DE2019176C3 - Process for the production of Alkylcoba la minen - Google Patents

Description

Alkylcobalamins have heretofore been made by first adding cobalamins, such as hydroxocobalamin and cyanocobalamin, in solution, e.g., with sodium borohydride, zinc dust and dilute acetic acid or zinc dust and 10% ammonium chloride solution reduced under anaerobic conditions has. The reduced cobalamin was then treated with an alkylating agent such as an alkyl halide or alkyl sulfate alkylated. The formation of the cobalamin compound, which contains a monovalent cobalt atom is determined by visually observing the color change of the cobalamin solution from red to a gray-green confirmed.

These conversions are usually of education accompanied by by-products. Due to the similarity of the by-products in terms of physical and chemical properties it is difficult to distinguish this from the alkylcobalamines by the conventional separation and purification methods, such as extraction, crystallization or lyophilization remove completely.

If zinc is used as a reducing agent and methyl iodide as an alkylating agent, that's it Deposition of a considerable amount of a solid substance has been observed. This solid is believed to be Dimethyl zinc. For these reasons, the isolation of the methylcobalamin obtained is quite good difficult.

Methylcobalamin, which is produced by reducing cobalamin with zinc dust and 10% ammonium chloride solution and formed by methylation with dimethyl sulfate, contains a large amount, about 30 up to 40%, a by-product that gives a yellow stain in a place that is slightly different from the original closer than that of methylcobalamin when using descending paper chromatography and secondary butanol-water-ice-liquid as the solvent system (100: 50: 3) is used. The yellow color of the stain will gradually change red when exposed to light. The by-product that is believed to be a compound that by excessive methylation up to the N-3 site of the 5,6-dimethylbenzimidazole portion of the cobalamin molecule is formed is difficult to remove from the desired product.

It has now been found that the previous reduction to a cobalamin with monovalent cobalt does not take place can if a direct alkylation of the hydroxocobalamin in an alkaline medium is made which contains a hydrazino compound.

The invention therefore relates to a process for the preparation of alkylcobalamines with 1 to 5 Carbon atoms in the alkyl group attached to the cobalt atom, which is characterized by

ίο that you can use hydroxocobalamin or its salts in alkaline Medium in the presence of a compound containing hydrazino groups with the corresponding Reacts alkyl chloride, broimide or iodide. By the method of the invention it is possible to pure

^{Obtain 1} S alkylcobalamine in high yield without the presence of undesired by-products.

The following compounds can be used as hydrazino compounds suitable for the process of the present invention to be named:

Hydrazine compounds such as hydrazine per se, phenylhydrazine and the corresponding salts, e.g. B. the hydrochloride; Semicarbazides and their salts such as hydrochloride; Hydrazides of saturated, aliphatic Monocarboxylic acids such as acetic acid hydrazide, propionic acid hydrazide, butyric acid hydrazide and Valeric hydrazide; Hydrazides of saturated aliphatic monocarboxylic acids, such as acrylic acid hydrazide and crotonic acid hydrazide; Hydrazides of saturated dicarboxylic acids, such as oxalic acid iono- and di-

Johydrazide, succinic acid dihydrazide and malonic acid dihydrazide. Hydrazides of unsaturated dicarboxylic acids such as maleic acid dihydrazide and fumaric acid dihydrazide; Hydrazides of hydroxycarboxylic acids, such as Miiehsärehydrazid, ß-Hydroxypropionsäurehy-

drazid, malic acid dihydrazide, tartaric acid mono- and Dihydrazide. Citric acid hydrazide and gluconic acid hydrazide. Hydrazides of aminocarboxylic acids, such as glycine hydra / iti, N-monomethylglycine hydrazide, N, N-dimethylglycine hydra / id (Girard's Reagent D),

