepared as in 2 above,
Add 50 μl of fluorescent labeling reagent prepared as in 1 above,
After reacting at 80 ° C. for 1 hour, 0.9 ml of the reaction stop solution was added, and separation was performed by liquid chromatography as in Reference Example 1, and fluorescence was detected. Reaction with optical isomer amines 5. Preparation of fluorescent labeling reagent (S)-(-)-NBD-Pro-COCl 3.0 mg
Was dissolved in benzene so that the total amount was 1.0 ml. 6. Preparation of test sample solution 17.1 mg of 1- (1-naphthyl) ethylamine was dissolved in benzene so that the total amount was 10 ml. Then, 0.1 ml was taken from this, and 0.1 ml of a 20% pyridine solution of pyridine prepared separately was added to make the total volume 1.0 m.
It was set to l. Similarly, test sample solutions were prepared for 1-cyclohexylethylamine and 1-phenylethylamine. 7. Preparation of reaction stop solution Total amount of 30% methylamine aqueous solution 3.33 ml is 100
It was dissolved in acetonitrile so that the amount became ml. 8. In 50 μl of the sample solution prepared as in 6 above,
Add 50 μl of fluorescent labeling reagent prepared as in 5 above,
After reacting at 50 ° C. for 1 hour, 0.9 ml of a reaction stop solution was added, and separation was performed by liquid chromatography as in Reference Example 2, and fluorescence was detected. The elution times of the peaks of these fluorescently labeled optical isomer alcohols or amines were measured, and the separation coefficient (α) and the resolution (Rs) were calculated according to the following formulas. The results are shown in Tables 3 and 4. α = k ₁ ′ / k ₂ ′ Rs = 2 (t _R2 −t _R1 ) / (W ₁ + W ₂ ) k ₁ ′, k ₂ ′: Fluorescently labeled (R) − and (S)
-Retention ratio of alcohols or amines _tR1 , _tR2 : fluorescently labeled (R)-and (S)
Elution time of alcohols or amines W ₁ , W ₂ : fluorescently labeled (R)-and (S)
-Peak width of alcohols or amines Device: Shimadzu LC-10A liquid chromatographic column: Inertosyl SIL (150 x 4.6 m)
m, i. d. , 5 μm) Column temperature: 40 ° C. Eluent: ethyl acetate / n-hexane (20/80)
(When separating alcohol derivative) Ethyl acetate / n-hexane (45/55) (When separating amine derivative) Injection volume: 5 μl Flow rate: 1.0 ml / min Fluorescence detector: Shimadzu RF-10A Detection wavelength: Excitation Wavelength 470nm, fluorescence wavelength 540nm

[0017]

[Table 3]

[0018]

[Table 4]

[0019]

The compound of the present invention has a chloroformyl group for reacting with alcohols and amines under mild conditions, and the chloroformyl is directly bonded to the asymmetric carbon atom. Therefore, it reacts with optically active alcohols and amines to form diastereomers that separate very well on HPLC. Furthermore, it has strong fluorescence due to the 2,1,3-benzoxadiazole skeleton, and its excitation wavelength and fluorescence wavelength are both long wavelengths. Therefore,
It can be used as an excellent fluorescent labeling reagent for separating and quantifying optical isomers of optically active alcohols and amines.

[0020]

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 (S)-(-)-4- (2-chloroformylpyrrolidine)
-1-yl) -7-nitro-2,1,3-benzoxa
Diazole [hereinafter (S)-(-)-NBD-Pro-C
OCl] synthesis (S)-(−)-4- (2-carboxypyrrolidine-1
-Yl) -7-nitro-2,1,3-benzoxadiazole 2.6 g was dissolved in 200 ml dehydrated dichloromethane, oxalyl chloride 10 ml and dimethylformamide 0.2 ml were added to this solution, and the mixture was allowed to stand at room temperature for 1 hour. Stir to react. The solvent is distilled off under reduced pressure to obtain crystals. The obtained crystals were dissolved in 100 ml of dehydrated benzene, the insoluble material was filtered through a glass filter, and the filtrate was evaporated under reduced pressure. The residue was dried under reduced pressure in the presence of phosphorus pentoxide (S)-(-)-NB.
2.4 g of red-orange crystals of D-Pro-COCl were obtained.
The main physical properties of (S)-(-)-NBD-Pro-COCl are as follows. Melting point 103-104 ° C (decomposition); [α] _D ²⁰ = -204.2 ° (c = 0.43).
Chloroform); IR (KBr) 1794, 1615,
1555, 1495, 1447, 1325, 1154
1111, 999, 959, 708, 673 cm ^-1 ;
¹ H-NMR (deuterated chloroform) δ 2.14 to 2.39
(2H, m), 2.56 to 2.70 (2H, m), 3.
86 (2H, br), 5.67 (1H, br), 6.1
7 (1H, d), 8.44 (1H, d); Elemental analysis value (%) C44.48, H2.87, N18.30 (C
Theoretical value of ₁₁ H ₉ ClN ₄ O ₄ C44.53, H3.0
6, N18.89) (R)-(+)-4- (2-chloroformylpyrrolidin-1-yl) -7-nitro-2,1,3-benzoxadiazole is (R)- (+)-4- (2-Carboxypyrrolidin-1-yl) -7-nitro-2,1,
It could be similarly obtained from 3-benzooxadiazole.

[0022]

[Effect] As described above, by using the compound of the present invention, diastereomers can be produced under milder conditions as compared with the conventional method, and the detection limit was as high as subpicomolar. The resolution Rs between the optical isomers of the optically active alcohols and amines was 2.9 or more, which was good. Further, by selectively using (+)-and (-)-of the compound of the present invention, the elution position of the optical isomer [(+)-form or (-)-form] of the optically active alcohol or amine can be reversed. Therefore, it was possible to elute one of the optical isomers present in a very small amount on the chromatogram earlier than the other optical isomer present in a large amount. Therefore, it is possible to accurately measure one of the extremely small amounts of optical isomers, which could not be separated and quantified due to the interference of a large amount of optical isomers. In addition, the excitation wavelength and the fluorescence wavelength of the derivative of the compound of the present invention are both long wavelengths, and are not easily affected by the impurities in the actual sample. Furthermore, it is also possible to detect with a laser fluorescence detector, which enables higher sensitivity. Thus, the compound of the present invention can be applied not only to the qualitative and quantitative analysis of trace amounts of individual optically active alcohols or amines, but also to a wide range of alcohols, amine-containing proteins and peptides, and amines in enzymes. Of the functions of alcohols and alcohols, separation and detection of optically active alcohols and amines in the living body of cells, membranes, tissues, etc., or examination of the relationship between the structure and function of biological components, various secretion fluids, Metabolism based on the determination of alcohols and amines in other clinical samples, clinical analysis, etc., the basis of their automation, etc. It has a very wide range of applications in biochemistry, physiology, and basic and clinical medical research. is there.

Claims (1)

[Claims] 1. The following structural formula: (However, * represents an asymmetric carbon atom.) New optically active (R)-(+)-and (S)-(-)-4-
(2-chloroformylpyrrolidin-1-yl) -7-nitro-2,1,3-benzoxadiazole.