JP2962755B2 - Novel Vitamin B (lower 1) (lower 2) derivative, its production method and its use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION INDUSTRIAL FIELD The present invention relates to novel vitamin B ₁₂ derivatives, their preparation and their use. More specifically, the general formula (I) (Wherein L is a cyano group, a substituted or unsubstituted adenosyl group or an adenylylalkyl group, a hydroxyl group,
One or two identical or different ligands selected from a water molecule and a linear or branched alkyl group having 1 to 8 carbon atoms, B represents a base having a heterocyclic structure,
Represents a substituted or unsubstituted hydrocarbon group. Vitamin B ₁₂ derivative represented by), a method for manufacturing the same, and vitamin B ₁₂ antagonist containing it as an active ingredient, suppression or inhibitors of cell proliferation, relates to an anti-tumor agent.

PRIOR ART Vitamin B ₁₂ is one of the essential trace nutrient factors for animals such as humans, in mammals, especially contained in many liver. Neither animals nor plants can biosynthesize this substance, only microorganisms produce it. vitamin
The deficiency of B _12, is particularly pernicious anemia is typical,
Megaloblastic hematopoiesis, methylmalonic aciduria, homocysteinuria, neuropathy and the like are seen. Vitamin B ₁₂ is, when absorbed into the body, are converted metabolically active form in which vitamin B ₁₂ coenzyme (adenosyl cobalamin) and methyl cobalamin, the former, for example hydrogen transfer, such as methylmalonyl CoA mutase And the latter function as a coenzyme in an enzymatic reaction involving the transfer of a methyl group such as methionine synthase. Particularly methylcobalamin, by relating to C ₁ metabolism via the folate coenzymes, indirectly involved in the biosynthesis of thymidylate,
Plays an important role in cell proliferation. Therefore, a compound that antagonizes the vitamin B ₁₂ group, that is, vitamin B ₁₂
Antagonists are thought to inhibit or block cell growth by inhibiting DNA synthesis, and to act effectively on tumor cells (cancer cells) as antitumor agents (anticancer agents). In addition, also in microorganisms, vitamin B ₁₂ group is important for its growth, vitamin B ₁₂ antagonist,
It is considered to have an action as an antibacterial agent. Conversely, vitamin B ₁₂ antagonist, is believed to be helpful in screening for microbial mutants having vitamin B ₁₂ higher productivity as an index for resistance to this.

Conventionally, various vitamin B ₁₂ derivatives have been synthesized, for example, from cobyric acid, was synthesized using chemical or microbial, isopropanolamine part of vitamin B ₁₂ group _{(-NHCH 2 CH (CH 3)} O- ), for _{example, -NHCH (CH 3) CH 2} O
_{-, - NHCH 2 CH 2 CH} 2 O -, - NHC (CH 3) 2 CH 2 O -, - NHCH 2 (C
₆ H ₅₎ O-, or _{-NHCH 2 CH (CH 2 F)} O- as vitamin B ₁₂ derivatives substituted on the, E. coli (Escherichia
coli ) 113-3, Lactobacillus reichmani ( Lactoba
cillus leichmannii ) (Friedrich, Vitamin B ₁₂ und verwa)
ndte Corrinoide (Georg Thieme Verlag, Stuttgard),
289-308, 1975). It is also known that a heterologous metal corinoid obtained by introducing, for example, rhodium, copper, or zinc into a cobalt-deficient corinoid isolated from a microorganism or, for example, rhodium, copper, or zinc exhibits an antagonistic action against the above-described bacteria. (Friedrich, Vitamin B ₁₂ und ver
wandte Corrinoide (Georg Thieme Verlag, Stuttgar
d), pp. 289-308, 1975, and Copenhagen, B ₁₂
(John Wiley & Sons, New York), II, 105-149, 1
982).

However, when using cobyric acid, its preparation is complicated and it is difficult to supply it in large quantities. When using a microorganism, there is a problem in practicality in terms of isolation.

Further, bromine C-10 position of the corrin ring of vitamin B ₁₂ derivative or to introduce a nitro group, also, the corrin ring surrounding the side chains, for example, a carboxyl group, ethylamide, anilides, also vitamin B ₁₂ derivatives substituted hydrazide chemistry (Friedrich, Vitamin B ₁₂ und v
erwandte Corrinoide (Georg Thieme Verlag, Stuttgar
d), pages 289-308, 1975), but these derivatives are still inadequate in antagonism.

Therefore, excellent vitamin B ₁₂ antagonism, and large supply can be vitamin B ₁₂ antagonist has been desired.

Problems to be Solved by the InventionThe present inventors have conducted intensive studies to solve these problems of the prior art, and as a result, obtained by modifying the ribose portion of the nucleotide portion of the vitamin B ₁₂ group. vitamin B ₁₂ derivatives are possible large supply, and a vitamin B ₁₂ antagonist, found to have an excellent effect and reached to the present invention.

Means for Solving the Problems That is, the present invention provides the following formula (I) (Wherein L is a cyano group, a substituted or unsubstituted adenosyl group or an adenylylalkyl group, a hydroxyl group,
One or two identical or different ligands selected from a water molecule and a linear or branched alkyl group having 1 to 8 carbon atoms, B represents a base having a heterocyclic structure,
Represents a substituted or unsubstituted hydrocarbon group. ) Is a vitamin B ₁₂ derivative represented by the.

In vitamin B ₁₂ derivative represented by the general formula (I), L is a cyano group, a substituted or unsubstituted adenosyl group or adenylyltransferase alkyl group (the alkyl group is a straight chain of 1 to 8 carbon atoms or branched chain alkyl, Group), a hydroxyl group, a water molecule and one or two identical or different ligands selected from linear or branched alkyl groups having 1 to 8 carbon atoms. As such an alkyl group having 1 to 8 carbon atoms, a methyl group, an ethyl group, and a propyl group are preferable. The ligand L is generally coordinated above the choline ring, but may be on one side of the choline ring or on both sides thereof.

In the general formula (I), R is a substituted or unsubstituted hydrocarbon group, for example, a substituted or unsubstituted linear or branched C1-C8 linear or branched carbon atom. Examples thereof include an alkylene group and a cyclic hydrocarbon group. Among them, an alkylene group having 1 to 8 carbon atoms is preferable.

In the general formula (I), B represents a base having a heterocyclic structure, and examples thereof include a substituted or unsubstituted imidazole group, a pyridine group, and derivatives thereof. Examples of such a derivative include a benzimidazole group in which a benzene ring is condensed with an imidazole group, and a derivative thereof such as a 5,6-dimethylbenzimidazole group. Such a base B is usually coordinated below the cobalt of the choline ring, but the present invention includes the case where it is not coordinated.

