JPH029028B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing N-acyl-L-carnosine. N-acyl-L-carnosine is N-acyl-β
It has the structure of -alanyl-L-histidine, and in particular, N-acetyl-L-carnosine, like L-carnosine, has a strong inhibitory effect on the limbic system and is useful as a medicine. The aluminum salt also has excellent anti-ulcer properties. Conventionally, A.Coll and G.
City is L in J.Org.Chom., 26 , 617 (1961))
It is reported that N-acetylcarnosine was obtained by acetylating -carnosine using acetic anhydride in glacial acetic acid. However, when the present inventor tried this known method again, as is clear from Reference Example 1 below, not only the raw material L-carnosine was racemized, but also the nitrogen atom in the imidazole nucleus of histidine was acetylated at the same time. It was confirmed that there are drawbacks such as easy generation of by-products. Therefore, as a result of a detailed study of the acylation conditions of L-carnosine, the present inventors conducted an acylation reaction on L-carnosine in an alkaline aqueous medium while maintaining a pH value of approximately 9 to 13.5 and a reaction temperature of approximately 5 to 25°C. When carried out, side reactions due to racemization and acylation of the nitrogen atom on the imidazole nucleus in histidine are completely suppressed, and the desired N-acyl-L-carnosine can be obtained in high yield. They discovered this and completed the present invention. That is, the present invention involves the production of N-acylcarnosine by reacting L-carnosine with an acyl donor, while maintaining the pH at about 9 to 13.5 and the reaction temperature at about 5 to 25°C in an alkaline aqueous medium. This is a method for producing N-acyl-L-carnosine, which is characterized by carrying out a reaction. In the present invention, the general formula (RCO) ₂ O (wherein R
is an alkyl, aryl or aralkyl group) or a carboxylic acid anhydride with the general formula RCOCl (however,
Carboxylic acid chlorides represented by the formula (in which R represents an alkyl, aryl or aralkyl group) are preferably used, and representative examples thereof include acetic anhydride, propionic anhydride, formic acid-acetic acid mixed acid anhydride, acetyl chloride, propionyl chloride. , benzoyl chloride, phenylacetyl chloride and the like. In addition, L-carnosine, which is one of the raw materials, may be in the form of a hydrochloride in addition to the free form. Generally, the reaction is carried out by adding the acyl donor as it is or dissolved in an organic solvent to an aqueous solution of L-carnosine or a mixed solution of water and an aqueous organic solvent under basic conditions. As the water-soluble organic solvent, for example, acetone, acetonitrile, methanol, etc. are used, and as the organic solvent, for example, acetone, acetonitrile, chloroform, dichloromethane, ethylene dichloride, methyl isobutyl ketone, etc. are used. As basic conditions, the pH of the reaction medium is maintained at 9.0 to 13.5, preferably 11.5 to 13.5 using an inorganic base such as sodium hydroxide or potassium hydroxide or an organic base such as triethylamine, tri-n-butylamine, or pyridine. By doing so, side reactions are suppressed as much as possible, and the desired N-acyl-L-carnosine can be obtained in high yield. In order to maintain the pH within the above range during the reaction, it is preferable to add the acyl donor and the base solution to the L-carnosine solution at the same time. The reaction temperature is preferably 25°C or lower, preferably 5 to 10°C. Since L-carnosine is generally more expensive than the acyl donor, an excess of the acyl donor is used to drive the reaction to completion. Preferably 1.1 to 1.5 times the molar amount of acyl donor is used. After the reaction is complete, the reaction mixture is diluted with e.g.
SK-1B, Amberlight IR-120, DOWEX
If treated with a strongly acidic ion exchange resin (H-type) such as 50W, the target substance will be adsorbed to the resin, so it will be effectively removed from the carboxylic acid and inorganic substances generated by hydrolysis of the unreacted acyl donor. Separated. Next, the resin was eluted with aqueous ammonia, concentrated and deammoniated, and then passed through a weakly acidic ion exchange resin (H-type) such as Amberlite IRC-50 to remove a small amount of unreacted L-carnosine and By removing ammonia, a highly purified target product can be easily obtained. Desalting can also be easily carried out by electrodialyzing the reaction mixture or by extracting the target product from the residue with a water-soluble solvent such as methanol after concentration. Thereafter, the resin treatment as described above may be performed as necessary. The N-acyl-L-carnosine thus obtained has a sufficiently high purity as it is, but it can be made into a purified product by further crystallizing it from a water-isopropanol mixed solvent. The present invention will be explained in detail below using Examples and Reference Examples. Example 1 90.4 g (0.40 mol) of L-carnosine was dissolved in 200 ml of 2N aqueous sodium hydroxide solution, and added to this solution with stirring at a reaction temperature of 5°C to 10°C and a pH of 13.0 to
48.6g of 96% acetic anhydride while maintaining the range of 13.5
(0.44 mol) and 2N sodium hydroxide aqueous solution 260
ml was added dropwise at the same time. After the dropwise addition was completed, the reaction product was stirred at the same temperature for 1 hour and analyzed by silica gel thin layer chromatography.
