A process for the synthesis of coenzyme tetrahydrate

Abstract

The application discloses a synthesis method of tetrahydrate coenzyme, which comprises the following steps: reacting thiamine monophosphate with a condensing agent in an organic solvent to obtain an intermediate I; reacting the intermediate I with a phosphate to obtain coenzyme; and refining the coenzyme to obtain tetrahydrate coenzyme. The method uses a by-product thiamine monophosphate recovered in a traditional preparation process as a starting material to prepare the tetrahydrate coenzyme, has high reaction selectivity, and improves the material utilization rate. The method avoids high-temperature reaction conditions of the traditional process, does not discharge hydrogen chloride gas, reduces the use of organic solvents and toxic reagents, reduces the pressure on environmental protection, and improves the safety of the reaction.

Description

TECHNICAL FIELD

[0001] The technical field belongs to the field of pharmaceutical synthesis, and particularly relates to a synthesis method of cocarboxylase tetrahydrate. BACKGROUND

[0002] Cocarboxylase tetrahydrate, also known as thiamine pyrophosphate tetrahydrate, is one of raw materials of multivitamin injection (12). It is a form of vitamin B1, participates in oxidation decarboxylation of pyruvic acid and alpha-ketoglutaric acid in sugar metabolism in vivo, and is deficient in oxidation, pyruvic acid, lactic acid accumulation, and energy supply of the body. Its symptoms mainly manifest in the nervous system, cardiovascular system and digestive system.

[0003] There are many preparation methods of cocarboxylase tetrahydrate reported in the literature. In 1937, Stern and Hofer prepared thiamine pyrophosphate by reacting vitamin B1 and phosphorus oxychloride [Science, Vol. 85, p. 483 (1937)]. J. Weij1ard and H. Tauber prepared thiamine pyrophosphate by using vitamin B1 and a mixture of sodium pyrophosphate and phosphoric acid. Thiamine pyrophosphate was obtained by treating with phosphotungstic acid solution and a large amount of organic solvent, and the yield was about 10% [Journal of the American Chemical Society, Vol. 60, p. 2263 (1938)]. In 1940, it was reported that vitamin B1 analog 5-bromovinylthiazole and silver phosphate were reacted to prepare chlorinated vitamin B1 pyrophosphate, which was precipitated in the form of silver salt, and then silver ion was removed by hydrogen sulfide and treated with hydrogen chloride to obtain chlorinated thiamine pyrophosphate [Biochemical Journal, Vol. 34, p. 980 (1940)]. The above method needs to use toxic reagents or a large amount of organic solvent, or the temperature is high, which is not conducive to environmental protection.

[0004] US5047223, US2991284, CN1887891A, CN101787048B disclose that vitamin B1 is used as raw material, and reacts with phosphating reagent (phosphorus pentoxide and phosphoric acid, phosphorus pentoxide and pyrophosphoric acid), the treated reaction liquid is purified by a weakly basic anion resin column and then a weakly acidic cation exchange resin column, the qualified components are concentrated at 35℃, and ethanol is added to obtain the product by crystallization. This method is a commonly used method for producing tetrahydrate coenzyme I at present, but the method needs to be carried out at high temperature, hydrogen chloride gas is released during the reaction process, and a large amount of organic solvent is used, which is not conducive to environmental protection. Moreover, the reaction yield is very low, the highest yield is about 40% (CN101787048B), the reaction selectivity is poor, and the by-product thiamine monophosphate accounts for 60%-70% (US5047223) during the production process, which is directly treated as waste, causing material waste and increasing production cost. Therefore, a method for converting thiamine monophosphate into tetrahydrate coenzyme I is urgently needed. SUMMARY

[0005] In view of the above deficiencies in the prior art, in order to improve the material conversion rate and reduce the cost, the present application uses the by-product thiamine monophosphate recovered in the traditional preparation process as the starting material, first reacts with a condensing agent to obtain intermediate I in the first step, then reacts intermediate I with a phosphate and a catalyst to obtain coenzyme I in the second step, and finally refines coenzyme I to obtain tetrahydrate coenzyme I. The synthesis route is as follows:

[0006]

[0007] characterized in that it comprises the following steps:

[0008] Step one, in solvent A, the temperature is 15-30℃, thiamine monophosphate reacts with a condensing agent under the action of a base to obtain intermediate I:

[0009]

[0010] Step two, in solvent B, the temperature is -5-30℃, intermediate I reacts with a phosphate under the action of a catalyst to obtain coenzyme I;

[0011]

[0012] Step three, coenzyme I is refined to obtain tetrahydrate coenzyme I.

