Vitamin B.sub.12 derivative, preparation process thereof, and use thereof

Vitamin B.sub.12 compounds, salts and pharmaceutical compositions containing said compounds represented by formula (I): ##STR1## wherein L, a ligand to the cobalt of the corrin ring, is selected from the group consisting of a cyano or adenosyl group, B is an imidazole group or a 5,6-dimethylbenzimidazole group, and R is a straight alkylene group having 1 to 8 carbon atoms, are disclosed. Methods for preparing compounds of Formula (I) as well as methods for using the compounds in in vitro assays, in cell growth studies and in in vivo studies using transplanted murine tumor cells are disclosed.

TECHNICAL FIELD 
The present invention relates to a novel vitamin B.sub.12 derivative and a 
salt thereof, a process for producing the same, and a use thereof. More 
specifically, it relates to a vitamin B.sub.12 derivative and a salt 
thereof represented by the formula (I): 
##STR2## 
wherein L represents a ligand to the cobalt of the corrin ring, B a base 
having a heterocyclic structure, and R a substituted or nonsubstituted 
hydrocarbon group, a process for preparing the same, a vitamin B.sub.12 
antagonistic agent, and a cell growth inhibiting or interfering agent 
containing the same as the active ingredient. 
BACKGROUND ART 
Vitamin B.sub.12 is an essential nutrient factor required for human and 
animals in a minute amount. In mammals, it is contained relatively 
abundantly in the liver. Neither animals nor plants can biosynthesize this 
vitamin, which can be produced only by microorganisms. As a disease due to 
a lack of vitamin B.sub.12 , pernicious anemia is particularly 
representative thereof, and megaloblastic hemopoiesis, methylmalonic acid 
uria, homocystinuria, and neuropathy, etc., are caused thereby. When 
absorbed into the body, vitamin B.sub.12 is converted to a metabolically 
active vitamin B.sub.12 coenzyme (adenosylcobalamin) and methylcobalamin; 
the former functioning as a coenzyme in an enzymatic reaction accompanied 
by a movement of hydrogen, such as methylmalonyl CoA mutase, and the 
latter functioning as a coenzyme in an enzymatic reaction accompanied by a 
movement of a methyl group, such as methionine synthetase. Particularly, 
methylcobalamin functions in a C.sub.1 metabolism involving a folic acid 
coenzyme, thereby participating indirectly in a biosynthesis of thymidylic 
acid and playing an important role in cell growth. Accordingly, a compound 
exhibiting an antagonistic activity against the vitamin B.sub.12 group, 
i.e., a vitamin B.sub.12 antagonist, may be considered to suppress or 
inhibit cell growth by interfering with the DNA synthesis, and to be 
effective as an antitumor agent (anticancer agent) against tumor cells 
(cancer cells). Also, the vitamin B.sub.12 group is important for the 
growth of microorganisms, and the vitamin B.sub.12 antagonist is 
considered to have an activity as an antimicrobial agent. Conversely, the 
vitamin B.sub.12 antagonist may be considered to be applied for screening 
a microorganism mutant strain having a high vitamin B.sub.12 productivity, 
with a resistance thereto as the index. 
In the prior art, various vitamin B.sub.12 derivatives have been 
synthesized and, for example, it is known that the vitamin B.sub.12 
derivatives synthesized chemically or microbiologically from cobyric acid 
and having the isopropanolamine moiety (--NHCH.sub.2 CH(CH.sub.3)O--) 
converted to, for example, --NHCH(CH.sub.3)CH.sub.2 O--, --NHCH.sub.2 
CH.sub.2 CH.sub.2 O--, --NHC(CH.sub.3).sub.2 CH.sub.2 O--, --NHCH.sub.2 
CH(C.sub.6 H.sub.5)O-- or --NHCH.sub.2 CH(CH.sub.2 F)O--, exhibit an 
antagonistic activity against Escherichia coli 113-3 and Lactobacillus 
leichmannii (Friedrich, Vitamin B.sub.12 und verwandte Corrinoid (Georg 
Thieme Verlag, Stuttgart), p. 289-308, 1975). Also, cobalt-free corrinoid 
isolated from a microorganism, or a different kind of metal corrinoid in 
which, for example, rhodium, copper, and zinc, are introduced into a 
cobalt-free corrinoid, is known to exhibit a similar antagonistic activity 
against the above-mentioned microorganisms (Friedrich, Vitamin B.sub.12 
und verwandte Corrinoid (Georg Thieme Verlag, Stuttgart), p. 289-308, 
1975, and Copenhagen, B.sub.12 (John Wiley & Sons, New York), vol. II, p. 
105-149, 1982). 
Nevertheless, the preparation of cobyric acid is complicated and it is 
difficult to obtain same in a large amount, and when a microorganism is 
used, a problem arises in practical application due to the isolation 
thereof. 
Also, vitamin B.sub.12 derivatives having a bromine or nitro group 
introduced into the C-10 position of the corrin ring, and vitamin B.sub.12 
derivatives in which the surrounding side chains of the corrin ring are 
derivatized to, for example, carboxyl group, ethylamide, anilide, and 
hydrazide, have been chemically synthesized (Friedrich, Vitamin B.sub.12 
und verwandte Corrinoid (Georg Thieme Verlag, Stuttgart), p. 289-308, 
1975), but these derivatives are still unsatisfactory due to a low 
antagonistic activity thereof. 
Accordingly, there is a need for a vitamin B.sub.12 antagonist having an 
excellent antagonistic activity, and which can be supplied in a large 
amount. 
DISCLOSURE OF THE INVENTION 
Accordingly, an object of the present invention is to provide a novel 
vitamin B.sub.12 antagonist having an excellent antagonistic activity, and 
which can be supplied in a large amount. 
Another object of the present invention is to provide a process for 
preparing a vitamin B.sub.12 derivative having an excellent B.sub.12 
-antagonistic activity, and which can be supplied in a large amount. 
A further object of the present invention is to provide a novel vitamin 
B.sub.12 antagonistic agent. 
A still further object of the present invention is to provide a novel cell 
growth inhibiting or interfering agent. 
A still further object of the present invention is to provide a novel 
antitumor agent. 
A still further object of the present invention is to provide a method of 
screening a microorganism mutant strain of high vitamin B.sub.12 
productivity. 
Other objects and advantages of the present invention will be apparent from 
the following descriptions. 
In accordance with the present invention, there is provided a vitamin 
B.sub.12 derivative and a salt thereof represented by the formula (I): 
##STR3## 
wherein L represents a ligand to the cobalt of the corrin ring, B a base 
having a heterocyclic structure, and R a substituted or nonsubstituted 
hydrocarbon group, a vitamin B.sub.12 antagonistic agent, a cell growth 
inhibiting or interfering agent, and an antitumor agent containing the 
same as the active ingredient.

BEST MODE OF CARRYING OUT THE INVENTION 
The present inventors made intensive studies of the problems of the prior 
art as described above, and found that the vitamin B.sub.12 derivatives of 
the formula (I) obtained by a modification of the ribose moiety of the 
nucleotide portion of the vitamin B.sub.12 group can be supplied in a 
large amount, and have a very good vitamin B.sub.12 antagonist activity. 