The present invention also relates to a vitamin represented by the general formula (I)
A method for producing B ₁₂ derivatives.

That is, cyano aquacobinamide or dicyanocobinamide is converted into a compound represented by the following formula (II): BR-O-PO ₃ H ₂ ... (II) (wherein B and R are the same as defined in the above formula (I)) To produce a corresponding compound represented by the general formula (I) wherein L is a cyano group.

The reaction between the compound represented by the general formula (II) and cyano aquacobinamide or dicyanocombamide is as follows:
For example, using a condensing agent, preferably dicyclohexylcarbodiimide, in an organic solvent, preferably pyridine and N,
It can be performed in a mixed solution of N-dimethylformamide. The reaction temperature is lower than the boiling point of the solvent used, for example, preferably around room temperature. Cyano Aqua cobinamide vitamin B ₁₂ (cyanocobalamin), or are readily prepared from dicyano cobinamide. The compound obtained by the reaction and represented by the general formula (I) wherein L is a cyano group can be purified by, for example, extraction, column chromatography, and / or high performance liquid chromatography.

Alternatively, the compound represented by the general formula (I) in which L is a cyano group is reduced and then a) re-oxidized, or b) alkylated and then photo-decomposed, so that L is a hydroxyl group or water. A compound represented by the general formula (I) which is a molecule, or a compound represented by the general formula (I) wherein L is a cyano group, a hydroxyl group or a water molecule, for example, sodium borohydride, zinc and ammonium chloride , Zinc and acetic acid, or divalent chromium, and then, for example, a halogenated alkane (an alkyl group is a linear or branched alkyl group having 1 to 8 carbon atoms, preferably a methyl group, an ethyl group, Propyl group) or halogenated or tosylated substituted or unsubstituted adenosine or adenylylalkane (the alkyl group has 8 linear or branched alkyl groups) for example preferred the reacted with 5'-halo-5'-deoxyadenosine, can be prepared corresponding compounds of the formula (I). These compounds can be obtained as purified products by, for example, extraction and column chromatography.

The obtained compound represented by the general formula (I) can be optionally formed into a salt with, for example, sulfuric acid or the like.

The phosphate ester derivative represented by the general formula (II) and a salt thereof are the vitamin B of the present invention represented by the general formula (I)
_It is a useful intermediate used in the production of ₁₂ derivatives and can be obtained by the following method.

That is, the phosphoric ester derivative represented by the general formula (II) and a salt thereof include a free base B ′ having a heterocyclic structure and the following formula (III): X—R—OH (III) Represents a leaving group, and R is the same as defined in the above formula (I)), and is reacted with a compound represented by the general formula (IV) BR-OH (IV) wherein B and R are A compound of the formula (I) is obtained, which is then phosphorylated, preferably by phosphorylation using 2-cyanoethylphosphate pyridine.

The free base B ′ having a heterocyclic structure is, for example, substituted or unsubstituted imidazole, pyridine, or a derivative thereof.
For example, benzimidazole, 5,6-dimethylbenzimidazole and the like can be mentioned. These compounds B '
Can be obtained as a commercial product or can be produced by a known method.

In the compound represented by the general formula (III), X is
For example, a leaving group consisting of a halogen atom, preferably chlorine. The compound represented by the general formula (III) can be obtained as a commercial product or by a known method.

A free base B ′ having a heterocyclic structure and a compound represented by the general formula (II
The reaction of the compound represented by I) is carried out in the presence of a base, preferably sodium hydride or potassium carbonate, at a reaction temperature of preferably around room temperature, or may be carried out under reflux. This reaction is carried out in an organic solvent,
It is preferably carried out in N, N-dimethylformamide or dioxane. The compound represented by the general formula (IV), which is a reaction product, can be used as a crude product in the following reaction, but is preferably separated from the reaction mixture by a known method, for example, washing, extraction, or column chromatography. It is used after purification.

The phosphorylation of the compound represented by the general formula (IV) is carried out, for example, in the presence of a condensing agent, preferably by using pyridine salt of 2-cyanoethylphosphate, followed by reaction with lithium hydroxide. As the condensing agent, dicyclohexylcarbodiimide is preferred, the reaction is carried out in an organic solvent, preferably in pyridine, and the reaction is carried out at a temperature not higher than the boiling point of the solvent used, preferably at about room temperature. 2-cyanoethyl phosphate pyridine salt can be easily prepared from barium 2-cyanoethyl phosphate according to the method of Tenor (J. Am. Chem. Soc., 83, 159-168, 1961). . Compound represented by the reaction product general formula (II), as a crude product, it can be used in the middle of vitamin B ₁₂ derivative prepared represented by the general formula (I), optionally, a known method, for example, It can be separated and purified from the reaction mixture by filtration, extraction, or column chromatography, or, if desired, can be obtained by known methods, for example, in the form of a salt with lithium, sodium, potassium, barium or pyridine. it can.

Vitamin B ₁₂ derivative represented by the general formula (I) has a very valuable pharmacological properties. These compounds have activity as vitamin B ₁₂ antagonists, Vitamin B
₁₂ known vitamin B that can be used as an antagonist
_It shows excellent antagonism even compared to ₁₂ antagonists. further,
The vitamin B ₁₂ derivative represented by the general formula (I) uses cyano aquacobinamide or dicyanokobinamide as a raw material according to the above-mentioned production method, so that it is simpler and more abundant than known vitamin B ₁₂ antagonists. Can be supplied. Also, vitamin B ₁₂ derivative represented by the general formula (I) of the present invention, at least one of which comprises, as an active ingredient cytostatic or inhibitor, for example, when cells of a microorganism, as antimicrobial agents, or When the cells are animal cells, particularly tumor cells (cancer cells), they can be used as antitumor agents (anticancer agents). When used as such cytostatic or inhibitors of the general formula (I) vitamin B ₁₂ derivative and the at least one active ingredient and a pharmaceutically acceptable carrier and a pharmaceutically acceptable salt thereof represented by (Or) may be a pharmaceutical preparation comprising the necessary additives.

Vitamin B ₁₂ derivative represented by the general formula (I) of the present invention, to act antagonistically against vitamin B ₁₂ group,
For example, for the microorganisms to high productivity of vitamin B _12,
Growth inhibitory effects of vitamin B ₁₂ derivative represented by the general formula (I) is weakened, or not show the inhibitory effect. Accordingly, the present invention for the purpose of screening for vitamin B ₁₂ highly productive mutant strain of microorganism, vitamin B ₁₂ of the present invention
The use of derivatives is also covered.

