DE2019176A1 - Alkylcobalamine prepn - Google Patents

Abstract

Process comprises reacting hydroxocobalamine or its salt with alkyl halide in the presence of a cpd. contg. hydrazino gp.

Description

Process for Making Alkylcobalamin The invention relates to refer to a process for the preparation of alkylcobalamin and in particular to a improved process for the preparation of alkyloobalamin by reacting hydroxocobalamin with an alkyl halide in the presence of one containing the hydrazino group -NH-NH2 Compound hereinafter referred to as "hydrazino compound" for the sake of simplicity shall be.

Alkylcobalamin has heretofore been made by first Cobalamins, such as hydroxocobalamin and cyanocobalamin in solution with a reducing agent, such as sodium borohydride, zinc dust, and dilute acetic acid or zinc dust and 10 show ammonium chloride solution in anasrobic conditions, for example has reduced under nitrogen so that it is in the corrin core of the cobalamin molecule contained trivalent cobalt atom has been reduced to monovalent cobalt. Then was the reduced cobalamin with an alkylating agent such as an alkyl halide or alkyl sulfate alkylated. The formation of the cobalamin compound, which is a monovalent Containing cobalt atom is determined by visually observing the change in color of the Cobalamin solution confirmed from red to gray-green.

However, it was known that these reactions, which in the known Procedures take place, usually from the formation of a by-product in quantities were accompanied by about 4%. Due to the similarity of the by-product to the aimed alkylcobalamin in terms of coloration as well as other physical ones and chemical properties it is difficult, the by-product of the alkylcobalamin by the conventional separation and purification methods, such as extractions and such as to be completely removed.

The alkylcobalamin product obtained by known methods Crystallization or lyophilization is obtained, has only a low purity, what is down to the contamination with the by-product. The presence of the by-product in the alkylation product obtained can be determined by paper chromatography Investigations are founda So, for example, results in a descending order Paper chromatography, when using a solvent system consisting of secondary butanol, water and Glacial acetic acid in a ratio of 100: 50: 3, the by-product is a red spot at a point more distant from the original. is than that of the alkylcobalamin.

In the known method in which zinc is used as a reducing agent and methyl iodide are used as the alkylating agent, is the deposit a considerable amount of a solid substance, upon which by the addition of ether and water to the phenol extract of the reaction mixture was observed to separate been. This solid is believed to be dimethyl zinc. For these reasons, the Isolation of the obtained methylcobalamin rather 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 to 40%, of a by-product that is a yellow spots in a place slightly closer to the original than the one of methylcobalamin when using descending paper chromatography and the above-mentioned solvent system is used. The yellow color of the stain gradually changes to red upon exposure to light.

The by-product that is believed to be a compound produced by excessive methylation up to the N-3 site of the 5,6-dimethylbenzimidazole moiety of the cobalamin molecule is formed is difficult from the desired product removable, so that a poor yield and a poor purity of the intended end product in must be accepted.

It has now surprisingly been found that the alkylcobalamin can advantageously be prepared when the direct alkylation of the Hydroxocobalamins made with an alkylating agent in an alkaline medium containing a hydrazino compound.

This requires the cobalamin intermediate, which is a monovalent cobalt atom contains not to be used.

However, the use of this intermediate has been established in the known Proceedings considered inevitable.

The process of the invention makes it possible to obtain pure alkylcobalamin to be obtained with high yield, without the presence of undesired by-products.

Suitable hydrazino compounds for the process according to the invention the following compounds can be mentioned: hydrazine compounds such as hydrazine per se, phenylhydrazine and the corresponding salts, e.g. the hydrochlorides; 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 acid hydrazide; Hydrazides of unsaturated, aliphatic monocarboxylic acids, such as acrylic acid hydrazide and crotonic acid hydrazide; Hydrazides of saturated Dicarboxylic acids, such as oxalic acid mono- and dihydrazides, 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 lactic acid hydrazide, ß-hydroxypropionic acid hydrazide, Malic acid dihydrazide, tartaric acid mono- and dihydrazide, citric acid trihydrazide and Gluconic acid hydrazide; Hydrazides of aminocarboxylic acids, such as glycine hydrazide, N-monomethylglycine hydrazide, N, N-Dimethylglycine hydrazide (Girardts Reagent D) trimethylamino-acetohydrazide chloride (Girardts Reagent T) and serine hydrazide; Hydrazides of aralkyl carboxylic acids, such as phenylacetic acid hydrazide, Ph-enXlpropionic 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, piperadinoacetohydrazide-imidazolino-acetohydrazide, Pyrazolino-acetohydrazide and its quaternary salts, piridinium-acetylhydrazide-chloride (Girardts Reagent P) pyrazinium acetyl hydrazide chloride, pyrimidium acetyl hydrazide chloride and pyridazinium acetylhydrazide chloride; as well as hydrazides of heterocyclic carboxylic acids, such as isonicotinic acid and nicotinic acid hydrazide, picolinic acid hydrazide and their quaternary Salts.