In the vitamin B.sub.12 derivative represented by the formula (I), L 
represents a ligand to the cobalt of the corrin ring. Examples of the 
ligand represented by L include a cyano group, substituted or 
nonsubstituted adenosyl group or adeninylalkyl group (alkyl group is a 
straight or branched alkyl group having 1 to 8 carbon atoms), hydroxyl 
group, water molecule, or a straight or branched alkyl group having 1 to 8 
carbon atoms, and further, L can be represented by two of these groups, 
which may be the same or different. Alkyl groups having 1 to 8 carbon 
atoms, are preferably a methyl group, ethyl group, and propyl group. The 
ligand L is generally coordinated above the corrin ring, but can be on 
either side of the corrin ring, or can exist on both sides thereof. 
In the formula (I), R is a substituted or nonsubstituted hydrocarbon group, 
and is exemplified by an alkylene group having 1 to 8 carbon atoms, which 
is substituted by an aromatic group or a halogen atom or is 
nonsubstituted, or a cyclic hydrocarbon group. An alkylene group having 1 
to 8 carbon atoms is preferred. 
In the formula (I), B represents a base having a heterocyclic structure, as 
exemplified by a substituted or nonsubstituted imidazole group, pyridine 
group, or derivatives thereof. As such derivatives, a benzimidazole group 
having a benzene ring fused to imidazole group, or a 
5,6-dimethylbenzimidazole group, which is a derivative thereof, are 
included. This base B is coordinated generally below the cobalt of the 
corrin ring, but in the present invention, the case wherein it is not 
coordinated is included. 
Also, the present invention provides a process for preparing the vitamin 
B.sub.12 derivative represented by the formula (I). 
More specifically, (1) cyanoaquacobinamide or dicyanocobinamide is allowed 
to react with a phosphoric acid ester derivative or a salt thereof 
represented by the following formula (II): 
EQU B--R--O--PO.sub.3 H.sub.2 (II) 
wherein B and R are the same as defined in the above formula (I), to 
prepare a corresponding compound of the formula (I) wherein L is a cyano 
group. 
The reaction between the compound represented by the formula (II) and 
cyanoaquacobinamide or dicyanocobinamide can be carried out by using, for 
example, a condensing agent, preferably dicyclohexylcarbodiimide, in an 
organic solvent, preferably a mixed solution of pyridine and 
N,N-dimethylformamide. The reaction temperature must not be higher than 
the boiling temperature of the solvent employed, and is, for example, 
preferably around room temperature. Cyanoaquacobinamide is prepared easily 
from vitamin B.sub.12 (cyanocobalamin) or dicyanocobinamide. The compound 
represented by the formula (I), wherein L is a cyano group obtained as a 
result of a reaction, can be purified by extraction, column 
chromatography, and (or) high performance liquid chromatography. 
Alternatively, (2) the compound represented by the formula (I) wherein L is 
a cyano group can be reduced and then subjected to a) reoxidation, or b) 
alkylation followed by photolysis to be converted into a compound 
represented by the formula (I) wherein L is a hydroxyl group or water 
molecule, or (3) the compound represented by the formula (I) wherein L is 
a cyano group, hydroxyl group or water molecule can be reduced with, for 
example, sodium borohydride, zinc and ammonium chloride, zinc and acetic 
acid, or divalent chromium, and then reacted with, for example, a 
halogenated alkane (the alkyl moiety is a straight or branched alkyl group 
having 1 to 8 carbon atoms, preferably a methyl group, ethyl group, or 
propyl group), or a halogenated or tosylated substituted or nonsubstituted 
adenosine or adeninylalkane (the alkyl moiety is a straight or branched 
alkyl group having 1 to 8 carbon atoms), for example, preferably 
5'-halo-5'-deoxyadenosine, to prepare a corresponding compound represented 
by the formula (I). These compounds can be obtained as purified products 
by, for example, extraction or column chromatography. The obtained 
compound represented by the formula (I) can be also reacted with, for 
example, sulfuric acid to form a salt. 
Further, the present invention concerns a phosphoric acid ester derivative 
represented by the formula (II) and a salt thereof, and a method of 
preparing same. These phosphoric acid ester derivatives and salts thereof 
are useful intermediates for the preparation of the vitamin B.sub.12 
derivatives of the present invention represented by the formula (I), and 
can be obtained by the methods described below. 
Namely, the phosphoric acid ester derivative represented by the formula 
(II) and a salt thereof is prepared by reacting a free base B' having a 
heterocyclic structure with a compound represented by the following 
formula (III): 
EQU X--R--OH (XXX) 
wherein X represents a leaving group, and R is the same as defined in the 
above formula (I) to obtain a compound represented by the following 
formula (IV): 
EQU S--R--OH (XV) 
wherein B and R are the same as defined in the above formula (I), and 
subsequently, phosphorylating this compound, preferably by using a 
2-cyanoethylphosphoric acid pyridinium salt. 
The free base B' having a heterocyclic structure can be, for example, a 
substituted or nonsubstituted imidazole, pyridine, or a derivative 
thereof, and examples of the imidazole derivatives include benzimidazole 
or 5,6-dimethylbenzimidazole. These compounds B' are commercially 
available, or can be prepared by known methods. 
In the compound represented by the formula (III), X represents a leaving 
group which is, for example, a halogen atom, preferably a chlorine atom. 
The compound represented by the formula (III) is commercially available or 
can be obtained by known methods. 
The reaction between the free base B' having a heterocyclic structure and 
the compound represented by the formula (III) may be carried out in the 
presence of a base, preferably sodium hydride or potassium carbonate, at a 
reaction temperature of preferably around room temperature, or the 
reaction may be carried out by heating under reflux. This reaction is 
carried out in an organic solvent, preferably N,N-dimethylformamide, or 
dioxane. The compound represented by the formula (IV), which is the 
reaction product, can be used as crude product for the subsequent 
reactions, but preferably is separated and purified from the reaction 
mixture by known methods such as washing, extraction or column 
chromatography, before use. 
A phosphorylation of the compound represented by the formula (IV) is 
practiced in the presence of, for example, a condensing agent, preferably 
2-cyanoethylphosphoric acid pyridinium salt, followed by the reaction with 
lithium hydroxide. As the condensing agent, dicyclohexylcarbodiimide is 
preferable, and the reaction is carried out in an organic solvent, 
preferably pyridine, at a reaction temperature not higher than the boiling 
point of the solvent employed, preferably around room temperature. The 
2-cyanoethylphosphoric acid pyridinium salt can be easily prepared from 
2-cyanoethylphosphoric acid barium salt, according to the method of Tener 
(J. Am. Chem. Soc., vol. 83, p. 159-168, 1961). The compound represented 
by the formula (II), which is the reaction product, can be used as the 
crude product for the intermediate for the preparation of the vitamin 
B.sub.12 derivative represented by the formula (I), or if desired, can be 
separated and purified from the reaction mixture by known methods such as 
filtration, extraction or column chromatography, or if preferable, in the 
form of a salt such as lithium, sodium, potassium, barium or pyridine, by 
known methods. 
