JPH06184141A - New optically active@(3754/24)+)-and (-)-4-(n,n-dimethylaminosulfonyl)-7-(2-chloroformylpyrrolidin-1-yl)-2,1,3-benzoxadiazole - Google Patents

Abstract

PURPOSE:To provide the subject optically active new compound useful as a fluorescent labeled reagent to be used for the separation and determination of optical isomer of alcohols or amines required in the field of medicine, pharmacology, agriculture, clinical chemistry, etc. CONSTITUTION:The optically active (+)- and (-)-4-(N,N- dimethylaminosulfonyl)-7-(2-chloroformylpyrrolidin-1-yl)-2,1,3-benzoxa diazole of formula I (* represents asymmetric carbon atom). The compound of formula I can be produced e.g. by reacting 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3- benzoxadiazole of formula II with an optically active 2-carboxypyrrolidine and reacting the obtained optically active 4-(N,N-dimethylaminosulfonyl)-7-(2- carboxypyrrolidin-1-yl)-2,1,3-benzoxadiazole of formula III with phosphorus pentachloride, etc.

Description

Detailed Description of the Invention

[0001]

The present invention relates to (+)-and (-)-4- (N, N-dimethylaminosulfonyl) -which can be used as a fluorescent labeling reagent which reacts quantitatively with a hydroxyl group or an amino group. It relates to 7- (2-chloroformylpyrrolidin-1-yl) -2,1,3-benzoxadiazole and is required in the fields of medicine, pharmacy, agriculture, clinical chemistry and other fields. It is used to separate and quantify 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 diastereomeric 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 limits. . (+)-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 a high detection limit, but the reaction conditions for producing the 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.)

(+)-And (-)-4-represented by
(N, N-Dimethylaminosulfonyl) -7- (2-
(Chloroformylpyrrolidin-1-yl) -2,1,3-
It relates to benzooxadiazole. 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, optically active 4- (N, N-dimethylaminosulfonyl) according to the following reaction formula -7- (2-Carboxypyrrolidin-1-yl) -2,1,3-benzoxadiazole is reacted with phosphorus pentachloride, phosphorus trichloride, phosgene, triphosgene, thionyl chloride or the like to give the compound of the present invention. Is optically active 4- (N, N-dimethylaminosulfonyl)-
7- (2-chloroformylpyrrolidin-1-yl)-
2,1,3-benzoxadiazole 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 and dimethylformamide. 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 organic solvent such as ether, benzene, dichloromethane, chloroform, ethyl acetate, recrystallization, or silica gel, activated carbon, ion exchange resin, dextran cross-linked polymer, styrene or acrylic ester porous polymer, etc. It can be isolated and purified by performing various kinds of chromatography used. 4- (N, N-dimethylaminosulfonyl) starting material
-7-Fluoro-2,1,3-benzoxadiazole, optically active 4- (N, N-dimethylaminosulfonyl) -7- (2-carboxypyrrolidin-1-yl)-
2,1,3-Benzoxadiazole is a known compound, for example, the method of Toyooka et al. [Analyst, 114 ,
413 (1989), Analyst, 114 , 123.
3 (1989)].

