A kind of synthetic method of N-acetyl-L-carnosine
By reacting liquid carrier with Fmoc-L-His-OH, combined with deprotection and condensation steps, the existing N-acetyl-L-carnosine synthesis method has been solved, and the problem of long steps and the use of hazardous chemicals is achieved, achieving an efficient and low-cost synthesis process, suitable for industrial production.
Patent Information
- Application Number
- CN202411629910.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2025-04-01
AI Technical Summary
The existing chemical synthesis method of N-acetyl-L-carnosine has long steps, complex post-processing, uses hazardous chemicals, and has low yields, resulting in high development costs and is not suitable for industrial production.
Reacted with Fmoc-L-His-OH using a liquid phase carrier, and after deprotection, condensation and purification, N-acetyl-L-carnosine was obtained. This method requires no hazardous chemicals, is simple to process, has fast reaction speed and high yield, and is suitable for industrial production.
It has achieved efficient synthesis of N-Berma-L-carnosine, with a product purity of more than 99%, and a yield of more than 80%, which simplifies the process flow, reduces development costs, and is suitable for industrial production.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of organic synthesis, and in particular to a method for synthesizing N-acetyl-L-carnosine. Background Art
[0002] The English name of N-acetyl-L-carnosine is N-Acetyl-L-Carnosine, its chemical name is N-Acetyl-β-Alanyl-L-Histidine, and its chemical formula is C 11 H 16 N4O4, with a relative molecular mass of 268.12 and a CAS number of 56353-15-2, is a white crystalline powder with the following structural formula:
[0003]
[0004] N-acetyl-L-carnosine is not only a very effective treatment for senile cataracts, but also studies have shown that N-acetyl-L-carnosine has the effects of anti-wrinkle, anti-aging and repairing skin damage, and has no toxic side effects. It can effectively delay, prevent, and even partially reverse the aging process of human cells. In addition, the aluminum salt of N-acetyl-L-carnosine can be used as a preventive and healing agent for peptic ulcers. Surveys show that it has become an indisputable fact to use N-acetyl-L-carnosine in beauty products, and as its beauty effects become more obvious, the market demand has further increased. Therefore, the development of this product has great market value.
[0005] At present, there are four main methods reported for the chemical synthesis of N-acetyl-L-carnosine:
[0006] 1. β-alanine is aminoacetylated to obtain N-acetyl-β-alanine, which is then reacted with an acylating agent in a non-polar solvent to obtain N-acetyl-β-alanyl chloride. L-histidine is reacted with organosilane under the catalysis of an acid to obtain organosilane-protected L-histidine, N-acetyl-β-alanyl chloride is condensed with organosilane-protected histidine to obtain N-acetyl-L-carnosine, a polar solvent is added to remove the protecting group, and N-acetyl-L-carnosine is obtained after separation and purification.
[0007] 2. Using β-alanine methyl ester as the starting material, the amino group is modified by 2,4,6-trimethoxybenzylation, further aminoacetylation, hydrolysis of the methyl ester, and then condensation with histidine, hydrolysis of the methyl ester, strong acid deprotection, resin absorption, and ammonia elution to obtain N-acetyl-L-carnosine.
[0008] 3. Add the active ester solution in the presence of acetyl chloride and an acid-binding agent to the aqueous solution of L-carnosine and a protective agent. After the reaction is completed, separate the layers, decolorize, concentrate under reduced pressure, add acetic acid, stir, cool, filter, and dry to obtain N-acetyl-L-carnosine.
[0009] 4. Using polyethylene glycol as a solvent, β-alanine undergoes a first reaction with a condensation reagent; under alkaline conditions, the obtained first reaction product undergoes a second reaction with histidine; the obtained second reaction product undergoes a third reaction with an acetylation reagent to obtain N-acetyl-L-carnosine.
[0010] The above-mentioned method for synthesizing N-acetyl-L-carnosine reported at present has long synthesis steps, inconvenient post-processing, or repeatedly adding and removing protecting groups, complicated post-processing, dangerous chemicals used, and low yield. Therefore, it is of great significance and market prospect to develop a synthetic process of N-acetyl-L-carnosine with low cost, high yield and easy industrialization. Summary of the invention
[0011] In view of this, the present invention provides a method for synthesizing N-acetyl-L-carnosine. The synthesis method provided by the present invention has simple post-processing operation, does not require the use of hazardous chemicals, has a fast synthesis speed, a high yield, and is suitable for industrial production.
