USOO6657057B2

(12) United States Patent (10) Patent No.: US 6,657,057 B2

Hisatake et al. (45) Date of Patent: Dec. 2, 2003

(54) PROCESS FOR PRODUCTION OF 3,789,211 A 1/1974 Mervyn et al.

METHYLCOBALAMIN 3,928,320 A 12/1975 Boige ......................... 540/452
(75) Inventors: Yoshihiko Hisatake, Ibaraki (JP); FOREIGN PATENT DOCUMENTS
Hiroshi Kuroda, Ibaraki (JP) DE 2019 176 11/1971
(73) Assignee: Eisai Co., Ltd., Tokyo (JP) FR 2240232 3/1975
GB 1306958 2/1973
(*) Notice: Subject to any disclaimer, the term of this GB A 1355899 6/1974
patent is extended or adjusted under 35 GB 1355899 A 6/1974
JP 45-38059 12/1970
U.S.C. 154(b) by 0 days. JP A 45-38059 12/1970
(21) Appl. No.: 10/130,797 JP A 49-47899 5/1974
JP A 50-14900 4/1975
(22) PCT Filed: Dec. 7, 2000 JP A 50-38120 12/1975
JP 8-143590 6/1996
(86) PCT No.: PCT/JP00/08675
S371 (c)(1), OTHER PUBLICATIONS
(2), (4) Date: May 23, 2002
(87) PCT Pub. No.: WO01/42271 Rachkus et al., Leituvos Tsr Mokslu Akademijos Darbai, C.
Serija, Biologijos Mokslai, Mokslas, Vilnius, Lt., No. 1, pp.
PCT Pub. Date:Jun. 14, 2001 133-141 (1979).
(65) Prior Publication Data Haglund et al., Chem. Res. Toxicol., Vol. 13, No. 22, pp.
US 2002/0183511 A1 Dec. 5, 2002
253–256, (2000).
(30) Foreign Application Priority Data
Primary Examiner John M. Ford
Dec. 9, 1999 (JP) ........................................... 11/350683 (74) Attorney, Agent, or Firm-Birch, Stewart, Kolasch &
Jun. 23, 2000 (JP) ....................................... 2000-188619 Birch, LLP
(51) Int. Cl................................................. C07H 11/04 (57) ABSTRACT
(52) U.S. Cl. ...................... 540/452; 536/117; 536/17.1;
536/26.41 The present invention provides an industrially excellent and
(58) Field of Search .......................... 540/452; 536/117, novel process for producing methylcobalamin useful as
536/17.1, 26.41 medicineS. Namely, it provides a process for producing
methylcobalamin, which comprises the Step of methylating
(56) References Cited cyanocobalamin or hydroxocobalamin in the presence of a
reducing agent and a water-Soluble methylating agent.
U.S. PATENT DOCUMENTS
3,773,756 A 11/1973 Penasse et al. ............. 540/452 10 Claims, No Drawings
 US 6,657,057 B2
1 2
PROCESS FOR PRODUCTION OF highly pure methylcobalamin by the method of using methyl
METHYLCOBALAMIN iodide, operation for purification by one or more kinds of
column chromatography is usually necessary, which is a
This application is the national phase under 35 U.S.C. Serious problem from operational viewpoint and viewpoint
S371 of PCT International Application No. PCT/JP00/08675 of production cost. In addition, the quantity of organic
which has an International filing date of Dec. 7, 2000, which Solvents for use in the column purification is large and also
designated the United States of America. waste liquid quantity tends to be enormous.
Thus, an industrially excellent process for producing
FIELD OF THE INVENTION methylcobalamin is not completely established yet and
hence a novel excellent method has been desired.
