Method for preparing glutathione and cysteine ​​derivatives

Abstract

The present invention relates to a method for synthesizing acyl derivatives of glutathione and cysteine, which is highly environmentally advantageous since it allows the derivative compounds to be synthesized efficiently without using organic solvents that are harmful to the environment.

Description

[Technical Field]

[0001] The present invention relates to a simple and novel method for preparing compounds derived from glutathione and cysteine.

[0002] In particular, the present invention relates to a method for synthesizing acyl derivatives of glutathione and cysteine, which is highly environmentally advantageous since it does not require the use of environmentally harmful organic solvents. [Background technology]

[0003] Glutathione, a tripeptide composed of three amino acids (cysteine, glycine, and glutamine), plays an important role in numerous physiological processes. Reduced glutathione (GSH), the major non-protein thiol compound present in animal cells, is a powerful endogenous antioxidant that protects cells from damage caused by free radicals and other reactive oxygen species, helping to maintain cellular health and suppress inflammation. Glutathione also plays a key role in detoxification processes, helping to neutralize and eliminate toxins, pharmaceuticals, heavy metals, and other toxic compounds.

[0004] Glutathione also aids the immune system by contributing to the production and activation of T lymphocytes, which are important agents in the defense against infection, and protects DNA from oxidative damage.

[0005] Due to the role of glutathione in many metabolic processes, glutathione deficiency has been linked to the onset and progression of various diseases.

[0006] For example, individuals with glutathione deficiency exhibit hemolytic anemia, metabolic acidosis, bacterial infections, and progressive dysfunction of the central nervous system. Furthermore, in humans, inherited deficiencies in γ-glutamyl cycle enzymes result in reduced glutathione production, making cells particularly vulnerable to oxidative stress and promoting apoptotic and necrotic processes. The resulting damage is a critical aspect in the development and progression of many diseases, as low levels of GSH are found in a wide range of conditions, including neurodegenerative diseases, cystic fibrosis, and various viral infections.

[0007] Therefore, glutathione or its precursors have been used as active ingredients in pharmaceutical and nutritional supplement formulations. Such pharmaceutical compositions can be administered intravenously or orally.

[0008] Intravenous administration is commonly used in hospitals to reduce the effects of chemotherapy treatments for cancer, Parkinson's disease, diabetes, dialysis-related anemia, atherosclerosis, and male infertility disorders.

[0009] Because of the therapeutic potential of reduced glutathione (GSH), a convenient method for synthesizing acylglutathione derivatives, compounds that can enhance GSH absorption, protect GSH from oxidation during the absorption process, and simultaneously release GSH efficiently into the human body, is highly desirable.

[0010] For example, U.S. Patent No. 5,382,679 reports the preparation of various S-acyl glutathione derivatives, particularly those with acetic acid (SAG), pivalic acid, benzoic acid, phenylacetic acid, and thienylcarboxylic acids. WO 2011 / 081715 and WO 2011 / 081716 describe S-acylated GSH derivatives with both saturated and polyunsaturated long-chain fatty acids, primarily designed for skin use.

[0011] Another synthesis of S-acyl glutathione is described by Francesca Bartoccini et al. in Org. Process Res. Dev. 2019, 23, 9, 2069-2073 and in DE 10018098 A1.

[0012] Alan R. Katritzky et al., J. Org. Chem. 2009, 74, 18, 7165-7167, describe the synthesis of N-acyl cysteines using N-acyl 1H-benzotriazoles.

[0013] Known methods for synthesizing S-acyl glutathione derivatives have certain drawbacks due to the need to use organic solvents. In particular, known methods require: - the use of the appropriate acid chlorides or the corresponding anhydrides in the presence of organic bases, always using various organic solvents; - Use of the corresponding carboxylic acid in the presence of 1,2,3-benzotriazole as coupling agent only in the presence of an organic solvent; - The use of organic carboxylic acids and trifluoroacetic acid, a highly toxic pollutant.

[0014] Specifically, the synthesis of S-acetylglutathione (SAG) involves the use of acetic acid as a solvent, acetic anhydride as a drying and acylating agent, and perchloric acid. The product is isolated from a water / acetone mixture, generating a significant amount of highly polluted aqueous wastewater.

[0015] Therefore, there is a need for a method for preparing acyl glutathione derivatives without using organic solvents and / or reagents that are difficult to dispose of in an environmentally friendly manner. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] U.S. Patent No. 5,382,679 [Patent Document 2] International Publication No. 2011 / 081715 [Patent Document 3] International Publication No. 2011 / 081716 [Patent Document 4] DE 10018098 A1 [Non-patent literature]

[0017] [Non-Patent Document 1] Org.Process Res.Dev.2019, 23, 9, 2069―2073 [Non-patent document 2] J.Org.Chem.2009, 74, 18, 7165―7167 Summary of the Invention [Problem to be solved by the invention]

[0018] The object of the present invention is therefore to provide a process for the preparation of S-acyl glutathione derivatives which overcomes the drawbacks associated with the use of organic solvents in the synthesis steps mentioned above.

