JPS6139320B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to cobalt-R-corrinoid [R is β-
represents a carbalkoxy group which is located at the cobalt atom of the corrinoid nucleus and is bonded to the cobalt atom of the corrinoid nucleus through a C _1-5 alkyl, hydroxyalkyl, carboxyalkyl, aralkyl, acyl or carbon atom,
or R is a nucleoside residue or a sugar alcohol residue, or R can represent an inorganic anion such as a hydroxide anion or a bisulfite anion.

The term "nucleoside" refers to N-glycosides or deoxyglycosides of heterocyclic bases, including natural nucleosides (e.g. adenosine, cytidine, inosine, guanosine or uridine and their deoxy derivatives), natural or synthetic sugars formed from synthetic glycosides (eg hexoses, pentoses or their deoxy derivatives) as well as natural or synthetic heterocyclic bases. The term "nucleoside residue" refers to a nucleoside group attached to a cobalt atom by a carbon-cobalt bond. The heterocyclic base can be natural or synthetic and can be, for example, pyridine, quinoline, isoquinoline, benzimidazole, azapurine, azapyrimidine, and the like.

The reactive groups of the nucleoside must be protected throughout the reaction, except for one group attached to the carbon atom that is to be attached to the cobalt atom of the cobamide molecule. For this purpose, customary protective groups can be used, which are removed after the reaction according to methods known per se.

Sugar alcohol residues include, for example, residues of mannitol derivatives, such as 1,6-bis[(2-
chloroethyl)-amino]-1,6-dideoxy-D-mannitol [Degranol] or 1,6-dibromo-1,
It can be the residue of 6-dideoxy-D-mannitol (Myelobromol).

Most of the compounds of general formula () are known and have useful pharmacological properties, e.g.
Has _B12 action and modified vitamin _B12 action. They also have significant enzymatic activity.

Many methods are known for the production of cobalt-R-corrinoids.

British Patent No. 963373 and German Patent No.
According to the method described in No. 1213842, cobalt-R-cobamide containing an unsubstituted or substituted aliphatic hydrocarbyl group, an acyl group or an alkyl, aralkyl or arylsulfonyl group is used as an anion, especially The cobamide with the cyanide anion or the water molecule bound to the cobalt atom is reduced and the resulting derivative, in which the initially trivalent cobalt atom has been reduced to monovalent, is treated with a suitable alkylating agent, acylating agent or sulfonylation. It is produced by condensation with an agent.
When using hydrooxocobalamin as starting material, this compound is first purified by passing through an ion exchange column. Unconverted hydrooxocobalamin appears as an impurity in the product. When using cyanocobalamin as a starting material, part of the cyanide groups eliminated in the reduction step leaves the system, while another part is bound to the zinc used as reducing agent. In slightly acidic media, cyanide tends to be liberated slowly and some of the product is converted back to cyanocobalamin. Therefore, the crude product contains cyanocobalamin and hydroxocobalamin as impurities. These impurities are difficult to remove because they crystallize under similar conditions to the product. Also,
Cyanocobalamin is difficult to remove by column chromatography because it passes through the column almost together with the final product. In this reaction, other separation products of cobamide are also formed as intermediates and these also have to be removed.

Belgian Patent No. 759,614 describes a method for removing cyanide ions from a solution of Co ¹⁺ -cobamide obtained during the reduction of cyanocobalmin. According to this specification, various metal salts are used for this purpose. These separate in the form of metal hydroxides from the reaction mixture, which is strongly alkaline as a result of the use of sodium borohydride as reducing agent, and bind the free cyanide ions in the complex. This method is disadvantageous in that the reaction proceeds slowly and thus increases the amount of hydrooxocobalamin impurities. Ferrocyanide, another product of this reaction, forms a stable precipitate with hydroxocobalamin, causing a decrease in yield. Furthermore, the crude product must be purified by chromatography, since acidification of alkaline solutions can liberate cyanide.

Also, a large number of patent specifications deal with the production of methylcobalamin, a substance useful as a medicine.

