JPH0586092A - Glutathione monoester salt and its production - Google Patents

Abstract

PURPOSE:To provide the title novel compound useful as a readily absorptive active substance for glutathione, good in crystallinity, easy to purify, thus capable of easily giving in high yield the corresponding glutathione monoester free from the corresponding highly toxic diester. CONSTITUTION:The objective salt, a lower alkyl ester alkaline earth metal chloride for the glycine carboxyl group of gamma-L-glutamyl-L-cysteinylglycine (glutathione), for example, ethyl l-Lglutamyl-L-cysteinylglycinate calcium chloride. The present compound can be obtained by reaction between glutathione and an alcohol of formula R-OH (R is lower alkyl) such as ethanol in the presence of hydrochloric acid and an alkaline earth metal chloride (e.g. calcium chloride) followed by pref. neutralizing the reaction liquor with triethylamine and then crystallization.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lower alkyl ester (hereinafter abbreviated as glutathione monoester) / alkaline earth of a novel γ-L-glutamyl-L-cysteinylglycine (glutathione) glycine carboxyl group. The present invention relates to a metal chloride salt and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Glutathione is a major reducing agent in cells widely existing in tissues in the living body and plays an important role in cell protection and the like, and has pharmacological actions such as antioxidation action, detoxification action and radiation protection action. have. Therefore, when the tissue glutathione is considerably reduced due to disease, aging, etc., the tissue becomes easily damaged. In such a case, it is important to restore tissue glutathione to a normal value, and it is thought that even in normal cases, increasing tissue glutathione can further enhance the cytoprotective function. There is. However, even if glutathione itself is administered,
Glutathione monoesters have been reported as compounds that have the disadvantage that they are not efficiently taken up by tissues, and that they migrate to various cells at high concentrations in experimental animals and are superior to glutathione in tissue glutathione-elevating ability. Therefore, glutathione monoesters are useful as easily absorbable active forms of glutathione. Glutathione monoester is produced by esterifying glutathione with an alcohol in the presence of an acid catalyst, and its acid addition salt (hydrochloride, sulfate) is also known, but other salts are not reported.

[0003]

Glutathione monoesters and acid addition salts thereof are produced by a general esterification reaction of glutathione (see, for example, JP-A-60-).
No. 123497, No. 61-249997, No. 62-8
9695). However, in the process of this esterification, 5
About 20% of glutathione diester is produced as a by-product. Since this is a highly toxic compound unlike monoester, its removal must be completely performed.
It has drawbacks such as complicated operation and reduced yield.

[0004]

DISCLOSURE OF THE INVENTION The inventors of the present invention have conducted extensive studies to overcome such drawbacks, and as a result, glutathione has the formula: R
In the method for producing glutathione monoester by esterification in the presence of hydrochloric acid with an alcohol represented by —OH (wherein R represents a lower alkyl group), and then neutralizing the same, an alkaline earth metal chloride is present. By doing so, it was surprisingly found that only glutathione monoester / alkaline earth metal chloride salt was precipitated as crystals in good yield, and the present invention was completed. That is, the present invention provides a novel glutathione monoester / alkaline earth metal chloride salt which has not been described in the literature, and since this compound has good crystallinity and is easy to purify, it can be produced by the production method of the present invention. Thus, glutathione monoesters which can easily provide a good yield and do not contaminate the diester can be provided. The salts of the present invention are compounds in which the molar ratio of glutathione monoester and alkaline earth metal chloride is approximately 3: 1. In addition, in the present specification, the lower alkyl ester represents methyl, ethyl, propyl, and isopropyl ester, and the alkaline earth metal chloride is calcium chloride,
Represents magnesium chloride.

Glutathione monoester / alkaline earth metal chloride salt is a formula containing glutathione and hydrochloric acid:
In the step of reacting with an alcohol represented by R-OH (wherein R represents a lower alkyl group) to form an ester, and then neutralizing and then crystallizing, an alkaline earth metal chloride is added before the crystallization step. It is obtained by making things exist. Examples of the alcohol represented by the formula: R-OH include methanol, ethanol, propanol, and isopropanol. The esterification reaction is carried out by a known method. As the solvent used in the reaction, an alcohol represented by the above formula: R-OH is used in an excess amount, preferably 2 to 20 times (weight ratio) with respect to glutathione. The reaction is carried out in the presence of hydrochloric acid. The amount of hydrochloric acid added is 0.2 to 10 N, preferably 0.5 to 2 N, as the concentration in the reaction system. The reaction temperature is 0 ° C. to room temperature, and the reaction time is about 2 hours to 2 days, varying depending on the type of alcohol, the acid concentration, the reaction temperature and the like. After the esterification reaction, the reaction solution is neutralized with a tertiary amine. Examples of the tertiary amine used include triethylamine and pyridine, but it is preferable to use triethylamine, and neutralization is performed at pH
It is desirable to carry out the treatment so that the pH is in the range of 4.5 to 7.0, preferably 5.0 to 6.5.

The present invention is carried out in the presence of an alkaline earth metal chloride. The alkaline earth metal chloride may be added in any step, but if neutralized without addition, neutralization may occur during neutralization. It is desirable to add during the esterification reaction or after the esterification reaction and before the neutralization step because a precipitate is formed and the operation becomes complicated. Examples of the alkaline earth metal chloride to be used include calcium chloride and magnesium chloride. Use of about 0.1 to 1.0 times mol, preferably 0.2 to 0.5 times mol of the added glutathione. However, if the amount used is large, the salt of the diester is contaminated.
The reaction product is easily isolated by cooling the neutralized solution and collecting the precipitated crystals by filtration.
It can be recrystallized from water or the like.