Preference is given to semicarbazide, acetic acid hydrazide, propionic acid hydrazide, Acrylic acid hydrazide, crotonic acid hydrazide, lactic acid hydrazide, oxalic acid dihydrazide, Malonic acid dihydrazide, maleic acid dihydrazide, malic acid dihydrazide, N-monomethylglycine hydrazide, N, N-dimethylglycine hydrazide, mandelic acid hydrazide, morpholino-acetohydrazide, pyrrolidinoacetohydrazide and pyridinium acetyl hydrazide chloride.

Semicarbazide, malonic acid dihydrazide, isonicotinic acid hydrazide are particularly preferred, Maleic acid hydrazide, mandelic acid dihydrazide, acetic acid hydrazide and phthalic acid hydrazide.

Regarding the appropriate alkyl portion of that used as the alkylating agent Alkyl halides can be named: methyl, ethyl, propyl, butyl, pentyl and the like.

As halogen atoms contained in the alkyl halide, chlorine, bromine and iodine are called. Alkyl iodides show the highest reactivity towards the Hydroxycobalamin.

The reactivity then decreases via the bromide after the chloride down. Thus, alkyl iodide is best suited as hydroxocobalamin, which Hydroxocobalamin is suitable per se as well as its starting material Salts with organic and inorganic acids, e.g. the acetate and the hydrochloride.

The method of the invention can be carried out in the manner that one can hydroxocobalamin, a hydrazino compound, and an alkyl halide in any Sequence in a suitable LUsmittel to dissolve and this then an alkali solution adds. Any solvent can be used for this purpose assuming that it is capable of doing so, at least partially to dissolve both the hydroxocobalamin and the hydrazino compound. As suitable Solvents can be e.g. water and lower alcohols such as methanol and ethanol as well as their mixtures in any proportions.

When the method of the invention is carried out in a non-aqueous medium then the required alkalinity of the system is maintained by that you have an alcoholic alkali metal alcoholate, such as sodium and potassium methoxide or ethoxide is added, while when carrying out the process in an aqueous medium the alkalinity of the system is maintained by using an aqueous alkali metal hydroxide, like sodium or potassium hydroxide added. When using hydrazine as such there is naturally no need to add a further alkaline Substance into the reaction system, since the hydrazine has a moderately basic nature, which is sufficient to bring the system to the necessary alkalinity. The reaction can generally at temperatures from room temperature (about 25 ° C) to about 50 ° C be made. A reaction temperature of about 35 ° C is preferred.

It has also been found that at temperatures below room temperature the rate of reaction is considerably reduced and becomes a reaction product which contains unreacted hydroxocobalamin while at temperatures above about 500C, the tendency to form unusual by-products consists.

The alkylation reaction. as done according to the invention, takes only for a very short period of time, i.e.

usually from 1 to 5 minutes to complete when the reaction temperature is kept within the above range.

It is particularly noteworthy that the running according to the invention The reaction can advantageously be carried out under atmospheric pressure without that, as in the known methods, an inert gas such as nitrogen are used got to.

The separation of the resulting alkylcobalamin from the reaction mixture and the purification of the separated product can after, in particular in the Chemistry of vitamin B12 known processes happen.

The reaction mixture is thus poured into water with dilute Mineral acid neutralized and extracted with phenol. The phenolic extract is made shaken vigorously with ether and a small amount of water to obtain the alkylcobalamin to transfer into the aqueous phase. If necessary, the aqueous phase becomes purified by treatment with cation and anion exchange resins. As The aqueous phase obtained from the stream 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 a cold place in order to crystallize out the alkylcobalamin.

Lyophilization can also be carried out.

It should be noted that the above overall levels, the include alkylation, separation and purification, in the dark or under a weak red light, as it is in the chemistry of the vitamin Coenzyme-type B12 is common.