According to the method described in Belgian Patent No. 773144, under strong light irradiation and nitrogen atmosphere,
Hydroxocobalamin is reduced with stannous chloride,
The reduced cobamide is then alkylated in the dark. However, it is known that other methyl groups in the choline skeleton are eliminated by a radical mechanism when exposed to strong light, and this side reaction product contaminates the final product.

According to Japanese Patent Publication No. 45-36911, hydrooxocobalamin is reduced with half-carbazide hydrochloride and the resulting product is chromatographed on a series of ion exchange columns with different properties.

According to the method described in German Patent No. 2255203 and Belgian Patent No. 817937, hydrooxocobalamin or cyanocobalamin is simultaneously reduced and methylated by using a metal salt and monomethyl oxalate, The crude product obtained is then purified by chromatography. This method requires a large ion exchange column and gives pure product in 65% yield.

As described in Japanese Patent Publication No. 47-34720, alkyl Cobalamin is obtained in much better yields.

According to the method disclosed in Hungarian Patent No. 163770, iodocobalamin, hydrooxocobalamin or B ₁₂ coenzyme is reacted with methylmercury halide or ammonium-methyl-hexafluorosilicate. however,
The product of this selective methylation carried out in a single step also contains some degradation products. These are those that form when the mixture is heated to 65° C. for 6 hours in the presence of large amounts of iodine or when the mixture is evaporated to dryness. The nature of the reagents applied in this method limits the number of derivatives obtained.
Additionally, large-scale production is difficult due to difficulties in manufacturing the reagents.

An object of the present invention is to provide a new method for producing cobalt-R-corrinoid that is easy to implement even under large-scale conditions.

The present invention utilizes the starting cobalt-X-corrinoid (X
is an anion bonded to a cobalt atom that can be bonded to an aldehyde or ketone).If the anion X of X is bonded to an aldehyde or ketone in the reduction step, side effects of the anion can be eliminated and β- of the cobalt atom can be bonded. It is based on the knowledge that substitution, ie, formation of a carbon-cobalt bond, can easily proceed.

According to the invention, the general formula (): [In the formula, R is at the β-position and C _1-5 alkyl, hydroxyalkyl, carboxyalkyl,
represents an aralkyl, acyl or carbalkoxy group bonded to the cobalt atom of the corrinoid nucleus via a carbon atom, or R is a nucleoside or sugar alcohol residue, or R is a hydroxide or bisulfite anion; A method for producing a cobalt-R-corrinoid [which can represent an inorganic anion such as , sulfite ion, rhodanide ion, nitrite ion or ammonium ion] in the presence of an aldehyde or ketone in the absence of oxygen and light, and the resulting reduced corrinoid is treated with an alkylating agent, hydroxyalkyl cobalt-R--, which comprises reacting a reactive derivative of a nucleoside, a sugar alcohol derivative, or an inorganic salt with a oxidizing agent, a carboxyalkylating agent, an aralkylating agent, an acylating agent, or a carbalkoxylating agent, a reactive derivative of a nucleoside, a sugar alcohol derivative, or an inorganic salt.
An object of the present invention is to provide an improved method for producing corrinoids. This method is characterized in that the reduction of cobalt-X-corrinoids is carried out in the presence of aldehydes or ketones. This aldehyde or ketone binds by addition to the groups X liberated during the reaction and any primary amine impurities present, thereby removing them from the reaction mixture. As a result, no unreacted or reconverted starting materials appear as impurities in the final product, and there is no possibility of the formation of metabolically resistant substances.

For example, if the group X is a cyanide anion or an anion exhibiting cyanide-like behavior, such as a cyanide ion or a rhodanide ion, it can be attached to an aliphatic or aromatic ketone or an aldose or ketose. These compounds together with the groups X listed above form adducts.

As the aliphatic aldehyde, for example, formaldehyde or acetonaldehyde can be used, and as the aromatic aldehyde, for example, benzaldehyde or p-dimethylaminobenzaldehyde can be used.

Substances applicable to this method include aromatic ketones such as phenylethyl ketone, and aliphatic ketones such as acetone or methyl ethyl ketone.

Examples of aldoses include D-ribose, arabinose, glucose, mannose, galactose, etc.; examples of ketoses include:
Full course or sorbose can be used.