The vitamin B.sub.12 derivatives represented by the formula (I) have highly 
beneficial pharmacological properties. Namely, these compounds have 
vitamin B.sub.12 antagonist activities, can be used as vitamin B.sub.12 
antagonistic agents, and exhibit better antagonistic activities than known 
vitamin B.sub.12 antagonists. Further, since the vitamin B.sub.12 
derivative represented by the formula (I) makes use of cyanoaquacobinamide 
or dicyanocobinamide as the starting material, according to the 
preparation method as described above, it can be also supplied more simply 
and in a larger amount than known vitamin B.sub.12 antagonists. Also, the 
vitamin B.sub.12 derivatives represented by the formula (I) of the present 
invention can be used as a cell growth inhibiting or interfering agent 
containing at least one thereof as the antimicrobial agent when the cell 
is a microorganism, or as the antitumor agent (anticancer agent) when the 
cell is an animal cell, particularly a tumor cell (cancer cell). When used 
as such a cell growth inhibiting or interfering agent, the compound can be 
formulated into a pharmaceutical preparation comprising at least one 
active ingredient of the vitamin B.sub.12 derivatives represented by the 
formula (I), and a pharmaceutically acceptable carrier and (or) necessary 
additives. 
The vitamin B.sub.12 derivatives represented by the formula (I) of the 
present invention act antagonistically against the vitamin B.sub.12 group, 
and therefore, growth of microorganisms producing a high quantity of 
vitamin B.sub.12 will be suppressed weakly by the vitamin B.sub.12 
derivatives represented by the formula (I), or will not be suppressed at 
all. Therefore, a subject matter of the present invention is also the use 
of the vitamin B.sub.12 derivative of the present invention, for screening 
a microorganism mutant strain of high vitamin B.sub.12 productivity. 
As described above, the vitamin B.sub.12 derivative of the present 
invention has a vitamin B.sub.12 antagonistic activity, and is very useful 
for the study of a vitamin B.sub.12 group during an enzymatic reaction, 
for example, a coenzyme function thereof. Further, the vitamin B.sub.12 
derivative of the present invention exhibits a very good antagonistic 
activity, and the cell inhibiting or interfering agent containing such a 
vitamin B.sub.12 derivative as the active ingredient is useful as, for 
example, an antimicrobial agent or antitumor agent (anticancer agent). 
Also, the vitamin B.sub.12 derivative of the present invention can be used 
for screening a microorganism mutant of high vitamin B.sub.12 
productivity. Further, the vitamin B.sub.12 derivative of the present 
invention can be supplied simply and in a large amount, because 
cyanoaquacobinamide or dicyanocobinamide is employed as the starting 
material. 
The vitamin B.sub.12 derivative of the above formula (I) and a 
pharmaceutically acceptable salt thereof may be administered individually, 
but if necessary or desired, can be administered orally or parenterally in 
a desired dosage form admixed with pharmaceutically acceptable general 
purpose carriers, excipients, solvents, and vehicles, etc. Oral 
administration agents may be in the form of tablets, pills, granules, 
powders, solutions, suspensions, or capsules. Parenteral administration 
agents may be in the form of subcutaneous and dermal ointments, creams, or 
gels. Intratracheal agents may be administered intratracheally by use of 
an appropriate spraying agent such as an aerosol. 
The tablet of the composition containing the vitamin B.sub.12 derivative of 
the above formula (I) of the present invention or a pharmaceutically 
acceptable salt thereof can be prepared by mixing an excipient such as 
lactose, starch, or crystalline cellulose, optionally with a binder such 
as carboxymethyl cellulose, methyl cellulose, and polyvinyl pyrrolidone; 
and/or disintegrating agent such as sodium alginate and sodium hydrogen 
carbonate, with the active ingredient, and molding the mixture in a 
conventional manner. For a preparation of solutions or suspensions, for 
example, glycerol esters such as tricaprylin and triacetin, and alcohols 
such as ethanol, may be mixed with the active ingredient, and a 
conventional method may be applied to the mixture. For the preparation of 
capsules, granules, powders or solutions may be mixed together with the 
active ingredient and capsule forming materials such as gelatin, etc., and 
a capsule forming method applied thereto. 
The injections may be prepared by dissolving the active ingredient in, for 
example, physiological saline, ethanol, or propylene glycol, depending on 
the form of aqueous or nonaqueous solution agent, and adding, if 
necessary, preservatives and stabilizers, etc. 
As the suppository, conventional dosage forms such as gelatin soft capsules 
containing the active ingredient may be employed. 
Ointments, creams and the like may be formed in a conventional manner from 
the active ingredient and the required carrier. 
The aerosol administration agent can be prepared by using a 
pharmaceutically acceptable surfactant prepared from a fatty acid having 6 
to 22 carbon atoms, a fatty acid polyhydric alcohol or a cyclic anhydride 
thereof, a propellant such as an alkane or a fluorinated alkane having not 
more than 5 carbon atoms, and the active ingredient. 
The concentration of the vitamin B.sub.12 derivative of the formula (I) and 
a pharmaceutically acceptable salt thereof, in such a pharmaceutical 
preparation, is not particularly limited, but is suitably about 0.001 to 
50% by weight, preferably about 0.01 to 10% by weight, in the preparation. 
Also, the dose is not particularly limited, but is suitably 0.01 to 500 
mg/day/human, preferably 0.1 to 100 mg/day/human, generally in 1 to 4 
doses per day. 
EXAMPLES 
The present invention is now described in more detail, with reference to 
Examples, which in no way limit the present invention. 
Example 1 
Synthesis of 2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide phosphoric 
acid 
In Example 1, the following processes were carried out. 
1) Synthesis of 1-(2-hydroxyethyl)-5,6-dimethylbenzimidazole 
A 1.46 g amount of 5,6-dimethylbenzimidazole was dissolved in 50 ml of dry 
N,N-dimethylformamide, 0.96 g of NaH was added to the solution, and the 
mixture was stirred in an ice-water bath for 30 minutes. Then to the 
mixture was added dropwise 5 ml of ethylene chloride, the mixture was 
stirred at room temperature for 15 hours, and the reaction was further 
carried out for 24 hours while adding 0.78 g of NaH. The reaction was 
stopped by an addition of 50 ml of water, and the obtained reaction 
mixture was washed with n-hexane, diluted with water, adjusted to a pH of 
2.5 with HCl, and then applied to an ion exchange column (Dowex 50 
(hydrogen ion form)). An elution was effected successively with water, 30% 
ethanol, and ammoniacal 30% ethanol, and the fractions eluted with 
ammoniacal 30% ethanol were concentrated to dryness under a reduced 
pressure to give crude 1-(2-hydroxy-ethyl)-5,6-dimethylbenzimidazole. The 
conversion to this compound from 5,6-dimethylbenzimidazole was found to be 
75% by an assay according to thin layer chromatography. 