Trimethylamino-azetohydrazide chloride (Girard's

Reagent T) and serine hydrazide; Hydrazides of aralkylcarboxylic acids, such as phenylacetic acid hydrazide, phenylpropionic acid hydrazide and mandelic acid hydrazide; Hydrazides of aromatic carboxylic acids, such as benzoic acid hydrazide, salicylic acid hydrazide, o-aminobenzoic acid hydrazide, Phthalic acid mono- and di-hydrazide; Hydrazides of heterocyclically substituted acetic acid, such as morpholinoacetohydrazide, pyrrolinoacetohydrazide. Pineridinoaeetohvdrazid. Imidazolinoacetohydrazide, pyrazolirioacetohydrazide and their . quaternary salts, pyridinium acetylhydrazide chlo- • ride (Girard's. Reagent P) pyrazinium acetyl hydrazide chloride, pyrimidinium acetyl hydrazide chloride, and pyridazinium acetyl hydrazide chloride; as well as hydrazides of heterocyclic carboxylic acids such as isonicotinic acid and nieotinic acid hydrazide, picolinic acid hydrazide and their quaternary salts.

The alkyl radicals which can be introduced into the cobalamin are methyl, ethyl, propyl, butyl and pentyl radicals. The corresponding alkali iodides show the highest reactivity towards hydroxycobalamin. The reactivity then decreases via the bromide towards the chloride. Thus the are alkyl iodides most suitable.

As the hydroxocobalamin which is used as the starting material, hydroxocobalamin per se suitable as well as its salts with organic and inorganic acids, e.g. the acetate and the hydro-

chloride.

The process of the invention can be carried out in such a way that hydroxocobalamin, a hydrazino compound and the respective alkyl halide dissolves in any order in a suitable solvent and for this purpose then add an alkali solution. Any solvent can be used for this purpose provided that it is capable of at least in part both the hydroxocobalamin and the hydrazino compound dissolve. Suitable solvents can, for. B. water and lower alcohols such as methanol and ethanol, as well as their Mixtures in any proportions can be mentioned. * 5

Depending on the solvent, the alkaline agent used is alkali metal alcoholate, such as sodium and potassium methoxide or ethoxide or an alkali metal hydroxide such as sodium or potassium hydroxide. Naturally, when using hydrazine as such, there is no need for its addition Another alkaline substance in the reaction system, since the hydrazine is moderately basic in nature sufficient to bring the system to the necessary alkalinity. The reaction can in general »5 mean to be made at temperatures from room temperature (about 25 "C) to about 50 ° C. A reaction temperature of about 35 "C is preferred.

The alkylation reaction as it is according to the invention takes place, usually takes 1 to 5 minutes to complete when the reaction temperature is within of the above range is maintained.

It is particularly noteworthy that the reaction proceeding according to the invention does not take place in the absence of air needs to be carried out.

The reaction mixture is poured into water, neutralized with dilute mineral acid and extracted with phenol. The phenolic extract is vigorously to transfer with ether and a small amount of water overall \ o shaken the resulting Alkylcobalamin in the aqueous phase. If necessary, the aqueous phase is purified by treatment with cation and anion exchange resins. The aqueous phase obtained as an effluent is then concentrated, whereby a solution is obtained which contains about 10 mg / ml alkylcobalamin. Acetone is then added to this solution and this is then placed in the cold in order to crystallize out the alkylcobalamin. Lyophilization can also be carried out. so

It should be noted that the above-mentioned total stages including alkylation, separation and purification, to be carried out in the dark or under a weak red light, as is customary in the chemistry of vitamin B ₁₂ from the coenzyme-type.

The invention is illustrated in the examples.

example 1

Manufacture of methylcobalamin

a) Implementation:

100 mg of hydroxocobalamin and 50 mg of semicarbazide hydrochloride were in 10 ml of methanol dissolved. The resulting solution was left to stand at a temperature of 35 ° C. for 25 minutes calmly. At this temperature, 1 ml of a solution of methyl iodide in methanol with a Concentration of 500 mg / ml and 3 ml of a sodium methoxide solution in methanol of 16.5 mg / ml was added to the solution while stirring gently. The reaction mixture became Leave to stand at 35 ° C for 5 minutes to complete the reaction.

b) Separation and purification of the reaction product:

To. 100 ml of water was added to the above reaction mixture. It was neutralized with dilute hydrochloric acid. The neutralized solution was extracted twice, initially with 30 ml and then with 5 ml of phenol. The phenolic extracts were combined and washed 3 times with 5 ml of water each time.
The phenol layer was 8 times the volume *. Ether and 8 ml of water. The whole was shaken vigorously and allowed to sit for a while in order to cause the aqueous layer to separate out.