If X represents a bisulfite anion, it can also be bound to an aldehyde in the form of an aldehyde bisulfite.

As already mentioned above, the main advantage of the process according to the invention is that relatively pure end products are obtained. Also, only small amounts of impurities are formed, and the impurities that are formed can be removed without using a chromatographic step or an ion exchange column or a previously inactivated diethylaminoethyl- or carboxymethyl cellulose column.

The present invention will be explained in detail with reference to the following examples, but these are not intended to limit the invention.

Example 1 5.0 g of crystalline cyanocobalamin is placed in a 500 ml four-necked round-bottomed flask with the necks ground together, and then 100 ml of distilled water and 2 ml of a 40% formaldehyde aqueous solution are added thereto. Attach a dropping funnel, a small rubber balloon, and a short joint with a stop and a long joint with a stop to each of the necks that are attached to the flask.

The mixture is stirred magnetically, the system is degassed by suction, and the system is then filled with argon through a long joint. This operation is repeated two more times, and then the short joint is connected to a gas washer filled with distilled water.

Thereafter, a solution of sodium borohydride (2 g) in distilled water (1.0 ml) was added to this mixture via an addition funnel, and rapid effervescence and boiling began, then the red crystalline mixture initially turned brown; Then it turns greenish blue. From this point on, all operations are performed in weakly scattered light.

After stirring for 1 hour, a further 0.5 g of sodium borohydride, dissolved in 5 ml of distilled water, is added to the crystal-free mixture. Care must be taken to avoid introducing air into the system during this addition. The mixture is stirred for a further 15 minutes and then 10 ml of methanol containing 1 ml of methyl iodide are charged. When the reagents are added, the greenish-blue solution suddenly turns red. After stirring for 20 minutes, an additional 3 ml of methanol containing 0.5 ml of methyl iodide is added to the mixture to complete the reaction.

The mixture is stirred for 20 minutes, then 1.2-15 ml of 10% aqueous hydrochloric acid is slowly added dropwise to the mixture to adjust the pH to 6. When no more hydrogen is evolved, check the PH of the mixture and charge the contents of the flask to a separatory funnel. Wash the flask with distilled water and add the wash liquid to the mixture as well.

10 g of crystalline phenol were dissolved in about 200 ml of the resulting solution and the mixture was then mixed with 75, 50 and 25 g of a 1:1 v/v mixture of phenol and chloroform.
Shake with ml. The organic phases are combined, washed with an equal volume of distilled water, and the phases are separated from each other.

150 ml of acetone, 60 ml of distilled water and 500 ml of chloroform are added to the organic phase, the organic phase is separated again and then washed with 25 ml of distilled water.

The aqueous phases are combined, 10 parts of acetone are mixed to 1 part of the aqueous phase, and the mixture is left overnight at room temperature to crystallize.

The precipitated needle-shaped crystals are collected using a glass filter and dried at 60°C until the moisture content is 10%. 4.92 g of the crude methylcobalamin obtained is recrystallized as follows: 20.0 g of crude methylcobalamin is dissolved in 500 ml of warm (40-45° C.) distilled water containing 20% by volume of ethanol. Freshly activated diethylaminoethyl cellulose, washed to neutrality, is added to this solution and the mixture is stirred for 15-20 minutes. The still warm mixture (approximately 45° C.) is passed through a porcelain filter under jet pump vacuum. Wash the cellulose remaining on the filter with warm (approximately 45° C.) distilled water containing 20% ethanol by volume until a colorless wash is obtained. Freshly activated carboxymethylcellulose, washed to neutrality, was added to the liquor, the mixture was treated as before, and the resulting clear liquor was dried in a rotary evaporator for 40 min.
Concentrate to a final volume of approximately 300 ml at a temperature below °C. The concentrate is left at 17-22°C overnight to crystallize. The precipitated crystals were collected using a glass filter, washed with acetone, and then dried in the air.
After that, it is placed in a dryer at 60℃ until the moisture content is 10%.
Dry in . 15.2 g of crystalline methylcobalamin are thus obtained.