As described above, the vitamin B ₁₂ derivative of the present invention
It has an action as a vitamin B ₁₂ antagonist, and is very useful for studying the function of the vitamin B ₁₂ group in the enzymatic reaction, for example, coenzyme function. Further, the vitamin B _{12 of the} present invention
Derivatives show excellent antagonism, cytostatic or inhibitor containing as an active ingredient according vitamin B ₁₂ derivatives are useful as for example, antimicrobials, or anti-tumor agent (anticancer agent). Further, the vitamin B ₁₂ derivative of the present invention,
It can be used to screen for vitamin B ₁₂ high producing microorganism mutants. Further, the vitamin B _{12 of the} present invention
Derivatives can be easily supplied in large quantities because cyano aquacobinamide or dicyanocobinamide is used as a raw material.

Vitamin B ₁₂ derivatives and pharmaceutically acceptable salts of the formula (I) of the present invention may be administered per se alone, necessary or desirable by a general purpose being other conventional pharmaceutically acceptable And a mixture thereof with a carrier, excipient, solvent, diluent or the like, and can be administered orally or parenterally as a desired dosage form. The oral preparation may be any of tablets, pills, granules, powders, solutions, suspensions, capsules and the like. Parenteral administration includes subcutaneous and dermal ointments, creams,
Any shape such as gel may be used. Trans-respiratory administration is administered via the aerosol or other suitable means of nebulization.

Tablets of the vitamin B ₁₂ derivative or a composition comprising a pharmaceutically acceptable salt thereof as an active ingredient of the general formula (I) of the present invention include lactose, starch, and excipients such crystalline cellulose, needs Accordingly, carboxymethylcellulose,
Binders such as methylcellulose and polyvinylpyrrolidone;
And / or a disintegrating agent such as sodium alginate or sodium hydrogen carbonate is mixed with the active ingredient, and the mixture is molded by a usual method. In order to produce a solution or suspension, for example, glycerin esters such as tricaprylin and triacetin, alcohols such as ethanol and the like are mixed with the active ingredient, and a normal method may be applied to the mixture. In order to produce capsules, granules, powders, liquids, and the like are mixed with an active ingredient together with a capsule-forming material such as gelatin, and the capsule-forming method is applied thereto.

Injectables may be in the form of aqueous or non-aqueous solutions and the like, and the active ingredient may be dissolved in a solvent such as physiological saline, ethanol, or propylene glycol, and if necessary, preservatives and stabilizers may be added. Manufactured.

As the suppository, those of usual dosage forms such as gelatin soft capsules containing the active ingredient are used.

Ointments, creams and the like are formed from the active ingredient and the required carrier by an ordinary method.

Aerosol administration agents include fatty acids having 6 to 22 carbon atoms,
Manufactured using a pharmaceutically acceptable surfactant, a propellant such as an alkane having 5 or less carbon atoms or a fluorinated alkane, and an active ingredient, which is made from a fatty acid polyhydric alcohol or a cyclic anhydride thereof. Can be.

It is not particularly limited to vitamin B ₁₂ derivative and the concentration of the pharmaceutically acceptable salts of the general formula of such a pharmaceutical formulation (I), generally about 0.001 to 50 wt% in the formulation, preferably 0.01 to About 10% by weight is appropriate. The dose is not particularly limited, but is suitably 0.01 to 500 mg / day / person, preferably 0.1 to 100 mg / day / person, and the number of administration is usually 1
1-4 times per day.

Examples Next, the present invention will be described specifically with reference to Examples.
The present invention is not limited by the examples.

Example 1 Synthesis of 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid The following operation was performed in Example 1.

1) Synthesis of 1- (2-hydroxyethyl) -5,6-dimethylbenzimidazole 1.46 g of 5,6-dimethylbenzimidazole was dissolved in 50 ml of dry N, N-dimethylformamide and 0.96 g of NaH
Was added and the mixture was stirred in an ice water bath for 30 minutes. To this was added dropwise 5 ml of ethylene chlorohydrin, and the mixture was stirred at room temperature for 15 hours, and further reacted for 24 hours while appropriately adding 0.78 g of NaH. The reaction was stopped by adding 50 ml of water. The resulting reaction solution was washed with n-hexane, added with water, adjusted to pH 2.5 with HCl, and then ion-exchanged with a column (Dowex 50). (Hydrogen ion type)). Elution is carried out sequentially with water, 30% ethanol and ammoniacal 30% ethanol, and the fraction eluted with ammoniacal 30% ethanol is concentrated to dryness under reduced pressure to give crude 1-.
(2-Hydroxyethyl) -5,6-dimethylbenzimidazole was obtained. The conversion of this compound from 5,6-dimethylbenzimidazole was 75% as determined by thin-layer chromatography.

Furthermore, the obtained crude 1- (2-hydroxyethyl)
After dissolving -5,6-dimethylbenzimidazole in 50% methanol and removing insolubles, purification was performed by reversed-phase high-performance liquid chromatography, followed by concentration to dryness under reduced pressure to obtain purified 1- (2- (Hydroxyethyl) -5,6-dimethylbenzimidazole was obtained. ¹³ C-NMR (CDCl ₃ , δ ppm) 20.13, 20.51, 48.11, 60.43, 109.54, 119.59, 130.82, 131.83. 2) Preparation of 2-cyanoethylphosphoric acid pyridine salt Tenor method (J. Am. Chem. Soc. 83 Volume, pp. 159-168, 1961
), Pyridine 2-cyanoethylphosphate was prepared. That is, 1.61 g of barium 2-cyanoethylphosphate and 10 g of an ion exchange resin (Dowex 50 (hydrogen ion type)) were suspended in water, stirred at room temperature for 30 minutes, and then 2 ml of pyridine was added to the supernatant and the washing solution with water. , And concentrated to dryness under reduced pressure.
A mol / ml 2-cyanoethylphosphate pyridine salt solution was obtained.

3) Synthesis of 2- (5,6-dimethylbenzimidazolyl) ethyl phosphoric acid 0.19 g of crude 1- (2-hydroxyethyl) -5,6-dimethylbenzimidazole and 2 ml of pyridine salt solution of 2-cyanoethyl phosphate (1 mmol) / ml) was dissolved in 20 ml of dry pyridine and concentrated to dryness under reduced pressure. This operation is repeated two more times, and after sufficiently drying with a vacuum pump,
This was dissolved in 20 ml of dry pyridine, 1.67 g of dicyclohexylcarbodiimide was added, and the mixture was stirred at room temperature for 2 days. Water was added thereto, and the mixture was concentrated to dryness under reduced pressure. Then, 40 ml of a LiOH aqueous solution (0.5 N) was added, and the mixture was added for 10 minutes for 45 minutes.
The reaction was performed at 0 ° C. The reaction solution was passed to obtain a solution, water was added to the solution, the pH was adjusted to 2.5 to 3 with HCl, and the solution was applied to an ion exchange column (Dowex 50 (hydrogen ion type)). Elution was carried out sequentially with water and 2N pyridine, and the fraction eluted in the latter was concentrated to dryness under reduced pressure to obtain 0.27 g of crude 2- (5,6-dimethylbenzimidazolyl) ethyl phosphoric acid.