Methylcobalamin is a coenzyme-type Vitamin B2 exist
ing in blood and cerebroSpinal fluid and is excellent in DISCLOSURE OF THE INVENTION
migrating ability to nervous tissueS as compared with other The present inventors have extensively studied for the
B homologS. Biochemically, it exhibits a pharmacological purpose of improving the above problems. As a result,
action of accelerating metabolism of nucleic acids, proteins 15 Surprisingly, they have found that aimed methylcobalamin
and lipids by methyl group rearrangement and thereby can be conveniently, Safely, and inexpensively obtained in
restoring damaged nervous tissues. Based on these high yields by the below-mentioned method, and thus
properties, it has been clinically employed for preventing, accomplished the present invention.
treating or improving peripheral neuropathy Such as diabetic Accordingly, the present invention provides an industri
neuropathy and polyneuritis, particularly numbness, pain ally excellent process for producing methylcobalamin, par
and paralysis, and is also effective in megaloblastic anemia ticularly a novel process using no methyl iodide and no
owing to Vitamin B deficiency, and thus, it is an important purification by column chromatography.
Vitamin.
Accordingly, the present invention relates to an industri The following will explain the present invention in detail.
ally excellent and novel process for producing methylcobal 25
The present invention relates to a proceSS for producing
amin useful as medicines. methylcobalamin (V), which is represented by the following
chemical reaction formula:
PRIOR ART Cobalamin-CN or Cobalamin-OH->Cobalamin-CH
Cyanocobalamin (I), hydroxocobalamin (II), and methyl
Methylcobalamin has been hitherto produced mainly by cobalamin (V) according to the present invention are
the following synthetic methods: known natural compounds and are represented by the
(1) a method of reacting hydroxocobalamin with a dicar following chemical formula:
boxylic acid monomethyl ester in the presence of a Cyanocobalamin, CAS Res. No.: 68-19-9
metal powder (JP-A 49-47899); Hydroxocobalamin, CAS Res. No.: 13422-51-0
(2) a method of reacting cyanocobalamin with monom 35 Methylcobalamin, CAS Res. No.: 13422-55-4
ethyl oxalate in the presence of a metal powder in
hydrous methanol (JP-A 50-41900); NH3COCHCH GHCHCONH,
(3) a method of reacting hydroxocobalamin with meth NHCOCH -
ylmercury iodide or ammonium methylhexafluorosili HC
cate (JP-B 50-38120); and 40 HC
(4) a method of reacting cyanocobalamin with methyl
iodide in the presence of sodium borohydride (JP-B
45-38059).
However, dicarboxylic acid monomethyl esterS Such as
monomethyl oxalate to be used in the methods (1) and (2) 45
are not commercially available and hence are necessary to
prepare in use, So that it is impossible to utilize them H HO
industrially. Furthermore, Zinc powder to be used as the
metal powder is a heavy metal and hence it is inevitable to H-CHCONHCH-i-o--o H ---CHOH
take measures for preventing its contamination into products 50 dH, O
and for protecting the environment, So that the powder is
industrially not preferable.
Moreover, methylmercury iodide to be used in (3) is a R=CN: Cyanocobalamin (I)
pollutant and hence cannot be employed industrially. R’=OH: Hydroxocobalamin (II)
Furthermore, ammonium methylhexafluorosilicate is also 55 R=CH: Methylcobalamin (V)
not commercially available and hence is necessary to pre The characteristic feature of the present invention is that
pare in use, So that it is impossible to utilize it industrially. a highly pure methylcobalamin equal to or Superior to the
On the other hand, the synthetic method (4) is a very product purified by column chromatography can be conve
excellent method in View of yield and product purity, but is niently obtained in high yields only by methylating cyano
not satisfactory as an industrial proceSS because methyl 60 cobalamin (I) or hydroxocobalamin (II) in the presence of a
iodide has an extremely low boiling point (41 to 43 C.) and reducing agent (III) and a water-Soluble methylating agent
is difficult to handle. Furthermore, from the viewpoint of (IV) usually in an aqueous Solution or a hydrous organic
protecting working environment or natural environment, the Solvent, if necessary, precipitating the reaction product
use of methyl iodide assigned as a specified chemical which is hardly Soluble in water as crystals or precipitates,
Substance and having toxicity Such as possibility of carci 65 and then Separating and treating it.
nogenicity is by no means preferable in View of industrial The water-soluble methylating agent (IV) in the present
health of factory workers. Moreover, in order to obtain invention is not limited as far as it's solubility in water is 2%
 US 6,657,057 B2
3 4
or more, and Specifically includes trimethylsulfur deriva is necessary to use ferrous Sulfate in an amount of at least
tives (VI) represented by the following formula, for 30% by weight or more relative to cyanocobalamin (I) or
example. hydroxocobalamin (II).