Further, the crude 1-(2-hydroxyethyl)-5,6-dimethylbenzimidazole was 
dissolved in 50% methanol, and after a removal of insolubles, purified by 
reverse phase liquid chromatography and concentrated to dryness under a 
reduced pressure, to give purified 
1-(2-hydroxyethyl)-5,6-dimethylbenzimidazole. 
.sup.13 C--NMR (CDCl.sub.3, .delta. ppm) 20.13, 20.51, 48.11, 60.43, 
109.54, 119,59, 130.82, 131.83. 
2) Preparation of 2-cyanoethylphosphoric acid pyridinium salt 
According to the method of Tenor (J. Am. Chem. Soc., p. 159-168, 1961), 
2-cyanoethylphosphoric acid pyridinium salt was prepared. Namely, 1.61 g 
of barium 2-cyanoethylphosphate and 10 g of an ion exchange resin (Dowex 
50 (hydrogen ion form)) were suspended in water, stirred at room 
temperature for 30 minutes, 2 ml of pyridine was added to the supernatant, 
the mixture was washed with water, and the mixture then concentrated to 
dryness under a reduced pressure, followed by an addition of pyridine to 
give 1 mmol/ml of 2-cyanoethylphosphoric acid pyridinium solution. 
3) Synthesis of 2-(5,6-dimethylbenzimidazolyl)ethylphosphoric acid 
A 0.19 g amount of the crude 1-(2-hydroxyethyl)-5,6-dimethylbenzimidazole 
and 2 ml of 2-cyanoethylphosphoric acid pyridinium salt solution (1 
mmol/ml) were dissolved in 20 ml of dry pyridine, and the solution was 
concentrated to dryness under a reduced pressure. This process was 
repeated twice more, and further, the dried material was thoroughly dried 
by a vacuum pump, dissolved in 20 ml of dry pyridine, 1.67 g of 
dicyclohexylcarbodiimide was added thereto, and the mixture was stored at 
room temperature for 2 days. After an addition of water to the product, 
the mixture was concentrated to dryness under a reduced pressure, followed 
by an addition of 40 ml of an aqueous LiOH solution (0.5 N) to carry out 
the reaction at 100.degree. C. for 45 minutes. The reaction mixture was 
filtered to give a filtrate, which was diluted with water, adjusted to a 
pH of 2.5 to 3, and then the mixture was applied to an ion exchange column 
(Dowex 50 (hydrogen ion form)). Elution was effected successively with 
water and 2 N pyridine, and the fractions eluted with the latter were 
concentrated to dryness under a reduced pressure to give 0.27 g of crude 
2-(5,6-dimethylbenzimidazolyl)ethylphosphoric acid. 
4 ) Preparation of cyanoaquacobinamide 
A 1 g amount of cyanocobalamin (vitamin B.sub.12) was dissolved in 144 ml 
of water, and to the solution were added 76.8 mg of an aqueous 0.33 M 
Ce(NO.sub.3).sub.3 solution and 51.2 ml of 1 N NaOH solution. 
Subsequently, to the mixture was added 1.77 g of KCN, and after stirring 
at 90.degree. to 100.degree. C. for one hour, the mixture was cooled to 
room temperature, adjusted to a pH of 8.5, and then left to stand at 
4.degree. C. overnight. The mixture was then filtered, and the filtrate 
desalted by phenol extraction and applied to an ion exchange column 
(diethylaminoethyl cellulose (acetate form)). To the fractions eluted with 
water was added a small amount of acetic acid, and the mixture was 
concentrated to dryness under a reduced pressure to give crude 
cyanoaquacobinamide. This was further applied to a phosphocellulose column 
(pH 7), eluted successively with 50% ethanol and a 50 mM NaCl solution, 
and the fractions eluted with the latter were desalted by phenol 
extraction to give 800 mg of crude cyanoaquacobinamide. 
5) Synthesis of 2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide 
phosphoric acid 
A solution of 0.27 g of crude 2-(5,6-dimethylbenzimidazolyl)ethylphosphoric 
acid and 0.2 g of purified cyanoaquacobinamide dissolved in 5 ml of 
pyridine was concentrated to dryness under a reduced pressure. This 
process was repeated twice more, and further, the dried product thoroughly 
dried by a vacuum pump, followed by addition of 15 ml of dry 
N,N-dimethylformamide, 10 ml of dry pyridine, and 1.5 g of 
dicyclohexylcarbodiimide thereto. The mixture was stirred at room 
temperature for 12 days, and the reaction was stopped by an addition of 
water and KCN. The reaction mixture was desalted by phenol extraction, 
then applied to a phosphocellulose column (pH 3), then the fractions 
passed as such were applied to an ion exchange column (diethylaminoethyl 
cellulose (acetate form)). Further, the fractions passed as such were 
purified by reverse phase high performance liquid chromatography to give 
50 mg of purified 2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide 
phosphoric acid as a concentrated dry product. The 
2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide phosphoric acid was 
confirmed by reverse phase high performance liquid chromatography and thin 
layer chromatography to be single. Further, it was confirmed by thin layer 
chromatography and reverse phase high performance liquid chromatography 
that 1-(2-hydroxyethyl)-5,6-dimethylbenzimidazole was obtained 
qualitatively and quantitatively by cerium hydrolysis of 
2-(5,6-dimethylbenzimidazolyl)-ethylcyanocobinamide phosphoric acid 
obtained. 
Example 2 
Synthesis of 3-(5,6-dimethylbenzimidazolyl)propylcyanocobinamide phosphoric 
acid 
In Example 2, the following processes were carried out. 
1) Synthesis of 1-(3-hydroxypropyl)-5,6-dimethylbenzimidazole 
To a solution of 1.46 g of 5,6-dimethylbenzimidazole dissolved in 50 ml of 
dry N,N-dimethylformamide was added 0.76 g of NaH, the mixture was stirred 
at room temperature for 30 minutes. To the mixture was added dropwise 5 ml 
of 3-chloro-1-propanol, and the reaction was carried out by stirring at 
room temperature for 2 days, followed by an addition of water to stop the 
reaction. Subsequently, according to the same processes as in Example 1, 
crude 1-(3-hydroxypropyl)-5,6-dimethylbenzimidazole was obtained. The 
conversion to this compound from 5,6-dimethylbenzimidazole was found to be 
90%. 
Further, the crude 1-(3-hydroxypropyl)-5,6-dimethylbenzimidazole was 
dissolved in 50% methanol, purified by high performance liquid 
chromatography, and concentrated to dryness under a reduced pressure to 
give purified 1-(3-hydroxypropyl)-5,6-dimethylbenzimidazole. 
.sup.13 C--NMR (CDCl.sub.3 , .delta. ppm) 20.04, 20.40, 31.71, 41.16, 
58.47, 109.97, 120.38, 131.21, 132.28. 