[0019] The same method can be used under similar operating conditions to prepare N-acyl derivatives of cysteine, again without the need for organic solvents in the synthesis step. [Means for solving the problem]

[0020] The present invention is directed to a method for preparing an acyl glutathione derivative, comprising the steps of: A) Mass reaction of carbonyldiimidazole with carboxylic acid R-COOH (R=alkyl or phenyl) to give N-acylimidazole:

[0021] [ka]

[0022] B1) Reaction of N-acylimidazole obtained in step A) with glutathione in aqueous solution to form S-acylglutathione:

[0023] [ka]

[0024] Here, both reactions A) and B1) are carried out in the absence of organic solvents.

[0025] The method of the present invention is novel and overcomes the drawbacks associated with the use of toxic and / or polluting organic solvents.

[0026] The applicant has surprisingly found that the use of carbonyldiimidazole as a coupling agent in the preceding step A) allows the synthesis of acyl glutathione derivatives without the use of organic solvents.

[0027] As mentioned above, cysteine ​​is one of the basic components of reduced glutathione, along with glutamic acid and glycine.

[0028] The synthesis method of the present invention can be effectively used to prepare not only S-acyl glutathione derivatives but also N-acyl cysteine ​​derivatives.

[0029] Therefore, a second object of the present invention is to provide a method for preparing an acylcysteine ​​derivative, which comprises the following steps: A) Bulk reaction of carbonyldiimidazole with carboxylic acid R-COOH (R=alkyl or phenyl) to give N-acylimidazole:

[0030] [ka]

[0031] B2) Reaction of the N-acylimidazole obtained in step A) with cysteine ​​in aqueous solution to form an intermediate S-acylcysteine, which spontaneously undergoes intramolecular rearrangement to give N-acylcysteine:

[0032] [ka]

[0033] Here, both reactions A) and B2) are carried out in the absence of organic solvents.

[0034] In the case of acylation of cysteine, the acylcysteine ​​derivative can be synthesized without using an organic solvent by using carbonyldiimidazole as a coupling agent in the preceding reaction A).

[0035] The term "alkyl" refers in particular to straight-chain or branched alkyl groups, preferably C1-C 18 It refers to a group, more preferably a C1-C6 alkyl group.

[0036] When R is phenyl, it may be substituted with 1 to 3 identical or different substituents selected from a halogen atom, a C1-C3 alkyl group, a C1-C3 alkoxy group, nitro, and cyano. R is preferably alkyl.

[0037] The products obtained by the process of the present invention can be advantageously used in dietary supplement formulations, which can be utilized in the supplement industry as a source of glutathione or cysteine. DETAILED DESCRIPTION OF THE INVENTION

[0038] The synthetic method of the present invention for preparing acyl derivatives of glutathione or cysteine ​​comprises a first reaction step A) in which an N-acylimidazole is formed.

[0039] [ka]

[0040] The intermediate reaction compound is water soluble, which has been found to be a great advantage in simplifying the subsequent acylation reaction B1) or B2).

[0041] In fact, the acylation reaction B1) or B2) can be carried out in the absence of organic solvents by simply adding N-acylimidazole to an aqueous solution of glutathione (reaction B1) or cysteine ​​(reaction B2).

[0042] The acylation reaction occurs spontaneously, and the reaction product precipitates out of solution after simply cooling the aqueous solution to change its pH, allowing for the synthesis of glutathione- or cysteine-derived compounds without the use of organic solvents, thereby saving raw materials and reducing environmental pollutants.

[0043] The preparation of the intermediate N-acylimidazole (Reaction A) is carried out in bulk in the absence of a solvent at a temperature close to room temperature.

[0044] Regarding the operating conditions, the acylation reactions B1) and B2) are usually carried out in aqueous solution at a temperature in the range of 0°C to 60°C, and the pH is generally set to a value in the range of 6.5 to 11, preferably in the range of 7.0 to 10.

[0045] The residence time required for the acylation reaction B1) to occur is generally 20-60 minutes. In the case of reaction B2), the reaction time is increased to at least 3 hours to allow the rearrangement to complete and produce the N-acyl cysteine.

[0046] After the reaction is complete after this time has elapsed, the pH of the aqueous solution is adjusted to a value in the range of 2 to 5, preferably 2.5 to 3.5, by adding an inorganic acid such as hydrochloric acid, to promote spontaneous precipitation of the glutathione and / or cysteine ​​derivatives.