However, in the present invention, it is possible to obtain
(VI) highly pure methylcobalamin in high yields because methy
lation proceeds even when no ferrous Sulfate is used as a
cyan ion-trapping agent.
Furthermore, in the case that ferrous Sulfate is used in a
Small amount as a cyan ion-trapping agent, the reaction
proceeds more rapidly and highly pure methylcobalamin can
wherein X represents a halogen atom or methoxySulfony be obtained in high yields by the same post-treatment as in
loxy group; and n represents 0 or 1. the case that no ferrous Sulfate is used. Moreover, in the case
Examples of the trimethylsulfur derivatives (VI) include that cobalt chloride is used in a Small amount, highly pure
the following compounds but they are not limited thereto. methylcobalamin can be also obtained in high yields
(1) Trimethylsulfoxonium iodide, CAS Res. No.: 1774 15 because the methylation proceeds highly Selectively and
47-6 hence the production of impurities is inhibited.
(2) Trimethylsulfonium iodide, CAS Res. No. 2181-42-2 Therefore, the present invention also relates to a process
for producing methylcobalamin (V), which comprises the
(3) Trimethylsulfoxonium chloride, CAS Res. No. 5034 Steps of methylating cyanocobalamin (I) or hydroxocobal
O6-0
amin (II) in the presence of a cyan ion-trapping agent, a
(4) Trimethylsulfonium chloride, CAS Res. No. 3086 reducing agent (III) and a water-Soluble methylating agent
29-1 (IV) in an aqueous Solution or a hydrous organic Solvent;
(5) Trimethylsulfoxonium bromide, CAS Res. No. 3084 and then precipitating the reaction product as crystals or
53-5 precipitates.
(6) Trimethylsulfoxonium bromide, CAS Res. No.: 25 In the present invention, in the case that a cyan ion
25596-24-1 trapping agent is used, examples of the cyan ion-trapping
(7) Trimethylsulfonium methyl sulfate, CAS Res. No.: agent include metals or metal Salts Such as ferrous Sulfate,
2181-44-4 iron powder, Mohr’s salt, ferrous chloride, cobalt chloride,
All these compounds are known products and, in nickel chloride and Zinc chloride, and particularly preferred
particular, trimethylsulfoxonium iodide, trimethylsulfonium are ferrous Sulfate and/or cobalt chloride. These metals or
iodide, trimethylsulfoxonium chloride, trimethylsulfoxo metal Salts may be used Solely or in combination.
nium bromide and trimethylsulfonium bromide are inexpen The cyan ion-trapping agent may be used in a Small
Sive and available as industrial starting materials. Moreover, amount, and the amount is usually from 1 to 30% by weight
trimethylsulfonium chloride can be easily Synthesized and and more preferably from 1 to 10% by weight to cyanoco
available by the method described in Tetrahedron Lett., 27, 35 balamin (I) or hydroxocobalamin (II).
1233 (1986) (B. Byrne et al.). Finally, the use of a reaction Solvent is not particularly
Among the trimethylsulfur derivatives (VI), trimethylsul limited, and in the case of using a Solvent, it is not particu
foxonium bromide, trimethylsulfonium bromide, trimethyl larly limited as far as it is inert to cyanocobalamin (I),
sulfoxonium chloride and trimethylsulfonium chloride par hydroxocobalamin (II), trimethylsulfur derivative (VI) or
ticularly exhibit a high solubility in water and have a 40 methylcobalamin (V). The reaction solvent is usually an
characteristic that the use in a Smaller amount affords highly aqueous Solution or a hydrous organic Solvent. AS the
pure methylcobalamin in high yields. organic Solvent, preferred is usually a water-Soluble one, and
The amount of the trimethylsulfur derivative (VI) to be examples thereof include lower alcohols Such as methanol,
used is not particularly limited, but it is used in an amount ethanol, propanol, isopropanol, butanol, isobutanol, Sec
of usually 1.0 to 5 equivalents, preferably 1.1 to 4.5 equiva 45 butanol and t-butanol; various esterS Such as methyl formate,
lents and more preferably 1.2 to 4 equivalents to cyanoco ethyl formate, methyl acetate, ethyl acetate and isopropyl
balamin (I) or hydroxocobalamin (II). acetate; various ketones Such as acetone, 2-butanone and
The reducing agent (III) according to the present inven 3-methyl-2-butanone; cyclic ethers such as THF and diox
tion is not particularly limited as far as it is a reducing agent ane; acetonitrile, DMF, DMSO, pyridine etc.; and mixtures
employable in the Synthesis of cyanocobalamin (I) or 50 containing one or more of them.