[0012] The present invention provides a method for synthesizing N-acetyl-L-carnosine, comprising the following steps:
[0013] A) reacting the liquid phase carrier represented by formula (1) with the compound Fmoc-L-His-OH represented by formula (2) to obtain the compound represented by formula (3);
[0014] B) deprotecting the compound represented by formula (3) to obtain the intermediate 1 represented by formula (4);
[0015] C) reacting the intermediate 1 represented by formula (4) with N-acetyl-β-alanine represented by formula (5) to obtain the intermediate 2 represented by formula (6);
[0016] D) removing the carrier from the intermediate 2 shown in the formula (6), and then performing purification and post-treatment to obtain N-acetyl-L-carnosine;
[0017]
[0018]
[0019] Preferably, in step A), the reaction temperature is 25±5°C and the reaction time is 0.5 to 4h;
[0020] The reaction is carried out in a solvent medium; the solvent is dichloromethane.
[0021] Preferably, in step A), the reaction is carried out in the presence of HOBt, EDCI and DIEA.
[0022] Preferably, step B) specifically comprises: mixing the compound represented by formula (3) with DBU solution to obtain intermediate 1 represented by formula (4).
[0023] Preferably, the reaction temperature in step B) is 25±5° C. and the reaction time is 0.5 to 2 h.
[0024] Preferably, in step C), the reaction temperature is 25±5° C. and the reaction time is 0.5 to 4 h.
[0025] Preferably, in step C), the reaction is carried out in the presence of HOBt, EDCI and DIEA.
[0026] Preferably, step D) specifically comprises: mixing the intermediate 2 represented by formula (6) with a trifluoroacetic acid solution for reaction, separating the solid from the liquid, and removing the solvent from the separated liquid to obtain an oily substance; and then purifying and post-treating the oily substance to obtain N-acetyl-L-carnosine.
[0027] Preferably, the post-purification treatment comprises: adding water to dissolve the oily substance, then adding alkaline substances to adjust the pH value, then adding ethanol, cooling and crystallizing, and solid-liquid separation to obtain an N-acetyl-L-carnosine solid product.
[0028] Preferably, the adjusted pH value is 5.
[0029] The synthesis method provided by the present invention is based on the liquid phase carrier shown in formula (1), condensation, deprotection, and re-condensation are performed on the carrier, and the final product is cut from the carrier and purified and post-processed. The reaction conditions of the above method are mild, the post-processing operation is simple, no hazardous chemicals are required, the reaction speed is fast, the synthesis speed is fast, the raw materials used are simple, there are no impurities, and the purity does not need to be monitored in the middle. After drying, it is directly thrown down, and the final product is monitored together when it is obtained. In general, the above method is a synthesis method with mild reaction, simple process and equipment, easy operation and no damage to the environment, and can ensure product purity and yield.
[0030] The test results show that the product purity of the method of the present invention is above 99% and the yield is above 80%. DETAILED DESCRIPTION
[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.
[0032] In this article, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
[0033] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.
[0034] Herein, when it comes to numerical ranges, unless otherwise specified, the numerical ranges are deemed to be continuous and include the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.
[0035] In this article, when referring to the units of a data range, if only the right endpoint is followed by a unit, it means that the units of the left and right endpoints are the same.
[0036] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and likewise any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.
[0037] The present invention provides a method for synthesizing N-acetyl-L-carnosine, comprising the following steps:
[0038] A) reacting the liquid phase carrier represented by formula (1) with the compound Fmoc-L-His-OH represented by formula (2) to obtain the compound represented by formula (3);
[0039] B) deprotecting the compound represented by formula (3) to obtain the intermediate 1 represented by formula (4);
[0040] C) reacting the intermediate 1 represented by formula (4) with N-acetyl-β-alanine represented by formula (5) to obtain the intermediate 2 represented by formula (6);
[0041] D) removing the carrier from the intermediate 2 shown in the formula (6), and then performing purification and post-treatment to obtain N-acetyl-L-carnosine;
[0042]
[0043]
[0044] [About Step A]:
[0045] A) The liquid phase carrier represented by formula (1) reacts with the compound Fmoc-L-His-OH represented by formula (2) to obtain the compound represented by formula (3).
[0046] The reaction route of step A) is as follows:
[0047]
[0048] In the present invention, the chemical name of the liquid phase carrier shown in formula (1) is 4'-hexadecyloxy-4-biphenylmethanol, and its source is not particularly limited, and it can be prepared according to a preparation method known in the art, see the document Polym.Chem., 2016, 7, 3520-3529. The present invention uses the above compound as a connection carrier, which is stably connected with the intermediate and the target product during the process and is easy to elute, has a high comprehensive yield, and has a good shape when precipitated, which is easy to operate.