Although the presence of acetylcarnosine was recognized, the raw material carnosine and the byproduct N-acetyl-N ^im
*-The presence of acetylcarnosine was not observed (*indicates the nitrogen atom on the imidazole nucleus in histidine). This reaction solution was passed through a strongly acidic ion exchange resin Diaion SK1B to adsorb N-acetylcarnosine. After that, wash with water until neutral, then elute with 1N aqueous ammonia,
The eluate was concentrated to 200ml. After adding acetic acid to this concentrated solution and adjusting the pH to 4.90, the temperature of this solution was adjusted to
Isopropyl alcohol 780 while keeping at 50℃
Added ml. After stirring at the same temperature for 3 hours and further stirring at 20°C for 1 hour, the precipitated crude N-acetyl-L
- Carnosine was dried to obtain 86.6 g. Its specific optical rotation was [α] ²⁰ _D =+26.10 (C=3, water). 86.6g of crude N-acetyl-L-carnosine in water
After dissolving in 104ml and decolorizing by adding 0.8g of activated carbon,
While maintaining this decolorizing solution at 60° C., 780 ml of isopropyl alcohol was added. Stir at the same temperature for 3 hours,
After further stirring at 20° C. for 1 hour, the precipitated N-acetyl-L-carnosine was dried to obtain 82.4 g. The thus obtained N-acetyl-L-carnosine was analyzed by IR and NMR, and as a result, it completely matched with the standard product. Furthermore, analysis by silica gel thin layer chromatography gave a single spot. Also, its specific rotation is [α] ²⁰ _D = +26.20
(C=3, water) and matched the standard sample, confirming that no racemization occurred during acetylation in this method. Silica gel thin layer chromatography analysis used a Merck silica gel 60 thin layer plate, and after developing with a mixed solvent of ethyl acetate: acetic acid: water = 2:1:1,
Detection was performed using iodine. Under these conditions, the Rf values of diacetylcarnosine, acetylcarnosine, carnosine, and histidine are as follows:
They were 0.43, 0.36, 0.22, and 0.27. Example 2 22.6 g (0.10 mol) of L-carnosine was dissolved in 55 ml of a 2N aqueous sodium hydroxide solution, and the pH was adjusted to 13.3. Add 14.0 g of 96% acetic anhydride to this solution while stirring.
(0.132 mol) and 90 ml of 2N sodium hydroxide solution
were simultaneously added dropwise over 1.5 hours. During this time, the pH after the reaction is 12.5 to 13.5, and the reaction temperature is 18 to 20℃.
was held at After stirring at the same temperature for 1 hour, the reaction product was analyzed by silica gel thin layer chromatography, and it was found that the target product, N-acetylcarnosine, was produced, but the starting material, carnosine, and the by-product, N- Acetyl-N ^im -acetylcarnosine was not observed. This reaction solution was used in Example 1.
Similarly, strongly acidic ion exchange resin treatment and PH adjustment,
A water-isopropanol crystallization treatment was performed to obtain 21.2 g of N-acetyl-L-carnosine. Its specific optical rotation was [α] ²⁰ _D =+26.10 (C=3, water), so no racemization occurred. Example 3 33.9 g (0.15 mol) of L-carnosine in 100 ml of water
and 100 ml of acetone, and about 83 ml of 2N aqueous sodium hydroxide solution was added to adjust the pH to 12.5. To this solution, 100 ml of an acetone solution containing 25.2 g of benzoyl chloride and a 2N aqueous sodium hydroxide solution were simultaneously added dropwise at a reaction temperature of 10° C. while stirring. During the dropwise addition, the pH after the reaction was maintained at 11.5 to 12.5. After the dropwise addition was completed, the mixture was stirred at room temperature for 30 minutes, and then the acetone was distilled off under reduced pressure, 100 ml of water was added, and the mixture was treated in the same manner as in Example 1 to obtain 38.0 g of N-benzoyl-L-carnosine crystals. This stuff has a melting point of 216
~218°C, and the specific optical rotation is [α] ²⁵ _D = +10 (C =
3, H ₂ O), and no racemization was observed. Also, both IR and NMR were consistent with the standard sample. Furthermore, thin layer chromatography analysis revealed only a single spot. Reference example 1 A.Coll and G.City.J.Org.Chem., 26 , 617
(1961) L-carnosine 10.5
g in 30 g of glacial acetic acid, and added acetic anhydride with stirring.