4) Preparation 1g cyano Aqua cobinamide cyanocobalamin (the vitamin B ₁₂₎ was dissolved in water of 144 ml, this, 76.8Ml and 1 Ce (NO _{3) 3} aqueous solution of 0.33M
51.2 ml of N NaOH solution was added. Next, 1.77 g of KCN was added to the mixture, and the mixture was stirred at 90 to 100 ° C. for 1 hour, cooled to room temperature, adjusted to pH 8.5, and left at 4 ° C. overnight. After that, the solution is desalted by phenol extraction, and then subjected to ion exchange column (diethylaminoethylcellulose (acetate type)).
To A small amount of acetic acid was added to the fraction eluted with water, and the mixture was concentrated to dryness under reduced pressure to obtain crude cyanoaquacobinamide. This was further applied to a phosphocellulose column (pH 7) and eluted sequentially with 50% ethanol and 50 mM NaCl solution. The fraction eluted in the latter was desalted by phenol extraction to give crude cyanoaquacobinamide.
800 mg were obtained.

5) Synthesis of 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid 0.27 g of crude 2- (5,6-dimethylbenzimidazolyl) ethylphosphoric acid and 0.2 g of purified cyanoaquacobinamide were converted to pyridine. It was dissolved in 5 ml and concentrated to dryness under reduced pressure. This operation was further repeated twice, and after further drying sufficiently with a vacuum pump, 15 ml of dry N, N-dimethylformamide, 10 ml of dry pyridine, and 1.5 g of dicyclohexylcarbodiimide were added, and the mixture was stirred at room temperature for 12 days.
The reaction was stopped by adding water and KCN. The reaction mixture was desalted by phenol extraction and then applied to a forphocellulose column (pH 3), and then the passed fraction was applied to an ion exchange column (diethylaminoethylcellulose (acetate type)). Further, the flow-through fraction was purified by reversed-phase high-performance liquid chromatography,
As a concentrated and dried product, 50 mg of purified 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid was obtained. The obtained 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid was confirmed to be single by reversed-phase high-performance liquid chromatography and thin-layer chromatography. Furthermore, the obtained 2
Cerium hydrolysis of-(5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphate yields qualitatively and quantitatively 1- (2-hydroxyethyl) -5,6-dimethylbenzimidazole. This was confirmed by thin-layer chromatography and reversed-phase high-performance liquid chromatography.

Example 2 Synthesis of 3- (5,6-dimethylbenzimidazolyl) propylcyanocobinamidophosphoric acid The following operation was performed in Example 2.

1) Synthesis of 1- (3-hydroxypropyl) -5,6-dimethylbenzimidazole 1.46 g of 5,6-dimethylbenzimidazole was dissolved in 50 ml of dry N, N-dimethylformamide and 0.76 g of NaH
Was added and stirred at room temperature for 30 minutes. In addition, 3-chloro-
After 1 ml of 1-propanol was added dropwise and reacted at room temperature for 2 days with stirring, water was added to stop the reaction. Hereinafter, crude 1- (3-hydroxypropyl) -5,6-dimethylbenzimidazole was obtained in the same manner as in Example 1. The exchange rate of this compound from 5,6-dimethylbenzimidazole was 90%.

Further, crude 1- (3-hydroxypropyl) -5,6
-Dimethylbenzimidazole is dissolved in 50% methanol, purified by reversed-phase high performance liquid chromatography, concentrated to dryness under reduced pressure, and purified 1- (3-hydroxypropyl) -5,6-dimethylbenzimidazole is purified. Obtained. ¹³ C-NMR (CDCl ₃ , δ ppm) 20.04, 20.40, 31.71, 41.16, 58.47, 109.97, 120.38, 131.21, 132.28. ¹ H-NMR (CDCl ₃ , δ 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 0.2 g of crude 1- (3-hydroxypropyl) -5,6-dimethylbenzimidazole as starting material and 2
-2 m of cyanoethyl phosphate pyridine salt solution (1 mmol / ml)
Using the same procedure as in Example 1, the crude 3-
(5,6-dimethylbenzimidazolyl) propyl phosphoric acid
0.3 g was obtained.

3) Synthesis of 3- (5,6-dimethylbenzimidazolyl) propylcyanocobinamidophosphoric acid As starting materials, 0.3 g of crude 3- (5,6-dimethylbenzimidazolyl) propylphosphoric acid and purified cyanoaquacobinamide Using the same procedure as in Example 1, 33 mg of crude 3- (5,6-dimethylbenzimidazolyl) propylcyanocobinamidophosphoric acid was obtained. However, the reaction time was 3.5 days. In addition, it was confirmed that this compound was a single compound in the same manner as in Example 1. Further, by cerium-hydrolyzing the obtained 3- (5,6-dimethylbenzimidazolyl) propylcyanocobinamidophosphoric acid, 1- (3-hydroxypropyl) -5,6-dimethylbenzimidazole is qualitatively converted. And quantitatively confirmed by thin-layer chromatography and reverse-phase high-performance liquid chromatography,
The ¹³ C-NMR and ¹ H-NMR also corresponded to those of 1- (3-hydroxypropyl) -5,6-dimethylbenzimidazole obtained in 1) of Example 2.

Example 3 Synthesis of 4- (5,6-dimethylbenzimidazolyl) butylcyanocobinamidophosphoric acid The following operation was performed in Example 3.

1) Synthesis of 1- (4-hydroxybutyl) -5,6-dimethylbenzimidazole 1.46 g of 5,6-dimethylbenzimidazole was dissolved in 50 ml of dry N, N-dimethylformamide, and 0.9 g of NaH was dissolved. The mixture was stirred at room temperature for 30 minutes. In addition, 4-chloro-1
-8 ml of butanol was added dropwise and after stirring at room temperature for 1 day,
Further, 0.65 g of NaH and 2 ml of 4-chloro-1-butanol were appropriately added to carry out a reaction for 8 days. Hereinafter, crude 1- (4-hydroxybutyl) -5,6-dimethylbenzimidazole was obtained in the same manner as in Example 1.
The conversion of this compound from 5,6-dimethylbenzimidazole was 50%.

