AU2006226577A1 - Process for the production of methylcobalamin - Google Patents

Description

WO 2006/100059 PCT/EP2006/002635 PROCESS FOR THE PRODUCTION OF METHYLCOBALAMIN Field of the invention 5 The present invention relates to a new process for the production of methylcobalamin (I), a coenzyme type vitamin B 12 useful in the biosynthesis of methionine and widely employed in medicine for the management of peripheral nervous system diseases. 10 Background of the invention Japanese Patent 45038059 discloses the preparation of methylcobalamin (I) by reduction methylation of cyanocobalamin (II) with methyl iodide and sodium 15 borohydride, in the presence of iron salts and the resulting cyano ion is removed. German Patent 2,058,892 (Offen.) discloses the preparation of methylcobalamin (I) by methylation of cyanocobalamin reduced with sodium borohydride through 20 methyl p-toluenesulphonate. The reaction occurs in the presence of metallic salts (copper or iron) which result in stable complexes with the cyano ion and in the absence of oxygen and light. German Patent 2,149,150 (Offen.) discloses the 25 preparation of methylcobalamin (I) by methylation of hydroxocobalamin (II) with methyl mercuric iodide [MeHgI] or ammonium methylhexafluorosilicate

(NH

4 ) SiF 6 Me] . German Patent 2,255,203 (Offen.) discloses the 30 preparation of methylcobalamin (I) by methylation and reduction of hydroxocobalamin (III) through oxalic acid WO 2006/100059 PCT/EP2006/002635 2 methyl monoester and powdered Zn, in the presence of cobalt salts that catalyze the reaction. German Patent 2,434,967 (Offen.) discloses the preparation of methylcobalamin (I) by methylation and 5 reduction of cyanocobalamin (II) through methyl acid oxalate and powdered Zn, in the presence of cobalt salts that catalyze the reaction. Belgian Patent 889,787 discloses the preparation of methylcobalamin (I) by methylation with methyl 10 iodide after reducing cyanocobalamin (II) with sodium borohydride; the reaction occurs in the presence of an aldehyde in order to sequester the released cyano ion. European Patent 1236737 discloses the preparation of methylcobalamin by reduction methylation of 15 cyanocobalamin or hydroxocobalamin through sodium borohydride and trimethylsulfonium or trimethylsulfoxonium halides in the presence of iron or cobalt salts. All of these processes have several disadvantages 20 because of using either commercially ~ unavailable reagents (oxalic acid monoesters, ammonium methylhexafluorosilicate) or toxic reagents (methyl iodide, methyl p-toluenesulfonate) or environment unfriendly reagents (methyl mercuric iodide). 25 Methylation with trimethylsulfonium or trimethylsulfoxonium halides produces dimethyl sulfur as a by-product, which is deleterious and stinking. The formation of dimethylsulfoxide as a by-product can also occur, which, although non toxic, is not easily removed 30 from the aqueous phase because of its high solubility and high boiling point (1890C).

WO 2006/100059 PCT/EP2006/002635 3 In conclusion, an industrial process for the production of methylcobalamin that employs a non-toxic and/or environment-friendly methylating agent is not currently available. 5 Description of the invention The present invention provides a new process for the production of methylcobalamin (I) that comprises using dimethyl carbonate (IV), a non-toxic and 10 environment-friendly reagent, as a methylating agent. 0 Il MeO- OMe IV In recent years dimethyl carbonate has arisen as a 15 methylating agent in organic chemistry, especially in the field of the so-called green or sustainable chemistry. The advantages of dimethyl carbonate are its innocuousness, low commercial cost and the formation of only carbon dioxide and methanol as by-products. 20 However, due to its poor reactivity and insolubility in water, dimethyl carbonate usually requires anhydrous and vigorous conditions. Working conditions are usually at high temperatures and in strongly basic media. In the present invention, dimethyl carbonate, 25 despite being an insoluble reagent in water (Merck Index, 13th Edition, #6065), has proved to be an excellent methylating agent of cobalamin Co, reduced form (B 1 2 3) obtained by reduction of either cyanocobalamin (II) or hydroxocobalamin (III) . That is why the so-called 30 supernucleophilic