8.5g was gradually added dropwise to 20-25°C. After that,
The reaction was stirred at room temperature overnight to obtain a homogeneous and clear solution. The reaction solution was concentrated under reduced pressure, and 100 ml of water was added to the resulting syrup, which was further concentrated under reduced pressure.
This operation was performed twice. When the obtained aqueous solution was analyzed by silica gel thin layer chromatography, the presence of the raw material L-carnosine was not recognized, but in addition to the target N-acetylcarnosine, the by-product N-acetyl-N ^im -The presence of acetylcarnosine was observed. This reaction solution was treated with a strongly acidic ion exchange resin Diaion SK1B and water-isopropanol crystallization treatment in the same manner as in Example 1.
7.8 g of acetylcarnosine was obtained. This product showed no specific rotation ([α] ²⁰ _D =O (C=3, water)) and was completely racemic. Reference example 2 22.6g (0.10mol) of L-carnosine in 200ml of water
42.5 g (0.40 mol) of 96% acetic anhydride was added dropwise to this solution with stirring at 5 to 10° C. without adjusting the pH. After the dropwise addition was completed, the mixture was stirred at the same temperature for 2 hours.
When the reaction product was analyzed by silica gel thin layer chromatography, it was found that in addition to the target N-acetylcarnosine, unreacted carnosine and by-product N
-Acetyl-N ^im -acetylcarnosine was observed to be produced. This reaction solution was treated with a strongly acidic ion exchange resin Diaion SK-1B in the same manner as in Example 1 to remove the by-product N-acetyl-N ^im -acetylcarnosine. Furthermore, in order to remove unreacted carnosine, the eluate was passed through a weakly acidic ion exchange resin Amberlite C-50, and then washed with water until it became neutral. Combine the penetrating liquid and washing liquid and heat under reduced pressure for 30 minutes.
Concentrated to ml. After that, the same operation as in Example 1 was carried out, and 11.9 g of N-acetyl-L-carnosine was obtained.
Obtained. This has a specific optical rotation [α] ²⁰ _D = +24.10 (C = 3,
water), which was lower than that of the standard product. (Standard [α] ²⁰ _D = +
26 (C=3, water)) Therefore, it is considered that partial racemization occurs during the acetylation step. Reference example 3 22.6g (0.10mol) of L-carnosine in 100ml of water
to this solution under stirring at a reaction temperature of 5 to
50 ml of an aqueous solution containing 16.6 g of 96% acetic anhydride and 15.9 g of sodium carbonate was simultaneously added dropwise while maintaining the temperature at 10° C. and the pH of the reaction solution in the range of 7.0 to 7.4. After finishing dropping,
After stirring at the same temperature for 1 hour, the pH was adjusted to 5.0 with acetic acid. When the reaction product was analyzed by silica gel thin layer chromatography, it was found that in addition to the target product N-acetylcarnosine, unreacted carnosine and a by-product N-acetyl-N ^im -acetylcarnosine were produced. This reaction solution was subjected to the same operation as in Reference Example 2, and 18.5 g of N-acetyl-L-carnosine was obtained.
I got it. The N-acetylcarnosine thus obtained had a specific optical rotation [α] ²⁰ _D =+21.45 (C=3, H ₂ O), which did not match that of the standard product. Therefore, it is considered that partial racemization occurs during the acetylation step. The reaction conditions and results obtained in Examples 1 to 3 and Reference Examples 1 to 3 are collectively shown in Table 1. As is clear from the table, according to the method of the present invention,
It will be understood that the reaction proceeds smoothly and the desired N-acyl-L-carnosine is obtained in good yield without any racemization.

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【table】

Claims (1)

[Scope of Claims] 1. When producing N-acylcarnosine by reacting an acyl donor with L-carnosine, in an alkaline aqueous medium, the pH is about 9 to 13.5, and the reaction temperature is about 5 to 25°C.
1. A method for producing N-acyl-L-carnosine, which comprises carrying out an acylation reaction while maintaining 2 The acyl donor has the general formula (RCO) ₂ O or
The method according to claim 1, which is a carboxylic acid anhydride or carboxylic acid chloride represented by RCOCl (wherein R represents an alkyl, aryl, or aralkyl group). 3. The production method according to claim 1, wherein the acyl donor is acetic anhydride. 4. The production method according to claim 1, wherein the acyl donor is benzoyl chloride. 5. Claims 1, 2, 3, or 4, wherein the alkaline aqueous medium is an aqueous sodium hydroxide solution or a mixed solvent of an aqueous sodium hydroxide solution and a water-soluble organic solvent.
Manufacturing method described in section.