JPS6115870A - Production of glutathione monoester - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as an absorption activating agent for glutathione having antidotal activity and radiophylactic activity, etc., in a state free of glutathione diester, by treating a glutathione monoester with a strong organic acid. CONSTITUTION:A protected glutathione mono-(lower alkyl) ester of formula I (X and Y are amino- and carboxyl-protective groups; R<1> and R<2> are lower alkyl) (abbreviated as glutathione monoester) is treated with a strong organic acid (e.g. mixture of trifluoromethanesulfonic acid and trifluoroacetic acid) to obtain the objective compound of formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field of the Invention) The present invention relates to a method for producing a mono-lower alkyl ester of glycine carboxyl group of glutathione (hereinafter abbreviated as glutathione monoester).

(Prior Art and Problems) Glutathione monoester has been reported in animal experiments as a glutathione derivative that migrates to the liver and kidneys in high concentrations, undergoes hydrolysis, and reverts to glutathione.

Therefore, the object compound of the present invention is useful as a glutathione absorption active substance that effectively exhibits the effects recognized for glutathione, such as detoxification and radioprotection in vivo.

The object compound of the present invention can be obtained from the Proceedings of the National Academy of Sciences of the United States of America (Proc., Natl., Aca.
d, Set, U, S, A,) Volume 80.

1983, p. 5258. but,
The same document describes glutathione monoester production method 51-
54. (1933)), it merely states that the corresponding hydrochloride salt was obtained. Physiol.

According to Ahem, 22151-54 (1933), the method of Bergmann and Zervas is a method for producing γ-ethyl ester of glutamic acid by esterifying glutamic acid with ethanol/hydrochloric acid.

The above Proc, Nat1. Acad, Set, U, S,
Who reported A.?

When producing glutathione monoester.

It is presumed that glutathione is used as a raw material and is directly monoesterified with ethanol (or methanol)/hydrochloric acid.

However, this production method has the drawback of contamination with glutathione di-lower alkyl ester. Glutathione diester is a highly toxic compound unlike monoester, so it must be completely removed.

An object of the present invention is to provide a method for producing glutathione monoester that does not contain such diesters.

(Means for solving problems) The production method developed by the present inventors reduces glycine.

The reaction formula for the production method of the present invention is as follows.

(In the formula, R1 and R2 are lower alkyl groups I, XI and X2 are amino group protecting groups, and Y is a carboxyl group protecting group.) Each step will be explained below.

1st step This step consists of combining φlysine lower alkyl ester (1) and cysteine (2) with protected amino and mercapto groups.
) and the partial elimination step of the protecting group.

Examples of the ester (1) include linear or branched lower alkyl esters having 1 to 5 carbon atoms, such as glycine methyl ester, ethyl ester, flobyl ester, isopropyl ester, methyl ester, tert-butyl ester, etc. is used. These esters remain in the target compound.

Among the protecting groups of compound (2), examples of amino group include tert-butoxycarbonyl group, tertiary amyloxycarbonyl group, p-biphenylisoglobyloxycarbonyl group, and protection of mercapto group. Examples of the group include p-lower alkoxybenzyl group. This protecting group for the mercapto group is important in the present invention. In the present invention, protecting groups commonly used for protecting mercapto groups such as benzyl group and nitrobenzyl group 2 are unsuitable. When these protecting groups are used, lower alkyl esters of glycine are also affected because their elimination requires stronger reaction conditions.

For the condensation of compounds (1) and (2), methods commonly used for forming peptide bonds can be employed. A typical method is to use a common condensing agent such as dicyclohexylcarbodiimide (hereinafter abbreviated as DCC), but there is also an acid conversion method in which (2) is converted into its reactive derivative. The physical method, active ester method 2, etc. are also used.

Cysteinylglycine lower alkyl ester with protected amine and mercapto groups (3)
This is then treated with trifluoroacetic acid, hydrofluoric acid, etc. in a conventional manner to obtain cysteinylglycine lower alkyl ester (4) from which only the protecting group of the amine group has been removed.