B

12 s anion is known to be one of the most powerful nucleophilic reagents in organic chemistry WO 2006/100059 PCT/EP2006/002635 4 (N.N. Greenwood and A. Earnshaw, Chemistry of the elements, Pergamon Press, Oxford, England, 1984, pp. 1321-1322). In fact, the aforesaid nucleophilic form Col (B 12 s) 5 results from the reduction of cobalamin B 12 according to the following scheme (D. Autisser et al., Bull. Soc. Chim. France 1980, part 2, 192): CN 00_Il__+_-__ C(l) +e- Co (1)~ - CN

B

1 2 B12r B12s 10 and, later, from the nucleophilic attack of B12, on the methyl group of dimethyl carbonate, methylcobalamin is obtained. Consequently, a major embodiment of the present 15 invention is the production of methylcobalamin (I) by reduction of cyanocobalamin (II) or hydroxocobalamin (III), and subsequent methylation with dimethyl carbonate (IV): WO 2006/100059 PCT/EP2006/002635 5

NH

2

COCH

2

CH

2

CH

3 CH

CH

2

CH

2 CNH-03 2 CHCONH2O NH2COCH2 CH2CH2CONH2 H3C R CH N + N N N'- fN CH , O CH3 NH2COCH2 CH3 CH3 CH3 CH2CH2CONH2 RH CH2CH2CONH-CH2-CH-0-P- H CH13 0 HCH2OH R R = CN : Cyanocobalamin (11) RR = OH : Hydroxocobalamin (111) R= CH3 : Methylcobalamin (1) R The contribution of the present invention is therefore based on the preparation of methylcobalamin with dimethyl carbonate under non-anhydrous mild 5 conditions, which allow the reaction to be carried out under very selective conditions. As a matter of fact the methylation reaction occurs in the presence of a reducing agent (V), in aqueous medium, with or without an organic co-solvent, at nearly 10 ambient temperatures and in slightly basic medium. Under these conditions, methylcobalamin is obtained with a good yield and quality. Moreover, the methylation with dimethyl carbonate does not form by products that may give rise to safety, toxicity or 15 environmental problems.