[0013]

[0014] The synthesis method of the above tetrahydrate coenzyme I is characterized in that the condensing agent in step one is one of dicyclohexyl carbodiimide-morpholine, N,N'-carbonyldiimidazole;

[0015] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the molar ratio of the condensing agent to thiamine monophosphate in step one is (3-4):1;

[0016] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the reaction temperature of step one is 15-30℃;

[0017] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the solvent A in step one is one of acetonitrile, N,N-dimethylformamide and N,N-dimethylacetamide; and the mass ratio of the solvent A to thiamine monophosphate in step one is (8-10):1;

[0018] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the phosphate salt used in the reaction of step one and step two is one of triethylamine phosphate, tributylamine phosphate and tetrabutylammonium phosphate;

[0019] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the molar ratio of the phosphate salt to the intermediate I in step two is (3-5):1;

[0020] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the solvent B in step two is one of acetonitrile, acetone and tetrahydrofuran; and the mass ratio of the solvent B to the intermediate I in step two is (10-15):1;

[0021] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the reaction temperature of step two is -5-30℃;

[0022] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the catalyst in step two is one of magnesium sulfate, manganese chloride and zinc chloride; and the mass ratio of the catalyst to the intermediate I is (0.04-0.06):1;

[0023] The synthesis method of the tetrahydrate coenzyme of the above-mentioned auxiliary carboxylase is characterized in that the refining process of step three is as follows: the auxiliary coenzyme obtained in step two is added into purified water, heated and dissolved, activated carbon is added, stirred, filtered while hot, the filtrate is cooled, ethanol is added dropwise until white solid is gradually precipitated in the filtrate, stirred, suction filtered and dried to obtain the tetrahydrate coenzyme of the auxiliary carboxylase;

[0024] The mass ratio of the purified water to the auxiliary coenzyme in step three is preferably 9:1; and the mass ratio of the ethanol to the auxiliary coenzyme is preferably 15:1.

[0025] Compared with the prior art, the present application has the following advantages:

[0026] 1. The present application uses the by-product thiamine monophosphate recovered in the traditional preparation process as a starting material to prepare the tetrahydrate coenzyme of the auxiliary carboxylase, thereby improving the material utilization rate.

[0027] 2、The preparation process of the present application avoids the high-temperature reaction conditions of the traditional process, eliminates the emission of hydrogen chloride gas, reduces the use of organic solvents and toxic reagents, relieves the pressure on environmental protection, and improves the safety of the reaction.

[0028] 3、A new preparation route of tetrahydrate co-carboxylase is developed, the reaction selectivity is high, and the product yield is significantly improved to 70.6%; the liquid phase detection result of the product shows that the purity is greater than 99.5%.

[0029] 4、The raw materials used in the route of the present application are easy to obtain, the process is simple, the preparation steps are short, the reaction process is easy to control, and it is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A synthesis route for preparing tetrahydrate co-carboxylase from thiamine monophosphate as a starting material.

[0031] Figure 2 A liquid phase diagram of tetrahydrate co-carboxylase prepared according to patent US5047223.

[0032] Figure 3 A liquid phase diagram of tetrahydrate co-carboxylase obtained from Example 1 of the present application. DETAILED DESCRIPTION

[0033] The present application will be further described in conjunction with the specific embodiments below, and the following description is only for the purpose of explaining the present application, and the protection scope of the present application is not limited to these embodiments, and those skilled in the art should understand that equivalent replacements or corresponding improvements made to the content of the present application still fall within the protection scope of the present application.

[0034] (1) Sample source:

[0035] Thiamine monophosphate was self-made, synthesized and separated according to the method described in patent US5047223;

[0036] Phosphonium salt, phosphonium salt, phosphonium salt, and other reagents are commercially available.