2) Synthesis of 4- (5,6-dimethylbenzimidazolyl) butyl phosphoric acid As starting material, crude 1- (4-hydroxybutyl)
Using 0.2 g of -5,6-dimethylbenzimidazole and 2 ml of a pyridine salt solution of 2-cyanoethylphosphate (1 mmol / ml), the crude 4- (5,
0.3 g of 6-dimethylbenzimidazolyl) butyl phosphoric acid was obtained.

3) Synthesis of 4- (5,6-dimethylbenzimidazolyl) butylcyanocobinamidophosphoric acid As starting materials, 0.3 g of crude 4- (5,6-dimethylbenzimidazolyl) butylphosphoric acid and purified cyanoaquacobinamide were used. Using 0.2 g, 15 mg of purified 4- (5,6-dimethylbenzimidazolyl) butylcyanocobinamidophosphate was obtained in the same manner as in Example 1 below. However, the reaction time was 6 days. The identification of this compound was carried out by thin layer chromatography in the same manner as in Example 1.

Example 4 Synthesis of 6- (5,6-dimethylbenzimidazolyl) hexylcyanocobinamidophosphoric acid The following operation was performed in Example 4.

1) Synthesis of 1- (6-hydroxyhexyl) -5,6-dimethylbenzimidazole 1.46 g of 5,6-dimethylbenzimidazole was dissolved in 50 ml of dry N, N-dimethylformamide and 0.84 g of NaH
Was added thereto, and 8.2 ml of 6-chloro-1-hexanol was further added. The mixture was stirred at room temperature for 1 day to react, and water was added to stop the reaction. Hereinafter, crude 1- (6-hydroxyhexyl) -5,6-
Dimethylbenzimidazole was obtained. To this compound,
The exchange rate from 5,6-dimethylbenzimidazole is 95
% Or more.

2) Synthesis of 6- (5,6-dimethylbenzimidazolyl) hexyl phosphoric acid As starting materials, 0.2 g of crude 1- (6-hydroxyhexyl) -5,6-dimethylbenzimidazole and 2
-2 m of cyanoethyl phosphate pyridine salt solution (1 mmol / ml)
Using the same procedure as in Example 1, crude 6-
(5,6-dimethylbenzimidazolyl) hexyl phosphoric acid
0.35 g was obtained.

3) Synthesis of 6- (5,6-dimethylbenzimidazolyl) hexylcyanocobinamidophosphoric acid As a starting material, 0.35 g of crude 6- (5,6-dimethylbenzimidazolyl) hexylphosphoric acid and purified cyanoaquacobinamide were used. Using 0.2 g, 20 mg of purified 6- (5,6-dimethylbenzimidazolyl) hexylcyanocobinamidophosphoric acid was obtained in the same manner as in Example 1 below. However, the reaction time was 5 days. This compound was confirmed by the same operation as in Example 1 using thin-layer chromatography.

Example 5 Synthesis of 3-imidazolylpropylcyanocobinamidophosphoric acid The following operation was performed in Example 5.

1) Synthesis of 1- (3-hydroxypropyl) imidazole 1.7 g of imidazole was dissolved in 125 ml of dioxane, 17.25 g of potassium carbonate was added thereto, and 13.8 ml of 3-chloro-1-propanol was further added dropwise. And heated at reflux for 7.5 hours. After removing potassium carbonate by passing the obtained reaction mixture, water was added to stop the reaction, and the pH was adjusted to 2.
Adjust to 5 and then use an ion exchange column (Dowex 50
(Hydrogen ion type)). Elution is performed sequentially with water, 30% ethanol, and 30% ethanolic ammonia. The fraction eluted with 30% ethanolic ammonia is concentrated, and the concentrate is applied to a phosphocellulose column (pH 8). The residue was concentrated and dried under reduced pressure to obtain 2.5 g of purified 1- (3-hydroxypropyl) imidazole. ¹³ C-NMR (D ₂ O, δ ppm) 34.86, 45.75, 60.51, 122.54, 130.05, 140.26. 2) Synthesis of 3-imidazolylpropyl phosphate 0.13 g of purified 1- (3-hydroxypropyl) imidazole as a starting material Then, 0.2 g of crude 3-imidazolyl propyl phosphoric acid was obtained by the same operation as in Example 1 using 2 ml of a pyridine salt solution of pyridine and 2-cyanoethylphosphate (1 mmol / ml).

3) Synthesis of 3-imidazolylpropylcyanocobinamidophosphoric acid As starting materials, 0.2 g of crude 3-imidazolylpropylphosphoric acid and 0.2 g of purified cyanoaquacobinamide were used. 20mg purification 3
-Imidazolylpropylcyanocobinamidophosphoric acid was obtained. However, the reaction time was 3 weeks. This compound was confirmed by reversed-phase high-performance liquid chromatography, ¹³ C
-NMR and ¹ H-NMR were performed in the same manner as in Example 2.

Example 6 Preparation of 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid 10 mg of 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid was dissolved in 3 ml of water. , to this was added NaBH ₄ of 100mg, and sealed. This was left to reduce for 15 minutes, and then, in the dark, 25 mg of 5'-iodo-
5'-Deoxyadenosine was poured into the sealed container together with 3 ml of N, N-dimethylformamide, and left for 30 minutes while cooling with water. The following operations were performed in a dark place. Water and a 1M potassium phosphate buffer (pH 5.0) were added to the obtained reaction solution, which was then applied to an adsorption column (Amberlite XAD-2), and eluted with water and 80% ethanol sequentially. The fraction eluted in the latter was concentrated to dryness under reduced pressure, dissolved in a small amount of water, applied to an ion exchange column (carboxymethylcellulose (hydrogen ion type)), and eluted sequentially with water and a 50 mM NaCl solution. . The fraction eluted in the latter is desalted by an adsorption column (Amberlite XAD-2), further purified by an ion exchange column (phosphocellulose column (pH 6)), and concentrated to dryness under reduced pressure. Purified 2- (5,6-dimethylbenzimidazolyl) ethyladenosylcobinamidophosphate was obtained.

Example 7 Preparation of 3- (5,6-dimethylbenzimidazolyl) propyladenosylcobinamidophosphoric acid In Example 7, 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamide of Example 6 was used. 3- (5,6-dimethylbenzimidazolyl) propylcyanocobinamidophosphoric acid was used in place of phosphoric acid, and the same operation as in Example 6 was carried out. Binamidophosphoric acid was obtained.

