JPS6256491A - Production of high-purity methylcobalamin - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a remedy for peripheral neuropathy, etc., by methylating cyanocobalamin, adjusting the pH, bringing the resultant reaction mixture into contant with silica gel and then divinylbenzene/styrene based polymer chromatographic column one after another, eluting the adsorbed substance, concentration the eluate and crystallizing the compound. CONSTITUTION:Cyanocobalamin is methylated by a conventional method, e.g. reduction and methylation, etc., to give a reaction mixture consisting essentially of methylcobalamin. The resultant reaction mixture is then adjusted to 2.5-7.0 pH, passed through a silica gel column and adsorbed. A lower aliphatic alcohol-water mixture or aliphatic ketone-water mixture is discharged to the adsorbed substance and a lower aliphatic alcohol-water mixture is discharged to the adsorbed substance to distill away the adsorbed substance. The resultant distillate is then brought into contact with a divinylbenzene/styrene based copolymer chromatographic column. In the process, the concentration of the lower aliphatic alcohol-water, etc., is increased from the lower to the higher concentration stepwise to elute the adsorbed substance. The adsorbed substance is finally desorbed with a lower aliphatic ketone-water mixture and the distillate is then concentrated. A lower aliphatic ketone is then added thereto to crystallize the aimed compound.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing highly pure methylcobalamin that can be practically used as a medicine.

BACKGROUND OF THE INVENTION Methylcobalamin, as a coenzyme type BI2 present in blood and cerebrospinal fluid, has excellent migration properties into tissues such as nerve cells. Biochemically, it is involved in methyl group transfer reactions, and exhibits the highest in vivo activity among B1□ homologs in nucleic acid, protein, and lipid metabolism. Clinically.

It is effective not only for macroblastic anemia but also as a therapeutic agent for peripheral neuropathies such as diabetic neuropathy and polyneuritis.

Various methods are known for the production of methylcobalamin.9 A typical method is, for example, a method using cyanocobalamin as a starting material (Japanese Patent Publication No. 1973-
38059).

Problems to be Solved by the Invention The methylcobalamin-containing solution obtained by this synthesis method contains trace amounts of cyanocobalamin, hydroxocobalamin, yellow substances, salts, and the like. Therefore, the problem is how to remove these interfering components other than methicobal and obtain highly pure methylcobalamin.

As methods for removing these interfering components, since these substances have similar properties except for salts, there are two methods that take advantage of the difference in adsorption properties, such as a method using ion exchange resin chromatography and a water-based method. A seven-ton crystallization method is already known.

However, these methods have the following drawbacks. That is, the method using ion-exchange resin receiving side chromatography has the drawback that the ion-exchange resin is expensive and requires separate equipment for reuse.
Since the chemical properties are similar in the a7ton crystallization method, 2
Methylcobalamin has the disadvantage that it is difficult to separate and purify it.

Means for Solving the ProblemsThe inventors of the present invention have conducted research for many years in order to obtain high-purity methylcobalamin that does not have these drawbacks, and have found that the problem can be solved by the following means.2The present invention has been made. completed. Namely.

Methylate cyanocobalamin using a conventional method.

A reaction mixture containing methylcobalamin as the main component was obtained.

This reaction mixture was used as a starting material to purify it.

In the process of obtaining high purity methylcobalamin.

A method for producing highly pure methylcobalamin characterized by combining the following five steps. (1) A step of adjusting the pH of the reaction mixture to about 2.5 to 7.0.

(2) A step of bringing into contact with silica gel and causing lower aliphatic alcohol-water or lower aliphatic ketone-water to flow out into the adsorbed material.

(2) Next, lower aliphatic alcohol-water is allowed to flow out into the adsorbent, and the adsorbate is distilled out.

(2) A step in which the distillate is brought into contact with divinylbenzene/styrene copolymer resin chromatography, and lower aliphatic alcohol-water or lower aliphatic ketone-water is eluted from a low concentration by increasing the concentration stepwise. .

■ A step of draining the lower aliphatic ketone-water from the adsorption section to desorb the adsorbate, concentrating the distillate, adding lower aliphatic ketone to crystallize it, and obtaining highly purified methylcobalamin from the crystallization section. The reaction mixture containing methylcobalamin as a main component used as a starting material is obtained by methylating cyanocobalamin by a conventional method. According to the method described and two methods, in addition to methylcobalamin as the main component.

Impurities such as cyanocobalamin and hydroxycobalamin:
/, Me-Comp, NaC1! It contains salts such as.

The present invention is a method for obtaining highly pure methylcobalamin by using this as a starting material and removing impurities. The above steps will be described in more detail as follows.

Step (2) This step is to adjust the pH of the reaction mixture to about 2.5 to 7.0.2 It is usually carried out using a dilute aqueous hydrochloric acid solution, preferably dilute hydrochloric acid of 0.03N or less. The pH is most preferably around 5. Adjusting the pH to within this range has a significant effect on increasing the amount of adsorption and reducing the difficulty of elution. If the pH exceeds 7.0, it will not be separated, which is disadvantageous.

(This step is a step in which the methylation reaction solution is brought into contact with silica gel. For example, silica gel-40 (manufactured by Merck & Co., Ltd.) is used as the silica gel. Silica gel-40 has a specific surface area of 650 m / ! i' is much more advantageous for adsorption than other silica 1 gels.