The invention is illustrated in the examples.

All procedural stages were in the dark or under a weak Red light carried out.

Example 1 Preparation of Methylcobalamin a) Reaction: 100 mg of hydroxocobalamin and 50 mg of semicarbazide hydrochloride were dissolved in 10 ml of methanol.

The resulting solution was kept at a temperature of 35 ° C for 25 minutes ditched. 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 at 16.5 mg / ml was added to the solution while stirring gently. The reaction mixture was left at 350 ° C. for 5 minutes to complete the reaction ditched.

b) Separation and purification of the reaction product: the above 1.00 ml of water was added to the reaction mixture. It was made with dilute hydrochloric acid neutralized. The neutralized solution was 2 times extracted , first with 30 ml and then with 5 ml of phenol. The phenolic extracts were combined and washed 3 times each time with 5 ml of water.

Eight times the volume of ether and 8 ml of water were added to the phenol layer given. The whole thing was shaken violently and allowed to sit down for a while to bring the aqueous layer to the deposition.

The supernatant ethereal solution then became violent with 3 ml of water shaken and allowed to settle for a while to add the second aqueous layer Bring deposition. The combined aqueous layers of approximately 10 ml were Washed 2 times each with 5 ml of ether to remove the phenol that is in the aqueous solution could have been left behind to remove.

Thus it became an aqueous solution of the aimed product obtain.

c) Treatment with ion exchangers: the aqueous solution obtained above Solution was successively passed through a column (1 cm x 10 cm) of carboxymethyl cellulose and then through a column (1cm x 3 cm) of an acetic acid type exchanger (Dowex 1-X2).

The columns were then eluted with distilled water. 30 ml of the first effluent from the latter column were collected.

As by paper chromatography, electrophoresis and the absorption spectrum the absence of impurities in the effluent was confirmed to be the The effluent was lyophilized, which was done in the usual manner. There were, 88 mg of methylcobalamin obtained in the form of a red powder with a yield of 88%.

The product showed maxima in the absorption spectrum in aqueous solution at 268 m / u. 317 m / u, 342 m / u, yr4 m / u and 522 mµ. In descending paper chromatography with a filter paper (Toyo Filter Paper No. 50) and using a solvent system , which consisted of secondary eutanol / water / glacial acetic acid in a ratio of 100: 50: 3, a single orange-red spot with an Rf value of 1.45 to 1.5 was obtained. That of the cyanocobalamin was 1.0.

The electrophoresis of the product on a filter paper (Toyo filter Paper No. 50) with a 0.5 m acetic acid solution under an electrical potential of 13 V / cm performed for 2.5 hours became a relative mobility from 1 to 1.4 compared to 1.0 and 2.0 for cyanocobalamin and hydroxocobalamin established.

This is done by treating a fraction of the product with an aqueous one Cyanocobalamin obtained from a solution of potassium cyanide showed a sharp absorption maximum at 368 m / u. All values given above stand with those for authentic Methylcobalamin pattern in line.

Example 2 Preparation of thylcobalamin 100 mg and hydroxocobalamin 50 mg of semicarbazide hydrochloride were dissolved in 10 ml of ethanol. The received The solution was left to stand at 350 ° C. for 20 minutes.

The solution was made 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) were added. The whole thing took 5 minutes at 55 ° C for reaction. 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 was obtained. To this solution 100 ml of acetone were added. The whole thing was left to stand in a refrigerator for 3 hours left to crystallize the product. In this way there was 82 mg of ethylcobalamin obtained in the form of red needles.

The aqueous solution of the product showed absorption maxima at 266 m / u, 345 m / u and 511 m / u. In the case of paper chromatography of the product made under the conditions of Example 1 was carried out, the relative Rf value was 1.63 to 1.66 (yellow) received. In paper electrophoresis, the relative mobility was or migration 1.68 to 1.72. Both values were identical to those of cyanocobalamin compared. The values were in agreement with those of authentic ethylcobalamin samples.

Example 3 Preparation of n-butylcobalamin Those indicated under a) and b) The procedures of Example 1 were repeated except that instead of the methanolic solution of methyl iodide 1.5 ml 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 obtained. 100 ml of acetone was added to the solution. The whole was kept in a refrigerator for 3 days. In this way there were 84 mg of red needles obtain.