Example 8 Preparation of 4- (5,6-dimethylbenzimidazolyl) butyladenosylcobinamidophosphoric acid In Example 8, 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamide of Example 6 was used. Using 4- (5,6-dimethylbenzimidazolyl) butylcyanocobinamidophosphoric acid instead of phosphoric acid, the same operation as in Example 6 was carried out to obtain 4- (5,6-dimethylbenzimidazolyl) butyladenosylcophosphate. Binamidophosphoric acid was obtained.

[Example 9] Preparation of 6- (5,6-dimethylbenzimidazolyl) hexyl adenosylcobinamidophosphoric acid In Example 9, 2- (5,6-dimethylbenzimidazolyl) ethyl cyanocobinamide of Example 6 was used. 6- (5,6-Dimethylbenzimidazolyl) hexylcyanocobinamidophosphoric acid was used in place of phosphoric acid, and 6- (5,6-dimethylbenzimidazolyl) hexyladenosylcophosphate was obtained in the same manner as in Example 6. Binamidophosphoric acid was obtained.

Example 10 Preparation of 3-imidazolylpropyladenosylcobinamidophosphoric acid In Example 10, 3- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid was used in place of 2- (5,6-dimethylbenzimidazolyl) ethylcyanocobinamidophosphoric acid in Example 6. Using imidazolylpropylcyanocobinamidophosphoric acid, by the same operation as in Example 6,
3-Imidazolylpropyl adenosylcobinamidophosphate was obtained.

[Example 11] coenzyme activity of vitamin B ₁₂ derivative prepared in the measurement examples 1 to 10 of coenzyme activity of vitamin B ₁₂ derivatives were determined by two methods shown below.

1) Measurement of coenzyme activity using 3-methyl-2-benzothiazolinone hydrazone The method of Toraya et al. (J. Biol. Chem., 252, 963-970, 1977)
Year).

Diol dehydrase was used as an enzyme. The enzyme adenosyl cobalamin, i.e. an enzyme and coenzyme vitamin B ₁₂ coenzyme, show no effect in the absence of vitamin B ₁₂ coenzyme (adenosylcobalamin). Diol dehydrase is prepared by the method of Poznanskaya et al. (Arc
h. Biochem. Biophys., 194, pp. 379-386, 1979), and Klebsiella pneumoni ( Klebsiella pneumoni).
ae ATCC 8724) and purified to a 0.3 Unit / ml solution with 0.05M potassium phosphate buffer (pH 8.0). As a substrate for this enzyme, a 1M aqueous solution of 1,2-propanediol was used.

In an ice bath, 0.1 ml of 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
ml of 0.05M potassium phosphate buffer (pH 8.0) were mixed, to which, in the dark, 0.2 mM vitamin B ₁₂ coenzyme (adenosylcobalamin), vitamin B ₁₂ (cyanocobalamin), or Example 1 It was added the vitamin B ₁₂ derivative 0.1ml prepared in 10. After keeping this at 37 ° C. for 10 minutes in the dark, the enzyme reaction was stopped by adding 1 ml of 0.1 M potassium citrate buffer (pH 3.6), and 0.1% of 3-methyl-2-
Add 0.5 ml of benzothiazolinone vidrazone aqueous solution,
The incubation was continued at 37 ° C. for another 15 minutes. To this, add 1 ml of water, and use Shimadzu Double Beam Spectrophotometer UV-140-02 to add 3 ml.
The coenzyme activity (k _cat ) was calculated by measuring the absorbance at 05 nm.

2) Measurement of coenzyme activity using alcohol dehydrogenase and reduced nicotinamide adenine dinucleotide The method of Toraya et al. (Biochemistry, 18, 417-426, 1979)
Year).

The diol dehydrolase used was adjusted to 0.15 Unit / ml with a 0.05M potassium phosphate buffer (pH 8.0), and a 1M 1,2-propanediol aqueous solution was used as the substrate. In addition, with a 0.05 M potassium phosphate buffer (pH 8.0), 0.5 mg / ml of alcohol dehydrogenase and 2 mM
Reduced nicotinamide adenine dinucleotide was prepared. 0.1 ml of a substrate solution, 0.1 ml of an alcohol dehydrogenase solution, 0.1 ml of a reduced nicotinamide adenine dinucleotide solution, and 0.5 ml of a 0.05 M potassium phosphate buffer (pH 8.0) were mixed. Of diol dehydrase was added. After keeping this at 37 ° C. for 5 minutes, in the dark place, 0.2 mM vitamin B ₁₂ coenzyme (adenosylcobalamin), vitamin B ₁₂ (cyanocobalamin), or each vitamin B prepared in Examples 1 to 10 0.1 ml of ₁₂ derivative was added, and using a union spectrophotometer SM-401,
By tracking the decrease in absorbance at 340 nm over time,
Coenzyme activity (k _cat ) was calculated.

Table 1 shows the coenzyme activity (k _cat ) calculated by the above method 1) or 2). A larger k _cat indicates stronger coenzyme activity. Table 1 also shows the relative activity (%) of the vitamin B ₁₂ coenzyme (adenosylcobalamin) with respect to the coenzyme activity.
It was shown to. Vitamin B ₁₂ derivative prepared in Example 6 and Example 9 showed no coenzyme activity.

Example 12 Measurement of Michaelis Constant and / or Inhibition Constant of Vitamin B ₁₂ Derivative In Example 11, vitamin B ₁₂ showing coenzyme activity
For derivatives, the Michaelis constant (K _m ) was determined. That is, in the method 1) of Example 11, by varying the concentration of vitamin B ₁₂ derivatives appropriately, the respective enzyme activity was measured to determine the Michaelis constant (K _m) by Lineweaver-Burk plot. The results are shown in Table 1. K _m affinity with the enzyme the smaller (binding) indicates that strong.

Further, in Example 11, for the vitamin B ₁₂ derivative showed no coenzyme activity was determined inhibition constants (Ki). That is, in the method 1) of Example 11, against the concentration of each vitamin B ₁₂ derivatives of a certain amount, by adding varying concentrations of vitamin B ₁₂ coenzyme (adenosylcobalamin) as appropriate, an enzyme of the time Each activity was measured, and the inhibition constant (Ki) was determined by Lineweaver-Burk plot. The results are shown in Table 1. The smaller the Ki, the stronger the inhibitory activity.

Example 13 Vitamin B ₁₂ derivatives of Escherichia coli (Escherichia coli) growth promotion for 215 activity and growth inhibitory activity E. coli (Escherichia coli) 215 is a vitamin B ₁₂ auxotrophic mutant, seen by Ikeda et al. issued, it is used in bioassays of vitamin B ₁₂ (vitamins, 10, pp. 268-279, 1956). That is, the bacteria can not grow in the absence of vitamin B _12, shows growth vitamin B ₁₂ dependent manner.