[0049] In the present invention, the compound Fmoc-L-His-OH represented by formula (2) is commonly known as Fmoc-L-histidine, and its chemical name is (((9H-fluoren-9-yl)methoxy)carbonyl)-L-histidine, wherein Fmoc (9-fluorenylmethoxycarbonyl) is a protecting group. The present invention has no particular limitation on the source of the compound represented by formula (2), and it can be a commercial product or one prepared according to a preparation method known in the art.
[0050] In the present invention, the mass ratio of the liquid phase carrier represented by formula (1) to the compound Fmoc-L-His-OH represented by formula (2) is preferably 100:(97.76-115.53), and more preferably 100:97.76.
[0051] In the present invention, the reaction temperature is preferably 25±5°C, specifically 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C. The reaction time is preferably 0.5-4h, specifically 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h, 3.5h, 4.0h.
[0052] In the present invention, the reaction is preferably carried out in a solvent medium. The solvent is preferably an organic solvent, more preferably dichloromethane. Using dichloromethane as a solvent, the solubility of the raw material is good, but the solubility of the intermediate and the product is poor. By using this solubility difference, the intermediate or the target product can be obtained by a simple solid-liquid separation operation; dichloromethane is immiscible with water, and the product can be purified by extraction; the intermediate is evenly dispersed in dichloromethane and has good properties, which is convenient for the operation of the suction filtration step; therefore, the use of dichloromethane is conducive to simplifying the overall process and improving the convenience of operation, and is helpful for the product yield. In the present invention, the amount ratio of the liquid phase carrier shown in the formula (1) to the solvent is preferably 100g: (500-2000) mL, specifically 100g: 500mL, 100g: 1000mL, 100g: 1500mL, 100g: 2000mL, and more preferably 100g: 1000mL.
[0053] In the present invention, the reaction is preferably carried out in the presence of HOBt (1-hydroxybenzotriazole), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and DIEA (N,N-diisopropylethylamine). The above three substances act as activators, condensing agents and bases. Among them, the mass ratio of HOBt to the liquid phase carrier represented by formula (1) is preferably (35.00-41.36):100, specifically 35.0:100, 41.36:100. The mass ratio of EDCI to the liquid phase carrier represented by formula (1) is preferably (58.68-67.71):100, specifically 58.68:100. The mass ratio of the liquid phase carrier represented by the DIEA formula (1) is preferably (45.65-76.08):100, specifically 45.65:100, 60.87:100, or 76.08:100.
[0054] In the present invention, the step A) preferably specifically comprises: mixing the liquid phase carrier shown in formula (1) with a solvent, then adding the compound Fmoc-L-His-OH, HOBt, EDCI and DIEA shown in formula (2), reacting, and then filtering under reduced pressure to obtain the compound shown in formula (3). Wherein, after the reduced pressure filtering, it is preferably treated as follows: pulping the filter cake with a solvent, filtering, and then repeatedly pulping with a solvent to collect the filter cake. Wherein, the solvent is preferably the same as the solvent used in the reaction, and is preferably dichloromethane. The amount ratio of the solvent used for each pulping to the liquid phase carrier shown in formula (1) is preferably (300-800) mL: 100 g, and can be specifically 300 mL: 100 g, 400 mL: 100 g, 500 mL: 100 g, 600 mL: 100 g, 700 mL: 100 g, 800 mL: 100 g, and more preferably 500 mL: 100 g. The beating time for each time is preferably 10 to 60 minutes, specifically 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, and more preferably 10 minutes. After beating, the substance in the filter cake finally collected is the compound represented by formula (3).
[0055] [About step B]:
[0056] B) Deprotecting the compound represented by formula (3) to obtain the intermediate 1 represented by formula (4).
[0057] The reaction route of step B) is as follows:
[0058]
[0059] In the present invention, the deprotection of the compound represented by formula (3) is to remove the Fmoc group in FmocHN- in formula (3) to convert FmocHN- into H2N-.
[0060] In the present invention, step B) preferably specifically comprises: mixing the compound represented by formula (3) with a DBU solution to obtain an intermediate 1 represented by formula (4).