Second Step The second step is a condensation step of the above compound (4) and glutamic acid (5) in which the amino group and the α-carboxyl group are protected.

Among the protecting groups for glutamic acid used here, as the amine group, those used for cysteine described above can be used. Furthermore, as a protecting group for carboxyl group, benzyloxy group is used.

A benzyloxy group substituted with a nitro group or a lower alkoxy group can be used.

The reaction in this step can be performed in the same manner as the condensation method in the first step. Preferred conditions include dichloromethane,
Chloroform, dichloroethane.

Ru.

In this way, a glutamylcysteinylglycine lower alkyl ester (6) in which the amine group, carboxyl group, and mercapto group are protected can be obtained.

Third step: Next, by treating compound (6) with a strong organic acid, 1
This can lead to the target compound of the present invention. A typical strong organic acid used here is trifluoromethanesulfonic acid.

Compound (6) was prepared using this acid in the coexistence of trifluoroacetic acid.
The protective groups for the amine group, carboxyl group, and mercapto group can be simultaneously and efficiently removed by treatment, and at this time, the lower alkyl ester of the glycine moiety is not affected. It was unexpected that such conditions were used with good results in the preparation of mono-lower alkyl esters of glutathione on glycine.

In this step, trifluoromethanesulfonate of glutathione monoester is obtained, which can be purified to obtain a pure target compound. Purification is
After treating with trifluoromethanesulfate to obtain free glutathione monoester, if necessary, it is further purified using activated carbon or the like.

Purification using this copper salt is much more advantageous in comparison to purification methods using ion exchange resins or the like.

(Effects of the Invention) The present invention has been described in detail above, and in addition to the synthetic route being different from the conventional method mentioned at the beginning, this method is completely free from the risk of contamination with glutathione diester, and is a method for producing glutathione monoester. It is useful as a manufacturing method.

(Example) Next, two examples will be given to explain the production method of the present invention. -(p-methoxybenzyl)cysteinyl]crysinate was produced by the methods of Reference Examples 1 and 2 below, respectively.

Reference example 1 0'-tert-butylhydrodiene N-tert
-Production of butoxycarbonyl-L-glutamate 05-
Benzylhydrodiene N-tert-butoxycarbonyl-glutamate 50g dichloromethane 400g
m1 solution in a dry ice/acetone bath.

Add 400 mZ of chloroform, 400 mt of water, and 50 g of sodium chloride to separate the layers. The organic layer was washed 6 times with 400 ml of water, dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel column chromatography. n-hexane-chloroform (1:1
volume ratio).

The eluent was n-hexane-chloroform (1:1 volume ratio).
- ethyl acetate (9:1 volume ratio) and elute with 05-benzyl O' -tert-butyl N -t
ert-butoxycarbonyl-glutamy)22.
5 g are obtained as crystals.

05-benzyl O' -tert-butyl N -te
rt-butoxycarbonyl-L-glutamate 45.0
g l 8 g of 10% palladium on carbon is added to a solution of 180 mZ of tetrahydrofuran and 180 mt of methanol, and when hydrogen is passed through the solution with stirring at room temperature, about 2.6 ml of hydrogen is absorbed and the reaction is completed.

After removing the catalyst, the solvent was distilled off to obtain 33.6 g of O'-tert-butylhydrodiene (N-tert-butoxycarbonyl-L-glutamic acid) as crystals.

Nuclear magnetic resonance spectrum (CDCl2) δ: 1.46 (18H, s) 1.80
~2.24 (2H, m)2.24~2.60
(2H, m)4.00~4.40 (IH,
m) Reference Example 2 Production of methyl N-[5-(P-methoxybenzyl)-L-cysteinyl]glycinate 13.55 g of glycine methyl ester hydrochloride was added to a solution of 135 mZ of dichloromethane under water cooling while stirring.

