JP2005068067A - Method for purifying 5,5-dimethylpyrroline-n-oxide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a purification method for efficiently highly purifying 5,5-dimethylpyrroline-N-oxide (DMPO) usable as a spin trapping agent for an electron spin resonance technique (ESR) by carrying out a series of operations at a temperature not higher than room temperature to reduce the effects of heat. <P>SOLUTION: The method for purifying the DMPO comprises a step for dissolving a crude DMPO in an organic solvent at ≤35°C, and a step for recrystallizing the DMPO at ≤6°C from the obtained solution. The solvent is ethyl acetate, a mixed solvent of the ethyl acetate, or the like, and the amount of the used solvent is 1-3 times as much as that of the crude DMPO. As a result, a colorless columnar crystal is obtained. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for purifying DMPO from crude 5,5-dimethylpyrroline-N-oxide containing impurities (hereinafter, 5,5-dimethylpyrroline-N-oxide is referred to as DMPO).

  Free radicals are known as substances that exhibit various physiological activities in vivo. Free radicals are related to many diseases such as ischemic diseases, digestive diseases, cancer, cranial nerve diseases with neurodegeneration, inflammation, and organ damage caused by drugs. Research is ongoing.

  As a method for detecting such free radicals, there is an electron spin resonance method (ESR) that directly detects unpaired electrons in the molecule. However, free radicals usually have a very short life and are difficult to detect directly under physiological conditions using the ESR method. For this reason, a spin trap method is generally used in which the short-lived free radical is chemically captured using a radical scavenger (spin trap agent) and detected as a stable radical (spin adduct). DMPO is used as a kind of spin trapping agent and is often used for analysis of hydroxy radicals, superoxides, and the like.

  By the way, since the ESR method is a high-sensitivity analysis method, if high-purity DMPO is not used, an ESR spectrum derived from impurities is shown, which hinders measurement of the intended free radical. Therefore, in order to obtain high-purity DMPO, crude DMPO is purified by vacuum distillation or precision distillation.

  As a DMPO purification method, Patent Document 1 discloses a purification method characterized in that a crude DMPO aqueous solution is washed with a nonpolar organic solvent, extracted with a polar organic solvent, and purified by distillation. (Patent Document 1). Patent Document 2 discloses a purification method characterized by including an oxidation process in the purification of crude DMPO (Patent Document 2).

Japanese Patent Laid-Open No. 1-153675 Japanese Patent Laid-Open No. 1-305061

  However, it is difficult to completely remove impurities using these methods. Further, even if a high-purity product is obtained, the purification efficiency is poor because the amount obtained is very small compared to the whole or it is necessary to repeatedly perform distillation. Furthermore, since DMPO is not a heat-stable compound, when performing distillation, it is necessary to reduce the temperature applied from outside by setting the vacuum as high as possible.

  Furthermore, there is a method of adding activated carbon to a DMPO aqueous solution to adsorb impurities and then using it for analysis, but it is not suitable for use in quantitative analysis because the exact concentration is unknown.

  On the other hand, a recrystallization method is known as a method for purifying a substance. Recrystallization is usually a method of precipitating crystals by dissolving a composition containing a substance to be purified in a heated solution until saturated, and then cooling the saturated solution. However, DMPO is a low-melting-point organic compound (melting point: 25 ° C.), and is usually an oily substance at room temperature. Therefore, recrystallization has been considered difficult.

  In view of the above problems, an object of the present invention is to provide a purification method for efficiently purifying DMPO by reducing the influence of heat by performing a series of operations at room temperature or lower. Another object of the present invention is to provide a highly accurate free radical measurement by providing a reagent containing high-purity DMPO.

  As a result of intensive studies to solve the above problems, it has been found that the above problems can be solved by performing the recrystallization method at a specific temperature or lower. That is, it has been found that DMPO is dissolved in a normal temperature or a solvent cooled from normal temperature, and then cooled to a specific temperature or lower and recrystallized to efficiently obtain high-purity DMPO.

  Specifically, (1) a step of dissolving crude 5,5-dimethylpyrroline-N-oxide in an organic solvent at a temperature of 35 ° C. or lower, and recrystallizing DMPO from the resulting solution at a temperature of 6 ° C. or lower. A process for purifying 5,5-dimethylpyrroline-N-oxide; and (2) in the above (1), a solid 5,5-dimethylpyrroline-N-oxide in the solution A DMPO purification method characterized by further comprising the step of adding

  By employing the purification method of the present invention, it has become possible to obtain highly pure DMPO by a simple procedure. In particular, the purification method of the present invention not only makes it possible to further purify DMPO by repeating the process, but also has the advantage of easily and efficiently recovering the remaining crude DMPO. Furthermore, the high purity of DMPO helps to more accurate measurement of free radicals and the like, and the manufacturing cost can be reduced by increasing the manufacturing efficiency of high purity products.