[0061] in,
[0062] The DBU solution is a solution formed by dissolving DBU (1,8-diazobispiro[5.4.0]undec-7-ene, also known as bicyclic amidine) in a solvent. The solvent is preferably an organic solvent, more preferably dichloromethane. The mass percentage concentration of the DBU solution is preferably 10% to 40%, specifically 10%, 20%, 30%, 40%, and more preferably 20%. The amount ratio of the DBU solution to the liquid phase carrier represented by formula (1) used in step A) is preferably (300 to 800) mL: 100 g, specifically 300 mL: 100 g, 400 mL: 100 g, 500 mL: 100 g, 600 mL: 100 g, 700 mL: 100 g, 800 mL: 100 g, and more preferably 500 mL: 100 g. The temperature of the reaction is preferably 25±5°C, specifically 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C. The reaction time is preferably 0.5-2h, specifically 0.5h, 1.0h, 1.5h, 2.0h. The reaction process is preferably accompanied by stirring. After the reaction is completed, it is preferably further treated as follows: adding a solvent for pulping and filtering. Among them, the solvent is preferably dichloromethane. The amount ratio of the solvent used for each pulping to the liquid phase carrier shown in formula (1) is preferably (300-800) mL: 100g, specifically 300mL: 100g, 400mL: 100g, 500mL: 100g, 600mL: 100g, 700mL: 100g, 800mL: 100g, more preferably 500mL: 100g. The time of each beating is preferably 10 to 60 minutes, specifically 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, and more preferably 10 minutes. The operation of adding solvent for beating and filtering can be repeated, and the present invention is preferably performed twice. After the above treatment, the intermediate 1 shown in formula (4) is obtained. The intermediate does not need to be tested for purity and is directly used for the next step of feeding.
[0063] [About Step C]:
[0064] C) The intermediate 1 represented by formula (4) reacts with N-acetyl-β-alanine represented by formula (5) to obtain the intermediate 2 represented by formula (6).
[0065] The reaction route of step C) is as follows:
[0066]
[0067] In the present invention, the source of N-acetyl-β-alanine represented by formula (5) is not particularly limited, and can be a commercial product or prepared according to a conventional preparation method known in the art. In the present invention, the mass ratio of N-acetyl-β-alanine represented by formula (5) to the liquid phase carrier represented by formula (1) used in step A) is preferably (18.40-21.7):100, more preferably 18.4:100.
[0068] In the present invention, the reaction temperature is preferably 25±5°C, specifically 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C. The reaction time is preferably 0.5-4h, specifically 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h, 3.5h, 4.0h.
[0069] In the present invention, the reaction is preferably carried out in a solvent medium. The solvent is preferably an organic solvent, more preferably dichloromethane. In the present invention, the amount ratio of the solvent to the liquid phase carrier represented by formula (1) in step A) is preferably (500-2000) mL: 100 g, specifically 500 mL: 100 g, 1000 mL: 100 g, 1500 mL: 100 g, 2000 mL: 100 g, more preferably 1000 mL: 100 g.
[0070] In the present invention, the reaction is preferably carried out in the presence of HOBt (1-hydroxybenzotriazole), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and DIEA (N,N-diisopropylethylamine). The mass ratio of HOBt to the liquid phase carrier represented by formula (1) in step A) is preferably (26.46-31.27):100, specifically 26.46:100, 31.27:100. The mass ratio of EDCI to the liquid phase carrier represented by formula (1) in step A) is preferably (31.78-36.67):100, specifically 31.78:100. The mass ratio of the liquid phase carrier represented by formula (1) in the DIEA step A) is preferably (24.73-41.21):100, specifically 24.73:100, 32.97:100, or 41.21:100.
[0071] In the present invention, the step C) preferably specifically comprises: mixing the intermediate 1 shown in formula (4) with a solvent, then adding N-acetyl-β-alanine, HOBt, EDCI and DIEA shown in formula (5), reacting, and then filtering under reduced pressure to obtain the intermediate 2 shown in formula (6). Wherein, after the reduced pressure filtering, the following treatment is preferably performed: pulping the filter cake with a solvent, filtering, and then repeatedly pulping with a solvent to collect the filter cake. Wherein, the solvent is preferably the same as the solvent used in the reaction, and is preferably dichloromethane. The amount ratio of the solvent used for each pulping to the liquid phase carrier shown in formula (1) used in step A) is preferably (300-800) mL: 100 g, and can be specifically 300 mL: 100 g, 400 mL: 100 g, 500 mL: 100 g, 600 mL: 100 g, 700 mL: 100 g, 800 mL: 100 g, and more preferably 500 mL: 100 g. The beating time for each time is preferably 10 to 60 minutes, specifically 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, and more preferably 10 minutes. After beating, the substance in the filter cake finally collected is the intermediate 2 shown in formula (6).
[0072] [About Step D]:
[0073] D) removing the carrier from the intermediate 2 represented by the formula (6), and then performing purification and post-treatment to obtain N-acetyl-L-carnosine.