.sup.1 H--NMR (CDCl.sub.3, .delta. ppm) 2.0-2.2 (2H, m), 2.35 (3H, s), 2.37 
(3H, s), 3.58 (2H, t, J=6.0 Hz), 4.30 (2H, t, J=7.0 Hz), 7.18 (1H, s), 
7.54 (1H, s), 7.78 (1H, s). 
2) Synthesis of 3-(5,6-dimethylbenzimidazolyl)propyl phosphoric acid 
Using 0.2 g of the crude 1-(3-hydroxypropyl)-5,6-dimethylbenzimidazole and 
2 ml of 2-cyanoethylphosphoric acid pyridinium salt solution (1 mmol/ml) 
as the starting substances, according to the same processes as in Example 
1, 0.3 g of crude 3-(5,6-dimethylbenzimidazolyl)propylphosphoric acid was 
obtained. 
3) Synthesis of 3-(5,6-dimethylbenzimidazolyl)propylcyanocobinamide 
phosphoric acid 
Using 0.3 g of crude 3-(5,6-dimethylbenzimidazolyl)propylphosphoric acid 
and 0.2 g of purified cyanocobinamide as the starting substances, and 
subsequently carrying out the same processes as in Example 1, 33 mg of 
purified 3-(5,6-dimethylbenzimidazolyl)propylcyanocobinamide phosphoric 
acid was obtained. The reaction time was made 3.5 days, and the 
confirmation of the compound as a single product was similar to that of 
Example 1. Further, it was confirmed by thin layer chromatography and 
reverse phase high performance liquid chromatography that 
1-(3-hydroxypropyl)-5,6-dimethylbenzimidazole was obtained qualitatively 
and quantitatively by cerium hydrolysis of the 
3-(5,6-dimethylbenzimidazolyl)propylcyanocobinamide phosphoric acid, and 
the .sup.13 C--NMR and .sup.1 H--NMR thereof were found to be identical 
with those of the 1-(3-hydroxypropyl)-5,6-dimethylbenzimidazole obtained 
in 1) of Example 2. 
Example 3 
Synthesis of 4-(5,6-dimethylbenzimidazoyl)butylcyanocobinamide phosphoric 
acid 
In Example 3, the following processes were carried out. 
1) Synthesis of 1-(4-hydroxybutyl)-5,6-dimethylbenzimidazole 
To a solution of 1.46 g of 5,6-dimethylbenzimidazole dissolved in 50 ml of 
dry N,N-dimethylformamide was added 0.9 g of NaH, the mixture was stirred 
at room temperature for 30 minutes, 8 ml of 4-chloro-1-butanol was added 
dropwise, and after stirring at room temperature for one day, 0.65 g of 
NaH and 2 ml of 4-chloro-1-butanol were further added to carry out the 
reaction for 8 days. Following the same processes as in Example 1, crude 
1-(4-hydroxybutyl)-5,6-dimethylbenzimidazole was obtained. The conversion 
to this compound from 5,6-dimethylbenzimidazole was found to be 50%. 
2) Synthesis of 4-(5,6-dimethylbenzimidazolyl)butylphosphoric acid 
Using 0.2 g of the crude 1-(4-hydroxybutyl)-5,6-dimethylbenzimidazole and 2 
ml of 2-cyanoethylphosphoric acid pyridinium salt solution (1 mmol/ml) as 
the starting substances, according to the same processes as in Example 1, 
0.3 g of crude 4-(5,6-dimethylbenzimidazolyl)butylphosphoric acid was 
obtained. 
3) Synthesis of 4-(5,6-dimethylbenzimidazolyl)butylcyanocobinamide 
phosphoric acid 
Using 0.3 g of the crude 4-(5,6-dimethylbenzimidazolyl)butylphosphoric acid 
and 0.2 g of purified cyanoaquacobinamide as the starting substances, 
according to the same processes as in Example 1, 15 mg of purified 
4-(5,6-dimethylbenzimidazolyl)butylcyanocobinamide phosphoric acid was 
obtained. The reaction time was 6 days, and the compound was confirmed 
according to the same processes as in Example 1, by thin layer 
chromatography. 
Example 4 
Synthesis of 6-(5,6-dimethylbenzimidazolyl)hexylcyanocobinamide phosphoric 
acid 
In Example 4, the following operations were carried out. 
1) Synthesis of 1-(6-hydroxyhexyl)-5,6-dimethylbenzimidazole 
To a solution of 1.46 g of 5,6-dimethylbenzimidazole dissolved in 50 ml of 
dry N,N-dimethylformamide was added 0.84 g of NaH, and further, 8.2 ml of 
6-chloro-1-hexanol. After the reaction was carried out by stirring the 
mixture at room temperature for one day, the reaction was stopped by an 
addition of water. Subsequently, according to the same processes as in 
Example 1, crude 1-(6-hydroxyhexyl)-5,6-dimethylbenzimidazole was 
obtained. The conversion to this compound from 5,6-dimethylbenzimidazole 
was found to be 95% or higher. 
2) Synthesis of 6-(5,6-dimethylbenzimidazolyl)hexylphosphoric acid 
Using 0.2 g of the crude 1-(6-hydroxyhexyl)-5,6-dimethylbenzimidazole and 2 
ml of a 2-cyanoethylphosphoric acid pyridinium salt solution (1 mmol/ml), 
and subsequently following the same procedures as in Example 1, 0.35 g of 
crude 6-(5,6-dimethylbenzimidazolyl)hexylphosphoric acid was obtained. 
3) Synthesis of 6-(5,6-dimethylbenzimidazolyl)hexylcyanocobinamide 
phosphoric acid 
Using 0.35 g of the crude 6-(5,6-dimethylbenzimidazolyl)hexylphosphoric 
acid and 0.2 g of purified cyanoaquacobinamide, and subsequently following 
the same processes as in Example 1, 20 mg of purified 
6-(5,6-dimethylbenzimidazolyl)hexylcyanocobinamide phosphoric acid was 
obtained. The reaction time was 5 days, and this compound was confirmed 
according to the same processes as in Example 1, by thin layer 
chromatography. 
Example 5 
Synthesis of 3-imidazolylpropylcyanocobinamide phosphoric acid 
In Example 5, the following processes were carried out. 
1) Synthesis of 1-(3-hydroxypropyl)imidazole 
To a solution of 1.7 g of imidazole dissolved in 125 ml of dioxane was 
added 17.25 g of potassium carbonate, and further, 13.8 ml of 
3-chloro-1-propanol was added dropwise, followed by heating under reflux 
for 7.5 hours. The reaction mixture obtained was filtrated to remove 
potassium carbonate, the reaction stopped by an addition of water, and the 
pH adjusted to 2.5 before the reaction mixture was applied to an ion 
exchange column (Dowex 50 (hydrogen ion form)). Elution was effected 
successively with water, 30% ethanol, and ammonical 30% ethanol, and the 
fractions eluted with ammonical 30% ethanol were concentrated, the 
concentrate was applied to a phosphocellulose column (pH 8), and the 
fractions passed as such were concentrated to dryness under a reduced 
pressure, whereby 2.5 g of purified 1-(3-hydroxypropyl)imidazole was 
obtained. 