14.9 mZ of triethylamine is added dropwise. into this solution.

Next, 24.3 g of N-tert-butoxycarbonyl-3- (hereinafter abbreviated as DCC) was added and washed with 50 ml of dichloromethane. Remove for 15 minutes, stop cooling with water and only stir, and continue overnight. After removing the precipitated dicyclohexylurea from the furnace, it was separated into 250 ml of 5% citric acid solution,
The dichloromethane layer is further saturated with chloride.) It is washed twice with a lithium solution and twice with 250 mZ of water, and then dehydrated with anhydrous magnesium sulfate.

After distilling off dichloromethane, it crystallizes when left overnight at room temperature. To this, 150n+Z of a mixed solution of ether-n-hexane (1:3 volume ratio) was added, stirred, and made into fine particles.
After removing P, washing with 200 mt of a mixed solution of ether-n-hexane (1:3 volume ratio) and drying, methyl N-(N-
tert-Butoxycarbonyl-3-(P-meth)oxybenzyl)-L-cysteinyl]glycinate 34.5
g is obtained as crystals.

390 ml of methyl N-[N-tert-butoxycanibonyl trifluoroacetic acid is added dropwise over 15 minutes. After continuing this state for an additional 30 minutes, the water cooling was stopped and the mixture was stirred at room temperature for an additional hour. After distilling off volatile components under reduced pressure, it was subjected to silica gel column chromatography, eluted with chloroform, and then the eluent was purified with methanol.
When the elution was changed to chloroform (1:10 volume ratio), methyl N-[8-(p-methoxybenzyl)-L-cysteinyl]glycinate trifluoroacetate 38.9
g is obtained.

Methyl N-[5-(p-methoxybenzyl)-L-cysteinyl]glycinate trifluoroacetate 38.9
To 600 mZ of chloroform solution, add 250 t of 0.5 N hydrogen carbonate solution and separate into 9 parts.

The chloroform layer was washed with 200 mZ of water and 200 mZ of saturated sodium chloride solution, and then dehydrated with anhydrous magnesium sulfate.
The solvent was evaporated to dryness under reduced pressure to give methyl N-[8-(p-
26.85 g of methoxybenzyl)-L-cysteinyl]glycinate are obtained.

Nuclear magnetic resonance spectrum (CDC13) δ: 2.48-3.12 (2H, m) 3.40-3.
60 (IH,q) 3.68 (2H,s) 3.76 (3H,s) 3.80 (3H,s) 4.04 (2H,d) 6.86 (2H,d, J = 9. 0H
z) 7.26 (2H, d, J = 9.
0Hz) Example 1 0'-tert-butylhydrodiene N-tert
-butoxycarbonyl-L-glutamy) 26.1g
and.

To a solution of 26.85 g of methyl N-CS-(p-methoxybenzyl)-L-cysteinyl]glycinate in 150 ml of dichloromethane is added 180 g of DCC, and the mixture is stirred at room temperature for 1.5 hours.

After removing the precipitated dicyclohexylurea, the mixture was separated with 100 mt of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure.

The residue was subjected to silica gel column chromatography and eluted with ethyl acetate-chloroform (1:1 volume ratio) to give methyl N-(N-(N-tert).
-butoxycarbonyl-〇'-tert-butyl-γ-
L-glutamyl)-8-(p-methoxybenzyl)-L
43.2 g of -cysteinyl]glycinate are obtained.