[0074] The reaction route of step D) is as follows:
[0075]
[0076] In the present invention, step D) preferably specifically comprises: mixing the intermediate 2 represented by formula (6) with a trifluoroacetic acid solution for reaction, separating the solid from the liquid, and then removing the solvent from the obtained separated liquid to obtain an oily substance; then, purifying the oily substance to obtain N-acetyl-L-carnosine.
[0077] Wherein, the trifluoroacetic acid solution is a solution formed by dissolving trifluoroacetic acid in an organic solvent. The organic solvent is preferably dichloromethane. The mass percentage concentration of the trifluoroacetic acid solution is preferably 20% to 60%, specifically 20%, 30%, 40%, 50%, 60%, and more preferably 30%. The amount ratio of the trifluoroacetic acid solution to the liquid phase carrier shown in formula (1) used in step A) is preferably (300 to 800) mL: 100 g, specifically 300 mL: 100 g, 400 mL: 100 g, 500 mL: 100 g, 600 mL: 100 g, 700 mL: 100 g, 800 mL: 100 g, and more preferably 500 mL: 100 g. The temperature of the reaction is preferably 25 ± 5 ° C, specifically 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C. The reaction time is preferably 4 to 8 hours, specifically 4 hours, 5 hours, 6 hours, 7 hours, and 8 hours. The reaction is preferably accompanied by stirring. After the reaction is completed, solid-liquid separation is performed. The solid-liquid separation method is preferably suction filtration, and the filtrate is collected after solid-liquid separation. After suction filtration, a solvent is added to rinse the filter cake, and the filtrate is continued to be collected. The solvent used for elution is preferably dichloromethane. After the filtrate is collected, the solvent is removed. The method for removing the solvent is preferably rotary evaporation to remove the solvent. After removing the solvent, an oily substance is obtained.
[0078] After obtaining the oily substance, the present invention performs a purification post-treatment on it. In the present invention, the purification post-treatment preferably includes: adding water to dissolve the oily substance, then adding an alkaline substance to adjust the pH value, then adding ethanol, cooling and crystallizing, and solid-liquid separation to obtain an N-acetyl-L-carnosine solid product. Wherein, the amount ratio of the water to the liquid phase carrier shown in formula (1) used in step A) is preferably (30-60) mL: 100 g, specifically 30 mL: 100 g, 40 mL: 100 g, 50 mL: 100 g, 60 mL: 100 g, and more preferably 50 mL: 100 g. The alkaline substance is preferably at least one of sodium carbonate, potassium carbonate, sodium bicarbonate and potassium bicarbonate. The pH value is preferably adjusted to 5. At this pH value, crystallization can reduce losses and ensure a high crystallization yield. If the pH is too high or too low, it may cause a low crystallization yield. The method of adding ethanol is preferably dropwise addition. The amount ratio of the liquid phase carrier shown in formula (1) used in the ethanol step A) is preferably (200-1000) mL: 100 g, specifically 200 mL: 100 g, 300 mL: 100 g, 400 mL: 100 g, 500 mL: 100 g, 600 mL: 100 g, 700 mL: 100 g, 800 mL: 100 g, 900 mL: 100 g, 1000 mL: 100 g, more preferably 600 mL: 100 g. After adding ethanol, cool and crystallize. The cooling is preferably reduced to -10-10°C, specifically -10°C, -5°C, 0°C, 5°C, 10°C, more preferably 10°C. After cooling to the above target temperature, wait for crystallization. The crystallization process is preferably accompanied by stirring. The crystallization time is preferably 4-8h, specifically 4h, 5h, 6h, 7h, 8h. The present invention adopts a water-ethanol system for crystallization, and its crystallization purification effect is better, the crystal form is better, and the pollution is less. After crystallization, solid-liquid separation is performed. The method of the solid-liquid separation is preferably suction filtration. After the solid is separated to obtain a solid, it is preferably washed with ethanol to finally obtain an N-acetyl-L-carnosine solid product.
[0079] The synthesis method provided by the present invention is based on the liquid phase carrier shown in formula (1), condensation, deprotection, and re-condensation are performed on the carrier, and the final product is cut from the carrier and purified and post-processed. The reaction conditions of the above method are mild, the post-processing operation is simple, no hazardous chemicals are required, the reaction speed is fast, the synthesis speed is fast, the raw materials used are simple, there are no impurities, and the purity does not need to be monitored in the middle. After drying, it is directly thrown down, and the final product is monitored together when it is obtained. In general, the above method is a synthesis method with mild reaction, simple process and equipment, easy operation and no damage to the environment, and can ensure product purity and yield.