.sup.13 C--NMR (D.sub.2 O, .delta. ppm) 34.86, 45.75, 60.51, 122.54, 
130.05, 140.26. 
2) Synthesis of 3-imidazolylpropylphosphoric acid 
Using 0.13 g of the purified 1-(3-hydroxypropyl)imidazole and 2 ml of 
2-cyanoethylphosphoric acid pyridinium solution (1 mmol/ml), and 
subsequently following the same processes as in Example 1, 0.2 g of crude 
3-imidazolylpropylphosphoric acid was obtained. 
3) Synthesis of 3-imidazolylpropylcyanocobinamide phosphoric acid 
Using 0.2 g of the crude 3-imidazolylpropylphosphoric acid and 0.2 of 
purified cyanoaquacobinamide, and subsequently following the same 
processes as in Example 1, 20 mg of purified 
3-imidazolylpropylcyanocobinamide phosphoric acid was obtained. The 
reaction time was 3 weeks, and the compound was confirmed by high 
performance liquid chromatography, .sup.13 C--NMR and .sup.1 H--NMR 
according to the same processes as in Example 2. 
Example 6 
Preparation of 2-(5,6-dimethylbenzimidazolyl)ethyladenosylcobinamide 
phosphoric acid 
To a solution of 10 mg of 
2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide phosphoric acid 
dissolved in 3 ml of water was added 100 mg of NaBH.sub.4 , and the 
mixture was sealed, left to stand for 15 minutes to effect reduction, 25 
mg of 5'-iodo-5'-deoxyadenosine together with 3 ml of 
N,N-dimethylformamide were injected into the sealed vessel in the dark, 
and further left to stand under water cooling for 30 minutes. The 
following processes were carried out in the dark. To the reaction mixture 
were added water and 1 M potassium phosphate buffer (pH 5.0), and 
subsequently, the mixture was applied to an adsorption column (Amberlite 
XAD-2) and successively eluted with water and 80% ethanol. The fractions 
eluted with the latter were concentrated to dryness under a reduced 
pressure, the product dissolved in a small amount of water, and the 
solution applied to an ion exchange column (carboxymethyl cellulose 
(hydrogen ion form)) and successively eluted with water and 50 mM NaCl 
solution. The fractions eluted with the latter were desalted with an 
adsorption column (Amberlite XAD-2), further purified by an ion exchange 
column (phosphocellulose column (pH 6)), and concentrated to dryness under 
a reduced pressure to give purified 
2-(5,6-dimethylbenzimidazolyl)ethyladenosylcobinamide phosphoric acid. 
Example 7 
Preparation of 3-(5,6-dimethylbenzimidazolyl)propyladenosylcobinamide 
phosphoric acid 
In Example 7, using 3-(5,6-dimethylbenzimidazolyl)propylcyanocobinamide 
phosphoric acid in place of 
2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide phosphoric acid in 
Example 6, 3-(5,6-dimethylbenzimidazolyl)propyladenosylcobinamide 
phosphoric acid was obtained according to the same procedures as in 
Example 6. 
Example 8 
Preparation of 4-(5,6-dimethylbenzimidazolyl)butyladenosylcobinamide 
phosphoric acid 
In Example 8, using 4-(5,6-dimethylbenzimidazolyl)butylcyanocobinamide 
phosphoric acid in place of 
2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide phosphoric acid in 
Example 6, 4-(5,6-dimethylbenzimidazolyl)butyladenosylcobinamide 
phospholic acid was obtained according to the same procedures as in 
Example 6. 
Example 9 
Preparation of 6-(5,6-dimethylbenzimidazolyl)hexyladenosylcobinamide 
phosphoric acid 
In Example 9, using 6-(5,6-dimethylbenzimidazolyl)hexylcyanocobinamide 
phosphoric acid in place of 
2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide phosphoric acid in 
Example 6, 6-(5,6-dimethylbenzimidazolyl)hexyladenosylcobinamide 
phosphoric acid was obtained according to the same procedures as in 
Example 6. 
Example 10 
Preparation of 3-imidazolylpropyladenosylcobinamide phosphoric acid 
In Example 10, using 3-imidazolylpropylcyanocobinamide phosphoric acid in 
place of 2-(5,6-dimethylbenzimidazolyl)ethylcyanocobinamide phosphoric 
acid in Example 6, 3-imidazolylpropyladenosylcobinamide phosphoric acid 
was obtained according to the same procedures as in Example 6. 
Example 11 
Assay of coenzyme activity of vitamin B.sub.12 derivative 
The coenzyme activities of vitamin B.sub.12 derivatives prepared in 
Examples 1 to 10 were assayed according to the two methods shown below. 
1) Assay of coenzyme activity by using 
3-methyl-2-benzothiazolinonehydrazone 
The assay was carried out according to the method of Toraya et al. (J. 
Biol. Chem., vol. 252, p. 963-970, 1977). 
As the enzyme, diol dehydrase was employed. This enzyme utilizes 
adenosylcobalamin, the vitamin B.sub.12 coenzyme as the coenzyme and 
exhibits no activity if the vitamin B.sub.12 coenzyme (adenosylcobalamin) 
is not present. Diol dehydrase was highly purified from Klebsiella 
pneumoniae ATCC 8724 according to the method of Poznanskaya (Arch. 
Biochem. Biophys., vol. 194, p. 379-386, 1979) and adjusted to a solution 
of 0.3 Unit/ml with 0.05 M potassium phosphate buffer (pH 8.0). As the 
substrate of this enzyme, a 1 M aqueous 1,2-propanediol solution was 
employed. 
In an ice-water bath, 0.1 ml of the substrate solution, 0.1 ml of 0.5 M 
aqueous potassium chloride solution, 0.1 ml of diol dehydrase solution, 
and 0.6 ml of 0.05 M potassium phosphate buffer (pH 8.0) were mixed, and 
to the mixture were added, in the dark, 0.1 ml of 0.2 mM vitamin B.sub.12 
coenzyme (adenosylcobalamin), vitamin B.sub.12 (cyanocobalamin) or each 
vitamin B.sub.12 derivative prepared in Examples 1 to 10. After the 
mixture was incubated in the dark at 37.degree. C. for 10 minutes, 1 ml of 
0.1M potassium citrate buffer (pH 3.6) was added to stop the enzyme 
reaction, and then 0.5 ml of 0.1% aqueous 
3-methyl-2-benzothianolinonehydrazone was added, followed by a 
continuation of the incubation at 37.degree. C. for 15 minutes. To the 
mixture was added 1 ml of water, and the absorbance at 305 nm was measured 
by a Shimazu Double-beam Spectrophotometer Model UV-140-02 Model and the 
coenzyme activity (k.sub.cat) was calculated. 
2) Assay of coenzyme activity using alcohol dehydrogenase and reduced 
nicotinamide adenine dinucleotide 
The assay was carried out according to the method of Toraya et al. 
(Biochemistry, vol. 18, p. 417-426, 1979). 