[0007] Glutathione monoester / alkaline earth metal chloride salt can be easily converted to glutathione monoester in free form. For example, as shown in Reference Examples, copper is removed by treatment with an ion exchange resin such as HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.) or after conversion into glutathione monoester / copper salt, followed by hydrogen sulfide or the like. It leads to a free form.

[0008]

The present invention will be described in detail below with reference to examples. Example 1 5 g of glutathione was added to 30 ml of ethyl alcohol containing 1.3 g of hydrogen chloride, and the mixture was stirred at 0 to 5 ° C overnight. Triethylamine was gradually added dropwise to this solution with stirring under cooling to adjust the pH.
It was set to 0.5. Then, a solution prepared by dissolving 0.6 g of calcium chloride in 60 ml of methanol was added, and triethylamine was gradually added dropwise with stirring under cooling to adjust the pH to 6.0. This solution was left overnight in the refrigerator, and the precipitated crystals were collected by filtration and washed with cold ethanol to obtain ethyl γ-L-glutamyl-.
L-cysteinyl glycinate / calcium chloride salt 4.
5 g was obtained. A portion of this was taken from ethanol-water (4:
It was recrystallized from 1) and used as a sample for analysis. Melting point 170-172 ° C. TLC Rf = 0.66 (silica gel 60F ₂₅₄ (Merck), n-PrOH: AcOH: H ₂ O = 10:
1: 5) IR (KBr, cm ^-1 ) 3312, 1732, 164
0,1551, 1213, 1023. Elemental analysis (C ₁₂ H ₂₁ N ₃ O ₆ S ・ 1 / 3CaCl ₂・
½ C ₂ H ₅ OH · H ₂ O) Calculated value (%) C: 37.77 H: 6.34 N:
10.16S: 7.75 Ca: 3.23 Cl:
5.71 Measured value (%) C: 37.73 H: 6.13
N: 10.28 S: 7.51 Ca: 3.07 C
l: 5.41

Example 2 To 30 ml of ethyl alcohol containing 1.3 g of hydrogen chloride, 0.45 g of magnesium chloride was added and dissolved, then 5 g of glutathione was added, and the mixture was stirred at 0 to 5 ° C overnight. 60 ml of ethanol was added to this solution, and triethylamine was gradually added dropwise with stirring under cooling to adjust the pH to 6.0. This solution was left overnight in the refrigerator, and the precipitated crystals were collected by filtration and washed with cold ethanol to obtain 3.8 g of ethyl γ-L-glutamyl-L-cysteinyl glycinate / magnesium chloride salt. Melting point 168 to 169 ° C. TLC Rf = 0.66 (silica gel 60F ₂₅₄ (manufactured by Merck), n-PrOH: AcOH: H ₂ O = 10:
1: 5) IR (KBr, cm ^-1 ) 3302, 1747, 164
0,1538,1209,1024.

Example 3 30 ml of isopropyl alcohol containing 1.3 g of hydrogen chloride
Glutathione (5 g) was added thereto, and the mixture was stirred at 0 to 5 ° C for 2 hours and at room temperature overnight. Stirring triethylamine was gradually added dropwise to the solution under cooling to adjust the pH to 0.5. Next, a solution prepared by dissolving 0.6 g of calcium chloride in 60 ml of methanol was added, and triethylamine was gradually added dropwise while stirring with cooling, and p
It was set to H6.0. This solution was allowed to stand in a refrigerator overnight, and the precipitated crystals were collected by filtration and washed with cold methanol to obtain 4.2 g of isopropyl γ-L-glutamyl-L-cysteinyl glycinate / calcium chloride salt. A part of this was recrystallized from ethanol-water (4: 1) to obtain a sample for analysis. Melting point 177 to 179 ° C. TLC Rf = 0.70 (silica gel 60F ₂₅₄ (manufactured by Merck), n-PrOH: AcOH: H ₂ O = 10:
1: 5) IR (KBr, cm ^-1 ) 3304, 1730, 164
3,1538,1220,1107. Elemental analysis (C ₁₂ H ₂₁ N ₃ O ₆ S ・ 1 / 3CaCl ₂・
½ C ₂ H ₅ OH.H ₂ O) Calculated value (%) C: 39.34 H: 6.60 N:
9.83S: 7.50 Ca: 3.13 Cl:
5.46 Measured value (%) C: 39.47 H: 6.62
N: 10.05S: 7.48 Ca: 2.88 C
l: 5.09

Reference Example 2.0 g of the unpurified calcium chloride salt obtained in Example 1 was dissolved in 50 ml of water, 50 ml of HP-20 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) was adsorbed, washed with water and discharged with 50% methanol. , A fraction containing the desired product was obtained. This fraction was concentrated and freeze-dried to obtain ethyl γ-L-glutamyl-L.
-1.4 g of cysteinyl glycinate are obtained. This compound, by comparison of infrared absorption spectrum with a standard sample,
The structure was confirmed.

[0012]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a novel glutathione monoester / alkaline earth metal chloride salt, which has not been described in the literature, is provided. Glutathione monoesters which do not contaminate the diester in good yield can be produced.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location A61K 37/02 AGZ 8314-4C

Claims (3)

[Claims] 1. A lower alkyl ester / alkaline earth metal chloride salt of the glycine carboxyl group of γ-L-glutamyl-L-cysteinylglycine. 2. The salt of claim 1, wherein the alkaline earth metal chloride is calcium chloride. 3. γ-L-Glutamyl-L-cysteinylglycine is reacted with an alcohol represented by the formula: R—OH (wherein R represents a lower alkyl group) in the presence of hydrochloric acid, and then, A lower alkyl ester / alkali relating to a glycine carboxyl group of γ-L-glutamyl-L-cysteinylglycine, which is characterized in that an alkaline earth metal chloride is present in the method of crystallization after soaking. A process for producing earth metal chloride salts.