The supernatant ethereal solution was then vigorously shaken with 3 ml of water and a Allowed to settle for a while to cause the second aqueous layer to deposit. The United aqueous layers of about 10 ml were washed twice with 5 ml of ether each time remove the phenol that may have remained in the aqueous solution. To this Thus, an aqueous solution of the aimed product was obtained.

c) Treatment with ion exchangers:

The aqueous solution obtained above was successively through a column (1 cm x K) cm) from carboxymethyl cellulose and then through a column (1 cm X 3 cm) of a polystyrene-trimethylbenzylammonium exchanger of the acetic acid type.

The columns were then eluted with distilled water. 30 ml of the first effluent from the latter column was collected.
When the absence of impurities in the effluent was confirmed by paper chromatography, electrophoresis and absorption spectrum, the effluent was lyophilized, which was done in the usual manner. This gave 88 mg of methylcobalamin in the form of a red powder with a yield of 88%.

In aqueous solution, the product showed maxima in the absorption spectrum at 268 ιτιμ, 317 ιημ, 342 ιημ, 374 πιμ and 522 ιπμ. Descending paper chromatography with filter paper and a solvent system consisting of secondary butanol / water / glacial acetic acid in a ratio of 100: 50: 3 resulted in a single orange-red spot with an Rf value of 1.45 to 1.5. That of the cyanocobalamin was 1.0.
Electrophoresis of the product on filter paper with a 0.5 M acetic acid solution under an electric potential of 13 V / cm, carried out for 2.5 hours, found a relative mobility of 1.35 to 1.4 compared to 1.0 and 2.0 were found for cyanocobalamin and hydroxocobalamin.
The cyanocobalamin obtained by treating a fraction of the product with an aqueous solution of potassium cyanide showed a sharp absorption maximum at 368 μm. All up

values reported are consistent with those for authentic methylcobalamin samples.

Example 2

Manufacture of ethylcobalamin.

100 mg hydroxocobalamin and 50 mg semicarbazide hydrochloride were dissolved in 10 ml of ethanol. The resulting solution was at 35 ° C for 20 minutes ditched.

The solution was treated with 1.2 ml of a solution of ethyl iodide in methanol (500 mg / ml) and 3 ml of a solution of sodium methoxide in methanol (16.5 mg / ml). The whole was reacted at 35 ° C for 5 minutes. The reaction mixture was purified according to the methods of Example 1 described under b), whereby about 10 ml of an aqueous solution of the product were obtained. 100 ml of acetone were added to this solution. The whole was left to stand in a refrigerator for 3 hours to crystallize the product. In this way, 82 mg of ethylcobalamin in the form of red needles was obtained.

The aqueous solution of the product showed absorption maxima at 266 ιτιμ, 345 πιμ and 511 ΐημ Bei a paper chromatography of the product carried out under the conditions of Example 1, a relative Rf value of 1.63 to 1.66 (felb) receive. For paper electrophoresis, the relative mobility or migration was 1.68 to 1.72. Both values were compared with those of cyanocobalamin. The values were in agreement with those of authentic ethylcobalamin patterns.

Example 3

Manufacture of n-butylcobalamin.

The procedures of Example 1 given under a) and b) were repeated, with the exception that instead of the methanolic solution of methyl iodide 1.5 nnl of a solution of n-butyl iodide in methanol (50 mg / ml) was used. About 10 ml of an aqueous solution of the reaction product was obtained. 100 ml Given acetone. The whole was kept in a refrigerator for 3 days. In this way they became 84 mg of red needles obtained.

The product obtained showed a spectrum very similar to that of ethylcobalamin. Absorption maxima at 266 τημ, 345 πιμ and 511 ιπμ were obtained in aqueous solution. In paper chromatography and paper electrophoresis carried out under the conditions of Example 1, the relative Rf values were 1.80 to 1.86 and the relative mobility or migration was 1.68 to 1.72. These were obtained in comparison with those of cyanocobalamin. It has been shown that these values are completely in agreement with those of authentic n-butylcobalamin samples.