The diol dehydrase employed was diluted with 0.05 M potassium phosphate 
buffer (pH 8.0) to 0.15 Unit/ml, and 1M aqueous propanediol was used as 
the substrate solution. By using a 0.05 M potassium phosphate buffer (pH 
8.0), 0.5 mg/ml of alcohol dehydrogenase and a 2 mM reduced nicotinamide 
adenine dinucleotide were prepared. To a mixture of 0.1 ml of the 
substrate solution, 0.1 ml of the alcohol dehydrogenase solution, 0.1 ml 
of the reduced nicotinamide adenine dinucleotide solution, and 0.5 ml of 
the 0.05 M potassium phosphate buffer (pH 8.0) was added 0.1 ml of the 
diol dehydrase solution. After the mixture was incubated at 37.degree. C. 
for 5 minutes, there were added, in the dark, 0.1 ml of 0.2 mM vitamin 
B.sub.12 coenzyme (adenosylcobalamin), vitamin B.sub.12 (cyanocobalamin) 
or each vitamin B.sub.12 derivative prepared in Examples 1 to 10, and the 
coenzyme activity (k.sub.cat) was calculated by continuously monitoring 
the decrease in absorbance at 340 nm, by a Union Spectrophotometer SM-401. 
The coenzyme activity (k.sub.cat) measured according to the method 1) or 2) 
is shown in Table 1. The coenzyme activity is higher when the k.sub.cat is 
larger. The activity relative to the vitamin B.sub.12 coenzyme 
(adenosylcobalamin) is also shown in % in Table 1. The vitamin B.sub.12 
derivatives prepared in Example 6 and Example 9 exhibited no coenzyme 
activity. 
Example 12 
Assay of Michaelis constant and (or) inhibition constant of vitamin 
B.sub.12 derivative 
The Michaelis constant (K.sub.m) was determined for the vitamin B.sub.12 
derivatives which exhibited a coenzyme activity in Example 11. Namely, in 
the method 1) in Example 11, the respective enzyme activities were assayed 
by suitably varying the concentration of the vitamin B.sub.12 derivative, 
and Michaelis constant (k.sub.m) was determined by the Lineweaver Burk 
plot. The results are shown in Table 1. The affinity (binding ability) for 
the enzyme is higher when the K.sub.m is smaller. 
The inhibition constant (K.sub.i) was determined for the vitamin B.sub.12 
derivatives which exhibited no coenzyme activity in Example 11. Namely, in 
the method 1) in Example 11, by adding adequately varied concentration of 
the vitamin B.sub.12 coenzyme (adenosylcobalamin) to a fixed concentration 
of each vitamin B.sub.12 derivative, the enzyme activity was respectively 
assayed and the inhibition constant (K.sub.i) was determined by the 
Lineweaver-Burk plot. The results are shown in Table 1. The inhibitory 
activity is stronger when the K.sub.i is smaller. 
TABLE 1 
______________________________________ 
K.sub.cat K.sub.m K.sub.i 
Compound (s.sup.-1) 
(%) (.mu.M) 
(.mu.M) 
______________________________________ 
Control Example 
337 100 0.80 -- 
(Vitamin B.sub.12 
coenzyme) 
Example 6 -- -- -- 52 
Example 7 199 59 0.82 -- 
Example 8 167 50 11.2 -- 
Example 9 -- -- -- 38 
Example 10 3 0.9 1.1 1.0 
Vitamin B.sub.12 
-- -- -- 24 
Example 1 -- -- -- 1.9 
Example 2 -- -- -- 0.9 
Example 3 -- -- -- 3.0 
Example 4 -- -- -- 43 
Example 5 -- -- -- 3.7 
______________________________________ 
Example 13 
Growth promoting activity and growth inhibitory activity of vitamin 
B.sub.12 derivative for Escherichia coli 215 
Escherichia coli 215 is a vitamin B.sub.12 requiring mutant strain, 
discovered by Ikeda et al., and is employed for the bioassay of vitamin 
B.sub.12 (Vitamin, vol. 10, p. 268-279, 1956). That is, this microorganism 
cannot grow if vitamin B.sub.12 is not present, and exhibits a growth 
depending on the presence of vitamin B.sub.12. 
The medium composition for Escherichia coli 215 used in this experiment is 
shown in Table 2. 
TABLE 2 
______________________________________ 
KH.sub.2 PO.sub.4 0.6 g 
K.sub.2 HPO.sub.4 1.4 g 
Sodium citrate 0.1 g 
MgSO.sub.4 0.01 g 
(NH.sub.4).sub.2 SO.sub.4 
0.2 g 
NaCl 0.1 g 
D-Glucose 2.0 g 
Water 200 ml 
______________________________________ 
Into the medium shown in Table 2 were pipetted vitamin B.sub.12 
(cyanocobalamin) or each vitamin B.sub.12 derivative synthesized in 
Examples 1 to 5 at various concentrations in the range of 0.1 to 100 
ng/ml, and the mixture was steam-sterilized at 120 degrees for 5 minutes. 
Escherichia coli 215 microorganism cells previously cultured in a 
preculture medium with the same composition as the medium shown in Table 2 
containing 1.5 .mu.g/ml of L-methionine were harvested and washed 
thoroughly with physiological saline, and then inoculated into the test 
media, and stationarily cultured at 37.degree. C. overnight. By measuring 
the turbidity of these culture broths at 660 nm using a Shimazu 
Double-beam Spectrophotometer Model UV-140-02, the growth degree of 
microorganism cells was determined. The molar concentration of vitamin 
B.sub.12 (cyanocobalamin) or each vitamin B.sub.12 derivative which gives 
1/2 of the maximum growth was defined as the growth promoting activity 
(K.sub.1/2(g)), and the results are shown in Table 3. The growth 
promoting activity is higher when the K.sub.1/2(g) is smaller. The vitamin 
B.sub.12 derivatives synthesized in Examples 1 to 5 exhibited no growth 
promoting activity. 
Also, to the medium shown in Table 2 was added vitamin B.sub.12 
(cyanocobalamin) at a concentration of 0.1 ng/ml, and in addition, each 
vitamin B.sub.12 derivatives synthesized in Examples 1 to 5 were pipetted 
at various concentrations in the range of 1 to 80 ng/ml, and the mixture 
was steam-sterilized at 120.degree. C. for 5 minutes. To these media were 
inoculated the microorganism cells precultured, harvested, and washed 
according to the same methods as described above, and after stationary 
cultivation at 37.degree. C. overnight, the growth degree of the 
microorganism cells was determined by the turbidity (660 nm) of the 
culture broth. Also, the same experiment was conducted without an addition 
of the vitamin B.sub.12 derivative, and the molar concentration of each 
vitamin B.sub.12 derivative which gives 1/2 of the growth attained in its 
absence was defined as the growth inhibitory activity (K.sub.1/2(i)). 