[0047] The acylation reaction is exothermic, so the temperature of the aqueous solution increases during the reaction. Therefore, once the acylation reaction is complete, it is advisable to reduce the temperature of the aqueous solution to a value in the range of 0°C to 30°C, preferably 0°C to 10°C, to promote spontaneous precipitation of the reaction product as a solid residue.

[0048] The pH and temperature changes promote spontaneous precipitation of the acylated derivatives of glutathione and / or cysteine, which are recovered by filtering the solids from the aqueous solution, followed by washing with water and finally drying under vacuum.

[0049] The acylglutathione derivatives, in particular S-butyrylglutathione, obtainable by the method of the present invention are particularly advantageous for the preparation of medicaments and / or supplements for the treatment of GSH deficiency disorders.

[0050] Further advantages and features of the present invention will become apparent from the following examples. [Example]

[0051] [Example 1] Preparation of S-butyryl glutathione Step A) Preparation of the intermediate N-butylyl imidazole 9.0 g (102 mmol) of butyric acid is reacted with 16.5 g (102 mmol) of carbonyldiimidazole (C3H3N2)2CO at room temperature. The bulk reaction yielded 14.1 g of N-butylimidazole CH 10 N2O is produced.

[0052] Step B1) Synthesis of S-butyrylglutathione Dissolve 32 g (104 mmol) of glutathione in 300 mL of water and cool the resulting solution to 10° C. Adjust the pH of the resulting solution to 10.0 by adding caustic soda NaOH. Next, 14 g (102 mmol) of N-butylimidazole CH 10N2O is added to the previously prepared aqueous solution of glutathione. The reaction mixture is maintained for 30 minutes under constant stirring and at a temperature of 10°C. The reaction is monitored by HPLC analysis and once the reaction is complete, the pH of the solution is adjusted to 3.0 by adding 20% ​​hydrochloric acid HCl, keeping the temperature at 10°C at all times. The reaction product, S-butyrylglutathione, spontaneously precipitates from aqueous solution, allowing the compound to be synthesized without using solvents, leading to savings in raw materials and reduction of environmental pollutants. After about 30 minutes, the resulting solid precipitate corresponding to the desired product is filtered, washed with water and dried under vacuum to give 37 g of S-butyrylglutathione.

[0053] [Example 2] Preparation of S-butyryl glutathione Step A) Preparation of the intermediate N-butylyl imidazole 9.0 g (102 mmol) of butyric acid is reacted with 17.5 g (108 mmol) of carbonyldiimidazole (C3H3N2)2CO at room temperature. The bulk reaction produces 14.1 g of N-butylilimidazole.

[0054] Step B1) Synthesis of S-butyrylglutathione Dissolve 32 g (104 mmol) of glutathione in 350 mL of water at room temperature. Adjust the pH of the resulting solution to 7.0 by adding NaHCO3. Subsequently, 14 g of N-butylilimidazole prepared in step a) are added to the aqueous solution of glutathione GSH. The reaction mixture is stirred for 30 minutes while maintaining the temperature at 20°C, and 65 mL of 20% HCl is added to adjust the pH to 3. The temperature is still maintained unchanged, and after about 30 minutes the resulting solid precipitate corresponding to the desired product is filtered, washed with water and dried under vacuum to give 27.4 g of S-butyrylglutathione C 14 H 23 N3O7S is obtained.

[0055] [Example 3] Preparation of S-acetylglutathione Step A) Preparation of the intermediate N-acetylimidazole 6.1 g (102 mmol) of acetic acid are reacted with 16.5 g (102 mmol) of carbonyldiimidazole at room temperature. The bulk reaction produces 22 g of N-acetylimidazole.

[0056] Step B1) Synthesis of S-acetylglutathione Dissolve 32 g (104 mmol) of glutathione in 100 mL of water and cool the resulting solution to 5° C. Adjust the pH of the resulting solution to 8.0 by adding NaOH. 11.7 g of N-acetylimidazole previously prepared in step a) is added to the aqueous solution of GSH, the temperature of which rises to about 45° C. in this slightly exothermic reaction, and the reaction mixture is stirred for 30 minutes while maintaining the same temperature. The resulting solution is cooled to 5°C and the pH is adjusted to 3.0 by adding 20% ​​hydrochloric acid HCl. Once the reaction is complete, the resulting solid precipitate, corresponding to the desired product, is filtered, washed with water, and dried under vacuum to give 26 g of S-acetylglutathione.

[0057] Example 4: Preparation of S-acetylglutathione Step A) Preparation of the intermediate N-acetylimidazole 15 g of acetic acid is reacted with 40 g of carbonyldiimidazole (C3H3N2)2CO at room temperature. The bulk reaction produces 27.5 g of N-acetylimidazole.