Escherichia coli used in this experiment
The culture medium composition of E. coli ) 215 is shown in Table 2.

The medium shown in Table 2, vitamin B ₁₂ (cyanocobalamin), or each vitamin B ₁₂ derivatives synthesized in Examples 1-5, respectively dispensed with varying concentrations in the concentration range of 0.1~100ng / ml, This was steam sterilized at 120 ° C. for 5 minutes. In these media, L-methionine 1.5 μg / ml was previously added.
Escherichia coli 2 cultured on a preculture medium having the same composition as the medium shown in Table 2 containing Escherichia coli
After 15 cells were collected, the cells were thoroughly washed with physiological saline, inoculated, and cultured at 37 ° C. overnight. The turbidity of these cultures was measured using a Shimadzu Double Beam Spectrophotometer UV-140-02.
By measuring at 60 nm, the degree of growth of the bacterial cells was determined. The molar concentration of vitamin B ₁₂ (cyanocobalamin), which gives half the maximum growth, or each vitamin B ₁₂ derivative was defined as growth promoting activity (K _{1/2 (g)} ). The results are shown in Table 3. K
_A smaller _{value of 1/2 (g)} indicates a stronger growth promoting activity. Vitamin B ₁₂ derivative synthesized in Example 1-5,
It did not show growth promoting activity.

Further, vitamin B ₁₂ (cyanocobalamin) was added to the medium shown in Table 2 at a concentration of 0.1 ng / ml, and the vitamin B ₁₂ derivatives synthesized in Examples 1 to 5 were added to The solution was dispensed at different concentrations within a concentration range of 80 ng / ml, and this was steam sterilized at 120 ° C. for 5 minutes. Precultured in the same manner as described above, inoculated with the collected and washed cells, and cultured at 37 ° C. overnight. 660 nm). Also, it performed a similar experiment without addition of vitamin B ₁₂ derivatives provide one-half the growth of the in this case, the vitamin B ₁₂ derivative added molarity growth inhibitory activity Defined. Further, the following equation (V) (Wherein C is vitamin B ₁₂ using (cyanocobalamin)
Are shown. ) Gave as an indication of competitive inhibition activity of vitamin B ₁₂ derivatives were calculated ID _50. The results are shown in Table 3.

Growth inhibitory activity The smaller indicates that strong antagonistic inhibitory activity as ID ₅₀ is small.

Example 14 Vitamin B ₁₂ derivatives Lactobacillus Raihimani (Lactobacillus leichmannii) growth promotion for ATCC 7830 activity and growth inhibitory activity Lactobacillus Raihimani (Lactobacillus leich
Mannii), due Sukeggusu et al, found that a vitamin B ₁₂ requirement (J.Biol.Chem., 184, pp. 211-221
P. 1950), Lactobacillus leichmannii ) ATCC 7830
Vitamin B ₁₂ kit for quantification using are readily available as commercial products. In other words, Raihimani vitamin B ₁₂ for quantification basal medium "Nissui" for the Nissui Pharmaceutical, Lactobacillus Raihimani (Lactobacillus leichmannii) ATCC
A medium was prepared according to the method described in the instruction manual, and vitamin B ₁₂ (cyanocobalamin) or the vitamin B ₁₂ derivative synthesized in Examples 1 to 5 was used in an amount of 0.1 to 100 ng / each. The solution was dispensed at different concentrations within the ml concentration range and sterilized by steaming at 120 ° C. for 5 minutes. These media are inoculated with bacterial cells according to a predetermined method, and 37
The culture was allowed to stand at room temperature overnight. The growth of the cells was measured in the same manner as described in Example 13, and the growth promoting activity (K
_{1/2 (g)} ). The results are shown in Table 4. Against this bacterium, vitamin B ₁₂ derivatives synthesized in Examples 2-4 showed growth promoting activity but was very weak. Also, vitamin B ₁₂ derivatives synthesized in Examples 1 and 5 showed no growth promoting activity.

In addition, vitamin B ₁₂ (cyanocobalamin) was added to the above basal medium to a concentration of 0.05 ng / ml, and the vitamin B ₁₂ derivatives synthesized in Examples 1 to 5 were further changed in concentration as appropriate. And add it for 5 minutes
Steam sterilization at 120 ° C. The cells were inoculated into these mediums according to a predetermined method, and cultured at 37 ° C. overnight, and the growth of the cells was measured in the same manner as described in Example 13. Also, it performed a similar experiment without addition of vitamin B ₁₂ derivative according to the method described in Example 13, growth inhibitory activity And antagonistic inhibitory activity (ID ₅₀ ). Table 4 shows the results.

Example 15 Vitamin B ₁₂ Derivative, Mouse Leukemia L ₁₂₁₀
Murine leukemia L ₁₂₁₀ cells used in in vitro growth inhibitory activity present experiments on cells, those grown and maintained in the peritoneal cavity of DBA mice aseptically removed with ascites, fetal calf serum (5v / v%) and ethanolamine (1.2 mg / l) and cultured from RPMI-1640 medium for animal cell culture adapted to the medium to remove the vitamin B ₁₂ (cyanocobalamin) containing. In addition, 100,000 units of penicillin and 100 m of streptomycin as antibacterial agents
g was added. L ₁₂₁₀ cells grown adapted in In vitro yet Fujii et al's method (J.Biol.Chem., 256, pp. 10329-10334,
1981), the medium was adapted to the above-mentioned medium containing bovine serum albumin at a concentration of 7.0 g / l instead of fetal bovine serum, and then subjected to experiments.