The compound of the present invention rapidly reacts with amines. Further, it selectively reacts with alcohols by a deoxidizing agent or the like to form a compound exhibiting remarkable fluorescence. Therefore, by utilizing this, after reacting the optically active amines or optically active alcohols in the sample with the compound according to the present invention, the resulting fluorescent compound is separated by HPLC and measured to obtain the sample in the sample. It is possible to detect the optically active amines or alcohols separately, and to quantify them using a calibration curve prepared with a standard substance. Among the compounds of the present invention, (−)-4
-(N, N-Dimethylaminosulfonyl) -7- (2
-Chloroformylpyrrolidin-1-yl) -2,1,3
-Benzoxadiazole [hereinafter (-)-DBD-Pr
o-COCl] is taken as a representative example, and the reactivity with optically active alcohols and amines [(+)-and (-)-forms] and the resolution (Rs) of the produced fluorescent derivative by liquid chromatography An example of the experiment conducted is shown below. Reference Example 1 (-)-DBD-Pro-COCl and 1-heptanol
Study of reactivity with 1. Preparation of fluorescent labeling reagent (-)-DBD-Pro-COCl (3.6 mg) was dissolved in tetrahydrofuran so that the total amount was 1.0 ml. 2. Preparation of test sample 11.6 mg of 1-heptanol was dissolved in tetrahydrofuran so that the total amount was 10 ml. Next, 0.1 ml was taken from this, 0.1 ml of a 20% pyridine solution in tetrahydrofuran prepared separately was added, and then tetrahydrofuran was added to bring the total volume to 1.0 ml. 3. 0.5 ml of the sample prepared as in 2 above was added with 0.5 ml of the fluorescent labeling reagent solution of 1 at 15, 30, 45, 60, 90, 120, 18 at room temperature.
After standing for 0,240 minutes, an aliquot of these reaction solutions was separated by HPLC under the conditions shown below, and fluorescence was detected. The results are shown in Table 1. Device: Shimadzu LC-9A liquid chromatograph column: Inertosil ODS-2 (150 × 4.6)
mm, i. d. , 5 μm) Eluent: Water / acetonitrile (30/70) Injection volume: 10 μl Flow rate: 1.0 ml / min Fluorescence detector: Shimadzu RF-550 Detection wavelength: Excitation wavelength 450 nm, fluorescence wavelength 560 nm

[0011]

[Table 1]

The reaction was almost completed after 1 to 2 hours, and a slight increase in fluorescence intensity (peak area) was observed thereafter. Reference Example 2 (-)-DBD-Pro-COCl and (+)-and
(-)-Produced by reaction with alcohols and amines
Examination of the separation degree (Rs) of the formed fluorescent derivative Reaction with optically active alcohols (-)-DBD-Pro-COCl 3.6 mg was dissolved in acetonitrile so that the total amount was 10 ml. 0.1 ml of this liquid contained (+)-and (-)-alcohol (2
-Hexanol, 2-heptanol and 1-phenylethanol) 5 μl and 20% pyridine in acetonitrile 10 μl were added, and the mixture was reacted at room temperature for 1 hour.
The reaction solutions were treated under the same conditions as in Reference Example 1 under the following conditions:
Separation by PLC was performed and fluorescence detection was performed. 2. Reaction with optically active amines (-)-DBD-Pro-COCl 3.6 mg was dissolved in acetonitrile so that the total amount was 10 ml. To 0.1 ml of this solution were added (+)-and (-)-1-cyclohexylethylamine (5 µl) and 20% pyridine in acetonitrile (10 µl), and the mixture was reacted at room temperature for 1 hour. Under the following conditions,
Separation by HPLC and fluorescence detection were performed. Table 2 shows the results obtained by measuring the elution times of the peaks of these fluorescently labeled optically active alcohols or amines and calculating the separation coefficient (α) and the resolution (Rs) according to the following formulas. α = k ₁ ′ / k ₂ ′ R _S = 2 (t _R2- t _R1 ) / (W ₁ + W ₂ ) k ₁ ′, k ₂ ′: Fluorescently labeled (+) − and (−)
- retention ratio of alcohols or amines _{t R1,} t _R2: a fluorescently labeled (+) - and (-) - elution time of alcohols or amines _{W 1,} W _2: fluorescently labeled (+) - And (-)
-Peak width of alcohols or amines Device: Shimadzu LC-9A liquid chromatograph column: Inertosyl SIL (150 x 4.6 m)
m, i. d. , 5 μm) (when separating alcohol derivative) Inertosyl ODS-2 (150 × 4.6 mm, i.p.
d. , 5 μm) (when separating amine derivatives) Eluent: ethyl acetate / n-hexane (20/80)
(When separating alcohol derivative) Water / acetonitrile (55/45) (when separating amine derivative) Injection amount: 10 μl Flow rate: 1.0 ml / min Fluorescence detector: Shimadzu RF-550 Detection wavelength: Excitation wavelength 450 nm, Fluorescence wavelength 560nm

[0013]

[Table 2]

[0014]

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.