hydroxocobalamin (II). More specifically, examples thereof The reaction temperature in the present invention is also
include Sodium borohydride. not particularly limited, but the reaction is conducted at a
The amount of the reducing agent (III) to be used is not temperature of usually 0 to 90° C., preferably 10 to 70° C.
particularly limited, but it is used in an amount of usually 5 and more preferably 15 to 50° C.
to 30 equivalents, preferably 8 to 25 equivalents and more 55 A more preferred result is obtained by conducting the
preferably 10 to 20 equivalents to cyanocobalamin (I) or reaction under a stream of an inert gas Such as nitrogen
hydroxocobalamin (II). and/or in the dark place (under infrared ray).
The process according to the present invention enables the In order to explain the present invention Specifically,
production of highly pure methylcobalamin in high yields Examples will be described in the following, but the inven
using no metal ion or using only a Small amount thereof as 60 tion is by no means limited thereto.
a cyan ion-trapping agent, and the process exhibits an EXAMPLES
extremely excellent effect in View that no problem arises at
removal of metal ion products, which is difficult to filter, Example 1
from the System. Synthesis of Methylcobalamin
Generally, when and methyl iodide is used as a methy 65
lating agent, ferrous Sulfate is used as a cyan ion-trapping The present Example was conducted in the dark place
agent in combination with those agents in most cases, and it (under infrared ray).
 US 6,657,057 B2
S 6
To 260 ml of ion-exchanged water were added 20 g of filtration and dried, to give 123 g of a crude product of the
cyanocobalamin, 6.02 g of trimethylsulfonium iodide and title compound. Thereto was added 1 1 of a 50% acetone
800 mg of iron(II) sulfate heptahydrate. The mixture was aqueous Solution, and the mixture was heated at 35 C.,
heated in a water bath and, after replacing the atmosphere of adjusted to pH 7.0 with concentrated hydrochloric acid and
the System by nitrogen, a Solution of Sodium borohydride (8 5 then filtered. 2.81 of acetone was added dropwise thereto,
g)/2N sodium hydroxide (0.2 ml)/water (40 ml) and 15 ml followed by stirring overnight. Precipitated crystals were
of 2-butanone were added dropwise thereto under Stirring at collected by filtration and dried, to give 93.2 g of the title
an inner temperature of 40 C. over 20 minutes, respectively. compound (yield 94%).
After Stirring for 15 minutes as it was, the mixture was Example 4
cooled to 15 C. Further, 15 ml of 2-butanone was added
thereto, followed by Stirring overnight. The precipitates Synthesis of Mecobalamin
were collected by filtration and dried, to give 21.4 g of a The present example was conducted in the dark place
crude product of the title compound. Thereto was added 200 (under infrared ray).
ml of a 50% acetone aqueous Solution, and the mixture was To 390 ml of ion-exchanged water were added 30 g of
heated, adjusted to pH 6.5 with concentrated hydrochloric 15
cyanocobalamin, 14.61 g of trimethylsulfoxonium iodide,
acid and then filtered. After washing with 40 ml of a 50% 900 mg of iron (II) sulfate heptahydrate, 900 mg of cobalt
acetone aqueous Solution, 630 ml of acetone was added chloride hexahydrate and 22.5 ml of 2-butanone. After
dropwise to the filtrate, followed by stirring at 15 C. replacing the atmosphere of the System by nitrogen, the
overnight. Precipitated crystals were collected by filtration mixture was heated in a water bath and a Solution of Sodium
and dried, to give 17 g of the title compound (yield 86%). borohydride (12 g)/2N sodium hydroxide (1 ml)/water (60
Physical Properties of Mecobalamin Obtained ml) was added dropwise thereto under Stirring at an inner
The hydrochloride buffer (pH 2.0): UV was detected at temperature of 20° C. After stirring for 3 hours as it was, the
264-266, 303-307 and 459–462 nm. mixture was cooled to 10° C. and then stirred overnight.
The phosphate buffer (pH 7.0): UV, was detected at Thereto was added 24 ml of 3-methyl-2-butanone, followed
266-269, 341–344 and 520–524 nm. 25
by Stirring for 2 hours. Then, the precipitates were collected
Referential values of UV (Merck Index, 12th edition) by filtration and dried, to give 35 g of a crude product of the
Example 2 title compound. Thereto was added 300 ml of a 50%
methanol aqueous Solution and the mixture was heated at
Synthesis of Mecobalamin 35 C, filtered and washed with 120 ml of a 50% methanol
aqueous Solution. After the mixture was adjusted to pH 7.0
The present example was conducted in the dark place with concentrated hydrochloric acid, 1365 ml of acetone
(under infrared ray). was added dropwise thereto and the mixture was Stirred at
To 1.3 l of ion-exchanged water were added 100 g of 10° C. overnight. Precipitated crystals were collected by
cyanocobalamin and 32.46 g of trimethylsulfoxonium 35 filtration and dried, to give 25.9 g of the title compound
iodide. After replacing the atmosphere of the System by (yield 86.3%).
nitrogen, the mixture was heated in a water bath and a Example 5
solution of sodium borohydride (40 g)/2NSodium hydroxide
(2 ml)/water (200 ml) was added dropwise thereto under Synthesis of Mecobalamin
stirring at an inner temperature of 40 C. over 30 minutes. 40 The present example was conducted in the dark place
After Stirring for 1 hour as it was, the mixture was cooled to (under infrared ray).
room temperature and then Stirred overnight. The precipi To 130 ml of ion-exchanged water were added 10 g of
tates were collected by filtration and dried, to give a crude cyanocobalamin, 3.83 g of trimethylsulfoxonium bromide,
product of the title compound. Thereto was added 11 of a 700 mg of iron (II) sulfate heptahydrate and 7.5 ml of
50% acetone aqueous Solution, and the mixture was heated, 45 2-butanone. After replacing the atmosphere of the System by
adjusted to pH 6.5 with concentrated hydrochloric acid and nitrogen, the mixture was heated in a water bath and a
then filtered. After washing with 400 ml of a 50% acetone solution of sodium borohydride (4 g)/2N sodium hydroxide
aqueous Solution, 2.8 l of acetone was added dropwise (0.2 ml)/water (20 ml) was added dropwise thereto under
thereto and the mixture was stirred at 17 C. overnight. stirring at an inner temperature of 35 C. After stirring for 3
Precipitated crystals were collected by filtration and dried, to 50 hours as it was, the mixture was cooled to 15 C. and then
give 90 g of the title compound (yield 91%). stirred overnight. Thereto was added 7.5 ml of 2-butanone,
Example 3 followed by stirring for 2 hours. Then, the precipitates were
collected by filtration and dried, to give a crude product of
Synthesis of Mecobalamin the title compound. Thereto was added 140 ml of a 50%
55 acetone aqueous Solution, and the mixture was heated at 45
The present Example was conducted in the dark place C., filtered and washed with 60 ml of a 50% acetone aqueous
(under infrared ray). solution. After the mixture was adjusted to pH 6.5 with
To 1.3 l of ion-exchanged water were added 100 g of concentrated hydrochloric acid, 475 ml of acetone was
cyanocobalamin and 32.46 g of trimethylsulfoxonium added dropwise thereto and the mixture was stirred at 20° C.
iodide, 4 g of iron(II) sulfate heptahydrate and 100 ml of 60 overnight. Precipitated crystals were collected by filtration
2-butanone. Under a nitrogen Stream, the mixture was and dried, to give 8.86 g of the title compound (yield
heated in a water bath and a solution of sodium borohydride 89.3%).
(40 g)/2N sodium hydroxide (1 ml)/water (200 ml) was Example 6
added dropwise thereto under Stirring at an inner tempera
ture of 40 C. over 30 minutes. After stirring for 30 minutes 65 Synthesis of Mecobalamin
as it was, the mixture was returned to room temperature and The present example was conducted in the dark place
further stirred overnight. The precipitates were collected by (under infrared ray).
 US 6,657,057 B2
7 8
To 650 ml of ion-exchanged water were added 50 g of title compound. Thereto was added 140 ml of a 50% acetone
cyanocobalamin, 19.51 g of trimethylsulfoxonium bromide, acqueous solution. The mixture was heated at 45 C., filtered
3.5 g of cobalt chloride hexahydrate and 37.5 ml of and washed with 60 ml of a 50% acetone aqueous solution.
2-butanone. After replacing the atmosphere of the System by After the mixture was adjusted to pH 6.5 with concentrated
nitrogen, the mixture was heated in a water bath and a hydrochloric acid, 475 ml of acetone was added dropwise
solution of sodium borohydride (20g)/2NSodium hydroxide thereto and the mixture was stirred at 20° C. overnight.
(1 ml)/water (100 ml) was added dropwise thereto under Precipitated crystals were collected by filtration and dried, to
stirring at an inner temperature of 35 C. After stirring for 2 give 8.92 g of the title compound (yield 89.9%).
hours as it was, the mixture was cooled to 15 C. and then What is claimed is:
stirred overnight. Thereto was added 37.5 ml of 2-butanone, 1. A process for producing methylcobalamin (V), which
followed by stirring for 1 hour. Then, the precipitates were comprises the step of methylating cyanocobalamin (I) or
collected by filtration and dried, to give a crude product of hydroxocobalamin (II) represented by the following for
the title compound. Thereto was added 700 ml of a 50% mula:
methanol aqueous Solution, and the mixture was heated at
40° C., filtered and washed with 300 ml of a 50% acetone 15
NH3COCH2CH2 (HSCH.CONH,
aqueous Solution. After the mixture was adjusted to pH 6.5 NH3COCH2 -
w

with concentrated hydrochloric acid, methanol was evapo HC

rated. To the residue was added dropwise 2250 ml of HC
acetone, followed by stirring at 20° C. overnight. Precipi
tated crystals were collected by filtration and dried, to give H
45.0 g of the title compound (yield 90.7%).
Example 7
Synthesis of Mecobalamin 25
H HO
The present example was conducted in the dark place
(under infrared ray). ?H-CHCONHCH--O--O
d H. O
H ---CH2OH
To 130 ml of ion-exchanged water were added 10 g of
cyanocobalamin, 3.48g of trimethylsulfonium bromide, 700
mg of cobalt chloride hexahydrate, and 7.5 ml of R=CN: Cyanocobalamin (I)
2-butanone. After replacing the atmosphere of the System by R=OH: Hydroxocobalamin (II)
nitrogen, the whole was warmed on a water bath and a R=CH: Methylcobalamin (V)
solution of sodium borohydride (4 g)/2N sodium hydroxide in the presence of a reducing agent (III) and a water-soluble
(0.2 ml)/water (20 ml) was added dropwise thereto under 35 methylating agent (IV) wherein the water-soluble methylat
stirring at an inner temperature of 35 C. After stirring for 3 ing agent is represented by the formula (VI)
hours as it was, the mixture was cooled to 15 C. and then
stirred overnight. Thereto was added 7.5 ml of butanone, (VI)
followed by stirring for 2 hours. Then, the precipitates were
collected by filtration and dried, to give a crude product of 40
the title compound. Thereto was added 140 ml of a 50%
acetone aqueous Solution and the mixture was heated at 45
C., filtered and washed with 60 ml of a 50% acetone aqueous
solution. After the mixture was adjusted to pH 6.5 with
concentrated hydrochloric acid, 475 ml of acetone was 45
and wherein X means a halogen atom or methoxySulfo
added dropwise thereto and the mixture was stirred at 20 C. nyloxy group;
overnight. Precipitated crystals were collected by filtration and n means 0 or 1.