BEST MODE FOR CARRYING OUT THE INVENTION

  The crude DMPO referred to in the present invention refers to a composition in which DMPO accounts for most. For example, a known synthesis method (for example, EG, Janzen, LTJandrisits, RVShetty, DLHaire, JWHilborn, Synthesis and purification of 5,5-Dimethyl-1-pyrroline-N-oxide for biological applications, Chem. Biol. , Interactions, 70, 167-172 (1989)), and DMPO obtained by further purification by chromatography or the like. In this case, it is preferable to remove the solvent and the like. Furthermore, crude DMPO more suitable for recrystallization can be obtained by performing simple vacuum distillation before recrystallization to remove the colored components.

  As the organic solvent used in the present invention, those which dissolve and recrystallize DMPO can be widely used. Moreover, in this invention, since recrystallization is tried at low temperature, it is preferable to satisfy the following requirements. (1) An organic solvent having a freezing point lower than the recrystallization temperature, preferably −40 ° C. or lower, more preferably −70 ° C. or lower is preferable. (2) A solvent having a large difference in solubility between DMPO in a cooled state and at room temperature. Specifically, ethyl acetate, ethyl acetate / n-hexane mixed solvent, diethyl ether, acetone, ethanol or the like can be used. Preferably, a mixed solvent of ethyl acetate and ethyl acetate / n-hexane can be used. The amount of the solvent used is not limited, but is preferably 1 to 3 times the volume of the crude DMPO.

  Dissolution of the crude DMPO in the organic solvent is usually performed at room temperature. For example, dissolution is performed within a temperature range of 0 to 35 ° C, preferably within a temperature range of 4 to 25 ° C. At the time of dissolution, stirring and shaking may be performed. Further, at the time of dissolution, it is preferable to dissolve the crude DMPO while gradually adding the minimum necessary solvent while observing the dissolution state of the crude DMPO. After dissolving the crude DMPO, it is preferable to proceed to the next step without raising the temperature above the melting temperature.

  The cooling temperature for recrystallizing the crude DMPO solution is not particularly limited as long as the DMPO is recrystallized. The temperature that satisfies this condition is 6 ° C. or less, more preferably 0 ° C. or less, and most preferably −5 ° C. or less. By adopting such means, it becomes possible to purify while preventing the DMPO from being affected by heat more effectively.

  During cooling, solid DMPO may be added as necessary. Solid DMPO plays a role as a nucleus for promoting recrystallization of DMPO. Therefore, the solid DMPO to be added is preferably in the form of fine particles. The DMPO fine particles are added during or during cooling of the solvent that dissolves the crude DMPO. As the DMPO fine particles, widely known ones can be adopted as long as the object of the present invention is achieved, including the shape and size thereof. Therefore, the DMPO fine particles are not limited to a specific shape.

DMPO is obtained in the form of a columnar or acicular crystal by cooling. The obtained crystal is taken out by suction filtration or decantation using a glass filter, and the organic solvent is completely removed by suction and pressure reduction with a desiccator. It is desirable to perform the above operation under a low temperature condition such as a cold room.
It is desirable to perform a series of operations from dissolution of crude DMPO in an organic solvent to acquisition of crystals in an inert gas atmosphere under light shielding. It is also desirable to perform a series of operations under low temperature conditions such as a cold room.

  The residue remaining in the organic solvent without crystallization can be recovered by distilling off the solvent under reduced pressure. Since the chemical change of DMPO can be suppressed as much as possible in the purification method of the present invention, DMPO that has not been recrystallized is contained in the residue recovered at this time. Therefore, DMPO with high purity can be further recovered by applying the purification method of the present invention or a known purification method to the residue. For this reason, according to the purification method of the present invention, expensive DMPO contained in crude DMPO obtained by synthesis can be efficiently used without being decomposed.

  According to the purification method of the present invention, DMPO having high purity can be efficiently obtained. In addition, the reagents and equipment required for carrying out the purification method of the present invention can be prepared at a lower cost than the conventional DMPO purification method. For this reason, if the refining method of the present invention is used, industrial applicability is high in that high-purity DMPO can be obtained easily and inexpensively.

  DMPO purified by the purification method of the present invention can be effectively used as various reagents. Since the reagent using DMPO purified by the purification method of the present invention can easily recognize the exact concentration of DMPO, it is particularly preferably used as a reagent for quantitative analysis. For example, if it is used as a reagent for measuring free radicals, quantitative analysis with higher accuracy becomes possible. DMPO purified by the purification method of the present invention can also be used effectively in a synthesis reaction using DMPO as a starting material.