[0037] (2) The purity of tetrahydrate co-carboxylase was determined by high performance liquid chromatography:

[0038] The chromatographic column is Ultimate@AQ-C18, 5μm, 4.6×250mm;

[0039] The mobile phase is 0.2 mol / L KH2PO4 buffer solution-methanol (99.7:0.3);

[0040] The wavelength is 235nm.

[0041] The following examples are provided to further illustrate the application, but not to limit the patent.

[0042] Example 1

[0043] 1. Preparation of intermediate I:

[0044] Put thiamine monophosphate (34.4g, 1.0eq), dicyclohexyl carbodiimide (61.8g, 3eq), morpholine (26.14g, 3eq) into a reaction flask, add 300ml N,N-dimethylacetamide, start stirring, control temperature at 30°C, drop in triethylamine (35.5g, 3.5eq), after dropping is completed, continue to control temperature at 30°C, reaction is completed by liquid phase detection, add purified water and ethyl acetate into the reaction solution, stir and separate into two phases, concentrate the organic phase, and obtain 37.2g of intermediate I.

[0045] 2. Preparation of coenzyme IV tetrahydrate:

[0046] Put intermediate I (37.2g, 1.0eq) and zinc chloride (2.23g, 0.06eq) obtained in the above step into a reaction flask, add 500ml acetone, start stirring, control temperature at 30°C, slowly add tributylamine phosphate (76.5g, 3.0eq), continue to control temperature at 30°C, filter after reaction is completed by liquid phase detection, and rinse the filter cake with acetone to obtain 27.7g of coenzyme IV, which is directly used in the next step.

[0047] Put the wet product again into a reaction flask, add purified water (9V / W), heat to 60°C, add activated carbon after the solid is completely dissolved, stir for 1 hour, filter while hot, cool the filtrate to 40°C, and drop in ethanol (15V / W) to gradually precipitate white solid in the filtrate, continue to stir for 1 hour, suction filter, rinse the filter cake with ethanol, and dry at 40°C to obtain 32.4g of coenzyme IV tetrahydrate (moisture 15.5%), total yield 65.0% (calculated based on the amount of thiamine monophosphate), and purity 99.84%. The following table is the liquid phase detection result of coenzyme IV tetrahydrate prepared according to the patent US5047223 and coenzyme IV tetrahydrate prepared according to Example 1. The liquid chromatogram is shown in the attached Figure 1 and attached Figure 2 .

[0048] Name Retention time Peak area percentage Coenzyme A tetrahydrate prepared according to patent US 5047223 8.496 99.83 Coenzyme A tetrahydrate prepared according to Example 1 8.367 99.84 .

[0049] Example 2

[0050] 1. Preparation of intermediate I:

[0051] Put thiamine monophosphate (34.4 g, 1.0 eq), N,N'-carbonyldiimidazole (65.0 g, 4 eq) into a reaction bottle, add 344 ml of acetonitrile, start stirring, control the temperature at 20°C, drop in triethylamine (35.5 g, 3.5 eq), after the drop is completed, continue to control the temperature at 30°C, after the reaction is completed by liquid phase detection, filter, wash the filter cake with acetonitrile, and dry at 30°C under reduced pressure to obtain 31.5 g of intermediate I.

[0052] 2. Preparation of tetrahydrate co-carboxylase:

[0053] Put the intermediate I (31.5 g, 1.0 eq) obtained in the above step and magnesium sulfate (1.58 g, 0.05 eq) into a reaction bottle, add 315 ml of acetonitrile, start stirring, control the temperature at -5°C, slowly add tetrabutylammonium phosphate (81.3 g, 3.0 eq), continue to control the temperature at -5°C, filter after the reaction is completed by liquid phase detection, wash the filter cake with acetonitrile, and obtain 31.1 g of co-carboxylase, which is directly used in the next step.

[0054] Put the wet product above into a reaction bottle again, add purified water (9V / W), heat to 60°C, add activated carbon after the solid is completely dissolved, stir for 1 hour, filter while hot, cool the filtrate to 40°C, and add ethanol (15V / W) dropwise to the filtrate to gradually precipitate white solids, continue to stir for 1 hour, filter, wash the filter cake with ethanol, and dry at 40°C to obtain 33.8 g of tetrahydrate co-carboxylase (moisture 15.3%), with a total yield of 68.0% (calculated based on the amount of thiamine monophosphate).