Example 4

Manufacture of methylcobalamin

100 mg hydroxocobalamin and 65 mg malonic acid dihydrazide were dissolved in 10 ml of a mixture of distilled water with methanol (1: 1). The resulting solution was left to stand at 35 ° C. for 15 minutes.

Then 1 ml of a methanolic solution of methyl iodide (500 mg / ml) and 3 ml of a 3n-w ^; added aqueous sodium hydroxide solution.

The whole was left to stand at 35 ° C for 5 minutes to complete the reaction. For this were 20 ml of water were added and the mixture was neutralized with dilute hydrochloric acid. The solution was carried out in accordance with the working method specified under b)

sen of Example 1 purified by phenol extraction. To 10 ml of the aqueous solution containing the reaction product was added 100 ml of acetone. The whole was left to stand in a refrigerator to cause the product to crystallize. Included 80 mg of methylcobalamin were obtained in the form of red needles.

Example 5

Manufacture of propylcobalamin

The procedures of Example 1 were repeated with the exception that 130 mg of malonic acid dihydrazide and, instead of methyl iodide, 1 ml of a methanolic solution of propyl iodide (500 mg / ml) were used. In this way, 80 mg of propylcobalamin in the form of red needles was obtained.

Example 6

Manufacture of methylcobalamin

The procedures of a), b) and c) of Example 1 were repeated with the exception that instead of the 50 mg of the semicarbazide hydrochloride 120 mg isonicotinic acid hydrazide and instead of 100 mg Hydroxocobalamin 80 mg were used. 35 ml of an effluent were obtained. The outflow was then purified by phenol. The aqueous solution obtained was reduced to 8 ml in vacuo concentrated. 90 ml of acetone was added to the aqueous concentrate and the whole was in one Left in the refrigerator for 3 days. In this way, 6? mg of methylcobalamin in the form of red Needles received.

Example 7

Manufacture of methylcobalamin

The procedures of Example 4 were repeated with the exception that in place of the 100 mg Hydroxocobalamin 100 mg hydroxocobalamin acetate and instead of 65 mg malonic acid dihydrazic 72 mg of maleic acid dihydrazide were used. In this way, 79 mg of methylcobalamin in Preserved the shape of red needles.

Example 8

Manufacture of methylcobalamin

The procedures of Example 4 were repeated, with the exception that 100 mg hydroxocobalamin acetate instead of 100 mg hydroxocobalamin and instead of 65 mg of malonic acid dihydrazide, 140 mg of phthalic acid dihydrazide were used. In this way, 76 mg of methylcobaiamine was obtained in the form of red needles.

Example 9

Manufacture of methylcobalamin

The procedures described under a), b) and c) of Example 1 were repeated, with the exception that instead of hydroxocobalamin and des

Semicarbazids 100 mg hydroxocobalamin hydrochloride and 83 mg mandelic acid hydrazide are used
became. The further purification of the reaction product by phenol was carried out, whereby 9 ml of an aqueous concentrate of the reaction product were obtained. 90 ml of acetone were added to the concentrate ''. The whole thing was in
stand in a refrigerator for 3 days to crystallize
calmly. This gave 75 mg of methylcobalamin io in the form of red needles.

Ii

Example 10

Making Methylcobalamin '- ^ *

■ The working methods of the example, 4 were again- '.' with the exception that 60 mg acetic acid hydrazide were used in place of the 65 mg malonic acid hydrazide. This gave 82 mg of methylcobalamin in the form of red needles.

Claims (3)

Patent claims: 1. Process for the preparation of alkylcobalamin having 1 to 5 carbon atoms in the alkyl group bonded to the cobalt atom, thereby characterized in that one hydroxocobalamin or its salts in an alkaline medium in the presence a compound containing hydrazino groups with the corresponding alkyl chloride, -bromide or iodide converts. 2. The method according to claim 1, characterized in that the corresponding alkyl iodides implements. 3. The method according to claim 1 or 2, characterized in that one semicarbazide hydrochloride used.