  The features of the present invention will be described more specifically with reference to the following examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

(Example)
1. Synthesis of crude DMPO 1) Synthesis of 4-methyl-4-nitropentan-1-al -10 with stirring a 7% sodium methoxide methanol solution (250 mL) in 2-nitropropane (211.5 g) under nitrogen atmosphere C. to -15.degree. C. was added dropwise. Then, a mixed solution of acrolein (21.0 g) and 2-nitropropane (49.1 g) was added dropwise so as to keep the temperature at −10 ° C. to −15 ° C. with stirring. After completion of the dropwise addition, the mixture was further stirred for 30 minutes, and the reaction solution was returned to room temperature. Then, the reaction solution was acidified by adding concentrated hydrochloric acid, filtered with suction, and the filtrate was evaporated. The residue was extracted with ether and dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the desired product was obtained (38.4 g, 82%) by distillation under reduced pressure (boiling point 90-93 ° C./6 mmHg).

2) Synthesis of 2- (3-methyl-3-nitrobutyl) -1,3-dioxolane 350 mL of 4-methyl-4-nitropentane-1-al (32.7 g) and ethylene glycol (16.0 g) were dehydrated It melt | dissolved in benzene, p-toluenesulfonic acid (0.5g) was added, and it heated and refluxed for 7 hours, removing water. After allowing to cool, the reaction solution was washed with a saturated aqueous sodium hydrogen carbonate solution and water, and then dried over anhydrous magnesium sulfate. After the solvent was distilled off under reduced pressure, the desired product was obtained (31.3 g, 74%) by distillation under reduced pressure (boiling point 85-90 ° C./4 mmHg).

3) Synthesis of 5,5-dimethylpyrroline-N-oxide (DMPO) Mixing 5% aqueous ammonium chloride solution (42 mL) of 2- (3-methyl-3-nitrobutyl) -1,3-dioxolane (7.0 g) Zinc powder (9.8 g) whose surface was washed and activated with hydrochloric acid so as to keep the solution light-shielded and stirred under nitrogen atmosphere and kept at 10 ° C. to 15 ° C. was added. After further stirring for 30 minutes, suction filtration and washing with hot water at 60 ° C., the filtrate was acidified with concentrated hydrochloric acid and allowed to stand for 12 hours. The solution was then heated to 70 ° C. for 40 minutes, allowed to cool and then made alkaline with sodium carbonate, and the solvent was distilled off under reduced pressure and extracted with chloroform. After drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure. The product was purified by silica gel column chromatography (chloroform: methanol = 20: 1), and the solvent was distilled off under reduced pressure to obtain the desired product (3.5 g, 84%).

2. Recrystallization of DMPO 5 g of the crude DMPO was dissolved in 2.5 mL of ethyl acetate at 25 ° C. Furthermore, 2.5 ml of n-hexane was added, and the mixture was allowed to stand in a -12 ° C freezer. A small amount of solid DMPO was added to the cooled DMPO solution, and the solution was allowed to stand in a freezer. Crystallization started when solid DMPO became a nucleus. Then, when the crystal growth stopped, the crystal and the solution were separated by decantation, and the organic solvent was completely removed by sucking and reducing the pressure of the crystal with a desiccator. The obtained crystals were colorless columnar crystals (2.5 g). On the solution side, the crude DMPO was recovered by distilling off the solvent under reduced pressure.

3. Measurement of impurity-derived ESR signal:
The crude DMPO before recrystallization 1 and DMPO obtained by recrystallization 2 were each diluted 20 times with ultrapure water. This solution was sucked into a flat cell (manufactured by Labotech) and an ESR spectrum was measured (manufactured by JEOL Ltd., product number: JES-FR80). The result is shown in FIG. The signals at both ends present in each ESR spectrum are signals derived from manganese markers used as a reference in ESR measurement. Here, (a) shows the ESR spectrum of the crude DMPO of 1 above, and (b) shows the ESR spectrum of DMPO obtained by the recrystallization of 2 above. Here, in (a), a large amount of impurity-derived ESR signal exists, but in (b), it was recognized that the impurity was lost. From this result, it was confirmed that the DMPO purification method using the method of the present invention was effective.

The ESR spectrum (a) of DMPO before purification and the ESR spectrum (b) of DMPO after purification are shown.

Claims (2)

A step of dissolving crude 5,5-dimethylpyrroline-N-oxide in an organic solvent at a temperature of 35 ° C. or lower, and a step of recrystallizing DMPO from the resulting solution at a temperature of 6 ° C. or lower. To purify 5,5-dimethylpyrroline-N-oxide. The purification method according to claim 1, further comprising a step of adding solid 5,5-dimethylpyrroline-N-oxide to the solution.

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