A further 4.12 g of crystalline final product are separated by evaporating the mother liquor. The amount of active drug in the final product is 99.4% (based on absorbance measurements). The amounts of impurities determined according to the British Pharmacy Act are as follows: cyanocobalamin 0%, acidic impurities 0.3%, free hydroxocobalamin 0.6%.

The final product is obtained with a yield of 98.6%.

Example 2 50 g of crystalline cyanocobalamin was charged into a 500 ml four-necked round-bottomed flask with the necks rubbed together,
Then 100 ml of a 1:1 v/v mixture of methanol and distilled water and 2 ml of 40% formaldehyde aqueous solution are added. The reduction was carried out analogously to Example 1 by first charging a solution of 2 g of sodium borohydride in 10 ml of distilled water and then a further 0.5 g of sodium borohydride in 5 ml of distilled water. do.

After the second charge of sodium borohydride, the mixture is stirred for 15 minutes and then the reagent prepared as described below is added thereto.

Freshly prepared 5'-O-mesyl-2',3'-isopropylidene-adenosine from 2.5 g of adenosine with a water content of less than 0.1% is evaporated in a round-bottomed flask until a resinous material is obtained. To this resinous substance, add methanol and 0.3N hydrochloric acid solution 1:1v/v.
65 ml of the mixture are added and the reaction mixture is refluxed for 30 minutes. The mixture was rapidly cooled to room temperature, neutralized using 2N aqueous sodium hydroxide solution to pH 7.2-8.2, and the resulting mixture was dissolved into a greenish solution containing hydridocobalamin prepared as described above. Gradually drip into the blue mixture. When the reagents are added, the reaction mixture suddenly turns red. moreover
Stir the mixture for 45 min, then acidify to PH6.0 with 10% aqueous hydrochloric acid, and add methanol to 40 °C.
Evaporate under reduced pressure at the following temperature. The residue is extracted with a 1:1 v/v mixture of phenol and chloroform. The organic phase is washed with water, then the salts are removed,
Afterwards the product is transferred back to the aqueous phase. Acetone is successively added to the mixture to crystallize it and the mixture is left overnight at 0-5°C. In this way, 5.4 g of crude vitamin B ₁₂ coenzyme is obtained. The product is recrystallized as in Example 1 to obtain 5.10 g of crystalline vitamin B ₁₂ coenzyme with an active drug content of 96.1%. Yield is 87.5
%. The amounts of impurities determined according to the British Pharmacy Act are: 0% cyanocobalamin, 0.3% acidic impurities, 0.5 free hydroxocobalamin.
%.

Example 3 5.0 g of crystalline Coα-[α-(5-hydroxy-benzimidazolyl)]-Coβ-cyanocobamide (vitamin B ₁₂ factor) was mixed with methanol and distilled water at 1:1v/1 in the same manner as in Example 1. v mixture
Reduce to hydridocobalamin in 100 ml.

Thereafter, 1 ml of acetaldehyde and 0.5 g of glucose were added to this mixture as cyanide binding substance.
and 5′-O-tosyl-adenosine 2.5
A solution containing g is charged as a nucleoside reagent. The reagent solution consisted of 2.8 g of 5'-O-tosyl-2',3'-isopropylidene-adenosine and 65 ml of a 1:1 v/v mixture of ethanol and 0.3N aqueous hydrochloric acid.
Reflux at 80°C for 30 minutes, then adjust the pH of the alcohol solution to 8.0 with 2N aqueous sodium hydroxide solution.

After salt removal, concentration and recrystallization, 5.06 g (86.8%) of vitamin _B12 factor coenzyme is obtained.

Example 4 Cyanocobalamin 5.0g in the same manner as Example 1
is reduced to hydridocobalamin. After that, the same method as in Example 1 is carried out. However, using 1 ml of benzaldehyde as the cyanide binder,
1.5 ml of ethyl bromide was used as the alkylating agent.
Ethyl bromide is charged in two portions. the result,
4.46 g (88.9%) of ethylcobalamin are obtained.