[0015]

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 (-)-4- (N, N-dimethylaminosulfonyl)
-7- (2-chloroformylpyrrolidin-1-yl)-
2,1,3-benzoxadiazole [hereinafter (-)-D
BD-Pro-COCl] and (+)-4- (N, N
-Dimethylaminosulfonyl ) -7- (2-chloroformylpyrrolidin-1-yl) -2,1,3-benzoo
Xadiazole [hereinafter (+)-DBD-Pro-COC
1] Synthesis of (−)-4- (N, N-dimethylaminosulfonyl)
-7- (2-Carboxypyrrolidin-1-yl) -2,
Dissolve 55 mg of 1,3-benzoxadiazole in 55 ml of anhydrous ether and cool with ice. 110 mg of phosphorus pentachloride is added to this solution, and the mixture is reacted at 5 ° C or lower for 1 hour. The undissolved crystals were 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, and (-)-
20 mg of tan needle crystals of DBD-Pro-COCl were obtained. In addition, (+)-4- (N, N-dimethylaminosulfonyl) -7- (2-carboxypyrrolidine-1-
) -2,1,3-benzoxadiazole as a starting material to give (+)-DBD-Pro-COC
I was able to obtain 1. (-)-DBD-Pro-CO
The main physical properties of Cl are as follows. Melting point 116-11
7 ° C. (decomposition); [α] _D ²⁰ = −44.7 ° (c =
0.894, chloroform); IR (KBr) 179
4,1601,1555,1416,1337,114
8,968,716 cm ⁻¹ ; ¹ H-NMR (deuterated chloroform) δ2.18 to 2.28 (2H, m), 2.54
-2.63 (2H, m), 2.88 (6H, s), 3.
74 to 3.84 (2H, m), 5.59 to 5.63 (1
H, m), 6.13 (1H, d) 7.90 (1H,
d); Elemental analysis value (%) C43.08, H3.70, N
_{15.00 (C 13 H 15 ClN 4} O 4 S of theory C4
3.52, H4.21, N15.62)

[0017]

[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 suppicomolar. The resolution Rs between the optical isomers of optically active alcohols and amines was as good as 1.4 or more. Further, by selectively using (+)-and (-)-of the compound of the present invention, the elution position of the optically active alcohol or amine optical isomer [(+)-form or (-)-form] can be reversed. It was possible, and it was possible to elute one of the optical isomers present in a very small amount earlier in the chromatogram 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. Further, detection with a chemiluminescence detector or a laser fluorescence detector is also possible. Thus, the compound of the present invention can be applied not only to the qualitative and quantitative analysis of trace amounts of individually optically active alcohols or amines, but also to the determination of proteins and peptides containing alcohols and amines widely,
Studies on the functions of amines and alcohols in enzymes, cells,
Separation and detection of optically active alcohols and amines in the living body such as membranes and tissues, or examination of the relationship between the structure and function of biological components, alcohols and amines in various secretory fluids and other clinical samples It has a very wide range of applications in the medical research, including biochemistry, physiology, and basic and clinical studies, based on the quantitative analysis of metabolism and clinical analysis.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical display area C09K 11/06 Z 9159-4H // C07C 29/88 31/00 8930-4H G01N 33/533 8310-2J ( C07D 413/04 207: 00 8314-4C 271: 00) 9283-4C

Claims (1)

[Claims] 1. The following structural formula: (However, * represents an asymmetric carbon atom.) New optically active (+)-and (-)-4- (N, N-dimethylaminosulfonyl) -7- (2-chloroformylpyrrolidine-1) -Yl) -2,1,3-benzoxadiazole.