Nuclear magnetic resonance spectrum (CDC13) δ: 1.42 (18H, s) 1.80~
2.40 (4H, m) 2.72-2.96
(2H, (1) 3.74 (5H, s) 3.80 (3H, II) 4.02 (2H, d) 4.08°~4.40 (IH, m) 4.40~4
．． 70 (IH,Q)6.86 (2H
, d, J=9.0Hz) 7.28 (2H,
d, J=9.0 ratio) OOH ■, CF35o3H Methyl N -(N-(N -tart-butoxycarbonyl-〇''-tert-butyl-γ-L-glutamyl)
-s-(p-methoxybenzyl)-L-cysteinyl]
Glycinate 19.2 g, dichloromethane 80
mZ, to a solution of 8 ml of anisole, add 80 mt of trifluoroacetic acid while stirring under water cooling; t* then add 4.8 ml of trifluoromethanesulfonic acid. Stirring was continued under water cooling for 15 minutes, the water bath was removed, and stirring was continued for an additional 2 hours.

After concentrating under reduced pressure, it was decanted twice with 800 mZ of ether and dried under reduced pressure. 20.4 g of ether-containing methyl r-L-glutamyl-cysteinylglycinate trifluoromethanesulfonate are obtained.

(1) Nuclear magnetic resonance spectrum (DMSO-d6) δ:
1.80-2.12 (2H, m) 2.16-2.
40 (2H, m) 2.60~3.00 (2
H, m)3.60 (3H,s)3
．． 84 (2H, d) 3.88~4
．． 08 (IH, m) 4.28-4.56
(IH, m) (11) Infrared absorption spectrum (
KBr). □-1: 1740.1650.1330.
12401170, 1050 OOH Copper salt methyl γ-L-glutamyl-L-cysteinylglycinate trifluoromethanesulfonate 20.4g
, 95% sulfuric acid diluted 20 times in a solution of 80 ml of water
Add 0ml and 2.2g of cuprous oxide. Shake well and warm in a 40°C water bath for about 30 minutes, then leave in an icebox overnight.

The deposited precipitate is placed in a centrifuge and washed until the aqueous layer becomes neutral, yielding methyl γ-L-rutamyl-L-cystinyl crucinate copper salt in a wet state.

Infrared absorption spectrum (KBr) cm-': 1735.1635 Methyl γ-L-glutamyl-L-cysteinylglycinate copper salt was dissolved in 50 mZ of water, and hydrogen sulfide gas was dissolved in 3
Passes for 0 minutes.

After removing the copper sulfide from the furnace, activated carbon is added and filtered.

After replacing the hydrogen sulfide gas with argon gas, the mixture is freeze-dried to obtain 1.65 g of methyl γ-L-glutamyl-L-cysteinylglycinate.

(1) Nuclear magnetic resonance spectrum (DMSO-d, +C
D30D) δ: 1.72 to 2.04 (2H, m
)2.20-2.40 (2H, m)2.64
~2.88 (2H, m)3.16~3.40
(IH, m) 3.60 (3H, s) 3.80 (2H, s) 4.20~4
．． 60(IH,m) (11) Infrared absorption spectrum (KBr)Cm':
1740.1635.1520.1400.1210
(iii) Optical rotation

Claims (4)

[Claims] (1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, X and Y represent a protecting group for an amino group and a carboxyl group, respectively, and R^1 and R^2 each mean a lower alkyl group. ) The general formula is characterized by treating the protected mono-lower alkyl ester of glutathione represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Manufacturing method (2) The production method according to claim 1, wherein the strong organic acid is trifluoromethanesulfonic acid. (3) The production method according to claim 1, wherein the strong organic acid is a mixed acid of trifluoromethanesulfonic acid and trifluoroacetic acid. (4) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, X and Y mean a protecting group for an amino group and a carboxyl group, respectively.) Glutamic acid or its reactive derivative at the γ-carboxyl group and mercapto represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R^1 and R^2 each mean a lower alkyl group.) A protected mono-lower alkyl ester of glutathione represented by the general formula ▲mathematical formula, chemical formula, table, etc.▼ is produced by reacting the lower alkyl ester of cysteinylglycine with a protected group, and then the compound is converted into an organic compound. 1. A method for producing a mono-lower alkyl ester, which comprises treating with a strong acid to obtain a mono-lower alkyl ester related to the glycine carboxyl group of glutathione represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼.