JP2014510134A - Synthesis process of methylcobalamin - Google Patents

Abstract

The present invention uses a chelating agent using sodium borohydride as a reducing agent, as dimethyl sulfate as an alkylating agent, alone or as a Vilsmeier complex containing unalkylated, monoalkylated and dialkylated amides. Without disclosing the process of synthesizing and purifying methylcobalamin. The purification is achieved in two different ways by slurrying in water and acetone-water or by resin.

Description

  Methylcobalamin is a cobalamin (MeCbl or MeB12) used in the treatment of peripheral neuropathy, diabetic neuropathy and as an initial treatment for amyotrophic lateral sclerosis. Methylcobalamin is a form of vitamin B12 and differs from cyanocobalamin in that the cyano group is replaced with a methyl group. This vitamer is one of two active coenzymes used by vitamin B12-dependent enzymes, specifically 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), also known as methionine synthase. Is the form of vitamin B12 used by Although the intermediate Ni-CH3 has been proposed in the final stage of methanogenesis, methylcobalamin is notable as one of the few examples due to its intrinsic organometallic nature. Methylcobalamin is also equivalent to vitamin B12 for the treatment of lesions caused by lack of vitamin B12, such as pernicious anemia.

Animals plants unable to produce vitamin B _12, is only a portion of the microorganisms can be produced. These microorganisms that inhabit sewage have large amounts of this vitamin, but are not available to people. The only useful sources for human diet are several elements of animal origin, especially ruminant meat and viscera. Because ruminants, because fastening the microorganism to produce vitamin B ₁₂ in the rumen. This microorganism is also present in certain fish and shellfish, mollusks and fish.

  There are various processes for the synthesis of methylcobalamin, and almost all processes are carried out in the absence of light using a solvent containing less than 1 ppm oxygen (removed with nitrogen bubbling).

  In other processes, the pH (6.5 or 7.0) is adjusted with concentrated hydrochloric acid. In this case, a resin for purifying methylcobalamin is not necessary. On the other hand, in a process that does not adjust the pH, it is necessary to use an ion exchange resin for purification.

  The yield is between 80-90%, whether by pH adjustment and recrystallization or by purification with resin.

  JP-A-62-056491 named “PRODUCCION DE METILCOBALAMINA DE ALTA PUREZA” describes that cyanocobalamin is denatured and adjusted to pH 2.5 to 7.0, and the resulting reaction mixture In a silica gel chromatography column, eluting one after another with a polymer of divinylbenzene and styrene, concentrating the eluate and crystallizing the compound to obtain substantially metolcobalamin. Yes.

  According to Japanese Patent Laid-Open No. 8-143590, a reaction mixture of hydroxycobalamin and cyanocobalamin is obtained by subjecting cyanocobalamin to conventional methylation using an inert gas in the absence of oxygen. Specifically, cyanocobalamin is reduced with a deionized aqueous solution of sodium borohydride and involves a post-reaction that is methylated by adding molle salt, ferric oxide and methyl iodide to the system. It is recommended that oxygen dissolved in deionized water be removed using an inert gas, and its concentration be 0.1 ppm or less. An inert gas is also used in the isolation process to reduce the amount of oxygen present.

  Inventive MX 632, a procedure for producing methylcobalamin from cyanocobalamin (PROCEDIMINENTO PARA LA FABRICATION DE LA METILCOBALAMINA A PARTIR DE LA CIANOCOBALAMININA), in which the procedure for producing methylcobalamin from cyanocobalamin is described. And release hydrogen by acting on mestil oxalate and methyl oxalate in a solution containing at least 40 g cyanoeobalamin per liter in a mixture of methanol and water The step of adding a metal powder that can be made, the purpose of which is to set the reaction environment at 28 ° C. Maintaining the temperature between 32 ° C., the reduction of cyanoeobalamin and the methylation of the reduced cobalamin to methicobalamin are realized simultaneously.

  US Pat. Nos. 6,657,057 and 7,220,855 are both by Hisatake et al., In which cyanocobalamin or hydroxocobalamin is reduced in the presence of a reducing agent and then water-soluble. A process is provided for the modification by the addition of a sex modifier to produce methylcobalamin.

  Mexican patent application MX / 2007/011649, PROCEDIMIENTO DE FA BRICACION DE LA METILCOBALAMINA, describes a process for the production of methylcobalamin, in which cyanocobalamin or hydroxo with dimethyl carbonate in the presence of a reducing agent. It includes the methylation of cobalamin.

  Mexican Patent No. 126161, an improved procedure for preparing methylcobalamin (PROCEDIMIENTO PARA PREPARAR METILCOBALAMINA) describes a procedure for preparing methylcobalamin by reduction and methylation of hydroxocobalamin. , Characterized in that it is carried out simultaneously in a hydroalcocobalamine hydrous alcohol solution by the action of a methyl monoester of carboxylic diacid in the presence of a metal powder, preferably the monoester is methyl oxalate, Is a zinc powder and the methylcobalamin formed is precipitated by addition of 3-5 volumes of acetone.

  In another process for obtaining methylcobalamin, the substitution of a cyano group with a methyl group has been described, which includes chelating agents (eg iron, nickel, cobalt, zinc chlorides or sulfides) and reducing agents (its Due to water solubility, in the presence of sodium borohydride), trimethylsulfonium or halogenated derivatives of trimethylsulfoxonium (iodide, bromide, chloride) and some other methyl such as monomethyl oxalate and dimethyl carbonate Arising from the group imparting agent. It is carried out in an aqueous solvent or a water-solvent mixture, ie 2-butanone assisted water / acetone or water / methanol.

  This document uses a chelating agent using sodium borohydride as a reducing agent, as a alkylating agent, dimethyl sulfate alone or as a Vilsmeier complex containing unalkylated, monoalkylated and dialkylated amides. Without presenting the form to obtain methylcobalamin.

  The purification is achieved in two different ways by slurrying in water and acetone-water or by resin.

[Preparation of crude methylcobalamin]
To a glass three-necked round bottom long neck flask equipped with a stirring mechanism and thermometer, add 30 mL of deionized water at ambient temperature, add 0.35 mL (1 equivalent) of dimethyl sulfate and 5 g of cyanocobalamin. Heat to 30 ° C. and replace the air atmosphere with nitrogen. A mixture of 2.0 g sodium borohydride, 0.16 mL 2N sodium hydroxide and 10 mL deionized water is prepared in advance. This mixture is added over 30 minutes to the cyanocobalamin suspension with care so that the temperature does not exceed 40 ° C. When addition is complete, hold at 40 ° C. for 3 hours. Cool to 10 ° C. and hold at this temperature for 60 minutes. Filter and wash with 10 mL of acetone. Dry in vacuum at 40 ° C. until moisture is below 13%.

  5-6 grams of crude methylcobalamin (evaluation value 78-82%) is obtained.

[Preparation of crude methylcobalamin]
To a glass three-necked round bottom long neck flask equipped with a stirrer and a thermometer, add 30 mL of deionized water at ambient temperature and add 0.35 mL (1 eq) dimethyl sulfate, 0.18 mL dimethylformamide and 5 g Add cyanocobalamin. Heat to 30 ° C. and replace the air atmosphere with nitrogen. A mixture of 2.0 g sodium borohydride, 0.16 mL 2N sodium hydroxide and 10 mL deionized water is prepared in advance. This mixture is added over 30 minutes to the cyanocobalamin suspension with care so that the temperature does not exceed 40 ° C.

  When addition is complete, hold at 40 ° C. for 90 minutes. Cool to 10 ° C. and hold at this temperature for 60 minutes. Filter and wash with 10 mL of acetone. Dry in vacuum at 40 ° C. until moisture is below 13%. 5-6 grams of crude methylcobalamin (evaluation value 78-82%) is obtained.

[Preparation of crude methylcobalamin]
To a glass three-necked round bottom long neck flask equipped with a stirrer and a thermometer, add 30 mL of deionized water at ambient temperature and add 0.35 mL (1 eq) dimethyl sulfate, 0.18 mL dimethylformamide and 5 g Add cyanocobalamin. Heat to 30 ° C. and replace the air atmosphere with nitrogen. A mixture of 2.0 g sodium borohydride, 0.16 mL 2N sodium hydroxide and 10 mL deionized water is prepared in advance. This mixture is added over 30 minutes to the cyanocobalamin suspension with care so that the temperature does not exceed 40 ° C. When addition is complete, hold at 40 ° C. for 90 minutes. Cool to 10 ° C. and hold at this temperature for 60 minutes. Filter and wash with 10 mL of acetone. Dry in vacuum at 40 ° C. until moisture is below 13%.

  5-6 grams of crude methylcobalamin (evaluation value 78-82%) is obtained.

[Preparation of crude methylcobalamin]
To a glass three-necked round bottom long neck flask equipped with a stirrer and a thermometer, add 30 mL of deionized water at ambient temperature and add 0.35 mL (1 eq) dimethyl sulfate, 0.18 mL dimethylformamide and 5 g Add cyanocobalamin. Heat to 30 ° C. and replace the air atmosphere with nitrogen. A mixture of 2.0 g sodium borohydride, 0.16 mL 2N sodium hydroxide and 10 mL deionized water is prepared in advance. This mixture is added to the suspension of cyanocobalamin over 30 minutes, taking care not to exceed a temperature of 40 ° C. When addition is complete, hold at 40 ° C. for 90 minutes. Cool to 15 ° C. Add 100 mL of acetone, taking care not to exceed 20 ° C. Cool to 10 ° C. and hold at this temperature for 60 minutes. Filter and wash with 10 mL of acetone. Dry in vacuum at 45 ° C. until moisture is below 13%.

  4.5 to 4.7 active grams of crude methylcobalamin are obtained.