[0058] Step B1) Synthesis of S-acetylglutathione Dissolve 76 g (250 mmol) of glutathione GSH in 250 mL of water at room temperature. Adjust the pH of the resulting solution to 8.0 by adding 50 g of NaHCO3. 27.5 g of N-acetylimidazole previously prepared in step a) is added to the aqueous solution of glutathione, and the reaction mixture is stirred for 30 minutes while maintaining the temperature at 5°C. While maintaining the temperature unchanged, the pH is adjusted to 2.8 by adding 20% ​​HCl. Once the reaction is complete, the resulting solid precipitate, which corresponds to the desired product, is filtered, washed with water and dried under vacuum to give 61 g of S-acetylglutathione.

[0059] Example 5: Preparation of N-acetylcysteine Step A) Preparation of the intermediate N-acetylimidazole 6.1 g (0.102 mol) of acetic acid are reacted with 16.5 g of carbonyldiimidazole (0.102 mol) at room temperature. The bulk reaction produces 11.2 g of N-acetylimidazole.

[0060] Step B2) Synthesis of N-acetylcysteine 12.1 g (100 mmol) of cysteine ​​is dissolved in 40 mL of water and the resulting solution is cooled to 5° C. The pH of the resulting solution is adjusted to 8.0 by adding NaOH. 11.2 g of N-acetylimidazole previously prepared in step a) is added to the aqueous solution of cysteine ​​and maintained at 10°C. The resulting reaction mixture is stirred at room temperature for 3 hours. The mixture is cooled to 0°C, and 20% HCl is added to adjust the pH to 2.5. The mixture is kept under stirring at 0°C for 1 hour, after which the resulting solid precipitate corresponding to the desired product is filtered, washed with water, and dried under vacuum to obtain 11.6 g of N-acetylcysteine.

[0061] Example 6: Preparation of N-butyrylcysteine Step A) Preparation of the intermediate N-butylyl imidazole 9.0 g (102 mmol) of butyric acid are reacted with 16.5 g (102 mmol) of carbonyldiimidazole at room temperature. The bulk reaction produces 14.1 g of N-butylilimidazole.

[0062] Step B2) Synthesis of N-butyrylcysteine 15.7 g (100 mmol) of cysteine ​​is dissolved in 60 mL of water and the resulting solution is cooled to 5° C. The pH of the resulting solution is adjusted to 8.0 by adding NaOH. 14.1 g of N-butylilimidazole previously prepared in step a) is added to the aqueous solution of cysteine ​​and maintained at 10°C. The resulting reaction mixture is stirred at room temperature for 3 hours. The mixture is cooled to 0°C, and 20% HCl is added to adjust the pH to 2.5. The mixture is kept under stirring at 0°C for 1 hour, after which the resulting solid precipitate corresponding to the desired product is filtered, washed with water, and dried under vacuum to obtain 14.6 g of N-acetylcysteine.

Claims

1. A method for preparing an acyl glutathione derivative, comprising the steps of: A) bulk reaction of carbonyldiimidazole with carboxylic acid R-COOH (R is alkyl or phenyl) to form N-acylimidazole; 【Chemical 1】 B1) reaction of the N-acylimidazole obtained in step A) with glutathione in aqueous solution to form S-acylglutathione; Including, 【Chemistry 2】 A process for preparation characterized in that reactions A) and B1) are carried out in the absence of organic solvents.

2. A method for preparing an acyl derivative of glutathione, comprising the steps of: A) Bulk reaction of carbonyldiimidazole with carboxylic acid R-COOH (R=alkyl or phenyl) to form N-acylimidazole; 【Chemistry 3】 B2) reaction of the N-acylimidazole obtained in step A) with cysteine ​​in aqueous solution to form an intermediate S-acylcysteine, which undergoes spontaneous intramolecular rearrangement to give N-acylcysteine; Including, 【Chemistry 4】 A process for the preparation characterized in that both reactions A) and B2) are carried out in the absence of organic solvents.

3. 3. The process according to claim 1 or 2, wherein the acylation reactions of steps B1) and B2) are carried out while maintaining the temperature in the range of 0°C to 60°C.

4. 4. The process according to any one of claims 1 to 3, characterized in that, once the acylation reactions of steps B1) and B2) are complete, the temperature of the aqueous solution is reduced to a value ranging from 0°C to 30°C to promote spontaneous precipitation of the reaction product as a solid residue.

5. 10. A process according to any one of the preceding claims, characterized in that the acylation reactions of steps B1) and B2) are carried out in an aqueous solution having a pH in the range of 6.5 to 11.

6. 10. A process according to any one of the preceding claims, characterized in that once the acylation reactions of steps B1) and B2) are complete, the pH of the aqueous solution is lowered to a value ranging from 2 to 5 by adding an inorganic acid.

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