and dried, to give 8.94 g of the title compound (yield 2. A process for producing methylcobalamin (V), which
90.1%). comprises the step of methylating cyanocobalamin (I) or
50
hydroxocobalamin (II) in the presence of a reducing agent
Example 8 (III) and a water-Soluble methylating agent (IV) in an
aqueous Solution or a hydrous organic Solvent;
Synthesis of Mecobalamin wherein the water-Soluble methylating agent is repre
The present Example was conducted in the dark place sented by the formula (VI)
(under infrared ray). 55
To 130 ml of ion-exchanged water were added 10 g of (VI)
cyanocobalamin, 2.85 g of trimethylsulfoxonium chloride,
700 mg of iron (II) sulfate heptahydrate and 7.5 ml of
2-butanone. After replacing the atmosphere of the System by
HC-Sl
3
()
/N X
HC CH
nitrogen, the mixture was heated in a water bath. A Solution 60
of sodium borohydride (4 g)/2N sodium hydroxide (0.5
ml)/water (20 ml) was added dropwise thereto under stirring and wherein X means a halogen atom or methoxySulfo
at an inner temperature of 35 C. After stirring for 3 hours nyloxy group;
as it was, the mixture was cooled to 15 C. and then stirred and n means 0 or 1.
overnight. Thereto was added 7.5 ml of butanone, followed 65 3. A process for producing methylcobalamin (V), which
by Stirring for 2 hours. Then, the precipitates were collected comprises the Steps of methylating cyanocobalamin (I) or
by filtration and dried, to give 35 g of a crude product of the hydroxocobalamin (II) in the presence of a reducing agent
 US 6,657,057 B2
9 10
(III) and a water-soluble methylating agent (IV) in an 5. The process for producing methylcobalamin (V)
aqueous Solution or a hydrous organic Solvent; according to any one of claims 1 to 4, wherein the trimeth
wherein the water-Soluble methylating agent is repre ylsulfur derivative (VI) is at least one selected from trim
sented by the formula (VI) ethylsulfoxonium iodide, trimethylsulfonium iodide, trim
ethylsulfoxonium bromide, trimethylsulfonium bromide,
(VI) trimethylsulfoxonium chloride and trimethylsulfonium
O

HC
()7\ CH
X
chloride.
6. The process for producing methylcobalamin (V)
according to any of claims 1 to 4, wherein the reducing agent
(III) is sodium borohydride.
7. The process for producing methylcobalamin (V)
and wherein X means a halogen atom or methyoxySulfo according to claim 4, wherein the cyanion-trapping agent is
nyloxy group; at least one Selected from ferrous Sulfate and cobalt chloride.
and then precipitating the reaction product as crystals or 8. The process for producing methylcobalamin (V)
precipitates. 15
according to claim 4 or 7, wherein the amount of the cyan
4. A process for producing methylcobalamin (V), which ion-trapping agent is from 1 to 30% by weight relative to
comprises the Steps of methylating cyanocobalamin (I) or cyanocobalamin (I) or hydroxocobalamin (II).
hydroxocobalamin (II) in the presence of a cyan ion 9. The process for producing methylcobalamin (V)
trapping agent, a reducing agent (III) and a water-soluble according to any of claims 1 to 4, wherein the trimethyl
methylating agent (IV) in an aqueous Solution or a hydrous sulfur derivative (VI) is at least one selected from trimeth
organic Solvent ylsulfoxonium iodide, trimethylsulfonium iodide, trimeth
wherein the water-Soluble methylating agent is repre ylsulfoxonium bromide, trimethylsulfonium bromide,
sented by the formula (VI) trimethylsulfoxonium chloride and trimethylsulfonium
(VI) 25
chloride; and the reducing agent (III) is Sodium borohydride.
O 10. The process for producing methylcobalamin (V)
HC-S."
/N
i) X
according to claim 4, wherein the trimethylsulfur derivative
(VI) is at least one selected from trimethylsulfoxonium
HC CH iodide, trimethylsulfonium iodide, trimethylsulfoxonium
bromide, trimethylsulfonium bromide, trimethylsulfoxo
nium chloride and trimethylsulfonium chloride; the reducing
and wherein X means a halogen atom or methoxySulfo agent (III) is Sodium borohydride; and the cyanion-trapping
nyloxy group; agent is at least one Selected from ferrous Sulfate and cobalt
and n means 0 or 1; chloride.
and then precipitating the reaction product as crystals or
precipitates.