For elution, use lower aliphatic alcohol-water or lower aliphatic ketone-water, preferably methanol.

Performing with sv O,7 gives good results. Methanol is preferably about 596 acetone.

By this operation, the target substance is present in the adsorption part, and sodium chloride, hydroxycobalamin, etc. are removed as non-adsorbed parts.

Step (2) This is a step in which lower aliphatic alcohol-water is further allowed to flow out into the adsorbate containing methylcobalamin, which is the target substance, and the adsorbate is distilled out. At this time, it is preferable that sv is in the range of 0.5 to 1.0.

Examples of lower aliphatic alcohols include methanol, ethanol, propatool, butanol, and the like, with methanol being the most preferred. The concentration of methanol is 1596
Before and after gives favorable results.

Through this process, the target substance, methylcobalamin, is present in the non-adsorbed portion, and what is adsorbed is the yellow impurity. It is thought that cyanocobalamin is present in the non-adsorbed portion in addition to methylcobalamin.

Step (2) This is a step in which the distillate is brought into contact with a divinylbenzene/styrene copolymer resin.

The divinylbenzene/styrene copolymer is polystyrene-based and structurally has the following structure (I).

Specific examples include Diaion HP-20 and Diaion HP-40.

Elution is carried out using lower aliphatic alcohol-water or lower aliphatic ketone-water, but most preferably a 10% acetone aqueous solution with an SV of about 1.0.

Through this operation, cyanocobalamin is present in the non-adsorbed area.
The target substance, methylcobalamin, is present in the adsorption part.

Step (2) This is a method for obtaining the target substance methylcobalamin from the adsorption section.

Specifically, a lower aliphatic ketone, preferably a 20% acetone aqueous solution, is passed through the above column at s v 1.0.

After the target product is desorbed, it is further crystallized with a lower aliphatic ketone, preferably acetone, and the target product, methylcobalamin, can be obtained by a conventional method.

The advantages of the present invention are summarized as follows.

(1) It is an industrial method for obtaining highly purified methylcobalamin in high yield.

(2) In the original process, the repurification process of silica gel chromatography and divinylbenzene/styrene copolymer resin chromatography is performed by elution and removal within the same column using an aqueous solution of common salt, lower alcohol, and lower ketone, which significantly shortens the time. No purification equipment is required.

Next, two representative examples of the present invention will be listed, but it goes without saying that the present invention is not limited to these only.

Example 1 According to the method for producing methylcobalamin from cyanocobalamin (Japanese Patent Publication No. 45-38059), the reduction and methylation reaction (cyanocobalamin 8.0.9) was carried out. 2 The remaining iodomethyl was concentrated and distilled off from the reaction solution.
6 Methylcobalamin-containing methanol-aqueous solution 600 c
I got c.

Adjust the solution to pH with dilute aqueous hydrochloric acid solution (0.03N or less).
After adjusting to 5,0, silicage/l/ (Kiesel
It is adsorbed onto a column packed with 500 cc of gel-40 (manufactured by Merli) at a flow rate of s v Q, 5. Then 5%
Elute 500 cc of methanol aqueous solution with 5VO97 to remove hydroxocobalamin, common salt, and metal complex salt (solubilized material). Next, 1500 cc of 15% methanol aqueous solution was poured at s v 0.5, and 9996
1000 ml of a solution containing methylcobalamin and trace amounts of cyanocobalamin was eluted. Add this solution to divinylbenzene/styrene copolymer resin (Diaion HP)
-20) Guided to a column packed with 1000cc and adsorbed at svO,5.

Subsequently, sv
Run at 1.0 to remove cyanocobalamin, followed by 2
Pour 1500 cc of 0% aqueous solution at Svl, 0,
1000 cc of methylcobalamin was eluted. This is 6
Depressurize below 0℃.

It was concentrated to make 100 cc of an aqueous solution. Add 1 part of acetone to this
Add 00 cc and dissolve at 40℃, then add acetone 800C
C was gradually added dropwise to effect crystallization to obtain methylcobalamin q (4 pc, yield 9245%). The content of the crystals was determined by HPLC and was found to be 99.9% methylcobalamin.

Cyanocobalamin was not detected.

Claims (1)

[Claims] (1) Cyanocobalamin is methylated by a conventional method to obtain a reaction mixture containing methylcobalamin as a main component, and this reaction mixture is used as a starting material to purify it to obtain highly pure methylcobalamin. A method for producing high-purity methylcobalamin characterized by combining the following steps: [1] Adjusting the pH of the reaction mixture to about 2.5 to 7.0; [2] Bringing the reaction mixture into contact with silica gel to remove the adsorbent. a step of causing lower aliphatic alcohol-water or lower aliphatic ketone-water to flow out, [3] a step of then allowing lower aliphatic alcohol-water to flow out to the adsorbent, and distilling the adsorbent, [4] a step of distilling the adsorbent. Contacting the distillate with divinylbenzene/styrene copolymer resin chromatography, at which time lower aliphatic alcohol-water or lower aliphatic ketone-water is eluted from a low concentration by increasing the concentration stepwise; [5] The lower aliphatic ketone-water is drained from the adsorption part to desorb the adsorbed matter, the distillate is concentrated, and the lower aliphatic ketone is added to crystallize it, and highly purified methylcobalamin is extracted from the crystal part. process of obtaining