[0080] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.
[0081] Example 1
[0082] A) Preparation of the compound represented by formula (3)
[0083] In a 2000mL reactor, 100g of the liquid phase carrier represented by formula (1) and 1000mL of dichloromethane were added, and after stirring evenly, 97.76g (1.1eq) of the compound Fmoc-L-His-OH represented by formula (2), 35.0g (1.1eq) of HOBt, 58.68g (1.3eq) of EDCI, and 45.65g (1.5eq) of DIEA were added in sequence, and the mixture was reacted at 25±5°C for 4h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500mL of dichloromethane for 10min, filtered, and 500mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the compound represented by formula (3).
[0084] B) Preparation of Intermediate 1
[0085] Add 500 mL of DBU-dichloromethane solution (mass percentage concentration of 20%) to the filter cake obtained in step A), and stir at 25±5°C for 2 h. After the reaction is completed, add 500 mL of dichloromethane to slurry for 10 min, filter under reduced pressure, then add 500 mL of dichloromethane again to slurry for 10 min, filter under reduced pressure, collect the filter cake, and obtain the intermediate 1 shown in formula (4), which is directly used for the next step.
[0086] C) Preparation of Intermediate 2
[0087] The intermediate 1 and 1000 mL of dichloromethane were added to a 2000 mL reactor, and after stirring, 18.4 g (1.1 eq) of N-acetyl-β-alanine, 26.46 g (1.1 eq) of HOBt, 31.78 g (1.3 eq) of EDCI, and 24.73 g (1.5 eq) of DIEA were added in sequence, and the mixture was reacted at 25°C ± 5°C for 4 h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500 mL of dichloromethane for 10 min, filtered, and then 500 mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the intermediate 2 shown in formula (6), which was directly used for the next step.
[0088] D) Preparation of N-acetyl-L-carnosine
[0089] Add 500mL of trifluoroacetic acid-dichloromethane solution (mass percentage concentration is 30%) and the above intermediate 2 into a 1000mL reactor, and stir and react at 25±5℃ for 4h. After the reaction is completed, the reaction solution is filtered, and the filter cake is rinsed twice with 100mL of dichloromethane, and the filtrate is collected. The filtrate is evaporated to remove the solvent to obtain an oily substance. Add 100mL of water to the oily substance to dissolve it, then add sodium carbonate solid to adjust the pH value to 5, add 600mL of ethanol dropwise, cool to 10℃, stir and crystallize for 4h, filter under reduced pressure, rinse the solid with 100mL of ethanol once, and obtain 33.85g of N-acetyl-L-carnosine finished product with a purity of 99% and a yield of 85%.
[0090] Example 2
[0091] A) Preparation of the compound represented by formula (3)
[0092] In a 2000mL reactor, 100g of the liquid phase carrier represented by formula (1) and 1000mL of dichloromethane were added, and after stirring, 97.76g (1.1eq) of the compound Fmoc-L-His-OH represented by formula (2), 41.36g (1.3eq) of HOBt, 58.68g (1.3eq) of EDCI, and 45.65g (1.5eq) of DIEA were added in sequence, and the mixture was reacted at 25±5°C for 4h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500mL of dichloromethane for 10min, filtered, and 500mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the compound represented by formula (3).
[0093] B) Preparation of Intermediate 1
[0094] Add 500 mL of DBU-dichloromethane solution (mass percentage concentration of 20%) to the filter cake obtained in step A), and stir at 25±5°C for 2 h. After the reaction is completed, add 500 mL of dichloromethane to slurry for 10 min, filter under reduced pressure, then add 500 mL of dichloromethane again to slurry for 10 min, filter under reduced pressure, collect the filter cake, and obtain the intermediate 1 shown in formula (4), which is directly used for the next step.
[0095] C) Preparation of Intermediate 2
[0096] The intermediate 1 and 1000 mL of dichloromethane were added to a 2000 mL reactor, and after stirring, 18.4 g (1.1 eq) of N-acetyl-β-alanine, 31.27 g (1.3 eq) of HOBt, 31.78 g (1.3 eq) of EDCI, and 24.73 g (1.5 eq) of DIEA were added in sequence, and the mixture was reacted at 25°C ± 5°C for 4 h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500 mL of dichloromethane for 10 min, filtered, and then 500 mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the intermediate 2 shown in formula (6), which was directly used for the next step of feeding.