[0055] Example 3

[0056] 1. Preparation of intermediate I:

[0057] Put thiamine monophosphate (34.4 g, 1.0 eq), dicyclohexyl carbodiimide (82.4 g, 4 eq), and morpholine (34.9 g, 4 eq) into a reaction bottle, add 276 ml of N,N-dimethylformamide, start stirring, control the temperature at 15°C, drop in triethylamine (35.5 g, 3.5 eq), after the drop is completed, continue to control the temperature at 15°C, add purified water and ethyl acetate to the reaction liquid after the reaction is completed by liquid phase detection, stir and separate the liquid, test the concentrated organic phase, and obtain 38.8 g of intermediate I.

[0058] 2. Preparation of tetrahydrate co-carboxylase:

[0059] The intermediate I (38.8 g, 1.0 eq) and manganese chloride (1.55 g, 0.04 eq) obtained in the previous step were placed in a reaction bottle, 582 ml of tetrahydrofuran was added, stirring was started, and triethylamine phosphate salt (79.8 g, 5.0 eq) was slowly added at 30°C. The reaction was continued at 30°C, and after the reaction was completed by liquid phase detection, filtration was performed, the filter cake was washed with tetrahydrofuran, and 32.2 g of co-carboxylase was obtained, which was directly used in the next step.

[0060] The wet product was placed in a reaction bottle again, 9V / W of purified water was added, and the temperature was raised to 60°C. After the solid was completely dissolved, activated carbon was added, stirring was performed for 1 hour, and hot filtration was performed. The filtrate was cooled to 40°C, and 15V / W of ethanol was added dropwise. White solids gradually precipitated in the filtrate, and stirring was continued for 1 hour. Filtration was performed, the filter cake was washed with ethanol, and drying was performed at 40°C. 35.0 g of co-carboxylase tetrahydrate (moisture 15.1%) was obtained, and the total yield was 70.6% (calculated based on the amount of thiamine monophosphate ester fed).

[0061] The above is only some embodiments of the present application, and the protection scope of the present application is not limited to these embodiments. Those skilled in the art should understand that equivalent replacements or corresponding improvements made to the content of the present application still belong to the protection scope of the present application.

Claims

1. A method for synthesizing tetrahydrated prosthetic coenzyme, characterized by, The method comprises the following steps: Step one, in solvent A, the temperature is 15-30℃, thiamine monophosphate reacts with condensing agent under the action of base to obtain intermediate I; ； The solvent A is one of acetonitrile, N,N-dimethylformamide and N,N-dimethylacetamide; The condensing agent is one of dicyclohexyl carbodiimide-morpholine and N,N'-carbonyldiimidazole; The molar ratio of the condensing agent to thiamine monophosphate is 3:1; The volume ratio of the solvent A to thiamine monophosphate is (8-10):1; Step two, in solvent B, the temperature is -5-30℃, intermediate I reacts with phosphate under the action of catalyst to obtain coenzyme F; ； The solvent B is one of acetonitrile, acetone and tetrahydrofuran; The catalyst is one of magnesium sulfate, manganese chloride and zinc chloride; The phosphate is one of triethylamine phosphate, tributylamine phosphate and tetrabutylammonium phosphate; The molar ratio of the phosphate to intermediate I is (3-5):1; The mass ratio of the catalyst to intermediate I is (0.04-0.06):1; The volume ratio of the solvent B to intermediate I is (10-15):1; Step three, coenzyme F is refined to obtain coenzyme F tetrahydrate; ； The refining process is as follows: the coenzyme F obtained in step two is added into purified water, heated and dissolved, activated carbon is added, stirred, filtered while hot, the filtrate is cooled, ethanol is added dropwise until white solid is gradually precipitated in the filtrate, stirred, suction filtered and dried to obtain coenzyme F tetrahydrate.

2. The method of claim 1, wherein, The volume ratio of the purified water to coenzyme F is 9:1, and the volume ratio of the ethanol to coenzyme F is 15:1.

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