WO 2006/100059 PCT/EP2006/002635 6 The amount to be used of dimethyl carbonate can range from 1 to 25 equivalents, but preferably from 3 to 15 equivalents. As reducing agents (V), those which are capable of 5 reducing the atom of Co(III) to Co(I) can be used, specially hydrides and more specially sodium borohydride. The amount to be used of reducing agent (V) is not critical but from 4 to 25 equivalents, preferably from 10 to 20 equivalents are generally employed. 10 The reaction temperature can range from 5 to 600C, but a temperature from 20 to 400C is recommended. The reaction is carried out in the presence of sequestering agents of cyano ion under standard conditions described in the literature. Specially 15 efficient is the use of iron salts, such as ferrous sulfate, and cobalt salts, such as cobalt chloride (II). The amount to be used of sequestering agent of cyano ion is small, usually in the range between 0.1 and 1.0 equivalents, but preferably between 0.2 and 0.5 20 equivalents. The reaction can be carried out by adding sequentially or simultaneously the respective solutions of reducing agent and dimethyl carbonate under the aforesaid conditions. 25 The reaction is carried out in aqueous or aqueous organic medium. If an organic solvent is used, its polarity should be enough as to attain solubilization in the aqueous reaction medium. For example, alcohols, ketones, ethers, esters, nitriles, amides and ureas; 30 preferably alcohols and lower ketones are useful. Preferred solvents are Ci - C4-alcohols such as methanol, ethanol, n-propanol, i-propanol; ketones such as acetone WO 2006/100059 PCT/EP2006/002635 7 and methyl ethyl ketone; ethers such as tetrahydrofurane and dioxane; acetonitrile and dimethylformamide. The reaction is carried out in the absence of oxygen and light in order to avoid the photo-oxidative 5 degradation of methylcobalamin (R. J. Anderson, et al., Journal of Organic Chemistry 1992, 437, 227-337). The present invention will now be described in detail by the following non-limiting examples. 10 Example 1: Methylcobalamin (I) To a stirred mixture of 10 g (0.0074 mole) of cyanocobalamin, 0.65 g (0.0027 mole) of cobalt chloride (II) hexahydrate, 6.65 g (0.075 mole) of dimethyl carbonate in 125 ml of water at 35-40'C, in inert 15 atmosphere and red light was added over a period of 2 hours a solution of 4.2 g (0.11 mole) of sodium borohydride and 0.42 g of IN sodium hydroxide in 25 ml of water. After stirring for 4 hours at the same temperature, the mixture was cooled at 10'C, stirred for 20 further 3 hours and then filtered. The solid obtained was treated with 200 mL of acetone/water (60:40 v/v) and filtered. The resultant solution was concentrated at reduced pressure until the acetone was removed. The aqueous solution was chromatographied through 500 ml of a 25 non-polar resin (Amberlite XAD-2) . All the inorganic salts were removed by washing first with water until constant conductivity, and then eluting the methylcobalamin with a mixture of methanol/water (50:50 v/v) . The eluate with the pure product was concentrated 30 at reduced pressure and then precipitated with 10 volumes of acetone to give 8.4 g (85%).

WO 2006/100059 PCT/EP2006/002635 8 UV spectrum, solution pH 2.0: maximum absorption at 262-266, 303-307 and 459-463 nm. UV spectrum, solution pH 7.0: maximum absorption at 265-269, 340-344 and 520-524 nm. 5 Purity by chromatographic assay (HPLC) was > 99%. Example 2: Methylcobalamin (I) To a solution of 13 g (0.0096 mole) of cyanocobalamin was added 0.91 g (0.0038 mole) of cobalt chloride (II) hexahydrate in 169 ml of water. The mixture 10 was heated at 35-37'C, and under nitrogen atmosphere and protected from light a solution of 4.75 g (0.053 mole) of dimethyl carbonate in 13 ml of methyl ethyl ketone and a solution of 5.2 g (0.138 mole) of sodium borohydride in 26 ml of water containing 0.26 ml of 2N sodium hydroxide 15 were simultaneously added over a period of 3 hours. After stirring for 1 hour at 35-37'C, the mixture was cooled at 120C, stirred for an additional 3 hours and filtered. The solid obtained was treated with 182 mL of acetone/water (50:50 v/v) at 35-40'C, filtered and the resultant 20 solution was concentrated at reduced pressure until the acetone was removed. The aqueous solution was chromatographied through 500 ml of a non-polar resin (Diaion HP20L). All the inorganic salts were removed by washing first with water until constant conductivity, and 25 then eluting the methylcobalamin with a mixture of methanol/water (50:50 v/v) . The fraction with the pure product was concentrated at reduced pressure and then precipitated with 10 volumes of acetone to give 11.6 g (90%). 30

Claims (5)

1. A process for the production of methylcobalamin that comprises the methylation of cyanocobalamin or 5 hydroxocobalamin with dimethyl carbonate in the presence of a reducing agent.

2. A process according to claim 1 wherein the methylation occurs in the presence of a sequestering 10 agent of cyano ion in an aqueous or hydro-organic medium.

3. A process according to claim 1 or 2 wherein the reducing agent is sodium borohydride. 15

4. A process according to any one of claims 1 to 3, wherein the sequestering agent is iron sulfate (II) or cobalt chloride (II). 20

5. A process according to any one of claims 1 to 4 wherein the reaction occurs by adding sequentially or simultaneously sodium borohydride and dimethyl carbonate.