[0097] D) Preparation of N-acetyl-L-carnosine
[0098] Add 500mL of trifluoroacetic acid-dichloromethane solution (mass percentage concentration is 30%) and the above intermediate 2 into a 1000mL reactor, and stir and react at 25±5℃ for 8h. After the reaction is completed, the reaction solution is filtered, and the filter cake is rinsed twice with 100mL of dichloromethane, and the filtrate is collected. The filtrate is evaporated to remove the solvent to obtain an oily substance. Add 100mL of water to the oily substance to dissolve it, then add sodium carbonate solid to adjust the pH value to 5, add 1200mL of ethanol dropwise, cool to 10℃, stir and crystallize for 8h, filter under reduced pressure, rinse the solid with 100mL of ethanol once, and obtain 31.8g of N-acetyl-L-carnosine finished product with a purity of 99% and a yield of 80%.
[0099] Example 3
[0100] A) Preparation of the compound represented by formula (3)
[0101] In a 2000mL reactor, 100g of the liquid phase carrier represented by formula (1) and 1000mL of dichloromethane were added, and after stirring, 97.76g (1.1eq) of the compound Fmoc-L-His-OH represented by formula (2), 41.36g (1.3eq) of HOBt, 58.68g (1.3eq) of EDCI, and 60.87g (2.0eq) of DIEA were added in sequence, and the mixture was reacted at 25±5°C for 4h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500mL of dichloromethane for 10min, filtered, and 500mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the compound represented by formula (3).
[0102] B) Preparation of Intermediate 1
[0103] Add 500 mL of DBU-dichloromethane solution (mass percentage concentration of 20%) to the filter cake obtained in step A), and stir at 25±5°C for 2 h. After the reaction is completed, add 500 mL of dichloromethane to slurry for 10 min, filter under reduced pressure, then add 500 mL of dichloromethane again to slurry for 10 min, filter under reduced pressure, collect the filter cake, and obtain the intermediate 1 shown in formula (4), which is directly used for the next step.
[0104] C) Preparation of Intermediate 2
[0105] The intermediate 1 and 1000 mL of dichloromethane were added to a 2000 mL reactor, and after stirring, 18.4 g (1.1 eq) of N-acetyl-β-alanine, 31.27 g (1.3 eq) of HOBt, 31.78 g (1.3 eq) of EDCI, and 32.97 g (2.0 eq) of DIEA were added in sequence, and the mixture was reacted at 25°C ± 5°C for 4 h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500 mL of dichloromethane for 10 min, filtered, and then 500 mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the intermediate 2 shown in formula (6), which was directly used for the next step of feeding.
[0106] D) Preparation of N-acetyl-L-carnosine
[0107] Add 500mL of trifluoroacetic acid-dichloromethane solution (mass percentage concentration is 30%) and the above intermediate 2 into a 1000mL reactor, and stir and react at 25±5℃ for 8h. After the reaction is completed, the reaction solution is filtered, and the filter cake is rinsed twice with 100mL of dichloromethane, and the filtrate is collected. The filtrate is evaporated to remove the solvent to obtain an oily substance. Add 50mL of water to the oily substance to dissolve it, then add solid sodium carbonate to adjust the pH value to 5, add 1200mL of ethanol dropwise, cool to 10℃, stir and crystallize for 8h, filter under reduced pressure, rinse the solid with 100mL of ethanol once, and obtain 34.07g of N-acetyl-L-carnosine finished product with a purity of 99% and a yield of 86%.
[0108] Example 4
[0109] A) Preparation of the compound represented by formula (3)
[0110] In a 2000mL reactor, 100g of the liquid phase carrier represented by formula (1) and 1000mL of dichloromethane were added, and after stirring, 97.76g (1.1eq) of the compound Fmoc-L-His-OH represented by formula (2), 41.36g (1.3eq) of HOBt, 58.68g (1.3eq) of EDCI, and 76.08g (2.5eq) of DIEA were added in sequence, and the mixture was reacted at 25±5°C for 4h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500mL of dichloromethane for 10min, filtered, and 500mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the compound represented by formula (3).
[0111] B) Preparation of Intermediate 1
[0112] Add 500 mL of DBU-dichloromethane solution (mass percentage concentration of 20%) to the filter cake obtained in step A), and stir at 25±5°C for 2 h. After the reaction is completed, add 500 mL of dichloromethane to slurry for 10 min, filter under reduced pressure, then add 500 mL of dichloromethane again to slurry for 10 min, filter under reduced pressure, collect the filter cake, and obtain the intermediate 1 shown in formula (4), which is directly used for the next step.