The product obtained showed a spectrum similar to that of ethylcobalamin was very similar. In aqueous solution, absorption maxima were found at 266 m / u, 345 m / u and 511 received. In paper chromatography and paper electrophoresis, the were carried out under the conditions of Example 1, the relative values were Rf values 1.80 to 1.86 and the relative mobility or migration 1.68 to 1.72. These were obtained in comparison with those of the cyanocobalamin. It could it can be proved that these values are completely consistent with those of authentic n-butylcobalamin samples To be in harmony.

Example 4 Preparation of Methylcobalamin 100 ml of flydroxocobalamin and 65 mg of malonic acid dihydrazide were distilled in 10 ml of a mixture Water dissolved with methanol (1: 1). The resulting solution was 15 minutes at Left to stand at 35 ° C.

1. ml of a methanolic solution were then added to the solution with stirring Solution of methyl iodide (500 mg / ml) and 3 ml of a 3N aqueous sodium hydroxide solution given.

This took 5 minutes to complete the reaction at 350C ditched. To this, 20 ml of water was added and the mixture was diluted with Hydrochloric acid neutralized. The solution was according to the working procedures given under b) of Example 1 purified by phenol extraction.

To 10 ml of the aqueous solution containing the reaction product were added 100 ml of acetone added. The whole thing was left to stand in a refrigerator to cause the product to crystallize. This gave 80 mg of methylcobalamin obtained in the form of red needles.

Example 5 Preparation of Propyleobalamin The procedures of the 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) was used became.

In this way there was 80 mg of propylcobalamin in the form of red needles obtain.

Example 6 Preparation 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 hydroWplorld 120 mg isonicotinic acid hydrazide and instead of 100 mg Bydroxocobalamin 80 mg were used. 35 ml received an outflow. The effluent was then purified by phenol.

The resulting aqueous solution was concentrated to 8 ml in vacuo. 90 ml of acetone was added to the aqueous concentrate and the whole was in one Left refrigerator for 3 days. In this way there was 62 mg of methylcobalamin obtained in the form of red needles.

Example 7 Preparation of Methylcobalamin The procedures of the example 4 were repeated with the exception that instead of 100 mg hydroxocobalarnin 100 mg ZTgdroxocobalamin acetate and instead of 65 mg malonic acid dihydrazide 72 mg of maleic acid dihydrazide were used. In this way there was 79 mg of methylcobalamin obtained in the form of red needles.

Example 8 Preparation of Methylcobalamin The procedures of the example 4 were repeated with the exception that instead of 100 mg, hydroxocobalamin 100 mg hydroxocobalamin acetate and instead of 65 mg mdonsäure-dihydrazfd 140 mg Phthalic acid dihydrazide were used. In this way there was 76 mg of methylcobalamine obtained in the form of red needles.

Example 9 Production of Methylcobalamin The under a), b) and c) the procedures described in Example 1 were repeated, with the exception that instead of hydroxocobalamin and semicarbazide 100 mg hydroxocobalamin hydrochloride and 83 mg of mandelic acid hydrazide were used. The further purification of the reaction product by phenol was made using approximately 9 ml of an aqueous concentrate of the To the concentrate was added 90 ml of acetone. The whole was left to crystallize in a refrigerator for 3 days. This gave 75 mg of methylcobalamin in the form of red needles.

Example 10 Preparation of Methylcobalamin The procedures of Example 4 was repeated with the exception that 60 mg of acetic acid hydrazide was used instead 5 mg of malonic acid bydrazide were used. Thereby / 82 mg methylcobalamin obtained in the form of red needles.

Claims (4)

Claims 1. Process for the production of alkylcobalamin, thereby g e k e n n z e i to c h n e t that one hydroxocobalamin or its salts in the alkaline Medium in the presence of a compound containing at least one hydrazino group with an alkyl halide. 2. The method according to claim 1, characterized in that g e k e.n n -z e i c h n e t that the alkyl halide is methyl, ethyl, propyl, butyl and / or pentyl iodide used. 3. The method according to claim 1 or 2, characterized in that g e k e n n z e i c h n e t that one can use hydroxocobalamin as hydroxocobalamin per se or its hydrochloride or acetate is used. 4. The method according to any one of the preceding claims, characterized g e k It is noted that the at least one hydrazino group is included Compound semicarbazide hydrochloride, malonic acid d-hydrazide, isonicotinic acid hydrazide, Maleic acid dehydrazide, phthalic acid dihydrazide, mandelic acid hydrazide and / or acetic acid hydrazide used.