Example 5 Cyanocobalamin 5.0g in the same manner as in Example 1
is reduced to hydridocobalamin. After that, the same method as in Example 1 is carried out. However, 1 ml of methyl-ethyl-ketone and 0.5 g of fructose are used as the cyanide binder, and a solution of 1.5 g of sodium pyrosulfite in 10 ml of distilled water is used as the reagent. As a result, 5.12g of crystalline sulfitocobalamin
(98.4%).

Example 6 5.0 g of cyanocobalamin is reduced to hydridocobalamin in the same manner as in Example 1 in the presence of formaldehyde. Co-β- using a solution of 1.5 g of sodium nitrite in 1 ml of distilled water as a reagent.
Perform a replacement. After stirring for 10 minutes, adjust the pH of the solution to 5.5-6.0 with 20% aqueous acetic acid solution, and add 4.5 g of hydroxyamine hydrochloride and ammonium chloride.
Add a solution of 3.0 g in 30 ml of distilled water. The reaction mixture is stirred occasionally. After about 2 hours, the mixture is extracted with a mixture of phenol and chloroform to remove the salts and the product is converted to hydrooxocobalamin base and then to hydrooxocobalamin formate according to known methods. Thus, 4.8 g of crystalline hydrooxocobalamin formate free of cyanocobalamin and nitricobalamin impurities
(94.0%).

Example 7 100 ml of distilled water and 6 ml of liquid phenol are added to 5.0 g of crystalline nitrocobalamin. Thereafter, the same procedure as in Example 1 was carried out to obtain 4.7 g of crystalline methylcobalamin. Yield is 96.1%.

Example 8 The same method as in Example 1 is carried out. However, 2.0 g of crystalline rhodana tocobalamin is used as the starting material. As a result, methylcobalamin 1.86g (95.8
%).

Example 9 The same method as in Example 2 is carried out. However, crystalline Coα-[α-(aden-9-yl)] and Coβ-cyanocobalamide (shudo-vitamin B ₁₂ ) is used as the starting material. As a result, crystal Coα-
[α-(aden-9-yl)]-Coβ-adenosyl-cobamide (Syudo-vitamin B ₁₂ coenzyme)
Obtain 0.86g.

Example 10 1.0 g of crystalline vitamin B ₁₂ -monocarboxylic acid (substance with the "e" position) is reduced to hydridocobalamin with sodium borohydride in the presence of 20 ml of distilled water and 0.4 ml of 40% aqueous formaldehyde solution.
The reaction is carried out in the same manner as in Example 1.

After that, 1,6-bis[(2-chloroethyl)-amino]-1,6- in ethanol (10 ml)
A solution of dideoxy-D-mannitol (0.5 g) is added to the reaction mixture. The mixture is stirred for 45 minutes and then the PH of the mixture is adjusted to 5.5-6.0 with 10% aqueous hydrochloric acid. The mixture is briefly distilled under reduced pressure, then salts are removed and concentrated using a mixture of phenol and chloroform. Traces of organic solvent are removed from the aqueous solution and the active agent is bound to a column packed with activated dimethylaminoethylcellulose. The product is eluted with a 2% sodium chloride solution formed using distilled water and the pH of the eluent is adjusted to 8.0 with 2N aqueous sodium hydroxide solution. The effluent is acidified to PH5.5 with 10% aqueous hydrochloric acid, after which the salt is removed from the solution and passed through a column packed with carboxymethylcellulose.
The column is washed with a 0.05 molar acetic acid solution until a colorless wash is obtained, and then the product is eluted with a 0.8 molar aqueous acetic acid solution. 10 g of crystalline phenol is dissolved in the resulting solution, which has a volume of approximately 200 ml, and the mixture is then diluted with phenol and chloroform in a ratio of 1:1 v/v.
Shake with 75, 50 and 25 ml of mixture. The organic phases are combined, washed with an equal volume of distilled water and the phases are separated from each other.

150 ml of acetone, 60 ml of distilled water and 500 ml of chloroform are added to the organic phase, the organic phase is separated again and then washed with 25 ml of distilled water.