[0113] C) Preparation of Intermediate 2
[0114] The intermediate 1 and 1000 mL of dichloromethane were added to a 2000 mL reactor, and after stirring, 18.4 g (1.1 eq) of N-acetyl-β-alanine, 31.27 g (1.3 eq) of HOBt, 31.78 g (1.3 eq) of EDCI, and 41.21 g (2.5 eq) of DIEA were added in sequence, and the mixture was reacted at 25°C ± 5°C for 4 h. After the reaction was completed, the mixture was filtered under reduced pressure, and the filter cake was slurried with 500 mL of dichloromethane for 10 min, filtered, and then 500 mL of dichloromethane was added again to repeat the slurrying once, and the filter cake was collected to obtain the intermediate 2 shown in formula (6), which was directly used for the next step.
[0115] D) Preparation of N-acetyl-L-carnosine
[0116] Add 500mL of trifluoroacetic acid-dichloromethane solution (mass percentage concentration is 30%) and the above intermediate 2 into a 1000mL reactor, and stir and react at 25±5℃ for 8h. After the reaction is completed, the reaction solution is filtered, and the filter cake is rinsed twice with 100mL of dichloromethane, and the filtrate is collected. The filtrate is evaporated to remove the solvent to obtain an oily substance. Add 50mL of water to the oily substance to dissolve it, then add sodium carbonate solid to adjust the pH value to 5, add 600mL of ethanol dropwise, cool to 10℃, stir and crystallize for 8h, reduce pressure and filter, and rinse the solid with 100mL of ethanol once to obtain 33.47g of N-acetyl-L-carnosine finished product with a purity of 99% and a yield of 84%.
[0117] The synthesis method provided by the present invention is based on the liquid phase carrier shown in formula (1), condensation, deprotection, and re-condensation are performed on the carrier, and the final product is cut from the carrier and purified and post-processed. The synthesis method of the present invention uses only one condensing agent, only removes one protecting group, and the post-processing is simple and convenient. The product purity and yield are high, the solvent is non-toxic, green and environmentally friendly, and suitable for industrial production.
[0118] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas, including the best mode, and also enable any technician in the field to practice the present invention, including making and using any device or system, and implementing any combined method. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The scope of patent protection of the present invention is defined by the claims and may include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements similar to the literal expression of the claims, or if they include equivalent structural elements that are not substantially different from the literal expression of the claims, then these other embodiments should also be included in the scope of the claims.
Claims
1. A method for synthesizing N-acetyl-L-carnosine, characterized in that, The following steps are involved: A) reacting the liquid carrier represented by formula (1) with the compound Fmoc-L-His-OH represented by formula (2) to obtain the compound represented by formula (3); B) deprotecting the compound represented by formula (3) to obtain the intermediate 1 represented by formula (4); C) reacting the intermediate 1 represented by formula (4) with N-acetyl-β-alanine represented by formula (5) to obtain the intermediate 2 represented by formula (6); D) removing the carrier from the intermediate 2 shown in the formula (6), and then performing purification and post-treatment to obtain N-acetyl-L-carnosine; 2. The preparation method according to claim 1, characterized in that: In step A), the reaction temperature is 25±5°C and the reaction time is 0.5 to 4 hours; The reaction is carried out in a solvent medium; the solvent is dichloromethane.
3. The preparation method according to claim 1, characterized in that: In step A), the reaction is carried out in the presence of HOBt, EDCI and DIEA.
4. The preparation method according to claim 1, characterized in that: Step B) specifically comprises: mixing the compound represented by formula (3) with DBU solution to obtain the intermediate 1 represented by formula (4).
5. The preparation method according to claim 4, characterized in that: The reaction temperature in step B) is 25±5°C and the reaction time is 0.5 to 2 hours.
6. The preparation method according to claim 1, characterized in that: In step C), the reaction temperature is 25±5° C. and the reaction time is 0.5 to 4 h.
7. The preparation method according to claim 1 or 6, characterized in that: In step C), the reaction is carried out in the presence of HOBt, EDCI and DIEA.
8. The preparation method according to claim 1, characterized in that: Step D) specifically comprises: mixing the intermediate 2 represented by formula (6) with a trifluoroacetic acid solution for reaction, separating the solid from the liquid, and removing the solvent from the separated liquid to obtain an oily substance; and then purifying and post-treating the oily substance to obtain N-acetyl-L-carnosine.
9. The preparation method according to claim 8, characterized in that: The post-purification treatment comprises: adding water to dissolve the oily substance, then adding alkaline substances to adjust the pH value, then adding ethanol, cooling and crystallizing, and solid-liquid separation to obtain an N-acetyl-L-carnosine solid product.
10. The preparation method according to claim 9, characterized in that: The adjusted pH value is 5.