Examples 2 and the purpose of examining the L ₁₂₁₀ growth inhibitory activity of the synthesized vitamin B ₁₂ derivative in 5, first ⁴ 5 × 10 in the same medium containing L ₁₂₁₀ cells adapted to albumin folic acid 10μM concentration cells / Inoculate at a density of ml and wet C
Preculture was performed at 37 ° C. for 3 days under O ₂ / air (5% / 95%). After the grown cells were centrifuged, they were washed twice with the same medium containing no folate to prepare an inoculated cell suspension of about 5 × 10 ⁶ cells / ml. The main culture was performed by adding 10 μl of 5-methyltetrahydrofolate instead of folic acid to the above basic medium containing albumin.
M and vitamin B ₁₂ (cyanocobalamin) were added to a concentration of 0.5 nM, and the washed cells were inoculated at a density of 5 × 10 ⁴ cells / ml, and the culture was continued for up to 9 days under the above conditions. L of vitamin B ₁₂ derivative synthesized in Examples 2 and 5
In ₁₂₁₀ during cell growth inhibitory activity present culture, methotrexate was added for the purpose of amplifying the vitamin B ₁₂ requirement (MT
X) 200 nM and vitamin B ₁₂ derivative of Example 2 (50 nM), vitamin B ₁₂ derivative of example 5 (50 nM, was investigated by the coexistence respectively 5000 nM). MTX, and results of growth inhibitory activity against murine leukemia L ₁₂₁₀ in the case of vitamin B ₁₂ derivatives presence revealed by measurement of cell density of 3, 5, 7 and 9 days by Coulter instrument MTX and the present invention Is shown in FIG. Incidentally, it is shown in Figure 1 at the same time growth measurement results of both cases no addition of vitamin B ₁₂ derivatives of MTX and the present invention as a control example (Control). From FIG. 1, it is clear that the compound of the present invention has a growth inhibitory effect on _L1210 .

9 day cultured cells shown in FIG. 1 - a (in the figure, × vitamin B ₁₂ derivatives of the present invention obtained in Example 5 indicated by × (5000 nM) added to those cultured) trypan for comparison in the same viability determined by staining with blue, in the case of MTX added alone whereas only 20% of the cells are killed; coexisted vitamin B ₁₂ derivative of example 5 with MTX In some cases 90% of the cells were dead.

[Brief description of the drawings]

Figure 1 is a vitamin B ₁₂ derivatives of the present invention, showing the in vitro growth inhibitory activity against murine leukemia L ₁₂₁₀ cells. In the figure, ●-● indicates a control, and ○-○ indicates MT.
X (200nM), △ ... △ is MTX (200 nM) and vitamin B ₁₂ derivative obtained in Example 2 (50nM), ● ... ● , × - × is, MTX
They are each 50 nM, the results obtained by adding 5000nM of vitamin B ₁₂ derivative obtained in Example 5 (200 nM).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 1/20 C12N 1/20 A // (C12N 1/20 C12R 1:01) (72) Inventor Katsuhiko Fujii Hino-shi, Tokyo 4-3-2 Asahigaoka Tokyo Research Center, Teijin Limited (56) References JP-A-54-115375 (JP, A) JP-A-61-85388 (JP, A) JP-A-52-31091 (JP) U.S. Pat. No. 4,531,004 (US, A) U.S. Pat. No. 4,386,079 (US, A) Hoppe-Seylers Z .; Physiol Chem. (1981), Vol. 362, no. 8, pages 1079-1090 (58) Fields investigated (Int. Cl. ⁶ , DB name) C07F 9/00-19/00 CA (STN) REGISTRY (STN)

Claims (11)

(57) [Claims] 1. The compound of the general formula (I) (Wherein L is a cyano group, a substituted or unsubstituted adenosyl group or an adenylylalkyl group, a hydroxyl group,
One or two identical or different ligands selected from a water molecule and a linear or branched alkyl group having 1 to 8 carbon atoms, B represents a base having a heterocyclic structure,
Represents a substituted or unsubstituted hydrocarbon. Vitamin B ₁₂ derivative represented by). 2. The method according to claim 1, wherein R is a linear or branched alkylene group having 1 to 8 carbon atoms or a cyclic hydrocarbon group which is substituted or unsubstituted with an aromatic group or a halogen atom. vitamin B ₁₂ derivatives. Wherein R is selected from the group consisting of vitamin B ₁₂ derivative according to claim 1, wherein a linear alkylene group having 1 to 8 carbon atoms. Wherein B is a substituted or unsubstituted, vitamin B ₁₂ derivative according to claim 1 wherein the imidazole group or pyridine group. 5. A compound of the formula (I) starting from cyano aquacobinamide or dicyanocobinamide. (Wherein L is a cyano group, a substituted or unsubstituted adenosyl group or an adenylylalkyl group, a hydroxyl group,
One or two identical or different ligands selected from a water molecule and a linear or branched alkyl group having 1 to 8 carbon atoms, B represents a base having a heterocyclic structure,
Represents a substituted or unsubstituted hydrocarbon. In vitamin production method B ₁₂ derivative represented by), cyano Aqua cobinamide or dicyano cobinamide and the following formula (II) B-R-OPO 3 H 2 (II) ( wherein, B and R are the formula ( a phosphoric acid ester derivative represented by the same.) to the definition of I) is subjected to condensation reaction, that in the formula (I) L to produce a vitamin B ₁₂ derivative is a cyano group, wherein obtained in, by the above formula (I) L is subjected to reduction of vitamin B ₁₂ derivative is a cyano group, followed by a) or subjected to an oxidation reaction, b) to halo decomposed and alkylated, the L is a hydroxyl group in the formula (I), or the production of vitamin B ₁₂ derivative is a water molecule, or obtained the or at, L in the formula (I)
There cyano group, a hydroxyl group or a vitamin B ₁₂ derivative is a water molecule subjected to reduction, and then the alkane or halogenated or tosylated a linear or branched C1-8 halogenated By reacting with a substituted or unsubstituted adenosine or adenylyl alkane, L in the above formula (I) has 1 to 1 carbon atoms.
Straight or branched chain alkyl group having 8, the manufacturing method of the vitamin B ₁₂ derivative, characterized in that, to produce a vitamin B ₁₂ derivative is a substituted or unsubstituted adenosyl group or adenylyltransferase alkyl group. Wherein B is a substituted or unsubstituted, a manufacturing method of the vitamin B ₁₂ derivative according to claim 5 wherein the imidazole group or pyridine group. 7. The method according to claim 5, wherein R is a linear or branched alkylene group having 1 to 8 carbon atoms or a cyclic hydrocarbon group which is substituted or unsubstituted with an aromatic group or a halogen atom. method for producing a vitamin B ₁₂ derivatives. 8. Vitamin B ₁₂ antagonist containing claim 1 Vitamin B ₁₂ derivative or a pharmaceutically acceptable salt thereof represented by the general formula (I) as an active ingredient. 9. The method of claim 1 formula vitamin B ₁₂ derivatives or cytostatic or inhibitor containing as an active ingredient a pharmaceutically acceptable salt thereof represented by the general formula (I) according. 10. antitumor agent containing claim 1 Vitamin B ₁₂ derivative or a pharmaceutically acceptable salt thereof represented by the general formula (I) as an active ingredient. 11. Using the vitamin B ₁₂ derivative represented by the general formula of claim 1 wherein (I), methods for the screening of vitamin B ₁₂ high producing microorganism mutants.