The aqueous phases are combined and concentrated to a volume of about 25 at a temperature not exceeding 40°C. 9-10 parts by volume of acetone are added to this concentrate and the resulting mixture is kept at a temperature below 5° C. for 6-8 days. As a result, 0.76 g of crystalline product is obtained. The product is vitamin B ₁₂ monocarboxylic acid 1,6-bis[(2-chloroethyl)-
Amino]-1,6-dideoxy-D-mannitol derivative.

Example 11 The same method as in Example 10 is carried out. However, 1,
6-bis[(2-chloroethyl)-amino]-
1,6-dibromo-1,6-dideoxy-D- instead of 1,6-dideoxy-D-mannitol
Mannitol is used as a reagent. As a result, 0.70 g of the product 1,6-dibromo-1,6-dideoxy-D-mannitol derivative of vitamin B ₂ monocarboxylic acid is obtained.

Example 12 The same method as in Example 10 is carried out. However, 1,
6-bis[(2-chloroethyl)-amino]-
0.5 g of p-chloromethyl-benzaldehyde is used as a reagent instead of 1,6-dideoxy-D-mannitol. As a result, products, vitamins
0.55 g of p-methylene-benzaldehyde derivative of B ₁₂ monocarboxylic acid is obtained.

Example 13 The same method as in Example 1 is carried out. However, _NH3
- 50 ml of an ammonia solution containing 1.0 g of cobamide (cobalichrome) at a pH of 9.1 are used as starting material. As a result, 0.94 g of crystalline methylcobalamin is obtained.

Example 14 The same method as in Example 1 is carried out. However, 50 ml of a solution containing 2.1 g of dicyanocobinamide is used as starting material, and 0.5 g of glucose, 1 ml of acetone and 0.1 g of sorbose are used as agents for binding cyanide. As a result, 1.77 g of aco(methyl)-cobinamide is obtained.

Claims (1)

[Claims] 1 General formula (): [In the formula, R is at the β-position and C _1-5 alkyl, hydroxyalkyl, carboxyalkyl,
represents an aralkyl, acyl or carbalkoxy group bonded to the cobalt atom of the corrinoid nucleus via a carbon atom, or R is a nucleoside residue or a sugar alcohol residue, or R can represent an inorganic anion. A method for producing a cobalt-R-corrinoid, comprising a cobalt-X-corrinoid corresponding to the general formula () except that it has an X group in place of the substituent R [wherein, X is an anion bonded to a cobalt atom] R] is reduced in the presence of an aldehyde or ketone in the absence of oxygen and light, and the resulting reduced corrinoid is treated with an alkylating agent. , a hydroxyalkylating agent, a carboxyalkylating agent, an aralkylating agent, an acylating agent, or a carbalkoxylating agent, a reactive derivative of a nucleoside, a sugar alcohol derivative, or an inorganic salt. . 2 Cobalt-X-corrinoid [X is cyano group,
or a group exhibiting cyanide-like behavior], an aliphatic or aromatic aldehyde, an aliphatic or aromatic ketone, an aldose or a ketose is applied as the agent for binding the group X. Manufacturing method described. 3. The manufacturing method according to claim 2, wherein formaldehyde or acetaldehyde is used as the aliphatic aldehyde, and benzaldehyde or p-dimethylamino-benzaldehyde is used as the aromatic aldehyde. 4. The manufacturing method according to claim 2, wherein the aliphatic ketone is acetone or methyl-ethyl-ketone, and the aromatic ketone is phenyl-ethyl-ketone. 5. The production method according to claim 2, wherein D-ribose, arabinose, glucose, mannose, or galactose is used as the aldose, and fructose or sorbose is used as the ketose. 6. The production method according to claim 1, in which an aliphatic or aromatic aldehyde is used as the agent for binding the group X for the conversion of cobalt-X-corrinoid [X is bisulfite]. 7. As cobalt-X-corrinoid, use vitamin B ₁₂ , vitamin B ₁₂ factor, vitamin B ₁₂ ψ or vitamin B ₁₂ factor A, which is complete cyanocobalamin, or cyano(aquo)- or dicyanocobinamide, which is incomplete cobinamide. The method according to any one of claims 1 to 6, wherein a mono-, di- or tricarboxylic derivative of corrinoid is used.