Further, from the following formula (V), ID.sub.50 was calculated as the 
index of the competitive inhibition activity of the vitamin B.sub.12 
derivative: 
EQU ID.sub.50 =K.sub.1/2(i) /C (V) 
(wherein C represents the molar concentration of the added vitamin B.sub.12 
(cyanocobalamin)). 
The results are shown in Table 3. The growth inhibitory activity is higher 
when the K.sub.1/2(i) is smaller, and the competitive inhibition activity 
is higher when the ID.sub.50 is smaller. 
TABLE 3 
______________________________________ 
K.sub.1/2(g) K.sub.1/2(i) 
Compound (nM) (nM) ID.sub.50 
______________________________________ 
Vitamin B.sub.12 
0.18 -- -- 
Example 1 -- 58.0 785 
Example 2 -- 4.6 62 
Example 3 -- 7.2 98 
Example 4 -- 8.0 108 
Example 5 -- 35.2 477 
______________________________________ 
Example 14 
Growth promoting activity and growth inhibitory activity of vitamin 
B.sub.12 derivative for Lactobacillus leichmannii ATCC 7830 
Lactobacillus leichmannii was found to be vitamin B.sub.12 -requiring by 
Skeggs et al. (J. Biol. Chem., vol. 184, p. 211-221, 1950), and the kit 
for B.sub.12 assay with Lactobacillus leichmannii ATCC 7830 is readily 
available as a commercial product. That is, using the basal medium 
"Nissui" of Nissui Seiyaku for a vitamin B.sub.12 assay with leichmannii 
as the medium for Lactobacillus leichmannii ATCC 7830, the medium was 
prepared following the method described in the instructions, and to the 
medium was pipetted vitamin B.sub.12 (cyanocobalamin), or the vitamin 
B.sub.12 derivative synthesized in Examples 1 to 5, each at various 
concentrations in the range of 0.1 to 100 ng/ml, followed by stream 
sterilization at 120.degree. C. for 5 minutes. To these media were 
inoculated the microorganism cells, following a prescribed method, which 
were stationarily cultured at 37.degree. C. overnight. The degree of 
growth of the microorganism cells was measured in the same manner as 
described in Example 13, to determine the growth promoting activity 
(K.sub.1/2(g)). The results are shown in Table 4. The vitamin B.sub.12 
derivatives synthesized in Examples 2 to 4 exhibited a very weak growth 
promoting activity for this microorganism. The vitamin B.sub.12 
derivatives synthesized in Example 1 and Example 5 exhibited no growth 
promoting activity. 
Also, to the above-mentioned basal medium was added vitamin B.sub.12 
(cyanocobalamin) at a concentration of 0.05 ng/ml, and in addition, the 
vitamin B.sub.12 derivatives synthesized in Examples 1 to 5 each at 
various suitable concentrations, and the resultant mixture was 
steam-sterilized at 120.degree. C. for 5 minutes. To these media were 
inoculated the microorganism cells, following a prescribed method, 
cultured stationarily at 37.degree. C. overnight, and the degree of growth 
of the microorganism cells was measured in the same manner as described in 
Example 13. Also, the same experiment was carried out without adding the 
vitamin B.sub.12 derivative, and the growth inhibitory activity 
(K.sub.1/2(i)) and competitive inhibition activity (ID.sub.50) were 
determined according to the method described in Example 13. The results 
are shown in Table 4. 
TABLE 4 
______________________________________ 
K.sub.1/2(g) K.sub.1/2(i) 
Compound (nM) (nM) ID.sub.50 
______________________________________ 
Vitamin B.sub.12 
0.047 -- -- 
Example 1 -- 86 2330 
Example 2 1.5 -- -- 
Example 3 0.5 -- -- 
Example 4 10 -- -- 
Example 5 -- 213 5772 
______________________________________ 
Example 15 
In vivo growth inhibitory activity of vitamin B.sub.12 derivative against 
mouse leukemia L.sub.1210 cells 
The mouse leukemia L.sub.1210 cells employed for this experiment were grown 
and maintained intraperitoneally in DBA mice, taken out together with 
ascites and aseptically cultured as adapted for RPMI-1640 medium for 
animal cell culture supplemented with fetal bovine serum (5 v/v %) and 
ethanolamine (1.2 mg/liter), but devoid of vitamin B.sub.12 
(cyanocobalamin). Also, 100,000 units of penicillin and 100 mg of 
streptomycin were added as the antimicrobial agent, per liter of this 
medium. The L.sub.1210 cells grown and adapted in vitro were further 
adapted to the above-mentioned medium containing 7.0 g/liter of bovine 
serum albumin in place of the fetal bovine serum, following the method of 
Fujii et al. (J. Biol. Chem., vol. 256, p. 10329-10334, 1981), and then 
used for experiment. 
To examine the L.sub.1210 growth inhibitory activities of the vitamin 
B.sub.12 derivatives synthesized in Examples 2 and 5, first L.sub.1210 
cells adapted to albumin were inoculated at a density of 5.times.10.sup.4 
cells/ml into the same medium containing folic acid at 10 .mu.M 
concentration, and a preculture was carried out under a humidified 
atmosphere of CO.sub.2 /air (5%/95%) at 37.degree. C. for 3 days. The 
cells grown were centrifuged, then washed twice with the same medium 
containing no folic acid, to prepare an inoculum cell suspension of about 
5.times.10.sup.6 cells/mi. Experimental culture was carried out by adding 
5-methyltetrahydrofolic acid at 10 .mu.M and vitamin B.sub.12 
(cyanocobalamin) at 0.5 nM in place of folic acid to the above-mentioned 
basal medium, followed by inoculation of washed cells thereto at a density 
of 56.times.10.sup.4 cells/ml, and continued under the above conditions 
for 9 days. The L.sub.1210 cell growth inhibitory activities of the 
vitamin B.sub.12 derivatives synthesized in Examples 2 and 5 were examined 
by permitting 200 nM of methotrexate (MTX) and each of the vitamin 
B.sub.12 derivative of Example 2 (50 nM) on the vitamin B.sub.12 
derivative of Example 5 (50 nM, 5000 nM) to be co-present, respectively. 
The growth inhibitory activity of MTX, or a combination and of MTX and the 
vitamin B.sub.12 derivative of the present invention against mouse 
leukemia L1210 cells was demonstrated by measurement of the cell density 
on days 3, 5, 7 and 9, by a Coulter counter as shown in the results in 
FIG. 1. As a control, the results of growth measurements when MTX and the 
vitamin B.sub.12 derivative of the present invention were not added are 
also shown in FIG. 1. From FIG. 1, it is evident that the compound of the 
present invention exhibits a growth inhibitory activity against L.sub.1210 
, and thus has an antitumor activity. 
When the cultured cells on day 9 shown in FIG. 1 (those cultured in the 
presence with addition of the vitamin B.sub.12 derivative of the present 
invention obtained in Example 5 (5000 nM) shown by the curve E in the 
Figure) was examined for their viability by staining with Trypan blue, 90% 
of the cells were found to be dead when both MTX and the vitamin B.sub.12 
derivative of Example 5 were co-present, while only 20% of the cells were 
dead when MTX above was added.