[Purification of methylcobalamin by slurrying]
To a three-necked round bottom long neck flask equipped with a stirring mechanism and a thermometer, add 25 mL of deionized water at ambient temperature. Add 5 g of crude methylcobalamin obtained by any of the above examples. The suspension is stirred for 60 minutes at ambient temperature. Filter and wash with 5 mL of acetone. The slurried methylcobalamin is again added to 25 mL of acetone / deionized water (4: 1) mixture. The suspension is stirred for 60 minutes at ambient temperature.

  Filter and wash with 5 mL of acetone. Dry in vacuum at 40 ° C. until moisture is below 13%. 3.78 g of pure methylcobalamin was obtained. The overall yield of methylcobalamin is 75-78% p / p relative to the starting cyanocobalamin.

[Purification of methylcobalamin by adsorption to resin (SEPABEADS (registered trademark) SP207)]
The methylcobalamin obtained in Example 4 is used. Suspend 5 active g of crude methylcobalamin in 89 ml of deionized water. Adjust to pH 2.3-2.6 with 9% HCl. A column containing 100 mL of the adsorbent SEPABEADS (registered trademark) SP207 which has been previously activated with methanol and washed with deionized water is prepared. Methylcobalamin dissolved in water is added at a flow rate of 7 mL / min. When finished, wash with deionized water using silver nitrate reagent until evidence of the absence of chloride is obtained. Prepare three 178 mL methanol / 20 mL water methanol / water mixtures, one each and a fourth 99 mL methanol / 99 mL water in advance. When the water wash is complete, the methanol / water mixture is added at a flow rate of 10 mL / min and the fraction rich in methylcobalamin coming out is collected. Concentrate this rich fraction in vacuo at a temperature of 45-50 ° C. until a volume of about 10 mL is obtained. Add 120 mL of acetone to the methylcobalamin concentrate within 30 minutes. The mixture is stirred at ambient temperature for 4.0 hours. The product is filtered in vacuo. Wash with 10 mL of acetone. Dry in vacuum at 40 ° C. for 6 hours until moisture content is below 13%.

  The overall yield of methylcobalamin is 88-92% p / p relative to the starting cyanocobalamin.

[result]
Methylcobalamin was identified by HPLC method.
Equipment: Waters
Product Name: Empower PRO
Type: 2487-2695
Reference: Mecobalamin Catalog number: MECO107, Japanese Pharmacopoeia Analysis method: Official research paper (Japanese Pharmacopoeia (14th).

Claims (9)

The following steps:
a) mixing deionized water, dimethyl sulfate, dimethylformamide and cyanocobalamin;
b) heating to 30 ° C. and replacing the air atmosphere with nitrogen;
c) preparing a mixture of sodium borohydride, 2N sodium hydroxide and deionized water;
d) adding the mixture of step c) over 30 minutes to the suspension of cyanocobalamin of step a) at a temperature below 40 ° C. and holding at 40 ° C. for 1.5-3 hours;
e) cooling to 10 ° C. and holding at this temperature for 60 minutes,
f) Filtering, washing with acetone, drying in vacuum at 40 ° C. to a moisture content of less than 13%, and g) a purification process comprising methylcobalamin.   Process according to claim 1, characterized in that step a) comprises a mixture of deionized water, dimethyl sulfate, dimethylformamide and cyanocobalamin.   The process according to claim 1, characterized in that the yield is 78-82% methylcobalamin.   The process according to claim 1, characterized in that the purification process of step g) is carried out by slurrying. The following steps:
a) adding the resulting crude methylcobalamin to deionized water;
b) stirring for 60 minutes at ambient temperature;
c) filtering and washing with acetone;
d) adding a 4: 1 mixture of acetone / deionized water to slurried methylcobalamin and stirring for 60 minutes at ambient temperature;
5. Process according to claim 4, characterized in that it comprises the steps of e) filtering, washing with acetone and drying in vacuum at 40 [deg.] C. to a moisture content of less than 13%.   5. Procedure according to claim 4, characterized in that the overall yield of methylcobalamin is 75-78% p / p relative to the starting cyanocobalamin.   Process according to claim 4, characterized in that the purification process of step g) is carried out by adsorption onto a resin. The following steps:
a) suspending crude methylcobalamin in deionized water and adjusting to pH 2.3-2.6 with 9% HCl;
b) preparing a column containing 100 mL of adsorbent resin previously activated with methanol and washed with deionized water;
c) adding methylcobalamin dissolved in water at a flow rate of 7 mL / min;
d) When finished, wash with deionized water using silver nitrate reagent until evidence of the absence of chloride is obtained;
e) preparing three methanol / water 9: 1 mixtures in advance and a fourth methanol / water 1: 1,
f) When the washing with water in step d) is completed, a methanol / water mixture is added at a flow rate of 10 mL / min, and the fraction that comes out is collected.
g) concentrating in vacuo at a temperature of 45-50 ° C.
h) adding acetone within 30 minutes;
i) stirring at ambient temperature for 4.0 hours, filtering the product in vacuo, washing with acetone and drying in vacuo at 40 ° C. for 6 hours until less than 13% moisture. The process of claim 7.   8. Procedure according to claim 7, characterized in that the overall yield of methylcobalamin is 88-92% p / p relative to the starting cyanocobalamin.